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Protozoan infections (Toxoplasma gondii, Neosporacaninum and Sarcocystis spp.) in sheep and goats:

recent advancesDavid Buxton

To cite this version:David Buxton. Protozoan infections (Toxoplasma gondii, Neospora caninum and Sarcocystis spp.) insheep and goats: recent advances. Veterinary Research, BioMed Central, 1998, 29 (3-4), pp.289-310.<hal-00902530>

Review article

Protozoan infections (Toxoplasma gondii, Neosporacaninum 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 sheepand goats. Oocysts, the parasite stage responsible for initiating infection, arc produced following ga 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 andhistopathology. Development of the polymerase chain reaction (PCR) specific for T. gondii mayalso 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 thelatter 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 insheep 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 goodmodel of the bovine infection. Furthermore clinical signs and pathological lesions in sheep andgoats 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 suchas the indirect immunofluorescent antibody test and the enzyme linked immunosorbent assayand the PCR. Sarcocystis, which exists as numerous species, undergoes a coccidian-like lifecycle 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 orgoats. However, infection is extremely common throughout the world and follows ingestion offood 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: buxtd@mri.sari.ac.uk

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 andcats or by controlling access of young susceptible stock to contaminated land. Relatively little isknown 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 islikely that sarcocystiosis is underdiagnosed as a problem and that better diagnostic methods areneeded to show the true extent of the losses caused. Neosporosis on the other hand would appearnot to be so common in sheep and goats. The value of experimental infections in these animalsmay 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 lemouton et la chèvre : avancées récentes. Le parasite protozoaire Toxoplasma gondii est une causesérieuse 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. Uneprimo-infection chez la brebis ou la chèvre gestante peut provoquer une infection placentaire etfoetale pouvant résulter en une mort et résorption foetale, un avortement, ou une mortinatalité. Lediagnostic se fait à l’aide de la description clinique, de la présence de petits foyers nécrotiquesblancs caractéristiques dans les cotylédons placentaires, de la présence éventuelle d’un foetusmomifié, et de la sérologie et de l’histopathologie foetale. Une technique d’amplification enchaîne par polymérase (PCR) spécifique de T. gondü, qui tend à se développer, est également unoutil 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 desmoutons avec le vaccin vivant est une mesure de prévention efficace, et l’utilisation de décoquinatedans 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 pass’il possède également un cycle sexué similaire dans l’hôte définitif. Neospora est une causeimportante 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’il il

existe de subtiles différences histopathologiques. Ces différences peuvent aider à faire un diagnosticprécis, de même que des tests sérologiques spécifiques tels que la détection d’anticorps parimmuno-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 celuides coccidies, chaque espèce ayant un hôte définitif particulier (généralement carnivore) quiexcrète des sporocystes dans l’environnement. La sarcocystose clinique est diagnostiquée bienmoins 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 pardes 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 symptômesliés à l’atteinte du système nerveux central (faiblesse des pattes arrières, ataxie, parésie), myopathieaiguë et mort peuvent se produire. Le diagnostic est difficile car l’infection est très fréquente, etles 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 causede la mort. Le contrôle de la maladie repose sur la prévention de la contamination des pâturageset de l’eau par les chiens, les renards et les chats, ou sur le contrôle de l’accès des cheptels jeuneset 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, apparaîtraitcomme 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 mêmemanière que la compréhension de la toxoplasmose chez le mouton a permis de développer desmesures de contrôle utiles de cette infection importante, et de fournir un modèle de la toxoplas-mose humaine. &copy; Inra/Elsevier, Paris

Toxoplasma / Neospora / Sarcocystis / mouton / chèvre

1. INTRODUCTION

Toxoplasma gondii, Neospora caninumand Sarcocystis spp. are all apicomplexanprotozoan parasites in the family Sarco-

cystidae. While the former two are con-sidered to exist as single species, Sarco-cystis occurs as many species. All threeorganisms have been recorded as causingclinical disease and mortality in sheep andgoats.

2. TOXOPLASMOSIS

Toxoplasma gondii, first described in1908, has a worldwide distribution and iscapable of infecting all warm-blooded ani-mals, in which it has a simple two-stageasexual life-cycle. Clinical toxoplasmo-sis occurs in sheep and goats when theysuffer a primary infection while pregnant(Dubey and Beattie, 1988).

2.1. Life cycle

The life cycle of Toxoplasma can bedivided into two parts; an asexual cyclewith little host specificity and a sexualcycle, confined to the enteroepithelial cellsof cats, which results in the production ofoocysts (Dubey and Beattie, 1988) (fig-ure 1). In the asexual cycle two develop-mental stages are involved, the tachyzoiteand the bradyzoite. Each crescent-shapedtachyzoite (about 5 pm by 1.5 pm) canactively penetrate a host cell, where itbecomes surrounded by a parasitophorousvacuole in which it multiplies by endodyo-geny (two daughter cells form within themother cell). Multiplication continues untilthe host cell ruptures when the organismsare released to parasitize further cells. Thisprocess continues until the host dies, or

more usually, develops immunity to theparasite. In the latter case a persistentinfection is established, extracellularorganisms are thus eliminated, intracellu-lar multiplication slows and tissue cystsdevelop. A small cyst contains only a fewbradyzoites (the second stage of the asex-ual cycle) but a large one may containthousands. Cysts are found most fre-quently in brain and skeletal muscle andrepresent the quiescent stage of the para-site within the host. When a cyst rupturesthe bradyzoites are released and, trans-formed into tachyzoites, enter other cellsto complete the asexual cycle (Dubey andBeattie, 1988).

