HHIWIHilfil‘lhbMyl ) [ 8H # I I—im Inn _m.l>- ESCH ERECHEA COL! iNFECTiON {N CHICKENS Thesis for the Define of M. S; MICHEGAN STATE COLLEGE Cheng, Ming-Ying 1948 THESIS This is to certify that the thesis entitled "Eschericia c011 Infection in Chickens“ presented by Ming ling Chang has been accepted towards fulfillment of the requirements for Master's degree in__B_a__g§_eriology m/IQVW Major professor] Date May 27, 1948 31-795 “ 4 .. - —— w.-.-—mq——-—-—--‘ .4 H 4-4 ~— JV." ESCHERICHIA COLI INFECTION IN CHIC KENS CHEN G, MING-YING IQ ' A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology 1 948 THesxs yykt ACKNOWLEDGMENT The writer is indebted to Dr. Henrik J. Stafseth for his direction and criticism ofthis work; to Dr.Char1es Cunningham for his suggestion; to Dr. Saul Norotsky for his continuous advice and assistance, and to Mrs. Ruth Gunn for her technical assistance. 199396 ESCHERICHIA COLI INFECTION IN CHIC KENS C ONTENTS Review of Literature . . . . . . . . ExperimentalWork ................ Identification of the Organism . . . Pathogenicity Tests . . . Cross Agglutination Tests . Discussion . . . . . Sumrnary..... Bibliography . . . . . . .12 15 20 2.2 23 ESCHERICHIA COLI INFECTION 2i CHICKENS On July 4, 1947 it was reported by the Regional poultry Research Laboratory, United States Department of Agriculture, East Lansing, Michigan, that a few chickens had died suddenly from which a bipolar organism resembling Pasteurella avicida morphologically had been isolated. Very little was known about this case except that the birds appeared to be depressed; they showed no diarrhea or other intest- inal disturbance. These birds died three days later and on aut0psy, petechiae on the heart muscle and the gizzard were the only lesions Observed. Nine broth cultures containing the isolated bacteria were brought to the Department of Bacteriology, Michigan State College. Cultures I 266VTRWS, I40 96 liv and I409 HT were submitted first; and later six cultures FllllS, F4680, F266B, FZSSWZIV liv, H107SGIP and H113L2 liv. The purpose of this work was: 1. to identify the organism, Z. to determine its pathogenicity for chickens and 3. to ascertain whether it had any antigenic relationship to Salmonella pullorum. REVIEW OF LITERATURE Klein (1889) described an infectious disease of grouse (Lag0pus scoticus), caused by Bacterium coli, characterized by congestion of the lungs, necrotic areas in the liver and patchy redness of the intestines. These findings were verified by Smith. Ligniers (1894) examined a number of hens which died showing diarrhea but no other symptoms. At aut0psy he observed generalized inflammation of the intestinal tract and a quantity of serous fluid in the body cavity together with enlargement of the spleen and liver. From the Spleen he isolated a culture which appeared to be of the _B_. 5:31} type. Sanfelice (1895) reported a disease among pigeons in Sardinia from which an organism resembling 2.213 was isolated. In the dead birds the prominent patho- logical features included fibrinous exudate in the body cavity, and over the surface of the intestines, also enlargement of the spleen. However, he did not present data on the biochemical features of the organism. Fiorentini (1896) isolated from a swan affected with hemorrhagic septicemia an organism resembling E. avicida but differing from it. He stated that it grew like Bacterium coli communis on solid agar. His description suggested that it was a virulent colon bacillus. Martel (1897) isolated a virulent colon bacillus from hens and turkeys. 