£)i[iiiiiliiliiiiiiiiiiirjiii ItlllllllllllllllflFlllllllllJIII lUllllllllllllllltllllllllll' LIBRARY NEW YORK STATE VETERINARY COLLEGE ITHACA, N. Y. ^iiiiiprrrriiiLiiiiiiiniiiiiiMiiiiiiitiiiiiiiiiiiiiiNJiiJiHiiiiiiiiiiittiiiiiiiiiiJjiiiiimiii iiiiiiJiiiiiriiiiiiiJiiMiiiii Hllltl llllllllllllllllillFI The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000277016 VETERINARY BACTERIOLOGY A TREATISE ON THE BACTERIA. YEASTS, MOLDS, AND PROTOZOA PATHOGENIC FOR DOMESTIC ANIMALS BY ROBERT EARLE BUCHANAN, Ph.D. PROFESSOR OF BACTERIOLOGY IN THE IOWA STATE COLLEGE OF AGRICITLTURE AND MECHANIC ARTS; f BACTERIOLOGIST OF THE IOWA AGRICULTURAL EXPERIMENT STATION AND CHARLES MURRAY, B. Sc, D. V. M. PROFESSOR OF VETERINARY RESEARCH, IOWA STATE COLLEGE THIRD EDITION THOROUGHLY REVISED PHILADELPHIA AND LONDON ■' W. B. SAUNDERS COMPANY 1922 Hn s. Copyright, igii, by W. B. Saunders Company. Revised, reprinted, and recopyrighted September, 1916. Reprinted July, 1917. Revised, reprinted, and recopyrighted January, 1922. Copyright, 1922, by W. B. Saunders Company PREFACE TO THE THIRD EDITION A SECOND revision of the Veterinary Bacteriology has been necessitated by the advances in this field in the last several years. A more adequate discussion of the classification and re- lationships of microorganisms has been included. The newer conceptions of acidity on the basis of hydrogen ion concentra- tion has received increased emphasis. Several of the chapters on specific pathogenic bacteria have been completely rewritten, and others extensively revised. R. E. Buchanan. Charles Murray. Iowa State College, Ames, Iowa. January, 1922. PREFACE The present volume is a revision of the lectures on veterinary bacteriology given during the past six years to classes in the Division of Veterinary Medicine in the Iowa State College. It constitutes a serious attempt to put in usable form that fund of knowledge concerning bacteriology which the student of veterinary medicine should master. It is in no sense a text on pathology, and discus- sion of purely pathological subjects has been minimized as much as possible. The intention has been to confine attention as far as practicable to those topics that unquestionably lie in the province of bacteriology. This has been defined to include a discussion of immunity and of the pathogenic bacteria, yeasts, molds, and protozoa. The book is not intended to serve as a manual of laboratory practice, hence detailed discussion of methods and technic has been omitted. Methods of significance in diagnosis or treatment are given in greater detail in the discussion of specific organisms. Several organisms causing diseases of man not transmissible to lower animals have been included. In all cases they are closely related to organisms having significance to the veterinarian, they cause diseases which are commonly confused with somewhat similar diseases of lower animals, or they are valuable as illustrations of methods of immunization, treatment, or diagnosis. Such organisms are relatively few in number. A group system of discussion of the pathogenic bacteria has been adopted. The classification used has proved very helpful in my own classwork. The groupings used are not entirely satis- factory, in part due to the fact that some of the species have not been adequately described and differentiated in the literature. An effort has been made to point out the deficiencies in our present knowledge, both to give a better balanced presentation of the sub- ject and to stimulate interest in the solution of the problems. 9 10 PREFACE The pathogenic protozoa constitute a group which is particu- larly difficult to treat adequately, largely due to the rapid growth of the subject. Relatively few of the forms, moreover, are of immediate interest to the North American studer^t. R. E. Buchanan. Iowa State College, Ames, Iowa. CONTENTS SECTION I MORPHOLOGY, PHYSIOLOGY, AND CLASSIFICATION OF BACTERIA PAGE Chapter I. — Introduction 17 Definition of Bacteriology, 17. — Scope of Baoteriology,17. — Scope of Veterinary Bacteriology, 19. — The Microscope and Ita Influence, 19. — Nature and Classi- fication of Microorganisms , 21. — Spontaneous Generation, 21. — Relationships of Microorganisms to Fermentation and Decay, 22. — Belationships of Microor- ganisms to Disease, 22. — Development of Laboratory Methods, 23. — Develop- ment of Theories of Immunity, 24. — Development of Sanitary Science and Pre ventive Medicine, 24. Chapter II. — Morphology and Relationships op Microorganisms Concerned in Disease Production 26 Position of Pathogenic Microorganisms, 26. — Differentiation of Animals and Plants, 26. — Subdivisions of the Thallophytes, 27. — Morphology of Bacteria, 28. — Shape of Bacteria, 28. — Grouping of Bacterial Cells, 29. — Size of Bacteria, 31. — Histology and Structure of Bacteria, 32. — Reproduction in Bacteria, 36. — Morphology of the Yeasts, Saccharomycetes, and Blastomycetes, 38.— Form, Size, and Grouping of Yeasts, 39. — Histology and Structure of the Yeasts, 39. —Yeast Protoplasm and Cell Inclusions, 39. — Reproduction in Yeasts, 40. — Morphology oj the Hyphomycetes or Molds, 41. — Form and Size of Hyphomy- cetes or Molds, 41. — -Histology and Structure of Molds, 42. — Reproduction of Molds, 43. — Morphology of the Protozoa, 44. — Form and Size of Protozoa, 45. — Histology, 45. — Reproduction, 45. Chapter III. — Physiology op Microorganisms 46 Food Relationships of Microorganisms, 46. — Composition of the Cell, 46. — Sources and Kinds of Foods, 46. — Moisture Relationships of Microorganisms, 47. — Respiration of Microorganisms, 48. — Growth Rates and Death Rates, 49. — Rates of Growth, 49. — Rates of Death, 60. — Temperature Relations of Miaro- organisms, 51. — Optimum Temperature, 51. — Minimum Temperature, 52. — Maximum Growth Temperature, 52. — Growth Temperature Range, 52. — Thermal Death-point, 52. — Light Relationships of Microorganisms, 53. — Effect of Electricity on Bacteria, 54. — Relationships of Microorganisms to Chemicals. 54. — Chemotaxy, 55. — Tropisms, 56. — Influence of Reaction of Medium on Growth, 56; — Antiseptics and Disinfectants, S6. — Theories of Action of Anti- septics and Disinfectants, 57. — ^Characteristics of an Ideal Disinfectant, 57. — Disinfectants and Antiseptics in Common Use, 59. — -Alkalies, 60.— Salts of the Heavy Metals, 60. — Adjustment of Organisms to Osmotic Pressure, 62. — Syr»- biosis, Antibiosis, and Commensalism, 63. — Pigment Production by Microorgarv- isms, 63. — Light Production by Microorganisms, 64. — Fermentation and Enzyme Production, 64. Chapter IV. — Changes of Economic Significance Brought About BY Non-pathogenic Organisms 68 Production of Alcohol, 69. — Production of Acids, 69. — Decay and Putrefac- tion, 71. — Reduction Processes in Inorganic Compounds, 73. — Oxidation of Inorganic Compounds, 74. — Miscellaneous Changes, 81. Chapter V. — Classification of Microorganisms. 82 Discussion of Principles of Nomenclature, 82.— Classification of Bacteria, 83. — Order Eubacteriales. The True Bacteria, 84. — Key to the Famihes of the Eubacteriales, 85. — The Family Coccacese. The Spherical Bacteria, 85.— Key to the Genera of the Coocacese, 85. — The family Bacillacese. The Spore Bearing Bacteria, 87. — The Family Bacteriacese, 88. — Key to the Genera of Bacteriacea, 88. — The Family Pseudomonadacese, 89. — The Family Spiril- laoeffi, 89. — Order Actinomyoetales. The Thread Bacteria, 90. — Key to the Genera of Actinomycetales, 90. — ^Order Spirochsetales, 92. — Classification of Yeasts, 93. — Classification of the Molds, 93. 11 12 CONTENTS SECTION II LABORATORY METHODS. AND TECHNIC FAOS Chapter VI. — Sterilization 94 Sterilization by the Flame, 94. — Sterilization by Hot Air, 94. ^Sterilization by Streaming Steam, 95. — Sterilization by Steam Under Pressure, 96. — -Steriliza- tion at Temperatures Lower than BoiUng-point, 98. — Sterilization by Addition of Chemicals, 98. — Sterilization by Filtration, 98. Chapter VII. — Cttlture-media and Their Preparation 100 Adjustment of the Reaction of Media, 100. — Nature of Nutrients Required by Bacteria, 104. — Liquid Media, 104. — Bouillon or Beef Broth from Meat, 104. — Bouillon or Broth from Beef Extract, 105. — Sugar-free Broth, 105. — Sugar Broth, 105. — Glycerin Broth, 105. — Serum Broth, 105. — Dunham's Solution, 105. — Beerwort, 105. — Milk, 105. — Synthetic Media, 106. — Liquefiable Solid Media.lOQ. — Nutrient Gelatin,106. — Other Gelatin Media,106. — NutrientAgar 107. — Blood-serum Agar, 107. — Chocolate Agar, 107. — Hormone Agar, 107. — Other Agar Media, 108. — Endo-agar-fuchsin Medium, 108. — Simplified Endo Agar, 108. — Eosin-methylene-blue Agar, 109. — Non-Liquefiable Media, 110. — Potato, 110. — Other Vegetable Media, 110. — Blood-serum, 110. — Egg Medium, 111. Chapter VIII. — Biochemical Tests 112 Acid and Alkali Production. Determination of Changes in Hydrogen Ion Concentration, 112. — Determination of Acid Production, 113. — Gas Produc- tion, 114. — Reduction Processes, 115. — Alcohol Production, 116. — Aldehyde, 116. — Acetyl Methyl Carbinol, 117. — Determination of Oxygen Relationships, 117. — Indol Production, 118.— Thermal Death-point, 118. — Eflaciency of Dis- infectants, 119. — Phenol Coefficient, 119. Chapter IX. — Microscopic Examination and Staining Methods. . 121 Oil Immersion Objectives, 121. — Measuring Bacteria, 122. — Examination of Living Bacteria — Hanging Drops, 122. — Staining Methods, 123. — Mordants, 123. — Formulas of Some of the Commonly Used Stains, 124. — Preparation of a Stained Mount, 124. — Spore Stain, .125. — Stain for Acid-fast (.Acid-proof) Organisms, 126. — Wirth's Stain for Much Granules, 126. — Flagella Stain, 127. — Gram's Staining Method, 128. — Stabilized Gentian Violet, 128. — Capsule Stains, 128. — Muir's Stain, 128.^Johne's Stain, 129. — Raebiger's Stain, 129. — Blood and Protozoan Stains, 129. — Wright's Stain, 129. — Giemsa's Stain, 130. — Negri Bodies, 130. — Lentz Method, 130. — India-ink Method, 131. Chapter X. — Methods of Securing Pure Cultures of Bacteria. . 132 Dilution Method, 132.— Isolation by Smearing, 132. — Direct Isolation, 132. — Isolation by Plating, 133. — Isolation by the Use of Heat, 134. — Isolation by the Use of Differential Antiseptics or Disinfectants, 134. — Isolation by Animal Inoculation, 134. Chapter XL — Study op Bacterial Cultures 135 Cultural Characters, 135. — Agar Stroke, 135. — Potato, 135. — Blood-serum, 136. — Gelatin Stab, 136. — Nutrient Broth, 136.^Milk, 136. — ^Litmus Milk, 137. — Gelatin Plate Colonies, 137. — Colonies on Agar Plates, 138. — Physiologic Characters, 140. SECTION ill BACTERIA AND THE RESISTANCE OP THE ANIMAL BODY TO DISEASE Chapter XII. — Microorganisms and Disease 141 Infectious Diseases, 141. — Contagious Diseases, 142. — -Avenues of Infection, 142. — Virulence, 144. — Koch's Postulates, 144. — Animal Inoculation, 145. — Methods of Animal Inoculation, 146. — Types of Infectious Diseases, 147. Chapter XIII. — Immunitt. General Discussion 149 Immunity, 149. — External Resistance to Infection, 149. — Variations of Indi- viduals in Susceptibility to Disease. Predisposing Factors, 150. — Types of Im- munity, 150. — Natural Immunity, 150. — Acquired Immunity, 151. — Active Acquired Immunity, 151. — Acquired Passive Immunity, 153. — Theories of Im- munity, 153. — Theory of Exhaustion, 153. — Noxious Retention Theory, 154. — Metchnikoft's Theory of Phagocytosis, 154. — Ehrlich's Humoral Theory, 154. — Duration of Immunity, 155. — Antigens and Antibodies, 155. — Antibodies as Factors in Acquired Immunity, 155. CONTENTS 13 PAGE Chapter XIV. — Antitoxins and Related Antibodies 157 Antibodies of Ehrlich's First Order, 157. — Toxins, 157. — Antitoxins, 159. — Constitution o£ the Toxin, 162. — Constitution of Antitoxin, 162, — Diagram- matic Representation of Toxin and Antitoxins, 163. — Preferential Union of Toxins with Bodj^-oells, 163. — Antitoxins of Commercial Importance, 164. — Manufacture of Diphtheria Toxin and Antitoxin, 164. — Preparation of Tetanus Toxin and Antitoxin, 171. — Preparation of Other Toxins and Antitoxins, 173. Chapter XV. — Agglutination and Precipitation 174 Antibodies of Ekrlich*s Second Order, 174. — Differentiation of Precipitation and Agglutination, 174. — Agglutination, 174. — Precipitins, 182. Chapter XVI. — Cytolysins, Including Bacteriolysins, and Hemol- ysins 185 Antibodies of Ehrlich's Third Order, 185. — Cytolysins, 185. — Group Cytolysins, 187. — Bacteriolysins, 188. — Hemolysins, 189. — Fixation of Complement and Its Utilization, 190. — Cytotoxins, 192. — Conglutination, 192. Chapter XVII. — Opsonins and Phagocytosis 194 Opsonins, 195. — Opsonic Index, 197. — Autogenic Vaccines, 201. — Passive Op- sonic Immunization, 203, — Leukocytic Extracts, 203. — Aggressins, 203. — Sen- sitized Vaccines, 204. Chapter XVIII. — Anaphylaxis and Hypersusceptibility 206 Phenomenon of Arthus, 206. — Serum Sickness in Man, 206. — Theobald Smith Phenomenon, 207. — Vaughn's Split Proteins, 207, — Mechanism of Anaphy- laxis, 207. — Relationship of Anaphylaxis to Certain Body Reactions, 210. — Bacterial Anaphylaxis, 210. SECTION IV PATHOGENIC MICROORGANISMS EXCLUSIVE OF THE PROTOZOA Chapter XIX. — Groups op Pathogenic Microorganisms 212 Chapter XX. — The Group op Streptococci. The Genus Strepto- coccus 214 Classification of Streptococci. 214. — Holman's Classification of Species of Streptococci, 216. — Streptococcus Pyogenes, 216.— Streptococcus Equi, 224. — Streptococcus Gallinarum, 227. — Streptococcus Vaginitidis, 229. — Strepto- coccus Mastiditis Sporadicse, 231. — Streptococcus Sp., 231. — Streptococcus Lacticus, 231. Chapter XXI. — The Group op Specific Pyogenic Cocci. The Genera Diplococcus and Neisseria , . 235 Diplococcus Pneumoniae, 235 — Neisseria Meningitidis, 238. — Neisseria Intra- cellularis Equi, 241. — Neisseria Gonorrhceae, 241. Chapter XXII. — Staphylococcus Group 244 Staphylococcus Aureus. 244. — Staphylococcus Albus, 248. — Staphylococcus Citreus, 249. — Staphylococcus tPyogenes) Bovis, 249. — Staphylococcus Asco- formans, 249. Chapter XXIII.— Anthrax Group. The Genus Bacillus 252 The Group of ASrobic Spore-producing Bacilli, 252. — Bacillus Anthracis, 253. Chapter XXIV.— Blackleg — Tetanus Group. The Genus Clos- tridium 264 Key to the More Important Pathogenic Members of the Tetanus-Blackleg Group of Bacteria, 265. — Clostridium Tetani, 265. — Clostridium Chauvsei, 271. . Clostridium Gastromycosis Ovis. 277.— Clostridium Welchu, 278,— Clostridium (Edematis, 282, — Clostridium of Ghon-Sachs, 285. — Clostridium Sporogenes Metchnikoff, 286. — Clostridium Botulinum, 286. Chapter XXV — Pasteurella, or Hemorrhagic Septicemia Group 290 Common Characters of Group, 291..— Pasteurella Cholera Ganinarum, 293 — Suisepticus, 297. — Pasteurella Bovisepticus, 301. — Pasteurella Cunicuhcida, 302. — Pasteurella Pestis, 302, Chapter XXVI.— Glanders Group. The Genus Pfeifferella. . 306 PfeifiFerella Mallei, 306. — Bacilli of Selter, Babes, and Kutscher, 317. 14 CONTENTS FA.GE Chapter XXVII. — Intestinal or Colon-typhoid Group. The Genus Bacterium 318 Subgroup I. — Colon Subgroup, 319. — Bacterium Coli, 321. — Bacterium Acidi Lactici, 325. — Bacterium Communior, 325. — Bacterium Coscoroba,325. — Bac- terium Aerogenes, 325. — Bacterium Cloacse, 327. — Organisms Associated with Calf Scours or Calf Diarrhea, 327. — Capsulated Pathogenic Organisms, 328. — Bacterium Pneumonise, 329. Subgroup II. — Intermediate Hog-cholera, Enteriiidis, or G&rtner Subgroup, 330. — Bacterium Enteritidis, 330. — Bacterium Cholerse Suis, 333. — Bacterium Typhi Suis, 337. — Bacterium Para-typhi, 338. — Bacterium Typhi Murium, 339. — Bacterium Pullorum, 340. Subgroup 111. — Typhoid — Dysentery Subgroup, 342. — Bacterium Typhosum, 342. — Bacterium Dysenterise, 345. Chapter XXVIII. — Abortion — Malta Fever Group. The Genus Brucella 349 Brucella (Bacterium) Abortus, 349. — Brucella (Bacterium) Melitensis, 353. Chapter XXIX. — Dog Distemper Group. The Genus Bacterium. 356 Bacterium Bronchisepticum l,Bronchicanis), 356. Chapter XXX. — Fluorescent Group. The Genus Pseudomonas. 358 Pseudomonas Pyocyanea, 358. Chapter XXXI. — Diphtheria-pseudotuberculosis Group. The Genus Corner acterium 361 Cornebacterium Pseudotuberculosis, 362. — Cornebacterium Diphtheria, 367. — Cornebacterium Hofifmanni, 372, — Cornebacterium Xerosis, 372. Chapter XXXII. — Swine Erysipelas Group. The Genus Erysi- pelothrix 373 Erysipelothrix Rhusiopathise, 373. — Erysipelothrix Muriaeptica, 377. Chapter XXXIII. — Hemophilic, or Influenza Group. The Genus Hemophilus 379 Hemophilus Pyogenes, 379. — Hemophilus InfluenzEe, 383. — Hemophilus Per- tussis, 384. Chapter XXXIV. — Acid-fast Group. The Genus Mycobacterium 385 Mycobacterium Tuberculosis, 385. — Mycobacterium Paratuberculosis, 406. — Mycobacterium Leprae, 407. — -Non-pathogenic Acid-fast Bacteria, 408. Chapter XXXV. — Vibrio Group 410 Vibrio Metchnikovi, 410. — Vibrio Cholerse. 412. — Vibrio Fetus, 414. — Non- pathogenic Spirilla, 416. Chapter XXXVI. — Spirochete Group. 417 Cultivation of Spirochetes, 420.^ — Classification of Spirochetes, 420. — Spiro- chaeta Obermeieri, 421, — Spirochaeta Duttoni, 423. — Spirochaeta Kochi, 425. — Spirochaeta Anserina (or gallinarum), 425. — Spirochaeta Theileri, 427. — Spiro- chaeta Pallida, 428. — Spirochseta Pertenuis, 431. — Other Spirochetes, 431. Chapter XXXVII. — Actinomyces Group 432 Classification of Actinomyces, 432. — Actinomyces Bovis, 434. — Actinomyces Nocardii, 437. — Actinomyces Caprae, 439. — Actinoniyces Necrophorus, 440. — Actinomyces Madurse, 445. — Actinomyces of Other Infections, 445. Chapter XXXVIII. — Blastomycetes. 446 Blastomyces Farciminosus, 447. — Blastomyces Dermatitidis, 449. — Blasto- myces Coccidioides, 452. Chapter XXXIX. — Mold or Hyphomycete Group. 454 Aspergillus, 455. — Aspergillus Fumigatus, 457, — Aspergillus Flavus, 460. — Aspergillus Niger, 461. — Other Species of Aspergilli, 462. — Penicillium, 462. — ■ Fusarium, 463. — Fusarium Equinum, 464. — Sporotrichum, 466. — Sporotrich- um Beurmanni, 466. — Trichophyton, 470. — Trichophyton Granulosum, 472. — Trichophyton Felineum, 472. — Trichophyton Equinum, 472. — Trichophyton Caninum, 473. — Microsporum, 473. — Microsporum Lanosum, 474. — Micro- sporum Felineum, 474. — Microsporum Equinum, 475. — Achorion, 475. — Acho- rion Muris, 475. — Achorion Gallinse, 475. — Oidium Albicans, 477. CONTENTS 15 SECTION V PATHOGENIC PROTOZOA Chapter XL. — Structure, Relationships, and Classification op THE Protozoa 478 Structure of the Protozoa, 478, — Claaaification of the Protozoa, 480. Chapter XLI. — Pathogenic Protozoa of the Flagellata (Exclu- sn^E op the Spirochetes) 481 The Genus Trypanosoma, 418. — Morphology, 482. — Cultivation of Trypano- somes, 484. — Method of Disease Production, 484. — Examination and Staining Methods, 484. — Trypanosoma Equiperdum, 485. — Trypanosoma Evansi, 487. — Trypanosoma Brucei, 488. — Trypanosoma Equinum, 490. — Trypanosoma Dimorphon, 492. — Trypanosoma Conelense, 492. — Trypanosoma Pecaudi, 49,'!. — Trypanosoma Cazalboui, 494. — Trypanosoma Theileri, 495. — Trypanosoma Gambiense, 495. — Trypanosoma I.ewisi, 496. — Trypanosoma Hippicum, 497. — rAe Genus Herpetomonas, 498. — Herpetomonas Donovani, 498. — Leishmania ^Herpetomonas?) Infantum, 409. — Leishmania Tropica, 499. Chapter XLII. — Pathogenic Protozoa of the Class Rhizopoda. . 500 The Genus Entamceba, 501 . — Staining Methods, 502. — Methods of Isolation and Cultivation, 502. — Entamceba Coli, 503. — Entamceba Histolytica, 504, — Enta- moeba Tetragena, 507. Chapter XLIII. — Sporozoa 508 Tlie Genus Piroplasma, or Babesia, 509. — Babesia Bigemina, 509. — Babesia Mutans, 511. — Babesia Eciui, 511. — ^Babesia Ovis, 512. — Babesia Canis, 512. — Babesia (Piroplasma) Gibsoni, 514. — Babesia (Piroplasma) Commune, 514. — Theileria, 516. — Theileria Parva, 516. — The Genus Plasmodium, 516. — Plas- modium Vivax, 516. — Plasmodium Malarice, 518. — Plaamodiuin Immacula- tumand Falciparum, .518. — The Genera Proteosoma, Halt&ridium, and Hemopro- teus, 519. — TheGenus Anavlasma. 519. — Anaplasma Marginale, 519. — TheGen- us Leiikocytozoon, 521. — Genus Saroooystis, 521. — The Genus Eimeria yCocci- dium), 522.^Eimeria Avium, 523. — Eimeria Stiedffl, 525. — Eimeria Bovis, 525. — Eimeria Faurei, 526. — Isospora Bigemina, 527. Chapter XLIV. — Parasitic Protozoa of the Ciliata 528 Protozoan Commensals of the Rumen and Cecum, 528. — Balantidium Coli, 531. SECTION VI INFECTIOUS DISEASES IN WHICH THE SPECIFIC CAUSE IS NOT CERTAINLY KNOWN Chapter XLV. — Diseases Produced by Unknown Organisms 533 Bacterial or Protozoan Relationships of Ultramicroscopic Organisms, 534. — Virus^of Pleuropneumonia, 635. — Virus of Foot-and-mouth Disease, 536. — Virus of Rinderpest or Cattle Plague, 537. — Virus of Hog-cholera, 539. — Virus of Horse Sickness, 514. — Virus of Infectious Anemia of the Horse, 541. — Virus of Dog Distemper, 542. — Virus of Fowl Plague, 543. — Virus of Epithelioma Contagiosum. 544. — Virus of the Poxes, 545. — Virus of Yellow Fever, 546. — ■ Virus of Rabies, 546. — Virus of Infectious Agalactia of Sheep and Goats, 550. — Virus of Guinea-pig Plague, 551. — Virus of Fowl Leukemia, 551. — Virus of Cer- tain Chicken Tumors, 551. SECTION VII BACTERIA OF WATER AND FOOD Chapter XLVI. — Bacteria op Water and Water Purification. . . 552 Water Purification, 557. Chapter XLVII. — Milk. Its Constituents, Contamination, and Examination 561 Influences Which Determine the Ultimate Bacterial Content, 567. — Standards for Production and Distribution of Certified Milk, 569. — rHygiene of the Dairy Under the Supervision and Control of the Veterinarian, 570. — Transportation, 575. — Veterinary Supervision of the Herd, 575. — Bacteriologic Standards, 577. Bibliographic Index 579 Index 585 VETERINARY BACTERIOLOGY SECTION I MORPHOLOGY, PHYSIOLOGY, AND CLASSIFICATION OF BACTERIA CHAPTER I INTRODUCTION Bacteriology is commonly defined to include a considera- tion of three distinct groups of minute living organisms: the true bacteria, the molds, and the pathogenic protozoa. Some authorities have objected to the inclusion of a discussion of forms other than the true bacteria under the heading of bacteriology, and have proposed that the term microbiology be used instead. In accord- ance with this latter conception, the term microbe includes the bacteria, the molds, and the protozoa, and the science of microbiology includes the corresponding subdivisions, bacteriology, mycology, and protozoology. As most commonly used, however, the term "bacteriology" is regarded as a synonym of "microbiology," and in this text this usage will be followed. The reasons for including the bacteria, the molds (particularly those pathogenic to animals), and the pathogenic protozoa under bacteriology may be stated briefly as follows: All three groups must be studied by very similar methods. AH three contain or- ganisms which are capable of bringing about pathologic conditions in man and animals. The microscopic unicellular animals, known as protozoa, are known to produce disease in some cases, and have been studied largely by means of the laboratory technic developed 2 17 18 VETERINARY BACTERIOLOGY by the bacteriologist. The boundaries between the groups of the bacteria on the one hand and protozoa on the other are like- wise far from distinct. In many cases it would prove impossible from a clinical examination alone of a new disease to determine whether it is caused by the invasion of true bacteria or of protozoa. It is evident that there are many practical difficulties in differen- tiating the bacteria and protozoa. Furthermore, the line of demarcation between the bacteria and fungi (including the molds, mildews, smuts, rusts, toadstools, puffballs, etc.) is very poorly defined. Particularly is it easy to find all intergradations between the bacteria and those groups of fungi commonly known as yeasts and molds. It appears, there- fore, that because of difficulties in differentiation and because of the relationships indicated that a consideration of bacteria, yeasts, molds, and protozoa may all be included under the title of bac- teriology. Parasitology is that branch of science commonly defined to in- clude a discussion of animal parasites, such as the mites, lice, nematodes, and similar types. Sometimes it is held to include con- sideration of the pathogenic protozoa and the parasitic molds. To this extent the fields of bacteriology and parasitology would therefore overlap. Although bacteriology is one of the youngest of the sciences, nevertheless it has grown so rapidly that many divisions and sub- divisions have come to be recognized. Medical bacteriology may be defined as that branch of bac- teriology which treats of those microorganisms (including the true bacteria,. molds, and protozoa) that produce disease in the animal body or are related directly or indirectly to the maintenance of health. Agricultural bacteriology, with its subdivisions — soil bac- teriology, dairy bacteriology, and plant bacteriology — includes a con- sideration of all organisms of importance in soil fertility, in pro- duction and distribution of dairy products, in disease causation in plants, and to a certain degree in domestic animals. Sanitary bacteriology has for its field the methods of disease prevention based upon the knowledge of the organisms causing disease and the man- ner in which they spread. Systematic bacteriology discusses the classification and relationships of organisms. Immunology con- INTRODUCTION 19 siders the resistance and susceptibility of the body to disease and the means which may be used to increase such resistance. It is evident from the preceding definitions that the various divisions of bacteriology overlap to a considerable degree. Veterinary bacteriology may be defined to include that por- tion of medical bacteriology which concerns those microorganisms (bacteria, yeasts, molds, or protozoa) which affect the health of domestic animals. The history of veterinary bacteriology is closely linked with that of general medical bacteriology, for many of the diseases of man are transmissible to animals and vice versa. It should be remembered that both are merely subdivisions of a great science, concerning which it is important that the student should gain something of a perspective view, particularly with ref- erence to its history and development. This development has 'Cisi Fig. 1. — Leeuwenhoek's drawings of bacteria: A, B, Bacilli, probably; C-D, path of movement; E, cocci; F, Leptotrichia, probably; G, spirillum. been so rapid, and so many of the important discoveries have been made within recent years, that it is frequently difficult to deter- mine their relative importance. However, certain facts and personalities stand out so conspicuously that they are deserving of brief consideration. The Microscope and its Influence. — The existence of living plants or animals smaller than can be seen by the unaided eye was conjectured by several of the Greek philosophers and phy- sicians who used such theories in their speculations on the origin and cause of fermentation and disease. Until the discovery of the microscope such speculations were without any basis in fact. Leeuwenhoek (1632-1723), in the course of his examinations of a great variety of natural objects by means of the somewhat 20 VETERINARY BACTERIOLOGY crude lenses of his own manufacture, chanced to observe the presence of motile and motionless microorganisms in the tartar from teeth and in various decaying organic materials. His correspondence with the Royal Society of London and the figures published leave no doubt but that he actually observed bacteria. These drawings are of such historic interest that they are here reproduced. Each advance in the efiiciency of the microscope was followed by an advance in our knowledge of the microorganisms, although speculation frequently outran the ability to see clearly. The com- pound microscope has proved to be indispensable in the study of these forms. Since the introduction of this instrument the degree of magnification, the clearness of definition, and the mechanic ar- rangements for accurate focusing have been gradually improved until at the present time the homogeneous oil immersion objective, the compensating ocular, and the Abb6 condenser are in constant use in the laboratory, and enable us to secure readily magnifica- tion to 1500 diameters or more. During the last several decades there has been little increase in magnification, due to two reasons. The greater the magnification the more convex and consequently the smaller must be the lenses used in the objectives, and the more difficult becomes their grinding and adjustment. Furthermore, the physicist tells us that a clear view, with determination of the size and shape of microscopic objects, cannot be obtained when the objects examined are smaller than one-half the wave length of the rays of light in which they are examined. There is thus a seemingly insurmountable barrier set to an indefinite increase in magnification. A recent advance has been made through the development of the ultramicroscope. This has made visible objects much smaller than those which had been observed previously. A bright gleam of light from an arc or similar source is passed across the darkened field of the microscope, and the light is reflected to the eye from any particles that may be in suspension. These objects are seen in the same manner that minute particles of dust are made visible in a bright ray of light that enters a darkened room. The use of the ultramicroscope has not as yet added many facts of value to our knowledge of the bacteria. INTRODUCTION 21 Nature and Classification of Microorganisms. — Leeuwenhoek, whom we have seen to be the first observer of tacteria, contributed very little to a knowledge of their essential nature. F. Miiller (1786) worked out a simple classification, but did not differentiate between bacteria and protozoa. To him we owe several of the group names appUed to bacteria, such as bacillus, vibrio, spirillum. Ehrenberg (1795-1875), with the improved microscope and lenses at his command, prepared the first logical classification of bacteria. Cohn (1828-1898) elaborated and modified Ehren- berg's classification. He differentiated the true bacteria from the protozoa, and his arrangement is the basis for the- classi- fication used most extensively at present. With the continued im-. provement in the microscope and laboratory technic, more careful studies of structure, form, and relationships have been rendered possible, and many classifications and groupings for bacteria have been suggested. The difficulty in finding morphologic characters that are accurate indices to true relationships has made the subject a troublesome one. In recent years several attempts have been made to secure a satisfactory classification of the genera of bacteria. The one used here is an adaptation of that suggested by a committee on nomenclature of the Society of American Bacteriologists. Spontaneous Generation. — In ancient times and even during the middle ages it was generally held by the philosophers and scientists that hving things, animals, and plants, could arise de novo. Among the first observations that created doubt in man's mind as to the validity of this belief was that of Francisco Redi, who covered meat with gauze to protect it from flies, and found that maggots did not develop in it spontaneously, but arose from the eggs which the flies deposited on the screen. This pointed the path for other similar studies, and it was not long before the idea of spontaneous generation of the higher forms of life was aban- doned. When the microscope revealed the presence of myriads of microorganisms in all decaying or putrefying materials, it was concluded that these organisms arose without progenitors of their own kind, but directly from the organic materials of their sur- roundings. Boiling was believed to certainly destroy all fife, 22 VETERINARY BACTERIOLOGY yet it was found that boiled decoctions would not always remain free from microorganisms. The theory of spontaneous generation of these bacteria was opposed by some and supported vigorously by others of the best scientists of the time. Experiments were care- fully planned and a great variety of materials used, paving the way for the development later of the laboratory technic of the bacteriol- ogist. The sterihzing action of heat, the antiseptic action of certain chemicals, and the value of the cotton plug as a bacterial filter were demonstrated. The theory of spontaneous generation as a topic of contention practically disappeared about 1860. This was largely due to the efforts of Pasteur, who, by a long series of ingenious experiments, overthrew the last defense of the supporters of the theory. The dictum, omne vivum ex vivo (all life from life), is universally accepted at the present time, and the controversy has little but historic interest". Relationships of Microorganisms to Fermentation and Decay. — As has been previously noted, some of the early philosophers hazarded the opinion that decay might be caused by invisible living beings of some kind. The causal relationship of micro- organisms to decay and particularly to fermentation was first definitely established by the work of Louis Pasteur (1822-1895). He found the production of alcohol and carbon dioxid from sugar was due to a yeast, that milk soured because of the activity of bacteria, and that many of the familiar changes in organic sub- stances were accomplished by microorganisms. His conclusions were strenuously opposed and ridiculed by the great German chemist, Liebig. Doubtless the necessity for meeting the attacks of the latter and of establishing his points beyond possibility of refutation led him to devise and develop many of the laboratory methods in common use at the present time. As a result of Pasteur's work the fundamental importance of bacteria in the trans- formation of nitrogen and carbon compounds in nature, the disposal of waste, the purification of water, the enriching of the soil, and many of the changes in the manufacture of foods have been estabhshed. Relationship of Microorganisms to Disease. — The probable causal relationship of microorganisms of some kind to disease was argued as long ago as 1762 by Plenciz, of Vienna. His INTRODUCTION 23 theories were not generally accepted, and it was not until 1840 that Henle proposed what we have come to call the germ theory of disease. He never succeeded in proving his point satisfactorily because of the lack of proper methods and technic. Many other writers within the next few years discussed the theory and numerous facts were adduced in favor of it. The majority of medical practitioners, however, put very little faith in it. The argument that certain organisms were always present was met with the statement that these organisms were the result and not the cause of the disease. Davaine (1863) practically demon- strated by inoculation experiments the causal relationship of a bacillus he foimd in the blood of diseased animals to anthrax. Pasteur (1865) proved the cause of a silkworm disease to be a protozoan parasite. Koch and Pasteur later cultivated the anthrax organism in the laboratory and showed beyond a doubt its relationship to the specific disease. Improved laboratory technic cleared up the cause of many disea'ses within the next de- cade or two. The discovery of the bacillus of tuberculosis by Koch (1882) marks the real beginning of bacteriologic science. The knowledge of protozoa as a cause of disease lagged somewhat behind that of bacterial infections. Evans (1880) described the trypanosome of surra and transmitted the disease by inocula- tion experiments. In 1882 Laveran observed the Plasmodium malarice, the cause of malaria. Development of Laboratory Methods. — Progress was delayed in the study of objects as minute as the bacteria because of the lack of proper methods for their isolation, observation, and identi- fication. Culture-media in which the pathogenic microorganisms could be grown were used by Pasteur and Koch. To the latter we are indebted (1882) for our knowledge of the solid media which can be used for the isolation of organisms from mixed cultures. The importance of this contribution can hardly be overestimated, for the use of pure cultures lies at the very foundation of all modern bacteriologic investigation. This one discovery accounts in large measure for the rapid advance made during the next two decades in the identification of the organisms producing disease. The use of aniHne dyes in rendering cells and their struc- ture more plainly visible under the microscope we owe to Weigert, 24 VETERINAEY BACTERIOLOGY but the application to bacteriology was made by Koch. Since their introduction successive advances in staining technic have in every instance been followed by the discovery of new organisms related to disease. The microscope, liquefiable media, and anilin dyes constitute the trio of most important factors in the development of the science of bacteriology. Development of Theories of Immunity. — Knowledge that one attack of certain diseases generally prevented a recurrence and that diseases could not be indiscriminately transferred to all species of animals has existed ever since the foundation of medi- cine. Many theories have been advanced to account for this phenomenon. A few of the names of investigators who have put the facts into logical form for presentation and study should be considered. Metchnikoff conceived that the white blood-cor- puscles and some other body cells acted as scavengers and des- troyed microorganisms in the blood. This theory in modified form furnishes to-day the logical basis for many of the operations of the practitioner. Von Behring (1890) published results of stud- ies on the diphtheria bacillus in which he recounted the discovery of the specific toxin of this organism and a specific antitoxin. He laid the foundation for the present-day " humoral " theory of immunity, which supplements so well the phagocytic theories of Metchnikoff. Ehrlich has correlated and coordinated the vari- ous facts of the humoral theory and has made substantial additions to it. He has created a terminology which has been quite generally accepted and has proved most useful in discussion of the sub- ject. So extensive have been researches in the field of immunity during the last two decades (since 1890) that it has assumed almost the rank of a coordinate science. Development of Sanitary Science and Preventive Medicine. — In 1858 Murchison formulated the so-called pythogenic theory of disease — i.e., that disease is caused by the emanations arising from decaying or putrefying organic matter, and by the consumption of such materials in water and food. This theory was quite com- monly accepted, and its practical applications form the basis for our modern sanitary science. The disposal of sewage and refuse, the purification of drinking-water, and adequate systems of plumb- ing were advocated and adopted before the germ theory of disease INTRODUCTION 25 had been well formulated and established. The rapid accumula- tion of evidence in favor of the germ theory gave rise to the art of preventive medicine. Lister (1875) advocated the use of antiseptics in the treatment of wounds and wound infection as a direct method of combating the activity of undesirable bacteria. The discovery of the means of transmission in most of the infectious diseases has enabled man to take measures for their eradication. Yellow fever, malaria, diphtheria, bubonic plague, and many other diseases are now kept in check by the use of preventive measures indicated by our knowledge of the manner in which they spread. It is probable that greater advance will be made within the next few years in the domain of preventive medicine, for mankind is fast coming to reaHze. that prevention is better than cure. CHAPTER II MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS CONCERNED IN DISEASE PRODUCTION Position of Pathogenic Microorganisms Differentiation of Animals and Plants. — The distinctions we commonly recognize as differentiating plants from animals in large measure disappear among the microscopic forms of life. It is worth while, therefore, to discuss the factors that are taken into consideration in the assignment of a particular microorgan- ism to the animal or the plant kingdom. The difficulty arises particularly in the differentiation of the bacteria and the protozoa. Bacteria, as will be shown later, probably have in all cases a cell- wall which in function is closely related to that of plants. A cell-wall is frequently absent in protozoa, the limiting mem- brane usually consisting of the ectoplast (see below) alone. The composition of the cell-wall is in some cases like that of many animals (chitin), in others like plants (cellulose). In shape and habit of growth and reproduction the bacteria resemble very closely certain undoubted plants among the blue-green algffi and the mold fungi much more than they do animal forms. On the other hand, there are some types which intergrade with the protozoa, so that there is at present doubt as to their correct systematic position. In the matter of food supply and food utilization some bacteria resemble higher plants, others resemble animals. The possession of organs of motion by bacteria has no direct bearing on the subject, inasmuch as undoubted plants of the group of algse and many of the protozoa have them. The evidence, on the whole, is much in favor of classij^cation of the true bacteria with plants. Before beginning a study of the morphology or structure of these microorganisms, their position and relationships to the other groups of plants and animals should be understood. Fol- 26 MOEPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 27 lowing is a discussion of these various groups, with particular emphasis upon the groups or subgroups of significance in medical bacteriology. The plant kingdom is generally divided into four great groups, the Spermatophytes, or seed-bearing plants, the Pteridophytes, or ferns and fern-like plants, the Bryophytes, or moss plants, and the Thallophytes, including all plants low in the scale of evolution, that have not become highly differentiated and that never have roots, stems, and leaves. All of the plant-like organisms to be con- sidered belong to this lowest group, Thallophytes. The animal kingdom may be divided into the Metazoa, or higher differentiated types, made up of many cells, and the Pro- tozoa, or unicellular forms. To the latter group belong all the animal-like organisms to be considered. Subdivisions of the Thallophsrtes. — FolloAving is a key or out- line of the principal subgroups of the Thallophyta : A. Unicellular forms, multiplying only by splitting of the cells to form two equal daughter-cells. Never any sex cells. I. Cells containing blue-green coloring-matter. 1. Schizophycece (Cyanophycese or blue-green algae). II. Cells not containing blue-green coloring-matter. 2. Schizomycetes (Bacteria), B. Unicellular or multicellular, multiplication by some method other than simple fission. Frequently sexual repro- duction occurs. I. Cells containing green coloring-matter (chlorophyll). 3. Algce (sea-weeds, pond scums, etc.). II. Cells without green coloring-matter. 4. Fungi (yeasts, molds, mildews, rusts, smuts, toadstools, puffballs, mushrooms, etc.). Of the last group (Fungi) only two subgroups are of especial pathogenic significance for the veterinarian, the yeasts and the molds. These two, with the bacteria, constitute the three types of microorganishis belonging to the plant kingdom that contain forms pathogenic for animals. 28 VETERINARY BACTERIOLOGY Morphology of Bacteria Shape of Bacteria.— Bacteria may be classified according to shape into spheres, straight rods, bent or spiral rods, and filaments. A spherical form is called a coccus. Although the cocci are theoretically spherical, .there are many that appear somewhat flattened or ovoid when in groups or chains. Fig. 2. — Types of bacteria: Cocci, bacilli, and spirilla (Jordan). A straight rod is called a bacillus. A curved or spiral rod is called a spirillum. The filamentous bacteria are those in which the organism is greatly elongated. The name trichobacteria has been given to such forms. Frequently the filamentous type exhibits branching and in other ways resembles the mycelium of the higher fungi or molds. Fig. 3. — Involution forms of bacteria: 1, Rhizobium leguminosarum. a, Normal rods; b, bacteroids. 2, Mycobacterium tuberculosis. 3, Bacillus subtilis. 4, Acetobacter aceti. 5, Pasteurella pestis. 6, Actinomyces sp. 7, Spirillum sp. forms. 8, Staphylococcus aureus: a, Normal forms; b, involution When grown under unfavorable conditions, or as a result of the action of certain stimulation, many bacteria assume unusual and abnormal shapes. Cells of this type are called involution forms. Such cells are not necessarily incapable of continued growth and MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 29 reproduction of the more usual or normal type when brought under favorable conditions. Frequently, however, these cells soon die and can no longer reproduce. Various bacteria differ considerably with respect to the ease with which they produce involution forms. Grouping of Bacterial CeUs,— The cells of bacteria are frequently surrounded by a gelatinous material (capsule, see below) which causes them to chng together in groups. This grouping in the various types is so constant that it is used to differentiate various genera from each other, and in some- cases the species within © © 0® GK© QQQQ ®®®® ©©O0CSC© ^ &) ©& Fig. 4. — Development of groups of cocci' 1, Development of strepto- coccus; 2, development of micrococcus; 3, development of sarcina; 4, •development of staphylococcus. the genus. Bacterial cells divide normally at right angles to the longest axis of the cell. This allows of but Httle variation in the grouping of elongated tjrpes, but as the cocci have no "longest axis" they divide in various planes. Some cocci divide constantly in the same plane or in a plane parallel to the first. This results in the formation of a chain of cocci. An organism showing this grouping is called a strepto- coccus (pi. streptococci). Other cocci divide alternately in two planes at right angles to each other. Such an organism will be found in twos (diplo- •coccus) or in fours (tetrad or tetracoccus) . Diplococci may also 30 VETERINARY BACTERIOLOGY be produced by the breaking up of chains of streptococci into pairs. Cocci sometimes divide in three planes at right angles to each other. This results in the formation of cubes or packets of cocci. A packet of this kind is called a sardna (pi. sarcince) . ©^ Z Fig. 5. — Shapes and groups of cocci: 1, Single coccus (micrococcus); 2, cocci in an irregular mass (staphylococcus); 3, spheric diplococci; 4, flat- tened diplococci; 5, coffee-bean-shaped diplococci; 6, lanceolate diplo- cocci; 7, streptococcus with short chains; 8, streptococcus with long chains; 9, a streptococcus made up of diplococcus elements; 10, cap- sulated micrococci; 11, sarcina. A staphylococcus (pi. staphylococci) is a coccus whose planes of division are not at right angles, or which divides at different intervals with a consequent irregular grouping of the cells much resembling grapes in a cluster. cc^ i>^ ^^ ^^iitL^S^^1IS^i..MiJi^UaJ^^f Q© @ ^3^3 '.J % Fig. 6.— Types of bacilli. BacilU occur either singly or in chains. The latter are some- times known as streptobacilli. Spirilla usually occur singly, although short chains of two or three individuals are sometimes observed. In a few bacteria the gelatinous envelop of the cell is greatly MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 31 thickened, and the bacteria, either cocci or bacilli, are embedded in the mass. Such a mass of cells is called a zooglea. Size of Bacteria. — The unit of microscopic measurement is the micron and is abbreviated by the Greek letter fi. It is the Fig. 7. — Types of spirilla. YFTo part of a miUimeter or, approximately, the 25^ part of an inch. Bacteria vary considerably in size, from forms 0.1 ^ or less in diameter, barely visible under the microscope, to forms Fig. 8. — Types of filamentous bacteria: A, Leptotriohia; B, Cladothrix; C, Nocardia; D, Actinomyces or Streptothrix. 100 /w or more in length. Most bacteria are between 0.5 and 5 [I in diameter and 0.5 fi and 10 (i in length. Some bacteria are undoubtedly too small to be seen with the highest powers of our microscopes, hence less than 0.1 /tf in diameter. We know of the existence of these organisms by their effects. The organism caus- 32 VETERINARY BACTERIOLOGY ing hog cholera, for example, is so small that it will pass through the pores of a fine porcelain filter, and will cause disease when in- jected into a healthy pig. Such an organismjs frequently spoken of as ultramicroscopic or, preferably, as a filterable virus. Histology and Structure of Bacteria. — This topic may be treated under four subheads, the cell wall with its rekted sheaths and cap- sules, the protoplasm, the cell inclusions, and the fiagella. Cell Wall. — The bacterial cell is in all cases surrounded by a definite membrane that morphologically resembles the cell wall of higher plants. When tested chemically with various reagents and examined microscopically, it is sometimes found to give the reactions characteristic of chitin, the material which makes up the hard outer shell of insects, and is found as a cell mem- Fig. 9. — Capsulated bacteria. brane in many animals. Chemically chitin is an amino-sub- stitution product of a carbohydrate. The fact that the cell walls in bacteria so frequently resemble in composition those of certain animals has been used as an argument for the animal relationships of the bacteria. This is negatived, however, by the fact that in numerous molds and other fungi, undoubted plants, the cell walls are made up of a similar substance. The cell wall in bacteria is usually covered by a layer of mucil- aginous material, in most cases so thin that the most careful technic must be employed in its demonstration, in other cases a thick coating or capsule. The nature of this capsular substance has been a fertile subject for dispute. A few bacteriologists have claimed that it is composed of living protoplasm, the majority, MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 33 with seeming justification, believe that this is either an excreted material or merely an outer swollen and differentiated portion of the cell wall. Chemically this material differs in the various capsulated bacteria. In some cases it is composed of mucin, a slimy material made up of a protein-like substance united with a carbohydrate, and resembhng the mucus secreted by certain body cells. In other cases the capsule is made up of pure carbo- hydrates and is closely allied to certain of the vegetable gums, such as gum arable and gum tragacanth. The capsule of some bacteria is partially or wholly soluble in water. When such an organ- ism grows in suitable nutrient solution it renders the medium slimy or gelatinous. Slimy milk, bread and whey are caused by the luxuriant growth of such organisms. Fig. 10. — Bacteria showing sheaths. Some of the filamentous bacteria produce a firm sheath or tube outside of the cell, this sheath usually inclosing an entire filament or chain of cells. In composition it probably closely resembles the cell wall. In some cases it is impregnated with iron oxid. It is possible that the sheath is a modified type of capsule. Cell Protoplasm. — The hving material within the cell wall is called the protoplasm. Structurally it may usually be dif- ferentiated into two layers, an outer thin layer lying closely appressed to the cell wall, and an inner portion. The outer layer or edoplast performs one of the most important functions of the cell, as this is the membrane, and not the cell wall, that determines what materials in solution may enter and what may leave the cell; through it must pass by diffusion all the food that enters the cell. When certain bacteria, as the cholera spirillum, are placed in a strong solution of some salt which does not readily pass through 3 34 VETERINARY BACTERIOLOGY this ectoplast, the water from the cell in part passes out, the protoplasm shrinks away from the cell wall, and the cell is said to be plasmolyzed (noun, plasmolysis) . Such a plasmolyzed cell shows clearly the ectoplast separated from the cell wall. When a cell of certain species is placed in distilled water or a concentra- A Fig. 11. — Plasmolysis and plasmoptysis of bacterial cells: A, Plasmo- lyzed bacterial cells; B, cells showing plasmoptysis, the protoplasm has burst the cell wall and is escaping. (Adapted from Fischer.) tion of salt considerably less than that to which it has been accus- tomed, the cell takes up water, the cell wall bursts, and part of the protoplasm escapes. This phenomenon is called plasmoptysis. The protoplasm of the cell is commonly homogeneous in appear- ance, and stains best with the basic aniline dyes. Either a definite 'oOo 'Ofc ^1 , glycogen granules; E, meta- chromatic granules; F, sulphur granules. nucleus is not present, or the nuclear material is so scattered as to make the entire mass functionally a nucleus. Some bac- teria have been described as possessing a primitive type of differentiated nucleus, but such structures cannot be discerned in others. MORPHOLOGY AND RELATIONSHIPS OP MICROORGANISMS 35 Cell Inclusions. — Bacterial cells sometimes contain vacmles, or spaces in the protoplasm filled with cell sap or some non-staining or non-refractive substance. A large vacuole near the center of the cell may crowd the protoplasm to the ends of the cell, and such organisms, when stained, are said to show polar staining. In other forms, as the diphtheria bacillus, granules are formed that stain much more intensely with the basic aniline dyes than does the remainder of the protoplasm. These are called meta- chromatic granules. The function of these granules is not clear. Certain species of bacteria living in water containing hydrogen sulphid are found to contain granules of free sulphur in their protoplasm. Still others have food materials in the form of oil globules or granules of glycogen. Fig. 13. — Distribution of the flagella of bacteria: A, Non-motile or atrich- ous bacilli, spirilla, and cocci; B, monotrichous flagella of bacilli, spirilla, and cocci; C, lophotrichous flagella of bacilli and spirilla; D, amphitrichous flagella of bacilli and spirilla; E, peritrichous flagella of bacilli and spirilla. Flagella. — Many bacteria are motile by means of whip-like threads of protoplasm which extend from their surfaces. These threads are known as whips or flagella (sing, flagellum). These flagella are observed with difficulty in the living organism .ex- cept with dark field illumination and require peculiar stain- ing technic and careful treatment to make them visible in a stained mount. Comparatively few cocci, many of the bacilli, and most of the spirilla are flagellated. The distribution of flagella on the surface of the cell has been used as a basis for grouping. Atrichous bacteria have no flagella; monotrichous bacteria have a single flagellum at One end; lophotrichous, a group of flagella at one pole; amphitrichous, flagella at both eiids; and 36 VETERINARY BACTERIOLOGY peritrichous, flagella on all sides. Old cells and cells transferred from one medium to another are very apt to loose their flagella. A young culture is most suitable for determination of motility. True motility must not be confused with Brownian movement, which is a vibrating or oscillating motion of finely divided par- ticles of almost any kind suspended in water, and visible when examined under the microscope. This motion has not been satisfactorily explained, but it is probably due to rapid changes in surface tension of the liquid at the point of contact with the particles. Reproduction in Bacteria. — ^Multiplication in all bacteria is a simple process. The cell commonly elongates or enlarges, a cell wall develops across the middle, and the two cells separate. This operation may occur with considerable rapidity. Some organ- isms in favorable medium can grow to their full size and divide to form two individuals in the course of twenty minutes to an hour. If the organism could multiply in this geometric ratio for a short time the number of resultant organisms would be practically incalculable. For example, if a bacterium should divide every half -hour, at the end of two days the progeny would be represented by 2°°, a number having twenty-eight figures. Such rapid multi- pHcation is never long continued, for food supply is never long favorable, and waste products of the bacterial growth tend to accumulate and diminish the rate. Nevertheless, the rapidity of increase of bacteria accounts in a large measure for the consider- able changes they bring about in a short time, as in the souring of milk or invasion of the body in disease. Many bacteria also reproduce by means of spores. These are of two types: endospores, produced inside the bacterial cell, and arthrospores, consisting of entire differentiated cells. The former aire produced by certain bacilli and spirilla, the latter by certain of the filamentous forms or trichobacteria. Endospores are formed by many bacilli, and possibly by some spirilla. They are produced in response to some definite stimulus, such as unfavorable conditions of the environment, accumulation of waste products, or change in reaction of the medium. The spore is essentially a portion of the protoplasm of the cell which has expelled most of its water and shrunken in MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 37 size until it occupies a portion only of the space within the cell wall, and has then surrounded itself with a heavy wall, probably chitinous in nature. In practically all cases there is but one spore in a cell. The spore may be equatorial or polar in position, and of less or greater diameter than the cell which produces it. The term Clostridium is sometimes used to indicate a spore-bearing Fig. 14. — Development of endospores in a bacillus. " (After Fischer.) rod in which the spore is equatorial and of greater diameter than that of the cell, resulting in a spindle. Endospores contain only about 20 per cent, of water as compared with 80 to 90 per cent, in the cells which produced them. An organism without a spore is usually differentiated by the term vegetative rod or vegetative cell. Spores are much more resistant to desiccation, heat, light, and chemicals than the vegetative cells. They are of use in Fig. 15. — Bacterial spore types: A, Equatorial spores of a diameter less than the cell; B, polar spores of a diameter less than the cell; C, equatorial spores of a diameter greater than the cell (Clostridium type) ; D, drumstick or polai- spores of a diameter greater than the cell. tiding the organism over unfavorable conditions. Spore-bear- ing bacteria are abundant in the soil, where they often are exposed to great ranges of moisture, temperature, and light. When a spore again comes under favorable conditions for growth, it germinates and produces a cell typical of its species. Germina- tion is accomphshed either by stretching or bursting the spore wall. 38 VETERINARY BACTERIOLOGY Arthrospores are bacterial cells set apart for purposes of repro- duction, and are usually differentiated appreciably from the normal cell. Several investigators have claimed that they are produced by some of the cocci, but this has never been satisfactorily estab- Fig. 16. — Germination of spores: A, Bacillus subtilis (Prazmowski) ; B, Bacil- lus anthracis (deBary) ; C, Clostridium sp. (deBary) . lished. The filamentous bacteria or trichobacteria produce arthrospores or conidia, as they are sometimes called, by the dis- integration of some of the filaments into short rods or spheres which are capable of reproducing the parent type or by a process BuU C| Fig. 17. — Arthrospores: A, Crenothrix polyspora (Cohn); B, Galliohella ferru- ginea, showing conidia formation (Ellis) ; C, Leptothrix ochracea (Ellis) . of budding. In many cases the threads which break up into the spores are somewhat differentiated from the normal cells of the plant, and are aerial, resembling closely some of the molds. Morphology of the Yeasts, Saccharomycetes, and Blasto- MYCETES Yeasts, from the standpoint of the systematic botanist, are placed at some distance from the bacteria, for there are many differences between typical yeasts and typical bacteria. On the other hand, there are forms which intergrade between the two. MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 39 and are sometimes assigned to one group, sometimes to another. The yeasts and the molds also show intermediate types. Form, Size, and Grouping of Yeasts.— Yeast cells are usu- ally spherical, oval, eUipsoid, or cyUndrical. For the most part they are larger than bacterial cells, although there are excep- tions. The true yeasts multiply not by fission, but by a process of budding. The cells commonly remain united for a time, giv- ing rise to masses consisting of many individuals. Sooner or later they break apart. The relative shape, size, and groupings of the yeast cells are used in the differentiation of species. In some species part of the cells become considerably elongated and form a kind of false mycelium resembling that of the molds. This character is not always constant in a given species, it may Fig. 18. — Types of yeast cells and groupings. appear when the organism is growing in one kind of medium and not appear in another. Histology and Structure of the Yeast. — The very young cell has no cell wall, but by the time it is one-third grown the wall appears as a delicate membrane. In old cells it is sometimes of considerable thickness. Its composition has not been certainly determined, probably it is a carbohydrate or related compound, and not chitinous, as are the walls of bacterial and mold cells. To this substance the name yeast cellulose has been given. It has not been prepared free from nitrogen, so that it is possible that it may be nitrogenous in nature. It is sometimes surrounded by a gelatinous excretion or capsule, as is the case with bacteria. The yeast cells are never motile. Yeast Protoplasm and Cell Inclusions. — The contents of the 40 VETEEINAEY BACTERIOLOGY yeast cell are more highly differentiated than are those of bacteria. The edoplast, or limiting membrane of the protoplasm, is easily demonstrated by plasmolyzing the cell. This edoplast (German Hautschicht) is the only membrane of the young cell, and the cell wall is probably secreted by it. The protoplasm is differ- entiated definitely into a nudeus and cytoplasm. The nucleus is hot as easily demonstrated as in the higher plants and animals, Fig. 19. — Diagram of budding yeast cells and their contents: a, Glycogen granules; b, vacuoles; c, nucleus; d, dividing nucleus in bud formation. but may be shown by proper staining methods. The cytoplasm usually contains one or more vacuoles, spaces filled with cell sap and not taking the stain as does the cytoplasm. Older cells may also show oil globules or glycogen granules. Reproduction in Yeasts. — Yeasts commonly multiply vege- tatively by budding. A bit of the protoplasm protrudes on one side of the mother cell, the nucleus divides, and one part goes to Fig. 20. — Spores (ascospores) of the yeast (Hansen). the bud, the other remains within the cell, the bud enlarges, develops a cell wall, and is constricted off as a distinct individual. Many yeasts may also under favorable conditions produce spores. The development of the spores in the yeast cell differs considerably from that in the bacteria. The latter typically have but one spore developed within the cell, while a yeast cell usually produces two, four, six, or even eight spores. The nucleus divides several times to form a number of nuclei, each of which, together with the MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 41 protoplasm lying in contact with it, becomes surrounded by a membrane. A cell of the yeast (and certain other fungi) when filled with spores is called the ascus (pi. asci) or sac. The spores are called ascospores (Fig. 20). This method of spore production relates the yeasts quite definitely to some of the higher fungi. In some cases there is a primitive type of sexual reproduction or fertilization associated with the development of the spores. The spores are more resistant to an unfavorable environment than the vegetative cells. When brought under favorable conditions they germinate and develop into the typical yeast plant. In old yeast cultures some cells develop heavy cell walls, are 'filled with granular reserve food materials, and become potentially spores. Such cells are likewise probably resistant to unfavorable conditions, and serve to tide the yeast over periods of desiccation or poor food supply. They resemble the chlamydospores produced by many molds. Morphology of the Hyphomycetes or Molds The molds or hyphomycetes do not constitute a homogeneous group in the eyes of the systematic botanist, but belong to vari- ous subdivisions of the group of fungi. Some are related to the algse and are grouped imder the Phycomycetes, others belong to the sac fvmgi or Ascomycetes, others are related to the smuts, rusts, and toadstools, or Basidiomycetes, and the largest number belong to the group of imperfect fungi or Fungi Imperfedi. From the viewpoint of the bacteriologist these botanic relationships are not significant; all the fungi, regardless of kinship, that agree in having the plant body made up of threads usually more or less branched, and forming more or less loose or cottony masses, in short, those that answer to the popular conception of molds, are grouped together as Hyphomycetes. Such a classification is scientifically justifiable only because of the great complexity of the various members of the family of fungi, and the fact that it is not the systematic position but the economic importance of the forms that is of significance. Form and Size of Hyphomycetes or Molds.— A mold may be differentiated from the yeasts and bacteria in that it is multi- cellular, with the cells united to form more or less branched 42 VETERINARY BACTERIOLOGY threads called hyphce (sing, hypha) . The whole mass of threads or hyphse which go to make up the plant body of the mold is called the mycelium. In most molds certain threads are differentiated for the production of spores. The mycelium of the mold may extend over a considerable area, growing deep into the substratum for food or into the air to develop spores. Histology and Structure of Molds. — The mycelium in some molds is continuous throughout its length, possessing no cross walls which might separate the cells from each other. In the majority of forms, and in all those of economic importance to the veterinarian, the hyphse are divided by cross walls or septa Fig. 21. — Mold hyphae: A, B, Non-septate hyphse of the Phycomycetes; C, tip of a non-septate hypha, showing numerous nuclei and vacuoles; D, septate branching hyphse; E, a single cell of a septate hypha, showing nucleus and vac- uoles. (sing, septum). The cell wall is composed of true cellulose in a few molds, in the majority it is chitinous as in the bacteria. The almost universal presence of chitin in the cell walls of the fungi is frequently lost sight of by those who regard its presence in the cell walls of bacteria as evidence of animal affinities. The protoplasm of molds, as in the yeasts, is made up of cytoplasm and nucleus. The outer layer of the cytoplasm or ectoplast is readily demonstrated in most molds by plasmolysis. In the forms that do not have septa, dividing the hyphse into cells, the numerous nuclei are imbedded in the common cytoplasm. Functionally each nucleus with its bit of surrounding cytoplasm constitutes a MORPHOLOGY AND HELATIONSHIPS OF MICROORGANISMS 43 cell, although the statement is often made that the entire mold filament in the non-septate type is a single cell. Reproduction of Molds.^It is impracticable to go into detail concerning the reproduction of molds. Spores of many different types are produced (Fig. 22), sometimes three or four kinds by a single species. The spores exhibit every conceivable shape and coloring, are sometimes unicellular, at other times multiseptate. Hundreds of genera and thousands of species are known. The names applied to the different parts of the molds concerned in reproduction and the manner in which the spores are borne in some of the commoner molds may, however, be briefly discussed. Molds may be divided, for convenience, into those which bear the spores enclosed in a spore case or sporangium and those in which they are not so inclosed. This sporangium is commonly Fig. 22. — Types of mold spores. borne at the tip of a hyphal thread differentiated for the pur- pose, called a sporangiophore. Spores not produced inside of a sporangium and not the result of fertilization (i. e., asexual spores) are termed conidia (sing, conidium). Conidia are usually developed at the tip of specialized branches called conidiophores. Sometimes they are formed by the breaking up of the mycehal threads or hyphae, and are then called oldia (sing, oidium), in other cases they develop within the hyphae and are surrounded by it as by a sheath. When one of the cells in a hypha becomes enlarged and surrounded with a heavy wall it is called a chlamydo- spore. Some molds develop spores as a result of the union of sex cells (sexual spores). These are called ascospores when pro- duced in sacs (asd) and zygospores when formed by the union of two like cells as in certain Phycomycetes. Spores of the molds are commonly born on hyphs that extend 44 VETERINARY BACTERIOLOGY into the air away from the moist surface of the medium in which they are growing. This faciUtates their dispersal by the air Fig. 23. — Types of the spores and the spore-bearing organs of the molds — ■ 1, Sporangium of the Mucor: a, columella; 6, sporangiophore; c, spores; d, sporangium wall. 2, Sporangia of Sporodinia: a, sporangiophore; b, sporangia containing spores. 3. Ascus of an ascomycete, Peziza: a, ascus or spore sac; b, spore; c, sterile threads or paraphyses. 4, Oidium spore formation; the hyphae are segmenting to form spores or oidia. 5, o, Chlamydospores formed in the hypha of a Chlamydomucor. 6, Zygospore of a Mucor: a, hypha; 6, suspensor; c, zygospore. 7, Conidiophore and conidia of Penicillium: a, con- idiophore; 6, verticillate branches of the conidiophore; c, chains of spores or conidia. 8, Aspergillus: a, conidiophore; 6, inflated tip of the conidiophore; c, sterigmata; d, chain of spores. 9, a, Hypha; b, poorly differentiated conidio- phore; c, chain of conidia. currents. When they fall upon a suitable medium they ger- minate and soon develop the typical mold plant. Morphology of the Protozoa The protozoa are unicellular and bear much the same relation to the higher animals or metazoa that the bacteria do to the higher plants. Notwithstanding that they are reckoned among the simplest forms of life, they are, nevertheless, greatly diversified in shape, size, and structure. Only the barest outline of their structure can be given here. For a more detailed account the student is referred to the section on Protozoa. MORPHOLOGY AND RELATIONSHIPS OF MICROORGANISMS 45 Form and Size of Protozoa. — The pathogenic protozoa vary- in size from those visible to the naked eye to those barely visible with the highest powers of the microscope. Some are pos- sibly ultramicroscopic, the organism causing yellow fever, for example. In form and shape the greatest diversity is to be noted. Some are without definite shape, and are apparently only lumps of protoplasm, others are highly differentiated and have as great variety of organs (organella) as some of the higher animals. Histology. — A true cell wall, as found in the bacteria, yeasts, and fungi, is frequently not present in the protozoa. When found, it is chitinous in nature. The edoplast forms the limit- ing membrane of the cell in the majority of cases. The protoplasm is differentiated into nucleus (sometimes two, a micronucleus and a macronucleus) and endoplasm or cytoplasm. Power of move- ment is possessed by many forms. This may be due to the development of pseudopodia, of flagella, or of dlia. Reproduction.^Asexual reproduction is accomplished in many cases by a simple process of fission, in others the procedure is much more complex. Sexual reproduction is quite general, but here again the complexity is so great as to render brief treatment impracticable. The relationship and structure of these forms will be considered in greater detail under the heading of Patho- genic Protozoa in Section V. CHAPTER III PHYSIOLOGY OF MICROORGANISMS Physiology has been defined by Barnes to include " a study of the behavior of plants (and animals) of all sorts, and of the ways in which this is affected by external agents of every sort." In our discussion of the physiology of microorganisms we shall have to deal principally with the interrelationships existing between these microorganisms and their environment. FOOD Relationships of Microorganisms A food is any substance which a living organism may make a part of its hving material or use as a source of growth energy. The term is frequently used very loosely to include all the sub- stances of which an organism may make any use. For example, a distinction is sometimes made between green plants and animals on the basis of food used. The former are said to live on inorganic foods and the latter on organic. This distinction is erroneous. The difference is simply that green plants can manufacture their own foods out of inorganic material by the aid of the energy secured from the sun's rays through the green coloring-matter or chlorophyll, while animals make use of food already prepared. The materials of which some microorganisms make use are no more foods than the rays of the sun are a food for green plants. Composition of the Cell. — The food utiUzed by any micro- organism must contain the elements needed for the building up of the cell substance. The analysis of such cells shows them to be made up of the same elements as those of higher plants and animals, namely, carbon, oxygen, nitrogen, hydrogen, and smaller amounts of phosphorus, iron, magnesium, calcium, and some other elements. The foods utilized by organisms must, therefore, contain these elements likewise. Sources and Kinds of Foods. — Some bacteria, like the green plants, are capable of manufacturing their own food. For this purpose a source of energy is necessary. Some species utilize the 4fi PHYSIOLOGY OF MICROORGANISMS 47 energy of the rays of sunlight in much the same manner probably as do green plants. The coloring-matter in these forms, however, is purple or red {baderiopurpurin) . Other forms living in water which contains hydrogen sulphid, as in the sulphur springs, oxidize the hydrogen sulfid to free sulphur and even sulphuric acid and gain energy for the manufacture of their foods from carbon dioxid, water, and other compounds by this process. Still other forms are believed to make use of iron, ammonia, nitrites, and other inorganic substances, and by their oxidation secure the necessary energy. Organisms which can manufacture their own food out of inorganic substances are said to be prototrophic. The proto- trophic microorganisms so far as known are all bacteria or molds. Most microorganisms utilize organic matter in a dead or living condition for food. Those which utilize dead organic matter are called metatrophic, while those requiring living material or complex protein foods are called paratrophic. The latter are frequently dis- ease producing. It must not be supposed that these division lines are strictly drawn. For example, certain bacteria seem to make use of the oxidation of carbohydrates and other organic substances to enable them to take up the nitrogen from the air and to convert it into usable form. Such are both prototrophic and metatrophic. The peculiar food requirements of the different species must be kept in mind in the preparation of nutrient media for their growth. Some organisms will not grow in the presence of organic materials, while others require such specialized media as blood- serum or hemoglobin. A second grouping of microorganisms commonly used is based upon the relationship to other Hving organisms. Those which do not require a living host (animal or plant) are called sapro- phytes if bacteria, yeasts, or molds, and saprozoites if protozoa; those which require a living host are called parasites. Those parasites which do not produce disease are termed commensals. Moisture Relationships of Microorganisms Microorganisms require considerable amounts of water for their development. The optimum condition for growth in most cases is saturation. There is great variation in ability to resist desiccation. The spores of some bacteria and fungi 48 VETERINARY BACTERIOLOGY and the encysted cells of some protozoa will live for years, while other forms are destroyed if allowed to become completely dried. Respiration of Microorganisms Respiration is frequently defined as the taking up of oxygen and the elimination of carbon dioxid. This definition is entirely inadequate when we come to a discussion of microorganisms, if, indeed, it can be applied in any case even to higher animals and plants. Respiration seems fundamentally to be the process whereljy energy is generated in the cell. Energy when evolved in the cell always originates from chemical changes in the compounds within the cell. Whether or not this energy may be gained by the oxidation of food materials when taken into the cell, or whether they must be first built up into protoplasm and this then broken down, is a matter of dispute at present among scientists. In any event the presence of free oxygen is certainly not neces- sary to this release of energy, for many bacteria as well as other plants and animals live in the absence of free oxygen. Organ- isms that grow only in the presence of oxygen are called aerobic; those which will grow only in the absence of fi:ee oxygen, anaerobic, and those which will grow either with or without free oxygen, facul- tative. It is probable that most of the so-called anaerobes grow better in the presence of minute quantities of oxygen. The end-products of respiration are found to differ with the type, aerobic bacteria usually produce carbon dioxid and water; anae- robic forms, less highly oxidized substances, such as alcohol and butyric acid. The oxygen requirements of anaerobic bacteria must be recog- nized in the laboratory if they are to be successfully cultivated. The air of the culture-tube or flask may be removed by a stream of hydrogen, nitrogen, or some other inert gas, the oxygen may be absorbed by the use of alkaline sodium pyrogallate, the air may be exhausted by an air-pump, the oxygen may be excluded by covering the medium with oil or some similar material, or the organism may be mixed with some aerobic form which will use up the oxygen and allow growth of the anaerobe. Probably the latter is the common method whereby anaerobes are able to grow in nature. An organism may be an obligate aerobe under certain conditions or in certain media and facultative under PHYSIOLOGY OF MICBOORGANISMS 49 different conditions. For example, many bacteria will grow in the absence of oxygen providing there is available some compound from which oxygen may be readily obtained. Some will use nitrates for this purpose, others may use sulphates, reducing them to nitrites and sulphids respectively. Many will grow under anaerobic conditions providing suitable carbohydrates which they can ferment are present. Growth Rates and Death Rates The various physical and chemical agencies to be discussed, manifest their action upon microorganisms by their effect upon the rate of growth on the one hand and the rate of death on the other. Before they can' be discussed adequately, therefore, certain general considerations concerning rates of growth and death must be presented. Rates of Growth. — When bacteria or other organisms are planted in a suitable culture medium, and optimum conditions for growth maintained, the bacteria soon reach a maximum rate of growth. This can best be measured by determining the average length of time required for an organism to grow to its full size, divide, and form two individuals. This has been termed the generation time. It is the average length of time elapsing between cell divisions. It may be most conveniently determined in any given case, not by watcjiing the bacteria under the microscope, but by counting the number of living cells present in a given amount of culture after varying lengths of time. It is evident that the shorter the generation time is found to be, the more rapid is the rate of multiplication. Anything therefore which tends to shorten the generation time increases the rate of growth. The actual length of the generation time may be determined if it is certain that the bacteria are multiplying at a uniform rate, that is, that the cultures are not too young or too old, by count- ing the bacteria at the beginning and at the end of a given period. It is evident that if one starts with a single organism, at the end of the first generation period there will be two, at the end of the next four (2^), at the end of the next eight (2^) and so on. If n represents the number of generations, then there will be at the end of the nth generation period 2" bacteria. If B 50 VETERINABY BACTERIOLOGY represents the number of bacteria at the beginning, and b the number at the end, then b = 52". If b and B are known, their values may be substituted into the equation, and the value of n determined. If t is the total time, and g is the generation time, it is evident that the generation time must equal the total time divided by the number of generations t ^ = n Careful studies of bacteria growing in culture media show that the length of the generation time does not remain a constant throughout the time of cultivation. When bacteria are first planted in a culture, particularly if they have been taken from an old culture, they frequently grow slowly at first. As they become •accustomed to their environment they multiply more rapidly, that is, the value of g decreases. After a time the maximum rate of growth occurs, and the value of g is at a minimum. Still later the bacteria become crowded and the rate of growth slows down, g increases and finally the bacteria cease to grow at all and begin to die off. It is evident that a fair comparison of effect of any influence upon bacteria can be determined by comparing the values of g under different conditions. Rates of Death. — It is equally important to know something of the laws which govern the rates at which bacteria die off under unfavorable conditions. In most of the cases which have been carefully studied, the bacteria die off in accordance with a definite law, which may be stated as follows : with a given kind of organisms under uniform unfavorable conditions, the number of bacteria present in a culture will always be reduced by one half in equal periods of time, that is no matter how many bacteria there are at the beginning of a definite period of time, one half that number wUl always be aUve at the end of the proper interval. For example, suppose that two cultures of the same organism one containing a million bacteria and the other a thousand bacteria are subjected to the same unfavorable conditions. It is PHYSIOLOGY OF MICROORGANISMS 51 found that at the end of a definite period of time, say ten minutes, the bacteria in the less concentrated suspension number five hundred. It will be found that in the same period of time the number in the other culture has also been halved, that is, there are 500,000 bacteria left. Another way of stating is this, during each equal interval of time a definite percentage of those bacteria living at the beginning of the period will be killed. If we wish to compare unfavorable conditions in their effect upon the death of microorganisms we may compare the length of time required to reduce the numbers of bacteria by a definite percentage, say one half. If at one temperature, for example, half of the bacteria are killed in ten minutes, and at another temperature one half • the bacteria are killed in five minutes, it is evident that the second temperature is far more destructive than the first. It will be noted that time required to kill half the bacteria is mathe- matically the converse of the generation time. In summary it may be emphasized that all effects of environ- ment upon microorganisms may be manifested in growing cul- tures by changes in the length of the generation time, changes in morphology, and in the physiological and cultural reactions. Likewise the effect of environment will be noted upon the rate of death by comparing the length of time necessary to kill a definite percentage of the microorganisms present. Temperature Relations of Microorganisms Optimum Temperature. — The optimum growth temperature is that which most favors the development of the microorgan- isms, that is, that temperature which gives the shortest generation time. The optimtmi varies with the species. A few organisms found in the ocean, in cold waters, alpine regions, etc., prefer a low temperature, fxom 0° to 15°. These are called psychrophilic. Those which prefer a somewhat higher temperature are called mesophilic. These latter may be again subdivided into those that prefer a "room" temperature of 18° to 25°, and those that prefer blood heat (man 37.5°) for the most parasitic forms. Tem- peratures such as are found in hot springs, interior of compost heaps (50° to 70°) favor the development of thermophilic bacteria. 52 VETERINARY BACTERIOLOGY Minimum Temperature. — The lowest temperature at which an organism will continue growth is said to be its minimum. This temperature varies for different species. Some organisms will multiply in brine held at temperatures lower than the freez- ing-point of water. Maximum Growth Temperature. — The highest temperature at which an organism will multiply is called its maximum. This must not be confused with the thermal death point (see below). The majority of bacteria cannot grow above 45°. Growth Temperature Range. — The differences between the minimum and maximum growth temperatures vary within rather wide limits. Those organisms which exhibit considerable adap- tability and are able to grow through a wide range of temperature are called euryihermic. Most of the saprophytic organisms belong here. The parasitic types which have minima and maxima varying but little from the optima are stenoihermic. Thermal Death Point. — The thermal death point of an organism is usually defined as that temperature which under given conditions will certainly destroy all the cells. It has been noted above that bacteria do not die off instantaneously. The following factors must be taken into consideration in the deter- mination of the rate of death of bacteria: 1. The Absence or Presence of Spores. — Spores are much more resistant to high temperatures than are the vegetative cells. Forms having spores, therefore, have two rates of death, one for the vegetative cells and the other for the spores. 2. Presence or Absence of Moisture. — Bacteria are more resis- tant to dry than to moist heat. The thermal death point is probably the temperature at which incipient coagulation of the albuminous protoplasm occurs, resulting in an inability to function. Water is necessary for this coagulation. The follow- ing table from Frost and McCampbell illustrates this point: Egg albumen + 50 per cent, water coagulates at 66° C. Egg albumen + 25 " " " 74-80° C. Egg albumen + 18 " " " 80-90° C. Egg albumen + 6 " " " 145° C. Egg albumen + no water " 160-170° C. PHYSIOLOGY OF MICHOORGANISMS 53 This fact is emphasized by the laboratory methods of sterilization. The autoclave, with live steam at temperature of 120°, will destroy in ten minutes the most resistant spores, while in the hot-air oven a temperature of 150° to 170° for an hour is necessary. 3. Reaction and Composition of Medium. — The reaction and composition of the medium has been found to exert a marked influ- ence on the thermal death point. Particularly should the hydro- gen-ion concentration of the medium, that is, the actual acidity, be noted. It is a well known fact, for example, that acid fruits are much more easily sterilized than are the more nearly neutral vegetables. 4. Time of Exposure. — In general, the higher the temperature the shorter the period required to destroy life in the cells. Math- ematic formulas have been developed giving the time as a func- tion of temperature for some forms. Ten minutes' exposure is the usual standard. 5. Specific Character of Organism. — Intrinsic variations in the character of protoplasm of different species make it necessary to determine the thermal death point for each species. Light Relationships of Microorganisms A few bacteria possessing bacteriopurpurin require light for their development. For most other microorganisms, particu- larly the bacteria and the pathogenic protozoa, darkness is the optimum hght condition. Light, particularly the direct rays of the sun, will destroy all but the most resistant bacteria if exposed for a sufficient length of time. Sunlight when passed through a prism is readily broken up into its constituent colors, the least refracted rays, or the reds and yellows, at one end, and the more highly refracted rays, the blues and the violets, at the other. Exposure of bacteria to these various colored rays has shown the blues and violets to be most powerful, while the reds and yellows of the other end have little or no effect. It will be re- membered that the blue and violet rays are the ones which affect the photographic plate most intensely. The germicidal action of light on the pathogenic bacteria is of the greatest practical importance. It renders infection through the medium of the air in most cases a remote possibility. 54 VETERINARY BACTERIOLOGY Even more powerful than the visible blue and violet light rays are the invisible ultra-violet rays. Lamps have been devised which give a maximum of ultra-violet hght, and these have been found to be quite efficient in certain types of sterilization. The lamp com- monly used is a modification of the Cooper-Hewitt mercury vapor arc, in which the glass has been replaced with quartz, for experi- ment has shown that most types of glass which permit of the pas- sage of visible hght rays are at least partially opaque to the ultra- violet, while quartz will allow the free passage of such rays. When clear liquids are exposed in relatively thin layers to the rays from such a lamp, the bacteria present are quickly killed; the liquid is steriUzed. The process is not efficient, however, when the liquid to be treated is quite turbid, or opaque, or opalescent, as milk. It is probable that the ultra-violet hght produces a coagulation or some other irreversible change in the protoplasm of the bacterial cells, for solutions of proteins exposed are precipitated. Sterilization of water by ultra-violet light has been used suc- cessfully in the purification of water supphes, and of water used in swimming-pools, etc. Apparently where properly used it is eSi- cient. Efiect of Electricity on Bacteria Strong direct currents of electricity passed through a solution containing bacteria will sterilize it. It is difficult, however, to dissociate the physical effect of the current directly upon the bacteria from the action of the chemicals produced by electrolysis. No practical use has been made of the destructive action of electricity upon microorganisms, as the method is difficult to apply and is inefficient at best. The Rontgen rays (x-rays) do not destroy bacteria even when the latter are exposed for consid- erable periods. Relationships of Microorganisms to Chemicals Microorganisms are profoundly affected both in growth and movement by the chemicals with which they come in contact. They may be attracted or repulsed, stimulated to increased growth, their development inhibited, or they may be destroyed when certain substances are present. PHYSIOLOGY OF MICROORGANISMS 55 Chemotaxy. — Motile microorganisms are attracted or repulsed by certain chemicals. The first is known as positive chemotaxy, the latter as negative. Certain protozoa and bacteria are attracted by oxygen and may be observed to swim about air bubbles under '■^M> ' FJg- 24. — Chemotaxy: a, Spirilla attracted by a green algal cell which is giving off oxygen, aerotaxis; 6, a leukocyte containing several bacteria which it has enguHed; c, capillary pipette containing a solution of beef extract, and at 2 an air bubble, placed in a drop of water containing motile bacteria. The latter are attracted in large numbers to the mouth of the tube; d, an air bubble surrounded by two concentric circles of organisms, the inner one bacteria, the outer protozoa. Each remains in the concentration of oxygen most favorable to its growth. the microscope. From their movements it is evident that differ- ent species prefer varying amounts of oxygen. This results in a grouping of the different kinds in concentric circles about the bubble. This type of chemotaxy is called aerotaxy (not aero- tropism) . The avidity of certain bacteria for oxygen has been used in the laboratory for their isolation from water, particularly the Asiatic cholera organism. Peptones and meat extractives at- tract many kinds of bacteria. This phenomenon may be readily demonstrated by introducing the tip of a minute capillary tube filled with such a solution into water containing numerous bacteria. These will be found to congregate in great numbers about the mouth of the tube and to enter it. Probably chemotaxis accounts for the flocking of the leukocytes or white blood-corpuscles to any part of the body attacked by certain bacteria. Micro- organisms are not always attracted by food stuffs and repelled by harmful substances. A mixture of peptone and mercuric ehlorid will attract bacteria and then destroy them. 56 VETERINAEY BACTEBIOLOGY Tropisms. — Organisms which are not free to move in response to a chemotactic stimulus may nevertheless be influenced in their direction of growth. Such a response in the direction of growth to an external stimulus is called a tropism. Mold hyphae will often grow toward a moist medium, while the conidiophores which they bear rise at right angles to its surface and seek to produce the spores as far as possible from a moist surface. These phenom- ena are known respectively as positive and negative hydrotropism. -mxu Fig. 25. — Chemotropism: a, b, Mold hyphse and conidiophores, showing the negative hydrotropism of the latter; c, an air bubble in a medium with four germinating mold spores. The hjrphse are growing toward the air, showing positive aerotropism. The influencing of the direction of the growth by the action of chemicals is called chemotropism. The forms of mold and bacterial colonies, when growing upon artificial media, are largely determined by this factor. For example, many molds radiate in practically straight lines from their point of origin in a medivun,and every branch quite exactly bisects the angle between the two filaments on either side. This mutual repulsion of the hyphffi is doubtless due to certain of the products excreted by the cells. Heliotropism, or the influence of hght on the direction of growth, is also observed in some forms. Influence of Reaction of Medium on Growth. — Many organisms are quite exacting in their requirements as to the reaction of the medium in which they are grown. Some forms grow best in a medium slightly acid, others refuse to develop except in one which is slightly alkaline. The majority of bacteria, however, grow well in a medium that is neutral to litmus. Antiseptics and Disinfectants An antiseptic is usually defined as anything that will inhibit the growth of microorganisms without necessarily destroying PHYSIOLOGY OF 'MICROORGANISMS 57 them. Inasmuch as anything which completely stops growth of bacteria must also kill them more or less rapidly an antiseptic, strictly speaking, is any substance which produces a relatively slow rate of death in bacteria. In the broadest sense, this term would include such physical agencies as the action of cold and heat, but in practice it is generally confined to chemical sub- stances. A disinfectant is a substance that will destroy patho- genic bacteria. Inasmuch as pathogenic and non-pathogenic bacteria are both destroyed by the same substances, there is little real difference in the meaning of disinfectant and germicide (something that will destroy all bacteria). A disinfectant, as distinguished from an antiseptic, kills bacteria rapidly. A deodorant is any substance that masks disagreeable odors or eliminates them entirely by removing their cause. A deodorant may or may not be a disinfectant or antiseptic. These latter terms are relative ones only, for any disinfectant if sufficiently dUuted becomes an antiseptic. Theories of Action of Antiseptics and Disinfectants. — Germ- icides may destroy microorganisms by forming compounds with the protoplasm, by dissolving or coagulating the protoplasm, or by oxidation and complete destruction of the cells. Our most efficient disinfectants are those which destroy in the manner first named. The activity and efficiency of many disinfectants depend upon the ionization of the compound in solution. This is particularly true with the salts of heavy metals, such as mercuric chlorid (corrosive sublimate). It is the ionized mercury that is poisonous to bacteria. Mercuric chlorid does not ionize in pure alcohol, hence it is not poisonous to bacteria when in solution in that substance. The addition of various other chemicals may increase or decrease the ionization of the disinfectant and enhance or diminish its destructive action. Characteristics of an Ideal Disinfectant. — Certain character- istics may be listed as belonging to an ideal disinfectant. To the extent that a particular disinfectant measures up in its character- istics to those of the ideal, it is valuable for general use. The more important of these characteristics are as follows: 1. Germicidal Power. — The ideal disinfectant should possess high germicidal power, that is, it should kill bacteria in dilute 58 VETERINARY BACTERIOLOGY solution. The ability of a disinfectant to kill microorganisms is usually compared with that of phenol. In making such compari- sons it is customary to use the Bacterium typhosum, the cause of typhoid fever, as the test organism. Most commercial disinfectants, particularly the coal tar products, are sold upon the basis of their phenol coefficient. 2. Stability.— The disinfectant to be most valuable should be relatively stable in the presence of organic matter. Some of the most powerful of the disinfectants combine with organic matter forming insoluble compounds and pass out of solu- tion relatively completely. The strength of the disinfectant may be thereby rapidly decreased to a point where it no longer destroys microorganisms. 3. Homogeneity. — Disinfectants should be homogeneous in composition. Substances which may be bought in pure condi- tion or in crystalline form such as mercuric chlorid are ideal from this point of view. Many of the commercial disinfectants, particularly those prepared from coal tar, may vary considerably in their composition from time to time, and conseq^lently in their germicidal value. 4. Solubility. — The ideal disinfectant is one which will dis- solve in all proportions in water, as alcohol, for example. 5. Non-toxic to Higher Life. — An ideal disinfectant would be non-poisonous to man and to the higher animals. Obviously disinfectants which will kill one kind of cell and not injure another are not the most common. Most of the valuable disin- fectants are more or less injurious to tissues. Certain disinfec- tants, however, may be injected into the blood, exerting a more harmful influence upon microorganisms than upon tissues of the body, destroying the former without seriously injuring the latter. Such, for example, is the arsphenamine used in the treatment of syphilis. 6. Non-corrosive. — Inasmuch as disinfectants must frequently be used in contact with metals, fabrics, etc. it is highly desirable that they should not attack metal, injure fabrics, leave stains or bleach color. 7. Penetration. — Disinfectants differ decidedly in their power PHYSIOLOGY OF MICROORGANISMS 59 to penetrate materials to be sterilized. An ideal disinfectant is one which penetrates rapidly and eflBiciently. 8. Economy. — The ideal disinfectant should be low in cost. Certain valuable disinfectants, because they are so expensive, can be used only in limited quantities and for special purposes. The high cost of salts of silver for example limits the use of silver practically to medicine. If the entire water supply of a city is to be sterilized obviously a disinfectant must be chosen which is relatively inexpensive. 9. Power to- Remove Dirt and Gfease. — A film of oil or grease over the surface of certain materials may wholly prevent the action of many disinfectants. Those which have power to dis- solve or remove grease and all kinds of dirt are naturally more efficient. 