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 Spirochaeta suis; its significance as a p 
 
 3 1924 000 225 742 
 
The original of this book is in 
 the Cornell University Library. 
 
 There are no known copyright restrictions in 
 the United States on the use of the text. 
 
 http://www.archive.org/details/cu31924000225742 
 
SPIROCHAETA SUIS, ITS SIGNIFICANCE 
 
 AS A PATHOGENIC ORGANISM 
 
 STUDIES ON HOCV CHOLERA 
 
 (WITH PLATE 4) 
 
 WALTER E. KING and GEORGE t. HOFFMANN 
 
 Reprinted from 
 The JotTENAi, OT Infectious Diseases, Vol. 13, No. 3, November 1913, pp. 463- 
 
 CHICAGO 
 
SPIROCHAETA SUIS, ITS SIGNIFICANCE AS A 
 
 PATHOGENIC ORGANISM.* 
 
 STUDIES ON HOG CHOLERA. 
 
 Walter E. King and George L. Hotfihann. 
 {From the Research Laboratory, Parke, Davis and Co., Detroit, Michigan.) 
 
 (with plate 4.) 
 introduction. 
 
 Since results of the dark field study of the blood of animals 
 suffering from hog cholera were reported,' the work has been con- 
 tinued and several new phases of the problem have been investigated. 
 In the former article it was reported that a relatively large spiro- 
 chete and characteristic granules were present in the blood of 
 40 hogs infected with cholera, representing 12 different strains 
 of virus, while the blood of 48 normal hogs was observed to be 
 free from spirochetes and granules. The spirochetes observed in 
 specimens of blood from cholera hogs were very few in number, 
 but the characteristic granules were apparently always present 
 during the fastigium and were regarded as important findings. 
 
 In conjunction with the later studies of this spirochete in the 
 blood, many observations were made by means of the dark field 
 of material taken from the intestinal ulcers of hogs dead from hog 
 cholera. Dark field examinations have been made of the necrotic 
 tissue from the intestinal ulcers of all animals dead from hog 
 cholera during the course of our experiments of the last six months. 
 Scrapings from the intestinal mucosa and contents of the cr3^ts in 
 the ceca of normal hogs, some of these being susceptible to hog 
 cholera, others immune to the disease, were also examined at every 
 opportunity. In all, the study with the dark field of material from 
 the crj^ts and ulcers of normal and diseased hogs has included 
 about 140 cases. As a result of this work, the details of which will 
 be pubhshed separately, we are inclined to believe that the following 
 observations are true. 
 
 • Received for publication August ii, 1913. 
 
 ' King, Baeslack, and Hoffmann, Jour. Infect. Dis., 1913, 12, p. 206. 
 
 463 
 
464 Walter E. King and George L. Hoffmann 
 
 1. The mucosa of the large intestine, particularly that of the 
 cecum, of normal hogs, includes in its flora non-pathogenic spirilla 
 and spirochetes, relatively large forms predominating. 
 
 2. In the ulcerated patches of cecal mucosa and in the crypts, 
 near the ileo-cecal valve, of hogs dead from cholera is localized a 
 constant species of spirochete, which is pathogenic for swine and 
 which plays an important part in the production of hog cholera. 
 
 3. The crypts and healed ulcers of hogs actively immunized 
 against hog cholera may contain, for a variable period of time 
 after immunization, in addition to non-pathogenic spirochetes, the 
 same species of pathogenic spirochete referred to above. 
 
 There are many factors entering into the above tentative con- 
 clusions which cannot be discussed in detail in this paper, but 
 which will follow in future reports of our studies on this disease. 
 Among the more important observations, however, are the results 
 obtained from the experimental curative treatment of cases of hog 
 cholera with arsenical and mercurial preparations. This work 
 resulted, not in cures, but, usually, in prolongation of life, which, 
 in most cases, was coincident with an apparent decrease in number 
 or total disappearance of the spirochetes in question from the 
 necrotic tissue in the cecal ulcers. Positive dark field findings of 
 spirochetes in intestinal ulcers of cholera hogs are in accordance 
 with the results reported by von Riither,' Uhlenhuth and Haendel,^ 
 and Betegh.^ The presence of spirochetes in the bile of cholera 
 hogs, as reported by Uhlenhuth and Haendel, has not been found 
 in the cases examined thus far. 
 
 On account of the fact that efforts to secure cultures or satis- 
 factory stained preparations of Spirochaeta suis from the blood 
 of cholera hogs were not successful, and because of the large amount 
 of bacterial contamination present in the intestinal lesions, attempts 
 were made to find localized foci of the organism elsewhere in the 
 diseased body. Microscopical preparations and cultures were 
 made from the spleen, Kver, lymphatic glands, spinal cord, cerebro- 
 spinal fluid, bile, and urine. These attempts were unsuccessful 
 except in one instance. In one case (Hog 615, inoculated subcu- 
 
 ' Berl. tierar2.iL Wchnschr,, 1.911, 27, p. 191. 
 ' Handbuch d. path. Mikroorg., 1913, 6, p. 337. 
 3 Berl. tierarztl. Wchnschr., 1912, 28, p. g68. 
 
Spirochaeta Suis, a Pathogenic Organism 465 
 
 taneously with exudate from local lesion, Hog 599), the dark field 
 revealed in the inguinal lymph gland a spirochete resembling the 
 assumed pathogenic form. In many of the dark field preparations 
 numerous granules were observed, but it was impossible to draw 
 any definite conclusions as to their origin and relationship to 
 Spirochaeta suis or to tissue elements. 
 
 Finally, when an opportunity presented itself, dark field 
 examinations were made of external lesions on the leg and ear of a 
 case of cholera of the chronic type (Hog 583, May 8, 1913) and they 
 were found to contain spirochetes, presumably of one species, in 
 large numbers. So far as we know, similar observations have never 
 been reported except by Dodd' of Pretoria, Transvaal. In describ- 
 ing "a disease of the pig due to a spirochete," Dodd states that he 
 found a spirochete in the cutaneous lesions of a number of pigs. 
 The disease could not be transmitted experimentally by the inocula- 
 tion of blood from an infected animal, but was transmitted, how- 
 ever, by actual contact. The infection was fatal to several pigs. 
 Autopsy revealed nothing except the local skin lesions and general 
 anemia. The disease appeared to be some cutaneous infection 
 due to the spirochete found in the local lesions. Scrapings from 
 the skin of healthy hogs and from the normal skin of the affected 
 pigs were found to contain no spirochetes. 
 
 Since the first observation we have not only found that all 
 external local lesions examined, including 11 cases of hog cholera, 
 were constant foci for Spirochaeta suis, but we have also been able 
 to produce typical hog cholera, with local external lesions, by 
 inoculating a small bit of the tissue or a small amount of exudate 
 from local lesions of the affected animal into a healthy pig. Scrap- 
 ings from the healthy skin of hogs and from the areas of discolora- 
 tion on the ventral surface of the body of animals suffering from 
 cholera have been submitted to dark field examination with nega- 
 tive results. The results of this work, which is still in progress, 
 will be published later. 
 
 In our former paper, the spirochete observed in the blood of 
 hogs infected with hog cholera was described as follows: 
 
 " In the specimens of blood from all infected hogs, which have been observed by 
 means of the dark field, a relatively large spirochete has been found. It averages 
 » Jour. Comp. Path, and Ther., igo6, ig, p. 216. 
 
466 Walter E. King and George L. Hoefmann 
 
 from five to seven microns in length and one micron in width. The body of the organ- 
 ism is iiexible and round at its ends. It presents no knobbed appearance at its poles 
 Actively motile, it revolves about its longitudinal axis. Its motility is undulating in 
 character and its spirals are fixed. A few of these organisms have been observed 
 dividing longitudinally. In one permanent microscopical mount, prepared by india 
 ink fixation, one of these organisms apparently shows a polar flagellum. On the dark 
 field this spirochete is readily distinguished from bacteria on account of its lack of 
 rigidity and its characteristic motility, and from 'blood filaments' by its greater 
 refractive properties and characteristic morphology. 
 
 " This spirochete has not been found in large numbers; in any of the blood prepara- 
 tions. However, in nearly every specimen examined, more than one has been observed 
 and in many cases five or six have been found with httle difiiculty. As a rule the 
 organisms have been found to be more numerous at the height of the disease. The 
 specimens of blood examined have been diluted in the proportion of about one to 
 ten or fifteen with sterile sodium citrate solution, which factor should be considered 
 in contemplating the number observed in a given positive specimen. Moreover, it is 
 suggested that this organism, when observed as a spirochete form, constitutes only 
 one stage of its development." 
 
