‘JHE STUDY OF AN ORGANISM ESGIATED IN PURE CULTURE MOM DISEASE!) POULTRY T313335 for Degree. of M. 5. Helen J. Ellis 392.5 .~.p,l»..a . ‘ , . .13‘. 5 AB . . . .. . , . 7- . f .V .. . . J . . i. U . f .. , . v, V. i .t. .F. . x . . . .. , .L. .. .. .,. ., . L. . .V! 3. .2... {3.123 . . ...,..:. :......1.,v.v(.:u.wuh,r. ....... . 3.1.» 2;..wa 1533“.“112... K. Tufuuuh . . .5 v, Luz... (I It .._ ,4 . an,» if. .. ...,3. , ‘ Infzifi. Ezlnwzft .2: IE. n4... .w “half. ur—r. .. H; r.. 7X2P£i 1'. htxr. r7754: » 5!va . cunt-11¢ «.v.»¢.~m..er....au:‘vLAt‘uEUHVEE. . J . .J w~ 3?.» . . .. a .31. 1 ‘9 v r) ,. . , JV , mum ‘ 7 l~h l .‘l V ..... .II {“ ‘ . ‘ l u . , I a p . . 3 ‘V r > f . I \ u I I f ’0) I. .t . 14v l .- .- . \ l a. a ml: .3; . . _ l . t- I r 4. . z nuns m SMY OF AN ORGANISM ISOLATED IN PURE CULTURE FROM DISEASE!) POULTRY THE STUDY OF AN ORGANISM ISOLATED IN PURE CULTURE FROM DISEASED POULTRY Thesis Submitted to the Faculty of the Michigan Stute college of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of Master of science. BY 4 Helen J.‘§llia 1925. I. II. III. IV. V. VII. VIII. THE STUDY 9}: g; ORGANISM ISOLATED g1 HIRE CULTURE FROM DISEASED POULTRY. gable or contents. Introduction Review of Literature History of Gases Methods of Investigation, and Results A. Source and Isolation B. Morphology 0. Cultural characteristics D. Physiological characteristics E. Serclcgical studies F. Pathogenicity Discussiun of Results Summary Acknowledgment BibliOgrsfihy 96064 I. INTRODUCTION The incompleteness of knowledge of the cause of diseased conditions is well known. Poultry diseases have received much less study than those of other domestic animals and man. In only twenty percent of the cases coming to the attention of Michigan State College of Agriculture and Applied Science last year was positive diagnosis made. A variety of organisms are encountered, the significance of which is unknown. Among them is the organism here discussed. From July 1924 to May 1925, it was isolated in pure culture from thirty-five autOpsies performed in this laboratory. As in no case any Specific well known organism explaining the lesions could be discovered, it was thought worth while to make a thorough study of this organism and to attempt to show whether or not it is capable of producing pathogenic effects in birds. II. REVIEW OF LITERATURE . nonquet, in an article entitled "Deuxieme note sur la typhoae aviare", in Bul. Soc. centr de Med. Vet.. Bar. 1922, 98, 65-70, tells of the isolation of a Gram-positive organism.(aingie or diplo-), oval or round, isolated from blood, liver, spleen, bone marrow, peritoneal fluid of birds. It is impossible to tell from the description Ihether the organism is the same as the one here discussed. III. HISTORY OF CASES It seems advisable, in the absence of historical background, to include reviews of the routine autopsies of the birds from which the organism.studied was isolated, especially since there was great variety in.the lesions reported. no effort is made to give the complete autOpsy -report, only positive pathological findings being listed. All organs and tissues not mentioned are to be considered normal. §§l - Legweakness: as: 368 an 372 Liver: small. Heart: flabby, small hemorrhages on fat. Duodenum: marked hemorrhagic inflammation. Liver: a few pinhead sized white spots. Heart: flabby, chronic pericarditis. Duodenum: chronic catarrhal inflammation. Liver: enlarged. Gall bladder: much extended with thick bile. Spleen: enlarged. Proventriculous: hemorrhages around openings of glands. Ovary: chronic lesions (tumor formation). Mesentery: thickened with tissues like those found in every. Peritonitis. Chronic suppurative peritonitis with adhesions. Ovary: diseased, inactive, lesions suggesting early tumor~formatiou. Lesions like 367 and 368, except that changes are more along the posterior part of the intestines. Ovary: very large, like 367 and 368. Liver: "nests“ of'necrotic foci. - Liver: shows seven or eight circular necrotic spots suggesting blackhead lesions: however, with calcification. 9.2. an Ovary: gelatinous degeneration. Intestines: inflammation in upper half. Geeum: wall thickened and resembling lesions seen in 367 and 368. Spleen: small and anemic. Intestines: slight oatarrhal enteritis. Retained yolk, sise of hazelnut, oaseous peritonitis. Spleen: small. Heart: flabby with minute hemorrhages in fat. Intestines: oatarrhal condition in upper part. Oecum: areas of superficial necrosis. Legweakuess. Pancreas: much enlarged and hard. Ovary: tumor formation. Heart: flsbbYe. Intestines: oatarrhal condition. Abcess on sternum. Heart: sero-fibronous pericarditis and tumor in muscle. Retained yolk. Ovary: tumor formation. Spleen: congested. Rigth lung: small abcess. Gissard: ulcerations. Intestines: general hemorrhagic inflammation. 