Confirmation of Sarcocystis jamaicensis Sarcocysts in IFN-γ Gene Knockout Mice Orally Inoculated with Sporocysts from a Red-Tailed Hawk ( Buteo jamaicensis).

Here, we report confirmation of sarcocysts of Sarcocystis jamaicensis in an experimental intermediate host, IFN-γ gene knockout (KO) mice orally inoculated sporocysts from its natural definitive host, a red-tailed hawk ( Buteo jamaicensis) (RTH). A RTH submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because it could not be rehabilitated and released. Fully sporulated sporocysts from intestinal scrapings of the RTH were orally fed to 2 laboratory-reared outbred Swiss Webster mice (SW; Mus musculus) and to 2 KO mice. The sporocysts were infective for KO mice but not to SW mice. Both SW mice remained asymptomatic, and neither schizonts nor sarcocysts were found in their tissues when euthanized on day 54 post-inoculation (PI). The KO mice developed neurological signs and were necropsied 38-54 days PI. Schizonts/merozoites were found in both KO mice euthanized and they were confined to the brain. The predominant lesion was meningoencephalitis. Microscopic sarcocysts were found in muscles of both KO mice. When viewed with light microscopy, the sarcocyst wall appeared thin (<1 µm thick) and smooth. Ultrastructural details of sarcocysts are described.

Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.

Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals.

An intronic polymorphism in the porcine IRF7 gene is associated with better health and immunity of the host during Sarcocystis infection, and affects interferon signalling.

Interferon regulatory factor 7 (IRF7), as a key regulator of type I interferon response, plays an important role during innate response against viral infection. Although well conserved across species, the structure of IRF7 and its function during parasite infection are not well documented in farm animals, such as the pig. To bridge this gap, we have determined the porcine IRF7 gene structure and identified two intronic single nucleotide polymorphisms (SNPs), SNP g.748G>C and SNP g.761A>G, in commercial pig breeds. The distribution of SNP g.761A>G in multiple breeds suggested that it was in Hardy-Weinberg equilibrium and allowed us to map it at the top of SSC2. We found that during Sarcocystis miescheriana infection, the G allele was associated with high lymphocyte levels (P < 0.02), reduced drop in platelet levels (P < 0.002) and IgG1-Th2-dominated response (P < 0.05). This suggests that the G allele was associated with better health and immunity of the host during Sarcocystis infection. Furthermore, we have also provided suggestive evidence that the G allele of SNPc.761A>G enhances the transactivation activity of IRF7, possibly by improving IRF7 transcript splicing of intron-3. These findings would suggest that IRF7, as a transcriptional regulator, is involved in the defence mechanism against a larger spectrum of pathogens, and in more host species, than initially anticipated.

Ultrastructural and molecular confirmation of the development of Sarcocystis neurona tissue cysts in the central nervous system of southern sea otters (Enhydra lutris nereis).

In 2004, three wild sea otters were diagnosed with putative Sarcocystis neurona-associated meningoencephalitis by histopathology and immunohistochemistry. Schizonts, free merozoites and tissue cysts were observed in the brains of all three infected animals. Tissue cysts walls from sea otter 1 (SO1) stained positively using anti-S. neurona polyclonal antiserum. However, positive staining does not preclude infection by closely related or cross-reactive tissue cyst-forming coccidian parasites. Two immature tissue cysts in the brain of SO1 were examined using transmission electron microscopy. Ultrastructural features included cyst walls with thin villous projections up to 1 microm long with tapered ends and a distinctive, electron-dense outer lining layer composed of linearly-arranged, semi-circular structures with a "hobnailed" surface contour. Small numbers of microtubules extended down through the villi into the underlying granular layer. Metrocytes were short and plump with an anterior apical complex, 22 sub-pellicular microtubules, numerous free ribosomes and no rhoptries. Some metrocytes appeared to be dividing, with two adjacent nuclear profiles. Collectively these ultrastructural features were compatible with developing protozoal cysts and were similar to prior descriptions of S. neurona tissue cysts. Panspecific 18S rDNA primers were utilized to identify protozoa infecting the brains of these otters and DNA amplification and additional sequencing at the ITS1 locus confirmed that all three otters were infected with S. neurona. No other Sarcocystis spp. were detected in the brains or skeletal muscles of these animals by immunohistochemistry or PCR. We believe this is the first ultrastructural and molecular confirmation of the development of S. neurona tissue cysts in the CNS of any animal.

