PERINATAL LAMB MORTALITY IN WESTERN AUSTRALIA 6. Listeric Infection S . M. DENNIS,* B.V.Sc., Ph.D., F.R.C.V.S., M.A.C.V.Sc. Department of Agriculture, Jarrah Road, Perth, Western Australia Introduction

Results

Listeric abortion in sheep was first reported in Listeriosis was diagnosed on 14 occasions and Australia in New South Wales by Hindmarsh and 4 of these were associated with other pathogens: Blumer (1932) and has since been diagnosed in Cumpylobacter fetus subsp. intestinalis (l), Bruall the States of Australia: New South Wales cellu ovis (l), Tomplasma gondii (l), and C . fetus (Charles 1950; Diplock 1957; Hughes et a1 1964; and Br. ovis (1). Listeriosis was diagnosed on 12 Kater 1965; Haughey et al 1967; Plant et a1 farms in 1963 and suspected on another 9. It was 1972), Victoria (Gorrie 1962; McDonald 1967; diagnosed once in 1964 and in 1965. All outBroadbent 1972), Queensland (Moule 1954), breaks were characterised by abortion, perinatal South Australia (Moule 1963), Tasmania (Mun- losses, and lack of clinical signs in pregnant or day et a1 1966), and Western Australia (Dennis postparturient ewes. Abortions usually occurred 2 1963). Apart from the recent Victorian reports weeks before the lambing season and the estifew details of foetal and placental findings were mated abortion rate varied from 5 to 22%. No recorded (McDonald 1967; Broadbent 1972). The cases of neuro-listeriosis were observed. epizootiology of the outbreaks was unknown. Epizootiology This paper records the findings in outbreaks of The outbreaks were scattered sporadically over listeric abortion in Western Australia during an an area of approximately 200 miles south of investigation into the causes of perinatal lamb Perth, 60 miles east, and 80 miles south-east. The mortality during 1963-65. disease was originally diagnosed in a recently developed small War Service Settlement area in Materials and Methods General procedures followed in this investigation were the Denbarker district and subsequent investigadescribed previously (Dennis 1974). tions revealed a number of farms to be infected. Abomasal contents and infected cotyledons from Each farm consisted of 1,000-1,150 acres of suspected cases of listeric abortion were inoculated onto which approximately two-thirds were cleared and Difco phenylethanol blood agar (PEA). Initially, the had subterranean clover pastures. The area had liver, heart blood and brain were cultured but this was discontinued when it was found that Listeria monocyto- been developed for 1 to 4 years and the sheep had been purchased from many parts of Western genes was readily recovered from abomasal contents and placenta. The meconium was cultured when it was yellow- Australia in small lots of 50-150 which made orange and mucoid. The plates were incubated in an tracing the origin of the disease almost impossible. atmosphere of 10% Coz in air for 48 hours and if no Almost all these new flocks were affected with growth was evident, they were discarded. clover disease. Serums were tested for L . monocytogenes antibodies Six farms reported abortions in 1963 in the by a macroscopic, stained-antigen slide agglutination test. The antigen was a suspension of L . inonocyiogenes Denbarker district. They were found to be inserotype 5 cells in buffered formal saline stained with fected with listeriosis; 2 were mixed infections. haematoxylin. One drop of serum was added to a drop of antigen, mixed, allowed to stand and read at 5-10 Later investigations revealed that another 3 properties had abortions, and serological examminutes. ination of a random group of 10 ewes revealed a With the cooperation of the Agricultural Protection Board of Western Australia, a limited survey was under- number of positive reactions to the stained-antitaken for evidence of listeriosis in native fauna in the gen slide agglutination test for L. monucytogenes. southern areas and in particular, the Denbarker district. A small serological survey of 10 ewes selected at Vermin control officers were requested to randomly kill a number of rabbits and submit sections in 10% buffered random from a number of other farms in this neutral formalin from any livers with scattered small isolated compact area of 25-30 farms gave a numwhite foci. In addition, livers, kidney and lung speci- ber of positive reactions to the stained-antigen mens were submitted from foxes, rabbits, hawks, mice test for listeriosis. and kangaroos caught in the Denbarker area. There was circumstantial evidence that preda'Present Address: College of Veterinary Medicine, Kansas State tors - foxes (Vulpes vulpes) and/or crows (CorUniversity, Manhattan, Kansas, United States of America, 66502. Australian Veterinary Journal, Vol. 51, February, 1975

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vus spp) - may have spread infection to at least 2 farms. The first farm on which genital listeriosis was diagnosed in 1963 was triangular in shape and was surrounded by a road and a double fence on 2 sides, and a thick forest on the third. One neighbouring farm lambed early and had no trouble; 2 others had abortions but only in paddocks adjacent to the first affected farm. Another farm situated a mile away from the first farm was the second known to be involved. A farm between these 2 infected farms became infected towards the end of lambing. The only known contact between these farms was predators. The outbreaks in the Denbarker district occurred during winter and spring lambing of 1963. No abortions were reported from any of these farms during the succeeding two years. In other districts listeric abortions occurred in 2 closed flocks following outbreaks earlier on neighbouring farms. There was no direct animal contact. An infected twin was born dead and L. monocytogenes was recovered in pure culture from the liver and meconium; the other twin survived. Listeric abortion has not been diagnosed in Western Australia since 1965 (M. E. Nairn, personal communication 1972).