422 - Ovary: poorly developed. Oviduct; appeared tp be absent. i522- Reart: serous pericarditis. Intestines: hemorrhagic inflammation. Some legweakness, blindness and congestion in the middle part of the small intestines. Liver: enlarged, with a few small yellow spots; also hemorrhagic spots. Spleen: somewhat enlarged, with.nodules. Kidneys: somewhat enlarged, yellowish grey in.color. Tubercle bacillus was demonstrated in spleen, liver and kidneys. Rabbit, inoculated for diagnostic purposes, October 31, 1924., (died Bov. 11, 1924) with culture of the organism.under discussion from autoPsy 210, case 381. Peritonitis and marked pleurisy. Liver: enlarged with yellowish grey spots, perhaps indicating chronic hepatitis. Gall bladder: distended with bloody thickly coagulated bile. ‘ Spleen: enlarged, swollen around edge. Kidneys: right one showed . whitish area about 6 an. in diameter, which on the cut surface had shape of infarct. Stomach: tore very easily. Lungs: numerous hemorrhages. Bronchi: edema 522 - Some legveahiess. 50'? Eli 5.1.9. 3532 Liver: small yellowish grey spots, pin point to pinhead in size, over entire organ Bones :brittle Intestines: severe enteritis, crcupcus at middle portion. The organism was isolated from liver and spleen. B. pullorum was isolated from the heart. B. sanguinarium from liver and heart. Legweakness, emaciated, had cold and retained yolk. Spleen: small. Intestines: inflamed. Legwealmess, emaciated, may wsrms. convulsions or spasms. Liver: very light in color, large and fatty. Heart: hemorrhages on fat. Liver: liecrctic spot, size of pinhead, surrounded by hemorrhagic area. Spleen: small hematmaa, size of peel Heart: fibrincus pericarditis, with large hemorrhages. Lungs: oaseous pneumonia. Liver: small and anemic. Spleen: very small Heart: hemorrhages. Proventriculus: mmiy hemorrhages around openings of glands. Intestines: hemorrhagic duodenitis. L. U. 654 - Liver: anemic. IS |§ 2:2 557 584 2.52 763 Small retained yolk. Larynx: hemorrhages. Liver and.heart: congested. Liver: yellowed, slightly congested. Spleen: mmsll. Intestines: slight enteritis in a few places. Liver: chronic necrotic lesions. Spleen: small Larynx: hemorrhages. Pharynx' and trachea: diphtheritio membrane. Heart: chronic pericarditis. Spleen: small. Larynx and trachea: congested. Intestines: inflammed. Died forty days after being injected with culture from case 488 for laboratory diagnosis. Emaciated. Liver: enlarged, pale, few white nodular lesions. Spleen: greatly enlarged, soft. Heart: flabby pericarditis. Kidneys: enlarged. All inner organs comeshat atrophied. Heart: flabby. Ovary: diseased. Intestines: pronounced chronic inflammation. ill organs rather small. Ovary: diseased. Retained yolk. 769 - Spleen: enlarged. Liver: enlarged. Intestines: oatarrhal enteritis. Retained yolk. Larynx occluded with canker. Heart: numerous hemorrhages on fat. I. Liver: s meshat congested. Spleen: mottled.. Intestines: hemorrhagic enteritis. Lungs: pneumonia. Died thirty days after being injected with culture from case 763 for laboratory diagnosis. Liver: enlarged, ruptured, mottled showing*many small yellowish spots. Spleen: enlarged, pale. Intestines: sane hemorrhages. 115. O :05 14 . 19 I 32 ‘ 65 69 ’, _ 98 61. IV. METHODS OF INVESTIGATION AND RESULTS. In studying the organism.culturally the descriptive chart recommended by the society of American Bacteriologists was used, with some additions. All strains were invigor; ated five days in broth before they were grown on the standard media and all experiments were performed at least three times Mn duplicate, with intervals of some weeks between the repetitions. Media were prepared according to instructions in Giltner's "Microbiology" unless otherwise specified. -12.. A. Source and psolation of Cultures. The strains used in the culture study were isolated in the spring and early summer of 1924. . Those used in serological studies and.for injection, were isolated in the fall of'1924. The method used in isolation was that used in the laboratory in the routine bacteriological diagnosis of chicken diseases. Organs of the chickens that are autopsied are plated out on beef infusion agar. .