0n autopsy he found pericarditis, with an abundant false membrane without much liquid, congestion and friability of the spleen, inflammation of the small intes- tine and the ceca and suppurative conjunctivitis. From the blood, liver, false pericardial membrane and the conjunctival exudate he isolated a 3 bacillus, and upon studying it culturally and biochemically, proved it to be a colon bacillus. Dawson (1898) investigated a coli-like organism, isolated from sick birds in the vicinity of Washington, D. C., which produced acid and gas in lactose, dextrose and sucrose broth. The symptoms of the disease were: emaciation and voracious appetite lasting about three months before fetal issue. ”Going light" or infectious asthenia was the name he gave to this disease. Mazza (1899) observed an outbreak of hemorrhagic enteritis among hens that was probably caused by _B_. _<_:_o_l_i_. Joest (19oz) isolated 3. con and _B_. intestinale gallinarum from the heart, blood, spleen and liver of dead hens. Morse (1906) studied a contagious disease of quail caused by _B_. 5213. The post-mortem revealed slightly congested lungs, and a few areas of superficial necrosis in the liver, the intestines being studded with minute ulcers, the spleen was always congested. The organism was usually cultivated from the liver in which lesions existed. Claussen (1907) found pure cultures of colon bacilli in the blood of dead birds. This organism was very similar to Past. cholera gallinarum but appeared about twice its size. Upon dissection, the heart was found to be filled with black, partially clotted blood and the epicardium was covered with small petechiae. The intestinal mucosa appeared slightly inflamed and the parenchyma of‘the liver was slightly darkened. Hadley (1910) isolated 3.391} from the Spleen, liver and heart blood of sick birds affected with a cholera-like disease. High tempera- ture and yellowish diarrhea were the symptoms observed. Upon examina- tion, the liver was hyperemic and soft, the spleen was enlarged and the hemorrhagic inflammation extended through the entire intestine. Zeiss (1914) 4 reported two cases of coli septicemia in hens. The organism isolated from this case exhibited oval, elliptical and polar-stained rods which were very pathogenic for canary birds. Baudet (1922) isolated from the carcass of a hen a virulent strain of __B_. coli which killed mice and pigeons in two days after subcutaneous injection of 1 loonul of the cultures and chickens in a few days after intramuscular injection. Palmer and Baker (1922) found that Bacterium coli communis was the causative organism of an infectious enteritis of chickens, ducks and turkeys in Northern and Central Delaware. May and Tibbetts (1923) reported two bacterial cultures isolated from diseased poultry in Rhode Island. One of them originally came from Rahn of the Michigan Agricultural College. No further in- formation accompanied the culture as to the symptom of the disease and the extent of the epidemic. They suggested that this organism might be a strain of E. coli. In 1928, an acute generalized E. coli infection in chicks was reported by Kansas Agricultural Experiment Station. The symptoms were sudden onset of pulmonary dyspnea, accompanied by marked depression. Lewis and Hitchner (1936) described a slow lac- tose fermenting organism pathogenic for chickens. This organism was proved distinctly pathogenic for young chicks when given orally or ' subcutaneously. Davis (1938) encountered colibacillosis in chickens in Maryland. Upon autOpsy, the livers were about 50% larger than normal, mottled and contained numerous small white spots. The heart was slightly enlarged and the pericardial sac was markedly distended with amber colored fluid. He attributed the etiology of this disease to faulty incuba- tion, insufficient moisture and impr0per ventilation. Twisselmann (1939) reported that seventeen birds of a well isolated group of two hundred and fifty pullets were attacked by a rapidly fatal disease of which he stated, _E_J. £211 was the etiological agent. Autopsy showed. the crops of several birds to be partially filled with grain. The pectoral muscles had a reddish congested appearance. The myocardium was degenerated and in some there was a fibrinous