THESIS This is to certify that the thesis entitled Antigenic Coupon-on of T101" Strains of Vibrio Fetus presented by Loyd Wayne flung has been accepted towards fulfillment of the requirements for flfflw / Major professfi Date septa» 16, 1952 0-169 AN ANTIGENIC COMPARISON OF TWELVE STRAINS 0F VIBRIO FETUS By LOYD WAYNE TIFFANY 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 and Public Health 1952 ,’ was“; I gratefully acknowledge the directive work of Dr. Jack J. Stockton. TABLE OF CONTENTS PAGE Introduction ........................................ 1 Historical Review ................................... 5 Characteristics of Vibrio fetus ..................... 6 Photomicrograph of Vibrio fetus ..................... 8 Materials and Procedures ............................ 9 Table I: Results of preliminary titer determinations of the serum of rabbits against respective strains of Vibrio fetus .............. 15 Table II: A protocol of the scheme of dilutions for agglutination and cross-agglutination re- actions .............................. 14 Results and Discussion .............................. 16 Table III: Resulting titers of the agglutination and cross- agglutination reactions of the twelve strains of Vibrio fetus ........................ 17 Graphs: Graphical Illustrations of results recorded in Table III ......... 19 summa-ry 0.0.00.0...OOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOI 24 Bibliography OOOOOOOOOOOOOOOOOOOOCOCOOOOOOOOOOOOOOOOO 25 INTRODUCTION Because of the greater importance of brucellosis and the difficulty in growing Vibrio fetus, vibriosis (infection with X; £3333) has received little attention until the present decade. Though it is not a new disease, having been first reported in 1915, it is nevertheless becoming of importance today because of the role it plays in sterility and abortion in cattle and sheep. According to McFadyean and Stockman (1915), this organism is responsible for 20 to 25 per cent of the abortions and 10 per cent of the sterility among sheep. Studies done by Smith and his associates (1919) showed that of 109 cases of abortion, 5? per cent were associated with EEE‘ cella abortus and 25.8 per cent with E;_f§tggt In cattle, the annual abortion rate may vary from 4 to 20 per cent. (W.N. Plast- ridge, 1945-1951a:b) Methods of diagnosis are based, in part, on the absence of brucellosis, but depend to the greatest extent upon isolation and identification of the causative organism. Smith gt_gl_(1920) suggested the possibility of using the agglutination test to aid in the diagnosis of vibriosis and found that blood serum from cows which had been infected with X; fetug would agglutinate cells of X;_fgtgg, Barger (1928) and Brookle- huret (19#6) reported confirmatory results to this agglutination procedure. Since the organism may be present in the host for several weeks prior to actual abortion and as some animals may become - 1 - carriers of the organism, the agglutination test has come to as- sume a great importance in the testing of herds and flocks for evidence of the presence of this organism. This makes prOphy- lactic measures of some value in protecting the healthy members of the group against infection and, further, aids in the therapy of infected animals. In considering the use of the agglutination test, several factors must be considered: (1) selection of cultures which are specifically agglutinable, (2) methods for maintaining the spe- cific agglutinability of cultures selected, (5) cultural methods for growing large volumes of cells, and (4) information on the significance of reactions obtained. It is with the first of these factors that I am herewith concerned. Since it has not been definitely established that each strain is antigenically homologous and since information on the significance of a specific reaction is questionable, it has become of interest to determine whether a higher titer can be ob- tained by the cross—agglutination test. Also, it is desirable to know whether one antigen excels the others in such a cross-agglu— tination reaction and if any antigenic differences actually exist. This thesis records the results of cross-agglutination tests on 12 strains of Eghfgtgg and the conclusions which may be drawn therefrom. HISTORICAL REVIEW In 1909, McFadyean and Stockman were appointed by the Board of Agriculture and Fisheries to investigate a number of epizootic abortions which were occurring throughout England. In 1915, they made their report in which they described an organism, Spirillum 23333, and pointed out the connection of this organism.with abor- tion in ewes, from which animals they had first isolated it. They also made brief statements on the finding of a similar organism in two herds of cattle in Ireland and Wales. Some five years thence, Theobald Smith (1918) and Smith and associates (1919-1925) made the first report in the United States of having isolated an organism which was morphologically similar to the one described previously by McFadyean and Stockman. They named the organism Vibrio fetus. This isolation in New Jersey was of bovine origin. Since Smith's work, reports of isolations of and infections with this organism.have seldom appeared in the literature until the last decade. Reports of infections with E:_fgtgg in the Unit- ed States have appeared as follows: Schroeder (1920), Traum (1925), Berger (1928), Plastridge and Williams (1941 and 1945), Rhoades and Hardenbrook (1947), and Bell (1950). These reports were based on infections occurring in California, New York, Connecticut, Illinois, Michigan, Montana and Wyoming. 