‘Colonies are picked from the plates after twenty-four hour incubation, and are grown on beef infusion agar slants, (pH 6.8) for twenty-four hours. Durham fermentation tubes of one percent lactose, sucrose, dextrose, maltose and mannit with Andrade's indicator are inoculated from the slants and read in twenty-four hours, forty-eight hours, and at the 316.01 a week. Microscopic examination is made of the organisms that do not produce acid in mannit but do in the other four sugars used. The sources of cultures studied: Culture 1006 This culture was isolated, May 8, 1924, from a hen brought in while still alive, by a local farmer. The bird was dumpy, and died in four days. The clinical symptoms noted were drooping of’head and tail, sleepiness. The droppings were yellowish and.watery in the early stages, greenish in later stages of the disease. The, lesions observed were numerous petichial hemorrhages - 13 - in the heart muscle. The liver was yellowish. The colonies of the organism were small and white on beef infusion agar. Cultures 955 and 956. These cultures were isolated from two large and three small chicks sent in from Bangor, Michigan. The autopsy of the larger ones showed no abnormalities except a re- tained yolk in one. There were no lesions in the three small ones except that the liver of one was yellowish in color, and one had a retained yolk. The organisms were isolated from heart and liver. Culture 1087 This culture was isolated from five small chicks sent in from Stockbridge, Michigan. Three had ochre colored livers, one had a large retained yolk, three had smaller retained yolks. The colonies of the organism were small and bluish on infusion agar. The culture was from a yolk. Cultures 624 and 625 These cultures were isolated from two live hens and one dead one. One live one showed signs of legweakness. in were amaciated. one showed discolored liver and inactive abnormal ovary. The colonies were small and bIUIBh ‘hitOe -14- B. Morphology and staining. Vegetative Cells. Twenty-for hour cultures of beef infusion agar and nutrient broth were used in the study of cell forms. The organism seems to be a coccus. Many seam to be very diort rods,but the oceans form predoninates. Pleomorphism is not uncommon, spindle and club shapes being most often found. During the serological studies a rod with bipolarly staining bodies we encountered. Serum of injected animals had bem mixed with a very dilute suspension of the organism, incubated for sane hours, and a mall amount of tlm mixture plated on agar. A smear of the colonies that grew was stained with dilute carbol fuchsin and showed a predaninanoe ofthese firms with large deeply stained polar bodies. (photograph, page 1 ) Ho effort was made to explain mesa seeming inconsistencies. Such an attempt would involve an intense study of life cycles. Arrangement. The organism is found in a great variety of groupings. There seems, However, to be a large prOportion of diplococcus fears, the individuals showing a tendency to flatten on the adjacent sides. flag. The average diameter of the orgmimn is .97 microns with a few measuring one micron. There was very little variation. Staining characteristics. The organic stains readily with carbol fuchsin, diluted one to ten with Loeffler's methylene blue, with aqueous alcoholic solution of saffranin and Bismark brown. It is positive to Gram stain. It is not acid fast. no capsules or spores were demonstrated. - 16 - C. Cultural Characteristics. Agar Slant. Beef infusion agar was used. When first isolated this organism.grew well, appearing much like Becterium.pullorum. After it had been transferred two or three times it grew very poorly. On agar after several days incubation at 37°C the colonies were beaded, raised, gflistening, smooth, and transparent. There was no chromegenesis, odor or gas formed. The color of the medium was not altered. Gelatin Stab. The organismgdid not grow in gelatin. Cultures were incubated both at twenty and thirty seven degrees for three weeks. Due to the feet that it did not grow, its ability to liquify gelatin could not be determined. ' Nutrient Broth. After repeated transfers in broth the growth became scarce or disappeared entirely. Broth made with chicken meat seemed to stimulate growth. This was made according to instructions for the preparation of nutrient broth in Giltner's."Microbiology", page 27,except that chicken meat was substituted for chopped beef. The medium was clouded, with no surfece growth, sedimmnt or odor. Agar Plates. Beef infusion agar was used. Observa- tions were made at the end of 24 hours incubation at 37°C. The colonies were small, circular, smooth, raised and entire. When examined under the microscope they appeared to be bursting at several points, with organisms exuding at these places. When first isolated the colonies were -17.. about a millimeter in diameter and bluish white. Gelatin Plates. no growth. Potato Slant. The growth on potato medium was very poor. There was no chromogenesis nor any omer change in the medium. Blood plates. Two percent sterile sheep blood was added to beef infusion agar, plates were poured and allowed to harden. They were then streaked from a broth culture. MethcmOglobin was formed except by culture 625, which failed to grow. The amount of growth was moderate. glucose agar. The organism grew fairly well on sugar agars, its appearance being much like that on beef infusion agar when first isolated. D. Physiological Characteristics. Sugar Permentations. Sugar free broth containing one percent sugar, one-half percent sodium chloride, and one percent Andrade's indicator was used. The tubes were inoculated with one leap of twenty-four hour broth culture, and wereiread at the end of twenty-four and forty-eight- hours, and seven days.