Br. vet. 1. (1992) . 148, 339
EXPERIMENTAL YERSINIA ENTEROCOLITICA PLACENTITIS IN SHEEP
M . J . CORBEL*, BARBARA ELLIS, CAROL . RICHARDSON and R. BRADLEY
Ministry o` Agriculture, Fisheries and Food, Central Veterinary Laboratory, Weybridge, Surrey KT 15 3NB
SUMMARY A strain of Yersinia enterocolitica of O serogroup 6,30 isolated from the liver of an aborted ovine fetus was inoculated intravenously into a group of pregnant ewes at about 90 days gestation and produced placentitis with abortion or delivery of infected lambs about 50 days later . Y. enterocolitica of the same serogroup was recovered from the necrotic placental cotyledons and most other fetal tissues and could be isolated from vaginal discharges of the ewes for a least 2 weeks after abortion . Histological changes were consistent with an acute bacterial necrotizing placentitis and systemic infection of the fetus . Subsequent pregnancies in the ewes proceeded to term without evidence of infection .
INTRODUCTION Numerous studies, including several conducted in Britain, have shown that strains of Yersinia enterocolitica and related species are frequently present in the intestinal tract of farm animals (Leistner et al., 1975 ; Hurvell et al., 1979 ; Brewer & Corbel, 1983 ; Hunter el al., 1983) . Most of the isolates obtained have belonged to biovar l and O serogroups which are rarely implicated as primary pathogens . Nevertheless, Brewer and Corbel (1983) described strains isolated from cases of abortion in cattle and sheep . More recently Y. enterocolitica has been isolated from a substantial number of aborted ovine fetuses examined in the North of England (Corbel et al., 1990) . Although circumstantial evidence has implicated these isolates as the probable cause of' abortion, their role as primary pathogens is still uncertain . In an attempt to determine whether such strains could produce ovine abortion, the course of experimental infection in pregnant ewes was examined .
'Corresponding author, present address : Division of Bacteriologn, Aationat Instituic fir Biological Standards and Control, Blanche tand, South Mininis, Putters Bar . 1lerts . ENG 3Qi ; .
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MATERIALS AND METHODS Yersinia strains
Y. enterocolitica strain 3047 was supplied by Mr D . Hunter, Veterinary Investigation Centre, Leeds and was originally isolated from the liver of an aborted ovine fetus . It had been maintained on nutrient agar slopes held at 4 °C . On receipt, the strain was subcultured on sheep blood agar incubated at 22 °C for 3 days and the resulting growth used to inoculate gerbils by the intraperitoneal route (Corbel, 1981) . The growth produced on sheep blood agar at 37 °C by culturing the liver and spleen tissue of gerbils dying 3-4 days after intraperitoneal injection was used as the inoculum for sheep . Viable counts were performed on sheep blood agar immediately before use . Y. enterocolitica 0 :3 strain M8542, a virulent, plasmid-bearing strain, was obtained from Dr A . P . Phillips, Chemical Defence Establishment, Porton Down, Wiltshire .
Strain characterization The biovar of the strain was checked according to Bercovier & Mollaret (1984) and matched according to the computer programme of Brewer (1984) . 0 group serotyping (Winblad, 1978) was performed by agglutination tests with specific antisera kindly provided by Dr Huang Jian, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China . Examination for in vitro correlates of virulence included tests for autoagglutination in liquid medium (Laird & Cavanaugh, 1980), colonial size (Mazigh et al., 1983) growth on magnesium oxalate medium (Schiemann et al., 1981) and colonial morphology on buffered Congo red medium (Prpié et al., 1983) . In vivo tests were performed by intraperitoneal inoculation of gerbils (Corbel, 1981) . Examination for plasmids was done using the extraction method of Birnboim & Doly (1979) followed by agarose gel electrophoresis (Meyers et al., 1976) . The plasmid-bearing strain Y. enterocolitica 0 :3 strain M8542, was used as positive control in these tests .
Experimental infection in sheep Six ewes (Numbers 304-309) of mixed breed which had previously given negative results to cultural and serological tests for infection with Y. enterocolitica were served by a single ram . At approximately 75 days after the service date they were transferred to individual loose boxes and kept isolated . Stringent precautions were taken against the introduction of extraneous or cross-infection . Pregnancy was confirmed by clinical examination and ultrasonic screening in five of the ewes (304-308) . The sixth ewe (309) was retained as a non-pregnant control . From 7 days before inoculation, rectal temperatures were recorded daily and samples of blood and faeces collected for culture . At an estimated 85-90 days gestation, the ewes (304-308) were injected via the jugular vein with about 10`' colony forming units of Y. enterocolitica strain 3047 . Subsequently they were examined twice daily and rectal temperatures recorded on each occasion . Daily blood and faeces samples were also collected for culture . Immediately after parturition or abortion, samples of blood, vaginal discharge, faeces and placenta were collected .
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Vaginal swabs were then collected daily for the next 21 days ; blood and faeces samples were collected daily for the first 14 days and then subsequently at weekly intervals . Lambs born alive were killed by intravenous injection of sodium pentobarbitone and these and all stillborn lambs and aborted fetuses were necropsied . The ewes were kept under observation with periodic cultural examination for yersinia until the next breeding season approximately 9 months later . Oestrus was then synchronized with intravaginal Estrumate sponges (Imperial Chemical Industries, Macclesfield) and artificial insemination performed with semen from a healthy ram . Pregnancy was detected by assay of plasma progesterone concentrations (English et al., 1986) and confirmed by clinical and ultrasonic examination . Pregnancy was allowed to proceed to full term when all ewes and lambs were killed and subjected to a complete necropsy including culture of all major organs .
Pathological examination Fetuses, lambs and ewes were examined according to standard necropsy procedures devised by the Pathology Department of this laboratory . Findings were interpreted in relation to a database established from observations over an extended period on large numbers of uninfected pregnant ewes (Richardson et al., 1976) . A full range of tissues was collected for microbiological examination taking precautions to prevent extraneous or cross-contamination of individual tissues . Smears of body fluids, placental cotyledon and fetal viscera were heat-fixed and stained by the modified Ziehl-Neelsen method (Stamp et al., 1950) . A full range of tissues was also collected for histopathology, fixed in neutral, phosphatebuffered, 10% formalin and embedded in paraffin wax . Sections were cut and stained with haematoxylin and eosin or special stains, including immunospecific staining for IgG . Fetal development was assessed according to established data (Richardson et al., 1976) based on service date, stage of pregnancy, fetal organ weight, body weight, crown-anus length and radiological appearance of the long bones .
