Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) in sheep and goats: recent advances David Buxton

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David Buxton. Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) in sheep and goats: recent advances. Veterinary Research, BioMed Central, 1998, 29 (3-4), pp.289-310. ￿hal-00902530￿

HAL Id: hal-00902530 https://hal.archives-ouvertes.fr/hal-00902530 Submitted on 1 Jan 1998

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Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) in sheep and goats: recent advances

David Buxton

Division of Virology, Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 OPZ, Scotland, UK

(Received 14 October 1997; accepted 18 December 1997)

Abstract - The protozoan parasite Toxoplasma gondii is a serious cause of fetal mortality in sheep and goats. Oocysts, the parasite stage responsible for initiating infection, arc produced followingg a primary infection in cats. A primary infection in pregnant sheep and goats can establish a pla- cental and fetal infection which may result in fetal death and resorption, abortion or stillbirth. Diag- nosis is aided by the clinical picture, the presence of characteristic small white necrotic foci in pla- cental cotyledons, the possible presence of a mummified fetus and on fetal serology and histopathology. Development of the polymerase chain reaction (PCR) specific for T. gondii may also provide a valuable diagnostic tool. Measures to control abortion include improved man- agement of farm cats, fodder and water. Vaccination of sheep with the live vaccine is an effec- tive preventive measure and the use of decoquinate in feed may be useful in some situations. Neo,spora caninum is related to T gondii and while its asexual life cycle is similar to that of the latter it is currently not known whether it has a similar sexual life cycle in a definitive host. Neospora is an important cause of fetal loss in cattle and parallels that of T. gondii infection in sheep and goats. While it does not appear to cause frequent losses in these latter animals, exper- imental infection is readily induced in them and if initiated during pregnancy provides a very good model of the bovine infection. Furthermore clinical signs and pathological lesions in sheep and goats are similar to those induced in them by T. goiidii, although there are subtle histopatholog- ical differences. These changes will aid possible diagnosis as will specific serological tests such as the indirect immunofluorescent antibody test and the enzyme linked immunosorbent assay and the PCR. Sarcocystis, which exists as numerous species, undergoes a coccidian-like life cycle with each having a distinctive definitive (usually carnivore) host which excretes sporo- cysts into the environment. Clinical sarcocystiosis is much less commonly diagnosed than tox- oplasmosis and neither is it normally associated with fetal infection or abortion in either sheep or goats. However, infection is extremely common throughout the world and follows ingestion of food or water contaminated with sporocysts. Clinical signs, when seen, include fever, anaemia, inappetance and weight loss or reduced weight gain. Central nervous signs (hind limb weak- ness, ataxia, paresis), acute myopathy and death may occur. Diagnosis is difficult as infection is

Tel.: (44) 131 664 3262; fax: (44) 131 664 8001; e-mail: [email protected] so common and clinical signs absent, mild or non-specific. Serology may be useful in some sit- uations and histopathology/immunohistochemistry is valuable for confirming the cause of death. Control relies on preventing contamination of pasture and water with faeces of dogs, foxes and cats or by controlling access of young susceptible stock to contaminated land. Relatively little is known of the immunity induced by infection with Sarcocystis spp. but research indicates that pro- tective immunity does develop and that cell-mediated mechanisms are probably important. It is likely that sarcocystiosis is underdiagnosed as a problem and that better diagnostic methods are needed to show the true extent of the losses caused. Neosporosis on the other hand would appear not to be so common in sheep and goats. The value of experimental infections in these animals may be to provide a comparative model of the infection in cattle in the same way that our under- standing of toxoplasmosis in sheep provides a superior model of human toxoplasmosis. @ Inra/ Elsevier, Paris

Toxoplasma / Neospora / Sarcocystis / sheep / goats

Résumé - Protozooses (Toxoplasma gondii, Neospora caninum et Sarcocystis spp.) chez le mouton et la chèvre : avancées récentes. Le parasite protozoaire Toxoplasma gondii est une cause sérieuse de mortalité foetale chez le mouton et la chèvre. Les oocystes, stade du parasite respon- sable de l’initiation de l’infection, sont produits après une primo-infection chez le chat. Une primo-infection chez la brebis ou la chèvre gestante peut provoquer une infection placentaire et foetale pouvant résulter en une mort et résorption foetale, un avortement, ou une mortinatalité. Le diagnostic se fait à l’aide de la description clinique, de la présence de petits foyers nécrotiques blancs caractéristiques dans les cotylédons placentaires, de la présence éventuelle d’un foetus momifié, et de la sérologie et de l’histopathologie foetale. Une technique d’amplification en chaîne par polymérase (PCR) spécifique de T. gondü, qui tend à se développer, est également un outil de diagnostic précieux. Les mesures visant à contrôler les avortements incluent une amé- lioration, dans les fermes, de la gestion des chats, des fourrages, et de l’eau. La vaccination des moutons avec le vaccin vivant est une mesure de prévention efficace, et l’utilisation de décoquinate dans la nourriture peut être utile dans certaines situations. Neospora caninum est apparenté à T. gondii, mais bien que son cycle asexué soit similaire à celui de ce dernier, on ne sait toujours pas s’il possède également un cycle sexué similaire dans l’hôte définitif. Neospora est une cause importante de perte de foetus chez les bovins, et est analogue à l’infection par T. gondü chez le mou- ton et la chèvre. Alors que Neospora ne paraît pas causer de pertes fréquentes chez ces derniers, l’infection expérimentale est facilement induite chez eux, et si elle est initiée durant la gesta- tion, elle produit un très bon modèle de l’infection bovine. De plus, signes cliniques et lésions patho- logiques chez la chèvre et la brebis sont similaires à ceux induits chez eux par T. gondü, bien qu’ilil existe de subtiles différences histopathologiques. Ces différences peuvent aider à faire un diagnostic précis, de même que des tests sérologiques spécifiques tels que la détection d’anticorps par immuno-tluorescence indirecte, l’Elisa, ou la réaction en chaîne par polymérase. Sarcocystis, qui existe sous la forme de nombreuses espèces, a un cycle de développement semblable à celui des coccidies, chaque espèce ayant un hôte définitif particulier (généralement carnivore) qui excrète des sporocystes dans l’environnement. La sarcocystose clinique est diagnostiquée bien moins couramment que la toxoplasmose, et n’est normalement pas non plus associée à une infec- tion foetale ou à un avortement chez la brebis ou la chèvre. Toutefois, l’infection est très fré- quente dans le monde entier, et se produit après l’ingestion de nourriture ou d’eau contaminée par des sporocystes. Les signes cliniques, lorsqu’ils sont visibles, incluent de la fièvre, une anémie, un manque d’appétit et une perte de poids ou une diminution de la prise de poids. Des symptômes liés à l’atteinte du système nerveux central (faiblesse des pattes arrières, ataxie, parésie), myopathie aiguë et mort peuvent se produire. Le diagnostic est difficile car l’infection est très fréquente, et les signes cliniques absents, légers ou non spécifiques. La sérologie pourrait être utile dans cer- taines situations, et l’histopathologie/l’immunohistochimie est précieuse pour confirmer la cause de la mort. Le contrôle de la maladie repose sur la prévention de la contamination des pâturages et de l’eau par les chiens, les renards et les chats, ou sur le contrôle de l’accès des cheptels jeunes et sensibles aux terres contaminées. On connaît peu de choses sur l’immunité induite par l’infec- tion par Sarcocystis spp., mais des études indiquent qu’une immunité protectrice se développe, et que les mécanismes cellulaires sont probablement importants. Il est probable que la sarco- cystose soit un problème sous-estimé, et que de meilleures méthodes de diagnostic soient néces- saires pour montrer l’étendue réelle des pertes subies. La néosporose, en revanche, apparaîtrait comme n’étant pas si fréquente chez les moutons et les chèvres, et l’intérêt des infections expé- rimentales chez ces animaux est de fournir un modèle de l’infection chez les bovins, de la même manière que la compréhension de la toxoplasmose chez le mouton a permis de développer des mesures de contrôle utiles de cette infection importante, et de fournir un modèle de la toxoplas- mose humaine. © Inra/Elsevier, Paris