- The sugars used were inulin, dextrin, dextrose, dulcit, levulose, sucrose, mannit, rhamnose, adonite, salacin, gelactose, arebinose, maltose, xylos, reffinose, glycerin, and lactose. no gas was formed in in any of these sugars. Acid was produced in dextrose, sucrose, maltose, levulose, lactose, and salacin, very slowly in raffinose and dextrin. Cultures 624. and 625 died before the studies were completed, but showed no inconsistencies in the fermentations in which they were used. Ithas been found that the fermentation of lactose is sometimes slow, in the routine laboratory work, in which peptone broth is used in making the sugars. Litmus Milk Reaction. Acid reaction was produced in milk at the end of twenty-four hours incubation at 37°C. Coagulation was found in the lower part of the tubes in forty-eight hours and was complete in four days. A small amount of whey was extruded at the end of seven days. The litmus was reduced in sixteen hours. lie-oxidation took place to the extent of 10 percent in twenty-four hours and was complete in most cases at the end of seven days. Strains 625 and 626 were more rapid in all these processes than the other cultures. The first time the -18- experiment was made, re-oxidation of the litmus‘had taken place only in the upper half of the tubes at the end of seven days. \ Indcl Production. For the indol test, seven-day cultures in Dunhamfs peptone solution were used, Ehrlioh's method being followed. Indcl was produced by all strains studied. Nitrate Reduction. Pour day old nitrate-peptone solutions incubated at 37°C were used, following the method explained in Ciltner's "Microbiology", page 131. Presence of both ammonia and nitrites was demonstrated. Hydrogen-sulphide Production. The organism was streaked on plates of agar, to which one gram of lead carbonate had been added. Blackening 'of the media (caused by the formation of lead sulfid)denotes the production of H23. Hydr0gen sulfid was not produced by my of the strains studied. proteolytic snags. Heither'Loeffler's coagulated blood serum medium, milk, nor gelatin.showed the presence of proteclytic enzymes. nggen Requirements. The organism grew an anaerobic plates as well as on aerobic plates of the same medium (beef innision agar). Anaerobic conditions were obtained by the pyrogallic acid and HaOH method. (Giltner's "Microbiology", page 168). gydrogen-ion Requiremntg. The organism grew on agar varying in m from 6.2 to 7.8. -19- Thermal Death Point. For detailed description of method used see Ciltner's "Microbiology", page 198. This is Sternberg's method. In general, it consists of drawing a lull amount of broth culture. into a capillary tube, sealing the end, exposing the tube at the desired temperature a definite length of time, breaking off the ends of the tube and dropping it into broth. Growth or lack of growth is noted after twenty-four hours incubation. The organism died at 58°C in ten minutes. R. Serological Studies. Bacteriolysins: Tests for the presmce of bacteriolyoins were made in a great variety of'proportions of immune serum am suspension of crgmism, using the method explained in "A Laboratory Course in Serum.Study', by Zinsser, Hopkins and Ottenburg, page 58. In gmeral the method consists of incubating 1 cc. of 1-500 dilution of twmity-four hour broth culture of the organism, with varying mounts (.01 to .0001 cc) of immune serma. Two loopfulls of mixtures are added to tubes of agar that have been melted and cooled, and plates are poured. These are examined mr colonies after twenty- four hours incubation. The experiments were carried out with serum of guinea pigs that had been injected with live md attenuated organisms two months befbre. Serums of'two control pigs were also used. Bacteriolysins were not demonstrated in say dilution. The autOpsy of these guinea pigs seemed to show that the animals are not susceptible to 111s «guism, in which case it is not surprising that the presence of the antibody could not be proven. Agglutination tests were run, using'the seams of guinea pigs injected with live and attenuated organises, and controls (not injected): the serum of chickens injected with live organism, and with filtrates, and cmtrols: on the serum of birds showing lameness: and on flocks in iiioh more was no reason to suspect the presence of the orgmism. The antigen used in making the agglutination tests was made by growing the organism for ty-eight hours on dextrose agar in .Kolle flasks. Dextrose agar was used because the '21- organism grew much more abundantly on this medium. The growth was washed off with physiological salt solution to mich five-tenths percent phenol was added, and diluted to a turbidity of tube number one of McFarland's nephalometer with the one solution. One cubic centimeter was placed in Wesserman tubes. .05, .025, .01 and .005 cubic centimeters of serum were generally used, making dilutions of 1-20, 1,-4.0, l-lOO, and 17200. The tests were read after incuba- tion at 37°C for twenty-four and forty-eight hours, am were recorded as follows: 4-, i, - according to degree of agglutination; C if there was cloudiness obscuring the test, U if huuolysis or presence of cellsmade the test unreliable. Tests were first run using this antigen and serums reacting positive, negative and partial to the test for bacillary mite diarrhea (B. pullorum antigen employed), in an effort to determine the specificity of the reaction. In every case they were negative. Later there was reason to doubt the dependability'of this work, which was not very extensive. Several snples sent in for bacillary white diarrhea testing gave "partial" results, several experimental birds from the animal room also. It is possible that these birds were infected; however, it does not seem probable. Guinea Pig Serums. Guinea pigs were injected with live organism (5 cc. of the turbidity of number three of McFarland's nephalometer suspension) and repeatedly with attenuated organism. For D. -22- details see studies on pathOgenicity. The results of. all the agglutination tests using guinea pig serum were negative. Chicken Semm. The results or the agglutination tests that were made with serum of. the chickens that were injected with the organism are given in the following table. For details of the injections see "Patagonicity" page 25. The antigen used was a mixture or four strains. AGGLUTINATION TESTS OF INJECTED BIRDS 48 hour readings. Table 19 0e II e 6th 4' 4th 5th 7th 8th Bir Injected weer week week week 3 3°. “ith 1-20 -40 1-20 -40 1-201-40 1-201-40 1-23u-4o 1 Strain 488 + + 3; 1; 1 - + - + +' 8 Strain 505Y i + + + + + 3; L -_o-_ 13 Strain 584 U U U U + + U + 1'... 2’. 14 Strain 505Y + + + + + 4-2 v + + + 18 filtrate :- 2 - - - - i 1.- : :- 3‘ Str.1n 584 ‘ - a n :- t. u - a o 4.2 strain 488 ‘ u u - - - 1 a U - .- 48 not injected U U U U U U U U U , 59 Piltrate 4 - 4- + 1; 1 - - 3; _e_. 61 not injected U U - - 3; - U U - - 6'! Piltrate U U U U U_ U U U U U 91 not injected U U - 4- - U U U U . 92 Strain 4'75 4- - e 3; + + + - 4- _+_ 9? Strain 4'75 + 1 e 1'. + 1’. + 3; + 1 (Ton-- trol (no serum used - " ’ ' "' " " ' " " -24- OROS S AGGLUTI NATI (NS .05 .03 .01 .005 .05 .025 .01 .005 Injected with Injected with culture 5841. culture 4888 Antigen 433 g 3; - Antigen 488 - - .. .. Antigen 584 - '- 1'- Ant 18011 584 II - II I- Antigen 4'75 1’. 4_-_ 2’. Antigen 475 a. - 1 .- Chicken no. 9'? hicken no. 8 Injected with Injected with culture 475]! culture 505! Antigen 488 C C C Antigen 488 + + + Unfit Antigen 584 C C C Antigen 584 1" 2': .t. " Antigen 4'75 C C C Anti gen 4‘75 .. .. 1'; 1'. Chicken no. 91 hicken No. 1 Bot injected Injected with culture 4883 Antigen 488 + + 1 Antigen 488 C C C Antigen 584 3.. :0; + Antigen 584 C C C O Antigen 475 + + - a Antigen 4’75 C C C C Chicken lie. 61 Chicken No. 92 not injected Injected with culture 475E Antigen 488 + + 3; 4w Antigen 488 z 1’. + + Antigen 584 + + + I Antigen 584 1 3 - - Antigen 475 E E _-._._ ': Antigen 4'75 1; 1 + 4- Chicken no. 48 Chicken no. 15 not injected Injected with Culture 5841. Antigen 488 + + 1 : Antigen 488 1'. - L". 1'. Antigen 584 3 '3; + + Antigen 584 .t z - Unfit Antigen 4'75 1 :0 1; 1 Antigen 4‘75 3-. 1’. + + Chicken no. 45 Chicken ‘ Bot injected Injected wit toxin Antigen 488 + + + 3 Antigen 488 z. 1'. 1'. 1'. Antigen 584 + + I Antigen 584 1 I. 3; L Antigen 4'75 4» + 3‘. Antigen 475 Li— : z 1 All these birds were kept together in one colony house. P. Bathogenicity. The chickens used in the study of the patcgenicity of this organism were injected January 25rd, 1924. Eight received 2 cc of twenty-four hour broth cultures of different strains, intraperitoneally, three received respectively one and one-half, three, and five cubic centimeters of filtrate of a five-day peptic digest broth culture of strain 5841.. The birds were killed March 30th and were autopsied two hours after they were killed. In some cases parasites were found, consisting chiefly of round worms, usually in small numbers. Ovary, liver, heart md spleen, in most cases, were plated.in an attempt to reisolate the organism. A large number of colonies were picked and run on sugars after growing twenty-four hours on agar slants. Microscopic examination was made. The organism was isolated from only six birds, 9’7, 14, 34, l, 48, and 67. Bird no. 1. Injected with strain No. 4883. 