Seroprevalence of sarcocystosis in the local communities of Pangkor and Tioman Islands using recombinant surface antigens 3 (rSAG3) of Sarcocystis falcatula.

OBJECTIVE: To investigate the seroprevalence of Sarcocystosis in the local communities of Pangkor and Tioman islands, Malaysia, by using antigenic recombinant surface antigens 2 and 3 from Sarcocystis falcatula (rSfSAG2 and rSfSAG3) as the target proteins via Western blot and ELISA assays. METHODS: SfSAG2 and SfSAG3 genes were isolated from S. falcatula and expressed in Escherichia coli expression system. A total of 348 serum samples [volunteers from both islands (n = 100), non-Sarcocystis parasitic infections patients (n = 50) and healthy donors (n = 100)] were collected and tested with purified SfSAGs in Western blot and ELISA assays to measure the seroprevalence of human sarcocystosis. RESULTS: None of the sera in this study reacted with rSfSAG2 by Western blot and ELISA. For rSfSAG3, relatively high prevalence of sarcocystosis was observed in Tioman Island (75.5%) than in Pangkor Island (34%) by Western blot. In ELISA, the different prevalence rate was observed between Tioman Island (43.8%) and Pangkor Island (37%). The prevalence rate in other parasitic infections (amoebiasis, cysticercosis, filariasis, malaria, toxocariasis and toxoplasmosis) was 30% by Western blot and 26% by ELISA. Only 8% (by Western blot) and 10% (by ELISA) of healthy donors showed reactivity towards rSfSAG3. CONCLUSION: This is the first study reporting a seroprevalence of sarcocystosis in Pangkor and Tioman Islands, Malaysia. The combination of Western blot and ELISA is suitable to be used for serodiagnosis of sarcocystosis. With further evaluations, SfSAG3 can potentially be used to confirm infection, asymptomatic screening, surveillance and epidemiological studies.

Immunoprotective responses against murine sarcocystosis by ß - Irradiated sporocysts.

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using ß-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 µSv ß-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4+ and CD8+ T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8+ T, NK1.1+ and FasL+ cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1+ & FasL+ cells, serum IFN-γ and intestinal IgA, while CD4+ and CD8+ T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. ß-irradiated sporocysts with 1.84 µSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver.

Seropositivity to Sarcocystis infection of llamas correlates with breeding practices.

Production of llama (Lama glama) meat in rural communities of the Andean regions is largely affected by Sarcocystis spp. infection. Macroscopic cysts develop in muscles as a consequence of S. aucheniae parasitism, often resulting in meat downgrade or condemnation. Llama meat production is informal in Argentina but has broad perspectives for improvement, and would significantly benefit from the development of standardized control methodologies. This work analyzes whether the presence of anti-Sarcocystis spp. antibodies in llamas is influenced by factors such as geographic region and/or herd management practices. To this aim, an indirect ELISA was set up based on a ~23kDa soluble immunogenic protein fraction (Sa23), isolated from S. aucheniae macrocysts (Sa23-iELISA). Serum samples (n=507) were collected from llamas bred under three different conditions: (i) with no sanitation controls and in the presence of pastoral dogs by small producers of different localities of the Argentine Puna (Group I, n=237); (ii) with sanitation controls and no pastoral dogs, in fenced fields of an experimental agricultural station in the Argentine Puna (Group II, n=167); and (iii) with sanitation controls and no pastoral dogs in fenced fields of farms of the humid Pampas (Group III, n=103). Results of the Sa23-iELISA were expressed as percentages of positivity with respect to a reference Sarcocystis-positive serum. Notably, the percentage of sera that fell above the cut-off (31.5% positivity) in group (i) was significantly higher (p<0.001) than those of groups (ii) and (iii) (50% vs 23% and 26%, respectively). These results indicate that herd management practices constitute a critical risk factor for sarcocystiosis in llamas. Differences in these practices include feeding of dogs with raw Sarcocystis-infected llama meat, with the consequent maintenance of the parasite life cycle by the contamination of pastures and water with fecal-derived infective oocysts/sporocysts. Additionally, the itinerancy of llama herds in search for pastures and water sources possibly exposes animals to a higher number of infective foci. On the other hand, percentages of seropositive llamas kept under controlled conditions in the Puna or the humid Pampas were not significantly different, suggesting that climate, altitude, and/or pasture characteristics do not influence Sarcocystis-infection. Male gender and older age of llamas were found to be propensity factors for sarcocystiosis in llamas bred in La Puna under controlled conditions. Availability of diagnostic tools, as well as increased knowledge on the parasite and its epidemiology, will allow the design of control strategies for SAC sarcocystiosis.

Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil / Prevalência de anticorpos contra Neospora spp e Sarcocystis neurona em jumentos do nordeste do Brazil

Abstract Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples tested by IFAT (cut-off ≥50) and 21% (69 donkeys) by the direct agglutination test (SAT ≥50). The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051). This is the first report ofNeospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

Resumo Sarcocystis neurona e Neospora hughesi são protozoários coccídios que infectam equídeos e são responsáveis por doenças neurológicas nessas espécies. Neste estudo, a soroprevalência de infecção porS. neurona e Neospora spp. foi determinada em amostras de 333 soros de jumentos da Região Nordeste do Brasil. Anticorpos contra Neospora spp. foram detectados em 2% (7 jumentos) dos 333 animais examinados pela reação de imunofluorescência indireta (RIFI), com ponto de corte de 40. Anticorpos contra S. neurona foram detectados em 3% (10 jumentos) das amostras pela RIFI (ponto de corte de 50) e em 21% (69 jumentos) pela técnica de aglutinação direta (SAT - ponto de corte de 50). SAT e RIFI, para diagnóstico de S. neurona, apresentaram uma baixa concordância (Kappa = 0,051). Essa é a primeira observação de anticorpos anti-N. caninum em jumentos brasileiros e a primeira detecção de anticorpos contra S. neurona nessa espécie.

[Muscular sarcocystosis after travel to Malaysia: a case series from Germany]. / Muskuläre Sarkozystose nach Malaysiareise: eine Fallserie aus Deutschland.

BACKGROUND: Since 2011, about 100 travellers to the island of Tioman, Malaysia, have been diagnosed worldwide with suspected muscular sarcocystosis, a previously only sporadically observed parasitic disease. Source of infection and therapy remain unclear. Final diagnosis requires microscopic identification of cysts in muscle biopsies. The study objective was a systematic description of characteristic symptoms, laboratory investigations and treatment response. METHODS: Systematic case series. RESULTS: The 26 cases of 5 centers for tropical medicine in Germany showed a characteristic biphasic development: symptoms began in general 2 weeks after mid-holidays (min. 7.5, max. 22 days) with unspecific fever and headaches lasting for almost 1 week. After an asymptomatic period of 2 weeks, severe myalgia (6.5, scale 0-10) and fever developed and lasted for about 6 weeks (min. 7, max. 207 days), accompanied by creatin-phosphokinase(CK)-elevation (up to 3.5 times), and eosinophilia (2.9 times). One out of two muscle biopsies revealed a cyst typical for sarcocystosis. In 6 out of 7 patients an increase in Sarcocystis-specific antibody concentration could be demonstrated by ELISA. Treatment with systemic steroids and albendazole, or ivermectin resulted in significant symptomatic improvement in most of the patients. One patient was treated early with cotrimoxazole and subsequently did not develop a second phase of the disease. All patients had stayed in the North-West of the island Tioman. CONCLUSIONS: Muscular sarcocystosis develops in a biphasic pattern with initial fever and later prolonged myalgia, eosinophilia, and CK-elevation. Steroids achieve symptomatic relief in the late phase. Early cotrimoxazole-therapy could possibly prevent parasitic muscle invasion. In fever after travel to Malaysia differential diagnosis should include sarcocystosis. The source of infection appears to be located in North-West of Tioman. Further studies are needed, including addressing early diagnosis and treatment.

Experimental transmission of Sarcocystis muris (Apicomplexa: Sarcocystidae) sporocysts from a naturally infected cat (Felis catus) to immunocompetent and immunocompromised mice.