Initiation of the sexual cycle occurswhen a non-immune cat ingests food con-taminated by oocysts or containing tachy-zoites or tissue cysts. In the case of thelatter the cyst wall is dissolved by prote-olytic enzymes in the stomach and smallintestine and the released bradyzoites pen-etrate the epithelial cells of the small intes-tine. While the parasite spreads to brainand muscles where tissue cysts will

develop (asexual cycle), simultaneouslytoxoplasms also undergo gametogeny(sexual cycle) in enteroepithelial cells.Here, in the small intestine (most com-monly the ileum) gametocytes develop,over 3-15 days after infection. Microga-

metes form and are released to penetratemature macrogametes triggering the for-mation of an oocyst wall around each fer-tilized gamete. The oocysts (10 x 12 pm indiameter), each almost filled by thesporont, are then discharged into theintestinal lumen to pass out in the faeces.

Sporulation occurs within 1-5 days(depending on aeration and temperature) toproduce two ellipsoidal sporocysts, eachcontaining four sporozoites within eachoocyst (Dubey and Beattie, 1988).

Thus, during the 4-12 days after ingest-ing tissue cysts the cat is capable of shed-ding millions of oocysts in its faeces, afterwhich it will not normally excrete the par-asite again, although stress can trigger therecrudescence of infection (Dubey andFrenkel, 1974). Unrelated illness maytherefore lead to the excretion of oocystsin smaller numbers and for a shorter timethan in a primary infection. Cats may alsobecome infected by ingesting oocysts ortachyzoites but in this case they tend toproduce oocysts after 19 or 20 days, foronly a day or two and in relatively smallnumbers and even then about half of theseanimals will not excrete oocysts (Dubeyand Beattie, 1988).

2.2. Clinical disease

Sporulated oocysts ingested by a sus-ceptible pregnant sheep excyst in the smallintestines, each able to release the eightsporozoites. Four days later tachyzoitescan be found in the mesenteric lymphnodes, where they multiply (Dubey, 1984),and they in turn are released into the bloodto cause a parasitaemia, which may lastfrom the 5th until the 12th day after infec-tion (Dubey and Sharma, 1980a; Reid etal., 1982; Wastling et al., 1993), dissemi-nating infection to many tissues. The ces-sation of the parasitaemia coincides withthe onset of a protective immune responseand infection then persists as bradyzoites

within tissue cysts. However, in the preg-nant ewe infection may establish in the

gravid uterus where maternal immuno-logical responses may be altered and theability of the fetus, with its placenta, torecognize and respond to the parasite isnegligible in the early stages of gestationbut develops progressively with time sothat lambs are born immunocompetent(Salami et al., 1985). Toxoplasms initiallyparasitise the caruncular septa, the mater-nal tissues of the placentome, beforeinvading the adjacent trophoblast cells ofthe fetal villi and from there the rest of thefetus (Buxton and Finlayson, 1986). Thus,the outcome of infection early in gesta-tion can result in fetal death and resorp-tion/abortion while infection in the latter

part of gestation, when fetal immunity isrelatively well developed, may have noclinical effect, the offspring being bornnormal but infected and immune. In sheeptypical clinical signs of toxoplasma abor-tion usually result following infection inmid gestation, with ewes producing still-born and/or weakly lambs often accom-panied by a small, mummified fetus.Cotyledons on the accompanying pla-centa(s) will also show lesions visible tothe naked eye (Buxton, .1991). Abortionand neonatal mortality in goats is essen-tially similar to that seen in sheep whetheroccurring naturally (Munday and Mason,1979; Chhabra and Gautam, 1984; Dubey,1981a; Dubey et al., 1981; Nurse andLenghaus, 1986) or produced experimen-tally (Dubey et al., 1980; Dubey, 1988).During an acute infection in goats toxo-plasms may be excreted in the milk(Dubey, 1980; Skinner et al., 1990) andbe a possible source of human infectionif drunk unpasteurised (Skinner et al.,1990). Also, experimentally at least, tox-oplasms may be present in goat semen fora variable time after infection (Dubey andSharma, 1980b) but the epidemiologicalsignificance of this, as in sheep (Blewett etal., 1982), may be very slight.

2.3. Diagnosis

2.3.1. Pathological changes

Characteristically the placental cotyle-dons appear bright to dark red and speck-led with white foci of necrosis 2-3 mm indiameter which may be sparse or sonumerous that they can become conflu-ent, while the intercotyledonary allanto-chorion appears normal (Hartley andKater, 1963; Beverley et al., 1971 b). Vis-ible changes in lambs and kids vary, themost obvious being the mummified fetus,a small chocolate brown miniature of a

lamb/kid, often with its own small

grey-brown placenta. Fetuses dying laterin gestation are born in various stages ofdecomposition often with clear to bloodysubcutaneous oedema and a variableamount of clear to bloodstained fluid in

body cavities (Hartley and Kater, 1963).However, while these latter changes indi-cate an intrauterine infection they are notspecific to infection with Toxoplasma.

The most obvious histopathologicalchanges are the necrotic foci, visiblemacroscopically in the cotyledons. Micro-scopically they appear as large foci ofcoagulative necrosis, remarkably free ofinflammatory cells, which may becomemineralised with time. Sometimes smallnumbers of intracellular and extracellular

toxoplasms are visible, usually on theperiphery of the necrotic lesions or in avillus which is in the early stages of infec-tion (Buxton and Finlayson, 1986). In thefetal brain both primary and secondarylesions develop. Glial foci, typically sur-rounding a necrotic and sometimes min-eralised centre, often associated with amild lymphoid meningitis, represent a fetalimmune response following direct dam-age by local parasite multiplication. Tox-oplasms are only rarely found,.usually atthe periphery of the lesions. Focal leuko-malacia, seen most commonly in cerebralwhite matter cores, is also common and

is probably due to fetal anoxia in late ges-tation caused by advanced necrosis in theplacentome preventing sufficient oxygentransfer from mother to fetus (Buxton etal., 1982).

