fowl. typhi, b. paratyphi a, b. paratyphi b, b. gallinarium, and the bacterium of barbone....

THE BACTERIOPHAGE IN RELATION TO SALMON-ELLA PULLORAl INFECTION IN THE DOMESTIC

FOWLNORMAN J. PYLE

Department of Veterinary Science and Animal Pathology, Massachusetts Agri-cultural Experiment Station, Amherst, Maseachusetts

Received for publication December 9, 1925

It has been observed by Hankin (1896), Twort (1915), d'Herelleand his associates (1922), and others that Berkefeld filtrates ofstools, tissues, and other cultures would kill and dissolve youngcultures of dysentery and many related organisms. A transferof one of these dissolved cultures to a new culture would cause itin turn to dissolve. Thus this lytic or dissolving principle couldbe transferred from generation to generation. These bacterio-lytic filtrates or their subsequent generations are called, in ourpresent literature on the subject, bacteriolytic agents, bacterioly-sants, or bacteriophages. The process by which these filtratesdissolve many organisms is known as the d'Herelle phenomenonor bacteriophagy.

Since d'Herelle's (1922) early work on the bacteriophage hadso much to do with the identification of the lytic principle inrelationship to the organisms of the intestinal groups of bacteria,orignating in disease and in healthy individuals of both thehuman and animal subject, together with the study of specificavian pathogens, it was considered of importance from the stand-point of comparative pathology, to seek for a lytic principle inthe domestic fowl against Salmonella pullora (Bacterium pullorumRettger). This organism is of great economic importance to thepoultry industry and shouldbestudied and eliminated from layinghens, thus protecting the progeny and improving the food supplyfor human consumption.

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The organisms especially studied were those of dysentery(d'Herelle, 1917), typhoid and paratyphoid, (d'Herelle, 1919a)fowl typhoid, (d'Herelle, 1919b, c) and pest and barbone(d'Herelle, 1921). D'Herelle found, when examining the stools ofdysentery patients, especially from convalescents, that a sus-pension of Shiga bacilli became limpid, and later found that thisunusual condition was due to a lytic principle, whidh could betransmitted in series, its lytic activity or virulence, as d'Herellestyled it, being increased from generation to generation. He wasof the opinion that the recovery of these dysentery patients wasdue to the action of this bacteriophage upon the intestinal infec-tion in vivo. From this work and from the work of McKinley(1922, 1923), Gratia and de Namur (1922), Piorkowski (1922),Watanabe (1922), Davidson (1922a), Kuttner (1923), Beckerichand Hauduroy (1923), Le Blaye (1923), Pauron (1923), King(1923), Lehndorff (1924), Callow (1922), although data andopinions vary, there are indications that the bacteriophage maybe used therapeutically. In the hands of many European investi-gators, especially Beckerich and Hauduroy (1923) and Bruynogheand Maisin (1921), the bacteriophage has given great promiseas a therapeutic reagent. Bruynoghe and Maisin applied thebacteriophage in staphylococcic infections. After injections of0.5 to 2 cc. of lysate, improvements were noted in twenty-fourto forty-eight hours. Gratia claims that in cases of abscesses andfuruncles the process of cure is noticeably accelerated by theadministration of the lysate (bacteriophage).The work of Beckerich and Hauduroy is in accord with that of

Bruynoghe and Gratia and in conclusion they state that bacteri-ophagy applied therapeutically will be extensive and varied. Onthe other hand Callow (1922) finds that a bacteriophage principletransmissible in series against Staphylococcus aureus has beendemonstrated in the pus of a series of sixteen staphylococcicinfections. In six, it proved lytic for one or more heterologousstrains, but not for the homologous strain, in spite of repeatedtests. This fact would indicate that since the bacteriophage asisolated from the boils is not active against homologous strains ofstaphylococci, bacteriophagy in this direction as a therapeutic

