A STUDY OF THE PATHOLOGY OF EEBRZONATING CHICKEN EGJS INOCULATED WITH VIBRIO FETUS BY Harris Duanewfigbster 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 Animal Pathology 1951 I. II. III. IV. VI. VII. VIII. TABLE OF CONTENTS ACKNOWLEDGMENTS INTRODUCTION HISTORICAL REVIEW MATERIALS AND METHODS RESULTS A. Mortality B.. Macrosc0pic Lesions C.. MicroscOpic Findings DISCUSSION SUMMARY AND CONCLUSIONS LITERATURE CITED FIGURES ll 17 17 20 23 26 31 32 34 ACKNOWLEDG ZENTS' The author wishes to express his sincere appreciation to Dr. Frank Thorp, Jr. for his thoughtful guidance through- out the course of this investigation.. Grateful acknowledgement is extended to my colleagues in the Department of Animal Pathology, Michigan State Chl- lege who have rendered most unselfish assistance.. Hearty thanks are given to Mr..M. L. Gray for assistance with the photographic work and his helpful suggestions on other phases of the experiment.. The helpful suggestions and tech- nical assistance of Drs. J. A..Williams, R..F..Langham,.R.. L..Johnston and Miss S. L. Laine were also deeply appreci- ated.. The encouragement offered by Dr..R..A. Runnels, Head of the Department of Animal Pathology, is gratefully acknowl- edged.. -1- INTRODUCTION Vibrio fgtgg infection of the genital tract of cattle. has been known for 38 years. The disease received little. attention until recently due to the difficulty encountered in the isolation and cultivation of the micro-organism. 1.. £223; has been classified as second only to Brucella abortus asethe cause of abortion in untested herds; and as the pri- mary etiological factor in abortions from brucellosis-free herds.. Brucellosis has been and still is the greatest sin- gle cause of abortion in the cattle population as"a whole. Improved methods of diagnosis and the increased number of brucellosis-free herds have accounted for the present in- creased interest in vibriosis.. Emphasis has been placed on the clinical and diagnostic aspects of the disease. Gross and microscopic lesions of the aborted bovine 'fetus and fetal membranesswere described in early reports on the condition. Contemporary workers usually slight these aspects“of the disease or refer to the literature.. The ex- tensive degenerative changes usually occurring in the con- ceptus prior to its premature expulsion generally make the tissues unfit for intensive study.. This deterioration and the prohibitive costs involved in using cattle as experimen- tal animals enhanced the desirability of utilizing a suitable laboratory animal in preliminary studies on the pathology of vibrionic abortion. The common laboratory animals have been generally regarded as refractory to infection with 1..£§£g§. Embryonating chicken eggs were selected for this study be- cause they are a good eXperimental medium and had been shown by Plastridge and Williams to be susceptible to E..fetus.. HISTORICAL REVIEW The first authentic.record of the recovery of a vibrio from cases of abortion was contained in the Report of the. Departmental Committee Appointed by the Board of Agriculture and Fisheries to Inquire into Epizotic Abortion (1909).. This vibrio had been repeatedly isolated from outbreaks of abortion in ewes.. Committee members were able to experimen- tally infect other ewes, pregnant for the first time, with this organism. Unsuccessful attempts were made to infect pregnant cows.. A further report by the same committee (McFadyean (1913)). however, stated that infective material obtained from ewes was administered to seven pregnant bovines intra- venously, through natural orifices; or by both channels and that one cowvbecame infected.. Unsuccessful attempts were made to infect other species. One goat aborted four days after being inoculated with viru- lent material, but no vibrios were found in the cotyledons, discharges,.or uterus.. They speculated that the period be- tween inoculation and abortion was too short to permit much increase in the number of organisms.. These investigators also found that guinea pigs and rabbits did not suffer any illness when inoculated with vibrios; but in one of the few? casesrwhere pregnant guinea pigs were used abortion occurred 12 days post injection.. These workers reported that two natural outbreaks of vibrionic abortion in cows,.one in Ire-u land and one in Wales, had been investigated.. McFadyean and Stockman (1913) in an appendix to this report stated that the cow that became infected was given two intravenous injections of virulent ovine material 39 days apart. A dead hairless fetus was aborted 18 days after the last injection.. Vibrios were observed in stained smears of stomach contents and cotyledons.. A contaminated culture was obtained from fetal stomach contents. Smith (1918) observed that the lesions associated with a spirillum infection of cattle were largely, if notLexclu- sively,.restricted to the fetal membranes; and that the fe- tus suffered secondarily from a gradually increasing inter- ference with the placental circulation in much the same man- ner as with Brucella abortus.. It was found impossible to foretell whether a given fetus would yield cultures of bru- cella,.the vibrio or none at all.. The main fetal lesions were edema of the subcutaneous‘ tissues and effusions into the peritoneal and pleural