2.3.2. Detection of Toxoplasma gondii

While the most direct and establishedmethod of demonstrating Toxoplasmainfection in cases of abortion is to transmitthe parasite from aborted material (fetalbrain and placental cotyledons) to labo-ratory mice (Fleck and Kwantes, 1980) itis slow and expensive. Toxoplasma mayalso be grown in tissue culture in virtu-

ally any mammalian cell line and whilemore rapid than mouse inoculation it israrely used for routine diagnosis as it isexpensive and test samples may frequentlybe heavily contaminated. A more rapidbut less sensitive method of isolation isthe direct demonstration of T. gondii tissuecysts by centrifugation of lamb brainhomogenate on a discontinuous densitygradient of 30 and 90 % colloidal silicasolution (Blewett et al., 1983). ).

Immunohistochemical techniquesallowing visualisation of both intact T.gondii and antigenic debris in tissue sec-tions of aborted materials are convenientand sensitive methods and have the advan-

tage, when compared with attempts at iso-lation, of detecting toxoplasma antigeneven in decomposed tissues. The ABCindirect immunoperoxidase method (Vec-tor Laboratories, USA) and the peroxi-dase anti-peroxidase (PAP) technique(Uggla et al., 1987) are equally good.

Both viable and non-viable toxoplasmsmay be identified in tissues with the poly-merase chain reaction (PCR). Both the P30and the B 1 gene of T. gondii have beenused as PCR targets for the detection ofToxoplasma in various clinical specimenscollected from infected humans. Limitedstudies have also been carried out usingovine samples such as aborted placental

material, brain and peritoneal fluid fromaborted fetuses and lymph, blood andlymph nodes from artificially infected ewes(Wastling et al., 1993). Detection of T.gondii by amplification of the B 1 genewould seem to be more sensitive than bythe P30 gene due to the repetitive natureof the B 1 gene of which 25-50 copies arepresent in the genome of T. gondii com-pared with only a single copy of the longerP30 gene. Currently the technique is notused in routine diagnosis but the potential ofthe PCR to identify DNA in paraffin sec-tions from histopathological tissue blocksmay broaden its applicability (Ellis, 1997).

2.3.3. Serology

This is an important tool in the diag-nosis of ovine and caprine toxoplasmaabortion. The presence of specific anti-bodies in serum or tissue fluid from still-born lambs or kids or in precolostral serumfrom live offspring indicates uterine infec-tion. However, high toxoplasma antibodytitres in sera taken from ewes and nannyswithin a few weeks of abortion or pro-duction of stillborn lambs or kids can onlysuggest toxoplasmosis as titres remain rel-atively high for long periods after initialinfection. Serology will also indicate thedegree of exposure to infection in a groupof animals. The first method to be devel-

oped was the dye test (DT) of Sabin andFeldman (1948) but it is expensive, timeconsuming and not without hazard as it

requires live tachyzoites as antigen. Theindirect immunofluorescent antibody test(IFAT) gives titres comparable with theDT but is safer as it uses killed tachyzoites(Maley et al., 1997) and the latex aggluti-nation test (LAT) also performs well(Trees et al., 1989; Maley et al., 1997).The modified agglutination test (MAT)(Desmonts and Remington, 1980) hasbeen shown to perform particularly wellwith goat sera (Dubey et al., 1985)although for epidemiological studies theindirect haemagglutination test (IHA) (Pat-

ton et al.,1990), IFAT and LAT (Opel etal., 1991 ) are adequate. Both the IHA andLAT are easy to perform and the latter isavailable in kit form (Eiken Chemical Co.,Japan) and neither test requires species-specific antisera or conjugates.

The enzyme linked immunosorbent

assay (ELISA) for T. gondii antibodieshas been adapted for use in most domesticanimals including sheep (Buxton et al.,1988) and goats and can be made to dis-tinguish IgM and IgG antibodies and, as itis readily automated, it is suitable for han-dling large numbers of test sera.

2.4. Control

2.4.1. Cats as a source of infection

Sheep are frequently maintained in anenvironment significantly contaminatedwith oocysts and infection follows inges-tion of contaminated food or water

(Blewett, 1983; Blewett and Watson,1983), with pasture perhaps being the mostcommon source of infection, althoughwater can be a real threat not only to ani-mals but also to people (Bowie et al.,1997). Certainly, fields treated withmanure and bedding from farm buildingswhere cats live can cause infection (Faullet al., 1986) and cats defaecating in farmfeeds, such as hay and stored grain, willpose a risk (Plant et al., 1974).

Thus in the hypothetical situation inwhich a cat, actively shedding oocysts inits faeces, defaecates in 10 tonnes of grainstored on a farm, then the potential for theparasite being spread to livestock could beconsiderable. A single defaecation may con-tain as many as 10 000 000 oocysts. If fur-ther processing of the feed dispersed theseoocysts evenly throughout the grain theneach kilogram would contain between fiveand 25 sheep infective doses (McColgan etal., 1988). In this way cat faeces can createa large, potent, long-lasting source of infec-

tion for sheep. Thus, oocyst contaminationof farm feeds and bedding, as well as pas-ture, is a threat to susceptible, pregnantsheep and goats and is closely related to thenumber and distribution of cats.

Cats are born free of toxoplastna infec-tion and excretion of oocysts by them fol-lows establishment of a primary infection.Although it has been calculated that lessthan 1 % of all cats are shedding oocysts atany one time (Dubey and Beattie, 1988)this figure is probably higher for youngcats. Female feral cats can produce twoto three litters a year, each of up to eightkittens, and may rear their young com-munally (Macdonald, 1980). Numbers ofyoung cats are also dependent upon thedensity of breeding adults. In rural areasmale cats may have territories of 60-80hectares (250-200 acres) while femalesusually only occupy a tenth of this area(Macdonald, 1980). In an urban environ-ment these territories are considerablysmaller (Tabor, 1980). The area occupiedby feral cats is influenced by the supplyof food, which includes mice, voles,shrews, rats, rabbits and small birds (Mac-donald, 1980). Such animals persistentlyinfected with T. gondii are an importantsource of infection for cats (Jackson andHutchison, 1989; Peach et a]., 1989). Inaddition mice (Eichenwald, 1948; Bever-ley, 1959; De Roever-Bonnet, 1969), butprobably not rats (Dubey et al., I 997) areparticularly important because they canpass the parasite in utero without causingovert clinical disease or fetopathy. In thisway a reservoir of T. gondii tissue cystinfection for cats can exist in a particularpopulation of mice for a long time.