 This description appears to be correct, except that we have never 
 verified the presence of a polar flagellum. 
 
 It must be conceded that any successful dark field investigation 
 requires patient work and much practice in interpretation. There- 
 fore, one unacquainted with the dark field should not expect to 
 obtain immediate satisfactory results from the study of blood. 
 An intimate course of study is necessary to enable one to differenti- 
 ate between "filaments," artefacts, and organisms of the morpho- 
 logical nature of the spirochete described in the blood of cholera 
 hogs.' 
 
 The last statement quoted we now accept as a conclusion rather 
 than a suggestion. Results lead us to believe that the spirochete 
 observed in few numbers in the blood of cholera hogs is a transitional 
 form. 
 
 The spirochete, which is constantly observed in the ulcerated 
 mucosa and crypts in the ceca and in the external local lesions of 
 cholera-infected animals, is an actively motile, comparatively long, 
 spiral-shaped organism, of slight thickness. Measurements show 
 that it is approximately 6-10 microns in length, averaging about 
 
 ■ In this comection we were especially fortunate in the collaboration of our colleague, Dr. F. W. 
 Baeslack. Because of his experience with the dark field in his study of Spirochada pallidum (see Jour. 
 Infect. OK.,1913, 12, p. ss) and in his experimental observations of certain Trypanosomes, our dark 
 field study of hog cholera blood was placed to great advantage. It is a pleasure to acknowledge his 
 valuable assistance in that work, as well as the many helpful suggestions he has offered in our present 
 investigation. 
 
Spirochaeta Suis, a Pathogenic Organism 
 
 467 
 
 7 or 8 microns, and o , 3-0 . 6 micron in width. The convolutions vary 
 from three to ten in number, rarely more than eight, and the spirals 
 are fixed. It has been observed to divide longitudinally, and 
 morphological variations, such as described by Gleitsmann' as a 
 part of the life cycle of the Sudanspirochaetae, have been observed. 
 In our former report on the dark field study of the blood, 
 observations relative to the presence of granules were summarized 
 as follows: 
 
 " Certain types of granules appear to be characteristic of blood from cholera hogs. 
 It usually contains many granules, some very fine yet more distinct than blood dust, 
 some larger still, and some very dis- 
 tinct highly refractive bodies. In 
 many specimens of cholera blood were 
 observed innumerable small granules, 
 which were much more definite and 
 distinct in outline than blood dust, 
 and easily differentiated from the 
 whitish, partially refractive granules 
 from ruptured leukocytes and from 
 the more highly refractive and larger 
 bodies composed of debris, bacteria, 
 and filaments. In this work the pres- 
 ence of these granules appeared to be 
 so characteristic of blood from cholera 
 hogs that it became an invaluable aid 
 in finding the spirochetes. While 
 these particular bodies may be dis- 
 integrated blood elements resulting 
 from disease processes, yet it may 
 properly be suggested that some of them may represent certain stages in the life 
 cycle of the spirochete which has been observed." 
 
 As the result of continued investigations, we believe that the 
 granules represent an important factor in the Hfe history of Spi- 
 rochaeta suis. References to similar observations in the study of 
 various spirochetes are found in the writings of Balfour,^ Miihlens,' 
 Ross.'' McDonagh,5 Button,"* Mayer,' Mackinnon,^ Schellack,' 
 Bosanquet,'" Marchoux and Couvy," and others. 
 
 I Centralbl. f. Bakleriol., I, Orig., 1913, 68, p. 31. ^ Report IV, Wellcome Research Lab., p, 76. 
 
 3 Haiidbuch d. Path. Frotozoen, Prowazek, Leipzig, 1912, p. 361. 
 
 * Brit. Med. Jour., 1912, p. 1651. 
 
 s Lancet, Proc. Roy. Soc, TQ13, 6, p. 86. ^ Jour. Trop. Med., 1907, 10. p. 385. 
 
 ^ Arch.f. Schijjs.- u. Tropenhyg., 1908, 12, Beihefte i, p. i. 
 
 8 Parasitology, 1909, 2, p. 267. ' Arb. a. d. k. Gsndhlsamte, 1909, 30, p. 351. 
 
 ^0 Spirochaeies, Saunders, 1911. " Ann. de VInst Past., 1913, 27, p. 450. 
 
 Fig. I. — Photomicrograph of Spirochaeta suis in 
 exudate, ear lesion. Hog 626. X1200. (Darlt field 
 
 preparation.) 
 
468 Walter E. King and George L. Hoffmann 
 
 In cultures made from material containing Spirochaeta suis the 
 presence of small granular forms is quite constant. As these appear 
 in dark field preparations from cultures, they resemble those which 
 were observed in the blood, and are seen in the exudate from local 
 lesions, of cholera-infected hogs. They are relatively highly refrac- 
 tive, smaller than cocci, peculiarly distinct in appearance from 
 foreign material, and possess more than the characteristic Brownian 
 movement. The assumption that these granules form an integral 
 part of the biologic processes of the organism, Spirochaeta suis, is sub- 
 stantiated by the results of the detailed experiments which follow. 
 
 Recognizing the difficulties involved in attempting to study 
 Spirochaeta suis, and in determining its possible significance as a 
 pathogenic form, our efforts have been directed toward the culti- 
 vation of the organism. This work has included the inoculation 
 of artificial culture media with blood, intestinal ulcers, urine, con- 
 tents of local external lesions, and other tissues from cases of hog 
 cholera, followed by careful dark field examinations. 
 
 CULTIVATION OF SPIROCHAETA SUIS.^ 
 
 A great many plantings on a variety of artificial culture media 
 have been made from the blood, intestinal ulcers, and external 
 local lesions of cholera-infected animals. These cultures have 
 been made chiefly in deep horse serum media, to which have been 
 added various chemicals in different proportions. All have been 
 grown under strict anaerobic conditions in special electric incubators. 
 Various means were used in attempting to eliminate contaminating 
 organisms, which were always present in cultures made from the 
 ulcers and, to a lesser extent, in those made from material in local 
 lesions. 
 
 The culture medium which proved most successful consisted 
 of a mixture of horse serum and pyrogalHc acid, in which was placed 
 
 * As the proof of this paper is being read we note an article in ctirrent literature by Pfeiler and Lentz 
 IBerl. Uerarztl. Wchnschr., 1913, 29, p. 690), who present the case histories of 15 pigs which died after 
 having received injections of cultures made from the Berkefeld filtrates of serum and tissue exudates from 
 hog cholera infected animals. These cultures represented transfers in the first, second, third, and fourth 
 generations. The authors carefully avoid any statements which might indicate the nature of the cul- 
 tures. No mention is made of the character of the organism m their cultures, the composition of their 
 culture medium, or how long or under what conditions the cultures were incubated. On account of the 
 conspicuous lack of such important data one can form no conclusion as to their mode of procedure and 
 hence, the results which they have published must be regarded with some skepticism. 
 
Spirochaeta Suis, a Pathogenic Organism 469 
 
 fresh rabbit kidney at the time of inoculation. The pyrogalHc 
 medium of Proca, Danila, and Stroe' was made up in lots of one 
 liter each by adding to 900 c.c. of horse serum 100 c.c. of pyrogalHc 
 acid mixture. The pyrogalUc solution should be somewhat aged. 
 A stock solution was therefore kept, which consisted of pyrogalHc 
 acid, 10 gm.; sodium hydroxid, 20 gm., and water, 1,000 c.c. After 
 the addition of this mixture to horse serum and distribution in 
 tubes the medium was heated one hour at 60° C. on each of three 
 consecutive days. 
 
 While this pyrogalHc servun medium does not eUminate all 
 bacterial contamination, at the same time aUowing the spirochete 
 to grow, it was found to give better results than media containing 
 formol, gentian violet, phenol, or other inhibitory substances. 
 For uncontaminated material, such as Berkefeld filtrates of impure 
 cultures of spirochetes, plain or ascitic horse serum media were 
 utiHzed. Fresh rabbit kidney was added to all media used and care 
 was exercised that the horse serum was only partially coagulated 
 and soft in consistency when ready for use. 
 
