Toxoplasma gondii (T. gondii) is a common protozoan parasite of endotherms to which sheep are highly susceptible (Dubey, 2009; Stelzer et al., 2019). Felids are the definitive host, whilst all other endotherms are potential intermediate hosts (Frenkel et al., 1970). Prevalence estimates in sheep flocks globally vary from 3% to 96% with risk factors including climate, cat activity and feed and water source (Afonso et al., 2013; Dubey, 2009). Toxoplasmosis is both an important cause of reproductive loss in ewes and a significant zoonosis (Dubey, 2009; 2010). The consequence of infection is greatest when primary infection occurs during pregnancy with the outcome dependent on ewe gestation at infection. Early to mid-gestation infection results in foetal death and resorption, mummification and/or abortion (Benavides et al., 2017). The zoonotic consequences are greatest if primary infection occurs during pregnancy and in the immunocompromised (Montoya & Liesenfield, 2004). Toxoplasmosis of sheep is of zoonotic importance because undercooked sheep meat is a potential route of human exposure and Australian sheep meat is commonly contaminated with T. gondii (Dawson et al., 2020; Opsteegh et al., 2016).
The classic case history and clinical lesions consistent with ovine toxoplasmosis together with the magnitude of the outbreak make this case a salient reminder of the potential importance of T. gondii as an agent of ovine abortion.
The owner of a small flock of Wiltipoll sheep on the outskirts of Kempsey contacted the district veterinarian in early July 2020 after five out of six ewes aborted. Two of the five ewes that aborted were maidens, whilst three were on their second pregnancy having lambed successfully on the same property in 2019. The only ewe that did not abort was six years old, and successfully carried twins to term.
The sheep were grazing rye grass, sown into kikuyu pasture with water supplied in troughs from the river. The region had been very dry up until early 2020, and the sheep had been supplementary fed a commercial grain mix and hay up until the ram was put in on 10 February 2020. The first ewe aborted a single foetus on 26 May 2020, while three subsequent ewes aborted twins on 17, 21 and 30 June 2020.
The owner advised that there was a small paddock on the property where he had found several dead birds, and there had been some issues with feral cats in this area. The ewes had grazed this paddock when pregnant. He subsequently trapped a feral cat a few days later.
Twin foetuses were presented for autopsy on 30 June 2020. One foetus had a crown rump length of 15cm and was mummified, with no fluid present. The other had a crown rump length of 30cm, with marked subcutaneous serosanguinous, gelatinous fluid. There were numerous small focal white plaques on the placenta between cotyledons, though cotyledons themselves appeared normal.
The pericardial fluid tested negative for T. gondii (latex agglutination test) and Brucella ovis (compliment fixation test). Pestivirus testing (antigen capture ELISA) was also negative. Culture of abomasum content for Campylobacter species was negative, and routine culture of liver and lung produced no significant growth.
Histopathology findings revealed a non-suppurative, mild, diffuse chronic placentitis with mineralization. The brain was found to have a necrotising, moderate, multifocal chronic encephalitis with mineralization and gliosis. Histopathology of the heart revealed a non-suppurative, mild, multifocal chronic epicarditis with interstitial oedema. These changes were most suggestive of toxoplasmosis, though other agents including Neospora, or a bacterial infection could not be excluded.
Two of the ewes that aborted were blood tested on 13 July 2020. The toxoplasma latex agglutination test returned high positive results, 3072 and 1536 international units. The ram tested negative for ovine brucellosis.
Cats recently infected with T. gondii shed millions of environmentally tolerant oocysts in their faeces, contaminating terrestrial and aquatic environments (Dabritz et al., 2007; Frenkel et al., 1970; Hutchinson, 1965). On re-exposure, shedding continues for years (Zulpo et al., 2018). The ingestion of feed (including pasture, hay and grain) or water contaminated with oocysts is the most common route of infection in sheep (Munday, 1972). A dose of just 100-200 oocysts is sufficient to cause abortion in naïve ewes (Kirkbride et al., 1992; McColgan et al., 1988; Plant et al., 1974). Ingested oocysts excyst and the sporozoites invade host cells to transform into rapidly replicating tachyzoites. Parasitised cells rupture, releasing more tachyzoites that invade more host cells and so on, stimulating a strong inflammatory response (Dubey et al., 1998). The host's immune response stimulates tachyzoites to become bradyzoites that multiply slowly in brain and muscle tissue cysts (Montoya & Liesenfield, 2004). These cysts persist lifelong and are the infective stage for potential intermediate or definitive hosts that ingest infected tissue (Montoya & Liesenfeld, 2004). Bradyzoites may transform back into tachyzoites in immunocompromised individuals, resulting in recrudescence of infection.
Pyrexia is associated with early infection in sheep and may be detected 3-6 days after infection for up to 10 days (Dubey, 2009; Stelzer et al., 2019). There are few other clinical signs in non-pregnant ewes. The suppressed inflammatory cascade at the maternal-foetal interface provides a favourable environment for tachyzoite replication, which causes non-purulent inflammation and necrosis in the placenta and foetus (Innes et al., 2009). Subsequent abortion typically occurs four weeks after infection with multiple white foci in the cotyledons the most suggestive gross lesion (Benavides et al., 2017; Innes et al., 2009). Histopathology on foetal and placental tissue is required to confirm a diagnosis. Toxoplasma specific antibodies are elevated from day 10-12 after infection and are enduring (Dubey, 2009; Innes et al., 2009). High titres detected in recently exposed ewes and in late-gestation foetuses from infected ewes support a diagnosis (Dubey et al., 1987).