10. Deodorizing Power. — A disinfectant which can combine with and destroy malodorous substances is preferable to one which does not. Disinfectants and Antiseptics in Common Use. — The addition of acid to any solution containing bacteria with the consequent increas'e in hydrogen ion concentration increases markedly the rate of death. Bacteria which grow well in a relatively high hydrogen ion concentration are termed acidophiles. The ability of these or- ganisms to grow in the presence of high concentrations of acid is sometimes utilized in their isolation. Acids are most commonly used as preservatives. The more important are the acetic (usually in the form of vinegar) and the lactic acids, the latter the preservative agent in sour milk, sauer kraut, and in silage. Strong acids are those which ionize most completely. Weak acids are those which ionize poorly. Lactic and acetic acids are relatively weak acids, and must be present in relatively large amounts in order to secure a high concentration of hydrogen ions. For example, tenth normal hydrochloric acid contains about ten times as many hydrogen ions as normal acetic acid. In a few acids either the anion or undissociated acid molecule may also exert a disinfection action. In some, this action is much more important than that of the hydrogen ion. For 60 VETERINARY BACTERIOLOGY example, benzoic acid is a comparatively weak acid and yet it has relatively high antiseptic or disinfecting value. Alkalies. — High alkaUnity, that is high concentration of hydroxyl ions, likewise exerts a destructive action upon micro- organisms. Lime owes its value as a disinfectant to this fact. Unslaked Ume (calcium oxide) added to water is converted into calcium hydrate and when used in strong solutions, as in white wash, it has a marked disinfecting action. It is a valuable disinfectant to mix with stools or body excreta in order to destroy disease-producing bacteria. It should be noted, however, that upon exposure to air calcium hydrate is gradually converted into calcium carbonate which is no longer germicidal. Some bacteria will grow in relatively alkaline solutions. For example, the organism causing Asiatic cholera, the Vibrio choleroe, wUl grow in much higher hydroxyl ion concentration, that is, in more alkaline solutions, than will most other species of bacteria. Use is made of this fact in isolating this organism from feces, the medium being made so alkaline as to inhibit the growth of most other microorganisms rendering the securing of this organism in pure culture comparatively easy. Salts of the Heavy Metals. — The salts of gold, silver, copper, and mercury are all active disinfectants. Copper sulphate is sometimes used in an effort to free water reservoirs and other city supphes from growths of objectional algse. Mercury is the most efficient of the metals and its salts are most commonly used. It acts by forming a mercuric albuminate of the protoplasm. When used in any solution containing consider- able quantities of protein or similar materials, as in feces, it must be added in excess and thoroughly mixed, for it is apt to form an insoluble coating over the surface of the sohd particles and protect the bacteria in the interior from destruction. Mercuric chlorid is usually used in solutions of 1:1000 or 1:2000. Phenol, or carbolic acid, CsHsOH, and the methyl phenols or cresols, C6H4CH3OH, either pure or in the trade mixtures, such as kreso, tricresol, creolin, etc., are among the most efficient and useful of disinfectants. They are most frequently used in. PHYSIOLOGY OF MICROORGANISMS 61 1 to 5 per cent, solutions, and will destroy bacteria even in the presence of quantities of organic matter. Sulphur Dioxid and Sulphurous Add. — When sulphur is burned it yields sulphur dioxid, a gas that has been much used in fumigation. It is powerless to destroy bacteria imless moisture is present, with which it may unite and form sulphurous acid. The latter is a very active bleaching and corrosive agent, hence it should not be used except where it can do no harm. One pound of water (about 1 pint) should be vaporized in a room for every 5 pounds of sulphur burned. This amount should efficiently disinfect 1000 cubic feet. Insects and other vermin are destroyed by the sulphur fumes. Alcohol is frequently used as an antiseptic, and sometimes as a disinfectant. The efficacy of this material is dependent upon the concentration. Beyer has shown that 70 per cent, alcohol is most powerful, proving to be thirty times as efficient as 60 per cent, and forty times as powerful as 80 per cent. Concentrations below 60 per cent, and above 80 per cent, are apparently almost valueless. Formaldehyd, HCHO. — Formaldehyd is the gas used most widely in fumigation and disinfection. It is very soluble in water and is commonly sold as formalin, a 40 per cent, solution of formaldehyd. Like sulphur dioxid, formaldehyd is efficacious only in the presence of moisture, but, unlike it, does not bleach fabrics or injure materials. Formaldehyd may be evolved in gaseous form for disinfection in a variety of ways. Incomplete combustion of methyl alcohol according to the reaction 2CH3OH + O2 = 2HCH0 + 2H2O is utilized in a number of lamps upon the market. When properly carried out the method may be efficient, but it has several disadvantages, i. e., expense and presence of a fire in a closed room. Heating the formalin over an open flame will liberate a part of the formaldehyd readily, but under these conditions it polymerizes and some of the polymers (paraformaldehyd) are insoluble. If the evaporation is continued to dryness, all of these will again be broken up and given off as formaldehyd. ■* The same result can be reached more quickly by the addition of glyc- erin or some salt which will raise the boiling-point of the solu- 62 VETERINARY BACTERIOLOGY tion above the dissociation temperature of the paraformaldehyd. An autoclave or closed vessel in which the solution is heated con- siderably above the boiling-point of water will serve the same pur- pose. Twelve ounces of formalin should be used for every 1000 cubic feet to be fumigated. A convenient method for fumigating small rooms is to pour formalin over crystals of potassium per- manganate in an earthen vessel that is a poor conductor of heat. The permanganate is an active oxidizing agent and converts part of the formaldehyd into carbon dioxid and water, with the liberation of sufficient heat to vaporize a large portion of the remainder. The soUd paraformaldehyd or paraform may be heated and is thereby converted into formaldehyd gas. On account of its cheapness and effectiveness formaldehyd is used much more commonly at present than any other of the gaseous disinfectants. Adjustment of Organisms to Osmotic Pressure.^Any crystal- loid in solution behaves within the limits of the solution hke a gas, and the same laws of diffusion and diffusion pressures are applicable. Every organism when growing is surrounded by water containing substances in solution, and it also contains certain salts dissolved in the " cell sap " or water in the pro- toplasm. The ectoplast or limiting membrane of the protoplasm lying just within the cell wall is certainly in most, probably all, cases a semipermeable membrane, i. e., it will allow some sub- stances to pass through readily, as water; others pass through slowly, and still others, although in true solution, cannot pass at all. This ectoplast, in short, serves as an osmotic membrane and determines what substances may enter and leave the cell. An active cell always maintains within its sap a greater con- centration of solutes than the surrounding medium, the pressure on the inside of the membrane is greater than on the outside, and the cell is said to be in a state of turgor. When such a cell is placed in water containing a greater percentage of solutes than does the cell sap, water leaves the latter until the concentra- tion on the inside and outside again becomes the same. This means a shrinking of the protoplasm; it withdraws from the cell wall and. the cell is said to be plasmolyzed. After a time the cell may readjust the amount of solutes in the cell sap and regain PHYSIOLOGY OF MICROORGANISMS 63 its turgor. For every cell, however, there is a Hmit beyond which the organism cannot go. Some yeast cells have been found to develop slowly in a solution containing 35 per cent, of cane- sugar. A solution of this concentration exerts a pressure of more than 350 pounds per square inch. It is apparent that such a cell must be profoundly modified. The fact that concentration of solutes inhibits the growth of microorganisms is utilized in the preservation of many food stuffs. Such foods as syrups, jellies, and candied fruits are preserved by the high osmotic pressure of the solutes. The action of sugars, salts, etc., is in the nature of a physical antiseptic. Physiological salt solution is one having the same concentration of salt as do the body cells of the particular organism to be studied. It usually contains .85 per cent, of sodium chlorid. Symbiosis, Antibiosis, and Commensalism Two organisms that live together and which are mutually beneficial are said to live in symbiosis. Each organism is called a symbion or symMont. The symbionts are not necessarily closely related forms and may belong to the most widely separated groups of plants, as, for example, bacteria and members of the bean family of the flowering plants. Antibiosis is that condition which obtains when organisms prove inimical to each other's development. The growth of one species of organism in a culture-medium may completely inhibit the development of some other type. For example, the organism (Streptococcus ladicus) which ordinarily sours milk prevents the development of most other species. An organism which uses the by-products of another as food, in other words, is parasitic without producing disease, is called a commensal. Many of the bacteria found on the skin, in the mouth, and in the intestinal tract of man and animals are of this character. All degrees of intergradation between symbiosis, true para- sitism, and commensalism have been described for different species. Pigment Production by Microorganisms Molds, yeasts, and bacteria are frequently found to be chromo- genic, that is, capable of producing pigments or coloring-matter. 64 VETERINARY BACTERIOLOGY The colors produced range through all the colors and even shades of color of the spectrum. A few only of the pathogenic forms produce pigments. Many organisms, particularly molds and bacteria, excrete a pigment-forming substance which diffuses through the culture-medium and colors it. Such an organism is the Pseudomonas pyoqjanea, which produces a diffuse pigment, one that changes the medium first to a green, then to a brown. Some organisms produce pigment granules outside the cell, such are said to be chromoparous, for example, Erythrobacillus prodi- giosus, which produces a red pigment. The cell walls of some bacteria (such as B. violaceus) and of many of the molds are colored. Pigments are generally produced only in the presence of free oxygen. Cultivation at high temperatures causes some organ- isms to lose the power of pigment production. Some pigments are soluble in water, others in alcohol, and others in ether and various fat solvents. They are of little economic importance, but are of value to the systematic bacteriologist in the separation and identification of species. Light Production by Microorganisms Several species of bacteria and fungi are known that give off light. These are said to be photogenic. Bacteria of this type are found commonly in the water of the ocean, and are easily isolated from salt fish. When grown in a test-tube, they are sometimes sufficiently luminous, so that they may be photo- graphed by their own light. Fermentation and Enzyme Production All microorganisms have their protoplasm bounded by the ectoplast, a semipermeable membrane, as previously shown. The cell wall, when present, seems to be a mechanical protection and support, and is readily permeable to most substances in solution, so may be disregarded in discussion. Whatever food or other mate- rials are taken into the protoplasm must pass by diffusion through the ectoplast. This membrane, on the other hand, must prevent any valuable constituent of the cell from leaving by diffusion. Its action is, therefore, selective.. Microorganisms do not always find the potential food materials with which they are surrounded PHYSIOLOGY OF MICROORGANISMS 65 suitable for food, for they may be in a solid form or, if in solution, of such a character that they cannot pass through the ectoplast. Many organisms find it necessary to so change this food and digest it that it may be assimilated. Once inside the cell, it usually is not of such a character that it can be built up directly into the protoplasm and further changes are necessary; or if the Pig. 26. — ^A bacterial lamp. The inner wall of the flask is coated with a medium on which there is growing Bacterium phosphoreum. Photographed by- its own light (Molisch). material is used simply as a source of energy and not incorporated into the cell substance, it is essential that the cell have some means of developing this energy. All cells accomplish these changes by means of enzymes (Gr. en, within, zyme, leaven). A dis- tinction was once made between the so-called organized and unorganized ferments. The former was held to be living cells which could bring about a change or fermentation, the latter any cell secretion which could bring about such changes. In other 66 VETERINARY BACTERIOLOGT words, organized ferments were supposed to owe their activity directly to the protoplasm, the unorganized, to substances secreted by the protoplasm. This distinction is no longer maintained, as it seems altogether probable that fermentative changes of whatever kind are brought about by the secreted enzymes or unorganized ferments. Enzymes may be intra- or extracellular. The intracellular enzymes are extracted from the protoplasm with difficulty, and during the hfe of the cell do not leave it. Such an enzyme is that of bread or brewer's yeast (the zymase), which converts dextrose into alcohol and carbon dioxid. Extra- cellular enzymes are usually digestive in their action. Different kinds attack different substances. Microorganisms are known which produce enzymes that will break down cellulose, starch, sugars, fats, and proteins into simpler substances. The action of an enzyme is said to be specific; a given enzyme will in gen- eral change only one type of material. Enzymes are said to be organic catalysts (Gr. kata, down, lyo, to dissolve), that is, they bring about changes without themselves becoming part of the final product. Many inorganic catalysts, such as finely divided platinum, are known to the chemist. Although catalysts do not form a part of the final products, they certainly are a part of some of the intermediary- products in many cases, but become free again when the action has been completed. Enzymes, then, are peculiar in that they are not used up in the using. Theoretically the amount of change that can be brought about by a given enzyme is limited only by the time and conditions under which it must act. Most enzymes produce changes that are hydrolytic in nature, that is, they bring about the incorporation of water into the organic molecule with resultant disintegration. The digestion of gelatin by the bacterial enzyme gelatinase, the conversion of starch into maltose by ptyalin and diastase, the digestion of proteins by pepsin, the conversion of saccharose or cane-sugar into invert sugar by invertase, and the clotting of milk by rennet are a few examples of such hydrolytic changes. The following reaction illustrates the hydrolytic cleavage of saccharose by the invertase produced by yeast : CizHjjOu + HjO + Invertase 5i=T CeHijO, + C,HuO, + Invertase. Saccharose. Dextrose. Levulose. PHYSIOLOGY OF MICROORGANISMS 67 Other enzymes are active oxidizers. Changes of color in dead or injured plant and animal tissues are sometimes due to such oxidases. For example, potatoes turn black and apples become brown when the cells are bruised. Some other enzymes are said to be splitting. One of the best examples of these is the zymase of yeast, which converts dextrose into alcohol and carbon dioxid. CHuOe + Zymase = 2C2H5OH + 2CO3 + Zymase. Dextrose. Alcohol, Carbon dioxid. Although alcohol and carbon dioxid represent the end-products, it is by no means certain that intermediate hydrolytic products are not formed, and this splitting action may be essentially hydro- lytic. Reducing enzymes have also been demonstrated in plant and animal tissues and imdoubtedly occur in microorganisms. The autolytic (Gr. auto, self, lyo, dissolving) enzymes de- serve particular mention. Enzymes are known to occur in most animal and plant cells that will, at least partially, digest the cells in which they occur. The rigor mortis or stiffening of the tissues of an animal after death is due to such an autolytic enzyme which coagulates the muscle protoplasm. The softening of the tissues which occurs later, the so-called " ripening " of meat, is due in part to the action of another proteolytic enzyme which carries the digestion somewhat further. Microorganisms contain such en- zymes, and when the cells die, as in an old culture, they are then partially digested. This autolytic action we shall find to be of some practical significance in a discussion of disease production and immunity, as by it certain poisonous substances may be released from the cell. CHAPTER IV CHANGES OF ECONOMIC SIGNIFICANCE BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS Microorganisms bring about many changes, both analytic and synthetic, in the media in which they are cultivated. In any such medium growth products of many kinds will be found. These fermentative products may originate from the activity of extracellular enzymes, may consist of substances excreted from the cell as the product of intracellular enzymes, or as a result of the metabolic activity of the cell; they may be products of syn- thetic action (as the slimes and gums produced by a solution of the bacterial capsule), or they may be substances produced by the autolytic activity of intracellular enzymes after the death of the cell. Naturally, the products will vary greatly with the species of organism, the medium in which it is grown, and the character of the physical environment. From the standpoint of the veterinarian, microorganisms are of most importance because many of them produce disease. It would, however, give a false impression of the place and function of microorganisms in nature to neglect at least a brief consid- eration of some of the other changes which they can bring about. In this chapter some of the more important will be considered. The well-nigh universal distribution of bacteria and other microorganisms should be emphasized. They are to be found in the soil in great numbers, rich surface soil containing from 100,000 to 5,000,000 bacteria to every dry gram. Their dried bodies and spores are constantly present free or attached to dust particles in the air. They are to be found in all surface waters in considerable numbers and are present even in the water from deep wells. They grow upon the surface of the skin of animals, and the mouth and digestive tract support a large and varied flora. It is apparent that whenever conditions are favorable for the growth of micro- organisms they will be present to begin growth. 68 CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 69 The changes brought about by bacteria in nature are of such importance that but for their continuance plant and animal life on earth would quickly cease to exist. The fertility of the soil and the consequent production of all food stuffs is directly due to certain of the microorganisms present. Production of Alcohol. — The various alcohols, but more par- ticularly ethyl alcohol, are produced by certain bacteria, yeasts, and molds. It has been shown that in the yeast the ability to bring about this change is resident in the intracellular enzyme, zymase. Probably similar enzymes are present in the alcohol- producing bacteria and molds. The common bread or brewer's Fig. 27. — Brewer's yeast, Saccharomyces cerevisice. yeast is the form most commonly used in the manufacture of alcohohc beverages, but certain molds have been found very useful in the production of alcohol for industrial purposes. Alco- hol is commonly produced by the fermentation of one of the hexose monosaccharids, such as dextrose. The reaction may be given as follows : CeHi^O, = 2C,H50H + 2CO2. Dextrose. Alcohol. Carbon dioxid. Yeast is utilized for its other product of fermentation, carbon dioxid, by the baker in bread making. Higher alcohols, such as butylic and amylic, are also formed. The yeasts which produce this change are quite widely distributed in nature, being par- ticularly abundant on the surface of fruits and in saccharine liquids. Fruit juices and other solutions containing sugar if allowed to stand, therefore undergo " spontaneous " fermenta- tion, with production of ciders, wines, and similar beverages. Production of Acids.— Several of the organic acids are com- monly produced by fermentative organisms. Three of these are 70 VETEHINAHY BACTERIOLOGY of particular economic importance, namely lactic, acetic, and butyric. A great variety of others are occasionally produced, usually in small quantities only. Kg. 28.— Lactic acid bacteria: A, Streptococcus lacticus; B, LactobacUlus bidgaricus. Lactic Add.— Dextrose and some other monosaccharids are converted into lactic acid by several common organisms. The reaction may be empirically represented as follows: C„Hi,Oe = 2C,H,-0H-C00H. Dextrose. Lactic acid. The reaction occurs most frequently in milk which is allowed to stand. In this case the lactose or milk-sugar is first broken down into the monosaccharids before being converted into lactic acid. C,,H,,Ou + H,0 = C,Hi,Oe + CeHi,0„. Lactose. Dextrose. Galactose. The formation of lactic acid in milk is of the greatest economic importance, as the organisms which produce this acid are the ones Fig. 29. — Acetic acid organism, Acetobacter aceti: a, Normal individuals; b, involution forms. (Adapted from Hansen.) which are necessary to the development of proper flavors and quality in butter and cheese. This acid is also produced in the manufacture of sauer-kraut and to some extent in silage. The lactic acid formed in milk is instrumental in preventing the growth of putrefactive and other undesirable bacteria. CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 71 Acetic Add. — Acetic acid is the most important and the characteristic constituent of vinegar. It is produced by several species of bacteria by the oxidation of ethyl alcohol according to the following reaction: CH^OH + Oj = CH3COOH + H,0. Alcohol. Aoetio acid. Any solution containing alcohol, if left in contact with free oxygen, will commonly undergo this fermentation spontaneously, as Acetobacter aceti, the organism usually responsible, is ubiquitous. To insure rapid and efficient fermentation the cider or other alcoholic solution is sometimes inoculated with mother of vinegar, a mass of the organism which commonly forms a mat upon the surface of the fermenting liquid. %t Fig. 30. — ButjTic acid bacteria, Clostridium butyricum. (Adapted from Fischer.) Butyric Add. — Under anaerobic conditions saccharine solu- tions are apt to undergo butyric acid fermentation as a result of the development of the Clostridium butyricum or a related form. The reaction may be represented as follows: C,H,,Oe = CjHjCOOH + 2CO2 + 2H,. Dextrose. Butyric acid. Butyric acid has an exceedingly disagreeable odor and taste, hence the growth of this organism in any saccharine or starchy food substance renders it unfit for use. Inasmuch as these organisms are all spore producers, they resist heat well, and as they are anaerobic they will grow when sealed in a can and all air excluded. Rancidity in butter is sometimes due, in part at least, to the development of butyric acid. Decay and Putrefaction. — A distinction is sometimes made between the terms decay and putrefaction. The former is said to 72 VETERINARY BACTERIOLOGY be decomposition of organic matter brought about by the aerobic bacteria, the latter by the anaerobic types. This distinction is not always acknowledged and adhered to, however. The ' substances produced by the decomposition by bacteria depend, of course, quite largely upon the nature of the material to be de- composed. The carbohydrates and fats break down iiito alcohols, acids, and carbon dioxid, but the proteins are split into a great variety of substances. Other agents, as acids and alkalis, will break up the proteins in a similar manner and into many of the same substances as do bacteria. Chemists have in recent years demonstrated that proteins are made up of large numbers of molecules of the a-amino acids linked together. An a-amino acid is an organic acid that has the NHj group in the alpha position, that is, next the carboxyl. For example, the amino acid corre- sponding to C2H5CH2COOH, butyric acid, is C2H5CHNH2COOH. When these constituent links of the protein molecule are forced apart, they usually appear in the form of one of about twenty com- pounds which have been grouped as primary protein derivatives. Some of these' normal derivatives are further altered by bacteria. Among them have been found certain compounds called ptomains, some of which are known to be very poisonous. The splitting usually continues until much of the organic matter is reduced to comparatively simple compounds, such as HjS, CO3, CH4, and NH3. The process of protein disintegration is called proteolysis. It usually occurs in several distinct stages. The proteins are first broken down into relatively complex substances called prote- oses, these then are broken down into peptones. This is called peptonization, as it is essentially the change that may be brought about by the enzyme pepsin from the stomach. The process con- tinues and the peptones become amino acids and at last ammonia is liberated. From an economic point of view this liberation of ammonia has the greatest significance, for from this transformation comes all the nitrogenous material used by plants and indirectly by animals as food. This is the essential transformation that all organic nitrogenous fertihzers, such as barnyard manure and dried blood, will undergo before they can be of any use to higher plants. By such changes water contaminated by sewage purifies itself. CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 73 Some of the nitrogenous products of bacterial decomposition are worthy of note, inasmuch as they are used in the laboratory in the differentiation of certain species. The most important of these are indol and skatol. They are organic compounds having the following formulas: CeH.< )CH C.h/ .)cH. Indol. Skatol. Indol is produced by certain bacteria when growing in a solution of peptone. It is identified by the addition of nitrous acid, with which it combines to form nitroso indol, a bright red compound. In making the test in the laboratory it is customary to add a Fig. 31. — Some decay-producing and putrefactive bacteria. few drops of concentrated sulphuric acid, followed by a dilute solu- tion of nitrite. The sulphuric acid breaks up the nitrite, with the formation of free nitrous acid, which then unites. with the indol. Indol and skatol are also formed in the intestines by the activity of certain of the bacteria found there and are of considerable physiological significance. Reduction Processes in Inorganic Compoiuids. — Changes similar to those just discussed are sometimes brought about by bacteria in inorganic compounds. When nitrates are in solution together with organic substances and under anaerobic conditions, the bacteria present in many cases will reduce the nitrates to nitrites and the nitrites to free nitrogen, apparently in order to utilize the oxygen. This process is usually called denitrification because the medium loses nitrogen, but is more correctly a reduction or deoxidation. Sulphates are reduced to sulphites and even to sulphids under similar conditions. For example, the sewage from 74 VETERINARY BACTERIOLOGY a city whose water supply contains a large percentage of sulphates will develop hydrogen sulphid in considerable quantities if it is put under anaerobic conditions. Other reductions of a similar nature have been described for chlorates. B t-^ A Fig. 32. — Denitrifying bacteria : A, Bacterium coli, which changes nitrates into nitrites; B, Pseudomonas denitrificans, which produces free nitrogen from nitrates. Oxidation of Inorganic Compounds. — Bacteria and other microorganisms that live in the presence of oxygen are sometimes active oxidizers of inorganic compounds, securing in this manner the energy that is necessary for their various growth processes. iy Fig. 33. — Sulphate reducing spirillum, Spirillum desulfuricans. Oxidation of Hydrogen Sulphid. — Waters containing hydrogen sulphid, as do many of the so-called mineral springs, usually contain bacteria which gain their energy for food manufacture B Fig. 34.— Microorganisms that oxidize hydrogen sulphid: A, B, Beggiaioa sp.; C, Thiophysa volutans (Hinze). and growth from the oxidation of this substance. The slimy black and white deposit commonly found in such waters, when examined microscopically, will be seen to be made up of masses of CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 75 Beggiatoa and similar organisms whose cells will be found packed with sulphur granules. Probably the following reaction accounts for this formation of free sulphur: 2H,S + O2 = 2H,0 + S,. The process is carried still farther if there is any deficiency of the hydrogen sulphid, and the free sulphur is converted into sulphuric acid and sulphates. S2 + 3O3 = 2SO3 H,0 + SO3 = HjSO^. The sulphuric acid is, of course, at once neutralized by the bases present in the water. Oxidation of Iron. — Many natural waters contain ferrous car- bonate or some similar salt of iron. Certain bacteria oxidize Fig. 35. — Microorganisms that oxidize ferrous to ferric iron: a, Lep- tothrix ochracea; b, Gallionella ferruginea; c, Spirophyllum ferrugineum. (Adapted from Ellis.) this to ferric hydrate, and deposit this insoluble material in their sheaths. The reaction may be represented as follows: 2Fe2C03 + 3H,0 + O = Fe,(OH)e + 2C0,. Probably these organisms make use of the energy obtained by this reaction in the same manner that the sulphur bacteria do the oxidation of the sulphur, to secure energy for the formation of their foods and to gain the energy needed for growth and development. These organisms are particularly apt to occur in well water or spring water laden with iron, and have in some cases caused considerable trouble by clogging the water pipes with their growth. It is known that the bog iron ore of Sweden and probably the 76 VETERINARY BACTERIOLOGY great iron beds of northern Minnesota have been deposited by the activity of such organisms. Oxidation of Ammonia.— In most soils there are numerous bacteria that oxidize free ammonia to nitrous acid, and by neutral- ization with the soil bases form nitrites. These organisms do not develop well in the laboratory in the presence of organic matter. It seems evident that they utilize the energy secured from the Fig. 36. — Bacteria that oxidize ammonia and nitrous acid to nitrous and nitric acid respectively: a, Nitrosomonas europea; b, N. javensis; c, Nitrobacter (Winogradsky). oxidation of the ammonia to build up their protoplasm out of simple materials. They are among the best examples of the strictly prototrophic bacteria. Organisms capable of bringing about this change are called nitroso-hacteria. The reaction may be represented as follows : NH3 + 2O2 = HNO3 + H2O. This is the first of the two steps in the process called nitrification in the soil. Oxidation of Nitrous Acid. — The nitrous acid formed in the soil and in water, etc., by the preceding group is further changed by another group of organisms called the nitrate bacteria. Like the preceding, they are widely distributed in water and soil, and complete the process called nitrification or, better, oxidation of nitrogen. The nitrates produced by their activity are the source of nitrogen for green plants. A few of the latter are able to make use of nitrogen in the form of ammonia compounds, but in nature this rarely occurs. The reaction may be represented as follows: 2HN0, + 02 = 2HNO3. It is probable that the fertility of the average soil is more largely determined by the maintenance of conditions favorable to the CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 77 development of these nitrifying organisms than by any other single factor. Their importance in nature as essentials for the growth of the higher plants and, therefore, of animals can scarcely be overestimated. Nitrogen Fixation. — The free nitrogen of the air is so inert that very few living plants are capable of making use of it in the building up of their bodies. None of the green plants can bring this about of themselves. Certain molds and bacteria are able to make use of this source of nitrogen, however, and are, therefore, of the greatest economic importance. The fer- tility of the soil is largely dependent upon the fixed nitrogen that it contains, and the taking up of the free nitrogen by these organisms ultimately renders it available to other forms of plants. This does not mean that the bacteria take up the nitrogen from the A. /^|B^ Fig. 37. — Free living or non-symbiotic nitrogen-fixing bacteria: A, Clostridium ■pasteurianium; B, Azotdbacler; a, A. chroococcum; b, A. agilis. (A after Winogradsky, B after Beyerinck.) air and immediately transform it into nitrates for the use of the higher plants, but that it is built up into their protoplasm and ultimately is set free by the death and decomposition of the organ- isms. Microorganisms which make use of free nitrogen commonly utilize carbonaceous materials as a source of energy. Organisms capable of fixing nitrogen are subdivided into two general groups, those which live free in soils and those which live in or on the roots of certain plants in a kind of symbiosis. The free living organisms which fix nitrogen belong to three general groups: first, certain anaerobic types belonging to the general group of butyric acid bacteria; second, certain aerobic species; third, a few molds. The anaerobic organism known to fix the nitrogen is Clostridium pasteurianium. Probably this organism is not of the greatest importance, as the conditions for 78 VETERINARY BACTERIOLOGY its development do not often obtain. Bacteria of the nitrogen- fixing aerobic type belong to the group called Azotobacter. These organisms are abundant in many soils and fix considerable quan- tities of nitrogen, gaining energy therefor by oxidizing the car- bonaceous materials from dead plant tissues. The addition of straw, for example, to a soil will furnish sufficient food so that these bacteria will bring about an appreciable increase in the nitrogen content. The importance of the molds in this connection is not fully understood, but several species have been described which are capable of fixing nitrogen. The microorganisms which fix nitrogen in symbiosis with higher plants may be divided into two groups, those bacteria which grow upon the roots of legumes and the molds which grow on the roots of certain other plants. All plants belonging Fig. 38. — Bhizobium leguminosarum: a, Normal bacillar form; 6, bacteroids or involution forms. to the legume or pulse family, such as clover, alfalfa, peas, beans, etc., usually bear upon their roots tubercles or nodules which,, when opened, are found to be made up of cells tightly packed with bacteria. It has been shown experimentally that these organisms growing within the roots in some way take up free nitrogen from the air and eventually turn it over to the host plant, so that the legumes are not dependent for their development upon nitrogen which may be present in the soil, but can make use of the free nitrogen of the air as well. These plants are, therefore, very important in agriculture in the maintenance and increase of soil fertility. This organism, known as Rhizobium legumino- sarum (Fig. 38), enters the young growing root through a root hair and causes a kind of tumor formation in the tissues of the root, resulting in the development of the nodule. The organ- ism is at first a straight rod, but later, when growing inside the CHANGES BBOUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 79 cells of the host, it becomes much enlarged and shows many- involution forms. The other organisms growing symbiotically within or upon the roots of plants are all molds. They develop either upon the surface of the root, forming a white, cottony, floccose covering, or they grow in the tissues just below the epidermis. They sometimes cause nodules to develop, as is the case with the Russian olive and the alder, or they produce no characteristic overgrowth of tissues at any one point, but are Fig. 39. — Nodules on the root of a legume, Soy bean. (Moore, U. S. Dept. Agr.). found quite uniformly present upon the young growing roots. Certain trees, such as the oak and the pine, particularly when growing in nitrogen poor soils, show the development of this mycorrhiza (Gr. fungus and root). It has been shown that these molds are quite active in taking up free nitrogen from the air and are of benefit to the plant upon which they occur. 80 VETERINARY BACTERIOLOGY The Nitrogen Cycle— The relationship of microorganisms to nitrogen and its compounds has been noted in the preceding pages. These changes may be summarized as follows: Certain bacteria break down organic compounds containing nitrogen with the ulti- mate liberation of ammonia. Other species change the ammonia to nitrous acid and nitrites. Still other species transform the nitrites to nitrates, and these the higher plants take up from the soil and transform again into complex organic substances. These eventually again decay or are eaten by animals and converted into animal tissues. The nitrogen of both plant and animal tissues Proiiojts, \,p^^f,„u,.-ncn. ^'iTlCOJH plants. />> *ro. /ft 5"^ Fig. 40. — Nitrogen cycle. Changes brought about by bacteria indicated by solid lines, other changes by dotted lines. ultimately undergoes the change first noted and the nitrogen again appears as ammonia. This series of changes is called the nitrogen cycle. It is to be noted in addition that some bacteria are found which decompose nitrates with the formation of nitrites and liberate free nitrogen in the so-called process of denitrification. Other species, either alone or in symbiosis with higher plants, take up and fix free nitrogen from the air and eventually convert it into a form available for higher plants. These changes may be better imderstood by reference to the accompanying diagram. CHANGES BROUGHT ABOUT BY NON-PATHOGENIC ORGANISMS 81 Miscellaneous Changes. — ^Many changes are brought about by bacteria other than those which are here discussed. Micro- organisms are of importance in tanning, curing of tobacco, the preservation of food-stuffs, such as silage and sauer-kraut, the retting of flax and hemp, the curing of the so-called burnt or heated hay, and in many other ways. It is to be remembered that probably in all cases these changes are brought about by the enzymes produced by the bacteria. CHAPTER V CLASSIFICATION OF MICROORGANISMS It is necessary in a consideration of organisms belonging to either the plant or animal kingdoms to divide or separate them into groups, with their apparent relationships as the basis for the grouping. Microorganisms of pathogenic significance we have previously divided into the four groups — bacteria, yeasts, molds, and protozoa. A discussion of the classification of the last will be reserved to the chapter on Diseases Produced by Protozoa. The classification of micro-organisms is by no means in a satis- factory state. Many bacteriologists and others who have inves- tigated diseases have failed to recognize the importance of simple classifications and have introduced many new names needlessly. It is a principle of nomenclature accepted since the time of Lin- naeus that every plant and animal belonging to a distinct type or species shall receive a Latin name, this name to be made up of two words only. The second of these words is the species name, and is peculiar to the particular kind under consideration; the first is called the genus or generic name. For example, among higher plants there is the genus Quercus, or oak, which is subdivided into many species, such as white oak, red oak, swamp oak, etc. (Quercus alba, rubra, etc.) . The generic name is applied to all those species which resemble each other, as do all of the oaks. Species of plants and ani- mals are given names which are understood to serve as convenient terms for their designation. It is an established principle that the name first given to a plant or an animal is the one which should always be used whenever that name is in accordance with certain rules. Some bacteriologists have made the mistake of believing that a scientific name should be a description or even a descriptive term. It is no more necessary that the species name of a bacterium should describe that bacterium than that the ^ven or Christian name of an individual should describe him. Disregard of this rule has resulted in some very unwieldy names being given to micro- 82 CLASSIFICATION OP MICROORGANISMS 83 organisms, for example, names such as the following have been applied to bacteria: Bacillus membranaceous amethystinus mobilis, Bacillus argenteus phosphorescens liquefaciens, and even the fol- lowing. Bacillus saccharobutyricus flvx)rescens liquefaciens im- mobilis. Such names are given under the mistaken idea that the specific name should be a description of the species. This is not customary in naming any of the higher plants and animals, and it is certainly not more desirable in bacteria. The yeasts, molds, and protozoa much more commonly than the bacteria have been studied by those who have had technical training in nomen- clature, consequently the classification of these forms is on a much more satisfactory basis. The only justification for a specific name made up of more than two words is that the two words taken to- gether express but a single idea. In the classification of plants and animals, as has been noted, it is customary to unite species into genera. Related genera are likewise united into tribes, the tribes into families, the families into orders, and the orders into classes. Sometimes other groups are interpolated, for example, families are sometimes divided into subfamilies and these into tribes. In both botany and zoology these group names are characterized by certain terminations. All names of plant orders end in -ales; of families, in -acece; and of tribes, in -ece. Classification of Bacteria Many different classifications have been proposed for bacteria, but not one of these has come into general use. A careful exam- ination of different texts in bacteriology, particularly those devoted to the pathogenic bacteria, will show that different sys- tems and schemes of classifications are used in dealing with closely related organisms. Not only have the groups been fre- quently changed, but many different names have been applied to almost every one of the pathogenic bacteria. The consequence is that in studying any pathogenic organism it is necessary to give not only the name preferred by the author but also a list of the synonyms which have been used by others. It seems probable that a satisfactory system of nomenclature is yet to be devised. The system of bacterial classification which is here 84 VETERiNABY BACTEHIOLOGT outlined is that suggested by the Committee on Nomenclature of the Society of American Bacteriologists. The complete classification of bacteria from the standpoint of the botanist contains many forms of Httle or no economic importance, most having no sanitary or medical significance. In the classification here given most of these unimportant forms have been ehminated. The bacteria or Schizomycetes constitute a class which may be divided into five orders: the shme-mold bacteria (Myxo- baderiales), the true bacteria (Eubacteriales) , the sulphur bacteria (Thiobacteriales) , the thread bacteria or mold bacteria (Actinomycetales), and the spirochetes (SpirochcEtales) . The first and third of these orders contain no organisms of im- portance in veterinary medicine; they will be omitted in the classification. The order Eubacteriales or true bacteria includes those organ- isms which are least differentiated and least specialized. They are never filamentous and branched (except rarely in involution forms) and are never fiexuous though some are spiral. The order Actinomycetales or mold bacteria comprises those forms which normally produce elongate cells, tending to become fila- mentous, sometimes branched, and mycelum like. The order Spirochcetales includes protozoan like spirals, usually relatively slender and fiexuous. These orders will be discussed in order and the genera of veterinary significance noted. Order Eubacteriales. The True Bacteria Most of the common bacteria belong in this group, including the majority of the forms of medical significance. The cells may be spherical, cyhndrical or spiral, but not commonly filamentous and never branched, except in the so-called involutioa forms. They may be motile by means of flagella or non-motile. When spiral they are never notably fiexuous. In other words, they are neither mold-like nor protozoan-like. Altogether seven families have been described in this order of which five are of medical importance. They may be differen- tiated by reference to the following kej'-: CLASSIFICATION OF MICROORGANISMS 85- Key to the Families of the Eubacterialesi A. Cells spherical 1. Coccacese AA. Cells not spherical B. Cells cyhndrical. C. Producing endospores 2. Bacillacese CC. Not producing endospores D. Flagella when pres- 3. Bacteriaceae ent peritrichous DD. Flagella polar 4. Pseudomonadacese BB. Cells spiral 5. Spirillacese I. The Family Coccacese. The Spherical Bacteria The cells of the organisms belonging to this family are usually spherical when free, though during division they may be some- what elMptical. If the cells remain in contact after division they are frequently flattened at the plane of contact. They may form chains, packets or irregular masses. Very few cocci are motile; none of the motUe species are of economic importance. Spores are not produced. Most of the genera of this family are parasitic, many species are disease producing. A few are of importance because of the fermentative changes which they bring about. The most important genera are Neisseria, Strepto- coccus, Diplococcus, and Staphylococcus.^ These genera may be differentiated by the following key: Key to the Genera of the Coccaceas A. Cells occurring in chains 1. Streptococcus AA. Cells not in chains B. Cells in irregular masses, Gram positive 2. Staphylococcus BB. Cells in pairs C. Gram positive 3. Diplococcus CC. Gram negative 4. Neisseria ^ The other family Nitrohacleriacem includes a number of genera of agriculT tural importance. Among them may be noted NUrosomonas including soil forms oxidizing ammonia to nitrites, Nitrobacter including forms which oxidize nitrites to nitrates, Acetobacter including the acetic acid or vinegar bacteria, Bhizobium including the nitrogen-fixing bacteria of the roots of leguminous plants, and Azotobacter, free living nitrogen fixers. ^ Other genera are: Leuconostoc forming large gelatinous masses in sugar solutions; Rhodococcus including the cocci which produce a red pigment, Micrococcus including Gram negative cocci showing irregular masses and usu- ally yellow pigment and Sardna in which the cocci occur in regular packets. 86 VETEKINAEY BACTERIOLOGY 1. Streptococcus. — This genus includes those spherical bacteria whose cells occur normally in chains. For the most part the cells are Gram positive. Certain of the species are important in that they bring about lactic acid fermentation in milk. . Others are capable of producing diseases. Certain species, for example, are commonly associated with tonsilitis, rheumatism, with the disease strangles in the horse, wound infection, and pus pro- duction. Rg. 41. — Types of Streptococcus: a, d, Streptococci consisting of uniform ele- ments; 6, Streptococcus consisting of diplococcus elements; c, Diplococciis. 2. Staphylococcus. — In this genus the spherical cells are united in more or less irregular masses. They are Gram positive, and usually parasitic. They are commonly associated with pus production, wound infection, the development of boils, abscesses, and similar conditions in the bodies of man and animals. 3. Diplococcus. — The organisms belonging to this genus are strict parasites. Gram positive, occurring in pairs, the cells usually somewhat pointed and capsulated. The most important species is the organism usually associated with pneumonia. /a _. ^ 06©© a. Q Fig. 42. — Types of cocci: a, isolated cells; 6, siiowing tetrads, forming plates of cells or merismopedia; c, cells in an irregular mass — Staphylo- coccus. 4. Neisseria. — The organisms of the genus are strict parasites, usually growing rather poorly in artificial culture media. The cells usually occur in pairs, flattened at the proximal sides, usually coflfee bean shaped, without capsules and Gram negative. Two of the diseases produced by organisms of this group are of importance, namely, cerebral spinal meningitis and gonorrhea in man. CLASSIFICATION OF MICEOOBGANISMS 87 2. The Family Bacillaceae. The Spore Bearing Bacteria The bacteria of this family are all rod-shaped and aU: produce endospores. The cells may be motile or non-motile, when motile with peritrichous flagella. Two genera are of importance, Bacillus and Clostridium. The genus Bacillus includes those forms which are aerobic or facultative; the genus Clostridium those which are anaerobic. 1. Bacillus.— The organisms belonging to this genus are all rod-shaped, sometimes occurring in chains. Under the right conditions these bacteria are among the most active in nature producing decomposition of organic materials. One species pro- duces the disease anthrax in animals and man, and one species the disease termed foul brood of bees. Fig. 43. — Types of bacilli: a, b, Non-motile bacilli (Bacterium); c, mono- trichous bacillus (Pseudomonas); d, lophotrichous bacillus {Pseudomonas); e, f, peritrichous Bacillus. 2. Clostridium. — The organisms of this genus are all rod shaped, producing endospores but not growing in the presence of free atmospheric oxygen. Frequently the rods are swollen at time of spore production, producing spindle shaped or club shaped cells. Some of the bacteria of this group are among the most active of the putrefactive forms, particularly in the produc- tion of malodorous compounds such as butyric acid. Other species are capable of producing disease, especially when intro- duced into wounds. Among the diseases produced are gaseous gangrene in man, malignant edema, blackleg, and bradsot in animals and tetanus in man and animals. 88 VETERINARY BACTERIOLOGY 3. Tte Family Bacteriaceae This is the largest of the families of bacteria, including some nine genera. Here are found those rod shaped forms whose cells are usually regular, which do not produce endospores and which when motile have peritrichous flagella. In all of the genera containing disease producing forms the cells are Gram negative. Four of the genera are discussed here, three including pathogenic forms and one including species of importance in milk. These genera are Bacterium, Pasteurella, Hemophilus and Lactobacillus. Key to the Genera of Bacteriaceae' Cells Gram negative. B. Requiring hemoglobin or blood serum for growth in media 1. Hemophilus BB. Growing on ordinary culture media. C. Polar staining marked 2. Pasteurella CC. Polar staining not pro- 3. Bacterium nounced Cells Gram positive 4. Lactobacillus AA. 1. Hemophilus. — The organisms belonging to this genus are minute, parasitic, Gram negative rods, which grow in the labora- tory only in special media, preferably containing hemoglobin. The most important species are Hemophilus influenzoe, the or- ganism which has been supposed to cause influenza and which is quite certainly associated with many cases of the disease, Hemophilus pyogenes, a common cause of suppuration in animals, and Hemophilus pertussis, the cause of whooping cough. 2. Pasteurella. — The organisms of this genus are all rod shaped cells. Gram negative, and show bipolar staining. They are parasitic with very slight powers of fermentation. Members of ' Other genera belonging to this family are : — ErythrobadUus, producing red pigment. Chromobacterium, producing violet pigment. Erwinia, White, slimy forms pathogenic for plants. Proteus, closely related to Bacterium but liquefying gelatin rapidly. Zffpfius, Gram positive putrefactive forms. CLASSIFICATION OF MICROORGANISMS 89 this genus produce many diseases in man and animals including the bubonic plague of man and hemorrhagic septicemias of animals, such as fowl cholera, and related diseases in swine, sheep, cattle and horses. 3. Bacterium. — The organisms belonging to this genus are Gram negative rods, frequently motile, and easily cultivable. Most species ferment certain carbohydrates with the formation of acid and frequently of gas. They are typically intestinal para- sites in man and higher animals. Some species are not infre- quent in the soil. A few species are pathogenic, including the organisms causing typhoid fever, paratyphoid fever, dysentery and certain types of food poisoning. One species, the Bacterium coli, is frequently used as an index for the determination of the presence of sewage in water. 4. Lactobacillus. — The cells are rod shaped, often long and relatively slender. Gram positive, and non-motile. Some species have very short cells and are almost coccusJike. No endospores are developed. Acid, particularly lactic acid, is usually pro- duced in considerable quantities from carbohydrates. Many of the species prefer to grow in the absence of oxygen, although they are not strictly anaerobic. Included in this genus are many bacteria important in the souring of milk, the formation of lactic acid in silage, sauerkraut, etc. 4. Tte Family Pseadomonadaceae This family contains a single genus, Pseudomonas. The cells are rod shaped, and usually motile by means of polar flagella. Frequently a fluorescent green or brown pigment is produced in culture media. In other species an insoluble yellow pigment is formed. Among those producing a green fluorescent pigment is the Pseudomonas aeruginosa, an organism sometimes found in wounds. Many of the yellow species produce diseases in plants. 5. The Family Spirillaceae The organisms belonging to this group are curved rods. Two genera are included. Vibrio and Spirillum. The former only is considered as it alone has pathogenic species. 90 YETEKINABY BACTERIOLOGY Vibrio. — This genus included short curved rods, motUe by- means of one, two or three polar flagella. Many are intestinal parasites and some are capable of causing disease. The most important organism is the Vibrio cholerce producing Asiatic cholera in man, and certain species isolated from fowls. Fig. 44. — Types of spirilla: o, Non-motile spirillum; 6, monotrichous spirillum (Vibrio); c, lophotrichous spirillum with 2 or 3 flagella {Vibrio); d, lophotrichous spirillum (Spirillum). Order Actinomycetales, The Thread Bacteria The organisms belonging to this order usually have the cells somewhat elongated, frequently filamentous, and with a decided tendency to the formation of branches. In some genera a definite branched mycelium is developed. The cells frequently show swelUng, or are clubbed or irregular in shape. Endospores are not produced, although conidia are formed by some genera. Many of the genera are Gram positive and all are non-motile. Some species are aerobic. All of the eight genera contain species which are parasitic in man or animals, many of them pathogenic. Key to the Genera of Actinomycetales A. Typically producing long filaments B. Branched mycelium formed 1. Actinomyces BB. No true mycelium C. Cells more or less branched D. Gram negative 2. Actinobacillus DD. Gram positive 3. Erysipelothrix CC. Cells never branch. Gram positive threads later fragmenting in- to rods 4. Leptotrichia CLASSIFICATION OF MICROORGANISMS 91 AA. Not typically producing long fila- ments B. Acid fast BB. Not acid fast C. Cells elongate, fusiform CC. Cells not fusiform D. Gram positive DD. Gram negative 5. Mycobacterium 6. Fusiformis 7. Corynebaderium 8. Pfeifferella 1. Actinomyces. — The organisms belonging to this group form fine threads (or mycelium) which breaks up into short segments which function as spores or conidia. Some of the species are parasitic, one, the Actinomyces bovis, producing lumpy jaw in cattle. Many other species are widely distributed in the soils, apparently growing upon decaying roots and similar organic matter. Kg. 45. — A, Leptothrix; B, Cladothrix; C, Nocardia; D, Actinomyces. 2. Actinobacillus. — This genus differs from Actinomyces in that no true mycelium is formed. The cells are Gram negative, and fragment to form rods. The principal species is Actino- bacillxLS Ldgnieresi, the cause of the disease actinobadllosis in cattle. 3. Erysipelothrix. — This genus includes certain Gram positive bacteria in which the filaments break up into short rods. For the most part the species are microserophilic. Several species pro- duce disease in man and animals, notably Erysipelothrix rhusio- pathioe, the cause of swine erysipelas. 92 VETERINABY BACTEBIOLOGY 4. LeptotricMa. — The organisms of this genus have been largely described from the human mouth. The unbranched filaments are Gram positive, later fragmenting into rods. None are positively known to be pathogenic. 5. Mycobacterium. — These organisms are slender rods which are stained with difficulty, but when once stained are resistant to decolorization by acids, that is, they are termed add fast. The cells are sometimes swollen, showing club shapes or forked forms, occasionally the cells are branched. They are non-motile and Gram positive. Some of the species are present in the soil, a few are pathogenic to man and animals. The most important of the diseases caused are tuberculosis and leprosy in man, and para-tubercular dysentery in cattle. 6. Fusiformis. — These organisms are anaerobic or micro- aerophHic, the cells frequently elongate and fusiform, staining; somewhat unevenly. Filaments are sometimes formed. Several species have been found associated with disease in man as for example in Vincent's angina. 7. Corynebacterium. — The cells are slender, often slightly curved rods with a tendency toward the formation of clubs, and containing granules which give the cells when stained a. barred or irregular appearance. They are not acid fast but are Gram positive and aerobic. Most species are parasites, the most important being Corynebacterium diphtherice, the cause of diphtheria in man. 8. Pfeifferella. — These organisms are non-motile rods, slender and Gram negative, staining poorly, sometimes forming threads,: and showing a tendency toward branching. When grown upon potato in the laboratory they develop a characteristic honey-like growth. The most important species is Pfeifferella mallei, the organism which causes glanders in the horse. Order Spirochxtales The organisms belonging to this group in many respects resemble the protozoa, and may be regarded as intermediate between the true bacteria and the protozoa. They are all more or less curved rods, frequently very slender. Many species are motile but there has been no definite demonstration of flagella. CLASSIFICATION OF MICROORGANISMS 93 They multiply either by longitudinal or transverse fission. It is probable that in some cases they have a relatively complex life history. Several genera have been described, the most important being Treponema. Treponema.— The organisms belonging to this genus are exceedingly slender spiral rods, motile by means of flexous bend- ing of the body. The most important species is Treponema pallida, the cause of the disease syphilis in man. Figs. 46 and 47.— Types of spirochaetEB. Classification of Yeasts Mycologists recognize a number of different genera in the group of yeasts. It is probable that the yeasts do not constitute a homogeneous group. The genus Saccharomyces includes such forms as the common bread and brewer's yeast {Saccharomyces cerevisice) which produce spores and are active in alcoholic fer- mentation. The name Torula is sometimes given to similar yeasts that are not spore producing. This latter name, however, is incorrectly so apphed, as it was previously and is now used to indicate a genus of molds. The name Blastomyces has been commonly accepted to indicate the yeasts pathogenic for man and animals. It is probable that there is little reason for separation of Saccharomyces and Blastomyces on the basis of their morphology, but such a separation on the basis of pathogenesis seems to be advisable. Classification of the Molds Several hundred genera and many thousands of species have been described. Of these, a few genera only contain species that are pathogenic for man and animals. For a discussion of classi- fication the student is referred to Chapter XXXIX. SECTION II LABORATORY METHODS AND TECHNIC CHAPTER VI STERILIZATION Sterilization is the process whereby glassware, media, or any of the materials or apparatus used in the laboratory are entirely freed from living organisms. It is evident that in the study of bacteria it is necessary that we deal with pure cultures, that is, that one kind of organism only be present in the material which we are studying. It is quite impossible to determine from mixed cultures which of the organisms present bring about observed changes. Bacteria are present upon the surface of all laboratory apparatus, in the dust, in soil, upon the hands — they are ubiquitous, hence the necessity for sterilization. Sterilization may be accomplished by physical or chemical means. In practice the latter is generally called disinfection, and is rarely used in the laboratory. The term sterilization, there- fore, as commonly used, indicates the destruction of micro- organisms by physical processes. Sterilization by the Flame. — The platinum wire used in the transfer of bacteria in the laboratory is sterilized by heating to a red or white heat in the flame of the Bunsen burner. Similar methods are sometimes used in the sterilization of other small pieces of laboratory apparatus, such as cover-glasses and slides. Sterilization by Hot Air. — Glassware is commonly sterilized by subjecting it to a temperature of 150°C. to 170°C. in a hot-air oven for an hour. ■ AU bacteria will be destroyed at this tempera- ture providing the material to be sterilized is of a nature such that the heat can penetrate readily to all parts. This method cannot 94 STERILIZATION 95 be used, however, in the sterilization of liquids or of any organic material which might be decomposed at such a temperature. Sterilization by Streaming Steam.-It is found in practice that live steam is the most efficient sterilizing agent for many of the media used in the laboratory. Steam under atmospheric pressure at sea-level has a temperature of about 100°. Some type of apparatus is used such that the live steam comes in direct con- tact with the material to be sterilized. One type of the apparatus is called the Arnold steam sterilizer (Fig. 49). It consists essen- tially of a pan with a double bottom opening into the sterilizing Fig. 48. — Oven for sterilization by hot air (Jordan). chamber above. The water between the bottoms is quickly heated to boiling temperature and is automatically replaced from the supply on the exterior through small holes as rapidly as it boils away. A single exposure to live steam for fifteen minutes is sufficient to kill all vegetative bacteria, but spores are not thus destroyed. It is customary, therefore, to heat for fifteen minutes on one day, keep the medium for twenty-four hours at a temperature suitable for the germination and devel- opment of any spores present, then heat again for fifteen minutes in the same manner. Those spores which have germinated will 96 VETERINAHY BACTERIOLOGY be destroyed by this second heating. A third heating, twenty-four hours later, will quite certainly destroy all the bacteria which may have been present. This process is called intermittent steriliza- tion. It finds its principal application in the sterilization of materials which would be changed or broken down by heating at a higher temperature. Among such materials are media. con- taining sugars which undergo incipient caramelization when heated too hot. Sterilization by Steam under Pressure.— This is generally accomplished in the autoclave or digester, which consists essentially Fig. 49. — Arnold steam sterilizer (Fowler). of a chamber into which steam under pressure can be introduced (Fig. 50). Many different types of these autoclaves have been put upon the market. Live steam under a given pressure unmixed with air has a constant temperature; therefore, if the pressure of the steam is known, one can determine easily the temperature as well. It is necessary, however, that all air be first eliminated. This is accomplished by allowing the stop-cock, which is always present upon the steam chamber in the autoclave, to remain open until all the air has escaped and the steam issues in a constant stream. This cock is then closed and the pressure caused to STERILIZATION 97 rise as quickly as possible to 15 pounds to the square inch, or one additional atmosphere. This gives a temperature of about 121°. Material to be sterilized should be allowed to remain fifteen minutes usually. If large bulks, such as flasks of media, are to be sterilized, a longer period must be allowed in order that Fig. 50. — Autoclave for sterilizing by steam under pressure. the media may be completely heated through. If very small quantities of material are being sterilized, a shorter period may be used. When properly carried out, steriUzation by this method will certainly destroy all the bacteria present. Its principal disadvantage is that certain organic substances may be decomposed at this temperature. 98 VETEEINAHY BACTERIOLOGY Sterilization at Temperatures Lower than Boiling-point. — It is sometimes necessary to sterilize media, particularly blood- serum, at temperatures lower than the boiling-point of water. This is accomplished by placing the material to be sterihzed in an apparatus where it may be heated to the desired temperature, usually 70°-80° for one to two hours on each of five or more successive days. If large numbers of spores of. certain orga,nisms, such as Bacillus subtilis, are present, it is almost impossible to sterihze efficiently by this method. However, if care is used in securing the blood-serum to prevent the introduction of such organ- isms, sterilization may be easily accomplished at this tempera- ture. Sterilization by Addition of Chemicals.^It is only under excep- tional conditions that chemicals are used to sterilize media. Fig. 51. — Apparatus for sterilization by filtration (McFarland) . It has been found that the addition of soluble materials, such as lactose, in considerable quantities to media containing pure cultures of certain bacteria will destroy the organisms so that they may be used as a vaccine. This method does away with the destruction of any of the characteristic metabolic products by heat. Sterilization by Filtration. — Bacteria may be removed from a hquid by passing it through a filter with pores so fine that the organism cannot penetrate. Such filters are made up in a great variety of shapes and densities. Among the many used are the Berkefeld, the Pasteur, and the Chamberland. These are made of unglazed porcelain. In filtration through these it is necessary, of course, that all the apparatus used, particularly the vessel into which the filtrate runs and the filter itself, be sterilized before use. STERILIZATION 99 This method of steriUzation is commonly used for the removal of bacteria from culture-media when it is desired to study their soluble metabolic products, and in the removal of bacteria from sera which contain antitoxins and other antibodies. It is not commonly used in the sterilization of media intended for the cultivation of bacteria. Filters of this character have been used extensively in the filtration of water for drinking purposes. When first installed, they are quite efficient, but it is found that the organ- isms rapidly penetrate, and in the course of time are found in the filtrate. Such filters must, therefore, be sterilized at intervals if they are to remain efficient. CHAPTER VII CULTURE-MEDIA AND THEIR PREPARATION Microscopic examination alone is quite insufficient to differentiate species of bacteria. By the aid of a microscope one cannot readily recognize (the differences, for example, between the organisms which cause typhoid fever and certain of the normal inhabitants of the intestinal tract. It is necessary, therefore, in a study and differentiation of species, that we make use of different kinds of culture-media in which the bacteria may be grown. By the term medium is meant any nutrient substance or mixture upon which or in which bacteria will multiply. The bacteria in their development on the various media show certain growth reactions which are very useful in their differentiation. Some produce acids, others gas, alkalis, and proteolytic and coagulative enzymes. Adjustment of the Reaction of Media. — The reaction of a medium, that is its relative acidity or alkalinity, may be desig- nated in one of two ways; first, by the amount of normal^ acid or 1 A normal solution of a chemical may be defined as one in which there is one gram of replaceable (acid) hydrogen or its equivalent per liter of solu- tion. For example, if we wish to prepare a normal solution of HCl, we must so dilute the acid that it contains one gram of hydrogen per liter of solution. This is best accomplished in any substance that can be readily weighed by dissolving the molecular weight expressed in grams in sufficient water to make a liter of solution. If there is more than one atom of replaceable hydrogen in the molecule, it is necessary to divide the amount used by the number of such atoms. For example, the molecular weight of H2SO4 is approximately 98, but there are two replaceable acid hydrogen atoms. Therefore, half this molecular weight in grams, or 49 grams, of the H2SO4 is made up to a Kter of solution and contains one gram of acid hydrogen. The same principle is adopted in the preparation of a normal solution of an alkali; in this case, however it is necessary to divide the molecular weight by the number of atoms of the base present, which will replace hydrogen. For example, the molecular weight of NaOH is 40. It contains one atom only of sodium, and a normal solution, therefore, contains 40 grams to the liter. Dry sodium carbonate (Na2C02) has a molecular weight of 106. Two atoms of sodium are present; therefore it 'is necessary to divide by two, so that a normal solution of sodium carbonate contains 53 grams to the liter of solution. It is evident that a given volume of a normal solution of an acid will neutralize exactly an equal volume of a normal alkali. 100 CULTURE-MEDIA AND THEIR PREPARATION 101 normal alkali required to bring one hundred cubic centimeters of the medium to the neutral point of some particular indicator, or second, by the designation of the true hydrogen ion concen- tration or, conversely, of the true hydroxyl ion concentration of the medium. While the first method is still the more commonly used, the second method is in most instances preferable. Bacteria, yeasts, and molds are usually quite sensitive to the presence of an excess of acid or alkali. Some grow best in a medium which is strictly neutral, others prefer one which is somewhat on the acid side of neutrality, still others on the alka- line side of neutrality. Careful adjustment of reactions is there- fore necessary in many cases. Some organisms, for example, will not grow unless the medium has almost exactly the same reaction as has the blood or the tissues of the body in which they are accustomed to grow. The actual acidity of any solution is determined by the pres- ence of free hydrogen ions and alkalinity is determined by the presence of free hydroxyl ions. A solution is truly neutral when equal numbers of hydrogen and hydroxyl ions are present in a given volume. Pure distilled water is neutral, that is, when a molecule of water dissociates it breaks up into equal numbers of hydrogen and hydroxyl ions. The chemists has discovered, furthermore, that pure water and therefore any neutral solution of materials in water contain approximately one ten-miUionth of a gram of hydrogen ions to the liter. This may also be written 10"'' grams hydrogen ions per liter, or still more conveniently expressed in terms of normality of hydrogen ions. A normal solu- tion of hydrogen ions is one which contains one. gram of hydrogen ions per liter. A neutral solution, therefore, is one which has a concentration of hydrogen ions of 10"'' normal. It was noted above that in a neutral solution there is the same number of hydroxyl ions as hydrogen ions. The hydroxyl ion concentra- tion at neutrality must therefore be also lO"'' normal. The physical chemist has demonstrated that the product of the normality of hydrogen ions by the normality of hydroxyl ions in a solution is a constant number. What this number is for aqueous solutions may be determined by multiplying 10"' (nor- mality of hydrogen ions in a neutral solution) by 10"^ (normality 102 VETERINAHY BACTERIOLOGY of hydroxyl ions in a neutral solution) giving 10~^*. In other words, the product of the normality of the hydrogen ion con- centration of a solution by the normality of the hydroxyl ion concentration must always be approximately lO"". If either the hydroxyl or hydrogen ion concentration is known one can at once determine the concentration of the other. For example, if we are deahng with a solution having an hydrogen ion concentration of 10"^ normal its hydroxyl ion concentration must be 10"' normal. It is thus possible to arrange a scale to designate the acidity of any solution in terms of its hydrogen ion concentration as follows: 10" 10-1 10-2 10-^ 10-^ 10-5 10-6 10-' 10-» 10-' 10-" 10-" 10-12 10-1' 10-" On this scale it will be noted that the larger the numerical value of the exponent, the smaller is the hydrogen ion concentration. It will be recalled that IQ-' represents neutrality. Numbers to the right of lO-'' represent increasing values of alkalinity or decreasing hydrogen ion concentration. Numbers to the left represent increasing acidity. Inasmuch as this method of statement is somewhat cumbersome, it has been suggested by Sorensen that the exponents be used to make up a scale, using positive signs instead of negative. This gives the scale : 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. Each of these numbers is termed the Ph of a solution. A solu- tion having Ph of 0, for example, would have a normality of hydrogen ion concentration of 10° normal or 1. One having Pg value of 7 would have a hydrogen ion concentration of 10-' normal, that is, it would be neutral. It is evident, therefore, that the smaller the number in this scale the higher the hydrogen ion concentration, that is, the greater the acidity; and the larger the number the greater the alkalinity. Indicators are chemical substances which are of one color in a certain range of Pa values or hydrogen ion concentrations, and of another color in other ranges. The indicator most com- monly used is litmus, which has a lilac color at true neutrality, that is, at a Pg value of 7. At a Pg value of 8, that is in a more alkaline solution, it is blue. At a Pg value of 5, that is in a CULTURE-MEDIA AND THEIR PREPARATION 103 Stronger concentration of hydrogen ions, it is red. Other indica- tors change color at other Pt, values. For example, phenolph- thalein is colorless in all hydrogen ion concentrations having a Pb value less than 8.2, in more alkaline solutions, it is red. Many other indicators are known which change color at other points in the hydrogen ion scale. This change of color is not instantaneous. In adding alkah, for example, to an acid solu- tion containing litmus there is not an instantaneous transforma- tion of red into blue. By the use of standards whose hydrogen ion concentration is known it is possible to determine approxi- mately the hydrogen ion concentration of any material which it is desired to test, by the use of the intensity of color of appropriate indicators. Most bacteria grow in a hydrogen ion concentration of lO"' to 10-^ that is in solutions having a P„ value between 7 and 8. The Pb value of blood is usually about 7.35. This indicates therefore the hydrogen ion concentration most useful in culti- vating many species of pathogenic bacteria. Inasmuch as most media are on the acid side of true neutraUty it is necessary to add alkah, usually potassium or sodium hydrate, until test shows that the hydrogen ion concentration has become satisfactory. The hydrogen ion concentration of a medium or solution depends not only upon the actual concentration of the acid itself but upon the concentration of substances which are termed buffers. A buffer is any substance in a solution which tends to prevent rapid changes in hydrogen ion concentration upon the additions of alkalies or acids. Certain salts, particularly the phosphates, and many organic substances, particularly the amino acids and peptones, act in this manner. For example, the addition of a small quantity of an acid to distilled water will give a marked change in the hydrogen ion concentration, but the same quantity of acid added to solutions containing consider- able quantities of buffers may result in very slight differences in hydrogen ion concentration. It is evident since microorganisms are affected far more by differences in hydrogen ion concentra- tion than they are by the total amount of acid present, that heavily buffered media are preferred for the growth of micro- organisms. 104 VETEBINAEY BACTERIOLOGY It is noted above that media are sometimes standardized by determining the quantity of acid or alkali required to bring one hundred cubic centimeters to the neutral point of some indicator. The one usually chosen in bacteriology is phenolphthalein. A solution is said to be— 1, for example, when it will require one cubic centimeter of a normal solution of acid to bring it to the neutral point of phenolphthalein. Nature of Nutrients Required by Bacteria.— It is found that practically the same elements are necessary for the nutrition of bacteria as are essential for higher plants and animals, but they may be used in quite different proportions. It is particularly important that the disease-producing bac- teria be cultivated whenever possible. Cultivation outside the body is quite necessary to a satisfactory proof of pathogenicity, to differentiate species, and to secure the organism in quantities sufficient for preparation of vaccines, antitoxins, etc. A few standard media are commonly used in the laboratory for the growth of bacteria, and a great variety of special types have been devised for certain species that do not grow upon these. It is impracticable even to enumerate the many special media that have been employed. Liquid Media Bouillon or Beef Broth from Meat. — This is the commonest of laboratory media and serves as a basis for the preparation of many others. Place 500 gm. chopped lean beef in a liter of water and allow it to stand in a refrigerator over night. The juice is then pressed out with a meat press, boiled for half an hour, the coagulated albumins filtered out, the liquid made up to a liter with water, 10 gm. of peptone added, and heated sufficiently to dissolve. The reaction is adjusted to the proper point, usually + 1, by titra- tion, or the medium is simply neutralized by addition of nor- mal NaOH, using phenolphthalein paper as an indicator if a high degree of accuracy is not required. The broth is then autoclaved at 15 pounds pressure or boiled for fifteen minutes, allowed to cool, and then filtered. The cooling throws down a precipitate of magnesium ammonium phosphate, which may then be removed. CULTURE-MEDIA AND THEIR PREPARATION 105 In many cases this is not objectionable and filtration may be carried out while the solution is still hot. The finished bouillon or broth is placed in test-tubes and flasks, and sterilized in the autoclave under a pressure of 15 pounds for 15 minutes. Bouillon or Broth from Beef Extract. — It is customary, in much of the routine work of the laboratory, to substitute for the preceding a broth in which three grams of a beef extract, such as Liebig's, is substituted for the meat. Sugar-free Broth. — There is generally present in the preceding media a small amount of carbohydrate, largely dextrose. In some cases a sugar-free medium is required. Theobald Smith has devised a modification of the meat broth for this purpose which is commonly used. Several broth tubes containing vig- orous twenty-four-hour cultures of Bacterium coli are added to the meat infusion and kept at 37° for eighteen hours. In this time the bacteria will have used up all the sugar present. The broth is then prepared as above. Sugar Broth.^^ugar-free broth is generally modified by the addition of carbohydrates, such as dextrose, saccharose, and lactose, making 1 per cent, solutions. Such media must be subjected to intermittent sterilization in flowing steam and not in the autoclave, as the carbohydrates readily decompose. Glycerin Broth. — Five or 6 per cent, of glycerin added to broth makes it a much more favorable medium for many organisms. Serum Broth. — Blood-serum secured under strict aseptic pre- cautions may be added to sterile broth in various proportions. Tubes prepared in this manner should be incubated for a few days to determine whether or not the medium is sterile. Steril- ization can be effected only by filtration, as heating Would coagulate the serum. Dunham's Solution. — This is a solution containing 1 per cent, peptone and 0.5 per cent, sodium chlorid in water. It is used in growing organisms for the determination of indol. Beerwort.— Unhopped beerwort is frequently used for the growth of yeasts and molds. Milk.— Fresh separated milk is tubed and subjected to intermit- tent sterilization. Commonly, litmus is added in sufiicient quan- tities to make the milk a distinct blue. 106 VETERINARY BACTERIOLOGY Synthetic Media.— It is sometimes desirable to prepare a medium in which the exact chemical composition of every ingre- dient is known. The nature of all the changes brought about by bacteria can be studied chemically in such a medium, and the food requirements determined by changes in the composition. Most synthetic media contain as a basis an aqueous solution of certain salts, among them potassium phosphate and sodium chlorid. Special media of this kind are used extensively in the study of the soil bacteria; it is only occasionally that such a medium proves serviceable in the study of pathogenic forms. The most commonly used of the synthetic media is Uschinsky's solution. Water, distilled 1000 c.c. Asparagin ^ S™-; Ammonium lactate " S^^- > NajHPO^ 2gm.; NaCl 5gm.; This medium is known as albumin free. LiQUEFIABLE SOLID MEDIA Nutrient Gelatin. — This is prepared by the addition of 10-15 per cent, of gelatin to bouillon as prepared above. The gelatin should be the best "gold label." Care must be used in heating the solution while dissolving the gelatin or the latter will stick to the bottom of the vessel and bum. It is best to use an asbestos pad, a double boiler, or a rice-cooker. The gelatin is itself acid, so that it is necessary to adjust the reaction after it has dissolved. The medium is then cooled to 60°, and the white of an egg thor- oughly mixed with it. It is again heated to the boiling-point without stirring. The coagulation of the egg removes suspended particles and makes filtration easier. The nutrient gelatin is tubed and sterihzed in the autoclave at 120° for ten to fifteen min- utes. It should be cooled at once after removal from the sterilizer. . Care must be exercised not to heat the medium too long or it may fail to solidify when cooled. Other Gelatin Media. — Any of the liquid media already dis- cussed, with the exception of the milk and serum broth, may be made solid by the addition of 10 to 15 per cent, gelatin. Among the more commonly used are dextrose, lactose, and glycerin gelatin. CULTXTBE-MEDIA AND THEIR PREPARATION 107 Nutrient Agar.— This is prepared by the addition of 1.5 per cent, of shredded or powdered agar-agar to bouillon. Agar-agar is a carbohydrate-like material, probably related to the vegetable gums, which is prepared from certain of the seaweeds of the Pacific and Indian Oceans. The mixture must be boiled vigorously for half an hour to insure thorough solution of the agar. This medium does not burn as readily as does gelatin, and long- continued heating does not interfere with its solidification when cooled. The nutrient agar may be sterilized in the autoclave for fifteen minutes at 120°. Blood-serum Agar. — Liquid sterile blood-serum is heated to 40° to 50° C. and mixed with liquid agar cooled to 40° to 50° C, equal parts or in mixture of 1 : 2. It is then solidified by cooling. It may be poured into plates before solidifying or may be left in test-tubes. Ascites fluid and hydrocele fluid may be substituted for the serum. Chocolate Agar. — 10 c.c. fresh defibrinated sheep blood are added to every 100 c.c. of neutral beef infusion agar. It is important that the blood be added while the agar is hot; that is, immediately after the latter is taken from the Arnold sterilizer (95 F.). The medium should have a dark chocolate color. This medium is valuable for culturing material from cases of pneu- monia, broncho-pneumonia, etc. Hormone Agar. — (Huntoon.) Chopped beef heart or steak (must be comparatively fresh) 500 Water Peptone (Bacto peptone gives the best results) 10 Agar (Bacto or thread agar that has been soaked) 16 Salt 5 Whole Egg : 1 All of these ingredients are placed in an ordinary enamel- ware vessel, preferably a large coffee pot, and heated over an open flame with constant stirring until the red color of the meat infusion changes to brown, at a temperature about 68° C. Care should be taken not to run the temperature much above this point as the medium then begins to clot, which is undesirable at this time. The medium is now titrated by the addition of normal sodium hydrate until it is slightly alkaline to litmus , paper, and 108 VETERINARY BACTERIOLOGY then 1 c.c. per liter is added in addition. The vessel is covered and placed in the Arnold sterilizer or in a water bath at a temper- ature of 100° C. for 1 hour, removed, and the firm clot which has formed separated from the sides with a rod and the vessel re- turned to the sterilizer or water bath at 100° C. for 1}4 hours. It is now removed and allowed to stand at room temperature for about 10 minutes in a slightly inclined position; during this time the fluid portion separates and may be removed by pipetting or, in the case of the coffee pot, by simply pouring it off carefully. If it is poured through a fine wire sieve many small particles of meat clot may be caught. The product is allowed to stand in tall cylinders for 15 to 20 minutes until the fat present has risen to the surface when it can be removed. The medium is now tubed and sterilized by the intermittent method. Autoclaving is to be avoided. If the medium, although usually clear enough for practical purposes, seems too turbid, further clearing may take place by filtration through glass wool, asbestos wool, sedi- mentation or centrifugation. Other Agar Media. — Agar may be used as the solidifying agent for any of the liquid media described. It has the advan- tage over gelatin that it may be kept at blood heat, while gelatine under such conditions would liquefy. Endo-agar-fuchsin Medium : Beef infusion agar (3 per cent., neutral;, made alkaline by addition of 10 c.c. 10 per cent, soda solution . . . 1000 gm. Lactose 10 gm. Puchsin (saturated alcoholic) 5 c.c. Sodium sulphite (10 per cent, solution) 25 c.c. In plates this medium should have the natural agar color or light rose. Typhoid and paratyphoid colonies are colorless or light rose; coli colonies are an intense red. Simplified Endo Agar. — (Levine) Distilled water 1000 c c Pepton (Difco) 10 gm. Dipqtassium phosphate 2 to 5 gm Agar. .,..'....,.! 15 to 30 gm'. ' ' ' 'i ' ' CULTURE-MEDIA AND THEIR PREPARATION 109 Boil ingredients until dissolved and make up loss due to evaporation. Place measured quantities in flasks or bottles and sterilize in autoclave at 15 pounds for 15 minutes. Just prior to use add to each 100 c.c. of the melted agar Lactose 1 gram or 5 c.c. of 20 per cent, lactose solution Basic fuchsin (16 per cent, alcoholic solution) i^ c c Sodium sulphite (10 per cent, solution freshly prepared and brought to a boil over flame) 2)4 c.c. Pour plates and allow to harden in incubator. Inoculate in streaks. Bacterium coli forms colonies 4 mm. or more in diameter, deep red and buttonlike showing a greenish metallic sheen in reflected light. Bacterium aerogenes colonies do not show the metallic luster, are lighter colored, markedly convex and con- siderably larger; colonies of the intermediate and typhoid group are transparent, colorless or faintly yellow or amber colored. Eosin-methylene-blue Agar.— (Le vine) Distilled water 1000 c.c. Peptone (Difco) 10 gm. Dipotassium phosphate 2 gm. Agar 15 gm. Boil ingredients until dissolved, and make up loss due to evaporation. Place measured quantities in flasks or bottles and sterilize in autoclave at 15 pounds pressure for 15 or 20 minutes. Just prior to use add to each 100 c.c. of the melted agar the follow- ing: Lactose 1 gram or 5 c.c. of 20 per cent, lactose solution. Eosin (2 per cent, yellow aqueous solution) 2 c.c. Methylene blue (0.5 per cent, aqueous solution) 2 c.c. No adjustment of reaction is necessary. Pour medium into sterile petri dishes and allow to harden. Cultures to be tested are streaked on the surface. Bacterium coli forms flat, dark centered, button like colonies 2 to 4 mm. in diameter, which by reflected light show a charac- teristic greenish metallic sheen. Bacterium aerogenes colonies are in general much larger, convex with brownish centers and rarely exhibit a metallic luster. 110 VETEHINAET BACTERIOLOGY NON-LIQUEFIABLE MEDIA Potato.— Cylinders are cut from potatoes by means of an apple- corer or special potato borer. These are divided by a diagonal longitudinal cut such that each half has one long sloping surface. Fig. 52. — Preparation of potato tubes: a, Potato cylinder out diagonally; h, side view in tube; c, front view. It is well to soak in running water for a few hours to prevent their turning dark when sterilized. They are placed with the sloping surface up in test-tubes with a bit of saturated absorbent cotton in the bottom, or in special potato tubes. The latter are tubes constricted a short distance from the bottom. The bulb thus formed is filled with water and the potato rests on the constriction above. This device enables one to keep the potatoes moist for considerable periods. They are sterilized in the auto- clave for fifteen minutes at 120°. Other Vegetable Media.— Carrots and other vegetables may be prepared in the same manner as potato. Blood-serum, — Solidified blood-serum has been found to be essential to the growth in the laboratory of certain of the patho- genic bacteria. It is best to avoid all the initial contamination of the serum possible, as it is difficult, by the methods used in sterilization, to rid the medium of all the spore producers when they are present in considerable numbers. The blood, usually from cattle, is allowed to clot, and the clear, straw-colored serum is removed. A clear, solidified serum may be prepared by heating the slanted tubes to 76° for an hour or more on five or six consecu- CULTURE-MEDIA AND THEIR PREPARATION 111 tive days. An opaque medium is secured by heating to a tempera- ture of 95°. The serum may be sterilized and yet remain liquid by placing in an incubator at 58° C, or in a water-bath at the same temperature for three or four hours on several consecutive days. If care has been used in drawing the serum the slight contamination that may have occurred will be overcome by this method. Loeffler's blood-serum is a mixture of three parts of the serum with one part of neutral 1 per cent, dextrose broth. It is soUdi- fied in the same manner as the simple serum. Egg Medium. — This medium was developed by Dorset, of the U. S. Bureau of Animal Industry. It has come into common use for the growth of the Bacillus tuberculosis and has been used in recent years as a satisfactory substitute for blood-serum. Dorset's description of the method of preparation follows: "The egg shell is broken carefully, and the entire contents dropped into a wide-mouthed sterile flask. The yolk may be broken with a sterile platinum wire. Gentle shaking of the flask will serve to mix the white and yolk of the egg quite thoroughly. Care should be taken, however, not to shake the flask so that a foam will be produced, otherwise an uneven and unsatisfactory surface will be obtained when the medium is hardened. When the mixing is complete, the egg is poured into tubes, care being taken to avoid foaming, and the tubes containing about 10 c.c. of the medium are then inclined in a blood-serum oven and hardened at a temperature of 70° C. This hardening will usually require two days, four or five hours each day. Sterihzation will be accomplished at the same time. A higher temperature may be used and the medium will be hardened more quickly. The growth of tubercle bacillus seems to be more vigorous when the egg is hardened at 70° to 74° C, and, in addition, the prolonged heating probably insures a more certain sterilization. The medium after hardening is opaque and yellowish in color, and usually dry, there being practically no water of condensation in the tube. The egg tubes should be kept in an ice-box to prevent further drying. Just before inoculation, three or four drops of sterile distilled water should be added to each tube to supply the moisture required for the satisfactory development of the tubercle bacillus." CHAPTER VIII BIOCHEMICAL TESTS The physiological characteristics of bacteria are of consider- able importance in the differentiation of species. A knowledge of such characteristics is of assistance in the isolation and recog- nition of certain species, as in the detection of sewage bacteria in water. Acid and Alkali Production. Determination of Changes in Hydrogen Ion Concentration. — Many bacteria when grown in nutrient solutions, particularly those containing carbohydrates, bring about marked changes in hydrogen ion concentration. The amount of such change accomplished by a given organism depends upon several factors, the most important being the following: first the amount of acid produced, second the kind of acid produced, third the amount of buffer present in the nutrient medium, and fourth the amount of alkali (that is the concentration of hydroxyl ions) produced at the same time. Alkali may be developed either by the production of ammonia or by the trans- formation of the salt of a strong acid to the salt of a relatively weak or little dissociated acid. For example, certain bacteria may transform sodium citrate into sodium carbonate, the latter being decidedly alkaline in its reaction. Two methods are in common use for detecting changes in hydrogen ion concentration. The first is by a determination of the electric conductivity. The second is a colorimetric method. For usual laboratory routine the colorimetric method is the simpler and is the only one which will be discussed. In this method it is customary to add a suitable indicator either to the solution to be tested or to a portion of this solution diluted somewhat with distilled water. The color secured is then com- pared with the color produced by similar addition of indicator to standard solutions whose hydrogen ion concentration is known. The indicators most used in the bacteriological laboratory for 112 BIOCHEMICAL TESTS 113 this purpose are those developed by Clark and Lubs. In the following table the name of each of these indicators is given followed by the color in its acid range, next the color in its alka- line range, and finally the range of Ph values through which it changes color. Color Changes of Clarfc and Labs Indicators Indicators Full acid color FuU alkaline color Sensitive range. The in- dicator changes from the acid color to the alkaline color between the following Ph values Thymol blue (acid range) Brom phenol blue Red Yellow Red Yellow Yellow Yellow Yellow Yellow Colorless Colorless Yellow Blue Yellow Purple Blue Red Red Blue Red Red 1.2-3.8 3 0-4 6 Methyl red 4 4-6 Brom cresol purple 5 2-6 8 Brom thymol blue 6.0-7.6 Phenol red 6.8-8.4 7.2-8.8 Thymol blue (alkaline range) . Phenolphthalein 8.0-9.6 8.0-9.8 Cresolphthalein 8.2-9,8 It is apparent that an approximate idea can be secured of the change in hydrogen ion concentration by using different indi- cators and determining which gives an acid color and which an alkaline color. For example, if it is found that phenol red gives a yellow reaction the medium is acid and must be below the Ph value of 6.8. If methyl red, on the other hand, gives an alkaline color it must be of a Ph value above 6. The exact Ph value can be determined then by the use of Brom thymol blue, comparing the intensity of the color change from yellow to blue with stand- ard solutions whose Ph values are known. For discussion of the methods of preparing standard solutions and colors for accurate determination for hydrogen ion concen- tration a suitable laboratory manual should be consulted. Determination of Acid Production.— What has been stated above concerning determination of hydrogen ion concentration will indicate the method of determining whether or not acid has actually been produced by an organism. It should be noted, however, that the determination of hydrogen ion concentration 114 VETEEINAKY BACTERIOLOGY is not a determination of the total amount of acid produced. This can be determined only by a comparative titration. It is customary to titrate to a definite tint a sample of the sterile medium retained as a check, using phenolphthalein as an indi- cator. For this purpose, it is usual to place 5 c.c. of the sterile material mixed with 45 c.c. of distilled water in a porcelain evaporating dish, add a drop or two of alcoholic solution of phenolphthalein and add twentieth normal (N/20) alkaline until a definite pink color has been established. This is kept as a standard, and the amount of alkali required noted. A similar titration is made using the medium in which the organism to be studied has been grown. This is treated in exactly the same fashion and the twentieth normal alkali added until the same tint has been secured as with the check. The differences between the amounts of alkali required for bringing to the same tint of red will vary directly with the amount of acid formed. In some cases it is desirable to determine the kind of acid which has been produced. Simple chemical tests for the various organic acids have not in most instances been developed. Some- thing of their nature, however, may be determined by separating them into volatile and non-volatile. If a solution containing an acid is acidified with sulfuric acid and the material dis- tUled, certain acids, particularly acetic, propionic and butyric will pass over, while other acids, particularly lactic, will not. This makes it possible by titration of the distillate to determine the relative proportion of volatile and non-volatile acid. Gas Production. — A few pathogenic bacteria produce gas from proteins, but most gas-producing species require the presence of a carbohydrate. The gases most commonly formed are carbon dioxid and hydrogen. One or more species of cellulose-fermenting organisms can also produce methane, and some of the denitrifiers free nitrogen. The ability to produce gas may be determined by inoculation of the organism into a dextrose agar or gelatin tube. Gas-bubbles will appear in the medium if the organisms can ferment dextrose. This sugar is generally used, as it is more easily fermented than most other carbohydrates. The fermentation tube is commonly used for the study of gas production. The closed arm is entirely BIOCHEMICAL TESTS 115 filled, and the open arm partly filled, with broth containing the sugar to be tested. The gas found after inoculation col- lects in the closed arm and may be conveniently measured by means of a Frost gasometer. The approximate composition of the gas may be determined by filling the open arm with normal sodium hydrate and securely closing the opening with the thumb, mixing the gas with the alkaline solution by passing it several times from one arm to the other, finally returning it to the closed arm and removing the thumb. The Uquid will then rise in the Fig. 53.-Fermentation tube and Frost gasometer (Heinemann). Closed arm to replace the carbon dioxid absorbed. The remammg gas may be transferred to the open arm and ^-ted ^^ tl^e^f ^^ Hydrogen is indicated by a slight explosion. The ^l^tive pro po'rtion of carbon dioxid and hydrogen is sometimes of ij ta- I the differentiation of species. More ^-P^^^^* ^*^^^^^^^^^^ ability of a species to ferment different kmds of -rbohydra e • Some ferment dextrose, but not lactose or saccharose-some "^t:r£r:r^-!^cteria,whenlivingintheabsence 116 VETEEINABY BACTERIOLOGY of free oxygen, can reduce certain chemicals, evidently securing oxygen for growth processes by this means. Litmus, methylene- blue, and other pigments may be decolorized. Nitrates are frequently reduced to nitrites. For this determination a broth made from 0.1 per cent, peptone and 0.02 per cent, potassium nitrate is inoculated and incubated for four days. It is then tested by the following reagent for the presence of nitrites: a. 5 Naceticacid lOOOc.c. Sulphanilic acid 8 gno- b. 5 N acetic acid 1000 c.c. Alpha-amidonaphthylene 5 gm. Add 2 c.c. of each solution to the tube to be tested. A red or rose color will indicate the presence of nitrite. A control in check tubes of uninoculated broth should always be tested at the same time. In some cases denitrification goes still further and the nitrogen is liberated in the free state. Other reduction processes have been described. Among the more important are the reduction of sulphates to sulphids, and of chlorates to chlorites. Alcohol Production. — Yeasts produce considerable quantities of alcohol, and smaller amounts are formed as a result of the growth of a few bacteria and molds. Occasionally smaller amounts of other alcohols may be developed such as amyl, butyl and propyl. With yeasts, alcoholic fermentation is accom- panied by the production of carbon dioxide. The presence of ethyl alcohol may be detected by placing a few cubic centimeters of the material to be tested in a suitable container such as a test tube, and adding a small crystal of iodine and several cubic centimeters of strong solution of sodium hydrate. If alcohol is present heating this material over a Bunseh flame will give a distinct odor of iodoform. The amount of alcohol developed may be determined by distillation and the determination of the specific gravity of the distillate. Aldehyde. — Certain microorganisms, particularly when grow- ing in carbohydrate solutions, produce aldehyde in sufficient quantity to give a distinctive reaction. The detection of the presence of aldehyde is best accomplished by the use of a fuchsin BIOCHEMICAL TESTS 117 indicator. If a solution of basic fuchsin is decolorized by the addition of sodium sulphite (or better sulphurous acid) until the color has just disappeared or until the material is of a very light pink, the color of the fuchsin is restored upon the addition of aldehyde. This is the principle made use of in the so-called Endo medium. Certain bacteria when growing upon this medium form red colonies; the fuchsin turns red because of the development of aldehyde and acid. Other species of bacteria grown upon this medium do not change the color at all. Acetyl Methyl Carbinol.— This compound is produced by certain bacteria growing in the presence of carbohydrates. It is recognized by the addition of strong alkali such as sodium hydrate or potassium hydrate. When allowed to stand for a few hours an eosin pink or red color will develop, particularly near the surface, providing there is some peptone present. This is fre- quently called the Voges-Proskauer reaction, after the men who first noted it. Determination of Oxygen Relationships. — Bacteria are fre- quently divided into two groups, those which require free oxygen of the air for their development and those which will grow with- out. The first are termed aerobes, and the second anaerobes. Those bacteria which can grow either in the presence or the absence of free oxygen are termed facultative anaerobes, and those which win not grow in the presence of free oxygen are termed obligate anaerobes. It is frequently necessary in the laboratory to determine accurately the oxygen preferences of a microorganism. The bacteria which will grow only upon the surface of a medium, but never grow in the closed arm of a fermentation tube under any conditions are the obligate aerobes. For the most part these are organisms which are actual oxidizers, changing carbonaceous materials' to carbon dioxide and water, for example. The faculta- tive anaerobes are those which grow in contact with air, but at least under certain conditions will grow in the absence of free atmospheric oxygen. Most of the facultative organisms are obligate aerobes on certain media and facultative on others. Many bacteria, for example, which usually require atmospheric oxygen can grow in its absence providing nitrates are present to 118 VETERINARY BACTERIOLOGY furnish an available supply of oxygen, though not in the free form. Other bacteria will grow in the absence of oxygen pro- viding they have suitable carbohydrates. For example, the organism known as Bacterium coli will grow only in the open arm of a fermentation tube if sugar is absent, but in the presence of a suitable sugar such as dextrose it grows both in the open and in the closed arm. The obligate anaerobes are those which will grow only in the absence of free oxygen or are definitely injured by its presence. It is evident that special cultural conditions are necessary for their study. A few bacteria have been termed microcBrophiles because they tend to grow in a definite concentration of oxygen. When mixed with melted agar in a test tube and the agar allowed to^solidify, they will grow in a definite zone some distance below the surface of the medium. Indol Production. — Indol is one of the products of protein decomposition formed by bacterial action. It is of importance principally because it may be demonstrated readily and because of the economic importance of some of the bacteria which produce it. It is not formed in the presence of sugars. Dunham's solu- tion is inoculated with the organism to be tested and incubated for several days. To the tube are added a few drops of concentrated sulphuric acid and a cubic centimeter of a 0.1 per cent, solution of sodium nitrite. The sulphuric acid decomposes the nitrite, freeing nitrous acid, which unites with the indol to form a bright red compound known as nitrosoindol. The appearance of this characteristic red color is evidence, therefore, of indol production. Indol is an organic compound of the empirical formula, CgH^N, and the structural formula c„h/ Sen. It is one of the products formed in intestinal putrefaction, and is the principal product which gives rise, under these conditions, to the characteristic " fecal " odor. Thermal Death-point. — The accurate determination of the exact temperature that is necessary to destroy various species of bacteria is frequently of great economic importance. Efficient steriliza- ' tion and pasteurization can be accomplished only when these facts BIOCHEMICAL TESTS 119 are known. Many methods have been suggested. In the labora- tory the determination is frequently made by subjecting freshly inoculated tubes of broth to different temperatures in a water-bath for ten minutes each. For reliable results more accurate methods are needed. One of the commonest and best is the use of the Sternberg bulb. This is blown of thin glass. A definite amount of culture is introduced and the neck sealed in the flame. The bulbs are completely immersed in a water-bath and suspended by wires or by some other method, so that they do not come in contact with the walls of the bath, and heated. A number of bulbs are prepared and one heated five minutes, another ten minutes, at 50°. The temperature is raised two degrees and two more bulbs are ex- posed. For sporeless bacteria the test should be made to 70°, and still higher for those that produce spores. The bulbs are cooled quickly after their exposure, and their contents mixed with agar in a Petri dish, or added to a tube of other suitable medium. This is then incubated for several days. The minimum temperature required to destroy the bacteria can readily be determined by a comparison of the tubes. Efficiency of Disinfectants.