 The cultures have been examined at different intervals of time, 
 following inoculation and incubation, in order to detect any facts 
 which might come to Hght concerning the Ufe history, morphology, 
 and physiological habits of the organism. The method of examin- 
 ing cultures consists simply in withdrawing, with a sterile capil- 
 lary pipette, a small portion of culture from the lower layers of the 
 medium and examining the same in microscopical mount by means 
 of the dark field. The Giemsa method of stairung is employed 
 whenever permanent mounts are desired. After some practice one 
 can readily detect, by the dark field, spirochetes and granules in 
 fresh cultures in distinction, from cocci, other bacterial contami- 
 nation, crystals, and small particles of culture medium. 
 
 NOTES ON TRANSMISSION OF HOG CHOLERA BY MEANS OF CULTURES 
 OF SPIROCHAETA SUIS. 
 
 I. Original Culture. 
 Culture ssi-A was secured from the ulcerated cecum of Hog 551. The account 
 of this case may be found in the chart for Hog SSI-^ 
 
 » Compt. rend. Soc. de hiol., igi2, 72, p. 895. 
 
 " Positive and negative signs indicate the presence or absence of Spirochaeta suis in the blood of the 
 animal on dates indicated. 
 
47° 
 
 Walter E. King and George L. Hoffmann 
 
 Cultures were made from the ulcerated cecum, spleen, and lymphatic glands of 
 Hog 551. Those from the spleen and lymphatic glands developed no growth, while 
 some of those from the cecum, after five days' incubation under anaerobic conditions, 
 showed the growth of spirochetes which varied in form and size. Fig. 3, p. 212, in our 
 former publication' shows a photomicrograph of a stained preparation made from this 
 culture. When this culture was studied, our attention was directed especially' to the 
 larger forms of spirochetes which were found in it. This was due to the fact that we 
 were familiar with the form which had been observed in the blood of cases of hog 
 cholera. At this time no special attention was gi\'en to smaller forms representing 
 more typical spirochetes which were present in this culture, as was shown, some weeks 
 after the study of the culture, by careful re-examinations of the permanent stained 
 preparations. 
 
 Fig. 2. — Ulcers in cecum of Hog 569. 
 
 Transfers were made from Culture ssi-A and grown both aerobically and 
 anaerobically. The species of bacteria which contaminated Culture 5S1-A were very 
 carefully isolated and identified as follows: B. pyocyaneiis, three strains; B. coH com- 
 munis, five strains. These cultures, which contaminated SSi-A, representing B. coli 
 communis and B. pyocyaneus, were mixed and suspended in physiologic salt solution. 
 Six cubic centimeters of this suspension on March 14 were injected intravenously into 
 Hog 570. This animal showed no disposition to develop hog cholera, but did react 
 to the injection of live B. pyocyaueus and B. coli communis, as the chnical chart for 
 Hog 570 will show. 
 
 Culture S51-A was suspended in sterile physiologic salt solution and an intra- 
 venous injection of 10 c.c. given to Hog 559. The results of this inoculation are re- 
 corded in the chart for Hog 559. 
 
 * Jour. Inject. Dis., 1913, I3, p. 206. 
 
Spirochaeta Suis, a Pathogenic Organism 
 
 471 
 
 Hog SSI. 
 
 , ,««. 
 
 1913. JANUARY. TRunir.nY 1 
 
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 10 
 
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 21 
 
 1; 
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 13 
 
 14 
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 liieli? 
 
 2526|27 
 
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 January 10. Received from Rochester, Mich. 
 
 January 16. Intramuscular injection 8 c.c. of virus, Berkefeld filtered, 1:4 saline. Ohio (Pettigrew) 
 
 strain. 
 January 31. Animal very weak. 
 February 5. Animal moribund, bled and examined. 
 
 Typical lesions of cholera: lungs, large areas of hepatization; kidney, petechiated, capsule non- 
 adherent; lymphatic and inguinal glands, greatly enlarged and hemorrhagic; spleen, friable and engorged 
 with blood, normal in size; cecum, large area of ulcers. 
 
 Hog 570. 
 
 '--'—<' 1913. MAP.C 
 
 H. 
 
 irnji. .,.,,. 
 
 
 
 14 15 16 17 18 
 
 19 20 21 22 23 24 25 
 
 26 27 26 29 30 3 
 
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 March 14. Intravenous injection of 6 c.c. physiologic salt suspension cultures from cecum of Hog SS9- 
 
 " ~ Slightly indisposed, appetite not good, otherwise condition practically normal. 
 
 March 
 
 18- 
 
 2S 
 
 March 
 
 .31- 
 
 
 April 
 
 2. 
 
 
 April 
 
 9. 
 
 
 April 
 
 30. 
 
 
 Apparently in good condition. Released. 
 
 A little "off feed." 
 
 Fully recovered, released and put on other work April 14. 
 
 Animal found dead, result of intramuscular injection of 5 c.c. II Cycle virus. 
 
 lesions of hog cholera. 
 
 Typical 
 
472 
 
 Walter E. King and George L. Hoffmann 
 
 Hog 559 was in a perfectly healthy condition at the time of inoculation, February 
 10, and weighed approximately 75 pounds. Four days after inoculation, the tempera- 
 ture of the animal began to rise and on the fifth daj' symptoms of hog cholera appeared. 
 The animal was slightly "off feed" and inclined to be inactive. Darlc field examination 
 of the blood at this time showed the presence of a few spirochetes and characteristic 
 granules. After 48 hours the temperature dropped somewhat and for several days 
 ranged between 103° and 104°. About 12 days after the inoculation the animal 
 appeared to be brighter and seemed to be rapidly improving, but 15 days after inocula- 
 tion a remission occurred, the temperature was elevated 2° during one afternoon, and 
 
 Fig. 3.— Hog 559. 
 
 from this time the animal gradually grew worse until death, which occurred 22 days 
 after inoculation. The later symptoms consisted of weakness, inability to use hind 
 quarters, bloody urine, mucous diarrhea, anorexia, and general malaise. On March 4 
 the animal was moribund and, after being photographed, was bled from the carotid 
 artery and examined (see photograph of Hog 559). On autopsy typical lesions of hog 
 cholera were found. 
 
 Blood extravasations were present in the subcutaneous tissue, especially in the 
 axillary region and lower abdominal region. Both the auricles and ventricles of the 
 heart showed ecchymosis. The lungs contained numerous hemorrhagic infarcts and 
 a few areas of hepatization. The liver appeared to be somewhat enlarged and con- 
 tained areas of fatty degeneration. The spleen was shghtly atrophied, tissue firm, but 
 
Spirochaeta Suis, a Pathogenic Organism 
 
 473 
 
 engorged with blood. Both kidneys were full of petechiae. The mucosa of the cecum 
 and the large intestine was congested and marked with hemorrhagic spots throughout. 
 Two typical button ulcers were found in the cecum. In the small intestine there were 
 present two enlarged areas, consisting of necrotic intestinal wall surrounded by exten- 
 sive hemorrhages. The peritoneum in the lower abdominal region showed hemor- 
 rhagic areas. The mesenteric, inguinal, and intraperitoneal lymph glands were 
 enlarged and hemorrhagic (see Plate 4). 
 
 A specimen of blood serum from Hog 559 was filtered through Berkefeld, after 
 being diluted 1:4 with physiologic salt solution, and 10 c.c. of the filtrate injected 
 intramuscularly into Hog 569. This material proved to be virulent, as Hog 569 showed 
 severe symptoms of hog cholera five days after inoculation and became moribund 
 on the eleventh day. The autopsy showed classical lesions of hog cholera (see chart 
 for Hog 569). 
 
 Hog 559. 
 
 Hog 569. 
 
 
 1913. FEBBUAHY. MARCH. i 
 
 .. 
 
 
 10 
 
 1 
 11 
 
 2 
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 13 
 
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 February lo. Intravenous injection lo c.c. suspension 
 physiological salt solution of Culture SSi-A. 
 
 February 15. Clinical symptoms present, appetite poor. 
 
 February 23. Animal improved in condition. 
 
 February 25. Remission. 
 
 March 4. Animal moribund, and bled from carotid 
 artery. Tj^iical lesions: heart, lungs, kidney, 
 cecum, and lymphatic glands. 
 
 ,. — 
 
 1913. MARCH. 
 
 ■• 
 
 
 IIS 4l5|«l7|aL9|lO|ll 
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 March 7. 
 March 7, 
 
 March 12. 
 March 18. 
 