Cats serve as definitive hosts for zoonotic Toxoplasma gondii , a protozoan that threatens human reproductive health, but they also excrete sporocysts of related protozoan that pose no known human health risk. Here we provide the first definitive evidence for natural infection with the enzootic parasite Sarcocystis muris, one such enzootic parasite. Sporulated Sarcocystis sp. sporocysts were found in rectal contents of an adult feral cat ( Felis catus ) in Giza, Egypt. After these sporocysts were orally inoculated into 2 Swiss Webster mice, sarcocysts were found to have developed in skeletal muscles 114 days later. As observed through transmission electron microscopy, the cyst wall corresponded to Type 1, and the parasitophorous vacuolar membrane had tiny outpocketing of blebs (<200 nm thick) that were not invaginated into the interior of the cyst; these structures were identical to the sarcocyst wall described for a Costa Rican isolate of S. muris that has served as an experimental model for nearly 4 decades. Two parasite-free cats fed sarcocyst-infected muscles developed patent infections; fully sporulated sporocysts (10-11 × 7.0 µm) were found in the lamina propria of small intestines of cats killed 6 and 7 days postinoculation (PI). Interferon gamma gene knockout (KO) mice were orally inoculated with sporocysts from experimentally infected cats, and their tissues were examined histologically; sarcocysts were found in 5 KO mice killed 87, 115, 196, 196, 196 days PI, but no stages were seen in 5 KO mice 10, 14, 14, 18, and 39 days PI. Bradyzoites were released from intramuscular sarcocysts of a KO mouse killed 115 days PI and orally inoculated into 5 KO mice. No stage of Sarcocystis was found in any organ (including intestinal lamina propria) of KO mice killed 4, 8, 81, 190, and 190 days PI, confirming that the definitive host is required to complete the life cycle even in the case of immunodeficient mice. This is the first confirmation of S. muris infection in a naturally infected cat anywhere.

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi.

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.

Lambs infected with UV-attenuated sporocysts of Sarcocystis ovicanis produced abnormal sarcocysts and induced protective immunity against a challenge infection.

The present study surveyed the prevalence of natural infection of the sheep esophagus muscle with sarcocysts of Sarcocystis ovicanis and examined induction of protective immunity using UV-attenuated sporocysts. The overall prevalence of natural infection of the sheep was 95%. Infectivity of the collected sarcocysts was confirmed by shedding of sporulated oocysts after feeding infected esophageal tissues to dogs. To induce protective immunity, lambs were immunized 3 times (once a week) with 1.5 x 10(4) sporocysts exposed to UV-light for 30 min (UV-30 group) or 60 (UV-60 group) min and then challenged with 1.5 x 10(4) normal sporocysts at the 3rd week post the 1st vaccination. These lambs showed high survival and less clinical signs of sarcocystosis than normal infected lambs. The attenuated sporocysts produced abnormal cysts; small in size and detached from the muscle fiber. These abnormalities were more obvious in UV-60 group than UV-30 group. Also, the IFN-gamma level and lymphocyte percentage were increased while the total leukocyte count was decreased in the UV-60 group compared with other groups. The high level of IFN-gamma may be an evidence for the induction of Th1 responses which may have protective effect against a challenge infection.

Immunoglobulin subclass responses of wild brown rats to Sarcocystis singaporensis.

Immunoglobulin subclass responses of wild brown rats (Rattus norvegicus) from southeastern Asia to the endemic cyst-forming coccidian Sarcocystis singaporensis were characterised. The antibody response of brown rats to wild-type parasites (high reproductive capacity) showed a Th1 profile during acute infection, namely elevated concentrations of parasite-specific IgG2b and IgG2c and absence of IgG1. Chronic infection (bradyzoite development) resulted in a mixed Th1/Th2 pattern whereby significant concentrations of IgG1 appeared. A primary infection with 1000 sporocysts eight days before challenge induced protection, accompanied by significant concentrations of IgA and IgG2, particularly IgG2a. Western blot analysis of rat sera, using sporozoite and bradyzoite-extracts as antigen, revealed that IgG1, IgG2a, and IgG2b predominantly recognised molecules between 70-80 kDa in one or the other stage. Some of the antibodies were possibly directed against a 79 kDa heat shock protein of sporozoites. An apparent unresponsiveness to molecules in the low molecular weight range, particularly of bradyzoite antigens, was observed. This was abrogated by infection of rats with an avirulent strain of S. singaporensis (low reproductive capacity) indicating that a parasite that was less adapted to its host provoked a stronger immune response. These results suggest the existence of an immune evasion strategy used by Sarcocystis and show that wild rodents may be suitable as immunological research objects, reflecting the natural situation.

Sarcocystis muris possesses both diheteroxenous and dihomoxenous characters of life cycle.