— The efficiency of disinfectants is determined by testing their action on pure cultures of bacteria. Koch's method, which has been commonly used, consists in drying the organism on silk threads, immersing them for varying lengths of time in the disinfectant to be tested, washing in sterile water, and placing the threads upon the surface of agar to determine growth. Hill's method is a modification of that of Koch, and is rather more accurate and relatively simple. Sterilized glass rods are coated at the tip with the bacteria to be tested by dipping them into a broth culture to a depth of an inch. These are placed in test-tubes and carefully dried in a thermostat. They may then be immersed to a somewhat greater depth in the disinfectant to be tested for definite. periods of time, rinsed carefully in sterile water, and placed in tubes containing broth. Phenol Coefficient.-lt has in recent years become standard practice to rate disinfectants by comparing their disinfecting power with that of phenol. The method was first proposed by Rideal and Walker. They prepared various dilutions of the 120 VETERINAEY BACTERIOLOGY disinfectant to be tested, and of phenol, and determined the ratio of the dilution of the former which killed Bacterium ty- ■phosum in a certain length of time to the corresponding dilution of the latter. This ratio they termed the phenol coefficient. This method was modified by Anderson and McClintock of the Hygienic Laboratory. The following procedure is used : 1. Various dilutions of phenol and the disinfectant to be tested are prepared. 2. Each tube is inoculated with -^ c.c. of a broth culture of Bacterium iyphosum, so-called "Hopkins" strain. 3. Transfers of one standard loopful are made at intervals of two and one-half minutes to tubes of +1.5 meat extract broth, and these are in- cubated at 37° C. for forty-eight hours. 4. The average of the ratios between the lowest concentration of the disinfectant being tested and of phenol required to kill in two and one- half minutes, and fifteen minutes is termed the hygienic laboratory phenol coefficient. CHAPTER IX MICROSCOPIC EXAMINATION AND STAINING METHODS Objectives having a higher power than those commonly used in other work are required for the examination of bacteria. A ^^inch or 1.8-2 mm. oil-immersion objective is most commonly used. This lens differs from the low-power dry lenses in that it requires a layer of cedar oil between it and the object to be exam- ined. This oil is used upon the lens for the following reasons. In general, the higher the power of the objective, the smaller the Fig. 54.— Diagram showing the function of an oil-immersion objective (adapted from Gage). opening through which light may come to the eye. It is necessary, therefore, that all the light possible shall enter the lens m order that a well-illuminated field may result. The accompanymg ex- aggerated diagrammatic representation of the objective and the stage of the microscope may be helpful in understandmg the use of the oil. ^ -1 1 • Let C represent the microscopic slide, H the drop of oil havmg the same refractive index as glass, and L the tip of the objective 122 VETEKINAKY BACTEKIOLOGY with the opening F'F. The rays of light are focused upon the ob- ject to be examined by the mirror or Abbe condenser. Those rays of light, such as BN, that strike the glass perpendicularly pass through and enter the lens without any deflection. A ray of light, such as AB, striking the glass at a considerable angle, is refracted upward and toward the normal or in the direction of BD. Upon entering the air it would again be refracted and leave the glass in a direction parallel to the original ray, or DE, and would not enter the lens. If, on the other hand, a drop of oil having the same refractive index as the glass intervenes, there will be no refraction at D, but the ray will pass through to the opening of the lens. This is represented by the ray A'BD'F'. The use of the oil, therefore, results in a more brilliantly illuminated field and a clearer definition of the objects to be examined. Measuring Bacteria. — Bacteria may be measured under the microscope in one of several ways. A micrometer scale ruled on glass may be inserted in the ocular, and the distance between the lines determined by examination of a micrometer scale ruled upon the slide or cover-glass examined under the microscope. When the calibration has been effected, the ocular micrometer may be used to measure the bacteria directly. To illustrate the method of measuring bacteria by means of a micrometer proceed as follows : (o) Insert eye-piece micrometer in the microscope. (b) Examine bacteria on a slide and record their lengths in divisions of the eye-piece micrometer. (c) Remove the slide of bacteria and substitute for it the stage micrometer. (d) Determine the relation of the divisions of the eye-piece micrometer and those of the stage micrometer. (e) The divisions on the latter are of fixed length, usually ^^ mm. The unit of measurement of bacteria is the micron (symbol ;U), the ttsW part of a miUimeter, therefore the length stated above (xiff mm.) = lO^l. Suppose the adjustment is such as to show two divisions of the eye-piece micrometer equal to one division of the stage micrometer. This means that each eye-piece division represents one-half division on the stage micrometer or ^^ mm. = 6/x. Examination of Living Bacteria. — Hanging Drops. — The deter- mination of the motility of bacteria can best be accomplished by the examination of the Uving cells under the microscope. A hang- ing-drop preparation is commonly used for the purpose. A loop- MICKOSCOPIC EXAMINATION AND STAINING METHODS 123 f ul of broth culture of the organism to be tested is placed upon the center of a carefully cleansed and flamed cover-glass. Growth from an agar or other culture may be used by substituting a drop of physiological salt solution or sterile bouillon and introduc- ing a minute quantity of the growth on a platinum needle. This drop is then carefully inverted over the cavity in a hollow ground slide, and sealed with a little vaselin. It may be examined with a high-power dry lens or with the oil-immersion objective. The drop may most easily be brought into focus at its margin. The light must be carefully regulated by means of the mirror and the iris diaphragm of the Abb6 condenser to make the bacteria most clearly visible. Quite as effective an observation may be made in many cases by placing a drop of the culture upon a glass slide and dropping a cover-glass upon it, using care to include a few air-bubbles. A film of liquid sufficiently thick for the free movement of the bac- teria will remain between the two glasses. The edges of the air- bubbles furnish a convenient object upon which to focus. STAINING Methods Bacteria as well as the pathogenic protozoa are generally so transparent when examined in a living condition that the details of their morphology can be made out only with difficulty. It is customary to stain these organisms with various anilin dyes which render them distinctly visible. The stains used in biological work are, for the most part, known as anilin dyes, because they are derivatives of anilin, aUNH^. They are grouped as acid or basic, depending on whether the acid radical or the base possesses the tinctorial powers. Fuchsm for example, is a basic stain, while ammonium picrate is an acid stain. The basic stains are the more useful in the study of bacteria; the acid stains are sometimes used as counterstains Particularly • for tissues in which the organisms may be embedded The anilin dyes are of all the colors of the rainbow. The most com- monly used are gentian-violet, methylene-blue, thiomn blue, fuchsin, and Bismarck-brown. x +„ „„ Mordants.-Anything which will cause a stam to penetrate an ■organism better or which causes it to set is termed a mordant. 124 VETERINARY BACTERIOLOGY For example, carbolic acid or anilin added to certain stains makes them more intense. A solution of iodin in potassium iodid, a mixture of tannic acid and iron sulphate, and many other solu- tions are used under various conditions as mordants. Formulas of Some of the Commonly Used Stains.— There are a few stains which find constant use in the laboratory. The formulas of these will be given. There are, in addition, a great many others which have special appHcations. Loffler's methylene-hlue: Saturated alcoholic solution of methylene-blue 15 C.C. Solution of potassium hydrate (1 : 1000) 50 e.c. Aqweous solution of gentian-violet: Saturated alcoholic solution of gentian-violet 2.5 CO. Distilled water 47.5 ce. Anilin gentian-violet (Ehrlich's) : Saturated alcoholic solution of gentian-violet 6 C.C. Absolute alcohol 5 c.c. Anilin water 50 c.c. Anilin water is prepared by adding 2 c.c. of anilin to 98 c.c. of distilled water and shaking vigorously for several minutes. It should then be filtered until clear. Carbol or phenol fuchsin (Ziehl's) : Saturated alcoholic solution of fuchsin 5 c.c. Solution of phenol, 0.5 per cent 45 c.c. Bismarck-brown: This is commonly used as a saturated aqueous solution. Gabbett's methylene-blue: Methylene-blue, dry 2 gm. Sulphuric acid 25 c.c. Distilled water 75 c.c. Preparation of a Stained Mount. — ^A drop of water, blood-serum,, or broth about the size of a pinhead is placed upon a clean cover- glass. With a sterile platinum needle remove a small portion of the material to be examined and mix thoroughly in the drop. When the bacteria are in bouillon or other Uquid media the drop of liquid is unnecessary. This is then spread in a thin film over the MICROSCOPIC EXAMINATION AND STAINING METHODS 125 surface of the glass and dried. The film is next fixed by passing the cover-glass, film up, through the flame of the Bunsen burner three times. The stain is placed upon the glass and allowed to act for a few seconds to ten minutes, depending upon the organism and the stain used. This is then washed in water until no more stain comes off. It is dried between filter-paper and placed film down upon a drop of water on a sUde and examined under the microscope. If satisfactory, it may be floated off with water, dried, and placed film down on a drop of Canada balsam on the sUde. In many laboratories the use of the cover-glass is largely dispensed with, and certain routine examinations of many kinds can be more conveniently made by means of films prepared directly upon the glass microscopic slides. The procedure is practically identical with that detailed above for cover-glass preparations except that the immersion oil may be placed directly upon the stained film and no cover-glass used. Spore Stain. — Bacterial spores stain with difficulty, but once stained, do not yield up the stain readily. Any one of the follow- ing methods will be found to give good results : Hansen Method. — 1. Prepare a film, fix, and stain with steam- ing hot carbol-fuchsin for five minutes. 2. Decolorize with 5 per cent, acetic acid until the film is a light pink, and wash in water. 3.. Stain three minutes with Loffler's methylene-blue. 4. Examine. Mailer's Spore Stain. — 1. Air dry smear. 2. Fix in flame, or for two minutes in absolute alcohol. 3. Place in chloroform (to remove fat). 4. Wash in water. 5. Stain in carbol-fuchsin, heating for one minute. 6. Decolorize in 5 per cent, sulphuric acid. 7. Wash. 8. Contrast stain with methylene-blue. 9. Wash, dry mount, and examine. By either method the spores appear red and the cell-body blue. Klein's Method of Staining for Spores.— 1. Prepare a suspension of sporulating material in physiologic salt solution. Place in a small watch-glass or test-tube. 126 VETEBINARY BACTERIOLOGY 2. Mix this with an equal quantity of filtered Ziehl carbol- fuchsin. 3. Heat the mixture until it steams for six minutes. 4. Place small loopful on slide and spread. 5. Air dry and fix in flame. 6. Decolorize with 1 per cent, sulphuric acid a few (one to two) seconds. 7. Wash in water. 8. Counterstain in dilute methylene-blue three to four minutes. Stain for Acid-fast (Acid-proof) Organisms.— Certain bacteria are stained with difficulty, but when once stained, they resist decolorization with acids. The most important of these organisms is Mycobacterium 4uberculosis. Add Alcohol Method. — 1. Prepare film, fix in flame, and stain with hot carbol-fuchsin for two minutes. 2. Wash in 2 per cent, hydrochloric acid in 95 per cent, alcohol until there is no color visible in the thinner portions of the film. 3. Wash in water and stain with methylene-blue for contrast. 4. Wash and examine. Gdbbett's Method. — 1. Prepare film and stain as above with car- bol-fuchsin. 2. Wash in water. 3. Stain with Gabbett's methylene-blue for one-half to one minute. 4. Wash and examine. The acid-fast organisms will be red in a blue field. Herman's Stain for Tubercle Bacilli in Tissues. — Solutions required: 1. One per cent, ammonium carbonate in distilled water. 2. Three per cent, crystal violet in 95 per cent, ethyl alcohol. Sections to be stained are placed on a sHde or cover-glass and the water removed. About 7 drops of a mixture of 3 parts of solution 1 with 1 part of solution 2 are added, and placed on water-bath for one minute. Ten per cent, nitric acid is allowed to act for a few seconds, then the section is placed in 95 per cent, alcohol. The tissue may be counterstained with dilute fuchsin or eosin. The bacteria are stained purple. Wirth 's Stain for Staining Much 's Granules of Mycobacterium MICEOSCOPIC EXAMINATION AND STAINING METHODS 127 tuberculosis.— \. Stain in carbol methyl-violet twenty-foUr hours at room-temperature: Saturated alcoholic solution methyl-violet 10 c.c. Two per cent, carbol water 100 c.c. 2. Place in iodin solution (Lugol's) five to fifteen minutes. 3. Wash in 5 per cent. HNOs one minute. 4. Wash in 3 per cent. HCl ten seconds. 5. Differentiate in a mixture of acetone and absolute alcohol. For a counter-stain use highly diluted carbol-f uchsin 1 drop in 50 c.c. water. The mount is not permanent. Flagella Stain. — The flagella of bacteria are not visible in ordinary stained mounts, and can be demonstrated only by a special technic. Young, twelve- to eighteen-hour cultures of bacteria should be used for their demonstration. A tube con- taining a few cubic centimeters (5) is inoculated with sufScient quantity of the growth carefully removed from the agar surface to produce a slight turbidity. Incubate for an hour in the ther- mostat. Drop two or three drops without mixing or spreading on a clean cover-slip. Dry and then fix in the flame. Many methods of staining flagella have been suggested; the two follow- ing are probably the best: Van Ermengem's Method. — :1. Place the film for one hour in the following solution: Osmic acid, 2 per cent 1 part Tannin, 10-25 per cent, solution 2 parts 2. Wash in water, then absolute alcohol, then place in the following solution for a few seconds only: Silver nitrate, 0.05 per cent, in distilled water. 3. Wash in the following solution for a few seconds: Gallic acid 5 gm. Tannin 3 gm. Fused potassium acetate ^^ §™- Distilled water 350 c.c. 4. Wash in silver nitrate solution until film turns black. 5. Wash in water and examine. 128 VETERINARY BACTERIOLOGY Loffler's Method. — 1. Prepare film, fix, and apply the following mordant, heating for five minutes over a water-bath: Tannic acid (25 per cent, aqueous solution) 10 parts Saturated solution ferrous sulphate 6 parts Fuchsin (saturated alcoholic solution) 1 part 2. Wash and blot with filter-paper. 3. Stain with hot anilin-gentian-violet or carbol-fuchsin over a water-bath for five minutes. 4. Wash and examine. Gram's Staining Method. — This method was first used to demonstrate bacteria in tissues, the bacteria retaining and the tis- sues losing the stain. It was later found that not all bacteria could be stained by this method, and it has in consequence come into general use for separating bacteria into two groups, termed respectively gram-positive and gram-negative, the former retain- ing the stain and the latter losing it. 1. Prepare film, dry, and fix. 2. Stain one and one-half minutes in anilin-gentian-violet. 3. Treat with Gram's iodin solution one and one-half minutes. lodin 1 gm. Potassium iodid 2 gm. Water 300 c.c. 4. Decolorize with 95 per cent, alcohol for five minutes. 5. Wash, dry, and mount. Stabilized Gentian Violet.' Anilin 28 c.c. Gentian violet. 8 gm. 95 per cent, alcohol 100 c.c. N hydrochloric acid g g p Dist. water q.s 1000 Dissolve gentian violet in alcohol. Add HCl to the anilin and dissolve in water to make 900 c.c. Filter the aqueous solu- tion and add alcoholic stain. Filter. Capsule Stains.— Mwir's Capsule Stain.— 1. Prepare film and dry. 2. Apply carbol-fuchsin (hot) for thirty seconds. 3. Wash quickly in 95 per cent, alcohol, then in water. 1 Stovall and Nichols, Jour. A.M.A., 1916, 66, 1620-1621. MICROSCOPIC EXAMINATION AND STAINING METHODS 129 4. Apply following mordant for five or ten seconds: (a) Saturated aqueous solution HgCl2 2 parts. (b) Twenty per cent, aqueous tannic acid 2 parts. (c) Saturated solution potash alum 5 parts. 5. Wash in water and apply 95 per cent, alcohol one minute. 6. Wash in water and apply methylene-blue for thirty seconds. 7. Dry and examine. Johne's Capsule Stain. — This is particularly suited to the demonstration of the capsules of the anthrax bacillus in smears from blood or tissues. 1. Dry in air. 2. Fix by passing three times through the flame. 3. Stain with 2 per cent, aqueous gentian-violet, heating slightly for one-quarter to one-half minute. 4. Wash quickly in water. 5. Wash in 1 to 2 per cent, acetic acid six to ten seconds. 6. Wash in water; mount in water to examine. Raebiger's Capsule Stain. — This has been used for demonstra- tion of the capsules of the anthrax bacillus in tissue and blood- smears. The formahn gentian-violet is prepared by adding 15 to 20 gm. of gentian-violet to 100 to 150i gm. of 40 per cent, solution of for- maldehyd (commercial formalin). This should be thoroughly mixed and allowed to stand for some hours. The solution is filtered. 1. Prepare thin smear of material. 2. Air dry (do not fix). 3. Stain twenty seconds. 4. Wash in water. 5. Mount in balsam. The capsules should appear reddish violet, the bacilli dark violet. Blood and Protozoan Stains.— Many special stains have' been devised for demonstrating the blood elements and protozoa in the blood and in tissues. The chief of these are the Romanowsky and Giemsa, each with numerous modifications. These may most profitably be purchased ready for use from a reUable dealer. Wright's Stain.— One of the most satisfactory blood stains for 9 130 VETERINARY BACTERIOLOGY routine work is that of Wright. This stain can be purchased in hquid form ready for use, or in powder form from which a satu- rated solution is made up as a stock solution. This is prepared for use by adding to 20 c.c. of the filtered stock solution 5 c.c. of methyl alcohol. A blood-smear is made and allowed to air dry. The sUde is then flooded with the Wright stain and allowed to stand one minute. Distilled water is then added drop by drop until a metallic luster appears on the surface. The stain is per- mitted to act for five minutes, when it is washed off with distilled water, dried, and examined with or without oil immersion. Giemsa's Stain. — 1. Apply the following fixing agent to moist films for twelve hours. 95 per cent, alcohol 1 part. Saturated aqueous HgCl^ 2 parts. 2. Wash in water for a few seconds. 3. Apply Lugol's solution for five minutes. 4. Wash in water, then in .5 per cent, sodium thiosulphate. 5. Stain with Giemsa stain one to ten hours. 6. Wash and mount. A modification of this method consists of mixing 1 drop of concentrated Giemsa in 20 drops of distilled water. After fixing the smear for five minutes in methyl alcohol it is immersed in the dilute stain and placed in a 37|° incubator for ten to twelve hours. It is then washed off with distilled water until the film has a slight pink tinge. This method is recommended for staining of brain tissue for Negri bodies. Negri Bodies.— Lentz Method. — Smears upon clean glass slides are made from (a) Ammon's horn, (b) the cerebellum, or (c) the cerebral cortex. Without allowing to dry, the smears are fixed for about 10 seconds in neutralized methyl alcohol (alcohol 500 c.c. to which about 0.25 gm. of sodium carbonate has been added) to which 0.1 per cent, picric acid has been added. The excess of fixative is removed by blotting .with fine filter paper. The fixed smears are stained in the following solution : Saturated alcoholic solution of fuehsin 0.3 gm. Saturated alcohoUc solution of methylene blue 2.0 gm. Distilled water 30.0 gm. MICROSCOPIC EXAMINATION AND STAINING METHODS 13X This solution, which is a modification of the one proposed by Van Gieson, changes rather quicUy at room temperatures, but kept in the ice box gives good results for an indefinite time. ' The stain is poured on the smear and held over the flame until it steams. The smear is then washed in tap water and dried with fine filter paper. Negri bodies appear magenta, the nerve cells blue, and red blood cells yellow or salmon. 3. Stain in methylene-blue solution one minute. 4. Wash in water. 5. Dry between layers of filter-paper. 6. Differentiate in alkahne alcohol until only a sUght pink can be recognized. 7. Differentiate in acid alcohol until the thinner part of the preparation shows no blue. 8. Wash short time in absolute alcohol. 9. Dry and examine with oil immersion. The Negri bodies appear crimson as distinct from the vermilion blood-corpuscles, and show within them one or niore blue inclu- sion bodies. India-4nk Method for Bacteria and Protozoa.— Mix. the fluid containing the organisms to be examined with an equal quantity Of India ink. Make a thin smear, dry, and fix. The organisms do not stain with the ink, but appear as transparent bodies in the black field. CHAPTER X METHODS OF SECURING PURE CULTURES OF BACTERIA Bacteria must be studied in pure culture if one is to deter- mine with certainty their cultural, physiological, or pathogenic characters. One of the first efforts made in the study of a disease or any other process brought about by bacteria is to separate its causal organism from all others. Many methods have been devised for this purpose, not any one of them applicable to every case. Dilution Method. — This method of securing pure cultures is of historic interest only. In the beginnings of the cultivation of microorganisms the culture-media commonly used were liquids, such as infusions from meat and vegetables, and beerwort. This method was used most commonly in securing pure cultures of yeasts. A long series of flasks was prepared with sterile media. The impure culture or mixture of organisms was mixed thoroughly with the contents of the first flask, and a definite amount transferred from this to another flask, from this to each of several others, from each of these into another group, and so on. The last dilution would, in general, remain sterile, but among some of the dilutions would be a group in which some flasks would show growth and others of the same dilution would not. The inference was that such a flask had been planted with but a single organism, and the flask contents, therefore, constituted a pure culture. This method is cumbersome, uncertain, and is rarely used. Isolation by Smearing. — If a loopful of a mixed culture of microorganisms be drawn across the surface of a solid medium in parallel streaks, the first portion will generally show a solid line of mixed growth, but farther along the growth is discontinuous. Many of the isolated colonies here will be found upon examination to consist of pure cultures. This method is used for the isolation of bacteria from the mouth and throat in some cases. Direct Isolation. — Barber has devised a capillary pipette method whereby it is possible to pick up a single bacterial cell and transfer it to a nutrient medimn without any other organisms being carried 132 METHODS OF SECURING PURE CULTURES OF BACTERIA 133 over. This method has been found useful in the study of develop- mental and evolutionary problems, but is not practicable for routine laboratory isolations. Isolation by Plating.-The development by Koch of the lique- hable media furnished a ready means for the isolation in pure Fig. 55.— Isolation by successive streak cultures on an agar or gelatin plate: A, First streak solidly grown; B, second streak, discontinuous; C, third streak, having many isolated colonies. culture of most species of bacteria. Nutrient agar or gelatin or one of their modifications may be used. The medium is lique- fied by heat, then cooled in a water-bath to about 43°. The mixed culture of organisms from which it is desired to isolate* pure cultures is inoculated into one of the tubes. From this Fig. 56.— Petri dish (McFarland). transfers are made by means of a sterile platinum loop to a second tube; this is thoroughly mixed and transfers made to a third tube, and from this even to a fourth. Each of these tubes of media is then poured into a sterile, flat, glass, covered dish called a Petri dish. These Petri dishes or " plates " are allowed to stand until the medium has sohdified; they, are then incubated and examined 134 VETERINARY BACTERIOLOGY from time to time. The organisms are separated from each other by this process of dilution, and are held fast by the solidifica- tion of the medium. In most cases the conditions are favorable for growth, and development begins. Within a few days sufficient multiphcation takes place, so that the mass of organisms that has developed from the single isolated individuals has reached a size that can be easily seen with the unaided eye. Such a mass of organ- isms is termed a colony. Transfers from such colonies will show only a single kind of organism present, and by making isolations from each type of colony, pure cultures may be secured of each species present. Isolation by the Use of Heat. — When it is desired to isolate a spore-producing organism from non-sporulating forms, the culture may be heated to 80° for fifteen minutes. This will not destroy the spores, but will eliminate all other cells. If one species of spore-forming organism only is present, this results in a pure cul- ture at once; if more than one species, plating becomes necessary. Isolation by the Use of Differential Antiseptics or Disinfect- ants. — Not all species of bacteria are affected alike by a given antiseptic or disinfectant, and it is sometimes possible to add a substance that will prevent the growth or kill one form without interfering seriously with the growth of others. A small amount of phenol added to bouillon will inhibit the growth of most bacteria, with the exception of certain members of the intestinal group. A still better example of such substance is antiformin, which, when mixed with sputum or other materials containing tubercle baciUi, destroys all other organisms thaii these, and enables one to secure a pure culture at once. This will be discussed in greater detail under the heading of Tuberculosis. Isolation by Animal Inoculation. — Some species of pathogenic bacteria develop very slowly upon artificial media, or require a special medium for their growth. When these occur mixed with other organisms, it is sometimes difficult to secure them in pure culture. This difficulty may in some cases be overcome by animal inoculation. The injection of the organism into a suitable susceptible animal results in the destruction by the body of the other bacteria injected at the.same time, and the characteristic organism may later be isolated in pure cultures from the lesions of the disease. CHAPTER XI STUDY OF BACTERIAL CULTURES Species of bacteria are frequently separable from each other on the basis of differences in cultural characters alone. It is, therefore, important that careful descriptions should> be kept of the cultural characteristics of each of the species. For assistance in such descriptions the Society of American Bacteriologists has adopted a standard descriptive chart from which the following are adapted: Cultural Characters Agar Stroke. — This is prepared by drawing an inoculated needle from the base to the top of the slanted surface of an agar tube that has soUdified in the sloping position. In this culture are to Is* Fig. 57. — Typas of growth on agar sla.uts. be noted the abundance, form, elevation, luster, surface, and optical characters of the growth, its pigment production, odor, consistency, and any changes that have occurred in the medium. Potato.— The potato is inoculated and the growth character- istics studied in the same manner as the agar stroke. 135 136 VETEEINARY BACTERIOLOGY Blood-serum.— This is inoculated in the same manner as the agar slope, and the same characteristics are to be noted, with the addition of liquefaction or digestion of the medium. Gelatin Stab.— This is prepared by running an inoculated plat- inum needle in a straight line from the surface of an erect tube of nutrient gelatin nearly to the bottom, and withdrawing it with- out cutting the medium by any lateral motion of the needle. Fig. 58. — Potato slant culture (Page, Frothingham and Paige, in "Journal of Medical Research"). The characters to be noted are abundance and uniformity of growth along the line of the stab, the form of growth, Hquefaction, and other changes in the medium. Nutrient Broth. — This is inoculated by shaking an infected platinum needle in the medium. The characters to be noted are abundance arid character of surface growth and character of sediment. Milk. — Milk is inoculated in the same manner as the nutrient STUDY OF BACTERIAL CULTURES 137 broth. The characters to be noted are presence or absence of coagulation, type of curd produced, whether or not whey is extruded peptonization or digestion of the casein, acid production, con- sistency, and changes in color of the medium. Litmus Milk.— In addition to the preceding, acid or alkali production and reduction of the litmus are to be noted. » M M X viy t r $ mm 9 10 n P' T .;-.;.v..'.>/0.*:-;--": V J \ J \i''^A J QJ Fig. 59. — Types of growth in stab cultures: A, Non-liquefying: 1, Fili- form {Bact. coli) ; 2, beaded (Sir. pyogenes) ; 3, echinate {Bact. acidi lactici) ; 4, villous {Erysipel. murisepticum) ; 5, arborescent {B. mycoides). B, Lique- fying; 6, Crateriform (Pr. vulgaris, twenty-four hours); 7, napiform (B. subtilis, forty-eight hours); 8, infundibuliform (Erythrobadllus prodigiosus); 9, saccate (Vibrio Finhleri); 10, stratiform {Ps. fluorescens) (Frost). Gelatin Plate Colonies. — Two or three tubes of gelatin are melted, cooled to 40°, and one inoculated with a small amount of the organism to be studied. The tube is rolled until the bacteria are thoroughly distributed, and with a platinum loop a transfer is made to a second tube, and from this to a third. The con- tents of each tube are then poured into a Petri dish and allowed 138 ■ VETERINARY BACTERIOLOGY to solidify. The plate showing a small number of colonies devel- oping is the one chosen for examination. The characters to be Fig. 60. — Portion of an agar plate culture showing a mold colony and five bacterial colonies. noted are rapidity of growth, form, elevation, and edge of the colony and type of liquefaction if it occurs. Fig. 61. — Ameboid colony on an agar plate (Lewton-Brain and Deerr). Colonies on Agar Plates. — Plates containing nutrient agar are prepared in the same manner as the gelatin plates described. The STUDY OF BACTERIAL CULTURES 139 Fig. 62.— Spreading colony on an agar plate (Lewton-Brain and Deerr). Fig. 63. — Colonies in an agar plate culture (Lewton-Brain and Deerr). characters to be noted are rapidity of growth, form, surface ele- vation, edge, and internal structure of the colony. 140 VETERINARY BACTERIOLOGY Physiological Characters It is customary to determine gas and acid production in car- bohydrate media, development of ammonia, reduction of nitrates to nitrites, indol production, temperature relations, including optimum growth temperature and thermal death-point, resistance to desiccation and disinfectants, and pathogenic characters. The methods of study of these characters have already been discussed. SECTION III BACTERIA AND THE RESISTANCE OF THE ANIMAL BODY TO DISEASE CHAPTER XII IVnCRCX)RGANISMS AND DISEASE Infectious Diseases. — An infectious disease is one which is caused by some microorganism. The mere presence of micro- organisms in the body, however, does not constitute infection. In general, this is not regarded as occurring unless the organisms grow or multiply in the body and produce pathological changes in the tissues and symptoms of disease. An individual thus infected is termed the host. Both infective and infected have been used to describe inanimate objects which harbor pathogenic organisms and which may aid in their distribution; of these, the term infective is preferable. Infective objects, such as the manger of a glandered horse or the clothing of a person with a readily transmissible disease, are called fomites. The term virus is commonly used to designate the causal organism of an infectious disease when such organism is not known. Sometimes it is used in a broader sense to designate any disease-producing organism. Diseases, that is, pathological conditions of the body, may be either infectious or non-4nfectious. Among the infectious diseases of animals and man are the following : Fistula, boils, abscesses and similar pyogenic infections, strangles, pneumonia, meningitis, Malta fever, gonorrhea, diphtheria, tuberculosis, paratuberculosis, pseudotuberculosis, swine erysipelas, glanders, dysentery, typhoid, hemorrhagic septicemia, plague, chicken-cholera, anthrax, milk sickness or trembles, infectious abortion, foot-rot of sheep, tetanus, blackleg, malignant edema, botulism, Asiatic cholera, actinomy- cosis, blastomycosis, aspergilloses and related mycoses, ring- 141 142 VETERINARY BACTERIOLOGY worm, amebic dysentery, trypanosomiasis, LeJshmaniosis, relap- sing and tick fevers, Texas fever and related piroplasmoses, ana- plasmoses, malaria, yellow fever, coccidiosis, pleuropneumonia, foot-and-mouth disease, rinderpest, hog-cholera, horse sickness, equine infectious anemia, fowl plague, the poxes, such as small-pox and chicken-pox, infantile paralysis, rabies, and others. Non-infectious diseases are those which are caused by some injury to the body or by some physiologic disturbance not due to microorganisms. Among such diseases may be mentioned cer- tain types of tumors and cancers, diabetes, azoturia, certain types of neuritis, arteriosclerosis, cardiac hypertrophy, etc. In some cases the infectious or non-infectious origin of a disease has not been satisfactorily determined. Such a disease is cancer. Contagious Diseases. — Any disease in which the causal organ- ism may be readily transferred from one individual to another by direct or indirect contact is said to be contagious. The terms in- fectious and contagious have been used very loosely, sometimes, even interchangeably. The best usage, however, is strictly to limit the terms as here defined. Infecticus has to do with the cause of a disease, and contagious with its ease and method of transmission. All infectious diseases, therefore, which are readily transmitted by contact, direct or indirect, are said to be contagious. All contagious diseases are necessarily infectious, but the reverse is not true; for example, malaria in man and Texas fever in cattle cannot be regarded as contagious, as they are transmitted only through the bites of certain insects. Every gradation between highly contagious and non-contagious dis- eases is known. It is customary to indicate the degree of contagiousness by use of modifiers. We speak, therefore, of highly contagious, slightly contagious, etc. Avenues of Infection.— The avenue through which an organism gains entrance to the body is called its portal of entry or its infec^ tion atrium. An infection arising from contact with infective external objects is termed exogenous; one caused by organisms con- stantly or normally present in the body or on it is termed endogen- ous. Traumatic infection frequently occurs through a break in the continuity of the skin or mucous surfaces. Wounds caused by weapons, instruments, and similar objects, the bites of animals. MICROORGANISMS AND DISEASE 143 and sucking insects, such as the mosquito, flea, tick, or bedbug, may introduce a pathogenic organism. Ordinarily, the skin is an efficient barrier against infection. Microorganisms may occa- sionally enter through the glands or hair-follicles. Some bacteria apparently may injure the unbroken mucous surface, as, for example, the diphtheria bacillus. Certain disease organisms enter through the digestive tract. Bacteria have been shown to pass unharmed through the intestinal walls and to enter the lymph- vessels and the thoracic duct. To what extent this is the common source of infection in certain diseases, such as tuberculosis, is at present a matter of dispute. The lungs constitute the infection atrium in pneumonia, prob- ably in many cases of tuberculosis and aspergillosis, and possibly in certain diseases whose cause has not been determined, such as small-pox. The genital organs are the common infection atria in the so- called venereal diseases, such as syphilis, chancroid, and gonorrhea in man, dourine in the horse, and possibly in contagious abortion in cattle. Disease organisms rarely pass from the blood of the mother through the placenta to the blood of the fetus. In a strict sense, none of the infectious diseases present at birth are inherited. Cryptogenic infections are those which occur without the possi- bility of determining the infection atrium in a particular case. Tetanus, for example, sometimes occurs when careful search fails to show the channel through which the organism reached the tissues. The ability of an organism to multiply in the body or produce disease frequently depends upon the channel through which it enters; the organism which produces Asiatic cholera in man, for example, evokes this disease with its characteristic symptoms only when entering through the alimentary tract. The type of disease caused by a particular organism may also vary with the infection atrium; the disease caused by the organism of human plague shows differences depending on whether the infection occurs through the skin, the alimentary or respiratory tracts. An organism may pro- duce no lesions at the point of entry, but invade some other tissue; the bacillus of tuberculosis is known to pass through the mucous 144 VETERINARY BACTERIOLOGY membranes of the intestines without visible injury to them and to produce tuberculosis of adjacent glands. Virulence. — The virulence of an organism is its relative ability to combat the defences of the body against infection, to invade the tissues, and to produce disease. The degree of virulence on the part of the organism and the relative resistance to infection on the part of the animal determine whether or not disease will be pro- duced, as well as its course and termination. The virulence of organisms shows great variations even within the same species. It may be modified in various ways. Methods of immunization are in many cases based upon the possibility of attenuation. Proof of the Causal Relationship of a Microorganism to a Dis- ease — Koch's Postulates. — The proof of the germ theory of disease may be dated from 1876, when Koch succeeded in demonstrating the causal relationship of Bacillus anthracis to the disease anthrax. He later formulated the rules (known as Koch's rules or postulates) for the determination of the specific relationships of an organism to a disease. They may be stated as follows : 1. The suspected organism must be found in every case of the disease under consideration. 2. The organism must be isolated and grown in pure culture. 3. Inoculation of the organism into suitable animals should reproduce the disease. 4. The organism must again be isolated from such animals. Unfortunately, the demonstration by means of these rules of the causal relationships of specific organisms to many diseases has proved impossible, and is unsatisfactory in others. .There are several reasons for this: (a) The organisms in some cases have been shown to be very small, perhaps even ultramicroscopic,- and capable of passing through a fine porcelain filter, as, for example, those which cause fowl plague and hog-cholera. (6) Some organisms, although evidently not ultramicroscopic, have never been satisfactorily demonstrated under the microscope, possibly from lack of proper staining methods. (c) Some organisms are specific for man and do not reproduce disease of the same type when inoculated into animals. With MICROORGANISMS AND DISEASE 145 some of these, accidental or intentional inoculation into man has supplied the needed evidence. (d) The organism may be demonstrated microscopically, but will not grow upon the culture-media of the laboratory. Such are some of the protozoa. With a few of these the proof has been perfected by the study of the growth of the organism in an intermediate host, for example, the malarial parasite in the mosquito. Evidence of the relationship of an organism to a disease may frequently be secured by using the agglutination, precipitation, or some of the other tests discussed in the following chapters. Im- provements in staining technic and culture methods continually reveal new organisms. Anim al Inoculation. — Experimental inoculation and injections are made in the bacteriological laboratory for a number of reasons: 1. To determine the causal relationships of a specific organism to a disease in accordance with Koch's rules. 2. To diagnose certain diseases. For example, one of the methods of diagnosing glanders is to inject some of the nasal discharge from a suspected animal into a male guinea-pig and note the development of acute orchitis and subsequent general body reaction. 3. To isolate certain pathogenic bacteria. For example, if it is desired to isolate the organism causing tuberculosis from sputum or from milk, it may be accomplished most readily by inoculating the infective material into a suitable animal. All the non-patho- genic organisms will be destroyed and the specific organism may be isolated in pure culture from diseased tissue or lesions of the ani- mal so infected. The animal body is used in a sense as a filter for the removal of the non-pathogenic bacteria. 4. To determine the strength or concentration of certain bio- logical products. As will be seen later, the only way that has been devised for the determination of the strength or potency of certain poisons, such as toxins and of their antitoxins, is animal injection. The animal is used by the bacteriologist in much the same manner as an indicator is used by the chemist in determining the acidity or alkahnity of a solution. 5. For the production of certain so-called antibodies, such as 10 146 VETEEINAHY BACTERIOLOGY antitoxins, and for the demonstration of certain characteristics of the blood-serum in studies of immunity. The animal most frequently used in experimental work is the guinea-pig or cavy; next in importance is the rabbit. Mice and rats, particularly the white varieties, are often used. When birds are necessary, the pigeon and domestic fowl are generally utilized. Some of the larger animals, as the goat or horse, are used for the production of serum where it is required in considerable quanti- ties, as in the manufacture of antitoxins. The monkey has been used to some extent in the studj'^ of diseases peculiar to man. Heifers are utilized in the preparation of the vaccine against small- pox. Swine are used in the preparation of hog-cholera antiserum. Methods of Inoculation. — Animals may be inoculated just beneath the skin, or subcutaneously. The hair is shaved from the Fig. 64. — Ear veins of a rabbit: a, Posterior vein; b, point at which injections may be most easily made; c, median vein (adapted from Frost). area selected, the skin is washed with an antiseptic, and a hypo- dermic needle inserted into the subcutaneous tissue. In the inoculation of a solid material a little incision may be made in the skin, the material inserted, and the opening closed. Usually stitches to hold the skin are unnecessary. Intravenous inocula- tion is accomplished by inserting the needle into a vein. Usu- ally a rabbit is selected for this purpose. Reference to Fig. 64 will show the vein on the posterior edge of the ear, into which in- jections are usually made. The large median vein is not suitable, as it is situated in loose connective tissue and, therefore, difficult to enter. The posterior vein, on the other hand, is embedded in firm connective tissue and cartilage, so that it does not give before the needle-point. Intraperitoneal injection is accomplished by thrusting the hypo- MICROORGANISMS AND DISEASE 147 dermic needle through the abdominal wall. Some care must be used not to penetrate too rapidly, as there is danger of injuring the intestines. Intrathoracic inoculation is rarely practised. Injec- tion directly into the heart (intracardiac) may be successfully car- ried out if suflBcient care is used. Inoculation by scarijication is accomplished by scraping off the outer layers of skin without draw- ing blood and rubbing the organism on the surface moistened by the exuded serum. /n inverted fir tree. Tiie gelatin is liquefied slowly. The growth on potato is creamy in color and rather dry in consistency. Blood- serum is slowly Uquefied. Milk is rendered slightly acid, curdled by a lab ferment, and the casein digested. In bouillon the organ- ism frequently forms a pellicle which readily settles to the bottom. Clouding of the medium does not usually occur. As will be noted from the preceding descriptions, the Bacillus anthrads grows readily on .practically all the culture-media, par- ticularly if they are neutral or have a weakly alkaline reaction. All of the character- istics of the Bacillus an- thrads on culture-media can be duplicated in the growths of other mem- bers of the group which are strictly sapropiiytic. Care should, therefore, be used in the diagnosis of this organism from cultural characters alone. Physiology. — Bacil- lus anthrads grows best in the presence of oxy- gen, and produces spores pjg only under such condi- tions. Under anaerobic or semi-anaerobic conditions, as in lackmus molke and litmus agar, it decolorizes or acts as a reducing agent. The optimum growth temperature is 30-37°. Good growth may be secured at room-temperature or even below. At low tem- peratures the spores are not produced, the optimum for spore de- velopment being 25-32°. The maximum growth temperature is about 45°. The vegetative rods are readily destroyed by heat, five and one- half minutes at 65° sufficing. The spores are much more resistant, requiring 100° for from three to twelve minutes. When dried they have been heated to 140° for three hours before being killed. They likewise exhibit great resistance to desiccation. When dried upon 96. — Bacillus anthrads, with spores (Frankel and Pfeiffer). 256 VETERINARY BACTERIOLOGY threads they have been known to retain their vitality for many years. Because they are probably the most resistant of the spores of pathogenic bacteria, they have been commonly used as test objects in determining the efficiency of disinfectants. Five per cent, phenol kills the spores only after prolonged contact, ac- cording to some investigators even forty days. Several other species of bacteria, particularly the Pseudomonas pyocyanea, inhibit the growth of B. anthracis. Pyocyanase (q. v.), a preparation made from cultures of Pseudomonas pyocyanea, will dissolve B. anthracis in vitro. Fig. 97. — Bacillus anthracis colony (Gtinther). Fig. 98. — Bacillus anthracis, stab culture in gelatin (Gunther). Indol is not produced. Small quantities of hydrogen sulphid develop in peptone solutions. The enzyme gelatinase, which digests gelatin, has been demonstrated in the bacteria-free filtrates from cultures. A rennet-like enzyme and others which digest ca- sein and blood-serum are produced. Some acid is formed in certain media, but it is not marked. The organism causes hemolysis when grown in blood-bouillon or on blood-plates. Pathogenesis. — Mice and guinea-pigs are highly susceptible to infection with anthrax, rabbits only slightly less so. These animals usually succumb in from twenty-four to forty-eight hours after inoculation. Rats are less susceptible, but show considerable variations. They usually die in about three days, but the disease sometimes assiunes a subacute or chronic form and the animal may ANTHRAX GROUP. THE GENUS BACILLUS 257 live for several days to as many weeks. Sheep die quickly after the injection of minute doses of the organism, cattle are shghtly more resistant, but usually succumb. Horses, swine, and goats may also be infected, but do not contract the disease spontaneously as frequently as do the other species. Dogs and cats are also some- what susceptible. Certain birds may sometimes be infected, but, in general, are relatively immune. Cold-blooded animals are re- fractory. Man is relatively susceptible. Character of Disease and Lesions Produced. — The type of dis- ease and the lesions produced vary somewhat with the animal infected. In cattle the disease is usually an acute febrile infection with no external localization. The temperature goes to 41 to 42°, there are difficulty in respiration, hematuria, and bloody discharges from the body openings. It is very quickly fatal usually; the animal sometimes dies within a few minutes or hours after the first symptoms; in other cases the animal may live for several days. Occasionally the disease manifests itself as ah; anthrax carbuncle, usually on the shoulder, breast, or neck. This type is not quite so uniformly fatal. Sheep die very quickly; usually the sjonptoms are not manifest more than a few hours. Carbuncles are very rare. Horses usu- ally have the disease in acute form, djang in a day or two. Occa- sionally in localized anthrax (carbuncles) they live for somewhat longer periods. Swine, being decidedly more resistant, show car- buncles of the mucous membranes and locahzed glandular infec- tions not infrequently chronic or terminating in recovery. Anthrax in the dog is much as in swine. Three methods of infection in man determine the three types of anthrax which may develop : Pulmonary anthrax or woolsorters' disease is contracted by the inhalation of anthrax spores. The possible presence of anthrax spores in hair, wool, and on hides neces- sitates careful disinfection. The disease runs its course as an atypical pneumonia, practically always fatal. Intestinal anthrax results from the ingestion of living anthrax bacilli. The disease is very rare, but usually fatal. In its earlier stages it is probably localized. Cutaneous anthrax, or malignant carbuncle, is the result of infection through scratches or cuts in the skin. At first 17 258 VETERINARY BACTERIOLOGY the lesion resembles that of a common furuncle. Soon there is necrosis of tissues near the primary pustule, and blood infection and death frequently follow. However, in many cases the infection remains localized and eventually heals. The disease is in most animals a rapidly fatal septicemia. The most characteristic change to be noted upon autopsy is the great enlargement of the spleen, with distention of the capsule and soft- ening of the pulp, congested and usually dark in color. In the vicinity of areas of localized infection there are generally hemor- rhages and hemorrhagic exudate. The liver, kidneys, and lungs are usually congested and ecchymotic. The organism is found in great numbers in the blood-stream. Figs. 99 and 100. — Bacillus anthracis, stained mount of blood, showing the capsules of the bacilli (Preisz). Immunity. — No true toxin has been demonstrated for the anthrax bacillus; in fact, it is difficult to account for the patho- genicity of the organism on the basis of specific poison formation. Immune serum is claimed by some investigators to have a con- siderable specific agglutinative power, but others have failed to demonstrate this. It seems that this reaction is inconstant and may be entirely absent, even though the serum have a high im- munizing value. Certain normal bloods, as from the dog, show bacteriolytic power, but specific bacteriolysins cannot be demon- strated in most immune sera. Opsonins, both normal and immune, have been demonstrated. No wholly satisfactory explanation of the factors which de- termine immunity in animals naturally insusceptible and those ANTHRAX GROUP. THE GENUS BACILLUS 259 which have acquired immunity has been developed. Among the facts which apparently must 'be taken into consideration are the following: 1. Immunity cannot be wholly due to the natural bactericidal power of the seriun, for the serum of the highly susceptible rabbit has as much anthracidal power as that of the relatively resistant wild rat. There is apparently no direct relationship between the natural anthracidal power of a serum and the resistance of the animal. 2. There is apparently some other factor in determining im- munity than phagocytosis, for the white blood-cells of susceptible animals may rapidly engulf virulent anthrax bacilli in vitro. 3. The Bacillus anthrads when growing in the animal tissues usually produces capsules. This same character may be developed by growing the organisms in sera in- the laboratory. Such organ- isms have been termed "animalized." It has been claimed by' some authors that these capsulated organisms are far more resist- ant to the phagocytic and bactericidal action of immune blood than are the uncapsulated. Both points have been disputed by other writers. It seems doubtful if the development of the capsule really can account for the ability of the organism to invade the body of an animal and produce a disease. 4. The work of Bail and others seems to show that in infection of a susceptible animal two distinct stages may be noted. The intraperitoneal injection of considerable numbers of anthrax bacilli into a guinea-pig is followed by a rapid diminution of these num- bers, due to phagocytosis and bacteriolysis. Some organisms ap- parently persist, and after a time begin multiplying rapidly in the tissues and blood-stream. Possibly these organisms are more resistant, or the defences of the body have been broken down. Bail explains the phenomenon by the assumption that the persist- ing bacteria produce an aggressin which effectively neutrahzes the immune bodies of the tissues. The bacteria are thereupon free to multiply. He claims that a true active immunity may be developed only by causing the body to form an anti-aggressin, an immune body which will effectually neutrahze the aggressins of the bacillus, thus enabling the other antibodies to destroy the bacteria. 260 VETERINARY BACTERIOLOGY Active immunization by vaccination has been extensively prac- tised. The organism may be attenuated in a variety of ways. The first method developed was that of Pasteur, and it is still the one most commonly used. The organism is grown at a temperature of 42° to 43° for varying lengths of time. The pathogenicity gradu- ally decreases until injections no longer kill the rabbit; longer growth attenuates it until the guinea-pig is not susceptible, and finally, even the mouse will not succumb. The exact length of time required for attenuation in each instance can be determined only by experimentation, as there are many unknown or uncon- trolled factors involved. In Russia the Zenkowsky modification of the Pasteur method has been used. The organisms are attenuated until 0.2 gm. of Vaccine I will not kill a 350-gm. guinea-pig when injected sub- cutaneously, but 0.01 gm. is regularly fatal for the white mouse. Vaccine II kills a guinea-pig on the third day, while 0.5 gm. will not kill an 800-gm. rabbit. The organisms are grown on agar slants until spores are produced. These are then mixed with 30 to 40 per cent, glycerin. The advantage of this method of pre- paring lies in the fact that it may be kept for a longer period with- out deterioration. The immunity lasts about a year. Attempts to use killed cultures of anthrax bacilli or their sterile products in producing immunity have not yielded satisfactory results in practice. Bail claims that an active immunity may be established by the use of aggressins. The material used is the serous fluid from animals dead from anthrax. This is sterilized by phenol and injected into an animal to be immunized. The aggressin should preferably be secured from the same species of animal as the one to be immunized. The immunity is not established until after the lapse of ten days or more. When established, it is claimed that the animal will resist infection with fully virulent cultures. This method of im- munization with aggressins has not been thoroughly tested out in practice. Passive immunization by means of antisera has been tried by numerous investigators. Cattle, the horse, ass, and sheep have been used in the production of such sera. The animals are first immunized by Pasteur's method of vaccination, and in ten days ANTHRAX GROUP. THE GENUS BACILLUS 261 or two weeks from xwo" *o TSTT o^ ^ loop of a fully virulent culture is injected. Two or three weeks later a somewhat larger dose is given, and the dosage is gradually increased until many loopfuls — then entire cultures — are injected. In three to four months an immunity is developed such that the subcutaneous injection of several cultures from agar slants may be made without noteworthy reactions. The blood is drawn about two weeks after the last injection. The animal may then, after the lapse of two weeks, be again injected and again bled. The serum is pipetted off and preserved with 0.5 per cent, phenol. Exact methods of standardization such as are used with antitoxins cannot be employed. An approximation of the potency can be reached by the intravenous injections of varying amounts of serum into rabbits, followed five to ten minutes later by inoculating each subcutaneously with yoVo of a loop of a virulent culture. A serum is regarded as usable if 2 out of 5 animals injected with 2, 3, 4, 5, and 6 c.c. of the serum survive, and the remainder live longer than control animals. The serum shows in vitro no higher bacteriolytic power than serum from normal animals of the same species. The potency of the serum cannot be estimated by com- plement fixation reaction; nor can the activity of the serum be ascribed to its opsonin constant. Bail claims the activity to be due to the anti-aggressins present. Agglutinins are present, but cannot be used in determining the titer of the serum. The serum may be used both in prophylaxis and cure. It is of therapeutic value in man also. Ten c.c. is a prophylactic dose for sheep. The serum is of greatest use where it is necessary to immunize large numbers of animals quickly, as in a herd of sheep in which anthrax has made its appearance. Bacteriologic Diagnosis. — The disease may be diagnosed by the following bacteriologic methods: 1. Microscopic examination. 2. Cultures. 3. Animal inoculations. 4. Precipitation. Microscopic Examination. — Fresh blood from animals having septicemic anthrax will show the organisms in the hanging drop. Mounts from blood or infected tissues, particularly the spleen, may be stained with methylene-blue, by Gram's method, or by a suitable capsule stain. 262 VETERINARY BACTERIOLOGY CuUures. — ^Agar plates may be poured or streaked. The colonies showing the characteristic curled-hair margins should be fished and used for animal inoculation. Animal Inoculation. — A suspension of the suspected material should be injected into mice or guinea-pigs. Precipitation Test.— This test, first worked out by Ascoli and usually termed the Ascoli thermoprecipitation test, is based upon the fact that it is possible to secure precipitating sera of high titer from immunized animals for the anthrax bacillus audits soluble con- stituents. The test is most commonly employed in the recognition of anthrax in tissues and organs submitted for diagnosis. The task of securing a suitable precipitating serum is not always an easy one, as animals immunized in the same manner will show great differ- ences in the precipitin content of the serum. A portion (1 to 2 gm:) of the organ to be examined (usually the spleen) is heated for five minutes in a tube with 5 c.c. physiologic salt solution, cooled, and filtered. The clear filtrate is placed in a narrow tube, and 0.5 c.c. of the precipitating serum placed in the bottom by means of a capiUarjr pipette. A positive test is evidenced by the formation within fifteen minutes of a whitish cloud near or at the point of contact. Reactions which develop after two hours are not to be regarded as specific. This test is a useful adjunct to the other methods discussed. A reaction may often be secured even when putrefaction of the tissues is far advanced. The isolation of anthrax bacilU from soil, wool, hides, etc., is relatively difficult. The material may be heated to 60-70° for one-quarter to one-half hour to kill the vegetative cells, and the material used for animal inoculation. Transmission.— Anthrax is usually transmitted from one ani- mal to another by ingestion, more rarely through skin lesions. Cutaneous infection and infection by inhalation are most common in man. The organism does not sporulate within the body. Dead animals should be burned or buried deeply. The excretions from an infected animal, the feces in particular, contain many bacteria which can form spores on leaving the body. Pastures once in- fected may remain so for many years, as the spores are not readily destroyed by desiccation and may persist in the soil for a long time. Blood-sucking flies sometimes spread the disease from one ANTHRAX GBOUP. THE GENUS BACILLUS 263 animal to another by direct inoculation. Care should always be used in dealing with infected animals, as the disease is fatal to man as well. CHAPTER XXIV BLACKLEG— TETANUS GROUP. THE GENUS CLOSTRIDIUM The bacteria of this group include all the anaerobic spore- producing bacilli. Most of the organisms are obligate anaerobes, some are micro-aeropholic, but none are aerobic. With few ex- ceptions the organisms are motile. They are, in general, Gram- positive, though there is considerable variation in the rapidity with which decolorization takes place. The following are the more important forms which have been described as species of this group: Clostridium tetani, causing tetanus in man and animals; CI. chauvoei, causing blackleg in cattle; CI. welchii, causing an infectious edema in man and animals; CI. gastromycosis ovis, causing bradsot in sheep; CI. botulinum, causing botulism or meat-poisoning in man; CI. aedematis, causing malignant edema in animals and man; CI. sp. of Ghon-Sachs, causing an emphysematous edema in swine and man, and the CI. sp. of Hibler, causing gaseous edema. In addi- tion, CI. sp. of Npvy and the CI. enteritidis'sporogenes are capable of producing disease in experimentally infected laboratory ani- mals. Several non-pathogenic putrefactive and soil species of this group have been isolated, among them CI. putrificus, CI. amylobactef, and others. The separation of the various pathogenic and closely related non-pathogenic species is a matter of considerable difficulty. The first attempt to find criteria for adequate differential diagno- sis was that of Hibler.' More recently the British committee appointed to study the bacteriology of war wounds have greatly clarified the problem. They showed that many of the irregu- larities and discrepancies of the older literature were due to the failure to have pure cultures. The work of Meyer, Heller and their associates has also served to indicate the organisms of this group which are of most importance in animal pathology. lUntersuchungen tiber die pathogenen Anaeroben, Jena, 1908. 264 blackleg — tetanus group. the genus clostridium 265 Key to the More Important Members of the Blackleg -tetanus Group of Bacteria A. Spores terminal and spherical, giving drumstick appearance to the cell. Clostridium tetani. AA. Spores not both spherical and strictly apical. B. Non-motile. Spores only in sugar free media. Clostridium welchii. BB. Motile. C. Sucrose fermented. Clostridium chauvoei. CC. Sucrose not fermented. D. Galactose and salicin fermented, glycerol not fermented. Clostridium oedematis. DD. Galactose and salicin not fer- mented, glycerol fermented. Clostridium hotulinum. Clostridium tetani Synonjmis. — Bacillus of Nicolaier; Plectridium tetani; Bacillus tetani. Disease Produced. — Tetanus or lockjaw in man and animals. (German, Starrkrampf.) Nicolaier, in 1885, observed the Clostridium tetani in pus from laboratory animals that had died following subcutaneous inocula- tion with small amounts of garden-soil. He cultivated the organ- ism, but did not succeed in securing it in pure culture. Kitasato, in 1889, succeeded in growing the organism in pure culture, and in transmitting the disease experimentally. Kitasato and Veyl, in 1890, described the production of the tetanus toxin. Distribution. — The organism is found in all parts of the world. It is particularly common in street-dust and fertihzed garden-soil, and is found iquite constantly in the alimentary tract of herbivo- rous animals. Lukas found it present in the excrement of 16 out of 17 horses which he examined. It is possible that it may for a time maintain a saprophytic existence and multiply in the soil under certain conditions. Morphology and Staining. — Clostridium tetani is a rather long, slender rod, 0.5 by 2 to 5 m with rounded ends, usually single, rarely in short chains. It is motile by means of numerous peritrichic flagella. Capsules are not produced. Spores are formed abundantly. Their size and position are so character- istic as to be practically diagnostic. They are spherical, two or three times the diameter of the rod, and terminal, giving 266 VETEKINARY BACTERIOLOGY the organism the appearance of a drumstick. The organism stains readily with the ordinary anilin dyes and is Gram- positive. Isolation and Culture. — The isolation in pure culture of Clostri- dium tetani is attended with considerable difficulty, largely on account of its being an obligate anaerobe. Kitasato first succeeded in isolating it by producing tetanus in experimental animals, then inoculating broth, and, after growth had taken place, heat- ing to a temperature of 80° for half an hour. This temperature should destroy all but spores. The broth may then be inoculated into agar or gelatin and kept under anaerobic conditions. If spores of other anaerobes are present, it may be necessary to make several consecutive animal inoculations and iso- lations. The colonies of the te- tanus bacillus upon gelatin plates show minute radiating lines of growth from a central nucleus resembling somewhat those of Bacillus subtilis. Gelatin stabs show an arbor-, escent growth. The gelatin is slowly liquefied. Growth is favored by the presence of reducing substances, such as dex- trose or lackmus solution. Radiating filaments are also pro- duced in glucose agar stabs. Bouillon is clouded and a sediment forms. Blood-serum is liquefied. Milk is more or less com- pletely peptonized; it may or may not show coagulation. Hibler's brain medium is made alkaline and blackened as the result of the formation of iron sulfid. Physiology.— The optimum growth temperature is 37.5°, but the organism multiplies rapidly at room-temperatures, though not below 14°. It is an obligate anaerobe, and in pure culture requires practically complete exclusion of oxygen. It will develop, however, under aerobic conditions when in mixed cultures with Pig. 101. — Clostridium tetani, rods and spores (Gunther). BLACKLEG — TETANUS GKOUP. THE GENUS CLOSTRIDIUM 267 aerobes. The spores resist desiccation indefinitely. They are also much more than usually resistant to the action of disinfectants. Likewise, resistance to heat is so marked that Theobald Smith found in one case exposure to live steam for seventy minutes failed to destroy the organism, although usually a shorter period suffices. Acid is produced from carbohydrates, and a small amount of gas, consisting of methane and carbon dioxid from dextrose. En- zjTnes which liquefy gelatin and blood-serum have been demon- strated. Fig. 102. ^Clostridium tetani, colo- nies in dextrose gelatin (Frankel and Pfeiffer). Fig. 103. — Clostridium tetani, deep stab culture in dextrose gelatin (Frankel and Pfeiffer). Pathogenesis. — Experimental Evidence. — Injection of pure cul- tures of Clostridium tetani into experimental animals causes the development of a typical tetanus. The white mouse is among the most susceptible of animals to inoculation. Infection of the animal is within one to three days followed by tetanic convulsions and death. Birds are not ordinarily susceptible to infection. The 268 VETEBINAEY BACTEBIOLOGY disease is most common in men and in the horse, although no mammalia are immune. The disease is not transmitted by inges- tion. The injection of the characteristic toxin is followed by the symptoms of the disease. Character of Disease and Lesions Produced. — The disease is a typical toxemia. There is rarely, if ever, a general invasion of tbp' tissues. The organism remains locaUzed at the seat of inoculation, and produces the toxin which brings about the characteristic symp- toms. The entrance of the organism into a wound is not always followed by the development of tetanus, for anaerobic conditions must obtain, and it has been found that tetanus spores entirely freed from toxin cannot germinate when introduced into the tissues in moderate numbers. It is evident that the organism has little initial pathogenic power. Frequently considerable amounts of dirt are introduced into the wound simultaneously with the organism in natural infections, and produce proper conditions for rapid de- velopment. When conditions are not favorable to the germination of the spores at the site of inoculation, they may remain alive in situ for considerable periods of time. They may also be taken up by leukocytes and transported in the body fluids to other tissues where they may remain dormant indefinitely, being incited to growth only by some tissue injury at their point of lodgment. Such localization and subsequent activation doubtless account for the so-called cryptogenic infections. The tetanus toxin produced is in part absorbed by the end-organs of the motor nerves, and travels to the nerve-cells of the central nervous system by way of the axis- cylinders of the peripheral nerves, and in part is carried to the central gangUon-cells by the blood-stream. The incubation period noted is due to the time required for toxin to be produced and for it to reach the central nervous system. That the toxin has a special affinity for nervous tissue, and may be bound by it, has already been noted in the discussion of toxins and antitoxins. The period of incubation in man averages about nine or ten days. In the horse it varies from four to twenty days. Under exceptional conditions this period may be much longer. Mortality is over 90 per cent, when there is a short period of incubation, and over 50 per cent, where the period is prolonged. The characteristic symptom in all ani- mals is a tetanus, or stiffening of the muscles. The muscles at the BLACKLEG — TETANUS GROUP. THE GENUS CLOSTRIDIUM 269 site of inoculation may be the first, and in mild cases they may be the only ones, affected. In the horse the appearance of the tetanus or lockjaw, the retraction of the eyes and protrusion of the nicti- tating membrane, spasmodic contraction of other muscles of the head, and those of other parts of the body are diagnostic. A postmortem examination usually shows absence of gross lesions. Certain degenerative changes in the motor cells of the cord may be observed in stained sections. Hemorrhages in different organs are an inconstant accompaniment of the disease. Immunity.— For the preparation of toxin the organism is grown in bouillon under anaerobic conditions, i. e., in an atmosphere of hydrogen, with surfaces of the medium covered with paraffin or paraffin oil or with oxygen excluded in some other manner. After incubation for a period of one or two weeks the broth is filtered through porcelain. The toxin may be prepared in dried form by precipitation with an excess of ammonium sulphate. After stand- ing overnight the brown scum is removed and dried, first between hardened filter-paper, then in a desiccator, pulverized, and pre- served in a darkened refrigerator. Various methods of purifica- tion have been devised, such that a dried toxin may be prepared of which 0.00000025 gm. will prove quickly fatal to a white mouse. As has been said, this toxin has a peculiar affinity for the cells of the central nervous system. Two poisonous constituents of the toxin have been differentiated — tetanolysin, which lakes the red blood-cells, and tetanospasmin, which gives rise to the characteristic tetanus symptoms. In the preparation of antitoxin the unprecipitated toxic broth or a solution of the dried toxin is used. The smallest amount of toxin that will certainly kill a 350-gm. guinea-pig in three to four days is taken as the unit of toxicity. Increasing amounts of the toxin are inj ected at intervals into a horse. The blood-serum of the immunized horse contains the specific antitoxin. Many methods of standardization of the antitoxin have been used. In the United States it is titrated by guinea-pig injections against a standard toxin sent out by the Hygienic Laboratory of the Public Health and Marine Hospital Service. It is used in both human and veter- inary medicine, principally as a prophylactic. The tetanus anti- toxin has not taken the place in the treatment of tetanus that is 270 VETERINARY BACTERIOLOGY occupied by the antitoxin specific for diphtheria in the treatment of that disease. It seems that the symptoms of the disease are ex- hibited only after the union of the toxin with nerve-cells; that is, after much of the damage has already in large measure been ac- complished. The injection of antitoxin at this time will doubtless neutralize any toxin present in the blood, but cannot remove the toxin already bound to the nerve-cells. The antitoxin is generally injected subcutaneously, but in severe cases intravenous, intra- neural, and intraspinal injections are made to insure the contact of antitoxin with the toxin present. Its use is doubtless indicated in all cases. As a prophylactic it has been found quite certainly to prevent the development of tetanus when injected before the ap- pearance of symptoms. In human medicine it is customary to make injections following severe wounds into which dust and dirt have gained entrance, such as Fourth-of-July wounds. The same may be said with reference to severe wounds, nail-punctures, and similar traumata in the horse. Bacteriologic Diagnosis. — The organism may sometimes be recognized in stained mounts of the pus from the wounds.. The drumstick shape of the sporulating cells is quite characteristic. Isolation in pure culture and animal inoculation may also be used. The symptoms of tetanus are so distinctive, however, that these methods are rarely called into use. Transmission. — ^Tetanus is one of the best examples of a non- contagious, infectious disease. Infection occurs almost invariably directly through the skin. The almost universal presence of the organism about stables renders infection easy. Nail-punctures are particularly apt to result in tetanus, as they introduce the organism, deep into the tissues; superficial healing and exclusion of air quickly take place, and conditions are then right for rapid multiplication. In some localities tetanus is a common disease following castration of domestic animals. Infection through the umbilical cord in the newborn sometimes occurs. It should again be emphasized that it seems very difiicult for the tetanus bacillus to gain a foothold and proliferate except in tissues that have been injured. The constant presence of these organisms in the intestines does not produce dis- ease. So-called cryptic infections are not of uncommon occurrence, particularly in the horse. In these the point at which the organ- BLACKLEG— TETANUS GROUP. THE GENUS CLOSTRIDIUM 271 ism gains entrance to the body frequently cannot be determined. Usually this comes either from the wound having healed super- ficially, so as to be difficult of recognition, or from the wound having been originally so insignificant as to have escaped notice. Some investigators befieve that the organism may occasionally gain en- trance to the blood-stream from the intestines, but is unable to pro- duce an infection except when it lodges in tissue traumatically or otherwise injured, such as a broken bone or a bruise. Spores may be carried from a wound to other parts of the body, and develop only when the tissue has been injured. Clostridiuin chauvasi Synon3mis. — Bacillus feseri; Bacillus chauvcei; B. chauvaei; B. chauveaui; B. anthrads symptomatici. Diseases Produced. — Blackleg, symptomatic anthrax, quarter evil, quarter ill, Rauschbrand, charbon symptomatique ; in cattle and rarely, in sheep and goats. Arloing, Cornevin, and Thomas, in 1889, described the Clostri- dium chauvaei as the cause of blackleg, and proved its etiologic relation to the disease. Kitasato (1889) first grew the organism in pure culture. It has also been studied extensively by Grass- berger and Schattenfroh and by Hibler. Morphology and Staining. — Clostridium chaupcei is a large bacil- lus with rounded ends, usually single, but occasionally in pairs, . 0.5 to 0.6 by 3 to 5 m. When stained smears from fresh serous exudates or muscles are examined the organism is found never to occur in long chains or filaments. This fact is of value in the differentiation of this organism from the related CI. cedematis of Koch and from the Ghon-Sachs bacillus. It is motile by means of peritrichic flagella. Involution forms, consisting of greatly enlarged rods, are frequently encountered, particularly in old cultures. Capsules when produced in the body fluids are relatively thin. Spores are produced, sometimes central, but more frequently near a pole, rarely quite terminal. They are long ellipsoidal in shape and are not generally more than twice the diameter of the rod. The ability of the organism to produce spores in media containing carbohydrates is of use in differentiat- ing it from CI. welchii. Spore production is not abundant 272 VETERINARY BACTERIOLOGY when the medium becomes acid. The organism is easily stamed by the common aqueous anilin dyes and is Gram-positive. There is frequent occurrence of Gram-negative cells in old cultures. Smears prepared from muscles and treated with iodin potassium iodid show the presence in many cells of red stained granules termed ' ' erythrogranulose . ' ' Isolation and Culture.— The organism may in many cases be isolated in pure culture directly from the tissues infected. Growth in culture-media is quite de- pendent upon the reaction of the medium, and to a less de- gree upon the presence of carbo- hydrates. For the best growth I per cent, soda content beyond BH^i HHH eI' HH B^A .^^B 11 H ^'^' qH pi S^^^^H Bl*''':-^ ■^^f^^^^^HH^^I wM ■Hsp^H ■ '4 Sgfl:'";B ^ i hSI>'''~H H^lr'^H^"' ' '--'.f^^l j ^^^^^^H^K ^L-. v^^M^H 7 ! .3^B&ji..u:ll^iiflB Fig. 104. — Clostridium chauvad (KoUe and Wassermann). fig. 105. — Clostridium chauvcei, colonies in a dextrose gelatin shake culture (Frankel and Pfeiflfer). neutrality is advisable. Body fluids or tissues except as they may act as reducing agents or contain carbohydrates do not increase the suitability of media containing them. Inoculation of bits of infected tissue into broth containing a bit of paren- chymatous tissue to act as a reducing agent will generally give a pure culture. Plates may be poured and kept under anae- robic conditions. The colonies are spherical, or somewhat irregular, with microscopic radiations. Dextrose gelatin is an exceptionally favorable medium. In a shake culture the BLACKLEG — TETANUS GROUP. THE GENUS CLOSTRIDIUM 273 colonies appear in the lower portion of the tube, each usually with its gas bubble, and surrounded by a liquefied area. These colonies show numerous radiating threads, or papilla, giving the appearance of a chestnut burr. Bouillon is clouded, gas is produced, and a flaky white deposit forms. Growth in Hibler's brain medium results in the development of a permanent acidity and no blackening, but with the evolution of considerable quantities of gas. Milk is not particularly favorable as a medium without the addition of serum or tissues. Little gas is formed in milk. Usually a flocculent curd is formed, but this is not digested. Physiology'. — Serum media are not peptonized. The optimum growth temperature is about blood-heat, but good growth occurs at room-temperatures. The organism is a strict anaerobe. Con- cerning its other physiologic characters there is considerable dis- agreement among investigators. This may be due to the fact that there are strains which react very differently, and may constitute distinct varieties, or still more probably to the fact that impure cultures have been studied. Grassberger and Schattenfroh claim that the organism shows considerable variability, and that certain characters are easily lost. The spores are quite resistant to desiccation, living in this condition for years. Heating for six minutes at 100°, according to Hibler, is necessary certainly to destroy them. The dry spores are quite heat resistant. Gas is produced from many carbohydrates including glucose, levulose, maltose, lactose, sucrose, but not from mannitol, glycerol, dulci- tol, salicin and inulin. It is also produced in meat medium. Alkaline carbohydrate media is first neutralized and then becomes acid. Pathogenesis. — Experimental Evidence. — Inoculation of pure cultures into laboratory animals results in death, with production of many of the characteristic symptoms of blackleg, particularly the edema about the point of inoculation. Intramuscular injection of the guinea-pig is followed by the first symptoms in about fourteen hours. A soft inflamed swelling develops at the site of inoculation. In twenty-four to thirty hours the inflammation has spread to other muscles, and these have become emphysematous. Inoculation is usually fatal; occasionally the disease runs an atypical course and 18 274 VETERINABT BACTERIOLOGY the animal recovers. Upon section, in typical cases, the tissues are found to be edematous and hemorrhagic, and the muscles to contam many gas bubbles. The body cavities usually show an abundant serous exudate. The disease may also be produced experimentally in cattle, so that there is no doubt as to the etiologic relationship of this organism to the disease. It has not been found in man. Character of Disease and Lesions Produced. — Rabbits, white mice, and white rats may be infected. The gray rat appears to be relatively immune. Blackleg in cattle is characterized by a swelling, edema, and emphysema of the muscles and the sub- cutaneous tissues of the infected part. Infection appears most commonly in the shoulder or hindquarter. The swelling increases rapidly in size, and the emphysema soon manifests itself by the crackhng sound produced when the thumb is drawn firmly across the part. After death the organisms continue to grow and the body becomes distended with gas. The subcutaneous tissues of the infected part are edematous, even gelatinous, with blood and gas bubbles. The underlying muscles are dark brown or even blackish, whence the name, blackleg. The disease usually results fatally in cattle in from one to three days after the first appearance of the symptoms. Immunity. — The production of true toxins by Clostridium chauvcei is not well understood. According to some authors no toxin can be demonstrated. Others believe that there is a relatively thermostabile toxin produced which will endure a temperature of even 115°. Grassberger and Schattenfroh, who have made the most careful study of this problem, claim to have succeeded in producing broth cultures containing a toxin that, in doses as small as those employed with diphtheria toxin, will kill laboratory animals. This toxin is produced by certain strains of the organism only, but these they believe are the more patho^ genie. This toxin they have shown to be thermolahiU. They have worked out methods of standardization closely resembling those of Ehrlich for diphtheria. Antitoxin may be produced by the injection of increasing doses into suitable animals, particu- larly cattle, and this antitoxin has a protective influence when injected into other animals. This method of immunization has never come into general use. BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 275 Animals that have recovered from an attack of the disease acquire immunity to a recurrence. Very young cattle and aged cattle have a considerable degree of natural immunity. To what the immunity developed may be due is not well understood. Probably it is in part opsonic. Active immunization of animals by vaccination is extensively practiced. Many methods of attenuation of the organism for the vaccine have been developed. The one so long in common use in the United States is the one adopted by the Bureau of Animal Industry, and is essentially that developed by Kitt. Fresh ma- terial is secured by macerating in a mortar the muscle tissue from a blackleg tumor and squeezing the fluid through a linen cloth. This is spread in a thin layer and dried to a brown scale at a temperature at about blood-heat. This dried virus retains its virulence for several years at least. The vaccine is prepared by mixing 1 part of this material with 2 parts of water and placing in a hot-air oven at a temperature of 95° to 99° for six hours. This dries the material and attenuates the organism. It is then pulverized and put up in packages containing a definite number of doses. Before use a cubic centimeter of water for each dose is added, and the material mixed and then filtered. The injection then is made. with 1 c.c. The dried material, pressed into the form of tablets, is sometimes inserted under the skin without sus- pending it in water. In some cases threads soaked in suspen- sions of the attenuated organisms are drawn into the subcu- taneous tissues by means of a needle. Vaccination has proved quite satisfactory. Improved methods of more recent time include the use of aggressins and filtrates. Schobl showed by experiments' that the edematous fluid derived from blackleg lesions when made sterile by filtration through porcelain, possessed remarkable immunizing properties. Following his method of treatment the Kansas Experiment Station tested the germ free fluid on many thousands of cattle and found it highly efiicient. The affected tissues from blackleg calves were placed in a press and the juices expressed. These were then filtered, preservative added and used in doses of 8 to 15 c.c. and found to produce sufficient immunity to protect calves against doses of virus which promptly killed non-vaccinated calves. 276 VETERINARY BACTERIOLOGY At present there is in successful use a product known as black- leg filtrate, which consists of the medium on which blackleg bacteria have been grown, passed through porcelain filters to make it germ free. The results obtained have been very satis- factory. The chief advantage claimed for these new immunizing agents is that there is no possibility of the disease being produced, a thing which frequently occurs when the attenuated vaccine is used. Bacteriologic Diagnosis. — A presumptive determination of the organisms may be made by smear preparations from the in- fected tissues. The lack of chains and filaments in smears from serous effusions is particularly characteristic, according to Hibler. Anaerobic cultures will demonstrate the specific organism in pure culture if inoculated with a bit of the tissue before decomposition has begun and before putrefactive baciUi have gained entrance. Animal inoculation, particularly subcutaneous inoculation into the guinea-pig, may prove useful. Usually the symptoms of the disease .are so characteristic that a bacteriologic test is wholly unnecessary. Transmission.— It is believed that Clostridium chauvcei is widely distributed in nature. The disease occurs only in certain localities. There are districts which are never affected. Attempts have been made to correlate the topography of the country, such as character of soil, presence of marsh land, etc., with the prevalence of the dis- ease without marked success. Organisms closely related to CI. chauvcei may be found widely in the soil, but, for the most part, they do not possess the pecuUar pathogenic characters of this form. Infection is beUeved to occur through wounds. The dis- ease is rarely, if ever, contracted directly by one animal from another. It is not always possible to locate the point at which the organisms gained entrance— in fact, these cryptic infections con- stitute a considerable proportion of the cases. It is possible that the explanation sometimes offered for similar infections in tetanus will hold good here also; that is, that the organisms may occasion- ally gain entrance to the blood from the intestinal tract or from old wounds and that they cannot produce disease except when they lodge in some tissue that has been injured, as from a bruise. BLACKLEG — TETANUS GROX7P. THE GENUS CLOSTRIDIUM 277 Clostridium gasttomycosis ovis Disease Produced. — Braxy or bradsot in sheep. Distribution. — The disease is recorded from the northern por- tion of Europe, particularly in Ireland, the Faroes, the Shetlands, Scotland, portions of England, Norway, and North Germany. Gilruth claims the disease is also present in Tasmania. Historical. — The organism was first described in 1888 by Niel- sen, who clearly differentiated the disease from anthrax, with which it had previously been confused. The Highland and Agricultural Society and the British Board of Agriculture and Fisheries have instituted investigations as to the disease and its causal organism. Among the most careful studies have been those of Jensen. Morphology and Staining. — The organism is a large bacillus, about 1 by 2 to 6 M, with rounded ends. The cells are usually single, but in smears made from serous effusions chains and fila- ments are common, in this respect differing from the organism of blackleg. Spores are produced both in tissues and in cultures. They are ellipsoidal, usually central, only occasionally polar, and cause but httle enlargement of the cells. They are motile by means of peritrichous flagella. Capsules are not recorded. The cells stain readily and are Gram-positive. Isolation and Culture. — This organism grows readily in most media under anaerobic conditions, particularly if some sugar is present. Agaf-plate colonies, at first smooth and lens shaped, in forty-eight hours become covered with outgrowths resembling those of Clostridium chauvoei, giving the colony a felted appearance. Gelatin colonies are gradually surrounded by an area of liquefied medium. The addition of serum to the medium favors the growth of the organism. It grows well in milk, causing an acid coagulation, but no peptonization of the casein. Physiology. — The optimum growth temperature is between 35° and 40°, although good growth takes place at room-tempera- tures. The spores are resistant to desiccation, but are readily killed by boihng. It resembles the bacillus of blackleg in that the most favorable reaction of the medium is slightly alkaline, little or no growth occurring in an acid medium. While growth is favored by the presence of sugar, it is soon stopped by the acids developed. I 278 VETERINARY BACTERIOLOGY Gas and acid are formed (according to Bahr) from the fol- lowing sugars: Dextrose, mannose, galactose, fructose, lactose, maltose, and glycerin, but not from saccharose, raffinose, sorbose, arabinose, xylose, rhamnose, mannite, dulcite, adonite, and ery- thrite. The ability of the organism to produce gas from pure proteins is not well demonstrated. Gelatinase is produced; other proteo- lytic enzymes are seemingly not formed. ' Pathogenesis. — The disease is primarily one of infection through the walls of the omasum. It is characterized by the presence of reddish slimy fluid in the abomasum, edema and swelling, and frequently extensive necrosis of the mucous membranes and sub- mucosa. Generally there is a marked hemorrhagic infiltration in the region of the pylorus. Edema and necrosis and often em- physema of other tissues may be noted. Subcutaneous inoculation of sheep gives rise to an infection closely resembhng symptomatic anthrax or blackleg. The organ- ism has been shown to be pathogenic for sheep, goats, swine, calves, guinea-pigs, pigeons, and fowls, while mice and rabbits are more resistant. Immunity. — Small quantities of a toxic substance are present in culture filtrates. Whether true toxins are present has apparently not been conclusively demonstrated. Jensen has used several methods of immunization. The vac- cine most used is prepared similarly to that for blackleg by drying spore-bearing broth culture, then heating thirty minutes at 100° C. Suspensions of this material are injected subcutaneously. This vaccine has been used quite extensively and successfully. His serovaccine consists of a mixture of the above vaccine with dried immune serum. Transmission.— The site of the principal lesions of the disease leads to the assumption that the infection follows inge^ion of the organism. Feeding experiments in general, however, have failed to produce the disease. A completely adequate explanation of sea^ sonal prevalence, distribution, and method of infection has not been given. Clostridiom welchii Synonyms.— 5aCT7ZMs aerogenes capsulatus; Bacillus welchii- Bacterium welchii; B. phlegmonis emphysematosa; B. enteritidis BLACKLEG — TETANUS GROUP. THE GENUS CLOSTRIDIUM 279 sporogenes; B. perfringens; Granulobacillus saccharobutyricus immohilis; B. anaerobicus cryptobutyricus; B. cadaveris butyricus; B. emphysematis vaginoe. Disease Produced. — Gaseous edema in man, a secondary in- vader in various animal diseases. Welch and Nuttall, in 1892, described Bacillus aerogenes capsulatus from the body of a man who died from an aortic aneurysm. The internal organs and sub- cutaneous tissues showed considerable emphysema. Since that time it has been repeatedly isolated in Europe and America. It was independently described by Frankel in 1893 as B. phlegmonis emphysematosoe from .4 cases of gas gangrene. Distribution. — The organism is common in garden-soil, par- ticularly that contaminated with excreta. Morphology and Staining. — Clostridium welchii is a rod, 1 to 1.5 by 4 to 8 fi, with usually rather square ends. Very short, almost coccal forms, and long filaments may be found under certain growth conditions. Some strains show curved rods. Involution forms (club shapes, filaments, tadpole forms, granu- lar types, etc.) are found particularly in old cultures upon coagu- lated serum. It frequently occurs in chains, but may be found in pairs and small groups. The organism does not appear in serous infusions in chains or filaments. It is non-motile. In this respect it differs from the other members of this group. Spores are sometimes found in infected wounds, but are most readily formed in such media as casein broth, alkaline egg broth and coagulated serum, free from fermentable carbohy- drates. They are rarely seen in fermentable media. Individual strains vary in their spore producing ability. The spores are large, oval in shape and with slightly flattened ends, subterminal or central in position. They are central, and the cells develop as Clostridia. Capsules may be demonstrated in the body fluids and in media containing serum. The organism stains readily with the common anilin dyes and is Gram-positive in young cultures with frequent occurrence of Gram-negative individuals in old cultures. Involution forms are frequent in artificial media. Isolation and Culture. — McCampbell has described a modifica- tion of Welch's method of isolation as follows: "1 gm. of soil is shaken in sterile NaCl solution (0.85 per cent.), and inoculated 280 VETEBINARY BACTERIOLOGY into sterile neutral litmus-milk tubes, which are covered with 25 mm. of neutral paraffin oil, for the purpose of securing anaero- biosis, and then incubated for twenty-four hours at 37°. At the end of this time the milk in the tubes is coagulated and shows acids and gas. A subculture is made in a second litmus-milk tube under oU, and incubated for twelve hours in order to pre- vent the possible overgrowth of other bacteria. At the end of this time the milk usually shows coagulation, acid and gas production, as in the first instance ('stormy fermentation'); 0.5 c.c. of the whey in the subculture is then injected into the posterior au- ricular vein of a rabbit. In three or four minutes the animal is killed by a blow on the head, and the body is incubated at 37° for eight to ten hours, at the end of which time the abdomen is markedly distended with gas. Fig. 106.-^^^;;^™ ^eUhu T^^"" ^^!'^*^^' *^^' explodesand (Jordan). burns with a hydrogen flame. The thorax of the animal is carefully opened, and cultures made from the heart blood in dex- trose broth, covered by neutral paraffin oil. In from eight to twenty-four hours the culture tubes show a marked cloudiness, abundant gas production, and, in most instances, an odor of butyric acid." The colonies upon agar and gelatin plates are round, grayish, semitranslucent; they are usually nucleated, and resemble those of Clostridium teiani. Upon agar slants a thin, coalescent, yellowish-white growth occurs. Gelatin may or may not be slowly liquefied. BouiUon is clouded with a heavy precipitate. .' Little or no growth occurs on potato. Upon blood-serum the growth resembles that upon agar with no change in the medium. MUk is quickly coagulated, with gas and acid production. ^ Physiology.— Growth occurs best at 37°. The thermal death- point for non-sporulating culture is 50° for ten minutes, for spores 100° for fifteen minutes. Gas is produced from dextrose, levulose. BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 281 galactose, maltose, lactose, and saccharose, but not from mannitol, dulcitol, or salicin. Probably some differences are to be found in various strains. Gas is likewise produced from pure proteins, such as recrystallized egg-white. Butyric and lactic acids have been detected. Pathogenesis. — Experimental Evidence. — Intravenous injec- tion of the rabbit frequently, though not always, causes death, but subcutaneous inoculations are without effect. Guinea-pigs are susceptible, as are also pigeons and mice. Character of Disease and Lesions Produced. — Infections with Clostridium welchii among the lower animals have been noted in a few instances only, and then only in the rabbit and in the dog, as a result of severe injuries. However, it may quickly invade tissues after death, and give opportunity for mistaken diagnosis. It has been isolated from cattle dead of anthrax, swine that have suc- cumbed to cholera, together with CI. chauvcei from blackleg lesions in cattle, etc. It has not been shown satisfactorilv that it ever invades the tissues generally before death. It is a secondary invader in practically every instance of natural infection. It has been found in emphysema of many organs in the human body. Herter believes that the presence of large numbers of this organ- ism or its varieties in the intestines is responsible for the produc- tion of primary pernicious anemia, particularly in children. From the veterinary standpoint the organism is of principal interest, not so much because of its slight pathogenic power, as the fact that it may be confused upon isolation with other spore- bearing anaerobes. ^ Immunity. — Frankel failed to secure immunity in laboratory animals by injections of killed cultures, though an immune serum was secured from a dog after a process of immunization. CI. welchii produces a specific toxin which when injected into animals stimulates the production of a specific antitoxin. Well washed emulsions of the organism when introduced into experi- mental animals produce no infection. Combined, however, with a sub-lethal dose of toxin a fatal gas gangrene rapidly develops. Bull and Pritchett have proved that the necrosing and hemolytic properties of filtrates of CI. welchii are due to the presence of an exotoxin, which is destroyed by heating to between 282 VETERINARY BACTERIOLOGY 60° and 70° C, and which is capable of stimulating the produc- tion of antitoxin. This antitoxic serum when employed under proper conditions inhibits and arrests infection with CI. welchii. Opsonins are present in normal and in increased quantities in immune sera, as are also specific bactericidal substances. Bacteriologic Diagnosis. — The organism can be recognized certainly from tissues only by isolation, and a study of its mor- phology, physiology, and effect upon animals. Its Gram-positive staining characters, lack of motility, and the difficulty with which spores may be demonstrated are significant. By the use of specific antitoxin the bacteriologic diagnosis may be confirmed. A liquid culture of the organism under study may be mixed with varying doses of the specific antitoxin before injection into a suit- able laboratory animal. If infection is inhibited the organism is CI. welchii. Or if an injection of the antitoxin renders an animal passively immune to infection with a particular organism, then that organism is CI. welchii. Transmission. — The organism probably gains entrance to the body through' wounds or after death invades the tissues from the intestines. Clostridium cedematis Synon3rms. — Vibrion septique; Bacillus cedematis maligni, Koch. Diseases Produced. — Malignant edema; Malignes Edem, cedeme malin, septicemie gangreneuse, in various animals and in man. Pasteur, in 1877, found that the injection of putrid flesh into a rabbit was followed by an edema at the point of inoculation, and ultimately by the death of the animal, with changes in many of the internal organs. That these changes were due to a specific organism and not to the poisons of the putrid flesh alone, was shown by transfers from one animal to another, and by the isola- tion of an anaerobic bacterium. Koch later (in 1881) studied the disease. Morphology and Staining.