2.4.2. General management

During pregnancy a flock/herd, inwhich the majority are seronegative to T.gondii, could be at risk if it was allowedaccess to an environment contaminatedby cat faeces and so all food and water

should be kept free from soiling as far aspractically possible. Other measures toreduce environmental contamination byoocysts should be aimed at reducing thenumber of cats capable of sheddingoocysts. These would include selectiveculling of aged and diseased cats andattempts to control future breeding. If malecats are caught, neutered and returned totheir colonies the stability of the colonyis maintained; fertile male cats do not chal-lenge the neutered males (Tabor, 1980)and breeding is controlled. Thus the main-tenance of a small healthy population ofmature cats will reduce oocyst excretion aswell as help control rodents.

It is likely that a flock/herd, in whichseroconversion to T. gondii can be demon-strated in a majority of animals, is beingmaintained in an environment significantlycontaminated with T. gondii oocysts. Asyoung, seronegative, replacement stockwill be at risk, there is a case to be madefor attempting to expose them to a con-taminated environment before mating.Identification of such an environment isdifficult but is likely to be an area in oraround farm buildings where cats live, aswell as pasture spread with manure fromsuch buildings. While the foregoing is use-ful practical advice it does not guaranteesuccess and there is a clear need for more

precise methods of control.

2.4.3. Vaccination

Natural infection with T. gondÜ stimu-lates protective immunity in both sheep andgoats (McColgan et al., 1988) but inacti-vated toxoplasma tachyzoites, either alone(Beverley et al., 1971a) or in Freund’s sincomplete adjuvant (Wilkins et al., 1987)do not protect pregnant sheep against exper-imental challenge with the parasite. Simi-larly a preparation consisting of surface anti-gens of T. gondii combined with Quil Aprovided little protection against experi-mental challenge (Buxton et al., 1989).

The failure of these killed preparationsin sheep may be partly because, in naturalinfections, persistence of the parasite intissues continually stimulates immunity,as suggested in human toxoplasmosis(McHugh et al., 1997). However experi-ments in which mice and hamsters wereinfected with a live temperature-sensitivemutant of T. gondii, which does not persistin the host, as it cannot form bradyzoitesand cannot therefore form tissue cysts didinduce protective immunity (Waldelandand Frenkel, 1983; McLeod et al, 1988;Suzuki and Remington, 1990).

In 1988 the Ministry of Agriculture andFood (New Zealand) launched a live tox-oplasma vaccine for the control of ovinetoxoplasmosis (O’Connell et al., 1988;Wilkins et a]., 1988) and in 1992, afterfurther study (Buxton et al., 1991), it wasmarketed in the UK and Eire (Toxovax,Intervet UK) as a tissue-culture-grownvaccine. The vaccine consists of live S48

tachyzoites which were originally isolatedby mouse injection from a case of ovineabortion in New Zealand. After around3 000 passes twice weekly, in laboratorymice it was shown to have lost its ability todevelop bradyzoites in tissue cysts andunpublished data indicate that neither canthe tachyzoites initiate the sexual life cycleof the parasite in cats (Bos, pers. comm.).

Studies at the Moredun Research Insti-tute (Edinburgh, UK) showed that whensusceptible pregnant sheep were eachorally dosed with 2 000 sporulated T.gondii oocysts, a relatively severe chal-lenge, less than 18 % of lambs were bornlive and viable, whereas with vaccinatedewes similarly challenged 75 % of lambswere born live and viable. The placentallesions in vaccinated ewes, following chal-lenge, were much less frequent and/orsevere than in unvaccinated, challengedewes and the viable lambs born to the for-mer had significantly higher birth weightsthan those born to the unvaccinated ewes,thus enhancing their chances of survival

(Buxton et al., 1991, 1993a). Protectionafforded by one injection of the vaccinewas just as good after 18 months as it wasafter 6 months. Antibody may impede cellinvasion by tachyzoites and, with comple-ment, limit phagocytosis by macrophagesbut protective immunity in sheep given thelive vaccine is largely cell mediated(reviewed by Innes and Wastling, 1995)with CD4+ and CD8+ T cells (Innes et al.,1995a) and interferon gamma (Oura et al.,1993; Innes et al., 1995b) playing animportant role in suppressing T. gondii par-asitaemia in sheep (Buxton et al., 1994)thereby protecting the developing fetusfrom a maternal infection.

In field studies around half of 1 1 000

1-year-old female sheep in 10 1 flocks werevaccinated. Subsequently 20 flocks wereexposed to significant natural challengefrom the parasite and in vaccinated ani-mals abortions were reduced and lambingpercentages significantly improved whencompared with unvaccinated sheep in thesame flocks (Spence et al., 1992) thus con-firming the findings of field trials carriedout in New Zealand.

The manufacturers recommend that in nthe first instance the whole flock is vacci-nated at least 3 weeks before mating and insubsequent years newcomers to the flock,usually young replacement stock, are vac-cinated. Only one injection is required inthe life of the sheep. The vaccine has ashelf life of 7-10 days and is capable ofinfecting people so it must be handled withcare strictly according to the manufactur-ers recommendations.

As with sheep, the majority of goatspreviously exposed to infection with T.gondii develop a protective immunity tothe parasite so that they are protectedagainst subsequent challenge during preg-nancy (Obendorf et al., 1990), althoughrepeat abortions have been recorded(Dubey, 1982). Toxovax is not licensedfor use in goats. Immunity induced ingoats by experimental infection with the

related coccidian parasite Hammondiahammondi has been shown to offer some

cross-protection against challenge with T.gondii both in non-pregnant (Dubey,1981 b) and pregnant animals (Dubey,198 Ic; Munday and Dubey, 1988) but thisavenue of research has not been pursued.

