aiatmll Imtiwattg ffitbrarg BOUGHT WITH THE INCOME OF THE SAGE ENDOWMENT FUND THE GIFT OF HENRY W. SAGE 1891 DATE DUE ^J^AY \ 1989 \av '>i^' It It t* 3J a 1 i<)1l*» _ so f » f as „ o 3^ 8S « ^ /\ z; I « - <. "-« ., - — _ '-- 'T — r --- - P = - = = — — f "*"*■ ++ ^ f y^ ^ \ ^^, \ e - -- \ /' I J / <.l \ \ 1 1 \ ^ // \-- --' \ 1 1 \ \ \ / -- — - - - - - - \ ESS \ \ . s= Fig. 2. Marie Leb (26 years) Dysentery (Shiga) (B. dysenteriae from the patient B. dysenteriae, stock strain B. coli Stools contained blood Therefore, with Germaine Mel. . . . there was a bacteriophage of maximum activity, even from the beginning of the disease. Recovery took place within twenty-four hours. 2. Marie Leb (twenty-six years, fig. 2). This case was one with a mild dysentery, due to B. dysenteriae Shiga. The stools were typical, containing blood and mucus. Entrance to the Hospital took place on the eighth day of the disease. The first stool containing blood had been passed the day before. Upon entrance to the Hospital the feces contained a bacterio- THE BACTERIOPHAGE IN DISEASE 179 phage active for the Shiga organism (+), extremely active for B. coli (+ + ++), and but very sUghtly active for the dysentery bacillus found in the patient (+). Against this last bacillus the virulence increased during the course of the three following days, reached its maximum activity (+ + ++), fell away somewhat (++), and then definitely regained its full virulence (+ + + +). These fluctuations in virulence were reflected in the condition of the patient. At the end of convalescence there remained only a slight activity (+) of the bacteriophage against B. coli. Day of the Disease 4) U 'J lift i'tjf tqnu\inmi}i''j\ilijmtium.nmp£Wifk\u '~ »- ^ - \._.- I — J-. "~--^_ -:::^;^^;j:'::"/5i;:::::: : i^l l-y' ^. ::::/:::::.::::::.^;r::::::::::"" _.j h Fig. 3. Victor Kee (6 years) Dysentery (Shiga) (B. dysenteriae from the patient B. dysenteriae, stock strain B. coli Stnnk contained blood 3. Victor Ker. . . . (5 years, fig. 3). The dysentery was due to the Shiga baciUus, was of moderate severity, and was contracted by contact with the patient next discussed. When admitted to the Hospital, on the third day of the disease, the intestinal bac- teriophage already manifested an average virulence (++) for the stock Shiga strain as well as for the strain isolated from the patient. This virulence increased rapidly and maintained a high value up to the time of complete convalescence (+-| — [-). It then abruptly disappeared. 180 THE BACTERIOPHAGE Day of the Disease 3 U .S3 «" i' i 5 1- i fc 1 -i J .> .. .L 13 jJiI iil.^ It J I- I* ^VO w >(|iH7 tt tj J. SI j»l>-,i^li( >a ..__.^ ^______ " ■"■ \";::; "" - -|--,, j--^^ ^'^ ;^~ ~--, " " f \ i_ii c- :, •■■ \ ___ ,.^ ..,__:. ^,1, ■1; ' - -I' Z ; J ^ ' V... r Fig. 4. Jean Ker (6 years) Dysentery (Shiga) f B. dysenteriae from the patient Virulence for j B. dysenteriae, stodi strain [ B. coU Stools contained blood 4. Jean Ker. . . . (six years, fig. 4). This patient was a brother of the foregoing. The general condition was poor when admitted to the Hospital on the third day of the disease. There were from twenty to thirty bloody stools a day; a severe dysen- tery due to the Shiga baciUus. On the fourth day of the disease there were twenty-four bloody stools. The bacteriophage was feebly active {+) for B. coli and was inactive for the Shiga bacillus. The record shows the following: VIRULENCE OF THE BACTEKIOPHAGE AGAINST DISEASE NUMBEB OF BLOODT STOOLS B. dysenteriae (patient) B. dysenteriae (stock) B. coli 5th 23 + + 6th 13 + +++ + + 7th 9 + + + + + + + 8th 12 + + + + + + 9th 11 + + + + + 10th 12 + +++ + + + + 11th 12 +++ ++ + + + + + 12th (4 of 6 stools without blood) +++ + + + + + THE BACTEKIOPHAGB IN DISEASE 181 From this time on improvement became more and more marked. The activity of the bacteriophage did not disappear after con- valescence had been established. In the first three of these cases the dysentery was mild. The bacteriophage was active at the onset, the bacterium did not acquire a resistance, and its growth was quickly suppressed. In the last case there was a struggle and the bacillus acquired a resist- ance which was finally overcome. The condition of this patient was much more serious. 5. Lans. . . . (seventy years, fig. 5). This case illustrates an extremely severe dysentery due to the Shiga bacillus. The patient entered the Hospital on the second day of the disease. In this case the struggle was prolonged, with fluctuations due to the mixed cultures formed in the intestine. The condition of the patient registered faithfully the changes in the struggle. It may be noted particularly that the bacteriophage manifests a transitory activity on the eleventh day of the disease and the stools temporarily lose their bloody character. But the bacillus increases its resistance and this permits it to develop, and blood reappears in the stools. The disease is only definitely overcome at a time when the virulence of the bacteriophage is sufficiently high to dominate the resistance of the bacterimn. Aside from the five cases cited as examples others have been followed, both in France and in Indo-China. Seventeen other cases differing in severity were examined daily, and twenty-nine more were observed less frequently. In all of the cases the activity of the bacteriophage was manifested in an identical manner: 1. In case of recovery, the virulence of the bacteriophage com- mences to manifest itself in a marked manner toward B. coli. 2. The virulence next extends to the type strain of the Shiga bacilluS; that is to say, toward a strain which has been for a long time under artificial cultivation and which, for this reason, has been deprived of much of its resistance. 3. It manifests itself next, more or less quickly, toward the Shiga bacillus isolated from the patient himself at the onset of the disease.^ ' Obviously it is necessary to preserve this strain without replanting. The isolated colonies obtained on the original plates are planted on several 182 THE BACTERIOPHAGE m .1 ; 1 : MM "H 1 ! '1 •, 1 <' ;i ^ ^^ :i J ^ .^■ t ^ ,^' ^ ,.- J; < t ,^ ',,-- > ^ ;,.- ^1 <- -^ ^ .-- :l / ■' --• : ;il / - :;1 1 i Hi \ ) fl < =^l ■V :rl ( rz sr \ IZ s. SI ) iz . ,' S\ < ±L ■ SI > \ ■■ .-' / ■• ■• SI ) ,.• '■ Pfl 1 ! Pil J X 1 < ^^v^ 1 01 3f \ ; IS 1 t; } \ 1 S ^ c ^ ^ >. 1 'M - > S 1 5; ■A 3 - ^ /" . 1 ■- 1 O ) 1 / ■ . 1 ^ Z 1 ■1 p tt^ 1 y : 1 1 :a 1 zz l ' 1 iJl 1 ' 1 31 1 , ,,-1 ?l 1 - 1 il si 1 "j JS /■ 1 ,-i ( 1 |i — > _j_- 1 =-l f 1 s 1 ^1 > j Hi 1,, 1 ■=1 > ; '-■- . — r ■^ y r "^ 1 € — ^^^ »■ \- =- •^ 1 ». -^^ -4f j ^1 . I j ' 1 — f- . — - -^^4J- ^~ -ri - 1 ! , 1 1 1 a . J . , . O OS •S -S • il If -t ..|, U] -- -- ^ r > ■^ ^ \/ - >w •v N 1 -; z z Z _ z z z z p - J ^ z - - ^ \ ._ __ - = - - -- 'A I ''I - ■v ... -. •■] - "1 - - f '^- ' ' *, ■\ -V ; '^ / v C >. .^ 1 J. ^ '/ \ ^ ^ / ^ -% s " ^ ^ \ L- ... L._ __ Fig. 6. Maeie Mo (55 years) Clinically, Typhoid Fever Virulence for • B. typhosus B. coli B. paratyphosus A — B. paralyphosus B - B. dysenteriae Shiga of the bacteriophage for the strain of B. typhosus from the patient himseK, isolated either by stool culture or by blood culture. In order to use bacilli as comparable as possible with those found in the body of the patient the strains were transplanted as infrequently as possible. In each case an agar tube was inocu- lated with a colony taken from the primary culture, and each time that a fresh culture was needed for the preparation of sus- pensions against which the filtrates containing the bacteriophage from the patient were to be tested, it was always taken from this tube. In this way, the bacteriophage throughout the course THE BACTEBIOPHAGE IN DISEASE 191 of the disease was tested against a culture as nearly constant as possible, uniform especially from the point of view of the resistance of the bacterium. For the first three curves only (figures 6, 7, and 8) the organism of the patients had not been isolated (they had fevers which appeared benign) and the curves of the virulence of the bacterio- phage against the bacillus of the patient is, of course, lacking. For these three cases the virulence of the bacteriophage against Day of the D sease ■»i- 40 59- 38- sr If It It i{ ii H u u li U K 5 j( n !■ Jl Jl !! « sr u 'I M il h 11 It 1,] 'iu iin «[« iiUo 51 U. !1J>I__ « ■ 1 s \ r V / -^ \ H \ J a ^ - ^ ^ d J \l / % ^c = "*■ ^S i ■^ ? 1 =!: ';> -^ - - "- ■~ - '.- ^ i = ^= i ^ ■, ;■ -, ._ CL ' >^2 . \ ,--V - • > .'■ - \ -vT a ".> ::: ^.., / *i3 y \ \ y r ' '■ ■- -4 \ *, / om - - u - - _ - _ - - _ - _ - - - - - \ \\ 1 ... , -^ , ,., .,^ Fig. 7. Louis Pi (17 years) Clinically, Typhoid Fever Virulence for B. typhosus B. paratyphosus A — B. coli B. paratyphosus B - B. dysenteriae Shiga a Shiga dysentery strain, and against the paratyphoids A and B are given. We will select as examples cases of different severity. 1. Mild infections These were cases of typhoid fever or paratyphoid fever with a mild course. Chnically they were typhoid fever but the blood and stool cultures were negative. The curves for these three cases are given on pages 190, 191 and 192. 192 THE BACTEEIOPHAGE 1. Marie Mo. . . . (fifty-five years, fig. 6). 2. Louis Pi. . . . (seventeen years, fig. 7). 3. Frangois Jod. . . . (thirty-four years, fig. 8). In these cases the virulence of the intestinal bacteriophage was determined for B. coli, B. typhosus, B. paratyphosus A and B, and B. dysenteriae Shiga. It is needless to comment on these observations, since examination of the curves is more instructive than would be an explanation. Day of the Disease ? 1 iC fi A 1] m l! k a ([ n Ic y u U I* ir u 2 Lt 1 io Jl 3L U W )r it ij j< It .o ti/ n 11 'l* ,r v. ,T r 41" «o SO" . 3/ a> 3 t r ■^ J ^ ^ B ■V 7 \ \ E^ I— ^ -\ ^ I -% s. C: ~ ~ ~ ■~ ~ ~ ~ ~ ~ ~ ~ "" ~ ~ "' " ■ 5| ■: / .^ ^ - '■ ~\ 4 '3'^ 1 A \. ■V A 1 \ II ; C - - ~ "■ t:v \ \ \ (-1 -# .i r' I > 13 1 ( Zf 1 1 >. S 1 "s 1. Zt p > 1 / 1 ( I 1 \ ,' cr -I ^ "--- «. 1 < "i t; i v y S5 1 ^\ ^ 1 J~~ / ■ 5 1 <, 1 / 1 s ^ 1 / •^ :? 1 y '^ \ J 1 r" ,^' -{ r J ! > / r <, - ■> \ ^ ) r / V ■^ > _L_ / .' . 1 ) / / / V / / * ~ i ' 59- 9 o M t i2 13 .2 (u •p »-< as .a " §§ : 0\ if "s ajmBjaduiax oSnqdouaiD-eg aqi JO AjApDV THE BACTERIOPHAGE IN DISEASE 195 1 '^~ 1 1 ^ 1 1 1 1 1 1 1 1 1 il =1 -1 =1 il SI fcl > tl ^t i ^ ia t J •^1 > / r-.l r ■-il ) / ill / f s 1 ^l ' / 1 •51 / 1 5f n / SI 1 1 ^1 \ / SI - 31 \ ?l ' Jl " •k ll_ ^> sn Vn ■^ \?i ., SH i \ 5i ! \ ^ t .' s) / =?l / ril ( / =sl / .^1 { / n / "ai K / SI > / si < VI ) / 1 el / -' 31 > / ^ -> ■s?l ^ { ~> .91 ( \ y •ai ; \ -' 2d / ^ < [^ ^ -Si ^ : ?5, a o M H O 1- u % , ' o *" OS u > C § ^ O ■^ "►< sq; JO £?iAjpv 196 THE BACTERIOPHAGE ?r ___ f? ?- ^ ■* !t^ i; ' 5" ■ 3 t: 5" IT ~ IT * ~z / tr X ■ 3; I Ji 1 ; I^ ; 1 e; i c ^ 3t i ' c; X ^ iz / £ iz 1 •K 7 ~2 1 \ y^ 1 > m * < / nH ■ \ '" .a I 5 > (A — X _^ u _^ IJ K ^ d / ^ — " -K T^ ■ sC ^ 1 ST ^ " IJ5 S 1 ,, Sr 1 / \, zj; 1 1 \ t: ^ \ 3? , .. v: \ w ) 1 / =s 2~ \ s / N, ^ 1- ^ / 1 -~ w' ^ ::* / J E ^ V. 1 rj N, : ^ — _, ^ r' — o- ■i a S iqtqai b C! e aan^-BJSduigx aSBqdoTJspBa 3^1 JO J?;iApDV THE BACTERIOPHAGE IN DISEASE 197 1 1 ™=j.. '^'-- ' ^1 1 1 1 1 1 ; 1 1 1 1 |! 1 1 1 1 1 1 1 1 i ' 1 1 ■ z v I \ _J_, ^ '1 z r 1 -^ _3 ,P ( :; ' 1 "- 1 1 ' 1 t '-' n \ .2 _S ■- 1 >5 s ■ '1 /' ^■- h\ -'■> / M L , ' II ""S'i' \ s J 1 1 -■-■- 1 !;; / 1 1 , — >^; 1 :; ■ f 1 1 r' r' - ^ J 'I' 'V K '"' ' A <' III ----. ^ X, |> J .> ^ 1 '^ i| ^ \ '1 1 ^ ' ~ ii i " 1 ----- 2 _ ^ 1 / — 1 :; ■^^^i ii / t; 1 — ___ II \ ^ ■ •5 / t '-- ■ ^ / ' •; ■^ *z ■- ) r± J \ .t^ 2: <, c t; / = ^ 9 -'' if ^ ^ is &•' V , ' w fa a o w >< H o H Bi H TO 'r-J a<3 ?* (h cqcqpq ^ U gin^l-Ejaduraj, attj jo itjiAijoy 198 THE BACTEEIOPHAGE 4. Typhoid fever of extreme severity 1. Andr^e Dess. . . . (thirty years, fig. 13). 2. Jeanne Cot. . . . (twenty-four years, fig. 14). In these two cases strains of B. typhosus were isolated at different times during the course of the disease. These bacilli presented a marked resistance to the action of a very active strain of anti- typhoid bacteriophage and lost this resistance only after about ten transfers on agar. It is to be noted that at their isolation from the organism these bacilli were inagglutinable (this fact has frequently been observed) and that they did not become agglutinable until after a series of cultures. This transitory inagglutinability is, as we have seen, associated with resistance to the action of the bacteriophage. Examination of the curves shows clearly the struggle which was carried on within the organism between the bacterium and the bacteriophage, and the repercussions of this campaign upon the state of the patient. We find then, in typhoid fever, — an intestinal infection compli- cated by a septicemia — the same facts as seen in bacillary dysentery. The virulence of the bacteriophagous ultramicrobe isolated from the stools of the typhoid patient is not Hmited, in general, to a single pathogenic bacillus; at one and the same time it ex- tends, in some degree, to some or all of the baciUi of the colon- typhoid-dysentery group. This fact is particularly noted in mild cases or those of average severity. In the severe cases the bactericidal action is more specific and is often Hmited to the specific pathogenic organism and to B. coli, the latter always being attacked. In certain very severe cases the specificity becomes such that up to the beginning of actual improvement only the bacillus isolated from the patient is attacked, whether it has been secured by stool or by blood culture, to the exclusion of other bacilli, taken either from old laboratory cultures or from strains recently isolated from other patients. It seems, then, that in the course of their struggle each of the two organisms present, — bacteriophage and bacterium — acquires an individual personality, which differentiates them from other organisms of THE BACTERIOPHAGE IN DISEASE IS !« ^^ .„ Mmm. 1 i H 1 ° XI Xf < 1 ^ ■?; > ^ St fe. iL :s s ■^ V £i \ ^ r: -(' :; ) ^ /< <' ■^ / 1 ■^ * < , ^ :::l '' 1 ™ i 1 t^ , r- ^ ^ P-. < y tf > / f, v .t / ::: I. / \ ^ 1 \ >l y- " \ ■ ' \ 1 a ( j \ i j t; s , ,=?; f' 1 (, : ^ ■^j _-#■ ■? -; '^ i / -./ '^^ +3 w fe / /: S-3 s» / / " .- \ < / !:;: ! S r V S o 1 , o B -a "I s -. 1 ^■ / N 1 » r" / li j:- ( II ^ II •§■■&' "S y > < II ■g>-S>8 fi S <■ "-**. , ' £QB5BQ H " •> ^ ' y < ■- bj - < .-. ■> o "3 5; ■fe -^ < / ^ ) / g CJ / ^ — l> -f- ; ) / ^ y - > 1 ii f" / s; ^ \ ^ V rf \ / ^ / J ) ■ < 1 ^ V ^1 \ ^ ' k- ?: •±- '•'^ ^ r- \ \ -=■ J M \ ,' ^ ■ . /' s ( < .^_ / -f T \ t J> 1 J F ■ 4 if S S, am ■BJSC ^UI3 L aq; JO iiXAi;DV 200 THE BACTERIOPHAGE t — /IT 1 i 1 1 n f c (i 1 J 1 1 ■■ ^ ^ : . ■ 1 ■ ] 1 /- V / Jl / ^1' f' ./ I( M ^ 1 1 1 1 /■ 1 - : J / h .1 \ - h 1 1 1 J ~iz~ > h T .* *=± ]_ -J— ^ 1 -1 "^ — ^ ^~i r f > 1 1 E ;l ■ . / -1 ^ 1 1 ~/ '— — [-;- s ~7\ < ^ 1 1 -■ C: 1 ^ (, ^ I ■^ 1 il_ ' > 1 Si •1 ;| 1 ^ '~— ;i N. 3^ N ) 1 il^ , '^ .1 P / n 1' 1 1 ^ ■ -^ "i ■'I ^ 1 ;l ::J Ic i l\ \ -1 < ^ f ;l « ^ — -! H--1 1^ — — _| — p_ — :») =rt ,\ ') i ■:- . As- — 7^ ^1" S 1 (" ^ W -^ \\ -^ < a ^ S ^ V S /] O s^ . -"t I if — ■ pq - — - ■! '^1 j1 ! I ^— ^U- ---^ O) a o g O O G- >« " i£^-»* S ID . u BQCqtQ § ' 5 1 i! s ^ sScqdoiispvg; wn^BJOdinsx stH P ^^liAiPV THE BACTERIOPHAGE IN DISEASE 201 the same species rendered banal as a result of cultivation. How- ever, this individuality is effaced by cultivation, both in the case of the bacteriophage, which, after a few passages at the expense of the bacillus to which it is sensitive may develop activity toward any strain of B. typhosus, and of the typhoid bacillus which is then able to be attacked by any strain of antityphoid bacteriophage. Typhoid fever is not a purely intestinal infection as is dysentery. In the latter it can be understood how, when all of the pathogenic bacteria of the intestine or of the mucosa, that is, those in proximity to the ultramicrobes, have been destroyed the disease ends ipso facto. In typhoid fever there is in addition a septicemia and even though the destruction of the bacilli contained in the intestinal contents is sufficient to delay the appearance of the disease or to restrain it from the beginning, it may not be adequate to overcome the infection once the pathogenic bacilli have invaded the organism. We will see later that the protective action of the bacteriophage is not limited to the intestine. The intervention of the bac- teriophage, even in the same organism, may manifest itself in different ways. In the chapter dealing with the properties of the bacteriophage we have seen that the products which it secretes are possessed of an extremely high opsonizing power. A culture of an antity- phoid bacteriophage is precipitated by the addition of four vol- umes of 96 per cent alcohol. The precipitate is allowed to remain in contact with the alcohol for forty-eight hours, a time adequate to ensure the complete destruction of all of the bacteriophagous germs. One centigram of this moist precipitate is dissolved in ten cubic centimeters of saline. In determining the opsonic index, it will be seen that under the action of the lysin the leucocytes become so loaded with typhoid bacilli that it is impossible to count the num- ber of organisms ingested. The opsonic index is certainly higher than fifty. It is possible that the lysin secreted in the intestine as soon as the bacteriophage has acquired a virulence to dissolve the typhoid baciUi may be resorbed and pass into the circulation, and thus assure the destruction of the bacilli by phagocytosis. On the other hand, the bacteriophagous ultramicrobe itself does not remain strictly locaUzed in the intestine; at times it 202 THE BACTERIOPHAGE passes into the circulation. This has not been demonstrated in man for it is not practicable to carry out on man the repeated blood examinations which such a study requires.^ However, in paratyphoid fever in the rat induced by the ingestion of a very virulent strain of B. typhi murium a transitory appearance of the ultramicrobe in the blood has been demonstrated by cardiac puncture made between the fourth and sixth days after the inges- tion of the infectious material. All of the rats in which this phenomenon occurred were protected. At this time a bacterio- phage active for the pathogenic bacillus was present in the intestine and the rats resisted infection. In the third place we will see experimentally that the dissolved products found in the cultures of the bacteriophage provoke, after an incubation period, the development of an "organic im- munity" so potent that it borders on a refractory state. These dissolved products hkewise form in the intestine of the patient, and even within the body, since it is possible for the bacteriophage to pass into the circulation in a septicemia. Twenty-eight more non-fatal cases of typhoid fever were studied in order to determine the influence of the bacteriophage on the course of the disease. Three fatal cases were observed. In these three cases, at no period of the disease could the presence of a bacteriophage be demonstrated active for B. typhosus, either for a stock strain or for the bacillus from the patient. Furthermore, examination of the strains from the intestinal contents from five individuals who had died of typhoid failed to show any activity for the typhoid bacillus. But the bacteriophage was not entirely absent, since in six of these eight cases a bacteriophage of moder- ate activity for the colon bacillus was found. This bacteriophage did not, however, show any activity for the pathogenic organisms. Death in typhoid fever results, usually, because of a failure of the bacteriophage to adapt itself for the bacteriophagy of the in- vading bacillus. May death occur because of the acquisition of a resistant con- dition by the typhoid bacillus, which protects it from the action * Beckerich and Hauduroy have very recently found it in blood cultures. It is essential to examine them systematically for the bacteriophage; the negative cultures in particular. THE BACTERIOPHAGE IN DISEASE 203 of the bacteriophage, as we have seen in the case of dysentery? There has been no opportunity to estabhsh this up to the present but it is the more probable, since, in vitro as in vivo, the tendency toward resistance is certainly more marked for the typhoid bacillus than for B. dysenteriae. In any case, this cause of death is cer- tainly the exception, even in typhoid. It must necessarily accompany a septicemia when it occurs. In typhoid, as in dysentery, the investigation of the virulence of the bacteriophage is of prognostic significance. It is sufficient to verify simultaneously the virulence of the intestinal bacterio- phage of the patient toward B. coli, toward the pathogenic bacillus taken from the patient, and toward a stock culture of B. typhosus. A comparison of these three results furnishes the information desired. The detection of resistance in the pathogenic bacterium would indicate a poor prognosis, and that in proportion as the resistance is the more pronounced. The establishment of a re- fractory state in the bacterimn, resulting in the formation of a mixed culture in the intestine accompanying a septicemia, im- pHes a fatal outcome with a quick maturity. To summarize: in aU of the cases of typhoid fever studied, whatever may have been their severity, the appearance in the bacteriophagous ultramicrobe of virulence for the pathogenic bacillus has been preceded by an increase in virulence for B. coli, which has always begun in the course of the second week and has rapidly attained great intensity. This activity is maintained during the entire course of the infection and appreciably decreases only during convalescence, sometimes even later. On the con- trary, the development of virulence for the pathogenic bacillus has varied according to the severity of the disease. In cases that were mild or of average severity the activity of the bacteriophage for this bacillus appears before the end of the second week and disappears toward the end of convalescence. The activity for B. coli and for B. typhosus is there parallel. In the severe cases the activity for the typhoid bacillus only commences to manifest itself in an energetic manner towards the beginning of definite improvement. It persists for a greater or less length of time, in some cases up to the middle of the period of convalescence. In the forms with relapse and recrudescence the struggle is complicated by the fact of the acquisition of a resistance by the 204 THE BACTERIOPHAGE bacteria, and it is only toward the decline of this relapse or of the recrudescence that the virulence of the bacteriophage is sufficient to definitely control the resistance of the bacterium. Here, the activity of the bacteriophage is maintained up to complete re- covery, that is to say, up to the moment when, because of a total destruction of the pathogenic bacteria, the ultramicrobe is no longer able to develop at their expense. In all cases, the condition of the patient faithfully registers the vicissitudes of the struggle taking place within the body between the bacteriophage and the invading bacterium. AVIAN TTPHOSIS The disease Avian typhosis is a disease affecting principally the Gallinaceae. Despite its frequency it for a long time remained undetected, confounded with chicken cholera. This last disease is, in reality, very rare. In 1919, in investigating epizootics for the purpose of testing on domestic animals, which allow of experimentation, the conclusions reached as to the role of the bacteriophage in human dysentery and typhoid, an extended focus of "chicken cholera" was found in the Department of the Aube. In the first examinations the error which had been made became apparent; it was the disease known in the United States as "fowl typhoid," whose existence in France had up to that time been unrecognized. Shortly after this numerous foci throughout the surrounding territory were discovered. Fowl typhoid, which will here be called fowl typhosis, is a very interesting disease. Its study is complicated by the existence of several " paratyphoses" which resemble stiU more the human typhoid. The pathogenic agent, B. gallinarum Klein, studied by Moore under the name of B. sanguinarium, presents, with the exception of motility, all of the characteristics of the bacillus of Eberth {B. typhosus). It is even agglutinated to titre by an anti- typhoid serum. Aside from this type bacillus there are often found, in the same foci, bacilli presenting different agglutinative and biochemical reactions. The clinical type of the infection which they provoke does not differ from that caused by the THE BACTERIOPHAGE IN DISEASE 205 typhoid type. These differing species of bacteria have up to the present been studied only by American workers; Ph. Hadley among others, who describes B. pullorum A, B. pullorum B, B. jeffersonii, B. rettgerei, and B. pfaffi. A discussion of the distinctive char- acters of these different bacilli will not be presented here since it would not be germane to the study with which we are concerned.' It is sufficient to know that in France in the epizootic of 1919 the most frequent pathogenic agent was of the B. gallinarum type (found in 57 of 73 examinations). Along with B. gallinarum other forms have been found: — B. pull&rum A (once), B. pullorum B (6 times), B. jeffersonii (4 times), and B. pfaffi (4 times). In a single focus, of which the centre was found in the village of Trainel (Aube), a paratyphosis infection occurred due solely to B. pfaffi without admixture with baciUi of the true typhosis type. The clinical picture hardly varies whatever may be the causa- tive bacillus. A typical observation follows. On the evening of May 24 the chicken appeared perfectly well. On the morning of May 25 it remained apathetically on the ground of the poultry-yard and took no measures for its defense. The next day, toward noon, it appeared somnolent, the plumage rough, the eyes half-closed, the crest sHghtly violet colored. It did not eat or drink, and remained humped up "in a ball." The inspirations were deep, twenty-five per minute. There was a greenish yellow diarrhea with portions definitely yellow. The condition became worse in the afternoon. It fell on its side at about 8 o'clock and died a few minutes later. The necropsy showed the crest to be violet in color, with spots of the same nature over the skin. The liver was voluminous, congested, and presented foci of degeneration. There was a pericarditis. By direct microscopic examination the blood at first appeared negative, but a very careful search revealed three baciUi in a whole smear. The blood and tissues when cultured gave a pure growth of B. gallinarum, and this organism was also found, very abun- dantly, in the intestinal contents. ' Readers who are interested in tlie subject will find much useful informa- tion in the contribution by Ph. Hadley "The Colon-Typhoid Intermediates as Causative Agents of Diseases in Birds." Bulletin No. 174, Bhode Island Agrie. Exper. Sta., 1918. 