.— “MFL... _r_ .27-:— A- - ’ H H I H I“ I “‘ SALMONELLA PULLORUM INFECTION IN ADULT TURKEYS Thai: for flu Dam of M. 5‘ MICHIGAN STATE COLLEGE Esperanza Bonilla I948 4:" -' "I :1 / 9'“; I TREES. ‘ LL This is to certify that the thesis entitled Salmonella Pullorum Infection in Adult Turkeys presented by Esperanza Bonilla has been accepted towards fulfillment of the requirements for J_o So degree ill—E‘I-CteriOlogy major profesfi Date April 2, 1918 11-795 FALI'C‘NFTLIA Pmmmrrs II-IW‘ECTTCN IN ADULT TURKEYS I CGLPARATIVE STUDY OF TETRATHIONATE BROTH LANDELIC ACID BROTH AND SELFRITE F BHOTH ENEICHMLNT NEDIA IN THE ISO- LATION OF SALMONELLA PULLORUM II RELATIONSHIP BETWEEH THE AGGLUTINATION REACTION AND THE .1. OPEN CARRIER STATE RV It Esperanza Bonilla __‘ A THESIS Submitted to the School of Graduate Studies of yichigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 193.18 \ {THESIS TABLE 03 COHTLHTS Introduction......................................... Previous'Work........................................ Formulas of the Media................................ Naterials and Methods................................ TaOle ICOOOOOOOOOOOOOOCOOOOO0.0.00.0...OOOOOOOOOOOOO. ‘ Ta‘Dle 110......OOOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOO. '1 Table IIIOOOOOOOOOOOQOOOOOOO0.0000000000COOOOOOOOOOCO 1 Table IVCOOOCO0.0.0.0...OIOOOOOOOOOOOOO0.00.00.00.00. Table VOOOOOOOOOOOOOOOCOOOOO...OOOOOOOOOOOOOOOOOOCOO. Table VIOOCOCOOOOOOOOOOOOO0.000000000000COOOOOOOOOOOC Table VIII..OOOOOOOOOOOOOOCOO00.0.00...O00.000.00.000. DiSCUSSiOHOOOocococoooooooooooooocococoon-0000.00.00. Conclusions..........q.o..................o......a... Part II - Materials and Methods...................... Table 1.00.00.00.00.0.00.0.00000000000000.00.00.00.00. Table 3.0.0.0000000000IOOOOOOOOOOOOOOOOOQOOOOOOOOOOOC Table h000000000000‘0000000.0oooooooooooooooooooooooo mwu-J‘YT 7'- zsv‘—.— . , . ‘ ‘» ' O ' l' . - _. f ,:,, ,. __t DiSCUSSionooococoooooooooooooooooooooooooooodocooocoo sunwaIyOococoon.00.000000000000000.00.000000000000000, BibliograIIhy...0.000000000000000.COCOOOOOOOOOOOOOO... \ ...- . 3..-“; _;§. '__’. 53222:..a_ 195,390 Ta.ble 2.0.0.0000000000. ..... 0....OOOOOOOOOOOOOCOOOOCC~ INTRODUCTION The question as to whether pullorum disease spreads more or less readily among adult.turkeys is often brought up by turkey raisers. This question has not been answered yet by adequate experi- mental work. Obviously, as is the case with other infectious dis- eases, pullorum disease could not spread from bird to bird unless open carriers were present, disseminating a sufficient number of organisms to infect other birds. This thesis contains the records of a study of Salmonella pullcrum infection in 2h adult turkeys, which came from an infected flock. These birds were brought in for observation in June l9h6. S. pullorum was isolated from them at that tine. In the course of the work three other turkeys were infected artificially, and the period during which the germ'was found in the feces as well as the serological reactions were recorded and compared. The purpose of this work was: 1. To determine whether growing and adult infected birds discharge S. pullorum with their excreta; 2. whether the dissemination of this organism takes place continuously or intermittently, and 3. what relation may exist between positive agglutination reaction and the open carrier state. As a preliminary step in our attempts to detect the presence of S. pullorum in the living bird, a study was made of the comparative effectiveness of three enrichment media for the isolation of Salmonella. The comparison of the three media, tetrathionate broth, selenite F broth, and mandelic acid broth was made to determine their inhibiting power for the coliform organisms, and their adequacy for demonstrating the presence of Salmonella. -1- PART I PREVIOUS WORK Since 1923, when Killer first recommended tetrathionate broth for an enrichment medium in the isolation of members of the Salmonella, Shigella and Eberthella groups, systematic studies of the different media recommended, have appeared. Studies have been made on the effectiveness of the enrichment medium over plain media, as well as on the role played by the different constituents of the medium. An abundant amount of literature exists on tetrathionate which has been studied principally by three men, Pollok, Know, and Gell. Liefson (1936) and Gahar (l9h3) reported a new medium for the isolation of Salmonella, Shigella and Eberthella. Both workers used selenite salts and reported a great