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agent is not very encouraging. Kuttner (1923) found a bacterio-phage active against a homologous strain of typhoid, isolatedduring the convalescent stage of the disease, the patient laterdeveloping into a chronic carrier. This author states that it isnot easy to correlate with such data the supposed therapeuticvalue of the bacteriophage.According to Davidson (1922), d'Herelle's phenomenon as

related to human dysentery is apparently non-specific, and abacteriolysant from a patient's stool may not be as active againstthe organism causing that patient's disease as against otherstrains. He claims that it does not necessarily play a part inthe immunity or defense-mechanism of the body. These non-specific bacteriolysants had no therapeutic effect when adminis-tered to twelve young children suffering from bacillary dysentery(Davidson, 1922b).Davidson's work (1922a) suggests that the bacteriophage or the

lytic principle of d'Herelle's phenomenon is an enzyme. Theamount of the bacteriolytic enzyme may be increased in severalways by certain external influences which favor the developmentof lysogenic organisms at the expense of the non-lysogenic. Thisenzyme not only dissolves organisms but also favors the multi-plication of bacteria which produce the enzyme. In this way thebacteriolytic principle is carried from generation to generation.

D'Herelle (1922) made seventy examinations of the excreta offowls and tested the bacteriophage for virulence against eightdifferent bacterial strains. The cultures selected for avian bac-teriophagic action were B. coli, B. dysenteriae Shiga, Flexner, Hiss,B. typhi, B. paratyphi A, B. paratyphi B, B. gallinarium, andthe bacterium of barbone. Bacteriophage activity was demon-strated from all excreta studied, some samples showing markedactivity for all cultures treated.From observations made by d'Herelle (1924), in the course of

experiments upon avian typhosis he noted a chicken had con-tracted the disease, and on examination found that the virulenceof the intestinal bacteriophage increased quickly to a maximumand the bird recovered. The epizootic ceased abruptly andexperiment showed that in the feces of all chickens was to be found

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a bacteriophage of maximum activity. D'Herelle claims that hisexperiments with the bacteriophage, in relation to B. gallinariumor fowl typhoid infection, confirm the conclusion that immunityto a bacterium is assured at the time when the body contains abacteriophage virulent for this bacterium. The immunity isexogenous and is contagious.From Marshall's work (1925) on the d'Herelle bacteriophage,

it is noted that this author concurs in the theory that the agentresponsible for transmissible bacteriolysis is a living ultra-micro-scopic entity.Although there exists confusion in relation to the therapeutic

aspects of the bacteriophage problem, yet from the work of bothEuropean and American writers it may be inferred that bacteri-ophagy may be employed in therapeutics. Since d'Herelle andassociates have reported success in relationship to fowl typhoid,is it possible to find a bacteriophage lytic for Salmonella pullora(Bacterium pullorum Rettger)? Would such a lytic principlebe of any therapeutic value in the treatment of this disease inyoung chicks or mature domestic fowls?With the above information at hand, the writer has planned to

investigate materials and tissues of the domestic fowl to deter-mine the presence of a bacteriophage lytic for Salmonella pulloraand if present, in sufficient amount and activity, to apply it inthe treatment of bacillary white diarrhea of chicks, and Sal-monella pullora infection in the domestic fowl.

ISOLATION OF THE LYTIC PRINCIPLE

Prior to any attempt at isolation of a lytic principle from thefowl, two available strains of the bacteriophage, received fromother laboratories, were used. One strain was a portion ofd'Herelle's original Shiga bacteriophage, which did not proveactive against the Salmonella pullora organism after transmisionin series some fifty times, in an attempt to adapt the lytic prin-ciple to the specific organism. The same was true of a Coli Xbacteriophage obtained indirectly from d'Hereile's laboratory.Likewise a lytic principle isolated from the feces of a cow by thewriter, which was specific for the colon bacillus, typhoid and

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paratyphoid organisms, failed to demonstrate any lytic actionupon a suspension of the pullorum bacillus.While working with the above strains, daily attempts were

being made to isolate a "phage" from the feces of the domesticfowl. Several birds were obtained from a Massachusetts flockwhich had been affected with Salmonella pullora infection. Priorto testing the droppings of the specimens for the presence of thelytic principle, blood samples were taken and the serum sub-jected to an examination for the presence of agglutinins specificfor the organism of that disease.