cavi- ties.. These fluids, as a generaltrulc,.were'more or less heavily tinged with blood, and frequently associated with delicate,.loose, shreddy deposits of fibrin or more rarely with heavy,,whitish pseudomembranes in the abdomen and much less*abundantly on the pleura and pericardium.. The visceral lesions were chiefly autolytic changes following death.. Fo- cal lesions were not present.” The stomachs usually contained quantities of meconium apparently swallowed or possibly driven into the abomasum by antiperistaltic movements. The stomachs of normal fetuses contained a colorless, translucent, very thick, viscid fluid which in older fetuses might contain a few pellets of meco- nium and perhaps some hairs.» The diseased fetus, however, almost invariably had in the stomachs a very turbid, thick, yellowish, flaky fluid.. Not infrequently there were also found small, whitish, soft, disc-like masses which could be traced back to epithelial excrescences of the amnion which had sloughed off and been swallowed by the fetus.. The younger fetuses almost invariably showed congenital pulmonary atelectasis, however the lungs of the older abort- ed fetuses sometimes contained air.. In some, the air tubes contained fluid identical with that found in the stomachs. This fluid often filled the trachea and bronchi completely. A thick, bloody fluid usually collected in a thin layer under the renal capsule. The tissue surrounding the kidneys was also frequently distended with bloody fluid.. Histological sections of fetal tissue did not reveal anything characteristic of the infection.. The epithelial coverings of the digestive tract were partially or wholly desquamated which was interpreted as:a post—mortem change.. Smith and Taylor (1919) named the spirillum associated with disease of the fetal membranes of cattle Vibrio £233; 33.52.,.and speculated that it possibly was identical with -6- the organism associated with ovine abortion in England (McFadyean (1913)). Smith, Little, and Taylor (1920) described the lesions found in the placenta of one case of vibrionic abortion.- The chorion varied from a smooth, translucent, slightly con- gested.membrane to one Opaque, thickened and leathery.. The opacity was due to an infiltration which resulted in the formation of slightly elevated plaques not removable by gen- tle scraping.- In other places the infiltration was discrete in the form of whitish opacities, one-half to one mm in di- ameter.s There were also scattered minute tufts of adventi- tious villi, which were completely cheesy.. The subjacent tissue was edematous and varied in thickness.. Part of the cotyledons were normal and part were diseased.. Some of the latter were yellowish and pultaceous throughout.. The still normal cotyledons contained, usually on the margin, necrotic, yellowish villi.. More rarely such villi were scattered throughout the cotyledon.” Fixed sections from various regions of the placenta showed necrosis of villi, loss of surface epithelium with leucocytic infiltration of the underlying tissue in certain areas. Where the epithelium was present, no bacteria were found in the cells as with Sr..abortus. The endothelium of the capillaries had proliferated in places and it partially or nearly filled the lumina.. Bacteria resembling vibrios‘ were detected within these cells and in groups among necrotic villi. \[lIlllllIlllollll[‘[ Smith (1919) reported on additional cases of vibrionic abortion and observed that in most cases.the placenta was retained.. Four pregnant cows were given intravenous injec- tions of X. fgtg§.. The cultures were grown on sealed agar slants with or without added bits of guinea pig spleen and calf serum water for four to six days.. Although live calves were born, lesions were found in the placenta of two of the animals,.and the organism was observed in stained smears of placental tissue.. Smith (1923) injected six cows intravenously with 1. fgtgg and two of these animals received in addition a sub- cutaneous injection.. Two cows aborted and four were killed at varying periods after exposure. Five of the six showed lesions due to 1. fgtgg in the fetal membranes, and the or- ganism wastobserved in stained smears from various areas of the fetal membranes, fetuses,,and exudate in the uterochori- onic space.. Pure cultures of the organisms were obtained from three cases and a mixed culture of 1..§§tg§ and a strep- tococcus in a fourth case.. The cultures used in these exper- iments had been isolated within a four month period prior to the inoculations.. Rhoades and Hardenbrook (1947) were unsuccessful in at- tempts to prove the pathogenicity of a culture of 1..