2.4.4. Pharmaceuticals

Even though a live vaccine for toxo-plasmosis is available for use in sheepthere will always be a need for other meth-ods of preventing infection becomingestablished in unprotected ewes/nannysand for treating infection once it is foundin animals still to give birth. Chemopro-phylaxis with monensin given in the feedat the rate of 15 mg/animal/day duringpregnancy, can significantly suppress atoxoplasma infection in sheep (Buxton etal., 1988) but it is not licensed for this pur-pose. However the anticoccidial drugdecoquinate fed daily at 2 mg/kg bodyweight, can also significantly reduce theeffect of T. gondii oocysts ingested bypregnant sheep (Buxton et al., 1996) and islicensed for this use in sheep.

Both monensin and decoquinate workbest if they are already being fed to sus-ceptible ewes at the time they encounterinfection rather than after infection isestablished. However, it has been shownthat a combination of pyrimethamine andsulphamezathine, a well tried treatment inhuman medicine, which is effectivebecause it blocks folate synthesis, is effec-tive in the treatment of infected sheep(Buxton et al., 1993b). The drug combi-nation, baquiloprim and sulphadimidine(Zaquilan@, Schering-Plough AnimalHealth, UK) also blocks folate synthesisand has given promising results in a con-trolled pilot study in non-pregnant sheep(Buxton, unpublished data).

The control of toxoplasmosis in sheep(and goats) is now easier than before. Ourknowledge of its epidemiology permits

sensible management procedures to be fol-lowed to minimise the weight of infectionin the environment, pharmaceutical prepa-rations have been identified which maybe used to curb disease and there is aneffective vaccine for use in sheep.

3. NEOSPOROSIS

Neospora careinum has been recognisedonly since the 1980s and while it appearsto be a major cause of fetal loss in cattle(Dubey and Lindsay, 1996) it also maycause clinical loss in sheep and goats.

3.1. Life cycle

Neospora caninum naturally infects awide range of hosts including dogs, cat-tle, horses, deer (Dubey and Lindsay,1996) and foxes (Buxton et al., 1997a), aswell as sheep and goats (see below). Anasexual life cycle involving bradyzoites, intissue cysts, and tachyzoites are the onlylife cycle stages recognised at the time ofwriting (figure 2), although a sexual lifecycle in a definitive carnivore host, whichproduces oocysts in a similar way to thatin which cats produce T. gondii oocysts,has been predicted (Dubey and Lindsay,1993). The tachyzoites, which divide byendodyogeny, may be either crescentshaped or ovoid 3-7 pm long by 1-5 pmwide depending on their stage of division.The tissue cysts range from 20-100 pmin diameter and typically have a clearlydiscernible cyst wall 1-2 pm thick (Dubeyand Lindsay, 1996).

3.2. Clinical disease

Clinical neosporosis is most importantin cattle in which it is now recognised tobe a serious cause of abortion in manycountries around the world (Dubey andLindsay, 1996). Vertical transmission from

cow to developing fetus is likely to beimportant in the epidemiology of bovinedisease and is currently the only route oftransmission to have been demonstrated. Itis presumed that recrudescence of a per-sistent cyst infection in the mother occurs

during pregnancy permitting a tachyzoiteparasitaemia which allows the parasite toinvade the gravid uterus, the placenta andthen the fetus. Infection early in gestation,when the fetal immune system is little

developed can be fatal with fetal resorptionor abortion. Exposure of an older fetus,with its better developed immune systemmay result in the birth of a clinically nor-mal but congenitally infected calf (Dubeyand Lindsay, 1996).

While pregnant sheep have been shownto be very susceptible to experimentalinfection with N. caninum (Dubey andLindsay, 1990; McAllister et al., 1996;Buxton et al., 1997b) to date there is littleevidence of it being a significant fieldproblem (Otter et al., 1997) with only onereported case of natural, congenitalneosporosis in a weak, ataxic lamb whichdied when 1 week old (Hartley and Bridge,1975; Dubey et al., 1990). It is, however,noteworthy that the spinal lesions in thisanimal were similar to those seen in calves

congenitally infected with N. caninum.Neo.rpora has also been associated with

abortion in goats in the USA (Barr etal.,1992; Dubey et a]., 1992) and CostaRica (Dubey et al., 1996) but while only afew fetuses were lost there was evidenceof seroconversion to N. caninum in otheranimals in the Costa Rican herd.

3.3. Experimental neosporosis

Dubey and Lindsay (1990) showed inan uncontrolled pilot study with two ewesthe susceptibility of the ovine fetus toNeospora and McAllister et al. (1996) andBuxton et al. (1997b) confirmed this inlarger controlled studies. As with bovineneosporosis and ovine toxoplasmosis theyounger fetuses were shown to be more

susceptible than those challenged later ingestation (McAllister et al., 1996). Fetallesions were most commonly manifest as ameningoencephalitis with characteristicfoci of inflammation showing signs oforganisation, typically with a necrotic cen-tre surrounded by microglial and lymphoidcells. Light lymphoid vascular cuffs andmeningeal infiltrates and focal microgliosiswere also present. Milder lesions occurredless consistently in other tissues such as theheart and lungs (McAllister et al., 1996;Buxton et al., 1997b). Serial sampling offetuses from experimentally infected ewesshowed a progressive maturation of lymph

node structure with time and this correlatedwith the early production of IgM antibodyfollowed by an IgG response to Neo.spora(Buxton et al., 1997b).

In the placenta there were scattered fociof necrosis involving fetal villous tissueand adjacent caruncular septa with accu-mulations of necrotic debris apparently ofmaternal and chorionic epithelial origin.Typically a non-suppurative inflamma-tory infiltrate was present in surroundingmaternal and fetal tissue as well as chorio-allantoic membrane (McAllister et al.,1996; Buxton et al., 1997b). In one studyneospora organisms were readilydetectable (McAllister et al., 1996) whilein the other they were much less frequent(Buxton et al., 1997b). While the firststudy used a mixture of the Nc-Liverpooland NC-2 isolates the latter study usedonly the Nc-Liverpool isolate and so it ispossible that strain virulence is also impor-tant. Subsequent studies with the NC-1 Iisolate in pregnant sheep (Buxton et al.,1998) have shown it to be more virulentthan Nc-Liverpool and to produce greaterinflammation and more severe and fre-

quent focal necrosis, sometimes associ-ated with vasculitis and thrombosis.