D'Herelle's technic for isolation of the "phage" was carefullyadhered to. A portion of droppings weighing approximately 5

TABLE 1Agglutination test

SERUM DILUTIONBIRD NUMBER

1:50 1:100 1:200 1:400 1:800 1:1600

281 - - - - --

5651 ++++ ++++ ++++ ++++ ++++ +++7047 ++++ ++++ +++ +++

57315 - - + + + _9242 ++++ ++++ ++++ ++++ +++ -

grams was thoroughly agitated in a 250 cc. Erlenmeyer flaskcontaining 75 to 100 cc. of bouillon. The culture medium usedwas adopted after several trials of various formulae and hydrogenion concentrations. Witte's peptone was finally decided uponin preference to d'Herelle's suggestion of Martin's. It was usedin 1 per cent content as usual. The completed formula includedas well as the peptone 0.5 per cent NaCl, 0.3 per cent Liebig'sextract of beef, and distilled H20. Various hydrogen ion con-centrations were tried and that most suited to the optumgrowth of the pullorum organism (7.4 to 7.6) was selected andused exclusively.The flask and its agitated contents were incubated for four to

eight hours further to decompose the mass. Later on this pro-

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cedure proved unnecessary since results equally as good wereobtained with vigorous agitation alone.The contents of the flask were filtered through filter paper and

infusorial earth in order to remove the coarse material, and thenpassed through a Berkefeld filter of medium porosity into anotherErlenmeyer flask. If a bacteriophage were present it should becontained in this clear, "sterile" filtrate. A minute quantity ofthis supposed "phage" material was added to a fresh suspensionof Salmonella pullora organisms washed clear with bouillon froma twelve to eighteen-hour agar slant growth. This was incubatedfor a few hours to hasten the lytic action, again filtered and asmall amount of the filtrate added to a new fresh suspension ofthe organism. In this way the "phage" was transmitted in seriesthrough daily filtrations and inoculations.

Several modifications of this method were developed, but sincenone of them proved so satisfactory and simple, they will not bedescribed here.

Five strains of the bacteriophage were isolated from the drop-pings of the fowl, each exerting a marked lytic action againstthe pullorum organism. They demonstrated at first merely aninhibition in the growth of the suspension of organisms and it wasnot until the sixth series' generation that any definite lytic actionwas noted, and then it was of but weak potency. These strainswere designated by numbers 5651, 7047, 5568, 5735, and 9242corresponding to the leg band number of the birds (table 1) fromwhich they were isolated.

Strains 5568, 5735, and 9242 at no time showed a strong lyticaction even after daily passages through filters over a period ofseveral months. Often times lytic action seemed to be absent,or if present, it was very erratic. Again at irregular intervals thesuspensions would show complete dissolution of the organisms.Hope of developing these strains for therapeutic use was finallyabandoned after the one hundred and twentieth generation.These erratic actions of the lytic principles might not only bedue to a low virulence, but as well to the development of the"secondary cultures" mentioned by d'Herelle. Some few organ-isms of the suspensions were undoubtedly more resistant and

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withstood the action of the bacteriophage. There were instanceswhen strains of S. pullora in use appeared to develop markedresistance to bacteriophagy. This difficulty was eliminated byusing only young, actively growing organisms.

Strains 5651 and 7047 developed an unusual and marked degreeof lytic activity after passage through sixty generations. Anexplanation of this phenomenon is difficult to find. It might bethat the stubborn resistance of the Salmonella pullora strainsto bacteriophagy had been overcome by the lytic principles.Certainly there had been an apparent enhancement in thevirulence of the "phages." It is interesting to note that thesetwo strains, which ultimately became markedly potent, wereisolated from birds showing a high titre of their respective serafor agglutinins specific for the Salmonella pullora organism.