3113mm emaciated. Comb: sometat pale. Blood: not coagulated. very friable. 2: § Spleen: congested; Ovaries: active, sane ovules showing hemorrhagic condition, cone contain cheesy material and are collapsed. Kidneys: congested. Large Intestines: small areas of hemorrhage. Small Intestines: sliglt enteritis. Bird Bird Bird Bird -26- Provmutriculus: small hemorrhagic areas. Pancreas: hemorrhagic, necrotic areas in upper portion. No. 8. Injected wdth strain 505Y. Fair condition. Comb and settles: anemic. Liver: congested, very friable. Ovaries: active and hemorrhagic. large intestines: hemorrhagic. No. 13. Injected wiih strain 5843. Extremely emaciated. Liver: congested wi th necrotic foci, in general A strephic. Heart: strephied. Spleen: atrophied, anemic. Small Intestines: slight hemorrhagic enteritis. No. 14. Injected with strain 505Y. Slightly enciated. Comb: muio. Liver: friable, hemorrhagic, grayish discoloration similar to fatty degenerati a1. Ovary: active, slightly hemorrhagic. Intestines: few patechial hemorrhages. Proventriculus: hemorrhagic areas at posterior part. No. 34. Injected with strain 5841.. Fair cmduion. liver: congested, friable, degenerated.areas. Ovary: active, some ovules showing hemorrhagic oond iti on, others collapsed and containing cheesy material. Kidneys: slightly anemic. Bird no. 42. Injected with strain 4883. Slightly emaciated. Comb: pale Liver: congested in streaks, otherwise pale: friable Ovary: active, some ovules collapsed and contain cheesy material. Bird Ho. 61. Not injected. Emaciated. Liver: friable hauorrhagic; fatty and parenchymatous. de genera ti on. Heart: subacute pericarditis. Bird Ho. 6'7. Injected with 5 cc. of filtrate of peptic digest culture. Liver: cmgested, showing a few necrotic foci, in general of friable cmsistency. Heart: a few petechial hemorrhages on visceral part of pericardium. Ovaries: inactive, sane ovules collapsed, and containing cheesy material. Small intestines: ecchymotic hemorrhages. Proventriculus: few petechial hemorrhages. Bird no. 92. Injected with strain 4751!. Fair condition. Comb: somevmat manic. Liver: congested, friable, showing numerous very small yellowish spots. Heart: fatty degeneration of muscle. Kidneys: show a grayish yellow discoloration suggesting small necrotic foci. Gisaard: mucosa of reddish color in mall spots. Bird Ho. 9’7. Injected with strain 4'75H. Fair cmdition. Wattles: sunevhat anemic. Liver: hyperemia and parenchymatous degeneration, friable. Spleen: anemic Ovaries: active, congested. Pancreas: mall spots of congestion. Small Intestines: small hemorrhagic areas. par} 12'] -30- The rabbits used in the study of the pathogenioity of the orgmism were injected, January 25, 1925, with two cubic centimeters of twmty-four hour broth cul‘lnre, intra- venously. Two of them were killed in April. The lesions were so sligit wet it was not considered necessary to kill the other two, especially since they showed he symptoms of disease. Rabbit no. 166. Injected with strain 5841.. Liver: manual in sise and color, but on surface are several whitish spots about the size of a pin head, or a little larger. They are of tag: consistency, and sons may be found on the cut surface. They are seemingly of parasitic nature. Heart: chronic pericardi tis, probably due to bleeding. Cecum: glandular part was mlarged aid thickened. Rabbit no. 149. Injected with strain No. 475E. Liver: armic lesions, consisting of a number of small nodules which are probably parasitic in nature. Kidney (right): small pin point sized whitish spots. kidney (left): two small spots of the same nature. Cecum: thickened, but not so much as 166. Vila-ms. The pasibility that the organism loses virulence men grow on artificial nedia led to the injection of two more chickens. These two hens were each injected intramuscularly with 4 cc. of mspension from twenty-four hour agar cultures of a strain isolated seventy-two hours before. They were killed torty-fcur days after injection, and were autopsied at once. Bird no. 100. Liver: normal in size, and in general of mutual color. 0n the rigit lobe is a bluish discoloration on the capsule. The capsule is somewhat thickened at this point. near the border of the left and rigit lobes are some spots that are not sharply circum- scribed, of somwhat darhr reddish brown color. On to border of the left: lobe is a yellowish spot of the size of a pee, fine consistmicy of which is a little tough. Near this spot but on the lower surface is a larger spot showing partly dark brcm, partly a yellowidi color. On the lower surface of the right lobe is a projectionabozt the size of a pee, of dark red brown color, probably a hamstoma. Heart: chronic adhesive pericardi tie, a few very shall hemorrhages in the visceral part of the pericardium. - Ovary: some ovules quite normal, others