Historically, toxoplasmosis, campylobacteriosis and listeriosis were the most commonly reported infectious abortigens of sheep (Plant et al., 1972). In more recent case reports it is less commonly diagnosed than the latter agents. For example, toxoplasmosis was diagnosed in only 6% of 198 abortion investigations conducted by Agriculture Victoria from 2010 to 2017, compared to 25% listeriosis and 19% campylobacteriosis (Hallett & Rendell, 2018). Abortion presumably occurs in only a fraction of ewes infected with T. gondii as it requires ewes to be both naïve and pregnant at primary exposure. Sero-prevalence surveys provide an estimate of the extent of infection across the Australian sheep flock although most Australian studies are >30 years old and potential risk factors, including sheep management and cat dynamics have changed over this time. Historical investigations report 9% mean prevalence in rams in NSW, 7.4-25.2% (test dependent) prevalence in South Australian sheep and 16.9% (lambs) and 61.7% (adult sheep) prevalence in Tasmanian abattoir samples (Munday, 1975; O'donoghue et al., 1987; Plant et al., 1982). More recent estimates report 23.9% sheep seropositive with 97% positive flocks in South Australia, 6.7% mean prevalence with 60% of flocks positive in South-East NSW and 16.4% of lambs and 31.9% of sheep seropositive at an Australian export abattoir (Kiermeier et al., 2008; Lanyon & O'Handley, 2020; McGregor & Harvey, 2011). Preliminary results from 26 Victorian ewe flocks indicate that low level within-flock exposure is common (6.8% within-flock prevalence before adjusting for test sensitivity, 76% flocks with at least one positive; Glanville et al., 2018). The lower prevalence estimates in the eastern states suggest many flocks are relatively naïve, with a high risk of toxoplasmosis abortion if oocyst exposure occurs.
The expansive, largely international, body of literature describing risk factors associated with toxoplasmosis was recently reviewed by Stelzer et al. (2019). A general consensus on risk of T. gondii exposure based on this literature is complicated by differences in production systems, serological test, survey design and results.
Increasing ewe age is a fairly consistent risk factor (Blewett, 1983; Stelzer et al., 2019; Waldeland, 1977). Hence younger ewes are at greater risk of reproductive loss due to toxoplasmosis as they are less likely to have had prior exposure. The occurrence of abortion in all of the young ewes on this farm but not in the older ewe may be due to prior exposure. Interestingly, cat presence on farm is not a consistent risk factor. The importance of not just presence or absence of cats, but the likelihood of cats contaminating feed or water sources (significant in five of eight publications) and the type of cat population on farm (wild cats protective in one study but stray cats increased risk in another) is emphasised by Stelzer et al. (2019). An Australian pilot study of risk factors in NSW found a trend toward increased seroprevalence in sheep on farms where domestic cats had access to feed stores (McGregor & Harvey, 2011). In this clinical case, a cat was known to be active on paddocks where the ewes grazed when pregnant providing the most likely source of this outbreak.
Other environmental factors with a significant effect on T. gondii exposure include farming region or district (significant in 13 of 17 publications), altitude (significant in six of eight publications), mean monthly temperature and annual rainfall (direction of effect variable), land cover (forested and urban areas; one of one) and the presence of water-logged terrain (one of one; Stelzer et al., 2019). Important management factors include extensive versus intensive production systems (increased risk in extensive systems unless feed stores contaminated directly with cat faeces or indirectly with infected rodents increasing risk), flock size (variable findings) and access of rodents to feed. Feed- and water-related risk factors include feeding concentrate, uncovered feed stores, use of surface water and stagnant water. The more recent Australian survey found there was a trend toward higher prevalence on farms where sheep were supplementary fed hay and/or grain, bordering forest and rotationally grazed (McGregor & Harvey, 2011).
The disease course and outcome of infection for pregnant ewes is well described. Early gestation infection results in foetal death and resorption. Early-to-mid gestation infection results in foetal death and abortion at least four weeks after infection, with or without mummification. In multiple pregnancies, one foetus may be infected prior to the other(s) as was evident in this case. Infection becomes less abortigenic from mid-late pregnancy as foetal immunocompetence increases and ewes infected in late-gestation may birth congenitally infected stillborn, weak or clinically normal lambs (Benavides et al., 2017). The clinical signs in the submitted case are consistent with mid-gestation infection. Given the typical time between infection and abortion, the ewes were most likely infected in late April when the first ewe that aborted was 2-2.5 months pregnant. The rest of the ewes aborted 3-5 weeks after the first. The submitted case clearly shows one less-developed mummified foetus and one more developed non-mummified foetus. It is possible that all ewes were infected at a similar time, with one foetus dying and mummifying prior to abortion and death of the second foetus several weeks later. This could explain the differential development in the two foetuses and the subsequent abortions in the rest of the ewes over a period of two weeks.
The likely source of oocyst contamination, the cat, was promptly removed from this farm. However, oocysts from cat faeces are relatively hardy. Unsporulated oocysts can survive in the environment for at least three months, whilst sporulated oocysts remain infective in moist soils for up to 18 months (Frenkel et al., 1975; Lindsay et al., 2002) and in freshwater for several years (Lindsay & Dubey, 2009). This is an important consideration if this producer intends to run new ewes on the property in the next two years. As it is a small flock, it might be worthwhile advising that the T. gondii sero-status of any new ewes be determined and that new ewes be run on the paddock where the cat was active prior to pregnancy in the hope that natural protective immunity is stimulated prior to pregnancy. In terms of future reproductive potential from the aborted ewes, conception and pregnancy should proceed normally after the next joining. In most cases, both ewes infected prior to pregnancy and those that experience toxoplasmosis during gestation develop protective immunity against future pregnancy loss due to T. gondii (Innes et al., 2009). In New Zealand, where toxoplasmosis is a common cause of reproductive loss, a live vaccine provides an effective strategy to decrease the risk of ovine toxoplasmosis. No such vaccine is available in Australia.