Toxoplasma / Neospora / Sarcocystis / mouton / chèvre

1. INTRODUCTION cystidae. While the former two are con- sidered to exist as single species, Sarco- cystis occurs as many species. All three Toxoplasma gondii, Neospora caninum organisms have been recorded as causing and Sarcocystis spp. are all apicomplexan clinical disease and mortality in sheep and protozoan parasites in the family Sarco- goats. 2. TOXOPLASMOSIS more usually, develops immunity to the parasite. In the latter case a persistent Toxoplasma gondii, first described in infection is established, extracellular are thus eliminated, intracellu- 1908, has a worldwide distribution and is organisms capable of infecting all warm-blooded ani- lar multiplication slows and tissue cysts A small contains a few mals, in which it has a simple two-stage develop. cyst only asexual life-cycle. Clinical toxoplasmo- bradyzoites (the second stage of the asex- ual but a one may contain sis occurs in sheep and goats when they cycle) large thousands. are found most fre- suffer a primary infection while pregnant Cysts (Dubey and Beattie, 1988). quently in brain and skeletal muscle and represent the quiescent stage of the para- site within the host. When a cyst ruptures the bradyzoites are released and, trans- 2.1. Life cycle formed into tachyzoites, enter other cells to complete the asexual cycle (Dubey and The life cycle of Toxoplasma can be Beattie, 1988). divided into two parts; an asexual cycle with little host specificity and a sexual Initiation of the sexual cycle occurs cycle, confined to the enteroepithelial cells when a non-immune cat ingests food con- of cats, which results in the production of taminated by oocysts or containing tachy- oocysts (Dubey and Beattie, 1988) (fig- zoites or tissue cysts. In the case of the ure 1). In the asexual cycle two develop- latter the cyst wall is dissolved by prote- mental stages are involved, the tachyzoite olytic enzymes in the stomach and small and the bradyzoite. Each crescent-shaped intestine and the released bradyzoites pen- tachyzoite (about 5 pm by 1.5 pm) can etrate the epithelial cells of the small intes- actively penetrate a host cell, where it tine. While the parasite spreads to brain becomes surrounded by a parasitophorous and muscles where tissue cysts will vacuole in which it multiplies by endodyo- develop (asexual cycle), simultaneously geny (two daughter cells form within the toxoplasms also undergo gametogeny mother cell). Multiplication continues until (sexual cycle) in enteroepithelial cells. the host cell ruptures when the organisms Here, in the small intestine (most com- are released to parasitize further cells. This monly the ileum) gametocytes develop, process continues until the host dies, or over 3-15 days after infection. Microga- metes form and are released to penetrate within tissue cysts. However, in the preg- mature macrogametes triggering the for- nant ewe infection may establish in the mation of an oocyst wall around each fer- gravid uterus where maternal immuno- tilized gamete. The oocysts (10 x 12 pm in logical responses may be altered and the diameter), each almost filled by the ability of the fetus, with its placenta, to sporont, are then discharged into the recognize and respond to the parasite is intestinal lumen to pass out in the faeces. negligible in the early stages of gestation occurs within 1-5 Sporulation days but develops progressively with time so on aeration and to (depending temperature) that lambs are born immunocompetent produce two ellipsoidal sporocysts, each (Salami et al., 1985). Toxoplasms initially containing four sporozoites within each parasitise the caruncular septa, the mater- oocyst (Dubey and Beattie, 1988). nal tissues of the placentome, before Thus, during the 4-12 days after ingest- invading the adjacent trophoblast cells of ing tissue cysts the cat is capable of shed- the fetal villi and from there the rest of the ding millions of oocysts in its faeces, after fetus (Buxton and Finlayson, 1986). Thus, which it will not normally excrete the par- the outcome of infection early in gesta- asite again, although stress can trigger the tion can result in fetal death and resorp- recrudescence of infection (Dubey and tion/abortion while infection in the latter Frenkel, 1974). Unrelated illness may part of gestation, when fetal immunity is therefore lead to the excretion of oocysts relatively well developed, may have no in smaller numbers and for a shorter time clinical effect, the offspring being born than in a primary infection. Cats may also normal but infected and immune. In sheep become infected by ingesting oocysts or typical clinical signs of toxoplasma abor- tachyzoites but in this case they tend to tion usually result following infection in produce oocysts after 19 or 20 days, for mid gestation, with ewes producing still- only a day or two and in relatively small born and/or weakly lambs often accom- numbers and even then about half of these panied by a small, mummified fetus. animals will not excrete oocysts (Dubey Cotyledons on the accompanying pla- and Beattie, 1988). centa(s) will also show lesions visible to the naked eye (Buxton, .1991). Abortion and neonatal mortality in goats is essen- 2.2. Clinical disease tially similar to that seen in sheep whether occurring naturally (Munday and Mason, 1979; Chhabra and Gautam, 1984; Sporulated oocysts ingested by a sus- Dubey, ceptible pregnant sheep excyst in the small 1981a; Dubey et al., 1981; Nurse and intestines, each able to release the eight Lenghaus, 1986) or produced experimen- sporozoites. Four days later tachyzoites tally (Dubey et al., 1980; Dubey, 1988). an acute infection in toxo- can