 Blood negative, free from granules. 
 Intramuscular injection of 10 c.c. 
 Virus 559, diluted 1:4, physiological 
 salt and Berkefeld-filtered strain 
 (Culture 551-A). 
 Symptoms appeared. 
 Animal moribund and bled from |ca- 
 rotid artery. Lesions typical: heart, 
 lungs, spleen, kidney, inguinal glands, 
 and mesenteric lymph glands; ulcers 
 in cecum. 
 
 With reference to infection from other material than that which was used in the 
 inoculations, it should be stated that resuhs from Hog 559, as well as all others used in 
 this series of experiments, were carefully controlled by the presence of normal hogs in 
 the experimental stable. The animals used in each series of experiments^ as well as 
 the normal animals, were kept in separate rooms. The normal animals were placed 
 under exactly the same conditions and had the same care that was exercised in regard 
 to the treated animals. 
 
 An additional control on this work depended upon the use of Hog 570, which was 
 inoculated with the contamination present in Culture ssi-A. Furthermore, it has 
 been observed that the serum of Hog 559, filtered through Berkefeld, was capable 
 of producing typical hog cholera in Hog 569. 
 
474 
 
 Walter E. King and George L. Hoffmann 
 
 O 'U 
 
 no 
 
Spirochaeta Suis, a Pathogenic Organism 475 
 
 Among the criticisms which may be offered on this experiment, 
 the most important relates to the possibiUty of transferring the 
 original virus from Hog 551 through Culture SSi-A. This point 
 could not be controlled, altho an estimate was made that Hog 559 
 received less than 1/750 c.c. of material originally obtained from 
 the ulcer of Hog 551. It has been estimated by King and Wilson' 
 that the minimum fatal dose of hog cholera virus, diluted in physio- 
 logic salt solution, is approximately 1/300 c.c. The above approxi- 
 mation that Hog 559 received about 1/750 c.c. was obtained on the 
 basis of the utilization of about 1/15 gm. of ulcer from Hog 551 in 
 approximately 10 c.c, of culture media. However, these estimates, 
 including both the suggested minimum fatal dose of hog cholera 
 virus in physiologic salt solution and the amount of original ma- 
 terial transferred through Culture 551 -A to Hog 559, are open to 
 serious question in attempts to estimate whether or not any of the 
 original virus from Hog 551 was transferred to Hog 559. 
 
 II. Transfer i. Second Generation on Artificial Media. 
 
 This culture was secured from the strain of virus from Hog 577. This animal 
 received an intramuscular injection of 8 c.c. horse serum virus, diluted i :4 in physio- 
 logic salt solution and Berkefeld filtered. The animal developed typical symptoms 
 of hog cholera and after nine days, being moribund, was bled from the carotid 
 artery and examined. 
 
 Hog 587 on April 11 received an intramuscular injection of 5 c.c. of serum from 
 Hog 577. This animal promptly developed hog cholera of the acute type after five 
 days' incubation and was bled and examined eight days after inoculation. The 
 autopsy showed typical lesions of hog cholera of the acute type. Numerous blood 
 extravasations were present in the subcutaneous tissue, especially in the axillary region. 
 Both lungs were almost completely consohdated. The kidneys showed areas of cloudy 
 swelling and petechiae near the upper ends. The spleen was engorged with blood. 
 The mesenteric and inguinal glands were enlarged and hemorrhagic. The cecum con- 
 tained a few small ulcers around the valve (see chart for Hog 587). 
 
 On April 19 cultures were made from the material obtained from the small ulcers 
 in the cecum of Hog 587. These were incubated under anaerobic conditions for a 
 period of 17 days and on May s dark field examination showed the presence of Spiro- 
 chaeta suis. A variety of contaminating organisms was also present, both cocci 
 and baciUi being represented in the contamination. The culture medium in the tube 
 labeled "S87-A" showed no liquefaction from the growth of the contaminating bac- 
 teria, and from it a number of transfers were made on May 5. On May 27, after incu- 
 bation for 22 days, transfer labeled "Transfer 1-S87-A," when submitted to dark field 
 
 * Bull. 171, Kansas State Agric, Coll., p. 163. 
 
476 
 
 Walter E. King and George L. Hoffmann 
 
 Hog 577. 
 
 Hog 587. 
 
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 March 27. Intramuscular injection 8 c.c. acti- 
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 filtered. 
 
 April I. Symptoms appeared. 
 
 April 3. Animal very weak in hind quarters. 
 
 April 5. Animal moribund, bled and examined- 
 Typical lesions: heart, lungs, kidney, lymph 
 
 glands, cecum; small intestine infected with Ascari- 
 
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 Virus 577 I Cycle activated horse serum. 
 April i6. Symptoms of cholera appeared. 
 April 19. Animal moribund, bled from carotid 
 artery and examined. 
 Lesions: lungs, spleen, cecum, subcutaneous 
 tissue, and inguinal glands. 
 
 examination, showed a few Spirochaetae suis and numerous granules. All of the con- 
 taminating baciUi which were present in the original culture, 587-A, had disappeared 
 and the only contamination present in the transfer was a micrococcus. A portion of 
 this culture, Transfer 1-A-587-A, was broken up in sterile water and i . 5 c.c. of the 
 suspension of this culture were inoculated, intramuscularly, into each of three hogs, 
 Nos. 612, 613, and 614. Hogs 612 and 614, after eight days 'incubation, developed 
 symptoms of hog cholera. After a few days the disease assumed the chronic form. 
 
 Hog 612. 
 
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 1913. 3mm. 
 
 JULY. 1913, 
 
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 June 2. Intramuscular injection 1.5 c.c. suspension Culture Transfer 1-587. 
 
 June 10. Symptoms appeared. 
 
 June ID- July i. Chronic case of cholera. 
 
 July 4. Condition improved. 
 
 July 15. Animal had been fighting with strange hogs. 
 
 July 22. Released as healthy immune. 
 
Spirochaeta Suis, a Pathogenic Organism 
 
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 June 3. Intramuscular injection of i-S c.c. suspension Culture Transfer 1-587. 
 June 10. Symptoms appeared. 
 June i6-July 2. Subacute type of cholera. 
 July 2. Animal moribund, killed and examined. 
 Typical lesions. 
 
 The animals lost their normal appetite and, during a period of about three weeks fol- 
 lowing the initial symptoms, became somewhat emaciated. On the fifth week after 
 the inoculation of the animals, rapid convalescence took place and they fully regained 
 normal condition. Both animals had been confined with cholera hogs, but foEowing 
 the experiment they remained healthy and were released as active immunes. 
 
 Hog 613 developed a more acute form of the disease than Hogs 612 and 614. After 
 an incubation period of eight days, the temperature rapidly rose and maintained an 
 elevation of approximately io6°-io7° for a period of two weeks. During this time Hog 
 613 manifested the usual symptoms of hog cholera, consisting of anorexia, general 
 weakness and malaise, paralysis of hind quarters, emaciation, and constipation. On 
 July 2, 2g days after inoculation, the animal was killed; on examination, characteristic 
 
 Hog 614. 
 
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 June 4. Intramuscular injection i . 5 c.c. suspension Culture Transfer 1-578. 
 
 June 12. Animal "off feed." 
 
 June 15-30. Symptoms of chronic type of cholera. 
 
 July 4. General improvement noted. 
 
 July 15. Animal had been fighting, placed with strange hogs. 
 
 July 22. Released as healthy immune. 
 
Spirochaeta Suis, a Pathogenic Organism 479 
 
 Hog 620. 
 
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 June 30. Intramuscular injection of lo c.c. of blood of Hog 612, diluted 1:4, and Berkefeld filtered. 
 
 July 4-5. High temperature — ^may have been due to excessively hot weather. 
 
 July 8. Symptoms of cholera. 
 
 July 10-20. Gradual emaciation, constipation, anorexia, and malaise. 
 
 July 21. Gradual return of appetite and slow convalescence. 
 
 July 29. Apparently recovered. 
 
 July 30. Animal released as normal. 
 
 lesions of hog cholera were found. The heart was normal, the lungs were congested 
 and contained areas of hepatization. The spleen was enlarged, soft, and friable, and 
 engorged with blood. The kidneys were petechiated. The mesenteric and lymphatic 
 glands were enlarged and hemorrhagic, and the congested mucosa of the cecum con- 
 tained small tdcerated patches near the ileocecal valve. Spirochaeta suis was present 
 in the ulcers. 
 