Isolates L. monocytogenes was readily recovered in pure culture from all organs and sites cultured: cotyledons, abomasal contents, liver, pleural fluid, lung, heart blood, brain, meconium and fluid in the scrota1 sac. Listeria were numerous in smears from cotyledons, chorioallantois, abomasal contents, liver and meconium. All the Listeria isolates were markedly haemolytic. At 24 hours the colonies were round, slightly convex, translucent, 0.5 to 1 mm in diameter with a narrow zone of clear haemolysis surrounded by a larger incomplete zone of haemolysis. Small Listeria colonies were readily recognizable by the two distinctive zones of haemolysis. After 3-4 days, the colonies were 1-2 mm in diameter, surrounded by a clear zone of haemolysis up to 6 mm in diameter together with a further zone of incomplete haemolysis, making a total haemolytic area of 12-13 mm in diameter. The incomplete zone cleared and became complete. All the isolates were confirmed and typed L. rnonocytogenes serotype 5 Ivanov by Professor H. Seeliger, Institute of Hygiene and Microbiology, Wurzburg, Germany. L. monocytogenes serotype 5 had to be distinguished from the corynebacteria and streptococci. The important characteristics were motility 76

TABLE 1 Autopsy Findings in 40 Lambs Infected wirh Listeria

monocytogenes Serotype 5 in Western Australia Finding

% Infected

Lambs Ante-parturient deaths Parturient deaths Post-parturient deaths Subcutaneous oedema Excess fluid, serous cavities Focal liver lesions Hepatomegaly Necrotic foci, lungs Abomasal erosions Enlarged mesenteric lymph nodes Yellow-orenge mucoid meconium

20 4 16

50 10 40

62.5 77.5 74.5 17.5 12.5 32.5 37.5 37.5

at 25"C, acid but no gas from aesculin and salicin, and catalase positive. In addition, serotype 5 strains did not reduce nitrates; were Methyl Red and Voges-Proskauer positive, indole negative; produced acid but not gas from maltose, glucose, laevulose and trehalose; and did not grow on MacConkey's agar. Pathological Changes Listeriosis was diagnosed in 40 lamb carcases and from specimens submitted from another 5 lambs (Table 1). Three-quarters of the infected lambs had gross lesions and 50% had varying degrees of autolysis. Typically, lambs with congenital listeriosis had subcutaneous oedema, hydrothorax and hydroperitoneum, distended abdomen, enlarged pale bronze-red friable livers with small necrotic foci, small abomasal erosions, increased yellow-orange mucoid meconium and enlarged mesenteric lymph nodes. The fluid in the subcutaneous tissues and serous cavities varied from clear yellow to pink to dark blood-tinged depending upon the degree of autolysis. Numerous small white to yellow foci, 0.5 to 2 mm in diameter, were found in the liver of the majority of infected lambs (74.5%). The foci were circumscribed, not raised above the surface, and were mainly scattered throughout the right lobe and caudate lobe; only rarely was the left lobe involved. Microscopically, these necrotic foci contained numerous Listeria; neutrophils and macrophages were present or absent. The abomasal contents were mucoid and varied from yellow to reddish-brown. The abomasal mucosa of a number of lambs had scattered small greyish-white erosions, 1-3 mm in diameter. Microscopically, the erosions rarely extended beyond the lamina propria, contained numerous Listeria and were infiltrated with mononuclear Australian Veterinary Journal, Vol. 51, February, 1975