be found in the mesenteric lymph During goats nodes, where they multiply (Dubey, 1984), plasms may be excreted in the milk and they in turn are released into the blood (Dubey, 1980; Skinner et al., 1990) and to cause a parasitaemia, which may last be a possible source of human infection from the 5th until the 12th day after infec- if drunk unpasteurised (Skinner et al., tion (Dubey and Sharma, 1980a; Reid et 1990). Also, experimentally at least, tox- al., 1982; Wastling et al., 1993), dissemi- oplasms may be present in goat semen for nating infection to many tissues. The ces- a variable time after infection (Dubey and sation of the parasitaemia coincides with Sharma, 1980b) but the epidemiological the onset of a protective immune response significance of this, as in sheep (Blewett et and infection then persists as bradyzoites al., 1982), may be very slight. 2.3. Diagnosis is probably due to fetal anoxia in late ges- tation caused by advanced necrosis in the 2.3.1. Pathological changes placentome preventing sufficient oxygen transfer from mother to fetus (Buxton et al., 1982). Characteristically the placental cotyle- dons appear to dark red and speck- bright 2.3.2. Detection gondii led with white foci of necrosis 2-3 mm in of Toxoplasma diameter which may be sparse or so While the most direct and established numerous that they can become conflu- method of ent, while the intercotyledonary allanto- demonstrating Toxoplasma infection in cases of abortion is to transmit chorion appears normal (Hartley and the from aborted material Kater, 1963; Beverley et al., 1971 b). Vis- parasite (fetal brain and to labo- ible changes in lambs and kids vary, the placental cotyledons) mice and Kwantes, it most obvious being the mummified fetus, ratory (Fleck 1980) is slow and a small chocolate brown miniature of a expensive. Toxoplasma may also be in tissue culture in virtu- lamb/kid, often with its own small grown grey-brown placenta. Fetuses dying later ally any mammalian cell line and while more than mouse inoculation it is in gestation are born in various stages of rapid used for routine as it is decomposition often with clear to bloody rarely diagnosis and test subcutaneous oedema and a variable expensive samples may frequently A more amount of clear to bloodstained fluid in be heavily contaminated. rapid body cavities (Hartley and Kater, 1963). but less sensitive method of isolation is However, while these latter changes indi- the direct demonstration of T. gondii tissue of lamb brain cate an intrauterine infection they are not cysts by centrifugation on a discontinuous specific to infection with Toxoplasma. homogenate density gradient of 30 and 90 % colloidal silica The most obvious histopathological solution (Blewett et al., 1983).). are the necrotic foci, visible changes Immunohistochemical macroscopically in the cotyledons. Micro- techniques allowing visualisation of both intact T. scopically they appear as large foci of and debris in tissue sec- coagulative necrosis, remarkably free of gondii antigenic tions of aborted materials are convenient cells, which become inflammatory may and sensitive methods and have the advan- mineralised with time. Sometimes small when with at iso- numbers of intracellular and extracellular tage, compared attempts lation, of detecting toxoplasma antigen toxoplasms are visible, usually on the even in tissues. The ABC of the necrotic lesions or in a decomposed periphery indirect method (Vec- villus which is in the early stages of infec- immunoperoxidase tor Laboratories, USA) and the tion (Buxton and Finlayson, 1986). In the peroxi- dase (PAP) fetal brain both primary and secondary anti-peroxidase technique (Uggla et al., 1987) are equally good. lesions develop. Glial foci, typically sur- rounding a necrotic and sometimes min- Both viable and non-viable toxoplasms eralised centre, often associated with a may be identified in tissues with the poly- mild lymphoid meningitis, represent a fetal merase chain reaction (PCR). Both the P30 immune response following direct dam- and the B 1 gene of T. gondii have been age by local parasite multiplication. Tox- used as PCR targets for the detection of oplasms are only rarely found,.usually at Toxoplasma in various clinical specimens the periphery of the lesions. Focal leuko- collected from infected humans. Limited malacia, seen most commonly in cerebral studies have also been carried out using white matter cores, is also common and ovine samples such as aborted placental material, brain and peritoneal fluid from ton et al.,1990), IFAT and LAT (Opel et aborted fetuses and lymph, blood and al., 1991 ) are adequate. Both the IHA and lymph nodes from artificially infected ewes LAT are easy to perform and the latter is (Wastling et al., 1993). Detection of T. available in kit form (Eiken Chemical Co., gondii by amplification of the B 1 gene Japan) and neither test requires species- would seem to be more sensitive than by specific antisera or conjugates. the P30 due to the nature gene repetitive The enzyme linked immunosorbent of the B 1 of which 25-50 are gene copies assay (ELISA) for T. gondii antibodies present in the genome of T. com- gondii has been adapted for use in most domestic with a of the pared only single copy longer animals including sheep (Buxton et al., P30 the is not gene. Currently technique 1988) and goats and can be made to dis- used in routine but the of diagnosis potential tinguish IgM and IgG antibodies and, as it the PCR to DNA in sec- identify paraffin is readily automated, it is suitable for han- tions from tissue blocks histopathological dling large numbers of test sera. may broaden its applicability (Ellis, 1997).