Bacteriophage strains 5651 and 7047 were characteristic ofd'Herelle's lytic principle in every respect. They demonstratedvery well indeed the "watery, moth-eaten, or pellucid areas"described in his text. In plate 1 agar slant 4 and bouillon tube1 are controls. Agar slant 4 was produced by a streak inoculationwith a loopful of suspension from bouillon tube 1, containing 10cc. of suspension with 190,000,000 organisms per cubic centimeter,but no bacteriophage. Bouillon tubes 2, 3, and 4 each contained10 cc. of a suspension of Salmonella pullora, 190,000,000 organismsto the cubic centimeter with 0.01 cc. of bacteriophage added toeach tube. Immediately after the addition of the lytic principleto bouillon tube 2, a loopful of the suspension was streaked uponagar slant 1 and all tubes incubated. Following three hours'incubation another loopful from bouillon tube 2 was streakedupon agar slant 2 and incubated, and after five hours' incubationa third loopful from bouillon tube 2 was streaked upon a thirdagar slant 3 and then incubated. After sixteen hours' incubationagar slant 1 showed 8 to 10 watery areas, agar slant 2 numerous,watery areas, and agar slant 3 numerous coalescent wateryareas. This work supports the contention that the lytic principleis capable of proliferation.Moderately active strains of bacteriophage lytic for Salmonella

pullora were also isolated from 25 to 50 per cent glycerol-saline

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solution extracts of liver, intestines, and ovary of the fowl. Likeresults were obtained from an extract of mucous scrapings of theintestinal mucosa.The action of the lytic principle in relation to closely related

organisms corresponded very much to that of the group agglu-tinins. Lysis of the specific organism was more pronounced thanthat for closely allied organisms of the same grouping. Eberthella8angutnara was vigorously acted upon by the "phage" strains,while Eberthella typhi, Salmonella paratyphi, and Salmonella8ch6tmulle?i varied in reaction from a two plus dissolution of sus-pension to a bare inhibition of growth. Escherichia coli and theEberthella paradysenteriae and E. dysenteriae organisms did notdemonstrate bacteriophagy.

ENHANCEMENT OF VIRULENCE OR POWER OF BACTERIOPHAGY

There is little doubt but that the therapeutic value of the lyticprinciple would prmarily depend upon its potency. An attemptto' enhance the virulence of the bacteriophagic strains would,therefore, seem justified and would promise measurable success,for such a phenomenon had apparently already occurred. Thework as demonstrated in plate 1 indicates that the lytic principle,no matter what its nature might be, was capable of proliferation,and because of a close correlation, enhancement in lytic activityshould follow. If the results of such an attempt were positive,it would seem that d'Herelle's ultramicrobial theory as to thenature of the lytic principle would have some basis, but yet theenzyme, autolytic theory, which has been advanced by manyworkers in this country, would still have considerable foundation,and certainly should not be ignored. It is not the purpose of thispaper, however, to consider the explanation of that most com-plicated and difficult phase of the phenomenon.The plan of operation employed involved the attempt to obtain

complete lysis after sixteen hours' incubation of 10 cc. of bouillon,plus varying quantities of the lytic principle and specific organ-isms. The preliminary work of the experiment would, of course,be the determination of the quantitative virulence of the "phage,"or in other words, its minimum lytic dose. It would also be

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necessary to determine the maximum number of organisms thatwould be completely destroyed by this minimum lytic dose.Enhancement of the virulence could not be deduced until thesetwo quantitative factors were known."Phage" strains 5651 and 7047 were combined, as were also

five of the Salmonella pullora strains which had been in generaluse heretofore. Both polyvalent strains of "phages" and organ-isms were thus being used. At this point the work presentedconsiderable difficulty and there was doubt in the mind of thewriter as to whether or not it was practical to proceed. At thattime 0.0001 cc. of the "phage" caused complete lysis of a 10cc. bouillon suspension of 220,000,000 organisms per cubic centi-meter. The daily readings always presented a complete dissolu-tion of the suspensions following sixteen hours' incubation, if notimmediately upon removal from the incubator, a least within anhour or so after standing at room temperature.From these results it is indicated that a stock strain of the

avian bacteriophage can be carried indefinitely in the laboratoryas long as it is properly cared for by repeated transmission throughfilters and inoculations into media containing fresh, youngorgans.