206 THE BACTERIOPHAGE Sometimes death occurs more rapidly still, in certain cases in a striking manner. Epizootics of avian typhosis have a high mortality. In 1919 foci existed throughout the extent of France. In general, the epizootic begins quickly; within the space of three or four weeks a half, three-quarters, sometimes more, of the fowls on a farm succumb. Then the disease assumes a sporadic character, only an occasional animal dying during the course of a year. The disease may disappear for a few months and then reap- pear. The annual mortaUty amounts to forty to seventy per cent of the population of the infected poultry-yards. Young adults are the most susceptible, then the old animals; the chicks are in general spared. Epizootics of typhosis extend rapidly over large areas; cer- tain Departments were contaminated throughout in 1919. The establishment of a new focus begins by the importation of the organism from an infected region, either through the agency of a flock of sheep or herd of cattle, or by horsemen (this last mode of dissemination was particularly frequent during the war; this explains the extension of the disease during the years 1917 and 1918). The disease rages for a few days on a farm, passes to a neighboring farm, and then extends rapidly into the surrounding villages. The pathogenic bacillus remains alive and virulent during several months in the regions where the infection has been epi- demic. In several tests it has been shown that an isolated in- fected chicken-yard, cleaned and left unoccupied for six to eight months, still contains virulent germs, for, when repopulated with chickens from a region free of the disease, the infection breaks out again within a few days among the new occupants. Avian typhosis being a disease in general but httle known, I have thought it useful to consider it in some detail, since it will allow us the better to understand the facts now to be presented. The rSle of the bacteriophage in the course of the disease Because of the exceptional severity of the infection in avian typhosis it has been possible to follow only four cases which re- covered. In all, the picture has been identical. In the morn- ing the infected chicken remains on the ground, "balled up," THE BACTERIOPHAGE IN DISEASE 207 the feathers roughened, and with the characteristic diarrhea. The appearance is the same as in the fowls which succumb. At this stage of the infection examination of the feces gives results such as: B. gallinarum, present in abundance. Intestinal bacteriophage, virulent for B. coli + (in 2 cases) or H — \- (in 2 cases); for B. gallinarum (in the four cases). The blood culture was positive in the two cases in which it was done; the blood for culture being taken aseptically by puncture of the crest. During the course of the day the condition remains the same as that shown by animals which die. This state is prolonged and the next morning the chicken still appears the same. Examina- tion of the feces at this time shows: B. gallinarum present in three cases, absent in one. Intestinal bacteriophage virulent for B. coli + + + (4 cases); for B. gallinarum + (in 3 cases) + + + (in 1 case). Towards noon, in one case, in the course of the afternoon in the three others, blood cultures were negative. In three cases a bacteriophage active for B. gallinarum was found in the blood. The blood which was ultrasterile was that of the chicken whose condition was the best at this time and which had shown no pathogenic bacilli in the intestinal tract in the morning. The presence of the bac- teriophage in the blood is extremely transitory. On the morning of the third day the animals appeared normal, they drank a great deal, ate some grain, and the diarrhea was less profuse. Examination of the feces showed: B. gallinarum absent in the four cases. Intestinal bacteriophage active for B. coli + + + (4 cases), for B. gallinarum -\ — [-+ (3 cases) +-(- + + (1 case). Blood cultures were negative: no bacilU, no ultramicrobes. On the fourth day the animals were practically normal. In the four chickens which recovered the bacteriophage re- mained active for B. gallinarum for a very long time. After three months it showed the same degree of activity as at the time of recovery. In one of them, in which it has been possible to make an examination after five months, it was still as active as at first. We will see, from experimental observations that this 208 THE BACTEEIOPHAGE persistence of virulence depends solely upon the fact that the pathogenic bacillus, distributed in profusion in the exterior en- vironment, is frequently ingested by the animal and this maintains the virulence of the intestinal bacteriophage since it is able to grow at its expense. The feces of about one hundred chickens which had died of avian typhosis were examined. In no case was there a bacterio- phage active for B. gallinarum or for any of the bacillary agents of the paratyphoses. Nevertheless the bacteriophage had been present for it could be disclosed (91 times in 97 examinations) because of the activity shown for one or several species of the colon-typhoid-dysentery group. One sees clearly, then, that the lack of defense is not due to the absence of the bacteriophage, but solely to the fact that the intestinal bacteriophage remained passive because it failed to acquire a virulence for the pathogenic bacillus. To summarize: as in dysentery and in typhoid fever in himian beings, the acquisition of virulence by the intestinal bacteriophage for the pathogenic bacterium is the sine qua non of recovery. Rdh of the bacteriophage in the course of the epizootic Because of the dissemination and extent of the disease it was possible to study the r61e of the bacteriophage in the course of the epizootic as well as in the course of the disease in the individual infected animal. Let us consider first a fact bearing on the territory involved in the epizootic. During the last three years eighty-one examina- tions have been made upon the feces of barn-yard animals, not only in France but also in Indo-China, in regions where a^dan typhosis had not occurred in epidemic form among the fowls for several years. In each of these examinations a bacteriophage active for one or several of the bacilli of the colon-typhoid-dysen- tery group was isolated, but in no instance has the bacteriophage shown any detectable activity for B. gallinarum. In contaminated regions the situation is quite different. As an example, observations made on a farm located at Pougy-sur- Aube may be cited, where the disease was followed very closely. The disease appeared in 1917 in July. Within the period of a THE BACTERIOPHAGE IN DISEASE 209 month fifty-one of the ninety-eight fowls died; then the epizootic disappeared. In May, 1918 it reappeared in less violent form. Twenty-five of one hundred and four fowls died in the period from May to September, and it again disappeared. In 1919 it broke out again early in April. On the 21st of May, twenty-one of eighty had died. At this time I began my observations. On May 21, specimens of the excrement of thirty of the fifty- nine survivors were taken. Examination, made later in the lab- oratory, showed in twenty-six a bacteriophage of weak or moder- ate activity for B. galUnarum (23 were -t-, 3 were -1-+), in four it was absent. On May 22, two chickens contracted the disease. The strains taken the day before were numbered and examination showed that an active bacteriophage had not been found in these two animals. On May 23 one of the two chickens affected the day before died. On May 24, a third chicken, sick in the morning, died in the following night. Its excrement, collected on May 22, did not contain a bacteriophage active for B. galUnarum. On the morning of May 24 the chicken which had been taken sick on May 22 and which had resisted showed in its intestinal con- tents a bacteriophage of extreme activity (-|--|--F+) toward the pathogenic bacillus. On May 26 the fourth chicken, one of those whose feces had not showed an active bacteriophage when examined on May 22, was affected. It resisted, and on May 28 its symptoms had disappeared. The disease disappeared suddenly and during the next three months no new cases developed. On May 30 the feces of thirty chickens were examined and the following results were obtained: Virulence for B. galUnarum; in five +-|--f-f, in twenty-one -t--f-f-, in four -1- + . We see, then, on May 22, four animals among thirty in which the intestinal bacteriophage lacked activity for the pathogenic bacillus. These four animals contracted the disease during the four following days. In the twenty-six specimens collected on May 22 and showing positive results, the bacteriophage showed a relatively weak virulence. Nine days later this activity was very much greater, that is, at the time when the epizootic ceased. What, then, took place in this interval? The bird which became sick on May 22 and which resisted showed in its feces, when ex- 210 THE BACTERIOPHAGE amined on May 24, a bacteriophage endowed with a considerable activity for the pathogenic agent. Here is a second example of the same general nature, giving the results secured on farm M. . . . at V^ricourt (Aube). The epizootic first appeared among the flock of twenty-five chickens in May, 1919. The first animal died on May 18. On the next day twelve specimens of excreta were collected at random. Three only contained a bacteriophage, and that of feeble activity, for B. gallinarum. From May 19 to 26 twelve birds contracted the disease and of these eleven died. One, which became sick on May 23, showed on May 25 a strongly active bacteriophage (-|- + + for B. gallinarum) and recovered. The epidemic stopped abruptly. On May 27 twelve specimens were taken at random. In all a bacteriophage active for B. gallinarum was found (in 1 + + + + , in 9 + + +', in 2 ++). A third example may be mentioned, in which the infection was a paratyphosis.' On October 15 strains of B. pfaffi were isolated from two specimens of blood, taken from animals which had died in a chicken-yard where for about a month there had been an infection presenting the characters of typhosis. From specimens of the feces taken from two healthy animals Uving in the same yard two strains of bacteriophage were isolated, one showing a low virulence (-f-) for B. pfaffi, the other showing no activity for this bacillus. Towards the end of the month three chickens became sick, recovered after an interval of two or three days, and then the epizootic ceased. Six specimens of feces examined at this time all showed a bacteriophage of high virulence (-l--l--f-) for B. pfaffi. Against B. gallinarum four were inactive and two showed a weak virulence (-|-). B. pfaffi was therefore the cause, for when the epizootic broke out three months later the eighty chickens which had survived received a subcutaneous injection of 0.5 cc. of a culture of the anti-pfafii bacteriophage and the epidemic stopped abruptly and permanently from the time of the injection. We will see later that this abrupt cessation is the rule following immuniza- tion by means of a culture of the bacteriophage. ' These experiments were carried out with the assistance of M. Micheau, D. V. M. at Trainel fAube). THE BACTERIOPHAGE IN DISEASE 2U These facts can be explained in only one way. A weak or moderate activity of the intestinal bacteriophage for the patho- genic bacterium is sufficient to render the animal resistant to infection. The pathogenic bacteria which are able to penetrate into the intestine are destroyed before they can multiply. But it is not the same once the disease has appeared and the organism is invaded. The animal recovers, and this is very rare in typhosis, only because of a rapid adaptation of the bacteriophage and the acquisition of a high virulence which leads to an intensive destruc- tion. This bacteriophage with exalted virulence is distributed broadcast with the excreta of the recovered or convalescent ani- mals, and continues, indeed, during several months after recovery. This bacteriophage is necessarily ingested by the other animals of the barn-yard which become, in fact, "infected" by an ex- tremely active bacteriophage and by this means acquire a complete protection against the disease, in spite of the presence of the pathogenic organism in the environment, and in spite of its fre- quent ingestion, an ingestion which serves to maintain the viru- lence of the bacteriophage. These hypotheses are not simply idle speculation, for the interpretation given to these observed facts is confirmed by ex- periments which provide in a controlled manner the natural conditions of the epizootic. Furthermore, it will be seen that the role of defense assigned to the bacteriophage is confirmed by the immunization of several thousand animals by the adminis- tration of cultures of an active bacteriophage. Before discussing these control experiments I ought to mention that, thanks to the kindness of the veterinarians of different regions invaded by typhosis, I have been able to procure numer- ous specimens of blood and excreta taken from sick chickens, from chickens which had died or from those which had recovered, de- rived from eleven different foci scattered throughout all France. This allows me to generaUze from the facts that I have personally observed. Control experiments The control experiments have been conducted in Paris, that is to say, entirely outside of the epizootic area. 212 THE BACTEEIOPHAGE Six chickens, procured from a region free of infection, were placed under observation. Their excreta were examined daily for ten days for the purpose of estabUshing the complete absence of a bacteriophage active for B. gallinarum. Chicken no. 1 then received, per os, 1 cc. of a culture of a strain of bacteriophage very active for B. gallinarum (+ + ++)• Chicken no. 2 received 0.5 cc. of the same culture by subcutaneous injection. The next day examination of the feces of these two animals showed the presence of a bacteriophage strongly virulent for B. gallinarum. Therefore, the bacteriophage passed into the intestine, whether ingested or injected. This same fact has since been verified with man and with different animals. Chicken no. 1 next received per os daily for twenty-five days, 2 cc. of a bouiUon ciilture of B. gallinarum. The active bac- teriophage persisted in the intestine with its primary virulence {+ + ++) and maintained itself up to nine days after the last dose of the pathogenic organism. Chicken no. 2, which had received nothing after the inocula- tion of the active bacteriophage ceased to show an active strain for B. gallinarum within three days after the injection. In other words, chicken no. 1, subjected to repeated reinfections, retained an intestinal bacteriophage active for B. gallinarum for thirty- four days, while chicken no. 2, not infected, for only three days. It follows that the intestinal bacteriophage remains active only if it is able to develop in the intestine at the expense of this bacterium, but in such a case it remains active just so long as this condition is fulfilled. Inversely, the presence in the intestine of a bacteriophage possessing virulence for a given bacterium indicates that this bacterium was a short time previously in the intestine. In the course of the preceding experiment chickens nos. 3 and 4 were placed in contact with chicken no. 1. They all ate and drank from the same containers, the more so since they were changed about in the pens in such a manner as to simulate condi- tions of hfe analogous to those of the chicken-yard. Two days after the first contact, in the case of chicken no. 3, three days after with chicken no. 4, their exf^reta contained a bacteriophage very THE BACTERIOPHAGE IN DISEASE 213 virulent for B. gallinarum (-1 — 1- ++). From this time on they each received each day for twenty-one days, 2 cc. of a bouillon culture of B. gallinarum. At no time did they appear sick. The intestinal bacteriophage remained active for the bacillus through- out the entire period of the administration of the pathogenic bacillus, and even longer — seven days in no. 4 and ten days in no. 3. The intestinal bacteriophage did not then disappear, for as in the case of chickens nos. 1 and 2, it remained active for one or several members of the colon-typhoid-dysentery group. But the virulence for B. gallinarum did not persist when the ingestion of cultures of this last bacillus was stopped. The experiment with chickens nos. 3 and 4 shows clearly that the bacteriophagous ultramicrobe is infectious in exactly the same sense as is the pathogenic bacillus itself, since these birds were "contaminated" by contact with chicken no. 1. Chickens nos. 5 and 6, which had not been in contact with the other chickens, and which on repeated examinations were shown to be free of a bacteriophage active for B. gallinarum, each re- ceived per OS, on some bread, a single dose of 2 cc. of a bouillon culture of B. gallinarum. Three days after the infecting meal diarrhea appeared and they died two and three days later, after having shown all of the symptoms of the natural disease. Ne- cropsy showed the presence of the same lesions. Cultures of the blood gave pure cultures of the pathogenic bacillus, which was likewise found in abundance in the intestinal contents. Chickens nos. 1, 3, and 4, which had resisted repeated inges- tions of B. gallinarum culture without showing the least incon- venience, were therefore immunized; the first as a result of the ingestion of a bacteriophage active for the pathogenic bacterium, the two others by simple association with the first. About one month after the virulence of the bacteriophage for B. gallinarum had disappeared in chickens nos. 1, 2, 3 and 4 each of them was given on each of three days 2 cc. of a culture of the bacillus. In all the intestinal bacteriophage showed a new virulence for the pathogenic organism. None of them showed the sUghtest trouble. In all of these experiments the infections have been made with bouillon cultures of B. gallinarum prepared directly from the 214 THE BACTEBIOPHAGE blood of chickens dead of spontaneous natural infection. This is essential because of the loss in virulence of this organism which takes place under artificial cultivation. With chickens nos. 5 and 6 the ingestion of the pathogenic bacillus caused a fatal attack of typhosis. The intestinal bac- teriophage at no time manifested an activity for the causative organism. In chickens nos. 1, 2, 3, and 4, on the contrary, the ingestion of the same culture caused no disturbance and their intestinal bacteriophage which for about a month had showed no activity for the bacillus, rapidly recuperated its first activity. It had, therefore, not disappeared from the intestine, although its activity was no longer evident, but when it found itself again in contact in the intestine with the pathogenic organism it rapidly regained its potency. This "latent virulence" may be maintained for a very long time. In this connection I may recall the fact cited of a strain of bacteriophage stiU possessing after three years and more than 1000 passages in vitro, always with the Shiga bacillus, the power to attack B. coli and B. typhosus. It showed a weak power, but was capable of rapid augmentation by transfers at the expense of these organisms. This is exactly what this experiment shows us to take place in vivo in the chicken. Can a chicken contract typhosis in spite of the presence of an active bacteriophage in the intestine? It certainly can. As we have seen in many experiments the bacterium may develop a resistance to the action of the bacteriophage and this resistance is one of the factors comprising the virulence of the bacterimn. We have then, on the one hand, the bacterium, which when in- troduced into the organism may acquire a resistance to the action of the bacteriophage ranging from zero to absolute resistance, and on the other hand, the bacteriophage, which at the same time may possess a virulence rimning from zero to extreme activity. Infection occurs, or does not occur, according to whether the algebraic svun of virulence -|- resistance is in favor of the one or the other of the two organisms present. Once the disease has manifested itself, the virulence of the one and the resistance of the other become increased or attenuated according to the con- ditions of the moment and the aptitudes previously acquired THE BACTEEIOPHAGE IN DISEASE 215 favoring the one or the other of the two germs. The sequence in which the events of the struggle unfold determine the issue. Conclusions The observations made in natural disease and the experiments which confirm the deductions which these observations suggest, show that the bacteriophagous ultramicrobe is always present in the intestine of the chicken, whether it is healthy or sick, whether it lives in a locality free of infection or in an epizootic zone. Against a definite bacterium, B. galUnarum in so far as avian typhosis is concerned, the intestinal bacteriophage may be viru- lent or avirulent, and in the first case its virulence may be ex- ercised according to a scale which passes from the smallest degree capable of detection to one of extreme activity. Virulence of the bacteriophagous ultramicrobe for B. galU- narum is only observed in an infected locality. The absence of such a virulence is equally the rule with animals which are about to die and with those which have died. In a contaminated area animals which harbor in their intestine a bacteriophage endowed with sufficient virulence for the patho- genic bacteri\im are by this very fact protected against the dis- ease, and they remain so, provided the actual or latent virulence is maintained at a level sufficiently high to effect a rapid destruc- tion of the pathogenic bacilli ingested. The ingestion of pathogenic bacilli at sufficiently frequent intervals constitutes the principal factor in maintaining the virulence for the given bacterium. Among the factors which contribute to diminishing the virulence or causing the virulence of the bacteriophage for a pathogenic bacteriimi to disappear, I would place as most significant the introduction into the or- ganism of bacteria endowed with resistance to the action of the bacteriophage. We have clearly seen this fact in the course of the experimental study of the phenomenon of the resistance of bacteria. Another possible factor, influencing the activity of the bacteriophage is the reaction of the medium in the intestine, which may vary according to the accidental conditions of the moment, the type of food, etc. The importance of the reaction of the medium has already been shown for lysis in vitro. 216 THE BACTEBIOPHAGE A bacteriophage which has lost its virulence for the pathogenic bacterium lacks the power to exercise it because of the absence of this bacterium, but it possesses nevertheless, a latent viru- lence. When placed again after a greater or less length of time in the presence of this bacterium it regains its original virulence. The fact of the habitual virulence of the intestinal bacterio- phage for B. gallinarum in the infected regions indicates the frequency of the ingestion of these bacilli, and consequently the excessive contamination of the environment by the pathogenic organism. In contaminated regions the animal in which the intestinal bacteriophage does not enjoy any activity for B. gallinarum, quickly contracts the disease. It may resist and recover, but this is the exception, occurring only when the intestinal bacterio- phage quickly acquires a virulence for the infecting bacillus. In the contrary, and usual, case the animal succumbs. In a chicken which recovers, the intestinal bacteriophage ac- quires a considerable virulence against the pathogenic bacterium and maintains this for a very long time; in fact, as long as the exterior environment remains infected. This persistence of viru- lence is maintained by the frequent ingestion of pathogenic or- ganisms, which allow the bacteriophage to multiply at the expense of the particular organism. The resistant animal disseminates in its excreta the bacteriophage of enhanced virulence; the ani- mals which associate with it become "contaminated" and by this fact they enter the same class of resistant animals as those which have recovered. Recovery of one animal in a bam-yard often marks the end of an epizootic, or its arrest for a few months. The study of an epidemic of avian typhosis shows, in a word, that the history of the contagion reflects, in the last analysis, the story of the struggle between the two agents — the pathogenic bacterium and the bacteriophagous ultramicrobe — and since this last is transmissible from individual to individual the immunity is contagious in the same sense as the disease itseK. The be- ginning of an epizootic is marked by a diffusion of the bacteria, the end by a diffusion of a bacteriophage virulent for these bac- teria. We will encounter the same facts in another disease; in hemorrhagic septicemia in the buffalo. THE BACTERIOPHAGE IN DISEASE 217 HEMOBEHAGIC SEPTICEMIA OF THE BUFFALO (bARBONE)' Barbone, the disease Contrary to the diseases discussed up to this point, barbone does not present intestinal symptoms; it is of the hemorrhagic septicemia type. The pathogenic organism is a Pasteurella. Cultures of the organism in beef bouillon maintain their virulence for a considerable time — at least eighteen months. The inocula- tion of a buffalo or of a cow with 0.0002 cc. of a virulent culture kiUs the animal in between thirty-six and forty hours with all the symptoms of the spontaneously acquired disease. At ne- cropsy identical lesions are found and the pathogenic bacterium swarms in the blood and in the organs.