— The Clostridium cedematis closely resembles the Clostridium chauvcei morphologically, and was long considered closely related to it but the work of Meyer seems BLACKLEG— TETANUS GROUP. THE GENUS CLOSTRIDIUM 283 to prove that the two are quite distinct. The organism is a rod, 0.8 to 1 by 2 to 10 m, with rounded ends, single or in chains. Many of the cells are long and filamentous. This is particularly evident in smears made from serous effusions. It is motile, with numerous peritrichic flagella. Capsules have not been demonstrated. Spores are produced, usually polar, but some- times equatorial. The spore is short ellipsoidal. The rod is not greatly distended by the spore, although the snowshoe or Clos- tridium shape is usually evident. The organism is Gram-positive, though more readily decolorized by alcohol than are most Gram-positive forms, and stains readily with the common anilin dyes. Isolation and Culture. — The organism may be se- cured in pure culture with- out difficulty, under ana- erobic conditions, from the edematous tissues of the infected animal. Its cul- tural characters in many ways resemble those described for Clostridium chauvaei. In Hibler's brain medium alkalinity and blackening develop. Gelatin is digested, milk is curdled, and the casein digested is made alkaline. Coagulated serum is not liquefied. Physiology. — The organism is an obligate anaerobe. Growth is luxuriant at room-temperature as well as at blood-heat. The spores are resistant to desiccation and to heat. Hibler states that they can resist several hours' heating to 98°. The optimum tem- perature is about 37°, though good growth occurs at 18°. Gas is produced from dextrose, levulose, galactose, maltose, lactose and salicin, but not from sucrose, mannitol, dulcitol, glycerol or inulin, probably also from proteins. Enzymes that liquefy gelatin are present. Milk is clotted in 3 to 7 days. There is no proteolysis of meat or solid serum. Fig. 107. — Clostridium adematis , spores and rods from an agar culture (Frankel and Pfeiffer). 284 VETERINARY BACTERIOLOGY Fig. 108. — Clostridium oedematis, tissue smear showing rods without spores (Frankel and Pfeiffer). Pathogenesis.— Experimental Evidence. — Inoculation of pure cultures of the organism into the laboratory animals, and also into the horse and other domestic animals, will pro- duce a typical infection. The most infective mate- rial is 24-48 hour glucose broth. Character of Disease and Lesions Produced. — The tissues at the point of invasion, infiltrated with yellow or red serum, are usually hemorrhagic. The niuscle becomes an intense deep red and is softened, but there is no putrid odor. Hemorrhages are generally to be found in the subcutaneous tissues. Many cases of the disease have been, noted in man, and it is not uncommon in the horse and the sheep. It is probable that the cases of so-called symptomatic an- thrax in the horse have been, in reality, infections with this organism. Infection has also been observed in swine, dogs, and rabbits. Immunity.— Animals which recover from an infection are found to be thereafter im- mune. The organism is also known to produce a leukocidin which destroys white blood- cells. Antisera have been prepared, and have been shown to contain antibacterial substances and antitoxins. The antitoxic serum, unlike that of CI. welchii, does not protect if given after the infection is once established. Agglutinins are produced in Fig. 109. — Clostridium mdematis, dex- trose gelatin culture (Gunther). BLACKLEG — TETANUS GROUP. THE GENUS CLOSTEIDIUM 285 the blood of rabbits inoculated intravenously with heated washed cultures. Transmission. — The organism usually gains entrance through wounds, although the possibility of a cryptic infection, such as is claimed to occur in tetanus, should not be ignored. In man the disease has been known to occur following injections in which an unclean hypodermic syringe was used, and a case has been reported in which the organisms were believed to have gained entrance to the body through the intestinal ulcers of typhoid fever. Infection may follow delivery, castration, shearing of sheep, use of unclean syringes or instruments, or dirty wounds of any kind. Clostridium of Ghon-Sachs Ssmonyms. — Bacillus oedematis maligni of Ghon and Sachs. Disease Produced. — Gaseous edema in man and animals. Distribution. — Like the other members of this group, it is probable that this organism is widely distributed in the surface soils. Historical. — The organism was isolated from a case of gaseous gangrene in man by the above named investigators and they assuming that the CI. oedematis maligni was proteolytic, and finding that this non-proteolytic described it as a new organism. Recent investigation of the bacteria of war wounds seems to leave little doubt but that this organism was the same as the vibrion septique of Pasteur. It has been isolated from a variety of animals, particularly cows with puerperal infection, and from swine. Morphology and Staining. — The organism is a motile rod very similar in morphology to Clostridium chauvaei. In smears from serous effusions, however, it occurs in chains and long filaments. The sporulating cells are somewhat swollen. The spores are ellipsoidal in shape. No capsules have been demonstrated. The cells stain readUy and are Gram-positive. Culture. — The colonies in solid media resemble those of the blackleg bacillus. Milk is usually coagulated with gas and acid production, but with no digestion of the curd. Hibler's brain medium is acidified and not blackened. 286 VETERINARY BACTERIOLOGY Pathogenesis.— The organism is pathogenic for guinea-pigs, rabbits, white rats, gray rats, and mice. In experimental inocu- lation an emphysematous edema without much hemorrhage is produced. Clostridiam sporogenes Metchnifcofi This organism was found present in a large proportion of war wounds in acute cases of gas gangrene as well as in conditions in which the wound was progressing satisfactorily. It is generally in association with other organisms. In general it is not lethal for laboratory animals in doses up to 4 c.c. Some strains are,, however, capable of producing a putrid, perforating gangrene. The organism is of importance principally because of the possi- bility of confusion with other members of this group and because of its extraordinary capacity for persisting in the presence of other organisms. Clostridium botalinum Synonym. — Bacillus botulinus. Disease Produced. — Meat, sausage, and food poisoning in man, botulism (botulus, sausage). Van Ermengem, in 1896, isolated an organism from sausage, which he believed to be the cause of poisoning. The organism has since that time been several times isolated, and is, therefore, of some hygienic importance, particularly in meat inspection and in meat hygiene. This disease or poisoning should not be con- fused with that produced by the Bacterium enteritidis. Distribution.— A few well-authenticated reports of the isola- tion of the organism are on record, from European countries, many, within the past few years from the United States, from man, the horse, mule, cow and domestic fowls. The disease has- been reported as caused by the eating of sausage, fish, lobsters, oysters and vegetables such as preserved beans and peas and in animals from eating spoiled ensilage and grain. Morphology and Staining. — Clostridium botulinum is a large bacillus, with usually rounded ends, 0.9 to 1.2 by 4 to 6 m. It is commonly single or in pairs, sometimes in short chains. Involu- tion forms frequently occur. It is motile by means of four to BLACKLEG— TETANUS GROUP. THE GENUS CLOSTRIDIUM 287 eight peritrichic flagella. Capsules have not been demonstrated. SmaU oval spores, somewhat greater in diameter than the bacillus," are produced in a subterminal position. The organism stains readily with the anilin dyes and is Gram-positive. Isolation and Culture.— Growth is sparse in media which contain no sugar. The colonies on dextrose gelatin are at first circular, transparent, light yellow, and soon liquefy the gelatin. Under the low power of the microscope they appear to consist of granules in constant motion. Later the colonies become brown and opaque. According to Van Ermengem, milk is not curdled and the organism grows spar- ingly. Graham suggests the following method for the isola- tion and identification of the organism. "A sample of watery extract of the feed pre- ceding final filtration is diluted and seeded under conditions favorable for the development of B. hotulinus. Pork broth faintly alkaline plus 2 per cent, dextrose is then inoculated with various dilutions and placed in an atmosphere of hydrogen; or anaerobiosis may be ob- tained by covering the surface of the tubes with sterile parafiin oil. The pyrogallic acid or vacuum methods can also be em- ployed. Preceding the period of incubation, the inoculated tubes should be subjected to a temperature of 80° C. for fifteen or twenty minutes. After incubating in the dark at room tem- perature for ten days, pathogenicity of cultures is determined following oral administration of 0.5 mil of the mixed broth culture to guinea-pigs. The production of characteristic symptoms and death in guinea-pigs by the broth culture of obligate spore- bearing anaerobes, and facultative anaerobic spore-bearing bacUli is considered diagnostic. Immunological tests may be projected on guinea-pigs if toxic broth cultures are encoun- tered, antitoxic sera (A and B) being used to establish the Pig. HO. — Clostridium hotvlinum (van Ermengem in Kolle and Wassermann). 288 VETEBINAHY BACTERIOLOGY relation, if any, of the intoxication induced to B. botulinus. Pure cultures are obtained by seeding in plain agar and fishing colonies to pork agar shake cultures containing 2 per • cent, dextrose." Physiology. — The organism is an obligate anaerobe. Its optimum growth temperature is 25 to 30°. Some strains grow little, if at all, at blood-heat, and when developing at this tem- perature produce numerous involution forms. Gas is produced from dextrose and lactose but not from saccharose. Acid, in part butyric, is produced in dextrose media. Pathogenesis. — Injections of the organism into the body of laboratory animals have revealed the fact that some strains of the organism are pathogenic only by virtue of the toxins that are elaborated outside of the body. It does not apparently increase in numbers in the tissues. Probably this may in part be ac- counted for by its normal optimum growth temperature. The toxin produced, on the other hand, is very poisonous, whether injected or ingested. The use of raw or imperfectly cooked animal foods or of infective canned foods may give rise in man to the symptoms of botulism in the course of twenty-four to thirty-six hours, often with fatal termination. Immunity. — The toxin produced by Clostridium botulinum is among the most powerful known — 0.00005 to 0.0001 gm. is fatal in three to four days when injected subcutaneously into a guinea- pig, and 0.0001 to 0.0005 gm. will destroy a rabbit. A most striking characteristic of this toxin, and one which distinguishes it from those of diphtheria and tetanus, is its ability to produce poisoning when taken into the body by way of the alimentary tract, withstanding the gastric and intestinal digestive processes. Guinea-pigs and even apes are killed by the ingestion of 0.01 mil. of a dextrose broth-culture solution in which the organism has been grown. The toxin is destroyed by exposure to light and air. Heating to a temperature of 80° renders it non-toxic. Antitoxin has been prepared from the goat and from the horse by gradually increasing doses of the toxin. This antitoxin exerts both a prophylactic and a curative effect when injected. Immunologic tests with the antitoxin of Clostridium botulinum indicate that there are two types of the organism designated by Burke, Type BLACKLEG TETANUS GROUP. THE GENUS CLOSTRIDIUM 289 A and Type B. The latter type according to Graham is most frequently encountered in animals; the Type A from canned fruits and vegetables and from ensilage. Chickens are not sus- ceptible to Type B, but are to Type A. The antitoxic serum prepared against one strain is not effective in neutralizing the toxin from the other strain nor in checking the disease produced by the other strain. This would suggest the use of a polyvalent antitoxic serum in the treatment of spontaneous cases. Bacteriologic Diagnosis. — This can be accomplished only by isolation and cultivation of the specific organism by serological tests with known antitoxin against suspected toxin and by the agglutination test. Transmission. — The organism has been isolated from poi- sonous meat, canned vegetables and fruits, ensilage and grains and from normal swine feces. The disiease can be produced only by the ingestion of substances in which the organism has been growing. CHAPTER XXV PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP The organisms belonging to this group are all aerobic, non- motile, Gram-negative bacilli that do not produce spores, and that show a decided tendency to polar staining. They exhibit compara- tively slight powers of fermentation. The bacteria of this group were first recognized as closely re- lated by Hueppe in 1886. He included in his species Bacillus septicemicB hcBmorrhagiece, the causal organism of chicken cholera, rabbit septicemia, swine plague, hemorrhagic septicemia of cattle and wild animals, and several others. Trevisan included all of these organisms as separate species in a new genus which he named Pasteurella, after Pasteur, who had studied the causal organism of chicken cholera. The name pasteurellosis is, therefore, fre- quently used to designate a disease caused by an organism of this group. The classification proposed by Lignieres in 1901 has been extensively used, particularly by the French bacteriologists. The organisms which are to be grouped with certainty here are those which cause the following animal diseases : Hemorrhagic sep- ticemia of cattle, of sheep, septic pleuropneumonia of calves, fowl- cholera, rabbit septicemia, and the "Loffler-Schtitz" swine plague. Here is also to be included the organism which causes human plague. The great similarity of these various organisms to each other has led many writers to include them as varieties of a single species which has been variously named Bacillus bipolaris septicus, B. plurisepticus, Bacterium bipolare pluricida, Bacillus septicemiw hcemorrhagica, Coccobacillus, and Bacterium multicidum. There is still great need of careful comparative studies of the organisms belonging to this group. It is by no means certain how many species are included. It is probable that in certain of the diseases the organism is a secondary invader. Non-virulent strains and perhaps species are known to occur frequently in nature. 290 PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 291 Since it has thus far not proved possible to differentiate the organisms associated with the various diseases by means of their morphologic or biologic characters, it is necessary to adopt tentatively a pathologic classification, and group them with reference to the animals naturally infected and the diseases pro- duced. The following list includes only the more important types that have been described, and is not complete : A. Organisms which produce disease in lower animals only: 1. In birds. Pasteurella {aviseptica) cholerce gallinarum. 2. In swine. Pasteurella suiseptica. 3. In cattle. Pasteurella boviseptica. 4. In equines. Pasteurella equiseptica. 5. In rabbits. Pasteurella cuniculicida. 6. In rats and other rodents. Pasteurella pseudotuber- culosis rodentium. B. Organism producing disease in ma.n:- Pasteurella pestis. Because of the close resemblance among the organisms causing hemorrhagic septicemias of the lower animals, the discussion of morphologic, physiologic, cultural, and biologic characters common to all species will be given first, limiting the discussion under the species to the characters of value in differentiation and to problems of immunity and pathogenesis. Hemorrhagic Septicemia group Distribution. — Organisms having all the morphologic and bio- logic characters and sometimes even the pathogenesis of the dis- ease-producing members of this group have been isolated many times from the mouth, nose, and alimentary tract of many animals. They are undoubtedly common as secondary invaders, and in some diseases have served to obscure the real causal organism by the regularity of their presence. It is contended by some writers that the sporadic nature of these disea,ses is due to the sudden acquisi- tion of virulence by forms already present in the body. Not only the relative virulence of these ubiquitous organisms is in need of study, but also careful comparative studies of their cultural and physiologic characters. Morphology and Staining. — The organisms are non-motile rods. In body fluids they are usually about 0.5 by 1 n, and with aqueous 292 VETERINARY BACTERIOLOGY anilin dyes show intensive coloration at the poles, the central portion of the cell staining faintly or not at all. Giemsa's stain shows the polar granules well. In culture-media there occurs con- siderable variation in size. There do not seem to be well-marked differences in size among the different species. In pure cultures the organisms may be cocci, diplococci, or short rods. The polar staining character is much more difficult to demonstrate in cul- tures than in body fluids. By the use of carbol-fuchsin applied for one-half to one second many cells with polar granules may be demonstrated. The organisms are uniformly Gram-negative. Cultural Characters. — All species are readily cultivated on suitable artificial media. Gelatin colonies are small, white, and usually irregular, without marked distinctive characters. Gelatin slants show numerous small, round, flat, gray to translucent dewdrop-like colonies, which later become gray white and opaque. The gelatin is never liquefied. On agar plates the colonies resemble those on gelatin. ■ There is no tendency for the colonies to spread over the medium. The addition of blood-serum increases the luxuriance of growth, dex- trose and glycerin have no perceptible effect. The organism does not grow in Endo medium or on malachite green agar. On Drigalski's medium there is good growth without a change of color. Blood agar shows no hemolysis. Little or no growth occurs upon potato. In broth uniform clouding occurs with occasional clearing by sedimentation. Old broth cultures are often slimy. No change occurs in milk. Luxuriant growth occurs on Huntoon's hormone medium and on meat medium. Physiology. — The optimum temperature is about 37°, the minimum, 12° to 13°, and the maximum, 42° to 43°. The organisms are aerobic, growing little or not at all under anaerobic conditions. Gas is never developed in any sugars; small amounts of acid may be. No enzymes liquefying gelatin or blood-serum are produced. Indol is formed by some organisms, apparently not by others. Slight development of hydrogen sulphid and reduction of nitrates have been recorded. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 293 Pathogenesis. — The most susceptible of the laboratory animals is the rabbit. Intravenous injections usually prove fatal in a few- hours to a day. Autopsy reveals the development of a septicemia in which the lesions are not particularly characteristic. Petechial hemorrhages are commonly noted on the mucous membranes of the respiratory and digestive tracts,' and there may be swelling of lymph-nodes and spleen, congestion and edema of the lungs, and hyperemia of the kidneys. Practically all species are also pathogenic for mice and for spar- rows. Guinea-pigs are somewhat more resistant. Other species of animals are much more distinctive in their reactions. Fowls, for example, are quite susceptible to the bacillus of fowl cholera, but quite resistant to infection with mammalian types. No true toxin has been demonstrated and, in consequence, no antitoxin. The disease in its acute form in any animal is a bacter- emia in which enormous numbers of bacteria are present in the blood-stream. Immunity. — An endotoxin is produced which goes into solution slowly as a result of the autolysis of the cells. Broth filtrates of young cultures are innocuous, while autolyzed cultures are toxic. The toxicity of various strains has been shown to vary, but it is apparently a variable quite independent of virulence. MacFad- yean has also demonstrated the presence of intracellular poisons by grinding the cells at the temperature of liquid air and by ex- tracting with a dilute alkali. Weil and Bail have sought to explain the virulence of this organism and the pecuharities of the infection by the use of the aggressin hypothesis. It seems evident that certain strains are capable of paralyzing the antibacterial defences of the body and thereby prevent phagocytosis. Citron and others have shown that such inhibiting substances may be dissolved from virulent cells. Pasteurella cholerse gallinarom Sjnonyms. — Bacillus cholerw gallinarum; B. cholerm; Bacter- ium avicidum; Bacillus avisepticus. Diseases Produced. — Fowl or chicken cholera, in domestic fowls and other birds. 294 VETEKINABY BACTERIOLOGY Distribution.— The disease has a wide distribution in Europe and America. Historical. — Perroncito, in 1878, first discovered this organism. Pasteur, in 1880, succeeded in cultivating it, and with it performed many experiments on attenuation and immunization. , Consider- able historical importance is attached to it as marking the beginning of the study of experimental immunity. Morphology and Staining. — Typical of the group. Consider- able variations in size of the organisms from different strains- may occur. Isolation and Culture. — Characteristics of the group. Physiology. — Characteris- tics of the group. According to Hadley, indol may or may not be produced and nitrates are usually reduced. Some acid is produced from dex- trose, but not from saccharose, lactose, or mannite. f^^fi^jJiS**^ Pathogenesis. — Great vari- Fig. lU.-Pasteurella cholerm galli- ^tions in virulence have been narum, from an agar slant ( X 1000) noted among various strains. (Gunther). j^ j^^^y. prove pathogenic upon inoculation or in some cases upon ingestion to fowls, geese, pigeons, mice, and rabbits, producing very rapidly fatal septicemias. Guinea-pigs are relatively immune. The pigeon is among the most susceptible of experimental birds. The injection of minute quantities of a virulent culture into the breast muscle will prove fatal in from twelve to twenty-four hours. At the site of inoculation there develop an induration and thick- ening of the skin. The subcutaneous tissues show a straw-colored exudate. Usually there is found an acute pericarditis, enlarge- ment of the spleen, and severe hemorrhagic enteritis. In domestic fowls there appears congestion of the heart with an accumulation of serum in the pericardial sac. The liver is also congested, frequently showing petechise. The spleen is enlarged and softened. The lungs usually are congested and may show areas PASTEURELLA, OR HEMORRHAGIC SEPTICEMU GROUP 295 of hepatization. Enteritis is usually demonstrable, frequently hemorrhagic. Rabbits are likewise susceptible and succumb in ten to twenty hours with characteristic symptoms. Infection may also be secured in the larger animals, producing in many cases typical hemorrhagic septicemias. Immunity.— No true toxin has been demonstrated for this organism. Endotoxins are produced. One attack of the disease with recovery confers immunity. Agglutination in dilu- tions of 1 : 6000 has been shown with blood of animals artificially Pig. 112. — Pasteurella choleroe gallinarum, in pigeon's blood (Frankel and PfeifiFer). immunized. The nature of this immunity is not certainly known, although opsonins have been demonstrated. Pasteur, in 1880, worked out a method of prophylaxis by the use of vaccines prepared by attenuating the organisms by long-con- tinued cultivation upon artificial media. Broth cultures were allowed to stand from three to ten months. Under these condi- tions the virulence is gradually lost, and inoculation into the fowl is followed by a mild local reaction only. This immunizes against subsequent injections of the virulent form. Pasteur believed that the attenuating factor was the abundant supply of oxygen, for 296 VETEKINAKY BACTERIOLOGY cultures which he sealed from the free entrance of air he found to retain their virulence even after ten months. He also found that various strains showed great differences in their rate of attenuation. The Pasteur method of vaccination has never come into general use. Tests have shown that the use of the vaccine sent out by the Pasteur Institute was apt to produce typical cholera in some fowls. It has been shown that some degree of immunity is conferred by the injection of killed cultures of the organism. It has also been found that immunization against one of the members of the hemorrhagic septicemia group immunizes like- wise against others. Injections of the Pasteurellaboviseptica, for instance, will protect against subsequent injection with Past, cholerce gallinarum. Lignieres has prepared a polyvalent vaccine by growing at 42° to 43° organisms isolated from sheep, cattle, horses, dogs, hogs, and fowls in bouillon. When allowed to grow for five days it constitutes the Vaccine I; for two days only. Vaccine II. One-eighth c.c. of I is injected, and twelve to fifteen days later the same amount of Vaccine II. By this means he claims to be able to immunize against all types of hemorrhagic septicemia in animals other than fowls. This method has not been utilized in practice, although a few recorded tests have been favorable. Hadley has succeeded in isolating one strain (his No. 52) of a non-virulent organism which has very high immunizing value when used as a vaccine. He has found that vaccination with this strain confers an active immunity against all pathogenic strains tested. Hadley also concludes that the virulence of various strains of fowl-cholera organisms is subject to far less variation than has been supposed. Kitt and Mayr, in 1897, showed that it is possible to secure a protective serum from the horse and other animals, as goats and swine, by injections of living fowl-cholera organisms, and this serum, when injected in suitable quantities into susceptible animals, will protect them from injections of virulent bacilU. Schreiber, in 1899, elaborated upon an observation of the preceding investiga- tion that animals immunized against swine-plague bacilli (Pas- teurella suiseptica) were likewise immune to fowl cholera. In 1902 he gave the name "septicidin" to a polyvalent serum PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 297 which he prepared by immunization with Past, suiseptica and Pasteurella cholerce gallinarum. The reports relative to the effi- ciency of this serum are conflicting. It has not come intogeneral use. Hertel, in 1902, reported that by intravenous injections of dead bacteria followed by living bacteria into an ass he secured a serum which in injections of 0.5 c.c. protected pigeons against 10,000 times the normal lethal dose of the organism. Lignieres and Spitz have also prepared a polyvalent serum, using the Ligni^re vaccine noted above. There is no record of a practical utilization of this method. Kitt and others, in 1904, have described sera which - protect fowls experimentally inoculated with Past, cholerce gallinarum, but, like the others described, these have not come into general use. It may be concluded, therefore, that whUe immunization against fowl-cholera, either vaccination or the use of antisera, has been shown to be pos- sible, it has not been proved practicable. Bacteriologic Diagnosis.— Stained mounts of the blood which reveal the presence of Gram-negative bacilU showing prominent bipolar staining are diagnostic. The bacillus may be readily iso- lated in artificial media. Whether or not serum reactions, particu- larly agglutination, might be utihzed in diagnosis is not knoTiTi. Transmission. — The disease is supposed to be transmitted from bird to bird by ingestion of food or water fouled with excretions containing the specific organism. Pasteurella suiseptica Synonyms. — Bacterium suiddum; Loffler-Schutz bacillus; Ba- cillus suidda; Bacillus suiseptica. Disease Produced.— Swine-plague, Schweineseuche. Distribution.— The organism has been isolated from swine many times in Europe and America. LofHer and Schiitz, in 1886, published results which established the identity of swine-plague as a specific disease by the discovery of the causal organism. In the same year Smith isolated what proved to be the same organism from hogs in the United States. Since that time it has been isolated from animals in many parts of Europe and the United States. From the beginning the close relationship between the swine-plague and fowl-cholera bacilli was recognized. In the early . literature of swine diseases m America there is much confusion relative to the use of the terms 298 VETERINARY BACTERIOLOGY "swine-plague" and "hog-cholera." The discovery that hog- cholera is caused primarily by a filterable virus has made neces- sary a very careful retraversing of the knowledge relative to swine-plague, and it has been urged that probably the Pasteurella suiseptica is a secondary invader merely, as is the hog-cholera bacDlus, and that the two diseases differ not at all in their primary cause. The evidence at present seems to point, however, to a specific disease caused by Past, suiseptica and en- tirely distinct from hog- cholera. The question cannot be said to be satisfactorily settled at the present time. In the United States it seems probable that swine- plague, if such really exists, is relatively un- important in compari- son with hog-cholera. The situation is still fur- ther complicated by the fact that many writers have found in the respiratory tracts of normal swine numerous bacteria having all the characteristics of typical Past, suiseptica, even to pathogenicity. Morphology and Staining. — Typical of the group. Isolation and Culture. — Typical of the group. Physiology. — Typical. Slight acid production in dextrose and saccharose. Indol production and nitrate reduction appear to be variable. Pathogenicity. — The different strains show great fluctuations in virulence. In general, the pathogenicity for laboratory animals appears to be that of the group. The mouse and rabbit are particu- larly susceptible. The guinea-pig is somewhat more resistant. In fowls and pigeons large injections are usually required to produce infection. Experimental infection may also be secured in the horse, in cattle, sheep, dogs, and cats. The results in swine have proved quite variable. The injection of a highly virulent Fig. 113. — Past, suiseptica (after deSchwein- itz and McFarland). PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 299 culture may lead to the death of the animal within one to three days from septicemia, with an extensive hemorrhagic edema about the site of inoculation. The injection of less virulent cultures or the use of animals showing some degree of resistance may lead only to local edema followed by abscess formation, or to a more chronic infection which terminates fatally in a few weeks. In the latter, section shows extensive lung lesions including partial hepatization with necrotic areas, fibrinous pleuritis, and enlargement of the spleen. », o Intravenous injection of swine is " °^/^ ,^ usually followed by a fatal septicemia. *f^' ^^ '"p. A disease closely simulating the '„" ( j „ typical swine-plague has been produced ' " " ]r'^ in swine by intratracheal injections and ^ by inhalation. Fig. 114. — Pastewella sui- The spontaneous infection of swine f ^''"' '° ^^°''^ ^^^^^"^ ^^- , . , Schweinitz, Report Bureau may occur as a septicemia which proves ^f Animal Industry). very quickly fatal, as a pneumo-enteritis characterized by the involvement and hepatization of consider- able lung, areas, or as a chronic type. The evidence seems to point to the Past, suiseptica as a nor- mal inhabitant of swine. It is possible that, like the pneumo- coccus, it can produce infection and increase its virulence under certain conditions, decrease in body resistance, etc. Immunity. — As with the fowl-cholera bacillus, no true toxins have been demonstrated. Both active and passive immunization against the Past, suiseptica have been accomplished. Active immunization has been attempted in many different ways. The killed and living cultures have not, in general, proved satisfactory in immunizing the hog, although they have been successfully used in the preparation of antisera from the horse and other animals. Weil has elaborated the following technic, making use of the so- called "natural aggressins" for the establishment of immunity: A rabbit is injected intraperitoneally with 5 c.c. of bouillon con- taining a drop of twenty-four-hour culture of a highly virulent strain of the organism. The animal should die within the next twenty-four hours. The exudate, varying in amount from 1 to 20 c.c, is pipetted off and sterilized by the addition of 0.5 per cent, of ^ 300 VETERINAKY BACTERIOLOGY phenol, then heated to 44° for three hours, then its sterihty deter- mined by transfers to broth. If the broth shows no growth, the material is sterile and is ready for use. This may be used to inject laboratory animals and thereby establish immunity. The animal immediately after injection becomes more susceptible to the disease, presumably due to the presence of aggressin in the blood, but later a relatively permanent active immunity is produced. In practice it is found that in the immunization of hogs it is necessary that the exudate containing the aggressin be obtained from other hogS rather than from rabbits. Wassermann and Citron have de- veloped a somewhat similar method of immunization by the use of so-called "artificial aggressins" or bacterial extracts. These methods of immunization are of much more theoretic than prac- tical importance. Passive immunization by means of antisera has been studied by several investigators. A rabbit may be actively immunized by one of the preceding methods, and its serum may protect a mouse in doses of less than 0.1 c.c. against a fatal injection of a highly virulent organism. Wassermann and Ostertag and their pupils have shown that an antiserum specific for one strain of Past, suiseptica is not always effective for others. They, therefore, prepare serum by the systematic immunization of a horse against several strains of the organism until a serum of high potency is produced. Its strength is determined by injections into mice. Experiments upon young pigs with this serum are claimed to have been highly successful, but the method has not come into general use. Simultaneous injections of immune sera and of Past. suiseptica have also been advocated. In summary it may be said that immunization against swine- plague is still in the experimental stage, and that no completely satisfactory method has been evolved. Bacteriologic Diagnosis.— The identification of the causal organism by actual isolation is the only practicable method of bacteriologic diagnosis. Transmission. — The means by which the disease spreads natu- rally are not fully understood. It is possible that it is by ingestion probably sometimes by inhalation. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 301 Pasteurella boviseptica Synonyms.— Sactenwrn bovisepticum; B. bipolare multicidum; Bacillus hovidda, B. vitulisevticus: Bacillus bovisepticus. Diseases Produced.— Hemorrhagic septicemia in cattle, buf- falo, and related wild animals; septic pleuropneumonia of calves; Rinderseuche, Wildseuche. The early descriptions of the disease refer to it as attacking cattle, wild animals, and swine. Bolhnger, in 1878, first described it as Wild- and Rinderseuche, attacking wild boar and deer. Kitt, in 1885, isolated an organism belonging to the hemorrhagic septi- cemia group. Since that time numerous investigators have re- ported epizootics of the disease in many countries. Fernmore, in 1898, first noted its presence in the United States. It has been repeatedly found since that time, particularly in the Mississippi Valley. Morphology and Staining. — Typical of the group. Culture and Physiology.— Typical of the group. Pathogenesis. — The organism is usually pathogenic for the mouse, pigeon, rabbit, and sparrow. Highly virulent cultures produce a quickly fatal septicemia in calves. The lesions in the adult animals are characteristically petechise in many of the body organs, particularly in the serous surfaces. Hemorrhages are quite uniformly present also in the subcutaneous tissues. In some cases these are quite extensive and involve a con- siderable portion of the body surfaces. Septic pleuropneumonia in calves is one of the most frequent types of infection. In this disease the pleura is often partially covered with thick fibrin layers, the interstitial connective tissues of the lungs show a serohemorrhagic infiltration, the remainder of the lung tissue is hemorrhagic. According to Jensen, in those cases in which the infection progresses more slowly, the exudate laid down in the connective tissue is responsible for the development of firm white yellow layers which surround the thickened lung lobules, which are dark red or mottled in color. Immunity. — Repeated injections of animals at first with non- virulent and later with virulent organisms will produce a relatively high degree of active immunity. The serum from such animals has considerable power to produce passive immunity. Some inves- 302 VETERINARY BACTERIOLOGY tigators claim that there are many strains of hemorrhagic septi- cemia and, therefore, a polyvalent serum must be secured either by injecting many strains of organisms into the serum animal, or by mixing the serum secured from several animals each immun- ized against several strains. Bacterins prepared by killing cultures of Past, hoviseptica by heat or antiseptic have been used for prophylactic purposes with apparently good results. Such bacterins are frequently polyvalent. Other Hemorrhagic Septicemias of Animals Organisms belonging to this group have been isolated from a considerable number of animal diseases in addition to the ones which have already been described. Rabbit septicemia or rabbit plague (Pasteurella cunicuUcida) , pneumo-enteritis, or hemorrhagic septicemia of sheep and of the horse, infectious pneumonia of goats, Biififelseuche or pasteurellosis of the buffalo, dog typhoid or dog pasteurellosis, hemorrhagic septicemia of elephants, of geese, wild birds, and many other animals have been ascribed to Past, septicemice hwmorrhagicce. As has been before stated, the evidence in some of these cases seems to be inconclusive. Pasteurella pestis SYnonym.s.— Bacterium pestis; B. pestis bubonicoe; Bacillus pestis. Disease Produced.^Bubonic plague in man and rodents. Yersin and Kitasato, in 1894, independently described the organism which causes bubonic plague. Since that time it has been isolated and described by many observers in numerous outbreaks. Distribution. — The disease is endemic in parts of China and India. At various times it has spread as an epidemic over tha entire civilized world. Cases have been reported within recent years in most of the civilized countries. Morphology and Staining. — The Pasteurella pestis morphologi- cally resembles the other members of this group. Involution forms are produced so readily upon appropriate culture-media, such as partially desiccated agar and salt agar, that their develop- ment has been regarded as diagnostic. Capsules may sometimes be demonstrated on culture-media, but not in tissues. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 303 Isolation and Culture.— Growth in general is typical of the group. One character found useful in diagnosis is the "stalac- Fig. 115. — Pasteurella pestis (Wherry). tite" formation in broth covered with oil and allowed to remain without being disturbed. Under these conditions long deUcate threads are produced which hang from the oil and resemble the stalactites found in caves. Physiology. — Reactions in general are those typical of the group. Dextrose broth is acidified, but no gas is pro- duced. Indol is not formed. Pathogenesis. — Experi- mental Evidence. — The organ- ism readily infects mice, rats, guinea-pigs, rabbits, dogs, cats, and monkeys when they are experimentally inocu- lated. The symptoms and lesions produced are entirely typical of bubonic plague in man. Accidental infection of man resulting in 4 cases of plague occurred Fig. 116. — Pasteurella pestis, bacilli from a bubo (Gunther). 304 VETERINARY BACTERIOLOGY in a Vienna laboratory at a time when bubonic plague did not exist elsewhere in Europe. The causal relationship of the organism to the disease may be held to be fully established. Character of Disease and Lesions Produced.— The disease in experimental animals may be a rapidly fatal septicemia, or in those animals which are somewhat resistant, as the rat, typical buboes (enlarged and suppurating lymph-nodes) or abscesses in the spleen or liver are produced. The disease in man may be one of three types— septicemic, usually rapidly fatal with the organisms gen- erally distributed through the blood and various tissues of the body; the pneumonic, also rapidly fatal; and the bubonic, the most common, in which the lymph-nodes are infected, become enlarged, and ulcerate. The bubonic type is less fatal, recovery taking place in a small percentage of cases. The septicemic type of the disease is often accompanied by. extensive subcutaneous hemor- rhages, which gave the name "black death" to the epidemics of medieval Europe. Immunity. — No true toxin has been demonstrated for Pas- teurella pestis, although endotoxins have been shown to be present. Agglutinins for this organism may be found in the blood of ad- vanced cases and of convalescents, but they appear too late to be of any diagnostic value. The reaction is rather doubtfully specific. The reaction occurs only in low dilutions — rarely above 1 : 20. Agglutination in much higher dilutions may be secured with im- mune serum — sometimes as high as 1 : 1000 has been observed. Precipitins have also been noted in laboratory experimentation. Opsonins for Past, pestis have been demonstrated in normal human serum and in immune serum. Bactericidal substances are present in the serum of artificially immunized animals. Active immunization of man against bubonic plague has been quite extensively practised. The procedure consists in every case of injection of killed or attenuated bacilli or their products as a prophylactic measure. The use of the various substances has proved quite successful. Haffkine's vaccine has been used in India. It consists of a killed six-weeks' culture of plague bacilli in broth. Modifications of this method have been utilized by many investiga- tors. The immunity estabUshed is probably both opsonic and bactericidal. PASTEURELLA, OR HEMORRHAGIC SEPTICEMIA GROUP 305 Passive immunization by the injection of the serum of horses liyperimmunized against Pasteurella pestis has been highly suc- cessful, according to some, and of no material advantage accord- ing to others. Several procedures have been advocated. The following is that of Kolbe and Kumbein : A culture of Past, pestis is passed through rats to exalt its virulence. This is planted upon agar and incubated forty-eight hours at 30°, the growth washed off, and suspended in physiologic salt solution. The suspension is killed by heating to 70° for an hour and its sterility determined. The horse is injected at intervals of a few days with gradually increasing doses of the dead bacteria, until after seven or eight injections the bacteria from six or more culture-tubes are injected at one time. Injections of minute quantities of the living organ- ism are then begun, and finally, after repeated injections, the living organisms from 16 cultures are used. The interval be- tween injections is governed by the reaction of the animal. Usually it is from five to eight days. The animal is then bled, and the serum preserved by the addition of 0.5 per cent, phenol. Bacteriologic Diagnosis. — The organism may be recognized in stained moimts from the pus from a bubo as a small Gram-negative bacillus, with characteristic bipolar staining. It may also be isola- ted upon culture-media and identified by its growth characteristics. Transmission. — The pneumonic form of the disease may be transmitted by the inhalation of infectious droplets. Plague is not known to occur in the himian following ingestion of the organ- ism. The bubonic or most common type is probably transmitted toman by the bite of fleas (or from their excretions scratched into the skin), which have left rats dead of the disease. An epidemic of plague in the human is commonly preceded by an epizootic among the rats of the community. It has been shown experiment- ally that a flea may transmit the disease from an infected to. a non- infected individual. It is also known that the cannibalistic tend- ency of rats to eat their dead is in part responsible for the spread of the disease among vermin. The annihilation of the rat is the best prophylaxis known. The disease has in certain places, as about San Francisco, been found to spread to such rodents as the ground- squu-rels and wood-rats. When a region once becomes thoroughly infected it is, therefore, difficult to stamp out the disease. CHAPTER XXVI GLANDERS GROUP. THE GENUS PFEIFFERELLA One organism only, the Pfeifferella mallei, the cause of glanders and farcy in equines, is known to belong to this group. It should be noted that the so-called pseudoglanders and the causal organ- isms are treated under other chapter headings. These latter organisms are not related to the organism in question except in that they produce lesions which are sometimes confused with glanders clinically. Some of the pseudoglanders organisms be- long to such disease groups as the bacteria, the blastomycetes, and the hyphomycetes. Pfeifierella matfeS Synonjrms. — Bacterium mallei; Mycobacterium mallei; Bacillus mallei. Diseases Produced. — Glanders and farcy in equines; Rotz; morve. LofHer and Schiitz, in 1882, demonstrated the presence of a characteristic rod (Pf. mallei) in the nasal discharge of a horse affected with glanders. Kitt, in 1883, and Weichselbaum, in 1885, confirmed these results and added to our knowledge of the organism. Distribution. — Glanders is known in practically every civil- ized country. Morphology and Staining. — The glanders bacillus is a short rod, usually straight, but sometimes somewhat curved. The ends are rounded. It is usually single, more rarely in pairs or short chains in artificial media. In tissues the organism is generally in pairs. The cells are usually thicker and shorter in broth than on solid media. In stained mounts from the caseated pus from the lymph-gland of a guinea-pig, or from a glanders nodule from the liver of a field mouse, the cells are thicker and show a decided tendency to polar staining. , Involution forms are frequently pro- duced; enlarged cells, clubbed forms, filaments, and even branch- ing have been observed. This last fact has led to the grouping of 306 GLANDEHS GROUP. THE GENUS PFEIFFERELLA 307 this form with the higher fungi by some authors. The normal rods vary from 0.5 to 1.0 by 1.; to 5 /.. The organism is non- motile, and does not produce spores or capsules. It stains with the ordinary anUin dyes, and still better with stains containing a mordant. It sometimes shows some granular differentiation of the cytoplasm resembling the diphtheria bacillus. It is not acid- fast and is Gram-negative. Isolation and Culture.— The glanders bacillus is rarely in pure cultures in the nasal discharges, so that for its isolation from such sources a special technic is necessary. It is customary to inject intraperitoneally a male guinea-pig with a smaU quantity of the dis- charge from an ulcer, mixed with a little bouillon or physiologic salt solution. Within two to four days the testes swell and give evidence of acute inflam- mation. The animal is then killed, a testis re- moved and opened under aseptic conditions, and the contents of one of the small abscesses or foci of inflammation removed on a sterile platinum needle to suitable media. The glanders bacillus grows upon the ordinary culture-media, particularly upon those that contain glycerin, upon blood-serum, and potato. The colonies upon agar and glycerin-agar plates are whitish or yellowish, glistening, usually circular. Upon the slanted medium the, colonies are coalescent and form a moist, shining layer of a slimy consistency. In bouillon and glycerin bouillon it produces an initial turbidity, followed by sedimenta- tion; a shining white pellicle is likewise formed when the medium is not shaken. On blood-serum the colonies are first ,discrete, clear, yellowish, viscous, hemispheric drops which coaleste to form a transparent layer over the surface^, thi^s later becomes Kg. 117. — Pfeifferdla mallei from glycerin agar (X 1000) (Frankel and Pfeiffer). 368 VETERINARY BACTERIOLOGY gray and opaque. Gelatin is not liquefied. The growth upon potato is perhaps the most characteristic. It may be described as forming within forty-eight hours a yellow, honey-like, semitrans- parent growth that gradually becomes brownish or amber in tint. The potato itself is tinted greenish or greenish brown. This reaction is not characteristic if potatoes having too acid a reaction are used. They may be neutralized previously to inoculation by soaking in dilute sodium carbonate. Physiology.— The glanders bacillus is aerobic and facultative anaerobic. . Its optimum growth temperature is 30° to 40°, but its growth lirhits, at least in freshly isolated cultures, are about 25° and 42°. Its thermal death-point is 55°, with ten minutes' exposure. Pathogenesis. — Experi- mental Evidence. — There is an abundance of evidence to prove that Pf. mallei is the cause of glanders. All the lesions of the disease may be duplicated by the experimental inoculation of pure cultures into labora- tory animals and the horse. The guinea-pig is very sus- ceptible. A subcutaneous inoculation is followed within a few days by local swelling and in- duration, which soon ulcerates and discharges to the surface. The disease spreads largely through the lymph-channels, and the lymph-nodes enlarge and suppurate. Various metasta,tic infections of the joints, the lungs, liver, and other organs occur. Death seems to be due to exhaustion. Infection may similarly be transmitted to the rabbit. The horse may be readily infected, as may sheep, goats, the cat, and the dog. Cattle and the house-rat do not con- tract, the disease. It occurs in man through infection from gland- ered aninialls ) and through working with pure cultures in the laboratory. ' Fig. 118. — Pfeifferella mallei, in section from the spleen of a field-mouse (Frankel and Pfeiffer). GLANDERS GROUP. THE GENUS PPEIFFERELLA 309 Character of Disease and Lesions Produced. — The disease as found in equines may be either of an acute or a chronic type. The former is commoner in the ass and mule, and the latter in the horse. The acute type of disease is commonly ushered in with a chill, there is a mucopurulent discharge, and death usually occurs in from one to four weeks. The chronic type shows no marked characteristics in its early stages; the lymph-nodes in various parts of the body become infected and enlarge. This may exist for a long period in an animal, and may terminate finally in an acute attack. The lesions in the chronic type are generally present on the nasal mucosa, in the lungs, and in the Ijonph-glands. The- nodular glanders of the nasal mucosa is the most frequent type. The nodules, small at first, enlarge to the size of a pea, then break down, suppurate, and form chronic ulcers. When healing of the deeper ulcers occurs, the star-shaped scar resulting is quite characteristic. In the lungs lesions are almost invariably to be found; these may be nodular, or consist of infiltration of con- siderable areas of tissue. In farcy or cutaneous glanders the nod- ules form in the skin; the lymph- vessels become swollen and feel like a string of beads or a knotted cord. These nodules occasion- ally break through to the surface and ulcerate. In man the organ- ism commonly gains entrance through abrasions or wounds in the skin, or by inhalation, and the infection produced is practically always fatal. Immunity. — No toxins have been demonstrated for Pf. mallei, although endotoxins are produced. Agglutinins are present in the blood-serum of normal animals, but in much greater con- centration in the blood of infected animals. Precipitins may also be demonstrated. Of the bactericidal and opsonic nature of sera less is known. Active Immunization. — Immunization by the use of suspensions of dead bacteria or their products (mallein) has been attempted both in prophylaxis and cure. Although some favorable results have been reached, the subject needs further study. No method of vaccination or active immunization has as yet been shown to be practical and successful. Passive Immunization. — The blood-serum of animals, such as the ox, naturally iinmune to glanders has been claimed to possess 310 VETERINARY BACTERIOLOGY immunizmg power when injected into smaller laboratory animals, as the rabbit. Nocard and Prettner have each attempted im- munization by the administration of serum from cattle that had been repeatedly injected with virulent cultures of the glanders bacillus, but found the method ineffective. Galtier observed that treatment.with such serum only prolonged the course of the disease which had been induced by experimental inoculation. Bacteriologic Diagnosis. — A presumptive bacteriologic diag- nosis may be made by an examination of properly stained pus or sections of tissue, and a more positive diagnosis by the methods of animal inoculation, agglutination, precipitation, absorption of complement, and by the use of mallein. Examination of Pus and Tissues. — The nasal secretions and the pus from ulcers always contain a mixed bacterial flora, and are consequently unsatisfactory for microscopic examination. The bacilli of glanders are not readily recognizable and are, therefore, very difficult to differentiate from other bacteria. The nodules of the disease and the subcutaneous tissues are exceptions to the above rule, as are the foci in the submaxillary glands. These may be freshly incised and satisfactorily stained. For demonstration of the organism in tissues the method of Kiihne is recommended as giving good results. Carbol-methylene-blue (methylene-blue, 1.5 gm.; alcohol, 10 c.c, and 5 per cent, aqueous phenol or carbolic acid, 100 c.c.) is used to stain the sections, one-half hour; they are then washed in water, then in very dilute hydrochloric acid (10 drops to 500 c.c. of water), and quickly transferred to a solution of lithium carbonate (8 drops of a saturated solution to 10 c.c. of water), then to distilled water, dehydrated in absolute alcohol con- taining a little methylene-blue, then cleared in anilin oil. The bacteria should show plainly, but are few in number and not always readily discovered. Diagnosis by Animal Inoculation.— Strauss' Reaction— A male guinea-pig is inoculated intraperitoneally with a- small amount of the suspected material. This is preferably obtained from the interior of encapsulated nodules or abscesses, or from the base of fresh ulcers. The nasal discharge is less satisfactory because of the presence of large numbers of other varieties of bacteria, which may cause the death of the animal prematurely from peritonitis or sep- GLANDERS GROUP. THE GENUS PPEIFFERELLA 311 ticemia. In from two to four days, exceptionally not until the twelfth, the testes become enlarged and tender, the skin above them is reddened and shiny. The animal, in case of a positive reac- tion, should be killed and the contents of the testes examined mi- croscopically to determine the presence of a Gram-negative charac- teristic bacillus. If not killed, abscesses which discharge large numbers of bacteria develop, and the animal emaciates rapidly, dying within two weeks. Other organisms may give the orchitic reaction, but they are Gram-positive, with the exception of Pseudomonas pyocyanea. A culture should always be made from the pus in the scrotum to make diagnosis certain. Agglutination Test for Glanders. — The serum of a normal horse will frequently agglutinate the glanders bacillus when in dilutions of 1 : 100, 1 : 500, rarely more. The following general rule as men- tioned by Hutyra and Marek is quite applicable and reliable: Agglutination, if appearing in dilutions of 1 : 400 or less, with -few exceptions denotes freedom from infection; in dilutions of 1 : 1000 or more, also with few exceptions, agglutination indicates the presence of infection; in dilutions of 1 : 2000 agglutination signifies recent infection. The organisms used in the agglutination test may be either living or dead. The latter are commonly used, as it does away largely with danger of infection to man. The bac- terial suspension is prepared by removing the growth from a young culture on agar and suspending it in physiologic salt solution con- taining 0.5 per cent, phenol. This is heated at 70° for two to four hours; this kills the bacteria, but does not interfere with the ag- glutination reaction. Equal amounts of this suspension are placed in a series of small test-tubes, and to these are added equal amounts of different dilutions of the serum to be tested, and the final dilu- tions of the serum determined. Dilutions are usually prepared 1 : 100, 1 : 200, 1 : 400, 1 : 500, 1 : 800, 1 : 1000, and up to 1 : 4000 or more. The tubes are kept at 37° for from twenty-four to thirty- six hours, or the reading of the result may be hastened by centrif- ugation. A positive reaction is indicated by a fihn covering the entire bottom of the tube, a negative by no precipitate or a little sediment in the bottom of the convexity, not forming a film. Whether or not a positive reaction is accompanied by a complete clearing of the test fluid depends upon the concentration of the 312 VETERINARY BACTERIOLOGY suspension and the dilution and potency of the serum used. The fluid may remain somewhat cloudy in a positive reaction in the . higher dilutions, not all the organisms being agglutinated. The suspensions of killed organisms may be secured ready for use from some pharmaceutical houses, together with tubes and mate- rials for preparing the proper dilutions. The suspension when properly prepared and preserved in the dark will keep for a con- siderable time. The microscopic test for agglutination has not proved practicable, as normal serum agglutinates microscopically in high dilutions. When properly carried out the macroscopic test is claimed by some to be an even better diagnostic than mallein. Konew's Precipitation Test, or the Ring Test.— A solution of glanders baciUi prepared by adding 10 c.c. of an 8 per cent, anti- formini solution to the bacilU washed from the surface of a forty- eight-hour slant agar culture. The bacteria will go into solution within two hours. It is well to add even more of the organism if it appears to dissolve rapidly, as it is desirable to get as concen- trated a solution as possible. The solution must then be care- fully neutralized, preferably by the use of 5 per cent, sulphuric acid. This is then filtered through paper, then through a Berke- feld filter, to remove all undissolved bacteria. The filtered solu- tion is termed "mallease." A test-tube is filled to a depth of 3 cm, with mallease, and blood-serum from a suspected case is introduced by means of a pipette. The end of the pipette should be passed through the layer of mallease and should rest against the bottom of the tube before the serum is allowed to flow. A quantity of serum, about equal to the mallease is introduced. The pipette is with- drawn quickly and carefully to prevent any mixture of the twO' liquids. The serum has a higher specific gravity and remains at the bottom, with the mallease as a distinct superficial layer. If the serum is from an animal free from the disease, there will be no reaction. A positive diagnosis of glanders is indicated by a white cloudiness that appears along the line separating the two liquids. This is due apparently to precipitation by the specific precipitins formed in the serum. In acute or well-marked cases the reaction occurs almost immediately, and usually in all cases within fifteen 1 The composition of antiformin is given on p. 380 It is a patented dis- infecting solution, and may be purchased upon the market. GLANDBBS GBOUP. THE GENUS PFEIFFERELLA 313 minutes. Observations recorded up to the present indicate that this method is giving satisfactory results in most instances. The possibility of demonstrating precipitins in chronic cases presents an additional advantage for this method. Fixation of Complement Test. — Schiitz and Schubert^ have de- scribed a satisfactory method of adapting Wassermann's syphilis test by fixation of complement to the diagnosis of glanders. Mohler and Eichhorn^ have tested out the method and found it highly satisfactory. Hemolytic amboceptor is prepared, prefer- ably by a recent method described by A. F. Coca. Rabbits are given two intravenous injections of washed erythrocytes of the sheep of 1 c.c. or at most 2 c.c, at intervals of not less than four days. At the end of five days after the last injection the rabbits are bled and their blood-serum obtained. Such amboceptor is usu- ally highly potent and the potency remains uniform for a long time. It must be inactivated by heating to 56° for thirty minutes before it can be used. Fresh guinea-pig serum is used as complement. The antigen used is an extract of glanders baciUi prepared from the growth on slant glycerin-agar tubes. The growth is washed off with physio- logic salt solution and heated to 60° for four hours to kill the bac- teria. The suspension of organisms is then placed in flasks and shaken in a shaking apparatus for four days. It is then centrif uged, the clear liquid removed, and 10 per cent, of a 5 per cent, solution of phenol added. This antigen may be preserved without material deterioration for several months if kept in a cool, dark place. It is necessary to titrate the rabbit serum and likewise the antigen in order to determine the amounts most suitable for carry- ing out the test. For each set of determinations of diagnosis fresh guinea-pig serum must be used. Blood-serum from the animal that is suspected of having glanders must be inactivated by heat- ing to 58° for thirty minutes. The materials necessary for the test are — 1. Washed sheep corpuscles, 5 per cent, suspension (antigen 1). 2. Inactivated serum from rabbit immunized against 1 (am- boceptor 1). 1 Arch. f. Wiss. u. prakt. Tierheilkunde, Band 35, pp. 44-83, 1909. 2 Bull. 136, Bureau Animal Industry, U. S. Dept. of Agriculture. 314 VETERINARY BACTERIOLOGY 3. Fresh guinea-pig serum (complement). 4. Extract of glanders bacilli (antigen 2). 5. Inactivated serum from suspected animal (amboceptor 2). The test is carried out in test-tubes. In tubes 1 and 2 there is placed 0.1 c.c. of the serum (No. 5, above), and in tubes 3 and 4, 0.2 c.c. of the same. One c.c. of the established dilution of glanders baciUi (No. 4, above) is then added to tubes 1 and 3. To each tube' is then added 1 c.c. of the dilution of fresh guinea-pig serum that has been estabUshed by preliminary test. Each tube is now made up to 3 c.c. with physiologic salt solution. They are then placed in the thermostat at 37° for an hour. They are then removed and to each tube is added 1 c.c. of the previously standardized rabbit serum (No. 2, above) and 1 c.c. of the sheep corpuscles (No. 1, above). The tubes are shaken and incubated for ten hours. A positive diagnosis is indicated by lack of hemolysis in tubes 1 and 3 and complete hemolysis in tubes 2 and 4. Checks must be made to determine the hemolytic activity of each of the above con- stituents independently. This method is essentially a laboratory one and quite imprac- ticable for field work. There seems to be no reason why blood samples or, better, serum samples from suspected cases should not be sent to properly equipped laboratories for diagnosis and report. The method apparently is capable of giving good results, and seems to be more accurate than the mallein test. Conglutination Test for Glanders. — The conglutination reaction has been advocated by Anderson and others as a satisfactory method of diagnosis in glanders. The following materials are necessary in the test: 1. Suspension of killed glanders bacilli 0.05 c.c. 2. Inactivated serum of horse suspected, in various amounts, from 0.001-0.1 c.c. 3. Normal horse serum (containing complement) 0.1 c.c. 4. Inactivated cattle serum (containing conglutinin) . . 0.04 c.c. 6. Suspension of goat corpuscles in physiologic salt solution 0.5 c.c. Each volume is made up to 2.5 c.c. with physiologic salt salution. Numbers 1, 2, and 3 are first mixed and allowed to stand one-half hour at 37°, then numbers 4 and 5 are added. The reaction is GLANDERS GROUP. THE GENUS PFEIPFERELLA 315 determined after standing one hour at 37°. The test has been claimed to be quite specific, but it has not been extensively tried out. (See page 192.) Mallein Test for Glanders. — Mallein is a suspension of killed glanders bacilli, together with the products of their autolytic disin- tegration. What the active principles in bringing about the char- acteristic reaction in a glandered horse may be is not known. Probably they are the soluble bacterial proteins, possibly true endotoxins. The various laboratories use different methods of preparing mallein. The most important of these are worthy of note. The mallein of Roux is prepared by the Pasteur Institute as follows: The virulence of the glanders bacillus used is increased by passage through rabbits, and is such that mice and rabbits are killed in less than thirty hours by intravenous injections. Flasks containing 250 c.c. of glycerin bouillon are inoculated and incu- bated a month at 35°. The cultures are killed by exposure to a temperature of 100° for thirty minutes in an autoclave, then evaporated to one-tenth the volume, and filtered through filter- paper ("papier Chardin"). The final product is a dark-brown, syrupy liquid, containing 50 per cent, glycerin. For use this is mixed with nine times its volume of 0.5 per cent, carbolic acid. The diagnostic dose is 2.5 c.c. of this dilution. The mallein of Vladimiroff, used in the Russian Empire, is pre- pared by inoculating a considerable number of flasks, each con- taining 600 to 800 c.c. of beef-broth, with a vigorous culture of B. mallei, and incubating for eight months at 37°. The flasks are shaken from time to time to cause the shiny, gray-white pellicle which forms to sink to the bottom. The culture is then examined for purity, sterilized in the autoclave at 110°, and filtered. This is concentrated and again diluted until the diagnostic dose for the horse is 1 c.c. The mallem or morvin of Babes is prepared by inoculating potato paste with the glanders bacillus, and incubating six weeks. It then is heated at 68° for three and one-half hours, emulsified with water, filtered through a Witt filter, and precipitated with alcohol. This precipitate is washed in alcohol, then in ether, and dried. The diagnostic dose is 0.02 to 0.03 gm. It is prepared for injection by dissolving in a mixture of glycerin and water. 316 VETERINARY BACTERIOLOGY The malleinum siccum, or dried mallein, of Foth is prepared by growing the glanders bacillus in 4.5 per cent, glycerin broth. The cultures used are rendered virulent by passage through cats^ guinea-pigs, and field-mice. The material is incubated at 37.7° for three weeks. It is concentrated, and the organism killed by evaporation at a constant temperature of 76° to 80° to one-tenth of its former volume. This is filtered and poured into absolute alcohol, in which a precipitate immediately forms. This pre- cipitate is washed in alcohol and dried in a desiccator. The final product is a white powder which readily dissolves in water. The diagnostic dose for the horse is 0.045 to 0.05 gm. The mallein prepared in the laboratories of the Bureau of Animal Industry consists of glycerinated broth in which the P/. mallei has grown four to five months, has been heated, concen- trated, and filtered. It is diluted by the addition of one-half its volume of glycerin and one and one-half times its volume of 1 per cent, phenol. The diagnostic dose is 1 c.c. No practicable method of standardizing mallein has been worked out other than trial upon a considerable number of healthy and infected animals. The variations in the methods of produc- tion of mallein given above are due to a desire to secure a very uniform product. There are three methods of applying mallein in use at the present time. They are the subcutaneous, ophthalmic, arid cu- taneous. Subcutaneous Mallein Test. — Following subcutaneous injection of a suitable dose of ma;llein, animals suffering from glanders show a rise of temperature beginning usually between the fourth and eighth hour. From this time on to the fourteenth hour, in excep- tional cases later, the rise continues. 'After reaching the maximum the decrease is gradual until normal. Organic symptoms, such as increased respiration and heart action, muscular tremor, depression, dulness, and loss of appetite, may frequently be observed and are of value in diagnosis. The appearance of an inflamed, edematous swelling at the point of inoculation is also usually noted. In gen- eral, an increase of 2° C. rising above 40° C. is considered a positive reaction. Students are referred to texts on practice for complete discussion of this phase of the test. GLANDERS GROUP. THE GENUS PFBIFFERELLA 317 Ophthalmic Mallein Test. — This consists of introducing into the conjunctival sac of one of the eyes several drops of a spe- cially prepared mallein. This may be introduced either with the a.id of a camel's-hair brush or a medicine-dropper. The reaction begins from the fourth to the sixth hour after application and may continue for twenty-four to thirty-six hours. It consists of a conjunctivitis marked by swelling of the lids, redness of the membrane, and a purulent secretion which collects at the inner canthus and on the hair below the eye. The test is recognized by the Bureau of Animal Industry as a reliable one. Cutaneous Mallein Tests. — These may be carried out either by scarification of the skin, followed by application of concentrated mallein, or by intradermal injection of concentrated mallein. Either case is marked by edematous swellings whose extent depends upon the quantity of mallein injected. The limited number of tests of this character furnish a very unsatisfactory basis for con- clusions regarding the reliability and accuracy. Transmission. — The disease is transmitted from one animal to another through infected food, mangers, drinking troughs, etc.; rarely through wounds or skin abrasions. Veterinarians and horse- men sometimes become infected through the skin, rarely by inhala- tion. Bacilli of Selter, Babes, and Kutscher.— Organisms morpho- logically similar to the preceding have been isolated from pus by Selter and by Babes, and from the nostrils of a healthy horse by Kutscher. They may be differentiated readily by their lack of pathogenesis, and would rarely, if ever, lead to mistakes in diag- nosis. CHAPTER XXVII INTESTINAL OR COLON-TYPHOID GROUP. THE GENUS BACTERIUM The organisms belonging to this group may be characterized as plump, Gram-negative rods, frequently though not always motile; they produce no spores, do not in general liquefy gelatin, and in most cases ferment certain sugars, with acid and sometimes gas production. There is no distinct tendency to the formation of polar granules. The group contains many undoubted species that may be easily differentiated, but there are many intergrading types and forms showing similar morphologic and cultural charac- ters, but differing considerably in kind and in degree of virulence. These latter make a systematic presentation of the group as a whole difficult. The group name is given because of the prominence of these organisms in the intestinal flora in disease and health in both man and animals. They are, therefore, abundant in sewage and water contaminated thereby, and in soil, particularly that which has received additions of barnyard manure. They are less common in virgin soil and uncontaminated water. The members of this -group are divided, for convenience in study, into three subgroups. This arrangement seems to represent evident relationships. The fermentative powers of the organisms are used as a basis upon which to make the groupings. The first of these is known as the colon bacillus subgroup, the second as the intermediate, hog-cholera or enteritidis subgroup, and the third as the typhoid-dysentery subgroup. The principal points of differ- ence among these subgroups may be summarized in the following table, giving the fermentation reactions in dextrose and lactose broth: Subgroup I. Subgroup II. Subgroup III. Colon subgroup. Intermediate sub- Typhoid-dysentery sub- group. group. Acid. Gas. Acid. Gas. Acid. Gas. Dextrose + + Dextrose -|- -f- Dextrose ± — Lactose + + Lactose — — Lactose — 318 INTESTINAL OR COLON-TYPHOID GROUP 319 The differences may be summarized as fellows: The organisms of Subgroup I ferment both dextrose and lactose, with formation of both acid and gas; those of Subgroup II form acid and gas from dextrose, but not from lactose; and those of Subgroup III may or may not form acid from dextrose, but never from lactose, and gas from neither of the sugars. The fermentations of other carbohydrates and related com- pounds are used to differentiate species and varieties from each other. A few can be satisfactorily differentiated only by the ag- glutination reaction. Those organisms which produce gas in the fermentation tube grow in both the open and closed arm, as do those which produce acid, and those which do not ferment the sugar are usually confined to the open arm. The composition of the gas, that is, the relative proportion of CO2 and H2, is also of diag- nostic value. Subgroup L Colon Subgroup The organisms of the colon subgroup are characterized by their abihty to ferment both dextrose and lactose with formation of both acid and gas. A satisfactory classification of the species has not been worked out as yet. Even the approximate number of species to be recognized is uncertain. Many characters to be noted in separation of species have been proposed; those which have proved helpful are the following: 1. MotiUty and capsule production. 2. Indol production. 3. Carbon dioxid-hydrogen gas ratio. 4. Acid production, particularly hydrogen ion concentration in various sugars. 5. Gas production from different sugars. 6. Production of acetyl-methyl-carbinol (Voges-Proskauer reac- tion). 7. Utilization of uric acid as a source of nitrogen. The organisms of this subgroup are of interest because several are normal inhabitants of the intestinal tract, and their presence in water is, therefore, an evidence of fecal contamination and because some of the species are pathogenic. It is as yet im- possible to prepare a logical classification which includes all forms. The subgroup, for convenience, will be considered under three sections: 320 VETERINARY BACTERIOLOGY 1. Organisms primarily of sanitary significance. 2. Organisms associated with infectious diseases of animals, particularly calf diarrhea. 3. Organisms showing capsule formation, and sometimes patho- genic. These sections overlap; the same species in some cases must be considered under all three sections. The name Bacterium coli or Bacillus coli communis is often used to include all of the species here discussed, very often, in- deed, to include all forms of sanitary significance. Organisms of the Colon Subgroup Primarily of Sanitary Significance The organisms of this section are frequently used as an index of water pollution. They are all inhabitants of the intestinal tracts of man and most animals; certain species are not uncom- mon in soil, on grains, etc. The following key to some of the more important species is of assistance in their separation. It should be emphasized that the names Bacterium coli and B. coli communis are often used to in- clude all of these species. Key to the More Important Species of the Colon Group I. Not producing acetyl methyl carbinol (Voges-Proskauer reaction nega- tive); acid to methyl red, and carbon dioxide and hydrogen produced in approximately equal volumes from glucose. Cannot utilize uric acid as a source of nitrogen. A. Sucrose not attacked. 1. Salicin fermented with acid and gas. Coli Section. 1. Bad. coli. 2. Salicin not attacked. 2. Bad. acidi-ladici. B. Sucrose fermented with acid and gas. 1. Motile. 3. Bad. communior. 2. Non-motile. (a) Salicin fermented with acid and gas. 4. Bad. neapolitanum. (6) Salicin not fermented. 5. Bad. coscoroba II. Producing acetyl methyl carbinol (Voges-Proskauer reaction positive) • alkaline to methyl red, and forms two or more times as much carbon dioxide as hydrogen from glucose. Capable of utilizing uric acid as a source of nitrogen. INTESTINAL OR COLON-TYPHOID GROUP 321 A. Glycerol and starch fermented with acid and gas formation; non-motile, gelatin not liquefied. B. Glycerol and starch not fermented; motile, gelatin liquefied. Bacterium coli AfiEOGENEs Section. 6. Bact. Aerogenes. 7. Bact. cloacoe. Sjmonyms. — Bacillus coli communis; B. pyogenes fmtidus; Bacterium coli commune; colon bacillus. Emmerich, in 1885, isolated an organism which was later named Bacillus neapolitanus, from the feces of patients suffering from Asiatic cholera. Escherich, in 1886, isolated a related organism which he termed Bacterium coli commune, from normal feces. Since that time organisms of this group have been found to be constantly present in the intestines of man, most animals, and even some birds. The question of its occurrence in nature independent of fecal contamination is a moot one, but there is increasing ten- dency to consider that reports of its common occurrence in water and soils are due to confusion with Bacterium aero- genes. That it may maintain a saprophytic existence outside the body for some time seems to be well established, but the evidence that it does not usually long so maintain itself is increasing. Distribution.— Not all investigators are in agreement as to the proportion of lactose fermenting bacteria from the intestines which belong to the species Bacterium coli. MacConkey con- cluded about 38 per cent, from human feces and 25 per cent, from bovine feces were of this type.' Clemesha gives 17 and 9 per cent, respectively. Levine found 5 per cent, of Bad. coli in horse feces, 22 per cent, from the pig, 25 per cent, from the 21 Fig. 119. — Bacterium coli, stained preparation from a twenty-four-hour agar slant (X 650) (Heim). 322 VETERINARY BACTERIOLOGY COW, 6 per cent, from sheep, and 15 per cent, from man. The work of Ferriera, Horta, and Paredes indicated that this type is widely distributed in the feces of animals. Morphology and Staining. — The Bacterium colt is a rod, vary- ing from 0.4 to 0.7 by 2 to 4 m, sometimes shorter and almost coc- cus-like with rounded ends, usually single, but occasionally in short chains. It does not produce spores or capsules. It is rather sluggishly motile, at least in young cultures, usually with 2 to 8 flagella, rarely more. It stains readily with the ordinary anUin dyes, sometimes showing some vacuolization and polar granules. It is Gram-negative. Isolation and Culture. — Bacterium coli may be readily isolated from feces or sewage by plating the material in various dilutions in litmus-lactose agar and incubating at blood-heat. The colonies of Bact. coli appear surrounded by a zone of red, due to the formation of acids from the lactose. The colonies must be fished and tested in various carbohydrate solutions, so that those of true Bact. coli may be differentiated from other members of the subgroup. Upon gelatin plates the colonies are moist, grayish white, opaque, becoming darker and more coarsely granular. Gelatin is not liquefied. Stab cultures in gelatin show a filiform growth along the line of puncture and a spreading growth at the surface. The agar cultures resemble those on gelatin. Bouillon is quickly clouded, sometimes with formation of a pellicle. On potato a moist, spreading growth occurs, and the potato is dark- ened. Milk is coagulated by the formation of acids; the curd shrinks, but is not digested. The various media used in the recognition of Bact. coli in water analysis will be discussed under that heading. Physiology. — Bacterium coli is aerobic and facultative anaero- bic. Its optimum growth temperature is 37°, but growth is luxuriant at room-temperature and even below. The thermal death-point is 60° for fifteen minutes. The following carbohy- drates are fermented: levulose, galactose, dextrose, maltose, lactose, salicin, glycerin, dulcite, but not saccharose. The gas CO 1 formula from dextrose is approximately -=-? = -. When Ha 1 grown in dextrose (0.5 per cent.) phosphate medium the solution INTESTINAL OR COLON-TYPHOID GROUP 323 negative. becomes permanently acid to methyl-red. Indol is produced in Dunham's solution. Peptonizing and proteolytic enzymes have not been demonstrated. The Voges-Proskauer test is It cannot utilize nitrogen from uric acid. Pathogenesis.— The following quotation from Jordan epito- mized our present estimate of the pathogenicity of Bacterium coli: " The common occurrence of agonal or postmortem invasion of the body by the colon bacillus tends to diminish the value of the supposed evidence derived from finding the colon bacillus in the in- ternal organs after death, and there can be no doubt that the role in human pathology as- signed to the colon bac- illus by some investiga- tors, notably certain French bacteriologists, has been greatly exag- gerated. Failure to dis- tinguish between the true colon group and the group of meat-poisoning bacilli is doubtless re- sponsible for some of the statements attributing pronounced pathogenic properties to Bad. coli. The frequent ascription of various inflammatory processes, particularly those occurring in the appendix and peritoneum, to the unaided activities of Bact. coli appears to be without sufficient justification. Many of the cases reported rest on the evidence derived from simple aerobic cultivation, and the possible concurrence of anaerobic or other organisms not growing by ordinary methods has not been excluded." The preceding was written with pathogenesis for the human body in mind, but the conclusions are even more true with reference to its pathogenesis for ani- mals. Many diseases in domestic animals have been ascribed to infection with varieties of Bact. coli from insufficient evi- Fig. 120. — Bacterium coli showing the flagella (Migula). 324 VETERINARY BACTERIOLOGY dence. It has been shown that even in the normal body colon bacilli .sometimes escape from the intestines, and are to be found in the mesenteric lymph-nodes, and occasionally in some of the other internal organs. Experimental Evidence of Pathogenesis. — The intraperitoneal injection of broth cultures of Bad. coli into the guinea-pig results in the death of the animal, usually within three days. Animal experimentation has demonstrated quite conclusively that there are considerable differences in virulence of colon bacilli isolated from different animals or from the same animal at different times. Character of Lesions and Disease Produced. — Bacterium coli has been isolated from suppurations in pure culture. In man it is known occasionally to invade the gall-bladder, and is a common cause of cholecystitis. It may serve as a nucleus for gall-stones, and is probably instrumental in their formation by the precipita- tion of cholesterin. Inflammation of the ureters and of the urin- ary bladder is commonly caused in many by organisms that can- not be differentiated from typical Bact. coli. It has been reported as the cause of calf diarrhea or white scours, and from malignant catarrh in cattle. These will be discussed later. Usually the colon bacillus does not give rise to putrefactive prod- ucts, and must be regarded as a harmless or possibly even useful commensal. Immunity. — A considerable degree of immunity to Bacterium coli may be induced by injections of cultures, killed or living. Agglutinins are present in normal serum, but may be greatly increased by systematic immunization. Specific precipitins for the bacterial proteins are present in the immune serum. Opso- nins are present in normal serum. The body has naturally a high degree of immunity against the Bact. coli. This may be accounted for by the presence of Bact. coli in the intestines and the continued opportunity for infection. The toxic properties of the organism are probably due to the presence of endotoxins. ' Bacteriologic Diagnosis.— The isolation of the characteristic colonies upon litmus-lactose agar from Endo- or from Conradi- Drigalski plates are all simple and quick methods of determining the presence of Bact. coli. The recognition and isolation of this organism from water will be discussed at greater length under the heading of Water Analysis. INTESTINAL OR COLON-TYPHOID GROUP 325 BacteHum acidi lactici This organism was first isolated from milk by Hueppe. It has been frequently confused because of similarity of name, original source of isolation, and the production of lactic acid with Lactobacillus lactis acidi, a totally unrelated form. In all essen- tial characteristics this organism resembles the BacL coli except that it does not ferment dulcite with the production of gas. Organisms of this type are not uncommon in feces. Mac- Conkey found 34 per cent, in human feces and 16 per cent, in bo- vine, Clemesha found 53 and 40 per cent, respectively. Levine found 15 per cent, in horse dung, 45 per cent: from pig, 25 per cent, from cow, and 5 per cent, from sheep. It is possible that some of Jensen's calf-scours organisms be- long to this type. BacterSum commanior Synonyms. — B. coli communior; Bacillus communior. This organism was named Bacillus coli communior by Durham because he believed it to be even more common in the intestines than his B. coli communis. It differs from Bad. coli in its ability to ferment saccharose. MacCOnkey found about 15 per cent, of this type in human, and 48 per cent, in bovine feces. Clemesha gives 7 and 10 per cent, respectively. Levine found about 60 per cent, in the horse, 20 per cent, in the pig, 25 per cent, in the cow, and 45 per cent, in sheep. Its occurrence in water has the same significance as Bad. coli. It is probable that the most com- mon of the calf -scours bacterial types of Jensen belong to tjiis species. Bacteritim coscoroba This organism has been several times reported. It resembles Bacterium acidi lactid, but ferments saccharose. Bacteriam aerogenes S3monyms. — Bacterium lactis aerogenes; Bacillus pyogenes; Bacillus lactis aerogenes. The capsulated B. pneumoniae of Friedlander, B. rhinoscleromatis, and B. ozcenoe quite probably belong here or are closely related. 326 VETERINARY BACTERIOLOGY Escherich, in 1885, described this organism which he isolated from sour milk. The typical Bad. aerogenes seems to be quite widely distributed in nature; it has been found commonly present oil grains by several investigators, and has been found to be the predominant organism of this group in soils by Johnson and Levine. It is not so typically fecal in habitat as many other members of the group. Morphology and Staining. — Bacterium aerogenes differs mor- phologically from the Bad. coli principally by the lack of flagella and in the ability to produce capsules when grown in mUk. Isolation and Culture. — This organism may be isolated in the same manner as Baderium coli upon litmus-lactose agar. The colonies upon agar and gelatin are larger, thicker, and more slimy than those of the colon bacillus. Milk is curdled more rapidly. In gelatin stabs the growth along the streaks is filiform; that at the surface is thick, convex, and circumscribed. The whole stab culture is frequently described as "nail-like." Physiology. — In most respects this organism resembles Bac- ierium coli. It ferments dextrose, lactose, and saccharose and glycerol with production of both acid and gas. Many strains also ferment starch. Indol is not commonly produced in Dun- ham's solution. Dextrose (0.5 per cent.) phosphate medium becomes alkaline to methyl-red. The Voges-Proskauer reaction is positive, showing the development of acetyl-methyl-carbinol. This reaction is one of the most distinctive in the differenti- ation of this species from other coli-like organisms. Gelatin is not liquefied. Grows in media containing uric acid as sole source of nitrogen. Pathogenesis. — This organism is not known to possess patho- genic powers. It is of interest principally because of its close relationship to Baderium coli and association with it. In making water examinations in the past no distinction has ordinarily been made between Bad. coli and Bad. aerogenes, inasmuch as they re- semble each other so closely and it has been assumed that they come from the same sources. It seems probable as the result of more recent work that the presence of this organism has much less sanitary significance than the preceding species. INTESTINAL OR COLON-TYPHOID GROUP 327 Bacterium cloacas Synonjmi. — Bacillus cloacce. This organism was isolated from polluted water by Jordan, and has been repeatedly found by subsequent workers. It differs from Bad. aerogenes in the power to liquefy gelatin, is motile and glycerol is not fermented. It is not common in feces. Its sani- tary significance is uncertain. Organisms Assooated with Calf Scours or Calf Diarrhea Organisms belonging to the first subgroup of the intestinal group are generally regarded as the causal organisms of calf scours or calf diarrhea. The principal work has been that of Jensen in Denmark. Morphology and Staining. — These appear to be typical of the colon subgroup. Culture and Physiology. — ^Jensen has noted considerable varia- tion in the colony types of different races of the calf-scours organ- isms. The most marked differences, however, were found in sugar fermentations. He recognized two main groups (termed A and B) with three races in the first and four in the second. All the races agree in producing both acid and gas in the following sugars: Glucose, arabinose, xylose, rhamnose, maltose, and lactose. The races are differentiated on the basis of variations in sugar reactions in sucrose, sorbose, dulcitol, and adonite. The following key shows the groupings: A. Producing acid and gas from sucrose. Adonite negative. Group A. 1. Producing acid and gas from sorbose. (o) Producing acid and gas from dulcite. .' Eace A. I (6) Not producing acid and gas from dulcite. Race A. II 2. Not producing acid and gas from sorbose Race A. Ill B. Not producing acid and gas from sucrose. Adonite negative or positive. Group B. 1. Producing acid and gas from sorbose. (a) Producing acid and gas from dulcite Race B. I (6) Not producing acid and gas from dulcite Race B. II 2. Not producing acid and gas from sorbose. (o) Producing acid and gas from adonite. Dulcite negative. Race B. Ill (6) Not producing acid and gas from adonite. (1) Dulcite positive Race B. IV (2) Dulcite negative Race B. V 328 VETERINARY BACTERIOLOGY The one found most commonly (A. I) seems to be closely re- lated in fermentation reactions to Bacterium communior. Much work still remains to be done on the relationships of these organ- isms. It is possible that some of these types may constitute dis- tinct species. Jensen also records cases in which' apparently typical Bact. aerogenes was the causal organism. Jensen has noted that.it is usually the same race that causes trouble year after year in different animals on the same farm. Pathogenesis.— The characteristic symptoms do not seem to diifer as a result of infection with different races of coli. The disease usually appears soon after birth, frequently within forty-eight hours. The calf shows fever, loss of appetite, and coHc. The feces are liquid, usually yellowish, foul, and often mixed with gas. They may also show blood resulting from intes- tinal hemorrhage. Death may result promptly or there may be a more or less persistent diarrhea. Autopsy shows the mucous membranes of the abomasum to be congested or hemorrhagic, as also the intestinal mucosa. The mesenteric glands are often swol- len and red. The spleen is usually more or less swollen. Immunity. — Effective means of prophylaxis and cure by means of sera is complicated by the fact that so many races of colon bacilli may be responsible. Antisera may be prepared either against single strains or the sera may be polyvalent. The polyvalent serum is prepared by repeated intravenous injection of the horse with numerous races of colon bacilli isolated from cases of calf diarrhea. By combining the serum from differ- ent horses Jensen has prepared a polyvalent serum against as many as forty strains. No accurate method of determining the potency of the serum has been evolved. Bacterins, both univalent and polyvalent, have been prepared and used for prevention of the disease in districts or on farms where the annual losses are high. Capsulated Pathogenic Organisms Several species of capsulated bacteria belonging to this group have been described. The most important of these are Bacterium pneumonice, Bact. mucosum capsulatum, Bact. rhinoscleromatis, and Bact. ozcBncE. One only, the Bact. pneumonice, will be discussed. INTESTINAL OR COLON-TYPHOID GROUP 329 Bacterium pneumonis Synon3nais. — Bacillus pneumoniae; Bacillus capsulatus muco-- sus; pneumobacillus; pneumococcus of Friedlander. Friedlander, in 1883, discovered this organism in the sputum from a case of croupous pneumonia, and it was believed by him to be the cause of the disease. He failed to discover the real cause of pneumonia because the pneumococcus of Frankel does not grow readily upon plate cultures prepared by the method used. It has since been found repeatedly in normal saliva, and is still believed to be an occasional cause of pneumonia. It sometimes is found in the feces and in sewage. Morphology and Staining. — The organism as it occurs in the sputum is sometimes so short as to resemble a coccus. Usually it is single, rarely in chains. It is surrounded by a capsule in sputum and in milk. It is non-motile. It resembles the pre- ceding organisms closely in all other respects. Isolation and Culture. — Isolation is accomplished by plat- ing upon gelatin. Growth upon most media resembles that of the Bacterium aerogenes. Milk is not coagulated, although litmus milk is reddened. Physiology. — The organ- ism shows markedly less fermentative power than Bac- terium aerogenes, but other- wise closely resembles it. Dextrose, lactose, and saccha- rose are all fermented, but usually not vigorously. Growth occurs best at blood- heat, but the organism de- velops well at room-temperature about 56°. Indol is produced. Pathogenesis.— Bacfenitm pneumonias has a very low virulence —only exceptionally will it infect any of the lower animals. It has been isolated in pure culture from the vegetations upon the heart valve in endocarditis, from otitis media, and occasionally it Fig. 121. — Bacterium pneumonice, show- ing capsules (GUnther). The thermal death-point is 330 VETERINARY BACTERIOLOGY is believed to cause catarrhal or lobular pneumonia. It is noted here simply because of its obvious relationship to the preceding organisms of the group. Subgroup n. Intermediate, Hog-cholera, Enteritidis, or Gartner Subgroup The classification and relationships of the organisms belonging to this subgroup are much confused at present. Whether or not the various forms described are all distinct species is doubtful. The most important will be discussed under the names by which they are commonly known, but this uncertainty as to correct grouping must constantly be borne in mind. The different races or species belonging to this subgroup have been differentiated in many ways, but no classification has been worked. out that is entirely satisfactory. A grouping proposed by Harding and Ostenberg has been found useful by some workers. It is based upon the production of red in fuchsin sulphate agar when various carbohydrates are present. Jordan differentiates the more important types as follows; I. Xylose not fermented. Bact. paratyphosum (Paratyphoid A). II. Xyljose fermented with acid and gas. A. Arabinose and dulcitol rapidly fermented. Bact. enteritidis and Bad. schotmvUeri (Paratyphoid B). B. Arabinose and dulcitol not attacked or fermented very slowly. Bact. cholerw suis. Bacterium enteritidis Ssmonjrm. — Bacillus of Gartner. Bacillus enteritidis. Diseases Produced. — Meat-poisoning and enteritis in man and in cattle. Gartner, in 1888, studied an outbreak of meat-poisoning in a village in Saxony, and isolated from a fatal case and from the un- cooked flesh of a cow the organism now known as Bacterium enteritidis. This organism since that time has been found in similar outbreaks of meat-poisoning and associated with certain infections in cattle. The organism has been found in meat, or so- called "ptomain "-poisoning in the United States. INTESTINAL OR COLON-TYPHOID GROUP 331 Schmitz found as a result of routine investigations carried on four years on flesh of slaughtered animals that this organism had never been found in mature beef or pork, but that it had been found many times in veal. He regards the organism as having an etiologic relationship to calf diarrhea. Several investigators have isolated organisms of this type from rats. Morphology and Staining. — Bacterium enteritidis resembles B. coli morphologically. The organism is short and thick, some- times with a thin capsule, mo- tile by means of numerous or few flagella. It does not pro- duce spores. It stains well or irregularly with the anilin dyes and is Gram-negative. Isolation and Culture. — The organism has been isolated di- rectly from the blood-stream and the spleen, and from the intestinal contents by plate cultures. Malachite green „.,„„„,. , .,,. ^xr„ii„ -^uiuuiv, a Fig. 122. — Bactenum enteritidis (KoUe •dulcite broth has been used sue- ^n^ Wassermann). cessfully by several investiga- tors as an enrichment medium, as this seems to discourage the growth of colon bacilli. The cultural characters as reported vary with different authors, probably because different strains were studied. Colonies upon gelatin and agar resemble those of Bacterium coli. Bouillon is clouded, a delicate pellicle may form, and in a few days a whitish sediment collects. A yel- lowish, glistening layer forms on potato, frequently turning brownish with age. Growth in milk seems to vary with the organism studied. Some have been described as coagulating milk, but the typical form does not. Physiology. — Bacterium enteritidis is aerobic and facultative anaerobic. Its optimum growth temperature is between 30° and 40°, but it grows well at room-temperature also. The thermal death-point, as determined by Mohler and Buckley, is 58°,for twelve minutes. Dextrose and dulcite are fermented with produc- tion of acid and gas. Lactose, saccharose, salicin, and glycerm 332 VETERINARY BACTERIOLOGY are not fermented by the typical strains, although some strains have been reported by a few investigators to ferment lactose. Indol is not produced. Gelatin is not liquefied. Milk may show initial acidity, but this is followed by a permanent alkalinity. Pathogenesis.— Experimental Evidence.— Bacterium enteritidis is pathogenic f Or' the guinea-pig, mouse, and pigeon, but not for the cat. The guinea-pig may be fatally infected by intraperi- toneal of subcutaneous injections and by ingestion. The same is true of the rabbit. Mohler and Buckley produced a fatal infec- tion in young hoUse-rats, while other authors report the rat as immune. The same is true of the dog, though this animal is relatively resistant. Chickens are immune. Sheep are readily infected. The hog succumbs to intravenous injection, as well as through feeding. Type of Disease and Lesions Produced. — In man the infection is marked by a severe enteritis and enlargement of the lymph- foUicles and Peyer's patches, and by small hemorrhages. The mortality in infections is low — probably less than 5 per cent. The infection in cattle, as observed by Mohler and Buckley, was characterized by degeneration of the heart muscle (frequently fatty) and hemorrhages therein; in the liver parenchjmaatous degeneration was accompanied by localized hemorrhagic extrav- asations; the spleen was enlarged and hemorrhagic and the lesions of acute enteritis, with necrosis of the epithelium, were evident. Immunity. — The so-called " toxin " of the Bacterium enteritidis is probably an unusually soluble and potent endotoxin. It differs from true toxins in being exceptionally heat resistant. Meat which has been quite thoroughly cooked is sometimes found capa- ble of giving rise to toxic symptoms when ingested. The bac- teria-free filtrates from bouillon cultures and cultures in which the organisms have been killed by heat will kill guinea-pigs when injected in suitable quantities. It is probable that this endo- toxin is responsible for the quick development of symptoms in those who are poisoned by eating infected food. Specific agglu- tinins are developed in the blood of infected individuals, as are also coagglutinins for other members of the intestinal group. Some differences in agglutinability of the different strains isolated INTESTINAL OR COLON-TYPHOID GROUP 333 have been noted. It has been proposed that meat may be tested for the presence of Bacterium enteritidis by expressing the juice and determining its agglutinating power. This has not been proved practicable. It is probable that the baciUi abeady present in the meat would in some cases fix all the agglutinins present if stored for any length of time. Practicable methods of prophylactic pr curative immunization have not been demonstrated. Bacteriologic Diagnosis. — The organism may be demonstrated by inoculation of infected flesh into suitable enrichment medium, such as malachite green dulcite broth, followed by plating. In man the disease may be diagnosed by the agglutination test, al- though with difficulty, for, as has been noted above, the various strains agglutinate differently, and blood from a typhoid or a paratyphoid patient may show a marked capacity to agglutinate Bacterium enteritidis. Transmission and Prophylaxis.— Probably a large proportion of the cases of so-called ptomain-poisoning is due to infection with Bacferiuvi enteritidis and to the toxic products of metabolism of this organism. Such infection undoubtedly occurs frequently enough to justify rigorous measures for its prevention. Meat or milk from animals showing severe gastro-intestinal disturbances should never be used, as the infection in the human has in several well-authenticated instances been traced directly to such prac- tices. Probably most cases of meat-poisoning originate from use of flesh of diseased animals, but the possibility of infection with the organism after the animal has been slaughtered should not be ignored. Experiments have shown that when fresh meat is inoculated upon the surface with a culture of Bact. enteritidis the organism rapidly penetrates the tissues, even at low tempera- tures. Such infection might easily occiu- in unsanitary abattoirs through flies and careless handling. It should be noted that certain types of the paratyphoid bacil- lus are very similar to this form, if not identical with it, and doubtless are the cause of meat-poisoning as well. Bacteriam cholerze suis Synonsrms. — Bacillus suipestifer; B. cholera suis. B. saX- m,oni; Salmonella. 