 This experiment was controlled in the manner explained in the description of the 
 inoculation of Hog 559 with original culture. Normal hogs were kept in the stable 
 under the same conditions as were these animals. Hogs 612 and 614, which survived 
 
 Hog 617. 
 
 1913. JVHE. 
 
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 June 21. Intramuscular injection of 12 c.c. of blood of Hog 613, diluted 1:4, and Berkefeld filtered. 
 
 June 27. Symptoms appeared. 
 
 July i-is- Animal very weak, emaciated, constipated, anorexia, and malaise. 
 
 July 20. Some improvement noted. 
 
48o 
 
 Walter E. King and George L. Hoffmann 
 
 the experiment, possessed active immunity, as was demonstrated by the fact that 
 they were placed with hogs sick with cholera two weeks before being released. 
 
 During the period of infection, a sample of blood was obtained from the tail of 
 each of Hogs 612, 613, and 614, used in the experiment above in testing the patho- 
 genicity of Transfer 1-587-A. These samples were defibrinated, diluted 1:4 in sterile 
 water, and passed through the Berkefeld filter. Ten cubic centimeters of the diluted, 
 Berkefeld-filtered blood from Hogs 612, 613, and 614 were injected intramuscularly into 
 Hogs 620, 617, and 621, respectively. 
 
 Hog 620, which received the Berkefeld-filtered blood serum from Hog 612, 
 developed symptoms of cholera eight days after inoculation. This case assumed the 
 chronic type extending over two weeks' time after which the animal gradually became 
 convalescent. 
 
 Hog 617, which received the Berkefeld-filtered blood from Hog 613 (the animal 
 which developed the most acute form of the disease of the three cases described 
 
 Hog 621. 
 
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 June 30. Intramuscular injection of 10 c.c. of blood of Hog 614, diluted 1:4, and Berkefeld filtered. 
 
 July 6-9. Animal not normal, appetite poor, general malaise. 
 
 July 10. Condition improved. 
 
 July 14. Condition normal. 
 
 July 25. Released as healthy immune. 
 
 above), showed unmistakable signs of hog cholera about six days after inoculation. 
 The course of the disease in this animal was of the chronic type and death occurred 
 two months after inoculation. 
 
 Hog 621, inoculated with the Berkefeld-filtered serum of Hog 614, showed mild 
 symptoms of cholera eight days after inoculation. After being slightly "off feed" for 
 two or three days the animal regained its normal condition. 
 
 After the conclusion of this experiment. Hogs 620 and 621 were confined for several 
 weeks in an infected inclosure with cholera pigs. No evidences of disease developed, 
 thus establishing the fact that the animals possessed active immunity. 
 
 On July I one of the transfers, which was made from Strain 587 and which had 
 been kept in the incubator since the time of inoculation, May 5, was submitted to dark 
 field examination. It was found that the culture contained very few organisms which 
 could be recognized as spirochetes, but many granules were present and the smaU 
 amount of contamination consisted of a micrococcus. This culture, which had been 
 incubated for a period of 57 days, was designated "Transfer 1-587-3," and two hogs, 
 
Spirochaeta SuiSj a Pathogenic Organism 481 
 
 Hog 622. 
 
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 July 2. Intramuscular injection of loc.c. unfiltered suspension of positive culture No. 587 (May 5, 
 
 1913)— (July 2, 1913). 
 July 7-8. Appetite poor, general malaise, feces normal. 
 July 9. Apparently normal. 
 July 22. Exposed to hog cholera; placed with sick animals and injected intramuscularly with 2 c.c. 
 
 of virus from Hog 631. 
 July 29-August 2. Symptoms of paralysis and chorea. Appetite fairly good. Animal not emaciated. 
 August 3. Animal found dead and examined. 
 
 Typical lesions of hog cholera absent; intestinal mucosa normal. Culture made from edematous 
 fluid of muscular tissue shows Str. pyogenes present. 
 
 Hog 623. 
 
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 July 2. Intramuscular injection of 10 c.c. unfiltered suspension of positive culture No. 587 (May S, 
 
 1913)— (July 2, 1913)- 
 July 7-8. Appetite poor, general malaise, feces normal. 
 July 9. Apparently normal. 
 July 22. Exposed to hog cholera; placed with sick animals and injected intramuscularly with 2 c.c. of 
 
 virus from Hog 631. 
 July 29. Animal showed symptoms of paralysis and chorea. Could not walk. 
 July 30. Found dead and examined. 
 
 Mucous membrane of small and large intestine congested. No ulcers present in cecum. 
 Typical lesions of cholera absent. 
 
482 Walter E. King and George L. Hoffmann 
 
 Nos. 622 and 623, were each injected intramuscularly with 10 c.c. of the suspension 
 of this culture in sterile water. Six days after the inoculation of these animals, they 
 showed, in addition to slight temperature changes, mild symptoms of illness. The 
 appetite was poor and the animals were inclined to be listless and inactive. After 
 about two days of inactivity and loss of appetite, they again resumed a normal con- 
 dition and continued so imtil exposed to cholera. 
 
 On July 22 these animals were exposed to the disease by inoculation, natural ex- 
 posure, and access to carcass of hog dead from cholera. On July 28 both hogs, Nos. 
 622 and 623, developed symptoms of paralysis and chorea. On July 29 Hog 623 
 was found dead and autopsy did not show lesions of hog cholera. Dark field examina- 
 tion of the mucosa of the cecum failed to show any spirochetes. Hog 622, on July 30, 
 showed a moderate rise in temperature, but did not lose appetite or manifest other 
 symptoms of cholera. The right front leg became enormously swollen. On August 3 
 Hog 622 was found dead. No characteristic lesions of hog cholera were present and 
 cultures made from the serous fluid in the swollen leg developed Sir. pyogenes. Several 
 preparations were made from the mucosa of the cecum and were submitted to dark 
 field examination with negative results. 
 
 On account of the absence of tj^ical symptoms and lesions of hog cholera, also 
 the course of the disease in the control animal (see notes below on Hog 641) and the 
 presence of Str. pyogenes in the edematous tissue of Hog 622, it seemed probable that 
 Hogs 622 and 623 died from infection other than hog cholera. 
 
 in. Transfer 2. Third Generation on Artificial Media. 
 
 On May 27 a number of transfers were made from Culture Transfer 1-S87-A. 
 These cultures were incubated under anaerobic conditions from May 27 to July 2, 
 when they were removed from the incubator and examined by the dark field method. 
 One of these transfers, designated "Transfer 2-587-A," contained very Uttle contamina- 
 tion represented by one species of micrococcus and, while only a few spirochetes could 
 be found scattered through the medium, the culture contained many granules. This 
 culture. Transfer 2-S87-A, was macerated in sterile water, and on July 3, Hogs 627 
 and 628 were each inoculated, intramuscularly, with 10 c.c. of the suspension of this 
 transfer. Five days after inoculation both of these animals showed indications of 
 indisposition. From July 8 to July 12, the appetite was poor and the animals were 
 inactive. On July 14 the condition of both hogs was much improved and on July 16 
 both were pronoimced apparently healthy. 
 
 On July 22 these animals were exposed to hog cholera in the same manner as 
 Hogs 622 and 623, described above. After seven days' incubation both animals 
 developed symptoms of cholera. Hog 627 succumbed to the disease 22 days after 
 exposure. Hog 628, after suffering from the disease in chronic form, gradually became 
 convalescent and on September i was released. Both animals showed greater resistance 
 and maintained a better clinical condition than did the control Hog 641. 
 
 On July II, during the period in which Hog 628 was showing a general reaction 
 from the injection of Transfer 2-S87-A, a few cubic centimeters of blood were drawn 
 from the tail, defibrinated, diluted i :4 in sterile water, and filtered through a bacteria- 
 proof Berkefeld. On July 15 Hog 635 received intramuscularly 5 c.c. of the Berkefeld- 
 filtered serum, diluted i : 4, of Hog 628. This animal showed mild symptoms five days 
 after inoculation and soon developed hog cholera of the subacute type. Four weeks 
 after inoculation Hog 635 was found dead. Autopsy revealed typical lesions. 
 