The cystozoites of Sarcocystis muris were infective to other mice after peroral inoculation. They transformed into gamonts and after fertilization underwent sporulation with the production of infectious oocysts/sporocysts in the lamina propria of the small intestine. The present study demonstrated that S. muris possesses both diheteroxenous and dihomoxenous life cycle and can be transmitted by the cannibalism among mice.

Underlying disease stress augments plasma and tissue adrenomedullin (AM) responses to endotoxin: colocalized increases in AM and inducible nitric oxide synthase within pancreatic islets.

Rapid onset metabolic impairments accompany the initiation of the acute phase response to many disease stresses, whereas more chronic metabolic perturbations may prolong the recovery period. In the present experiment the application of a mild endotoxin challenge [lipopolysaccharide (LPS)] alone or additive to a chronic subclinical parasitic infection (Sarcocystis cruzi; LPS + PI) in calves was used as a model to investigate and define a dynamic axis coordinated between adrenomedullin (AM) and nitric oxide in response to immune challenge. Plasma AM and NO2/NO3 concentration responses after LPS (0.45 microg/kg, iv) were rapid in onset and of higher magnitude and longer duration in PI + LPS calves than in those challenged with LPS alone. The post-LPS increase in plasma insulin was significantly greater in PI + LPS than in LPS; following refeeding of calves, insulin secretion was most blunted in PI + LPS calves, consistent with the inhibitory effects of NO and AM on insulin secretion. A more chronic response to the immune challenge (organ specific) was in evidence in tissues harvested 24 h after LPS challenge. Where lung and liver showed no immunostaining for inducible nitric oxide (iNOS), iNOS immunostaining was present in the pancreas, localized to islets only. The percentages of iNOS-immunopositive cells in islets were 1.7%, 21%, 6.7%, and 24% for control (C; saline infused), PI, LPS, and PI + LPS calves, respectively. AM immunostaining was not evident in the liver and was present, but not differentially affected by treatment, in airway epithelium in the lung. The number of islet cells with positive immunostaining for AM was increased in LPS, PI, and PI + LPS calves. The percentages ofAM-immunopositive cells in islets were 8%, 27%, 20%, and 33% for C, PI, LPS, and PI + LPS, respectively. Immunostaining for AM and iNOS was colocalized with cells positive for pancreatic polypeptide. By triple label confocal fluorescence immunocytochemistry, colocalization of intense AM and iNOS immunostaining was confirmed in peripheral islet cells. A weaker, more diffuse iNOS signal was also apparent in insulin-containing cells in PI + LPS. We conclude that chronic low level infection potentiates the severity of metabolic perturbations that arise with additive sudden onset immune challenge, as can occur with bacterial toxins. These metabolic disturbances are reflected in and possibly mediated by early onset increases in plasma tumor necrosis factor-alpha, insulin, and AM and up-regulated iNOS activity. These acute complications rapidly progress into a more chronic state characterized by diminished insulin response to feeding stimulus and colocalized increases in pancreatic islet AM and iNOS. The pancreatic responses in AM and iNOS may play a major role in mediating prolonged disturbances in nutrient use by tissues through their influences on temporal patterns of pancreatic hormone secretion during chronic illness.

Endocrine modulation of physiological responses to catabolic disease.

Disease or endotoxemia alters the plasma concentrations of anabolic hormones, particularly growth hormone (GH) and insulin-like growth-factor I (IGF-I). In general, these hormones are inhibited during the catabolic disease state. A hypothesis has evolved that anabolic hormones might be useful in patients' recovery under these and other catabolic circumstances. The treatment of cattle with GH has provided significant improvement in the physiological response of the animals to the subsequent injection of bacterial lypopolysaccharide (LPS), perhaps via inhibition of tumor necrisis factor (TNF) release. However, this improved response to disease was not observed with animals treated with GH and infected with one of two parasitic organisms, Sarcocystis cruzi or Eimeria bovis. Recent attempts with other anabolic hormones, estradiol and progesterone, have proven remarkably effective in improving the adaptive physiological responses of calves to either E. bovis infection or to the injection of LPS. All animals displayed signs of infection, but the intensity and duration of symptoms were reduced. Although a mechanism is not yet known, there were no effects on TNF; cortisol; the percentages of lymphocytes expressing CD2, 4, or 8 antigens; or the production of antibodies.

Class-specific antibody responses in cattle following experimental challenge with sporocysts or merozoites of Sarcocystis cruzi.