334 VETERINARY BACTERIOLOGY Salmon and Smith, in 1885, described this organism as the cause of the disease called by them swine-plague. In the fol- lowing year Smith discovered another organism associated with a different disease of swine. This led to a revision of terminology which has since come into common use, and the organism first described is now known as the hog-cholera bacillus. Smith re- covered this organism from the spleens of about 500 hogs affected with hog-cholera. It was quite generally accepted as the cause of the disease until deSchweinitz and Dorset reported an outbreak of hog-cholera in which the Bad. cholerae suis was not the primary infecting agent. This was shown by the trans- mission of the disease by blood filtered through fine-grained porcelain bougies, a procedure which removed the bacillus completely, as shown by the fact that the filtrate was quite incapable of infecting culture-media. By the subsequent work of Dor- set, Bolton, and Mc- Bryde it was shown quite conclusively that the Bad. choleroe suis is not the cause of hog-cholera in the Mississippi Valley, and but a secondary invader at most. Other investigators in the United States and in Europe have confirmed these results. Hog- cholera and its virus will be considered, therefore, under the heading of Diseases Caused by Filterable Virus. The Bad. cholercB suis, however, doubtless plays some part in the disease as a secondary invader, and is, therefore, worthy of consideration. Uhlenhuth and his collaborators in a study of 600 swine found organisms indistinguishable from this form in the intestines of 8.4 per cent. Fig. 123. — Bacterium cholerae suis, organisms from a young culture (deSchweinitz, Bureau Animal Industry). INTESTINAL OR COLON-TYPHOID GROUP 335 Morphology and Staining.— This organ-ism differs morpho- logically in no essential character from Bacterium enteritidis. Isolation and Culture. — Bacterium cholerce mis may frequently be isolated at once in pure culture from the organs of infected animals, particularly from the spleen. It has likewise been iso- lated by plating the intestinal contents of normal and infected animals. The organism grows upon agar and gelatin, forming a grayish, glistening, non-viscid growth, which is not particu- larly characteristic. Upon Conradi-Drigalski agar blue colonies are formed. On LofHer's malachite green clouded but translu- Fig. 124. — Bacterium choleras suis, showing flagella (deSchweinitz, Bureau Animal Industry). cent colonies appear, in whose vicinity the agar is made yellowish. Endo plates give colorless colonies. No growth occurs upon potatoes having a decided acid reaction, but upon those which are neutral or alkaline a thin, glistening, usually yellowish layer is formed. Bouillon is uniformly clouded; a slight pellicle may- appear in time. A grayish, friable sediment is formed. Milk shows a slight initial acidity, but soon becomes alkaline, and gradually becomes opalescent, and finally translucent. In Pe- truschky's lackmus molke the medium is reddened, then it turns blue and becomes intensely alkaline. It will be noted that there 336 VETERINARY BACTERIOLOGY are no marked cultural differences between Bad. enteritidis and Bad. cholerce suis. Physiology. — Bacterium cholera suis is aerobic and facultative anaerobic. The optimum growth temperature is about 37°; it grows also, but more slowly, at room-temperature, and it will de- velop also at 45° C. The thermal death-point is 58°, with ten minutes' exposure. The organism will remain viable for several days when dried. Gas and acid are produced in dextrose broth, also from arabinose, xylose, fructose, galactose, mannose,_ maltose, dulcite, mannite, and sorbite. Lactose and saccharose are not fermented, and no growth occurs in the closed arm of the fermenta- tion tube containing these sugars. Glycogen, inulin, adonite, starch, erythrite, and raflBnose are not attacked. Indol is not or- dinarily produced. Gelatin is not hquefied. Hydrogen sulphid is formed from peptone. Pathogenesis. — It should again be emphasized that the Bac- terium cholerce suis is not the primary cause of hog-cholera, but that it is a secondary invader of importance, and may be occasion- ally the primary cause of disease in hogs, but this disease probably would possess, according to Dorset, Bolton, and McBryde, a low , degree of contagiousness. Experimental Evidence of Pathogenesis. — Some differences in virulence have been observed in cultures obtained from different sources. Rabbits succumb to septicemia in five to eight days when inoculated with yV cc. of a virulent bouillon culture. Guinea-pigs are more refractory, and die after seven to twelve days. Subcu- taneous and intravenous injections and feeding experiments rarely produce death in the hog. The animal may sometimes show fever and depression, particularly after intravenous inoculation, but the infection is rarely fatal unless 1 or 2 c.c. or more of culture are used. However, some highly virulent cultures have been described. Feeding with large quantities of culture or long-continued feeding sometimes proves fatal. Undoubtedly this organism sometimes causes spontaneous in- fection of swine independently of the filterable virus. Schem and Stange have suggested for this type of disease the name parapest. Character of Disease and Lesions Produced. — An examination of a rabbit killed by injections of Bact. cholerce suis shows lesions INTESTINAL OR COLON-TYPHOID GROUP 337 differing in no material respect from those discussed under Bact. enteritidis. To just what extent the characteristic lesions in hog- cholera, particularly in the chronic types, are due to infection by this organism is uncertain. It is probable that in many cases, at least, it is responsible for the development of intestinal ulcers. Inasmuch as it is sometimes found in the blood of animals infected by hog-cholera, it is probable that death may be due directly to their activity. Immunity. — The topic of immunity against hog-cholera will be considered under that heading. The Bacterium cholerce suis pro- duces no true toxin, but there is considerable formation of endo- toxin. Agglutinins are present normally in the blood of the hog, and immune agglutinins may be produced by the systematic im- munization of animals by killed cultures. Group agglutination with other members of this subgroup has been demonstrated. It has also been shown that immunization of the hog against true hog-cholera results in a considerable increase of agglutinins for BacL cholerce suis in the blood. Opsonins for Bact. cholerce suis have been shown to be present in normal serum. Since the dis- covery of the filterable virus efforts at immunization by the use of vaccines and sera prepared by the use of Bact. cholerce suis have been practically abandoned. Bacterium typhi suis S3mon3rms. — Bacillus of Glasser, probably also Bacillus suipes- tifer of Voldagsen. These are perhaps best regarded as strains of the Bacterium cholerce suis isolated by Glasser and by Voldagsen, and showing some special characteristics. Generally these organisms give a somewhat more delicate growth on culture-media than the typical Bact. cholerce suis. In milk frequently there is no change, or at least the change to alkahnity is relatively slow. Uhlenhuth and Haendel have concluded that the differences from Bact. cholerce suis are insignificant. The men who first worked with these organisms concluded that they have unusually high pathogenicity for swine particularly for animals not more than four months old. They ascribe to them a causal relationship to pig typhoid. 22 338 VETERINABY BACTERIOLOGY Bacteriam paratyphi Synonyms. — Paratyphoid or paracolon bacillus. Bacillus paratyphosus. Disease. Produced^ — Paratyphoid in man, possibly similar infections in animals. Gwyn, in 1898, isolated from a clinical typhoid case an organ- ism which belonged to the intermediate subgroup of intestinal organisms rather than to the typhoid-dysentery subgroup. In 1900 Schottmuller isolated two types of organisms from some- what similar cases. He termed these paratyphoid A. and paraty- phoid B. Similar organisms have been isolated repeatedly since that time — in some instances from an atypical typhoid case, in others from cases that had all the chnical symptoms of typhoid, bat that did not give the agglutination reaction. Morphology and Staining. — This organism corresponds closely in morphologic and staining characters to the Bacterium enteritidis and the Bad. cholera suis. Isolation and Culture. — The organism has been isolated in pure culture directly from the blood, and by plate cultures from the internal organs in disease, and particularly from the intesti- nal contents of man and of the lower animals. In general cul- tural characters the organism resembles the Bacterium enteritidis. It has been found in practice that two varieties may be differ- entiated, termed A and B respectively. Type A does not produce a terminal alkalinity in milk and dissolve the casein, and in that respect differs from Bact. enteritidis; it produces an almost invisi- ble growth on potato, lactose whey is made permanently acid. Type B grows more luxuriantly, even on potato, milk is made strongly alkaline and cleared, lactose whey is also made alkaline. Physiology. — Not markedly different from Bacterium cholerce suis, but according to Harding and Ostenberg producing red colora- tion on fuchsin sulphite agar containing either arabinose or xylose. Pathogenesis. — Paratyphoid fever in man has been attended by a low mortality; in consequence few autopsies have been re- ported. In both animals and man infection partakes more of the nature of an acute enteritis than does typhoid fever; the lympha- tics are not generally invaded as in typhoid, and the Peyer's patches are not swollen and ulcerated. INTESTINAL OR COLON-TYPHOID GROUP 339 Immunity.— Probably an endotoxin, less soluble, but in some respects similar to that of Bacterium enteritidis, is produced by these organisms. Agglutinins are produced in infected individ- uals. . The agglutination reactions of types A and B differ markedly. It was this difference which first suggested the exist- Fig. 125. — Bacterium typhi murium (Migula). ence of the two types. No method of practical immunization against the disease is known. Bacteriologic Diagnosis. — The differentiation of paratyphoid may be made clinically by the specific agglutination tests. The absence of a test in a case of clinical typhoid calls for a repetition with the two types of paratyphoid bacilU. Transmission. — It is probable that certain gastro-intestinal infections in cattle may be caused by organisms of this type, and that meat and milk may become contaminated from these sources. Milk and meat are probably the most common sources of infection, although water has been clearly shown, in some instances, to be the source of epidemics. Bacterium typhi muriam Synon3rm. — Bacillus typhi murium. Loffler, in 1889, described an organism as the cause of an epidemic among the mice kept for experimental purposes. Danysz, in 1900, described a similar, probably identical, organism, and 340 VETERINARY BACTERIOLOGY recommended its use in the destruction of rats. These forms have all the morphologic and cultural characteristics of the enteritidis group, but show some differences in pathogenicity and in formation of specific agglutinins. Cultures of these organisms have been widely exploited as specific for mice and rats, producing a rapidly fatal disease, but as harmless to the higher animals. Reports as to their efficacy when fed to the vermin are conflicting; the results seem in some cases to have been favorable. It evidently is true that the virulence of the organism is subject to considerable varia- tions, for the careful work of Rosenau showed the culture which he possessed to be worthless in the extermination of rats. On the other hand, there is some evidence that the organism is not so free from harmful effect on the human as has been supposed. Fatal infections in man have been reported from Japan. Bacterium pullorum Synonjons. — Bacillus pullorum. Disease Produced. — White diarrhea of chicks. Rettger and Harvey have described the Bacterium pullorum as the specific cause of a white diarrhea in young chicks. The adult fowl is also affected but the mortality is lower. As a source of infection of the young chick through the egg or by con- tact the diseased adult bird is of great importance. The type of organism found in the young stock differs slightly from that found in adults. Type A, found in young stock, shows a greater tendency to form acid in several sugars than does type B (adult stock), and is aerogenic (Hadley). Morphology and Staining. — Bacterium pullorum is a rod, 0.3 to 0.5 by 1 to 2.5 /u, with rounded ends. It occurs singly or very rarely in chains. It is non-motile, does not produce capsules or spores. It stains readily and uniformly with ordinary aqueous anilin dyes and is Gram-negative. Isolation and Culture. — The organism may be isolated from the infected chicks by opening the body with aseptic precautions, and making streaks upon the surface of agar slants with blood or the pulp of the spleen or liver. Upon the agar slant the colonies are discrete, and at first resemble the pin-point, translucent colonies of the Streptococcus. They enlarge later. Upon gela- INTESTINAL OR COLON-TYPHOID GROUP 341 tin the colonies resemble those of the typhoid bacillus. Little growth occurs upon potato. Milk is a suitable medium; but there is little change, no coagulation, and no proteolysis. Physiology. — The organism is aerobic and facultative anaerobic. The optimum growth temperature is about 37°. Dextrose and mannite are fermented, with the production of both acid and gas (Rettger) . Maltose, lactose, and saccharose are not fermented. Indol is not produced. Pathogenesis. — Experimental Evidence. — Rettger has isolated the specific organism in several outbreaks of the disease from the internal organs, particularly the livers of chicks that had died of the disease or were showing symptoms. He also isolated it from abnormal egg-yolks in the ovaries of hens, from freshly laid eggs, and from the yolk-sacs of fully developed chicks within the shell. He also succeeded in infecting chicks by feeding, but the disease was. not always contracted. Subcutaneous injections always proved fatal. The disease has appeared as a highly fatal one in several flocks of adult chickens, the organism recovered being agglutinable by immune serum of the type found in young stock but differing from the latter mainly in its reactions in sugar as above mentioned. Characteristics of Disease and Lesions. — The most noticeable antemortem characteristics are emaciation and wasting of the chick, and the white diarrhea. The lesions are confined princi- pally to the intestines, and are particularly evident in the cecum. The Hver is sometimes congested in areas. In the adult the ova present are irregular in appearance, instead of showing the normal spherical shape, while the color is greenish yellow to green. Immunity. — Practicable methods of immunization have not been evolved. Bacteriologic Diagnosis. — The organism may be isolated in pure culture from the internal organs, particularly the liver. To determine whether organisms resembUng this organism are common Rettger, Hadley and others have found diagnosis by means of the agglutination reaction accurate and reUable and by the appHcation of this test have succeeded in eliminating the infected birds of a flock, thereby eradicating the disease. Transmission and Prophylaxis.— Rettger claims that th^ 342 VETERINARY BACTERIOLOGY disease is sometimes present before hatching, the organism being present in the ovaries and oviduct, and that contamination of the food Hkewise results in infection. Subgroup III. Typhoid -dysentery Subgroup The three important organisms belonging to this subgroup — Bacterium typhi, Bad. dysenteric, and Bad. fcecalis alkaligenes — are not ordinarily pathogenic for the lower animals. They are, however, pathogenic for man, and since many of our diagnostic methods for other diseases have been discovered through their study, they are discussed briefly. Bacterium typhosum Synonyms. — Bacillus typhi; B. typhi abdominalis; Eberth or Eberth-Gaffky bacillus. Bacillus typhosus. Disease Produced. — Typhoid fever in man. Eberth, in 1880, discovered the Bacterium typhosum in the spleen and other internal organs of the body of persons who had died of typhoid fever. GafEky, in 1884, cultivated the organism. It is now generally conceded to be the cause of typhoid fever, although the experimental animals cannot ordinarily be infected. Distribution. — Typhoid fever is widely distributed throughout temperate and tropical countries. It is constantly present, fre- quently in epidemic form, in the United States. Morphology. — Bacterium typhosum is a short, plump rod, usu- ally varying between 0.5 and 0.8 /Ji in diameter, and 1 to 3 m in length. It is motile by means of numerous fiagella. It does not produce capsules or spores. It stains readily with aqueous anilin dyes. Granular staining is sometimes observed, although the cells usually stain uniformly. It is Gram-negative. Isolation and Culture. — The desirability of isolating Bacterium typhosum from contaminated water has led to the development of many media in an effort to accomplish this. A quantitative estimation of the typhoid bacillus from such sources does not seem to be practicable, but the qualitative determination of presence may be carried out. The methods used are to inhibit the growth of the purely saprophytic organisms present by the use of antiseptic substances, such as malachite green, caffein INTESTINAL OR COLON-TYPHOID GROUP 343 and crystal violet, and to incubate such media at blood-heat, These media do not inhibit, in general, the growth of either Bact. typhosum or Bact. coli, and dependence is placed upon differences in colony characters and media reactions to separate them upon subsequent plating. The colonies upon gelatin are somewhat smaller and more delicate than those of the Bacterium coli. This organism was^ originally described as producing a thin, " invisible growth" upon potato. This is true upon potato with an acid reac- tion, but upon alkahne or neutral potato the growth is relatively abundant. Physiology. — Bacterium typhosum develops best at a temperature of 37°, but will grow at room-tempera- ture. It is an aerobe and facultative anaerobe. No indol is produced. Acid, but no gas, is formed from dextrose. Neither acid nor gas is produced from lactose or saccharose. Some strains ferment xylose. There may be slight initial acidity in milk, but there is never coagulation of the casein. Proteolytic enzymes are not devel- oped in cultures. Pathogenesis. — Experimental Evidence. — The lesions typical of typhoid in man are not produced either by injection or feeding experiments upon most laboratory animals. The symptoms after intraperitoneal injection of a guinea-pig do not differ greatly from those produced by the Bacterium coli. Feeding experiments with anthrapoid apes within recent years have shown the possibility of producing the typical lesions of the disease in these, animals. In- fection with typhoid bacilli in laboratory workers has several times occurred following the accidental ingestion of pure cultures of the organism. Fig. 126. — Bacterium typhosum, clump in a section of a spleen (Frankel and Pfeiffer). 344 VETERINARY BACTERIOLOGY Character of Lesions and Disease Produced.— Clmical diagnosis of typhoid is frequently difficult, as the characteristics of the disease are often not well marked. The organism invades the intestinal lymph-system, and particularly the Peyer's patches. The latter become ulcerated, and perforation of the intestinal wall is not an uncommon result. The spleen is swollen. The bacteria are usu- ally found in the blood, though not commonly in large numbers, but are abundant in the spleen. Cystitis, cholecystitis, and bone metastases are not uncommon sequelae to the infection. Immunity. — No true toxin has been demonstrated for Bac- terium typhosum, but an endotoxin is present. Agglutinins and Fig. 127. — Bacterium typhosum, Fig. 128. — Bacterium typhosum, col- showing flagella (GUnther). ony on agar (Giinther). precipitins specific for the organism are likewise produced. Bac- teriolysins may be demonstrated in the blood of animals that have been artificially immunized by injections of the typhoid bacillus. There is developed in the body of an individual that has recovered from typhoid a certain degree of immunity, but this disappears, so that it is not unusual for a person to have seVeral attacks of the disease. This immunity is probably both bacteriolytic and opsonic in nature. The use of antisera in passive immunization against typhoid and in curing the disease has not proved successful. The injection of such sera has not been shown to have either an immunizing or a curative effect in man. Active immunization by the injection of dead or living bacteria or their products has, on the contrary, been INTESTINAL OR COLON-TYPHOID GROUP 345 quite successful. Usually the organisms are scraped from the sur- face of agar cultures, suspended in physiologic salt solution, and killed by heat, or a broth culture may be used. Bacteriologic Diagnosis.— The Widal or agglutination test is commonly used in the diagnosis of typhoid. A dilution of 1 : 40 and higher is generally made to minimize the effect of the normal agglutinins which may be present in the blood. Both microscopic and macroscopic tests are used; the former is the more dehcate, but the latter somewhat more reliable. The agglutinins often appear early in the course of the disease — usually by the fifth day or rarely later. Blood or serum for making the test may be either liquid or dried. It is received in the latter condition by many of the state and municipal bacteriological laboratories. The bacteria may be cultivated directly from the blood of a patient. Frequently the organisms can be found in the blood somewhat before the serum exhibits a marked agglutinating power. Isolation of the organisms directly from the feces is sometimes resorted to in an effort to determine the occurrence of the so-called "bacillus carriers." Transmission. — Typhoid fever is contracted from contami- nated drinking-water, milk and other foods, and by contact, the frequency being in about the order named. FUes probably are commonly instrumental in carrying the organism from dejecta of tjrphoid-fever patients to food materials. The term bacillus carrier, or germ-carrier, is used to designate an individual who still harbors a pathogenic organism in the body after convalescence. Such germ-carriers are particularly dangerous, as they may give rise to an epidemic that is almost impossible to trace to its source. The danger of milk infection is probably the greatest from indi- viduals that are employed in dairies. Several epidemics have been traced to this origin. Bacterium dysenteriae Synonyms. — Bacillus of Shiga; Bacillus of Flexner. Bacillus dysenterice. Disease Produced. — Bacillary dysentery in man. Shiga, in 1898, discovered in the feces of patients suffering from dysentery a bacillus which he beUeved to be the specific cause of the disease. Previous to this it had been shown that 346 VETERINARY BACTERIOLOGY amebffi may cause dysentery, and it was when examining stools for these protozoa that Shiga discovered this organism. In 1900 Flexner published the results of work in Manila and described another type of organism. Since that time many epidemics have been studied, and it is generaUy believed that the type described by Shiga is the more common, but that the bacillus of Flexner occurs in a certain proportion of the outbreaks, more particularly in the tropical countries. Other authors. Hiss in particular, have differentiated even more groups Morphology . — Bacterium dysenteries and Bact. typhosum are practically indistinguishable under the microscope in stained mounts. The Bact. dysenterioe, however, is non-motile. Spores and capsules are not produced. It stains uniformly and is Gram-negative. Isolation and Culture. — The organism may be isolated di- rectly from the dejecta by plat- ing. The cultural characters in general closely resemble those Milk is rendered permanently alkaline. Fig. 129. — Bacterium dysenterice (KoUe and Wassermann). of Bacterium' typhi. however. Physiology. — The physiologic characters of Bacterium dysen- terioe closely resemble those of Bact. typhosum. The ability to produce acid in solutions of various carbohydrates and of the related alcohols is used as a means of differentiation of the varieties. Otho listed some fifteen different types by this means. A more conservative and valuable classification is that of Hiss, as modified by Shiga. Recent work seems to indicate that the dysentery group may be divided into a number of species which may be differ- entiated by the following key. Key to the Dysentery and Allied Bacilli I. Mannitol not fermented. ' A. Indol not formed: rhamnose not fermented. 1. Bact. shigce. B. Indol positive; rhamnose fermented with acid. 2. Bact. ambiguum. INTESTINAL OR COLON-TYPHOID GROUP 347 II. Mannitol fermented forming acid. A. Xylose not fermented. 1. Lactose not fermented; milk slightly acid then alkaline; indol usually positive; rhamnose usually not fermented. 3. Bact. flexneri. 2. Lactose fermented with acid; milk rendered strongly acid and usually coagulated; indol not formed, rhamnose fermented ^itli acid. 4. Bad. sonnei. B. Xylose fermented with acid. 1. Lactose and sucrose fermented forming acid; milk acid and clotted; dulcitol not attacked. 5. Bact. dispar 2. Lactose and sucrose not fermented; milk alkaline; dulcitol attacked with acid production. 6. Bact. alkalescens. Pathogenesis.— Experimental Evidence.— The belief that the varieties of Bacterium dysenterice are the important etiologic factors in the disease is based upon the following facts : 1. This organism in some one of its varieties has been shown to be present with great constancy in the patient's excreta. 2. Injections of the organisms and their products kill labora- tory animals, particularly rabbits, although typical dysentery is not readily induced by feeding experiments. 3. The blood-serum of a patient will, in general, agglutinate in high dilution the strain isolated from the feces. 4. Antiserum has been successfully used in the prevention and cure of the disease. Character of Disease and Lesions Produced. — The intestine, particularly the colon, is inflamed and is sometimes ulcerated, and may even show diphtheritic necrosis. With the exception of this there is little that is characteristic of the disease. Unlike the typhoid bacillus, it does not commonly invade the blood or the internal organs, with the exception of the mesenteric glands. The disease is rather a toxemia than a bacteremia. Immunity. — ^A soluble toxin has been demonstrated for the Shiga type, but repeated efforts have failed to show that any such is produced by the Flexner type. This poison was at first believed to be an unusually potent endotoxin. Conradi first demonstrated the toxin by growing the organism upon agar, then suspending it in physiologic salt solution, and allowing the bacteria to undergo 348 VETERINARY BACTERIOLOGY autolysis. Later, Rosenthal and others showed that toxin will be produced in considerable quantities in an alkaline bouillon, but not in one that is neutral or acid. This bouillon is either filtered through porcelain filters, or 0.5 per cent, phenol is added" and al- lowed to stand, and then filtered through paper until clear. The toxin may be precipitated by ammonium sulphate, and after dial- ysis and drying of such, 1 to 2 gm. may be a lethal dose for a kilo of rabbit. It is weakened by prolonged heating at 70°, and de- stroyed at 80 to 100°. The rabbit is very susceptible to the in- jection of the toxin, while the guinea-pig is relatively resistant. The effect upon the rabbit may be characterized as a hemorrhagic necrotic enteritis. Shiga first used antisera in the treatment of dysentery. He regarded its curative properties as wholly bactericidal. Todd, Koram, Doerr, and others have, by systemat'ic immunization of a horse, secured a serum that neutralizes the toxin actively. This has been used with very favorable results in the treatment of dys- entery caused by the Shiga bacillus. Bacteriologic Diagnosis.— The disease may be recognized by the Widal or agglutination test, and the several types of organisms differentiated in the same manner. The organism may likewise be isolated directly from the stools by plating. Transmission. — Dysentery is spread in much the same manner as typhoid, and the same preventive measures must be used. CHAPTER XXVIII ABORTION— MALTA FEVER GROUP. THE GENUS BRUCELLA Brucella {Bacterium) abortus of Bang and Bacterium melitensis are placed in this group. It should be noted that other organi sms besides the Bact. abortus one here discussed are doubtless occa- sionally responsible for abortion in cattle and other animals. Brucella (Bacterium) abortus Synonyms. — Abortion bacillus of Bang; Bacillus abortus; Bacillus abonionis. Disease Produced. — Infectious abortion disease in the cow. Bang, in 1897, described a bacillus as the probable cause of infectious abortion in the cow. The specific organism was iso- lated with difliculty. It has been isolated since that time in* Europe several times, and more recently in the United States. Several investigators in the United States have described mem- bers of the colon group and of other groups as present in infectious abortion, but it is doubtful whether in many cases appropriate cultural methods have been utilized for the isolation of this organism. Distribution. — Contagious abortion has been reported from many localities on the continent of Europe and in Great Britain. It is known to occur in many sections of the United States. Morphology and Staining. — Bacterium abortus is very small, 1 to 2 M long, 0.3 to 0.8 m broad, and, according to Nowak, resem- bles the bacillus of chicken-cholera. Nowak, on the basis of its morphology, groups it with the Pasteurellas or hemorrhagic sep- ticemia bacilli. It is polymorphic in culture-media. Involution forms occur as branched and clubbed types. It is non-motile, and neither capsules nor spores have been demonstrated. It stains readily by the aqueous anilin dyes, frequently showing polar granules. Carbol thionin, methylene-blue, or borax methylene-blue and Giemsa stain furnish beautiful pictures of the organism. It is Gram-negative. 349 350 VETEKINAEY BACTERIOLOGY Isolation and Culture. — The isolation and cultivation of Bao- terium abortus are attended with peculiar difficulties. The or- ganism may be frequently obtained'at once in pure culture from the heart blood or the intestines of an aborted fetus. Bang used a medium consisting of nutrient agar (f per cent.) and hquid gelatin (5 per cent.) to which was added an equal quantity of sterile liquid blood-serum. These tubes were inoculated with suspected mate- rial, mixed well, and kept at blood-heat. In the course of three days numerous small, punctiform to pin-form colonies developed in a definite stratum a few millimeters below the surface. As will be noted under the discussion of physiology, the organism has an unusual relationship to oxygen, and the amount of oxygen needed Fig. 131. — Bacterium abor- tus. Culture in serum agar sliowing the definite stratum in which the colonies Fig. 130.^-Bacterium abortus (Nowak). develop (Nowak). for its development is to be found at the depth at which the colonies form. These colonies are compact, rounded, or somewhat irregular, sometimes showing a dense nucleus surrounded by a lighter zone. When the organism is present in impure culture, as in the vagina of the cow, other methods are necessary for its isola- tion. Nowak has described a procedure which has proved satis- factory in the hands of several investigators. Probably simpler methods will be devised in time, but this appears to be the best thus far developed. The material is smeared over the surface of suc- cessive tubes of serum agar or over the surface of this medium in Petri dishes. These are allowed to stand for several days, and the colonies which develop are marked, as they are not Bad. abortus. ABORTION — MALTA FEVER GROUP. THE GENUS BRUCELLA 351 The plates are then placed in a desiccator whose cubic contents of air has been determined, and plates of agar thickly seeded with B. subtilis are introduced, so that about 16 sq. cm. of surface is present per every 240 c.c. of space. It has been found experi- mentally that this wiU diminish the oxygen pressure to a point where the Bact. abortus will develop. After three days the plates are examined and search made for the Bact. abortus in the spaces between other colonies on the original plates. This same device, of reducing oxygen pressure by means of cultures of B. svhtilis, may be used in the study of growth-characters on other media. Schroeder and Cotton, Evans and Steck have isolated the Bact. abortus directly from milk in ordinary petri dishes, even in gelatin medium. Traum has isolated it directly from the amniotic fluid and from the liver of aborted pigs in open unsealed slants of the beef-liver-peptic-digest agar of Meyer. After primary culture the organism soon adapts itself to the ordinary oxygen tension and grows readily. The organism will grow in agar without addition of serum, particularly at the surface, but the addition of serum is decidedly beneficial. The colonies develop at the surface in about three days at 37° as small, usually discrete, transparent dots. In shake cultures in serum agar they appear in about four days in a well-defined stratum about 10 to 20 mm. below the surface. The individual colonies may reach a diameter of 1 mm. when well separated from each other. Growth in gelatin at room-temperature is slow. Bouil- lon cultures show development some millimeters below the surface, the medium above this remaining clear. Milk is not coagulated. Little or no growth takes place on potato. Physiology. — The optimum growth temperature is 37°, although the organism is found to multiply slowly at room-tem- perature. The relationship to oxygen, which has already been discussed, classifies the Bacterium abortus as a micro-aerophile rather than a strict anaerobe. Strangely enough, Bang reports that the organism will also grow in an atmosphere of pure oxygen; two oxygen optima are, therefore, evident. More recent work has shown that the organism may be cultivated under strict aerobic conditions after a few transfers on laboratory media. It is relatively resistant to desiccation. It will remain alive for 352 VETERINARY BACTERIOLOGY months in a retained mummified fetus, and for a year or more in a culture-medium. Acids and gas are not produced from carbohydrates. Pathogenesis. — Experimental Evidence. — Bang demonstrated the etiologic relationship of the organism to the disease by intra- venous injection, and by injection into the vagina and uterus of pregnant cows, also by feeding experiments. Sheep were also infected both by injection and feeding. Preisz did not succeed in transiriitting the disease by vaginal injections into the cow, guinea-pig, or rabbit. More recent work has shown that inocu- Fig. 132. — Bacterium abortus, colonies on serum agar (Nowak). lation of these animals results in a chronic infection characterized by specific changes in the liver and spleen, and by arthritis. Pregnant animals will abort. Nowak succeeded in producing typical abortion of dead feti in guinea-pigs and rabbits by intra- peritoneal and intravenous injections. He did not succeed by intravaginal injections. Character of Disease and Lesions. — There are few symptoms either preceding or following the expulsion of the fetus. It is characterized as a rule by an interference with the develop- ment of the fetus frequently resulting in its premature expulsion either dead or alive. There is also a frequent manifest inflamma- tion of the fetal membranes and of the maternal cotyledons to- gether with retention of the placental membranes. ABORTION MALTA FEVER GROUP. THE GENUS BRUCELLA 353 Im m unity. — It is known that cows that have aborted one or more times may become immunized against the disease. Vac- cination and serum treatments have been attempted, but their worth has not been thoroughly proved. Bang showed that it is possible to immunize sheep and goats by subcutaneous injections of increasing quantities of living cultures to such an extent that they can withstand severe infection through food. Killed cul- tures did not prove to be effective. His trials with heifers did not prove to be as successful, although vaccination in badly infected herds has been claimed to be successful in some cases. Passive immunization by the use of the serum from cows which have aborted two or more times and then calved normally has been attempted, but results are not conclusive. Bacteriologic Diagnosis. — This may be made certain by the isolation of the specific organism in culture as outlined above. A tentative diagnosis may be made by preparing stained mounts, and demonstrating the presence of a short, Gram-negative bacil- lus in the uterine exudate and in the blood and tissues of the fetus. The agglutination and complement fixation reactions have been extensively used in diagnosing this disease. Both have proved reliable. The German Imperial Board of Health recognizes as positive those tests in which agglutination occurs in serum dilu- tions of 1 : 100 or over; as negative those under 1 : 100. Pohle has shown that a precipitation test may be used. _ The presence of the organism in milk can best be recognized by injection of guinea-pigs, which will show the characteristic lesions. Transmission. — It has been proved that the Bad. abortus can enter the body through the digestive tract. It cannot be dis- puted that it may also enter through other channels, neither has it been definitely proved. Brucella (Bactericm) melitensis Synonym. — Micrococcus melitensis . Disease Produced. — Malta fever in the goat and in man; Mediterranean fever. Bruce, in 1887, discovered a microorganism in the spleen of men dead from Malta or Mediterranean fever. Since that time 23 354 VETERINARY BACTERIOLOGY it has been studied carefully by numerous investigators, and its relationship to the disease is well established. Distribution. — The disease is known to occur in all countries bordering on the Mediterranean, in southern Asia, South Africa, the Philippines, some of the islands of the West Indies, and in Mexico and Texas. Morphology and Staining. — Until the work of Meyer and Shaw in 1920, this organism was considered a coccus. These investigators found by a study of 21 cultures identified as Micro- coccus melitensis by various laboratories in the United States, England, Algiers and Italy that, confirming the repeated observations at previous times, the organism in smears made from young cultures and even from tissues is a typical short rod. Stained with gentian violet, the organism appears as short, oval rods frequently tapered at both ends. Identical smears stained with dilute carbol fuchsin show the organism more coccoid in morphology. Frequently in the water of condensation or liquid medium, short chains of 4-10 single, elongated coccoid elements can be recognized. The organism stains well with ordinary anilin dyes, but is Gram-negative. Isolation and Culture. — It may be isolated from the spleen during life or after death in pure culture, or by plating. On agar slants a fine granular film appears in from 24-36 hours. After 3 days a slight brownish tinge changes the moist well de- fined growth. Growth continues on peptic digest agar for weeks, even at room temperature, until a stringy, greasy, rather thick layer covers the surface. In gelatin the organism produces dark brownish granular colonies after from 10-30 days, but never liquefies the medium. In infusion broth, there is a slight initial turbidity, followed by clearing and the deposit of a stringy, tenacious sediment, occasionally a ring or pellicle formation. Milk is not changed. Meyer reports beef liver peptic digest agar admirably suited for isolation and growth of the organism. Physiology. — The Bacterium melitensis does not produce acid from any of the carbohydrates. Desiccation does not destroy it quickly, for the organism has been found to remain alive and virulent when dried for a considerable time. Pasteurization is fatal. ABORTION MALTA FEVER GROUP. THE GENUS BRUCELLA 355 Pathogenesis. — The disease is a true bacteremia. Inoculation of pure cultures reproduces the disease in the goat, cow, and monkey. Accidental laboratory infections have proved its power of producing disease in man. Infection probably usually arises through ingestion. The disease is characterized by its low mor- tality, its long duration in man, and the accompanying articular rheumatism. It is a dis- ease primarily of the goat, though it is possible that cattle may sometimes har- bor and transmit it. Horses have also been experiment- ally infected. In guinea- pigs and rabbits the disease runs a chronic course. Iminumty. — No toxins have been demonstrated. Agglutinins are present in the blood in infected indi- viduals, so that agglutina- tion may sometimes be secured with high dilutions — occasionally as high as 1 : 6000. This test is one of the readiest methods of diagnosing the disease. Bacteriologic Diagnosis. — Diagnosis may be made by isolation of the organism by a puncturp of the spleen, or by demonstrating the presence of specific agglutinins in the serum in dilutions of 1 : 50 or greater. Mohler and Eichhorn found that complement fixation is a satisfactory method of diagnosis in the disease, and more reliable than agglutination. Transmission and Prophylaxis. — The organism is excreted in the feces, urine, and milk from infected animals. Most cases in the human are acquired by drinking the milk of infected animals. The disease infects, in either the acute or chronic form, so large a proportion of the animals in some countries that the use of unheated milk is always attended with danger. Fig. 133.- -Bacterium melitensis ( X 1200) (Jordan). CHAPTER XXIX DOG DISTEMPER GROUP. THE GENUS BACTERIUM Bacterium bronchisepticum (bronchicanis) Diseases Produced. — Canine distemper and similar diseases in other animals. Ferry, in 1910, published a report of the bacterial findings in canine distemper in which he described an organism associated with the disease and which he believed to be the primary cause. He gave to this organism the name Bacillus bronchicanis, subse- quently changing this to B. bronchisepticus. His findings have been corroborated by M'Gowan, Torrey, and Rabe. Ferry' (1916) calls attention to the close morphologic, cultural and serological characters of the Bad. bronchisepticum and Bad. pertussis. Smith^ finds the Bad. bronchisepticum and Pseudo- monas pyocyanea closely related except in pigment production and action in gelatin. Morphology and Staining. — The organism is a short slender rod, 2.3 to 5 M long, usually found singly, but often in pairs. In primary inoculations in broth the organism may be larger and oval in shape. Here, too, filaments may be observed. No spores are produced. It stains best with methylene-blue, with a char- acteristic bipolar staining and is Gram-negative. Active and progressive motihty is noted. Isolation and Culture. — The organism is frequently in mixed infections with staphylococci, so that isolation is difficult. It is found in the nasal secretions, blood, and internal organs. Ferry reports positive findings in blood-cultures in 28.5 per cent, of cases. Others have found it less frequently. One to 5 c.c. of blood are planted in a flask containing 50 c.c. of broth. After twenty-four hours incubation plates or slant agar cultures in suc- cessive tubes are made. On agar the colonies appear after twenty-four hours as very fine, dewdrop-Hke, later enlarging and becoming opaque. iJourn. Bact., 1918, 3, p. 193. 2Joum. Med. Res., 1913, 29, p. 299. 356 DOG DISTEMPER GROUP. THE GENUS BACTERIUM 357 Bouillon is clouded with a heavy stringy growth in old cultures. Physiology.- — No gas is produced in any carbohydrates. Lit- mus milk remains strongly alkaline. No indol is produced. Pathogenesis. — Dogs, guinea-pigs, rabbits, and monkeys are susceptible to infection. Inoculations into the air-passages of young dogs produce the disease in its characteristic form. Older animals are resistant. Character of Disease: — The disease is marked by catarrh of the air-passages, intestinal catarrh, discharge from the eyes, pustules on the skin in a small percentage of cases, and nervous symptoms. Immunity. — Recovery from the disease confers an immunity. Ferry reports satisfactory results from bacterin and serum treat- ment and recommends the former as a prophylactic and the latter as a curative measure. Best results seem to be derived from a mixed bacterin of Bacterium bronchisepticum and staphylococci. Bacteriologic Diagnosis. — The serum of animals suffering from natural infection agglutinates in dilutions of from 1 : 40 to 1 : 800. Little use of the agglutination test for the diagnosis of the disease has been made. The finding of the characteristic organism in smears from the nasal mucous membranes is of value in diagnosis. Transmission. — The disease is readily transmitted from animal to animal, or through exposure in kennels where diseased animals have been kept. CHAPTER XXX FLUORESCENT GROUP. THE GENUS PSEUDOMONAS The group of fluorescent bacilli includes those forms which produce a water-soluble, diffusible bluish green or greenish pig- ment. AU of the species are Gram-negative, do not produce spores, are aerobic and facultative, and usually motile by means of one or more polar flagella. Several species belonging to this group have been described, the more important being Pseudomonas fluorescens, with several varieties, and Ps. pyocyanea. The latter is the only form which has distinct pathogenic properties. Pseudomonas pyocyanea S3monyms. — Bacillus pyocyaneus; Ps. aeruginosa; bacillus of green, or blue-green pus in man and animals. Gessard, in 1882, described the Pseudomonas pyocyanea from blue-green pus. Since that time it has been isolated and studied by numerous investigators both in Europe and America. Distribution. — This organism has been isolated from the feces of man and animals, from sewage and surface waters, from the soil, and from air and dust. It is usually saprophytic or com- mensal in its growth, and is only rarely pathogenic. Morphology and Staining. — Pseudomonas pyocyanea is a slender rod with rounded ends, about 0.6 by 2.6 m or smaller, usually single, rarely in chains of 2 to 6 individuals. It is motile by means of a single terminal flagellum. No spores or capsules are produced. It stains readily by the ordinary anilin dyes and is Gfam-negative. Isolation and Culture. — This organism is readily isolated by plating out pus which contains it, as the colonies are quite charac- teristic on account of the green pigment which they diffuse. It grows readily on all the common laboratory media. Upon agar and gelatin plates the thin, poorly defined colonies are charac- terized by the fluorescent pigment surrounding them. Upon 358 FLUORESCENT GROUP. THE GENUS PSEUDOMONAS 359 slant agar the color diffuses until the whole of the medium is a light green, then a darker blue green, and finally a brown or brown red. Gelatin is rapidly liquefied. Bouillon is clouded, a pellicle forms, and the fluorescent pigment diffuses from the top downward. Potatoes support a slimy growth and turn green, then brown. Milk is coagulated by a rennet-like enzyme and the curd pep- tonized. Physiology. — This organism is preferably an aerobe, and grows most luxuriantly in the presence of oxygen, but growth will continue under a,naerobic conditions. Proteolytic ferments which will digest gelatin, fibrin, and casein are produced. Two pigments are usually formed — one green and fluorescent (fluorescin), soluble ■ Pi..,r^ ^5a>>..„^_^ I r-^v.'?'-^^'-'" V'' :''\'.'-: ' .'- ' • •>' '. • * ■ . • ' j 1 \ '' '''' . / Fig. 134. — Pseudomonas pyocyanea (KoUe and Wassermann). in chloroform, the other (pyocyanin) bluish and insoluble i n chl.9 - roform . Pigment is not produced in the absence of oxygen. In old cultures the pigments become yellow or brown. Autolytic disintegration of the cells takes place in old cultures. Mucin, a compound made up of a protein and a carbohydrate, has been found present in cultures. To this may be ascribed its slimy con- sistency on agar or even in bouillon. The organism is resistant to desiccation. Pathogenesis. — Experimental Evidence. — Injection of cultures of Ps. ■pyocyanea subcutane'ously into the guinea-pig or rabbit causes rapidly spreading edema, suppuration, septicemia, and death within a day or two. Not all cultures are equally pathogenic. 360 VETEBINARY BACTERIOLOGY Character of Infection Produced. — The Ps. pyocyanea is usually a secondary invader, although in man it has been found causing primary infections. It has not yet been proved ever to cause suppuration alone in any of the domestic animals, but is not un- common in pus, to which it gives a green or blue-green color. In man it has been found in purulent otitis media, meningitis, broncho- pneumonia, infantile diarrhea, and generalized infections. Koske and others have ascribed to this organism an etiologic relationship to chronic rhinitis ("buUnose") in swine. Immunity. — A true toxin is produced by virulent cultures. Wassermann found 0.2 to 0.5 c.c. of this fatal for the guinea-pig. Poels has described a "pyocyaneus bacillosis" in calves in a single herd. The disease was characterized as an acute diarrhea. An antitoxin has been prepared for this pyocyaneus toxin. An endo- toxin has also been demonstrated. A leukocytic poison, leukoddin, and a hemolytic toxin, hemotoxin, have been differentiated. Im- munity has been experimentally produced by the injection of killed cultures. Emmerich and Low have proposed the name pyocyanase for the broth filtrate of old cultures of pyocyanea concentrated in a vacuum. This material has been found to be very high in proteolytic ferments and has been suggested for many clinical uses, among them the destruction of bacteria in vitro, the solution of diphtheritic membranes, the spraying of infected mem- branes to destroy the causal organisms, etc. While it has been extensively experimented with, it has never come into common use. Bacteriologic Diagnosis. — The organisms may be most readily determined by plating. CHAPTER XXXI DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP. THE GENUS CORYNEBACTERIUM The organisms which belong to this group are all rod-shaped bacteria of moderate size. They are non-motile, do not produce spores, are Gram-positive, and not acid fast. All are aerobic and facultative. They are all characterized by the possession of granules which cause irregular or banded staining of the cell, and all show a decided tendency to the production of branched and club-shaped cells. A number of species of these so-called "diphtheroid" bacilli have been described. Some of these are pathogenic, others are not. The latter are important principally because of the difficulty in differentiating them from the pathogenic types in disease diag- nosis. The important pathogenic organisms of this group are the Corynebacterium pseudotuberculosis, the cause of bovine caseous lymphadenitis, equine ulcerative lymphangitis and bovine lymph- adenitis, and pyelonephritis, and the Coryn. diphtherice, the cause of human diphtheria. Coryn. xerosis, from the eye, Coryn. hoffmanni, the pseudodiphtheria bacillus, and some other diph- theroids less well known, belong here. Methods of separation of the organisms of this group from each other are not entirely satisfactory, but in some cases are quite necessary because of importance in disease diagnosis, particularly in diphtheria. Acid production in carbohydrates and development of a true toxin are among the tests which have been used. Mor- phology is also of much assistance. The four most common members of the group may be differen- tiated by means of their reactions in Hiss's carbohydrate serum- water medium. Acid production in 1. per cent. Dextrose. . Glycerol. Saccharose. Dextrin. Corynebacterium diphtheria; + + — + Corynebacterium pseudotubercu- losis + — — — Corynebacterium xerosis + + + — Corynebacterium hoffmanni — — — — 361 362 VBTEHINABY BACTERIOLOGY All of the organisms named except Corynebacterium hoffmanni also acidify maltose. Morse ' has named two other non-patho- genic members of this group B. hoagii and B. flavidus. The former produces acid from dextrose, the latter from dextrose, mal- tose, and glycerin. The latter also produces a yellowish pigment. Morphologic and cultural differences among these species may be noted, and means of differentiation by their aid will be discussed under the several headings. Corynebacterium pseodotafaercolosis Synonyias.^Badllus pseudotuberculosis ovis, Preisz; Mycobac- terium pseudotuberculosis; Bacillus lymphangitidis ulcerosa; B. renalis bovis; B. pseudotuberculosis murium, Kutscher; Corynethrix pseudotuberculosis murium, Bongert; Bacillus furunculosis ulceroscB, Kitt; Bacillus of Preisz; Preisz-Nocard bacillus. To differentiate from the acid-fast bacteria resembling the tubercle bacillus, it has been suggested that the latter be called pseudotubercle bacilli, and the former pseudotuberculosis bacilli. There is not entire agreement among investigators as to the actual identity of many of the organisms listed above as synonyms. Some have believed that all constitute a single somewhat pleo- morphic species, others divide into two or more species upon the basis of pathogenicity, serum reactions, and culture. Diseases Produced. — The infections produced by this organism have received a variety of names depending upon the animals attacked and the lesions produced. In sheep the disease has been known as caseous lymphadenitis, pseudotuberculosis, and pyo- bacillosis; in equines, ulcerative lymphangitis (sometimes termed pseudofarcy and pseudoglanders) ; in cattle, caseous lymphadenitis, pyelonephritis, and pyobacillosis; and in rabbits and other rodents, pseudotuberculosis and lymphadenitis. Distribution. — The disease in sheep has been reported from France, Germany, the United States, Argentina, Australia. Somewhat similar infections in horses have been described from France and the United States. In Argentina 10 per cent, of sheep slaughtered and in Austraha even 15 per cent, are affected. 1 Morse, M. B., Jour. Inf. Dis., 1912, 11, p. 281. DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP 363 Historical. — This organism was first described from the lesions of sheep by Preisz and Guinard' and later by many other writers from France. Turski^ recorded the presence of the disease in Germany, and Norgaard and Mohler' in the United States. The organism as a cause of "pseudofarcy" in horses was first noted by Nocard^ in 1892. Later he concluded his organism to be identical with that of Preisz from sheep. Dunkel,^ as the result of comparative studies of Bacillus pseudotuberculosis avis and B. pyogenes suis, Grips, and B. pyogenes bovis, Kunnemann, came to the conclusion that the latter could be transformed into the former, and that they should all be classed as the same species. The more recent work of Priewe and of Glage indicates, however, that the last two species are quite distinct from the first and are far more closely related to the influenza group (q. v.). Morphology and Staining. — The organism is a non-motile bacillus about 0.4 by 1 to 3 ;u. It resembles the diphtheria bacillus in the formation on artificial media of thickened and club-shaped forms. It is non-motile and does not produce spores or capsules. It stains well with the ordinary anilin dyes and is Gram-posi- tive. Isolation. — Pure cultures may be obtained by smears upon suit- able culture-media directly from a caseous nodule. Growth is scant at first, but becomes better after a time as the organism adapts itself to artificial media. Cultural Characters. — On agar at 37° dry colonies are formed within two days, the maximum size being reached in six to eight days. The colony usually shows a folded or granular surface, fre- quently with concentric rings and a papillate center. Glycerin agar is unfavorable. On gelatin the organism grows sparingly when kept at 22°. Potato shows no growth according to some authors, others re- 1 Preisz and Guinard, Jour, de Med. Vet., 1891, 42, p. 503. 2 Turski, Zeitschr. f. Fleisch. u. Milchhygiene, 1897, p. 173. " Norgaard and Mohler, Sixteenth Annual Report Bureau of Animal Industry, 1899, p. 638. ■■ Nocard, Annales del' Institut. Pasteur, 1892. 5 Diss., Giessen, 1908. 364 VETERINARY BACTERIOLOGY port a grayish white, shghtly moist, irregular film, often scarcely visible. SoUdified serum shows characteristic colonies of creamy gold yellow or orange color, reaching a diameter of 1.5 mm. No change occurs in milk. Solidified egg-white is as suitable as blood-serum, though the yellow color does not appear. Sterile cattle serum gives abundant yellow flocculent growth, with decided clouding and yellowing of the serum. Finally, a heavy yellow sediment collects. Bouillon is not permanently clouded, a granular deposit forms, together with a dry surface peUicle. Figs. 135 and 136. — Corynebacterium pseudotuberculosis, colony and mount (Norgaard and Mohler in Report of Bureau of Animal Industry). Physiology.— A temperature of 65° for ten minutes will destroy the organism. Its optimum growth temperature is 37°, but some growth occurs even at 18°. It is relatively resistant to drying. In culture-media it remains viable for many months. Virulence ap- parently remains unimpaired for years. The organism is easily destroyed by disinfectants. Toxins (see below) may be developed in broth. Coryn. pseudotuberculosis is aerobic. Gas is not produced from sugar. Acid is formed from dextrose, but not saccharose, glycerin, or dextrin. DIPHTHERIA-PSEUDOTUBERCULOSIS GROUP 365 Pathogenesis. — The organism is pathogenic for mice, guinea- pigs, rabbits, goats, sheep, and probably for the horse. Fowls and pigeons are immune. Although toxins may be demonstrated in cultures, they probably are not sufficient in quantity to account for the development of the disease. Intravenous injection of the guinea-pig results in death in from four to ten days, with foci of suppuration and caseation in various internal organs, particularly the lungs and the liver. Subcutaneous injection is followed by enlargement and caseation or suppuration of the lymph-glands, with fatal termination in from fifteen to twenty-eight days. Particularly characteristic is the develop- ment of orchitis in male guinea-pigs following intraperitoneal injection. This resembles in many ways that following the injec- tion of glanders bacilli. Mice are even more susceptible than guinea-pigs. They suc- cumb to ingestion of the organism in from two to four weeks. Rabbits have about the same degree of susceptibility as guinea- pigs. This organism has most frequently been reported as the cause of ovine caseous lymphadenitis, but it is probably identical with organisms isolated from similar lesions in cattle and from equine ulcerative lymphangitis. The disease in sheep is found chiefly in breeding ewes. It pro- gresses slowly, and is frequently not recognized until the animal is slaughtered. The lymphatics are usually affected. The glands enlarge, caseate, and are often encapsulated. In more advanced cases the various internal organs are also infected, nodules some- what resembling those of tuberculosis appearing in the lungs, spleen, liver, and kidneys. The disease is not commonly found in young animals, probably because the lesions have not had time to develop sufficiently to cause enlargement of the glands noticeable on inspec- tion. The disease as described in the horse is an ulcerative lymphan- gitis, the subcutaneous lymph-nodes being chiefly affected. These enlarge and break through to the surface, producing a condition which may readily be mistaken for farcy. Involvement of deeper glands and of the internal organs may occur later in the progress of the infection. 366 VETERINARY BACTERIOLOGY Bacteriologic Diagnosis. — Because of the character of the lesions produced the disease may be confused with tuberculosis in the sheep or true farcy in the horse. Smears from the lesions in sheep show the Gram-positive, non-acid-fast organism which is very distinct from the tubercle bacillus. The glanders bacillus is Gram- negative; a mount of pus from equine lymphangitis stained by Gram's method should show the characteristic organism. Care in the differential diagnosis of farcy based upon the ability of the glanders bacillus to produce an acute orchitis in the male guinea-pig should be used, for this reaction is quite as characteristic of Corynebacterium pseudotuberculosis. Pure cultures secured are readily differentiated both morphologically and culturally. The Coryn. ■pseudotuberculosis resembles the diphtheria bacillus morphologically in many ways, but may be easily distinguished by annual inoculation and sugar fermentation reactions. Immunity. — This organism produces a true toxin when grown in broth culture. This reaches a sufficient concentration so that . 1 c.c. of the culture filtrate will kill a guinea-pig in twenty-four hours. Rabbits are likewise susceptible, but mice, cats, and dogs are more refractory. Intravenous injection of sheep with 10 c.c. is fatal within eighteen hours. The toxin resembles that- of the diphtheria bacillus in many respects. It is readily destroyed by heat. An antitoxic horse serum has been produced which will pro- tect sheep or laboratory animals against injection of the toxin. However, these animals are not protected against infection with the organism when introduced. This antitoxic immunity is, therefore, of little practical significance. It is also claimed that diphtheria antitoxin will at least in part neutrahze the toxin of the Coryn. pseudotuberculosis. Carr^ found that sheep that had re- covered from infection were not affected by injection of the toxin. This investigator also developed a method of vaccinating lambs against the disease by means of two vaccines of different degrees of virulence. While he claimed this to be successful, it has never come into use. Transmission. — In many- cases the disease foci are closed and the bacteria are not eliminated from the body. It is probable it usually takes place as the result of ingestion, or possibly the organ- ism may gain entrance through skin abrasions. DIPHTHERIA-PSEUDOTUBEKCULOSIS GHOUP 367 Corynebacterium diphtheriae Synonyms. — Klebs-Loffler bacillus; Bacillus diphtherice; Bac- terium diphtherice; Mycobacterium diphtherice. Disease Produced. — Diphtheria in man, rarely in some animals. Historical. — Klebs, in 1883, described an organism present in the false membrane of diphtheria, which Lofiler, in 1884, secured in pure culture and sho\yed to be pathogenic. A similar organism was isolated by him from a healthy child, so that he was reluctant to conclude that he had found the true cause of the disease. Roux and Yersin, in 1888-1890, showed that the various pathologic con- .^3^^^$^. Fig. 137. — Bacillm SipKtKeficB (Epstein in Journal of Infectious Diseases). ditions most characteristic of diphtheria could be duphcated in animals by injection of the broth filtrate containing the toxin. Distribution. — Occurs in epidemics, particularly among the young, in Europe and America. Morphology and Staining. — Corynebacterium diphtherice is closely related to some of the higher bacteria or even the fungi. It stains readily with the common anilin dyes and is Gram-positive. When stained with methylene-blue a smear, prepared directly from an infected mucous membrane, will show rods varying from 0.4 to 1 m in diameter and 1.5 to 3.5 yu in length, 368 VETERINARY BACTERIOLOGY frequently slightly curved, sometimes pointed or club shaped, some- times staining uniformly, but usually containing metachromatic granules, which stain more deeply than the remainder of the cell, and give a barred or granular appearance to the cell contents. These same variations may be observed in the organism taken from suitable culture-media, particularly Loffler's blood-serum. Occasionally branched forms may be observed. Demmy has shown that the diphtheria bacillus varies almost from hour to hour in its morphology when grown upon blood-serum. In five hours after the culture is made the cells take the stain uniformly; in eight hours some cells show vacuolization; in twelve hours the or- N*/ l, > ■4; ^ w L-jStSfS^ Fig. 144. — Mycobacterium tubercu- Fig. 145.^ — Mycobacterimn tubercu- losis in human sputum. Note the losis, human, mount from glycerin slender beaded character of the rods agar (Frankel and Pfeiffer). (X 1000) (Gunther). Morphology and Staining. — Mycobacterium tuberculosis is a slender rod, commonly somewhat bent, with rounded ends. It varies from 0.2 to 0.5 by 1.5 to 3.5 m, sometimes longer. Fre- quently the protoplasm takes the stain irregularly and gives a beaded appearance to the cell. No spores or capsules are pro- duced. The organism is non-motile. Branched and elongated forms resembling somewlia*-the actinomyces are sometimes observed. It is possible that these are involution forms, although many authors claim them to be developmental forms instead. The organism stains with difficulty, but when once stained is acid fast. It is possible that under certain conditions, 388 VETERINARY BACTERIOLOGY in the animal tissues in particular, this acid-fast property may be temporarily lost. In very young cultures the bac- teria are sometimes not acid fast. In tissues and cultures con- taining tubercle bacilli that do not show the acid-fast character Much has demonstrated Gram-positive granules, which are prob- ably a growth stage of the tubercle bacillus. The acid-fast charac- ter is apparently due to the presence of a wax-like substance in the bacterial cell. The cells from which this has been removed by ether and benzol are no longer acid fast. Certain observers have claimed that there are cer- tain morphologic differences commonly to be observed between bovine and human tubercle bacilli. They have stated that the former are shorter, straighter, and thicker than the latter, and are less apt to show the ir- regular or granular staining noted above. These charac- ters are, of course, not suffi- cient to differentiate isolated bacteria of the two types, but cultures can sometimes be identified by an experienced observer. Whether or not one type may be transformed into the other type by animal inoculation or by cultural methods is questionable. Some investigators claim to have isolated typical human bacilli from animals that have been inoculated with bovine bacilli; others hold that there is no evidence of the transformation of the one type to the other. However this may ultimately be decided, it is certain that each type retains its characters with a considerable degree of constancy. The existence of two well-marked varieties is unquestioned. The bacillus of avian tuberculosis resembles the bovine type closely in its morphology and staining reactions. Isolation. — The isolation of Mycobacterium tuberculosis from lesions is attended with considerable difficulty. This is even Fig. 146. — Mycobacterium tuberculosis, bovine, in a section of the peritoneum (Frankel and Pfeiffer). ACID-PAST GROUP. THE GENUS MYCOBACTEHIUM 389 more pronounced when an attempt is made to secure the organ- ism from the sputum or the feces where it exists in mixed culture. It may be isolated from infected organs by securing bits of the tissue and rubbing over the surface of inspissated blood-serum or other suitable medium. The method worked out by Theobald Smith has given excellent results in the hands of numerous in- vestigators. A dog is bled, using all aseptic precautions, from the femoral artery into a sterile vessel and the blood allowed to clot. The serum is removed by sterile pipettes to sterile test-tubes. These are slanted and heated to a temperature of 75° to 76° for about three hours, or until the serum is coagulated. The heating must be done in a saturated atmosphere and the medium stored so that there is no loss by evaporation. Bits of infected tissue are placed upon the surface and kept in a thermostat for several weeks. If no growth appears, the tissue is moved about and incubated again. A constant temperature of 37° and a saturated atmosphere must be maintained. A procedure somewhat simpler than the preceding has been described by Dorset and is found to give good results. The shell of fresh eggs is carefully broken, and the white and yolk dropped into a sterile flask, the yolk broken with a sterile rod or wire, and the contents of the flask shaken until the two are thoroughly mixed. Foaming is to be avoided. The mixture is placed in tubes, slanted, and heated at a temperature of about 70° for froin four to five hours on two days. This coagulates arid sterilizes the medium. The tubes should be stored where they will not lose water by evaporation. Several drops of distilled sterile water should be added to a tube just before inoculation. The isolation upon this medium is carried out as outlined above. A growth may generally be observed within ten days after inoculation with fresh tissue. Isolations from sputum or feces, milk, or other substances in' which the organisms occur mixed with other forms, is attended with some difficulty. It is usually accomplished by injecting the mate- rial or the sediment yielded by centrifugation directly into a guinea- pig. The bacilli may later be isolated in pure culture from the nodules produced. Within recent years the use of "antiformin" and similar substances has considerably simplified this procedure. 390 VETERINARY BACTERIOLOGY Antiformin is the trade-name given a disinfectant mixture having the following composition: Solution I. Sodium carbonate 12 gm Chlorinated lime 8 gm DistiUed water 80 gm Solution II. Sodium hydroxid 15 gm Distilled water 86 gm Equal quantities of the two solutions are mixed for use. The sputum or other material containing the tubercle organisms is placed in a centrifuge tube, and antiformin to about 20 per cent. of its bulk added. The tube is then corked, thoroughly shaken, and allowed to remain in a dark place for twenty-four hours. It is then centrifuged, the clear, supernatant liquid pipetted off, the tube filled with sterile physiologic salt solution, centrifuged, washed a second time, and the sediment smeared over the surface of serum slants. The antiformin destroys all other non-acid-fast bacteria present and dissolves the mucus and most of the cell elements, but when properly used seems to have little effect upon the tubercle bacilli, as they retain their vitality unimpaired. This is probably because of their chemical composition and waxy covering. Cultural Characters. — Mycobacterium tuberculosis does not grow readily when first isolated upon culture-media, and will grow only upon certain substances. After a few transfers it seems to become habituated to growth under these conditions and wUl develop through a much greater range of temperature and on other media. Development occurs best on media containing blood-serum, egg, or similar proteins, or to which glycerin has been added. The colonies upon blood-serum or glycerin agar appear in the course of ten days or two weeks as tiny grains barely visible to the naked eye. They gradually enlarge, and in subcultures may be- come confluent and cover the surface of the medium with a dry, rather mealy, wrinkled growth; the colonies direct from lesions do not coalesce usually. The growth is white and lusterless, or rarely in old cultures cream or brown. In glycerin bouillon the growth generally occurs as a more or less continuous, heavy, wrinkled, white peUicle that breaks into pieces and sinks to the bottom when the medium is shaken. Similar growths occur upon other media which contain glycerin. In no other case is the growth so rapid. ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 391 Cultural characters which may be used in the certain differen- tiation of the human, bovine, and avian tubercle bacilli are usually quite marked. The organisms isolated from the human and from the bird adapt themselves much more readily to arti- ficial media and grow more luxuriantly than does the bovine type. The former produce upon glycerin agar a relatively heavy wrinkled growth, the latter a much more delicate growth, frequently showing discrete colonies only. Physiology. — The tubercle bacillus is aerobic. Its optimum growth tempera- ture is about 37.5° for the human and the bovine types and somewhat higher for the avian. The growth temperature range is relatively narrow, no growth usu- ally being secured below 30° or above 42°. The thermal death-point is 60°^ for twenty minutes. This is, therefore, the minimum time and temperature for the efficient pasteurization of milk. Dry heat even at 100° does not kill the organism certainly within an hour. Sun- light destroys the organism quickly, but it is moderately resistant to desiccation. When dried in sputum cells have been known to live for at least two months. Most of the physiologic characters, such as acid, gas, pigment, and indol produc- tion, are negative. Theobald Smith has called attention to what appears to be a very constant differential character between human and bovine tubercle bacilli. He found that in glycerin bouillon (2 per cent.), acid to phenolphthalein the human bacillus causes a permanent acid reaction, while with the bacillus of bovine origin the acidity Fig. 147. — Mycobac- terium tuberculosis, gly- cerin agar slant (Curtis). 392 VETERINARY BACTERIOLOGY diminishes and the reaction becomes alkaline if the growth en- vironment of the culture is suitable. The tuberculin prepared from the human bacillus is acid, and from the bovine bacillus alka- line. This difference has been noted by other investigators since its first description, and seems to be one of the best methods of differential diagnosis. Pathogenesis. — Tuberculosis is characteristically a chronic disease. Even in experimental animals months are often required for it to run its course; As stated by Moore, "It does not destroy life by acute toxemia, but by a chronic and long-continued sys- temic poisoning and by the morbid changes brought about through the localization of these lesions in the organs necessary to life." Experimental Evidence of Pathogenesis. — The laboratory ani- mals are generally susceptible to infection with Mycobacterium tuberculosis. The constant presence of the organism in the lesions of the disease and its ability to reproduce the disease are sufficient evidence that it is the true etiologic factor. Important differences in pathogenesis are to be noted among the three varieties. The bovine bacillus is most pathogenic for laboratory animals, the human next, and the avian least (except for birds). Guinea-pigs inoculated subcutaneously with bovine bacilli generally succumb in less than fifty days; those inoculated with human bacilli generally live more than fifty days. Intra- peritoneal injection of the bovine type is fatal in seven to eighteen days, of the human type in frojn ten to thirty-eight days. The difference upon intravenous injection of the rabbit is even more marked— with bovine baciUi death occurs within three weeks, with human bacilli the animals usually live for several months and may even recover. The avian type ordinarily does not produce fatal infection in guinea-pigs, although rabbits succumb and fowls ■^nd pigeons contract the disease readily. Calves inoculated with bovine tubercle bacilli usually succumb promptly to generalized tuberculosis, while inoculation with human strains lead usually to local lesions which heal eventually. Character of Disease and Lesions Produced. — Almost any part of the body may be affected with tuberculosis. The disease, wherever found, generally involves the lymphatics. It is charac- terized by the development of nodules having an essentially similar ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 393 structure in all tissues. The presence of the tubercle bacilli in a tissue causes a proliferation of the fixed connective-tissue cells to form the beginning of a miliary tuherde. Lymphocytes are gener- ally attracted and are present in the surrounding tissues in con- siderable numbers. A more or less definite layer of "epithelioid" cells forms the boundary of the tubercle. Typical giant-cells with peripheral nuclei are found near the center. Coagulation- Figs. 148 and 149. — Tubercular hypertrophy of the intestinal wall in the bovine (Chauss^). necrosis proceeds and the interior caseates. Encapsulation with fibrous tissue may occur, and the whole may eventually become calcified. These calcareous grains persist in healed tuberculous areas. Tubercles frequently are formed in masses. In the cow tubercular lymph-glands sometimes may equal or exceed the size of an orange. The arrangement of the nodules frequently shows clearly the 394 VETERINARY BACTERIOLOGY path of the spread of the bacilli through lymphatic metastases. Direct growth through tissues with invasion of new areas probably rarely occurs. The organisms are not commonly found in the blood-stream, although they may sometimes be carried to other parts of the body by this means. Infection of the bones, joints, and meninges probably occurs in this manner. The organs most commonly the seat of lesions vary with the species of animal and the mode of infection. In the human, pul- monary infection (consumption) is most common, although in- testinal tuberculosis, infection of the lymphatics of the neck (scrofula), of the bones and joints (tubercular osteitis and arthritis), of the meninges, and of the liver, spleen, kidneys, and other organs of the body, and the serous membranes lining the cavities are not uncommon. Lupus or tuberculosis of the skin is of frequent occurrence in certain European countries. Cattle generally show nodules in the mesentery and in the peritoneum (Perlsucht or pearl disease). The lungs and the accompanying lymph-glands and the intestines commonly show lesions. In a certain small percentage of tuberculous cows, variously estimated from a frac- tion of 1 to 5 per cent., tuberculous lesions may be found in the udder. Any and all of the organs of the body may be infected. Swine are most commonly infected in the lymph-glands of the neck (swine scrofula), and in the abdominal organs and the lungs. Avian tuberculosis most frequently attacks the abdominal organs, particularly the liver and spleen, more rarely the lungs. Immunity. — No true toxin has been demonstrated for the tubercle bacillus. Endotoxins are produced. These are liberated from the cell with difficulty because of its composition and slow dissolution. Specific agglutinins and precipitins have been de- monstrated in the blood of infected individuals and in immune serum. Opsonins, both normal and immune, have been shown to occur. The development of bacteriolysins has not been satis- factorily demonstrated. Methods of active immunization are all dependent upon the use of killed or attenuated bacteria or their products. The name tuberculin is given to any suspension of dead tubercle bacilU or a solution of their products. As will be noted later, tubercuhn is not only of therapeutic significance but of great diagnostic value. ACID-FAST GROUP. THE GENUS MYCOBACTERIUM 395 Many types have been prepared by various workers. Some of the more important will be described before a discussion of their use in immunization and in diagnosis is undertaken. Koch's Old Tuberculin {Alt Tuberculin) .—FM-hoitomed flasks containing 5 per cent, glycerin-broth to a depth of 2 to 3 cm. are inoculated with Mycobacterium tuberculosis. For veteri- nary practice tuberculin may be prepared from either the bovine or human type. The latter grows far more rapidly on artificial media and for this reason is generally if not universally used. The inoculating material is carefully placed on the glass FT- A 3 0J ♦•(? ^^-^^ .« ®^ M^t'Ay^

^ 1^¥" Mux Fig. 164. — Spirochcela pallida in the lumen of a bronchus in congenital syphilis (Hedren). may be found in the internal organs of a syphilitic fetus. An infection has been produced in the cornea and the iris of the rabbit, and the organism shown to be present. The primary and secon- dary lesions of the disease have been produced in the monkey, particularly in the anthropoid apes, and the spirochetes found in each of the stages. The evidence is very strong, therefore, that Spirochceta pallida is the cause of syphilis. Until the organisms can be injected in pure cultures and produce the disease this evi- dence cannot, however, become indisputable. 430 ' VETiSRINARY BACTERIOLOGY Character of Disease and Lesions.— In man the primary lesion in the form of a chancre appears in about three weeks afler in- fection, usually on or near the external genitalia. It is followed by invasion of the neighboring lymphatics and by progressive en- largement of the lymph-nodes as the disease progresses. Usually about six weeks elapse between the appearance of the primary and secondary lesions. These latter are probably dependent upon an invasion of the blood, and consist of localized skin erup- tions, falling of the hair (alopecia) ■, and the symptoms of generalized infection, such as fever. This may last for several years and an immunity be established, which, however, may not be complete enough to prevent gradual sclerosis of blood-vessel walls and degenerations in the parenchymatous organs, and even the appear- ance of tertiary lesions. Immunity. — No practicable method of either active or passive immunization against the disease has been developed by the use of the organism or its products. Bacteriologic Diagnosis. — This may be accomplished by direct examination, stained mounts, the Wassermann test, or by chemical recognition of certain changes in the character and composition of the blood-serum. The organisms may be observed in the fluid expressed from fresh tissues by use of dark-field illumination. Smears may be prepared and stained with Giemsa's stain, or tissue sections may be used. The Wassermann test for syphilis has already been described in the discussion of fixation of complement in the section on Immunity.' As an antigen, extracts from the organs of a fetus are used, for in these organs the spirochetes are found in the greatest numbers. The blood-serum of the suspected patient is tested for its possible content of specific amboceptor with fresh guinea-pig serum for complement, sheep red blood-cells, and the serum from a. ra:bbit possessing hemolytic amboceptor for these erythrocytes. The test requires considerable care and must be checked at every step. It has been found in practice to give quite re- liable data. The test has been modified in many ways since first proposed. Noguchi has prepared an antigen from pure cultures of Spiro- chceta pallida. He has found that he could obtain positive tests SPIROCHETE GROUP 431 only in isolated cases of long-standing syphilis which had been treated, and that the positive reactions obtained in active cases were not due to antibodies that combine specifically with such antigen. Craig and Nichols have shown that serum from un- treated cases of syphilis give positive reaction against syphilitic liver extract, but negative against the pallida antigen. This material, which Noguchi terms liietin, has also been used in a dermal test for syphilis. Various substances, such as 1 per cent, solutions of lecithin, sodium oleate, sodium glycocholate, and taurin have been found to give more or less characteristic precipitates with the blood of syphilitics. Transmission. — The disease is transmitted usually through sexual congress, rarely through infective drinking-vessels, closets, and by direct inoculation, as sometimes happens in surgical work. The disease may be present at birth; the organism may possibly enter through the ovum or the sperm, or pass from the circulation of the mother to that of the fetus. SpirochaEta pertenais Synonym. — Treponema pertenioe. Disease Produced. — Yaws in man. Castellani, in 1905, reported the occurrence of spirochetes in a tropical disease known as yaws. The organism resembles that of syphihs, but is probably distinct, as shown by inoculation experi- ments and study of specific antigens and antibodies in comparison with those of syphilis. Other Spirochetes Dodd, in 1906, found a spirochete in a disease of the pig in South Africa. It was associated principally with dark, hemor- rhagic lesions of the skin. Spirochetes may be found in considerable numbers in the mouth, and, under certain conditions, in the intestinal tract, upon the skin, and about, the genitalia. They are, for the most part, believed to be harmless commensals. CHAPTER XXXVII ACTINOlVryCES GROUP The members of this group are often called Trichomycetes or thread fungi. In many of their morphologic characters they resemble bacteria. Frequently they occur as short rods that cannot be differentiated by examination from true baciUi. Usu- ally, however, they occur in threads, which in some genera may be branched. These threads may show more or less differentiation into parts, and certain portions may develop into conidia or spores. These organisms show a more complex life-history, therefore, than do the true bacteria. On the other hand, they can scarcely be grouped with the true molds, as they are much simpler in struc- ture. They may be considered as a group, therefore, related closely to both bacteria and molds and partaking of the nature of each. These organisms show such diversity of morphology in the animal body and in culture-media that a satisfactory classification into species and genera is a difficult problem. Many generic names have been proposed. They will all here be regarded as belonging to the genus Actinomyces. Several organisms are included in this genus, as here dis- cussed, that may be shown to belong to the bacilli and not to the trichomycetes. Many species of organisms of this group are known from the descriptions of a single author only. It is difficult to determine from these descriptions how many are valid species and how many merely synonyms. The facts seem to be that Actinomyces are widely distributed in nature. They may be isolated in abundance from most soils, and inay be found to develop upon almost any plate of medium exposed to the air. Only under exceptional conditions are they pathogenic, but it is probable that the species usually described as such are normally saprophytes that can upon occasion prohferate in the tissues of the body and produce disease. The fact that cattle become infected through the gums or tongue, where the awns of certain grasses penetrate, that the barley testers, who bite the barley grain to determine its brewing 432 ACTINOMYCES GROUP 433 quality, are most frequently infected among men in temperate climates, that injuries to the feet of natives of certain tropical countries (where no adequate protection is worn on the feet) are frequently followed by local infections, and that Actinomyces have -->■ •**.,: 33 ■■fe-'i^f ,1 lr< Fig. 165. — Actinomyces calicolor, a non-pathogenic trichomycete from the soil. A ring colony on a semisolid medium showing filaments and aerial hyphse (Mtiller). been found causing infections in practically all domestic animals by one investigator or another is evidence of the wide distribution of the members of the genus. The species to be described are Actinomyces bovis and A. nocardia in cattle, A. caprw in goats, A. maduroB, and A. eppingeri in man. The group, as a whole, may be characterized as consisting of slender, branching organisms, which may develop into colonies made up not only of threads but rods, cocci, and other cell forms. Frequently, in animal tissues, and sometimes upon artificial media, the ends of the threads may be clubbed. When grown upon the surface of artificial media some forms develop aerial hyphae, which segment into chains of conidia. All species retain the Gram 28 434 VETERINARY BACTERIOLOGY stain to a greater or less degree. Some are aerobic, others facul- tative, and still others obligate anaerobes. Pigments are produced by some species. Actinomyces faovis Synonyms.— (S^repfof/ina; bonis; Cladothrix adinomyces ; Strepto- thrix actinomyces ; Discomyces bovis. Fig. 166. — Actinomyces cmlicolor. Colony on agar. This colony structure is quite typical of many species (Miiller). Disease Produced. — Lumpy jaw and wooden tongue (actino- mycpsis) in cattle, and probably related infections in other animals and man. Harz, in 1878, gave the name Actinomyces to the ray-fungus, which Bollinger, in the preceding year, had found present in the characteristic tumor-like growths in cattle. Distribution. — The infection is known from Europe and North and South America. Morphology and Staining. — In the infected tissues the organism forms minute yellowish granules, sometimes large enough to be readily observed by the unaided eye. These granules are made up of compact masses of the organisms. Branched filaments, with a more or less radial arrangement, are to be observed occupying the central portion, commonly mixed with coccus-like degeneration products. The margin of the granule or rosette, when examined in cross-section, is found to consist of club-like enlargements of the threads, showing a marked ref ractivity to light. The filaments ACTINOMYCES GROUP 435 are slender, usually about 0.5 ;u in diameter. It is believed that the formation of the clubbed ends is correlated in some way with the resistance of tissue to invasion. They have been variously regarded as degeneration products, involution forms, and as indi- cating a thickening of the sheath to protect the organism against antibodies produced by the tissues. Young colonies on artificial media consist of interlacing, branched threads, which tend to form compact masses. These commonly break up into bacillus-like segments, in a manner not unlike the formation of certain spores among higher fungi, by segmentation of the hyphal threads. Whether or not these correspond to the oidial type of spore pro- duced in the higher fungi, or represent spores at all, is not known. The clubbed type rarely develops in artificial media. The organism stains readily with the common anilin dyes and is Gram-positive. It is not acid fast. Isolation and Culture. — The organism is not easily iso- lated in pure cultures, par- ticularly when it occurs in mixed cultures with pyogenic cocci in the lesions. Wright has described a technic which he found quite uniformly suc- cessful. Pus or tissues con- taining the organism in fila- mentous rosettes is preferable to that containing only the clubbed type, as in the latter degeneration has gone so far that frequently no growth will occur. The granules are washed in sterile water, crushed be- tween sterile sUdes, and inoculated in varying amounts into tubes of melted 1 per cent, dextrose agar, and incubated at 37°. In his experience the colonies developed characteristically from 5 to 12 mm. below the surface, but others have found them to form quite as well upon the surface of the medium. Isolated colonies may then be transferred to other media. In bouillon the organism forms distinct, solid, spherical, or mulberry-like Fig. 167. — Actinomyces boms, tissue section showing the radial arrangement and the clubbing of threads (Giinther). 436 VETERINARY BACTERIOLOGY masses at the bottom of the tube. Growth is secured with diffi- culty upon the surface of the medium, according to Wright, but other investigators have not experienced the same difficulty. It forms on agar and glycerin agar colonies, which at first resemble tiiiy drops of amber; these enlarge, and either remain discrete or coalesce to form a distinctly wrinkled, "Hchen-like" mem- brane, which frequently has a dusty appearance. Gelatin is slowly liquefied. Physiology. — The organism may be regarded as a facultative aerobe, as growth appears to take place best under anaerobic conditions. The optimum T^w«v growth temperature is 37°. The organism is resistant to desiccation and will live for a long period, probably months, in a dried condi- tion. Gelatin is liquefied. There is no gas- or acid- production. A brown to black pigment may be produced. Pathogenesis. — Ex-peri- mental Evidence. — In the great majority of cases ex- perimental inoculation, is without result. Many ani- mals have been used — cattle, sheep, swine, dogs, cats, rabbits, and guinea-pigs. In relatively a few cases significant lesions have been developed. Musgrave, Clegg, and Polk have produced extensive suppura- tive lesions by intraperitoneal inoculation of the monkey, the infection terminating fatally in about three weeks. The com- mon lack of pathogenesis may be due to differences in resistance, to a diminution of virulence due to cultivation, or to the manner of inoculation. In cattle it may gain entrance with a grass awn and this may protect it from the destructive agencies of the tissues until its pathogenicity is well established. Character of Disease and Lesions Produced. — A swelling or Fig. 168. — Actinomyces bovis (Strepto- thrix actinomyces), stained mount from culture-medium (Musgrave, Clegg, and Polk, in "Philippine Journal of Science"). ACTINOMYCES GROUP 437 tumor-like mass develops in cattle at the site of infection. This softens and ultimately discharges thick, yellowish pus. The discharge after the lesion has opened may become intermittent in character. When the tongue is the primary seat of infection it becomes swollen, indurated, and protrudes from the mouth in some cases. The bones of the jaw are often attacked. The in- fection is chronic. Animals rarely die from immediate effects. In a few instances metastatic infection of other parts of the body than the head and neck have been reported. In man the disease usually attacks the softer tissues, progresses more rapidly than in cattle, and is apt to terminate fatally from metastatic infection. Whether or not the organism isolated from human actinomycosis is the same as that found in cattle is uncer- tain. The same may be said of the forms that have been isolated from similar infections in other animals, among them the horse, dog, and pig. Immunity. — No method of immunization against the disease has been developed. Bacteriologic Diagnosis.^A microscopic examination of the imstained pus will usually reveal the characteristic granules, with the radial arrangement of clubs or of tangled bits of branched threads. A film stained by Gram's method will bring the latter out clearly when present in small numbers only. Transmission. — It is believed that the organism commonly enters the body through a trauma, through carious teeth, or by being carried into the tongue or the gum with the sharp awns of certain grasses and grains. So far as known the disease is wholly non-contagious. Actinomyces nocardii Synonyms. — Streptothrix nocardii; Actinomyces fardnica; Strep- tothrix fardnica; Nocardia fardnica. Disease Produced. — Bovine farcy. Farcin du bceuf . Nocard, in 1888, first described an Actinomyces or Strepto- thrix from the lesions of cattle in Guadeloupe suffering from a disease termed bovine farcy. The disease itself has not been adequately studied, although the organism has been investigated by several workers. There is no record of its occurrence in the United States. 438 VETERINARY BACTERIOLOGY Morphology and Staining. — The organism is slender, much branched, and interwoven. In culture-media -short, plump fila- ments with branches may occur, and in old cultures many ovoid cells are found. The organism is Gram-positive, and many por- tions, particularly in old cultures, are acid fast and also alcohol fast. Isolation and Culture. — It may be isolated in pure culture from lesions directly upon artificial media. The colonies upon agar ^r -^. Pig. 169. — Actinomyces nocardia, stained mount from culture (Musgrave Clegg, and Polk, in "Philippine Journal of Science"). are small, white, irregular, raised, and opaque. Upon glycerin agar they are at first deUcate, but soon coalesce and present a moist, meal-Uke growth. Bouillon is never clouded, but a grayish, flocculent mass forms at the bottom. Milk is un- changed. Physiology. — The organism is a facultative aerobe. It de- velops best at 37°. It is resistant to desiccation and maintains its virulence when cultivated. Pathogenesis.— Experimental Evidence.— Gumea.-pigs are easily infected by intraperitoneal injections. The organism produces numerous nodules resembling tubercles upon the peritoneum and the abdominal organs, particularly the liver, spleen, and ACTINOMYCES GROUP 439 kidneys. Intravenous inj ection gives rise to a condition resembling generalized miliary tuberculosis. Intraperitoneal injection of the monkey gives rise to similar lesions. Cattle and sheep develop, at the point of a subcutaneous inoculation, an abscess which dis- ' ' » I ' I ' ^ ' r " *: -I' Va -v, 'rf Fig. 170. — Actinomyces capros, stained mount from culture (Musgrave, Clegg, and Polk, in "Philippine Journal of Science"). charges, ulcerates, and may disappear, to reappear after an interval. Character of Disease and Lesions. — The disease in cattle is characterized by an enlargement of the superficial Ijnnph-nodes, which ulcerate and have much the appearance of farcy in the horse. The internal organs may be affected, with a resultant pseudo- tuberculosis. Immunity. — Methods of immunization have not been de- veloped. Bacteriologic Diagnosis. — The organism may be recognized in preparations from the lesions, but, for differentiation from other Actinomyces or Streptothrices, culture and animal inoculation are necessary. Transmission. — The disease is probably transmitted by wound infection, but this is not certainly known. Actinomyces caprss Synonyms. — Streptothrix caprce and possibly S. canis. 440 VETERINARY BACTERIOLOGY Disease Produced.— Actinomycosis (streptotliricosis) in goats, possibly in the dog. Silberschmidt, in 1899, publishes a description of an organism belonging to this group, which he isolated from a goat affected with a disease which closely simulated tuberculosis. It has been studied by several other investigators, who are in agreement that it should be regarded as a distinct species. Morphology and Staining. — Morphologically, it resembles the true bacteria more than other members of this group. The fila- ments are comparatively short and show httle tendency to form tangled masses, but separate easily. Both in culture and in the lesions rod forms and cocci are predominant. It stains with the anilin dyes rather irregularly, and is alcohol and acid fast. Isolation and Culture. — The organism grows rather readily upon most of the laboratory media, so that isolation is not a matter of difficulty. Upon agar the growth appears in two to three days as small, brownish colonies. It is somewhat more luxuriant upon glycerin and maltose agar, the colonies coalescing to give the growth a moist, mealy appearance. The colonies are light brown in color. Growth upon potato is similar. In bouillon the colonies develop upon the surface as fine dry disks, and form a pellicle, which finally settles as a sediment, the broth remaining clear. Physiology. — The organism is a facultative aerobe. Pathogenesis. — The organism produces tubercle-like lesions in the rabbit, guinea-pig, and monkey upon inoculation. It is not of any considerable economic importance. Actinomyces necropfaoras Synonyms. — Bacillus diphtherice vitulorum; B. filiformis; Streptothrix cuniculi; Actinomyces cuniculi; B. necroseos; Strep- tothrix necrophora; Bacillus necrophorus. Diseases Produced.— A large number of diphtheritic and ne- crotic pathologic conditions in animals, necrobacillosis. Loffler, in 1894, described this organism from calf diphtheria. Later Schiitz found it associated with the intestinal ulcerations of hog-cholera. It is now known to produce spontaneous disease in birds and in both domestic and wild animals, including cattle, sheep, goats, antelope, reindeer, horse, deer, roe, swine, kangaroo, guinea-pig, dog, monkey, and fowl. ACTINOMYCES GROUP 441 Distribution.— Bang succeeded in demonstrating the presence of this organism in the feces of normal hogs, but not in the intes- tinal contents of the cow. It is probably rather widely distributed in some locaUties. The infection has been described from various sections of Europe and America. Morphology and Staining.— The organism is a long, slender rod, usually bent more or less, although short rods and filaments may be observed. It is about 0.7 to 1.5 ju in diameter, and is generally thicker in cultures than in tissues. The filaments vary between 2 and 100 jti. In the tissues and colonies the filaments are matted together, but definite branching has not been satisfactorily demon- strated. The stained rods are usually beaded, giving rise to a very characteristic appearance. Involution forms, as long clubs, frequently occur. The organism is non-motile and does not pro- duce spores or capsules. It stains readily with the common anilin dyes, but is Gram-negative. Jensen has developed the following method of staining the organism in tissues: The piece of tissue is fiixed in Miiller's fluid, washed, and hardened in alcohol. The sections are stained some minutes in toluidin-safranin, dehydrated with a concentrated alco- holic solution of safranin, and decolorized in a concentrated solution of fluorescin in clove oil, then in clove oil, alcohol, counterstained in aqueous methyl-green, dehydrated by alcohol, xylol, and mounted in balsam. The necrosis bacilli are stained red, the tissue, green. None of the other bacilli studied by Jensen give this reaction. The pecuUarities of staining, and the possession of easily stained granules, or of a vacuolate protoplasm, have caused some authors to group this germ with the diphtheria bacillus, but these appear- ances are even more characteristic of certain of the Actinomyces, particularly those isolated from soil. Isolation and Culture. — Actinomyces necrophorus is most easily isolated in pure culture by inoculating diseased tissue into rabbits or white mice. The pure culture may then be secured from the infected organs. The cultural characters are all modi- fied by the fact that the organism is a strict anaerobe. Colonies may develop upon the surface of serum agar plates if the oxygen is removed by the alkaline pyrogallate method, but not, according to Mohler and Morse, in an atmosphere of hydrogen or in a vacuum. They appear in forty-eight hours as minute, 442 VETEKINABY BACTERIOLOGY dirty white, round, opaque colonies, with gas bubbles developing below the surface. In seventy-two hours the colony appears wooly, and the central portion, upon microscopic examination, is shown to be a felted mass of threads with a border of long, wavy filaments. The addition of serum to media increases materially the luxuriance of the growth. Bouillon becomes turbid, and then gradually clears, with subsidence of the organism. Gelatin is not liquefied. Physiology. — Actinomyces necrophorus is an obligate anaerobe. Its temperature growth limits are between 30° and 40°, with an optimum at about 35°. The organism is readily destroyed by Pig. 170a. — Actinomyces necrophorus (Mohler, Bureau of Animal Industry, Circular No. 160). disinfectants. No pigment is produced. A very characteristic odor, "between the odor of cheese and of glue," may be noted in both cultures and lesions. No enzymes capable of liquefying gelatin or blood-serum are produced. Gas is formed in bouillon. Milk is not coagulated nor are acids formed. Indol is produced. Pathogenesis. — Experimental Evidence. — Rabbits may be read- ily infected with the Actinomyces necrophorus. A subcutaneous injection of a small amount of necrosed tissue results in the death of the rabbit in about a week. The inoculated area is necrotic to some depth, and to a distance along the surface of J to 1, inch from the point of injection. The necrosis is complete and ACTINOMYCES GROUP 443 the tissues wholly disintegrated. In some cases gas bubbles may be observed. The inoculation of pure cultures results in death more slowly; frequently two weeks are required. The animal dies suddenly after a series of convulsions. Mice are readily infected. Guinea-pigs are much more refractory, but occasion- ally die as a result of inoculation. There seems to be abundant experimental evidence to connect the Actinomyces necrophorus with many types of necrosis in animals. There is no evidence that the organism enters the normal healthy unbroken skin. It is usually a secondary invader. Character of Disease and Lesions Produced. — Mohler and Wash- burn have given an excellent r^sum^ of the conditions under which this organism has been found. In many of these conditions it has not been satisfactorily established that this organism is the sole cause, for pyogenic cocci and other organisms may produce the same changes. More work is needed upon these infections. The possible presence of this organism in necrotic infections of all kinds must be borne in mind. The organism has been reported from the following infections, and probably in most cases is re- sponsible for the accopapanying necrosis: necrotic dermatitis, ne- crotic scratches in the horse, necrotic pox in horses, cattle, goats, and hogs, several types of necrosis in rabbits, necrosis of the hoof in the horse, necrosis of the mouth and esophagus, ulcerative and necrotic vulvitis, vaginitis, and metritis, foot-rot of cattle, lip and leg ulceration of sheep, necrotic omphalophlebitis, and joint ill in young animals, necrosis in the alimentary tract and other viscera in many animals, and possibly even avian diphtheria. It is some- times of considerable economic significance, particularly in the so-called lip and leg ulceration of sheep. Some of the affections, particularly this latter, are known to be contagious. Much work still remains to be done, however, on the different infections and possible variations in virulence. The lesions produced in all tissues have many common charac- ters. They are essentially coagulation necroses with caseation. Metastatic infection is very apt to occur. The local lesion is described by Mohler and Morse as a "sharply circumscribed patch of yellowish or dull brown, sometimes greenish-white, homogeneous, structureless, dry, crumbly tissue debris of soft, cheesy consistence. 