pericarditis. The liver showed congestion with small areas of degeneration and, in some cases, a slight greenish cast. Bunyea and MacDonald (1941) studied the pathogenicity of _E. 32E acidi lactic} for turkeys, and considered it responsible for young poult mortality. Osborne, Witter and Hitchner (1947) studied chronic colibacil- losis of fowls. The organism which they studied was proved to be very pathogenic for guinea pigs, rabbits and chicks. Durant and MacDougle (1947) found _E_. coli in the blood of adult fowl affected with the occular form of fowl paralysis. When 1 ml. amounts of the blood taken from the affected birds were injected into day old chicks, 84 of 85 chicks died. EXPERIMENTAL WORK The organism submitted by the Regional Poultry Research Lab- oratory was identified by its morphological characteristics and biochemical properties. For the determination of pathogenicity, chicks were fed a suspension of the living organism. Normal chicks were placed in the same cage with the exposed birds as controls. A normal rooster was immunized with a killed suspension of the isolated organism. Cross agglutination tests were made using the immune serum from the rooster against _S_ pullorum antigen, and _S_. pullorum immune serum against the organism being tested. For convenience, the procedure and results of each experiment will be presented separately. Identification of the Organism Morpholggy and cultural characters. Smears were made from the original cultures. By using Burke’s modification of Gram’s stain, the organism was found to be a gram negative, slender, pleomorphic, short rod. It stained unevenly, but was not bipolar. No spores or capsules were found. The size of this organism was about 0.5 n by 2 p. Single, isolated colonies for further identification were obtained by plating the original cultures on blood agar and S S agar plates. At the same time the three original cultures (1 266VTRWS, I 4096 liv. and I 409 HT) were injected into three chickens (three months old) designated by leg bands No. 27778, 13715 and the third without band. The routes used for injection were intraperitoneal, subcutaneous and intravenous respectively. Two days after injection all these birds appeared dull, however there was no other symptoms to be observed. Five days later the birds were destroyed and autopsied. Cultures were made from the heart and liver of each bird. The changes observed in these birds were: pericarditis, focal necrosis in the liver, congested kidneys and peritonitis. In addition bird No. 27778 showed petechiae in the intestines and bird No. 13715 showed diffuse enteritis. The original nine cultures and the cultures isolated from the three inoculated chickens produced pale, moist, medium size, convex, round, regular, non—hemolytic colonies on blood agar plates. Culture I 409 HT did not grow. On S S agar plates, small, scanty, pink convex colonies were occasionally found from cultures F111 IS, F468Q, H107SQIP liv. and I 266 V TRWS. Fourteen separate colonies were picked from the blood agar plates and inoculated into lactose motility agar tubes. Each one showed motility and fermented lactose after 2.4 hours incubation except Nos. 5 and 11 which showed beaded growth along the stab line. Table I shows the fermentation reactions of these organisms. TABLE I Fermentation Reactions in five common Sugars Culture .5 1 ml mn lumber 1 AG AG AG AG 2 AG AG AG AG 3 AG AG AG AG 4 AG AG AG AG 5 A A A .... 