An ELISA using antigen produced from merozoites of Sarcocystis cruzi was developed to monitor specific IgM and IgG antibody, following challenge of cattle with either merozoites or sporocysts of S. cruzi. This assay was compared with an ELISA using antigen produced from the cystozoite stage of the parasite. Both ELISAs were able to detect significant increases in levels of circulating IgM and IgG antibodies against Sarcocystis in all challenged cows; however, the magnitude of the titres was greater in the ELISA which used the antigen derived from the merozoites. This immunoassay also detected increases in the levels of IgG earlier than did the assay using antigen derived from cystozoites of S. cruzi. Since this rise coincided with the presence of clinical signs, and was persistent for several weeks, the IgG-ELISA using antigen derived from merozoites appears to be suitable for the diagnosis of acute sarcocystiosis in cattle. Furthermore, since significant increases in the levels of circulating IgM and IgG antibodies against Sarcocystis were detected in the cows infected with merozoites of S. cruzi, it is evident that merozoites of S. cruzi cultured in vitro maintain their capability to replicate in the natural intermediate host.

Production of a recombinant fusion protein of Sarcocystis tenella and evaluation of its diagnostic potential in an ELISA.

We have isolated a cDNA clone encoding an antigenic polypeptide of Sarcocystis tenella by screening a cystozoite-derived cDNA library in lambda gt11 with antibodies from sheep infected experimentally with S. tenella. This clone, termed STC29, was subcloned and expressed in the vector pGEX-3X as a soluble fusion protein with glutathione S-transferase having an apparent molecular mass of 46 kDa. Antibody raised against the recombinant fusion protein recognized a native polypeptide of 25 kDa in cystozoites of S. tenella. In an ELISA with sera from experimentally infected sheep, the recombinant STC29 antigen could differentiate infections with S. tenella from those with Sarcocystis arieticanis or Toxoplasma gondii. Hence, the research described here reports the identification of the first recombinant S. tenella antigen that may be useful for standardization of a serological test for the diagnosis of S.tenella infections in sheep.

Differentiating between Besnoitia besnoiti from cattle and Sarcocystis hoarensis from rodents.

To provide a biological basis for studies designed to establish the mode of transmission of the veterinary pathogen Besnoitia besnoiti, we compared salient features of this pathogen in cattle with those of Sarcocystis hoarensis in rodents. The cysts and cystozoites of these organisms can readily be distinguished morphologically. In contrast to S. hoarensis, which is well adapted to rodents, B. besnoiti fails to mature in jirds or mice and generally is lethal in jirds. Serological reagents discriminately detect these pathogens. B. besnoiti, therefore, can unambiguously be differentiated from S. hoarensis either by morphological or serological methods or on the basis of experimental comparisons of virulence in laboratory rodents.

Australian-German collaborative studies on the immunology of Sarcocystis infections.

This paper reviews a decade of collaborative studies performed between two laboratories in Germany and Australia on the life cycle, pathogenicity and immunology of infections by the cyst-forming sporozoan parasites Sarcocystis spp. in domestic animals. Experimental transmission studies demonstrated that certain parasite species could cause an acute clinical disease in sheep and pigs severely affecting their health, productivity and reproductivity. Despite the high prevalence of infections found in farm animals, few outbreaks of naturally-occurring clinical disease have ever been recorded. Host animals exposed to low levels of infection were found to develop a strong protective immunity against subsequent lethal challenge and clinical disease even though parasites were not completely eliminated nor prevented from further establishment (premunitive immunity rather than sterile immunity). Modern molecular biological techniques are currently being used to examine the parasite antigens and host immune responses involved in the acquisition of the protective immunity.

[Sarcocystosis in lambs]. / Sarkocystóza jehnat.