Bacteriological examination Replicate samples of blood, faeces, body fluids and tissue homogenates were cultured directly on to Y medium (Soltész et al., 1980), CIN agar (Schiemann, 1979) and sheep blood agar and also on these media after cold enrichment in PBS held at 4 °( : for up to 3 weeks . Any isolates were checked for identity as Yersinia biovars and serogroups according to established procedures (Brewer & Corbel, 1985) .
Serological examination Agglutination tests were performed with O antigen suspensions of Y. enterocolitica 0 :6,30 standardized to an OD'-', ; of 3 .30 . The procedure was as recommended by Winblad (1978) .
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RESULTS
Characterization of strain Y. enterocolitica strain 3047 behaved as a typical strain of biovar I in cultural and biochemical tests . Its matching coefficient was 0 .944 for biovar 1, compared with 0 .889, 0 .833, 0 .778 and 0 .611 for biovars 2, 3, 4 and 5 respectively . Its serogroup was confirmed as 0 :6,30 by slide agglutination tests with reference antisera . Strain 3047 did not autoagglutinate at either 22 °C or 37 °C in RPMI 1640 liquid medium . On RPMI agar it produced a mixture of large and small colony types, with the former predominating . No evidence of plasmids was found in strain 3047 although a 48 MDa plasmid was consistently detected in the virulent strain 8542 . Strain 3047 was of low pathogenicity for mice . Its pathogenicity for gerbils was similar to that of strain 8542, both producing death 4 days after inoculation of doses of 5x10' viable organisms .
Experimental infection of sheep About 24 h after intravenous injection of 10' Y. enterocolitica strain 3047 organisms the six ewes showed a transient rise in rectal temperature of up to I °C . One of the pregnant ewes also developed a cough at about the same time . However, these signs resolved rapidly and all animals appeared in normal condition by the third day after inoculation . Apart from one ewe (305) which gave a positive faecal culture of Y. enterocolitica 0 :6,30 on the third day after inoculation, all blood, faecal and vaginal cultures remained negative until near the end of the gestation period . At about the fiftieth day after inoculation ewes 305 and 306 each produced two dead lambs, 307 one dead lamb and 308 three weak lambs that failed to suck, experienced increased breathing difficulties and died within 12 h of birth . Ewe 304 produced two live full term lambs which sucked colostrum and appeared completely healthy . These were killed within 12 h of birth by intravenous injection of sodium pentobarbitone and subjected to the same necropsy procedures . Following parturition, moderate blood-stained vaginal discharge was seen . In
.,
4
Fig. 1 . Placenta, ewe 305 . Numerous weakly acid-fast short bacilli among cellular debris . Modified Ziehl-Neelsenx750 .
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the case of ewe 305 this became increasingly purulent over the next 2 days . This animal developed increasing signs of prostration and pyrexia caused by a mixed bacterial secondary metritis which resolved rapidly after tetracycline treatment . Examination of stained smears of the vaginal discharge from ewes 305, 306, 307 and 308 revealed a mixture of Gram-positive and Gram-negative organisms . However, pleomorphic Gram-negative rods with a tendency to bipolar staining were the predominant form . These organisms were much more abundant in smears of the placental cotyledons (Fig . 1) . Partial or complete placentas were recovered from all the ewes . Each showed a range of cotyledon morphology and extensive thickening of the intercotyledonary membranes . Dark red, apparently congested, cotyledons were present in all placentas . In those of ewes 305, 306, 307 and 308 many were yellowish-grey, friable and necrotic (Fig . 2) . Profuse growths of Y. enterocolitica 0 :6,30 in virtually pure culture were obtained from the cut surface of the cotyledons from ewes 305, 306, 307 and 308 . An identical organism was cultured from vaginal swabs collected from these ewes . For 3-5 days after parturition fairly profuse growth of the organism was obtained . This diminished rapidly although positive cultures were still obtained kw up to 3 weeks . Smears of the vaginal discharge contained bacteria of yersinia morphology for at least 2 weeks after abortion or infected parturition although the bacteria were noticeably more sparse after the first week .
Lxaminationn of lambs and f.tuses Gross examination . Lambs showed no significant gross external changes . Fetuses showed external changes consistent with intrauterine death sonic days previously . These consisted of blood and meconium staining of the fleece and skin . Radiographs .
Radiography of bones indicated that the skeletal structure and
Fig. 2 . Placenta, ewe 305 . Congested cotyledons . Thickened, necrotic intercotvledonan placenta
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mineralization of all lambs and fetuses was within normal limits. Only the liveborn lambs of ewe 304 showed fine compression lines in the neck of' the scapulae consistent with those produced as a result of walking . The remaining lambs and fetuses showed multiple growth retardation lines whose initial age of formation ranged from 90 to 124 days of gestation . Variability of these data within litters suggests a continuing fetopathic insult which affected littermates sequentially . The estimated developmental ages ranged from 113 days for one fetus to 153 and 158 days for the lambs of ewe 304 . The majority had a developmental age between 130 and 148 days, indicating significant retardation of growth relative to chonological age . Morbid anatomy . Examination of the internal organs did not disclose any abnormality in the lambs of ewe 304 . In all the other lambs and fetuses a range of macroscopic changes consistent with a response to microbial invasion were apparent . The abdominal and thoracic cavities contained excessive quantities of' serosanguineous fluid, which in the former was sometimes turbid . The major abdominal and thoracic organs and mesenteric lymph nodes were dark, congested or haemorrhagic and in some cases (e.g. liver) friable or swollen (e.g. kidneys and lymph nodes) . The spleen, adrenals and thymus were shrunken and sometimes haemorrhagic . The gut contained brownish or blood-tinged fluid . The walls were congested, with haemorrhages, especially in the ileum, jejunum and caecum . The lungs were congested, oedematous and marbled . The heart was sometimes normal, sometimes friable with myocardial haemorrhages . Meninges of the brain were congested . No consistent pattern of abnormalities was noted in the eye, pancreas, bladder, mammary gland or genital tract .