6 AC AC AG AG 7 AG AG AG AG 8 AG AG AG AG 9 AG AG AG AG 10 AG AC AC AG 1 1 A _1; r- '- - 12 AG AG AG AG 13 AG AG AG AG 14 AG AG AG AG S = dextrose - = no fermentation l = lactose A = acid ml = Maltose AG = acid and gas mn = mannitol :L = slight acid with s = sucrose insignificant pink color 9 These results show that the reactions of twelve of the fourteen cultures were identical. The twelve identical cultures were stained and found to be morphologically similar to the original cultures. Nos. 5 and 11 were gram positive streptococci and diplococci respectively, possibly contaminants and were discarded. For more exact identification, four cultures were selected for further studies, namely, 1, 3, 8 and 13 which were isolated from birds 13715, the bird without band, 27778 and original culture F 468 Q. Additional cultural and biochemical characteristics of the four selected cultures were studied using eosin—methylene-blue agar plates, Kligler’s iron agar slants, methyl-red tests. Vogtes - Proskauer tests, indol tests, nitrate reduction tests, gelatin liquefaction tests, citrate utilization tests, litmus milk coagulation tests and fermentation tests. The eosin—methylene-blue agar, Kligler iron agar and Simon citrate agar were Difco Bacto dehydrated media. The results of these tests are surmnarized in Table II. 10 TABLE II Biochemic a1 Characteristic s FCulture Number l 3 8 l3 Slant A A A A J" .‘3 ' 3 3" Butt A.G. A.G. A.G. A.G. so a a3. H25 production - - - - E.M.B. Agar M.S. M.S. M.S. M.S. Citrate Agar - - - - Litmus milk A.C.P. A.C.P. A.C.P. A.C.P. M.R. test + + + + V.P. test - - - - Indol test + + + + Nitrate Reduction test . + + + + Gelatin liquefaction test - - - - Adonite - - - - Arabinose A.G. A.G. A.G. A.G. Cellubiose — - - - Cellulose - - .. .. Dextrin A A A A very slight acid reaction Dextrose A.G. A.G. A.G. A.G. Dulcitol A.G. A.G. A.G. A.G. a little gas; pro- duced alkali 3 days after acid formation TABLE II (continued) Biochemical Characteri stic s 11 Culture Nurfiber 1 3 8 l3 Galactose A.G. A.G. A.G. A.G. Glycerol A.G. A.G. A.G. A.G.- slow gas forma- tion on the 3rd day after incubation ' Inosite - - - - Inulin: - - - - Lactose A.G. A.G. A.G. A.G. Levulose A.G. A.G. A.G. A.G. Maltose A.G. A.G. A.G. A.G. Mannitol A.G. A.G'. A.G. A.G. Mannose A.G. A.G. A.G. A.G. Raffinose - - - - Rhamnose A.G. A.G. A.G. A.G. a little gas Salicin - - .. .. Sorbitol A.G. A.G A.G. A.G. a little alkali pro- duced 2 days after the acid formation Starch - - - - Sucrose - - .. .. Trahalose A.G. A.G. A.G. A.G. Xylose A.G. A.G. A.G. A.G. A = acid A.C.P. = acid curd and G = gas peptonization A.G. '- acid and gas + = positive reaction M.S. = growth with metallic sheen - = negative reaction 12 The fermentation reactions in dulcitol, salicin and sorbitol were variable when the cultures were kept for a longer time. Culture 3 pro- duced a prominent quantity of alkali in three days. Culture 8 produced a rather abundant quantity of acid and gas in three days. All these four cultures reversed their action on dulcitol on the fourth day and at last neutralized the acid produced by the organism. Repetitions of the fer— mentation tests on dulcitol, salicin and sorbitol have been made, all showed the same results as before. In litmus Inilk they produced acid on the third day, and peptoniza- tion on the fifth day. The results when compared with the description of E. coli in Bergy‘s manual (5th edition) would indicate that the organism studied can be identified as Escherichia coli. Pathogenicity Tests on Chicks Cultures 1, 3, 7, 8, 10 and 14 were chosen as the test organisms. Among these, Nos. 1, 3,. 7 and 8 were reisolated from laboratory birds. Culture 1 was reisolated after subcutaneous injection. Culture 3 was reisolated after being given intravenously. Culture 7 was obtained from the pericardial sac of an intraperitoneally injected bird. Culture 8 was reisolated from the peritoneal fluid of an intraperitoneally injected bird. Twelve, normal two-day old chicks were selected as experimental birds. A saline suspension of each of the above mentioned cultures, standardized to a turbidity equal to that of Tube No. l MacFarland’s nephelometer, was prepared from a 24 hours tryptose agar slant culture. One ml. of each l3 suspension was fed to each chick as shown in table 111. These chicks were kept with 25 untreated chicks as controls. After