^ The buffalo is par excellence the beast of burden in the culti- vation of rice-fields; it replaces the ox in all southern Asia and in the islands of the Sunda Straits. It is utilized in certain regions of Italy, in Egypt, in Hungary, and in the Balkans. Wher- ever the buffalo hves there also wiU be foimd barbone, the most terrible, without doubt, of all the contagious diseases. The re- ports indicate a mortality of from 70 to 95 per cent. I was present during an epizootic which raged in June, 1920, in the Province of Bac Lieu (Cochin-China) where among the thirty thousand buffaloes of the region ten thousand died, and I did not have an opportunity to observe a single animal which recovered. Recovery may occur, but it is certainly rare, and the mortality in Cochin-China is certainly above 99 per cent of the animals affected. The average duration of the evolution of the disease is but eighteen to twenty-four hours; rarely thirty-six. Death some- times takes place without precursory symptoms. An animal yoked to a plow stops, remains motionless for a few moments with an ' The experiments on barbone have been performed in collaboration with G. Le Louet, Chief of the Veterinary Service in Cochin-China. ' In two different attempts I have proved that diluted blood or macera- tions of organs (liver and lung) taken from animals dead of spontaneous infection, filtered through a Chamberland filter (Lj) and inoculated in large amounts into the buffalo or into cattle do not cause the slightest dis- ease symptoms. 218 THE BACTERIOPHAGE haggard aspect and then falls as though struck by Ughtning. In typical cases, which can be reproduced in a perfect manner in experimental infection, the am'mal appears dejected, the eyes fixed, the head lowered. The temperature rapidly mounts to 41.5 to 42.5°C., the respiration, at first accelerated, becomes slowed and then dyspneic, the inspirations less and less frequent. The animal shows meteorism; it lies flat on the ground in complete lateral decubitus usually a short time before death which is pre- ceded by cramps and at times convulsions. Often tumefaction is to be observed, appearing usually in the region of the throat and extending back to the shoulder. The engorgement is produced by a gelatinous exudate of a yellow color within the connective tissue. At times the tumefaction appears in another part of the body, or it may be entirely lacking. This tumefaction, as shown in experimental infection, marks the portal of entrance of the pathogenic bacteria. Infection usually occurs by way of the digestive tract and the virus most frequently penetrates the tissues through some portion of the nasopharynx. A tumefaction on another part of the body — thigh, abdomen, rump — indicates a reinfection by the penetration of the virus through an excoriation. Examination of cadavers shows that the absence of tumefaction indicates an infection by way of the stomach and intestine. Bovines and the buffalo are equally susceptible, as was noted a long time ago by Plot in Egypt. The statistics of In do-China indicate, it is true, that the mortahty from barbone is but slight for cattle, but this is solely due to the fact that these animals are present in but small mmibers in the regions where barbone rages; regions which are extremely humid and admirably adapted to the buffalo, a semi-aquatic animal. The rare cattle found some- times in such regions contract the disease and die hke the buffalo, after having presented identical symptoms. The effect of low places and swamps on the contagion has been from time immemorial recognized by the natives. When it is possible, as soon as a case of barbone is detected in a neighborhood, they hasten to collect their animals and remove them to a more elevated region. It is known, moreover, that the organisms of the Pasteurella group remain virulent for a very long time in the mud of the marshes and in the slime of the streams. THE BACTERIOPHAGE IN DISEASE 219 R6le of the iacteriophage in the disease In Cochin-China barbone is always present in sporadic form causing each year numerous small epizootics which remain lo- calized in individual villages. A localized epidemic observed in Long Huu in the Province of Gocong may serve as an example. From May 5 to 13, 1920, seventeen buffaloes died: — on May 5, one; May 7, three; May 8, two; May 9, one; May 10, two; May 11, four; May 12, three; and May 13, one. Then the epizootic stopped and not a single case was detected during the next six months. Specimens of the feces of four of these animals were collected, either before death or from the cadaver. None contained a bac- teriophage active for the bacterium of barbone. On May 13 specimens of feces were collected from healthy animals, as follows : First. From a buffalo in a stable where two animals had died, one on May 12, the other on May 13. Second. From three buffaloes in a stable where one had died on May 5. Third. From two buffaloes in a stable where two had died, one on May 8, the other on May 11. Fourth. From four buffaloes in a stable which had not been invaded. Fifth. From one buffalo, living alone in a stable located at a distance of about five kilometers from the village of Long Huu. Sixth. From eight buffaloes in the surrounding villages, from eleven to nineteen kilometers distant. Of aU the specimens, those in the first, second, third and fourth groups gave a bacteriophage of weak or average activity (-1- or -t--|-) for the bacterium of barbone. An active bacteriophage was not foimd in the specimens from groups 5 and 6. Again on May 19 specimens were collected in Long Huu, as follows: First. From the buffalo which had furnished specimen no. 1 on May 13. Second. From two buffaloes living in a stable where three had died from May 7 to May 12. These specimens all gave a bacteriophage moderately virulent (++) for the bacterium of barbone. 220 THE BACTERIOPHAGE The animals which resisted, therefore, showed in their intestine a bacteriophage virulent for the pathogenic bacterium. The epizootic does not always remain localized in a village. At times it spreads rapidly from village to village and within a few days will extend over a very considerable territory. It is rarely possible to determine the primary focus, so great is the speed with which it spreads. The mortahty then becomes con- siderable, the losses often amount to tens of thousands of animals, as has been observed many times in China, in British India, and in the Dutch East Indies. Sometimes even, as actually happened in Java, the buffalo, as a race, is practically eliminated. In the first two weeks of June 1920 the epizootic became general in the Province of Bac Lieu and in certain parts of the adjacent provinces (western Cochin-China). It was possible to examine the blood of eleven animals which died in widely scattered parts of the area invaded, and in all the bacterium of barbone was found in considerable quantity.' The epizootic died out during the first fortnight of July. It had persisted for a month, killing a third of the animals in the district. The region of Thoi Binh was particularly affected, the loss amounting to more than fifty per cent of the buffaloes in the locality. From July 8 to 13, at the time when the epizootic was disappearing (the last animal to be affected died on July 12), twenty specimens of feces were collected. These were taken from buffaloes which had resisted the infection and which at no time showed any evidence of the typical symptoms of the disease. AH of the animals examined lived on the farms of the village of Thoi Binh or in the neighboring hamlets within a radius of fifteen kilometers. Tests for the virulence of the intestinal bacteriophage against the bacterium of barbone gave the following results: ' Bacteriological diagnosis is easy, even if the only available material is some blood or a fragment of an organ taken without any special pre- cautions in the field, as is usual in such countries. Even if the specimen is some days old it is only necessary to smear it over the shaved skin of a rabbit. If the bacterium of barbone is present the animal will die within 24 hours, and the organism will be found in pure culture in the blood, from which it may be readily isolated. This is also the best method for detecting the bacterium in soil or in fecal material. THE BACTERIOPHAGE IN DISEASE 221 Ngau 1 Ngau 2 Ngau 3 De Doil Doi 2 Lanh Tran The H. V. Chanh Hien Du Sam P. V. Chanh. Cu So No Phuc Gia Man MOBTALITT 2 5 9 1 3 2 2 6 1 3 5 8 8 1 THE LAST ANIMAL DIED ON July 7 June 10 June 28 July 2 July 4 July 11 July 2 July 2 June 30 July 2 July 6 June 30 July 3 June 29 June 30 NUMBER OF ANIMALS WHICH RESISTED 10 1 4 4 2 1 2 4 6 8 8 5 5 10 4 3 3 VIRULENCE OF THE BACTERIO- PHAGE + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -++ + + + + + + + + + + + + + + + + + + From this it appears that the intestinal bacteriophage is en- dowed with virulence for the bacterium of barbone in all the buffaloes which the disease had spared. In the course of different trips across Indo-China, I collected forty-one specimens of feces from buffaloes, each specimen col- lected in a different village in which no buffaloes had died of barbone for at least two years. In only three of these specimens could a bacteriophage active for the bacterium of barbone be demonstrated, and in these cases it was weak (+). Nevertheless, the intestinal bacteriophage was present in all; but although it was active for one or another of the intestinal organisms, its viru- lence was weak or lacking for the bacterium of barbone. We will see later, on the contrary, that in a contaminated area at the time when the epizootic dies out, the intestinal bacterio- phage of all of the buffaloes which escaped the disease is virulent for the bacterium, the causative agent of the epizootic. We find here, then, the same facts as in the previous disease studied; 222 THE BACTERIOPHAGE that the protection of the body in the case of barbone, a septicemic disease, is assured by the bacteriophage. In the buffaloes of a region ravaged by the disease the bacterio- phage preserves for a very long time its virulence for the patho- genic bacterium. This, the following example shows. In November, 1919, a localized epizootic of barbone occurred among the buffaloes of the village of Phuoc Thien (Province of Bien Hoa). On a farm having twenty-one buffaloes seven died — two adult animals and five aged from one to two years. The disease died out, or to speak more correctly, after this, two ani- mals recovered one after another. On the 12th of the following April, that is to say, five months later, specimens of the feces of eight of the surviving animals were collected. All contained a bacteriophage active for the bacterium of barbone (six were -I-4-, two were -|-). Two specimens of the mud of a water-hole where the animals were accustomed to remain immersed up to the neck during the hottest hours of the day were also examined. In both a bacterio- phage virulent (-|-+) for the bacterium of barbone was found. The destruction of the pathogenic bacterium in the external medium must often be effected by the bacteriophage, for it is certain that if the bacterium of barbone has once been introduced into a water-hole by a sick animal the bacteriophage present there must destroy it. Furthermore, this fact shows one of the modes of "contagion" of the active bacteriophage. A single buffalo, in the intestine of which the bacteriophage has acquired a virulence for the pathogenic bacterium, is sufiicient to "con- taminate" all the herd which frequent the water-hole. Localized epizootics are of short duration, but in spite of this we find that the pathogenic bacterium persists for several months in the ex- ternal world and that their ingestion by buffaloes is frequent, since the virulence of the bacteriophage maintains itself against this bacterium. The repeated ingestion of a bacterium is, as we have seen, essential for the permanence of the virulence of the bacteriophage toward this bacterium. The epizootic dies out, not because of an absence of pathogenic bacteria but because of the presence of a virulent bacteriophage in the intestine of all exposed animals. THE BACTERIOPHAGE IN DISEASE 223 All of the observations are therefore comparable, whether they deal with avian typhosis or with barbone in the buffalo. These epizootics of very different nature were investigated in- tentionally, that the general nature of the r61e of the bacterio- phage in immunity might be the better established. One may at first be quite astonished that the intestinal bac- teriophage, whose r61e can easily be conceived in infections with intestinal manifestations, constitutes a defense of the organism in septicemias. In reality, whatever may be the infection, the pathogenic bacterium always gets into the intestine. Let us take a localized disease, cerebrospinal meningitis, for example. We know that the initial symptom is a rhino-pharyngitis and that even healthy subjects who have been in contact with a patient often carry the specific germ in the nasopharynx. There can be no doubt but that a fair number of the meningococci present in the rhino-pharynx are swallowed and pass into the intestine. It is needless to insist on this, that, aside from a few rare exceptions to which we will later return, whatever may be the disease under consideration, the portal of entrance of the virus is either the buccal route or by way of the respiratory tract. In either case the ingestion of organisms is, it might be said, ob- ligatory. The pathogenic bacterium is always at some time in contact with the intestinal bacteriophage, this organism there- fore is thus able to adapt itself to the bacteriophagy of the bac- terium and to acquire a virulence. In the particular case of barbone the pathogenic bacterium is foimd freely disseminated through the exterior world in con- taminated regions. In an epizootic zone I have been able, in two different trials, to isolate it from the mud of a marsh where the buffaloes were accustomed to bury themselves. This is but natural since the bacterium of barbone is found in the intestinal tract of sick animals or of those which have succimibed. The ingestion of the pathogenic bacterium by the animals which re- main immersed for whole hours in a mire containing these or- ganisms is necessarily frequent. If the animal which ingests them has an erosion at any point in the digestive tract it is sus- ceptible to infection. Otherwise the bacteria reach the intestine and come within the range of the intestinal bacteriophage which 224 THE BACTEHIOPHAGE can then acquire a virulence for the virus. If this takes place the animal is thenceforth protected from the infection and becomes a carrier of the virulent bacteriophage. A diseased animal prop- agates his disease; an animal in a resistant condition propagates his immunity. BUBONIC PLAGUE Through a lack of favorable circumstances it has not been possible to follow the evolution of the intestinal bacteriophage in man affected with plague. The few cases that have been ex- amined have all been fatal, and at no time could the intestinal bacteriophage be shown to have the least virulence for B. pestis. The activity in these cases remained restricted to B. colt. How- ever, the stools of two convalescent individuals have been se- cured and examined. According to the physicians treating the cases the material was collected on the sixth and the eleventh days after the beginning of convalescence. Examination showed, in the first case, a bacteriophage of average virulence (-f-+), and in the second case, one of feeble virulence (+) for B. pestis. The virulence of the first of these strains has been enhanced in vitro and the bacteriophage has been maintained in culture. An attempt was made to find a bacteriophage active against this bacillus in the feces of twenty-two natives living in regions free of plague, but in no case could a strain be isolated. However, in view of the particular mode of infection in bubonic plague the study of its propagation in man offers only a matter of secondary interest, at least from the epidemiological point of view. We know that an epidemic of plague in man is only consequent to an epizootic among rats. That which it is interesting to study is, therefore, the epizootic, the primary cause of the epidemic. In order to attain a correct interpretation of results it is essential to follow the natmral order of things. From the point of view of man the epidemic is obviously the important fact; from the point of view of nature this is but a secondary incident, for if we were able to suppress the epizootic the epidemic would cease spontaneously. From what we actually know about the epidemiology of plague, it results that all of the rats living in a city where there has been THE BACTERIOPHAGE IN DISEASE 225 a case of plague in man are the animals which have resisted the contagion, either because they were infected and recovered, or because they remained unaffected. I have then, investigated the virulence of the intestinal bacteriophage of the rat toward B. pestis. First. Twenty-one specimens of the excrement of rats taken from towns in Indo-China free of plague were examined. The intestinal bacteriophage was found, active against one or another of the intestinal bacteria, but it never showed any virulence what- ever for B. pestis. Second. A small epidemic of plague (eleven fatal cases) occurred in the village of Bac Lieu, in the eastern part of Indo-China, during July, 1920. On the following 6th of November I procured in this town four specimens of the excreta of rats, each speci- men composed of some dozens of particles, and certainly derived from several individuals. The tests for virulence against B. pestis gave the following results: Specimen derived from a granary -1--|- Specimen derived from the embarkment quay + + 4- Speoimen derived from a decorticating mill -| — \ — f- Specimen procured in the house of a native -|--|- + + Those rats which have survived an epizootic, therefore, har- bor in their intestine a bacteriophage possessed of a high viru- lence for B. pestis. Plague has existed in the form of sporadic cases in the region of Phantiet, in southern Annam, for about twenty years. I obtained specimens of the excrement of rats in the infected vil- lages, each specimen being composed of the feces derived from several animals. The results of the tests for the virulence of the intestinal bacteriophage in these specimens were: Village Virulence Thien Due -1- + Hung Long + Due Hang -| — |--)- Duo Thang 4- + Tri Long + + PhuTay ++ + Cu Long -j- 226 THE BACTEKIOPHAGE The results are thus identical with those secured at Bac Lieu, although the virulence seems to be somewhat lower, but this can only be of relative importance since in the present case each specimen was composed of the excreta taken from several rats; the results then, indicate only an average. Is the bacteriophage present in all of the rats of an infected region or only in a certain number? At Phantiet I collected the excrement of six yoimg rats, according to their weight, aged from three to four weeks. Examination showed that four of the speci- mens contained a bacteriophage active for B. pestis (+) while two did not. These last two animals were therefore susceptible to plague. From the results given above one may conclude that, as for avian typhosis and for barbone, the cause of the resistance against B. pestis is the presence in the organism of a bacteriophage pos- sessing a virulence for this baciUus. How is the adaptation effected in the case of B. pestisf At different times it has been noted that the bacillus has been found in the intestinal contents of victims of plague. Thus, it is pos- sible for them to be disseminated by the feces throughout the external world where they may again be ingested. The bodies of dead rats constitute another mode of dissemination. These bodies are often devoured by the surviving rats and this extends the infection. In those animals which resist and which are infected the intestinal bacteriophage is maintained virulent for the pathogenic bacillus. But observation and direct experi- mentation have shown us that a bacteriophage is only possessed of a virulence for a bacterium when the ingestions of this bac- terium are frequent. The permanence of the virulence of the intestinal bacteriophage of the rat against the plague bacillus indicates the persistence of this bacillus in the external world, at least for several months after the last hiunan case has taken place. Moreover, the revival of the epidemic each year in cer- tain locahties, Bac Lieu for example, shows that it can not be otherwise." '" Demonstration of the presence of a bacteriophage active for B. pestis in the rats of a locality would in certain cases be very useful for it would indicate the presence of the bacillus in the exterior world and the possi- THE BACTERIOPHAGE IN DISEASE 227 FLACHEBIE A few experiments have been made on this disease, but only for the purpose of determining if defense against infection in invertebrates is also assured by the bacteriophage. In a breeding-place in Cochin-China a certain number of silk worms died of a disease presenting all of the characteristics of flacherie. Examination of the excreta of the sick worms, as well as of the cadavers, showed the presence of a cocco-bacillus, Gram-negative, which was not present in the dejections of healthy worms. The ingestion, on mulberry leaves, of some of the culture of this cocco-bacillus reproduced the disease; eleven out of twelve worms dying in from six to eleven days after the infecting feeding. Three filtrates were prepared from the excreta of healthy worms living in the baskets where the affected worms were found. These three filtrates contained a bacteriophage of moderate or high virulence {++, + ++, + ++) for the cocco-bacillus. On the other hand, two filtrates were prepared, the one with the intestinal contents of a sick worm, the other with the intestinal contents of a worm which had died of the infection. Neither contained a bacteriophage active for the coccobacillus. These experiments have not been carried further, since the desired end had been attained. They were adequate to show that the facts observed in infectious disease in mammals were reproduced in an infectious disease of an invertebrate. From this it seems logical to conclude that the defense of the organism by the bacteriophage must constitute a general fact throughout all animals. bility of a renewal of the epidemic. Such a demonstration might also be useful in establishing a retrospective or doubtful diagnosis. Suppose a few suspicious deaths have occurred in a group some time previously. The pres- ence in the rats of the neighborhood of a bacteriophage showing a virulence for B. pestis would eliminate all doubt; the deaths were due to plague. Or, the question of the nature of a epizootic among the rats may be in question. Was the mortality due to plague? The demonstration of a bacteriophage active for B. pestis either in the dead rats or in those that have survived provides the answer. 228 THE BACTERIOPHAGE CONCLUSIONS We may limit ourselves for the moment to the following points: Whatever may be the disease considered, the picture remains the same; when a pathogenic bacterium is introduced into an organism, one of two situations develops: First. The intestinal bacteriophage shows an activity for this bacterium, the latter is destroyed before it can develop, and disease does not appear. Second. The intestinal bacteriophage is inactive, the bac- terium develops, and disease results. In the course of the disease one of two things may happen: First, the bacteriophage in contact with the pathogenic bac- terium may acquire a virulence, or Second. The bacterium may acquire a Adrulence, in other words, may become resistant to the action of the bacteriophage. The vicissitudes in the struggle between these two factors are reflected in the condition of the infected individual. Convales- cence begins at the moment when the virulence of the bacterio- phage is sufficient to give it, definitely, the upper hand. The disease has a fatal outcome if the bacteriophage is inactive as a result of unfavorable conditions, or if the bacterium is able to acquire a refractory state. This last situation appears to be very infrequent, at least, in the diseases studied. In epidemics we find a large scale reproduction in a community of individuals of the struggle which takes place in a single indi- vidual between the ultramicrobe and the bacterium. The bacteriophagous ultramicrobe is transmissible from one individual to another just as is the bacterium itself. The his- tory of an epidemic is, in the last analysis, the story of an infec- tion with two microorganisms. The epidemic ceases at the moment when all susceptible individuals harbor a bacteriophage active for the causative organism of the epidemic. Either the bacteriophage has acquired virulence in the body of the indi- vidual who harbors it, or this individual has been " contaminated" by a bacteriophage which has acquired a virulence in another individual for the specific bacterium involved. CHAPTER II The Bacteriophage in the Healthy Individual The Bacteriophage in Man. The Bacteriophage in the Horse. The Bacteriophage in Fowls. The Bacteriophage in Diverse Animals. Conclusions. The experiments conducted on patients and on healthy in- dividuals exposed to infection have shown that a resistance to the infectious agent accompanies the presence in the intestine of an ultramicrobial bacteriophage possessing a virulence for the causative bacterium. On the other hand, as I have shown in Part I of this monograph, there is but a single species of bacterio- phage, capable, by adaptation, of acquiring virulence for the diverse bacteria which it attacks.