Histopathology . Significant lesions were absent in live-born lambs from ewe 304 and in the brain, pancreas, adrenal and uterus of all other fetuses and lambs . Tissues from two fetuses from ewe 308 were autolysed . Histological examination of the other tissues of the remainder disclosed a consistent pattern of response to infection . The congestion and haemorrhages in many organs recognized grossly were confirmed histologically . Depletion of lym phocytes occurred in spleens and was also seen in one thymus . Mesenteric lymph nodes were generally reactive resulting in frank suppurative lymphadenitis in one instance . Immunocytochemical detection of IgG was confirmed in lymphatic tissues . The small intestine and abomasum from the weak lamb showed suppurative and necrotic mucosal enteritis/abomasitis . Cardiac myocytes, including sonictimes Purkinje fibres, were vacuolated and in one instance contained an unidentified brown pigment . The lungs were congested with variable bronchiolar catarrh, suppurative bronchiolitis and alveolitis with occasional necrosis . Necrotic arteritis also occurred (Fig . 3) . The liver of one fetus from ewe 305 showed centrilobular necrosis and multifocal necrosis . Most others were congested with variable, nonbirefringment pigmentation (presumed to be haemosiderosis) and sinusoidal infiltration of leucocytes including mature and immature neutrophil polymorphonuclear leucocytes (Fig . 4) . These fetuses were also seen variably in other tissues. Apart from congestion, haemorrhage and focal polymorphonuclear infil-
YERSINIA PLACENTITIS IN SHEEP
tration in some kidneys, there was a non-suppurative pyelitis in one fetus of ewe 305 (Fig . 5) . The maternal caruncle and fetal cotyledons of placentas of the five fetuses examined showed consistent suppurative, and necrotic placentitis (Fig . 6) . Nec-
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Fig. 3. Lung, lamb . Necrotic arteritis . Haematoxvlin and eosin (HE)x210 .
Fig . 4. Liver, lamb . Leucocytic sinusoidal infiltration . Cells include mature and immature neutrophils . HEx840 .
BRI tIS11 VE4 FIRINARh,JOIJRN-AI . 148 .
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1
rotic vasculitis, thrombosis and plasma cell infiltration were also seen . Small Grainnegative bacilli morphologically resembling Yersinia spp . were seen in the placentas from ewes 305 and 308 . Microbiology . Cultural examination disclosed heavy growth of Y . enterocolitica from virtually all tissues of infected fetuses . The heaviest growth was from the liver, lungs, spleen, kidneys, ascitic and pleural fluids and heart blood . The tissues of
Fig. 5 .
Kidney, lamb . Non-suppurative pyelitis .
HEX 140 .
Fig . 6 . Placenta, ewe 305 . Suppurative placentitis . Bacterial colony on the left .
HEx187 .
YERSINIA PIACENTITIS IN SHEEP
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infected lambs tended to contain smaller numbers of yersiniae and isolates were not consistently obtained from the brain, eye, mammary gland, testis, ovary or uterus. Serological examination . None of agglutinated Y. enterocolitica
lation showed ition, 1 /80 . P ffect
the sera taken from the ewes before inocu0:6,30 to a titre greater than I to 10 . These little or no change during gestation . Immediately after abortion or parturthe sera of ewes 305, 306, 307 and 308 had agglutination titres of 1 /20 to These titres did not change in the following 4 weeks .
ofyersinial
infection on subsequent fertility
Because of the potential risk of transmission of infection, the ewes were not permitted to run with the ram . This made the determination of the date of resumption of oestrus difficult . Nevertheless, after synchronization of oestrus with Esn -umate, fertilization was achieved in all the ewes by artificial insemination . All pregnancies proceeded normally to term and all ewes delivered healthy lambs . Post-mortem examination of both lambs and ewes failed to disclose any lesions and Y. enterocolitica was not isolated from any tissue .
DISCUSSION Strains of Y. enterocolitica biovar I are frequently isolated from animal and environmental sources and are not usually associated with disease . Pathogenic strains usually belong to biovars, 2, 3, 4, and 5 and a limited range of O serogroups, partictilarly 0 :1,2,3 ; 0 :2,3 ; 0 :3; 0 :4,32 ; 0 :5,27 ; 0 :8; 0 :9; 0 :13a,ó ; 0 :18 ; 0 :20 ; 0 :21 (Bottone, 1977 ; Brewer & Corbel, 1985) . Virulence in Y. enterocolitica is associated with the presence of a 48 MDa plasmid which encodes genes determining calcium and temperature sensitivity, the production of the V and IN virulence-associated antigens (Portnoy et al., 1981) and at least 16 outer membrane proteins which promote intracellular survival (Skurnik, 1985 ; Rosqvist et al., 1990) . The presence of the virulence plasmid is associated with other properties such as the production of autoagglutination in liquid medium incubated at 37 °C but not 22 °C, production of V and W antigens (Skurnik, 1985), suppression of growth on magnesium oxalate medium at 37 °C (Schiemann et al., 1981) and production of lethal infection in mice . Although essential for full virulence, the 48 MDa plasmid is not the only determinant of pathogenicity and the capacity to invade cells is determined by two chromosomal loci, the inv and ail genes in Y. enterocolitica (Miller & Falkow, 1988 : Miller et al., 1989), deletion of which results in complete loss of pathogenicity (Pierson & Falkow, 1990) . Y. enterocolitica strain 3047 had the characteristics of a typical strain of biovar 1, did not contain any plasmids and did not display the full attributes of a virulent strain . This was shown by its failure to establish systemic iufection in adult ewes, even after intravenous inoculation of large doses . Nevertheless, it did possess some pathogenicity as shown by its capacity to produce lethal infections in gerbils from large inocula and by the production of placentitis in four out of five pregnant ewes . Bacteria which ltad no capacity for pathogenesis would not have persisted and established infection in immunocompetent individuals.