selective power of the new medium as well as a specific range of inhibiting power that permit- ted them to isolate with great ease, the Salmonella present. The Digestive Ferments Company, Difco, put out another medium, mandelic acid broth, that was reported as highly inhibitory by Teninga (19h7). Transfers were made from the enrichment media to McConkey and SS agar plates. The relation between the enrichment media and the secondary plating media is very important and has been pointed out by the majority of workers. Miller (1923) found that g. parathyphosum, §.,Salmonella and Proteus reduced tetrathionate while E. coli, B. dysenteriae and many others lacked that power. -2- Jones, (1936), working with thyphoid bacilli, stated that one million bacilli were necessary to produce growth on McConkey agar plates whereas E. coli and B. paratyphosus grew very well from small inocula. He pointed out the importance of the pH and the standardization of KcConkey's medium as necessary to obtain good results. Looking for a selective medium which would permit easy recognition of the parathyphoid group, he found that in tetrathio- nate the growth of the B. dysenteriae and E. coli was entirely suppressed after an inoculation of one million organisms, whereas other members of the same group grew satisfactorily. Knox (19h2), working with paracolon bacilli and B. paratyphosum on tetrathionate, found that with B. paratyphosum the period of rapid increase in cells was prolonged so that in five hours the population was two and one half times the number reached without tetrathionate. At the same time he reported that the growth of paracolon bacilli on the same medium was fairly slow. Attempting to explain this physiological effect, Knox found that similar re- sults were obtained when broth cultures in an ordinary medium were subjected to free airation. Thus be confirmed the belief of Wilson and Topley (1936) that tetrathionate acts as an alternative hydrogen acceptor to oxygen and in that way it acts as oxygen although not in the same degree. The practical conclusion was that in mixed cultures the salmonella group has an advantage over the coli group, since this is unable to reduce tetrathionate. Pollack and Gell (l9hS) reported the results of their studies concerning the efficiency of McConkey medium when used for direct and indirect plating. The report showed that the number of positives increased slightly but definitely when this medium was used in dir- ect plating, and in comparison with routine media. Using tetrathio- nate he found that indirect plating on.McConkey medium gave about three times as many positives as direct plating on McConkey's agar and two times as many as direct plating on other solid selective media. Nassey (l9h2) reported that tetrathionate gave a pure, heavy growth of Salmonella when the number of infecting organisms was 15 per cc., and found that in order to obtain the same growth by direct plating on McConkey's agar he needed five to ten times more organisms. He stated that the smallest number of organisms necessary to be de- tected in a feces suspension varied from 15 to 255 per cc. Mayfield and Sober (l9h0) had begun to use 88 agar because of unsatisfactory results with McConkey‘s agar. They found SS agar very efficient in Ithe isolation of Salmonella and reported that the morphology of the colonies was more characteristic in this medium. Cooper, Keller and Glesive (19h5) also reported a superiority of SS agar over McConkey's and desoxycholate citrate agar in the isolation of Salmonella. Mallok (19h2) reported his experiments with 58 agar and noted that from hO positive cases, 3h isolations of Salmonella were successful on SS agar only, 27 in tetrathionate followed.by McConkey's agar and 7 on McConkey's. In eight cases SS agar only was positive and in five instances only tetrathionate picked up the pathogens. In two of these cases SS agar and McCon- key's medium were overgrown by colon bacilli because too large an -h- inoculum was used. They reported that McConkey's medium did not give good results and that the combination of tetrathionate and SS agar allowed successful isolation in 97.5% of the cases studied. When tetrathionate was used followed by McConkey's agar only 72% of posi- tives were successful. In respect to the selenite F medium, Liefson (1936) began to work on selenite salts for the purpose of isolating Ebertella, Shigella and Salmonella. The toxicity of the selenium salts has been noted previously and thought to be useful because of its