Yersiniosis in sheep due to Yersinia enterocolitica

Br. vet.J. (1994). 150, 473

YERSINIOSIS IN SHEEP DUE T O YERSIN/A ENTEROCOLITICA

H. BIN-KUN*, X. DE-SHENG*, O. HONG-BI*, Z. SHI-XIANGt and K.J. SLEE~; Shaoyang A nti-epizootic Station of Domestic Animals*, Hunan 422004, China; Shaoyang Branch of Hunan Univt~sitfr, Shaoyang, Hunan 422004, China; Regional Veterinary Laboratory$, Department of Agriculture, Bairnsdale 3875, Victoria, Australia

SUMMARY AJ1 outbreak of acute yersiniosis o c c u r r e d amongst sheep transported fi'om Inner Mongolia to H u n a n Province in southern China. Morbidity was 41% and mortality o f affected sheep was 34%. Eleven apparently identical isolates of Yersinia enterocolitica were obtained from liver, lung and skin lesions of affected sheep and all were biotype 3. Isolates could not be serotyped with available antisera. Lesions were observed in the skin, intestine, liver and hmgs. T h e causative bacterium is apparently different from isolates previously identified as causing disease in sheep and goats in Australia, New Zealand, Norway and Great Britain. T h e source of infection could not be determined. Kexs~'okl)s: Yersiniosis; Yersinia enterocolitica; sheep; China.

INTRODUCTION A n u m b e r of bioserotypes of Yersinia enterocolitica are known e n t e r o p a t h o g e n s of h u m a n beings and animals (Swaminathan et aL, 1982; Robins-Browne, 1989). Y. enterocolitica was described as a cause of enteric disease in goats by Krogstad et al. (1972) in Norway. More recently, Y. enterocolitica has been shown to cause enteritis in sheep (McSporran et al., 1984; Slee & Button, 1990) and goats (Buddle et aL, 1988; Slee & Button, 1990) in Australia and New Zealand and abortion in sheep (Corbell et al., 1990) in Great Britain. Krogstand et al. (1972) identified their goat isolates as serotype 0 2 but did not biotype strains. Buddle et al. (1988) identified their goat isolates as biotype 5 but Correspondence to: Zhang Shi-xiang, Shaoyang Branch of Hunan University, Shaoyang, Hunan 422004, China. (11107/1935/94/050473-00/$08.00/0

© 1994 Bailli6reTindall

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did not serotype isolates. Slee and Button (1990) identified their sheep and goat isolates as biotype 5, serogroup 02,3. We r e p o r t here on aspects of an unusual outbreak o f generalized Y. enterocolitica infection in sheep shipped from Inner Mongolia to H u n a n Province in southern China.

MATERIALS A N D M E T H O D S

Flock histoTy In J u n e 1990, 722 sheep, including 300 adult ewes, 393 lambs and 29 rams were transported from Inner Mongolia to H u n a n Province in southern China. Transport was by train and took several days. T h e sheep were in p o o r condition prior to transport. Two days after arriving in H u n a n Province, occasional sheep were showing signs o f illness. Deaths started to occur from 7 days after arrival. A brief clinical examination was carried out on 334 sheep and a m o r e complete clinical examination on a f u r t h e r 18. Diseased sheep were submitted to the laboratory for examination.

Pathology Gross post-mortem examinations were p e r f o r m e d on 30 sheep. Eighteen o f these sheep had died o f the disease and 12 were killed in extremis. Samples were collected for bacteria and virus isolation and for histopathology.

Microbiology Liver, lung and skill lesions were cultured on rabbit blood agar and MacConkey agar and inoculated into Martin's serum broth and anaerobic meat-liver broth. Faecal and intestinal samples were not cultured. Culture plates were incubated at 37°C in both aerobic and anaerobic atmospheres for 2~ ~8 h. Suspect Yersinia sp. colonies were identified by the following: Gram reaction; motility at 22 and 37°C; fermentation of glucose, maltose, sucrose, xylose, fructose, inulin, mannitol, sorbitol, lactose, r h a m n o s e and raffinose; p r o d u c t i o n o f catalase, ornithine decarboxylase, urease, phenylalanine deaminase, lysine decarboxylase, indole, acetyi methyl carbinol and hydrogen sulphide; liquefaction o f gelatin; methyl red testing and citrate utilization. Biochemical testing was p e r f o r m e d at both 22 and 37°C as appropriate. Antimicrobial sensitivity testing was p e r f o r m e d on one isolate of Y. enterocolitica using the disc diffusion m e t h o d on rabbit blood agar plates incubated at 37°C.

Virology Necrotic tissues from the ears and lips, and p u r u l e n t material from lung lesions were e x a m i n e d for viruses by transmission electron microscopy and by culture and passage in 8 and 12 day e m b r y o n a t e d chicken eggs.