The focal necrosis visible macroscopi-cally as white spots scattered throughoutthe cotyledons and indistinguishable fromovine toxoplasmosis does differ micro-scopically from the latter. In toxoplasmo-sis the chorio-allantois is not involved,focal necrosis in placental cotyledons isremarkable for its relative lack of inflam-

mation, and vasculitis and thrombosis donot feature. Pathological changes in fetaland placental tissues from naturally occur-ring and experimentally producedneosporosis in goats would appear to besimilar to those seen in natural and exper-imental infections in sheep (Barr et al., .,

1992; Dubey et al., 1992, 1996; Lindsay etal., 1995). Vertical transmission to kidsdid not occur in a subsequent pregnancy(Lindsay et al., 1995).

In a preliminary report experimentalinfection with N. caninum before matingpartially protected ewes and their devel-oping fetuses against a second challengewith the parasite during pregnancy. Vac-cination against toxoplasmosis (Toxovax,Intervet UK) however did not protectagainst challenge with Neospora (Buxtonet al., 1997c). Thus it may be more diffi-cult to induce solid immunity in sheep toN. caninum than to T. gondii although pro-tection against both parasites probablyresults from stimulation of cell mediatedimmune mechanisms as in vitro studieshave shown that recombinant ovine inter-feron gamma (IFN-gamma) can signifi-cantly inhibit multiplication of N. can-inum in ovine fibroblast cell cultures

(Innes et al., 1995c) as is the case with T.gondii (Oura et al., 1993). Studies withmice have underlined the importance ofIFN-gamma in neosporosis and shownthat interleukin-12 also has a role to play(Khan et al., 1997).

3.4. Diagnosis

Serology in sheep and goats has largelybeen carried out with the IFAT to detect

IgM and IgG (Lindsay et al., 1995; Buxtonet al., 1997b), IgG alone (Dubey et al.,1996; McAllister et al., 1996) or undefinedimmunoglobulin (Otter et al., 1997).ELISA have been developed for use in cat-tle using soluble extracts of sonicatedneospora tachyzoites (Pare et al., 1995;Osawa et al., 1998), recombinant antigens(Lally et al., 1996a), extracted tachyzoiteantigen incorporated into an immuno-stimulating compound (ISCOM) (Bjork-man et al., 1997) or whole organisms(Williams et al., 1997). One of these assayshas been adapted successfully for use withsheep and goat sera (Osawa et al., 1998).

The PCR has been used both to make

comparisons between N. caninum and T.gondii (Ellis et al., 1994; Holmdahl et al.,

1994) and as a potentially powerful diag-nostic tool elegantly specific for N. can-inum (Holmdahl and Mattsson, 1996;Kaufmann et al., 1996; Lally et al., 1996b;Muller et al., 1996; Payne and Ellis, 1996;Yamage et al., 1996). Their use in the fieldstill remains to be demonstrated but their

application to sections cut from waxembedded pathological samples couldprove very valuable (Ellis, 1997).

3.5. Treatment

Dubey and Lindsay (1996) report thatclindamycin, sulphonamides and/orpyrimethamine and trimethoprim with sul-phadiazine have been used by variousauthors, often to good effect, in the treat-ment of clinical canine neosporosis. Theiruse at the appropriate dose rate could wellbe beneficial in the event of a diagnosisof clinical neosporosis being made ineither a sheep or goat considered suitableand worthy of treatment.

4. SARCOCYSTIOSIS

Sarcocystis (synonyms; sarcosporidia,Meischer’s tubules) exists as numerousspecies (Uggla and Buxton, 1990). In 1972Rommel and colleagues reported that Sar-cocystis underwent a coccidian-like life

cycle with each species having a distinctdefinitive host(s) (usually a carnivore) andan intermediate host (Rommel et al.,1979). Infections are recognised to occurin all parts of the world and farm animalsare intermediate (tangential) hosts for anumber of species (figure 3). Sheep arethe intermediate host for four species, twomicrocyst species (S. tenella [syn. S. ovi-canis] and S. arieticanis) and two macro-cyst species (S. gigantea [syn. S. ovifelis]and S. medusiformis) while goats are theintermediate host to S. capracanis, S.hir-cicanis and S. moulei.

4.1. Life cycle

Farm animals become infected follow-

ing ingestion of sporocysts released by thedefinitive host. Following excystation inthe intestinal lumen sporozoites penetratethe gut wall and multiply asexually by twoschizogonous cycles in endothelial cellsof small blood vessels (first and secondgeneration merogony), with an associatedparasitaemia. The merozoites releasedafter second generation merogony pene-trate muscle cells and form characteristic

sarcocysts filled with bradyzoites, althoughthey may also invade the central nervoussystem (Rommel, 1985). Ingestion ofinfected muscle by the definitive host trig-gers a sexual life cycle in the intestinal

lining with the formation of macro- andmicrogamonts leading to the developmentof oocysts and then sporocysts which areexcreted in the faeces.

4.2. Clinical disease

The parasite is ubiquitous and infec-tions, which are extremely common, occurfollowing ingestion of food or water con-taminated with sporocysts. While most

species appear to be non-pathogenic, afew, particularly those with a caninedefinitive host, may cause clinical illness(Uggla and Buxton, 1990). Symptomsinclude fever, anaemia and inappetanceand an associated reduction in productiv-ity. Fayer and Elsasser (1991) have sug-gested that the fever results from therelease of interleukin-1 (IL-1) andprostaglandin E2 while infection of

macrophages by Sarcocystis causes releaseof tumour necrosis factor-alpha whichi) causes inappetance, ii) together withIL- causes anaemia, iii) suppressesrelease of pituitary growth hormone, withresultant weight loss. It has also been sug-gested that retardation of growth in pigsinfected with S. meischeriana is due to the

parasite influencing an insulin-like growthfactor (IGF) and an IGF binding protein(Prickett et a]., l 992).