in- hibiting effects on certain groups of microbes. Liefson reported that selenite F medium exhibits differential inhibiting effects that permit one to differentiate very sharply among closely related bacteria. He found that selenite F was highly toxic for colon bacilli, but it was not sufficiently toxic to inhibit the growth of typhoid bacilli and enterococci. Attempting to explain the useful- ness of the medium in the isolation of Salmonella, he found that dur- ing the first 10 to 12 hours the growth of the colon bacilli de- creased followed by a rapid increase of the typhoid bacilli which soon outnumbered the colon bacilli. He reported that even when a few tpyhoid bacilli were present in a large amount of feces, they may be recovered with great ease and made to multiply to one million in 2b hours of incubation. He concluded that the sodium selenite F medium was superior to other existing media for the isolation of Salmonella and the typhoid bacilli. Gohar (l9h3) using selenite solutions in concentrations of 1/100 to 1/100,000 found that selenite was highly inhibitive for -5- 1‘. B. dysenteriae, Vibrio cholerae, and g. coli. On the other hand the salmonella group and Streptococcus fecalis were more resistant to the toxic effects of selenite salts. He also recommended the use ofsnflenite F medium and McConkey's agar tOgether in the isola- tion of Salmonella. Liefson (1936) observed, however, that the usefulness of selenite medium is conditioned by the pH, by the presence of phos- phates and that the use of the autoclave on selenite F medium, destroyed its effectiveness. In regard to mandelic acid medium I have not found any specific report aside from that presented by Teninga (l9h7). 'While the for— mulae of the tetrathionate broth and selenite F medium are well known, the formula for the mandelic acid medium has not been pub- lished. -6- Uifco tetrathionate Proteose peptone No. 2 Facto bile salts Calcium carbonate 1 Sodium thiosulfate 3 Iodine solution 2ml/lOOnl S or- 1 gm. 0 gm. 0 gm. e solution is prepared by dissolving 6 gm. of iodine crystals *n n 5 gm. potassium iodide in 20 ml of water. Difco selenite F. Sodium selenite Bacto tryptone Eacto lactose Disodium phosphate 1 Difco mandelic acid media Formula unknown SS Agar. Bacto beef extract Proteose peptone Difco Bacto lactose l Cacto bile salts No. 3 Sodium citrate Sodium thiosulfate Ferric citrate Bacto brilliant green Bacto neutral red KCConkey's Agar. H Bacto peptone Proteose peptone Bacto lactose Bacto bile salts No. 3 Sodium chloride Eacto agar Bacto neutral red Bacto crystal violet H H OO‘aJUll—‘Obd-J c>cr£rtn {IQ S O o (j\a) (3CDFJCD’N”DC)U1UI O UIUIUI NLQ \n o \n <3\» H o OO‘H em LJOUI. gm. gm. gm. 59.. mg. gm. gm. gm. gm. gm. gm. gm. m. -J MATERIALS AND METHODS In the spring of l9h6 the Poultry Clinic of the Department of Bacteriology and Public Health, Michigan State College, received 200 turkeys fran a flock infected with S. pullorum. One hundred and fifty of these died in two weeks. In October l9h6, 28 turkeys remained alive and were found to be pullorum negative by bacteriological examination. However, the kidneys and intestines of 2h turkeys, that died during the summer, yielded S. pullorum. The feces of these turkeys contained lactose fermenters, Esch. coli, Aurobacter aerogenes, and Proteus. These feces were used in testing the inhibiting power of the three media under investigation. The media used were all dehydrated products. (Difco). Tetrathionate broth. Tetrathionate Difco h.6 gm Distilled water 100. m1 This solution was heated to 95° C. and cooled to 60° C. At this point 2 ml of iodine solution was added to each 100 ml of medium. The medium must be used shortly after the iodine is added. Nandelic acid broth. Handelic acid Lifco 2.6 Distilled water 100. ml The solution was heated to 85° c. to avoid a possible precipi- tation of its components due to heat. Selenite broth. Selenite Difco 2.3 gm. Distilled water 100. ml The solution was heated to 850 0,. following the precautions recommended for mandelic acid medium. As secondary solid media, SS agar and McConkey's agar were SS agar Difco 60 gm. Distilled water 1000 ml The agar was dissolved by steaming for hS minutes and rapidly poured into regular Petri dishes. No sterilization is required. Autoclaving injures SS agar. McConkey's Agar Fcflonkey's agar Difco 50 gm. Oistilled water 1. ml The method of preparation was the same as for SS agar. Bacteriological examination of live turkeys for S. pullorum. 