£gtus.. Three cows between four and six months pregnant were used.. One cow received two intravenous injections and subsequent- ly delivered a live calf at term.. Another cow given an in- trauterine injection aborted four days after the inoculation.. -8- A streptococcus was isolated. All injections were made of 24-hour tryptose broth cultures of varying densities when compared to the McFarland nephelometric scale. A suspension of 15.3233; was mixed with the feed of a third cow at three- day intervals. A total of 1,290 ml of 24-hour.tryptose broth cultures were administered over a period of 25 days.. One month after the last exposure the cow was slaughtered. Cultures from various organs and tissues were negative fOr 1.,ggtgg. The authors speculated that attenuation of the. culture had occurred by growth on artificial media.. Sjollema, Stegenga,.and Terpstra (1949) concluded that 1. fetus infection in cattle was a venereal disease transa mitted at service.. The infection caused,,primarily,.a ca- tarrhal inflammation of the cervix and uterus., Infected cows failed to conceive, or the fertilized ovum soon died.. These animals sometimes continued to have regular heat peri- ods,.but in some the periods were considerably extended. Sometimes abortion occurred at a later stage of established pregnancy.“ These investigators found that usually an immunity was established within a three months period, and that the in+ fertility was thus of a temporary nature.. It was found that infected bulls could harbor the organisms for long periods» without showing any abnormality of the genital organs, and transmit the infection at the time of service.. The focus of infection in the bull was not determined. Roberts,.Gilman, and Larsen (1950) inoculated 1..g§tu§ into the conjunctival sac of three pregnant cows and the vagina of a fourth at the time of estrus without producing any evidence of disease. Plastridge (1951) isolated 1..£gtgg from scrapings of the uterine mucosa of two of three cows slaughtered because of failure to conceive after repeated services. Beveridge and Burnet (1946) reviewed the literature dealing with the experimental utilization of embryonating chicken eggs.. The method had been used in the study of chemotherapy,,immunological reactions with antisera,.and selective localization of the infective agent similar to that occurring in the natural disease.. Embryonating chicken eggs had also been used for growing bacteria otherwise not cultivable, for the production of antigen for compliment fixation tests, and for viral and rickettsial investigations.. These workers~credited the cultivation of spirochaetes in the chick embryo~by Levaditi in 1906 as being the first oc- casion on which the fertile egg was used to prOpagate an in- fective agent. Rous and Murphy (1911) reported that a cell-free ex- tract of a transmissible avian sarcoma caused tumor growth on the inner surfacerof the chorioallantoic membrane of de- veloping hens' eggs.. These workers were credited with being the first to use the chorioallantoic membrane for the study -10... of problems in experimental pathology and, more Specifically, virology. Woodruff and Goodpasture (1931) found that ectodermal and entodermal cells of the chorioallantoic membrane of the chick, as well as embryonic chick skin, were susceptible to infection with the virus of fowl-pox at an early stage in the develOpment of the embryo. Goodpasture and his associ- ates have been credited with the recognition of the potenti- alities of this method for virus research. Goodpasture and Anderson (1937) observed that Strepto- coccus viridans, Aerobacter aerogenes; Eberthella £1231: Brucella abortus and Mvcobacterigm tgberculosig agium were able to multiply within the protOplasm of mesodermal or epi- thelial cells, or both, of the chorioallantoic membrane of embryonating chicken sggss These, together with Staphylo- coccus aureus,.Streptococcus-haemolyticus and Cogynebacterium diphtheriae, would grow extracellularly in the presence of necrosis,.but apparently did not invade living tissues except? by the mechanism of phagocytosis.. The organisms were drapped on the exposed chorioallantoic membranes and the invasiveness studied. A polymorphonuclear and a mononuclear leucocytic response was generally noted.. Plastridge and Williams (1943) inoculated five embryon- ating chicken eggs with cultures of 1, fgtgg on the twelfth day of incubation.. All died within a period of five days. and growth of the organism occurred in the allantoic fluids. -11- MATERIALS AND METHODS‘ The culture of 1..£§tg§ used in this study was isolated from the abomasal contents of a bovine fetus aborted at an estimated three to three and one-half months after concep- tion.. HistOpathological changes typical of vibriosis occur- red in the fetal membranes (Smith, Little, and Taylor (1920)). ' Organismsihaving typical morphological characteristics were‘ observed in stained smears of the abomasal contents.. The tube agglutination test was positive in the 1-200 dilution (Plastridge, Williams, and Petrie (1947)). The organism was maintained in semisolid thiol medium for one month (Huddleson (1948)).. A tube of this medium was seeded from the original culture and incubated for 72 hours at 37 C. Seven tubes of the same medium were each inoculated with 0.1 ml of the growth obtained from the first transfer- These tubes,.plus one uninoculated tube were incubated for five days at 37 C. The culture was aspirated from the tubes using a sterile Pasteur pipette and diluted four times with a diluent consisting of 0.1 per cent tryptose and 0.5 per cent sodium chloride in distilled water (Wilson (1949)). The mixture was vigorously shaken and the resulting suspen- sion centrifuged for 35 minutes in an angle centrifuge* at *Ivan SOrvall,,Serveall Laboratory Aids, New York. - 12.. approximately 350 revolutions per minute.. The cloudy super- natant was removed with a Pasteur pipette