^ These facts being true, a question quite naturally arises. Does this bacteriophage which acquires virulence for diverse patho- genic bacteria make its appearance only at the exact moment when it is needed? Or is it, indeed, a normal inhabitant of the intestinal canal? Examination of the feces of numerous individ- uals belonging to very varied species permits an answer to this question. the bacteriophage in healthy men Variations in the virulence of the intestinal bacteriophage in healthy men have been followed. To this end, in a first series of experiments, specimens of the stool were collected every fifteen days. The study has been limited to testing the activity of the ultramicrobe against the following bacterial species: B. coli, B. dysenteriae Shiga, B. dysenteriae Flexner, B. dysenteriae Hiss, B. typhosus, B. paratyphosus A, and B. paratyphosus B. Later ' The possibility of a germ being virulent for a great number of different beings is not an exception peculiar to the bacteriophage. It is only neces- sary to mention B. tuberculosis, to which but few animals are insusceptible. 229 230 THE BACTERIOPHAGE where indicated, the tests were extended to other bacterial species of particular interest. With the first examinations a weak activity (+), especially for B. coli, was not detected or remained doubtful, but when the tests were repeated after several months, using a more satisfac- tory technic, the bacteriophage was clearly demonstrated. As will be seen upon examining the table where the results are re- corded (table 1), some of the examinations remained negative; the bacteriophage appeared to be absent. Would it have been the same if it had been possible to test the filtrate against all of the bacteria which may be found in the intestine? An answer to this question was sought. A specimen taken on July 1st was inactive toward the eight species of bacteria routinely employed, and it was tested against a different series of bacteria, selected at random. The filtrate showed a high activity for an organism of the Salmonella (hog cholera) group. When the same experi- mental tests were repeated on December 1st this filtrate was active for B. enteritidis. These two examples are sufficient to show that the absence of the bacteriophage is only apparent. It must be remembered that the ultramicrobe is recognized only by its activity, and con- sequently its presence in a filtrate can be detected only by test- ing it against a bacterimn for which it has a definite activity. On the other hand, it is not possible to carry out the examina- tion on strains of all bacteria which may be found normally or occasionally in the intestine. For this there are several reasons, the chief being the difficulty of numbers, for there is no species of bacteria which may not be found in the intestinal tract at one time or another. We have seen further that certain bacterial species are not "homogeneous" as regards the bacteriophage. B. coli is of this group. Certain strains are attacked while others remain unharmed. It would then be necessary to make tests with all varieties of a single species; a new impossibihty. Finally, it is known that there exist in the intestine certain bac- teria, revealed by the microscope, which it is impossible to iso- late and cultivate. May the bacteriophage not Uve in commensal- ism with these bacteria, with which they may form in the in- testine "mixed cultures?" It may even be that the impossibihty THE BACTEKIOPHAGE IN THE HEALTHY INDIVIDUAL 231 of isolating these bacteria may be due solely to this phenomenon of conamensalism, for we have seen that the agar on which mixed cultures are planted sometimes remains free of bacterial colonies. TABLE 1 January 15 February 1 February 15 ... . March 1 March 15 April 1 April 15 May 1 May 15 June 1 June 15 Julyl July 15 August 1 August 15 September 1 . . . . September 15 . . . October 1 October 15 November 1 . . . . November 15 . . . December 1 December 15... . VIRULENCE OP THE INTESTINAL BACTEBIC B. coli B. dysenteriae Bacillus a i 1 to 1 Ph >l Eh <1 OS + +++ + + +++ + ++ + + ++ +++ +++ + + +++ +++ +++ + +++ + +++ + +++ ++ + + ++ + other organisms Salmonena+4- + B. enteritidis++ However this may be, the table here given shows the results of the tests made on the normal man in question. The examina- tions are adequate to show that the bacteriophage is a normal inhabitant of the intestine (table 1). During the course of this same year this individual showed at two different times, July 3, and September 26, shght intestinal 232 THE BACTEEIOPHAGE disturbances lasting some hours. The first time there was no obvious cause; the second attack followed a suspected meal taken in a village tavern. On each occasion specimens of the stools were examined on the following days. The results are recorded in table 2. There can be no doubt that in the first case the cause was B. dysenteriae Flexner, and in the second B. paratyphosus B. Disease was aborted, the bacteriophage having quickly acquired virulence for the invading germ. TABLE 2 u. DTBENTEHIAE BACILLUS Shiga Flex- ner Hiss Typho- SU8 Para A ParaB +++ + + + ++ ++ + + + + + +++ + + + + ++ + + + +++ ++ + + + ++++ +++ + ++++ +++ ++ ++ +++ + +++ + + + July 4 July 5 July 6 July 7 July 8 September 27, September 28, September 29, September 30, October 1 October 2 October 3 We wiU complete this section by giving the results of tests made on the stools of three normal persons (aged 39, 22, and 17 years) during the period of the 15th to the 30th of July. Only the bacteria against which the bacteriophage showed a virulence in each of the three persons designated by the mmibers I, II, and III, are recorded (table 3). It is unnecessary to midtiply examples. AU of the experi- ments which have been conducted have given comparable results. One conclusion stands out; the bacteriophage is a normal in- habitant of the human intestinal canal. THE BACTERIOPHAGE IN THE HEALTHY INDIVIDUAL 233 THE BACTERIOPHAGE IN THE HORSE Sixty-two specimens of manure derived from horses living both in cities and in the country, in France and in Indo-China, have been examined and all contained an active bacteriophage. A list of the animals is given, and the results of the examination are recorded in table 4. No. 1. Horse No. 21 of the Pasteur Institute. This horse was used in the production of anti-dysentery serum. The ex- amination was made three days after the injection of Shiga toxin. TABLE 3 DATE I II III July 15 c+ c+ C+++H+++ C+++ H+ + C++H+++ C++H+ C+++ C++ C++ Sh.+++ C+ Sh.+ c+ C+++ C+++ C+ c+ C++ c+ c+ July 16 C+++ July 17 July 18 July 19 C++ B+ July 20 C+ B+ July 21 July 22 July 23 c+ July 24 July 25 July 26 July 27 C+++ July 28 July 29 July 30 C = jB. coK; T1 = B. dysenteriae Hiss ; Sh. = B. dysenteriae Shiga ; B = B. paratyphosus B. No. 2. Horse No. 21 (above), tested ten days later. No. 3. Horse No. 21 (above), tested four months later, the examination being made 48 hours after a toxin injection. No. 4. Horse No. 114. Used in the production of Shiga anti- dysentery serum. No. 5. Horse No. 18. Used in the production of Shiga anti- dysentery serum. No. 6. Horse No. 18. (above) tested four months later. The specimen was collected 48 hours after the injection of Shiga toxin. 234 THE BACTEBIOPHAGE No. 7. Horse No. 64. This horse had received injections of atoxic dysentery bacilli — Flexner and Hiss — for two years. No. 8. Horse No. 65. This horse had received injections of atoxic dysentery bacilli — Flexner and Hiss — ^for two years. TABLE 4 HORSE B.COLl B. DTSENTEBIAE BACILLUS B. GALLI- Shiga Flexner Hiss Typhosus Para A ParaB NASUM 1 + + + + + + + + + + _ 2 + + + — 3 + + + ++ + + — 4 + + + + + + + ++ 5 + + + + + +++ +++ 6 + +++ +++ 7 + + ++++ ++++ ++ — 8 + + + ++++ ++++ — 9 + + ++++ ++ ++ — 10 + + ++ ++ + — 11 + + ++ 12 + + +++ + ++ + 13 + + ++ ++ + + 14 + + ++++ ++ ++ 15 ++++ ++ +++ 16 ++ ++++ ++++ ++++ ++ +++ ++ ++ 17 ++ +++ ++ ++ + ++ ++ 18 +++ ++ +++ +++ +++ +++ ++ +++ 19 ++ ++++ + + ++ 20 +4- 21 + + + + ++ 22 ++ ++ 23 ++ +++ ++ ++ ++ 24 + ++++ +++ ++ ++ + 25 + ++ 26 +++ +++ ++ ++ +++ No. 9. Horse No. 68. This horse had received injections of atoxic dysentery baciUi — Flexner and Hiss — for two years. No. 10. This horse was receiving injections of B. anthrads. No. 11. This horse was receiving injections of B. anthrads. No. 12. A carriage horse in Paris. No. 13. A carriage horse in Paris. THE BACTERIOPHAGE IN THE HEALTHY INDIVIDUAL 235 No. 14. A carriage horse in Paris. No. 15. The same animal as No. 14 (above) but tested four days later. No. 16. A farm horse on a farm where avian typhosis was present. No. 17. A farm horse on a farm where avian typhosis was present. No. 18. A farm horse on a farm where avian typhosis was present. No. 19. A race horse at Chantilly. No. 20. A race horse at Chantilly. No. 21. The same animal as No. 19 (above) but tested eight days later. No. 22. The same animal as No. 20 (above) but tested eight days later. No. 23. A carriage horse at Saigon. No. 24. A carriage horse at Saigon. No. 25. A saddle-horse at Nha-Trang (Annam). No. 26. A saddle-horse at Phantiet (Annam). There is no point in adding to this list; the thirty-six other specimens gave entirely comparable results. Incidentally, horses No. 19 and No. 20, were examined to see if the bacteriophage presented a virulence for various other bac- teria, including the following organisms: 1. A cocco-baciUus (?) isolated from the nasal mucus of a horse in the same stable which showed the evening before an elevation of temperature: Horse No. 19 (++), horse No. 20 (-1--|-). 2. A cocco-bacillus isolated by C^sari from the blood of a horse slaughtered in the abattoir of Vaugirard: Horse No. 19 (+) horse No. 20 {++). 3. Salmonella (hog cholera): Horse No. 19 (-|--1-), horse No. 20 (++). 4. B. enteritidis: Horse No. 19 (0), horse No. 20 (+). These results show that at a single time the bacteriophage may show a virulence for a large munber of bacteria. It is sig- nificant that only in horses Nos. 16, 17, and 18, which lived in an environment contaminated by B. galUnarum, did the intestinal bacteriophage show a definite virulence for this bacteriimi. 236 THE BACTERIOPHAGE Examination was made of twenty-three specimens of sermn, of clot remaining after the decantation of the serum, and of the leucocytic layer on top of this clot, taken from horses harboring in their intestines a bacteriophage active for B. dysenteriae. In no case was a bacteriophage found. In all instances the speci- mens of blood were collected about two weeks after the last injection of toxin or of baciUi. All the specimens of blood ex- amined came from horses furnishing anti-dysentery sertun. It was therefore not determined whether the bacteriophage may not pass into the circulation immediately after the injection, espe- cially when living bacteria are used. As a matter of fact the pas- sage of the intestinal bacteriophage into the circulation has been observed in the rat, and in the fowl in cases of septicemia. In aU cases the demonstration of the presence, it might be said con- stant presence, in the excreta of the horse of a bacteriophage active for the Shiga bacillus, and the absence of this bacteriophage in the blood, shows in an unquestionable manner that the intestine is the only locahty where the bacteriophage normally grows. This fact alone is sufficient to demonstrate the error of the con- ception of Bordet, who has advanced the hypothesis that the bacteriophage is of leucocytic origin. THE BACTERIOPHAGE IN THE FOWL I have made seventy examinations of the excreta of fowls, and have tested the bacteriophage for virulence against the eight bacterial strains selected. It is needless to give all the results since they were all of the same nature. As examples, the results of only one or two tests in each lot wiU be given (table 6). Nos. 1 and 2 represent chickens living in France in regions free of avian typhosis (12 specimens examined). Nos. 3 and 4 represent healthy fowls living in regions where avian typhosis was present (19 other examinations carried out on the eight test bacteria gave comparable results, particularly as regards B. gallinarum). Nos. 6 and 6 represent chickens which had recovel-ed from avian typhosis (4 tests made). Nos. 7 and 8 represent chickens which died of avian typhosis (8 other tests gave similar results). The bacteriophage was pres- ent but was not virulent for the pathogenic bacillus. THE BACTERIOPHAGE IN THE HEALTHY INDIVIDUAL 237 Nos. 9 and 10 were chickens living in Cochin-China in regions free of both avian typhosis and barbone. Nos. 11 and 12 represent chickens living in Cochin-China in areas where barbone was present but free of avian typhosis (11 tests, all essentially the same). Nos. 13 and 14 were chickens in France living in regions where avian typhosis was present.^ TABLE 5 B.COLI B. DTSENTEBIAE BACILLUS B.GALLI- NABtTM BACT. NTJMBBH Shiga Flex- ner Hiss Typho- sus Para A ParaB BAB- BONE 1 + +++ — 2 ++ ++ + + — 3 + +++ +++ +++ + + ++ ++ — 4 +4- + + ++ ++ + + — 5 +++ ++++ +++ +++ ++ ++ ++ ++++ — 6 ++ +++ +++ +++ + +++ +++ — 7 + ++ ++ — 8 ++ + 4- — 9 ++ +++ + ++ 10 + + +++ ++ 11 ++ +++ ++ ++ ++ + 12 + + ++ + ++ ++ 13 +++ ++++ +++ +++ + ++ ++ ++ — 14 ++ ++ ++ ++++ +++ ++ - THE BACTEBIOPHAGE IN DIVERSE ANIMALS Examinations have been made of the excreta of the following animals (table 6). No. 1. A monkey, confined in a cage in Paris. Nos. 2 and 3. Cats in Paris. Nos. 4 and 5. Cattle Uving on a farm where avian typhosis was present. 2 1 would recommend that bacteriologists desiring to procure strains of the bacteriophage investigate principally the excreta of horses and chickens, particularly at the beginning of their work. It is from these animals that it is most easy to isolate strains of the bacteriophage having a high activity when taken from the body. These excreta are, moreover, more readily procured than the feces of convalescents. 238 THE BACTEBIOPHAGE Nos. 6 and 7. Cattle in France, in a region free of epizootic diseases. Nos. 8 and 9. Steers in Cochin-China, living in regions free of epizootics (42 other comparable tests). TABLE 6 «. DTaENTEHIAB BACILLUE BAC- TEBIUM B. NtTMBEB B. COLI OP QAU.I- Shiga Flexner Hias Typho- sus Para A-. Para B BAB- BOKE NABUM 1 + + + 2 + ++ + — — 3 + + + — — 4 +++ — + 5 ++ ++ + + — ++ 6 + ++ ++ — 7 ++ — 8 ++ +++ ++ + ++ — 9 + + — 10 + ++++ ++ — 11 ++ +++ — 12 +++ + ++ — 13 +++ ++ ++ +++ — 14 ++ +++ ++ + — 15 +++ + + — 16 + +++ + + + 17 ++ + + 18 + + ++ — 19 + ++ + — 20 ++ ++ +++ +++ +++ — ++ 21 +++ + + + + ++ — + 22 + ++ + +++ — — 23 + — _ 24 ++ + _ — 25 ++ - - Nos. 10 and 11. Buffaloes living in regions free of barbone (14 other comparable tests). Nos. 12 and 13. Healthy buffaloes hving in regions where barbone was present (24 other comparable tests). Nos. 14 and 15. (for comparison) Buffaloes sick (14) or dead (15) of barbone. Eight other tests have been made; five were THE BACTEBIOPHAGE IN THE HEALTHY INDIVIDUAL 239 comparable to those cited. In three others a bacteriophage was not found; if it was present it was inactive for the eight test organisms. Nos. 16 and 17. Swine in Cochin-China, in a barbone area. Nos. 18 and 19. Swine in Paris. Nos. 20 and 21. Swine in France, on a farm infected with avian typhosis (4 other comparable results). Nos. 22 and 23. Rabbits living in cages at the Pasteur Insti- tute (4 other comparable findings). Nos. 24 and 25. Goats in Paris. CONCLUSIONS In brief, in aU the healthy animals examined the presence of a bacteriophage possessing virulence for one or another of the intestinal bacteria selected as test organisms has been demonstrated. These examinations show that in an epizootic area the intestinal bacteriophage of refractory animals is generally possessed of viru- lence for the bacterium causing the epidemic; on the contrary, this is never observed outside of the foci of infection. The activity of the bacteriophagous ultramicrobe towards a given bacterium can only be explained as the result of growth at the expense of this bacterium. Tests of the virulence of the ul- tramicrobe in animals inhabiting an epizootic region or hving in an area free of the infection, against the bacterium considered as the cause of the disease, are, among other proofs, conclusive in this respect. The experiments in vitro are in accord with the facts observed in animals. All the facts agree in showing that in the body the bacteriophage ceases to be active for a bacterium a few days after the destruction of this bacterium. With ani- mals, the ingestion of typhoid, paratyphoid, and especially, dysentery bacilli, must be extremely frequent, since in animals the intestinal bacteriophage is, except in rare instances, virulent for one or another of these organisms. The sum totkl of the results suggests that the bacteriophage possesses "co-virulences" or "accessory virulences" extending to organisms belonging to the same group as the invading baciUus. For example, a bacteriophage increasing its virulence for the Shiga strain of B. dysenteriae must at the same time, although to a less 240 THE BACTERIOPHAGE degree, attack the bacillus of Flexner, or that of Hiss, or both at once. It is difficult to explain in any other manner the simul- taneous appearance of virulence for several baciUi of the same group. These co-virulences extend generally to the bacillary species which show among themselves the phenomenon of co- agglutination. With man, also, much less exposed to contagion because of his mode of life, the activity of the bacteriophagous ultramicrobe for the typhoid, paratyphoids, and dysentery bacilli is very fre- quent. Each time that it occurs it can only be the indication of an incipient infection, which usually passes undetected. The bacteriophage, as a result of its rapid adaptation destroys the invading baciUi before they can multiply.' The ultramicrobial bacteriophage is a normal inhabitant of the intestine of aU animals. Furthermore, as we have seen, it has a great vitahty. Thanks to its minuteness it is able cer- tainly to filter through soils which arrest bacteria. Everything shows that it must be extremely widely disseminated in the ex- ternal world. Everything which at any time may be contami- nated by the excreta of any animal must contain it. It must be particularly abundant where there are living organisms; in the soil, in rivers, and in the ocean.^ And everywhere it must be the principal factor in the destruction of bacteria. ' It seems then, even in animals considered refractory, that there occurs at times a delay in the adaptation of the bacteriophage. This is noted, for example, in cases of dysentery (Shiga bacillus) in horses in tropical countries. • I have isolated a strain of the bacteriophage active for B. coli from a specimen of sea-water taken off from the estuary of Mekong, and a second taken from the Mediterranean off Marseilles. I was unable to isolate a strain from a specimen of water from the Indian Ocean at a point approxi- mately 60° East longitude and 10° North latitude. Dumas has isolated strains from a specimen of garden soil and from the tap-water in Paris. Beckerich and Hauduroy, at the Institute of Hygiene at Strasbourg, have isolated a number of strains active for B. coli, for B. typhosus, and for dys- entery organisms, from different specimens of soil, from the water of the 111, and from Rhine water after sand filtration. These findings allow us to draw an important conclusion, which has a bearing on hygiene. The bacteriophage exercises a preponderant r61e in the defense of the organism. It is therefore of great interest that drinking CHAPTER III Immunization by Means of the Bacteriophage Immunization against Avian Typhosis. Immunization against Barbone- Immunization against Dysentery. Conclusions. The single test scientifically acceptable for a theory of im- munity can be furnished only by the reproduction of this im- munity in an animal naturally susceptible. That is, in placing this animal by experiment in the condition to which immunity is attributed. A strain of the bacteriophage active for a given bacterium can be isolated and cultivated in vitro indefinitely at the expense of this bacterium, thus maintaining its virulence. In this way any desired amount of culture can be obtained. If the theory of immunity by the bacteriophage which we have deduced from the investigations made on natural disease is correct we should be able at will to reproduce all the phenomena leading to recovery, provided there do not exist at the time of intervention organic lesions incompatible with life. We should likewise be able to place the exposed individual in the same re- fractory state enjoyed by the person who has passed through the epidemic period unaffected. It is for this reason that up to the present time I have confined myself almost entirely to a study of the role of the bacteriophage in animals, since these alone permit of experimental confirmation. water should contain the greatest possible number of bacteriophagous ultramicrobes virulent for the agents of contagious diseases. It is certain that to obtain such waters, containing at once the greatest possible number of virulent ultramicrobes and the smallest possible number of bacteria it is necessary to use filtered river water and not stored waters. As potable waters the first are at the same time superior, from both the hygienic and economic points of view. Attention may be called to the fact that in certain cases a search for a. bacteriophage virulent for a given bacterium may be of some significance in hydrological investigations. 241 242 THE BACTERIOPHAGE The immunization experiments by means of culttires of the bacteriophage have been conducted in two different ways: a. In an epizootic area of avian typhosis, where there would be an immunization against natural infection; and b. In a non-infected area, as in barbone, where the results could be checked against experimental controls. These last experiments have shown clearly the exact conditions under which immunization by means of the bacteriophage can be carried out. IMMUNIZATION AGAINST AVIAN TYPHOSIS Further mention need not be made of the immunization ex- periments conducted in the laboratory, which have been dis- cussed in the chapter dealing with the phenomena observed in the course of epizootics of avian typhosis. Imimimization experiments in a region where the epizootic was present, as in the case of avian typhosis, presented an especial difficulty, or rather, a complication. It has been mentioned that aside from the typical typhosis, due to B. gallinarum, there are several varieties of paratyphoses, each caused by a particu- lar species of bacterium. The differences which these bacterial species present from the biochemical and agglutinative points of view and which serve to differentiate them are of no particu- lar significance from the point of view of this study. In so far as the action of the bacteriophage is concerned for each of these, a straia of bacteriophage having an extreme virulence (+ + + +) for B. gallinarum possesses the same activity for aU the French and American strains as weU as for B. jeffersonii. The activity is less pronounced for B. pullorum A, still less for B. pullorum B, and is lacking for B. pfaffi and for B. rettgerei. With such a strain of bacteriophage the reactions are: B. gallinarum + + -1-+, B. jeffersonii + + + + , B. pullorum A + + , B. pullorum B +, B. pfaffi 0, and B. rettgerei 0. Inversely, a strain of bacteriophage secured from fowls resistant to paratyphosis due to B. pfaffi (focus at Trainel, Aube) had the following virulences: B. galli- narum 0, B. jeffersonii 0, B. pullorum A 0, B. pullorum B +, B. pfaffi + + + + , and B. rettgerei 0. The immunization experiments thus become singularly com- plicated, particularly since both typhosis and the paratyphoses IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 243 may be found in the same areas, as is also true for human enteric infections. In routine practise the solution is simple; it is sufficient to immunize the poultry with a mixture of cultures of different strains of the bacteriophage active against the diverse pathogens, the causes of typhosis and the paratyphoses. In the preUminary investigations this was not possible, for the differences be- tween the different diseases had not been recognized when I first undertook this study. The different bacilli, the agents of the paratyphoses, had been studied in the United States but their simultaneous presence in foci of typhosis had not been noted. And in so far as B. pfaffi is concerned, discovered by Pfaff in an epizootic in Vienna, it had not then been incriminated as capable of producing disease in the GaUinaceae. These facts have only been disclosed gradually in the course of these investigations. The cultures of bacteriophage used in the immunization ex- periments were prepared in the following maimer: A culture of B. gallinarum, in Martin bouillon, aged nine or ten hours, that is, very young but showing a definite turbidity, is inoculated with a bacteriophage isolated from the excreta of a recovered chicken and possessing a high virulence for the patho- genic bacillus. After about 12 hours the bacterial lysis is com- pletely finished and the bouillon is perfectly limpid. This cul- ture is filtered through a bougie^ and distributed into ampoules which are sealed. The dose employed for immunization has been in aU cases 0.5 cc, given subcutaneously. The point of injection is of no im- portance for the slightest local or general reaction has never been observed. Experiment I. The following experiments were conducted in 1919 and 1920 in the neighborhood of Agen with the assistance of M. Lambert, D.V.M. Barnyard 1. The epizootic began in August, 1919. By October 2, 110 of 160 fowls had died. The 50 survivors, of which 5 were already affected, were inoculated with the culture of bacteriophage. The 5 sick chickens 1 We have seen that whatever may be the virulence of the inoculated bacteriophage one may always obtain secondary cultures in a certain num- ber of tubes. Filtration is thus essential. 