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The course of the infection and its histopathology suggested that the organistns produced a relatively slowly evolving infection which only reached the stage of producing placental failure and fetal death when its effects were superimposed on the normal decline in placental function occurring in late gestation . These results indicated that strains of Y. enterocolitica biovar I of low natural pathogenicity could produce placental infection and abortion in pregnant sheep . This is consistent with observations from the field (Corbel et al., 1990) . Natural infection is likely to result from a maternal infection acquired by the oral route, presumably in association with an unknown factor which permits the establishment of 'a transient bacteraemia . The experimental results indicated that diagnosis could easily be based on isolation of the organism from placenta, a wide range of fetal tissues and the vaginal discharge . The limited serological studies performed here did not suggest that agglutination procedures would be of much diagnostic assistance . It is not clear why the antibody response was so limited . It is possible that most of the antigenic material was concentrated in the placenta and not available to the maternal immune system . However, more sensitive serological procedures or selection of different antigens might have indicated a more substantial antibody response . Follow-up of the experimentally infected sheep indicated that infection was not persistent and that there was no adverse effect on subsequent reproductive performance . Further studies would be required to determine whether a single infection confers immunity against reinfection with the same or heterologous serogroups .
REFERENCES BERCOVIER, H . & MOLIARET, H . H . (1984) . Genus XIV . Yersinia van Logherr, 1944, 15'" . In Bergey's Manual of Systematic Bacteriology, Vol . 1, eds . N . R . Krieg & J. G . Holt, p . 498 .
Baltimore : Williams & Wilkins . & Dote, J . (1979) . A rapid alkaline extraction procedure for screening recombinant plasmid DNA . Nun. Acids Res . 7, 1 .513 . BOTTONE, E . J . (1977) . Yersinia enterocolitica : a panoramic view of a charismatic microorganism . CRC crit. Rev . Mirrohiol. 5, 211 . BREWER, R . A. (1984) . A simple program for computer assisted biotyping of Yersinia enterocolitica and related species . Binary 3, 29-30 . BREWER, R. A. & CORBEL, M . J. (1983) . Characterization of Yersinia enterocolitica strains isolated from cattle, sheep and pigs in the United Kingdom . f. Hyg., Camb. 90, 425-33 . BREWER, R . A . & CORBEL,, M . J . (1985) . Yersinia enterocolitica and related species . In Isolation and Identification of Microorganisms of Medical and Veterinary Importance, eds . C . H . Collins & J . M . Grange, pp . 83-104 . London : Academic Press . CORBEL, M . J . (1981) . Observations on experimental infections with Yersinia enterocolitica in the gerbil . Ann. Sclavo 23, 1-19 . CORBEL, M . J ., BREWER, R . A. & HUNTER, D . (1990) . Characterization of Yersinia enterocoliticia strains associated with ovine abortion . Vet . Ren . 127, 526-7 . ENGLISH, J ., POUI :TON, A . L ., ARENOT, A. & SYMO Ns, A. M . (1986) . A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes . J. Reprod . Eo -t. 77, 321-7 . HUNTER, D ., HUGHES, S. & Fox, E . (1983) . Isolation of Yersinia enterocolitica from pigs in the United Kingdom . Vet . Ren . 112, 322-3 . BIRNBOIM, H . C .
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HLRVELE, B ., GIATTHARD, V . & THAL, E . (1979) . Isolation of Yersinia enterocolitica from swine at an abattoir in Sweden . Contrib . Microbiol. Immunol. 5, 243-8 . IAMRD, W . J . & CAVANAUGH, D . C . (1980) . Correlation of autoagglutination and virulence of
f
yersiniae . clin . Microbiol. 11, 430-2 . LraSTNER, L ., HECHELMANN, H ., KASHIWAZAKI, M . & ALBERTZ, R . (1975) . Nachweis von Yersinia enterocolitica in Faeces and Fleisch von Schweinen . Rindern and Geflügel . Fleischwirtschaft
55,1599-602 . MAZIGH, D ., ALONSO, J . M . & MOLLARET, H . H . (1983) . Simple method for demonstration of differential colony morphology of plasmid-associated virulent clones of Yersinia enterocoli-
tica . J clin Microbiol. 17, 555-7 . MEYERS, J . A., SANCHEZ, D ., ELWELL, L . P . & FALKOw, S . (1976) . Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid . J. Bacteriol. 127, 1529-37 . MILLER, V . L . & FAu .KOw, S . (1988) . Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells . Infect. Immun . 56, 1242-8 . MILLER, V. I . ., FARMER, J . J . III, HILL, W. E . & FAI.KOw, S . (1989) . The ail locus is found uniquely in Yersinia enterocolitica serotypes commonly associated with disease . Infect.
Immun . 57, 121-31 . PIERSON, D . E . & FALKOW, S . (1990) . Nonpathogenic isolates of Yersinia enterocolitica do not contain functional inv in homologous sequences . Infect . Immun . 58, 1059-64 . PORTNOY, D . A ., MOSELEY, S . L . & FAI .KOW, S . (1981) . Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis . Infect. Immun . 31,
775-82 . PRPiC, J . K ., RoBINs- BROWNE, R. M . & DAVEY, R . B . (1983) . Differentiation between virulent and avirulent Yersinia enterocolitica isolates by using Congo red agar . J. clin. Microbiol. 18, 486-90 . RICHARDSON, C ., HEBERT, C . N . & TERLECKI, S . (1976) . Estimation of the developmental age of the ovine fetus and lamb . Vet. Rec. 99, 22-6 . RosQvisi', R ., FORSBERG, A., RUMPILAINEN, M., BERGMAN, T . & WOLF-WATZ, H . (1990) . The cytotoxic protein YopE of Yersinia obstructs the primary host defence . Molex. Microbiol. 4, 657-67 . S( .HIF:Nr NN, D . A . (1979) . Synthesis of a selective agar medium for Yersinia enterocolitica . Can . f. Microbiol. 25, 1298-304 . SCHIEMANN, D . A ., DEVENISH, J . A. & ToMA, S . (1981) . Characteristics of virulence in human isolates of Yersinia enterocolitica . Infect. Immun . 32, 400-3 . SKURNIK, M . (1985) . Expression of antigens encoded by the virulence plasmid of Yersinia enterocolitica under different growth conditions. Infect . Immun . 47, 183-90 . SOLTFSZ, L . V., SCHAI .ÉN, C . & MARSH, P . A . (1980) . An effective selective medium for Yersinia enterocolitica containing sodium oxalate . Acta path . microbiol. scand. B88,1 1-16 . STOMP, J . T ., McEWEN, A . D ., WATT, J . A . A . & NISBET, D . I . (1950) . Enzootic abortion in ewes . Tranmission of the disease . Vet. Rec. 62, 251-4. WINBLAU, S . (1978) . Yersinia enterocolitica . In Methods in Microbiology, eds T . Bergan &J . R . Norris, Vol . 12, pp . 37-50 . London : Academic Press . (Accepledforpublieation 17 Fe&
ars 1992)
EXPERIMENTAL YERSINIA ENTEROCOLITICA PLACENTITIS IN SHEEP
M . J . CORBEL*, BARBARA ELLIS, CAROL . RICHARDSON and R. BRADLEY
Ministry o` Agriculture, Fisheries and Food, Central Veterinary Laboratory, Weybridge, Surrey KT 15 3NB
SUMMARY A strain of Yersinia enterocolitica of O serogroup 6,30 isolated from the liver of an aborted ovine fetus was inoculated intravenously into a group of pregnant ewes at about 90 days gestation and produced placentitis with abortion or delivery of infected lambs about 50 days later . Y. enterocolitica of the same serogroup was recovered from the necrotic placental cotyledons and most other fetal tissues and could be isolated from vaginal discharges of the ewes for a least 2 weeks after abortion . Histological changes were consistent with an acute bacterial necrotizing placentitis and systemic infection of the fetus . Subsequent pregnancies in the ewes proceeded to term without evidence of infection .