1. Rectal swabs were taken from each turkey. These were put into 5 ml of sterile peptone broth for half an hour. 2. One ml of the inoculated broth was placed into 10 ml of each of the three media using sterile pippettes. 3. The tubes were incubated for 17-20 hours at 37° C. The period of incubation is very important since after the twentieth hour the growth of Salmonella begins to decrease. h. Single loopfuls of the three media were smeared on SS agar plates marked off into two halves so that each plate received a sample from the same feces through two different enrichment media. 5. The plates were incubated h8 hours at 37° (3 and the colonies were then counted. The three media were tested four times with the same sample of feces. The results are shown in Taoles I, II, III, and IV. Tables V and VI compare the efficiency of the three media, where used in examining feces which contain Salmonella. The material used was feces of those same birds to which was added a pure 2h-hour culture of S. pullorum. The strain was iso- lated from the same flock. Procedure: 1. One gm. of feces was emulcified in 3 ml of sterile saline solution. 2. One tenth of a 2h hour culture of S. pullorum was added. 3. A loopful of the emulsion was placed in 10 ml of each of the three media, and the tubes incubated for 17-20 hours at 37° C. h. Plates of SS agar and FcConkey agar were used for plating and the SS agar plates were incubated for h8 hours and the KcConkey plates. 20 hours. 5. The colonies on the three media were counted. 6. The identification of the Salmonella was made by sugar fer- mentation. The experiment was repeated twice each time with 10 different samples. The results are tabulated in Tables V and VI. Tables VII shows the comparative ability of the three media in giving evidence of the presence S. pullorum in the intestinal tract of artificially infected turkeys. Three turkeys were infected orally, each withIUDml of a 2h-hour pure culture of the same strain used in the second experiment. The procedure followed was the same as in the first experiment. In this part new lots of selenite F and mandelic acid media were used, and, perhaps, this accounts for the results obtained. -10- The tests began 5 days after the turkeys were infected and were continued until one died and the others were killed for post mortem examination for evidence of pullorum infection. The results are shown in Table VII. -11- TABLE I Growth of Esch. coli on SS Agar following cultivation in selective enrichnent media. Plate No. Handelic Selcnite Tetrathionate l2 ++ 4 _ 13 - ++ H 114 - + «H 16 - ' ’ 17 - ++ ++ 20 - + +++ 2l - + ++ 22 - H - 23 - + + 25 - + - 26 - - 4 2? - - + ZR - - - 29 - - - 30 - + ++ 31 - - + 32 + + - 33 + - ++ 35 + ++ 44+ 37 - _ ++ 38 - - - hi - .. - b2 - + - 1o - - + 51 + - ++ 88 - - 4 89 + + ++ 90 - + - - negative i l—h colonies ++ 5-20 colonies fff more than 20 colonies -12- TABLE II Growth of Esch. coli on SS Agar following cultivation in selective enrichment media. Plate No. Mandelic Selenite Tetrathionate 12 13 1h 16 17 2o 21 22 23 25 26 27 28 29 3o 31 32 33 35 37 _ +++ +++ 38 -— - hl L2 h3 51 88 89 9o |+|++ 1.. II+I+IIIII+III+IIIT I I +-I it: + +Iill++++++l+++$+++i++tttt+ +l+| I - +++ -13- TABLE III Growth of Each. coli on SS Agar following cultivation in selective enrichment media. Plate No. Mandelic Selenite Tetrathionate l2 - 1 3 - + + 1h - i - 16 + - + 20 - - - 21 + +++ - 23 + + ++ 25 - + + 26 + - - 27 + -- + 2F - _ + 29 - - - 30 - + ++ 31 - ~ - 32 - - ++ 35 + - H 37 - - - 35 - i - t1 - - + 12 - - + 243 - - +++ hh - - +++ 51 + + +++ 87 - + +++ 88 - + - 89 - - + 90 + - - 91 - + + Note: A direct streaking from the suspension of feces on agar was made, obtaining good growth of different types of colonies. -114- Growth.2£ Esch. coli on SS agar following cultivation in selective _—enrichment media. Plate No. Mandelic Selenite Tetrathionate 12 - - - 13 - - + it - - ++ 16 - + ++ 20 - - + 21 - - - - 23 + - + 25 - - ++ 26 - - - 27 - - + 28 - + - 29 + - + 30 + — - 31 - - - 33 - - + 35 - - + 37 - - - t1 - - - t2 - - - Le - - - u + + - Note: Mandelic acid and selenite F media became darker, coinciding with an increase in inhibiting power. -15- TABLE V Growth ofq n. pullorum following cultivation in selective enrichment u.‘ --_... media when 0.1 ml of pure culture was inoculated into 5 m1 of emul- sion of feces. SS Agar McConkey's Agar Plate No. Man. Sel. Tetra. Man. Sel. Tetra. 