Mapping of quantitative trait loci for clinical-chemical traits in swine.

Clinical-chemical traits are diagnostic parameters essential for characterization of health and disease in veterinary practice. The traits show significant variability and are under genetic control, but little is known about the fundamental genetic architecture of this variability, especially in swine. We have identified QTL for alkaline phosphatase (ALP), lactate (LAC), bilirubin (BIL), creatinine (CRE) and ionized sodium (Na(+)), potassium (K(+)) and calcium (Ca(++)) from the serum of 139 F(2) pigs from a Meishan/Pietrain family before and after challenge with Sarcocystis miescheriana, a protozoan parasite of muscle. After infection, the pigs passed through three stages representing acute disease, subclinical disease and chronic disease. Forty-two QTL influencing clinical-chemical traits during these different stages were identified on 15 chromosomes. Eleven of the QTL were significant on a genome-wide level; 31 QTL were chromosome-wide significant. QTL showed specific health/disease patterns with respect to the baseline values of the traits as well as the values obtained through the different stages of disease. QTL influencing different traits at different times were found primarily on chromosomes 1, 3, 7 and 14. The most prominent QTL for the investigated clinical-chemical traits mapped to SSC3 and 7. Baseline traits of ALP, LAC, BIL, Ca(++) and K(+) were influenced by QTL regions on SSC3, 6, 7, 8 and 13. Single QTL explained up to 21.7% of F(2) phenotypic variance. Our analysis confirms that variation of clinical-chemical traits is associated with multiple chromosomal regions.

Heritabilities and quantitative trait loci for blood gases and blood pH in swine.

Maintaining pH and blood gases in a narrow range is essential to sustain normal biochemical reactions. Decreased oxygenation, poor tissue perfusion, disturbance to CO(2) expiration, and shortage of HCO(3)(-) can lead to metabolic acidosis. This is a common situation in swine, and originates from a broad range of medical conditions. pH and blood gases appear to be under genetic control, and populations with physiological traits closer to the pathological thresholds may be more susceptible to developing pathological conditions. However, little is known about the genetic basis of such traits. We have therefore estimated phenotypic and genetic variability and identified quantitative trait loci (QTL) for pH and blood gases in blood samples from 139 F(2) pigs from the Meishan/Pietrain family. Samples were taken before and after challenge with Sarcocystis miescheriana, a protozoan parasite of muscle. Twenty-seven QTL influencing pH and blood gases were identified on nine chromosomes. Five of the QTL were significant on a genome-wide level; 22 QTL were significant on a chromosome-wide level. QTL for pH-associated traits have been mapped to SSC3, 18 and X. QTL associated with CO(2) have been detected on SSC6, 7, 8 and 9, and QTL associated with O(2) on SSC2 and SSC8. QTL showed specific health/disease patterns that were related to the physiological state of the pigs from day 0, to acute disease (day 14), convalescence (day 28) and chronic disease (day 42). The results demonstrate that pH and blood gases are influenced by multiple chromosomal areas, each with relatively small effects.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.

Mapping of quantitative trait loci affecting resistance/susceptibility to Sarcocystis miescheriana in swine.

The outcome of infectious diseases in vertebrates is under genetic control at least to some extent. In swine, e.g., marked differences in resistance/susceptibility to Sarcocystis miescheriana have been shown between Chinese Meishan and European Pietrain pigs, and these differences are associated with high heritabilities. A first step toward the identification of genes and polymorphisms causal for these differences may be the mapping of quantitative trait loci (QTLs). Considering clinical, immunological, and parasitological traits in the above model system, this survey represents the first QTL study on parasite resistance in pigs. QTL mapping was performed in 139 F(2) pigs of a Meishan/Pietrain family infected with S. miescheriana. Fourteen genome-wide significant QTLs were mapped to several chromosomal areas. Among others, major QTLs were identified for bradyzoite numbers in skeletal muscles (F = 17.4; p < 0.001) and for S. miescheriana-specific plasma IgG(2) levels determined 42 days p.i. (F = 20.9; p < 0.001). The QTLs were mapped to different regions of chromosome 7, i.e., to the region of the major histocompatibility complex (bradyzoites) and to an immunoglobulin heavy chain cluster, respectively. These results provide evidence for a direct and causal role for gene variants within these gene clusters (cis-acting) in differences in resistance to S. miescheriana.