INTRODUCTION Numerous studies, including several conducted in Britain, have shown that strains of Yersinia enterocolitica and related species are frequently present in the intestinal tract of farm animals (Leistner et al., 1975 ; Hurvell et al., 1979 ; Brewer & Corbel, 1983 ; Hunter el al., 1983) . Most of the isolates obtained have belonged to biovar l and O serogroups which are rarely implicated as primary pathogens . Nevertheless, Brewer and Corbel (1983) described strains isolated from cases of abortion in cattle and sheep . More recently Y. enterocolitica has been isolated from a substantial number of aborted ovine fetuses examined in the North of England (Corbel et al., 1990) . Although circumstantial evidence has implicated these isolates as the probable cause of' abortion, their role as primary pathogens is still uncertain . In an attempt to determine whether such strains could produce ovine abortion, the course of experimental infection in pregnant ewes was examined .
'Corresponding author, present address : Division of Bacteriologn, Aationat Instituic fir Biological Standards and Control, Blanche tand, South Mininis, Putters Bar . 1lerts . ENG 3Qi ; .
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MATERIALS AND METHODS Yersinia strains
Y. enterocolitica strain 3047 was supplied by Mr D . Hunter, Veterinary Investigation Centre, Leeds and was originally isolated from the liver of an aborted ovine fetus . It had been maintained on nutrient agar slopes held at 4 °C . On receipt, the strain was subcultured on sheep blood agar incubated at 22 °C for 3 days and the resulting growth used to inoculate gerbils by the intraperitoneal route (Corbel, 1981) . The growth produced on sheep blood agar at 37 °C by culturing the liver and spleen tissue of gerbils dying 3-4 days after intraperitoneal injection was used as the inoculum for sheep . Viable counts were performed on sheep blood agar immediately before use . Y. enterocolitica 0 :3 strain M8542, a virulent, plasmid-bearing strain, was obtained from Dr A . P . Phillips, Chemical Defence Establishment, Porton Down, Wiltshire .
Strain characterization The biovar of the strain was checked according to Bercovier & Mollaret (1984) and matched according to the computer programme of Brewer (1984) . 0 group serotyping (Winblad, 1978) was performed by agglutination tests with specific antisera kindly provided by Dr Huang Jian, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China . Examination for in vitro correlates of virulence included tests for autoagglutination in liquid medium (Laird & Cavanaugh, 1980), colonial size (Mazigh et al., 1983) growth on magnesium oxalate medium (Schiemann et al., 1981) and colonial morphology on buffered Congo red medium (Prpié et al., 1983) . In vivo tests were performed by intraperitoneal inoculation of gerbils (Corbel, 1981) . Examination for plasmids was done using the extraction method of Birnboim & Doly (1979) followed by agarose gel electrophoresis (Meyers et al., 1976) . The plasmid-bearing strain Y. enterocolitica 0 :3 strain M8542, was used as positive control in these tests .
Experimental infection in sheep Six ewes (Numbers 304-309) of mixed breed which had previously given negative results to cultural and serological tests for infection with Y. enterocolitica were served by a single ram . At approximately 75 days after the service date they were transferred to individual loose boxes and kept isolated . Stringent precautions were taken against the introduction of extraneous or cross-infection . Pregnancy was confirmed by clinical examination and ultrasonic screening in five of the ewes (304-308) . The sixth ewe (309) was retained as a non-pregnant control . From 7 days before inoculation, rectal temperatures were recorded daily and samples of blood and faeces collected for culture . At an estimated 85-90 days gestation, the ewes (304-308) were injected via the jugular vein with about 10`' colony forming units of Y. enterocolitica strain 3047 . Subsequently they were examined twice daily and rectal temperatures recorded on each occasion . Daily blood and faeces samples were also collected for culture . Immediately after parturition or abortion, samples of blood, vaginal discharge, faeces and placenta were collected .
YERSINIA PLACENTITIS IN SHEEP
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Vaginal swabs were then collected daily for the next 21 days ; blood and faeces samples were collected daily for the first 14 days and then subsequently at weekly intervals . Lambs born alive were killed by intravenous injection of sodium pentobarbitone and these and all stillborn lambs and aborted fetuses were necropsied . The ewes were kept under observation with periodic cultural examination for yersinia until the next breeding season approximately 9 months later . Oestrus was then synchronized with intravaginal Estrumate sponges (Imperial Chemical Industries, Macclesfield) and artificial insemination performed with semen from a healthy ram . Pregnancy was detected by assay of plasma progesterone concentrations (English et al., 1986) and confirmed by clinical and ultrasonic examination . Pregnancy was allowed to proceed to full term when all ewes and lambs were killed and subjected to a complete necropsy including culture of all major organs .