LYTIC ACTIVITY OF SALMONELLA PULLORA BACTERIOPHAGE

Unless the lytic principle isolated from hens was specific for allSalmonella pullora strains, or proved bacteriolytic for homologousstrains, it would be of no practical therapeutic value.

This laboratory during the spring chick hatching season isactive in making diagnoses upon dead chicks. As each strain ofSalmonella pullora is isolated and identified, it is given an indexnumber, its history recorded, and the culture kept in the bac-teriological museum for a period of one year, at the end of whichtime it is discarded and replaced by an incoming new strain.Available material was thus on hand for the following experiment.Each strain of Salmonella pullora was subcultured several times

until it had so adapted itself that an eight to twelve hour agarslant growth produced abundant, fresh organisms for the sus-pension, to which the "phage" was to be added. A bacterial

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TABLE 2Lytic activity of avian bacteriophage, isolatedfrom domestic fowls for different strains

of Salmonella pullora

SALMONBLLA SALMONELLAF AMOUN'TPULLORA BOURCE OF CULTURE AS DAT ALMONLO A OF BACTERIO- DBGREE OFSTRAIN ISOLATED FROM CRIICK DINT1BCC.OF PHAGE LYSISNUMBER SUSPENSI.ON SOLUTION

mil7ion cc.

5 Unabsorbed yolk 3/11/24 195 0.01 ++++6 Liver 3/12/24 190 0.01 ++++7 Liver 3/13/24 200 0.015 +++8 Liver 3/13/24 195 0.01 +++9 Liver 3/13/24 185 0.01 ++++10 Liver 3/15/24 165 0.01 ++++11 Liver 3/15/24 175 0.01 +++12 Liver 3/16/24 180 0.01 ++++13 Liver 3/17/24 150 0.01 +++14 Unabsorbed yolk 3/25/24 155 0.01 ++++15 Liver 3/26/24 190 0.01 ++++16 Liver 3/26/24 190 0.01 +++17 Unabsorbed yolk 3/28/24 195 0.01 ++18 Liver 3/28/24 180 0.01 +++19 Unabsorbed yolk 3/28/24 175 0.01 +++20 Unabsorbed yolk 3/28/24 185 0.01 ++++21 Liver 3/28/24 160 0.01 ++++22 Unabsorbed yolk 3/29/24 173 0.01 +++23 Unabsorbed yolk 4/ 1/24 185 0.01 ++++24 Liver 4/ 1/24 190 0.01 ++++25 Unabsorbed yolk 4/ 2/24 185 0.01 ++++26 Liver 4/ 2/24 175 0.01 ++++27 Unabsorbed yolk 4/ 2/24 190 0.01 +++28 Unabsorbed yolk 4/10/24 180 0.01 ++29 Unabsorbed yolk 4/19/24 180 0.01 +++30 Unabsorbed yolk 4/23/24 180 0.01 +++31 Unabsorbed yolk 4/26/24 180 0.01 +++32 Unabsorbed yolk 5/12/24 185 0.01 +++33 Liver 5/17/24 190 0.01 ++++34 Liver 5/17/24 190 0.01 ++++35 Heart blood 6/ 1/24 190 0.01 ++++36 Heart blood 6/ 1/24 190 0.01 ++++

++++, complete lysis.

count was then made and 0.01 cc. of the bacteriophage added toa 10 cc. bouillon suspension of the organisms. Following anincubation period of sixteen hours at 37.5°C., and one to threehours standing at room temperature, the readings were taken


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and recorded as summarized below. Each lysed suspension wasthen passed through a Berkefeld candle and the filtrate used asthe bacteriophage for the test on the next strain of the organism.