2.3.3. Serology 2.4. Control

This is an important tool in the diag- 2.4.1. Cats as a source of infection nosis of ovine and caprine toxoplasma abortion. The presence of specific anti- are maintained in an bodies in serum or tissue fluid from still- Sheep frequently environment contaminated born lambs or kids or in precolostral serum significantly from live offspring indicates uterine infec- with oocysts and infection follows inges- tion. However, high toxoplasma antibody tion of contaminated food or water (Blewett, 1983; Blewett and Watson, titres in sera taken from ewes and nannys 1983), with the most within a few weeks of abortion or pro- pasture perhaps being common source of infection, duction of stillborn lambs or kids can only although water can be a real threat not to ani- suggest toxoplasmosis as titres remain rel- only for after initial mals but also to people (Bowie et al., atively high long periods with infection. Serology will also indicate the 1997). Certainly, fields treated manure and from farm degree of exposure to infection in a group bedding buildings where cats live can cause infection of animals. The first method to be devel- (Faull et al., 1986) and cats in farm oped was the dye test (DT) of Sabin and defaecating as will Feldman (1948) but it is expensive, time feeds, such hay and stored grain, pose a risk (Plant et al., 1974). consuming and not without hazard as it requires live tachyzoites as antigen. The Thus in the hypothetical situation in indirect immunofluorescent antibody test which a cat, actively shedding oocysts in (IFAT) gives titres comparable with the its faeces, defaecates in 10 tonnes of grain DT but is safer as it uses killed tachyzoites stored on a farm, then the potential for the (Maley et al., 1997) and the latex aggluti- parasite being spread to livestock could be nation test (LAT) also performs well considerable. A single defaecation may con- (Trees et al., 1989; Maley et al., 1997). tain as many as 10 000 000 oocysts. If fur- The modified agglutination test (MAT) ther processing of the feed dispersed these (Desmonts and Remington, 1980) has oocysts evenly throughout the grain then been shown to perform particularly well each kilogram would contain between five with goat sera (Dubey et al., 1985) and 25 sheep infective doses (McColgan et although for epidemiological studies the al., 1988). In this way cat faeces can create indirect haemagglutination test (IHA) (Pat- a large, potent, long-lasting source of infec- tion for sheep. Thus, oocyst contamination should be kept free from soiling as far as of farm feeds and bedding, as well as pas- practically possible. Other measures to ture, is a threat to susceptible, pregnant reduce environmental contamination by sheep and goats and is closely related to the oocysts should be aimed at reducing the number and distribution of cats. number of cats capable of shedding oocysts. These would include selective Cats are born free of toxoplastna infec- culling of aged and diseased cats and tion and excretion of oocysts by them fol- attempts to control future breeding. If male lows establishment of a infection. primary cats are neutered and returned to it has been calculated that less caught, Although their colonies the stability of the colony than 1 % of all cats are at shedding oocysts is maintained; fertile male cats do not chal- time and any one (Dubey Beattie, 1988) the neutered males this is for lenge (Tabor, 1980) figure probably higher young and breeding is controlled. Thus the main- cats. Female feral cats can produce two tenance of a small healthy population of to three litters a year, each of up to eight mature cats will reduce oocyst excretion as kittens, and may rear their young com- well as help control rodents. munally (Macdonald, 1980). Numbers of young cats are also dependent upon the It is likely that a flock/herd, in which density of breeding adults. In rural areas seroconversion to T. gondii can be demon- male cats may have territories of 60-80 strated in a majority of animals, is being hectares (250-200 acres) while females maintained in an environment significantly usually only occupy a tenth of this area contaminated with T. gondii oocysts. As (Macdonald, 1980). In an urban environ- young, seronegative, replacement stock ment these territories are considerably will be at risk, there is a case to be made smaller (Tabor, 1980). The area occupied for attempting to expose them to a con- by feral cats is influenced by the supply taminated environment before mating. of food, which includes mice, voles, Identification of such an environment is shrews, rats, rabbits and small birds (Mac- difficult but is likely to be an area in or donald, 1980). Such animals persistently around farm buildings where cats live, as infected with T. gondii are an important well as pasture spread with manure from source of infection for cats (Jackson and such buildings. While the foregoing is use- Hutchison, 1989; Peach et a]., 1989). In ful practical advice it does not guarantee addition mice (Eichenwald, 1948; Bever- success and there is a clear need for more ley, 1959; De Roever-Bonnet, 1969), but precise methods of control. probably not rats (Dubey et al., I 997) are particularly important because they can 2.4.3. Vaccination pass the parasite