Toxin detection O n e isolate of Y. enterocolitica was tested for tile ability to p r o d u c e thermostable endotoxin. T h e isolate was grown on rabbit blood agar at 22°C for 48 h. Growth

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was suspended in sterile saline, heated at 100°C for 1 h and tested for sterility on rabbit blood agar. T h e sterile suspension was injected intravenously into two rabbits and two goats. Rabbits received 0.5 ml o f suspension while goats received 1 ml.

Serology Serum samples from the five healthy, 30 diseased and 10 recovered sheep were tested for the presence of antibodies reacting with field isolates of Y. enterocolitica using a saline tube-agglutination technique.

Experimental infections Attempts were made to r e p r o d u c e disease in four 3-month-old goats of a native Chinese breed. Y. enterocolitica was grown in Martin's broth incubated at 22°C. A suspension containing 2 × lff' ml -~ bacteria was p r e p a r e d and the goats were inoculated into the peritoneal cavity with 1 ml o f this material. Two healthy goats were used as control animals. They received 1 ml sterile saline into the peritoneal cavity.

Vaccination A vaccine containing 2 × 10:' ml -~ Y. enterocolitica cells was p r e p a r e d by inactivation o f a culture with 0.4% formaldehyde. Two healthy 3-month-old goats were i m m u n i z e d with a single dose of 2 ml vaccine administered i.m. T h r e e weeks later these goats and two healthy control animals were challenged by injecting 2 x 10t' ml -~ Y. enterocolitica i.p. This challenge dose comprised 11 field isolates o f

Y. enterocolitica. 7)'eatment Fifty-one severely affected sheep were initially treated with penicillin G (1 million units) i.m. T r e a t m e n t was later changed to a combination of chloramphenicol (20-30 m g k g -I) and streptomycin (1 g) for severely affected animals while less affected animals were given streptomycin (0.5 million units) and sulphamethoxazole (0.5 g) orally.

RESULTS

Clinical findings In all, 295 sheep were ,affected (41%) and 101 (34%) of affected animals died. Lambs were most often affected; 246 (63%) showed symptoms and 81 (33%) of these died. Sick sheep a p p e a r e d depressed and t e n d e d to stand alone with a r c h e d backs. T h e y had p o o r coats, cough and muco-serous nasal discharges, tachypnoea and a fever of 40.1-41.5°C. T h e majority o f affected sheep had pustules on the skin at the angles o f the mouth, the base o f the ears and on the dorsum o f the nose but not on the muzzle. R e d d e n i n g and scab and ulcer formation o f the skin was observed in some sheep. Occasional sheep had diarrhoea. In many instances the condition o f affected sheep d e t e r i o r a t e d and death o c c u r r e d 5-7 days after the a p p e a r a n c e o f disease signs.

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Pathology Affected sheep were emaciated. T h e r e was usually an increased volume of cloudy fluid in the thoracic cavitT. T h e lungs c o n t a i n e d areas of r e d d e n i n g , necrosis and abscessation, and were covered with thick fibrinous exudates. T h e heart showed petechial h a e m o r r h a g e s and o e d e m a u n d e r tile epicardium. Some bleeding into tile intestinal tract was evident. Histologically, the skin showed necrosis, neutrophil infiltration a r o u n d blood vessels, nunlerous necrotic inflammatory cells and cocco-bacilli. Lung tissues were congested and there was thickening of alveolar septa and infiltration o f alveoli with o e d e m a fluid, lymphocytes and macrophages. Some bronchioles and alveoli contained neutrophils and necrotic cells, and walls of bronchi were congested and infiltrated with neutrophils and macrophages. T h e r e was o e d e m a o f the epicardium and myocardium with sequestration o f neutrophils ill blood vessels. T h e liver was congested and bepatocytes were swollen. Portal lymph nodes were o e d e m a t o u s with some increase in neutrophils and plasma cells. Intestines were not examined.

Microbiology Eleven isolates of Y. enterocolitica were recovered from lesions on tile lips, ears, lungs and livers o f the lambs and ewes examined. No o t h e r significant bacterial pathogens were isolated. MI isolates had identical cultural and biochemical features and belonged to biotype 3 (see Table I). Tile 11 isolates of Y. enterocolilica could not be serowped with O antisernms 1 to 33, 35 to 44 and 46 to 57 (Dr Chen Jian, personal c o m m u n i c a t i o n ) . T h e one isolate tested was sensitive to streptomycin and gentamicin, moderately sensitive to sulphonamides, kanamycin and chloranlphenicol, slightly sensitive to midecamycin, cefazolin and tetracycline but resistant to penicillin G, leucomycin tartrate, t r i m e t h o p r i m and filrazolidone.

Virology Virus particles or inchtsion bodies were not d e m o n s t r a t e d by electron microscopy of lips, skin and lung. Viruses could not be isolated fi'om these tissues on emb~Tonated eggs.