THE THERAPEUTIC VALUE OF THE AVIAN BACTERIOPHAGE

Since the avian bacteriophage, as isolated from hens numbered5651 and 7047, was actively lytic for all thirty-one strains ofSalmonella pullora cultures studied, and since such quantitiesas 0.0001 cc. caused lysis of a 10 cc. bouillon suspension containing220,000,000 organisms, material was at hand for applying thebacteriophage as an agent in treatment.Many factors must be considered when attempting to treat and

immunize with the bacteriophage, especially when dealing witha disease such as bacillary white diarrhea. Bacillary whitediarrhea infection is not only found in the intestines, but is en-encountered as a severe septicemia as well. The chronic formof this disease as seen in adult hens is usually organic and not toany great degree of an intestinal nature.

Therefore, our first problem was to consider the mode of ad-'ministration of the lytic principle. Injection of the agent wouldmore likely bring about an organic resolution and immunity,while ingestion could hardly be expected to accomplish suchresults.

EXPERIMENT I

A lot of forty chicks was obtained from a brood whose progeni-tors had been tested by means of the agglutination test forSalmonella pullora infection and pronounced free of that disease.They were treated as follows:

Pen 1. Ten chicks, controls on brooder management. Received0.5 cc. of sterile physiological salt solution subcutaneously over thepectoral region.Pen 2. Ten chicks, controls on pathogenicity of the pullorum cul-

ture. Received 0.5 cc. of the culture, subcutaneously, over the pectoralregion.

Pen 3. Ten chicks, received simultaneous injection of 0.5 cc. of cul-ture and 0.5 cc. of bacteriophage, subcutaneously, over pectoral region.

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Pen 4. Ten chicks, received subcutaneous injection over pectoralregion of 0.5 cc. of culture. Eight hours later after period of incuba-tion had been instituted, another subcutaneous injection over pectoralregion on opposite side of 0.5 cc. bacteriophage.

Results

All chicks in pen 1, serving as controls on brooder management,lived and thrived throughout the experiment and have since beenraised to maturity. All those of pen 2, which controlled thevirulence of the Salmonella pullora culture, succumbed to theinfection, beginning to die at the forty-fourth hour followinginjection. The chicks of pen 3, which received the simultaneousadministration of bacteriophage and culture, began to die at theone hundred and seventeenth hour following the injection, whilethose of pen 4, first having received the culture and, later, aftera short incubation period the bacteriophage, began to die at theone hundred and fifth hour.The strain of Salmonella pullora was recovered from all chicks

of pens 2 and 4, and from seven chicks of pen 3. It was im-possible to isolate the organism from the remaining three chicksof this pen, but a bacteriophage, presumably the one adminis-tered, was isolated from the intestinal tract of one of them. Fromthese results it might be interpreted that these three chicks ofpen 3 had reacted well to the bacteriophage, the infection beingentirely destroyed in vivo and death resulting from another cause,such asintoxication from necrotic tissue at the point of inoculation.In some cases this lesion was extensive and it undoubtedly playeda part in hastening death.The results indicate a slight postponement of death and possibly

a less acute form of bacillary white diarrhea following the injec-tion of the bacteriophage.

EXPERIMENT II

It was evident that administration of the culture and bacterio-phage by means of the hypodermic needle was very severe. Itwas found impractical to administer therapeutic treatment insuch a manner to numerous baby chicks. A second lot of chickswas accordingly treated in the following manner.

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Pen 1. Ten chicks, controls on brooder management, received 5 to10 drops of bacteriophage per orem 4:00 p.m. and same quantity 10:00a.m. the following day.Pen 2. Ten chicks, controls on pathogenicity of culture, received

5 to 10 drops of culture per orem.Pen S. Ten chicks, received simultaneously 3 to 5 drops of culture

and 5 to 10 drops of bacteriophage per orem.Pen 4. Ten chicks, received 5 to 10 drops of bacteriophage as a

preliminary or "immunizing" dose at 4:00 p.m., and the same amountnext morning at 10:00 a.m., simultaneously with 3 to 5 drops of culture.

Drinking water, medicated with bacteriophage, was kept beforethe birds of pens 3 and 4 at all times.

ResultS

The results were comparable to those of experiment I in thatall chicks of pen 1 lived and those of pen 2 began to die at the onehundred and first hour. The chicks of pen 3 began to die at theone hundred and forty-second hour and those of pen 4 at theone hundred and sixty-third hour.