444 VETERINARY BACTERIOLOGY resembling compressed yeast, and manifesting a characteristic stench. The line of demarcation between the living tissue and the dead mass is a narrow hyperemia zone." A false membrane is formed over the surface as a "result of coagulation necrosis of the inflammatory exudate and entanglement in its meshes of the hya- line degenerated tissue-cells and leukocytes." Immunity. — It has been suggested that the organism produces a true toxin because of its intense local destruction of tissue, and because of the death of laboratory animals with many of the symp- toms of a toxemia. No toxin has been isolated, however, in spite Fig. 171. — Actinomyces madurce, stained mount from culture (Musgrave, Clegg, and Polk, in "Philippine Journal of Science"). of many attempts. Probably an endotoxin is produced. It is stated that intravenous injections of the organism into the goat confer an immunity. No practicable method of immunization has been developed. Bacteriologic Diagnosis.— The organism may be observed in mounts prepared from the tissue just surrounding the necrosed area. Its appearance is characteristic enough to differentiate it from other forms that may be present. Animal inoculations, preferably into the rabbit, are generally necessary to secure pure cultures. Transmission.— It is improbable that the organism ever gains entrance through the unbroken- skin or mucous membrane. ACTINOMYCES GROUP 445 Scratches, wounds, abrasions, or injuries of other types supply an infection atrium. The disease, however, must be regarded as mildly contagious. Actinomyces madurse Synonym. — Streptothrix madurce. Disease Produced. — Madura-foot, mycetoma, streptothricosis in man. Vincent, in 1894, cultivated an Actinomyces from cases of mycetoma or madura-foot in man. This disease occurs in certain tropical countries, as southern Asia and the Philippines. It is undoubtedly a different species from those already described. It is not known to affect animals in nature, but will infect the monkey upon intraperitoneal inoculation. Actinomyces of Other Infections Probably about thirty or more other species have been described belonging to this genus. In most cases they have been reported but once or have been incompletely described. As has before been emphasized, careful work is still needed in order to deter- mine the true number of valid species and their relationship to disease. CHAPTER XXXVIII -v.. C" -Vji^ BLASTOMYCETES The genus name Blastomyces is used to designate a group of pathogenic fungi having many points in common with the members of the genera Saccharomyces and possibly Torula. It is not certainly known that the forms thus classified are closely related among themselves, for it is a well-known fact that many of the Hyphomycetes, when grown in certain culture-media, will assume a form indistinguishable from the yeasts. It is possible, therefore, that some of the forms described as members of the genus Blasto- myces may be only growth stages of higher forms. Here, again, as has been emphasized in other groups, there is need still for careful morphologic and cultural studies of the various species that have been described, for some of them are very imperfectly known. An understanding of the morphology of the Blasto- myces can best be obtained by a prehminary discussion of the Saccharomyces or true yeasts. The one character which sepa- rates this genus from the Hyphomycetes is the difference in the vegetative method of reproduction. This is accompUshed by budding. The mother-cell is usually oval or round, and, at various points on its surface, produces small buds, which enlarge and soon separate as independent cells. Occasionally these cells may remain together and become considerably elongated. By continued budding from the tip, a chain of cells is formed simu- lating a mycehal thread of one of the Hyphomycetes. The V. i^' l|i:^i^ Fig. 172. — Brewer's yeast, Saccharo- myces cerevisioe (Gunther). BLASTOMYCETES 447 cell differs from that of a bacterium by the presence of a definite nucleus, which may be demonstrated by careful staining technic. Spores are produced by some yeasts when the cells are brought under the right conditions of moisture, oxygen pressure, and temperature. Generally, two, four, or six are produced within a single cell. This type of spore formation relates such forms definitely to the higher fungi, known as Ascomycetes or sac fungi. In these fungi the spores are borne in a sac or ascus, and the cell of the yeast, with its contained spores, is supposed to represent a simple type of ascus. Resting cells, consist- ing of heavily walled or encapsulated cells filled with protein, glycogen, or oil-granules, are formed by many .yeasts. These granules may resemble spores and have doubtless many times been mistaken for them. When brought under favorable con- ditions the cell, as a whole, begins again to produce buds, showing conclusively that the granules cannot be regarded as spores. Among the true yeasts those which are not known to produce spores are sometimes placed in the form genus Torula. It is not cus- tomary to make this distinction among the pathogenic yeasts or Blastomyces, although it has been attempted by some authors. As here used, the term Blastomyces includes all those pathogenic forms which reproduce regularly by budding, and may or may not produce ascospores. The organisms belonging to this group are Blastomyces farci- minosus, B. dermatitidis, and B. coccidioides. Blastomyces farciminostss Synonyms. — Cryptococcus farciminosus ; Leishmania farcimi- nosa. Disease Produced. — Blastoipycetic epizootic lymphangitis or pseudofarcy in the horse. Rivolta, in 1873, first described the organism associated with this disease. Tokoshige, in 1897, cultivated the organism and determined its classification. It has been studied since that time by several investigators. Galli-Valerio contends that this organ- ism is a protozoan and not a Blastomyces. There is' a clinically 448 VETERINARY BACTERIOLOGY similar disease, since described in Europe and the United States as due to a member of the mold genus Sporotrichum. The organ- ism described by Tokoshige should be reinvestigated. It is pos- sible that it may prove to be a Sporotrichum also. Distribution. — The disease is known from Italy, Egypt, Tunis, England, France, northern Europe, Japan, India, the Philippines, and possibly the United States (North Dakota, Iowa). Morphology and Staining. — The organism as it occurs in tissues does not show budding forms ordinarily, but reproduces by a series of sporulations. In mounts prepared from the tissues it has a double refractive contour, which makes it stand out dis- tinctly from the remainder, even when unstained. It is usually spherical or ovoid, 3 to 4 ^u in diameter. The cell contents may be homogeneous or granular. In culture-media the organism consists of hyphal and spherical forms. Cells with buds may be found, identifying the organism definitely with the Blas- tomycetes. Cells containing granules, and resembling closely the resting cells of the yeasts, are common. It has not been conclusively shown that true sporulation takes place in culture- m.edia. The organism stains readily with aqueous anihn dyes and is Gram- positive. The latter method of stain- ing is useful in demonstrating the The alcohol must not remain too long in contact with the organism or it will lose color. Isolation and Culture. — The Blastomyces fardminosus is isolated upon culture-media with considerable difficulty. Several investi- gators, particularly those holding to the protozoan nature of the organism, deny that it has been accomplished. In view of the success which has attended the cultivation of an essentially similar organism in man, there seems no good reason to deny that Toko- shige and others have succeeded in securing it in pure cultures. A slightly acid medium is said to be more favorable than one which is alkaline. Growth is very slow in any event. Bouillon finally shows a white, flocculent deposit. Upon Fig. 173. — Blastomyces far- ciminosus, cells from culture- media (adapted from Toko- shige). organism in pus or tissues. BLASTOMYCETES 449 the surface of agar, gray-white granular colonies make their ap- pearance in the course of a month, and finally attain to a diameter of 1 to 4 mm. The colony is wrinkled, and can be removed only with difficulty. Growth upon gelatin is essentially similar. Potato seems somewhat more favorable, and growth occurs more rapidly, but is of the same character as on agar. Physiology. — The organism is aerobic. Growth occurs at room-temperature as well as at 37°. It does not liquefy gelatin. Sugars are not fermented with production of either acid or gas. Pathogenesis. — Experimental Evidence. — Guinea-pigs and rab- bits are not easily infected with pure cultures. Typical lesions have been produced in the horse by Tokoshige. They are readily produced by the injection of pus from natural infections. Character of Disease and Lesions. — The disease in the horse shows a marked superficial resemblance to farcy; The infection progresses through the subcutaneous lymphatics and forms dis- tinct nodules. These may suppurate. Metastatic infection of the internal organs occasionally occurs. Immunity. — ^No practicable method of immunization has been developed. Bacteriologic Diagnosis. — The organism may be readily ob- served in a mount of the pus from a lesion stained by Gram's method. Transmission. — It is supposed that infection is traumatic, that the organism gains entrance through cutaneous lesions. The disease not highly contagious. Blastomyces dermatitidis Synonyms. — Saccharomyces dermatitidis; O'idium dermatitidis. Disease. — Blastomycetic dermatitis in man. Busse, in 1894, first described an organism of this group as the cause of a fatal infection in man. Gilchrist, in 1896, found a similar organism as the cause of a dermatitis in man. Since that time the organism has been repeatedly isolated and studied. Distribution. — Blastomycetic dermatitis has been reported from the United States, the Philippines, and Europe. Morphology and Staining. — It is probable that several distinct 29 450 VETERINARY BACTERIOLOGY species have been grouped together; that is, not all cases have shown morphologically identical organisms to be present. They have not as yet been sufficiently studied to justify their separation as distinct species^ but will be treated rather as one polymorphic form. Careful morphologic and cultural studies are still needed. In the tissues the organisms appear almost invariably as budding forms. The cells are spherical or ovoid, from 10 to 17 /x in diameter. They are distinctly double ' contoured. Several investigators have observed what they believe to be sporulating forms. The cells are frequently granular or vacuolate, resembling \ Fig. 174:.— Blastomyces dermatiiidis. Budding forms and mycelial growth from glucose agar (Irons and Graham, in "Journal of Infectious Diseases"). typical yeast cells in this respect. Upon culture-media numerous hyphal threads and budding cells are produced. The organisms do not stain very readily with the aqueous anilin dyes. Isolation and Culture.— Isolation of the organism is usually attended with considerable difficulty. Blood-serum slants are usually employed and inoculated with material from the lesion. Repeated trials are sometimes necessary before a growth is secured. After once accustomed to growth on artificial media, no difficulty is found in getting the organism to develop upon most of the common culture-media. BLASTOMYCETES 451 Small white colonies showing a mold-like surface, due to the formation of numerous aerial hyphse, develop upon the surface of agar. The addition of dextrose to the medium somewhat in- creases the luxuriance of the. growth. In bouillon a fluffy, mold- like colony or a granular sediment develops without any e\'idence of the diffuse clouding generally found in yeast cultures. Gelatin is not liquefied, Milk may or may not show coagulation and slight digestion of the casein. Potato is a favorable mediimi. Fig. 175. — Blastomyces dermatitidis (Hamburger, in "Journal of Infectious Diseases"). Physiology. — Growth occurs at room-temperatures, but some- what more luxuriantly at 37°. The organism is aerobic and facul- tative anaerobic. Gas and acids are not produced in carbo- hydrate media. Pathogenesis. — Experimental Evidence. — Guinea-pigs and rab- bits may be infected, with production of either a local abscess or generalized blastomycosis. The lesions resemble in their essential characters those found in the human body. Character of Disease and Lesions. — In man a papule generally appears upon one of the extremities, the face, or more rarely, 452 VETERINARY BACTERIOLOGY elsewhere. A viscid pus is exuded, and there is commonly con- siderable enlargement. Healing with an abundant formation of cicatricial tissue gradually occurs. Usually the lymphatics are not involved, the disease differing in this respect from the lymph- angitis of the horse. The course of the disease is usually chronic, and it may persist for years, new ulcers appearing successively on various parts of the body. Generalization has been reported in a considerable number of cases. The skin lesions have sometimes been confused with those of syphihs and tuberculosis. Primary infection of the lungs has been shown in several cases. Immunity. — No method of estabhshing immunity has been developed. Bacteriologic Diagnosis. — This may be accomphshed by direct microscopic examination of the pus. Phalen and Nichols state that the organism may be most easily demonstrated by treating unstained sections with potassium hydrate and mounting in glycerin. Transmission. — It is supposed that infection sometimes occurs through wounds, but several instances have come to light in which the infection was primarily pulmonary and the skin lesions sec- ondary. Blastomyces coccidioides Synonym. — Oidium coccidioides. Disease Produced. — Blastomycosis, so-called coccidioidal gran- uloma in man. Posades and Wernecke, in 1892, first reported a case of so- called coccidioidal granuloma from Argentina. In the United States the disease has been recorded principally from California, particularly in the San Joaquin Valley. Morphology. — This form is of particular interest, because the budding or true blastomyces form very rarely occurs in the tissues and multipUcation is almost wholly through sporulation. The organism in the tissues is spherical and doubly contoured. It may reach a diameter of 30 fi or even more. Budding forms have been . recorded from pus. In artificial media the organism resembles a mold, but budding forms may be observed. The method of reproduction in tissues, by the formation of spores within the mother-cell and their liberation by a rupture of the cell membrane, BLASTOMYCETES 453 has led some investigators to believe that the organism is really a protozoan. However, sporulation of this general type occurs in the yeasts. The question seems to be definitely settled in favor of the plant hypothesis by the culture forms on artificial media. Pathogenesis. — ^Whether this organism should be separated from the preceding is uncertain. Frequently no cutaneous lesions are produced; the infection is systemic and probably always fatal. Meningeal involvement is common. CHAPTER XXXIX MOLD OR HYPHOMYCETE GROUP The term Hyphomycete, as usually interpreted, is one of con- venience only, for within this group are included members of the four great divisions of fungi generally recognized by botanists. The members of the group are many times not closely related. They all resemble each other in having a plant body or mycelium, which consists of threads or hyphce made up, in the majority of forms, of chains of cells. Reproduction is not generally by budding, although this may sometimes occur. The hyphae themselves break up into spores, or spores are borne at the tips of hyphse that have been differentiated for the purpose. The hyphae may unite to form a more or less solid mass, sometimes tissue-like in appearance. This mass may remain viable when dried for a considerable time, and may function in much the same manner as a resistant spore in tiding the organism over, unfavorable conditions. Such a structure is called a sclerotium. The names given to these various types of structures have already been discussed under the heading of Morphology in Section I. The Hyphomycetes, for the most part, belong to the division of fungi termed Fungi imperfecti by the botanist. The name is derived from the fact that these fungi are not known to produce perfect or sexual spores. Hundreds of genera and thousands of species have been described as belonging to this group. Many of these are doubtless simply developmental stages of forms that are known under other names. The life-history of some fungi has been found to be so complex, and consists of so many stages, that five or six names have been apphed and the different stages put in different groups of fungi, until it was found that all were the same polymorphic species. Unfortunately, careful mor- phological study has not been made of the pathogenic members of this group, and there is the greatest confysion in the nomen- clature. The pathologists and bacteriologists who have de- MOLD OR HYPHOMYCETE GROUP 455 scribed the organisms have rarely paid any attention to their botanical relationships, and the organisms themselves, for the most part, have been ignored by the botanist in his classification. As before stated, the possession . of a more or less definite mycelium, a more or less "mold-like" growth, and the general production of spores are all that is needed to.include an organism in the group. Many of the organisms of the group are very com- mon in nature and are pathogenic only under exceptional condi- tions, while others have so adapted themselves to a parasitic existence that they may be regarded as obligate parasites. Many, too, have been noted once or twice only in certain pathologic conditions, and it is by no means certain that they were more than accidental parasites. The genera of molds containing species of known pathogenicity are — Penicillium. Fusarium. Sporotrichum. Microsporon and Trichophyton. Achorion. O'idium or Oospora. The Genus Aspergillus The AspergiUi are widely distributed in nature. They are abundant in the soil and on decaying materials of all kinds. Their spores are common in the air, and cultures may readily be secured in most localities by simple plate exposure. They are not, however, present in such numbers as the genus next to be de- scribed, Penicillium. Several hundred species have been de- scribed, and by some authors the genus is subdivided into two genera, Aspergillus and Sterigmatocystis. Aspergillus is placed by the botanists among the Ascomycetes or sac fungi, because at one stage in the life-history sexual repro- duction occurs, resulting in the formation of sacs filled with spores. This phase of the life-history has been worked out in but few species ; however, it is probable that it occurs in all when grown under the right conditions. 456 VETERINARY BACTERIOLOGY The mycelium of Aspergillus is colorless and hyaline, much- branched, penetrating for a short distance into the substratum or medium, and usually sending up aerial hyphse, which give the colony a floccose or downy appearance. The hyphse are septate, that is, cross walls are formed and the cells are divided from each other by them. Asexual reproduction takes place by the forma- tion of enlarged, erect, spore-bearing hypha, called conidiophores. These conidiophores are inflated at the tip and become covered Fig. 176.— :-Morphology of the Aspergillus glaucus: a, Mycelia and perithecia on the surface of the medium, with a single conidiophore; d, a very young peri- thecium; e, cross-section through a perithecium, somewhat older; /, cross- section through a mature perithecium, showing the asci and the ascospores (as); bV-, isolated asci; cc^, ascospores ripe and germinating {c h^ d e f after deBary, ab c^ after Wehmer). with papillae, which develop into short stalks, called sterigmata (singular, sterigma). The sterigmata may branch once or many times, giving rise to bunches of secondary sterigmata, or they may remain unbranched. The species with branched sterigmata are frequently grouped together into a genus Sterigmatocystis. From the tips of these sterigmata spores or conidia are abjointed and hang together to form long chains. The spore mass at the tip of the conidiophore is termed a head. The spores are usually colored green, brown, yellow, or black, or in a few species they are colorless. MOLD OR HYPHOMYCETE GROUP 457 They are spherical or oval in shape. Their surfaces are not easily wetted; they are easily detached, and are readily carried about by currents of air. This explains the readiness with which birds and some animals become infected in the respiratory tract when fed on moldy grain or fodder. When the spores come under favorable growth conditions they germinate and reproduce the mold. If growth conditions are right, careful observation will enable one to discover the sexual stages in the reproduction. Two filaments, somewhat differentiated, begin to twist together until they form a typical cork-screw. The cell contents fuse and fer- tilization is effected. A tangled mass of threads arises about this cell, forming a compact layer or covering termed the peri- thedum. The enclosed cell grows rapidly, and produces a con- siderable number of enlarged cells, each of which eventually is found to contain spores, usually eight in number. These cells or sacs are called asd (singular ascus), and the spores are termed ascospores. These, like the conidia, when brought under favorable growth conditions, reproduce the mold. An Aspergillus may con- tinue to multiply indefinitely without the sexuaV stage developing; it is not improbable that some species have altogether lost the power of reproducing other than by means of the conidia. Several species of Aspergilli have been described as pathogenic. Doubtless these are normally saprophjrtes, and only produce disease under exceptional conditions. Aspergillus fumigatus Diseases Produced. — Aspergillosis of birds; pneumomycosis in man and many animals. The occasional presence of Aspergillus in lung infections has been known since early in the nineteenth century. Probably Mayer and Emmet, in 1815, were the first to note its presence in the lungs of a bird, in this instance a jay. Since that time the organism has been reported many times. In most cases no careful species determination was made, but the probabilities are greatly in favor of Aspergillus fumigatus being the species responsible. Aspergillosis has been reported from the stork, raven, flamingo, eider-duck, parrot, pigeon, chicken, hawk, bullfinch, plover, 458 VETEEINART BACTERIOLOGY pheasant, bustard, duck, goose, ostrich, swan, and turkey among birds, from the horse, dog, and cow among animals, and from man. It has been reported from Europe and the United States. Morphology.— In culture-medium it forms greenish or bluish gray or later brownish masses. The conidiophores are abundant, but short. The enlarged tip of the conidiophores is hemispheric, and 8 to 20 /x in diameter, bluish-green, and later brown. The Fig. 177. — AsTpergiUus fumigalus: 1, Optical section through a conidiophore; 2, conidiophore and conidia; 3, conidia; 4, a, perithecium; h, an isolated ascus; c, d, ascospores, front and lateral view; 6, swollen hyphse, hi, and conidiophores (4, a-d, after Grijns, remaining after Wehmer). perithecia with ascophores have been observed in culture-media. In the lung tissues the branching mycelium may be observed on microscopic examination, and the sporophores may be seen pro- jecting into the air-sacs, where the conidia are produced. These spores are never formed except in the presence of oxygen. Isolation and Culture. — The Aspergillus fumigatus may be readily isolated from the lesions upon almost any of the commonly used artificial media, particularly when spores are produced. For MOLD OR HYPHOMYCETE GROUP 459 the best development the medium should be shghtly acid. It develops readily upon potato and bread. Colonies become visible in a day, usually as tiny, white, cottony growths, which, within a few days, turn green, due to the formation of the spores of that color. Physiology. — The Aspergillus fumigatus is an aerobe. The optimum growth temperature is from 35° to 40°. According to Mohler and Buckley, growth occurs, but spores do not form below 20°. The spores are resistant to high temperatures. They Fig. 178. — Aspergillus fumigatus from a culture on agar (Prankel and Pfeiffer). have been found to survive an exposure of seven hours at 65°. Twelve hours' contact with a 5 per cent, solution of phenol is not sufficient certainly to destroy them. They can withstand desicca- tion indefinitely. Pathogenesis. — Experimental Evidence. — The organism pro- duces death within a few hours or days when injected intravenously or intrathoracically into the chicken. The pigeon is particularly susceptible to injections. Rabbits and guinea-pigs likewise suc- cumb, usually from a generalized infection. There is sufficient experimental evidence to justify the conclusion that Aspergillus fumigatus may produce a primary and fatal infection in many animals. 460 VETERINARY BACTERIOLOGY Character of Disease and Lesions Produced.— By far the greatest number of cases of aspergillosis have been reported from birds. The lesions are generally located in the lungs, air-sacs, and hollow bones, where the spores may readily lodge. In man and animals, particularly the horse, the usual picture is an infection of the lungs and air-passages, but occasionally of the mucous membranes of other parts of the body. Metastatic infection of other organs is not infrequent. The organism causes the development of nodules not unlike those of tuberculosis. In the lungs the tubes are fre- quently occluded by the green fructifications of the fungus. There is more or less necrosis of tissue immediately surrounding the or- ganisms. Immunity. — Several investigators claim to have produced toxic substances, if not true toxins, by the growth of the organisms in artificial media. These claims have not been sufl&ciently sub- stantiated, although there is considerable a priori evidence, from the character of the lesions and symptoms, that powerful toxic substances of some kind are produced. No method of suc- cessful immunization has been developed. Bacteriologic Diagnosis. — A diagnosis may usually be made by the character of the lesions and the appearance of the green spores. A microscopic examination of. the scrapings of the in- fected mucous membranes should reveal the spores and character- istic conidiophores without difficulty. Transmission. — The organism doubtless grows on decajdng organic matter outside the body. The feeding of moldy grain or fodder may give ample opportunity for infection by inhalation. The preponderance of pulmonarj' primary infections shows that inhalation probably is the common method of infection. Aspergillus flavus This organism has been described by various investigators, who beheved it to be, in part at least, responsible for blind staggers or meningo-encephalitis in horses. It occurs in great quantities on moldy corn and other grains. Although the complete data have not been presented, the work of Haslam indicates that it is of some pathogenic significance. Morphology. — The sterile hyphse are cobwebby and white. MOLD OR HYPHOMYCETE GROUP 461 The conidiophores are erect. Conidia are 5 to 7 /i in diameter, Fig. 179. — Aspergillus flavus: 1, 2, 3, 4, Various stages in the development of conidiophores; 5, section and surface of a hypha, showing the numerous colorless granules with which it is covered; 6, natural size of the fungus; 7, conidia (Wehmer). globose. Spore masses are yellow and yellowish green. Sclerotia are small and dark. Aspergillus niger This organism occurs under conditions similar to the preceding, and is believed also to be pathogenic to horses and other animals that consume grain infected with it. Morphology. — The mycelium is at first white, then darker, abundant, and penetrates the medium to a considerable distance. The conidiophores are long and the spores borne up at some dis- tance from the surface of the substratum. The sterigmata are branched. The conidia are 3.5 to 4.5 iJ, in diameter, roughened. The spore masses ultimately become black, and may be readily differentiated in this manner from the two preceding. This 462 VETERINARY BACTERIOLOGY Fig. 180. — Aspergillus niger: 1, 2, 3, 4, Stages in the development of the conidiophores; 5, conidia; 6, detail, showing the branched sterigmata; 7, 8, sclerotia; 9, natural size of the fungus (Wehmer). organism has also been found in the ear and in lesions in the lungs. Other Species of Aspergilli Several other species of Aspergilli have been reported as pathogenic. Among them are Aspergillus nigrescens, A. subfuscus, and A. glaucus. These produce nodular mycotic foci in the in- ternal organs of laboratory animals into which they have been injected. They do not commonly produce infection under natural conditions. The Genus Penicillium Penicillium is closely related to Aspergillus, the principal difference being the manner in which the asexual spores or conidia are borne. The conidiophores are erect and much branched at the tip, the branches arising in whorls and are not enlarged at the apex. From the end of each ultimate branch a chain of spores is abjointed,. MOLD OR HTPHOMYCBTE GROUP 463 giving to the organism under the microscope the appearance of being covered with httle brooms. The sexual stage is essentially similar to that of Aspergillus. PeniciUia are even more common than the AspergiUi. Thej' occur as blue or green molds upon fruit, and upon a great variety of decaying materials. Of the hundreds of species of Penicillium that have Vjeen described, the majority are green or bluish-green in color, but white, gray, yellow, orange, and brown forms are known. Fig. 181. — Penicillium glaucum: a, o', Tips of oonidiophores showing the characteristic method of branching and the chains of spores; b, perithecium; c, asci; d, ascospores (Brefeld). None of the species of Penicillium are known to be harmful, but their constant presence in moldy silage and grain which has poisoned anim-als makes it necessary to consider them in any discussion of forage poisoning. The Genus Fusarium The members of this genus are nearly all saprophytes or plant parasites. Fusarium is included among the Fungi imperfecti, as a sexual or perfect stage is unknown in the life-history of most 464 VETERINARY BACTERIOLOGY of the species. Webber has found an ascus stage in one species, and concludes that the genus Necomospora of the Ascomycetes is the perfect form; in other species it is the genus Gibberetta. Fusarium is characterized by its loose, spreading, cottony myceUum with numerous cross walls, i. e., septate. The conidio- phores are not markedly different from the sterile hyphse, and are usually branched. The conidia are borne at the tips of these branches. They are long, slender, sickle or crescent shaped usually, and divided into several or many cells by cross walls or septa. Several species of Fusarium are found commonly on grains and moldy corn. This fungus has been beheved by some investigators to be of significance in forage poisoning. It is one of the several forms which must be considered in a determination of the poisonous properties of forage. One species, the Fusarium equinum, is believed to produce dermatitis in the horse. Fosarium eqtiinum Disease Produced. — Itch disease, associated with sarcoptic dermatitis. Norgaard, in 1901, noted the presence of a Fusarium in a derma- titis of horses in the State of Oregon, and proposed the name Fusarium equinum for the fungus. Melvin and Mohler later studied the disease in greater detail. The disease has been re- ported only from this one locality, but in this instance affected several thousand horses on the Umatilla Indian reservation. Morphology. — The mycelium upon culture-media is septate and branched. Three forms of spores are produced. The microconidia are small and oval, one or two celled. The macroconidia are large, sickle shaped, three to five septate, and pointed at the ends. They are 25 to 55 /x long by 2.5 to 4.5 ix wide. Chlamydo- spores are formed in the myceUal threads by a cell rounding up to a diameter of 8 to 15 /x and becoming densely granular. The spores may be recognized in the hair-follicles of the diseased ani- mals. Isolation and Culture. — No difficulty was experienced in secur- ing a growth of the organism on artificial media. The more MOLD OR HYPHOMYCETE GROUP 465 favorable media are potato and bread, but good growth will take place on glucose or plain agar. The growth is white and cottony, and the spores are produced in abundance. Pathogenesis. — Inoculation experiments were unsuccessful, so that the evidence of pathogenesis rests entirely upon the constant occurrence of the organism in the disease in question. Itch- mites {Sar copies equi) were found, but the investigators believe, their numbers insufficient to account for the disease. It is entirely Fig. 182. — Fusarium eqwinum, mycelium and conidia (Melvin and Mohler, Bureau of Animal Industry). possible that the organism is a secondary invader, or produces the disease in a kind of symbiotic relationship with the Sarcoptes. The fungus seems to enter the hair-follicles, penetrates between the epidermal cells, and involves the surrounding skin, causing an intense itching. The body becomes covered with a crust or scurf, at first gray and afterward darker. The presence of the organism in the hair-follicles causes the hairs to fall out, resulting in an almost complete alopecia. 466 veterinaky bacteriology The Genus Sporotrichum Authorities differ greatly in the delimination of this genus. According to botanists, the genera Microsporon and Trichophyton are synonyms of Sporotrichum. Pathologists and bacteriologists in general, however, make a distinction between them. The classification of the latter will be adopted here and the term Sporo- trichum used in the narrow sense. Sporotrichum is distinguished by the production of definite hyphae, which are usually creeping and irregularly branched. Definite conidiophores are not developed, or consist only of small side branches. The conidia are borne either on the sides or ends of the hyphse, singly or in clusters. They are usually very numer- ous, ovoid or spherical in shape, and hyaline or rarely hghtly colored. The molds belonging to this group are in need of careful study and revision, as there is great uncertainty concerning many of the species. One, or possibly two, species of Sporotrichum have been shown recently to be of considerable pathogenic significance. Sporotricham beormanni Synonym. — Possibly Sporotrichum schenkii. Disease Produced. — Sporotrichosis in man and animals, one type of epizootic lymphangitis in horses. Schenk, in 1898, and Hektoen and Perkins, in 1900, described a species of Sporotrichum causing multiple abscesses in man. de Beurmann, in 1903, described similar fornis from France. The disease has been reported in man and rats from Brazil, from man in California, Argentina, Germanj'^, and in the horse in the United States and Madagascar. This disease is probably quite wide- spread, but has not been recognized, or has- been confused with others, imtil recently. It is to be differentiated sharply from the true epizootic lymphangitis of the horse. An excellen t discussion of the organism in its relation to disease in the horse was contributed by Page, Frothingham, and Paige in 1910. Morphology. — The examination of the material from culture is most easily made in a hanging drop. The hyphse are slender and septate. Spores or conidia are borne at the tips of side branches; usually a number are formed successively and are found then MOLD OR HYPHOMYCETE GROUP' 467 in clusters. The conidia are small, oval or spherical. They fre- quently bud to some extent, and resemble somewhat the cells of Blastomyces. The hyphse stain easily with the common anilin dyes and are Gram-positive. In using the latter stain the alcohol must not remain too long in contact, otherwise the stain will be removed. Whether or not the organism ever de- velops a perfect or sexual stage is not known, but it does not seem probable. Isolation and Culture. — The organism may readily be isolated from pus from the lesions. Potato is the most favorable medium. Fig. 183. — Sporotrichurk heurmanni, from culture showing the mycelium and spores (Page, Frothingham, and Paige, in "Journal of Medical Research"). Original isolations show at the end of a week, transplants at the end of two or three days, as white, filamentous colonies. These enlarge, become darker at the center, and finally turn dark brown or black, frequently surrounded by a rim of white. The colony becomes wrinkled. Upon gelatin the growth remains white. Liquefaction begins in from three to ten days or even later. The addition of dextrose causes the center of the colonies to darken. In agar, and particularly in neutralized glycerin agar, growth is good, and the colonies remain white. Blood-serum is not lique- fied. In litmus milk growth occurs with httle change in the 468 VETERINARY BACTERIOLOGY medium, or coagulation without acid production may take place after the lapse of- several weeks. In liquid media growth occurs in the form of more or less separated colonies, usually accompanied by a surface pellicle. Physiology. — The organism is an obligate aerobe. Growth occurs best at 25° to 28°, but is not prevented at 37°. The spores resist desiccation for considerable periods. Acid is produced from dextrose, but not from lactose, mal- tose, saccharose, mannite, dulcit, ado- nit, inulin, or raffinose. Gas is not produced from any sugar. No indol is formed. Gelatin is slowly liquefied. The organism is destroyed at a tem- perature of 60° for five minutes. Pathogenesis. — Experimental Evi- dence. — There is an abundance of evi- dence that Sporotrichum beurmanni is pathogenic for man and animals. Ac- cidental laboratory infections have taken place in man. Inoculation of pure cultures into mice and white rats gives rise to abscesses at the point of inoculation, and the infection gradu- ally extends. Guinea-pigs and rabbits are infected with greater difficulty. An infection somewhat resembling farcy develops upon inoculation into the horse. Character of Disease and Lesions Produced. — The infection is usually benign in character in the horse. There is no fever reaction during disease. Nodules develop which are gen- These nodules scarcely Fig. 184. — Sporotrichum beurmanni, culture and col- onies on potato (Page, Proth- ingham, and Paige, in "Jour- nal of Medical Research"). the course of the erally spherical and sharply delineated. rupture, but pus accumulates at the center, the skin above is thinned and softened, serum exudes from the surface, the hair is loosened, and a crust holding the hairs together is formed. The ulcers are crateriform, and usually contain a little creamy pus. MOLD OR HYPHOMYCETE GROUP 469 Healing is accomplished by granulation. Sections of nodules from laboratory animals reveal the organism in the tissues. Immunity. — No toxins have been demonstrated for this organ- ism. Widal has shown the presence of agglutinins for the spores in the blood of infected individuals. No method of immunization, based upon the organism or its prodycts, has been developed. Bacteriologic Diagnosis. — This may be accompHshed by animal in- oculation, thus securing the organism in pure culture. Widal claims that the agglutination of spores will take place in dilutions as high as 1 : 800, but the same reaction takes place in lower dilutions with the blood-serum of individuals having actinomycosis. Fig. 185.— Sporotrichum Bloch found that a bouillon filtrate beurmanni, in a section of a J. Ill, , 1 . • mesenteric abscess of a rat from an old culture would m man give ^^^^^^^ f^„^ ^-^-^^^y the von Pirquet cutaneous reaction, as was described in the chapter of Bacillus tuberculosis. Gougerot recommends for the skin reaction the use of sporotrichosin, a sterile suspension of killed Sporotrichum beurmanni in salt solution. Transmission. — The disease is transmitted by intimate contact usually. It has been found to be transferred from animals to man. Probably the organism usually gains entrance through abrasions of the skin. The Genera Trichophyton, Microsporum, Achorion, and OlDIUM The organisms belonging to the two genera. Trichophyton and Microsporon, are frequently included together under the single genus Sporotrichum. The two names. Trichophyton and Micro- sporon, are also used very loosely and interchangeably. A care- ful study of the relationships of the various forms is needed. No very clear differentiation of this group from the preceding can be given. Organisms belonging to these genera are the cause of many skin and hair infections in man and animals. 470 VETEKINAKY BACTERIOLOGY The Genus Trichophyton The species belonging to this genus and the two following genera have been studied most carefully by Sabouraud, and his classifica- tion has been extensively used. The various species described are all the causes of diseases in man and animals, known as Jierpes tonsurans and ringworms. AH invade the hair, compactly filling its interior with a mass of parallel hyphse extending longitudinallJ^ The hair may be ex- amined directly under the microscope by first immersing it for a few seconds in a warm solution of 30 grams of caustic potash in 70 c.c. of water. The filaments are composed entirely of quad- rangular cells 4 to 5 m in length and about the same width. These chains are quite regular in their arrangement, and are straight. When branching occurs it is by dichotomy, but is not abundant. These organisms may be cultivated readily on special maltose (or other sugar) agar. Isolation and Culture. — Pure cultures are secured with difficulty, as the skin and hair are generally filled with bac- teria which overgrow the mold. Krai has suggested pulverizing hairs with fine sand or silicon powder and pouring gelatin plates of various dilu- tions. Kitt has taken advantage of the resistance of the organism ' to destruction by alkahes by washing the hairs and scales from an infected area with a solution of KOH, which removes most of the bacteria without materially injuring the mold spores. Potato is a favorable medium for growth. A rather velvety, wrinkled, relatively heavy membrane forms which may be white or colored. Gelatin is slowly hquefied. Sabouraud states that there is great variation in the appear- ance of cultures in different media. A microscopic examination of a mount from culture-media shows the development of a dense Fig. 186. — Trichophyton tonsurans horn agar plate culture (Giinther). MOLD OR HYPHOMYCETE GROUP 471 mass of branched hyphse definitely septate, slender, and hyaline. The spores are borne laterally on the branches of the hyphse, usually sessile, one celled, and ovoid or spherical in shape. Some species also develop chlamydospores in the mycelium. The species of Trichophyton are divided into two principal groups, Endothrix and Ectothrix. In the former the chains of cells in the diseased hair are entirely within the hair, in the latter they are both within and without, forming a coating over the hair surface. Types of Endothrix. — The Endothrix group of species according to Sabouraud contains about fifteen species, all parasites on man. The best known of these are Trichophyton tonsurans (the Tr. crateriforme of Sabouraud), Tr. sulfurerum, and Tr. cerebriforme. Among 500 cases of dermatomycoses observed by Sabouraud the first named produced 115, the second 52, the third 39, the fourth 13 of the infections. The other species were rare. Ninety-five per cent, of all trichophytoses in man were found to be due to the first three species hsted. Types of Ectothrix. — The species of Ectothrix are divided into two groups termed Microldes and Megaspores. The spore-like cells which make up the mycelial threads as they affect the hair are small, 3 to 4 ;u in diameter in the former, and large, 8 jj,, in the latter. Species of Ectothrix microides. — Eight species of Microides are recognized by Sabouraud. They are placed in two subgroups, the type gypseum with six species, and the type niveum with two species. The former all produce chalky colonies on media, the latter downy colonies. The first group contains species which attack man primarily, though readily transferred to laboratory animals, as the guinea-pig. One species. Trichophyton granulosum, attacks the horse. The two species of the niveum type. are Tr. Jelineum (or Tr. radians) and Tr. denticulatum. The former is primarily a parasite of domestic animals transferable to man and will be discussed later, the latter is a human parasite primarily. ■ Species of Ectothrix megaspores. — Seven species have been de- scribed, of which the following are parasitic on animals: Tricho- -phyton eguinum, Tr. caninum, Tr. verrucosum, Tr. discoldes. The remainder are known only from man. 472 VETERINAKY BACTEBIOLOGY Trichophyton granalosum Synonym. — Trichophyton gypseum. This species was described by Sabouraud in 1908 as the cause of a dermatomycosis of the horse. Morphology.— Typical of the Microides group. Spores on hairs 3 to 4 /i in diameter. Culture.— On maltose agar a powdery yellowish colony is developed, with relatively large granules on its surface. The center is usually irregularly umbilicate. Pathogenesis. — The disease in the horse is characterized by a development of small areas on which the hair is roughened. The hair over these areas is stuck together in small pencils. The hair falls with a scaly crust. The areas from which the hair falls remain naked and dry for some time; finally hair grows again slowly. The plaques are numerous and small. The disease is quite persistent. Inoculation on horses and guinea-pigs is successful. The disease is apparently occasionally transmitted to man. Trichophyton felineum Synonyms. — Trichophyton niveum; Tr. radians. Disease Produced. — Ringworm of cat, perhaps also in horses, cattle, sheep, and swine. Morphology. — Typical of group. The base of a parasitized hair shows large spores, 7 to 9 ju in diameter, forming a collar ad- herent to the hair and to edge of hair-follicle. Culture. — On maltose agar a powdery white colony is formed, umbilicate and irregularly wrinkled on the interior and with a large number of slender rays about the margin. Pathogenesis. — According to Brumpt this organism attacks animals, producing a characteristic type of ringworm. It has been found by Sabouraud to produce disease in man as well. By some authors the cat is regarded as the principal host. Trichophyton eqtrinum This organism was first isolated by Matruchot and Dassonville, in 1898, from a dermatomycosis of the horse. It was later studied by Sabouraud and by Gedoelst. The former regards it as the com- monest type of Trichophyton attacking the horse. MOLD OR HYPHOMYCETE GROUP 473 Morphology.— In the crust observed on the lesions of the horse the filaments are not abundant, generally rather straight or un- dulating. In the hairs the morphology is typical of the group, the large spores surround the hair base. Culture. — On maltose agar a white colony is formed, but it is not powdery, but velvety in texture. On potato the growth is moist and of the color of yellow ochre. Pathogenesis. — The disease in the horse manifests itself in small areas more or less numerous on the rump, shoulder, and back. The largest patches are not more than 1| cm. in diameter. Man may also be infected, as may also the guinea-pig. Trichophyton canJnom Matruchot and Dassonville, in 1902, described a follicuhtis in the dog caused by this organism. The hair dropped from the infected areas. The organism is termed a true ectothrix large- spored Trichophyton. The Genus Microsporum The species of this genus all produce dermatomycoses in man or animals. In all types the hair appears frosted, encased for several millimeters with a delicate white sheath. Under the micro- scope this is found to be made up of small spores, 2 to 4 /x in di- ameter, not disposed in chains, but arranged as a mosaic over the surface. In culture-media the hyphse frequently show racquet- shaped cells arranged in rows, which eventually develop into inter- calary chlamydospores. The spores are borne much as in Tri- chophyton, usually they are somewhat more elongate. Many colonies show a peculiar multicellular, fusiform, relatively large spore, the septse of which are all parallel and transverse. These are also sometimes found in species of Trichophyton. Peculiar hyphse with outgrowths resembhng a comb, the so-called pectinate hyphse, are also formed. The various species may be cultivated in the same manner as Trichophyton. Four species are known which produce disease primarily in man. They are relatively slow growers upon laboratory media and are inoculable upon animals with difficulty or not at all. Seven 474 VETERINARY BACTERIOLOGY species primarily pathogenic for animals have been described. These grow readily upon laboratory media and often infect man. These species are : Mierosporum lanosttm Synonyms. — Mierosporum caninum; M. canis. Disease Produced. — A dermatomycosis of the dog, also occa- sionally attacking the horse and children. The disease was first studied and the organism recognized by Bo4in and Almy, in 1897. It is one of the common skin diseases of the dog. Morphology. — Relatively few of the hairs of the diseased area from the dog are found to be infected with the fungus. It may readily be found in the scales from the infected skin. The poly- hedric superficial spores on the hairs are of the characteristic type. Culture. — Growth occurs readily on maltose and other sugar media. The colony on maltose agar shows a central area which is glabrous and powdery, surrounding this a ring or annulus of velvety appearance. The central portion may be umbilicate. Pathogenesis. — Sabouraud described four distinct phases of the infection in the dog. The areas infected show first a roughening of the coat, followed by a loss of hair, leaving a smooth skin. Usu- ally there then appears a pustular folhculitis, followed finally by healing and recovery. Infection of man occurs readily, particularly in children, pro- ' ducing a ringworm of the scalp. The disease in man shows some clinical differences from that caused by the far more common Mierosporum adouini. Inoculations upon animals is successful; particularly suscep- tible are dogs and guinea-pigs. Mierosporum felineam This species was first recognized by Fox and Blaxall, in England, in 1896, as causing a disease of the cat. Transmission to man was noted by these investigators as well as by Mewborn, of New York, in 1902. 'Morphologically and culturally the organism is typical of the genus. It has been experimentally transferred to the cat, the dog, and the guinea-pig. MOLD OR HYPHOMYCETE GROUP 475 Microsporum equinum This species was described by Bodin and Delacroix, in 1896. The organism produces disease in the horse and in man, and may be developed in the guinea-pig by inoculation. The Genus Achorion This genus differs in minor details from Microsporum and Trichophyton. When growing inside a hair it seems to destroy the interior so that air enters, and when such hair is mounted for examination the air bubbles are quite characteristic, this fre- quently alone being sufficient to differentiate this causal organism as belonging to this genus. In the hair the filaments are not so closely massed as those of Trichophyton frequently are, and do not" show the straight rows of spore-like cells. Upon culture-media the organism produces numerous heavy walled chlamydospores, both intercalary and apical, on short side branches. The pectinate hyphse are often formed as in Micro- sporum. Spores both single celled and multilocular (likewise re- sembhng those of Microsporum) are also produced. The two genera are evidently closely related, if, in fact, they should be sep- arated at all. Culture upon media can be accomplished as for the two pre- ceding genera. One species, Achorion schonleinii is the common species attack- ing man. Three species, A. quinckeanum {A. muris), A. gypseum, and A. gallince attack animals or birds. Achorion maris Synonym. — Achorion quinckeanum. It has been recognized for more than a half-century that favus is a disease which may be transmitted from the mouse to man, producing an infection not unlike that of Achorion schon- leinii. Achorion gallinse Synonym. — Lopophyton gallince. Disease Produced. — Tinea cristse gallae. Fowl favus. The disease was first studied by Gerlach, in 1858. Since that ■date it has been described by numerous writers. 476 VETERINARY BACTERIOLOGY Morphology. — A microscopic examination of the fungus collar which surrounds the base of feathers from infected areas shows a mass of tangled threads of the mycelium, much resembling chains of spores. Culture. — The organism may readily be secured in pure culture from the infected feathers. The colonies on maltose-agar are white, usually showing concentric rings and radial foldings. If grown at 30° the colony takes on a rose color. Fig. 187. — Achorion schonleinii, section showing the hyphse (Frankel and Pfeiffer). Pathogenesis.— In the fowl the disease may attack the head, the comb, the wattles, or the ear lobes. It usually begins by the development of one or many small white points. It appears as a pelhcle adherent to the adjacent epidermis. The spots increase in size and fuse. When the lesion appears on a surface with feathers, a sort of collar or cone forms at the base of each feather, and the plumes fall, each carrying with it its fungus collar. The bird may recover, but the disease is commonly quite persistent. The disease may be inoculated into man, the rabbit, and the guinea-pig. MOLD OR HYPHOMYCETE GROUP 477 Oidium albicans Synonym. — Monilia Candida. Disease Produced. — Thrush in infants; sometimes in the young of animals. Berg, in 1840, described this organism as the cause of thrush. It has since that time been repeatedly isolated and cultivated. It is known from most civiHzed countries. Morphology. — The mycelium of this organism is poorly de- veloped; frequently the whole growth consists of budding yeast- like cells. These may be spherical, elliptical, oval or cylindrical, the shorter cells about 4 /x in diameter by 5 to 6 ju in length. The hyphal threads are much longer. There can be Uttle differentia- tion in many cases between the.conidia and the cells of the hyphae, but on artificial media the conidia are frequently definitely ad- jointed from the tip of the conidiophore. Chlamydospores may form in the hyphse. Isolation and Culture. — The organism may be isolated without difficulty from the lesions of the disease. A distinctly acid me- dium should be used. On most nutrient media it develops super- ficial, spherical, white, waxy to granular colonies. Gelatin is not liquefied, nor is blood-serum. Pathogenesis. — The organism, when injected intravenously into rabbits, produces a fatal infection not unhke a generalized infection with a Blastomyces. Typical thrush has been produced in the mouth of young animals and birds by inoculation. The disease generally occurs in the mouth of sucklings, usually as a benign infection. It is characterized by the formation of white patches on the mucous membrane, varying in size from points to considerable areas. The infection may extend to the pharyngeal or laryngeal mucosae; rarely metastatic infection of internal organs may occur. SECTION V PATHOGENIC PROTOZOA CHAPTER XL. STRUCTURE, RELATIONSHIPS, AND CXASSEFICATION OF THE PROTOZOA A PBOTOZOAN may be defined as a unicellular organism be- longing to the animal kingdom. Protozoa exist throughout their life-history as single-celled individuals, or as colonies of single cells; that is, the cells are not united to form tissues or organs, and never constitute a portion only of a multicellular form. The protozoa show some forms which intergrade with the bac- teria. The group is frequently defined as containing spiral forms, such as the Spirochsetse, some investigators believing that these have more bacterial than protozoan characteristics, others taking quite the opposite view. It is difficult, on the other hand, to draw a sharp line of demarcation between the protozoa and the multi- cellular animals or metazoa. Just when a group of cells ceases to be a mere group of independent units, and becomes a tissue which forms the whole or part of a multicellular- form, it is difficult to determine. Although the protozoa are regarded as the simplest and most primitive of living things, nevertheless many are complex in struc- ture and have the cell divided into specialized parts, sometimes termed organella, somewhat similar in function to the organs of the higher forms. They frequently undergo many changes in form during their life. Their life-history is, therefore, relatively com- plex as compared with that of the bacteria. Structure of the Protozoa.— The body substance of a protozoan may be divided into the ectoplasm, or outer layer, which comes into STRUCTURE, RELATIONSHIPS, CLASSIFICATION OF PROTOZOA 479 contact with the material environment, and the endoplasm, with- in. Within the endoplasm there is generally a nucleus (in some forms two nuclei, a large and a small), and frequenth^ inclusions of other sorts. The ectoplasm in some cases cannot be differentiated sharply from the endoplasm. These are the exception, however, and not the rule. The ectoplasm may become variously modified, and, by secretion, form shells or a heavy membrane for protection. Many of the pathogenic protozoa become encysted by the formation of a heavy, chitinous wall. The endoplasm is made up of a very delicate, foam-like or alveolar structure, the density of which varies greatly. It may enclose amorphous granules or crystals of different kinds, green granules or chromatophores, vacuoles, etc. None of the protozoa are certainly known to be entirely des- titute of nuclear material. The nucleus may be devoid of a nuclear membrane or distributed; generally a membrane is pres- ent. A single homogeneous nucleus is present in most forms, with the exception of the infusoria, which have, in general, a large or macronucleus and a small or microniocleus. At some stages in the life-history many nuclei may be present in a single cell. This is particularly true just before or during spore formation. The protozoa with few exceptions are motile, at least during certain stages in the life-history. The exceptions are among certain of the parasitic Sporozoa. Special organs of locomotion are frequently found. Pseudopodia are changeable processes of the protoplasm which are thrown out by the cell, the cell contents frequently flowing forward into the pseudopodia. Many or few may be present at one time. They may be short and blunt or long and slender. Usually the endoplasm as well as ectoplasm takes part in their formation. The protozoan flagella are derived from the ectoplasm; are usually unchangeable in shape, long, thin, and pointed; in general, longer than the cell itself. One, two, or many may be present. They propel the cell by a series of undulations or spiral or rotary motions. Cilia, when present, are found in considerable numbers. They are shorter, generally blunt, and, by striking the water in unison, resemble oars in their motion. 480 VETERINARY BACTERIOLOGY The life-histories of the various protozoa show such complexi- ties that they may better be treated under the separate subdi- visions. Classification of the Protozoa. — Authorities are not in entire accord with reference to the principal subdivisions of the protozoa. The classes here used are those proposed by Doflein in his "Lehr- buch." Key to Classes of Protozoa Subdivision I., Plasmodroma. — Protozoa motile by means of pseudopodia or flagella, with one or more vesicular nuclei, repro- duction isogamous or anisogamous, usually with two developmental cycles, in which sexual generations alternate with sexual. 1. Motile by means of flagella Class I. Mastigophora. 2. Motile by means of pseudopodia Class II. Rhizopoda. 3. Variously motile, usually reduced through parasit- ism. Metagamic multiplication by means of nimierous spores. Class III. Sporozoa. Subdivision II., Ciliophora. — Protozoa with numerous cilia, with one or more principal nuclei and one or more vesiculate ac- cessory nuclei (or occasionally the latter only). Reproduction by means of an isogamous fusion, or by means of interchange of nu- clear material without fusion of the cell-bodies. Multiphcation only as a result of simple division or by budding. 1. Cilia present throughout the life of the cell. Food taken up by osmosis or through a cytostome.. . .Class IV. Ciliata. 2. CiHa present only in the young cell; food taken up through funnel-like organella Class V. Suctoria. CHAPTER XLI PATHOGENIC PROTOZOA OF THE FLAGELLATA (Exclusive of the Spirochetes) The pathogenic forms of the class Flagellata differ from the Rhizopoda in that they do not possess pseudopodia. In most cases the organism has a relatively definite form. The cells are motile by means of one or many flagella. This class contains many hundreds of species distributed among many genera and families. Most of these are non-parasitic. Many species are commensals in the intestines of man and animals, and have been suspected of producing intestinal disorders. The genera, Spirochseta, Treponema, and Spiroschaudinnia, perhaps belong with the Flagellata, and show some distinct re- semblances to the genus Trypanosoma. Their position among the protozoa, however, is challenged by many bacteriologists. They are, therefore, treated as a separate group in a preceding chapter. An undulating membrane and terminal flagellum present Trypanosoma. Undulating membrane not present Herpetomonas. The Genus Trypanosoma The first recorded observations of trypanosomes are those of Valentine, who saw them in the blood of a salmon in 1841. Nu- merous observers found similar organisms in the blood of other fish, reptiles, and batrachia. Lewis, in 1878, observed the first trypanosome of mammahan blood in the rat. Evans, in 1880, noted them in the blood of animals infected with surra and be- lieved them to be the cause of the disease. Bruce, in 1894, de- scribed another form from Africa as the cause of nagana or tsetse-fly disease. Since that time the number of known species has in- o, 481 482 VETERINARY BACTERIOLOGY creased rapidly. A small proportion only of those described are known to be pathogenic. Among the pathogenic forms, however, are to be found those that are among the most serious hindrances to the development of a live-stock industry, and even to human habitation in certain countries. Morphology. — Trypanosomes usually are elongated cells, more or less spindle shaped, rarely almost as broad as long, but always tapering more or less to the ends. Most of the pathogenic forms that have been described are several times as long as broad when observed in the blood. The anterior end of the cell is tipped with a flagellum. Along one side, extending longitudinally on the cell, is a thin membrane. The flagellum extends along this membrane Fig. 188. — Trypanosoma equiperdum, morphology of the trypanosome : 1, A rather thick, short trypanosome — a, Blepharoplast; 6, protoplasm (cyto- plasm); c, nucleus; d, undulating membrane; e, flagellum attached to the edge of the membrane; /, anterior extension of the flagellum. 2, A longer cell. 3, 4, Trypanosomes in process of longitudinal division (adapted from Gonder and Sieber). and forms its outer edge. The flagellum finally terminates in the cell-protoplasm near a granule which stains deeply. This granule may be situated in various parts of the cell, but is usually in the posterior portion. Its relative position is one of the characters used in the differentiation of species. It has been called by various names, as micronucleus, kinetonucleus, motor nucleus, centrosome, and blepharoplast. This last name is to be preferred, as it is the one that is most frequently used in the descriptions, and is com- monly used for similar structures found in many other flagellated protozoa. The flagellum is, in part, therefore, embedded in the protoplasm, in part is attached to the edge of the undulating mem- brane, and in part is free at the anterior end. A nucleus, usually PATHOGENIC PROTOZOA OF THE FLAGELLATA 483 situated near the center or anterior end, may be demonstrated in stained preparations. It is relatively large and granular in struc- ture. The entire body of the trypanosome is mobile, and the or- ganism may vary its shape to some degree. It swims about with the flagellum in front. Multiplication is accomplished by a preliminary division of the blepharoplast followed by that of the nucleus, and this by a longi- tudinal splitting of the cell to form two individuals. In cultures the cells may frequently be observed in the form of rosettes or clusters, with the flagellar ends pointing out. These rosettes do not occur in the blood. Transverse division does not occur. No conjugation or fertilization process in trypanosomes has been cer- tainly detected. Some investigators have believed that trypanosomes in the animal body may have an ultramicroscopic stage in their develop- ment. Bruce and Bateman, in a series of careful experiments, have shown that this does not occur. The existence of a regular cycle of changes in the life of a try- panosome is at present a somewhat mooted question. Some inves- tigators believe that they have established the existence of a rela- tively complex life cycle. Kleine, Bruce, and others believe that certain species of insects which transfer the disease must be con- sidered true hosts, and that they do not become infective for some days — in the case of Trypanosoma gambiense about twenty — after biting an infected individual. Rodenwalt and others have found that certain developmental changes take place in the gut of the insect; others have failed to find them. At present it may be concluded that the occurrence of developmental changes has not been satisfactorily demonstrated, although there is good reason to believe that such may occur. Carimi, Schaudinn, and others have recognized what they believe to be an endoglobular stage in the development of the organism in the body. Battaglio claims to have demonstrated for Tr. hrucei, Tr. lewisi, and Tr. vespertilionis a developmental cycle in the blood, which includes sporulation of micro- and macrogametocytes, with formation of micro- and macro- gametes. These conclusions have been combated by others. Not all trypanosome infections are transmitted through insects. Certain transformations have been noted with some forms in the 484 VETERINARY BACTERIOLOGY body itself. There is no evidence that an insect can transfer the organism to its progeny; that is, hereditary transmission through insects play no part in the life-history. This is in marked con- trast to certain other diseases, such as the piroplasmoses. Cultivation of Tiypanosomes.^ — Novy and MacNeal, in 1903, gave an account of a method which they had successfully used in the cultivation of trypanosomes in artificial media. Equal parts of defibrinated rabbit blood and melted nutrient agar are mixed and the tubes are slanted and allowed to solidify. The water of con- densation is inoculated with a small amount of blood containing trypanosomes. The first culture of trypanosomes frequently develops slowly, but subsequent transfers more quickly. Not all trypanosomes can be cultivated in this manner with equal facility. Method of Disease Production. — The organisms are found typically in the circulating blood, and to a less degree in the other body-fluids. Anemia and emaciation are frequently associated with the various trypanosome infections. The spleen is quite commonly enlarged. Examination and Staining Methods. — Usually the examination under a cover-glass of a drop of blood from an infected animal will reveal the organism, particularly if made directly with the low- power objective of the microscope. The active motion of the organisms reveals their presence by movements of the blood-cells, and they may be thus located and then studied under the higher powers. Centrifugation of blood or body-fluids must be resorted to in some instances to concentrate the cells when they are present in small numbers only. Blood-films stained by Wright's method yield very satisfactory results. The vast number of tropical and subtropical diseases due to trypanosomes makes it impossible to discuss any except the most important of such parasites. Some of these trypanosomes are primarily important as the course of human diseases, secondarily ■as affecting animals. The most important of this class is the Try- panosoma gambiense, the cause of sleeping-sickness of certain African countries. Monkeys, dogs, cats, guinea-pigs, and rabbits are readily susceptible to this trypanosome. PATHOGENIC PROTOZOA OF THE FLAGELLATA 485 Trypanosoma equiperdum Synonym- Trypanosoma rougeti. Disease Produced. — Dourine : maladie du coit in horses (horse syphilis) . Rouget, in 1896, first described this trypanosome. Other investigators have conclusively established its etiologic relation- ship to the disease. It is of particular interest as the only trypanosome disease of importance in Europe and in North America. Distribution. — The disease is known from Germany, Austria, France, and southern European countries, northern Africa, western Asia and India, Chile, Java, and several local outbreaks have oc- curred in North America (Illinois, Nebraska, Wyoming, South Dakota, Iowa, and northwestern Canada). Morphology (See Fig. 188). — Moore and Bredini, in a study of this trypanosome as it occurs in artificially infected rats, con- cluded that it passed through certain developmental stages, fina,lly being converted into rounded bodies, with two long delicate flagella. The organism, as it occurs in the lesions and blood of the horse, is a slender cell, usually about 25 to 28 /x in length. There are no particular differentiating characters between this organism and the ones associated with other trypanosomiases of the horse. The blepharoplast is distinct, the membrane considerably folded, the nucleus central, and the free flagellum about ^ to -g- the length of the organism. Protoplasmic granules are never present in the organism directly originating from the horse. Cultivation. — Thomas and Breinl have succeeded in cultivating the organism in a modified Novy and MacNeal medium in one trial out of nineteen. Pathogenesis. — Experimental Evidence. — The disease may be transmitted experimentally to the horse, the ass, to fowls, and even to ruminants and apes, according to some observers, while according to others the latter animals are quite refractory. The disease occurs naturally only among the equines. Character of Disease. — The infected animal becomes emaciated, edematous swellings appear on the genitals, while whitish or chalk- like areas appear in the skin and mucosa of the external genitalia. Purulent foci occasionally develop in the testicular tissue. The dis- 486 VETERINARY BACTERIOLOGY ease usually runs a chronic course.- The animal frequently becomes gradually paralyzed. Recovery is infrequent. Immunity. — Animals that recover from the disease are thereby rendered immune to a second infection. They are not, however, rendered immune to infection with another trypanosome, such as that of surra. It appears that an immunity, acquired during preg- nancy may be transmitted to the offspring. No practical method of immunization based upon the organism or its products has been developed. Watson reports the use of serum from chronic cases of dourine in horses in large doses, up to 300 c.c. as protecting against virulent infection. Similar observations upon laboratory animals were made by Rouget, Nocard, Uhlenhuth, Zwick, and Fisher and others. The latter observed that such inoculations protected against the dourine trypanosome only, not against the nagana trypanosome. Bacteriologic Diagnosis. — This may occasionally be accom- pUshed bj' a microscopic examination of the fluids from the lesions and the identification of the characteristic trypanosome in stained mounts. The organisms can rarely be found in the peripheral blood. The blood-tinged fluid secured from the plaques when they first appear is the most favorable material for their identification. They may be found in the blood-stream of artificially infected laboratory animals. In doubtful cases inoculation of blood or the serous fluid from plaques may be made into dogs, and examination of the sweUings on such animals be subsequently made with positive findings. Agglutination Test. — ^Lange, Zwick, and Winkler and others report the agglutination test as satisfactory in determining virus carriers among stallions. They found that serum of infected animals would agglutinate dourine trypanosomes in homogeneous suspension in dilutions of from 1 : 400 to 1 : 25,000, while that of non-infected animals did not agglutinate in dilution higher than 1 : 50. The chief objection to this as a practical test is that other trypanosomes may be agglutinated, hence the test is of no value as a differentiating one. Complement-fixation Tesi.— Watson, of Lethbridge, Canada, makes report of 15,000 cases tested by this method. Concerning the reliabihty of the test he states that 100 per cent, of dourine- PATHOGENIC PROTOZOA OF THE FLAGELLATA 487 infected animals, whether in the active or latent stages of the disease, give positive reactions, provided two to three months incubation period has elapsed. In the less resistant animals one month is suflScient. He prepares his antigen by inoculating a number of white rats with Trypanosoma equiperdum, collecting blood from tliem when it is teeming with the trypanosomes, and centrifuging the blood at 1500 revolutions per minute to separate the trypanosomes from the blood-cells. Subsequent washing and centrifuging frees the trypanosomes from serum. Such antigen may be kept indefinitely if frozen solid. Conglutination Test. — This test has been used by a number of investigators. Wehrbein states that it is satisfactory, but is more sensitive to faulty technic, and consequently more difficult to employ than -the complement-fixation test. Transmission. — The disease is commonly transmitted from one animal to another through coition. Whether or not the organism can enter through the intact mucosa is not certainly known, but appears probable. Blood containing the organism will infect a rabbit if placed in the conjunctival sac. Sieber and Gonder claim to have succeeded in transmitting the disease through the medium of the fly Stomoxys caldtrans, but this certainly is not the com- mon method. No developmental stages could be observed in the fly. Trypanosoma evansi Synonym. — Spirochceta evansi. Disease Produced. — Surra in horses, mules, cattle, carabou or water buffalo, camels, elephants, dogs, goats, and sheep. The disease has been known for. a long time from southern Asia. The organism was first described from India by Evans in 1880. Distribution. — The disease is known from India, China, the PhiHppines, Africa, and Australia. Morphology. — The organism as it occurs in the blood is actively motile. It is usually between 22 and 30 m in length, and from 1 to 2 /x in diameter. It tapers to the anterior end, but the posterior is somewhat blunt. The undulating membrane and the free flagel- lum are well differentiated. This organism can scarcely be separ- ated from Trypanosoma hrucei on the basi-s of morphology. Laveran and Mesnil, however, differentiate on the basis of the former being 488 VETERINARY BACTERIOLOGY more slender, possessing a longer flagellum, and greater motility in hanging drop. Cultivation.— This organism has been cultivated, on Novy and MacNeal's medium, but only after repeated trials. Pathogenesis. — The disease may be readily transmitted to sus- ceptible animals by the injection of blood containing the try- panosome. The disease itself is characterized in the horse as a relapsing fever, with eruptions, either generalized or localized in the skin. Petechial hemorrhages of the mucosae are frequent. The subcutaneous tissues are infiltrated and edematous. It is almost invariably fatal in horses. Cattle are relatively resistant to the disease, and in these animals recovery usually occurs, but the blood may remain infective for a considerable period. Buffalo frequently succumb. Camels, elephants, and dogs are not infre- quently infected. This disease resembles nagana clinically, and the causal organ- isms can scarcely be differentiated. Animals immunized against the one disease are susceptible to the other, which would seem to establish specific differences sufficient to separate the organisms as distinct species. Bacteriologic Diagnosis. — The organisms gradually increase in numbers in the blood during the onset of the disease, and have been found as numerous as 350,000 per cubic millimeter. They are frequently not present in the blood between the periods of fever. Transmission. — Fraser and Symons state that in the Federated Malay States four species of the fly genus Tabanus, particularly Tabanus fumifer, are responsible for the spread of the disease. Probably other flies may carry the organism as well. Experiments seem to show that the transference in this case is merely mechan- ical, and that there is no developmental cycle in the intermediate host. Carnivorous animals may be infected by ingestion if there are lesions in the mucous membranes. Trypanosoma fartjcei Disease Produced. — Nagana or tsetse-fly disease in horses, cattle, camels, buffalo, antelopes, and related wild animals, pos- sibly the elephant. PATHOGENIC PROTOZOA OF THE FLAGELLATA 489 The fact that this disease follows the bite of the tsetse fly has long been known by African natives, and the early explorers con- firmed their belief. Bruce, in 1896, described the trypanosome which causes the disease. Distribution.— Known only in Africa, particularly in Zulu- land. Morphology.— The organism is sluggishly motile. According to Laveran and Mesnil, the organism from rats, mice, and guinea- pigs is 26 to 27 fj. long, including the flagellum, while in horses and mules it is 28 to 33 n. In breadth it varies between 1.5 and 2.5 fi. The free part of the flagellum is shorter than that of Trypanosoma evansi, while the breadth is usually greater. Granules may gen- erally be observed in the protoplasm. Irregular forms occur in the blood after death, and in the lymphatic; glands, spleen, bone- Fig. 189. — Trypanosoma brucei (adapted from Gonder and Sieber). marrow, liver, and lungs during life. Kleine believes, from his experiments with fly transmission, that there must be a develop- mental stage occurring in the insect. Isolation and Culture. — Novy and MacNeal succeeded in grow- ing the organism of nagana in the medium already described. Only a few tubes out of a large number were found to show growth. Pathogenesis. — Experimental Evidence. — Inoculation of the mouse, rat, dog, cat, and monkey results in an acute infection; of the rabbit, guinea-pig, equines, and swine, in a subacute infec- tion; and of cattle, goats, and sheep, in a chronic infection. Character of Disease and Lesions Produced. — The disease is of greatest importance in the equines. The incubation period is from three to twelve daj^s (Theiler). There is a continued or re- mittent fever and a watery discharge from the eyes and nose. The animal becomes much emaciated before death, which usually 490 VETERINARY BACTERIOLOGY occurs in from two weeks to three months. Edema of the ventral region is common. If the animal dips during a febrile attack the spleen is acutely swollen, otherwise there is generally present a chronic tumor of the organ. Sometimes the spleen is entirely normal. The lymph-glands are generally enlarged. Immunity.— Rodet and Vallet believe that the organism is rapidly destroyed in the spleen. No practicable method of im- munizing against the organism has been developed. It has been found that the injection of human serum into laboratory animals at intervals will greatly prolong their life, but will not cure. There is probably some relationship between immunity of man and the trypanicidal character of his serum. Goats, sheep, and cattle show a considerable percentage of cures and are thereafter immune. Their serum, however, has little immunizing power. Bacteriologic Diagnosis. — Stained mounts of the blood from infected animals will generally reveal the characteristic parasites. Centrifugation of the blood will frequently result in the collec- tion of the organisms in a layer just at the surface of the cor- puscles. Transmission — The disease is commonly transmitted from one animal to another by the bite of the tsetse fly (Glossina morsitans) , and, according to Koch, in those regions where this fly is unknown by the closely related fly G. fusca. The herbivorous animals native to sections of the country where the disease' is prevalent are almost invariably infected, and render infection of other animals easy. Kleine experimentally showed that another species of Glossina (G. palpalis) could transmit the disease. He found that the flies did not become infective until eighteen days had elapsed after biting an infected animal. Other experimenters have found that the fly loses its infectiveness within a day or two after feeding upon an infected animal, and have concluded that transmission is wholly a mechanical affair. Trypanosoma equinum Synonym. — Trypanosoma elmassiani. Disease Produced. — Mai de caderas of the horse (Spanish caderas = rump or hindquarter) . Elmassian, in 1901, announced his discovery of the specific PATHOGENIC PROTOZOA OF THE FLAGELLATA 491 trypanosome of this disease in Argentina. Voges and LigniSres confirmed this discovery in the following year. The disease is so prevalent in some sections that cattle exclusively are used for riding and driving. Distribution. — Parts of South America, particularly Brazil, Paraguaj', and Argentina. Morphology. — This trypanosome resembles those of surra and nagana, but the blepharoplast is so inconspicuous and stains so faintly that it may be readily overlooked. The cell is usually between 22 and 24 n in length and 2 to 4 ^ in width. Cells about to divide are somewhat larger. The difference in the blepharoplast of this and most other forms renders identification easy even in mixed infections. Pathogenesis— -Experimental Evidence. — Inoculation of the organism causes a fatal infection in the horse; the mule and donkey are somewhat more resistant, as are mice, rats, and other rodents, rabbits, and other laboratory animals. Birds cannot be infected. The pig, sheep, goat, and ox may show transitory symptoms, but are highly refractory. Character of Disease and Lesions. — The disease differs from surra and nagana in the almost complete absence of edema, and is characterized by a paralysis of the hindquarters. There is a progressive emaciation, fever, and the hindquarters become weak; the horse in walking scarcely raises the hoof above the ground. Finally, the animal supports itself by leaning or falls to the ground. There are no lesions upon the genital organs. Immunity. — No method of practicable immunization against the organism has been developed. Bacteriologic Diagnosis. — The organism may be found in the blood, particularly during the fever paroxysms. Transmission. — The disease is evidently enzootic in certain parts of South America in rodents or other animals. One of these, the capybara (Hydrocharus capybara), has been found to be in- fected, and it is said that stockmen can sometimes foretell an outbreak of the mal de caderas by the death of many of these ani- mals in the vicinity. The method of transmission is not certainly known. Flies have been supposed to act as carriers, but definite proof is lacking. 492 VETERINARY BACTERIOLOGY Trypanosoma dimorphon Disease Produced. — Gambian horse sickness, trypanosomiasis in horses and other equines, cattle, sheep, and goats. Button and Todd, in 1902, reported the discovery of a specific trypanosome in a disease of horses in Senegambia. The same, or very similar trypanosome, has since that time been reported from many African locaUties in other animals as well. Distribution. — Various localities in Africa (French Guinea, Zanzibar, Sierra Leone, Mozambique, Zululand). Morphology. — The organism is characterized by the absence of a free flagellum, the flagellum terminating with the undulating membrane. It is dimorphic, some of the cells being 20 to 25 fx in length, others only about 12 ju. Transitional forms between these extremes may be found. The undulating membrane is not well developed. Protoplasmic granules are very rare or are absent in the cell. Pathogenesis. — Experimental Evidence. — The disease has been experimentally produced by the inoculation of the organism into sheep, cattle, goats, rabbits, horses, and white rats. Character of Disease and Lesions. — The infection is acute in the rat, acute or chronic in the rabbit and dog, and chronic in the ox, sheep, and in equines. It produces a severe anemia, with changes in the red blood-cells. In the horse there is progressive emacia- tion. A marked edema is rarely produced. Death does not occur usually for months after infection. Recovery sometimes occurs. Immunity. — No practicable method of immunization by means of the organism or its products has been developed. Bacteriologic Diagnosis. The organism may be found in the blood at certain periods, but repeated examination is sometimes necessary. Inoculation of other animals with the blood will never- theless show it still to be infective. Transmission. — Transmission supposedly occurs through the Glossina palpalis. Other flies are considered to be occasional carriers. Trypanosoma congolense Broden described a disease of horses in the Congo Free State which closely simulated nagana, but the trypanosome resembled rather Trypanosoma dimorphon. It is also reported from Rhodesia. PATHOGENIC PROTOZOA OF THE FLAGELLATA 493 Asses and dromedaries also are infected. Laveran, as a result of inoculation experiments, has concluded that this is a distinct species. It has been reported from several localities in southern Africa. It IS fatal for cattle and sheep. The organisms are 10 to 20 M in length and 1.5 to 2.5 m wide. The flagellum shows no tree portion, hence animal inoculations are necessary to differen- tiate between this and Tr. dvmorphon. Transmission is through the Glossina morsitans. Trypanosoma pecaudi Disease Produced.— Baleri, or trypanosomiasis in horses, cattle, sheep, and goats. This organism was described by Laveran from the blood of in- oculated sheep brought to Paris by Cazalbou. Fig. 190. — Trypanosoma pecaudi (Laveran). Distribution. — The disease is known from the French Sudan. Morphology. — The organism closely resembles Trypanosoma dimorphon. Two forms are described : long slender cells, 25 to 35 H in length by about 1.5 ;u in width, with narrow, undulating mem- brane and fairly long flagellum, and short broad forms, 14 to 20 /z by 3 to 4 /i, with no free flagellum and a wide, undulating membrane. Pathogenesis. — Character of Disease and Lesions. — In the horse the disease is characterized by repeated attacks of a severe fever, swellings in various parts of the body, injection of the conjunctiva, and a considerable degree of emaciation. Rats, mice, guinea- pigs, and dogs are susceptible to infection. 494 VETERINARY BACTERIOLOGY Transmission. — Buff ard claims the Glossing, palpalis is the agent of its transmission, others that the G. longipalpis is the commoner. Trypanosoma cazalfooui Disease Produced. — Souma or soumaya in cattle, sheep, horses, and mules. Distribution. — Africa, from the French Sudan, French Congo, Upper Nile. Morphology. — The organism, including its free flagellum, is about 21 by 1.5 /i. The oval nucleus is centrally located. The undulating membrane is poorly developed and is little folded. The terminal portion of the flagellum is free. There are no marked characters differentiating the organism from Trypanosoma evansi Fig. 191. — Trypanosoma cazalboui (Laveran). Pathogenesis. — Experimental Evidence. — Dogs and small test animals seem to be relatively immune. The infection may be readily transmitted to horses and cattle and the smaller ruminants. Cross-inoculation experiments have shown the disease to be dis- tinct from surra. Character of Disease. — It generally attacks cattle. The disease leads to a progressive emaciation, weakness of hindparts, the skin is harsh, and there is a staring coat. In many individuals the lower surfaces of the body show marked edema. The temperature is variable. The disease may be acute lasting not over fifty days, while in other cases the course may be prolonged for a year or more. PATHOGENIC PROTOZOA OF THE FLAGELLATA 495 Bacteriologic Diagnosis. — The organisms are present in the blood, usually in small numbers only. Transmission. — Pecaud concludes that Glossina palpalis is responsible for the distant transmission of the organism, and that members of the fly-genera, Stomoxys and Tabanus, may produce immediate transmission. Trypanosoma tfaeileri Disease Produced. — Theiler at first beheved this organism to be the cause of galziekte or gall-sickness in bovines, but later investigation has led him to class it as a harmless commensal in the blood of cattle. This organism was first noted by Theiler and was described at greater length by Laveran. Schnitt in Germany, Peters in South America, and Crowley in America have described very similar organisms in the blood of cattle. Distribution. — A large part of South Africa, possibly also in India. Morphology. — The organism associated with this disease is of unusual size, 30 to 70 /u in length and 2 to 5 /x in width. This alone is sufficient to differentiate it from other forms. There is a long, free flagellum. The nucleus is central, but the blepharoplast is a considerable distance from the posterior end. Mq,ny proto- plasmic granules may usually be seen. Pathogenesis. — The organism seems to be inoculable only into ■cattle. It resembles the rat trypanosome in being thus limited to a single host. Bacteriologic Diagnosis. — The organisms are' quite common in the blood, but soon disappear. Transmission. — It has been found to be transmitted by the bite of the fly, Hypobosca rufipes. Trypanosoma gambiense Synonyms. — Trypanosoma ugandense; Tr. castellani. Disease Produced. — Human trypanosomiasis, or sleeping sickness. Distribution. — ^The disease is endemic upon the western coast of Africa and in certain of the central portions. 4&6 VETERINARY BACTERIOLOGY Morphology. — The organism is 10 to 28 by 1.4 to 2 /x. Forms undergoing division are somewhat larger. The free flagellum may be one-fourth to one-third the length of the body. Rarely no free portion of the flagellum can be demonstrated. The undulating membrane is narrow. The blepharoplast is near the posterior end. Protoplasmic granules that stain like chromatin are commonly observed. Pathogenesis. — Experimental Evidence. — The disease, with its characteristic cUnical symptoms, may be reproduced by the injec- tion of blood containing the organisms into the monkey. Dogs, jackals, cats, guinea-pigs, and rabbits are readily infected. Mice frequently recover and are thereafter immune. Goats and sheep are relatively refractory, but sometimes succumb. It may pro- duce a mild chronic infection in the horse and in cattle. Character of Disease. — The disease is insidious in its onset. Two distinct stages may be recognized. These were for a long time supposed to be different diseases. In the first stage the organisms appear in the blood; there may or may not be fever. In the sec- ond stage pains in the back, tremors, arid drowsiness supervene. Finally the patient dies in a comatose condition. In this second stage the organisms are present in numbers in the cerebrospinal fluid. The disease appears to be always fatal, but may run a chronic course, lasting several years. Immunity. — No practicable method of immunization has been developed. Bacteriologic Diagnosis. — The organisms are usually scanty in the blood, and centrifugation is necessary to find them. They may usually be demonstrated in the fluid secured by a lumbar puncture. They may also commonly be demonstrated by puncturing an en- larged Ijonphatic gland and examining the fluid secured. Transmission. — One of the tsetse flies, Glossina palpalis, has been found to transfer the disease. Trypanosoma lewisi Chaussat, in 1850, and Lewis, in 1877, noted the presence of a flagellate in the blood of rats. It is so commonly present in the blood of rats in many parts of the world that it has been frequently used in the laboratory for study and demonstration, although its PATHOGENIC PROTOZOA OF THE FLAGELLATA 497 pathogenic properties are almost nil. This trypanosome cannot be transmitted to any other genus of mammals so far as known; even the closely related genera of the rodents are refractory. It evidently is a highly speciaUzed commensal. The organism, with its flagellum, measures about 24 to 25 ju in length by 1.5 /i in width. Protoplasmic granules are frequently present. Trypanosoma hippicum Disease Produced. — A disease of equines, known as murrina and derrengadera. The organism was described by DarUng, in 1910, as the cause of disease in horses and mules. Distribution. — Panama, Venezuela, and Central America. Morphology. — In size the organism resembles Trypanosoma evansi and Tr. brucei. According to Darling, the length varies from 18 to 28 ju, breadth, 1^ to 3 ix. The flagellum is usually short and not entirely free, though sometimes free. Nucleus is located posteriorly. Coarse granules are numerous in the protoplasm. The posterior end is blunt, never attenuated as is that of Tr. kwisi. Pathogenesis. — Besides horses and mules , most of the labora- tory animals can be infected. In the dog it produces lameness in the posterior extremities. Swine may be infected, but calves are refractory. Cattle never acquire the disease. Character of Disease. — Progressive emaciation, edema, anemia, febrile paroxysms, and conjunctivitis. Postmortem Changes. — Slight enlargement of the spleen, pe- techiated kidneys, hemorrhages on both endocardium and epi- cardiimi. Immunity. — No method of immunization has been developed. Bacteriologic Diagnosis. — The clearness of the blepharoplast characterizes the organism. The parasite is found in the peripheral blood quite generally, but not constantly. As soon as the tem- perature of an infected animal reaches 101°, blood examination will usually reveal the parasite. During the latter part of the paroxysm it may not be demonstrable. Inoculation of the more suscep- tible laboratory animals is an aid to diagnosis. Cross inoculation with other trypanosomes has shown the Trypanosoma hippicum to be distinct. 498 VETERINARY BACTERIOLOGY Transmission. — This probably does not occur through the tabanids, as the disease occurs in locaUties where these flies are not found. Darhng concludes from observations that flies probably act as mechanical carriers by ahghting on gall sores and wounds of animals infected, and from these carrying the infectious material to non-infected animals. The Genus Herpetomonas This genus includes certain flagellates that have an essentially trypanosome-like structure without an undulating membrane. The cell is elongated in the typical Herpetomonas; the closely related genus Crithridia comprises those forms in which the body is' much shortened. The flagellum is anterior, the blepharoplast distinct, as in the trypanosomes, and the nucleus centrally located. Organisms of this genus have been frequently reported from the gut of mosquitoes and flies. Certain of the trypanosomes sometimes assume shapes that resemble closely the Herpetomonas. The genus assumes pathogenic significance, principally because of the tentative classification of certain protozoa known as the Leishman-Donovan bodies as members of this genus. Three species have been described. Herpetomonas donovani Synonyms. — Leishman-Donovan bodies; Leishmania donovani; Trypanosoma donovani. Disease Produced. — Kala-azar, cachexial or Dumdum fever in man. Leishman, in 1900, observed this parasite in smears from the spleen of a patient that had died of Dumdum fever. His account was pubhshed in 1903. Distribution. — Throughout southern Asia and northern Africa. Morphology. — The organism as it occurs in the body is com- monly intracellular. It is found principally in the spleen, liver, bone-marrow, and lymph-glands. It is oval, spherical, or pear shaped, usually between 2 and 3.5 p in length and 1.5 to 2 yu in width. Two staining granules occur in the interior, the larger spherical, and the smaller, and more deeply staining granule, somewhat elongated. The organisms multiply by a preliminary division of both of the chromatic granules (nucleus and blepharo- PATHOGENIC PROTOZOA OF THE FLAGELLATA 499 plast), followed by a constriction of the cell. Ln cultures typical flagellates are produced. The organism elongates somewhat, and a vacuole appears at one side of the blepharoplast, and from this a single flagellum develops. The body finally assumes an elongated form not unlike a trypanosome without an undulating membrane. This is the Herpetomonas stage. This may now di- vide longitudinally, frequently unequally, splitting off very slender cells. The systematic position of this organism is still somewhat in doubt. It may be that it should be regarded as belonging to a distinct genus, and the. name Leishmania used instead of Herpeto- monas. Pathogenesis. — The disease is characterized by enlargement of the spleen and by fever. Transmission. — It is believed that the parasite is transferred from the dog to man through some intermediate host. Leishmania (Herpetomonas ?) infantum NicoUe has described an organism similar to the preceding from a disease which he calls infantile kala-azar. The organisms re- Fig. 192. — Herpetomonas infantum: A, Organisms from the spleen of a child; B, a mononuclear containing the organisms, and C, an endothelial cell from the spleen; D, various stages in the development of the Herpetomonas form from- the Leishman-Donovan bodies (Nicolle). semble the preceding, but are believed to constitute a separate species. The disease is primarily one of dogs, which may be trans- mitted to children. Leishmania tropica Synonyms. — Ovoplasma orientale; Helcosoma tropicum. Wright has described a similar organism as the cause of oriental sore or Delhi boil in man. It is probably transmitted likewise by some biting insect. The dog may be infected and show clinical symptoms similar to man. CHAPTER XLII PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA The Rhizopoda are protozoa having during adult life movable or changeable processes of protoplasm called pseudopodia. Repro- duction is accomplished through simple fission and by spore formation. Among, the hundreds of genera and thousands of species of organisms belonging to this group that have been described there Fig. 193. — Ameba in culture (Schardinger). is one (possibly two or three) which has been found to be patho- genic. They are certainly known to be pathogenic in man only, but the frequent occurrence of the organisms of this genus is the 500 PATHOGENIC PROTOZOA OP THE CLASS RHIZOPODA 501 intestines of the lower animals, and the possibility of confusing the adult stage with certain developmental stages of the sporozoa renders a discussion of these forms advisable in a veterinary text. The possibility of any of the forms being pathogenic for lower animals has not been sufficiently investigated. The Genus Entamceba The normal inhabitant of the intestinal tract of man was first known as Amoeba coli. Later, Schaudinn renamed it Entamceba coll, and gave the name Entamoeba histolytica to the pathogenic type associated with the amebic dysentery. The genus Entamoeba was differentiated from Amoeba by the absence of a contractile vacuole and by the formation of multinucleated cysts. Authorities differ greatly in their estimates of the number of species of amebae present in the intestines of man. Walker and Sellards state that eighteen forms have been described as separate species. The best classification, and the one most commonly used now, is that of Schaudinn. He recognizes two species at least — one a normal non-pathogenic form, Entamceba coli, and one patho- genic for man, Entamoeba histolytica. More recently Hartmann and others have described a third species, E. tetragena, and Koid- sumi a fourth, E. nipponica, both from cases of dysentery. The possibility of any of the dysenteries of the lower animals being caused by amebae has not been sufficiently studied. Smith believes that certain diseases of turkeys and other fowls, particularly entero- hepatitis, are caused by an ameba termed Amoeba meleagridis, but , others have contended this to be but a developmental stage in the life-history of a Coccidium. Examination of Living Amebce. — The amebse may be examined in the stools in a living condition by placing a portion of the Uquid or a bit of the sohder material moistened with physiologic salt solution on a slide, and pressing down a cover-glass not too firmly. Craig advises the use of a very weak solution of neutral red to stain the living organisms when«they are not present in too great nimabers. For the specific determination of the amebse present an examination of this kind is frequently all that is neces- sary. The slide must be maintained at about blood-heat in order to detect motility. 502 . VETERINARY BACTERIOLOGY Staining Methods. — The organisms stain rather readily with the usual laboratory stains, but these are of Httle value in the differ- entiation of the parts of the cell and in separating species. Wright's stain in favorable specimens, if carefully used, gives the best differentiation of the parts of the cell. The organism in tissues is best stained by Heidenhain's iron-hematoxylon or Borrel's stain. The smears should be fixed from fifteen to twenty minutes in alcohol and ether, equal parts, for all stains but Wright's. For the latter no fixation is required. Methods of Isolation and Cultivation.