244 THE BACTERIOPHAGE recovered and tlie epizootic stopped abruptly and definitely on the same day as the immunization. Barnyard S. The epizootic began on about August 20. By October 6, 120 of 200 fowls had died. The 80 survivors, of which 7 were sick, received an injection of the antigallinarum bacteriophage. The 7 recovered; the epizootic immediately and permanently disappeared. Barnyard 3. The epizootic began October 10. By the 15th, 21 fowls had died. The 130 that were alive, of which 8 were already sick, were inocu- lated. The 8 recovered and the epizootic disappeared from the day of the inoculation. Barnyard ^. The epizootic began on about November 15. By December 1, 26 of 51 fowls were dead. The 25 survivors, among which were 4 which were infected, were inoculated. One of the sick animals died, the other 3 recovered. The mortality stopped from the date of the inoculation. Barnyard S. The epizootic began about November 25. By December 1, 7 of 60 chickens had succumbed. The 53 survivors were inoculated. Of these 4 were sick. The sick animals recovered and no new cases appeared. Barnyard 6. The epizootic began on December 16. On the 28th, 40 of 142 fowls had died. The 102 survivors, of which 3 were infected, were in- oculated. The sick recovered and the disease abruptly stopped. Barnyard 7. The epizootic began on January 2. By January 14, 15 of 50 animals had died. The 35 survivors were inoculated. No new cases developed from this time on. Barnyard 8. The epizootic began on about January 15 with a daily mortality of 4 to 6 fowls. On January 21 the 121 survivors, including 5 which were sick, were inoculated. The sick recovered and the epizootic stopped at once. Barnyard 9. The epizootic began on about February 10. By February 20, 14 chickens had died from among the original 84. The 70 survivors were inoculated and the disease disappeared at once. Barnyard 10. The epizootic began about February 25. By March 1, 20 chickens had died. The 120 survivors, of which 5 were sick, were inocu- lated. The 5 recovered and the epizootic stopped. Barnyard 11. The epizootic began on February 4. From February 4 to 10, 10 chickens died. On February 10 the 48 living fowls were inoculated in the wing -with 0.5 cc. of the antigallinarum bacteriophage, as had been all the chickens in the ten preceding experiments. The epizootic con- tinued its course and 5 chickens died from February 10 to 17. On Feb- ruary 17 the 43 fowls which remained were inoculated with 0.5 cc. of a mix- ture of four strains of bacteriophage : active against B. galUnarum, B. pullo- rum A, B. pullorum B, and B. pjaffi. The epizootic stopped immediateh' after this second inoculation. Barnyard IS. This barnyard was adjacent to the preceding and here the same facts were observed. A first inoculation made on February 9 on 80 chickens with a culture of the antigallinarum bacteriophage was without IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 245 effect. The epizootic stopped abruptly after an inoculation of bacterio- phage active for the bacillary agents of the paratyphoses, made on Feb- ruary 17. Examination of the blood of fowls dead in Barnyard No. 12 resulted in the isolation of a B. pfaffi type of bacillus. This organism, then, was responsible for the epizootics in groups 11 and 12. In this connection I will only mention the instance of the epizootic of paratyphosis at Trainel of which I have spoken in the chapter on avian typhosis. This outbreat was likewise due to B. pfaffi and was controlled by the inoculation of an anti- pfaflB bacteriophage. Experiment II. This was performed at Poully en Auxois with the assistance of MM. Voillot and Bouhier, D.V.M. Barnyard 1. On Januarys, 20 chickens were taken at random from a poultry-yard containing about 100 fowls where typhosis had appeared. These 20 were immunized with a culture of anti-gallinarum bacteriophage. On February 7 the immunized birds were all alive and in perfect condition, while the epizootic had continued to spread among the non-immunized animals, of which only about 20 remained. Barnyard 2. On February 23 the surviving chickens of a poultry-yard containing at that time 102 animals were immunized. The epizootic which began about 10 days previously, and which had resulted in a daily mortality of 4 or 5 chickens, stopped quickly and permanently from the time of the immunization. The epizootic continued, on the contrary, to ravage with the same intensity as formerly in all the neighboring poultry-yards which served as controls. Experiment III. This experiment was conducted at Provins with the aid of M. Sorriau D.V.M. , in an important poultry- yard where typhosis was present in endemic form. For several months the daily mortality had been 2 or 3 fowls. On Jan- uary 25 the 225 survivors were immunized. The epizootic immediately and permanently disappeared from the date of the immunization. Experiment IV. Performed at Rouillac, Charente, with the assistance of M. Chollet, D.V.M. On December 15, 100 fowls were immunized in a poultry-yard where typhosis had appeared about ten days previously. The daily mortality had been from 4 to 6 animals. With the immunization there was an imme- diate and permanent cessation of the epizootic. Typhosis continued to prevail on all the neighboring farms. Among the 100 chickens inoculated, about 12 were already affected. Of these only 2 died, 2 and 3 hours after the injection. 246 THE BACTEBIOPHAGB Experiment V. This test was conducted with the assistance of Dr. Ormi^res at Carcassonne. The epizootic began during the month of August. By October 1, 80 chickens had succumbed. The 120 survivors were immunized. The epizootic stopped immediately and no further cases appeared after the date of the immunization. Experiment VI. This experiment was conducted with the assistance of M. Mesnard, Departmental Veterinarian at Angou- llme. In these experiments the chickens were immunized by the ingestion, on bread, of about 1 cc. of an antigallinarum bacteriophage. A. On July 2 the 50 chickens surviving in a poultry-yard where typhosis had been prevalent for six weeks, with a daily mortality of 2 or 3 fowls, each ingested about 1 cc. of the bacteriophage culture. Seven months later no new case had developed since the time of the ingestion. B. The same test was performed on October 15 on about 100 chickens on a neighboring farm where typhosis had been present fro several months. The epizootic was immediately and completely checked. In both of these cases the disease continued to spread throughout the neighboring poultry-yards that were held as controls. It appears needless to multiply such examples. In all cases the picture has been the same. The epizootic disappeared from the time that the culture of bacteriophage virulent for the patho- genic bacterium, the cause of the epizootic, had been introduced into the organism of the susceptible animal, whether this was brought about by injection or ingestion. We will see later that this last mode of administration is somewhat less efficient than injection. On the contrary, injections of cultures of a bacteriophage active for B. gallinarum, the specific cause of typhosis, had in general, no effect when the epizootic was a paratyphosis, par- ticularly in the case of infections due to B. pjaffii. In practise, it is only necessary to inject a mixture of different strains of bacteriophage active for the various pathogenic bacteria that may produce the epizootic. This mixture should also include a strain active for chicken cholera. It will be very easy to accom- pUsh this, for the dose of 0.5 cc. which I have arbitrarily adopted IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 247 is indeed much larger than necessary, as we will see. Even in mixing five or six different strains of bacteriophage, the quantity necessary to effect immunization is not more than a fraction of a cubic centimeter. In the course of the experiments cited there has been no selection. All of the animals of the poultry-yard, even though they were moribund, received the immunizing injection. About 100 sick chickens have therefore been injected, and the mortahty among these has been 5 per cent. This is an appreciable reduc- tion since the mortality among affected animals varies from one hundred per cent at the beginning of the epizootic to 95 per cent, when, after some weeks, the disease appears in only sporadic cases. A culture of the bacteriophage, as we have shown in several ways, is composed of bacteriophagous ultramicrobes suspended in a medium containing the dissolved bacterial substance; the bacteria which have been destroyed by the action of the lysins secreted by the ultramicrobe. What, among these different principles, is the one which plays the active role in the protec- tion of the healthy animal or in the one aheady sick, under the conditions of the experiment, that is, in a contaminated area? Unquestionably it is the bacteriophagous germs themselves. The immediate protection assured by the injection or even by the ingestion of the bacteriophage culture suffices to demonstrate this. An organic immunity necessarily requires a certain time for its development. Other phenomena of immimity, organic in nature, are produced only after an incubation period, as the experiments on barbone wiU show. For the moment, let us conclude only that with sensitive animals immunized by the injection of a culture of the bacterio- phage active for the causative pathogenic bacterium, in a con- taminated area, that is to say, in an area where frequent reinfec- tions may take place as a result of the dissemination of the pathogenic bacteria in the external environment, the principal role of protection is played by the bacteriophage itseff. The other phenomena of immunity which may later develop, stimu- lated by the other substances contained in the cultures injected, play no r61e under such conditions, unless it be a very secondary 248 THE BACTERIOPHAGE r61e. We will see that this proposition becomes reversed when similar experiments are carried out in a non-contaminated area. IMMUNIZATION AGAINST BABBONE^ Thanks to the liberality of the Government of Cochin-China, which placed at our disposal all the animals, steers and buffaloes, which we needed, we have been able to study in detail certain of the conditions underlying immunization by means of the bac- teriophage. Barbone is, indeed, an ideal disease for a study of this type. The blood taken from an animal about to die of the disease can be preserved in sealed ampoules for at least six months without any loss in the virulence of the bacteria present. Bouil- lon inoculated with a drop of this blood yields a culture which regularly kills the steer or the buffalo in a dose of 0.0002 cc. With half this dose, 0.0001 cc, usually one out of two animals will be kiUed. Experimental infection reproduces the spontaneous disease in the most minute details; the same temperature curve, the same symptoms, the characteristic edema at the point of entrance of the virus. Like the natural infection, the disease is fatal; aU animals succumb and death occurs in the same length of time in the two cases, within twelve to eighteen hours of the appearance of the first symptoms. The lesions to be found at autopsy are identical. Immunization experiments conducted with such a disease provide, then, absolute results. I may state here, once for all, that each time that the immimity of one animal has been tested by the inoculation of a culture of the bacterium of barbone this test has been controlled by the injection of an equal dose into a control animal of the same weight, and never has the control resisted. Furthermore, although there can be no possible doubt concerning the cause of death, confirma- tion has always been made by microscopic examination, by blood culture, and by the demonstration of the lesions at autopsy. The temperature of the experimental animals was taken regularly, morning and evening, and the slightest reaction in the immu- nized animals could not have passed unobserved. ' These experiments were conducted at Saigon with the assistance of G. Le Louet, Chief of the Veterinary Service in Cochin-China. IMMUNIZATION BY MEANS OP THE BACTERIOPHAGE 249 The strain of bacteriophage employed for the preparation of the cultures destined for use in the immunization experiments had been isolated from the feces of a buffalo which had passed unaffected through the epizootic mentioned in the preceding chapter. This bacteriophage possessed, when derived from the organism, a strong virulence (+ + +) for the bacterium of bar- bone. After about ten passages in vitro the virulence became extreme (+ + + +), and at this time it was used. A fairly turbid bouiUon culture of the bacterium of barbone about 12 hours old received one drop of the previously described active (+ + + +) culture of anti-barbone bacteriophage. After about 12 hours the medium became perfectly limpid. This culture was filtered through a Chamberland filter (L3) and dis- tributed into ampoules, which were sealed. I would call atten- tion to the necessity of employing only cultures with which the lysis of the bacteria has been complete. Such cultures ought, moreover, to be filtered because of the fact tWat a secondary culture may develop in some of the tubes. The cultures of anti-barbone bacteriophage have been used after a variable length of time, — from twenty days to five months after their preparation. No difference fias ever been observed in their mode of action, whatever the time elapsed between the date of preparation and the time of use. AU of the experiments, except those deahng with the effect of the age of the animal upon the development of immunity, have been effected on steers of the indigenous race, in a perfect state of health, aged from twelve to eighteen months, and of an average weight of 100 kgms.,' and on buffaloes aged from one to twelve years. The bovine race and the buffalo are equally susceptible to barbone. In Egypt, Plot has seen the herds of cattle deci- mated to the same extent as the herds of buffalo. According to our observations the buffalo may be rather easier to immunize than cattle. Let us consider first the experiments conducted for the purpose of determining what conditions control the development of the immunity resulting from the injection of a culture of the bacterio- ' The race in Indo-China is of small size. 250 THE BACTERIOPHAGE phage. The size of the dose amd the age of the anunals are the two principal factors whose variation has the greatest influence on the result. To facihtate discussion, we may consider the effects of smaller and smaller doses, although in reality the chronologi- cal order of the experiments was somewhat different, since the tests were first made with the injection of a dose arbitrarily fixed at five cubic centimeters. In this experiment the animals all died, when the test injection was given twenty days later. Think- ing that the immunizing dose was inadequate it was increased in the next test to twenty cubic centimeters. Here again, the results were the same. It was only somewhat later, when smaller doses were employed, that the treatment proved to be efl&cacious. We have seen already that immunization by means of bacterio- phage cultures presents individual peculiarities. Determination of the immunizing dose I. Eight steers received 20 cc. of the bacteriophage culture subcutane- ously. Six of these were tested after a lapse of time varying from fifteen to forty days by the inoculation of a quantity of barbone culture repre- senting certainly 50 fatal doses. All died in the same length of time as the control animals. The remaining 2 were tested, also with 50 fatal doses, sixty days after the immunizing injection. They showed no obvious disturbance. The two controls died in nineteen and twenty-two hours after the inoculation of virulent material. II. Four steers received, subcutaneously, 5 cc. of the bacteriophage cul- ture. Three were tested after thirteen, fifteen and twenty-eight days by the inoculation of 50 lethal doses of virulent bacilli. All died in the same length of time as the controls. The fourth was tested on the fortieth day. It showed no reaction. The control died in twenty-two hours. III. Forty-one animals; 25 steers, 4 buffaloes aged from one to two years, and 12 adult buffaloes, received an injection of 0.25 cc. of the bacteri- ophage culture. A . Eight steers were tested between the third and twelfth days following the injection by the inoculation of virulent culture, representing, according to the weight of the animal, from 5 to 1000 surely fatal doses. All died. B. Twelve steers and one buffalo were tested between the 13th and 20th days, all by the inoculation of 1000 surely fatal doses of barbone culture. Five resisted, the others succumbed. The experiment is given in detail: IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 251 TEST INJECTION ANIMAL 1000 FATAL DOSES GIVEN AFTEH RESULT days Buffalo 13 Besisted without showing any reaction Steer no. 50 15 Died 26 hours after the inoculation Steer no. 52 15 Died 20 hours after the inoculation Steer no. 53 15 Died 23 hours after the inoculation Steer no. 55 15 Died 26 hours after the inoculation Steer no. 56 15 Died 25 hours after the inoculation Steer no. 38 15 Resisted, without showing any symptoms Steer no. 27 16 Died 68 hours after the inoculation Steer no. 20 16 Resisted, without showing any symptoms Steer no. 28 17 Besisted, without showing any symptoms Steer no. 30 17 Resisted, without showing any symptoms Steer no. 89 17 Died 34 hours after the inoculation Steer no. 90 17 Died 32 hours after the inoculation Two control steers died in 22 and 26 hours after the inoculation, and one buifalo, as control, died in 19 hours. C. Twenty animals; 5 steers, 3 young buffaloes, and 12 adult buffaloes were tested during the period from the twenty-first to the sixtieth day after the immunizing injection. All received 1000 surely fatal doses of culture. All resisted without showing any reaction. Five control animals died, all between 16 and 23 hours after the inoculation of culture. IV. Eight steers received 0.04 cc. of bacteriophage culture. They were tested after a variable number of days by the inoculation of 5 surely fatal doses of virulent culture. The results were : ANIMAL TESTED AFTER RESULT days Steer no. 107 1 Resisted, no reaction whatever Steer no. 103 1 Resisted, no reaction Steer no. 106 2 Died 36 hours after the inoculation Steer no. 101 3 Died 28 hours after the inoculation Steer no. 83 4 Resisted, no reaction Steer no. 84 4 Resisted, no reaction Steer no. 104 5 Resisted, no reaction The last steer, No. 102, was tested 60 days after the injection of the immun- izing dose by the inoculation of 50 surely fatal doses of culture. It resisted without showing any disturbance. V. A last experiment, as a control, was performed with a view to testing the practical application of immimization of buffaloes against barbone by M. Le Louet after my departure from Saigon. Twelve steers received by 252 THE BACTERIOPHAGE subcutaneous injection 0.25 cc. of bacteriophage culture. They were tested 25 days later by the inoculation of 2000 surely fatal doses of barbone culture. They resisted without showing the slightest reaction. The controls died in from 18 to 22 hours after the inoculation. The injection of the bacteriophage did not produce in any of the animals, even in twenty cubic centimeter doses, the slightest reaction, either local or general. The temperature curve follow- ing the immunizing injection could be superimposed throughout on the curves of normal untreated animals. From this it is clear that, contrary to general belief, an immunity bordering on the refractory state may be acquired without the manifestation of the slightest reaction. Dvuing the course of these experiments aphthous fever made its appearance at Saigon. The animals in the course of immuniza- tion contracted it but this complication in no instance exerted any influence upon the development of immunity to barbone. From these different experiments it may be deduced that with a large dose of bacteriophage culture the immunity is slow in being estabhshed; about forty to sixty days with a dose of twenty cubic centimeters, more than twenty-eight days with 5 cc. With 0.25 cc. it is not effective for all animals until about the twentieth day. It then permits them to resist without apparent discomfort two thousand surely fatal doses of the culture of barbone, that is to say, the immunity conferred borders on the refractory state. With the minimal dose of 0.04 cc, or less than a normal drop, a solid immunity is acquired by the fourth day. We are not con- cerned for the moment with the steers which have resisted after twenty-four hours; the immunity which they enjoy is of a differ- ent order, as we will see later. We have seen in Part I of this text that the serum of rabbits which have received four injections of bacteriophage culture possesses the property of sensitizing the animals against the bac- teria for which the bacteriophage injected was active. The de- lay in the establishment of the immunity as a result of the in- jection of large doses of antibarbone bacteriophage culture ought to induce this same phenomenon. The injection produces in the animals two phenomena of different orders: an immunity and a sensitization which varies in intensity according to the IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 253 dose inoculated. With a small dose the first surpasses the second which disappears quickly; with a large dose, on the contrary, the inhibitive action persists for a very long time — about sixty days for an injection of 20 cc. As we have seen in the rabbit the sensitization dominates and persists if the injections of the bac- teriophage are repeated.* The experiments further show that the immunity conferred by the injection of cultures of the bacteriophage is absolute when once established, and is negative during the period of incubation. There is no intermediary state. The animals, young or old, which receive the test inoculation during the period of incubation die, with very few exceptions, in the same time as the controls, even if this inoculation is made at a time very close to that where all the immunized animals resist. On the other hand, all those which are tested after the incubation period resist without pre- senting any apparent malaise, whatever the test dose may be. It seems indeed, as a result of these findings, that after an incuba- tion time, more or less protracted according to the amount of bacteriophage culture injected, a period during which the animal remains as sensitive as a normal animal, the immunity increases very rapidly once its manifestation has commenced. In a word, the release of immunity is abrupt. Effect of the age of the animals on the acquisition of immunity We have seen that thirty-two animals, steers, young buffaloes, or adult buffaloes of less than twelve years, have all acquired an immunity that approaches the refractory condition within twenty hours of the injection of 0.25 cc. of the bacteriophage cul- ture. We wished to see how this would compare with the results obtained in old animals. Three buffaloes between fourteen and sixteen years and five very old animals no longer working and certainly more than ' Anaphylaxis shows a phenomenon of the same order. The smaller the sensitizing dose, the shorter the period of time before the animal is sensitized. For example, with a dose of . 001 gc. of serum the guinea pig is sensitized after about fourteen days, with 5 cc. sensitization is present only after several months. 