2;.23333 infections in sheep were reported by Carpenter (1920), Baker and Stone (1959), Welsh and Marsh (1924), Graham and Thorp (1950), Ryff (1940), and Lee and Schrivner (1945). These reports -5- were made on infections occurring in New York, Montana, Illinois, Michigan and Wyoming. The limited amount of research on Xéufgtgg and vibrionic abor- tion up to the present day may be attributed to difficulties en- countered in cultivating this organism on laboratory media, lack of inexpensive, specific, diagnostic tests, and the greater importance of Brucella abortus in.the herds. However, with the lowering of the number of herds infected with §;_abortus and with the improved medium described by Huddleson (1948), a more promising future awaits in this field. Smith and his associates (1920) reported that blood serum from three cows agglutinated I: 22333 cells and suggested the pos- sibility of using this agglutination test in the diagnosis of vi- briosis in cattle. Berger (1928) also made a report on two cases of vibrionic abortion in which he reported findings similar to those of Smith 9; §_l_. Brocklehurst (1946), after examination of 255 blood samples submitted for brucellosis testing, reported that 7 per cent gave significant titers with.2;.fgtgg_antigen. Levi (1950) concluded, after an investigation of the cross— agglutinability of five strains of E;_§gtgg, that the results in~ dicated that the agglutination test was not a suitable procedure to be used as a routine laboratory method of diagnosis in vibrionic abortion in sheep. Evidence contrary to the above was presented by W.N. Plastridge (1951), who concluded that when the normal titer of the antigen was taken into consideration, the agglutination test was valuable in the diagnosis of vibrionic infection in cattle. Evidence of agglutinins for Z; fetus in the vaginal mucosa of -4- cows was demonstrated by Stegenga and Terpstra (1949). Following this, Terpstra and Elena (1951) described a procedure whereby these agglutinins could be detected. 0n the basis of this test, Terpstra (1951) bred cows with bulls which were known to be infected with E;_fgtggt His results indicated the bull to be a potential source of vibrionic infection. Similar findings were reported by Plastridge 9.15.21. (1951). Jepsen gt_gl (1951) reported a procedure whereby they made use of a tampon to collect the vaginal mucus for the determination of the presence of vibrionic agglutinins. This test has the advan- tage of showing positive results when a blood serum test would be negative. Agglutinins occurring in the vaginal mucosa are slower in developing and persist for longer periods of time than do agglu- tinins occurring in blood serum. CHARACTERISTICS OF VIBRIO FETUS In younger cultures of E;_fgtg§_the cells appear as short, curved rods, 1.5 to 2.0 microns long and 0.2 to 0.5 microns wide. The cells of older cultures may appear in rather long, spiralled filaments of from 1 to 5 windings. The gram negative cells are motile, having a single polar flagellum. The longer spirals may have a tuft of flagella at the end of the filament. The cells stain readily with dilute aqueous fuchsin, methy- lene blue, gentian violet and are decolorized by Gram's method. A 5 to 10 per cent carbon dioxide tension is necessary for the growth of the organism upon first isolation and may be used to enhance the growth of older cultures. No growth occurs on solid nutrient agar. Growth may be ob- tained on blood agar and solid "Thiol' medium (Plastridge's modi- fication) provided the proper conditions of temperature and car- bon dioxide tension are maintained. The best media are the semi- solid "Thiol" medium and liver infusion broth. The organism grows out in about 96 hours in "Thiol" medium at a temperature of from 55° C to 57° C, 57° C being the Optimum temperature for growth. No acid or gas formation has been reported from any of the common sugars (maltose, dextrose, sucrose, xylose, inulin, galac- tose, lactose). E; fetus is highly sensitive to light and is killed in 5 minutes at 58° C. The cells resist drying for two hours (labora- tory tests) and from 10 to 50 days (field tests), depending on the temperature. The longer survival period was at 6° C and the shorter - 5 - survival period was at 20° C. X; fetus has been shown to be pathogenic for pregnant guinea pigs, sheep, cows and for l2-day-old chick embryos. Its patho- genicity for man is uncertain. vrsaro serve x 5900 MATERIALS AND PROCEDURES The twelve strains of E;_fgtgg used in this work were ob- tained from a lyophilized stock supply furnished by Dr. Jack J. Stockton of the Department of Bacteriology and Public Health, Michigan State College, East Lansing, Michigan. The length of time which the individual strains were subjected to the 1y0philized state prior to their use in this work varied from one to four years. On one of the strains a second isolation had been made from the original source and ly0philized. This second isolation was re- vived and constituted a part of the twelve strains used in this work. The strains used in this work were identified as follows: l552-lst (lst isolation), 1552-2nd (2nd isolation), 1545, 1256, 3-24, 8-42, 490 (8/10/48): 490 (4/17/50): 322-50, Plastridge strain (4/17/50): Plastridge strain (10/8/48)* and Eggelston's strain. All strains except one were of bovine origin and had been isolated from aborted bovine fetuses or from the paracervical mu- cosa of cows in herds where vibriosis was known to exist. Isolations from the vaginal mucosa of cows were made on the following dates: 1552-1st, 12/9/49; 1552—2nd, 