1 + - +++ . + - H 2 - - +++ - - H 3 - - ++ - - ++ h - - +1- - - «H S + - +++ - — H 6 + - +++ - - H 7 + f +44- + - H 8 + -' H- - - H 9 + - ++ - - ++ 10 + - + + - ++ Note: 1. S. pullorum was recovered only from the 10th tube 3. in the series in which mendalic acid medium was used. 0n EcConkey's agar, as eXpected, Esch. coli colonies as well as Salmonella colonies developed. Selenite medium and mandelic acid medium appeared to be too inhibitory for Salmonella, the original sample had 0.1 of pure culture of S. pullorum, and it was recovered through tetrathionate. -16.. l V I- o- . .- 1» n ‘.a.. a ,. I .- ,; TABLE VI Growth of S. pullorum following cultivation in selective enrichment media whenIO.I 51 of pure culture was inoculated into 5 ml of emul- sion of feces. SS Agar McConkey's Agar Plate No. Man. Sel. Tetra. Nan. Sel. Tetra. 11 - — ++ - - ++ 12 - - ++ - - ++ 13 - - ++ — - + 1h - - ++ - - ++ 15 - - ++ - - + 16 - - 4+ 3 - - + 17 - - ++ — - + 18 - - ++ - - ++ 19 - - ++ - - ++ 20 - - ++ - + +1 Note: After the second experiment with mandelic acid and selenite F medii,‘two new samples of these media were requested. These media are very hygroscopic and must always be kept in tightly stOppered bottles, if not the media will oxidize and become excessively inhibitory. -17- TABLE VII Growth of S. pullorum following cultivation in selective enrichment media when feces of artificially infected birds were used. (fr-’1) SS Agar McConkey's Agar to. of Bird Man. Sel. Tetra. kan. Sel. Tetra. 11929 - + + E. coli E. coli E. coli ++ 11932 - — — _ - - 11950 + + + E. coli proteus E. coli Proteus - (#2) SS Agar McConkey's Agar Ho. of Bird Tan. Sel. Tetra. Han. Sel. Tetra. 11929 - + +++ - E. coli - + E. 001i E0 001.1 11932 - - - E. coli 11950 - + + E. coli + + Controls qulsion of S. pullorum 5 m1 of saline solution from pure slant culture 0.1 In]. - — 0.6 ml — - -4' Growth of S. pullorum - No growth 6f 8. pull orum J TABLE VII (Continued) (#3) SS Agar McConkey's Bird . No. Man. Sel. Tetra. Man. Sel. Tetra. 11929 + - ++ + - ++ 11932 E. coli E. coli - - E. coli E. coli 11950 - + + - - (#h) SS Agar thonkey's Bird’ No. Man. Sel. Tetra. Man. Sel. Tetra. 11929 +4 + 4 - + + 11932 - - - E. coli - — E. coli E. coli - 11950 1 — + .. + + (#5) SS Agar McConkey's gird V00 Turkey Man. Sel. Tetra. Man. Sel. Tetra. 11929 ++ ++ + E. coli + + 11932 E. coli E. coli - E. coli E. coli — + E.cdi E.cdi 11950 E. coli - - - - + -19- - 6‘ (Continued) (#6) SS Agar McConkey's §ird No. Han. Sel. Tetra. Van. Sel. Tetra. 11929 - H H E. coli + L 11932 E.-coli E: coli - E.-coli E: coli - 11950 E.’coli - - E.~coli E: coli - (#7) SS Agar EcConkey's Eird No. Fan. Sel. Tetra. Mans Sel. Tetra. 11929 - - - — - - 11932 - - - E. c o l i 11950 - - - - E. coli - Controls Emulsion of S. pullorum hiShfl ofsaline solution from pure slant culture S. pullorum - Note: + 1+ had become negative. - - + The results shown in Table VII (7) indicate that the birds The £€§Ult3 obtained with the new samples of selenite F media are very different from those shown in the Tables I, II, III, IV, V, VI. -2 O... DISCUSSION From the work done it was concluded that tetrathionate broth is superior to mandelic acid broth and selenite F broth for the isolation of S. pullorum. This conclusion was based on their com- parative ability to inhibit organisms other than S. pullorums and their adequacy for detecting é? pullorum. When S. pullorum was present, it was proved that tetrathionate broth was the only medium that consistently allowed the growth of this organism. In regard to the other two media it was observed that an increasing inhibitory power accompanied a change of color in the broth, which, in both cases, was much lighter when the medium.was prepared from newly received, bottled samples. This aroused the suspicion that some chemical change had occurred in the lots of selenite F, and mandelic acid media first received. In the case of selenite F broth the results shown in Table VII differ com- pletely from the result obtained when an old sample of the medium was used. One instance was recorded in which E. pullorum was re- covered from a tube containing 7 m1 of the medium instead of the 10 m1 used before. 