254 THE BACTERIOPHAGE twenty years old* received 0.25 cc. of the culture of bacteriophage. All eight were tested forty-three days later by the inoculation of 1000 surely fatal doses of bacterium barbone culture at the same time as a normal control animal. This last died in seventeen hours. One of the three youngest buffaloes showed no reaction other than a transitory edema at the site of the inoculation, the other two showed a voluminous edema and were obviously sick, but aU three recovered and could be considered normal six days after the test inoculation. The five very old buffaloes suc- cumbed after 48, 53, 54, 60, and 142 hoiKs; that is, after a time considerably longer than the control. Fifteen young animals immunized and tested at the same time failed to show any reac- tion to the test injection. It is evident that although the test dose was enormous, that did not alter the fact that in the old animals the acquisition of immunity was much more difficult, somewhat in proportion to the age. The relative immunity against an extremely severe experimental test is observed only in these old animals; with the young or with adults in the prime of life, the immunity, as we have seen, is absent dvrring the incubation period and complete once it has appeared at all. The duration of the immunity After my departure from Indo-China, my collaborator M. Le Louet, continued the experiments with a view to ascertaining the duration of the immunity produced by the inoculation of a culture of the bacteriophage. In January, 1921, he injected 15 steers, aged about one year, with a cubic centimeter of a culture of the bacteriophage that was about one month old, that is, a bouillon culture of the bacterium of barbone which had been ' The buffalo usually lives about twenty-five or thirty years. The Annamite never kills a buffalo ; old and no longer able to work, it is fed and cared for as well as are the younger animals. The attachment of the natives for these buffaloes is such that it is difiBcult to find a person who will sell one of these animals. Those which served in the experiments were procured, some through the agency of the Governor of Cochin-China, M. le Gallen; others by M. Priv(5, Director of the plantations of An Loc and Suzannah, without considering the possible loss. I offer them my sincere thanks. IMMUNIZATION BY MEANS OF THE BACTEKIOPHAGE 255 lysed by the bacteriophage one month before use. In March, 1922, all of the animals were tested, along with nine controls, by the inoculation of 0.1 cc. of a virulent culture of the bacterium of barbone. The virulence of this culture was such that in amounts of 0.002 cc. it regularly killed steers in less than thirty-six hours. Of the animals thus infected all of the controls died in from seventeen to twenty-three hours after the injection, while of the vaccinated animals ten resisted without any evident reaction and five died in from two to five days after inoculation. This experiment shows that foiirteen months after vaccination two-thirds of the animals possessed an inamunity sufficiently strong to enable them to withstand a massive dose of the patho- genic bacterium. The immunizing principle Under the conditions of the experiment, that is to say, in a non-contaminated area, what, in the culture of the bacteriophage, is the principle which brings about the immunization? A culture of the bacteriophage contains, as we know: 1. The bacteriophagous ultramicrobes, and 2. The soluble substances contained in the culture medium. These are the soluble substances derived from the bacterial bodies at the expense of which the bacteriophage has developed, the lysins secreted by these ultramicrobes and which remain in the medium once lysis has ended, and finally, somewhat later, the anti-lysins of defense secreted by the bacteria. The course of the phenomenon alone, has shown us already that the immunizing principle must be different according as the immunity is developed in a contaminated area, as was the case in the experiments made on typhosis, or in a non-contaminated area, as in those on barbone. In the first, the immunity is acquired immediately; in the second, it becomes effective only after an incubation period. However, direct experiment allows us to confirm this idea. 1. If one injects steers, by the subcutaneous route, with from 5 to 20 cc. of anti-barbone bacteriophage culture it is possible to isolate the active ultramicrobe from the blood throughout the first twenty-four hours after the injection. After this period 256 THE BACTERIOPHAGE they have disappeared. Experiment further shows that the ultramicrobes pass quickly into the intestine. They can be isolated from the intestine within about twelve hours after the injection and thfey persist there for a somewhat longer time than in the circulation: for two or three days (up to six days in a single case). In all instances they have disappeared long before the immunity is estabhshed. Let us repeat that this 'appUes only to the case where the introduction of the bacteriophage into the organism takes place in a territory free from the infection. We have seen, for example, that five months after the termination of an epizootic of barbone it is still possible to isolate a bacterio- phage active for the pathogenic bacterimn from the excreta of buffaloes which have resisted. On the other hand experimenta- tion in the chicken has shown us that the activity of the bacterio- phage for the pathogenic bacillus is maintained just as long as the experimental animal continues to ingest these bacteria. 2. Bablet has shown that the bacteriophagous germs are de- stroyed by preservation for a week in glycerine. We know that this substance exerts no destructive influence on either the dias- tases or the toxins. It may be assumed, therefore, that in a mixture of bacteriophage culture and glycerine the ultramicrobes alone will be destroyed while the immunizing substances con- tained in the mediimi wiU remain intact. Starting from this hypothesis, we mixed 0.5 cc. of a culture of the anti-barbone bacteriophage with 9.5 cc. of glycerine. After holding the mixture at incubator temperature (37°C) for ten days, and after we were assured that the bacteriophagous ultramicrobes were effectively destroyed, we inoculated two steers with this liquid, diluted in 500 cc. of sahne. Each steer received then 0.25 cc. of the original culture. Tests after forty-five days, respectively with 5 and 50 fatal doses of a culture of the bacterium of barbone, showed that these two animals resisted. They had acquired an immunity in spite of the destruction of the bacteriophagous ultramicrobes. In the case of experimental barbone the tests were made in a barbone-free region, and the principle which is responsible for the development of the immunity is most probably constituted of the substance of the bacterial cells. The r61e which the bac- teriophage plays here is to dissolve the bacteria, in which condi- IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 257 tion the bacterial substance is in a state particularly adapted to stimulating the cells of the body which enter into the production of organic immunity. The substance of the bacterial body dis- solves in the medium under the influence of the lysins secreted by the ultramicrobes, but it is not present in the same condition as in the body of the hving bacterium, for the bacteriophage does not simply produce a disintegration. This is shown by the fact that the culture medium becomes perfectly limpid, whereas the medium remains cloudy when a simple disintegration takes place. As we have seen in several tests, the destruction of the bacterium by the activities of the lysins — the diastases — is a process of solution. Indeed, it is rather the substances composing the bacterial body which are dissolved. This process is of necessity accompanied by a change in state. It is, then, not proper to speak of the bacterial substance as the principle which provokes the acquisition of immunity; it is in reality the products result- ing from the degradation, under the influences of lysins secreted by the ultramicrobes, of the substances composing the bacterial cells which are effective. It is obvious that this is yet only an hypothesis, experiment showing only that the principle which provokes the appearance of immunity is not, under the conditions of the experiment, the bacteriophage considered as a Uving being. Aside from the dissolved bacterial substance do the diverse principles present in the culture, namely, the bodies of dead ultramicrobes, the lysins, and eventually the anti-lysins, play any part in the pro- duction of immunity? In the present state of these investiga- tions it is impossible to affirm or deny this. We have tested the action of temperature on the immunizing element contained in the bacteriolysate. To this end, we have repeated the experiment of the culture of the bacteriophage treated with glycerine, with the difference that the culture has previously been subjected to a temperature of 56°C. maintained for a half hour. Two steers have each received a dose of this culture, heated and glycerinized, corresponding to 0.25 c.c. of the original culture. After forty-five days they were tested, the one with five, the other with fifty, fatal doses of barbone culture. The first re- sisted, the second died. The immunizing principle contained in the 258 THE BACTERIOPHAGE bacteriophage culttire is not destroyed but is sensibly weakened by heating for a half hour at 56°C. Although it is not yet possible to know with certainty the nature of the process which controls the development of organic inununity, we are at least able to recognize the result and to note the property which distinguishes the animal immunized by an injection of the bacteriophage from a normal animal. In the case of the immunity acquired as a result of an attack of a contagious disease the blood possesses preventive properties. The blood of immunized animals enjoys the same property, as the following experiments show. I. Steer no. 54 received on November 5, 0.25 cc. of an anti-barbone bac- teriophage culture. Fourteen days later 500 cc. of blood was taken into a flask containing 25 cc. of a 10 per cent solution of sodium citrate. The bldod was immediately injected into the jugular vein of steer no. 43. This last animal was tested twenty-three hours later by the injection of 1000 fatal doses of the bacterium of barbone culture. It failed to show the least evidence of infection. A control died in twenty-three hours. Steer no. 54 likewise resisted the inoculation of 1000 fatal doses, given on Decem- ber 1st. II. The experiment given above was repeated. Steer no. 112 received into the jugular vein 500 cc. of blood from steer no. 95. Both of them resisted the test injections. III. Steer no. 104 received on December 29 a subcutaneous injection of 0.04 cc. of a culture of the bacteriophage. Four days later 600 cc. of blood were taken, as before, and this was transfused into steer no. 108. The next day the two steers resisted the inoculation of five fatal doses, which killed the control animal in thirty-two hours. IV. The above experiment (III) was repeated. The steer which received the blood of the immunized animal was not tested by the inoculation of 50 fatal doses until forty-five days after the transfusion. It resisted, without showing any apparent disturbance, as did also the steer which was immu- nized directly. This last experiment does not, however, prove anything with regard to the duration of passive immunity conferred by the blood of an immunized animal, for it was performed with homologous blood, and we know that an immunity thus produced is of much longer duration than when produced with heterologous blood. In all cases, the immunity thus conferred is extremely powerful and these experiments open the way for further investigations IMMUNIZATION BY MEANS OF THE BACTEKIOPHAGE 259 on the production of therapeutic sera in animals immunized by a single injection of an active bacteriophage, not only for barbone, but for other diseases as well. One might conceive that the "principle" which is contained in the blood of the immunized animal and which confers the pas- sive immunity does not differ from the culture of the bacteriophage which persists for a certain length of time in the circulation. But this is impossible, for if the blood is taken at a time sufficiently close to the immunizing injection of the bacteriophage it is in no way effective. That is, blood taken during the incubation period confers no immunity to the transfused animal. Steers nos. 89 and 90 received on December 19, 0.25 co. of the bacte- riophage culture subcutaneously. Sixteen days later 500 cc. of blood were withdrawn from each animal and transfused into steers nos. 92 and 93. The four animals, tested the next day, died with no greater delay than the controls. Steer no. 46 received on November 5, 20 cc. of the culture of bacteriophage. On November 19, 500 cc. of blood were taken and trans- fused into steer no. 42. These two animals died in the same time as the control after a test injection. As is to be seen, the incubation period of immunity in animals which receive the immunizing injection of bacteriophage culture parallels the appearance of the protective power in their blood. Inamunity develops abruptly; in the same way the protective power of the blood manifests itself suddenly, and at the same moment. What then, is the immunizing principle which makes its sudden appearance in the blood at the moment when immunity is es- tablished, even in animals which have received only the minimal dose of a single drop of the culture of the bacteriophage? Can it be an amboceptor? By no means, for the complement fixa- tion reaction shows that the sera of animals immunized with cultures of the bacteriophage do not contain a specific ambo- ceptor in detectable quantity. In conducting the reaction of Bordet with even 0.5 cc. one obtains an exactly comparable he- molysis, of the same intensity and in the same time, as that which occurs in a control tube containing the same quantity of normal serum. 260 THE BACTERIOPHAGE Examination of the opsonic power of two of these sera gave indices of 0.3 and 0.4; indices which are essentially negative.' The serum containing the protective principle does not con- tain inhibiting substances or even substances delaying the growth of the bacterium of barbone. Bouillon, mixed with such a senun, in any proportion (from 0.05 cc. to 3 cc. per 10 cc. of bouillon), with or without the addition of fresh guinea pig serum, furnishes a mediimi which, when inoculated, gives luxuriant cultures of the bacterium of barbone. Finally, the serum contains no traces of agglutinins. Organic immunity, then, is not due to the presence of an am- boceptor, nor to the presence of an opsonin in the blood of the vaccinated subjects. The blood contains neither agglutinins nor inhibiting substances. The immunity is most probably antitoxic. We have seen in the experiments performed on avian typhosis, that, in an infected area, the protection of the animal is immediate and that this protection is assured only by the presence of bac- teriophagous ultramicrobes virulent for the pathogenic bac- terium. We have again found this immediate immunity in the Case of barbone. It is that which protected steers nos. 103 and 107 against the inoculation of five fatal doses of culture when given only twenty-four hours after the injection of the bacteriophage. In typhosis, this heterologous immunity has been permanent, for the daily reinfections which occur in the infected area allow the bacteriophage to multiply at the expense of the pathogenic bacteria ingested and thus to maintain its virulence for this bacterium. In barbone, this same thing takes place in an in- fected area, since we have seen that the bacteriophage virulent for the bacterium of barbone was present in the intestine of buffaloes five months after the complete disappearance of the epizootic. ' We have seen in Part I that the lysin secreted by the bacteriophage possesses a very high opsonic power. The organism must respond to an injection of lysin (certainly present in the culture of the bacteriophage) by the production of an anti-opsonic antibody. IMMUNIZATION BY MEANS OF THE BACTEHIOPHAGE 261 In a non-infected region, and this was the case in the experi- ments performed on barbone, the mechanism is not the same. In the absence of reinfection the bacteriophage active for the bacterium is ehminated very rapidly from the organism, since it is not able to multiply at the expense of this bacterium. The heterologous immunity disappears with it, — that is to say, after one or two days, — and the animal then becomes susceptible. It remains in this condition throughout the entire duration of the incubation of the organic immunity, which develops under the influence of the soluble products contained in the culture of the bacteriophage. Once this organic immunity is established the animal is refractory. We will see in connection with dysentery, that when a culture of the bacteriophage is injected the organism responds by the production of an antitoxin. It is probable that the same thing takes place in barbone and that the protective principle present in the blood, since it is neither an amboceptor nor an opsonin, is likewise an antitoxin; the response of the organism to the in- jection of the modified substance of the lysed bacterial cells contained in the culture of the bacteriophage. To summarize: the injection of the buffalo or of cattle, with a culture of bacteriophage active for the bacterium of barbone confers: 1. An heterologous immunity, solely due to the presence in the body of bacteriophagous ultramicrobes virulent for the bac- terium of barbone, which assures the destruction of the bacteria upon their introduction into the organism. This immunity terminates just as soon as the ultramicrobes are eliminated from the body. In the absence of frequent reinfections this ehmina- tion is very rapid, since the continued growth and the mainte- nance of virulence can not persist. 2. A!n homologous immunity, or organic and powerful immunity, induced by a reaction of the organism of the animal to the soluble principles contained in the culture of bacteriophage injected. This organic immunity is characterized principally by the appear- ance in the blood of an extremely potent immunizing substance — probably an antitoxin. The organic immunity estabhshes itself abruptly after an incubation period, which varies with the dosein- jected, being longer as the amount of injected culture is increased. 262 THE BACTERIOPHAGE A single injection of 0.04 cc, or less than a normal sized drop, into a steer of 100 kgms. weight places the animal within 4 days in a condition where it can withstand a test inoculation of five fatal doses. Sixty days later the animal resists a test inoculation representing fifty surely fatal doses. The blood of an immunized animal injected into a normal animal confers on the latter a passive immunity as soUd as that enjoyed by the actively immunized one itself, even if this last one has received but a single injection of 0.04 cc. of culture of the bacteriophage. And this passive immunity, under ex- perimental conditions at least, is still intact forty-five days after the injection of the blood. IMMUNIZATION AGAINST DYSENTERY "The cultures of Shiga lysed by the invisible microbe, which are in reality cultures of the anti-microbe, possess the property ctf immunizing the rabbit against a dose of Shiga bacilh which will kill the controls in five days." This statement is taken from my first communication on the bacteriophage. The experi- mental data upon which this affirmation was based are given in the following protocols. The rabbit, although naturally refractory to bacillary dysentery is, on the contrary, susceptible to the inoculation of dysentery toxin. This animal could, then, be utiUzed for the preliminary antitoxic immunization experiments. The following experiments showed at first that the culture of anti-Shiga bacteriophage, a short time after lysis, is toxic, although to a less degree than is a normal culture of Shiga baciUi. Rabbit no. 1. One cubic centimeter of a normal culture of Shiga bacilli was injected intravenously on August 10. The animal died on August 16. Rabbit no. 2. Two cubic centimeters of a normal culture of Shiga bacilli were injected subcutaneously on August 10. The animal died on August 16. Rabbit no. 3. One cubic centimeter of a Shiga bacillus culture which had been subjected to lysis for six hours was injected intravenously. (The amount of bacillary substance here was the same as in the preceding.) The rabbit lived. Rabbit no. 4. Two cubic centimeters of a Shiga culture which had been lysed for six hours were injected subcutaneously. The animal died on August 16. This rabbit had also been injected on August 10. IMMUNIZATION BY MEANS OF THE BACTEBIOPHAGE 263 Six days after the completion of the lysis the toxicity of the culture was markedly diminished, as the following tests show: Rabbit no. 5. Two cubic centimeters of a Shiga bacillus culture which had been lysed for six days were injected intravenously on August 10. The animal lived. Eabbit no. 6. Three cubic centimeters of the Shiga culture which had been lysed for six days were injected subcutaneously on August 10. This rabbit also lived. Eabbit no. 7. Five cubic centimeters of the Shiga culture which had been lysed for six days were injected intravenously on August 10. The rabbit died on August 21. When the tests were done with a Shiga culture which had been lysed for a month the toxicity had disappeared, as is shown by the following. Rabbit no. 8. Fifteen cubic centimeters of Shiga culture which had been lysed for one month were injected subcutaneously. The rabbit lived. The injection was given on August 10.. Rabbit no. 9. Ten cubic centimeters of this same culture were injected intravenously on August 10. This rabbit lived. The following protocol illustrates an immunization experiment. On August 23, eight rabbits received a subcutaneous injection of 0.25 cc. of a culture of the anti-Shiga bacteriophage, two months after the lysis- was completed. These animals were tested by the injection of 3 cc. of a twenty-four hour bouillon culture of Shiga bacilli. For the strain employed this represented two surely fatal doses. The strain of Shiga used in the test differed from that used to prepare the suspension lysed by the bacteriophage. Rabbit no. 10; tested after twenty-eight hours. Died six days later. Rabbit no. 11 ; tested after four days. Died five days later. Rabbit no. 12; tested after six days. Lived. Rabbit no. 13; tested after eight days. Lived. Rabbit no. 14; tested after ten days. Lived. Rabbit no. 15; tested after one month. Lived. Rabbit no. 16; tested after two months. Lived. Rabbit no. 17; tested after three months. Lived. All the control rabbits inoculated with half the dose, that is, with 1.5 cc. of the Shiga culture, died in from four to seven days. The rabbit is therefore, immunized against two surely fatal doses of B. dysenteriae Shiga culture by the injection of a quarter 264 THE BACTEKIOPHAGE of a cubic centimeter of a culture of the anti-Shiga bacteriophage. The antitoxic immunity is established six days after the injec- tion and persists for at least three months. In an experiment of this kind there can be no question of the nature of the process. The bacteriophage as a hving being can not be the cause of the immunity. The responsible agent must be the soluble principles contained in the culture medium." Before undertaking experiments on man I had to assure myself that the administration of cultures of the anti-Shiga bacterio- phage caused no reaction. First, I ingested increasing quantities of the cultures, aged from six days to a month, from one to thirty cubic centimeters, without detecting the shghtest malaise. Three persons in my family next ingested variable quantities several times without showing the least disturbance. I then injected myself subcutaneously with one cubic centimeter of a forty-day old ciiltiu-e. There was neither a local nor a general reaction. In all the cases, twenty-four hours after the ingestion or after the injection, I was able to isolate from thfe stools a bacteriophage possessing for the Shiga bacillus an activity equal to that of the ultramicrobe administered. More recently G. Ehava has re- ceived by subcutaneous injection 5 cc. of a culture of the anti- Shiga bacteriophage aged thirty days. No reaction, local or general, followed. It is known that the subcutaneous injection of Shiga bacilli, killed by any procedure whatsoever, can not be performed be- cause of the extremely violent reaction^ produced, and which are due to the toxicity of the germ. This is precisely the reason that vaccine prophylaxis is not apphed to dysentery as it is in the case of typhoid. The absolute innocuity of injections of ' Several immunizing experiments with the bacteriophage for B. typhosus and for the paratyphoid organisms have been performed upon laboratory animals, both rabbits and guinea pigs. In all cases these showed a perfect immunization;— provided it is permissible to employ the word immuniza- tion when the process is carried out in refractory animals. Not attributing any value to experiments of this type I have not included them in the monograph. In all cases the bacteriophage administered, either when given by subcutaneous injection or by the buccal route, has been isolated a few hours later from the intestinal tract. IMMUNIZATION BY MEANS OP THE BACTERIOPHAGE 265 the anti-Shiga bacteriophage cultures, which contain the sub- stance of the bacterial bodies in a dissolved state, shows indeed that these substances undergo profound modifications under the influence of the lysins secreted by the bacteriophagous ultrami- crobe. Nevertheless, these ntew substances possess a specific immunizing power much more potent than the original substance. The experiments on rabbits, and in particular the results secured in immunization against barbone, demonstrate this beyond pos- sible doubt. Prophylactic vaccination against baciUary dysentery by means of cultures of the anti-dysentery bacteriophage is therefore apphcable to man. In practice, quite naturally, the prophylactic injections should be performed with a mixture of bacteriophage cultures — anti-Shiga, anti-Flexner, and anti-Hiss. Such a mix- ture would constitute a polyvalent dysentery vaccine. The Shiga bacillus is one of the most toxic organisms known, and it may be assumed that the harmlessness of injections of such a culture indicates a general law, whatever may be the bacterium against Which the bacteriophage culture is prepared. In order to test this hypothesis, I injected myself, subcutaneously, with half a cubic centimeter of anti-plague bacteriophage. No reac- tion, either general or local, followed. Stool examination made twenty-four hours after the injection showed that the bacterio- phage, equal in virulence to that injected, was present. The inoculation experiment was repeated with anti-typhoid bac- teriophage. G. Ehava repeated it with the anti-staphylococcus bacteriophage, and the same results were secured in both cases. These observations are confirmed in part by another fact, ob- served in several tests, that following the adminWration of the bacteriophage, either by injection or by ingestion, the bacterio- phage passes in a short time into the intestine. It is ehminated rapidly if it fails to encounter the bacterium against which it has a virulence, that is to say, in an uninfected individual. On the contrary, it grows and maintains its virulence if it is in contact with this bacterium, a condition which, as we have seen in several instances, is produced in an infected environment among animals which remained healthy, or which had been in- fected and were recovered. 