Toxin detection Twenty-one to 24 h after inoculation o f killed Y. enterocolitica, rabbits and goats became progressively more depressed with anorexia, tachypuoea, urinal T incontin e n c e and heat tilt. Coma and finally death o c c u r r e d 98-114 h after inoculation. Post-mortem examination showed petechiae in tile meninges and urina~-y tract, and mineralization in the myocardium. No o t h e r lesions were seen and no bacteria were isolated fi'om the lesions.

Serolo~l Five adult sheep that r e m a i n e d normal during tile epizootic did not have antibodies reacting with Y. enl~'ocolilica. Thirty sick sheep had antibody titres ranging from l:10 to 1:80. T e n sheep that had recovered fi-om infection had antibody titres of 1 : 6 4 0 .

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Table I B i o c h e m i c a l reactions o f Y. enterocolitica isolates f r o m s h e e p

Test

Reaorion

Glucose Maltose

+

Sttcrose Xylose

+ +

Fructose Inulin Mannitol Sorbitol I.actose Rhamnose Raffinose

+ + + +

Catalase

+

Ornithine decarboxylase Phenylalanine deaminase I.ysine decarboxylase Nitrate reduction Methyl red test Hydrogen sulphide production Acetoin production (VP) U tease Aesculin hydrolysis Gelatin liquefaction Indole Citrate utilization

+

+

+

+ + +

Experimental infections Four goats inoculated i.p. with Y. enterocolitica b e c a m e sick and died 56-68 h after challenge. T h e major clinical signs observed post-challenge were lack of appetite, fever of 41.3-41.8°C and tachypnoea. Skin lesions did not develop. On post-mortem examination, o e d e m a o f epicardium and lymph nodes, hyperaemia, consolidation and abscessation of lungs and catarrhal inflammation o f intestine were ohserved. T h e two control goats remained normal for 15 clays. Vaccination Two goats challenged by inoculation with a live suspension 21 days after they had been vaccinated with a field isolate of 1: enterocolitica remained healthy while the two non-vaccinated control animals died within 78 h. Post-mortem examinations of the control goats showed similar lesions to o t h e r naturally and experimentally infected animals, and the organisms used in the challenge were re-isolated from liver and lung. 7"r:atmt Severely affected sheep treated with cllloramphenicol and streptomycin and less severely affected animals given streptomycin and stdphamethoxazole all recovered. Severely affected sheep treated with penicillin G died.

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DISCUSSION

The epizootic described in this report was apparently caused by a single strain of Y. enterocolitica belonging to biotype 3 but of unknown serotype. ): enterocolitica was consistently isolated fl'om affected animals, and no other viral or bacterial pathogens were identified. Antibodies to an isolate were present in dying and recovered animals but not uninfected animals. Challenge of healthy goats with isolates from affected sheep caused death with many similarities to the natural disease. Treatment trials showed recovery in animals given antibiotics to which the bacterium was sensitive, but no recovel-y in animals given antibiotics to which the bacterium w a s s h o w n t o be resistant. This disease outbreak was unusual because of its high morhidity and mortality and because of the generalized nature of infection, involving as it did the gastrointestinal tract, hmgs and skin. Previous reports of yersiniosis in sheep (McSporran et al., 1984; Slee & Button, 1990) and goats (I,h'ogstad et al., 1972; Buddle et al., 1988; Slee & Button, 1990) describe sporadically occurring disease with enteritis being the major clinical sign. Although skin infection with .1". ente)vcolitica has apparently not been previously described, conjunctivitis associated with Y. pseudotuberculosis infection has been noted previously in goats in Australia (Slee, tmpublished information). Toxin testing showed that the isolates of Y. enterocolitica probably produced thermostable toxins capable of producing bleeding from capillaries, myocardial degeneration and death. The disease outbreak described here occurred in animals that were stressed by poor feeding, long-distance transport and a sudden change in climatic conditions. All of these factors have been described previously as predisposing to epizootics of yersiniosis in a range of livestock species (McSporran et aL, 1984; Buddle et aL, 1988; Slee et aL, 1988;Jerrett et al., 1990). The lower incidence of disease in ewes and rams probably reflects previous exposure to K enterocolitica infection and the presence of protective immunity in many of these older animals (Slee & Skilbeck, 1992) although antibodies were not detected in the five healthy adult sheep tested. Diseased sheep treated with appropriate antibiotics were shown to recover and vaccination appeared to be effective in preventing the development of disease in experimentally infected goats, indicating that both prophylaxis and treatment are feasible.

ACKNOWLEDGEMENTS

The authors acknowledge the assistance of Professor Peng Lon-xiang of Hunan Medical University for examining samples for viruses, Professor Chen Ke-yi of Hunan Agricultural College for pathological examinations and Dr Chen Jian of Hunan Anti-epidemic Station for attempting the serotyping of Y. enterocolitica isolates. Dr Peter Mitchell of the Regional Veterinary Laboratory, Bairnsdale also assisted by reviewing drafts of this paper.

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(Accepledfor publication 23 December1993)