'S. pullorum was isolated on McConkey's Agar in the case mentioned but on SS agar the combined inhibitory effect of the two media evidently suppressed the growth of S. pullorum. When 0.1 ml of pure culture of S. pullorum.was inoculated into 5 ml of feces emulsion, S, pullorum was recovered in few cases, but the medium did not seem to be adequate for detection of S. pullorum in a high percentage of cases. When a new sample of selenite F -21- medium was used, S. pullorum was recovered in all but one case. On SS agar pure cultures of S. pullorum were obtained but on McConkey's agar occasionally mixed cultures were obtained. This serves to indi- cate the high selective action obtained when subcultures from selenite F broth cultures were made on SS agar. Through personal information, during the writing of this discus- sion, I learned that Dr. Bevins found selenite F to be the best medium for the isolation of S. pullorum. My own observations on the change in Selenite F medium with increased inhibitory action and the importance of the amount of inoculum in relation to the amount of medium indicate that selenite F broth offers possibilities of effec- tiveness provided the condition of the medium and every phase of the procedure are definitely determined and preperly controlled. Since these factors have not yet been definitely established the use of tetrathionate enrichment broth is recommended for the isolation of _S_. pull orum. handelic acid medium showed a relative effectiveness only the first time it was used. It was thought that the presence of oxygen, even in small quantities, altered the medium. This opens a field for a biochemical study of this medium. An understanding of the results obtained will be possible only when the medium has been tested under different conditions. These conditions should include quantity of the medium in relation to: 1. the size of the sample; 2. the number of organisms present in concentrated and diluted suspensions. -22... Another thing to be studied is the effect of air on these two media. Until this has been done no definite evaluation of mandelic acid medium can be made. -23- 1. 2. 3. CONCLUSIONS Tetrathionate broth inhibits coliform organisms and allows the growth of S. pullorum in the first 17 hours of incuba- tion. This medium.was most effective in the isolation of ‘S. pullorum from feces of turkeys. Tetrathionate broth followed by subcultures on SS agar is recommended for the isolation of S. pullorum. When McConkey's agar is used for subcultures the growth of coliform organisms is not complete- ly inhibited. Selenite F broth has a higher inhibitory action on coliforms than tetrathionate broth, which allows the growth of Proteus. Under certain conditions Selenite F broth proved too inhibi- tory and suppressed growth of S. pullorum. The combined inhibitory effects of Selenite F medium.and SS agar are too great to insure growth of Salmonella. When Selenite F medium.is used the subcultures should be made on McConkey's agar. Mandelic acid medium inhibited all kinds of organisms pres- ent in feces of turkeys. —2h- PART II This part represents some preliminary studies on the pullorum disease carrier state. NATERIALS AND METHODS The feces of twenty five turkeys from a flock infected with ‘S. pullorum contained S. pullorum in June 19h6, when they were two months old. At that time the blood serum of several of these birds was positive to the agglutination test with pullorum antigen. When this work began these twenty five turkeys were thought to be suitable subjects for a study of the pullorum infection car- rier state. Periodic examinations were made of drOppings from these birds to see if the birds discharged S. pullorum. At the same time agglutination tests were made in order to determine whether there might be any relation between the agglutination titer and posi- tive fecal cultures. The procedure for the bacteriological examination was the same as that employed in Part I of this work. For the agglutination test, pullorum antigen prepared by Dr. J. A. Bevins of the Department of Bacteriology and Public Health of Michigan State College. The dilution of the serum used was of 1/25. The tubes were incubated twenty hours at 50° C. in a water bath, and two hours at room temperature. -25- In view of the negative results obtained during the fall and winter l9h6-h7, (Table 1, page 27) three turkeys (not from the origg inal 25) were infected orally with a pure culture of g. pullorum. The length of time during which they discharged the organism in the feces and the agglutination titer of these birds were observed. The results are shown in Tables II, III, and IV. During the Spring l9h7, 100 eggs laid by these twenty five turkeys and SO laid by the three artificially infected birds were examined for g. pullorum. The whole egg was broken in 100 ml of tetrathionate broth, incubated for 17 hours at 37° C, and subcul- tures were made on SS agar. One of the 100 and five of the 50 eggs yielded 8. pullormi. TABLE 1 Table showing the results of the bacteriological examination for S. pullorum in the droppings of 25 birds from a flock naturally infected with S. pullorum five months earlier and results of the agglutination test. Bird No. Bacteriological Agglutination Exam Exam 12 — - 13 - - 1h - - 16 - - l7 - — 2O - - . 