^Amebse commonly use bacteria and related organisms as food. A culture of amebse must provide for the growth of bacteria, but this growth must not be so luxuriant as to overgrow and eliminate the amebse. The agar medium recommended by Musgrave and Clegg may be used. This contains agar, 20 gm.; NaCl, 0.03 to 0.05 gm.; beef-extract, 0.3 to 0.5 gm.; water, 1000 c.c, made 1 per cent, alkahne to phenolphthalein. Variations in the materials used are sometimes necessary for some specialized saprozoites. This medium is melted, poured into a sterile Petri dish, and allowed to solidify. The surface of the medium is streaked with the material containing the amebse. The bacteria and amebse will usually both multiply if the plate be kept at a suitable temperature. Two operations are necessary to secure a culture in which it is known that all of the amebse are of one species and all of the bacteria of one kind. The mixed culture is placed upon the medium in the center of the Petri dish. Concentric circles of the organism with which it is desired to grow the ameba are placed about this. The amebse, as they crawl through the successive circles, gradually lose the original organisms with which they started and come to feed on the one kind. After one or more transfers a growth of the amebse may be secured with the one species of organism desired. It is not always possible to secure growth with every species of organism, as the different amebse have been found to require various species of bacteria. In order to secure a culture containing a single kind of ameba it is necessary to isolate a single individual. Ordinarily this may be accomphshed by examination of the surface of an agar culture until an isolated ameba is found, and this is then transferred to a PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 503 fresh plate. Musgrave and Clegg recommend running the tip of the lens against such an organism and removing it, attached to the lens, and inoculating the new medium by running the lens down in contact with it. Entamoeba coli Synonym. — Amoeba coli. Disease Produced. — The organism is probably non-pathogenic. The Entamoeba coli was probably seen and recognized by Lambl in 1860. Since that time it has been repeatedly noted by many investigators. Schaudinn, in 1905, first clearly differentiated the organism and traced its life-history. His work has been con- firmed and extended by Craig in the United States. The organ- ism is present in a large percentage (50 per cent., according to Fig. 194. — Entamoeba coli: A, Non-motile form; B, cell showing psudopodia; D, E, stages in cell division; F, G, H, I, J, stages in encystment and sporula- tion (adapted from Craig). Schaudinn) of healthy individuals. It is most easily recognized in the feces after the administration of a saline cathartic. Morphology. — The Entamoeba coli is a mass of protoplasm not possessing a definite cell wall, but with a nucleus containing usually one or more nucleoli. One (rarely more) non-contractile vacuole is occasionally present. The organism varies from 8 to 50 /i in diameter, but in the majority of cases is between 10 and 20 /x. When encysted it is usually between 10 and 15 /i. The organism is approximately spherical when not in motion. It is sluggishly motile, and usually is not moving when observed in a hanging drop. In this it is of a dull gray color. The protoplasm cannot be differentiated into endoplasm and ectoplasm when the organ- ism is at rest. When in motion the ectoplasm may sometimes be seen. This fact is of considerable diagnostic value. The endo- plasm is finely granular, and rarely contains more than a single 504 VETERINAEY BACTERIOLOGY vacuole. The nucleus is definite, spherical, and contains chroma- tin granules and one or more nucleoh; it is 5 to 8 ^ in diameter. When stained by Wright's method, the ectoplasm and endoplasm may be easily differentiated. The ectoplasm stains blue, the endoplasm violet, and the nucleus red. Multiphcation is commonly accompUshed in the intestines by simple division. The nucleus divides and the protoplasm con- stricts to form two individuals. This process is common in the liquid stools, but when drier and firmer, cystic reproduction is more common. A refractive hyahne cyst forms about the spher- ical organism and thickens. The protoplasm becomes homo- geneous in appearance and clearer than before. The nucleus then undergoes complicated divisions and recombinations, which ul- timately result in the formation of eight nuclei. When the wall is ruptured, these nuclei, with bits of the protoplasm, escape and constitute eight young amebae. Pathogenesis. — Repeated efforts to produce disease by injec- tion of Entamoeba coli into the colon of cats and other animals have failed. It must be regarded as a normal inhabitant of the intes- tines of man. Similar organisms have been found in the intes- tines of animals. Bacteriologic Diagnosis. — Entamoeba coli may be recognized in the feces by a direct microscopic examination as an ameba showing very slight motility, rounded pseudopodia, little or no observable differentiation between ectoplasm and endoplasm, comparative lack of vacuoles in protoplasm, and by the production of eight daughter-cells from each cyst. Entamoeba histolytica Synonym. — Amoeba dysenterioe. Disease Produced. — ^Amebic dysentery in man. Loesch, in 1875, described Amoeba coli as the cause of a dysen- tery in man, and claimed that the rectal injection of feces con- taining the organisms into dogs produced dysentery. Robert Koch, in 1883, showed the organism to be present in the ulcerations of the intestines. Kartulis firmly established a probable etio- logic relationship by his studies in Egypt, pubUshed in 1886. Councilman and Lafleur studied the pathology of the disease in PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 505 great detail. Schaudinn gave to the organism its present name of E. histolytica, and described its morphology with accuracy. The disease has been reported from all sections of the world. It seems to be much more prevalent in tropical than in temperate climates, but has been identified repeatedly in the United States. Morphology.— According to Craig, the morphology of this organism varies so greatly in culture-media that only the forms found in the feces can be regarded as typical. The organism under these conditions varies in diameter to 50 fi or more, showing it to be larger than Entamoeba coli. At rest, the organism is spherical Fig. 195. — Entamasba histolytica: 1, Organism in motion — -a, Vacuoles; 6, red blood-cells; c, pseudopodium composed chiefly of ectoplasm. 2, Organ- ism showing nucleus a, and vacuoles, b. 3. Preliminary changes upon begin- ning sporulation — a, Vacuoles; 6, chromatin masses escaping from the nucleus and scattering through the cell. 4, Chromatin masses scattered through the cell, 6; c, vacuole. 5, Cell budding off spores, the chromatin masses or new nuclei passing through the ectoplasm and escaping as spores. 6, Free spores (adapted from Craig). or ovoid; when in motion, its shape is extremely variable. It is usually actively motile, throwing out pseudopodia much more rapidly than E. coli. Color is absent, and the organism appears clear, or there may be a greenish tinge, due to the presence of hemoglobin from the blood in the feces and the blood-corpuscles engulfed. In the larger cells, rarely in the smaller, the endoplasm and ectoplasm may be quite sharply differentiated. The latter is hyaline, glass-like, and refractive, much firmer than the comparatively delicate membrane of Entamoeba coli, and the two are relatively 506 VETERINARY BACTERIOLOGY easily distinguishable by this means. The ectoplasm apparently comprises about one-third of the protoplasm. One or more vacu- oles are always present, as many as ten sometimes being found. The nucleus is faint and difficult to distinguish in the unstained mount. It is about 5 to 6 m in diameter. The chromatin is relatively sparse. With Wright's stain the ectoplasm stains more deeply than the endoplasm, the opposite of what is true in E. coli. Reproduction is accomplished in two ways. The first consists of a division of the nucleus, followed by a constriction of the cell to form two individuals. This process is essentially the same as that in Entamoeba coli. The method of spore formation, gemma- tion, or budding is quite different. When conditions arise unfa- vorable to continued vegetative existence, spores are produced. The nucleus, by a process of fragmentation, throws out chromatin granules or chromidia, which gradually collect under the ecto- plasm, form new nuclei, and are finally thrown off from the ex- terior, together with some of the protoplasm, as a spore or bud. These spores are round or oval, have a yellowish membrane, and measure 3 to 6 ;u, usually about 4 ^i in diameter. The membrane which forms about these spores is resistant to the penetration and action of stains. Pathogenesis. — Experimental Evidence. — Schaudinn dried feces of dysentery patients after demonstrating that they contained Entamoeba histolytica, in the form of spores in large numbers, and that they were free from E. coli. These were fed to a kitten, and death resulted in fourteen days, with characteristic ulceration of the intestinal wall. Similar experiments have since been repeated many times. There is little or no doubt of the pathogenicity of the organism, and the disease produced may be considered as a clinical entity. Character of Disease and Lesions. — The disease produced is a chronic dysentery, marked by intestinal ulceration, and frequently by abscesses in the liver. The presence of the relatively firm ectoplasm is believed to account for tTie ability of the organism to force its way into the tissues between the cells. Immunity. — No method of immunization against the Entamoeba histolytica has been developed. PATHOGENIC PROTOZOA OF THE CLASS RHIZOPODA 507 Bacteriologic Diagnosis. — The Entamoeba histolytica may be recognized in the feces, and separated from the E. coli by its larger size, its distinct differentiation into a hyaline refringent ectoplasm and a more granular endoplasm, the presence of more than one vacuole usually, the common presence of erythrocytes in the pro- toplasm in the course of digestion, by the less prominent nucleus, and by formation of spores by a process of budding with encyst- ment. Entamceba tetragena Synonjrm. — Entamoeba africana. This species was first described by Viereck in dysentery in Africa. Since that time the same organism has been noted by Hartmann, Werner, and others. It has been found capable of producing dysentery in cats. It resembles morphologically the Entamoeba coli more closely than the E. histolytica, but differs by the formation of four spores instead of eight within the cysts. CHAPTER XLIII SPOROZOA The Sporozoa stand alone among the protozoa, in that all the species are parasites. Many are harmless commensals, but a considerable number are pathogenic. The sporozoan cell contains typically a single nucleus, except in the MjTcosporidia, which are multinucleate. Food is taken in by diffusion through the plasma wall. Gastric and contractile vacuoles are present. The adult form is non-motile; the young forms are frequently actively motile, either ameboid or flagellate. In most of the Sporozoa the differentiation of the protoplasm into ectoplasm and endo- plasm can be clearly made out. The reproduction of the Sporozoa is the principal character which differentiates this group from others. Spores are always produced in those forms in which the complete life-history is known. The details of spore formation vary greatly in the different forms, but the essentials are the same. The cell as a whole, in most forms, divides to form archispores or sporoblasts ; each of the archispores forms spores, and each spore then produces one or more sporozoites. Many forms show addi- tional methods of reproduction. Formation of sex cells or gametes, with fusion of hke or unhke cells, takes place in many forms, and serves to further complicate the life-history. Blood-smears may be stained by one of the Romanowsky stains or by Wright's method to demonstrate the protozoa of this group. Considerable care must be exercised in the examination of such blood mounts not to confuse the normal blood con- tents, such as blood-platelets, with developmental stages of the protozoa. The class Sporozoa contains many families and genera; only a few of the latter contain species that are of economic importance. The following artificial key will assist in differentiating the various genera of veterinary interest: 308' • \ SPOROZOA 509 A. Sporozoa found in the blood-cells. 1. In erythrocytes. (o) At some stage occupying a considerable proportion of the interior of the cell. (1) In mammalian blood. (a) Well-differentiated, often two in a cell. In animals. 1. Initial multiplication in the spleen, organisms in peripheral blood usu- ally rod shaped Theileria. 2. Constantly in blood-cells. Usually pear shaped Babesia (Piroplasma). (ft) Ameboid of first, finally filling the cell Plasmodium. (2) In avian blood Proteosoma (HaBmoproteus). (b) Forming minute dots, seemingly entirely of chromatin Anaplasma. 2. In leukocytes. (a) In mammals Leukocytogregarina. (6) In birds LeukocytozoBn. B. Sporozoa occurring in muscles Sarcocystis. C. Sporozoa occurring in membranes (mucous or serous) Eimeria (Coccidium) and Isospora. The Genus Piroplasma, or Babesia An organism belonging to this genus was first noted by Babes and called by him Hcematococcus. Theobald Smith, in 1889, made the first observation which related one of these organisms to Texas fever. He called the organism Pyrosoma. This was later changed to Piroplasma by Patton, and still later by Starcovici to Babesia. The organisms of this genus occur in the red blood-cells of various mammals and produce several distinct diseases. They do not show pigmentation. The life-history of all the species has not been satisfactorily worked out. Babesia bfgemina Synonyms. — Pyrosoma bigeminum; Apiosoma bigeminus; Piro- plasma bigeminum; Hcematococcus bonis; Ixidoplasma bigeminum. Disease Produced. — Texas fever, or tick fever in cattle- bovine piroplasmosis. Theobald Smith, in 1889, discovered the cause of Texas fever in cattle. His work was fundamental and remarkably complete. Since that time investigators have found the same organisms in many countries. 510 VETERINARY BACTERIOLOGY Distribution. — Southern United States, Australia, South Amer- ica, Europe, India, Philippines, and Africa. Morphology. — In the blood of infected animals the organisms are generally in pairs. They are commonly piriform, one end being rounded and the other somewhat pointed. The acute ends are usually pointed toward each other. The organisms vary from 0.5 to 2 /x in diameter, and 2 to 4 ^i in length. The reproductive stages have not been thoroughly worked out, nor has the develop- ment in the tick; The organism stains readily with such dyes as alkaUne methylene-blue and by Wright's method. Pathogenesis.^The relationship of the organism to the disease has been satisfactorily demonstrated by inoculation experiments. Fig. 196. — Babesia bigemina, infected red blood-cells with 1-4 parasites, a blood-platelet in the center (Sieber). The disease in cattle is characterized by fever and a hemoglobin- uria, with considerable destruction of red blood-corpuscles. In acute cases death often occurs in five to eight days after the symptoms are first noted. Those cases in which recovery takes place generally harbor still in their bodies the specific organisms, but remain perfectly well. Immunity. — ^As already noted, recovery from disease does not necessarily predicate the disappearance of the organism from the blood, and it may persist for years. It has been found that immunity against a fatal attack of this disease may be conferred by inoculation with the blood of immune animals. This results generally in a mild infection, which immunizes against one of a SPOROZOA 511 severer type. Such methods of vaccination are quite widely practised. Bacteriologic Diagnosis.— The organism may usually be demonstrated in the blood when stained with Loffler's methylene- blue or Wright's stain. Transmission. — Natural infection takes place only through the bite of infected cattle ticks {Rhipicephalus annulatus or Boophilus bovis) in the United States and closely related forms in other countries. The female tick becomes engorged with blood and falls to the ground, where, after a time, eggs are laid. They hatch in from nineteen days to five or six months, depending upon the tem- perature conditions. The young ticks crawl up the stems of grass and shrubs. They must get upon the body of an animal or die of starvation. The ticks from an infected mother are themselves infective, and may transmit the disease to the animal whose blood they suck. Babesia mutans Synonym. — Piroplasma mutans. Theiler has estabhshed the presence of a form of bovine piro- plasmosis in southern Africa, due to a prototozoan which he has natned Piroplasma mutans. It is smaller than the P. bigeminum, and animals immunized against one will contract disease caused by the other. Babesia eqai Synonym. — Piroplasma equi. Possibly Babesia asini. Disease Produced. — Equine biUary fever. Equine piroplas- mosis. Guglienni, in 1899, discovered this organism in Italy, and Theiler later elaborated an account of the disease and the organism as it occurs in South Africa. Distribution. — The disease has been noted from South Africa, Central Africa, Algeria, Italy, Sweden, Russia, India, and Vene- zuela. It is evident that further observation may show an exten- sive distribution. Morphology. — The organism is smaller than Piroplasma bigemi- num, but resembles it. It occurs singly or in pairs, or rarely in rosettes, in the red blood-cells. Occasionally it is free in the plasma. The disease was first supposed to be non-transmissible by 512 VETERINARY BACTERIOLOGY blood injection, but Theiler succeeded by intravenous injections of virulent blood. It also attacks the ass and the mule. Pathogenesis. — The disease is characterized by jaundice and a high fever. It may run an acute or a chronic course; frequently it is fatal within a few days. The lymph-nodes and the spleen are considerably enlarged. Animals born in infected districts are commonly immune. Transmission. — The disease is transmitted by ticks in South Africa by Rhipicephalus evertsi. The nymphs are infected and transmit the disease to other horses when they become adults. Babesia ovis Synonyms. — Piroplasma ovis; Hcematococcus ovis; Amcebospo- ridium polyphagum. Disease Produced. — Hemoglobinuria, malarial catarrhal fever, or icterohematuria in sheep. Babes, in 1892, first noted the parasites in the blood-cells of sheep in Rumania at the time that he made his observations on piroplasmosis of cattle. Distribution. — It has been noted from Italy, France, Turkey, Venezuela, and the West Indies, South Africa, Rumania, German East Africa, and probably in the United States (Montana). Morphology. — It is similar to Piroplasma bigeminum, but smaller (1 to 1.8 /x in diameter). It is commonly single, sometimes double, in the cells, and frequently occurs in the plasma. Pathogenesis. — The disease may be transferred by the injec- tion of blood containing the organisms into healthy animals. The incubation period is about eight to ten days. Other animals, including cattle, cannot be infected. The disease is commonly fatal. It is characterized by anemia, icterus, and frequently hemoglobinuria. Transmission. — The disease is transmitted through the bite of a tick (Rhipicephalus bursa) . The adult only transfers the disease. Babesia canis Synonyms. — Pyrosoma bigeminum yslt. canis; Piroplasma canis. Disease Produced. — BiUary fever or malignant jaundice of the dog. SPOROZOA 513 Prani and Galli-Valerio, in 1895, first described the blood parasite of canine piroplasmosis. Distribution. — It has been reported in China, India, Italy, France, Hungary, South Africa, East Africa, and possibly from the United States. Morphology. — The organisms are morphologically almost identical with Piroplasma bigeminum, but are larger. They are generally 2 to 4 ^i in diameter. They are sometimes found abun- dantly in the plasma, and in a single blood-cell there may be as many as sixteen of the organisms. The free organisms are spher- ical; those within the corpuscles are pear shaped or many angled. Multiplication is apparently by direct division. The life cycle is better known for this species than for other members of the genus. The pear-shaped bodies within the red Fig. 197. — Babesia cards: 1-11, Organisms in various developmental stages in red blood-ceUs in culture; 12, 13, organisms free in the plasma (Deseler). cells are relatively large, one end being regularly pointed. Usually two are found in a single cell, sometimes four, eight, or more. They multiply by longitudinal fusion. An ameboid stage occurs while the organism is on the exterior of the red cell, or while the organism is young. Breinl and Hindle showed that if a dog in an advanced stage of the disease is bled, the heart blood mked with 2\ per cent, of sodium citrate, motile cells with long flagella may be developed. These may be differentiated into gametes. Union of the gametes occurs in the ahmentary tract of the tick. The zygote is elongate, and makes its way into the body of the tick, eventually infecting the blood of an animal bitten by the tick. Culture .^Thomson and Fantham have succeeded in cultivating Babesia cards in vitro. Heart blood is secured, and xfr c.c. of 50 per cent, solution of glucose in water added to 10 c.c, defibrinated, 33 514 VETERINARY BACTERIOLOGY and kept at 37°. In the culture, oval, piriform, ameboid, and round parasites were found. Hemolysis occurs in all cultures. Pathogenesis. — The disease may be readily transferred by the injection of virulent blood. It cannot be transmitted to other species of animals. Nuttall and Graham-Smith did not succeed in reproducing the disease in the fox and jackal. .The period of incubation is three days or more. There is fever, and sometimes icterus and hemoglobinuria. Anemia is marked. The spleen is greatly enlarged, the gall-bladder is distended, and the kidneys are often ecchymotic. Chronic cases frequently recover. The acute cases are almost invariably fatal. Animals which have ap- parently recovered show the parasite in the blood for long periods and retain their infectivity. Bacteriologic Diagnosis. — Stained blood-films will demonstrate the organism if present. Transmission. — At least three species of tick, and probably one species of flea, have been found to act as carriers of the organism. Babesia (Piroplasma) gibsoni Patton has described an organism causing piroplasmosis in hounds in the Madras Hunt in India. Later it was discovered also in the blood of a native jackal. Its relationship to the Piro- plasma canis has not been satisfactorily determined. Babesia (Piroplasma) commune Phillips and McCampbell have described a species of Piro- plasma as the cause of an epizootic of dogs at Columbus, Ohio. Fig. 198. — Babesia commune, organisms in the red blood-cells (adapted from Phillips and McCampbell). The organisms found were similar to the Babesia canis, but these investigators were able to demonstrate the organism in the blood of guinea-pigs injected with virulent blood. The cat was also infected, but not the horse, cow, rat, or rabbit. This fact of SPOHOZOA 515 r" H Fig. 199. — Theileria parva, life cycle: 1, Agametes of the first generation (metagametes) ; 2, a and 6, agamonts with one nucleus; 3, a and 6, agamonts with several nuclei; 4, a and 6, medium-sized agamonts; 5, a and 6, large aga- monts with numerous nuclei; 6, a and 6, agamonts undergoing schizogony; 7, a and b, agametes; 8, a and 6, reduction fonns of agamonts; 9, a and 6, seg- mentation of reduction forms of agamonts; 10, a and b, young agamonts; 11, a and 6, medium-sized gamonts with several nuclei; 12 and 13, a and 5, large gamonts with numerous nuclei; 14, a and 6, gamonts undergoing schizogony; 15, free gametocytes; 16, gametocytes in the red blood-corpuscles; 17, micro- and macrogametes in the stomach of the tick; 18, copulation; 19, karyomjfjds; 20 and 21, formation of the ookinetes; 21, retort forms of ookinete; 22, ookinete (Gonder, in "Journal of Comparative Pathology and Therapeutics"). 516 VETERINARY BACTERIOLOGY transmissibility led to the tentative adoption of a new specific name, as the true Piroplasma canis is not known to be transmissible to any other animals. The organisms found were round or pear shaped. The round type were from 0.5 to 1.5 n in diameter, and the piriform 1.5 by 2.3 fx. Considerable pleomorphism was evident. Theileria parva Synon3mis. — Piroplasma parva; Babesia parva. Disease Produced. — East African coast fever, Rhodesian red- water, Rhodesian tick fever in cattle. The organism and the disease have been studied by Theiler, Koch, and others. This protozoan is the smallest of the Piro- plasmas known. In the red cells it forms a small rod that has a chromatin granule at one end. Frequently ring forms are observed, never the pear-shaped types of P. bigeminum. Gonder has worked out in detail the life-history of the organism. This disease is pecuhar, in that transference of blood containing the organism from one animal to another does not result in the transference of the disease. Repeated inoculations are without effect. It is trans- mitted by means of the brown tick {Rhipicephalus appendiculatus) and the black pitted tick {R. simus). The affected' animals show high fever and sweUing of the lymph-nodes. Anemia, icterus, and hemoglobinuria are rarely observed. Immunity to this disease does not immunize against Texas fever. What is probably the same disease has also been described from southern Russia and in Java. The Genus Plasmodium Malaria in man has been found to be due to three species of protozoa, usually placed in the genus Plasmodium. The organ- isms pass certain parts of their life-cycle in the blood-corpuscles in man, and the remainder in the gut and tissues of the mosquito. The organisms of malaria were first noted by Laveran in 1880, and the various types have been differentiated since that time. Plasmodium vivax Disease Produced. — Tertian malaria in man. Distribution. — This is the commoner malaria of temperate climates. SPOEOZOA 517 Morphology and Life-history.— The organism when first recognized in the blood is small, with ameboid movements. It penetrates the red blood-corpuscle, and develops until the interior Fig. 200. — Diagram illustrating the life-cycle of the malarial parasite: A, Sporozoites entering a red blood-cell; B, C, D, E, the organism in various stages of development; F, G, the formation of sporocytes and their division into spores which infect new red blood-cells. The series A to H represents the cycle through which the organism passes in the human body; /, infected red cell ingested by a mosquito. The organism may now develop through the series J', K', L', M', to form microgametocytes and microgametes or through J, K, L, M, to form a macrogamete which unites with a microgamete to form a fertiUzed ovum; P, the organism penetrates the stomach-wall of the mos- quito and develops through the stages Q, R, S, T, U. From U large ntmibers of slender spores are liberated into the body cavity. These pass to the salivary glands of the mosquito and are injected when the insect again bites (Rees). of the corpuscle is filled. When full grown, it may double the diameter of a blood-cell. The organism then segments to form a rosette of bodies, which round off to form small spores or merozo- ites. These are freed by the disintegration of the red blood-cell, and 518 VETERINARY BACTERIOLOGY attach themselves to other cells and begin development anew. This may be repeated several times. This is called the asexual phase of the life-history. The organism may be taken in by the mosquito (Anopheles), and here completes its life-cycle by passing through the sexual phase. Two types of cells are found to de- velop from the spores in the body of the mosquito. The male cell (known as microgametocyte) produces five to eight micro- gametes. The female cells (macrogametes) are larger and granu- lar. A microgamete fuses with one of the macrogametes to form what may be termed a fertilized "egg," copula, or ookinete. This burrows into the wall of the stomach of the mosquito, en- cysts, and enlarges greatly. The contents finally break up into a considerable number of spherical bodies known as sporo- blasts. These in turn produce great numbers of delicate fila- mentous bodies called sporozoites. These are liberated by the rupture of the cyst, and pass through the body cavity, and finally enter the poison or salivary gland, whence they are inoculated into the. next victim of the mosquito. This cycle in the insect is completed in from eight to ten days, and during this time the insect is not infective. Pathogenesis. — The disease is characterized by chills, followed by fever, which occur every forty-eight hours. The infection is usually benign; a fatal termination is very rare. The chills and fever develop at the time of formation of the merozoites and the infection of new cells. Transmission. — The disease is transmissible only through the bite of the mosquito. The elimination of the possibility of this transfer is the all-important factor in efficient prophylaxis. Plasffloditim malariae This organism produces the quartan malaria in which the interval between paroxysms of fever is seventy-two hours, and the asexual cycle is completed in this time. This disease, like the preceding, is benign, and yields readily to quinin treatment. Plasmodifim immaculatum and falciparum This type of malaria is usually tropical. It is malignant, and does not yield readily to treatment. Two types are known, SPOROZOA 519 a quotidian, which completes its asexual cycle in twenty four hours, and a tertian, which requires forty-eight. Whether or not these are distinct species is uncertain, but is probable. The Genus Proteosoma, Halteridium, and Hemoproteus These are genera of sporozoa which produce a malaria-like infection in birds. In some forms a part of the life-cycle is also spent in the body of the mosquito — in this case a Culex. None of the species are of any considerable economic importance. The Genus Anaplasma This genus was created by Theiler in 1910 to accommodate the organism described as "marginal points" in the erythrocytes of cattle. The protozoan consists of a tiny dot of chromatin- like material in the corpuscle, usually near the margin, never more than one-thirtieth to one-twentieth the size of the cell. The name Anaplasma comes from the apparent lack of any cyto- plasmic material. Anaplasma marginale Synonym. — Marginal points. Disease Produced. — Anaplasmosis, Galziekte, or gall sickness in cattle. This organism, according to Theiler, has been observed by several investigators, among them Smith and Kilbome, in their study of Texas fever. These observers have believed it to be a developmental stage of the Piroplasnm (Babesia) bigeminum. Theiler has succeeded in demonstrating the -distinction between the organisms, and defines Texas fever as a mixed infection of Anaplasma marginale and Piroplasma bigeminum. Distribution. — Known with certainty from South Africa; prob- ably widely distributed. Morphology and Staining.— The organism may be single in the corpuscles, or thertf may be several within a single cell. The para- sites usually lie near the periphery of the corpuscle, rarely free in the blood. They are small, spherical, rarely more than one- tenth of the diameter of the cell, frequently less. By appropriate staining methods the presence of a central granule surrounded by a less apparent capsule may be demonstrated. Sieber has ob- 520 VETEEINAKY BACTEBIOLOGY served multiplication of the organisms within the blood-cells by a simple type of division. Fig. 201. — Anaplasma tnarginale in red blood-cells. Note the irregularity in size and in the staining of these cells (Sieber). The Ana'plasma marginale does not stain readily with the usual anilin dyes, but can be demonstrated easily by a stain ?^J^ V ^ 1 Fig. 202, — Anaplasma marginale, stained by Heidenhain's hematoxylon: o, Single parasite; b, beginning of division, the diplococcus types; c, dumb-beU forms; d, completed divisions; e, free parasites (Sieber). such as Giemsa's. Heidenhain's iron hematoxylin also gives good results. It may be seen even in the living cells as a refrac- tive marginal granule. SPOROZOA 521 Pathogenesis.— The disease produced has an incubation period of sixteen to sixty days. The specific organisms may be first demonstrated from the spleen; later they become abundant in the blood. As many as 30 per cent, of the cells may be infected. The first evident reaction is irregularity and poikilocytosis of the red blood-cells, followed by more or less polychromasia and fragmenta- tion. The serum does not seem to acquire any hemolytic proper- ties. The febrile periods in the disease coincide with the presence of the greatest number of marginal points in the corpuscles. Cattle are susceptible to artificial infection. It is believed by Theiler and his coworkers that the primary symptoms of disease in Texas fever are due to Piroplasma Ugemina, but that the secondary symptoms are due to this Anaplasma marginale, which requires a longer incubation period. Transmission.— The organism is transmitted through the tick (Boophilus decolm-atus) . The Genus Leukocytozo6n Protozoa somewhat resembhng the malarial parasites have been found in the white blood-corpuscles or leukocytes in birds Fig. 203. — Hepatozo'on pemiciosum, a leucocytozoon from the blood of a rat: a, Free parasite; 6, parasites in the mononuclear leukocytes (adapted from Miller). by several observers, and in the domestic fowl and the dog in Tonkin by Marhis and Leger. They are not known to be of any economic importance. The Genus Sarcocystis These sporozoa are usually elongated, tubular, oval, or even spherical. Cysts with a double membrane are formed, and in these 522 VETEKINAKY BACTERIOLOGY are produced reniform or sickle-shaped sporozoites, with a polar capsule and a projectile thread. Species of this genus have been described from the muscles of a large number of vertebrates. In most cases the organism does not do any appreciable harm. Recently (1908) Watson has called attention to the prevalence of sarcosporidiosis in western Canada, particularly in animals suspected of loco-poisoning or infected with dourine. Six cases were found in cattle and two in horses suspected of being locoed, three in dourine-affected equines, and one in a filly showing cachexia. He concludes that these or- ganisms may sometimes be an important factor in disease. In some cases the entire musculature may be affected with serious and even fatal consequences. A close relationship between loco- poisoning and sarcosporidiosis is shown by the autopsy records. Recovery from this affection and from dourine may be prevented or retarded by the presence of the organisms. The species found in the horse is Sarcocystis bertrami; in sheep and goats, S. tenella; in swine, S. miescheriana; in man, S. lendemanni; in the mouse, *S. muris. The Genus Eimeria (Coccidium) This genus belongs to the sporozoan order Coccidiida. This order is characterized by having in the adult an oval or spherical form, not motile. Sporulation takes place within an endocellular cyst. The genus Eimeria is differentiated by the formation of four sporocysts or sporoblasts, each of which contains two sporozoites. The life-history is relatively complex, and varies in some details in different species. The organism is taken into the body with food in the form of a cyst, which ruptures and allows the escape of the spindle- shaped sporozoites. These penetrate the epithehal cells of the intestinal walls or other membranes. The sporozoite on entering the cells rounds up into a sphere, and then grows rapidly in size at the expense of the host cell. These growing organisms are called at this stage schizonts. The nucleus of the mature schizont fragments, and the protoplasm then breaks up into a considerable number of spindle-shaped cells, called merozoites, somewhat re- sembling the sporozoites. These break out of the mother schizont SPOROZOA 523 and infect new cells. They may then develop as schizonts and repeat the same cycle, or may develop into sexual reproductive cells. Some of these are of considerable size and correspond to an egg; these are termed the macrogametes. Others, called micro- gametocytes, develop similarly at first, then form a considerable number of very slender, thread-like cells called microgametes. A microgamete fuses with a macrogamete to form the oocyte. This then continues to enlarge, and secretes a chitinous wall; i. e., becomes encysted. When mature, the contents of the cyst divide to form four spherical bodies called sporoblasts. These become somewhat elongated and spindle shaped. In each of the sporo- blasts two still more slender fusiform sporozoites develop. The cyst is freed, and upon ingestion by a suitable host the cycle be- gins again. Coccidiosis occurs in many of the invertebrates, which do not seem to be seriously affected, but in the vertebrates serious and even fatal disease may be caused by the organisms. Eimeria avitnn Synonjrms. — Psorospermium avium; Cocddium revolta; C. per- foratum; C. tenellum. Disease Produced. — Coccidiosis in domestic fowls and in birds. These diseases have been studied by a great number of inves- tigators, and many theories of their causation have been de- veloped. Hadley and others have seemed recently to show quite conclusively that they are cases of avian coccidiosis, and the various organisms described 'by others as causal were secondary invaders or developmental stages of the organism in question. Distribution. — The disease probably has a very wide distribu- tion over the United States and Europe, but adequate data are not at hand for a determination. It is certainly known from many localities in the eastern States. Morphology and Life-history. — The life-history is typical for Coccidium as outlmed above. The adult coccidial cyst is oval or ellipsoidal. It measures about 14 by 21 fx. Pathogenesis. — A considerable number of infections in the domestic fowls have been ascribed to this organism. A mild in- fection may not result in marked symptoms. An intestinal and 524 VETERINARY BACTERIOLOGY cecal infection in the young chick has been found to be a potent, if not the principal, cause of a diarrhea, which causes such 4ar 79> 7 Fig. 204. — Eimeria avium, life-cycle: 1, Mature encysted Coccidium; 2, division into four sporoblasts; 3, sporoblasts elongated and spindle shaped: 4, two sporozoites have developed in each sporoblast; 4 a the cyst ruptured and the sporoblasts and the sporozoites escaping; 5, epitheUal cell of the in- testinal tract; 5 o, 5 6, 5 c, 6, 6 a, stages in development within the cell, the schizont stage; 7, schizont dividing to form merozoites; 7 o, cell and schizont ruptured and merozoites escaping. These again infest the epithelial cells and may repeat the cycle 5-7 a. Others produce the sexual stages 8 and 8 a to 11; 8, 9, infection of a cell with a merozoite and development of the macrogamete; 10, cell ruptured, exposing the macrogamete; 8 o, infection of a cell with a merozoite and development of the microgametocyte; 9 a, formation of the microgametes; 10 a, liberation of the microgametes; 11, fusion of the micro- gamete with the macrogamete; 12, 13, 14, 15, development of the mature encysted coccidium (Cole, Hadley, and Fitzpatrick). heavy losses in certain localities. A similar infection in adult chickens may also prove fatal. This is particularly true of the SPOEOZOA 525 turkey, which is unusually susceptible. The principal symptoms are three in number — diarrhea, progressive languor or stupor, and loss of appetite with pronounced emaciation. The disease may be acute or chronic, but is quite generally fatal. Some fowls may harbor the organisms for long periods without any apparent symptoms. Bacteriologic Diagnosis. — An examination of smears from in- fected membranes will show developmental stages of the organism. The cysts may usually be demonstrated in the feces. Transmission. — The disease is undoubtedly acquired by the ingestion of cysts which have been given off in the feces of diseased fowls. It is believed probable that wild birds may also become in- fected, and aid materially in the dissemination of the organism. Eimeria stiedas Synonyms. — Monocystis stiedce; Psorospermium cuniculi; Coc- cidium oviforme; C. perforans; Pfeifferia princeps. Disease Produced. — Coccidiosis of the rabbit. Rivolta, in 1878, first described this organism from the rabbit. It occurs in the intestinal epithelium. It may be present without evidence of disease, but is undoubtedly the cause of serious epi- zootics among tame and wild rabbits. Eimeria faovis Disease Produced. — Bovine coccidiosis. It has been re- ported most frequently from Switzerland. The organism was first noted by Zschokke in 1892. The dis- ease of young cattle characterized by enteritis with bloody feces has been reported by several European investigators. The organ- ism is morphologically typical of the genus, and has the same life- history. The disease has an incubation period of about three weeks. In the feces appear blood flecks of greater or less size; in severe cases there develops a bloody diarrhea. Occasionally the animal dies, but usually recovery takes place within a few weeks. The oocytes are abundant in the feces of affected animals, re- sembling those of Eimeria stiedce. The organisms in all stages of 526 VETERINARY BACTERIOLOGY development may be found in the epithelium of the large intestine and the rectimi. The mucous membranes may be inflamed, even purulent and covered with a diphtheritic membrane. Petechial and larger hemorrhages are to be observed. Fig. 205. — Eimeria stiedce. Schizonts, with production of mero- zoites which may repeat the cycle or develop the sexual stage; e, f, g, develop- ment of the macrogamete; h, i, j, s, development of the microgametocytes and microgametes; k, mature coccidium, which encysts and divides to form four sporoblasts; I, formation of the sporozoites and their hberation by a rupture of the cyst (Schaudinn). Eimeria faurei Disease Produced. — Ovine coccidiosis. Moussu and Marotel have described an ovine coccidiosis, par- ticularly in lambs. The developmental cycle was typical. The same or a similar coccidiosis has been reported in the United States. SPOHOZOA 527 Isospora bigemSna Disease Produced. — Coccidiosis of the dog and cat. Stiles, in 1892, described a Coccidium bigeminum, as a parasite occurring in the intestinal villi of the dog and cat. It has also been reported in man. The ripe oocysts of this form produce two spores, each with four sporozoites (in contrast to Eimeria). The oocysts are 22 to 40 by 19 to 38 ju. The spores are ellip- soidal, filling nearly the interior of the oocyte. The spores are 10 to 18 M in length. CHAPTER XLIV PARASITIC PROTOZOA OF THE CILIATA The Ciliata are differentiated from other protozoa by the pres- ence of ciha during the entire hfe of the cell. In most species there is also a cytostome or mouth, though a few species take up their food by osmosis. The cells are relatively constant in shape, that is, never ameboid. In many cases the cells are complicated in struc- ture, possessing many different types of organella. Most forms possess one or more contractile vacuoles which are useful in species differentiation. Multiplication is usually through a process of spHtting into two individuals, usually without the development of a resting stage. Many species become encysted to withstand unfavorable conditions. Only one or two species are known to be disease producers, but many are commensals in the alimentary tracts of animals, particularly in the rumen of the ruminants and in the cecum of the horse. Protozoan Commensals of the Rumen and Cecum Certain protozoa are quite constantly met in those parts of the alimentary tract of herbivorous animals where cellulose digestion occurs, in the rumen of cattle, sheep and goatSj and in the cecum of the horse. They are present in relatively large numbers. Some of the earher investigators believed that perhaps one-fifth of the contents of these organs were protozoan cells. This is probably an exaggeration, but indicates their abundance. They seem to be destroyed for the most part when the food masses pass to other parts of the alimentary tract. They probably feed on bacteria and plant fragments. These organisms are, so far as known, neither useful nor harmful, they are true commensals. The following key to the most common species may be useful in the differentiation of these commensals in the search for patho- genic organisms in disease: 528 PARASITIC PROTOZOA OF THE CILIATA 529 A. No spiral zone of cilia about th& mouth. 1. Cells oval or nearly spherical, anterior end trun- cate. Ectoplasm homogeneous, cuticle-like. Surface closely covered with very fine ciMa ar- ranged in relatively wide longitudinal rows. Near the anterior end the cilia are longer. One vacuole containing a refractile concretion is found near the anterior end. Principal nu- cleus is large, spherical. No secondary nucleus evident Genus 1. Butschlia. 2. Cells more elongate, ciliation very uniform. Front of cell rounded, posterior end somewhat pointed, slightly flattened on one side, no an- terior ring of longer cilia. Both principal and secondary nucleus present Genus 2. Isotrichia. B. Possessing a definite spiral zone of large cilia or membraneUa about the mouth. 1. Body with ciUa vmiformly distributed. Cell oval or eUipsoidal, the anterior end somewhat blunted, cells capable of changing form to some degree. Usually longitudinally striped. Principal nucleus is kidney shaped, secondary nucleus vesicular ; . . . Genus 3. Balantidium. 2. Ciha absent from major portion of body, mem- braneUa about the mouth or grouped at definite points on the cell. (a) Possessing two spiral rows of membraneUa on anterior end. Cells relatively large, long oval, somewhat flattened. At posterior end there are three rows of membranes passing around the cell, each membrane with three points Genus 4. Ophryoscoler. (b) Possessing a single spiral row of membraneUa at anterior end. (1) Posterior end of cell extended into three points, one of which is rudder-like and much longer than the other two. The peristome or ring or adoral membraneUa are retrac- tile Genus 5. Entodinium. (2) Posterior end of ceU entire. Adoral ring consists of twenty-four membraneUa, or large ciUa. The mouth is in a blunt cone within the peristome. Near posterior end there occur two short tubular bodies from which a cluster of membraneUa protrude. These are used in swimming Genus 6. Cycloposthium. Butschlia. — Two species, Butschlia parva and B. negleda, are common in the rumen of cattle. B. postdliata occurs in the cecum of the horse. 34 530 VETERINARY BACTERIOLOGY Isotrichia. — The species Isotrichia prostoma and I. intestinalis are very common in the rmnen of cattle, as is also a closely related form, Dasytricha ruminantium. Ophryoscolex. — Two species from the rumen of sheep are Ophryoscolex scolez and 0. inermis. Entodinium. — The species Entodinium caudatum, E. bursa, E. dentatum, E. rostratum, and E. minimum are all found in the paunch of ruminants. ■^-^^l^h "Vti ^^- Fig. 206. — Balantidium coli in a blood-vessel of the submucosa of the intes- tines (Bowman, in "Philippine Journal of Science"). Cycloposthium.— The species Cycloposthium bipalmatum occurs in the cecum of the horse. Balantidium. — One species of Balantidium produces disease in man. Other species of protozoa which have been described by Braune from the ruminants are Trichomastix ruminantium, Trichomonas ruminantium, Callimastix frontalis. Awerinzeff and Mutafoua have described Diplodinium fiorentinii, Ophryoscolex intermixtus, 0. fasciculus, and 0. lohiatus. PARASITIC PROTOZOA OF THE CILIATA 531 Trichomonas. — Hadley has reported the occurrence of tricho- monads in healthy and sick fowls throughout the intestinal tract. They are almost invariably present in the intestinal and cecal contents but are found in the greatest numbers in the mucous layers of the caeca, sometimes deep in the crypts, whereas cocci- dia multiply mainly in the duodenum and araoebEe in the epithe- lial layer of the lower intestine and caeca. He has presented evidence to prove that entero-hepatitis (black head) of turkeys is due to Trichomonas and that the Amceba meleagridis of Smith is simply one stage of this organism. He states that confusion with the amoebae arises when the Trichomonas becomes non-motile, loses its flagella, and becomes rounded or oval in shape. It was this stage which stains pink with eosin that he believes was mis- taken by Smith for an amoeba. This flagellate is described by Hadley' as "pear shape, elon- gate, half moon or globular shaped depending upon the stage of development. In the free swimming stage it has a length of 8 to 12 M. It manifests the usual Trichomonas characteristics, viz., three anterior and one posterior flagella, a vibratory mem- brane, axostyle, chromatic line, chromatin blocks, nucleus, vacuoles, peristome, blepharoplast and rhizoplast." Hadley finds that the pathological condition is complicated in the liver only rarely by amoebic infection, and in the caeca both by amoebae and coccidia. Balantidittm colS Disease Produced. — A rare fatal enteritis in man. This organism is oval in shape — 50 to 70 by 60 to 100 fi — and possesses a funnel-shaped peristome which terminates at the mouth. Cilia cover the surface. The two nuclei and two contractile vacu- oles may commonly be made out. An excretory apparatus is evi- denced by the extrusion of waste material at a definite point in the cell surface. The life-history is relatively complex. The cell commonly multipUes by direct division. Conjugation and encyst- ment may occur. The Balantidium coli appears to be a common inhabitant of the intestinal tract of swine. A large number of instances are recorded 'Bull. Rhode Island Agr. Exper. Sta., 166 and 168. 532 VETERINABY BACTERIOLOGY in which it has been found associated in man with a severe and even fatal type of diarrhea. The connection of this organism with the disease as a causal agent rather than as a commensal seems to be well authenticated. It appears that infection may follow the ingestion of the cysts produced by the organism. The disease has been reported from Europe, the United States, and the Philippines. SECTION VI INFECTIOUS DISEASES IN WHICH THE SPECIFIC CAUSE IS NOT CERTAINLY KNOWN CHAPTER XLV DISEASES PRODUCED BY UNKNOWN ORGANISMS Within the last two decades there have been described a number of diseases in which the causal organism is said to be ultramicroscopic. By this is meant that with the best powers of the microscope no definite organism can be distinguished. Such an organism is better called a filterable virus, because filtrates passed through porcelain filters retain their pathogenicity for sus- ceptible animals. It is a principle of optics that no object can be clearly differen- tiated that is smaller than one-half the wave-length of the light in which it is examined. This means that there is an apparently insuperable physical obstacle to the observation of some of these forms, as our best lenses approach moderately near to this limit in their magnification. Our reasons for believing that there may be organisms this small will be discussed under the heading of various diseases. There is in more or less general use an instrument known as the ultramicroscope, which renders visible objects more minute than had heretofore been observed. This instrument enables one to observe objects by making use of the principle worked out by Tyndall in determining the absence of floating dust particles in the air. He noted that when a ray of a very bright fight was admitted to a darkened room or box, this ray could be distinctly seen as long as there were any floating .particles, but became invisible when these dust particles had completely subsided. The motes or particles, even when smaller than can usually be seen by the un- aided eye, became visible when thus illuminated. This principle is 533 534 VETERINARY BACTERIOLOGY applied to microscopic examination by sending the rays from an arc light or similar source, so that they are concentrated in a powerful beam, which is passed through the hanging drop or similar preparation from side to side. Exceedingly minute particles may thus be made visible. The use of this instrument has been found to be less helpful in the fields of biologic research than had been hoped. Very few facts concerning the organisms that cause disease, and particularly the ultramicroscopic organisms, have been discovered by its aid. An attempt has sometimes been made to compare the relative size of ultramicroscopic organisms by the use of porcelain filters of different degrees of density. It has been found that the virus , of some diseases will pass through coarse filters, but not through the finer ones. It does not seem to be entirely a matter of the relative size of pores and organisms that may pass through the filter. It is probably a phenomenon analogous to an adsorption quite as much as mechanical filtration that removes the organ- isms. Bacterial or Protozoan Relationships of Ultramicroscopic Organisms. — There is no practicable method telling certainly whether or not a filterable virus should be 'grouped with the protozoa or with the bacteria. There are methods which may sometimes be used that will give inferences, however. It has been found possible in some cases to secure growth in culture-media, such as is used for bacteria. Such organisms are probably bac- teria. In others, the type of disease produced may resemble so closely some other infection produced by a known organism that a probable classification into protozoan or bacterial might be made. The type of immunity developed may likewise be of importance. In most instances these differences are not pronounced enough to allow of certainty in the classification. The more important diseases which have been described as due to filterable virus are contagious pleuropneumonia of cattle, rinderpest or cattle plague, foot-and-mouth disease, hog-cholera, horse sickness, dog distemper, fowl plague, equine anemia, fowl- pox, infectious agalactia in sheep and goats, fowl leukemia, guinea-pig plague, certain chicken tumors. DISEASES PRODUCED BY UNKNOWN ORGANISMS 535 Virus of Pletiropneamonia Disease Produced. — Pleuropneumonia, peripneumonia, lung plague of cattle and other bovines. Nocard and Roux described the causal organism in 1898. The disease itself has been known in Europe for several centuries. Distribution. — The disease is known from Europe, Africa, Aus- tralia, Asia, and has been imported into the United States, where, in 1886, it killed 10,000 animals in lUinois alone. Nature of Virus.^The causal organism is doubtless a bacterium that is just at the lunit of visibility. In suitable fluids it may be observed under very high powers as a tiny motile point. Bouil- lon inoculated with the serous exudate from the pleura or from one of the areas of consoUdation in the lungs, sealed in a collodion sac, and placed in the peritoneal cavity of a rabbit for two weeks, will show a slight clouding or opalescence. Transfers to new media similarly treated will likewise result in growth. This materia] may be shown to be infective. The organisni may also be culti- vated in a mixture of bouillon and blood-serum outside the animal body. In two to three days it shows a very faint clouding. Agar containing serum develops delicate, transparent, almost invisible colonies. The optimum temperature is 37°; no growth occurs under 30°. Pathogenesis.^ — Injection of pure cultures of the organism into cattle results in infection. Intrapleural injections or inhalation of the organism results in a typical clinical picture of the disease. The disease is characterized by more or less extensive areas of he- patization in the lungs and an inflammation of the pleura, accom- panied by a serofibrinous exudate. The amount of fluid which collects may be very considerable. Immunity. — Recovery from the disease results in a relatively permanent immunity. Immunization against the disease by vac- cination with serum from the pleural cavity of infected animals has been practised. The material is injected subcutaneously. Its use is attended with danger, as from 0.5 to 5 per cent, or even more of those vaccinated have been killed by the vaccine. The method in question has been of some use in immunization, but a stamping- out process would appear to be more efiicacious. Nocard and Roux have advised vaccination with pure cultures. 536 VETERINARY BACTERIOLOGY and claim to have secured more favorable results than by the. older method. Nocard has also produced a curative and pro- phylactic serum by the hyperimmunization of animals by the injection of increasing doses of pure culture until 6 liters have been used. In doses of 40 c.c. it served as an efficient prophylactic, and in larger amounts as a curative agent in the early stages of the disease. Transmission. — The method of natural spread of the disease is not certainly known. It is probably through inhalation of the causal organism. Virus of Foot-and-moath Disease Disease Produced. — Foot-and-mouth disease, aphthous fever, Maul- and Klauenseuche in cattle and other boyines, sheep, goats, swine, deer, and occasionally horses, dogs, cats, and man. The disease has been known for over a century in Europe. Loffler and Frosch, in 1897, Hecker, in 1898, and others since that time have shown that the virus of foot-and-mouth disease may pass through Chamberland and Berkefeld filters, but not through the thicker Kitasato filter. Distribution. — The disease is known from most of Europe, Asia, and Africa. It has been introduced several times into the United States, but has been stamped out. It has also been reported from Argentina. Nature of the Virus. — Recently Stauffacher has claimed to have demonstrated the causal organism to be a protozoan which he names Aphthomonas infestans. He places it close to the genus Leishmania. For demonstration of the organisms in tissue sec- tions and blood-smears he fixed in 70 per cent, alcohol, stained from two to six hours in 0.2 per cent, aqueous acid fuchsin, rinsed in distilled water, and stained in Ehrlich's fuchsin methylene-blue for six to ten hours, rinsed in distilled water and placed in abso- lute alcohol until color was no longer extracted, cleared in xylol, and mounted in balsam. Stauffacher succeeded in cultivating the organisms in the con- densation water of NicoUe's blood-agar both from the blood and from vesicles of infected animals. The protozoan here was found in two forms, one shorter and more plump, the body 20 to 25 fi in DISEASES PRODUCED BY UNKNOWN OEGANISMS 537 length and 3 (lin width, with long flagellum. The other type were long slender cells, even 120 n in length. A type of spore produc- tion was also observed leading to the production of cells probably small enough to pass a filter. The disease was transmitted by the use of pure cultures and the same organism isolated. Pathogenesis. — The disease may be produced by the inocula- tion of susceptible animals with the filtrate through a porcelain filter. It is characterized by an acute fever, the appearance of a vesicular eruption on the mucous membranes of the mouth, on the feet, and between the toes. It is commonly not fatal, but is so contagious, and leads to such losses in flesh and milk, that it is among the most feared of cattle diseases. Immunity. — Vaccination by intentional infection of animals has sometimes been practised in an effort to "get it over with" as quickly as possible when it breaks out in a herd. An animal recovered is relatively immune. Such methods are attended by considerable risk. An efficient and safe method of immunization or vaccination has not been developed. Transmission. — The organisms gain entrance through contact of healthy animals with the saliva or other secretions of an in- fected animal. They may be transmitted to young animals or to man in milk. Virus of Rinderpest or Cattle Plagae Disease Produced.— Cattle plague, rinderpest, or contagious typhus in cattle, rarely in sheep, goats, and camels. Nocard and, later, Tartakowsky observed that the body fluids in animals having this disease contained no visible microorgan- isms but were infective. NicoUe and Adelbey estabhshed that these fluids, if thinned with water, could be passed through the coarser Berkefeld filters, but not through a fine-pored Chamber- land, without losing their virulence. Distribution.— The disease has been reported from a large portion of the area of Europe. It is endemic in southern Asia, is known in the Phihppines, and has caused great losses m Egypt and in Southern Africa. It has not gained entrance to the United States. ' , , ^ u u- Character of Virus.— It is filterable and has not been culti- vated. Blood sealed hermetically in tubes is found to retain its 538 VETERINARY BACTERIOLOGY virulence for months. The virus is destroyed by desiccation or by heating to 58° to 60°. It may survive in putrefying flesh for considerable periods. It is easily destroyed by disinfectants. Pathogenesis.— The virus is present in all the body tissues and excretions. One one-thousandth of a gram of blood from an infected animal at the height of the disease has been found sufficient to reproduce the disease in a susceptible animal. The infection is an acute, highly fatal fever, in which there are croupous diphtheritic lesions of the intestinal tract. It is typically a cattle disease, but occasionally attacks other animals. Immunity. — Animals which recover spontaneously from the disease are highly immune, and the blood has some power of pas- sive immunization when injected into another animal. Vac- cination with the nasal secretion of sick animals into the tails of others has been practised, and has been found in some instances to result in a low mortality. The method has been practically abandoned. Injections of the bile from animals having the disease has been advocated by Koch and extensively practised in South Africa, with good results. The common method of immunization against rinderpest is that developed by KoUe and Turner. Animals which have recov- ered spontaneously from an infection, or that have been im- munized by injections of virulent blood and gall, are hyper- immunized by repeated injections of virulent blood. The first injection is of a liter of virulent blood. After the subsidence of the reaction an injection of 500 c.c. is given, and later a third injec- tion of a liter. A fourth injection may be made. The blood is drawn from the jugular vein at three intervals, a week apart. Another injection of a liter of virulent blood is given, and later the animal is again bled. The serum, in amounts of 20 c.c, should protect an animal against injection of 1 c.c. of virulent blood. An injection of 50 to 100 c.c. of the serum so secured will protect an animal against infection for a space of two to four months usually. A more permanent immunity may be established by the use of what is termed the "serum simultaneous" method. Thp animal is injected on one side with 8 to 25 c.c. of the immune serum, and on the other with 1 c.c. of virulent blood. Some animals react by a distinct fever, others show no effect. The latter are rendered DISEASES PRODUCED BY UNKNOWN ORGANISMS 539 immune for several months only, while the former for much longer periods. The blood of the animals reacting is infective during the period of fever. The vaccination mortality is about 1 per cent. Transmission.— The disease is readily transmitted by means of soiled food, water, and by direct contact. Virus of Hog-cholera Disease Produced.— Hog-cholera, Schweinepest, swine fever. From the time of the researches of Salmon and Smith on this disease, pubhshed in 1885, until 1904, the cause of hog-cholera was believed to be the Bacterium choleroe suis (Bad. suipestifer). In the latter year de Schweinitz and Dorset showed the typical hog- cholera in the United States to be due to a filterable virus. This has been confirmed by Hutyra, Uhlenhuth, and others in Europe. Distribution. — The disease is wide-spread in Europe and North America. Nature of Virus. — The organism causing hog-cholera is a filter- able virus. It passes readily through porcelain filters. It has never been cultivated. Fluid material will retain its virulence for a pe- riod of ten to fourteen weeks, at least when kept at room-tem- peratures. It is killed by exposure to 60° to 70° for an hour. Desiccation does not destroy it at once, but only after a lapse of several days. It may be destroyed by disinfectants^ but is rela- tively resistant. Pathogenesis.— The subcutaneous injection of 1 to 2 c.c. of filtered blood-serum or body fluids results in the production of the disease. The animals may die of a very acute type of the disease, or it may assume a chronic form. The acute cases generally reveal, on autopsy, hyperemia and acute swelling of the internal organs, and hemorrhages on the serous and mucous membranes, and fre- quently a serous transudate into the pericardium. In the more chronic type ulcerated and necrotic areas are commonly found m the intestines, together with pneumonia. The disease cannot be transferred to other animal species. The Bacterium cholerw suis is probably a secondary invader, but the lesions produced by this organism may in some cases be of the greatest importance. 540 VETEBINAKT BACTEBIOLOGY Immunity.— Animals that have recovered from an infection with hog-cholera are thereafter immune, and it has been shown that their blood has some immunizing power when injected into other susceptible individuals. Practical methods of immunization were developed through the work of Dorset, McBryde, and Niles in this country. The method devised by them and commonly used is as follows: It is necessary to start with an animal that has recovered from the disease or that has already been immunized. This animal is then hyperimmunized by the intravenous injection of virulent blood. Such blood must be of a strain of known virulence established by previous tests. It is preferably obtained from pigs weighing 60 to 90 pounds which have been inoculated seven to ten days pre- viously, and which show well-marked symptoms and subsequent postmortem lesions of acute cholera. About 5 c.c. of the defibrin- ated virulent blood is injected for each pound of body weight. Ten days later the animal thus hyperimmunized is bled from the carotid artery or the tip of the tail. The blood is defibrinated and the clot removed. This defibrinated blood is preserved by the addition of ^ of 1 per cent, carbolic acid. Before use it is tested on susceptible pigs to determine its potency. For testing it is customary to use young pigs weighing from 50 to 60 pounds. A serum of. satisfactory potency should protect, in a dose of 15 c.c. or less, one of these animals against an injection of 2 c.c. of virulent blood. Permanent immunity is conferred by the use of the serum simultaneous method. In this method virulent blood (1 to 2 c.c.) is injected at the same time as the immune serum. This results in the development of an active immunity, which is relatively permanent in comparison with the immunity of four to six weeks conferred by antiserum injection alone. The use of this serum has been found to be highly successful in practice. It is not known what property of the antiserum is thus effec- tive, whether it is antitoxic, opsonic, or bactericidal. There is some evidence that it is the last, but proof is difficult to secure. Transmission. — The virus may be demonstrated in the blood, the tissues, and the urine of infected animals. It is probable that infection commonly takes place through ingestion. DISEASES PRODUCED BY UNKNOWN ORGANISMS 541 Virus of Horse Sickness Disease Produced.— African horse sickness, or Pferdesterbe. This disease, known from southern Africa for more than a century, was first shown by MacFadyen, in 1900, and later, in 1901, by Nocard to be due to a filterable virus. The disease is characterized as an acute or subacute disease of solipeds, that ap- pears in epizootics during the hot months of the year. The prin- cipal lesions are edematous sweUings and hemorrhages of the inter- nal organs. The virus will pass through a Berkefeld or a Chamber- land porcelain filter if the serum is, diluted with physiologic salt solution. Immunization against the disease may be brought about by the use of serum from hyperimmunized animals. Koch hyperimmu- nized horses that had recovered from the disease by three to four injections of virulent blood at intervals of two to four weeks, as much as 2 liters being used for the last injection. Serum simul- taneous injections of this hyperimmunized serum and virulent blood into susceptible animals in correctly proportioned doses will immunize. Theiler reports the safest method to be the injection of 300 c.c. immune serum intravenously and | c.c. virulent blood subcutaneously. Animals thus treated will show a rise in tempera- ture, upon which a second injection of 50 to 100 c.c. of serum is made. The disease has been found to be contracted generally at night, and the first frost puts an end to the epizootic for the year. Con- siderable quantities of the virus must be fed before an infection is produced, showing that natural infection is probably in some other manner than bj'^ ingestion. It is probable that mosquitoes, possibly flies, act as carriers. The disease cannot be regarded, therefore, in the strictest sense as contagious. Virus of Infectious Anemia of tfie Horse Disease Produced. — Infectious anemia, pernicious anemia, mud fever, swamp fever of the horse. This disease has been known as a clinical entity in Europe for three-quarters of a century. Carre and Villee (1904-1906) and Ostertag and Marek (1907) have demonstrated the disease to be due to a filterable virus. The disease has been studied in North 542 VETERINARY BACTERIOLOGY America by several investigators, and the ultramicroscopic nature of the virus has been independently demonstrated. It is not certain that all the infections described under this name are identical, but there is considerable evidence tending to establish such as a fact. Distribution.— The disease is probably wide-spread, but has not always been clearly differentiated. It is known from Germany, France, Hungary, Switzerland, and Sweden in Europe; and from Saskatchewan, Manitoba, Minnesota, the Dakotas, Nebraska, Kansas, Colorado, Wyoming, Montana, Texas, and Nevada in North America. Nature of the Virus. — The causal organism is a filterable virus that cannot be differentiated by staining methods and has not been cultivated. It is found in the blood, the urine, and the feces of infected animals. It is destroyed at a temperature of 58°, It will withstand drying for several months, and liquids maintain their infectivity for months, even when decaying. Pathogenesis. — The virulent blood or blood-serum will infect another animal upon subcutaneous injections of small quantities. The incubation period after injection varies from five to nine days, or even more. The initial symptom is a fever. The disease is more apt to be acute when the organism is introduced by injections than by ingestion. The disease may be characterized as an acute or chronic anemia which has the appearance of a septicemia in which there is a great destruction of blood-elements. The anatomic findings are characteristic. Mack, in his work on cases in Nevada, notes profound cardiac and respiratory disturbances. There is a progressive destruction of the red blood-cells, parenchymatous degeneration of the kidneys and liver, and extensive changes in the vascular system. The spleen is engorged and frequently degener- ated, and the bone-marrow undergoes profound degeneration. Immunity. — No method of immunization has been developed. Transmission. — European writers are of the opinion that in- fection arises through the ingestion of food soiled by excretions of infected animals. The mode of dissemination has not been satis- factorily established. Virus of Dog Distemper Disease Produced. — Dog distemper, Hundstaupe. DISEASES PRODUCED BY UNKNOWN ORGANISMS 543 Bacteria belonging to several different groups, particularly to the colon-typhoid and to the hemorrhagic septicemia groups, have been described as the cause of dog distemper. Carr^, in 1905, attributed the cause to a filterable virus. Ferry and others concluded that Carr6 must have been in error in his conclusions, masmuch as they claim to have found Bacterium broncMsepiicum (q. V.) to be the primary cause. Distribution.— Europe and America; probably in other parts of the world. Nature of the Virus.— Little is known of the virus beyond the fact that it can be passed through a porcelain filter. It has not been cultivated. Pathogenesis.— The disease is a highly fatal, acute infection of young carnivorous animals, characterized by an acute catarrh of the mucous membrane, and frequently a catarrhal pneumonia. In a small percentage of the cases nervous symptoms develop. The discharge from the mucous membranes is highly infective. Secondary infection with bacteria is common, and is believed by some investigators to account for many of the deaths. Immunity. — ^Many attempts have been made to prepare an anti- or immune serum that would be satisfactory in preventing or curing the disease. Several have been placed upon the market, but none has been shown to be efficacious. It is possible that success might be attained by the use of hyperimmune blood. Transmission. — The disease is transmitted by direct or indirect contact with infected individuals. Virus of Fowl Plague Disease Produced. — Fowl plague, chickenpest of domestic fowls. This disease has generally been confused with chicken cholera, which it closely resembles clinically, although Perroncito un- doubtedly described it in 1878. Centanni and Savonuzzi, in 1901, showed that the virus could pass through a porcelain filter. This has been amply confirmed by other writers. Distribution. — The disease is known only from northern Italy, the Tyrol, Germany, and France. Character of the Virus. — The organism is a filterable virus, and 544 VETERINAEY BACTEEIOLOGY is probably ultramicroscopic, although Rosenthal, Kleine, and Schiffman have described bodies in the nerve-centers that are possibly protozoan in nature. The virus is found in the blood, the nasal secretion, and generally throughout the tissues. The blood retains its virulence for three months when sealed in tubes and kept in a dark place. The thermal death-point is 55° for thirty minutes or 60° for five minutes. The dried virus has been found to retain its virulence for two hundred days, and in glycerin and serum mixture for two hundred and seventy days. It is easily destroyed by disinfectants. More recent experimental work has shown that the virus of fowl plague is capable of passing the pores of fine-grained filters, and even the so-called ultra-filters. It is claimed by some authors that the virus must approach in size the protein molecule. Andriewsky and others contend that this is, an example of a Contagium vivum fluidum, that is, does not consist of organized cells at all, but a self-perpetuating fluid living material. It has been claimed that it shows many of the reactions of the serum globulin. Pathogenesis. — Injection of as small amount as 1,000,000 ^■^- of virulent blood or secretions is sufficient to infect. The virus is pathogenic for many birds besides fowls, but not for mam- mals. The disease is characterized by the hemorrhages into the serous membranes in acute cases, and in the less acute edema of the subcutaneous tissues and of the serous membranes, and the formation of a fibrous exudate upon the latter. Immunity. — -Practicable methods of immunization have not been developed. Transmission. — ^The disease is transmitted by the ingestion of food soiled with the infective feces, nasal secretion, or blood of infected birds. Virus of Epithelioma Gsntagiosam Disease Produced. — Fowl pox, epithelioma contagiosum in domestic fowls, sore head, probably fowl diphtheria. Marx and Sticker, in 1902, determined the cause of fowl pox to be a filterable virus. Several other investigators subsequently confirmed their results. Distribution. — The disease is known to occur in Europe and in the United States. It is doubtless widely distributed. DISEASES PRODUCED BY UNKNOWN ORGANISMS 545 Nature Of the Virus.— Marx and Sticker showed that when th fl .j^ \^°'^^le was triturated in physiologic salt solution Som ." ^^^^^ ^^^^^^ through a Berkefeld filter was infective. 9^ • A- ^^'^^ ^^^® •^^^^''"^sd tiny spherical granules, less than . M m diameter, in the emulsion of the virus, but it is by no means certain that these are the disease-producing organisms. The virus IS relatively resistant to unfavorable conditions. The nodules may be dried for weeks without death of the virus. It is destroyed by heating to 60° for eight minutes. Mixed with glycerin it re- tams Its mfectivity for many weeks. It is easily destroyed by disinfectants. Pathogenesis. — The disease is a chronic, contagious infection, characterized by an initial catarrh of the mucosa of the head, followed by wart-like growths (epithelial hyperplasia) of the skin, especially of the comb and naked skin of the head, sometimes associated with a croupous diphtheritic condition of the mucosa of the head. This latter condition is one of those grouped under the general name of fowl diphtheria. The disease commonly ter- minates favorably in three to five weeks. Hadley and Beach claim to have been successful in securing active immunity by triturating the crust which forms on the skin and the membranes from the mucous surfaces in physiologic salt solution, heating at 55° C, and using this as a vaccine. Transmission.— The disease is transmitted by direct contact with infected fowls. Virus of the Poxes Disease Produced. — Small-pox in man, cow-pox, sheep-pox, horse-pox, swine-pox, goat-pox. There is much doubt relative to the position of the virus of the various poxes. They are included in this group tentatively, as it is found that the contents of the vesicles of the eruptions may be filtered through a thin, coarse porcelain filter under pressure with- out losing their infectivity. The causal microorganisms are, at some stages at least, filterable and possibly ultramicroscopic. Certain cell inclusions have been described as being probably of protozoan nature, but the subject cannot be said at present to be 35 546 VETERINARY BACTERIOLOGY completely elucidated. The protozoan parasite has been named Cytorrhydes vacdnce in man. Immunity. — Recovery from an attack of variola is accompanied by a relatively permanent immunity. Vaccination is, therefore, commonly practised, particularly against small-pox in man. The attenuated virus in this instance is secured by passage through an animal, usually a heifer. The vaccinating material is the lymph from the vesicles produced on the animals. It is inoculated into the skin by scarification. The virulence is apparently very greatly decreased by this method of inoculation, so that a relatively mild type of disease is produced which terminates in immunity being established. To what this immunity may be due is not known. Virus of Yellow Fever Yellow fever in man has been shown to be due to a filterable virus, possibly an ultramicroscopic organism. All efforts at cultivation have failed. The disease is spread only through the bites of mosquitoes that have taken virulent blood. The organ- ism evidently undergoes a part of its Hfe-cycle in the blood of the mosquito (Stegomyia), for the latter does not become infective itself for several days. It is evidently more than a mere mechan- ical transfer of the organism by the mosquito; the latter serves as a true intermediate host. The recent work of Noguchi would seem to indicate that a spirochete (Leptospira) is the causal organism, this organism being cultivable and microscopic. Virus of Rabies Disease Produced. — Rabies in animals. Hydrophobia in man. Lyssa. The disease has been studied at great length by many in- vestigators, and there is still great disparity of opinion as to the nature of the cause. Remlinger and Riff at Bey in 1903 showed that the virus could be passed through a porous Berkefield filter. This has been substantiated since by several workers. As will be seen below, this does not satisfactorily settle the problem, as those who hold to the protozoan nature of certain bodies in the nerve-centers in the disease contend that extremely minute plas- tic stages in the life-cycle of the organism might easily pass through. DISEASES PBODUCED BY UNKNOWN ORGANISMS 547 Distribution. — The disease is worldwide in distribution. Nature of the Virus. — Students of the etiology of this disease may be divided into two groups — those who beheve in the presence of a specific ultramicroscopic organism, and those who believe m the presence of a protozoan with certain stages of development in which the organism is small enough to pass the pores of the filter. The latter theory has been developed by Negri. The organism has been named Neurorrhyctes hydrophobicp. In 1903 he demonstrated the presence of specific bodies, which have been termed Negri bodies, in the larger ganglia-cells of Ammon's horn, as well as in other parts of the central nervous system. There is little ques- tion but what these bodies are characteristic of the disease; the disputed point is whether they are specific organisms or degenera- tion products of the cell. Williams and Lowden summarize the evidence of the protozoan nature of these bodies as follows: "They have definite characteristic morphology; this mor- phology is constantly cyclic, i. e., certain forms always predominate in certain stages of the disease, and a definite series of forms in- dicating growth and multiplication can be demonstrated; the structure and staining qualities, as shown especially by the smear method of examination, resemble those of certain known protozoa, notably of those belonging to the suborder Microsporidia." The Negri bodies in suitably stained preparations are found to vary in size from less than 0.6 to 25 /*. This variation in size is markedly noticeable in different species of animals. In cattle they are very large and numerous in the hippocampus; in the horse very small and in limited areas; in cats mostly in the Purkinje cells of the cerebellum. In shape they may be spherical, ovoid, or ellipsoidal. The bodies shows a characteristic structure, a smooth hyaline margin, with inclusions of various kinds that resemble chromatin granules. They may be readily stained by Giemsa's method, or with eosin and methylene-blue. Pathogenesis. — The organism enters the body through wounds, usually bites of animals. It then passes slowly along the per- ipheral nerves to the central nervous system. The portion of this to which these nerves directly lead is the most seriousfy affected. Characteristic gross anatomic lesions are quite lacking in this disease. 548 VETERINARY BACTERIOLOGY The period of incubation is variable; it is usually several weeks. It probably represents the period necessary for the virus to reach the central nervous system and develop there. The dis- ease is commonly fatal. It affects most mammals, including man, but is primarily a disease of the carnivora, particularly the dog. Immunity. — The Pasteur method of treatment is essentially a method of vaccination at intervals with attenuated virus. The virus is found, on experimentation, to be rather variable in its power to produce disease. The virulence is exalted by repeated inoculations of rabbits until it becomes the "fixed" virus of Pasteur, and will kill rabbits in six to seven days. This is then Fig. 207. — A Negri body. Note the circle of chromatoid granules about the central body (X 2000) (Williams and Lowden). injected into a rabbit, and upon its death the spinal cord is care- fully removed with all aseptic precautions, and suspended in a desiccator over caustic potash. It is kept at a constant tempera- ture of 23° in the absence of light for two weeks. The vaccine consists of an emulsion of this cord in physiologic salt solution. Later, injections are made with a cord that has been dried for a shorter period. Repeated injections are made. The fact that the disease has normally a long incubation period gives an oppor- tunity in the human for the use of this method. The active immunity established by the injection of the attenuated virus is DISEASES PRODUCED BY UNKNOWN ORGANISMS 549 suflBcient to destroy the infecting organism. This method of treatment has been highly successful when commenced in time. It is still strictly an active immunity. To what principle it is due is not known. While the Pasteur method is extensively used in institutions for the treatment of rabies, as also by physicians, there have been developed other methods. One of these is coming into more general use. This is the " dilution method " of Hogges. A definite quantity of the spinal cord of a rabbit killed by fixed virus is emulsified in 100 c.c. of normal salt solu- tion. Dilutions of this solution are prepared and the patient is given first a dilution of 1 to 1000, and subsequent injections of increasing concentration until a dilution of 1-10 is reached. Fig. 298. — Removal of the spinal cord from a rabbit (Stimson, BuU. No. 65, Hygienic Laboratory). / Bacteriologic Diagnosis. — The disease may be diagnosed by animal inoculation and by microscopic examination. For the former it is customary to inject an emulsion from the brain into a rabbit. The inoculation is usually made subdurally. Sections or smears may be made from the brain and stained to show the characteristic Negri bodies. Small portions of the gray substance 550 VETERINARY BACTEBIOLOGY are removed from the cerebral cortex in the region of the crucial sulcus, the cerebellar cortex, and the hippocampus major. These are crushed on a slide and a smear made by means, of a cover-glass. These dried smears may be stained by Giemsa or other stains, perhaps most readily by the method described by Williams and Lowden: "To 10 c.c. of distilled water 3 drops of a saturated alcoholic solution of basic fuchsin and 2 c.c. of LofHer's solution of methylene-blue are added. The smears are fixed while moist in Fig. 209.— Method of drying the spinal cord of a rabbit for the purpose of attenuation (Stimson, Bull. No. 65, Hygienic Laboratory). methyl alcohol for one minute. The stain is then poured on, warmed until it steams, poured off, and the smear is rinsed in water and allowed to dry." Transmission.— The saliva of diseased animals is found to be infective, and the disease is transmitted commonly through the bite. Virus of Infectious Agalactia of Sheep and Goats Celli and deBlasi in 1906 determined the infectious agalactia of sheep and goats to be due to a filterable virus, and their con- DISEASES PRODUCED BY UNKNOWN ORGANISMS 651 elusions were verified by Carr6. The channel of infection as de- termined by experiments appears to be the alimentary tract. The disease results in a diminution or cessation of milk flow. Food is probably infected through the milk or from the abundant secretion from the eyes resulting from a keratitis characteristic of the disease. By hyperimmunization by repeated injections of virus it is pos- sible to secure a serum that possesses a decided immunizing power. Viras of Guinea-pig Plagtie deGaspari has described a guinea-pig disease characterized by loss of appetite, trembling, convulsions, and finally death. In the brain substance this investigator first found a filterable virus by the use of the Berkefeld filter. This reproduced the disease upon inoculation into healthy animals, the infection always proving fatal. The virus is present in the blood-serum and in all the body organs. The virus is relatively resistant. It may be destroyed by ex- posure for an hour to a temperature of from 70° to 72°. Six days in decaying material, fourteen days in pure glycerin, and ten days' drying did not suffice to destroy it. Five per cent, phenol killed the virus in half an hour. Other animals than the guinea-pig are not susceptible to infection. Viros of Fowl Letikemia EUemann has claimed that the leukemia of the domestic fowl is due to a filterable virus. Vires of Certain Chicken Tamors Rous, Peyton, and Murphy have described three distinct types of tumors in the domestic fowl, each due to a filterable virus. One of these was a spindle-celled sarcoma, another an osteochondro- sarcoma, and a third a spindle-celled sarcoma with numerous blood sinuses. Repeated inoculation gradually raised the virulence of each strain, but each continued to produce its particular type of tumor upon inoculation. A certain amount of tissue injury at the site of inoculation seems to favor the development of the tumor. In- jection of the infusorial earth, for example, increases the percentage of "takes." SECTION VII BACTERIA OF WATER AND FOOD CHAPTER XLVI BACTERIA OF WATER AND WATER PURinCATION Diseases of man and animals, particularly those of the ali- mentary tract, are frequently transmitted through contaminated or impure water. The impurity, so-called, arises from the presence of sewage or surface-wash. This does not mean that every water containing sewage is necessarily harmful, but that the presence of the sewage is an indication of the possible and probable occasional presence of pathogenic forms. Bacteriologic examination of water is important for several reasons. Much smaller quantities of contaminating organic matter may be determined by bacteriologic than by chemical means. Its methods may be used in the determination of the potabihty of water-supplies, in tracing a typhoid or similar epidemic to its source, and in determining the efficiency of filters for water-supplies and of different types of sewage-disposal systems. Water may be examined bacteriologically, either quantitatively or qualitatively. In the former a determination of the total num- ber of bacteria present in the water is made; in the latter the tests are designed to determine the abundance of certain specific disease- producing bacteria, as Bacterium typhosum. The former is the most useful examination made in determining the potability of a water; the latter is rarely used. Quantitative Examination of Water. — In general, the greater the quantity of organic and decomposing matter present in water, the greater will be the number of bacteria present. However, it must be noted that changes in the environment, such as tem- 552 BACTERIA OF WATER AND WATER PURIFICATION 553 perature, may cause great variations in bacterial content, even though the original water be contaminated. For example, water from a source quite above suspicion may have less than 100 bac- teria to the cubic centimeter. This same water, carefully sampled in a sterile bottle and allowed to stand at room-temperature, may show in twenty-four to forty-eight hours hundreds of thousands of bacteria to the cubic centimeter. It is important, therefore, that the sample taken for examination shall be typical, and that it be examined immediately, to prevent multiplication of the bacteria present. Media Used. — Either nutrient gelatin or agar may be used. It should be prepared according to the methods outlined by the American Pubhc Health Association. The gelatin will, in general, give a somewhat higher count than the agar, but, when many hquefying species are present, the count on the gelatin must be made before all the slower-growing species have had a chance to develop. Methods. — Various dilutions of the water to be tested are placed in a series of sterile Petri dishes, and the melted medium to be used is poured in and thoroughly mixed. After the medium has solidified, the plates may be kept at room-temperature or, better, placed in a thermostat which maintains a temperature of about 20° to 22°, or with agar, 37°. The number of colonies developing upon a given plate, multiplied by the dilutions introduced, will give approximately the number of organisms present per cubic centimeter in the original sample. The final count should be made after the lapse of forty-eight hours. Agar at 37° is counted in twenty-four hours. Discrepancies will usually be detected be- tween the numbers, as determined from the plates containing the lesser and the greater dilutions. It is customary to use the plate having the nearest to 200 colonies in making the final estimation. Where more than this number of colonies are present, it is probable- that many more have failed to develop at all, or to a size that can be readily detected, on account of the overcrowding. The numbers of bacteria can, of course, be determined only approximately by the higher dilution; it is, therefore, customary to follow the mode of expression suggested by the committee on standard methods of the American Public Health Association: 564 VETERINARY BACTERIOLOGY Numbers of bacteria from — 1-50 shall be recorded to the nearest unit. 51-100 ( tt it 5 101-250 I ti It 10 251-500 t it (( 25 501-1,000 1 ti it 60 1,001-10,000 I it tt 100 10,001-50,000 i (t a 500 50,001-100,000 t ii tt 1000 100,001-500,000 i ft it 10,000 500,001-1,000,000 t it It 50,000 1,000,001-5,000,000 I it if 100,000 Interpretation of Results.— No standard for the number of bacteria that may be present in potable water can be set because of the various factors which may determine a high count. Stern- berg, however, has suggested a standard that is in general appUc- able; water containing less than 100 bacteria per cubic centimeter is probably pure; one containing 500 bacteria is suspicious, and one with 1000 bacteria is quite certainly bad. The number of bac- teria normally present in unpolluted supplies of various kinds differs considerably; for example, that in the deep wells of a region from those of its lakes, and standards must, therefore, be es- tabUshed for each. Determination of numbers is probably most useful in systematic examination of the efficiency of filtration of pubUc water-suppUes. In some countries these tests are made daily, and the maximum bacterial content of the filtered water that may be used has even been fixed by law. When gelatin is used, a separate count may be made of the colonies which develop that are capable of liquefying the medium. Such organisms are particularly characteristic of the surface soil, and usually belong to the Bacillus subtilis or to the Proteus groups. The presence of such in large numbers is an index to the extent of the surface wash, and not in general of the extent of sewage pollu- tion. This determination may be of value in the examination of shallow wells. Agar plates may be incubated at blood-heat. The typical water bacteria develop very slowly, if at all, at this temperature. A count made in twenty-four hours of such a plate is a fair index of the amount of sewage contamination usually, as the organisms from this source thrive best at this temperature. This determination, BACTERIA OF WATER AND WATER PURIFICATION 555 liowever, is largely displaced by the use of the litmus-lactose-agar plates, as discussed under Qualitative Analysis. Qualitative Examination of Water. — As has before been stated, water may be examined for the specific pathogens it may contain, or for the presence of sewage and intestinal bacteria, particularly Bacillus coll. Isolation of Specific Pathogens. — Bacterium typhosum is the organism for which examinations have been most frequently made. It has been actually isolated from water in a few instances only. Frequently only a very small percentage of the colonies which develop from direct plating of typhoid stools are typhoid colonies. The chances of direct isolation by plating sewage or water from a supply is, therefore, remote, even though this organism be present in numbers such as to cause an epidemic of the disease among the consumers. Usually the search for the specific organism in the water-supply is not begun until there is an outbreak of the disease, and the probabilities are that the organism by that time has disappeared from the supply. Many methods of isolation have been devised, but are not commonly used. For the most part, they are dependent upon enrichment by placing the suspected water in broth contain- ing antiseptics which will inhibit the growth of other bacteria, but not of the intestinal forms. This material is then plated and the t5T)hoid-like colonies are fished out and tested one at a time, the crucial test usually applied being the ability to agglutinate with high dilution of typhoid antiserum. The specific organism of Asiatic cholera may be more readily isolated than that of typhoid. Flasks of peptone salt solution are inoculated with the suspected water, and incubated at blood- heat for twenty-four hours or less, and transfers are made to fresh flasks from the surface layer. The cholera spirillum has a consider- able avidity for free oxygen, and swarms just below the surface in much greater numbers than elsewhere in the medium. Plates made from this surface film should show the characteristic colonies. Isolation of Bacterium coli. — Advantage may be taken of the physiologic and cultural characteristics of the Bacterium coli to isolate it from water. In examination of large numbers of sam- ples it is often found useful to make what is termed a preliminary or 556 VETEEINARY BACTBBIOLOGY 'presumptive test for the presence of the colon bacillus. Fermenta- tion tubes containing 1 per cent, lactose broth are inoculated with varying amounts of the water to be tested. If gas is not produced in any of the tubes, it is evident that Bad. coli is not present, at least in any considerable numbers. A negative result is, therefore, good evidence of the purity of the water examined. A positive test makes it probable that the water contains the colon bacillus, although further tests are necessary to establish the fact; hence the name, presumptive test. The evidence that Bact. coli is present is much strengthened if gas is formed to the amount of 30 per cent, and not more than 80 per cent. An approximation of the number of colon baciUi present may some- times be made by observing the dilutions of the water in which gas is produced. For example, if gas is produced in dilutions of 1 : 0, 1 : 10, and 1 : 100, but not in higher dilutions, it may be inferred that there are between 100 to 1000 B. coli present per cubic centimeter in the original sample. Many investigators use lactose bile rather than broth in the presumptive tests. A determination of the number of Bacterium coli present in a given sample may be secured by plating different dilutions in agar containing 1 per cent, lactose, colored blue by litmus solu- tion, and incubating twenty-four to forty-eight hours at 37°. Organisms which can ferment lactose with acid production are surrounded by a red discoloration of the litmus. Such organisms are the Bact. coli, Bact. aerogenes, and Streptococcus. The first two may be considered together, as they are usually held to indi- cate the same facts, although recent work seems to indicate that Bad. aerogenes is not a true intestinal form, but found in soils, on grains, etc. The colonies of these organisms may usually be readily differentiated from those of Streptococcus by their larger size, their shiny appearance, and the frequent gas bubble accom- panying the colony if it lies below the surface. The Streptococ- cus colonies, on the other hand, are small, rarely larger than a pin-head, and never have gas bubbles. It is usually necessary to make transfers from colonies and carry them through the various media to complete the identification. A highly con- taminated water will commonly reveal acid colonies directly upon plating, but in presence of large numbers of other bacteria preliminary enrichment will often show presence of Bact. coli BACTERIA OF WATER AND WATER PURIFICATION 557 when direct plating would give negative results. Plates may be poured from fermentation tubes that show gas production. Bacterium coli is not uncommon in nature ; its constant presence in the feces of most animals makes it widely distributed. It is entirely probable that the presence of small numbers of Bad. coli in water may, therefore, be without significance from the stand- point of potability. It is generally regarded in America that if Bad. coli can be constantly demonstrated in 1 c.c. samples of the water, this is an indication of recent sewage contamination. When its presence may be demonstrated only by the use of larger samples than 1 c.c. the evidence must be regarded as inconclusive. Water Purification Self-ptirification of Natural Waters. — ^Natural waters, both running and impounded (as in lakes and reservoirs), gradually free themselves from organic and bacterial contamination. The rapidity and efl&ciency of this cleansing process depend upon many factors. Bacterial purification is most rapid in impounded waters, and chemical purification in running streams. Sedimentation is probably the most potent factor in freeing a contaminated water from bacteria. The bacteria themselves have a slightly greater specific gravity than water, and tend to go to the bottom under the influence of gravity. This occurs more rapidly when other and larger solid particles are in suspen- sion; flocculation and more rapid sedimentation then frequently occur. Advantage is taken of this fact in the artificial purification of water, and coagulants of different kinds are added, which carry down the bacteria, together with other suspended material. Dimi- nution of food supply with consequent disappearance of many bac- teria is likewise important. Probably light destroys some organ- isms, and others are ingested by protozoa. Some species do not develop in the presence of certain other forms, that is, they exhibit antibiosis. Water-plants, algce, and natural obstructions of all kinds to water-flow exert a filtering action. Changes in temperature may inhibit the growth and even destroy some bacteria. A con- taminated stream is constantly diluted by the influx of ground- water and of triijutaries. Purification of Drinking-water. — For domestic purposes water 558 VETEBINARY BACTERIOLOGY may be effectually purified by heating to the boiling-point for ar few minutes. All the pathogenic bacteria are eliminated by this- method. Berkefeld porcelain filters, if properly constructed, are also efficient. They must be cleaned and sterilized at short inter- vals, otherwise the organisms will grow through the pores of the filter, and the water passing through will be as contaminated as the original supply. Even more efficient is purification by means of the ultra-violet rays. Where a city supply must be purified, it is com- monly pumped into reservoirs and allowed to settle, with or without the addition of coagulants. It is then passed through filters of various types, usually sand. Passage through a properly con- structed filter of this type has been shown to be exceptionally effi- cient. Such a system requires careful supervision. An efficient system will remove over 99 per cent, of the bacteria originally present. Some city supplies are pumped under pressure through sand-ffiters. This does not seem to be as efficient a means of rid- ding the water of bacteria as the other; as a filter, in any case, to retain its highest efficiency, must remain for some time undisturbed, and filters of the latter type require frequent cleaning and washing. The installation of filtration plants for purification of city suppUes has in many cases resulted in a marked diminution of the death-rate from typhoid and other intestinal diseases. Sewage Disposal. — The question of proper disposal oif sewage is closely related to the topic of pure water for domestic purposes. Usually sewage is allowed to flow into a suitable stream, and is purified as it passes down stream. There is no valid objection to this, providing there is a sufficient and constant flow of water in the stream to insure dilution, and the water of this stream is not used as a city supply further down. Unfortunately, too little attention has been paid to this subject, and the high typhoid death- rates in some cities are due directly to the use of such sewage-pol- luted water. BerUn and Paris purify their sewage by using it in the irrigation of large tracts of land, and re-collecting the water in the underdrains. Such a system is highly efficient, but, as it requires a particular type of soil, large areas, and suitable conditions, it is not often practicable. For sewage disposal in small cities and towns, and even private residences or farms, some of the numerous modifications of the septic tank and filter-bed have been shown to BACTERIA OF WATER AND WATER PURIFICATION 559' be most efficient. The sewage is first carried to a septic tank, so- called— a large tanlc, usually of brick or concrete, and commonly- covered. This tank is planned so that the sewage flow of twelve to twenty-four hours will fill it, or in other words, that a given portion of sewage will require that time to pass through. Here much of the soHd material settles out. The dissolved oxygen, if any be present in the raw sewage, is quickly used up, and anaerobic condi- tions are established. Under such treatment the decomposition of the organic matter occurs rapidly. Most of the sediment of the septic tank is soon dissolved by bacterial action. Gases, particularly H2S, CH4, H2, and NHs, are produced. These rise to the top, and are there intercepted by the heavy scum which forms, and oxidized to H2SO4, or free S2, CO2, H2O, and HNO3;: hence there is but little disagreeable odor to be noted about such a plant. The organic material is, for the most part, broken down into soluble, easily oxidizable substances. The sewage must not be held under these conditions for too long a period, otherwise the decomposition will go too far. The sewage then usually passes to a dosing chamber. This is simply a chamber which automatically discharges through one or more siphons whenever it becomes filled. The sewage passes from here either into contact beds or into filter- beds. The former consist of beds of crushed rock usually with water-tight walls. The sewage may be sprinkled over the surface constantly and allowed to trickle through (the so-called trickling filter), or it may be poured on to the bed in bulk, and held in con- tact with the crushed stone for a time and then discharged. In either case the sewage becomes thoroughly aerated, and the aerobic bacteria rapidly oxidize the organic matter present. A filter-bed, on the other hand, is constructed of sand underlaid with gravel and stone. The sewage is spread out over the surface and is allowed to seep through. Opportunity for thorough aeration of the sand and gravel is given by the time elapsing between the discharges from the dosing chamber. Frequently several beds are used, and the sewage is discharged first upon one, then upon another. The or- ganic material is retained, probably by adsorption, and, as the sewage passes through, the bacteria are largely filtered out, and the organic material is, for the most part, quite completely oxidized. The water leaving the drains under these filter-beds is relatively 560 VETERINARY BACTERIOLOGY pure, in some cases quite as pure as water from the average shallow well. As has been stated, there are many modifications of the type of disposal plant. It has been adapted to use for the farm- house as well as the city. Recently a marked advance has been made in sewage disposal by the use of the sO-called activated sludge method. The sewage is run into tanks, either with intermittent or constant flow, and air is bubbled through rapidly. Gradually oxidizing bacteria multiply in the sewage, particularly in the sludge, and oxidation of the organic matter is comparatively rapid. When the tank has reached its maximum efficiency, sewage is purified within a few hours, being separated into two portions, a clear liquid which will prove stable and is comparatively free from bacteria and a flocculent sediment or sludge. This latter is in part removed from time to time, and may be used as a fertilizer. CHAPTER XLVII MILK. ITS CONSTITUENTS, CONTAMINATION, AND EXAMINATION Milk is a complex mixture consisting of dissolved substances forming a solution which contains suspended matter, this entire mixture in turn containing in emulsion certain undissolved sub- stances. The suspended and dissolved substances constitute the milk plasma which separates into coagulum and milk serum upon coagulation. The fat is in emulsion. Several salts and certain cells undissolved or precipitated are also contained. About 87 per cent, of milk is water and 13 per cent, solids, Approximately 4 per cent, of the solids is fat, the balance solids not fat. The con- tent of the latter is about 3.3 per cent, protein (nitrogenous com- pounds), 0.7 per cent, ash, and 5 percent, lactose. Caseinogen and albumen are the chief nitrogenous compounds, of which the former makes up about four-fifths of all. The following table, summarized from Van Slyke's analyses, shows the composition of mUk: (■Pct — An f Albumen=0.7 Q^i;^= ~ *■" r Proteins =3.3 -^ Casein =2.6 '°Sfat^9]MiJ.