observation for two months; four chicks were dead as shown in table 111. As a rule, one or two days before death, the birds refused to eat and drink, and walked with staggering gaits. Gram negative rods were isolated from venous blood of one bird before his death. The organism was identified as _E— _c_c_>_l_i_. Symptoms were always observed for two days except in the case of No. 3216 which showed sickness for two days and then seemed to have recovered. However, it died sud- denly five days later. Autopsies were made on each dead bird. Congestion of the lungs, peritonitis and cloudy swelling of the liver were the common patho- logical changes, except for slight inflammation of the jejunum in bird No. 3227. Cultures were made from lungs and livers. Through micro- sc0pic examination, EMB agar plates cultivation, -M.R. tests, V. P. tests and indol tests these organisms were found to be identical with No control birds died, except one which died from ruptured liver. Cultures from the liver of the chick yielded no organism. 14 TABLE III Results of Pathogenicity Tests by Feeding Cultures to Chicks ”We J m Number Chick being Date of Date of Survived Number fed feeding death days 3209 1 Oct. 24 ’47 60 3214 " “ 60 3215 3 ” 60 3216 " " Nov. 2 ’47 8 3226 7 ” Nov. 15 ‘47 21 3227 ” ” Oct. 28 ’47 3 3228 8 ” 60 3251 ” ” 60 3252 10 ” 60 3253 ” " 60 3254 14 ” 60 3255 ” ” Oct. 26 ’47 l 15 Cross Agglutination Tests Preparation of E. Coli Antiserum. Two strains of _S. pullorum, No. 1-137-5 isolated from a turkey and No. 119-3 from a baby chick, were obtained from Mrs. Ruth Gunn, Instructor of Bacteriology, Michigan State College. One rooster and samples of pullorum positive serum and pullorum negative serum were obtained from Dr. Saul Narotsky, Instruc- tor of Bacteriology, Michigan State College. Before carrying out the immunization, 8 ml of blood was drawn, the serum separated and placed in a refrigerator. Agglutination tests performed with these sera proved the bird to be negative to the pullorum test. _E_. coli No. 1_§. pullorum No. 1-137-5 and No. 119-3 were seeded on tryptose agar slants and in- cubated for 24 hours. The antigens were prepared by washing off the growth with saline (pH 8.25), heating in a water bath at 56° c for 1 hour and washing three times. The turbidity of all antigens used for the ag- glutination test was adjusted to equal that of Tube No. 1 of MacFarland’s nephelometer . In order to identify cultures No. 1-137-5 and 119-3 as _S_. pullorum and to prove that the serum of the rooster was free from pullorum anti- bodies and anticoli antibodies agglutination tests were performed with three kinds of sera and three kinds of antigens as shown in table IV. Preliminary Antigenic and Serological TABLE IV 16 Testing I Dilutions If Con- Sera Antigens 20X 40X 80X 160x 320x 640x trol Serum of S. pullorum - - - — .. .. - rooster which No. 1-137-5 was to be S. pullorum - - — _ .. - .. used for im- No. 119-3 rnunization E. coli — - - .. .. - - Pullorum S. pullorum ++++ ++++ ++++ +++ +++ +++ - positive No. 1-137-5 serum S. pullorum ++++ ++++ ++++ +++ +++ +++ - E. coli 1 - - — .. - .. Pullorum S. pullorum - - — — .. - _ negative No. 1—137-5 serum 5. pullorum - - - — .. .. .. E. coli :1; - - .. .. _ - ++++ = complete agglutination +++ = fairly strong agglutination i = doubtful reaction no reaction 17 The preliminary tests proved the rooster to be satisfactory for the purpose of immunization. Antigen of _E. 2213 was prepared as des- cribed above. The rooster was injected intravenously four times at one-day intervals starting with 1 ml of the suspension, followed by two injections of 2 ml at one-day intervals. The rooster was bled seven days after the last injection and the serum separated. Agglutination tests were made with E. 5313 antigen. This antigen was prepared by seeding £2. 