We studied the problems of sarcocystosis of lambs in regard to the transfer of antibodies over ewes to lambs. Antibodies in the blood serum of 26 ewes found out by help of indirect immunofluorescent reaction (NFR) varied within the range from 640 to 5120 titres, while in six cases their level was higher than in blood. In none of new-born lambs there were proved antibodies to the sarcocystosis before the drinking of colostrum but three days later we already detected antibodies in the dilution 1:4 to 1:128. Then we studied the titre dynamics of antibodies in weaned lambs. In the age of 10-18 weeks we proved only low titres of antibodies, from the 20th to the 24th weeks the titres gradually increased and from the 26th to the 30th weeks the level of antibodies increased significantly (5120 titres) with its maximum on about the 28th week. Animals had no clinic symptoms of disease. In the other part of the work we examined the muscular substance of 342 lambs, killed in the slaughter house, by a digestive method and we carried out the blood serologic examination (NFR). Microcysts were found out in 35.67% of lambs while macrocysts were so in none of them. Specific antibodies of sarcocystosis were detected in 94.44% of young animals. A high discrepancy of the both methods (61.69%) in young animals is a result of a significant antibody reaction at still undeveloped muscular cysts.

Immune responses against Toxoplasma and Sarcocystis infections in ruminants: diagnosis and prospects for vaccination.

Toxoplasma gondii and a variety of Sarcocystis species are common cyst-forming coccidian parasites of domestic ruminants worldwide. T. gondii infection is a major cause of ovine and caprine abortion and perinatal mortality, but does not appear commonly to cause significant disease in other ruminants. Sarcocystis infections are probably most important in growing ruminants in which they can cause subclinical anaemia and reduced weight gain. The life cycles, pathogenesis and current knowledge of immunity to these protozoa, in ruminants, are reviewed. Although clinical signs may suggest a diagnosis, confirmation requires the use of specific immunological methods such as serology and immunohistochemistry. These are discussed and the necessity for using characterised antigens and antisera is emphasised. Whereas live Toxoplasma "vaccines" may be effective in sheep and goats, the eventual preparation of a killed vaccine capable of inducing long-term protection is of the utmost importance. To achieve this goal it will be necessary to identify the specific antigens capable of stimulating protective immunity and to conduct further research on both the pathogenesis of toxoplasmosis and fundamental aspects of the immune response of pregnant ruminants to T. gondii.

In vitro and in vivo interaction between sporocysts of Sarcocystis muris and mouse peritoneal macrophages.

The interaction between the sporocysts of Sarcocystis muris and mouse peritoneal macrophages was studied both in vitro and in vivo in an attempt to determine whether or not resident peritoneal macrophages might effect the excystation of S. muris sporozoites from sporocysts injected intraperitoneally. Sporocysts of S. muris were phagocytosed by peritoneal macrophages both in vitro and in vivo. The addition of either unheated mouse serum or fetal calf serum did not significantly alter the level of phagocytosis. The percentage of phagocytosis in vivo and by thioglycolate-, proteose peptone- and BCG-elicited macrophages in vitro was greater than that shown by unstimulated macrophages in vitro. After 8 h incubation in vivo and in vitro a small proportion of sporocysts (less than 5%) was seen to have collapsed walls and up to 5% to have stained sporozoites, suggesting increased permeability of the sporocyst wall. The significance of increased permeability of the cyst wall in the process of sporozoite excystation is discussed.

Antibody development and cellular immune responses in sheep immunized and challenged with Sarcocystis tenella sporocysts.

Four specific-pathogen-free (SPF) sheep were experimentally infected with 10(3) or 10(4) Sarcocystis tenella (syn. S. ovicanis) sporocysts and another two sheep served as uninfected controls. All sheep were challenged 49 days later by infection with 2.5 X 10(5) sporocysts and their humoral and cellular responses to infection and challenge were assessed weekly by enzyme immunoassays and lymphocyte transformation assays. The control sheep died from acute sarcocystosis 29-30 days after challenge, whereas the immunized sheep survived and were protected against acute disease. Specific IgM and IgG antibodies were detected in the immunized sheep from 28 days after infection onwards. Lymphocytes collected before and after challenge did not exhibit any significant differences in their responses to stimulation with S. tenella cystozoite or sporozoite antigens. Furthermore, lymphocytes collected before challenge did not differ from the controls in their responses to stimulation with the mitogens lipopolysaccharide or phytohaemagglutinin. However, lymphocytes collected after challenge did exhibit increased blastogenic responses to stimulation with both mitogens from 21-28 days after challenge onwards. The infected sheep were necropsied 46 days after challenge, and histological and ultrastructural studies revealed numerous infiltrates of lymphocytes, histiocytes and plasma cells in the skeletal muscles, sometimes in association with degenerating parasitic cysts and macrophage myophagia. Parasites were not completely eliminated nor prevented from further establishment, therefore the protective immunity was not sterile but rather a state of premunition.