-sugar=4jl ^^■^ L 8.9 Various hydrolytic enzymes, such as oxidases, galactase, etc., are contained. Bacteria-free milk is very difficult to obtain without some process of sterilization. Bacteria capable of bringing about various changes in milk must, therefore, be considered. Changes from Normal to Decomposed Milk. — Koning distin- guishes seven types of important changes which may occur in milk. The milk will show most, if not all, of the following stages : 1. Germicidal stage. 2. Development of lactic-acid-producing microorganisms. 36 561 562 VETEBINAHY BACTEEIOLOGY 3. Neutralization of acid by alkali producers. 4. Putrefaction. 5. Lactic acid bacteria again multiply due to neutralization of acid by organisms in third stage. 6. Development of fungi {O'idium lactis and others). 7. Butyric acid production by bacteria and change of the food product into a stinking putrid fluid. The Germicidal Action of Milk.— Wolfhiigel and Riedel as early as 1886 claimed to have demonstrated bactericidal substances in milk by showing that the cholera vibrio multiplied much more rapidly in boiled milk than in unboiled. Fokker, in 1890, asserted that normal raw milk possessed germicidal properties. He cul- tured lactic acid bacteria in raw and in boiled milk, and showed that the former resisted spoiling for a longer period. Other investi- gators claimed that the bactericidal properties existed only for certain kinds of bacteria and not for others. These latter investi- gators maintained that the composition of milk favors certain bac- teria by creating conditions favorable for their development, while others are held in abeyance because of the lack of constituents favoring their growth, and also become injured by the products of the favored organisms. Bauer, Rullman, Tommsdorf, and others have proved that specific germicidal substances, such as amboceptors, leucins, alex- ins, etc., do exist in special kinds of milk, such as mammitis and colostral milk, while they contend that their existence in normal milk has not been satisfactorily demonstrated. Some would at- tempt to account for the apparent diminution in numbers of bac- teria by the fact that milk has an agglutinating action upon bacteria, and that, therefore, bacterial clumps rather than single bacteria originate the colonies on a plate. The decrease of bacteria in fresh milk as shown by bacterial counts seems, nevertheless, to be estabUshed. It is only after a few hours, generally, thait rapid multiplication of bacteria begins. The presence of leukocytes in milk, and the fact that for a few hours subsequent to drawing the milk they are capable of ingesting bacteria, suggests the possibility that germicidal properties of milk are due in part to phagocytic action. This action plus that of MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 563 the true bactericidal substances may account for the germicidal action of milk. The bactericidal property of milk persists for but a relatively short time. Under favorable cool temperatures it may last for some days, but in warm temperature it disap- pears after a few hours. A temperature exposure of 80° C. com- pletely destroys it. Under even the most favorable conditions no reliance should be placed upon it as a means of destroying all bacteria. Acid Production in Milk. — The organisms producing lactic acid primarily, belong for the most part to the genera Streptococ- cus and Lactobacillus. Organisms producing smaller amounts of lactic acid and larger amounts of volatile acids, proteolytic ferments, etc., in general belong to the genera Bacterium and Staphylococcus. Putrefactive bacteria are practically always present in milk. They rarely develop sufficiently, however, to become prominent because of the much more rapid growth of the lactic acid bacteria and the creation of an acid reaction unfavorable to putrefaction. Opportunity is therefore denied for the production of poisonous putrefactive substances. The complete destruction of all the lactic acid bacteria, as may occasionally occur in pasteurization, or the inhibition of their growth by holding milk for a long period at low temperatures may offer opportunities for the develop- ment of these putrefactive forms. It is probable also that the acid production inhibits to some degree the multiplication of pathogenic bacteria. The flavor and aroma of butter wiU depend very largely upon the type of organisms which have been growing in the cream; the butter fat absorbs considerable quantities of dissolved substances present in the fermenting cream; it is, therefore highly desirable that these should be of the proper character. If batches of cream are allowed to ripen spontaneously without the addition of any starter there is apt to be a marked lack in uniformity in the pro- duct. A much better and more uniform result is secured by inoculating the cream which is to be ripened with a considerable amount of desirable lactic acid bacteria. Such organisms are sold under the name of commercial starters. It is generally assumed that they are pure cultures, or practically pure cultures, of Strep- 564 VETERINARY BACTERIOLOGY tococcus lacticus or some very closely related organism. This is introduced into pasteurized milk and allowed to grow until the milk has reached a satisfactory state of acidity. With this a larger bulk of milk is inoculated and finally the whole mass used a starter in the cream which is to be churned. A heavy inocu- lation with the desirable microorganisms and their growth prod- ucts usually overwhelms undesirable types of bacteria and leads to the development of a satisfactory product. Recent studies have made it evident that the changes brought about in a satisfactory starter are not the result simply of the growth of Streptococcus lacticus. Another organism closely related to it must ordinarily grow with it. Cream which has been ripened by means of a pure culture of the ordinary Strepto- coccus lacticus does not produce butter with as satisfactory a flavor and aroma as that produced by cream which has been inoculated with a mixture of these organisms. The ripening pro- duced by a starter is therefore an excellent example of associa- tive action. The true Streptococcus lacticus apparently produces very little change in milk or cream other than the, development of the pure lactic acid from lactose. The associative organism, also a coccus {Streptococcus citrovorus) can by its own growth bring about very little change in milk. When grown in the presence of Streptococcus lacticus, however, the activity of this organism is greatly stimulated and small amounts of volatile acids are developed. It is to the absorption of these and perhaps of other growthoproducts that the butter owes its characteristic aroma and flavor. Neutralization of Acid.— Very little decomposition of sour milk occurs for several days if the milk be kept under anaerobic condi- tions, but upon exposure to air there occurs the development of ' molds (chiefly Oldium lactis) upon the surface, and the lactic acid is oxidized to carbon dioxid and water. Thus the acidity is de- stroyed and conditions favorable for the development of putre- factive organisms are created. Combination of acid with the caseinogen of the milk also assists in the neutralization. Putrefaction.— Putrefactive bacteria multiply rapidly as soon as excess of acidity is overcome. Proteus, Bacillus suUilis, Ps. fluorescens, B. mesentericus, and others, together with molds, complete the changes whereby the milk becomes putrescent. MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 565 Some of the unusual changes that occur in milk due to certain bacteria give rise to such terms as ropy, blue, red, soapy, bitter, etc. Ropy milk results from slime-producing bacteria which may either produce capsules which are dissolved, act upon proteins with the formation of mucin-like substances, or upon the carbo- hydrates forming gums. Blue and red milk are the result of pigment formation by Pseudomonas cyanogenes, Erythrobacillus erythrogenes, and others. B. lactis saponacei and B. sapolacticum cause a soapy condition of milk; a sharp, rancid, soapy taste develops, and upon shaking a tenacious foam forms. Such undesirable bacteria are usually present because of un- sterilized milking utensils, and the application of milk of lime and hot soda solutions, with cleaning and disinfecting of stables usually result in their elimination. Contamination of Milk with Bacteria. — The possibility of milk contamination is recognized as from the following sources: Mam- mary gland and ducts of the same, hair and skin of the cow, air of stables, hands and clothing of the milker, milking utensils, handling of milk subsequent to milking. Mammary Gland. — The healthy tissue of the udder, like other normal tissues, is free from bacteria. Milk, therefore, as it is being secreted is sterile. In the ducts of the udder and particularly in the milk cistern it may come in contact with bacteria, so that when it leaves the teat the foremilk particularly may show a low bac- terial count. This usually is not above 100 per cubic centimeter, rarely as much as 500. variations in different cows are noticeable, and some which appear normal may show a much higher bacterial content. Hair and Skin. — Lack of careful grooming results in the ac- cumulation on the'hair of masses of filth, largely fecal matter, which at time of milking may fall into the pail and serve as an important source of milk contamination. Daily grooming at a time as remote from milking time as possible is recommended. Experiments by ; the New York Experiment Station do not indicate that cUpping the hair is of advantage in preventing contamination; in fact, milk from- clipped cows had a higher germ content than that from un- dipped. This was supposedly due to the fact that whereas in undipped cows the dirt at the base of the hairs not removed by a 566 VETERINABY BACTERIOLOGY superficial grooming is held there by the hair, in clipped ones this dirt readily became detached and fell into the pails. Air of Stables.— Dmi particles which are floating in the air of the stable may play an important part in milk contamination. It is only under the most adverse conditions, such as during the handling of hay and feed or bedding or by continued exposure of the milk in the open pail, that contamination from air is to be consid- ered of great importance. These are the conclusions reached by the New York State Experiment Station, where it was found that "while the numbers of bacteria in stable air markedly exceeded those of city street or office air, the number faUing into the milk during the ordinary milking time under conditions permissible in any respectable dairy is so small as to be negligible." The bacteria from this source are usually those of the putre- factive or the Bacillus subtilis types. Hands and Clothing of Milkers. — Personal cleanliness of milkers and the use of clean outer garments have much to do with the purity of milk. The chance for infection from unclean milkers is great, and such contamination is very apt to lead to serious re- sults, since organisms from this source are much more apt to pro- duce disease in man than those coming from the animal. Milking Utensils. — These doubtless contribute a greater num- ber of bacteria to milk than any of the other sources. Poorly soldered seams, rusted areas, and sharp angles at edges furnish favorable lodgment for the accumulation of milk, which at once becomes a favorable medium for the growth of bacteria. Exposure to a heavy fall of dust particles and bacteria from the air is avoided by the use of pails with small tops, rather than the old-style pail with flaring top. Vessels should be cleansed with sal soda and hot water, scrubbed with a brush to remove all adhering masses, rinsed thor- ougly in clear water, and steamed for fifteen minutes in a chest. Before placing in the chest the tops should be covered with cloth, and this should not be removed until the pail is to be used. Careless Handling of Milk. — Although milk may show a very low bacterial count up to the time it is ready for storage, there is still ample opportunity for contamination. If allowed to stand open in the cans or to remain for long periods in warm temperature the entrance and multiplication of bacteria will be great. Dipping from the cans in dippers which have been neglected as to proper MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 567 sanitary care may result in the introduction of enormous numbers of bacteria not only with the milk thus removed, but into that remaining in the can. Influences Which Determine the Ultimate Bacterial Content There are at least five important factors to consider as influenc- ing the bacterial content of milk. They are: Initial contamination; the time elapsing between the drawing of the milk and its con- sumption; the temperature to which it was lowered immediately following withdrawal and at which it has been maintained since; the care with which it has been handled; and whether it has been pasteurized. Initial contamination is to be kept at the minimum by proper care of stables, cows, and milkers. Furnishing as it does such a favorable medium for the growth of bacteria, it is important that the time milk is held before consumption should be as short as possible, for given much time the increase of bacteria will be enor- mous. Closely associated with this factor is that of temperature. Held even for but a comparatively short time at a favorable tem- perature the increase will be rapid. Milk should be quickly cooled immediately after it is drawn, preferably to a temperature of 50° or less. It will keep thus for several days without serious deteriora- tion. Careful handling in transportation and in retail shops is im- portant in keeping the bacterial content low. Pasteurization results in a great reduction of the bacterial content, and pasteurized milk kept properly cooled will retain its favorable condition for a long time. Diseases Transmitted Through Milk. — There are two classes of disease of the human which may be transmitted through the agency of milk. The first includes those which come from the consumption of milk from animals suffering from mastitis, the sec- ond those due to specific microorganisms. At least three types of disease in the human traceable to con- sumption of milk from udders affected with infectious mastitis are recognizable: (1) Gastro-intestinal catarrh due to streptococci contained in such milk. This condition is characterized by fever, dulness, nausea, vomiting, diarrhea, fainting, and cramps. -Numerous well- authenticated cases of this kind are on record, and careful inves- 568 VETERINARY BACTERIOLOGY tigations in all reported cases trace the condition back to the in- fected udder. (2) Epidemics of sore throat accompanied by sweUing of the lymph-glands of the neck, fever, cohc, and diarrhea. Several such epidemics in this country have been investigated, and here again the cause has been found to be streptococci of milk from mas- titis cases. (3) Enteritidis and paratyphoid infections, in which the evidence points strongly to millc infection. Reports of such conditions are few, and none can be said to trace with certainty to enteritidis, or paratyphoid mastitis, but such origin is not improbable. The specific diseases whose transmission through milk is recog- nized include tj^hoid and paratyphoid fever, cholera, diphtheria, tuberculosis, and scarlet fever. The occurrence of these disease- producing organisms in milk may be traced to the handling of milk by affected persons or by healthy persons who are bacillus carriers, or to infective material gaining access to the utensils through the use of polluted water. Typhoid fever epidemics due to a con- taminated milk supply are usually easily differentiated from those of other organs due to the fact that the disease is confined to house- holds having a common milk supply. While Levy claims to have isolated Bacterium typhosum from an abscess of the udder, it is generally recognized that this organism is unable to produce disease in cattle, and its presence in milk is due to careless hand- ling, such as the use of contaminated water for washing vessels, or through bacillus carriers or convalescing patients. The possi- bility of transmission through bottles that have been returned unwashed from households where typhoid is present should also be recognized. Paratyphoid is transmitted in ways quite similar to typhoid. In additiion, -transmission through consumption of milk from cows with mastitis due to organisms of this type is held as possible and probable. Diphtheria and scarlet fever, while not so commonly traceable to contaminated milk supplies, are recognized as being transmitted in this way. The contamination in this case is from some infected individual who has handled the milk. Infection of milk with the Mycobacterium tuberculosis occurs readily and directly when the animal is suffering from tuberculosis of the MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 569 udder. It may occur also, in an indirect way, through con- tamination of the udder or skin of the cow with feces in pul- monary or intestinal tuberculosis, the organisms being thus introduced when the infective material falls into the pail. The recognition of the bovine type of tuberculosis in lesions of tuberculosis both of children and adults is mentioned in another chapter (see p. 405). This strongly indicates the possibility of the bovine tubercle bacillus being transmitted to man, and sug- gests the necessity of careful supervision of herds supplying milk to the public. Tuberculin-tested and tuberculosis-free herds should be the rule, and where this is impossible, pasteurization should be required. A few diseases, such as anthrax, Malta fever, and foot-and- mouth disease, which are transmissible from animal to man are occasionally spread through milk, but their occurrence is rare enough as to be considered of sligjit importance. Standards for Production and Distribution of Certified Milk Certified milk, in the strict sense of the term, iS milk produced under legal contract between a medical milk commission and a dairyman, and which conforms to the requirements of such com- mission. These requirements differ in different cities. Some cities have excellent milk ordinances which conform quite closely to the standards as adopted by the American Association of Medical Milk Commissions. Milk entitled to be certified is clean and wholesome, is obtained from healthy cows kept in sanitary quarters, fed wholesome feed, and given pure water. It is drawn from clean cows by clean healthy attendants into clean receptacles and in a clean atmosphere. It is handled in a clean manner, cooled quickly, put into sterile vessels, placed in cold storage, and iced in transporta- tion. Inspected milk comes from clean cows that are tuberculin- tested and is drawn and cared for under sanitary conditions, but does not meet all the rigid standards demanded for a certified milk. Pasteurized milk is milk heated for a time at a temperature below the boiUng-poiflt in order to destroy harmful bacteria. The 570 VETBEINAKY BACTERIOLOGY so-called holder process is the one in most general use. In this the milk is heated to 60° to 65° C. and this temperature is maintained for about twenty minutes. The following provisions contained in the methods and stand- ards for production and distribution of certified milk, as adopted by the American Association of Medical Milk Commissions, May 1, 1912, relate particularly to bacteriologic standards, veterinary inspection, and animal hygiene, and are of particular interest to veterinary students. Hygiene of the Dairy Under the Supervision and Control of the Veterinarian 1. Pastures or Paddocks. — Pastures or paddocks to which the cows have access shall be free from marshes or stagnant pools, crossed by no stream which might become dangerously contami- nated, at sufficient distances from offensive conditions to suffer no bad effects from them, and shall be free from plants which affect the milk deleteriously. 2. Surroundings of Buildings. — The surroundings of all build- ings shall be kept clean and free from accumulations of dirt, rub- bish, decayed vegetable or animal matter or animal waste, and the stable yard shall be well drained. 3. Location of Buildings. — Buildings in which certified milk is produced and handled shall be so located as to insure proper shelter and good drainage, and at sufficient distance from other buildings, dusty roads, cultivated and dusty fields, and all other possible sources of contamination; provided, in the case of unavoidable prox- imity to dusty roads or fields, the exposed side shall be screened with cheese-cloth. 4. Construction of Stables. — The stables shall be constructed so as to facilitate the prompt and easy removal of waste-products. The floors and platforms shall be made of cement or other non- absorbent material and the gutters of cement only. The floors shall be properly graded and drained, and the manure gutters shall be properly graded and drained, and shall be from 6 to 8 inches deep and so placed in relation to the platform that all manure will drop into them. 5. The inside surface of the walls and all interior construction shall be smooth, with tight joints, and shall be capable of shedding MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 571 water. The ceiling shall be of smooth material and dust-tight. All horizontal and slanting surfaces which might harbor dust shall be avoided. 6. Drinking and Feed TroMfif/is.— Drinking troughs or basins shall be drained and cleaned each day, and feed troughs and mixing floors shall be kept in a clean and sanitary condition. 7. Stanchions. — Stanchions, when used, shall be constructed of iron pipes or hard wood, and throat latches shall be provided to prevent the cows from lying down between the time of cleaning and the time of milking. 8. Ventilation. — The cow stables shall be provided with ade- quate ventilation either by means of some approved artificial device, or by the substitution of cheese-cloth for glass in the windows. Each cow to be provided with a minimum of 600 cubic feet of air space. 9. Windows. — ^A sufficient number of windows shall be installed and so distributed as to provide satisfactory light and a maximum of sunshine, 2 feet square of window area to each 600 cubic feet of air space to represent the minimum. The coverings of such win- dows shall be kept free from dust and dirt. 10. Exclusion of Flies, Etc. — ^All necessary measures should be taken to prevent the entrance of flies and other insects, and rats and other vermin into all the buildings. 11. Exclusion of Animals from the Herd. — No horses, hogs, dogs, or other animals or fowls shall be allowed to come in contact with the certified herd, either in the stables or elsewhere. 12. Bedding. — No dusty or moldy hay or straw, bedding from horse-stalls, or other unclean materials shall be used for bedding the cows. Only bedding which is clean, dry, and absorbent may be used, preferably shavings or straw. 13. Cleaning Stable and Disposal of Manure. — Soiled bedding and manure shall be removed at least twice daily, and the floors shall be swept and kept free from refuse. Such cleaning shall be done at least one hour before the miUdng time. Manure, when removed, shall be drawn to the field or temporarily stored in con- tainers so screened as to exclude flies. Manure shall not be even temporarily stored within 300 feet of the bam or dairy building. 14. Cleaning of Cows. — Each cow in the herd shall be groomed daily, and no manure, mud, or filth shall be allowed to remain upon 572 VETEEINABY BACTERIOLOGY her during milking; for cleaning, a vacuum apparatus is recom- mended. 15. Clipping. — Long hairs shall be cUpped from the udder and flanks of the cow and from the tail above the brush. The hair on the tail shall be cut so that the brush may be well above the ground. 16. Cleaning of Udders. — ^The udders and teats of the cow shall be cleaned before milking; they shall be washed with a cloth and water, and dry wiped with another clean sterihzed cloth-^a sep- arate cloth for drying each cow. 17. Feeding. — All foodstuffs shall be kept in an apartment sep- arate from and not directly communicating with the cow barn. They shall be brought into the barn only immediately before the feeding hour, which shall follow the milking. 18. Only those foods shall be used which consist of fresh, palat- able, or nutritious materials, such as will not injure the health of the cows or unfavorably affect the taste or character of the milk. Any dirty or moldy food or food in a state of decomposition or putrefaction shall not be given. 19. A well-balanced ration shall be used, and all changes of food ■ shall be made slowly. The first few feedings of grass, alfalfa, en- silage, green corn, or other green feeds shall be given in small rations and increased gradually to full ration. 20. Exercise. — All dairy cows shall be turned out for exercise at least two hours in each twenty-four in suitable weather. Exer- cise yards shall be kept free from manure and other filth. 21. Washing of Hands. — Conveniently located facilities shall be provided for the milkers to wash in before and during milking. 22. The hands of the milkers shall be thoroughly washed with soap, water and brush, and carefully dried on a clean towel im- mediately before milking. The hands of the milkers shall be rinsed with clean water and carefully dried before milking each cow. The practice of moistening the hands with milk is forbidden. 23. Milking Clothes. — Clean overalls, jumper, and cap shall be worn during milking. They shall be washed or sterilized each day, and used for no other purpose, and when not in use they shall be kept in a clean place, protected from dust and dirt. 24. Things to be Avoided by Milker s.^While engaged about the MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 573 dairy or in handling the milk employees shall not use tobacco or intoxicating hquors. They shall keep their fingers away from their nose and mouth, and no milker shall permit his hands, fingers, lips, or tongue to come in contact with milk intended for sale. 25. During milking the milkers shall be careful not to touch anything but the clean top of the milking stool, the milk pail, and the cow's teats. 26. Milkers are forbidden to spit upon the walls or floors of stables, or upon the walls or floors of milk houses, or into the water used for cooling the milk or washing the utensils. 27. Foremilk. — The first streams from each teat shall be re- jected, as this foremilk contains large numbers of bacteria. Such milk shall be collected into a separate vessel and not milked on to the floors or into the gutters. The milking shall be done rapidly and quietly, and the cows shall be treated kindly. 28. Milk and Calving Period. — Milk from all cows shall be excluded for a period of forty-five days before and seven days after parturition. 29. Bloody and Stringy Milk. — If milk from any cow is bloody and stringy or of unnatural appearance, the milk from that cow shall be rejected and the cow isolated from the herd until the cause of such abnormal appearance has been determined and removed, special attention being given in the meantime to the feeding or to possible injuries. If dirt gets into the pail, the milk shall be dis- carded and the pail washed before it is used. 30. Make-up of Herd. — No cows except those receiving the same supervision and care as the certified herd shall be kept in the same barn or brought in contact with them. 31. Employees Other than Milkers. — The requirements for milk- ers, relative to garments and cleaning of hands, shall apply to all other persons handling the milk, and children unattended by adults shall not be allowed in the dairy nor in the stable during milking. 32. Straining and Strainers. — ^Promptly after the milk is drawn it shall be removed from the stable to a clean room and then emp- tied from the milk pail to the can, being strained through strainers made of a double layer of finely meshed cheese-cloth or absorbent cotton thoroughly sterilized. Several strainers shall be provided for each milking, in order that they may be frequently changed. 574 VETERINARY BACTERIOLOGY 33. Dairy Building. — A dairy building shall be provided which shall be located at a distance from the stable and dwelling pre- scribed by the local commission, and there shall be no hog-pen,, privy, or manure pile at a higher level or within 300 feet of it. 34. The dairy building shall be kept clean and shall not b6 used for purposes other than the handling and storing of milk and milk utensils. It shall be provided with Ught and ventilation, and the floors shall be graded and water-tight. 35. The dairy building shall be well lighted and screened and drained through well-trapped pipes. No animals shall be allowed therein. No part of the dairy building shall be used for dwelling or lodging purposes, and the bottling room shall be used for no other purpose than to provide a place for clean milk utensils and for handling the milk. During bottling this room shall be entered only by persons employed therein. The bottling room shall be kept scrupulously clean and free from odors. 36. Temperature of Milk. — Proper cooling to reduce the tem- perature to 45° F. shall be used, and aerators shall be so situated that they can be protected from flies, dust, and odors. The milk shall be cooled immediately after being milked, and maintained at a temperature between 35° and 45° F. until dehvered to the con- sumer. 37. Sealing of Bottles. — Milk, after being cooled and bottled, shall be immediately sealed in a manner satisfactory to the com- mission, but such seal shall include a sterile hood which completely covers the lip of the bottle. 38. Cleaning and Sterilizing of Bottles. — The dairy building shall be provided with approved apparatus for the cleansing and steril- izing of aU bottles and utensils used in milk production. All bottles and utensils shall be thoroughly cleaned by hot water and sal soda, or equally pure agent, rinsed until the cleaning water is thoroughly removed, then exposed to live steam or boiling water at least twenty minutes, and then kept inverted until used, in a place free from dust and other contaminating materials. 39. Utensils. — ^AU utensils shall be so constructed as to be easily cleaned. The milk pail .should preferably have an eUiptical open- ing 5 by 7 inches in diameter. The cover of this pail should be convex, so as to make the entire interior of the pail visible and accessible for cleaning. The pail shall be made of heavy seamless MILK. ITS CONSTITUENTS, CONTAMINATION, EXAMINATION 575 tin, and with seams which are flushed and made smooth by solder. Wooden pails, galvanized-iron pails, or pails made of rough, porous materials are forbidden. AH utensils used in milking shall be kept in good repair. 40. Water-supply.— The entire water-supply shall be abso- lutely free from contamination, and shall be sufficient for all dairy purposes. It shall be protected against flood or surface drainage, and shall be conveniently situated in relation to the milk house. 41. Privies, etc., in Relation to Water-supply. — Privies, pig- pens, manure piles, and all other possible sources of contamination shall be so situated on the farm as to render impossible the con- tamination of the water-supply, and shall be so protected by use of screens and other measures as to prevent their becoming breed- ing-grounds for flies. Transportation 42. In transit the milk packages shall be kept free from dust and dirt. The wagOn, trays, and crates shall be kept scrupulously clean. No bottles shall be collected from houses in which com- municable diseases prevail, unless a separate wagon is used and under conditions prescribed by the department of health and the medical milk commission. 43. All certified milk shall reach the consumer within thirty hours after milking. Veterinary Supervision of the Herd 44. Tuberculin Test. — The herd shall be free from tuberculosis, as shown by the tuberculin test. The test shall be applied in accord- ance with the rules and regulations of the United States Govern- ment, and aU reactors shaU be removed immediately from the farm. 45 No animals shall be admitted to the herd without first having passed a satisfactory tubercuUn test, made in accordance ■fh the rules and regulations mentioned; the tuberculin to be bt ined and applied only by the official veterinarian of the com- mission. , . , 1- . 46. Immediately following the application of the tuberculin test to a herd for the purpose of eliminating tuberculous cattle, the cow stable and exercising yards shall be disinfected by the veterinary 576 VETERINABY BACTERIOLOGY inspector in accordance with the rules and regulations of the United States Government. 47. A second tuberculin test shall follow each primary test after an interval of six months, and shall be applied in accordance with the rules and regulations mentioned. Thereafter, tuberculin tests shall be reapphed annually, but it is recommended that the retests be applied semi-annually. 48. Identification of Cows. — Each dairy cow in each of the certi- fied herds shall be labeled or tagged with a number or mark which will permanently identify her. 49. Herd-book Record. — Each cow in the herd shall be registered in a herd book, which register shall be accurately kept, so that her entrance and departure from the herd and her tuberculin testing can be identified. 50. A copy of this herd-book record shall be kept in the liands of the veterinarian of the medical milk commission under which the dairy farm is operating, and the veterinarian shall be made re- sponsible for the accuracy of this record. 51. Dates of Tuberculin Tests. — The dates of the annual tuber- cufin tests shall be definitely arranged by the medical milk com- mission, and all of the results of such tests shall be recorded by the veterinarian and regularly reported to the secretary of the medical milk commission issuing the certificate. 52. The results of all tuberculin tests shall be kept on file by each medical milk commission, and a copy of all such tests shall be made available to the American Association of Medical Milk Com- missions for statistical purposes. 53. The proper designated officers of the American Association of Medical Milk Commissions should receive copies of reports of all of the annual, semi-annual, and other ofl&cial tuberculin tests which are made, and keep copies of the same on file and compile them annually for the use of the association. 54. Disposition of Cows Sick with Diseases Other than Tuberculo- sis. — Cows having rheumatism, leukorrhea, inflammation of the uterus, severe diarrhea or disease of the udder, or cows that from any other cause may be a menace to the herd shall be removed from the herd and placed in a building separate from that which may be used for the isolation of cows with tuberculosis, unless such build- miijiv. no (JUJNSTllxnBlNTS, COJSITAMINATION, EXAMINATION 577 ing has been properly disinfected since it was last used for this pur- pose. The milk from such cows shall not be used nor shall the cows be restored to the herd until permission has been given by the veterinary inspector after a careful physical examination. 55. Notification of Veterinary Inspector. — In the event of the occurrence of any of the diseases just described between the visits of the veterinary inspector, or if at any time a number of cows become sick at one time in such a way as to suggest the outbreak of a contagious disease or poisoning, it shall be the duty of the dairyman to withdraw such sickened cattle from the herd, to de- stroy their milk, and to notify the veterinary inspector by telegraph or telephone immediately. 56. Emaciated Cows. — Cows that are emaciated from chronic diseases, or from any cause that in the opinion of the veterinary in- spector may endanger the quality of the milk, shall be removed from the herd. Bacteriologic Standards 57. Bacterial Counts. — Certified milk shall contain less 'than 10,000 bacteria per cubic centimeter when delivered. In case a count exceeding 10,000 bacteria per cubic centimeter is found, daily counts shall be made, and if normal counts are not restored within ten days the certificate shall be suspended. 58. Bacterial counts shall be made at least once a week. 59. Collection of Samples. — The samples to be examined shall be obtained from milk as offered for sale, and shall be taken by a representative of the milk commission. The samples shall be received in the original packages, in properly iced containers, and they shall be so kept until examined, so as to limit as far as possible changes in their bacterial content. 60. For the purpose of ascertaining the temperature, a separate original package shall be used, and the temperature taken at the time of collecting the sample, using for the purpose a standardized thermometer graduated in the Centigrade scale. 61. Interval Between Milking and Plating. — The examinations shall be made as soon after collection of the samples as possible, and in no case shall the interval between milking and plating the samples be longer than forty hours. 62. Plating. — The packages shall be opened with aseptic pre- 37 . 578 VETEHINARY BACTEBIOLOGY cautions after the milk has been thoroughly mixed by vigorously reversing and shaking the container twenty-five times. 63. Two plates at least shall be made for each sample of milk, and there shall also be made a control of each lot of medium and apparatus used at each testing. The plates shall be grown at 37° C. for forty-eight hours. 64. In making the plates there shall be used agar-agar media containing 1.5 per cent, agar and giving a reaction of 1.0 to phenol- phthalein. 65. Samples of milk for plating shall be diluted in the proportion of 1 part of milk to 99 parts of sterile water; shake twenty-five times and plate 1 c.c. of the dilution. 66. Determination of Taste and Odor of Milk. — After the plates have been prepared and placed in the incubator, the taste and odor of the milk shall be determined after warming the milk to 100° F. 67. Counts. — The total number of colonies on each plate should be counted, and the results expressed in multiples of the dilution factor. Colonies too small to be seen with the naked eye or with slight magnification shall not be considered in the count. 68. Records of Bacteriologic Tests. — The results of all bacterial tests shall be kept on file by the secretary of each commission, copies of which should be made available annually for the use of the American Association of Medical Milk Commissioners. BIBLIOGRAPHIC INDEX Abbe, 122 Adelbey and Nicolle, 537 Almy and Bodin, 474 Alt, 395 Anderson, 314 Anderson and McClintock, 120 Anderson and Rosenau, 210 Andrewes and Herder, 215 Andriewsky, 544 Arloing, 271 Arning, 408 Arnold, 95 Ascoli, 262 Babes, 315, 509, 512 Babes and Selter, 317 Bahr, 278 Bail, 203, 205, 247, 259, 261 Bail and Weil, 293 Balfour, 426, 427 Bang, 349, 350, 351, 352, 353, 403, 407, 441 Banzhaf, 171 Banzhaf and Gibson, 170 Barber, 132 Barnes, 46 Bateman and Bruce, 483 Battaglio, 483 Bauer, 562 Beach and Hadley, 545 Behring, 166 Berg, 477 Berkefeld, 98 Besredka and Metchnikoff, 204 Beyer, 61 Beyerinck, 77 Blanchard, 417 Blaxall and Fox, 474 Bloch, 469 Bodin and Almy, 474 Bodin and Delacroix, 475 Bollinger, 249, 301, 434 Bolton, Dorset, and McBryde, 334, 336 Bordet, 190 Bordet and Gay, 192 Borrel, 419, 502 Bowman, 530 Braune, 530 Bredini and Moore, 485 Brefeld, 463 Breinl and Hindle, 613 Breinl and Thomas, 485 Broden, 492 Brown and Orantt, 380 Bruce, 353, 481, 483, 489 Bruce and Bateman, 483 Brumpt, 472 Buckley and Mohler, 331, 332, 459 Buerger, 215, 238 Bull and Pritchett, 281 Bumm, 241 Bureschello,- 224 Burger and Wolbach, 419 Burke, 288 Busse, 449 Calmette, 402, 403 Carimi, 483 Carrg, 366, 543, 551 Carr6 and Villee, 541 Castellani, 431 Celli and de Blasi, 550 Centanni and Savonuzzi, 543 Chamberland, 98 Charbon, 253 Chaussat, 496 Chauss6, 393, 395 Chillees, 582 Citron, 293 Citron and Wassermann, 300 Clark and Lubs, 113 Clegg and 'Musgrave, 502, 503 Clegg, Musgrave and Polk, 436, 438, 439, 444 Clemesha, 321, 325 Coca, 313 Cohn, 21 Cole, Hadley, and Fitzpatrick, 524 Conradi, 347 Cornevin, 271 Cotton and Schroeder, 351 Councilman, 195 Councilman and Lafleur, 504 Craig, 501, 503, 505 Craig and Nichols, 431 Crowley, 495 Curtis, 391 Damman and Manengold, 228 Danysz, 339 Darling, 497, 498 579 580 BIBLIOGRAPHIC INDEX Dassonville and Matruchot, 472, 473 Davaine, 23 de Bary, 38, 456 de Beurmann, 466 de Blasi and Celli, 550 Deerr and Lewton-Braiu, 138 de Gaspari, 551 Delacroix and Bodin, 475 Demmy, 368 Deneke, 416 de Schweinitz, 299, 334, 335 de Schweinitz and Dorset, 334, 539 de Schweinitz and McFarland, 298 Deseler, 513 Dodd, 431 Doerr, 348 Doflein, 480 Dorset, 111, 389 Dorset and de Schweinitz, 334, 539 Dorset, Bolton, and McBrvde, 334, 336 Dorset, McBryde and Niles, 540 Douglas and Wright, 195 Drigalski, 292 Dunham, 105 Dunkel, 363, 380 Durham, 325 Dutton and Todd, 423, 492 Ebeblena, 249 Eberth, 342 Ehrenberg, 21 Ehrlich, 24, 124, 154, 157, 159, 160, 161, 166, 167, 168, 170, 174, 175, 185, 186, 187, 274, 371 Ehrlich and Sachs, 192 Eichhorn and Mohler, 313, 355 EUemann, 551 Ellis, 38, 75 Elmassian, 490 Emmerich, 321 Emmerich and Low, 360 Emmerlich and Mastbaum, 376 Epstein, 367 Ernst, 222 Escherich, 321, 326 Evans, 23, 481, 487 Evans and Steck, 351 Fantham and Thomson, 513 Fehleisen, 217 Fernmore, 301 Ferriera, Horta and Paredes, 322 Ferry, 356, 357, 543 Fielitz, 469 Finkler and Prior, 416 Fischer, 34, 37, 71 Fisher, 486 Fitzpatrick, Hadley and Cole, 524 Flexner, 240, 346 Flexner and Jobling, 240 Fokker, 562 Foth, 316 Pox and Blaxall, 474 Frankel, 235, 279, 281, 329, 419 Frankel and Pfeiffer, 219, 223, 237, 247, 255, 267, 272, 283, 284, 295, 307, 308, 343, 369, 370, 374, 387, 388, 409, 411, 412, 459, 476 Eraser and Symons, 488 Freeman, 19.5 Friedberger. 379 Friedlanderj 235, 329 Friedmann, 398 Frosch and Loffler, 536 Frost, 146 Frost and McCampbell, 52 Frothingham and Johne, 406 Frothingham, Page and Paige, 136, 466, 467, 468 Fuchs, 253 Gabbett, 124, 126 Gabritschew.sky, 425 Gaffky, 342 Gage, 121 Galli-Valerio, 447 Galli-Valerio and Prani, 513 Galtier, 310 Gamal^ia, 410 Gartner, 330 Gay and Bordet, 192 Gedvelst, 472. Gengou, 190 Gerber, 418 Gerlach, 475 Gessard, 358 Ghon and Sachs, 264, 271, 285 Gibson and Banzhaf, 170 Giemsa, 129, 130, 426, 620 Gilchrist, 449 Glage, 250, 363, 379, 380, 382 Glage and Priewe, 380 Glasser, 337 Gonder, 417, 515, 516 Gonder and Sieber, 482, 487, 489 Gougerot, 469 Graham, 287, 289 Graham, and Irons, 450 Graham-Smith and Nuttall, 514 Gram, 128 Grassberger and Schattenfroh, 271, 274 Grijns, 458 Grips, 379 Gruber, 174, 179 Guglienni, 511 Guinard and Preisz, 363 Gunther, 242, 245, 253, 256, 284, 294, 303, 329, 344, 378, 410, 413, 435, 446, 470 Gwyn, 338 BIBLIOGRAPHIC INDEX 581 Hadley, 294, 296 340, 341, 523, 531 Hadley and Beach, 545 Hadley, Cole and Fitzpatrick, 524 Haendel and Uhlenhuth, 337 Haffkine, 304 Hamburger, 451 Hammer, 233 Hansen, 40, 70, 125, 407 Hansen and Neisser, 407 Harding and Ostenberg, 330, 338 Hartmann, 501, 507 Harvey and Rettger, 340 Harz, 434 Haslam, 460 Hata, 422 Hecker, 536 Heoker, Raebiger, and Hess, 229 Hedrin, 429 Heidenhain, 502, 520 Heim, 245, 321 Heinemann, 115, 217, 232, 233 Hektoen and Perkins, 466 Hektoen and Ruediger, 223 HeUer, 264 Henle, 23 Herman, 126 Hertel, 297 Herter, 281 Hess, Hecker and Raebiger, 229 Hejrman, 398 Hibler, 264, 266, 271, 273, 276, 283, 285 Hill, 119 Hindle and Breinl, 513 Hinze, 74 Hirschfelder, 396 Hiss, -346, 361 Hoffmann and Schaudinn, 428 Hogges, 549 Holman, 215, 216 Holth, 225 Hopkins, 120 Horder and Andrewes, 215 Home, 407 Horta, Ferriera and Paredes, 322 Hueppe, 290, 325 Huntoon, 107, 292 Hutyra, 539 Hut3a'a and Marek, 311 Ingeam and Twort, 406 Inman and Levaditi, 195 Irons and Graham, 450 Jbnnbe, 195, 199 Jensen, 227, 277, 278, 301, 325, 327, 328, 441 Jobhng and Flexner, 240 Johne, 129, 241, 249, 254 Johne and Frothingham, 406 Johnson and Levine, 326 Jordan, 28, 95, 180, 280, 323, 327, 330, 355 Karlinski, 220 Kartulis, 504 Kilborne and Smith, 519 Kitasato, 265, 266, 271 Kitasato and Veyl, 265 Kitasato and Yersin, 302 Kitt, 227, 275, 297, 301, 306, 375,470 Kitt and Mayr, 296 Klebs, 367 Klein, 125 Klein and MoUer, 254 Kleine, 483, 489, 490 Kleine, Rosenthal, and Schiffman, 544 Klett, 254 Klimmer, 398, 402 Klimmer and Wolff-Eisner, 397 Knapp and Novy, 419, 422, 424, 425, 426, 428 Koch, 23, 24, 119, 133, 144, 253, 271, 282, 377, 386, 395, 397, 412, 490, 504, 516, 538, 541 Koch and Pasteur, 23 Koch and Schutz, 398 Kolbe and Kumbein, 305 KoUe and Turner, 538 KoUe and Wassermann, 236, 272, 287, 331, 346, 359, 408 Konew, 312 Konig, 561 Koram, 348 Koske, 360 Krai, 470 Kraus and Levaditi, 167 Kretz, 167 Krumweide and Park, 404, 405 Kruse, 231 Ktlhne, 310 Kuhnemann, 413 Kumbein and Kolbe, 305 Kunnemann, 363, 379, 380, 382 Kutscher, 317 Lafletjk and Councilman, 504 Lafosse, 224 Lambl, 503 Lange, 376 Lange, Zwick and Winkler, 486 Laveran, 23, 493, 494, 495, 516 Laveran and Mesnil, 487, 489 Leclainche, 376, 377 Leeuwenhoek, 19, 21 Leger and Marhis, 521 Leishman, 231, 232, 418, 424, 425, 426, 498 Lentz, 130 Levaditi, 165, 166, 427 Levaditi and Inman, 195 Levaditi and Kraus, 167 Levine, 108, 109, 321, 325 Levine and Johnson, 326 Lewis, 481, 496 582 BIBLIOGRAPHIC INDEX Lewton-Brain and Deerr, 138 Liebig, 22 Lignilres, 290, 296, 297, 401 LigniSres and Spitz, 297 Ligniferes and Voges, 491 Lingelsheim, 214 Linnaeus, 82 Lister, 25 IjOGsch ^04 Loffler,' 124, 128, 339, 367, 373, 440 Loffler and Frosch, 536 Loffler and Schutz, 290, 297, 306 Lorenz, 376 Low and Emmerich, 360 Lowden and Williams, 547, 548, 550 Lubs and Clark, 113 Lucet, 220, 244, 248 Lugol, 127, 130 Lukas, 265 MacConkey, 321, 325 MacFadyean, 293, 541 Mack, 542 Mack and Moore, 228 MacNeal and Now, 484, 485, 488, 489 Madsen, 169 Magnusson, 228 Mandelbaum, 181 Manengold and Damman, 228 Marchoux, 418 • Marchoux and Salimbeni, 425, 427 Marek and Hutyra, 311 Marek and Ostertag, 541 Marhis and Leger, 521 Marmorek, 223 Marotel and Moussu, 526 Mastbaum and Emmerlich, 376 Matruchot and DassonviUe, 472, 473 Marx, 376 Marx and Sticker, 544, 545 Marxer, 227 Mayer, 235, 236 Mayer and Emmet, 457 Mavr and Kitt, 296 McBryde, Bolton and Dorset, 334, 336 McBryde, Dorset and Niles, 540 McCampbell, 199, 201, 279 McCampbell and Frost, 52 McCampbell and Phillips, 514 McClintock, 120 McFarland, 98, 133, 202, 368, 396 McFarland and de Schweinitz, 298 Melvin and Mohler, 464, 465 Mesnil and Laveran, 487, 489 Metchnikoff, 24, 1.54, 194, 204 Mewborn, 474 Meyer, 264, 282, 351, 354, 406, 407 Meyer and Shaw, 354 M' Go wan, 356 MiceUone and Rivolta, 249 Migula, 323, 339 Miller, 197, 198, 521 Milne and Ross, 423 Milzbrand, 253 Mohler, 442 Mohler and Buckley, 331, 332, 459 Mohler and Eichhorn, 313, 355 Mohler and Melvin, 464, 465 Mohler and Morse, 441, 443 Mohler and Norgaard, 227, 228, 363, 364 Mohler and Washburn, 443 Molisch, 65 MoUer, 125 MoUer and Klein, 254 Moore, 79, 221, 222, 392 Moore and Bredini, 485 Moore and Mack, 228 Morse, 362 Morse and Mohler, 441, 443 Moussu and Marotel, 526 Much, 126 Muir, 128 MuUer, 21, 226, 227, 433, 434 Murchison, 24 Murphy, Peyton and Rous, 551 Musgrave and Clegg, 502, 503 Musgrave, Clegg and Polk, 436, 438, 439, 444 Negri, 547 Neisser, 241 Neisser and Hansen, 407 Neisser and Wichsberg, 188 Nichols and Craig, 431 Nichols and Phalen, 452 Nichols and Stovall, 128 Nicolaier, 265 Nicolle, 499 NicoUe and Adelbey, 537 Nielsen, 277 Niles, Dorset and McBryde, 540 Nocard, 363, 437, 486, 536, 537, 541 Nocard and Prettner, 310 Nocard and Roux, 535 Noguchi, 420, 423, 429, 430, 431, 546 Norgaard, 464 Norgaard and Mohler, 227, 228, 363, 364 Novy, 246 Novy and Knapp, 419, 422, 424, 425, 426, 428 Novy and MaCNeal, 484, 485, 488, 489 Nowak, 349, 350, 352 Nuttall and Graham-Smith, 514 Nuttall and Welch, 279 Obehmeieb, 421 Ogston, 217, 244 Olt, 383 Orantt and Brown, 380 BIBLIOGRAPHIC INDEX 583 Ostenberg and Harding, 330, 338 Ostertag, 229, 241, 382 Ostertag and Marck, 541 Ostertag and Wassermann, 300 Ostertag and Weichel, 382, 383 Otho, 346 Page, Frothingham and Paiee, 136, 466, 467, 468 Paige, Frothingham and Page, 136, 466, 467, 468 Paredes, Ferriera and Horta, 322 Park, 164 Park and Krumweide, 404, 405 Parum, 194 Pasteur, 22, 23, 98, 217, 260, 282, 285, 290, 294, 295, 296, 316, 375, 548, 549 Pasteur and Koch, 23 Pasteur and Thuiller, 373 Patton, 509, 614 Perkins and Hektoen, 466 Perroncito, 294, 543 Peters, 495 Petruschky, 336 Peyton, Rous, and Murphy, 661 Pfaundler, 177, 181 Pfeiflfer, 185 Pfeiffer and Frankel, 219, 223, 237, 247, 265, 267, 272, 283, 284, 295, 307, 308, 343, 369, 370, 374, 387, 388, 409, 411, 412, 459, 476 PffeUer, 193 Pfuhl, 410 Phalen and Nichols, 462 Phillips and McCampbell, 514 Pirquet, 401 Plenciz, 22 Poels, 360, 379 Pohle, 363 Polk, Musgrave and Clegg, 436, 438, 439, 444 PoUender, 263 Porrey, 243 Posadas and Wernecke, 452 Prani and Galli-Valerio, 513 Preisz, 258, 362 Preisz and Guinard, 363 Prettner, 376 Prettner and Nocard, 310 Priewe, 363 Priewe and Glage, 380 Prior and Finkler, 416 Pritchett and BuU, 281 Proskauer and Voges, 117, 319, 323 Prowazek, 418 Rabe, 249, 356 Raebiger, 129, 264 Raebiger, Hecker and Hess, 229 Rayer, 253 Redi, 21 Rees, 617 Remlinger and Riff 646 Rettger, 341 Rettger and Harvey, 340 Ricketts, 149 Rideal and Walker, 119 Riedel and Wolfhugel, 662 Riff and Remlinger, 546 Rivolta, 447, 625 Rivolta and Micellone, 249 Rodenwalt, 483 Rodet and VaUet, 490 Romanowsky, 129, 199, 264, 422 Rosenau, 168, 340 Rosenau and Anderson, 210 Rosenbach, 217, 244, 249 Rosenthal, 348 Rosenthal, Kleine and Schiffman, 544 R«ss and Milne, 423 Rouget, 486, 486 Rous, Peyton and Murphy, 551 Roux, 316 Roux and Nocard, 536 Roux and Yersin, 367 Ruediger and Hektoen, 223 RuUman, 662 Sabouhaud, 470, 471, 472, 474 Sacharoff, 425 Sachs and Ehrlich, 192 Sachs and Ghon, 264, 271, 286 Salimbeni and Marchoux, 425, 427 Salmon and Smith, 334, 539 Savonuzzi and Centanni, 643 Schardinger, 500 Schattenfroh and Grassberger, 271, 274 Schaudinn, 483, 501, 603, 506, 506, 526 Schaudinn and Hoffmann, 428 Schenk, 466 Schern and Stange, 336 Schiffman, Rosenthal and Kleine,' 544 Schmitz, 331 Schnitt, 495 Sehobl, 275 Schottm tiller, 214, 338 Schreiber, 296 Schreuber and Schubert, 376 Schroeder and Cotton, 361 Schubert and Schreuber, 376 Schubert and Schiitz, 313 Schiitz, 224, 225, 226, 440 Schiitz and Koch, 398 Schiitz and Loffler, 290, 297, 306 Schiitz and Schubert, 313 Schutz and Voges, 376 Sellards and Walker, 501 Selter and Babes, 317 Shaw and Meyer, 354 584 BIBLIOGRAPHIC INDEX Shiga, 345, 346, 348 Sieber, 510, 519, 520 Sieber and Gonder, 482, 487, 489 Silberschmidt, 440 Smith, T., 105, 204, 207, 267, 297, 334, 356, 386, 389, 391, 410, 414, 501, 509, 531 Smith and Kilborne, 519 Smith and Salmon, 334, 339 SoUeysel, 224 Sorensen, 102 Spitz and Ligniere, 297 Stange and Schern, 336 Starcovici, 509 ■ Starrkrampf, 265 Stauffaohei\ 536 Steok and Evans, 351 Sternberg, 235, 554 Sticker and Marx, 544, 545 Stiles, 527 Stimson, 549, 550 Stovall and Nichols, 128 Stranigg, 193 Strauss, 310 Strenge, 193 Symons and Praser, 488 Tahtakowsky, 537 Theiler, 427, 428, 489, 495, 511, 516, 519, 541 Thomas, 271 Thomas and Breinl, 485 Thomson and Fantham, 613 Thuiller and Pasteur, 373 Todd, 226, 348, 424, 428 Todd and Button, 423, 492 Tokoshige, 447, 448, 449 Tommsdorf,,562 Torrey, 356 Toyoda, 423 Traum, 351 Trevisan, 290 Turner and Kolle, 538 Turski, 363 Twort and Ingram, 406 Tyndall, 533 Uhlenhuth, 183, 334, 486, 539 Uhlenhuth and Haendel, 337 Uhlenhuth and Weidanz, 172 Usohinsky, 106 Valentine, 481 Vallet and Rodet, 490 Van Ermengen, 127, 286, 287 Van Gieson, 131 Van Slyke, 561 Vaughn, 207 Vaughn and Wheeler, 207, 210 Veyl and Kitasato, 265 Viereck, 507 Villee and Carr6, 541 Villeman, 385 Vincent, 445 Vladimiroff, 315 Voges and Ligniferes, 491 Voges and Proskauer, 117, 319, 323 Voges and Schiitz, 376 Voldagsen, 337 Von Behring, 24, 397 Von Pirquet, 401 Walkbe and Rideal, 119 Walker and Sellards, 501 Ward, 380' Washburn and Mohler, 443 Wassermann, 191, 313, 360 Wassermann and Citron, 300 Wassermann and Kolle, 236, 272, 287, 331, 346, 359, 408 Wassermann and Ostertag, 300 Watson, 486, 522 Wehmer, 456, 458, 461, 462 Wehrbein, 487 Weidanz, 171 Weidanz and Uhlenhuth, 173 Weichel and Ostertag, 382, 383 Weichselbaum, 236, 238, 306 Weigert, 23, 161 Weil and Bail, 293 Welch\ 279 Welch and Nuttall, 279 Wernecke and Posades, 452 Werner, 507 Wertheim, 242 Wesbrook, 368 Wheeler and Vaughn, 207, 210 Wherry, 303, 407 . Wichsberg and Neisser, 188 Widal, 179, 469 Williams, 164 Williams and Lowden, 547, 548, 550 Winkler, Zwick and Lange, 486 Winogradsky, 76, 77 Witt, 315 Wolbach and Burger, 419 Wolff-Eisner, 402 Wolff-Eisner and'Klimmer, 397 Wolfhugel and Riedel, 562 Wright, 129, 130, 195, 199, 435, 436, 499 Wright and Douglas, 195 Yeksin and Kitasato, 302 Yersin and Roux, 367 Zbnkowsky, 260 Ziehl, 124 Zschokke, 525 Zwick, 486 Zwick, Lange and Winkler, 486 INDEX Abobtion, 349 bacillus of Bang, 349 infectious, 349 of cattle, 414 Abrin, 158 Absorption, 190 Acetic acid, 59, 71 as preservative, 59 Acetobacter, 85 aceti, 28, 70, 71 Acetyl metliyl carbinol, production of, 117 Achorion, 469, 475 gaUinse, 475 gypseum, 475 muris, 475 quinckeanum, 475 schonleinii, 475 Acid, acetic, 59, 71 as preservative, 59 alcohol stain for acid-fast bacteria, 126 as preservative, 59 butyric, 71 carbolic, 60 lactic, 59, 70 as preservative, 59 neutralization of, 564 nitrous, oxidation of, 76 production, 69, 112 determination of, 113 in milk, 563 of bacteria, determination of, 113 sulphurous, 61 Acid-fast bacteria, 92 non-pathogenic, 408 stain for, 126 group, 385 Acidophiles, 59 Acquired immunity, 151 active, 151 antibodies as factors in, 155 passive, 153 passive immunity, 153 Actinobacillosis, 91 Actinobacillus, 90, 91 lignieresi, 91 Actinomyces, 90, 91 bovis, 91, 433, 434 caprse, 433, 439 Antinomyees cuniculi, 440 eppingeri, 433 farcinica, 437 group, 432 madurae, 433, 445 necrophorus, 440 nocardii, 433, 437 of other infections, 445 Actinomycetales, 84, 90 Actinomycosis, 434 in goats, 440 Action of amboceptor, 186 of complement, 186 Active acquired immunity, 151 Aerobes, 117 Aerobic bacteria, 48 rods, 252 spore-producing bacilli, 252 Aerotaxy^ 55 Aerotropism, 55 African horse sickness, 541 Agalactia, infectious, 550 Agar, blood-serum, 107 chocolate, 107 endo, simplified, 108 eosin-methylene-blue, 109 hormone, 107 media, 108 nutrient, 107 plates, colonies on, 138, 139 stroke, 135 Agglutination, 156, 174 group, 178 precipitation and, differentiation, 174 test, 179, 181, 486 for glanders, 311 in disease diagnosis, 179 Agglutinin, 174 action, method of, 177 body, 176, 177 constitution of, 175 flagellar, 176, 177 group, 178 immune, 174 in immunity, significance of, 181 normal, 174 production of, Ehrlich's theory of, 175 somatic, 176, 177 I Agglutinogen, 175 585 586 INDEX Agglutinoid, 176 Agglutinometers, 181 Agglutinophore, 176 Aggressin hypothesis, 203 Aggressins, 203 artificial, 300 natural, 299 Agricultural bacteriology, definition, 18 Air, hot, sterilization by, 94 Albumin-free culture-media, 106 Alcohol, 61 production of, 69, 116 Aldehyde, production of, 116 Algae, 27 Alkali, production of, 112 Alkalies, 60 Alt tuberculin, 395 Amboceptor, 186, 192 action of, 186 formation of, Ehrlich's conception of, 187 specificity of, 186 Amebic dysentery, 504 Ameboid colony on agar plate, 138 Ammonia, oxidation of, 76 AmcBba coli, 501, 503 dysenteriae, 504 meleagridis, 501, 531 Amoebosporidium polyphragum, 512 Amphitrichous bacteria, 35 Anaerobes, 117 facultative, 117 obligate, 117 Anaerobic bacteria, 48 spore-producing bacilli, 264 Anaphylaxis, 156, 206, 207 bacterial, 210 mechanism of, 207 relationship of, to certain body reactions, 210 Anaplasma, 509, 519 marginale, 519 Anaplasmosis, 519 Anemia, infectious, 541 of horse, virus of, 541 pernicious, 541 Anginas, 221 AniHn dyes, 24, 123 gentian-violet stain, 124 water, 124 Animal body, resistance of, to disease, 141 inoculation, 145 Animals and plants, differentiation, 26 Anthrax, 87, 253 cutaneous, 257 group, 252 intestinal, 257 pulmonary, 257 Anthrax, sjrmptomatic, 271 Antiabrin, 173 Antibiosis, 63 Antibodies, 154, 155 and related antitoxins, 157 as factors in acquired immunity, 155 of Ehrlich's first order, 157 second order, 174 third order, 185 Antiformin, 312, 389, 390, 409 Antigens, 155 Antigonococcus serum, 243 Antiphthisin, 396 ■ Antiphymatol, 398 Antiricin, 173 Antiseptics, 56 in common use, 59 theories of action of, 57 Antitoxin, 155, 159 and related antibodies, 157 constitution of, 162 diagrammatic representation of, 163 diphtheria, 371 standardization of, 166 manufacture of, 164 for pollen, 173 of commercial importance, 164 production of, Ehrlich's theory of, 161 tetanus, preparation of, 171 Antituberculin reaction, 401 Aphthomonas infestans, 536 Aphthous fever, 536 Apiosoma bigeminus, 509 Apoplectiform septicemia, 227 Archispores, 508 Arthritis, 221 Arthrospores, 36, 38 Arthus, phenomenon of, 206 Artificial aggressins, 300 Asci, 43, 457 Ascococcus Johnei, 249 Ascoli thermoprecipitation test, 262 Ascomycetes, 41 Ascospores, 40, 41, 43, 457 Ascus, 41 Asiatic cholera, 412 Aspergillosis, 457 Aspergillus, 455 flavus, 460 fumigatus, 457 glaucus, 462 niger, 461 nigrescens, 462 subfuscus, 462 Atrichous bacteria, 35 Atrium, infection, 142 Autooytotoxins, 192 Autogenic vaccmes, 201 INDEX 587 Autolysins, 189 Autolytic enzymes, 67 Avenues of infection, 142 Avian coccidiosis, 523 tuberculosis, 385 Azotobacter, 85 Babesia, 509 asini, 611 bigemina, 509 canis, 512 equi, 511 mutans, 511 ovis, 512 parva, 516 (piroplasma) commune, 514 gibsoni, 514 Bacillacefe, 85, 87 Bacillary dysentery, 345 Bacillus, 28, 87, 252 abortionis, 349 abortus, 349 aerobic spore-producing bacilli, 252 aerogenes capsulatus, 278 anaerobic spore-producing, 264 anaerobicus cyptobutyricus, 279 a-nthracis, 252, 253 symptomatici, 271 avisepticus, 293 bipolaris septicus, 290 botulinus, 286 bovicidaj 301 bovisepticus, 301 bronchicanis, 356 bronchisepticus, 356 butter, 409 cadaveris butyricus, 279 capsulatus mucosus, 329 carrier, 345 ohauvsei, 271 chauveaui, 271 cholerse, 293 gallinarum, 293 suis, 333 clavatus, 372 cloacse, 327 coli communior, 325 communis, 320, 321 colon, subgroup, 318, 319 communior, 325 diphtheria, 367 vitulorum, 440 dung, 409 dysenteriae, 345 emphysematis vaginae, 279 emulsion of Koch, 397 enteritidis, 330 sporogenes, 279 erysipelatis suis, 373 feseri, 271 Bacillus filiformis, 440 flavidus, 362 furunculosis ulcerossB, 362 grass, 409 hay, 252 hoagii, 362 hoffmanni, 372 influenzae, 383 lactis acidi, 231 aerogenes, 233, 325 leprte, 407 lymphangitidis, ulcerosa, 362 mallei, 306 murisepticus, 377 mycoides, 137 neapolitanus, 321 necrophorus, 440 necroseos, 440 oedematis maligni, 282 maligni of Ghon and Sachs, 285 of Babes, 317 of Flexner, 345 of Gartner, 330 of Glasser, 337 of green pus, 358 of Johnes' disease, 406 of Kutscher, 317 of Nicolaier, 265 of Pfeiffer, 383 of Preisz, 362 of Selter, 317 of Shiga, 345 ozoenae, 325 paratuberculosis, 406 paratyphoid, 338 perfringens, 279 pertussis, 384 pestis, 302 bubonicae, 302 phlegmonis emphysematosae, 278 plurisepticus, 290 pneumoniae, 329 of Friedlander, 325 pseudodiphthericus, 372 pseudotuberculosis ovis, 362 puUorum, 340 pyocyaneus, 358 pyogenes, 326, 379, 380 bovis, 379, 380 fcBtidus, 321 suis, 379, 380 renalis bovis, 362 rhinoscleromatis, 325 rhusiopathiae, 373 suis, 373 salnioni, 333 septicemiae, haemorrhagicae, 290 subtilis, 137, 252 suicida, 297 suipestifer, 333 588 INDEX Bacillus suipestifer of Voldagsen,337 suiseptica, 297 tetani, 265 tuberculosis, 385 types of, 87 typhi, 342 abdominalis, 342 murium, 339 typhosus, 342 violaceus, 64 vitulisepticus, 301 welchii, 278 Bacteremia, 148 Bacteria, 17, 27, 212 acid production of, determmation of, 113 acid-fast,' 112 non-pathogenic, 408 aerobic, 48 amphitrichous, 35 anaerobic, 48 and disease, 141 and resistance of animal body to disease, 141 atrichous, 35 capsulated, 32 pathogenic, 328 causal relationship of, to disease, proof of, 144 cell inclusions of, 35 protoplasm of, 33 cell wall of, 32 cells of, grouping of, 29 changes in hydrogen ion concen- tration, 112 chromogenic, 63 chromoparous, 64 classification of, 82 composition of cell, 46 cultural characters, 135 decay-producing, 73 denitrifying, 74 differentiation of, 184 distribution of, 68 effect of electricity on, 54 eurythermic, 52 ' facultative, 48 filamentous, 28 types of, 31 flagella of, 35 distribution of, 35 food relationships of, 46 foods of, 46 generation time of, 49 growth temperature range of, 52 histology of, 32 in contamination of milk, 565 india ink method for, 131 influence of reaction of medium on growth of, 56 involution forms of, 28 Bacteria, light production by, 64 relationships of, 53 living, examination of, 122 lophotrichous, 35 measuring of, 122 mesophihc, 51 metatrophic, '47 moisture relationships of, 47 monotrichous, 35 morphology of, 28 nature and classification of, 21 of nutrients required by, 104 nitrate, 76 - nitroso-, 76 of food, 552 of water, 552 oxygen relationships, 117 paratrophic, 47 pathogenic, groups of, 212 position of, 26 peritrichous, 36 photogenic, 64 physiological characters, 140 physiology of, 46 pigment production by, 63 prototrophic, 47 psychrophilic, 51 rates of death, 50 of growth, 49 reduction processes of, 115 relationships of chemicals to, 54 to disease, 22 to fermentation and decay, 22 reproduction in, 36 respiration of, 48 shape of, 28 showing sheaths, 33 size of, 31 sources of foods, 46 spherical, 85 spore bearing, 87 staining of, 123 stenothermic, 52 structure of, 32 temperature relations of, 51 thermal death point of, 52 thermophilic, 61 thread, 90 true, 84 types of, 28 Bacteriacese, 85, 88 Bacterial anaphylaxis, 210 cells, grouping of, 29 inclusions, 34 plasmoptysis of, 34 content of milk, 567 cultures, study of, 135 lamp, 65 relationships of ultramicroscopic organisms, 534 spore types, 37 INDEX 589 Bacteridium anthracis, 253 Bacterins, 201 Bacteriology, agricultural, definition, 18 definition, 17 medical, definition, 18 sanitary, 18 veterinary, 19 Bacteriolysins, 156, 185, 188 Bacteriolytic sera used in practice, 188 Ba'cteriopurpurin, 47 Bacterium, 87, 88, 89 abortus, 349, 414 acidi lactici, 137, 325 aerogenes, 325 anthracis, 253 avicidum, 293 bipolare pluricida, 290 boyisepticum, 301 bronchisepticum (bronchicanis), 356 cholera suis, 333, 539 cloacae, 327 coli, 74, 89, 137, 320, 321 commune, 321 communior, 325 coscoroba, 325 diphtheriffi, 367 dysenterise, 342, 345 enteritidis, 330 f oecalis alkaligenes, 342 lactis aoidi, 231 aerogenes, 325 leprae, 407 mallei, 306 melitensis, 349 mucosum capsulatum, 328 multicidum, 290 ozoense, 328 paratyphi, 338 pestis, 302 pneumoniae, 328, 329 pseudodiphthericum, 372 puUorum, 340 rhinoscleromatis, 328 suicidum, 297 suipestifer, 639 tuberculosis, 385 typhi, 342 murium, 339 suis, 337 typhosum, 58, 342, 568 welchii, 278 Balantidium, 529, 530 coli, 531 Baleri, 493 Basidiomycetes, 41 Beef broth, 104 extract bouillon, 105 Beerwort, 105 Bile, 149 Biliary fever, 512 equine, 511 Biochemical tests, 112 Bipolare multicidum, 301 Bismarck-brown stain, 124 Black death, 304 head, 531 Blackleg, 87, 271 Blackleg-tetanus group, 264 Blastomyces, 93, 446 coccidioides, 447, 452 dermatitidis, 447, 449 farciminosus, 447 Blastomycetes, 38, 446 Blastomycetic dermatitis, 449 epizootic lymphangitis, 447 Blastomycosis, 452 Blind staggers, 460 Blood and protozoan stains, 129 parasites, 421 Blood-serum, 110, 136 agar, 107 Blood-stains, recognition of, 183 Blue milk, 565 Body agglutinins, 176, 177 Body-cells, preferential union of toxins with, 163 Bordet-Gengou phenomenon, 190 Botrycoccus ascoformans, 249 Botryomyces ascoformans, 249 Botryomycosis, 249 Botulism, 286, 581 Bouillon, 104 beef extract^ 105 Bovine coccidiosis, 525 farcy, 437 piroplasmosis, 509 Bovotuberkulol, 397 Bovovaccine, 398 Bradsot, 87, 277 Braxy, 277 Brewer's yeast, 69 Broth, beef, 104 beef extract, 105 glycerin, 105 nutrient, 136 serum, 105 sugar, 105 sugar -free, 105 Brownian movement, 36 Brucella (bacterium) abortus, 349 melitensis, 353 Bryophytes, 27 Bubonic plague, 89, 302 Buffers, 103 BuUnose, 360 Butschlia, 529 neglecta, 529 parva, 529 postciliata, 529 590 INDEX Butter bacillus, 409 Butyric acid, 71 Cachbxial fever, 498 Calcium oxide, 60 Calf diarrhea, 327 scours, 325, 327 Callimastix frontalis, 630 Canine distemper, 356 Capsulated bacteria, 32 pathogenic bacteria, 328 Capsule, 32 stain, Johne's, 129 Muir's, 128 Raebiger's, 129 Carbol or phenol fuchsin stain, 124 Carbolic acid, 60 Carbuncle, malignant, 253 Carrier, 345 Caseous Ijonphadenitis, 362 Catalysts, 66 Catarrhal fever, malarial, 512 Cattle plague, 537 Cecum, protozoan commensals of, 528 Cell inclusions, 35 of yeast, 39 nutrition, Ehrlich's theory of, 159 plasmolyzed, 34 protoplasm, 33 receptors, 160 vegetative, 37 wall, 32, 45 Cellulitis suppurative, 222 Cellulose, 26 yeast, 39 Cerebrospinal meningitis, epidemic, 238 Certified milk, 569 Charbon, 253 symptomatique, 271 Chemicals, relationships of, to bacteria, 54 sterilization by, 98 Chemotaxy^ 55 Chemotropism, 56 Chicken tumors, 551 Chickenpest, 543 Chitin, 26, 32 Chlamydospore, 41, 43 Chocolate agar, 107 Cholera, Asiatic, 412 Chromobaoterium, 88 Chromogenic bacteria, 63 Chromoparous bacteria, 64 Chronic enteritis, 406 Ciliata, 45, 480, 528 Ciliophora, 480 Cladothrix actinomyces, 434 Classification of bacteria, 82 Clostridium, 37, 87, 264 Clostridium amylobacter, 264 botulinum, 264, 286 butyricum, 71 chauvsei, 264, 271 enteritidis sporogenes, 264 gastromycosis ovis, 264, 277 oedematis, 264, 282 of Ghon-Sachs, 264, 285 of Hibler, 264 of Novy, 264 pasteurianium, 77 putrificus, 264 sporogenes Metchnikoff, 286 tetani, 264, 265 - welchii, 264, 278 Coagglutmins, 178 Coccacese, 85 Cocci, types of, 86 Coccidioidal granuloma, 452 Coccidiosis, 523, 525 avian, 523 bovine, 525 cat, 527 dog, 527 ovine, 526 rabbit, 525 Coccidium, 509 bigeminum, 527 oviforme, 525 perforans, 525 perforatum, 523 revolta, 523 tenellum, 523 Coccobacillus, 290 Coccus, 28 Coefficient, phenol, 119 Colon bacillus, 321 subgroup, 318, 319 Colonies, gelatin plate, 138 on agar plates, 138, 139 Colon-typhoid group, 318 Comma bacillus, 413 Commensalism, 63 Commensals, 47, 63 Complement, 186 action of, 186 fixation of, 190 test in glanders, 313 formation of, Ehrlich's conception of, 187 specificity of, 186 Complement-fixation test, 486 Conglutination, 192 test for glanders, 314 Conglutinin, 192 Conidia, 38, 43 Conidiophores, 43, 456 Conjunctival tuberculin test, 402 Contagious diseases, 142 typhus in cattle, 537 Corrosive sublimate, 57 INDEX 591 CorynebacteTium, 91, 92 diphtheria, 92, 361, 367 hoffmanni, 361, 362, 372 pseudodiphthericum, 372 pseudotuberculosis, 361, 362 xerosis, 361, 372 Corynethrix pseudotuberculosis mu- rium, 362 Cow-pox, 546 Cryptococcus farciminosus, 447 Cryptogenic infections, 143 Cultural characters of bacteria, 135 Culture-media, 23 acidity of, 101 adjustment of reaction of, 100 agar, 108 albumin-free, 106 beef broth from meat, 104 beerwort, 105 blood-serum, 110 agar, 107 broth from beef extract, 105 chocolate agar, 107 Dunham's solution, 105 egg. 111 endo-agar, simplified, 108 endo-agar-fuchsin, 108 eosin-methylene-blue agar, 109 gelatin, 106 glycerin broth, 105 hormone agar, 107 hydrogen ion concentration of, 103 liquefiable solid, 106 liquid, 104 milk, 105 non-liquefiable, IIC nutrient agar, 107 gelatin, 106 potato, 110 preparation of, 100 serum broth, 105 sugar broth, 105 sugar-free broth, 105 synthetic, 106 Uschinsky's solution, 106 vegetable, 110 Cultures of bacteria, methods of securing, 132 by dilution, 132 by direct isolation, 132 isolation by animal inocu- lation, 134 by plating, 133 by smearing, 132 by use of differential antiseptics or disin- fectants, 134 by use of heat, 134 study of, 135 stab, types of growth in, 137 Cutaneous anthrax, 257 mallein test, 317 tuberculin reaction, 401 CyanophycesB, 27 Cycloposthium, 529, 530 bipalmatum, 530 Cytolysins, 185 group, 187 Cytoplasm, 40, 45 Cytorrhyctes vaccinae, 546 Cytotoxins, 185, 192 Dairy bacteriology, 18 hygiene, 570 Dasytricha ruminantium, 530 Death, black, 304 rates of bacteria, 50 Decay, 71 relationships of microorganisms to, 22 Decay-producing and putrefactive bacteria, 7b Delhi boil, 499 Deneke's spirillum, 416 Denitrification, 73 Denys tuberculin, 396 Deodorant, 57 Deodorizing power of disinfectant, 59 Dermatitis, blastomycetic, 449 sarcoptic, 464 Dermatomycosis, 472, 474 Derrengadera, 497 Desiccation, 47 Dextrose, 69 Diarrhea, white, of chicks, 340 Differentiation of bacteria, 184 of meats, 183 Dilution method in treatment of rabies, 549 of securing pure cultures, 132 Diphtheria, 367 antitoxin, 371 fowl, 544 toxin and antitoxin, manufacture of, 164 Diphtheria-pseudotuberculosis group 361 Diphtheroid bacilli, 361 Diplococcus, 29, 85, 86, 235 gonorrhoese, 241 intracellularis equi, 241 meningitidis, 238 lanceolatus, 235 of Neisser, 241 pneumonise, 235 Diplodinium iiorentinii, 530 Direct isolation method of securing pure cultures, 132 Discomyces bovis, 434 equi, 249 Disease and bacteria, 141 592 INDEX Disease, contagious, 142 diagnosis, agglutination tests in 179 infectious, 141 types of, 147 non-infectious, 141, 142 predisposing factors to, 150 produced by unknown organisms, 533 proof of causal relationship of microorganism to, 144 relationship of microorganisms to, 22 resistance of animal body to, 141 susceptibility to, variation of individuals in, 150 transmitted through milk, 567 woolsorters', 253, 257 Disinfectant, 56 deodorizing power, 59 economy of, 59 efficiency of, standardization of, 119 germicidal power, 57 homogeneity of, 58 ideal, characteristics of, 57 in common use, 69 non-corrosive, 58 non-toxic to higher life, 58 penetration of, 58 power to remove dirt and grease, 59 solubihty of, 58 stability of, 58 theories of action of, 57 Distemper, 224 Dog distemper group, 356 virus of, 542 Dourine, 485 Dried mallein, 316 Drinking-water, purification of, 557 Drops, hanging, 122 Drug habituation, 158 Dumdum fever, 498 Dung bacillus, 409 Dunham's solution, 105 Dysentery, 89 amebic, 504 bacillary, 345 paratubercular, 406 East African coast fever, 516 tick fever, 425 Eberth-Gaffky bacillus, 342 Economy in disinfectants, 59 Ectoplast, 33, 40, 45 Ectothrix, 471 megaspores, 471 micro'ides, 471 Edema, gaseous, 279 malignant, 87, 282 Efficiency of disinfectants, stand- ardization of, 119 Egg medium. 111 Ehrlich's conception of formation of amboceptor and complement, 187 humoral theory of immunity, 154 theory of agglutinin production, 175 of antitoxin production, 161 of cell nutrition, 159 of immunity, 159 Eimeria, (coccidium), 509, 522 avium, 523 boviSp 525 faurei, 626 stiedse, 526 Electricity, effect of, on bacteria, 54 Endo agar simplified, 108 Endo-agar-fuchsin medium, 108 Endocardftis, ulcerative, 221 Endogenous infection, 142 Endolysin, 203 Endoplasm, 45 Endospores, 36, 252 development of, in a bacillus, 37 Endothrix, 471 Endotoxin, 157 Entamoeba, 501 africana, 607 coli, 601, 503 histolytica, 601, 604 nipponica, 501 tetragena, 501, 507 Enteritidis subgroup, 318, 330 Enteritis, 221, 330, 531 chronic, 406 Entodinium, 529, 530 bursa, 630 caudatum, 630 dentatum, 530 minimum, 630 rostratum, 630 Enzymes, 66, 66 autolytic, 67 production, 64 splitting, 67 Eosin-methylene-blue agar, 109 Epidemic cerebrospinal meningitis, 238 Epithelioma contagiosum, 644 Epizootic lymphangitis, 466 pleuropneumonia in equines, 231 Equine biliary fever, 511 piroplasmosis, 511 Erwinia, 88 Erysipelas, 220 swine, 373 Erysipelatis murisepticum, 137 Erysipelothrix, 90, 91 muriseptica, 373, 377 INDEX 593 Erysipelothrix porci, 373 rhusiopathise, 91, 373 Erythrobacillus, 88 prodigiosus, 64, 137 Erythrogranulose, 272 Eubacteriales, 84, 85 Eurythermic bacteria, 52 Exanthemata, 148 Exhaustion, theory of, 153 Exogenous infection, 142 External resistance, 149 Extracellular enzymes, 66, 68 Facultative anaerobes, 117 bacteria, 48 Farcin du bcBuf, 437 Farcy, 306 Fermentation, 64 relationships of microorganisms to, 22 Ferments, organized, 65 unorganized, 65 Fever, paratyphoid, 89 puerperal, 221 splenic, 253 typhoid, 89, 342 Filamentous bacteria, 28 types of, 31 Filterable virus, 32, 533 Filtration, sterilization by, 98 Fixation, nitrogen, 77 of complement, 190 test in glanders, 313 Flagella, 45 of bacteria, distribution of, 35 stain, 127 Flagellar agglutinins, 176, 177 FlageUata, pathogenic protozoa of, 481 Flame, sterilization by, 94 Fluorescent group, 358 Fluorescin, 359 Fomites, 141 Food, bacteria of, 552 poisoning, 89 in man, 286 relationships to bacteria, 46 Foot-and-mouth disease, 536 virus of, 536 Foot-rot, 222 Formaldehyd, 61 Formalin, 61 Foul brood of bees, 87 Fowl diphtheria, 544 favus, 475 leukemia, 551 plague, virus of, 543 pox, 544 septicemia, 227 sleeping sickness, 228 tumors, 551 Fungi,. 27 imperfecti, 41, 454 thread, 432 Pusarium equinum, 464 Pusiformis, 91, 92 Gabbett's method for acid-fast bacteria, 126 methylene-blue stain, 124 Gall-sickness, 495, 519 Galziekte, 495, 519 Gambian horse sickness, 492 Gangrene, gaseous, 87 Gartner subgroup, 330 Gas, production of, 114 , Gaseous edema, 279 gangrene, 87 Gastric juice, 149 Gelatin, nutrient, 106 plate colonies, 137 stab, 136 Generation, spontaneous, 21 tinie of bacteria, 49 Gentian-violet, aqueous solution of, 124 stabilized stain, 128 Genus achorion, 475 aspergillus, 455 bacillus, 252 bacterium, 318, 356 brucella^ 349 clostridmm, 264 corynebacterium, 361 entamoeba, 501 erysipelothrix, 373 fusarium, 463 hemophilus, 379 herpetomonas, 498 microsporum, 473 mycobacterium, 385 penicillium, 462 pfeifferella, 306 piroplasma, 509 Plasmodium, 516 pseudomonas, 358 sporotrichum, 466 trypanosoma, 481 Germ theory of disease, proof of, 144 Germ-carrier, 345 Germicidal action of milk, 562 power, of disinfectants, 57 Germicide, 57 Germination of spores, 38 Giemsa's stain, 129, 130 Glanders, 306 group, 306 Globules, oil, 85, 40 Glycerin broth, 105 Glycogen, 35 granules, 40 Goat-pox, 545 594 INDEX Gonococcus, 241 Gonorrhea, 241 Gram's staining method, 128 Granules, glycogen, 40 metachromatic, 35 Granulobacillus saccharobutyricus immobilis, 279 Granuloma, coccidioidal, 452 Grass bacillus, 409 Group agglutination, 178 agglutinins, 178 cytolysins, 187 of pathogenic bacteria, 212 precipitation, 182 Growth temperature range, 52 Gruber-Widal test, 179 Guinea-pig plague, 551 Gypseum, 471 Habituation, drug, 158 Ha;matococcus, 509 bovis, 509 ovis, 512 Haemoproteus, 509 Hair infections, 469 Halteridium, 519 Hanging drops, 122 Hansen's method for spore stain, 125 Haptophore, 162, 175, 182 Hautschicht, 40 Hay bacillus, 252 Hay-fever, antitoxin for, 173 Head, 456 Helcosoma tropicum, 499 Heliotropism, 56 Hemagglutinins, 181 Hemoglobinuria, 512 Hemolysins, 185, 189 Hemophilic group, 379 Hemophilus, 88 haemoglobinophilis canis, 379 influenza;, 88, 379, 383 pertussis, 88, 379, 384 pyogenes, 88, 379 Hemoproteus, 619 Hemorrhagic septicemia, 89, 301 group, 290, 291 Hemotoxin, 159, 360 Herman's stain for tubercle bacilli in tissues, 126 Herpetomonas, 498 donovani, 498 Heterologous serum, 175 Heterolysins, 189 Hoffmann's bacillus. 372 Hog-cholera, 298 group, 318, 330 virus of, 539 Homogeneity of disinfectants, 58 Homologous serum, 175 Hormone agar, 107 Horse sickness, 492 virus of, 541 syphilis, 485 Horse-pox, 545 Host, 141 Hot air, sterilization by, 94 Humoral theory of immunity, 24 Ehrhch's, 154 Hund'staupe, 542 Hydrogen ion concentration, deter- mination of changes in, 112 sulphid, oxidation of, 74 Hydrophobia in man, 646 Hydrotropism, 56 Hygiene of dairy, 570 Hyperimmunization, 188 Hypersensitiveness, '206 HypersusceptibUity, 206 Hyphse, 42, 454 Hyphomycetes, 41, 464 form of, 41 group, 454 histology of, 42 morphology of, 41 reproduction of, 43 size of, 41 structure of, 42 Hypothesis, aggress !n, 203 ICTEROHEMATURIA, 512 111, quarter, 271 Immune agglutinin, 174 opsonins, 196 Immunity, acquired, 151 active, 151 antibodies as factors in, 155 passive, 153 agglutinins in, significance of, 181 duration of, 155 general discussion, 149 natural, 150 racial, 150 side-chain theory of, 159 theories of, 153 development of, 24 types of, 150 Immunization, passive opsonic, 203 Immunology, definition, 19 Incubation, period of, 168 India ink method for bacteria and protozoa, 131 Indol„73 production of, 118 Infantile kala-azar, 499 Infected, 141 Infection, 141 atrium, 142 avenues of, 142 by ingestion, 147 by inhalation, 147 cryptogenic, 143 INDEX 595 Infection, endogenous, 142 exogenous, 142 mixed, 147 phlogistic, 148 secondary, 147 traumatic, 142 Infectious abortion, 349 agalactia, 550 anemia, 641 of horse, virus of, 541 disease, 141 in which specific cause is not known, 533 types of, 147 Infective, 141 Influenza, 383 group, 379 Ingestion, infection by, 147 Inhalation, infection by, 147 Injection, intracardiac, 147 intracranial, 147 intra-ocular, 147 intraperitoneal, 146 Inoculation, animal, 145 by scarification, 147 intrathoracic, 147 intravenous, 146 methods of, 146 subdural, 147 Inorganic compounds, oxidation of, 74 . , reduction processes in, 73 Inspected milk, 569 Intermediate subgroup, 318, 330 Internal resistance, 149 Intertransmissibility of human bovine and avian tuberculosis, 404 Intestinal anthrax,- 257 group, 318 Intracardiac injection, 147 Intracellular enzymes, 66, 68 Intracranial injections, 147 Intradermal tuberculin test, 401 Intra-ocular injections, 147 Intraperitoneal injection, 146 Intrathoracic inoculation, 147 Intravenous inoculation, 146 Iron, oxidation of, 75 Isolation by animal inoculation, for securing pure cultures, 134 by plating, for securing pure cultures, 133 by smearing for securing pure cultures, 132 by use of differential antiseptics or disinfectants, for securing pure cultures, 134 of heat for securing pure cultures, 134 direct, for securing pure cultures, 132 Isolysins, 189 Isospora, 509 bigemma, 527 Isotrichia, 529, 530 intestinalis, 530 prostoma, 530 Itch disease, 464 Ixidoplasma bigeminum, 509 Jaundice, malignant, 512 Jaw, lumpy, 434 Johne's capsule stain, 129 disease, 406 Kala-azab, 498 infantile, 499 Keuchhusten bacillus, 384 Klebs-Loffler bacillus, 367 Klein's method of staining for spores, 125 Koch's old tuberculin, 395 postulates, 144 Koch-Weeks bacillus, 379 Konew's precipitation test, 312 Laboratory methods, development of, 23 Lactic acid, 59, 70 "- as preservative, 59 Lactobacillus, 88, 89 bulgaricus, 70 lactis acidi, 325 Lateral chain theory of immunity, 159 Leishman-Donovan bodies, 498 Leishmania donovani, 498 farciminosa, 447 (herpetomonas) infantum, 499 tropica, 499 Lentz's stain for Negri bodies, 130 Leprosy^, 407 Leptotrichia, 90, 92 Leuconostoc, 85 Leukemia, fowl, 551 Leukocidin, 246, 360 Leukocytic extracts, 203 Leukocytogregarina, 509 Leukocytozoon, 509, 521 Light production by bacteria, 64 relationships to bacteria, 53 Lime as disinfectant, 60 Liquefiable solid media, 106 Liquid media, 104 Litmus milk, 137 Living bacteria, examination of, 122 Lockjaw, 265 Loffler's flagella stain, 128 methylene-blue stain, 124 LoSler-Schutz bacillus, 297 Lophotrichous bacteria, 35 Lopophyton gallinse, 475 596 INDEX Luetin, 431 Lumpy jaw, 434 Lung plague of cattle, 635 Lymphadenitis, 362 caseous, 362 Lymphangitis, blastomycotic epi- zootic, 447 epizootic, 466 ulcerative, 362 Lysins, 185 Maceogametes, 523 Macronucleus, 45 Macrophages, 194 Macroscopic Widal test, 179, 181 Madura-foot, 445 Maladie du coit, 485 Malaria, 516 quartan, 518 tertian, 516 Malarial catarrhal fever, 512 Mai de oaderas, 490 Malignant carbuncle, 253 edema, 282 jaundice, 512 Malignes edem, 282 Mallease, 312 Mallein, dried, of Foth, 316 of Babes, 315 of Roux, 315 of Vladimiroff, 315 test, cutaneous, 317 glanders, 315 ophthalmic, 317 subcutaneous, 316 Malleinum siccum, 316 Malta fever, 353 group, 349 Marginal points, 619 Mastigophora, 488 Mastitis, 222 Maximum growth temperature, 52 McCampbell's modification of opsonic index, 199 Measuring bacteria, 122 Meat, differentiation of, 183 Meat poisoning, 286, 330, 333 Media, 100. See also Culture-media. Medical bacteriology, definition, 18 Medicine, preventive, development of, 24 Megaspores, 471 Meningitis, epidemic cerebrospinal, 238 Meningococcus, 238 Meningo-encephalitis, 460 Mercuric albuminate, 60 chlorid, 60 Mercury, 60 Mesophilic bacteria, 61 Metachromatic granules, 35 •Metals, heavy, salts of, 60 Metatrophic bacteria, 47 Metazoa, 27, 44 Metchnikoff's theory of phagocy- tosis, 154 Microaerophiles, 118 Microbe, 17 de Coqueluche, 384 Microbiology, 17 Micrococcus, 85 ascoformans, 249 aureus, 244 botryogenus, 249 citreus, 249 gonorrhoese, 241 intracellularis equi, 241 lanceolatus, 235 melitensis, 3'53 pneumoniae, 235 pyogenes albus, 248 aureus, 244 bovis, 249 citreus, 249 weichselbaumii, 238 Microgametoeytes, 623 Microides, 471 Micronucleus, 45 Microorganisms. See Bacteria. Microphages, 194 Microscope and its influence, 19 Microscopic examination, 123 Widal test, 179 Microspira comma, 412 metchnikovi, 410 Microsporum, 469, 473 adouini, 474 cajninum, 474 canis, 474 equinum, 476 felineum, 474 lanosum, 474 Milk, 106, 136 acid production in, 563 bacteria in contamination of, 565 bacterial content of, 567 certified, 669 changes in from normal to decom- posed, 661 composition of, 561 constituents of, 561 contamination of, 561, 565 diseases transmitted through, 567 examination of, 661 germicidal action of, 562 inspected, 669 litmus, 137 pasteurized, 569 putrefaction in, 564 Milzbrand, 253 Minimum temperatiire, 52 Mixed infections, 147 INDEX 597 Moisture relationships to bacteria, 47 Mold group, 454 hyphsB, 42 spores, types of, 43, 44 Molds, 17, 212 chromogenic, 63 classification of, 93 form of, 41 histologj' of, 42 morphology of, 41 reproduction of, 43 size of, 41 structure of, 42 MoUer's spore stain, 125 Monilia Candida, 477 Monocystis stiedse, 525 Monotrichous bacteria, 35 Morbus maculosus, 222 Mordants, 123 Morvin of Babes, 315 Mouse septicemia, 377 Much's granules of mycobacterium tuberculosis, Wirth's stain for, 126 Mucin, 33 Mucous membranes, 149 Mud fever, 541 Muir's capsule stain, 128 Murrina, 497 Mycelium, 42, 91, 454 Mycetoma, 445 Mycobacterium, 91, 92 diphtherias, 367 leprae, 385, 407 maUei, 306 paratuberculosis, 385, 406 pseudotuberculosis, 362 smegmatis, 408 tuberculosis, 28, 126, 385, 568 Mycology, 17 Mycorrhiza, 79 Myxobacteriales, 84 Nagana, 488 Natural aggressins, 299 immunitv, 150 Navel ill, 221 Neerobacillosis, 440 Negative chemotaxy, 55 hydrotropism, 56 Negri bodies, Lentz's stain, 130 Neisseria, 85, 86, 235 gonorrhoeae, 235, 241 intracellularis equi, 235, 241 meningitidis, 235, 238 Nephrolysins, 186 Neurorrhyctes hydrophobise, 547 Neurotoxin, 159 Neutralization of acid, 564 New tuberculin, 397 Nitrate bacteria, 76 Nitrification in soil, 76 Nitrobacter, 85 Nitrobacteriacese, 85 Nitrogen cycle, 80 fixation, 77 Nitroso-bacteria, 76 Nitrosomonas, 85 Nitrous acid, oxidation of, 76 Niveum, 471 Nocardia farcinica, 437 Non-corrosive quality of disinfect- ants, 58 Non-infectious diseases, 141, 142 Non-liquefiable media, 110 Non-pathogenic acid-fast bacteria, 408 spirilla, 416 Non-toxic to higher life, quality of disinfectants^ 58 Normal agglutmin, 174 opsonins, 196 solution, definition of, 100 Noxious retention theory, 154 Nucleus, 40, 45 Nutrient agar, 107 broth, 136 gelatin, 106 required by bacteria, nature of, 104 Nutrition, cell, Ehrlich's theory of, 159 Obligate anaerobes, 117 (Edema malin, 282 Oidium, 43, 469 albicans, 477 coccidioides, 452 dermatitidis, 449 lactis, 564 Oil globules, 36, 40 Old tuberculin, 395 Omphalophlebitis, 221 Oocyte, 523 Ophryoscolex, 529, 530 fasciculus, 530 inermis, 530 intermixtus, 530 labiatus, 530 scolex, 530 Ophthalmic mallein test, 317 Ophthalmo-tuberculin test, 402 Opsonic immunization, passive, 203 index, 197 determination of, 199 McCampbell's modification of, 199 preparation of bacterial emul- sion for, 198 of leukocytes for, 197 of serum for, 198 technic of test, 198 Opsonins, 156, 194, 195 598 INDEX Opsonins, immune, 196 normal, 196 Optimum temperature, 51 Organella, 45, 478 Organisms, ultramicroscopic, 32 Organized ferments, 65 Oriental sore, 499 Osmotic pressure, adjustment of organisms to, 62 Ovine coccidiosis, 526 Ovoplasma orientale, 499 Oxidation of ammonia, 76 of hydrogen sulphid, 74 of inorganic compounds, 74 of iron, 75 of nitrous acid, 76 Oxidizers, 67 Oxygen relationships, determination of, 117 Papier Chardin, 315 Paracolon bacillus, 338 Paraformaldehyd, 61 Parasites, 47 blood, 421 Parasitic protozoa of ciliata, 528 Parasitology, definition, 18 Paratrophic bacteria, 47 Paratubercular dysentery, 406 Paratyphoid bacillus, 338 fever, 89 Passive immunity^ acquired, 153 opsonic immunization, 203 PasteureUa, 88, 290 bovteeptica, 291, 296, 297, 301 cholera; gallinarum, 291, 293, 296, 297 cunicuUcida, 291, 302 equiseptica, 291 pestis, 28, '291, 302 pseudotuberculosis rodentium, 291 suiseptica, 291, 296, 297 Pasteurellosis, 290 Pasteurized mUk, 669 Pathogenic Bacteria, groups of, 212 microorganisms, position of, 26 protozoa, 478 of class rhizopoda, 500 of fiagellata, 481 Penetrating quality of disinfectants, 58 PeniciUium, 462 Peptonization, 72 Period of incubation, 158 Peripneumonia, 535 Perithecium, 457 Peritonitis, 221 Peritrichous bacteria, 36 Pernicious anemia, 541 Pertussis, 384 Petechial fever, 222 Petri dish, 133 Pfaundler's reaction, 177, 181 Pfeifferella, 91, 92 mallei, 92, 306 Pfeifferia princeps, 525 PfeiflEer's phenomenon, 185 Pferdesterbe, 541 Phagocytes, 154, 194 Phagocytosis, 194 Metchnikoff's theory of,. 164 PhenoL 60 coefficient, 119 Phenomenon of Arthus, 206 Theobald Smith, 207 Phlogistic infections, 148 Photogenic bacteria, 64 Phycomycetes. 41 Phymatin, 397 Physiological salt solution, 63 Pigment production by bacteria, 63 Piroplasma, 509 bigeminum, 509 canis, 512 equi, 511 mutans, 611 ovis, 512 parva, 516 Piroplasmosis, equine, 511 Plague, bubonic, 89, 302 cattle, 537 fowl, 543 guinea-pig, 561 lung, of cattle, 536 Plant bacteriology, 18 Plants and animals, differentiation, 26 Plasmodium, 609, 516 falciparum, 518 immaculatum, 518 malarias, 518 vivax, 516 Plasmodroma, 480 Plasmolysis, 34, 42 Plasmolyzed cell, 62 Plasmoptysis, 34 of bacterial cells, 34 Plectridium tetani, 265 Pleuropneumonia, 536 epizootic, in equines, 231 septic, 301 virus of, 535 Pneumobacillus, 329 Pneumococcus, 235 of Friedlander, 329 Pneumomycosis, 467 Pneumonia, 221 stable, 231 Poisoning, food, 89 meat, 286, 330 ptomain, 330, 333 INDEX 599 Polar staining, 35 Pollantin, 173 Pollen, antitoxin for, 173 Polyarthritis, 379 Polyvalent vaccine, 201 Positive chemotaxy, 65 hydrotropism, 56 Potato, 110, 135, 136 Power of disinfectant to remove dirt and grease, 59 Precipitation, 156, 174 agglutination and, differentiation, 174 group, 182 test, Konew's, 312' uses made of, 183 Precipitin, 174, 182 Precipitogen, 182 Precipitoid, 182 Predisposing factors to disease, 150 Preisz-Nocard bacillus, 362 Preventive medicine, development of, 24 Proteins, salting out of, 177 split, Vaughn's, 207 Proteolysis, 72 Proteosoma, 509, 519 Proteus, 88 vulgaris, 137 Protoplasm, cell, 33 yeast, 39 Prototrophic bacteria, 47 Protozoa, 17, 27, 212 classification of, 480 form of; 45 histology of, 45 india ink method for, 131 morphology of, 44 parasitic, of ciliata, 528 pathogenic, 478 ' of class rhizopoda, 500 of flagellata, 481 reproduction of, 45 size of, 45 structure of, 478 Protozoan and blood stains, 129 commensals of rumen and cecum, 528 Protozoology, 17 Pseudofarcy, 362, 363 in horse, 447 Pseudoglanders, 362 Pseudomonadaceae, 86, 89 Pseudomonas, 87, 89 aeruginosa, 89, 368 denitrificans, 74 fluorescens, 137, 358 pyocyanea, 64, 256, 311, 356, 358 Pseudopodia, 45, 479 Pseudotubercle bacilli, 362 Pseudotuberculosis, 362 bacilli, 362 murium, 362 Psorospermium avium, 523 cuniculi, 525 Psychrophilic bacteria, 51 Pteridophytes, 27 Ptomain-poisoning, 330, 333 Ptomains, 72 Puerperal fever, 221 Pulmonary anthrax, 257 Purification of drinking-water, 557 of water, 552, 557 Putrefaction, 71, 664 Pyelonephritis, 362 Pyemia, 148, 220 PyobaciUosis, 362, 379 Pyocyanase, 266, 360 Pyocyaneus bacillosis, 360 Pyocyanin, 359 Pyogenic cocci, 235 Pyrosoma, 509 bigeminum, 509 var. canis, 512 Pythogenic theory of disease, 24 Qualitative examination of water, 565 Quantitative examination of water, 552 Quartan malaria, 618 Quarter evil, 271 ill, 271 Rabies in animals, 646 Racial immunity, 160 Raebiger's capsule stain, 129 Rauschbrand, 271 Reaction of media, adjustment of, 100 Receptors, cell, 160 Recognition of blood-stains, 183 Recurrent fever, 421 Red fever of swine, 373 milk, 565 Reduction processes ia inorganic compounds, 73 of bacteria, 115 Relapsing fever, 421 Reproduction in bacteria, 36 Resistance, 149 external, 149 internal, 149 Retention, noxious, theory of, 154 Rhizobium, 86 leguminosarum, 28, 78 Rhizopoda, 480, 500 Rhodesian redwater, 516 tick fever, 516 Rhodococcus, 85 Ricin, 168 600 INDEX Rigor mortis, 67 Rinderpest, 537 Rinderseuche, 301 Ring test, 312 Ringworm, 472 Robin, 158 Rods, aerobic, 252 vegetative, 37 Romanowsky stain, 129 Ropy milk, 665 Rumen, protozoan commensals of, 528 Saccharomyces, 93 cerevisiae, 69, 93 dermatitidis, 449 morphology of, 38 Salmonella, 333 Salt solution, physiological, 63 Salting out of proteins, 177 Salts of heavy metals, 60 Sanitary bacteriology, definition, 18 science, development of, 24 Sapremia, 148 Saprophytes, 47 Saprozoites, 47 Sarcina, 30, 85 Sarcocystis, 509, 521 bertrami, 522 lendemanni, 522 miescheriana, 522 muris, 522 tenella, 522 Sarcoptes equi, 465 Sarcoptie dermatitis, 464 Sausage poisoning, 286, 581 Scarification, inoculation by, 147 Schizomycetes, 27, 84 Schizonts, 522 Schizophycese, 27 Schweinepest, 539 Schweineseuche, 297 Sclerotium, 454 Scrofula, swine, 394 Secondary infections, 147 Self-purification of natural waters, 557 Sensitization, 156 Sensitized vaccines, 204 Septic pleuropneumonia, 301 Septicemia, 148, 220 hemorrhagic, 89, 301 group, 290 in fowls, 410, 425 mouse, 377 Septicemic gangreneuse, 282 Septic idin, 296 Septum, 42 Sera, bacteriolytic, used in practice, 188 Serobacterin, 204 Serum antidiphtheriticum, 166 antigonococcus, 243 broth, 105 heterologous, 175 homologous, 175 sickness in man, 206 simultaneous method, 538 Sewage disposal, 558 Sheath, 33 Sheep-pox, 545 Sickness, serum, in man, 206 Side-chain theory of immunity, 159 Side-chains, 160 Skatol, 73 Skin, 149 Sleeping sickness, 495 Small-pox in man, 545 Soapy milk, 565 Sodium chlorid, 63 Soil bacteriology, 18 nitrification in, 76 Solubility of disinfectants, 58 Somatic agglutinins, 176, 177 Sore head, 544 throat, septic, 222 Souma, 494 Spermatophytes, 27 Spherical bacteria, 85 Spirilla, 30 non-pathogenic, 416 types of, 31, 90 Spirillaceae, 85, 89 Spirillosis, 421, 425, 427 Spirillum, 28, 89, 90 anserina, 425 cholerae, asiaticae, 412 duttoni, 423 fetus, 414 metchnikovi, 410 obermeieri, 421 of Finkler and Prior, 416 ovina, 427 ovis, 428 pallidum, 428 phosphorescens, 416 theileri, 427 tyrogenum, 416 Spirochaita, 93, 420, 421 anserina, 421, 425 biflexa, 420 duttoni, 421, 423 elusa, 420 equi, 427, 428 evansi, 487 gallinarum, 421, 425 granulosa, 425 hyos, 421 kochi, 425 Marchouxi, 425 nicoUei, 425 novyi, 425 INDEX 601 Spirochaeta obermeieri, 421 ovina, 421 ovis, 427 pallida, 420, 421, 428 pertenuis, 421, 431 recurrentis, 421 theileri, 421, 427 Spirochete group, 417 Spirochetes, 431 of other infections, 431 Spirochetosis, 425 Spirochffitales, 84, 92 Spiroschaudinnia, 421 duttoni, 423 recurrentis, 421 Splenic fever, 253 Split proteins, Vaughn's, 207 Splitting enzymes, 67 Spontaneous generation, 21 Sporangiophore, 43 Sporangium, 43 Spore case, 43 stain, 125 Spore-bearing bacteria, 87 Spore-producing bacilli, anaerobic, 264 Spores, 36, 508 germination of, 38 of yeast, 40 Sporoblasts, 508 Sporotrichosin, 469 Sporotrichosis, 466 Sporotrichum, 466 beurmanni, 466 schenkii, 466 Sporozoa, 480, 508 Sporozoites, 508 Stab cultures, types of growth in, 137 Stability of disinfectants, 58 Stabilized gentian-violet, 128 Stable pneumonia, 231 Stain, 124 anilin gentian-violet, 124 aqueous solution of gentian-violet, 124 Bismarck-brown, 124 blood, 129 Giemsa's, 130 Wright's, 129 capsule, 128 Johne's, 129 Muir's, 128 Raebiger's, 129 carbol or phenol fuchsin, 124 Ehrlich's, 124 flagella, 127 Loffler's, 128 Van Ermengem's, 127 for acid-fast bacteria, 126 acid alcohol method, 126 Gabbett's method, 126 Stain for negri bodies, Lentz's, 130 Gabbett's methylene-blue, 124 Giemsa, 129 Gram's method, 128 Herman's for tubercle bacilli in tissues, 126 Loffler's methylene-blue, 124 Muir's capsule, 128 protozoan, 129 Romano wsky, 129 spore, 125 Hansen's method, 125 Klein's rriethod, 125 Holler's, 125 stabilized gentian-violet, 128 Wirth's for Much's granules of, mycobacterium tuberculosis, 126 Wright's, 129 Ziehl's, 124 Stained mount, preparation of, 124 Staining methods, 123 polar, 36 Stalactite, 303 Staphylococcus, 30, 85, 86, 244 albus, 244, 248 ascoformans, 249 aureus, 28, 244 (Botryomyces) ascoformans, 244 citreus, 244, 249 . pyogeneSj 248 albus, 248 aureus, 244 bovis, 249- citreus, 249 Staphylolysin, 246 Starrkrampf, 265 Starter, 233 Steam, streaming, sterilization by, 95 under pressure, sterilization by, 96 Stenothermic bacteria, 52 Sterigmata, 456 Sterigmatocystis, 455, 456 Sterilization, 94 at temperatures lower than boiling-point, 98 by addition of chemicals, 98 by filtration, 98 by flame, 94 by hot air, 94 by steam under pressure, 96 by streaming steam, 95 Strangles, 224 Strauss' reaction, 310 Streaming steam, sterflization by, 95 StreptobaciUi, 30 Streptococcus, 29, 85, 86, 214 acidi lactici, 231 agalactise contagiosae, 231 vaccarum, 217 602 INDEX Streptococcus anginosus, 215 articulorum, 216 brevis, 214 capsulatus gallinarum, 227 citrovorus, 234, 564 classification of, 214 coryzsB contagiosse equorum, 224 equi, 216, 224, 231 equinus, 215 erysipelatos, 215, 216 foecalis, 215 gallinarum, 216, 227 Holman's classification of, 215 lacticus, 63, 70, 216, 231, 232, 564 lactis, 231 longus, 214, 215 mastitidis, 217, 231 sporadicse, 231 meningitidis, 238 mitis, 215 mucosus, 215 of Norgaard and Mohler, 227 of Ostertag, 229 phlogogenes, 217 pneumonia, 235 puerperalis, 216 pyogenes, 137, 215, 216, 229, 231, 232 bovis, 217 malignis, 216 salivarius, 215 scavlatinosus, 216 septicus, 216 vaginitidis, 216, 229 viridans, 215 Streptothricosis, 440, 445 Streptothrix actinomyces, 434 bbvis, 434 canis, 439 caprae, 439 cuniculi, 440 farcinica, 437 madurse, 445 necrophora, 440 Subcutaneous mallein test, 316 Subdural inoculations, 147 Suctoria, 480 Sugar broth, 105 Sugar-free broth, 105 Sulphur dioxid, 61 Sulphurous acid, 61 Suppurative allulitis, 222 Surra, 487 Susceptibility, 149 to disease, variation of individuals in, 150 Swamp fever, 641 Swine erysipelas, 373 group, 373 fever, 539 red fever of, 373 Swine scrofula, 394 Swine-plague, 297, 298, 334 Swine-pox, 545 Symbion, 63 Symbiont, 63 Symbiosis, 63 Symptomatic anthrax, 271 Synthetic media, 106 Syphilis, 428 horse, 485 Wassermann test for, 191 Systematic bacteriology, 18 Taubuman, 398 Temperature lower than boiling- point, sterilization at, 98 relations to bacteria, 51 Tertian malaria,, 616 Test, agglutination, 179, 181 for glanders, 311 complement-fixation, in glanders, 313 conglutination for glanders, 314 cutaneous mallein, 317 Gruber-Widal, 179 mallein, for glanders, 316 ophthalmic mallein, 317 Pfaundler's, 181 precipitation, Konew's, 312 ring, 312 Strauss', 311 subcutaneous mallein, 316 thermoprecipitation of Ascoli, 262 Wassermann for syphilis, 191 Widal, 179 Tetanolysin, 269 Tetanospasmin, 269 Tetanus, 87, 265 toxin and antitoxin, preparation of, 171 Tetracoccus, 29 Tetrad, 29 Texas fever, 509 Thallophytes, 27 subdivisions of, 27 Theileria, 509 parva,, 516 Theobald Smith phenomenon, 207 Theories of immunity, 163 development of, 24 Ehrlich's humoral, of immunity, 164 of cell nutrition, Ehrlich's, 159 of exhaustion, 153 of noxious retention, 154 of phagocytosis, Metchnikoff's, ' 164 Thermal death point, 62, 118 Thermophilic bacteria, 51 Thermoprecipitation test of Ascoli, 262 INDEX 603 Thiobacteriales, 84 Thread bacteria, 90 fungi, 432 Thrush, 477 Tick fever, 509 Tinea crista; gallae, 475 Tongue, wooden, 434 Tonsillitis, 221 Torula, 93 Toxemias, 148 Toxin, 155, 157 characteristics of, 157 constitution of, 162 diagrammatic representation of, 163 diphtheria, manufacture of, 164 standardization of, 166 preferential union of, with body- cells, 163 sources of, 1'58 specificity of, 159 tetanus, preparation of, 171 Toxine, 167 Toxoid, 162 Toxone, 170, 371 Toxophore, 162 Traumatic infection, 142 Treponema, 93, 421 pallidum, 93, 428 pertenue, 431 Trichobacteria, 28, 36 Trichomastix ruminantium, 530 Trichomonas, 531 rurninantium, 530 Trichomycetes, 432 Trichophyton, 469, 470 caninum, 471, 473 cerebrifbrme, 471 crateriforme, 471 dentieulatum, 471 discoides, 471 equinum, 471, 472 felineum, 471, 472 granulosum, 471. 472 gypseum, 472 . niveum, 472 radians, 471, 472 sulfurerum, 471 tonsurans, 471 verrucosum, 471 Tropisms, 56 True bacteria, 84 Trypanosoma, 481 brucei, 483, 488 castellani, 495 cazalboui, 494 congolense, 492 dimorphon, 492 donovani, 498 elmassiani, 490 equinum, 490 Trypanosoma equiperdum, 485 evansi, 487 gambiense, 483, 484, 495 hippicum, 497 lewisi, 483, 491 pecaudi, 493 rougeti, 485 theileri, 495 ugandense, 495 vespertilionis, 483 Trypanosomiasis, 492, 493 human, 495 Tsetse-fly disease, 488 , Tubercle bacilli in tissues, Herman's stain for,, 126 Tuberculin, 394 Alt, 395 Koch's, 395 New, 397 of Denys, 396 test, conjunctival, 402 cutaneous, 402 intradermal, 401 ophthalmo, 402 T. R., 397, 398 Tuberculocidin, 396 Tuberculol, 396 Tuberculosis, 385 Tumors, chicken, 551 Turgor, 62 Typhoid fever, 89, 342 Typhoid-dysentery subgroup, 318, 342 Typhus, contagious, in cattle, 537 TlLCBRATrvE endocarditis, 221 lymphangitis, 362 Ultramicroscope, 20 Ultramicroscopic organisms, 32 bacterial relationships of, 534 Univalent vaccine, 201 Unorganized ferments, -65 Uschinsky's solution, 106 Vaccination, 153, 189, 200 Vaccines, autogenic, 201 isolation of organism, 201 preparation of, 201 standardization of, 202 polyvalent, 201 sensitized, 204 univalent, 201 Vacuoles, 35, 40 Van Ermengem's flagella stain, 127 Variation of individuals in sus- ceptibility to disease, 150 Vaughn's split proteins, 207 Vegetable culture-media, 110 Vegetative cell, 37 rod, 37 604 INDEX Veterinary bacteriology, definition, 19 supervision of herd, 575 Vibrio, 89, 90 cholerse, 60, 90, 410, 412 asiaticse, 412 fetus, 414 Finkleri, 137 group, 410 metchnikovi, 410 proteus, 416 Vibrion septique, 282 Virulence, 144, 203 Virus, 141 filterable, 32, 533 of dog distemper, 542 of epithelioma contagiosum, 544 of foot-and-mouth disease, 536 of fowl plague, 543 of guinea-pig plague, 551 of hog-cholera, 539 of horse sickness, 541 of infectious agalactia of sheep and goats, 560 of infectious anemia of horse, 541 of pleuropneumonia, 635 of poxes, 545 of rabies, 546 of rinderpest, 537 of yellow fever, 546 Voges-Proskauer reaction, 117 Wassebmann syphilis test, 191 Water analysis, 562 anilin, 124 bacteria of, 562 natural, self-purification of, 557 purification, 552, 557 quantitative examination of, 652 West African tick fever, 423 Whips, 35 White diarrhea of chicks, 340 Whooping-cough, 384 Widal test, 179 Wildseuche, 301 Wirth's stain for staining Much's granules of mycobacterium tuber- culosis, 126 Wooden tongue, 434 Woolsorters' disease, 253, 257 Wright's blood stain, 130 Yaws, 431 Yeasts, 212 brewer's, 69 cell inclusion of, 39 cells and groupings, types of, 39 cellulose, 39 chromogenic, 63 classification of, 93 form of, 39 grouping of, 39 histology of, 39 morphology of, 38 protoplasm, 39 reproduction in, 40 size of, 39 spores of, 40 structure of, 39 Yellow fever, 546 Zibhl's carbol or phenol fuchsia stain, 124 Zooglea, 31 Zoogloea pulmonis equi, 249 Zopfius, 88 Zygospores, 43 Zymase, 66, 69 Zymophore, 176, 182 Zymotoxic group, 176