5313 No. l on tryptose agar slants and incubating for 24 hours. The growth was washed off with physiological saline solution (pH 8.25) containing 0.5% phenol and ad- justed with physiological saline solution (pH 8.25) containing 0.3% phenol to the turbidity of Tube No. 1 MacFarland’s nephelometer. The results are shown in Table V. TABLE V Titration of Immune Serum Serum AntigerZOX 40X 80X 160x 320): 64ox12sox ZSSOX Cont. E. coli I'mr-rfii—ne E. coli ++++ ++++ +++ +++ ++ + + + - serum of — * rooster It was shown that the agglutinating titer of the rooster’s serum was 4 + up to 160X and fairly strong in 320x etc. The _E_. coli antiserum was therefore satisfactory for agglutination tests. Antigens of _S_. pullorum No. 1-137-5, 119-3 and _E_. coli No. 1 were then prepared by the same method as described before and cross agglu- tination tests were made with _E_. coli antiserum and pullorum positive serum. The results are shown in table VI. 18 TABLE VI Cross Agglutination Tests __Y Dilution I Con- Sera Antigens 20X 40X 80X 160X 320x 640K trol E. coli imm. S. pullorum + _+_ J; - — .. .. — serum 1-137-5 _E_. coli imm. S. pullorum + + - - .. - .. serum 119—3 Pullorum E. coli + + - - - .. - m positive serum No. 1 negative reaction doubtful reaction :1: + = weak positive reaction This procedure was repeated with greater care, because the re- sults obtained were so indefinite. The antigens were centrifuged and washed four times. Three clear, satisfactory suspensions of antigens were obtained and cross agglutination tests were made. The results are shown in Table VII. 19 TABLE VII Final Cross Agglutination Tests :========: Dilution Con- Sera Antigens 20X 40X 80X 160X 320x 640X trol E. coli imrn. S. pullorum + - - - .. .. - serum 1-137-5 E. coli imm. S. pullorum i - - - .. - .. serum 119—3 Pullorum E. coli No. 1 + + - - — - - Positive serum - - negative reaction 1 = doubtful reaction + = faint'positive reaction These results show that there was no significant antigenic rela- tionship between this strain of _E. coli and _S_. Pullorum. 20 DISCUSSION Strictly speaking, _E_. 5311 is but an opportunist. It can cause dysentery and navel infection in calves, pyelonephritis, cervicitis and mastitis in cows and wound infections in various animals when the en- vironment favors its growth. Also it has been recovered from eggs of hens affected with fowlparatyphoid. It was found in the liver of quail suffering from paratyphoid. Summing up the data from all the cases cited in the review of literature, concerning the nature of the disease caused by _E_. £213, it seems reasonable to assume that this organism has a predilection for the mucous membranes of the digestive tract and the parenchymatous organs, su'ch as the liver, spleen and heart. This organism also causes septicemia. The case encountered in our laboratory was very similar to that described in Maryland. According to the pathogenicity tests performed in this work, it has been shown that culture No. 7 will kill chicks when given orally or injected intraperitoneally. Considering the history of the case, this organism might be assumed to be very virulent, but it did not prove to be so by virulence tests. The reasons for this disagreement might be: 1. bacterial variation 2. natural field infection may differ from experimental infection 3. lack of necessary contributory factors 4. different susceptibility of individual birds. It is noteworthy that there has been no outbreak of bacterial disease in the stock of the Regional Poultry Research Laboratory in 21 East Lansing during its existence until this one occurred. Therefore some support is given to the thought that the strain of _E_. coli isolated from the affected birds may have been the cause. In regard to the serological relationship between _E_. coli and S. pullorum, some information is available. According to the Kauffmann- White schema “Bact. coli 3” possesses a part of the 0 antigen found in _S. pullorum, and numerous other coliform bacilli have the same antigen. 