Molecular identification of seven Sarcocystis species in red deer (Cervus elaphus) from Lithuania.

The diaphragm muscles of 77 free-ranging red deer (Cervus elaphus) were examined for Sarcocystis species in Lithuania. Sarcocysts were detected in 61 out of 77 (79.2%) animals investigated. A total of 60 isolated sarcocysts were identified to species using subunit I of cytochrome c oxidase (cox1) sequence analysis. Overall, seven species, S. entzerothi, S. hjorti, S. iberica, S. linearis, S. pilosa, S. truncata and S. venatoria, were confirmed in Lithuanian red deer. Sarcocystis entzerothi was reported in red deer for the first time. Previously this species was shown to use sika deer as well as roe deer and fallow deer as an intermediate host. Based on cox1, with the addition of the current data, altogether 13 Sarcocystis species have so far been shown to use red deer as an intermediate host. Species detected in red deer demonstrated considerable differences in intraspecific genetic variation at cox1. Genetic distances between different samples of S. hjorti and S. linearis were calculated using principal coordinates analysis (PCoA), implying molecular divergence of same Sarcocystis species using different hosts in the same geographical area and divergence of those employing same intermediate host species from different areas.

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy.

Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5µM range but interfere with intracellular division at 2.5µM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis.

Prevalence, morphology, and molecular characteristics of Sarcocystis spp. in domestic goats (Capra hircus) from Kunming, China.

Despite the importance of worldwide goat production, little is known about the prevalence of Sarcocystis spp. in domestic goats (Capra hircus) in China. The aims of the present study were to determine prevalence of Sarcocystis spp. in domestic goats in Kunming, China, as well as to identify parasite species based on morphological characteristics and DNA sequence analysis. Only microscopic sarcocysts of Sarcocystis spp. were detected in 174 of 225 goats (77.3 %). By light and transmission electron microscopy, two species, i.e., Sarcocystis capracanis and Sarcocystis hircicanis, were identified. Two sarcocysts from each of the two species were randomly selected for DNA extraction; the 18S rRNA gene (18S rRNA), the 28S rRNA gene (28S rRNA), and the mitochondrial cytochrome c oxidase subunit 1 (cox1) were amplified by the polymerase chain reaction (PCR) and subsequently sequenced. The results were compared with other previously sequenced Sarcocystis species retrieved from GenBank. There was little sequence variation between two isolates of the same species. S. capracanis was most closely related with Sarcocystis tenella; 18S rRNA, 28S rRNA, and mitochondrial cox1 sequences shared identities of 95.7-99.1, 95.3, and 92.3-93.2 % with those of S. tenella, respectively. Thus, mitochondrial cox1 sequences seem to perform better than 18S rRNA sequences or 28S rRNA sequences for identification of the two species. S. hircicanis was most closely related to Sarcocystis arieticanis, i.e., 18S rRNA and 28S rRNA sequences of the former species shared 97.2-97.4 and 95.6-96.1 % identities with those of latter, respectively. Phylogenetic analysis inferred from the three genetic markers yielded similar results and indicated the two species were within a group of Sarcocystis species with canines as known, or presumed, definitive hosts.

Domestic cats (Felis catus) are definitive hosts for Sarcocystis sinensis from water buffaloes (Bubalus bubalis).

The definitive hosts of Sarcocystis sinensis in water buffaloes have hitherto been unknown, but the close similarity of this species to the cat-transmitted Sarcocystis bovifelis in cattle suggested they were felids. In a previous study, two domestic cats were fed macroscopic sarcocysts of Sarcocystis fusiformis contained within or dissected from the esophageal muscles of water buffaloes, while no microscopic sarcocysts of S. sinensis were noticed. Both cats started shedding small numbers of sporocysts 8-10 days post infection (dpi) and were euthanized 15 dpi. Using a PCR-based molecular assay targeting the mitochondrial cox1 gene of S. fusiformis, both cats were shown to act as definitive hosts for this species. In the present study, DNA samples derived from oocysts/sporocysts in the intestinal mucosa of both cats were further examined by PCR for the presence of S. sinensis using 2 newly designed primers selectively targeting the cox1 gene of this species. All 6 DNA samples examined from each cat tested positive for S. sinensis. A 1,038-bp-long portion of cox1 was amplified and sequenced as 2 overlapping fragments from 5 of these DNA samples. The 5 sequences shared 99.3-100% identity with 7 previous cox1 sequences of S. sinensis obtained from sarcocysts in water buffaloes. Additionally, amplification of the ITS1 region with primers targeting various Sarcocystis spp., yielded amplicons of 2 different lengths, corresponding to those obtained from sarcocyst isolates of S. sinensis and S. fusiformis, respectively. This is the first study to show that cats act as definitive hosts for S. sinensis.

Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt.

A total of 33 macroscopically visible (3-11 × 1-5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3-99.9 %. In comparison, the sequence identity was 98.8-99.0 % among eight complete 18S rRNA gene sequences (1,873-1,879-bp long), 98.1-100 % among 28 complete ITS1 sequences (853-864-bp long) and 97.4-99.6 % among five partial 28S rRNA gene sequences (1,607-1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7-99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8-10 days post-infection (dpi) and were euthanized 15 dpi. Sporocysts isolated from the intestinal mucosa of both cats were identified molecularly as belonging to S. fusiformis through PCR amplification and sequencing of the partial cox1. The two sporocyst-derived cox1 sequences were identical with the most common sarcocyst-derived cox1 haplotype.

Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite.

Limited genetic diversity among Sarcocystis neurona strains infecting southern sea otters precludes distinction between marine and terrestrial isolates.

Sarcocystis neurona is an apicomplexan parasite identified as a cause of fatal neurological disease in the threatened southern sea otter (Enhydra lutris nereis). In an effort to characterize virulent S. neurona strains circulating in the marine ecosystem, this study developed a range of markers relevant for molecular genotyping. Highly conserved sequences within the 18S ribosomal gene array, the plastid-encoded RNA polymerase (RPOb) and the cytochrome c oxidase subunit 1 mitochondrial gene (CO1) were assessed for their ability to distinguish isolates at the genus and species level. For within-species comparisons, five surface antigens (SnSAG1-SnSAG5) and one high resolution microsatellite marker (Sn9) were developed as genotyping markers to evaluate intra-strain diversity. Molecular analysis at multiple loci revealed insufficient genetic diversity to distinguish terrestrial isolates from strains infecting marine mammals. Furthermore, SnSAG specific primers applied against DNA from the closely related species, Sarcocystis falcatula, lead to the discovery of highly similar orthologs to SnSAG2, 3, and 4, calling into question the specificity of diagnostic tests based on these antigens. The results of this study suggest a population genetic structure for S. neurona similar to that reported for the related parasite, Toxoplasma gondii, dominated by a limited number of successful genotypes.

Sarcocystis neurona: molecular characterization of enolase domain I region and a comparison to other protozoa.

Sarcocystis neurona causes protozoal myeloencephalitis and has the ability to infect a wide host range in contrast to other Sarcocystis species. In the current study, five S. neurona isolates from a variety of sources, three Sarcocystis falcatula, one Sarcocystis dasypi/S. neurona-like isolate, and one Besnoitia darlingi isolate were used to compare the enolase 2 gene segment containing the domain I region to previously sequenced enolase genes from Neospora caninum, Neospora hughesi, Toxoplasma gondii, Plasmodium falciparum, and Trypanosoma cruzi; enolase 2 segment containing domain I region is highly conserved amongst these parasites of veterinary and medical importance. Immunohistochemistry results indicates reactivity of T. gondii enolase 1 and 2 antibodies to S. neurona merozoites and metrocytes, but no reactivity of anti-enolase 1 to the S. neurona bradyzoite stage despite reactivity to T. gondii bradyzoites, suggesting expression differences between organisms.

[A comparative study on isoenzymes of Sarcocystis spp. from cattle and water buffaloes].

OBJECTIVE: To analyze the zymogram of peroxidase (PER) and phosphoglucose isomerase (PGI) of three species of Sarcocystis. METHODS: The collected parasites were homogenized and fragmented by ultrasonication. After centrifugation, the supernatants were analyzed by isoelectric focusing electrophoresis. RESULTS: The isolates of S. cruzi from infected water buffalo and cattle all showed identical enzyme profiles, 7 bands of PER at pH 4.44-6.98 and 6 bands of PGI at pH 4.66-6.53; and same with the isolates of S. hirsuta. 5 bands of PER at pH 4.97-7.15 and 4 bands of PGI at pH 4.70-6.51. The zymograms among S. cruzi, S. hirsuta and S. fusiformis were different considerably. CONCLUSION: The data support the hypothesis that both water buffalo and cattle are the natural intermediate hosts of S. cruzi and S. hirsuta at the gene level. S. cruzi, S. hirsuta and S. fusiformis are different species.

The identification of a sequence related to apicomplexan enolase from Sarcocystis neurona.

Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by Sarcocystis neurona, an apicomplexan parasite. S. neurona is also associated with EPM-like diseases in marine and small mammals. The mechanisms of transmission and ability to infect a wide host range remain obscure; therefore, characterization of essential proteins may provide evolutionary information allowing the development of novel chemotherapeutics that target non-mammalian biochemical pathways. In the current study, two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry were combined to characterize and identify an enolase protein from S. neurona based on peptide homology to the Toxoplasma gondii protein. Enolase is thought to be a vestigial, non-photosynthetic protein resulting from an evolutionary endosymbiosis event of an apicomplexan ancestor with green algae. Enolase has also been suggested to play a role in parasite stage conversion for T. gondii. Characterization of this protein in S. neurona and comparison to other protozoans indicate a biochemical similarity of S. neurona enolase to other tissue-cyst forming coccidians that cause encephalitis.

Immunohistochemical localisation of 3beta-hydroxysteroid dehydrogenase in Sarcocystis spp.

3Beta-hydroxysteroid-dehydrogenase (3beta-HSD) is an isoenzyme that catalyses an essential step in the synthesis of all classes of active steroid hormones. The presence of steroid hormones of the vertebrate type in invertebrates is acknowledged in addition to a group of steroid-like hormones called ecdysteroids that were present in arthropods and helminths. In the present study, 3beta-HSD was detected in the bradyzoites enclosed in sarcocysts of Sarcocystis spp. with immunohistochemistry. The results suggest that self-originating steroid hormones may play important roles in the development of Sarcocystis spp., and possibly in the regulation of the reciprocal immune interaction between the host and these parasites.

Characterization of a serine protease activity in Sarcocystis neurona merozoites.

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.

Isolation and characterization of a cDNA clone encoding a thiol proteinase of Sarcocystis muris cyst mero' zoites (Apicomplexa).

A cDNA library of Sarcocystis muris cyst merozoites was screened using a digoxigenin-labeled probe. This probe was derived from a 504-bp polymerase-chain-reaction fragment representing part of a thiol proteinase. Several cDNA clones were isolated, one of which (PH08) consists of a nucleotide sequence of 1694 bp and encodes the complete prepropolypeptide of a cathepsin L-like proteinase. PH08 contains an open reading frame of 394 amino acid (aa) residues with a 46-residue signal sequence, which is followed by a 129-residue propeptide and 219 aa residues of the mature enzyme. Two potential glycosylation sites and a putative polyadenylation signal were also identified. The occurrence of the highly conserved interspersed ERFNIN aa motif, not found in cathepsin B-like proteinases, suggests the classification of the enzyme as a cathepsin L-like proteinase. Results worked out in this study will enable production of the recombinant thiol proteinase of S. muris cyst merozoites necessary for study of the substrate specificity as well as other biochemical parameters of this enzyme.

Amplification of genomic DNA fragments of Sarcocystis muris (Apicomplexa) cyst merozoites encoding a thiol (cysteine) proteinase.

Parasites of the phylum Apicomplexa (Sporozoa) cause diseases such as malaria, toxoplasmosis, or intestinal coccidiosis. Invasive stages possess typical apical organelles such as dense granules that harbor a broad range of polypeptides that are believed to take part in the parasite-host cell interaction. In previous studies a 26-kDa polypeptide of dense granules from Sarcocystis muris cyst merozoites (bradyzoites) was characterized as a thiol (cysteine) proteinase. In this paper a method is demonstrated to amplify DNA fragments from genomic DNA of S. muris cyst merozoites by polymerase chain reaction, which probably code for the 26-kDa antigen.

[The localization and functional significance of arylsulfatases in parasitic sarcosporidian protozoa in the tissue-cyst life cycle phase]. / Lokalizatsiia i funktsional'noe znachenie arilsul'fataz u paraziticheskikh prosteishikh sarkosporidii v faze tkanevoi tsisty zhizhnennogo tsikla.

The hydrolytic enzymes arylsulphatases (AS) were detected in developing tissue cysts of Sarcocystis ovifelis, using two methods: the Goldfischer lead technique, with two different pH values-5.5 (AS-A) and 4.2 (AS-B), and the Hopsu-Havu barium technique (Gayer, 1974). The enzymatic activity was identified by the presence of an electron dense finely granulated precipitation. In cyst cells, lead sulphate precipitation was spotted only in the inner membrane complex (IC) of the pellicle, whereas barium sulphate marked, in addition, the plasma membrane. Besides, AS activity was detected in the endoplasmic reticulum, Golgi complex, lysosomes and micronemes of cyst cells. Of interest is the finding of AS in the outer membrane of IC and matrix of pellicular evaginations. In the cyst ground substance (CGS) of S. ovifelis AS activity is confined to the membrane and matrix of transport vesicles, originating from cyst cell pellicle evaginations. These cystic vesicles carry enzymes from the places of their synthesis, in the cyst cells, to the tissue cyst periphery near the cyst wall. In the CGS, the obvious precipitations of lead and barium sulphate, respectively, are seen around some cyst cells being in the state of destroying due to natural death, and around so-called apoptotic-like bodies made from the destroyed cells. AS activity is seen both in the cyst wall and in vesicles separating from the wall ("wall vesicles") that find eventually their way in the cytoplasm of infected muscle cells, the granulation being observed around destroyed organelles of such cells. The investigated dynamics of AS movement, by means of the transport cystic and wall vesicles, extends general knowledge of the distant metabolic interaction between cells of the host and the parasite in tissue cysts of Sarcocystis spp.