in utero without causing overt clinical disease or fetopathy. In this Natural infection with T. gondÜ stimu- way a reservoir of T. gondii tissue cyst lates protective in both and infection for cats can exist in a particular immunity sheep goats (McColgan et al., 1988) but inacti- population of mice for a long time. vated toxoplasma tachyzoites, either alone (Beverley et al., 1971a) or in Freund’ss 2.4.2. General management incomplete adjuvant (Wilkins et al., 1987) do not protect pregnant sheep against exper- During pregnancy a flock/herd, in imental challenge with the parasite. Simi- which the majority are seronegative to T. larly a preparation consisting of surface anti- gondii, could be at risk if it was allowed gens of T. gondii combined with Quil A access to an environment contaminated provided little protection against experi- by cat faeces and so all food and water mental challenge (Buxton et al., 1989). The failure of these killed preparations (Buxton et al., 1991, 1993a). Protection in sheep may be partly because, in natural afforded by one injection of the vaccine infections, persistence of the parasite in was just as good after 18 months as it was tissues continually stimulates immunity, after 6 months. Antibody may impede cell as suggested in human toxoplasmosis invasion by tachyzoites and, with comple- (McHugh et al., 1997). However experi- ment, limit phagocytosis by macrophages ments in which mice and hamsters were but protective immunity in sheep given the infected with a live temperature-sensitive live vaccine is largely cell mediated mutant of T. gondii, which does not persist (reviewed by Innes and Wastling, 1995) in the host, as it cannot form bradyzoites with CD4+ and CD8+ T cells (Innes et al., and cannot therefore form tissue cysts did 1995a) and interferon gamma (Oura et al., induce protective immunity (Waldeland 1993; Innes et al., 1995b) playing an and Frenkel, 1983; McLeod et al, 1988; important role in suppressing T. gondii par- Suzuki and Remington, 1990). asitaemia in sheep (Buxton et al., 1994) thereby protecting the developing fetus In 1988 the of and Ministry Agriculture from a maternal infection. Food (New Zealand) launched a live tox- oplasma vaccine for the control of ovine In field studies around half of 1 1 000 toxoplasmosis (O’Connell et al., 1988; 1-year-old female sheep in 10 1 flocks were Wilkins et a]., 1988) and in 1992, after vaccinated. Subsequently 20 flocks were natural further study (Buxton et al., 1991), it was exposed to significant challenge marketed in the UK and Eire (Toxovax, from the parasite and in vaccinated ani- mals abortions were reduced and Intervet UK) as a tissue-culture-grown lambing vaccine. The vaccine consists of live S48 percentages significantly improved when with unvaccinated in the tachyzoites which were originally isolated compared sheep same flocks et thus con- by mouse injection from a case of ovine (Spence al., 1992) abortion in New Zealand. After around firming the findings of field trials carried out in New Zealand. 3 000 passes twice weekly, in laboratory mice it was shown to have lost its ability to The manufacturers recommend that inn develop bradyzoites in tissue cysts and the first instance the whole flock is vacci- unpublished data indicate that neither can nated at least 3 weeks before mating and in the tachyzoites initiate the sexual life cycle subsequent years newcomers to the flock, of the parasite in cats (Bos, pers. comm.). usually young replacement stock, are vac- cinated. one is in Studies at the Moredun Research Insti- Only injection required the life of the The vaccine has a tute (Edinburgh, UK) showed that when sheep. shelf life of 7-10 days and is capable of susceptible pregnant sheep were each so it must be handled with orally dosed with 2 000 sporulated T. infecting people care according to the manufactur- gondii oocysts, a relatively severe chal- strictly ers recommendations. lenge, less than 18 % of lambs were born live and viable, whereas with vaccinated As with sheep, the majority of goats ewes similarly challenged 75 % of lambs previously exposed to infection with T. were born live and viable. The placental gondii develop a protective immunity to lesions in vaccinated ewes, following chal- the parasite so that they are protected lenge, were much less frequent and/or against subsequent challenge during preg- severe than in unvaccinated, challenged nancy (Obendorf et al., 1990), although ewes and the viable lambs born to the for- repeat abortions have been recorded mer had significantly higher birth weights (Dubey, 1982). Toxovax is not licensed than those born to the unvaccinated ewes, for use in goats. Immunity induced in thus enhancing their chances of survival goats by experimental infection with the related coccidian parasite Hammondia sensible management procedures to be fol- hammondi has been shown to offer some lowed to minimise the weight of infection cross-protection against challenge with T. in the environment, pharmaceutical prepa- gondii both in non-pregnant (Dubey, rations have been identified which may 1981 b) and pregnant animals (Dubey, be used to curb disease and there is an 198 Ic; Munday and Dubey, 1988) but this effective vaccine for use in sheep. avenue of research has not been pursued.