In some instances abortion and still-births (Uggla and Buxton, 1990; Mackieet al., 1992) or central nervous signs,including hind limb weakness, ataxia, pare-sis and death (Jeffrey et al., 1988; Dubey etal., 1989; Hamir et al., 1993; O’Toole etal., 1993) have been associated with sar-cocystiosis. Hind limb weakness, quadri-paresis and death have also been recordedin sheep suffering from acute myopathyassociated with a sarcocystis infection (Jef-frey et al., 1988) and in cattle eosinophilicmyositis is recognised (Granstrom et al.,1989; Saito et al., 1993). In experimen-tally induced disease the severity of clini-

cal symptoms is dose dependant (Rom-mel, 1985). Thus O’Donoghue and Wilkin-son ( 1988) gave lambs 104 S. tenella sporo-cysts but recorded no clinical signs whileMunday ( 1981 ) showed that pregnantsheep experimentally infected with 6 x 104S. tenella sporocysts developed a mildfever followed by anaemia and gave birthearly to small, weak lambs. After parturi-tion the ewes developed myositis,myocarditis and encephalitis and died.

4.3. Diagnosis

As infection is extremely common it isvery difficult to attribute the presence of

sarcocysts to a disease process. Clinical

symptoms are not commonly useful as ameans of diagnosis as the vast majorityof natural sarcocystis infections are sub-clinical and symptoms, when present, maybe vague and non-specific. In cases of fatalacute sarcocystiosis tissues, such as thetongue, may appear mottled due to capil-lary haemorrhages (Uggla and Buxton,1990). Histopathology is invaluable butonly evidence of an active infection per-mits the possibility of a diagnosis of acutesarcocystiosis. Inflammatory changes maybe confined to the central nervous system(CNS), often with the more prominentlesions in the spinal cord. Characteristi-cally a non-suppurative meningo-encephalitis, associated with sarcocyst-like merozoites and meronts, gliosis andassociated oedematous changes, is pre-sent (Jeffrey et al., 1988; Dubey et al.,1989; O’Toole et al., 1993). Mackie et al.( 1992) also report a stillborn goat kid witha multifocal, necrotising, non-suppurativeencephalitis with associated vascularendothelial cells containing sarcocyst-likemeronts containing merozoites. Outwiththe CNS the heart, tongue and liver mayshow marked mononuclear cell inflam-mation while tissues such as skeletal mus-

cle, lung and kidney are usually lessaffected (Uggla and Buxton, 1990),

although in a spontaneous outbreak ofacute myopathy in sheep, linked with asarcocystis infection, histopathologicallesions were confined largely to skeletalmuscles and consisted of a non-suppura-tive myositis associated with mature,immature and degenerate sarcocysts (Jef-frey et al., 1989). As with toxoplasmosisand neosporosis, immunohistochemistrymay be valuable (Jeffrey et al., 1988;O’Toole et al., 1993).

Serological methods most commonlyused are the IFAT (Tadros and Laarman,1982; Tenter, 1988; Uggla et al., 1987)and ELISA (Tadros and Laarman, 1982;Weiland et al., 1982; Gasbarre et al., 1984;O’Donoghue and Weyreter, 1984; Smithand Herbert, 1986; Tenter 1988). Duringthe first merogonous stage of a Sarcocys-tis infection it may be difficult to detectan antibody response. This may be becausethe serological test uses an antigen prepa-ration derived from cystozoites which maybe antigenically distinct from the first gen-eration merozoites (Uggla and Buxton,1990). In this respect good sensitivity wasreported for an ELISA using merozoiteantigen, developed by Savini et al. ( 1994).The detection of an IgM response will sug-gest an acute or recent infection while an

IgG response, in the absence of IgM anti-bodies, indicates a longer standing persis-tent infection (Uggla and Buxton, 1990).More recently PCR methods have beenreported for a number of Sarcocystis spp.(MacPherson and Gajadhar, 1994; Tenteret al., 1994; Guo and Johnson, 1995;Joachim et al., 1996; Marsh et al., 1996)and they will prove valuable in establishingmore accurate diagnostic procedures.

4.4. Control

Control is difficult but when a problemis perceived to occur attempts should bemade to prevent contamination of animalpasture and other feed by faeces from

dogs, foxes, cats and other possible defini-tive hosts, or prevent access of young sus-ceptible animals to such locations wherecontamination cannot be prevented.

4.4.1. Immunity

It seems likely that Sarcocystis spp.stimulate immune mechanisms similar tothose induced by T. gondii and N. can-inum. While little is known of the specificevents which occur, in broad terms exper-imentally induced infection with a ’low’dose stimulates protective immunity to asecondary, homologous, ’high’ (fatal)challenge dose (see below).

Goats experimentally infected with S.capracanis sporocysts resisted an other-wise fatal homologous challenge as earlyas 14 days later (Dubey, 1981 d). Howeverimmunity to one species does not appear totrigger protective immunity to anotherspecies. Thus Fayer ard Dubey (1984)showed that calves given 1 x 105 S. CrUZi

sporocysts developed non-fatal clinicalsarcocystiosis and, when challenged 91 1days later with 5 x 105 S. cruzi sporocysts,they again showed clinical signs of feverand anaemia but survived. Four controlcalves given only the second challengedied 27 (n = 2) and 28 (n = 2) days laterand a further group of four given an initialinfection of 1 x 105 S. hirsuta sporocystsand challenged 91 days later with 5 x 105S. ct-uzi sporocysts also died 27 (n = 2)and 28 (n = 2) days after the second chal-lenge. However Savini et al. ( 1996)showed that cows, considered to have been

naturally infected with Sarcocysti.s, werenot protected against a subsequent chal-lenge with sporocysts or merozoites of S.cruzi during pregnancy. The former treat-ment caused stillbirths while the cows

challenged with the latter produced livecalves which were significantly lighterthan control calves. These workers con-cluded that protective immunity from nat-ural infection was transient.