collapsed and grayish yellow in color. Sligit pritonitis. Bird no. 101. Liver: peculiar yellowish discoloration, tough of normal cansistmcy. Probably due to fatty degmeration. Heart: flabby, adhesive pericarditis. Ovary: sale ovules collapsed and of yellowish grayish di sc olorati on. -38- On February 13th four guinea pigs were injected intra- peritoneally with .5 cc of suspension of live organises (turbidity 3) in .85 percent salt solution. Four pigs were injected with suspension of orgmism (turbidity of 2) that had been heated 30 minutes at 55‘C for the purpose of attenuation. Thsynwere given four injections, at four day intervals, the size of the does being doubled each time. The first injection was 1/2 cc., the last one 4 cc. Strain number 488 was used for all guinea pig injections. Two control pigs were kept with the others. The entire group was killed March 9th. The lesions were slight and were found in the control pigs as well as in he injected ones. Therefore it is not considered worth while to include even a brief summary of the findings. The mimals’ probably are not susceptible. V. DISCUSSION OF REULTS. As may be concluded from the data of a number of postmortems reported before, the results Obtained were to a large extent inconsi stunt and inconclusive. Consequently it is not an easy matter to decide whether the orgmism in question represents a pathogenic parasite or a harmless saprOphyte or perhaps a intermediate form. The fact that the crganism he been repeatedly isolated in pure culture from birds that died and from which no other organia known'h be capable of causing disease was isolated, seems to suggest that we have to deal with an organism, the pathogenicity of mich is at least highly probable. This probability is supported by the fact that as it may be seen from Table III a number of he birds infected experimentally with this organism mowed more or less pronounced lesions and hat fra six out of ten so infected birds tie organism could be reisolated. The variation and in may cases the abaauce of lesions in a fairly large number of cases of the routine autopsies (Table I) as well as in the autOpsies of a number of injected birds tends to dies, however, that this again my, under as yet unknown circumstances, be capable of causing chmges in some of the «guns of the host. If such fava'able conditions are absent the organism seems to be harmless for either birds or rabbits. Guinea pigs are mtirely resistant. Of special importance so far as the patcgenicity of this .. i ill-I1; ‘2’. r‘ organism is concerned are the following cases: i Rabbit no. 488 (Inge 6 ) injected intravenously with culture freshly isolated by Dr. Stafseth. The rabbit died in twelve days, and any lesions were found. All the lesions noted have been found in chickens, thouQ none of the. are consistently found in all animals from which the organism had been isolated. Birds 906 aid 584 (pages 9 med 8) also injected with fremly isolated strains are evidence also of pathogenicity. Both died within a comparatively short time, and many lesions were noted. The autOpsies of birds 496 and 475 seam to point out that the preemies of the organism is coincidental and the cause of the diseased condition. In these cases, flougi the organism was isolated, other organisms that are known to be capable of causing death were also isolated. It would not be possible to say mich orgmism killed to bird. Firther the organin seen to be avirulent in most cases. This seems to be paved not only by its occassi onal pathOgenic effects, but also by its cultural peculiarities its ability tog-ow on artificial media decreases very rapidly. It might be due to this low virulence that in some of the experimental infections (100 and 101) no lesi we could be encountered and that even after heavy doses the ergmism very soon disappeared from the organism of the hat. 8 The low virulence is indicated farmer by the nature of the lesions of different orgms. Those usually mow a chronic character though all organs and especially the blood may lmrbor large numbers of this organism (bacteraemia). Whether or not the disease possibly caused by this organism is cmtagious, is a question of interest. The organism has been isolated from a number of birds of two flocks. Contagiousness might account for the finding of lesions in the cmtrol bird kept with those injected ft: the study of pathOgenicity, also in the ones that were injected with filtrate of peptic digest broth. It might be me cause of the positive reactions to the agglutination test obtained with the serum of the birds that were hpt in the animal room near those injected during the study of this organism. It is not known tether or not the organism produces toxin. The lesions found in the birds injected with filtrate of peptic digest cultures might have been caused