Sarcocystis fusiformis: some Krebs cycle enzymes in various fractions of sarcocysts of buffalo (Bubalus bubalis).

A biochemical investigation was carried out on the relative presence of some enzymes of the Krebs cycle and of the associated energy metabolism in various fractions (namely, cyst wall, cyst fluid and zoites) of sarcocysts of Sarcocystis fusiformis from the oesophageal muscles of naturally infected Indian water buffalo (Bubalus bubalis). Except for malate dehydrogenase, the activities of aconitase, isocitrate dehydrogenase, succinate dehydrogenase and fumarase were beyond detectable limits, pointing to a non-functional Krebs cycle in the cysts of this parasite. The activities of adenosine triphosphatase and cytochromes were lowest in cyst fluid and were maximally depicted by cyst wall and zoites.

Some enzymes of gluconeogenesis in various fractions of sarcocysts of Sarcocystis fusiformis of buffalo (Bubalus bubalis).

A biochemical study was conducted to assess the relative presence of some enzymes of gluconeogenesis in various fractions--the cyst wall, cyst fluid and zoites--of sarcocysts of Sarcocystis fusiformis obtained from the oesophageal muscles of naturally infected Indian water buffalo (Bubalus bubalis). The activities of fructose 1,6-diphosphatase and malic enzyme were beyond detectable limits. Phosphoenol pyruvate carboxykinase (PEPCK) activity was maximally present in the zoites, whereas glucose 6-phosphatase activity was highest in the cyst wall. PEPCK seemed to play a crucial role in carbon dioxide fixation metabolism.

Sarcocystis fusiformis: some protein metabolic enzymes in various fractions of sarcocysts of buffalo (Bubalus bubalis).

An investigation on the relative presence of some protein metabolic enzymes, namely aspartate aminotransferase (AST), alanine aminotransferase (ALT), NAD+ and NADP+ dependent glutamate dehydrogenase (GLDH) and arginase in cyst wall (CW), cyst fluid (CF) and zoite (ZT) fractions of the sarcocysts of Sarcocystis fusiformis in the oesophageal muscles of Indian water buffalo was carried out. Both the transaminases were present in all the fractions of the cyst, although in variable amounts. There was a higher level of AST activity than of ALT activity. AST activity was the highest in ZT, whereas ALT activity was at a maximum in the CF fraction. The levels of activity of NAD+ and NADP+ dependent GLDH and arginase remained beyond detectable limits. The study revealed that the intermediates of carbohydrate metabolism are linked to protein metabolism by transaminases. The possibility of concomitant removal of ammonia and its subsequent incorporation into the urea cycle is ruled out in this parasitic protozoan.

Some glucose metabolic enzymes in various fractions of sarcocysts of Sarcocystis fusiformis of buffalo (Bubalus bubalis).

A comparative biochemical study on some enzymes of glycogenolysis, glycolysis and the hexose monophosphate shunt pathway in various fractions (cyst wall, cyst fluid and zoites) of the sarcocysts of Sarcocystis fusiformis from the oesophageal muscles of naturally infected Indian water buffalo (Bubalus bubalis) was carried out. The pattern and the magnitude of enzymic activity differed markedly in these fractions. Phosphorylase, hexokinase, aldolase and pyruvate kinase showed their highest levels of activity in the zoites fractions, whereas lactate dehydrogenase was the highest in cyst fluid. Alcohol dehydrogenases were non-detectable. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were localized in the cyst wall only. Zoites were considered to be the most active metabolic sites for glucose breakdown.

Sarcocystis muris (Apicomplexa): a thiol protease from the dense granules.

Homogenates of Sarcocystis muris merozoites (cyst form) and the subcellular fraction of dense granules were assayed for protease activity with substrate-impregnated SDS-polyacrylamide gels. Four acidic and several basic proteases were detected in the merozoites. One of the basic proteases was further characterized as a thiol protease (EC 3.4.22). The activity of this protease was enriched in the dense granule fraction.