Lambs experimentally infected with S.tenella sporocysts developed specific IgMand IgG to the parasite but neither fol-lowing this primary challenge nor after agreater secondary challenge with the sameparasite did they respond in a lymphocytestimulation assay (LSA) to S. tenella anti-gen (either bradyzoite or sporozoite)(O’Dono!!hue and Wilkinson, 1988). Thisis in contrast to work by Gasbarre et al.( 1984) who demonstrated with a LSA ablastogenic response in sheep to,S. tenellaantigen between 24 and 52 days afterexperimental infection (dpi) with S. teuellnsporocysts. The authors also showed thatcalves infected with S. crrri developed anearlier and shorter ( 15-21 dpi) blastogenicresponse to S. cru:,i antigen in a similarLSA. Granstrom et al. ( 1989) suggestedthat this latter brief response may haveoccurred in the presence of continued anti-

genic stimulation and thus been associ-ated with some form of parasite inducedimmunosuppression. Experiments in micewith S. l71l/ris have shown that the para-site causes both cell-mediated and humoral

immunosuppression and that induction ofimmune memory appears to be affectedwhile expression of immune memoryremains unaffected (Gill et a]., 1988a).These workers also showed that S. lI1urisinfection of mice causes splenomegalywith increased numbers of B-cclls and, toa lesser extent, T-cells (Gill et al., 1988b).Whether this is linked to the observationthat a Sarcocystis lectin (from S. gigan-ten) has both mitogcnic properties as wellas being a B cell activator (Tietz et al.,1990) remains to be seen.

Eosinophilic myositis (muscular sar-cocystiosis) may be observed in cattle atslaughter. On inspection affected animalshave yellow-green rice-grain sized nod-ules throughout their muscles. Microscopicexamination has shown that each lesion

represents a focus of necrosis infiltratedby eosinophils as well as lymphocytes andmultinucleate giant cells (Saito et al.,

1993). Both sarcocystis antigen, IgG andIgE have been demonstrated within suchlesions and specific IgE antibody has beendemonstrated in the serum of affected cat-tle. It has been suggested that type-1 Ihypersensitivity is involved in the patho-genesis (Granstrom et al. 1989, 1990).

4.4.2. Pharmaceuticals

Treatment of clinical cases may be

attempted and good results are recordedby Moore ct al. ( 1997) who used trimetho-prim-sulphamethoxazole and pyrimetha-mine and vitamin E for the treatment ofhorses clinically affected with S. nouronu.

5. CURRENT AND FUTURERESEARCH

Sarcocv.sti,s spp. are capable of caus-ing both clinical and subclinical illness in

sheep, goats and other animals and sarco-cystiosis is probably underdiagnosed as acause of production loss. It must be a pri-ority to establish and deploy better diag-nostic procedures so as to accuratelydefine the true cost of these losses. Immu-

nity develops following infection and itwould seem to be both long lasting and tobe associated with persistence of a viable’quiet’ infection. Whether this quiet infec-tion repeatedly stimulates the immune sys-tem so maintaining an effective degree ofimmunity in the host is not known andneither is it clear to what extent recurrent

exposure to infection is important. Theseapparently simple questions need to beaddressed together with the more detailedmechanisms of host immunity and theirrole in protecting the host, as well as per-haps causing pathological change and clin-ical illness, in this very common infec-tion.

Neosporosis in sheep and goats, how-ever, would not appear to be as common afield infection as in cattle, even though

they often share a common environment,possibly because vertical transmissionoccurs much more readily in the latter.However, experimental ovine and caprineneosporosis is readily reproduced, offersa good model of the bovine infection andpermits a more detailed analysis of certainaspects of the host/parasite interaction thancould be achieved with cattle. At the same

time any discrepancies between the ‘modelinfection’ and bovine neosporosis, such asvertical transmission, could point out thecrucial differences in the respective hostimmune responses that could be exploitedin the development of control measuresfor neospora abortion in cattle.

Toxoplasmosis in sheep and goats isimportant in its own right but is also agood model for studies of human toxo-plasmosis as the latter more closely resem-bles the infection in sheep than it does themore extensively researched infection inmice (Innes and Wastling, 1995). The verysuccess of Toxoplasma as an intracellularparasite is that it would appear able toinfect any nucleated cell-type in anywarm-blooded vertebrate. This very ubiq-uity and the relative ease with which a liveinfection (and hence live vaccine) inducesimmunity is in indirect proportion to thedifficulty scientists have had in developinga non-viable vaccine. Success will requirea detailed knowledge both of the parasiteand the host response to it, including howtachyzoites bind to and subsequentlyinvade cells (Joiner and Dubremetz, 1993;Kasper and Mineo, 1994) and the role ofthe secretory organelles (rhoptries,micronemes and dense granules) in estab-lishing the tachyzoite in its para-sitophorous vacuole in the cell (Joiner andDubremetz, 1993; Cesbron-Delauw, 1994)where it multiplies. Equally it has beensuggested that as Toxoplasma naturallyinfects animals orally, research should notignore the potential role of mucosal immu-nity. In support of this experiments haveshown that the purified major surface

tachyzoite protein SAG 1 (30kDa/P30 anti-gen) in association with ’cholera toxin’presented to mice by the nasal route canvery substantially reduce subsequentdevelopment of T. gondii tissue cysts inbrain (Debard et al., 1996). Eventual suc-cess with these and other avenues ofresearch will lead eventually to a cheapvaccine to prevent human and animal tox-

oplasmosis (Araujo, 1994) but also theknowledge gained will provide valuableinsights into how to stimulate protectiveimmunity to other apicomplexan proto-zoa such as Neo,spora and Sarcocy.stis spp.

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