Pathological examination Fetuses, lambs and ewes were examined according to standard necropsy procedures devised by the Pathology Department of this laboratory . Findings were interpreted in relation to a database established from observations over an extended period on large numbers of uninfected pregnant ewes (Richardson et al., 1976) . A full range of tissues was collected for microbiological examination taking precautions to prevent extraneous or cross-contamination of individual tissues . Smears of body fluids, placental cotyledon and fetal viscera were heat-fixed and stained by the modified Ziehl-Neelsen method (Stamp et al., 1950) . A full range of tissues was also collected for histopathology, fixed in neutral, phosphatebuffered, 10% formalin and embedded in paraffin wax . Sections were cut and stained with haematoxylin and eosin or special stains, including immunospecific staining for IgG . Fetal development was assessed according to established data (Richardson et al., 1976) based on service date, stage of pregnancy, fetal organ weight, body weight, crown-anus length and radiological appearance of the long bones .
Bacteriological examination Replicate samples of blood, faeces, body fluids and tissue homogenates were cultured directly on to Y medium (Soltész et al., 1980), CIN agar (Schiemann, 1979) and sheep blood agar and also on these media after cold enrichment in PBS held at 4 °( : for up to 3 weeks . Any isolates were checked for identity as Yersinia biovars and serogroups according to established procedures (Brewer & Corbel, 1985) .
Serological examination Agglutination tests were performed with O antigen suspensions of Y. enterocolitica 0 :6,30 standardized to an OD'-', ; of 3 .30 . The procedure was as recommended by Winblad (1978) .
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BRITISH VETERINARY JOURNAL, 148, -1
RESULTS
Characterization of strain Y. enterocolitica strain 3047 behaved as a typical strain of biovar I in cultural and biochemical tests . Its matching coefficient was 0 .944 for biovar 1, compared with 0 .889, 0 .833, 0 .778 and 0 .611 for biovars 2, 3, 4 and 5 respectively . Its serogroup was confirmed as 0 :6,30 by slide agglutination tests with reference antisera . Strain 3047 did not autoagglutinate at either 22 °C or 37 °C in RPMI 1640 liquid medium . On RPMI agar it produced a mixture of large and small colony types, with the former predominating . No evidence of plasmids was found in strain 3047 although a 48 MDa plasmid was consistently detected in the virulent strain 8542 . Strain 3047 was of low pathogenicity for mice . Its pathogenicity for gerbils was similar to that of strain 8542, both producing death 4 days after inoculation of doses of 5x10' viable organisms .
Experimental infection of sheep About 24 h after intravenous injection of 10' Y. enterocolitica strain 3047 organisms the six ewes showed a transient rise in rectal temperature of up to I °C . One of the pregnant ewes also developed a cough at about the same time . However, these signs resolved rapidly and all animals appeared in normal condition by the third day after inoculation . Apart from one ewe (305) which gave a positive faecal culture of Y. enterocolitica 0 :6,30 on the third day after inoculation, all blood, faecal and vaginal cultures remained negative until near the end of the gestation period . At about the fiftieth day after inoculation ewes 305 and 306 each produced two dead lambs, 307 one dead lamb and 308 three weak lambs that failed to suck, experienced increased breathing difficulties and died within 12 h of birth . Ewe 304 produced two live full term lambs which sucked colostrum and appeared completely healthy . These were killed within 12 h of birth by intravenous injection of sodium pentobarbitone and subjected to the same necropsy procedures . Following parturition, moderate blood-stained vaginal discharge was seen . In
.,
4
Fig. 1 . Placenta, ewe 305 . Numerous weakly acid-fast short bacilli among cellular debris . Modified Ziehl-Neelsenx750 .
YERSINIA PIACENTITIS IN SHEEP
313
the case of ewe 305 this became increasingly purulent over the next 2 days . This animal developed increasing signs of prostration and pyrexia caused by a mixed bacterial secondary metritis which resolved rapidly after tetracycline treatment . Examination of stained smears of the vaginal discharge from ewes 305, 306, 307 and 308 revealed a mixture of Gram-positive and Gram-negative organisms . However, pleomorphic Gram-negative rods with a tendency to bipolar staining were the predominant form . These organisms were much more abundant in smears of the placental cotyledons (Fig . 1) . Partial or complete placentas were recovered from all the ewes . Each showed a range of cotyledon morphology and extensive thickening of the intercotyledonary membranes . Dark red, apparently congested, cotyledons were present in all placentas . In those of ewes 305, 306, 307 and 308 many were yellowish-grey, friable and necrotic (Fig . 2) . Profuse growths of Y. enterocolitica 0 :6,30 in virtually pure culture were obtained from the cut surface of the cotyledons from ewes 305, 306, 307 and 308 . An identical organism was cultured from vaginal swabs collected from these ewes . For 3-5 days after parturition fairly profuse growth of the organism was obtained . This diminished rapidly although positive cultures were still obtained kw up to 3 weeks . Smears of the vaginal discharge contained bacteria of yersinia morphology for at least 2 weeks after abortion or infected parturition although the bacteria were noticeably more sparse after the first week .
Lxaminationn of lambs and f.tuses Gross examination . Lambs showed no significant gross external changes . Fetuses showed external changes consistent with intrauterine death sonic days previously . These consisted of blood and meconium staining of the fleece and skin . Radiographs .
Radiography of bones indicated that the skeletal structure and
Fig. 2 . Placenta, ewe 305 . Congested cotyledons . Thickened, necrotic intercotvledonan placenta
3 44
BRITISH VETERINARY J( )t RNAI ., 148 . I
mineralization of all lambs and fetuses was within normal limits. Only the liveborn lambs of ewe 304 showed fine compression lines in the neck of' the scapulae consistent with those produced as a result of walking . The remaining lambs and fetuses showed multiple growth retardation lines whose initial age of formation ranged from 90 to 124 days of gestation . Variability of these data within litters suggests a continuing fetopathic insult which affected littermates sequentially . The estimated developmental ages ranged from 113 days for one fetus to 153 and 158 days for the lambs of ewe 304 . The majority had a developmental age between 130 and 148 days, indicating significant retardation of growth relative to chonological age . Morbid anatomy . Examination of the internal organs did not disclose any abnormality in the lambs of ewe 304 . In all the other lambs and fetuses a range of macroscopic changes consistent with a response to microbial invasion were apparent . The abdominal and thoracic cavities contained excessive quantities of' serosanguineous fluid, which in the former was sometimes turbid . The major abdominal and thoracic organs and mesenteric lymph nodes were dark, congested or haemorrhagic and in some cases (e.g. liver) friable or swollen (e.g. kidneys and lymph nodes) . The spleen, adrenals and thymus were shrunken and sometimes haemorrhagic . The gut contained brownish or blood-tinged fluid . The walls were congested, with haemorrhages, especially in the ileum, jejunum and caecum . The lungs were congested, oedematous and marbled . The heart was sometimes normal, sometimes friable with myocardial haemorrhages . Meninges of the brain were congested . No consistent pattern of abnormalities was noted in the eye, pancreas, bladder, mammary gland or genital tract .