21 - _ 22 - - 23 - - 2S - - 26 - - 27 - - 28 - - 29 - ~ - 3o - - 31 — - 32 - - 33 - - 3s - - 37 - - 36- - - hi - - u2 - - 1:3 - - 51 - - 88 - - 89 - - 9O - - Note: These birds were tested in the same way every two weeks during the fall and winter (l9h6-l9h7) With identical results. One hundred eggs laid during spring (19h?) were negative for S. pullorum with one single exception. -27- Comparative results of the bacteriological examination and agglutination test for pullorum infection in bird No. 11929, ir- fected orally on February 22, 19h?, with 10 ml of a 2h hour agar culture of S. pullorum suspended in saline. Date Bacteriological Agglutination Examination Test F‘Ebo 26. + z A March 6 + / 11; + % ,4 90 + . ¢ ,4 2“ + )l r4 April 2 ~ / ,Z 10 .. 15 - 23 _ tr 31 .. Note: Bird No. 11929 was killed on June 30th. It was found apparently normal on autOpsy and S. pullorum was not isolated from its organs. Key: — negative agglutination test and negative culture weakly positive agglutination test and positive culture /% marked positive agglutination test K%% strong, positive agglutination test TABLE 3 Comparative results of the bacteriological examination and the agglutination test for pullorum infection in bird No. 11932 infected in the same way as bird No. 11929 with S. pullorum on February 22nd. Date Bacteriological Agglutination Test Examination Feb. 26 — . - March 6 — - 1b - - 20 - - 28 - x ,1 ,1 April 2 - / / / lo - :1 2‘ 18 - ,z x 23 - x / Kay 3 - / / 10 - ,1 x 16 ' - x x 23 - x x 31 - ,z hote: Pird No. 1193? died on June 18th.. The autopsy did not show visible sign of pullorum infection. S. pullorum was iso- lated from the ovary and liver but not from other organs. Comparative results of the bacteriological examination and agglutination test for pullorum infection in bird No. 11950 in- fected in the same way as bird No. 11929 with S. pullorum on February 22nd. Date Bacteriological Agglutination Test April Q Lav I: Examination 26 + f 6 + ’ 1h + 20 + 28 + - 2 - - lO - - 18 - g ,z 23 - ,1 ,1 10 _ .. 1:? + ,1 23 .. ,z 31 - ,2 On June 17th bird No. 11950 died. On autopsy the following changes were found: hydropericardium, necrotic foci in the Spleen. The kidneys were found enlarged. Not a patho- logical change. The vitelene membrane of the ovary was thickened. There was severe peritonitis. S. pullorum was isolated from the heart, liver, lungs, intestines—End—ovary. -30- DISCUSSION Due to the limited sc0pe of the work no definite conclusions can be made. Each of the three birds reacted differently. Two of the birds discharged the organism through the intestine during the first four weeks following infection, while the other did not discharge S. pullorum at all. However, the organism was isolated from its liver and ovary and its serum was reactive after the fourth week of infec- tion. The organism was not recovered by taking rectal swabs and sub- jecting them to careful bacteriological examinations as described. When five months of age the original 28 turkeys did not dis- charge S. pullorum with their droppings as far as could be ascertained by cultures made from rectal swabs. They were also negative to the pullorum agglutination test. 'When they were two months old one of them discharged S. pullorum and the serum of five of them reacted to the agglutination test. S. pullorum was isolated from one of 100 eggs examined, showing that S. pullorum can be transmitted through eggs while no dissemination through drOppings was evident. -31- l. 3. SUWNfiRY Twenty five turkeys from a flock infected with g. pullorum were found to be consistently negative on bacteriological examination and to the agglutination test for pullorum in- fection when they were five months old. At two months of age one had been found to discharge g. pullorum in the feces and five were positive to the agglutination test. These turkeys were possible carriers since they had been in contact with the disease as poults. The feces and sera of three turkeys artificially infected orally with é. pullorum were also tested for pullorum infection. Two of them discharged the organism regularly for five weeks follow- ing the infection. After this time Pird No. 11929 remained nega- tive, the other one, No. 11950, again discharged