5/15/50; 1256, 5/16/50; 1595, 12/50/50. With the exception of Plastridge strain which was received from Dr. w.N. Plastridge, Storrs Experiment Station, Storre, Connecticut, in the spring of 1948, isolations were made from the stomach content of aborted bovine fetuses on the following dates: 490, summer 1948; 822-50, March 1950; 8-24, May 1950; Eggelston, May 1950; 8-42, July 1950. These isolations were made in the laboratories of the Depart- ment of Bacteriology and Public Health, Michigan State College. *Date of lyOphilization a _ In considering the proper medium for reviving the lyophilized organisms and maintaining them for use, there was little choice to be made, since 1; fgtgg_is very fastidious and can be cultivated on but one medium with any great degree of success. This ”Thiol" med- ium (Difco) was used to revive, carry and support the organism dur— ing its growth for antigen production. "Thiol" medium is a semi- solid preparation of the following formula: Bacto proteose peptone #5.....................10 Bacto yeast extract............................5 Bacto dextrose.................................1 Sodium.chloride................................5 ThiOl complex.00......O...OOOOOOOOOOOOOOOOOO‘OO.8 ‘B ‘E 'E ‘E ‘B ‘B Bacto agar.....................................l p amino-benzoic acid........................0.05 gm Thirty grams of this dehydrated powder was dissolved in 1000 ml of distilled water and the pH adjusted to 7.0. Ten ml amounts of the dissolved medium were placed in large (20 mm) tubes. The tubes were then gauze-plugged and sterilized in an autoclave at 250° F for 10 minutes. Using aseptic technique, the vials containing the lyophilized strains of'2;_fgtgg were opened and the contents of each introduced individually into separate tubes of the medium. Duplicate tube cul- tures were prepared for each strain. The inoculated tubes were incubated at 57° C and at the end of 96 hours, growth was found within the first 0.5 cm from the surface of the medium. A smear of each tube culture was made after the per- iod of incubation. It was then stained with Gram's stain and exa- mined under oil immersion for contamination. Those culture-tubes - 10 - showing contamination, as well as those cultures in which the or- ganisms failed to grow, were replaced by opening new inocula from the lyophilized stock supply and seeding into freshly prepared medium. After a good, non-contaminated growth was established in all the culture-tubes, 0.25 ml amounts were transferred to fresh "Thiol" medium and the inoculated cultures were incubated for 96 hours at 57° C. Following the initial revival, six subcultures were made in succession at 96-hour intervals. Sufficient tubes (eight) of "Thiol" medium were prepared, ino- culated and incubated. These eight tubes were used to inoculate 100 additional tubes of medium for each or the twelve strains. After four days of incubation at 57° C, the subsurface growth was removed from each tube (the strains being kept separate) by means of a water aspirator and placed in a large Erlenmeyer flask. To this harvest of organisms and some medium, an equal volume of formalinized saline (0.5% formalin and 0.85% sodium chloride at pH 7.0) was added. The suspension was centrifuged at 1400 r.p.m. (International Equipment Company #2 centrifuge, angle head) for 45 minutes to re- move the agar by sedimentation. The supernatant fluid containing the cells was decanted. The agar sediment was resuspended in for- malinized saline and again centrifuged. The supernatant fluids were combined and centrifuged for 55 minutes (approximate Relative Cen— trifugal Force of 4150 X G) in a refrigerated centrifuge (Interna- tional Equipment Company PR1 centrifuge) to separate the cells. The supernatant liquid was decanted, the cells resuspended in formalinized saline and again centrifuged. After this washing process, the super- - 11 _ natant fluid was discarded, the cells suspended in 40 cc of for- malinized saline and placed in the refrigerator to be used as a stock supply of antigen. All density determinations of the antigens were made with a Cenco Industrial Photelometer, Type B2, using a light wave of 550 mu. (green filter). The instrument was adjusted to read 100% trans- mission with formalinized saline as a blank. The standard antigen (originally obtained from Dr. W.N. Plast- ridge) gave a reading of 76% transmission which is equivalent to either 1.5 nephelometer or an optical density of 0.119. The anti- gens used in the periodical titer check and those used in the final cross-agglutination reactions were adjusted to 76% transmission in accordance with the standard antigen. Dilutions were made by adding 0.01 ml portions of concentrated antigen to 5.0 ml of formalinized saline. The suspension was thor- oughly shaken and the process repeated until the desired density was reached. The animals used in this work were male and female rabbits of varying ages. Each animal at the time of the eXperimental work was in normal health and received a similar diet of rabbit pellets through- out the period of antibody production. Prior to starting the injec- tions of the antigens, 10 cc of blood was removed from the heart of each rabbit and a check made for the presence of any titer against X;_§gtgg (Table I). The titer was checked against the respective rabbit which was to receive a particular antigen. Two rabbits were injected with each strain and received these injections on the first, third and fifth days of the week, except Sunday, for a period of six weeks. 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