Salmonella pullora was recovered from all chicks examined, butin no instance was the search for the bacteriophage successful.A slight postponement in death is again indicated. Each chick

received at least 75,000,000 organisms and the bacteriophage wascalled upon for action against a severe artificial infection. Noopportunity was given for experimental use of the lytic principleagainst a natural infection.

SUMMARY

The purpose of this work has been first to study the intestinalcontents, fluids and tissues from the domestic fowl, and tolocate and identify in these materials a bacteriophage, lytic forSalmonella pullora (Bacterium pullorum Rettger).

Secondly, methods have been employed for increasing thelytic activity of the avian bacteriophage in vitro, to the end ofusing it as a therapeutic reagent in avian therapeutics.From the data presented and the observations made, the follow-

ing conclusions appear to be justified.

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1. Tissues of the domestic fowl do contain bacteriophagescausing lysis of various strains of Salmonella pullora.

2. Up to the present time, although these avian "phages" arelytic for Salmonella pullora, it has not been demonstrated thatthey are specific only for this organism.

3. When agar slant cultures of Salmonella pullora are treatedwith liquid cultures of these avian bacteriophages which arelytic for Salmonella pullora, watery, moth-eaten or pellucidareas are demonstrated. This fact, together with the demon-stration of the transmission of bacteriophagy for Salmonellapullora in series would indicate that the transmissible bacteriolysisis a living ultra-microscopic entity. These reactions of theavian bacteriophage are characteristic of d'Herelle's "Bacterio-phagum Intestinale."

4. Although the bacteriophages isolated and studied havedemonstrated marked bacteriolysis in vitro, the evidence fromanimal experiments does not indicate that as now prepared andused that they have much therapeutic effect in freeing thebird's body of infection.

5. Bacteriophages actively bacteriolytic for Salmonella pullorahave been isolated from domestic fowls showing a high agglu-tinative titre for this organism.

REFERENCES

BECKERICB, A., AND HAUDUROY, P. 1923 Le bacteriophage de d'Herelle: Sesapplications therapeutiques. Jour. Bacteriol., 8, 163-171.

BRUTNOGRE, R., AND MAISIN, J. 1921 Au sujet de l'unite du principe bacterio-phage. Compt. Rend. Soc. de Biol., 85, 1122.

CALLOW, B. R. 1922 Bacteriophage Phenomena with Staphylococcus aureus.Jour. Inf. Dis., 30, 643-650.

DAVIDSON, W. C. 1922a Observations on the properties of bacteriolysants(d'Herelle phenomenon, bacteriophage, bacteriolytic agent, etc.).Jour. Bacteriol., 7, 475.

DAVIDSON, W. C. 1922b The bacteriolysants therapy of bacillary dysentery inchildren. Amer. Jour. Dis. Child., 23, 531-534.

D'HERLLE, F. 1917. Sur un microbe invisible antagoniste des facilles dysen-teriques. Compt. rend. Acad. Sci., 165, 373.

D'HERELLE, F. 1919a Du role du microbe filtrant bacteriophage dans la fievretyphoide. Compt. rend. Acad. Sci., 168, 631.

D'HERELLE, F. 1919b Sur une epizootic de typhose aviaire. Compt. rend. Acad.Sci., 169, 817.

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SAIMONELLA PULLORA INFECTION IN FOWL 259

D'HERELLE, F. 1919c Sur le role du microbe bacteriophage dans la typhoseaviaire. Compt. rend. Acad. Sci., 169, 932.

D'EHEIZELLE, F. 1921 Le microbe bacteriophage agent d'immunite dans la pesteet le barbone. Compt. rend. Acad. Sci., 172, 99.

D'HERELLE, F. - 1922 "The Bacteriophage: Its R6le in Immunity." (The Bac-teriophage in Disease, p. 177-228. The Bacteriophage in the HealthyIndividual, p. 229-245.) The Williams & Wilkins Co., Baltimore,287 pp.

D'HEzELLE, F., AND ASSOCIATES. 1922 Bibliographic Review. Bibliographylisted by W. C. Davidson. Abst. Bacteriol. 6, 174.