Genetic resistance to Sarcocystis miescheriana in pigs following experimental infection.

Clinical and parasitological traits of Sarcocystis miescheriana differ in Pietrain and Meishan pigs. For further description and characterization of the genetic basis of this variation a F(2) family based on Pietrain boars and Meishan sows as founders was generated. One hundred and thirty-nine F(2) pigs were challenged orally at an age of 100 days with 50,000 sporozysts to produce the typical clinical picture of a moderate dose Sarcocystis infection. Heritabilities were estimated for clinical and clinical-chemical traits, for specific antibody responses to the infection and for bradyzoite numbers found in skeletal (Musculus longissimus dorsi: M.l.d.) and heart muscles at necropsy 70 days post-infection (p.i.) Apart from several low to moderate heritabilities, high heritabilities were observed for bradyzoite numbers in the M.l.d. (0.68), IgM antibody levels (0.74) during the acute (14 days p.i.) and titres of specific IgG antibodies (0.42) in the early stage of cyst formation (42 days p.i.). Marked heritabilities of these traits, which are basic for acute phase of the disease (14 days p.i.) or chronic Sarcocystosis presume genes that explain sufficient shares of variance (QTL). The model is considered valuable for screening of gene variants associated with resistance/susceptibility to Sarcocystis infection. Such gene variants could then be used in susceptibility-scoring or selection programs in the future.

A genetically diverse but distinct North American population of Sarcocystis neurona includes an overrepresented clone described by 12 microsatellite alleles.

The population genetics and systematics of most coccidians remain poorly defined despite their impact on human and veterinary health. Non-recombinant parasite clones characterized by distinct transmission and pathogenesis traits persist in the coccidian Toxoplasma gondii despite opportunities for sexual recombination. In order to determine whether this may be generally true for tissue-cyst forming coccidia, and to address evolutionary and taxonomic problems within the genus Sarcocystis, we characterized polymorphic microsatellite markers in Sarcocystis neurona, the major causative agent of equine protozoal myeloencephalitis (EPM). Bayesian statistical modeling, phylogenetic reconstruction based on genotypic chord distances, and analyses of linkage disequilibrium were employed to examine the population structure within S. neurona and closely related Sarcocystis falcatula isolates from North and South America. North American S. neurona were clearly differentiated from those of South America and also from isolates of S. falcatula. Although S. neurona is characterized by substantial allelic and genotypic diversity typical of interbreeding populations, one genotype occurs with significantly excessive frequency; thus, some degree of asexual propagation of S. neurona clones may naturally occur. Finally, S. neurona isolated from disparate North American localities and diverse hosts (opossums, a Southern sea otter, and horses) comprise a single genetic population. Isolates associated with clinical neurological disease bear no obvious distinction as measured by these presumably neutral genetic markers.

Pathology of sarcocystis neurona in interferon-gamma gene knockout mice.

Pathologic changes were studied in 27 interferon-gamma gene knockout mice 34-54 days after being fed graded doses of Sarcocystis neurona sporocysts derived from a naturally infected opossum. The target tissue for S. neurona infection was the central nervous system. Characteristic histopathologic changes present in all mice consisted of an inflammatory infiltrate consisting of mostly neutrophils and macrophages, fewer eosinophils, and rare multinucleated giant cells. Intralesional protozoa and scattered subacute perivascular cuffs were present. Where the infiltrates were extensive, neuropil rarefaction was frequent. Pathologic changes were much more frequent and severe in the caudal portion of the brain, especially in the cerebellum, than in the middle and cranial portions. Changes were present in all spinal cords examined (10 of 10). Lesions were equally distributed in white and gray matter of the brain and spinal cord and their meningeal linings.

Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana.

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite interactions depend to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5x10(4) sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1-2 standard deviations. The study lays the foundation for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite interaction in protozoan infectious disease.

Mice lacking the gene for inducible or endothelial nitric oxide are resistant to sporocyst induced Sarcocystis neurona infections.

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. Little is known about the role of immunobiological mediators to this parasite. Nitric oxide (NO) is important in resistance to many intracellular parasites. We, therefore, investigated the role of inducible and endothelial NO in resistance to clinical disease caused by S. neurona in mice. Groups of interferon-gamma gene knockout (IFN-gamma-KO) mice, inducible nitric oxide synthase gene knockout (iNOS-KO) mice, endothelial nitric oxide synthase gene knockout (eNOS-KO) and appropriate genetic background mice (BALB/c or C57BL/6) were orally fed sporocysts or Hanks balanced salt solution. Mice were observed for signs of clinical disease and examined at necropsy. Clinical disease and deaths occurred only in the IFN-gamma-KO mice. Microscopic lesions were seen only in the brains of IFN-gamma-KO mice. Results of this study indicate that iNOS and eNOS are not major mediators of resistance to S. neurona infections. Results of this study suggest that IFN-gamma mediated immunity to S. neurona may be mediated by non-NO-dependent mechanisms.

The gamma interferon knockout mouse model for sarcocystis neurona: comparison of infectivity of sporocysts and merozoites and routes of inoculation.

The dose-related infectivity of Sarcocystis neurona sporocysts and merozoites of 2 recent isolates of S. neurona was compared in gamma interferon knockout (KO) mice. Tenfold dilutions of sporocysts or merozoites were bioassayed in mice, cell culture, or both. All 8 mice, fed 1,000 sporocysts, developed neurological signs with demonstrable S. neurona in their tissues. Of 24 mice fed low numbers of sporocysts (100, 10, 1), 18 became ill by 4 wk postinoculation, and S. neurona was demonstrated in their brains; antibodies (S. neurona agglutination test) to S. neurona and S. neurona parasites were not found in tissues of the 6 mice that were fed sporocysts and survived for >39 days. One thousand culture-derived merozoites of these 2 isolates were pathogenic to all 8 mice inoculated subcutaneously (s.c.). Of the 24 mice inoculated s.c. with merozoites numbering 100, 10, or 1, only 3 mice had demonstrable S. neurona infection; antibodies to S. neurona were not found in the 21 mice that had no demonstrable organisms. As few as 10 merozoites were infective for cell cultures. These results demonstrate that at least 1,000 merozoites are needed to cause disease in KO mice. Sarcocystis neurona sporocysts were infective to mice by the s.c. route.

Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (P<0.001, Fisher's exact test). The S. cruzi antibody-blocked Western blot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

Comparison of immunological and molecular methods for the diagnosis of infections with pathogenic Sarcocystis species in sheep.

Sheep may be infected by four species of Sarcocystis: Sarcocystis tenella and Sarcocystis arieticanis are pathogenic, Sarcocystis gigantea and Sarcocystis medusiformis are non-pathogenic. The two pathogenic species may cause abortion or acute disease during the early phase of infection and chronic disease during the late phase of infection. Thus far, diagnosis of sarcocystiosis has been limited, because traditional diagnostic tests based on the detection of Sarcocystis-specific antibodies are only genus-specific and, thus, cannot differentiate between pathogenic and non-pathogenic Sarcocystis species. In addition, most of these tests can only detect chronic sarcocystiosis. Therefore, diagnosis of acute sarcocystiosis or Sarcocystis-induced abortion has been based mainly on post-mortem examination, i. e. after the animal had succumbed to the disease. Recently, we have established species-specific PCR assays based on unique ribosomal RNA gene sequences of S. tenella and S. arieticanis. These assays enable the diagnosis and differentiation of infections with S. tenella and S. arieticanis in sheep intra vitam during the acute phase of the disease and, therefore, facilitate for the first time comprehensive studies on the epidemiology and importance of infections with pathogenic Sarcocystis species in sheep.