266 THE BACTERIOPHAGE After being assured of the innociiity of the ingestion of cultures of the anti-Shiga bacteriophage, this treatment was apphed for therapeutic purposes to patients affected with bacillary dysentery.' As in the experimental work, so also here in the clinical tests, the therapy has been hmited to those cases in which the etiology of the infection was proved by the isolation of the pathogenic organism, and where, in addition, the virulence of the intestinal bacteriophage was negative toward the different dysentery baciUi at the time of the administration of the culture of bacteriophage. It is evident that in routine practice it would not be necessary to investigate all these points, especially since the administration of the bacteriophage cultures is always inoffensive. In each of the following cases the only treatment instituted has been the ingestion of the culture of the bacteriophage. Bobert K. . . . (eleven years). This is a case of baoillary dysentery of moderate severity with from 5 to 7 bloody stools a day. August 1. The stool examination showed: B. dysenteriae Shiga present. The intestinal bacteriophage with virulences as follows: B. coli + + , Shiga 0, Flexner 0, Hiss 0. August 2. At 10 o'clock in the morning the patient ingested 2 cc. of a anti-Shiga bacteriophage culture. This culture had been lysed for thirty- five days. During the afternoon of this day there were 3 bloody stools, in the evening there was one stool and that was free of blood. August 3. During this day there was only the one formed stool. Exami- nation showed: B. dysenteriae Shiga absent. The intestinal bacteriophage with virulences as follows: B. coli -|--|--|--|-, Shiga +-t-|-f-, Flexner -f-l-+, Hiss ++-I-. * These experiments have been made with the assistance of M. Nadal, on the service of Pr. Hutinel, at the H6pital des Enf ants Malades. Tests have also been made in cases of toxic diarrhea of infants, but thej' will not be discussed here since a conclusion regarding them has not yet been reached. In those cases there is an especial difficulty, for the path- ogenic organism is still unknown. It was at first thought that this might be determined through the ability to isolate and cultivate an active strain of bacteriophage which might be used for curative purposes. It is indeed probable that there is, not one, but several diarrheas of infants caused by different bacterial types, as the experiments of Nob6court made during the past few years would also indicate. The solution of the problem is not impossible but it would be necessary to administer to the affected infants a mixture of cultures of diverse strains of the bacteriophage, active against the diverse bacterial types capable of inciting the diarrhea. It can readily be conceived that under such circumstances the investigation must be protracted. IMMUNIZATION BY MEANS OF THE BACTEHIOPHAGE 267 Augusl; 8. The intestinal bacteriophage was active as follows: B. coli +++, Shiga +, Plexner 0, Hiss +. August 9. The patient was discharged from the hospital. Andr6 B. . . . (ten years). A case of bacillary dysentery of moderate severity. During the period from August 25 to 29 inclusive there were 9 to H bloody stools a day. August 28. Stool examination showed: B. dysenteriae Shiga present. Intestinal bacteriophage active as follows: B. coli +, Shiga 0, Flexner 0, Hiss 0. August 29. At 4 p.m. the patient ingested 2 cc. of an anti-Shiga bac- teriophage culture. The culture had been lysed for two months. August 30. There was one bloody stool in the morning and during the afternoon and the night there were 5 stools, none of which showed any blood. August 31. There were 3 fluid, but not bloody, stools. Examination showed: Shiga bacilli not present. The intestinal bacteriophage active as follows: B. coli + + -!-+, Flexner ++, Hiss +++, Shiga +-1-1-+. September 1. There was one fluid stool, without blood. September 2. There was one fluid stool, without blood. September 3. There was one formed stool. Examination of the intes- tinal bacteriophage showed: B. coli +++, Shiga + +++, Flexner +++, Hiss +. September 8. Reactions with the intestinal bacteriophage were: B. coli ++, Shiga 0, Flexner 0, Hiss +. September 9. The patient was discharged from the hospital. Robert D. . . . (twelve years). This patient had a very severe dysen- tery, with vomiting, cold sweats, chilling of the extremities, and involun- tary and uncountable stools. September 8. The stools could not be counted. They were fetid, purulent, and streaked with blood. Examination showed: B. dysenteriae. Shiga present; about 1 out of every 10 colonies on the plates was the dysen- tery bacillus. The intestinal bacteriophage showed no virulence for B. coli, or for the Shiga, Flexner or Hiss organisms. September 9. Two cubic centimeters of an anti-Shiga bacteriophage culture were ingested at 11 o'clock. This culture had been lysed for three and one-half months. During the afternoon and the night the stools became less numerous but continued bloody. September 10. There were 6 fluid stools, without blood. Examination showed: B. dysenteriae Shiga, not present. Intestinal bacteriophage active as follows: B. coli +-| — | — h, Shiga ++ ++, Flexner ++ ++, Hiss + + + . September 11. There were 2 normal, formed stools. September 20. The patient was discharged from the hospital. 268 THE BACTERIOPHAGE Julien D. . . . (three and one-half years). This was a case of very severe dysentery. The general condition of the patient was very bad. A sister of the patient had died at home of dysentery on September 8. From the 11th to the 13th of September the number of stools, all of which were bloody, could not be counted. September 13. The patient entered the hospital. Examination showed: B. dysenteriae Shiga present, the dysentery bacilli constituting about 4 of every 5 colonies on the plates. The intestinal bacteriophage was without activity for either B. colt or the dysentery organisms. September 13. The patient ingested 2 cc. of anti-Shiga bacteriophage culture at 5 o'clock. This culture had been lysed for fifteen days. September 14. There were 6 bloody stools. The intestinal bacterio- phage showed virulences as follows: B. coli ■\--\ — f-, Shiga -1 — I — f-, Flexner ++, Hiss +. September 15. During the day there was one bloody stool. There were also 5 stools without blood. Examination showed: B. dysenteriae Shiga absent. The intestinal bacteriophage with activities as follows: B. coli -|- + H — l-i Shiga + + ++, Flexner +++, Hiss ++-1-. September 16. There were 4 stools, all without blood. The intestinal bacteriophage showed: B. coli ++++, Shiga +-!-++, Flexner ++, Hiss + ++. September 17. During the day there were one fluid stool and 2 formed stools. The intestinal bacteriophage showed: B. coli ++++, Shiga ++++, Flexner ++, Hiss +. September 18. There were 2 formed stools. The intestinal bacterio- phage was virulent as follows: B. coli ++++, Shiga ++++, Flexner +, Hiss ++. September 26. The patient was discharged from the hospital. On this date the virulence of the intestinal bacteriophage was: B. coli +-1-+, Shiga 0, Flexner 0, Hiss-|-. Emile D. . . . (seven and one-half years). This patient was a brother of the preceding case, and showed a very severe dysentery. On the 11th and 12th of September there were 20 to 25 fetid stools, fluid but not bloody. September 12. Examination showed that the Shiga bacilli were very abundant. The intestinal bacteriophage was inactive for B. coli or for the dysentery organisms. September 13. There were 25 bloody stools. At 5 o'clock the patient ingested 2 cc. of bacteriophage culture. The culture had been lysed for six and one-half months. September 14. There were 4 bloody stools in the morning and 2 stools without blood in the afternoon. Examination of one of the latter showed no Shiga bacilli. The virulences of the intestinal bacteriophage were: B. coli -1-+-I-1-, Shiga -(-f-+-|-, Flexner 4-+, Hiss -|-|-+. IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 269 September 15. There were 5 stools, all free of blood. September 16, 17, and 18. During these days there were 3 or 4 stools a day. None of these contained blood. September 19. There were 2 formed stools on this day. September 28. The patient was discharged from the hospital. In all of these cases the general condition of the patient has always coincided with the severity of the intestinal symptoms. Two other cases of dysentery due to the Shiga bacillus, treated in the same man\ier, but outside of the hospital, gave comparable results. In these there was a cessation of the bloody stools with improvement in the general condition in the twenty-four hours immediately following the administration of the culture of anti- Sfhiga bacteriophage. Obviously, seven cases are not sufficient to afford an absolute proof in favor of the specific therapy of bacillary dysentery by means of bacteriophage cultures. However, they do suffice to show that the ingestion of cultures of a virulent bacteriophage — virulent for the infecting bacillus— is as harmless for the sick as for the healthy person. They also show that the ingested bacteriophage traverses the upper digestive tract in man as it does in animals, and within a few hours will be found in the intestine where it grows at the expense of the bacterium for which it is active. Moreover, these seven cases acquire a significance from the fact that the cultures of bacteriophage restrained the disease, as was the case in avian typhosis, as is shown in the results of the experiments which have been recorded and in experiments bearing on about one hundred cases. All these facts authorize clinicians to continue the experimental treatment on a more elaborate scale, not only in bacillary dys- entery but in other infectious hiiman disease^ for which strains of the bacteriophage have been isolated — typhoid fever, the paratyphoid infections, and bubonic plague.' Whatever may be the nature of the disease under considera- tion, the isolation of a strain of the bacteriophage active for the pathogenic bacterium is easy once it appears in an acute disease " It may also be suggested that the bacteriophage may have an applica- tion in surgery, as in the treatment of wounds or in peritonitis, either as a preventive when an infection is to be feared, or as a therapeutic agent when infection has once appeared. 270 THE BACTERIOPHAGE where the bacterium is known and cultivable. It is only neces- sary to apply the principles which have been discussed in the course of this work.'" '" Since the publication of the French edition of this work Bruynoghe and Maisin at the Bacteriologic Institute of Louvain, and Beckerich and Hauduroy at the Institute of Hygiene at Strasbourg have confirmed these conclusions. Bruynoghe and Maisin have treated with success affections due to the staphylococcus and anthrax by the subcutaneous injection of cultures of the bacteriophage. Beckerich and Hauduroy have experimented with typhoid fever and with pyelocystitis. They have employed cultures of the bacteriophage virulent tor the causative bacterium, heated to 58°C. for thirty minutes. They record the following cases; 1. An adult with typhoid fever of moderate severity. The patient ingested 2 cc. of bacteriophage culture on the 18th day of the disease. Defervescence took place forty-eight hours later. 2. An adult with typhoid fever of moderate severity. The patient received the same treatment, with the same results. The culture was given on the ninth day of the disease. 3. An infant with severe typhoid fever. The patient was given 2 cc. of the bacteriophage culture by mouth and in addition the simultaneous injection of 1 cc. This treatment was given on the twentieth day. The temperature came down within forty-eight hours and the apyrexia was permanent. 4. An infant with a paratyphoid B infection, whose condition was grave. On the ninth day the bacteriophage was given by the simultaneous inges- tion and injection of the culture. After the next day the apyrexia was permanent. 5. An infant with paratyphoid B infection of average severity. On the 23rd day the bacteriophage was given by ingestion and injection. The apyrexia was permanent after the next day. Two adults affected with typhoid fever of the ataxo-adynamic form and with pronounced myocardial involvement were treated. The apyrexia which followed the treatment within forty-eight hours did not prevent death. Considering that in these two cases the failure might be due, either to a too delayed intervention, or to too small a dose in view of the severity of the cases, they decided to increase the quantity of bacteriophage culture administered by ingestion in such cases. 6. An infant affected with typhoid fever in very severe form. On the tenth day the patient was given 5 cc. of culture by mouth and a simul- taneous injection of 1 cc. Within forty-eight hours there was permanent defervescence with euphoria. 7. An infant with a very severe case of typhoid fever. The same treat- ment was given on the fourteenth day. Defervescence with euphoria occurred in forty-eight hours. IMMUNIZATION BY MEANS OF THE BACTERIOPHAGE 271 These authors confirm the absolute harmlessness of the administration of cultures of the bacteriophage, whatever may be the mode of introduction into the organism. The sole reaction which they observed consisted of a sudoral crisis which followed the administration in about two hours, even when the administration was effected by the oral route. They con- sider that this reaction is due to the lysis of the pathogenic bacteria within the body of the patient. This view is certainly correct for this sudoral crisis is not observed in the healthy individual, either after the injection or after the ingestion of cultures of the bacteriophage. This has been demonstrated in several instances. They note the constant coincidence which exists between the admin- istration of the culture and the apyrexia which follows within forty-eight hours. This relationship appears to be independent of the stage of the disease when the culture is given. In all the treated cases blood cultures made forty-eight hours before the intervention were positive, that is, the treatment was always applied at a period when the disease was fully active. Beckerich and Hauduroy have, moreover, treated two cases of puer- peral pyelocystitis due to B. coli by the subcutaneous injection of 1 cc. of an anti-coli bacteriophage culture. In both cases the sudoral crisis followed in two hours, and permanent apyrexia in forty-eight hours. CHAPTER IV The Bacteriophage and Immunitt summary and conclusions The bacteriophage, Bacteriophagum intestinale d'Herelle, 1918, an ultramicrobial parasite of bacteria, normally exists in the intestinfal tracts of animals, both vertebrates and invertebrates. The possibility of counting the ultramicrobes is a most important point in the study of the bacteriophage, for it makes it possible to follow its developmetot and to recognize its mode of action in vitro and in vivo. An obligatory parasite, the bacteriophage Uves only at the expense of living, normal bacteria, which con- stitute its sole cultute medium. Experiments and ultramicro- scopic examination agree in showing that the ultramicrobial bacteriophage penetrates into the interior of the bacterium, there forms a colony of fifteen to twenty-five elements within one to one and one-half hours; whereupon the bacteriimi bursts and liberates the young ultramicrobes. For its development the bacteriophage utilizes the substance of the bacteria which it dissolves by the aid of the lytic diastases which it secretes. The property possessed by the bacteriophage of secreting a lysin, active for a given bacterium, — that which permits it to penetrate this bacterium and to reproduce there — represents, in the strict sense of the word, its virulence for this bacterium. There is but a single species of bacteriophage, common to all animals, capable of acquiring virulence for different bacterial species, probably for all species. Just as for each pathogenic bacterium there is a scale of viru- lence for a given animal, so also for each bacteriophage there is an individual virulence. We are able to increase or attenuate the virulence of the pathogenic bacterium, and the same phenom- enon can be obtained with the bacteriophage. The parasitized superior organism defends itself against the bacterial secretions and is able to acquire an antitoxic immunity; the bacterium at- 272 THE BACTERIOPHAGE AND IMMUNITY 273 tacked by the bacteriophage does not remain passive. It resists, and is able to overcome the ultramicrobe and to acquire an anti- lytic immunity. All the vicissitudes of the struggle between the animal and the bacterium have their counterpart in the struggle between the parasitizing bacteriophage and the bacterium at- tacked. The resemblance is complete. It is simply a matter of descending a degree in the order of size of the beings involved. The existence of the bacteriophage in the intestine of all liv- ing beings, its exiguity which allows it to filter through soils impermeable to bacteria, its vitaUty and resistance to agents of destruction, explain its extreme diffusion in nature. When derived from the organism a single strain of the bac- teriophage is rarely active for but a single bacterial species. Usually it attacks several species at one and the same time, and for each it possesses a separate and variable virulence. There is but one bacteriophage but there is an ini&nity of strains, each possessing, when taken from the organism, the power of attack- ing a certain niunber of bacteria. A single strain is variable from time to time, both as to its intensity of action against each bac- terial species, and as to the extent of its action with regard to the number of bacterial species attacked. All combinations of virulence being possible in quantity as in quality, it can be im- derstood in view of the infinite nxunber of possible combinations, that there can exist no two strains of ultramicrobial bacterio- phage which can be exactly alike. In a bacterial suspension inoculated with a culture of an active strain of bacteriophage it is not always the latter which prevails. If the bacterium succeeds in acquiring a resistance a selection of more and more resistant bacteria occurs, resulting in the forma- tion of a state of equiUbrium between the resistant bacterium and the virulent bacteriophage which then coexist in the medium. A mixed culture results in which the equihbrivim is more or less stable but which can be overthrown in favor of the one or the other of the germs there present, according to the circumstances of the moment. The acquisition of resistance is accompanied by changes in morphology and in the properties of the bacteria; the baciUi take a coccoid aspect and become sxirrounded by a capsule. 274 THE BACTEBIOPHAGE They become inagglutinable. They resist phagocytosis. They are endowed with a very great vitaUty and a very high vinilence. Loss in resistance is accompanied by a retiirn to normal form and properties. Although the bacteriophage is capable of acquiring a virulence for the bacterium, the bacteriimi on its side is capable of acquir- ing a resistance to the bacteriophage. The virulence of the one and the resistance of the other are not fixed, but are essentially variables, being enhanced or attenuated according to the in- herited properties of each of the two germs, and according to the circumstances of the moment which favor the one or the other of the two antagonists. These two phenomena dominate the pathogenesis and the pathology of infectious diseases. The bacteriophagous ultramicrobe is a normal inhabitant of the intestine, an obhgatory parasite, and there maintains itself at the expense of saprophytic bacteria hereditarily endowed with a certain resistance, with which it lives in commensahsm. For any bacterium whatever, pathogenic or not when introduced into the intestine, the bacteriophage exalts its virulence toward the invader, and this so much the more rapidly when this bac- terium is lacking in resistance and when the bacteriophage is hereditarily the more adapted to the struggle. The more fre- quent the reinfections by a given bacterium, the more hkely is the bacteriophage to quickly acquire a degree of virulence suffi- cient to inhibit aU growth. If the bacterium which invades an individual is the agent of an intestinal infection or if the avenue of infection is intestinal, the infectious process is then prevented at its very inception and the disease aborts before morbid sj^nptoms appear. The rapid adaptation of the bacteriophage may be delayed or even prevented by unfavorable circmnstances. More sensitive than the bacteria to the action of acids and alkahes, the reaction of the medium is a principal factor which influences the develop- ment of the bacteriophage and its power of attack. On the other hand, its activity may be annihilated if the invading germ is de- rived from an organism in which it has been in conflict with the bacteriophage, a conflict which has allowed it to acquire some degree of resistance. In either the one or the other of these cases THE BACTERIOPHAGE AND IMMUNITY 275 the pathogenic bacterium grows and disease results. If the en- vironmental conditions remain unfavorable and inhibit the action of the bacteriophage, the bacterium develops freely and the invaded individual succumbs quickly, or the conflict may become estabUshed, with the virulence of the bacteriophage gradually increasing by selection and the resistance of the bac- terium likewise increasing as the result of a similar selective process. The condition of the individual in which this con- flict is taking place faithfully reflects the changes in the struggle. Convalescence is estabHshed only at the moment when the virulence of the bacteriophage effectively dominates the resistance of the bacterium. If the opposite results, if the bacterium acquires a refractory state, no further barrier is opposed to the invasion of the individual and death follows. In a word, recovery is always a result of the exaltation of the virulence of the bacteriophage, an increase sufficient to permit it to parasitize and destroy the pathogenic bacteria implanted in the body. Death takes place, either as a result of inertia in the bacteriophage, or because of the acquisition of a refractory state by the bacteria, conditions which, in either case, allow the latter to develop without hindrance. There is, however, a third possibility. One may isolate from the intestinal contents of "baciflus carriers," typhoid or dysen- tery, and indeed constantly, a resistant pathogenic bacterium and a bacteriophage virulent for this bacterium. There has been a commensality (as is the case with the normal saprophytes of the intestine), a mixed culture. Usually this equilibrium is quickly broken in favor of the bacteriophage and the carrier state ends. But in individuals who become carriers the conflict car- ried on in the intestine between the bacteriophage and the bac- terium is sufficiently long to permit the development of an organic immunity. The bacteria act on the organism only by means of their toxins. A bacterium whose toxin does not exercise any action on the cells of an animal is as inoffensive for it as a bacterium naturally atoxic. From the time when an organic immunity is acquired by the carrier the pathogenic bacterium becomes for him a saprophyte. One can comprehend, on the other hand, the danger of con- tamination from carriers, who, although they distribute a viru- 276 THE BACTERIOPHAGE lent bacteriophage also at the same time distribute a resistant bacterium, that is to say, a bacterium partictilarly apt to nega- tive the defense exercised by the intestinal bacteriophage of the susceptible individuals contaminated by the carrier. The observations made in pyelonephritis lead us to beUeve that the individual affected with a chronic infectious disease is in reality an internal carrier. Here also, the individual enjoys an antitoxic immunity. Nevertheless there is a struggle within the organism between the virulent bacteriophage and the re- sistant bacterium. For while in the intestine the bacteriiun is henceforth inoffensive and offers no great inconvenience to the host, the presence of a bacterial culture within the tissues is not an indifferent matter because of the inflammatory reactions which it provokes. In addition, phagocytosis is not able to play an active r61e, for we have seen that bacteria which have acquired a resistance to the bacteriophage are likewise resistant to the phagocytic phenomenon. The r61e of the bacteriophage is not confined to the intestine. Whatever may be the infectious disease under consideration, there is always the introduction of the pathogenic bacterivun into the intestine, either by the digestive path or by the hepatic route. Thus, the intestinal bacteriophage may come in contact with, and acquire a virulence for, the pathogenic bacterium. Further- more, experiment shows that the bacteriophage may enter the circulation in the case of a septicemia. Hence it may exert its action at any point in the body. The action of the