2.4.4. Pharmaceuticals 3. NEOSPOROSIS

careinum has been Even though a live vaccine for toxo- Neospora recognised since the 1980s and while it plasmosis is available for use in sheep only appears to be a cause of fetal loss in cattle there will always be a need for other meth- major ods of preventing infection becoming (Dubey and Lindsay, 1996) it also may established in unprotected ewes/nannys cause clinical loss in sheep and goats. and for treating infection once it is found in animals still to give birth. Chemopro- phylaxis with monensin given in the feed 3.1. Life cycle at the rate of 15 mg/animal/day during caninum infects a pregnancy, can significantly suppress a Neospora naturally toxoplasma infection in sheep (Buxton et wide range of hosts including dogs, cat- deer and al., 1988) but it is not licensed for this pur- tle, horses, (Dubey Lindsay, and foxes et as pose. However the anticoccidial drug 1996) (Buxton al., 1997a), decoquinate fed daily at 2 mg/kg body well as sheep and goats (see below). An asexual life in weight, can also significantly reduce the cycle involving bradyzoites, effect of T. gondii oocysts ingested by tissue cysts, and tachyzoites are the only pregnant sheep (Buxton et al., 1996) and is life cycle stages recognised at the time of a sexual life licensed for this use in sheep. writing (figure 2), although cycle in a definitive carnivore host, which Both monensin and work decoquinate produces oocysts in a similar way to that best if are fed to sus- they already being in which cats produce T. gondii oocysts, ceptible ewes at the time they encounter has been predicted (Dubey and Lindsay, infection rather than after infection is 1993). The tachyzoites, which divide by established. However, it has been shown endodyogeny, may be either crescent that a combination of and pyrimethamine shaped or ovoid 3-7 pm long by 1-5 pm a well tried treatment in sulphamezathine, wide depending on their stage of division. human medicine, which is effective The tissue cysts range from 20-100 pm because it blocks folate is effec- synthesis, in diameter and typically have a clearly tive in the treatment of infected sheep discernible cyst wall 1-2 pm thick (Dubey (Buxton et al., 1993b). The drug combi- and Lindsay, 1996). nation, baquiloprim and sulphadimidine (Zaquilan@, Schering-Plough Animal Health, UK) also blocks folate synthesis 3.2. Clinical disease and has given promising results in a con- trolled in pilot study non-pregnant sheep Clinical neosporosis is most important (Buxton, data). unpublished in cattle in which it is now recognised to The control of toxoplasmosis in sheep be a serious cause of abortion in many (and goats) is now easier than before. Our countries around the world (Dubey and knowledge of its epidemiology permits Lindsay, 1996). Vertical transmission from cow to developing fetus is likely to be abortion in goats in the USA (Barr et important in the epidemiology of bovine al.,1992; Dubey et a]., 1992) and Costa disease and is currently the only route of Rica (Dubey et al., 1996) but while only a transmission to have been demonstrated. It few fetuses were lost there was evidence is presumed that recrudescence of a per- of seroconversion to N. caninum in other sistent cyst infection in the mother occurs animals in the Costa Rican herd. during pregnancy permitting a tachyzoite parasitaemia which allows the parasite to invade the gravid uterus, the placenta and 3.3. Experimental neosporosis then the fetus. Infection early in gestation, when the fetal immune system is little Dubey and Lindsay (1990) showed in developed can be fatal with fetal resorption an uncontrolled pilot study with two ewes or abortion. Exposure of an older fetus, the susceptibility of the ovine fetus to with its better developed immune system Neospora and McAllister et al. (1996) and may result in the birth of a clinically nor- Buxton et al. (1997b) confirmed this in mal but congenitally infected calf (Dubey larger controlled studies. As with bovine and Lindsay, 1996). neosporosis and ovine toxoplasmosis the younger fetuses were shown to be more While pregnant sheep have been shown susceptible than those challenged later in to be very susceptible to experimental gestation (McAllister et al., 1996). Fetal infection with N. caninum (Dubey and lesions were most commonly manifest as a Lindsay, 1990; McAllister et al., 1996; meningoencephalitis with characteristic Buxton et al., 1997b) to date there is little foci of inflammation showing signs of evidence of it being a significant field organisation, typically with a necrotic cen- problem (Otter et al., 1997) with only one tre surrounded by microglial and lymphoid reported case of natural, congenital cells. Light lymphoid vascular cuffs and neosporosis in a weak, ataxic lamb which meningeal infiltrates and focal microgliosis died when 1 week old (Hartley and Bridge, were also present. Milder lesions occurred 1975; Dubey et al., 1990). It is, however, less consistently in other tissues such as the noteworthy that the spinal lesions in this heart and lungs (McAllister et al., 1996; animal were similar to those seen in calves Buxton et al., 1997b). Serial sampling of congenitally infected with N. caninum. fetuses from experimentally infected ewes Neo.rpora has also been associated with showed a progressive maturation of lymph node structure with time and this correlated In a preliminary report experimental with the early production of IgM antibody infection with N. caninum before mating followed by an IgG response to Neo.spora partially protected ewes and their devel- (Buxton et al., 1997b). oping fetuses against a second challenge with the parasite during pregnancy. Vac- In the there were scattered foci placenta cination (Toxovax, of necrosis fetal villous tissue against toxoplasmosis involving Intervet UK) however did not protect and caruncular with accu- adjacent septa with Neospora (Buxton mulations of necrotic debris of against challenge apparently et al., 1997c). Thus it may be more diffi- maternal and chorionic epithelial origin. cult to induce solid immunity in sheep to a inflamma- Typically non-suppurative N. caninum than to T. gondii although pro- infiltrate was in tory present surrounding tection against both parasites probably maternal and fetal tissue as well as chorio- results from stimulation of cell mediated allantoic membrane et (McAllister al., immune mechanisms as in vitro studies Buxton et In one 1996; al., 1997b). study have shown that recombinant ovine inter- neospora organisms were readily feron gamma (IFN-gamma) can signifi- detectable (McAllister et al., 1996) while cantly inhibit multiplication of N. can- in the other were much less they frequent inum in ovine fibroblast cell cultures (Buxton et al., 1997b). While the first (Innes et al., 1995c) as is the case with T. used a mixture of the study Nc-Liverpool (Oura et al., 1993). Studies with NC-2 isolates the latter used gondii and study mice have underlined the importance of isolate and so it is only the Nc-Liverpool in and shown that strain virulence is also IFN-gamma neosporosis possible impor- that interleukin-12 also has a role to studies with the NC-1I play tant. Subsequent (Khan et al., 1997). isolate in pregnant sheep (Buxton et al., 1998) have shown it to be more virulent than Nc-Liverpool and to produce greater inflammation and more severe and fre- 3.4. Diagnosis quent focal necrosis, sometimes associ- ated with vasculitis and thrombosis. Serology in sheep and goats has largely been carried out with the IFAT to detect The focal necrosis