cells and neutrophils (Figure 1). The underlying submucosa was hyperaemic and oedematous. The caecum and large intestines of a third of the listeria-infected lambs (37.5%) were enlarged and contained yellow-orange mucoid contents or meconium; the mesenteric lymph nodes were greatly enlarged and oedematous. Small necrotic foci were found in the lungs of 5 lambs (12.5%), and microscopically they were similar to the hepatic foci. One lamb had severe bronchopneumonia. Necrotic foci were observed in the myocardium of one lamb, and in the ski^ of another, especially over the scrotum. The brains were not examined. Placental Lesions Placental lesions varied from focal oedematous thickening of the chorio-allantois to widespread oedema to greyish-white leathery pericotyledonary areas 10-12 cm in diameter. The oedematous fluid was yellowish to reddish-brown. Most cotyledons were affected and were paler in color, being a mottled orange to a pale greyish-pink with areas of necrosis. Many cotyledons were convex and firm, and others were flat. Microscopically, there was extensive oedema and neutrophilic infiltration of the chorio-allantois, and vasculitis (Figure 2). There were necrosis, neutrophilic accumulations and calcification in the villous mesenchyme, and numerous Listeria in the chorionic epithelium. Fauna Survey Sixty-one specimens from 7 species of native fauna were submitted from 20 farms, mainly from the Denbarker district. Lisreria was not detected culturally or histologically in specimens from 35 rabbits, 8 mice, 1 rat, 4 hawks, 5 kangaroos, 5 crows and 3 foxes.

Figure 1. Photomicrograph of a small abomasal erosion i n an aborted lamb due to 1. monocytogenes infection. The lesion is infiltrated with numerous neutrophils and mononuclear cells. 100 x.

Australian Veterinary Journal, Vol. 51, February, 1975

Discussion

The interesting epizootiological aspect of ovine listeriosis in Western Australia was the number of reported outbreaks of the genital type over a wide area of the southern agricultural area in 1963, only one outbreak for both 1964 and 1965 and none since. The diagnosis in 1963 was the first time listeriosis had been found in domestic animals or man in the State. The apparent disappearance of the disease within the State is puzzling. Epizootiology of listeriosis is poorly understood. L . monocytogenes has a widespread distribution in nature, has remarkable resistance to environmental factors and may survive in soil and faeces for up to 2 years (Gray and Killinger 1966). Evidence is accumulating that healthy carriers exist in animals and man and play a predominant role in perpetuation and transmission of Iisteriosis. No explanation is found in the majority of outbreaks for the sudden occurrence of genital listeriosis in a flock (Young 1968). McDonald’s findings (1 967) indicated that lambs could become healthy carriers and that venereal spread was a possibility. Gorrie (1962) also envisaged the possibility of venereal transmission. Epizootiologically L. rnonocytogenes in Western Australia poses interesting questions. Where did it come from suddenly in 1963 and then apparently disappear? Why the high incidence in a small soldier settlement area in 1963? What is the mode of transmission between flocks and farms? Outbreaks are known to follow introduction of purchased animals (Gray and Killinger

Figure 2. Pt otomicrograph of the chorio-allantois from an aborted lamb due t o L. monocytogenes. Note the numerous neutrophils (l), vasculitis (2), and extensive oedema (3).