S. pullorum, twelve weeks following infection, and died two weeks after show- ing clinical symptans. The third one, No. 11,029 was killed for ' autopsy. '§. pullorum was not isolated from its organs. The serum of Bird No. 11950 was always positive to the agglutination test, with the exception of a period of two weeks following the dissapearance of the E. pullorum from the feces. The serum of Bird No. 11929 was positive during the entire period of this study. Bird No. ll§32 did not discharge §. pullorum through the intes- tine, but the serum became highly reactive the fourth week follow- ing the infection and remained positive for the remainder of the experimental period. When it was killed E. pullorum was isolated from the liver. -32- h. One of 100 eggs (1?) laid by 25 turkeys from a naturally infected flock yielded 9. pullorum. Five of 50 eggs (10“) laid by three artificially infected turkeys yielded S. pullorum. -33... REFEREKCES Cooper, K. E., Wood, N., Caswell, E., Eliot, and Small: The l9h2 Bacterium paratyphosun P from Faeces. Jour of Path. —T f — r..‘ “r . C-‘ A ' '5‘: aha pact. nu; jhb—gg3o _ Cohar, V. A.: Sodium Selenite as a Bacteriostatic Substance and lPhB its Use in the Isolation of Paratyphoid Bacilli. Jour. Trop. Med. and Hyg. ho. No. 3, 29—32. Jones, P. J., Knox R., Cell, P. C. H.: Experiments with B'illiant ...4 1936 Creen - Eosin Agar. Jour. of Path. and Pact. h2zh5. Knox, P. T.: The effect of Tetrathionate on Bacterial Growth. 19h5 Brit. Jr. of Exper. Path. 26 (3): 1h6-50. Knox, R., Cell, R. G. H., and Pollack, h. R.: Isolation of Intes- l?h2 tinal Pathogens, Comparative Study of Iedia. Jour of Path. and Bact. Eh, h69-h63. Knox, R., Sell, P. G. H., and Pollack, n. R.: Selective Action of l9h3 Tetrathionate in Bacteriological hedia J. Hygiene £3 (3): 1117-158 0 Leifscn, Einar: New Selenite Enrichment hedia for the Isolation of 1936 Paratyphoid (Salmonella) Eacilli. Am. Jour. of Hygiene 8h, h23. -_ Nassey, Kathleen M.: Notes on Tetrathionate Broth and McConkey's l9h2 agar;Meiia in the Isolation of Salmonella enteritides var dublin from Bovine Faeces afidfiEIIFT—_Jr. Comp. Path. and Therap. 53, No. 2, 151. Jan. fibstr. Eul. Hygiene 18, 867. — _ Vedical Research Council: A Brilliant Green Acid Fuchsin medium 19h2' for isolation of Salmonella. (Monthly Bull. Fmergency Pub. Health Lab. Service. harch V2, 26-3) Bull. Hygiene 19, SOS. Nedical Research Council: Growth of Typhoid Pacilli on Different lShZ Selective Nedia (Monthly Pull. Emergency Publ. Health Lab. Service. Sept. 7-?) Pull. Hygiene 18, 7h. Kollov, Nollie; Winter, Jeanette; Steinberg, Philip: SS agar for l9h2 the isolation of Eberthella, Salmonella and Shigella groups of feces. Jour. Lab. and Clin. Med. 28, l5§1-1027. Pollack, M. R.: The Influence of Temperature on Adaption of l9h5 Tetrathionate in Tashed SuSpensicns of B. paratyphosum B. Bri. Jour. cf Exper. Path. gé (6): th-lé. Buys, A., Charlotte, N. 9.: The Isolation of Typhoid,Paratyphoid 19h0 and Dysentery Bacteria from Paeces and Urine. Brit. fed. Jour. l9h0. Vol. 1, 606. Wynn, E. S. and william, C. B.: "rowth of Iberthella typhosa and l9h5 Aerohacter aerogenes in Association—in Tetrathionate Broth. UOur. of‘Bact. MB: 62i-632. Teninga, Grace.: Enrichment hedia in the Isolation of Pathogenic l9h7 Organisms Belonging to the Colon-Typhoid Group. Thesis for the Degree of 1. S.,Iichigan State College. ' rW'H. -" fr —, r- ‘v f /\ f '-’i " Um y.l/....\.,H .- L .-..:-_ is The writer wishes to thank hrs. Vera Bleil and Dr. James Pevins of the Department of Bacteriology and Public Health of Michigan State College for their ad- vice and assistance during the experimental work of this thesis. I want to thank Fichigan State College for the facilities for work provided to me during my studies. I am deeply grateful to Dr. H. J. Stafseth, Head of the Department of Bacteriology and Public Health and my major professor from whom I have learned every time I have met him. His advice during my residence here, his patience with the difficulties I found in my work, and his help in the writing of this thesis have made possible my degree. ‘Without him my study would not have been pos- sible. JUU'_‘L"~J’. {J'JL I. I‘ u I‘ n M .-(‘n l ‘; \ \ ewes-7.159 \ I" I‘- I‘ l . "c" ’1'. i”?- ‘", 3 ’ {'2 ’mr '7 Ease b ‘5 Y _ :Fusxéfin, fw‘u. I. . .\ I: 1'. a‘d Jr- 1 :uk’ 13 9.”! ‘5' =4“ 9““ ' if g. , cur r~ >. «,r Oct ‘0 a v» w: .x + -: i 4 ' -.; , 15.21 (L , : 1 "L ’ ' . n ‘K S s I . I Dog 5 No 1'53" k afiw FilighuGAN STAIE UNIVERSITY LIBRARIES v > I i I r ' l ‘ ”nu-1“.