1 have described the presence of a common Stamp and Stone antigen in many members of the coliform and paracolon group. It is more heat- stable than the H antigen and less stable than 0 antigen. It is inactivated at 100° c in 15 minutes but withstands a temperature of 75° c for 1 hour. It is associated with recently isolated strains, and tends to be lost on subculture. Thus it is doubtful whether this antigen was retained by the organism under investigation. The antigenic composi- tion of the coliform bacilli has not been completely determined. The cross agglutination tests revealed no significant antigenic relationship between E. coli No. l and _S. pullorum. Hut lWilson, G. S. and Miles, A. A. .. Tapley and Wilson’s Principles of Bacteriology and Immunity. 3rd. ed. The Williams & Wilkins Co., Baltimore, p. 666. .* “v a __...- n.‘ II "a. 7|- :TJ 6-.1- "Smh-.tr-—‘Tfl- _ ' - b. “I _‘ 'ul'lmn‘...‘ -. a... .. J _. - .l 9&- Aa an... .nur...__,_. :— _ s. .1- A';“.—1 L's ”'1‘; _.‘___._. m—‘I—a—A—n‘. ‘52 'TLJ'W. .. l' ‘5 n v - ‘u. . y- _ ‘ _ '4' . 4.x 1 ’.-E.’"' -."lI-..-f 3 ,. . a if“ nl'gfl.r_{_~’ ‘ . .ut L r' _-' ‘n—i I; II A . 'aum‘ '5‘. 22 SUMMARY Some strains of an organism which proved to be _E_J. coli were isolated from chickens affected with a cholera-like disease. This organism proved to be moderately pathogenic for chicks when administer ed orally. Cross agglutination tests with _S_. pullorum revealed no signifi- cant antigenic relationship between these two organisms. 23 BIBLIOGRAPHY Baudet, Y. 1922. Colibacillosis in chickens. Vet. Med., 17, 451. Beach, I. R. and Stewart, M. A. 1942. Colibacillosis. Univ. Calif. Agr. Expt. Sta., Bull. 674, 76. Bergy, D. H. 1939. Manual of Determinative Bacteriology. 5th. ed. The Williams 8: Wilkins Co., Baltimore, 388-394. Biester, H. E. and Deveries, L. 1943. Diseases of Poultry. The Iowa State College Press, Ames, Iowa. 225-236, 349-352, 511-522. Bunyea, H. and MacDonald, A. D. 1942. The pathogenicity of Aerobaiter aerggenes and _E_. coli acidi lactici for turkeys and their response to the agglutination test for pullorum disease. Poultry Science 21, Davis, C. R. 1938. Colibacillosis in young chicks. J. Am. Vet. Med. Assoc. 92, 518-522. Durant, A. I. and MacDougle, H. C. 1947. E. coli in the blood stream of adult fowl affected with the ocular form of fowl paralysis. Am. J. Vet. Research. 8, 213-215. Experimental Station Record, 1931. Work with diseases and parasites of poultry at Kansas Station. 64, 880-882. Hadley, P. 1918. The colon-typhoid intermediates as causative agents of disease in birds: I. The paratyphoid bacteria. R. 1. State COI. Agr. Expto Sta. B111]... 174, 1'9. Lewis, K. H. and Hitchner, E. R. 1936. Slow—lactose fermenting bac- teria pathogenic for young chicks. J. Infectious Diseases. 59, 225-235. May, H. G. and H. A. M. Tibbets. 1923. The colon-typhoid intermediates as causative agents of disease in birds: 11. The atypical organisms. Re I. State C01. Agr. EXPto Sta. B1111. 191, 1-42. Merchant, I. A. 1946. Veterinary Bacteriology 3rd. ed. The Iowa State College Press, Ames, Iowa. 307-312. Osborne, I. C., Witter, I. F. and Hitchner, E. R. 1947. A comparative study of cultures of microorganisms involved in chronic coliba- cillosis in fowl. M. S. C. Vet., 6, 25-29. 24 Palmer, C. C. and Baker, H. R. 1923. Studies on the infectious enteritis of poultry caused by Bact. coli communis. J. Am. Vet. Med. Assoc., 63, 85-96. I W Peluffo, C. A., Edwards, P. R. and Bruner, D. W. 1942. A group of coliform bacilli serOIOgically related to the genus Salmonella. I. Infectious Diseases, 70, 185-192. Twisselmann, N. M. 1939. An acute infectious disease of pullets ap- parently caused by _E_. coli communis. J. Am. Vet. Med. Assoc., 94, 235—236. H45 Wilson, G. S. and Miles, A. A.A Topley and Wilson’s Principles of Bacteriology and Immunity. 3rd. ed. The Williams & Wilkins 199396 HWIH 950 .1” i“ i 14.1