Histopathology . Significant lesions were absent in live-born lambs from ewe 304 and in the brain, pancreas, adrenal and uterus of all other fetuses and lambs . Tissues from two fetuses from ewe 308 were autolysed . Histological examination of the other tissues of the remainder disclosed a consistent pattern of response to infection . The congestion and haemorrhages in many organs recognized grossly were confirmed histologically . Depletion of lym phocytes occurred in spleens and was also seen in one thymus . Mesenteric lymph nodes were generally reactive resulting in frank suppurative lymphadenitis in one instance . Immunocytochemical detection of IgG was confirmed in lymphatic tissues . The small intestine and abomasum from the weak lamb showed suppurative and necrotic mucosal enteritis/abomasitis . Cardiac myocytes, including sonictimes Purkinje fibres, were vacuolated and in one instance contained an unidentified brown pigment . The lungs were congested with variable bronchiolar catarrh, suppurative bronchiolitis and alveolitis with occasional necrosis . Necrotic arteritis also occurred (Fig . 3) . The liver of one fetus from ewe 305 showed centrilobular necrosis and multifocal necrosis . Most others were congested with variable, nonbirefringment pigmentation (presumed to be haemosiderosis) and sinusoidal infiltration of leucocytes including mature and immature neutrophil polymorphonuclear leucocytes (Fig . 4) . These fetuses were also seen variably in other tissues. Apart from congestion, haemorrhage and focal polymorphonuclear infil-
YERSINIA PLACENTITIS IN SHEEP
tration in some kidneys, there was a non-suppurative pyelitis in one fetus of ewe 305 (Fig . 5) . The maternal caruncle and fetal cotyledons of placentas of the five fetuses examined showed consistent suppurative, and necrotic placentitis (Fig . 6) . Nec-
t ~'
!` :;..%
r4; ;sue
d
•~
+~,;
Fig. 3. Lung, lamb . Necrotic arteritis . Haematoxvlin and eosin (HE)x210 .
Fig . 4. Liver, lamb . Leucocytic sinusoidal infiltration . Cells include mature and immature neutrophils . HEx840 .
BRI tIS11 VE4 FIRINARh,JOIJRN-AI . 148 .
346
1
rotic vasculitis, thrombosis and plasma cell infiltration were also seen . Small Grainnegative bacilli morphologically resembling Yersinia spp . were seen in the placentas from ewes 305 and 308 . Microbiology . Cultural examination disclosed heavy growth of Y . enterocolitica from virtually all tissues of infected fetuses . The heaviest growth was from the liver, lungs, spleen, kidneys, ascitic and pleural fluids and heart blood . The tissues of
Fig. 5 .
Kidney, lamb . Non-suppurative pyelitis .
HEX 140 .
Fig . 6 . Placenta, ewe 305 . Suppurative placentitis . Bacterial colony on the left .
HEx187 .
YERSINIA PIACENTITIS IN SHEEP
317
infected lambs tended to contain smaller numbers of yersiniae and isolates were not consistently obtained from the brain, eye, mammary gland, testis, ovary or uterus. Serological examination . None of agglutinated Y. enterocolitica
lation showed ition, 1 /80 . P ffect
the sera taken from the ewes before inocu0:6,30 to a titre greater than I to 10 . These little or no change during gestation . Immediately after abortion or parturthe sera of ewes 305, 306, 307 and 308 had agglutination titres of 1 /20 to These titres did not change in the following 4 weeks .
ofyersinial
infection on subsequent fertility
Because of the potential risk of transmission of infection, the ewes were not permitted to run with the ram . This made the determination of the date of resumption of oestrus difficult . Nevertheless, after synchronization of oestrus with Esn -umate, fertilization was achieved in all the ewes by artificial insemination . All pregnancies proceeded normally to term and all ewes delivered healthy lambs . Post-mortem examination of both lambs and ewes failed to disclose any lesions and Y. enterocolitica was not isolated from any tissue .
DISCUSSION Strains of Y. enterocolitica biovar I are frequently isolated from animal and environmental sources and are not usually associated with disease . Pathogenic strains usually belong to biovars, 2, 3, 4, and 5 and a limited range of O serogroups, partictilarly 0 :1,2,3 ; 0 :2,3 ; 0 :3; 0 :4,32 ; 0 :5,27 ; 0 :8; 0 :9; 0 :13a,ó ; 0 :18 ; 0 :20 ; 0 :21 (Bottone, 1977 ; Brewer & Corbel, 1985) . Virulence in Y. enterocolitica is associated with the presence of a 48 MDa plasmid which encodes genes determining calcium and temperature sensitivity, the production of the V and IN virulence-associated antigens (Portnoy et al., 1981) and at least 16 outer membrane proteins which promote intracellular survival (Skurnik, 1985 ; Rosqvist et al., 1990) . The presence of the virulence plasmid is associated with other properties such as the production of autoagglutination in liquid medium incubated at 37 °C but not 22 °C, production of V and W antigens (Skurnik, 1985), suppression of growth on magnesium oxalate medium at 37 °C (Schiemann et al., 1981) and production of lethal infection in mice . Although essential for full virulence, the 48 MDa plasmid is not the only determinant of pathogenicity and the capacity to invade cells is determined by two chromosomal loci, the inv and ail genes in Y. enterocolitica (Miller & Falkow, 1988 : Miller et al., 1989), deletion of which results in complete loss of pathogenicity (Pierson & Falkow, 1990) . Y. enterocolitica strain 3047 had the characteristics of a typical strain of biovar 1, did not contain any plasmids and did not display the full attributes of a virulent strain . This was shown by its failure to establish systemic iufection in adult ewes, even after intravenous inoculation of large doses . Nevertheless, it did possess some pathogenicity as shown by its capacity to produce lethal infections in gerbils from large inocula and by the production of placentitis in four out of five pregnant ewes . Bacteria which ltad no capacity for pathogenesis would not have persisted and established infection in immunocompetent individuals.