Spirochaeta S.uis, a Pathogenic Organism 
 
 483 
 
 J 
 
484 
 
 Walter E. King and George L. Hoffmann 
 
 On July 24 Hogs 642 and 643 each received intramuscularly 5 c.c. of suspension 
 in physiologic salt solution of Culture Transfer 2-587-8. Eight days after inoculation 
 both animals manifested symptoms of mild cholera. They continued to show inac- 
 tivity, anorexia, arched back, mUd blepharitis, and roughened coat for a period of 
 five days, after which they apparently regained a normal condition, which was followed 
 by a recurrence in the case of both Hogs 642 and 643, in seven and twelve days, respec- 
 tively. Both cases terminated fatally. 
 
 Hog 627. 
 
 .,.__ 
 
 1913. JULY, 
 
 AUGUST. 
 
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 July 3. Intramuscular injection of ro c.c. unfiltered suspension culture No. 587 (May 27, 1913)— 
 
 (July 3. 1913)- 
 
 Poor appetite, inactive, eyes dull. 
 
 Nervous symptoms. 
 
 Condition improved. 
 
 Animal apparently normal. 
 
 Intramuscular injection 2 c.c. Virus Strain 631. 
 
 Had been fighting with strange hogs. 
 
 Animal suffering from a relapse, increasing emaciation; symptoms of acute type of cholera. 
 August 13. Animal dead. 
 
 Typical lesions of hog cholera, subcutaneous tissue, lungs, kidney, lymphatic glands, cecum. 
 
 Julys 
 
 -12. 
 
 July 
 
 12. 
 
 July 
 
 14- 
 
 July 
 
 16. 
 
 July 
 
 22. 
 
 July 
 
 23- 
 
 July 
 
 29. 
 
 IV. Filtered Cultuees. 
 
 a) Transfer i. — Transfer 1-587-B, described above, a suspension of which was 
 inoculated into Hogs 622 and 623, was also utUized in filtration experiments, which 
 were first suggested by our colleague. Dr. N. S. Ferry. This culture, which had been 
 incubated for a period of 57 days, after being macerated in sterile water in the pro- 
 portion of one part culture to ten parts water, and thoroughly shaken for about one 
 hour, was passed through a Berkefeld filter. The filter used was a bacteria-proof 
 Berkefeld filter, pressure less than one atmosphere, time of filtration about 20 minutes, 
 and volume of filtrate about 15 c.c. Cultures from this filtrate were made, both 
 aerobicaUy and anaerobically, and no bacterial growth resulted. Dark field examina- 
 tion of the filtrate revealed only relatively few granules. Hogs 624 and 625, on J uly 3, 
 each received intramuscularly 10 c.c. of filtered culture, Transfer 1-587-B, diluted 
 1:10 in sterile water. Five days after inoculation both of these animals gave evi- 
 dences of iUness. From July 8 to July 10, the appetite was poor, eyes dull, and animals 
 listless. On July 11 the condition of both hogs was improved and on July 16 they 
 were apparently healthy. 
 
Spirochaeta Suis, a Pathogenic Organism 
 
 485 
 
 These hogs, Nos. 624 and 625, were exposed with control Hog 641 (see p. 482), 
 by inoculation and by natural exposure. Three days after exposure Hog 624 showed 
 evidences of illness, apparently due to gastritis and autointoxication, resulting from 
 the ingestion of pus from an infected carcass. After a few days this animal fuUy 
 recovered and exhibited a thrifty condition throughout the experiment, thus proving 
 the existence of active immunity. Hog 625 showed scarcely any reaction as the 
 
 Hog 628. 
 
 ..^.u 1913. JULY. 
 
 AUGUST . 1 
 
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 — duly 3, 1913). 
 
 Inactive, anorexia. 
 
 Animal apparently normal. 
 
 Intramuscular injection 2 c.c. Virus Strain 631. 
 July 30-August s. Symptoms of chronic type of cholera. 
 August 6. Emaciated and growing progressively weaker. 
 September i. Animal normal, recovered and released. 
 
 July 
 July 
 July 
 
 8-13. 
 
 IS. 
 
 result of the exposure, and was considered as being actively immunized by the filtered 
 Culture Transfer 1-587-B. 
 
 On July 9, during the reaction following inoculation with filtered culture, a 
 specimen of blood taken from the tail of Hog 625 was defibrinated, diluted i : 4 in sterile 
 water, and filtered through the Berkefeld filter. On July 15, Hog 634 received intra- 
 muscularly 5 c.c. of the diluted, filtered blood from Hog 625. Five days after inocu- 
 lation of Hog 634 the temperature rose two degrees, the animal was "off feed" and 
 
486 Walter E. King and George L. Hoffmann 
 
 Hog 641. Hog 642. 
 
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 July 24. Intramuscular injection of 2 c.c. virus; 
 July 29. Typical symptouK of hog cholera. 
 August 5. Animal died. 
 
 Lesions: lungs, areas of hepatization; spleen, 
 enlarged, soft and friable, engorged with blood; kid- 
 neys, petechiated; lymphatic and inguinal glands, 
 enlarged and hemorrhagic; cecum, mucosa con- 
 gested, small ulcers near ileocecal valve. Spiro- 
 chaeta suis present. 
 
 »-- » 
 
 JULX AUGUST 1913, | 
 
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 filtered. 
 
 Symptoms of anorexia; malaise, slight blepharitis. 
 Animal normal. 
 
 Temperature normal, tho the appearance was not normal. 
 Indication of relapse; typical symptoms of cholera. 
 Animal found dead. 
 Typical lesions of hog cholera; subcutaneous tissue, heart, limgs, 
 spleen, kidney, lymphatic system, cecum; Spirochaeta suis present. 
 
 July 24. 
 
 August I. 
 August 5- 
 August ID. 
 August 14. 
 August 18. 
 
 Hog 635. 
 
 ;.^BM> 1513 ju I. AUGUST. 
 
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 July 15. Intramuscular injection of 5 c.c. blood serum of Hog 628, diluted 1:4, and Berkefeld filtered* 
 
 July 20-22. Animal "off feed," dull, inactive. 
 
 July 24. Condition improved; appetite poor. 
 
 July 29-August 5. Symptoms of subacute type of cholera. 
 
 August 6. Nearly moribund. 
 
 August II. Animal died. 
 
 Typical lesions of hog cholera: lungs, heart, spleen, kidney, cecum, and lymph glands. 
 
Spirochaeta Suis, a Pathogenic Organism 
 
 487 
 
488 
 
 Walter E. King and George L. Hoffmann 
 
 Hog 643. 
 
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 July 24. Intramuscular injection 5 c.c. physiologic salt suspension Culture 587 (May 27, 1913)— 
 
 (July 20, 1913), Berkefeld filtered. 
 August I. Symptoms of anorexia, malaise, slight blepharitis. 
 
 August 5. Animal normal. 
 August 10-17. Animal apparently normal. 
 August 23. Typical case of hog cholera. 
 August 26. Animal found dead. 
 
 Typical lesions of hog cholera: heart, lungs, spleen, kidney, lymphatic glands, subcutaneous tissue, 
 and cecum. 
 
 became constipated. This animal developed cholera of the subacute type and was 
 found dead about four weeks after inoculation. 
 
 6) Transfer 2. — Culture Transfer 2-587-A, described above, on July 2 was filtered 
 and treated in exactly the same manner as described concerning the Berkefeld filtra- 
 tion of Culture Transfer 1-587-B. Transfer 2-587-A had been grown in the incubator 
 for 36 days. On July 3 Hogs 629 and 630 were each injected intramuscularly with 
 
 Hog 624. 
 
 July 
 
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 Anorexia, dull, listless. 
 
 Condition improved. 
 
 Animal apparently normal. 
 
 Intramuscular injection 2 c.c. Virus Strain 631. 
 
 Animal had been fighting with strange hogs. 
 
 Animal normal. Released. 
 
 -(July 2, igi3)» diluted i:io, 
 
Spirochaeta Sxjis, a Pathogenic Organism 
 
 Hog 62s. 
 
 489 
 
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 1913. JUIY. AUGUST, 1 
 
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 July 3. Intramuscular injection of lo c.c. Culture 587 (May 5, 
 
 and Berkefeld filtered. 
 July 8-9. Poor appetite, eyes dull, inactive. 
 July 14. Animal apparently normal. 
 July 22. Intramuscular injection 2 c.c. Virus Strain 631. 
 August 4. Animal released. 
 