visible macroscopi- IgM and IgG (Lindsay et al., 1995; Buxton as white scattered cally spots throughout et al., 1997b), alone et al., the and from IgG (Dubey cotyledons indistinguishable 1996; McAllister et al., 1996) or undefined ovine does differ micro- toxoplasmosis immunoglobulin (Otter et al., 1997). scopically from the latter. In toxoplasmo- ELISA have been developed for use in cat- sis the chorio-allantois is not involved, tle soluble extracts of sonicated focal necrosis in is using placental cotyledons (Pare et al., 1995; remarkable for its relative lack of inflam- neospora tachyzoites Osawa et al., 1998), recombinant antigens mation, and vasculitis and thrombosis do (Lally et al., 1996a), extracted tachyzoite not feature. Pathological changes in fetal antigen incorporated into an immuno- and tissues from occur- placental naturally stimulating compound (ISCOM) (Bjork- ring and experimentally produced man et al., 1997) or whole organisms in goats would appear to be neosporosis (Williams et al., 1997). One of these assays similar to those seen in natural and exper- has been for use with et adapted successfully imental infections in sheep (Barr al.,., sheep and goat sera (Osawa et al., 1998). 1992; Dubey et al., 1992, 1996; Lindsay et al., 1995). Vertical transmission to kids The PCR has been used both to make did not occur in a subsequent pregnancy comparisons between N. caninum and T. (Lindsay et al., 1995). gondii (Ellis et al., 1994; Holmdahl et al., 1994) and as a potentially powerful diag- cycle with each species having a distinct nostic tool elegantly specific for N. can- definitive host(s) (usually a carnivore) and inum (Holmdahl and Mattsson, 1996; an intermediate host (Rommel et al., Kaufmann et al., 1996; Lally et al., 1996b; 1979). Infections are recognised to occur Muller et al., 1996; Payne and Ellis, 1996; in all parts of the world and farm animals Yamage et al., 1996). Their use in the field are intermediate (tangential) hosts for a still remains to be demonstrated but their number of species (figure 3). Sheep are application to sections cut from wax the intermediate host for four species, two embedded pathological samples could microcyst species (S. tenella [syn. S. ovi- prove very valuable (Ellis, 1997). canis] and S. arieticanis) and two macro- cyst species (S. gigantea [syn. S. ovifelis] and S. medusiformis) while goats are the 3.5. Treatment intermediate host to S. capracanis, S.hir- cicanis and S. moulei. Dubey and Lindsay (1996) report that clindamycin, sulphonamides and/or pyrimethamine and trimethoprim with sul- 4.1. Life cycle phadiazine have been used by various in authors, often to good effect, the treat- Farm animals become infected follow- ment of clinical canine Their neosporosis. ing ingestion of sporocysts released by the use at the appropriate dose rate could well definitive host. Following excystation in be beneficial in the event of a diagnosis the intestinal lumen sporozoites penetrate of clinical made in neosporosis being the gut wall and multiply asexually by two either a sheep or goat considered suitable schizogonous cycles in endothelial cells and worthy of treatment. of small blood vessels (first and second generation merogony), with an associated parasitaemia. The merozoites released 4. SARCOCYSTIOSIS after second generation merogony pene- trate muscle cells and form characteristic Sarcocystis (synonyms; sarcosporidia, sarcocysts filled with bradyzoites, although Meischer’s tubules) exists as numerous they may also invade the central nervous species (Uggla and Buxton, 1990). In 1972 system (Rommel, 1985). Ingestion of Rommel and colleagues reported that Sar- infected muscle by the definitive host trig- cocystis underwent a coccidian-like life gers a sexual life cycle in the intestinal lining with the formation of macro- and cal symptoms is dose dependant (Rom- microgamonts leading to the development mel, 1985). Thus O’Donoghue and Wilkin- of oocysts and then sporocysts which are son ( 1988) gave lambs 104 S. tenella sporo- excreted in the faeces. cysts but recorded no clinical signs while Munday ( 1981 ) showed that pregnant sheep experimentally infected with 64 x 10 4.2. Clinical disease S. tenella sporocysts developed a mild fever followed by anaemia and gave birth early to small, weak lambs. After parturi- The parasite is ubiquitous and infec- tion the ewes developed myositis, which are occur tions, extremely common, myocarditis and encephalitis and died. following ingestion of food or water con- taminated with sporocysts. While most to be a species appear non-pathogenic, 4.3. Diagnosis few, particularly those with a canine definitive host, may cause clinical illness As infection is common it is and Buxton, 1990). extremely (Uggla Symptoms difficult to attribute the of include fever, anaemia and inappetance very presence sarcocysts to a disease process. Clinical and an associated reduction in productiv- symptoms are not commonly useful as a ity. Fayer and Elsasser (1991) have sug- means of as the vast gested that the fever results from the diagnosis majority of natural infections are sub- release of interleukin-1 and sarcocystis (IL-1) clinical and when prostaglandin E2 while infection of symptoms, present, may be vague and non-specific. In cases of fatal macrophages by Sarcocystis causes release acute sarcocystiosis tissues, such as the of tumour necrosis factor-alpha which tongue, may appear mottled due to capil- i) causes ii) with inappetance, together and Buxton, IL- causes anaemia, lary haemorrhages (Uggla iii) suppresses is invaluable but release of pituitary growth hormone, with 1990). Histopathology only evidence of an active infection per- resultant weight loss. It has also been sug- mits the of a of acute gested that retardation of growth in pigs possibility diagnosis infected with S. meischeriana is due to the sarcocystiosis. Inflammatory changes may be confined to the central nervous system parasite influencing an insulin-like growth often with the more factor and an IGF (CNS), prominent (IGF) binding protein lesions in the cord. Characteristi- (Prickett et a]., l 992). spinal cally a non-suppurative meningo- In some instances abortion and still- encephalitis, associated with sarcocyst- births (Uggla and Buxton, 1990; Mackie like merozoites and meronts, gliosis and et al., 1992) or central nervous signs, associated oedematous changes, is pre- including hind limb weakness, ataxia, pare- sent (Jeffrey et al., 1988; Dubey et al., sis and death (Jeffrey et al., 1988; Dubey et 1989; O’Toole et al., 1993). Mackie et al. al., 1989; Hamir et al., 1993; O’Toole et ( 1992) also report a stillborn goat kid with al., 1993) have been associated with sar- a multifocal, necrotising, non-suppurative cocystiosis. Hind limb weakness, quadri- encephalitis with associated vascular paresis and death have also been recorded endothelial cells containing sarcocyst-like in sheep suffering from acute myopathy meronts containing merozoites. Outwith associated with a sarcocystis infection (Jef- the CNS the heart, tongue and liver may frey et al., 1988) and in cattle eosinophilic show marked mononuclear cell inflam- myositis is recognised (Granstrom et al., mation while tissues such as skeletal mus- 1989; Saito et al., 1993). In experimen- cle, lung and kidney are usually less tally induced disease the severity of clini- affected (Uggla and Buxton, 1990), although in a spontaneous outbreak of dogs, foxes, cats and other possible defini- acute myopathy in sheep, linked with a tive hosts, or prevent access of young sus- sarcocystis