D'HERELLE, F. 1924 Immunity in Natural Infectious Disease. English edi-tion, p. 286.

GRATIA, A., ANI DE NAMUR, M. 1922 Indivualite das principes lytiques staphy-lococciques de provenances differentes. Compt. rend. Soc. de biol.Paris, 87, 364-366.

HANKIN, E. H. 1896 L'action bactericide des eaux de la Jumma et du Gange surle vibrion du cholera. Ann. de l'Inst. Pasteur, 10, 511.

KING, D. S. 1923 The Twort d'Herelle phenomenon; its possible relationshipto therapeutics. Med. Clin. No. Amer., 7,883-908.

KUTTNER, A. G. 1923 Bacteriophage phenomena. Jour. Bacteriol., 8, 49-101.LzBLAYE, R. 1923 Le bacteriophage et ses applications therapeutiques. Arch.

Med. Chir. de Province Tours, 13, 177-188.LEEzNDORFF, H. 1924 Therapeutische Anwendung dee bacteriophagen Lysins

(Twort-d'Herelle) bei Kinderkrankheiten, Wien. Med. Wchnschr., 74,1051-1054.

MARSHALL, M. S. 1925 Observations on d'Herelle's bacteriophage. Jour.Infec. Dis., 37,126-160.

MCKINLEY, E. B. 1922 Notes on d'Herelle Phenomenon; adaptation of bac-teriophage antagonistic to Bacillus dy8enteriae and other bacilli tovarious cocci; development of a polyvalent bacteriolysant. Jour. Lab.and Clin. Med., 8, 311-317.

MCKINLEY, E. B. 1923 The bacteriophage in the treatment of infections.Arch. Int. Med., 32, 899-910.

PAURON, A. 1923 L'ultra-microbe bacteriophage: son role dans la prophylaxieet la therapeutique des infections. Arch. de med. et pharm. mil., 88,195-203.

PIORKOWSI, C. 1922 Beitrag zur Streptokokkenfrage. Anwendung des d'Her-elleschen Phanomens auf Streptokokken. Med. Klin., Berl., 18,474-476.

TWORT, F. W. 1915 An Investigation on the Nature of the UltramicroscopicViruses. Lancet, 2, 1241.

WATANABE, T. 1922 Ueber die Wirking von Staphylokokkenbacteriophagen.Wien. Klin. Wchnscher., 35, 603.

JOURNAL OF BACTURIOLOGY, VOL. XU, NO. 4


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260 NORMAK J. PYL

PLATE 1

AGAR SLANT AND BOUILLON CULTURES OF SALMONELLA PULLORA SHOWING" WATERY, MOTH-EATEN AREAS" AND COMPLETE LYSIS INDICATIVE OF TE

AVIAN BACTERIOPHAGIC ACTIVITYAgar slant 4 and bouillon culture 1 (reading from left to right) are controls, the

former being produced by a streak inoculation with a loopful of suspension fromthe latter. Bouillon cultures 2, 3, and 4 were each inoculated with 0.01 cc. ofthe avian bacteriophage and incubated. A loopful of suspension from bouillontube 2 was streaked upon agar slant 1 immediately after addition of the bacterio-phage. Agar slant 2 was produced from bouillon tube 2 in the same manner afterthree hours' incubation of bouillon tube 2, and agar slant 3 after five hours' incu-bation of bouillon tube 2. The agar slants and bouillon cultures after sixteenhours' incubation showed the following:

Agar slantsTube 1. 8 to 10 "pellucid" areasTube 2. Numerous, "pellucid" areas

Tube 3. Coalescent, "pellucid" areas

Tube 4. Control slant

Bouillon tubesTube 1. Control suspensionTube 2. Complete dissolution of or-

ganismsTubes 3 and 4. Supplementary to

tube 2.


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JOURNAL OF BACTERIOLOGY. VOL. Xll PLATE I_ r__ i

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261


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fowl. typhi, b. paratyphi a, b. paratyphi b, b. gallinarium, and the bacterium of barbone....

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