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1966). The source of the outbreaks and the reason strains reported by Ivanov (1957) were isolated for the sudden occurrence of listeriosis for the from aborted lambs (57 strains) and a neonatal first time in Western Australia in 1963 are un- lamb with enteritis (1 strain), These strains were known. A possible explanation was that with the characterised by pronounced beta-haemolysis, less rapid development of virgin land and subsequent biochemical activity, lower pathogenicity and a demand for more sheep, thousands of sheep were predilection for the gravid uterus. Serotype 5 had imported annually from the other States, particu- only been recorded from Bulgaria until the Auslarly South Australia, and that infection was tralasian isolations and only from sheep until introduced via carrier animals. The apparent dis- recovered from an aborted calf in New Zealand. appearance of the disease could be explained by As more investigations into perinatal lamb morits becoming subclinical in the affected flocks, tality are carried out in Australia the findings thereby providing a potential source €or future suggest that the incidence of listeriosis is higher outbreaks of listeric abortion if some of these than previously thought. It would be interesting sheep were introduced into susceptible flocks. to learn the incidence and distribution of L. None of the 7 species of native fauna from monocytogenes sterotype 5 in Australia. the infected Denbarker district had evidence of listeriosis and they were negative for Listeria. Summary McDonald (1967) also found crows (Corvus spp.) Listeric abortion was diagnosed in sheep on 14 and an ibis caught in the lambing paddock nega- occasions between 1963-65, and 4 of these were tive for Listeria. associated with other major abortion pathogens. Small necrotic foci in the liver were a constant Except for infection in one district, the outbreaks feature (74.5%) in the Western Australian out- were scattered sporadically over the southern breaks and were sufficiently characteristic to be agricultural area. The diagnosis in 1963 was the suggestive of listeric infection. These foci were not first time listeriosis had been found in animals or obscured by autolytic changes and were still obvi- man in Western Australia. ous. McDonald (1967) also found these lesions in L. monocytogenes was readily recovered in the majority of the lambs examined (75.6%). pure culture from all organs and sites cultured. Broadbent (1972) found that gross lesions were All the strains were serotype 5 Ivanov, with a often absent in infected lambs and reported that distinctive double zone of haemolysis. The timeonly 13% of 60 specimens from which L. mono- of-death of the 40 infected lambs was: antecytogenes was isolated had gross lesions. The parturient death 50%, parturient death 10% and yellow-orange meconium of mucoid consistency post-parturient death 40%. Three-quarters of the and the small abomasal erosions were found to lambs had gross lesions, the most consistent be useful gross guides; these changes were not being small necrotic foci in the liver. Typically, observed with the other abortion diseases. These lambs with congenital listeriosis had subcutaneous 2 findings together with small hepatic foci were oedema, hydrothorax, hydroperitoneum, distended almost pathognomonic for listeric abortion. The abdomen, enlarged pale bronze-red friable livers mucoid meconium contained a very high concen- with small necrotic foci, small abomasal erosions, increased yellow-orange mucoid meconium and tration of Listeria. enlarged mesenteric lymph nodes. The finding of All the Western Australian Listeriu isolates small yellowish, necrotic foci in the liver, small were serotype 5 Ivanov with a distinctive, wide abomasal erosions and yellow-orange meconium double zone of haemolysis. This haemolytic in an aborted lamb was considered to be almost phenomenon was useful for detecting suspected pathognomonic for listeriosis. colonies of L. monocytogenes. Characterisation of The origin of these outbreaks was unknown. 4 of the Western Australian strains has recently been reported (Cooper et a1 1973). Munday et a1 No evidence of Listeria was found in 61 speci(1966) commented that their ovine isolate was mens from 7 species of native fauna caught in the strongly haemolytic and that it differed in this infected areas. Epizootiological observations are aspect from their 4 bovine isolates. The Victorian reported. No cases of neuro-listeriosis were isolate reported by McDonald (1967) had a nar- observed. Acknowledgments row zone of beta haemolysis. Twenty-three of 25 later Victorian isolates were serotype 5 (BroadI wish to thank Mr D. Gooding, B.Sc. (Agric.), bent 1972). Hunter (1973) reported isolating 5 Mr J. Long, M.D.A. and the officers of the Agristrains of serotype 5 from aborted foetuses in cultural Protection Board of Western Australia New Zealand (4 ovine and 1 bovine). Serotype 5 for their cooperation with this investigation. 78

Australian Veterinary Journal, Vol. 5 1, February, 1975

References Broadbent, D. W. (1972)-Aust. vet. J. 48: 391. Insp. of Stk., N.S.W. Year Charles, G. (1950)-Inst. Book, p. 39. Cooper, R. F., Dennis, S. M. and McMahon, K. J. (1973)-Am. J. vet. Res. 34: 975. Dennis, S. M. (1963)-J. Dept Agric., West. Aust. 4: 461. Dennis, S. M. (1974)-Aust. vet. 1. In press. Diplock, P. T. (1957)-Aust. vet. 1. 33: 68. Gorrie, C. I. R. (1962)-Aust. vet. J. 38: 138. Gray, M. L. and Killinger, A. H. (1966)-BacterioZ. Rev. 30: 309. Haughey, K. G., Hughes, K. L. and HartIey, W. J . (1967)-Aust. vet. J. 43: 413. Hindmarsh, W. L. and Blumer, C. C. (1932)-Aust. vet. 1. 8: 149.

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Hughes, K. L., Hartley, W. J., Haughey, K. G. and McFarlane, D. (1964)-Proc. Aust. SOC. Anim. Prod. 5: 92. Hunter, R. (1973)-Med. lab. Tech. 30: 51. Ivanov, I. (1957)-Bull. Off. lnt. Epiz. 48: 571. Kater, J. C. (1965)-Vet. Notes, Dept Agric., N.S.W. 1: 4. McDonald, J . w. (1967)-A ~~t. vet. 1. 43: 564. Moule, G . R. (1954)-Aust. vet. J. 30: 153. Moule, G. R. (1963)-Fourth Meeting F.A.O. Expert Panel on Livestock Infertility, p 43. Munday, B. L., Ryan, F. B., King, S. J. and Corbould, A. (1966)-Ausf. vet. J . 42: 189. Plant, J. W., Beh, K. J. and Acland, H. M. (1972)Aud. vet. 1. 38: 558. Young, s. (1968)-"Abortion Diseases Of Livestock". Ed. Faulkner, L. C. Charles C. Thomas, Springfield, Illinois p 95. (Received for publication 1 November 1971)

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Perinatal lamb mortality in Western Australia. 6. Listeric infection.

PERINATAL LAMB MORTALITY IN WESTERN AUSTRALIA 6. Listeric Infection S . M. DENNIS,* B.V.Sc., Ph.D., F.R.C.V.S., M.A.C.V.Sc. Department of Agriculture,...
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