 C9i3)~(july 2, igi3), diluted 1:10, 
 
 10 C.C. of Culture Transfer 2-587-A, diluted i : 10, and filtered through bacteria-proof 
 Berkefeld. About eight days after inoculation, both animals showed unmistakable 
 symptoms of hog cholera. Hog 630, however, exhibited a milder case than Hog 62g. 
 Eleven days after inoculation Hog 630 quickly regained normal condition, rise in 
 temperature subsided by crisis, and on July 17 the animal was pronounced normal. 
 Subsequent exposure with control Hog 641 (see p. 482) demonstrated a condition of 
 active immunity. Hog 629 developed a subacute case of hog cholera. The course 
 of this disease lasted for weeks. This animal manifested typical symptoms pi the 
 disease and after July 1 2 the more important symptoms noted were anorexia, malaise, 
 
 Hog 634. 
 
 5|6 7|el9 |16|11|12|13 14|lbH6[17lie|19|E0 21|21]Eate4(2S] 2^27 28 !9 
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 July 15. Intramuscular injection of 5 c.c. blood serum of Hog 625, diluted 1:4, and Berkefeld filtered. 
 
 July 20-21. Inactive, poor appetite, somewhat constipated. 
 
 July 24. Apparently normal. 
 
 July 27-August s- Symptoms of subacute type of cholera. 
 
 August 6. Nearly moribund. 
 
 August 12. Animal found dead. 
 
 Typical lesions of hog cholera in subcutaneous tissue; heart, lungs, kidney, spleen, cecum, and 
 lymph glands. 
 
490 Walter E. King and George L. Hofemann 
 
 Hog 629. 
 
 .—- » 
 
 1913. JUtY. AUGUST. | 
 
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 Berkefeld filtered. 
 July g. Appetite poor. 
 July 10-20. Symptoms of subacute type of cholera: anorexia, blepharitis, malaise, roughened coat, 
 
 emaciation, and profuse diarrhea. 
 July 24. Improvement noted. 
 July 28. Relapse. 
 August 6. Nearly moribund. 
 August 7. Animal found dead. 
 
 Lesions: heart, ecchymosis on auricles; 
 friable; hemorrhagic; kidney, few petechiae; 
 
 lymph glands, enlarged, hemorrhagic; adhesions of ascending colon to peritoneum 
 subcutaneous tissue. Spirockaeta suis present. 
 
 lungs, complete consolidation; spleen, enlarged, soft, 
 cecum, ulcers near ileocecal valve; inguinal and other 
 
 extravasation in 
 
 Hog 630. 
 
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 July 8. Poor appetite. 
 
 July 9-14, General inactivity, poor appetite, no apparent emaciation or loss of general tone. 
 
 July 15. Appetite good. 
 
 July 17. Apparently normal. 
 
 July 31. Animal normal. Released. 
 
Spirochaeta Suis, a Pathogenic Organism 
 
 491 
 
 blepharitis, emaciation, and roughened coat. Hog 629 was found dead on August 7, 
 3S days after inoculation. 
 
 As a control on this experiment. Hogs 629 and 630, on July 11, were transferred 
 from the room which they had occupied in the experimental stable to another, and, after 
 disinfection of the room, normal Hogs 632 and 633 were immediately placed in the quar- 
 ters formerly occupied by sick Hogs 629 and 630. An examination of the dates on the 
 clinical charts wiU show that this rearrangement was made during the height of the 
 disease in Hogs 629 and 630. Hogs 632 and 633 were subjected to exactly the same 
 conditions as those attending the care and treatment of Hogs 629 and 630 except, of 
 course, that they received no inoculation with infectious material. These control 
 animals remained perfectly normal in every way and after being kept as controls for 
 some time were utilized in experiments. 
 
 Hogs 636 and 637 on July 15 each received an intramuscular injection of s c.c. of 
 the defibrinated, diluted, and Berkefeld-filtered blood serum of Hogs 629 and 630, 
 respectively. Both of these animals developed typical symptoms of hog cholera 
 
 Hog 632. 
 
 Hog 633. 
 
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 and both were moribund 26 days after inoculation. Practically the only difference 
 observed in their clinical appearance was that Hog 636, which had received the filtered 
 blood from Hog 629, showed more severe symptoms of the disease after a shorter incu- 
 bation period than those manifested in Hog 63 7, which had been inoculated with the 
 blood from Hog 630. This result was consistent, as the injection of the filtered culture 
 described above into Hog 629 produced the subacute type of hog cholera, while Hog 
 630, after a marked reaction, quickly regained normal condition. 
 
 In order to make the present experiment more complete Hogs 639 and 640 were 
 injected each intramuscularly with 4 c.c. of suspension of Culture Transfer 2-587-B, 
 which, after 53 days' incubation, had been macerated in sterile water, diluted i : 6, 
 and subjected to filtration through the Berkefeld. 
 
 From the fifth to the ninth day following inoculation. Hog 639 showed a marked 
 reaction. During this period the animal was somewhat inactive and had poor appetite. 
 Nine days after inoculation Hog 639 regained normal condition. 
 
 Hog 640 manifested no apparent reaction from the inoculation. This was re- 
 garded as an indication of natural immunity, as Hog 640 is a large brood sow and is 
 probably a "natural immune." • 
 
492 Walter E. King and George L. Hoffmann 
 
 Hog 636. 
 
 Hog 639. 
 
 —- — 1313. JULY. AUGUST . 
 
 
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 diluted 1:4, and Berkefeld filtered. 
 July 21. Animal "off feed" and constipated. 
 July 22-August 5. Typical symptoms of subacute type of cholera. 
 August 6. Animal nearly moribund. 
 August 1 1 . Animal killed. 
 
 Typical lesions of hog cholera in lungs, spleen, kidney, cecum, and 
 lymph glands. 
 
 July 
 
 July 24-28. 
 July 29. 
 
 Intramuscular injection of 4 c.c. suspen- 
 sion Culture 587 (May 27, 1913), dilute 
 1:6, and Berkefeld filtered. 
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 Hog 640. 
 
 Hog 637. 
 
 — — ll 
 
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 and Berkefeld filtered. 
 
 July 
 
 July 
 July 
 
 15. Intramuscular injection of 5 c.c. blood serum of Hog 630, 
 diluted 1:4, and Berkefeld filtered. 
 
 21. Appetite poor; constipated. 
 
 22. Anorexia, constipation and diarrhea, weakened condition, 
 emaciation. 
 
 August 6. Animal nearly moribund. 
 August 12. Animal killed. 
 
 Typical lesions of hog cholera; lungs, kidney, lymph glands, mucosa 
 of cecum congested. 
 
Spirochaeta Suis, a Pathogenic Organism 
 
 493 
 
 V. Unsuccessful Inoculations with Cultures, Control on Positive Results. 
 
 a) Strain S76. — ^Hog 576, on March 20, was inoculated intramuscularly with 5 c.c. 
 of hog cholera virus, strain Kansas III. This animal developed typical symptoms of 
 cholera and, being moribund 18 days after inoculation, was bled and examined. The 
 ordinary lesions of hog cholera were present and the material from the ulcers in the 
 cecum, when examined by the dark field method, showed numerous spirochetes and 
 spirilla of various forms. From this material in the ulcers, cultures were made, and 
 after three days' incubation some of these cultures were submitted to dark field 
 examination. On account of the fact that some large forms of spirochetes were present, 
 which at that time were regarded as possibly the same form which had repeatedly been 
 found in the blood of cholera hogs. Culture S76-A was macerated in physiologic salt 
 solution and 5 c.c. inoculated intravenously into Hog 584. 
 
 Hog 576. 
 
 ..^.^ 19 3. MARCH A RIL. 
 
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 Animal not eating, general symptoms 
 appearing. 
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 Moribund, bled from the carotid 
 artery. 
 Typical lesions: lungs, heart, spleen, lymph 
 glands; ulcers in cecum. 
 
 Intravenous injection of 5 c.c. physio- 
 logic salt suspension. Culture 576. 
 
 Hog 584, which received Culture S76-A, showed a sUght thermal reaction on April 
 18, seven days after injection with the culture. Again on April 21, the temperature 
 rose two degrees. These thermal reactions were possibly due to the presence of a 
 variety of contaminating organisms in the suspension, as no clinical symptoms were 
 apparent in the animal. On April 25 the animal was released as normal and subse- 
 quent inoculation with hog cholera serum showed that Hog 584 was susceptible to the 
 disease. 
 