by infection contracted from to bird injectedwith cultin-es, or the lesions of those injected with cultures may have been caused by toxins injected with the organism, since broth cultures were used. The two hens last injected were given suspensions fru agar slats, but the evidence from lesions in mass cases is not such as to warrant weight being given to it. The writer has made no effort to classify the a-ganism. Careful search has been made in Bergey's "Determinative Bacteriology", with the cmclusion that the orgmism has '-36- not ban studied befu'e. Characteristics about vhich fliers was no doubt, such as sugar fermentations, milk reaction, indol production, and Gram staining, were used as a basis of the search. The possibility that it is either a coccus or a rod was taken into consideration. 8. 3. 4. 5. -37- VI. SUMMARY The organism is a mall coccus or cocoo-baoillus, measuring about one micron, occuring most often in pairs, yelling poorly on artificial media, producing mthaemOglcbin on blood plates, fermenting dextrose, sucrose, maltose, levulose, lactose, salacin, reffinose, md dextrin, wit production of acid, but not gas, giving acid reaction in milk, with coagula- tion mid reduction of litmus, producing indol, reducing nitrates to nitrites and umonia. It is aerobic and facultative anaerobic. Its thermal death point is 58°C. Under as yet unknown circumstances this organism may be paid: ogenic for chickens mud rarely for rabbits. In most cases its virulence is very low and then its occurrence in chickens seems to be without any significance. If lesions are caused by this again they are of chronic nature. Infected birds apparm tly do not Ircdmre mcific agglutinins. Pb otographe,ehosing bi-polar bodies. t3 ‘ - ,0 009 o a t . . . ’9 O I g 0' a 0 ‘3 3!! ‘fi 4-:- O I Drawing showing characteristic groupings I 1" ’- 5‘ ‘\ I ‘ f “1.. \\\\\ // Involution forms,(drawn on much higher scale VII. ACKNOWIEDGM T. The writer wishes to acknowledge indebtedness to Dr. Ward Giltner, Dr. E. J. Stafseth, Dr. A. Kotlan, 111'. W. L. nallm md Miss Dorothy Yakeley 1hr assistance and helpful suggestions received during the course of these investigations. - 39 - VIII. BIBLIOGRAPHY Ila-so, 3. and 1!. Kapeloff 1922 - A simple nethod of Anaerobic Cultivation in Petri Dishes Abstracts of Bacteriology, p. 35 Rush, Jo Ea In! Go ‘a Palmer 1921 - On Decreasing the Exposure Necessary fer the Gelatin Jo BEOt. 6, 571 Bortm, J. 1'. and M. V. Sawyer 1921 - Indcl Production by Bacteria. .1. Beat. 6, 4'71 H‘u, we Ild. Ac Do ”8861' 1921 - The pH range for staphlococcus. Brit. J. Exper. Path., 2, 242 Graham, E. and E. A. Tunnicliff Coccidiosia of Poultry. U. of 111. E1. st. Circular no. 288 Graham, R. and E. A. runnicliff. Tuberculosis of Fowls Va 01 I11. Ex. 3‘. Circular NO. 285 Graham, R. an! E. A. Tunnicliff Fowl Typhoid U. of 1110 he Ste Ghoul” B0. 287 Graham, R. and E. A. Tunnicliff, Fowl Cholera He or 1110 Ex. Ste Circular NO. 286 Van Es, 1.. mid H. M. Mania. The More Important Poultry Diseases He 01 BOba he Ste Bulletin 195 Ray, Henry G. and Helena Tibbetts on-typhoid Intermediates as Causative Agents of Disease in Birds. II Atypical Orgaztm. A8. Exe Ste Kingstm, Ra Ia Giltner, Hard laboratory Hamel in General Microbiology. Harvey, W. 1'. mid K. R. K. Izengar Virulence of a Micro-organism, and its Dependence on the Culture Medium. Ban Reemsdi J]: 1921 - Du Kapselu der Bakterieu und eine neue memode diese enifach daraeistelleu. Centralbl. t. Baklirio, (etc) I, 5, 177 Hadley and Amison 1911 - Biological Study of Eleven PathOgmic Orgmisms from Cholera-like Diseases of Poultry. R. I. Bul. 146 Beaudette, Bushnell and Payne 1923 - study of an "Orgmism Resembling B. Eullorum from unabsorbed yolk of chicks Dead in S e J. of Inf. Dis., 32, 124 Three new Bacterial Species PathOgenic for Domestic Birds. no so Re, V01. 42, P. 478 Krumiviede au Kohn 1917 - Studies on Paratyphoid-enteritis Group Differentiation of the numbers of the Paratyphoid Enteritidis group from B. t hosus with Special Reference to anaerobic Strains an; UBservations on the Fermentation Characteristics of Avian Types. J. of Med. Res. 56, p. 505 '01:, Go Go Lo 1920 - The Influence of the Reaction of Media and of 111s Presence of Beeffer salts of the Metabolism of BBOtBrj-Io Brit. J. Exper. Pathol., 1, 288 36185013, 1!. 1921 - Uber Indcl-und Phenolbildenig durch Bakterieu. Munchen Med. ‘,Nchnsch 68,1384 Conn, He Jo 1921 - Standardization of; Strains. Report of Committee on Bacteriological Technique. December. 1921 - Technique: Scientific Proceedings of the Society of American Bacteriologists, Deceifirer.‘ Husker, G. 1‘. Comparison of Various Methods of Gram staining. *fi.tn ,. I...” A an; my ml. onus-n”. so»; ». .- -1v \.0 ‘a‘v-Ik-wues Vek‘ u- c" M" u "-z- ~.4~ Q r.a \‘afi‘. a-‘ua --‘-. a- . -x’rM'J'ev g‘.§ la ’49 .e 1