infection, histopathological ceptible animals to such locations where lesions were confined largely to skeletal contamination cannot be prevented. muscles and consisted of a non-suppura- tive myositis associated with mature, 4.4.1. Immunity immature and degenerate sarcocysts (Jef- frey et al., 1989). As with toxoplasmosis It seems that and immunohistochemistry likely Sarcocystis spp. neosporosis, stimulate immune mechanisms similar to may be valuable (Jeffrey et al., 1988; those induced T. and N. can- O’Toole et al., 1993). by gondii inum. While little is known of the specific Serological methods most commonly events which occur, in broad terms exper- used are the IFAT (Tadros and Laarman, imentally induced infection with a ’low’ 1982; Tenter, 1988; Uggla et al., 1987) dose stimulates protective immunity to a and ELISA (Tadros and Laarman, 1982; secondary, homologous, ’high’ (fatal) Weiland et al., 1982; Gasbarre et al., 1984; challenge dose (see below). and 1984; Smith O’Donoghue Weyreter, Goats infected with S. and Herbert, 1986; Tenter 1988). During experimentally capracanis sporocysts resisted an other- the first merogonous stage of a Sarcocys- wise fatal homologous challenge as early tis infection it may be difficult to detect as 14 days later (Dubey, 1981 d). However an antibody response. This may be because immunity to one species does not appear to the serological test uses an antigen prepa- trigger protective immunity to another ration derived from cystozoites which may species. Thus Fayer ard Dubey (1984) be antigenically distinct from the first gen- showed that calves 1 x 105 S. eration merozoites (Uggla and Buxton, given CrUZi sporocysts developed non-fatal clinical 1990). In this respect good sensitivity was sarcocystiosis and, when challenged 911 reported for an ELISA using merozoite later with 5 x 105 S. cruzi Savini et al. ( 1994). days sporocysts, antigen, developed by showed clinical of fever The detection of an will they again signs IgM response sug- and anaemia but survived. Four control gest an acute or recent infection while an calves given only the second challenge IgG response, in the absence of IgM anti- died 27 (n = 2) and 28 (n = 2) days later bodies, indicates a longer standing persis- and a further of four an initial tent infection and Buxton, 1990). group given (Uggla infection of 1 x 105 S. hirsuta More recently PCR methods have been sporocysts and challenged 91 days later with 5 x 10 reported for a number of Sarcocystis spp. S. also died 27 = (MacPherson and Gajadhar, 1994; Tenter ct-uzi sporocysts (n 2) and 28 (n = after the second chal- et al., 1994; Guo and Johnson, 1995; 2) days However Savini et al. ( 1996) Joachim et al., 1996; Marsh et al., 1996) lenge. showed that cows, considered to have been and they will prove valuable in establishing naturally infected with Sarcocysti.s, were more accurate diagnostic procedures. not protected against a subsequent chal- lenge with sporocysts or merozoites of S. cruzi during pregnancy. The former treat- 4.4. Control ment caused stillbirths while the cows challenged with the latter produced live Control is difficult but when a problem calves which were significantly lighter is perceived to occur attempts should be than control calves. These workers con- made to prevent contamination of animal cluded that protective immunity from nat- pasture and other feed by faeces from ural infection was transient. Lambs experimentally infected with S. 1993). Both sarcocystis antigen, IgG and tenella sporocysts developed specific IgM IgE have been demonstrated within such and IgG to the parasite but neither fol- lesions and specific IgE antibody has been lowing this primary challenge nor after a demonstrated in the serum of affected cat- greater secondary challenge with the same tle. It has been suggested that type-1I parasite did they respond in a lymphocyte hypersensitivity is involved in the patho- stimulation assay (LSA) to S. tenella anti- genesis (Granstrom et al. 1989, 1990). gen (either bradyzoite or sporozoite) (O’Dono!!hue and Wilkinson, 1988). This 4.4.2. Pharmaceuticals is in contrast to work by Gasbarre et al. ( 1984) who demonstrated with a LSA a Treatment of clinical cases may be in to,S. tenella blastogenic response sheep attempted and good results are recorded between 24 and 52 after antigen days by Moore ct al. ( 1997) who used trimetho- experimental infection (dpi) with S. teuelln prim-sulphamethoxazole and pyrimetha- The also sporocysts. authors showed that mine and vitamin E for the treatment of calves infected with S. crrri an developed horses clinically affected with S. nouronu. earlier and shorter ( 15-21 dpi) blastogenic response to S. cru:,i antigen in a similar LSA. Granstrom et al. ( 1989) suggested 5. CURRENT AND FUTURE that this latter brief have response may RESEARCH occurred in the presence of continued anti- genic stimulation and thus been associ- are of caus- ated with some form of parasite induced Sarcocv.sti,s spp. capable immunosuppression. Experiments in mice ing both clinical and subclinical illness in and other animals and sarco- with S. l71l/ris have shown that the para- sheep, goats is as a site causes both cell-mediated and humoral cystiosis probably underdiagnosed immunosuppression and that induction of cause of production loss. It must be a pri- to establish and better immune memory appears to be affected ority deploy diag- nostic so as to while expression of immune memory procedures accurately define the true cost of these losses. Immu- remains unaffected (Gill et a]., 1988a). These workers also showed that S. lI1uris nity develops following infection and it would seem to be both and to infection of mice causes splenomegaly long lasting be associated with of a viable with increased numbers of B-cclls and, to persistence infection. Whether this infec- a lesser extent, T-cells (Gill et al., 1988b). ’quiet’ quiet Whether this is linked to the observation tion repeatedly stimulates the immune sys- tem so an effective of that a Sarcocystis lectin (from S. gigan- maintaining degree in the host is not known and ten) has both mitogcnic properties as well immunity neither is it clear to what extent recurrent as being a B cell activator (Tietz et al., to infection is These 1990) remains to be seen. exposure important. apparently simple questions need to be addressed together with the more detailed Eosinophilic myositis (muscular sar- mechanisms of host immunity and their cocystiosis) may be observed in cattle at role in the as well as On affected animals protecting host, per- slaughter. inspection and clin- have yellow-green rice-grain sized nod- haps causing pathological change ical in this common infec- ules their muscles. illness, very throughout Microscopic tion. examination has shown that each lesion represents a focus of necrosis infiltrated Neosporosis in sheep and goats, how- by eosinophils as well as lymphocytes and ever, would not appear to be as common a multinucleate giant cells (Saito et al., field infection as in cattle, even though they often share a common environment, tachyzoite protein SAG 1 (30kDa/P30 anti- possibly because vertical transmission gen) in association with ’cholera toxin’ occurs much more readily in the latter. presented to mice by the nasal route can However, experimental ovine and caprine very substantially reduce subsequent neosporosis is readily reproduced, offers development of T. gondii tissue cysts in a good model of the bovine infection and brain (Debard et al., 1996). 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