 From the original culture, 576-A, transfers were made on April 11 and incubated 
 under anaerobic conditions. Transfer 1-S76-A, six days after inoculation, on being sub- 
 mitted to dark field examination, showed the presence of relatively large spirochetes, 
 similar to those which had been found in the original culture, 576-A. Therefore, 
 Transfer 1-S76-A was suspended in physiologic salt solution and 2 . 5 c.c. of the sus- 
 pension injected intravenously into Hog 585, on April 14. Five days after inoculation 
 
494 
 
 Walter E. King and George L. Hoffmann 
 
 the animal showed an irregular temperature ranging between 102° and 103°, and this 
 variation in thermal condition was present for several days following. However, no 
 symptoms of hog cholera were apparent and it was concluded that the fluctuating 
 temperature was possibly due to the injection of contaminating organisms which 
 were present in Transfer 1-S76-A. The animal was finally released, and on being 
 injected later with horse serum virus, hog cholera was produced and death eventually 
 resulted. 
 
 b) Strain $60. — On February 14 Hog 560 received 5 c.c. of Kansas HI virus 
 intramuscularly. Six days after inoculation the animal developed symptoms of acute 
 cholera and after a duration of the disease for five days, being moribund, was bled and 
 examined. Typical lesions of cholera were found on autopsy. Numerous spirochetes 
 of various forms were found in the necrotic material contained in the ulcers of the 
 cecum. Inoculations into culture media were made in the usual way and after five 
 days' incubation one of these cultures, designated as "Culture 560-A," after being 
 
 Hog 571. 
 
 
 
 Ivlght 
 
 Hog 
 
 200 
 
 16B. 
 
 
 
 
 
 
 
 
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 physiological salt suspension, Trans- 
 fer I, CxUture 576. 
 
 March 15. 
 March 15-24. 
 March 24. 
 
 March 31. 
 April 3. 
 
 Blood normal. 
 
 Animal normal. 
 
 Intravenous injection of 2 c.c. physiological salt 
 
 suspension, Culture i, Transfer 2, Formol (March 
 
 3, 1913), Hog 560. 
 
 Animal normal. 
 
 Animal a little "off feed." 
 
 examined on the dark field, was found to contain a growth of spirochetes. Contamina- 
 tion with various forms of bacteria and cocci, however, was present. In an attempt 
 to eliminate as much of this contamination as possible, Culture s6o-A was transferred 
 into various culture media containing different chemicals, for the purpose of exerting 
 slight germicidal action and the possible inhibition of the extraneous organisms. On 
 March 24, after 21 days' incubation, Transfer 1-560-A, which had been made in a horse 
 serum medium containing a small percentage of formol, was found, on dark field 
 examination, to contain a few spirochetes, but no granules were observed. Two cubic 
 centimeters of a physiologic salt suspension of Transfer 1-560-A were inoculated 
 intravenously, March 24, into Hog 571. This animal showed no apparent result 
 following the inoculation of this culture, except for a slight reaction which occurred 
 during the eighth and ninth days after the inoculation. Subsequent injection of Hog 
 571 with horse serum virus resulted in the death of the animal from hog cholera of the 
 acute type. 
 
Spirochaeta Suis, a Pathogenic Organism 495 
 
 This includes all of those experiments which proved unsuccessful, 
 and serve as admirable controls on the results given. It will be 
 noticed that the unsuccessful experiments included under the last 
 heading were conducted during the months of March and April. 
 At that time it had not been definitely determined that the crypts 
 of the ceca of normal hogs may contain spirochetes and spirilla. 
 In so far as our observations go, these organisms are usually 
 relatively large and altogether unlikely to be related to the form 
 which we have designated as Spirochaeta suis, the latter being found 
 in the intestinal ulcers and the external local lesions of cholera hogs. 
 No doubt, the small spirochete described in the first part of this 
 paper as Spirochaeta suis was present in the ulcers of Hogs 576 and 
 560, this material being used in the inoculation of the cultures 
 described under the present heading. However, all of the inocula- 
 tions into hogs, of cultures from these two strains, were made after 
 a comparatively short period of incubation. This explains the 
 failure to produce positive symptoms of hog cholera with the 
 ciiltures, as our later results reported above indicate that it is neces- 
 sary to incubate the cultures for several weeks in order to grow 
 Spirochaetae suis. 
 
 The most important bearing, however, of these unsuccessful 
 attempts to produce the disease with the cultures noted imder the 
 last heading pertains to the control which they give on the results 
 described in the preceding pages. The foregoing positive results 
 from inoculations of filtered and unfiltered cultures of Spirochaeta 
 suis are lacking in one detail. Without some means of control- 
 ling the results, it might be logically assumed that those animals 
 which had received hog cholera from the injection of cultures and 
 transfers did so as the result of the injection of a small amount of 
 original virus, which possibly might have been transferred through 
 the cultures. This we know to be untrue because of the failure 
 to produce hog cholera in animals inoculated with cultures and 
 transfers from Strains 576 and 560. Positive results followed the 
 inoculation of Hog 559 with the original culture, SSi-A, which had 
 an incubation period of only five days. The result may have 
 been due to some of the original virus having been transferred to 
 the animal through the culture. However, we believe that the 
 
496 
 
 Walter E. King and George L. Hoffmann 
 
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Spirochaeta Suis, a Pathogenic Organism 497 
 
 succeeding results are sufficiently controlled by the facts that 
 Hog 584 failed to develop hog cholera after inoculation with Culture 
 S76-A, and that Hogs 571 and 585 did not respond to the injection 
 of Transfers 1-576-A and 1-S60-A, respectively. 
 
 CONCLUSIONS. 
 
 Spirochaeta suis is an organism found in the intestinal ulcers, 
 crypts in the ceca, and external local lesions of animals suffering 
 from hog cholera. It is a typical spirochete, simulating in many 
 characteristics Spirochaeta pallida, Spirochaeta gallinarum, and 
 other forms whose morphology and hfe history are becoming better 
 understood. Spirochaeta suis appears to be capable of breaking 
 up into granules and these granules may play an important part in 
 the Hfe cycle and physiological functions of the organism. They 
 are present in the blood of cholera hogs, in cultures of the Spirochaeta 
 suis, and are capable of producing the disease in healthy hogs. 
 
 In the blood of hogs suffering from cholera the presence of a 
 relatively large spirochete in few numbers has been recognized. 
 As this organism has not been found in the blood of normal hogs 
 it may represent Spirochaeta suis in a transitional form. Its 
 morphological variation from Spirochaeta suis, as found in ulcers 
 and local foci, may be due to the unfavorable action of blood as 
 a medium or to its natural processes as a part of the life cycle of 
 the species. 
 
 Spirochaeta suis is an obhgatory anaerobic organism and usually 
 requires several weeks' incubation for growth to take place on 
 artificial culture medium. It may be transferred from generation 
 to generation on artificial culture medium. Cultures containing the 
 organism in the form of granules and spirochetes may be passed 
 through bacteria-proof filters and the spirochetes removed, the 
 few small granules which pass through being capable of producing 
 hog cholera or resistance to the disease. 
 
 Spirochaeta suis is capable of producing typical hog cholera 
 when injected into healthy hogs. This is true not alone of con- 
 taminated cultures made directly from the intestinal ulcers of 
 cholera hogs; second and third generations on artificial culture 
 media, containing the Spirochaeta suis, as well as the Berkefeld 
 
498 Walter E. King and George L. Hoffmann 
 
 filtrates of the same transfers, are capable of producing hog cholera 
 and marked reactions, which confer more or less protection against 
 the disease. The pathogenicity of these cultures does not appear 
 to be due to the passage of an unknown "invisible microorganism" 
 which is finally transmitted to healthy hogs by inoculation. Con- 
 trol experiments tend to show that the pathogenicity of the cultures 
 of Spirockaeta suis is due to the species itself in the form of 
 spirochetes or granules. 
 
 Finally, in those hogs which receive the disease from cultures of 
 Spirochaeta suis, the organism is present in the intestinal lesions or 
 local external lesions, as demonstrated by the dark field examination. 
 
 From the above results, which have practically fulfilled Koch's 
 laws, in so far as it is possible with an organism possessing the 
 biological characteristics of spirochetes, it may logically be con- 
 cluded that Spirochaeta suis is more nearly established as the 
 specific cause of hog cholera than any other known organism.