bacteriophage manifests itself in still another way. Growing at the expense of the bacteria it dissolves them. The bacterial substance, dissolved and modified under the action of the lysins secreted by the bacteriophage, is in a physical and chemical state particularly suited to act upon the cells of the organism which elaborate the antitoxins. Finally, experiment shows that the bacteriophage exercises a preponderant action on phagocytosis. On one side is the fact that the bacteriophage through its lysins possesses an ex- tremely high opsonic power* and on the other side, a bacterium > May not the lysins of the bacteriophage and the antilysins of the bac- teria be synonyms for opsonins and aggressins? THE BACTERIOPHAGE AND IMMUNITY 277 resisting the bacteriophage is, by this fact, also resistant to phago- cytosis. The bacteriophage, the direct agent of antimicrobial immunity which is by its nature heterologous, at least in the sensitive animal, is also indirectly an agent of organic immunity, by na- ture homologous. The history of the disease is in effect the history of the con- flict between the bacteriophage and the bacterium. We can observe that the same facts hold true, but on a larger scale, in the history of an epidemic. The virulent ultramicrobe, which is present in the intestine of all convalescents, is dis- seminated by them with their dejections. It is then capable of "contaminating" susceptible neighboring individuals quite regardless of whether the disease with which it is associated is intestinal, septicemic, or locaUzed in its nature. Observation shows that in the last analysis the history of an epidemic registers the variations in the struggle between the two agents, the pathogenic bacterium and the bacteriophagous ultramicrobe. It is also clear that the latter is transmissible from individual to individual. The immunity is contagious in the same degree as is the disease itself. The beginning of an epidemic is marked by the diffusion of a bacterium whose viru- lence is increased progressively by passages through susceptible individuals. Thus the epidemic extends. In its turn the ul- tramicrobial bacteriophage increases in virulence for the patho- genic bacterium, and extends equally. The epidemic ceases when all susceptible individuals have been infected by the viru- lent bacteriophage. We have seen that the bacteriophage may conserve for a long time a "latent" virulence for a given bacterium. These latent virulences, maintained moreover by accidental contaminations, explain the difference which exists between the mode of propaga- tion of sporadic diseases and of epidemic diseases. Against the bacteria, agents of the first, the bacteriophage is always ready to intervene, and it is only exceptionally that infection is followed by disease. In the second, on the contrary, particularly since the agent is most often imported, the bacteriophage does not at the beginning possess a specific virulence. The epidemic extends 278 THE BACTERIOPHAGE and only stops, or assumes a sporadic character, when there has been a diffusion of a bacteriophage virulent for the pathogenic bacterivun. The bacteriophagous ultramicrobe virulent for a given bac- terium is cultivable in vitro. It is therefore possible to obtain it in any desired amount. If its protective power is real a sus- ceptible individual should be rendered immune by inoculation, just as though he had naturally resisted the contagion. This has been demonstrated to be the case in the experiments made on avian typhosis and in hemorrhagic septicemia in the buffalo. The injection of an individual with a culture of the bacterio- phage virulent for a given bacterium is harmless and causes no reaction, even when the bacteriophage has developed at the ex- pense of a highly toxic bacterium — B. dysenteriae or B. pestis, for example. The injected ultramicrobes pass quickly into the intestine. The injection of a culture of the bacteriophage provokes two types of immunity, heterologous and homologous. The heterologous antimicrobial immunity is effective immediately. Indeed, it exists simply by virtue of the presence in the body of a bacteriophage active for the causative bacterium. In an un- contaminated or non-epidemic area this immunity is transitory. In a contaminated or epidemic area it persists as long as rein- fections occur. The homologous, or organic immunity, develops after an incu- bation period. It results from the production of specific anti- bodies, most hkely substances similar to the antitoxins. These antibodies are detectable in the serum of the immunized animal and persist there for a period at present imdetermined (more than seven months in the case of avian typhosis). The period of incubation in the homologous immunity is the longer as the dose injected is greater. The immunization experiments against barbone have shown us the importance of the question of dosage. The quantity of bacteriophage culture necessary and sufficient to provoke or- ganic immunity ought, in all cases, to be injected at a single time. As to the dose itself, it must certainly vary in accordance with the disease under consideration, and, consequently, with the THE BACTERIOPHAGE ANJ) IMMUNITY 279 bacterium for which the bacteriophage injected is virulent.^ In hemorrhagic septicemia of the buffalo the optimum single dose is a quarter of a cubic centimeter per hundred kilograms of body weight. This question of dosage must be fixed by prelimi- nary experiments for the other diseases.' With disease once declared, the introduction into the patient of the ultramicrobe virulent for the causative bacterium ought to place the affected individual in a condition analogous to that of the convalescent individual. The experiments in avian ty- phosis and in human dysentery show in effect that the ingestion or the injection of cultures of the bacteriophage exert a curative action. The administration to a patient of an active culture of bac- teriophage ought, as may be conceived, to be made at a time as near as possible to the beginning of the disease. For this there are two reasons. 1. We have seen that the acquisition of virulence in the bac- teriophage only represents one side of the question of recovery. The bacterium may acquire a state of resistance such that the action of the bacteriophage may be rendered inoperative. The administration of a culture of the active bacteriophage should have the more effect when the resistance of the bacterium is the least. On the other hand, the acquisition of resistance is the result of the conflict within the individual. Thus, the more rapid the intervention the less likely will be the formation of a resistant bacterial race. 2, If there exists at the time of intervention organic lesions incompatible with life the issue of the disease can not be other than fatal whatever the power of the bacteriophage.* ^ It should be noted that here we are dealing with injection only. The ingestion of cultures of the bacteriophage does not appear to be attended by the development of an organic immunity. Ingestions can be repeated without inconvenience, as I have demonstrated on myself. 3 It should be emphasized that the cultures of the bacteriophage used in immunization should be perfectly limpid; that is to say, the lysis ought to be complete. Filtration through a bougie is essential, for the reason which we have seen. If necessary, filtration may be replaced by heating at S8°C., but filtration is to be preferred. * The cases recently described by Beckerich and Hauduroy, which were mentioned in the note at the end of the preceding chapter, corroborate 280 THE BACTERIOPHAGE To summarize: Observation and experiment agree in showing that the bacteriophage is the direct agent of antibacterial immunity in the sensitive animal. It dissolves the bacteria at the expense of which it reproduces itself, and does this by means of lysins which it secretes and which remain in the solution once the bac- teria are destroyed. These lysins enjoy, furthermore, an extremely high opsonic power, which may hkewise contribute, in certain cases, to the destruction of the pathogenic bacteria. The bacteriophage also contributes to the estabhshment of organic immunity. The bacterial substance dissolved under the action of the lysins is in a physical and chemical state such that an extremely minute quantity suffices to provoke the forma- tion of a potent organic immunity. this statement. I state precisely then, and I insist on this point, that the treatment by the bacteriophage of any case of acute infection ought to be undertaken at once, without the loss of a minute. Whatever may be the disease it is useless and dangerous to await the results of laboratory examinations destined to confirm the clinical diagnosis. This last ought to be considered as sufficient to warrant the administration of the bacterio- phage. Such practice does not incur any risk whatever, even though there has been an error in the diagnosis, for the injection or the ingestion of cultures of the bacteriophage is in all cases absolutely innocuous. I would say further, even if the clinical diagnosis proves erroneous the adminis- tration of a bacteriophage avirulent for the causative bacterium may be useful. While in Indo-China, at three different times, I administered to cholera patients, per as, two cubic centimeters of an anti-Shiga bacterio- phage, and two of the three cases recovered. I do not affirm that this fortunate result could be referred to the administration of the anti-Shiga bacteriophage, although there is a strong presumption in favor of this hypothesis. In fact, of the 113 cases of cholera which I observed during my stay there, I did not see a single case recover spontaneously. In any event, even if it be but a coincidence, it is possible to affirm that the admin- istration of the bacteriophage caused no harm in the cholera cases. In so far as typhoid fever is concerned, for example, I would recommend the removal of the blood necessary for culture at the entrance of the patient into the hospital (or at the first visit of the physician treating the case) and the immediate administration of a culture of bacteriophage. Either two or five cubic centimeters may be given per as, or one cubic centimeter may be injected subcutaneously. In this way a bacteriologic diagnosis may be established without necessitating delay in treatment. In a word, whatever may be the disease, the absolute principle ought to be "never lose a minute." THE BACTEBIOPHAGE AND IMMUNITY 281 The immunity acquired as the result of a single injection of a small quantity of bacteriophage culture is accompanied by the appearance in the blood of a protective principle. The animal which receives this blood enjoys a solid inamunity, specific in nature, and identical with that possessed by the animal which received the immunizing injection of bacteriophage. The pro- tective principle is probably an antitoxin. It is possible that this new method of obtaining immunizing sera offers a means of intervening in liie course of a disease, even in cases where the administration of the active culture of bacteriophage may be without effect because of the previous acquisition of a resistance by the bacterium. Our knowledge of the bacteriophage, a cultivable agent of immunity, allows us to entertain the possibility of collective in- tervention in epidemics. Whatever may be the epidemic (provided, of course, the agent is known and cultivable) we have first the possibility of individual vaccination by means of a single injection of a small quantity of bacteriophage culture active for the causative bacterium. But we have seen that the presence in the intestine of active ultrami- crobes assures the protection of a susceptible individual. We are then able to consider the possibility of collective immuniza- tion of the population, for it would be easy to mix cultures of the bacteriophage with the drinking water, especially in urban centers. One might then be assured of an active bacteriophage in the intestine of all susceptible individuals throughout the critical period. The method offers no risk; the cultures can be ingested without inconvenience in any quantity. In spite of the fact that I have specified at several times in the course of this work that the experiments undertaken deal only with antibacterial immunity in the susceptible individual, I want in closing, in order to avoid confusion, to say a few words on the subject of phagocytosis, for it would be strange if, speaking of antibacterial immunity, I made no allusion to this mode of defense. I do not oppose any of the conclusions of Metchnikoff touching the r61e of phagocytosis in the natural immunity which character- izes the refractory state. In acquired immunity also, Metchni- 282 THE BACTERIOPHAGE koff and his collaborators affirm that phagocytosis plays a capital r61e. That the ehmination of bacteria is effected by phagocytosis, once organic immunity is established, would appear to be the proper interpretation. However, in the one or in the other case, the bacteriophage manifests its action. Its activity is not naturally Hmited to the bacteria pathogenic for a given animal; it is exercised without distinction against bacteria pathogenic and saprophytic, in all circumstances, and in all animals. Even if, in the immune animal, the bacteriophage should remain inert, the bacteria would none the less be eliminated by phagocj^osis. What then is the role of the bacteriophage in immunity? The defense of the susceptible individual exposed to infection, and the protection of the organism in the course of natural disease. Par- asitic of bacteria, the bacteriophage intervenes directly to destroy the pathogenic bacteria which venture to invade the organism; secreting lysins endowed with a powerful opsonic action, it ren- ders possible the education of the phagocyte and introduces the establishment of organic antibacterial immunity; dissolving the bacteria, it transforms the bacterial substance and places it in a physical and chemical state where it can stimulate the cells of the body which produce the antitoxic antibodies, it introduces thus, the establishment of organic antitoxic immunity. In other terms, the bacteriophage plays a preponderating r61e in all the phenomena of immunity which are accomplished in a susceptible individual. As a result of its presence it follows that, although exposed to infection it is possible to remain unharmed; and although sick, it is possible to recover. BIBLIOGRAPHY List of Communications on the Bacteriophage by F. d' Hebelle and His Associates (1) d'Hebelle, F. : Sur un microbe invisible antagoniste des bacilles dysenUriques. Compt. rend. Acad, sci., 1917, 165, 373. (2) d'Herelle, F. : Technique de la recherche du microbe fiUrant bacUrio- phage (Bacieriophagum intesiinale). Compt. rend. Soc. de biol., 1918, 81, 1160. (3) d'Herelle, F. : Sur le rdle du microbe fillrant bacteriophage dans la dysenterie bacillaire. Compt. rend. Acad, sci., 1918, 167, 970. (4) d'Herelle, F. : Du rdle du microbe fillrant bacteriophage dans la fiivre iyphoide. Compt. rend. Acad, soi., 1919, 168, 631. (5) d'Herelle, F. : Sur une epizootic de typhose aviaire. Compt. rend. Acad, sci., 1919, 169, 817. (6) d'Herelle, F. : Sur le rdle du microbe bacteriophage dans la typhose aviaire. Compt. rend. Acad, sci., 1919, 169, 932. (7) d'Herelle, F. : Sur le microbe bacteriophage. Compt. rend. Soc. de bid., 1919, 82, 1237. (8) d'Herelle, F. : Sur la resistance des bacteries li I'action du microbe bacteriophage. Compt. rend. Soc. de biol., 1920, 83, 97. (9) d'Herelle, F. : Le processus de defense contre les bacilles intestinaux et Vetiologie des maladies d'origine intestinale. Compt. rend. Acad, sci., 1920, 170, 72. do) d'Herelle, F. : Sur le microbe bacteriophage. Compt. rend. Soc. de biol., 1920, 83, 247. (11) d'Herelle, F. : Sur le microbe bacteriophage. Compt. rend. Soc. de biol., 1920, 83, 1318. (12) d'Herelle, F. : Sur la nature du principe bacteriophage. Compt. rend. Soc. de biol., 1920, 83, 1320. (13) Bablet, J. : Sur le principe bacteriophage de d'Herelle. Compt. rend. Soc. de biol., 1920, 83, 1322. (14) d'Herelle, F. : Le microbe bacteriophage, agent d'immunite dans la pest et le barbone. Compt. rend. Acad, sci., 1921, 172, 99. (15) d'Herelle, F. : Sur la nature du bacteriophage {Bacieriophagum intestinale de d'Herelle, 1918). Compt. rend. Soc. de biol., 1921, 84, 339. (16) d'Herelle, F. : Phinomhnes cmncidant avec V acquisition de la resist- ance des bacteries d I'action du bacteriophage. Compt. rend. Soc. de biol., 1921, 84, 384. 283 284 THE BACTERIOPHAGE (17 (18 (19 (2o: (21 (22; (23; (24 (25 (26: (27: (28: (29: (3o: (31 (32 (33 (34: (35: d'Heeelle, F. : R6le du bacteriophage dans I'immuniU. Compt. rend. Soc. de biol., 1921, 84, 538. d'Heeelle, F. and Eliava, G. : Sur le strum anti-bactiriophage. Compt. rend. Soc. de bid., 1921, 84, 719. Eliava, G. and Pozerski, E. : Sur les caractbres nouveaux prisentis par le bacille de Shiga ayani risisU A I'action du bactiriophage de d'Herelle. Compt. rend. Soc. de biol., 1921, 84, 708. d'Heeelle, F. : Sur I'historique du bactiriophage. Compt. rend. Soc. de biol., 1921, 84, 863. d'Heeelle, F. : Sur la nature du bactiriophage. Compt. rend. Soc. de biol., 1921, 84, 908. d'Heeelle, F.: Le bacteriophage: son rdle dans I'immuniti. Presse m6d., 1921, 29,463. Eliava, G. and Pozerski, E.: De I'action destructive des sels de quinine sur le bactiriophage de d'Herelle. Compt. rend. Soc. de biol., 1921, 86, 139. d'Herelle, F. : Le bactiriophage. La Nature, 1921, Oct. 1, p. 219. d'Herelle, F. and Eliava, G. : Uniciti de bactiriophage: sur la lysine du bactiriophage. Compt. rend. Soc. de biol., 1921, 85, 701. d'Heeelle, F. : L'ultramicrobe bactiriophage. Compt. rend. Soc. de biol., 1921, 85, 767. d'Herelle, F. and Pozerski, E.: Action de la tempirature sur le bactiriophage. Compt. rend. Soc. de biol., 1921, 85, 1011. d'Herelle, F. : Sur les anti-lysins d'origine baetirienne. Compt. rend. Soc. de biol., 1922, 86, 360. d'Heeelle, F. : Sur la presence du bactiriophage dans les leucocytes. Compt. rend. Soc. de biol., 1922, 86, 477. Communications by Othee Authors Damadb, E.: Le microbe filtrant bactiriophage de d'Herelle. Thfese (in medicine), Bordeaux, 1919. Kabeshima, T. : Becherches expirimentales sur la vaccination priventive contre le bacille dysentirique de Shiga. Compt. rend. Acad, sci., •1919, 169, 1061. Kabeshima, T. : Thirapie expirimentale des porteurs de germes. Compt. rend. Acad, sci., 1920, 170, 71. Kabeshima, T. : Sur un ferment d'immuniii bactiriolysant, du mican- isme d'immuniti infectieuse intestinale, de la nature du dit "microbe filtrant bactiriophage" de d'Herelle. Compt. rend. Soc. de biol., 1920, 83, 219. Kabeshima, T. : Sur le ferment d'immuniti bactiriolysant. Compt. rend. Soc. de biol., 1920, 83, 471. MfiTALNiKOv, S.: B. dysentirique et bactiriophage de d'Herelle chez les chenilles de Galleria mellonella. Compt. rend. Soc. de biol. 1920, 83, 667. BIBLIOGBAPHY 285 (36) BoRDET, J. AND CitTCA, M. : Exsudats leucocytaires et autolyse micro- bienne transmissible. Compt. rend. Soc. de biol., 1920, 83, 1293. (37) BoKDET, J. AND CiucA, M. : Le bactSriophage de d'Herelle, sa produc- tion et son interpretation. Compt. rend. Soc. de bid., 1920, 83, 1296. (38) Dumas, J. : Sur la presence du bactSriophage dans I'intestin sain, dans la terre et dans I'eau. Compt. rend. Soc. de biol., 1920, 83, 1314. (39) Debr£, R. and HAGTJENAtr: Quelques particularities du "phSnomkne de d'Herelle." Compt. rend. Soc. de biol., 1920, 83, 1348. (40) WoLLMANN, E. : A propos de la note de MM. Bordet et Ciuca (PhSno- mine de d'Herelle, autolyse microbienne transmissible de J. Bordet et M. Ciuca, et hypothbse de la panginkse de Darwin). Compt. rend. Soc. de biol., 1920, 83, 1478. (41) Salimbeni: Sur le bacteriophage de d'Herelle. Compt. rend. Soc. de biol., 1920, 83, 1545. (42) Salimbeni: Sur la nature du bacteriophage de d'Herelle. Compt. rend. Acad, sci., 1920, 171, 1240. (43) De Poorteb and Maisin: Contribution d, I'Hude de la nature du prin- cipe bacteriophage. Arch, internat. pharmocodyn., 1921, 25, 473. (44) WoLLMANN, E. : Sur le phSnomene de d'Herelle. Compt. rend. Soc. de biol., 1921, 84, 3. (45) Gbatia, a.: Influence de la reaction du milieu sur V autolyse micro- bienne transmissible. Compt. rend. Soc. de biol., 1921, 84, 275. (46) BoBDET, J. and Ciuca, M. : Determinisme de I'autolyse microbienne transmissible. Compt. rend. Soc. de biol., 1921, 84, 276. (47) BoEDBT, J. AND CiucA, M. : Spedficite de I'autolyse microbienne transmissible. Compt. rend. Soc. de biol., 1921, 84, 278. (48) BoBDET, J. AND CiuCA, M. : Autolyse microbienne et sirum antilytique. Compt. rend. Soc. de biol., 1921, 84, 280. (49) KuTTNEH, Anne: Preliminary report on a typhoid bacteriophage. Proc. Soc. Exper. Biol. & Med., 1920/21, 18, 158. (50) Maisin, J. : Au sujet de la nature du principe bacteriophage. Compt. rend. Soc. de biol., 1921, 84, 467. (51) Maisin, J.: Adaptation du bacteriophage. Compt. rend. Soc. de biol., 1921, 84, 468. (52) BoBDET, J. AND CiucA, M. I Evolution des cultures de coli lysogbne. Compt. rend. Soc. de biol., 1921, 84, 747. (53) Bordet, J. and Ciuca, M. : Remarques sur I'historique des recherches concernant la lyse microbienne transmissible. Compt. rend. Soc. de biol., 1921, 84, 745. (54) BoBDET, J. AND CiucA, M. : Guerison et retour d I'etat primitif par le serum antilytique du coli lysoghne. Compt. rend. Soc. de biol., 1921, 84, 748. (55) Gratia, A.: De I'adaptation hereditaire du Colibacille d I'autolyse microbienne transmissible. Compt. rend. Soc. de biol., 1921, 84, 750. 286 THE BACTERIOPHAGE (56) Gratia, A. : Dissociation d'une souche de Colihacille en deux types d'individus de propriites et de virulence differentes. Compt. rend. Soc. de bioL, 1921, 84, 751. (57) Gratia, A.: De la signification des "colonies de baclSriophage" ded'Her- elle. Compt. rend. Soc. de biol., 1921, 84, 753. (58) Gratia, A.: Sur la spicificite du principe lytique. Compt. rend. Soc. de biol., 1921, 84,755. (59) Maisin, J.: Au sujet du principe bactSriophage et des anticorps. Compt. rend. Soc. de biol., 1921, 84, 755. (60) Gratia, A.: Preliminary report on a staphylococcus bacteriophage. Proc. Soc. Exper. Biol. & Med., 1920/21, 18, 217. (61) KuTTNER, Anne : Ontheinfluenceof tissue enzymes on the bacteriophage principle. Proc. Soc. Exper. Biol. & Med., 1920/21, 18, 222. (62) BRtTTNOGHE, R. AND Maisin, J.: Au sujet des microbes devenus rSsistants au principe bacteriophage. Compt. rend. Soc. de biol., 1921, 84, 847. (63) Bail, O.: Das bakleriophage Virus von d'Herelle. Wien. klin. Wchnsclar., 1921, 34, 237. (64) BHtTTNOGHE, R. : Au sujet de la guSrison des germes devenus rSsistants au principe bacteriophage. Compt. rend. Soc. de biol., 1921, 86, 20. (65) Gratia, A.: L'autolyse transmissible du Staphylocoque et I'action coagulante des cultures lysies. Compt. rend. Soc. de biol., 1921, 85, 25. (66) Gratia, A. : Autolyse transmissible et variations microbiennes. Compt. rend. Soc. de biol., 1921, 85, 251. (67) BRtTTNOGHE, R. : Au sujet de la nature du principe bacteriophage. Compt. rend. Soc. de biol., 1921, 85, 258. (68) Gratia, A.: Studies on the d'Herelle phenomenon. J. Exper. Med., 1921, 34, 115. (69) Bail, O. : Bahteriophage-viirkungen gegen Flexner- und Koli-Bak- terien. Wien. klin. Wchnschr., 1921, 34, 555. (70) GiLDEMEisTEK, E. : Ueber das d'Herelle' sche Phanomen. Berl. klin. Wchnschr., 1921, 58, 1355. (71) Appelmans, R.: Le bacteriophage dans I'organisme. Compt. rend. Soc. de biol., 1921, 85, 722. (72) de Neckbb, J. : Au sujet de I'action inhibitive du principe bactSriophage sur le dSveloppement des microbes rSceptifs. Compt. rend. Soc. debioL, 1921,85,742. (73) WoLLMANN, E. AND GoLDENBERO, L. t Lc phSnomhnc de d'Herelle et la reaction de fixation. Compt. rend. Soc. de biol., 1921, 85, 772. (74) WoLLSTEiN, M. : Phenomenon o/ d'Herelle with Bacillus dysenteriae. J. Exper. Med., 1921, 34, 467. (75) Gratia, A. and Jaumain, D. : IdentitS du phSnomhne de Twort et du phSnomkne de d'Herelle. Compt. rend. Soc. de biol., 1921, 85, 880. BIBLIOGRAPHY 287 (76) Gratia, A. and Jaumain, D. : DualiU du principe lytique du Coli- bacille et du Staphylocoque. Compt. rend. Soc. de bioL, 1921, 85, 882. (77) BoHDET, J. AND CiXTCA, M. : SuT la rigeniration du principe actif dans I'autolyse microhienne. Compt. rend. Soc. de bioL, 1921, 85, 1095. (78) Appelmans, R. : De dosage du bacteriophage. Compt. rend. Soc. de biol., 1921, 85, 1098. (79) Brutnoghe, R. and Maisin, J. : Le principe bactSriophage du Staphy- locoque. Compt. rend. Soc. de biol., 1921, 85, 1118. (80) Brutnoghe, R. and Maisin, J.: Essais de tMrapeutique au moyen du bacteriophage du Staphylocoque. Compt. rend. Soc. de biol., 1921, 86, 1120. (81) Brutnoghe, R. and Maisin, J. : Au sujet de I'unite du principe bac- teriophage. Compt. rend. Soc. de biol., 1921, 85, 1122. (82) Beckerich, a. and Haudurot, P. : Au sujet du titrage du bacterio- phage. Compt. rend. Soc. de biol., 1922, 86, 165. (83) Beckerich, A. and Haudurot, P. Le bacteriophage dans le traite- ment de la fibvre typhmde. Compt. rend. Soc. de bioL, 1922, 86, 168. (84) Gratia, A.: La lyse transmissible du Staphylocoque. Sa production, ses applications thirapeutiques. Compt. rend. Soc. de biol., 1922, 86, 276. (85) Brutnoghe, R. and Maisin, J.: La phagocytose du bacteriophage. Compt. rend. Soc. de biol., 1922, 86, 292. (86) Brutnoghe, R. and Maisin, J. : Au sujet de la reaction consecutive d, I'injection du bacteriophage. Compt. rend. Soc. de biol., 1922, 86, 294. (87) BoRDET, J. AND CiucA, M. : Sur la tUorie du virus dans la lyse micro- bienne transmissible et les conditions de regeneration du principe actif. Compt. rend. Soc. de biol., 1922, 86, 295. (88) LisBONNE, BouLET AND CARRi;RE : Sur I'obtention du principe bacterio- phagique au moyen d'exudats leucocytaires in vitro. Compt. rend. 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