348
ItRV!ISH V°I !FRINARV' lot 'RNAL, I IS . I
The course of the infection and its histopathology suggested that the organistns produced a relatively slowly evolving infection which only reached the stage of producing placental failure and fetal death when its effects were superimposed on the normal decline in placental function occurring in late gestation . These results indicated that strains of Y. enterocolitica biovar I of low natural pathogenicity could produce placental infection and abortion in pregnant sheep . This is consistent with observations from the field (Corbel et al., 1990) . Natural infection is likely to result from a maternal infection acquired by the oral route, presumably in association with an unknown factor which permits the establishment of 'a transient bacteraemia . The experimental results indicated that diagnosis could easily be based on isolation of the organism from placenta, a wide range of fetal tissues and the vaginal discharge . The limited serological studies performed here did not suggest that agglutination procedures would be of much diagnostic assistance . It is not clear why the antibody response was so limited . It is possible that most of the antigenic material was concentrated in the placenta and not available to the maternal immune system . However, more sensitive serological procedures or selection of different antigens might have indicated a more substantial antibody response . Follow-up of the experimentally infected sheep indicated that infection was not persistent and that there was no adverse effect on subsequent reproductive performance . Further studies would be required to determine whether a single infection confers immunity against reinfection with the same or heterologous serogroups .
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Baltimore : Williams & Wilkins . & Dote, J . (1979) . A rapid alkaline extraction procedure for screening recombinant plasmid DNA . Nun. Acids Res . 7, 1 .513 . BOTTONE, E . J . (1977) . Yersinia enterocolitica : a panoramic view of a charismatic microorganism . CRC crit. Rev . Mirrohiol. 5, 211 . BREWER, R . A. (1984) . A simple program for computer assisted biotyping of Yersinia enterocolitica and related species . Binary 3, 29-30 . BREWER, R. A. & CORBEL, M . J. (1983) . Characterization of Yersinia enterocolitica strains isolated from cattle, sheep and pigs in the United Kingdom . f. Hyg., Camb. 90, 425-33 . BREWER, R . A . & CORBEL,, M . J . (1985) . Yersinia enterocolitica and related species . In Isolation and Identification of Microorganisms of Medical and Veterinary Importance, eds . C . H . Collins & J . M . Grange, pp . 83-104 . London : Academic Press . CORBEL, M . J . (1981) . Observations on experimental infections with Yersinia enterocolitica in the gerbil . Ann. Sclavo 23, 1-19 . CORBEL, M . J ., BREWER, R . A. & HUNTER, D . (1990) . Characterization of Yersinia enterocoliticia strains associated with ovine abortion . Vet . Ren . 127, 526-7 . ENGLISH, J ., POUI :TON, A . L ., ARENOT, A. & SYMO Ns, A. M . (1986) . A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes . J. Reprod . Eo -t. 77, 321-7 . HUNTER, D ., HUGHES, S. & Fox, E . (1983) . Isolation of Yersinia enterocolitica from pigs in the United Kingdom . Vet . Ren . 112, 322-3 . BIRNBOIM, H . C .
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HLRVELE, B ., GIATTHARD, V . & THAL, E . (1979) . Isolation of Yersinia enterocolitica from swine at an abattoir in Sweden . Contrib . Microbiol. Immunol. 5, 243-8 . IAMRD, W . J . & CAVANAUGH, D . C . (1980) . Correlation of autoagglutination and virulence of
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yersiniae . clin . Microbiol. 11, 430-2 . LraSTNER, L ., HECHELMANN, H ., KASHIWAZAKI, M . & ALBERTZ, R . (1975) . Nachweis von Yersinia enterocolitica in Faeces and Fleisch von Schweinen . Rindern and Geflügel . Fleischwirtschaft
55,1599-602 . MAZIGH, D ., ALONSO, J . M . & MOLLARET, H . H . (1983) . Simple method for demonstration of differential colony morphology of plasmid-associated virulent clones of Yersinia enterocoli-
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Immun . 57, 121-31 . PIERSON, D . E . & FALKOW, S . (1990) . Nonpathogenic isolates of Yersinia enterocolitica do not contain functional inv in homologous sequences . Infect . Immun . 58, 1059-64 . PORTNOY, D . A ., MOSELEY, S . L . & FAI .KOW, S . (1981) . Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis . Infect. Immun . 31,
775-82 . PRPiC, J . K ., RoBINs- BROWNE, R. M . & DAVEY, R . B . (1983) . Differentiation between virulent and avirulent Yersinia enterocolitica isolates by using Congo red agar . J. clin. Microbiol. 18, 486-90 . RICHARDSON, C ., HEBERT, C . N . & TERLECKI, S . (1976) . Estimation of the developmental age of the ovine fetus and lamb . Vet. Rec. 99, 22-6 . RosQvisi', R ., FORSBERG, A., RUMPILAINEN, M., BERGMAN, T . & WOLF-WATZ, H . (1990) . The cytotoxic protein YopE of Yersinia obstructs the primary host defence . Molex. Microbiol. 4, 657-67 . S( .HIF:Nr NN, D . A . (1979) . Synthesis of a selective agar medium for Yersinia enterocolitica . Can . f. Microbiol. 25, 1298-304 . SCHIEMANN, D . A ., DEVENISH, J . A. & ToMA, S . (1981) . Characteristics of virulence in human isolates of Yersinia enterocolitica . Infect. Immun . 32, 400-3 . SKURNIK, M . (1985) . Expression of antigens encoded by the virulence plasmid of Yersinia enterocolitica under different growth conditions. Infect . Immun . 47, 183-90 . SOLTFSZ, L . V., SCHAI .ÉN, C . & MARSH, P . A . (1980) . An effective selective medium for Yersinia enterocolitica containing sodium oxalate . Acta path . microbiol. scand. B88,1 1-16 . STOMP, J . T ., McEWEN, A . D ., WATT, J . A . A . & NISBET, D . I . (1950) . Enzootic abortion in ewes . Tranmission of the disease . Vet. Rec. 62, 251-4. WINBLAU, S . (1978) . Yersinia enterocolitica . In Methods in Microbiology, eds T . Bergan &J . R . Norris, Vol . 12, pp . 37-50 . London : Academic Press . (Accepledforpublieation 17 Fe&
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