A toxin isolated from Sarcocystis fayeri in raw horsemeat may be responsible for food poisoning.

Food poisoning has been reported after the consumption of raw horsemeat in Japan. Diarrhea with a short incubation period is a common symptom in such cases of food poisoning. Cysts found in horsemeat ingested by patients have been identified as Sarcocystis fayeri based on morphological and genetic evaluation and findings from experimental feeding of cysts to dogs, which resulted in the excretion of sporocysts. The extracts of the horsemeat containing the cysts produced a positive enterotoxic response in the rabbit ileal loop test. Intravenous injection of a 15-kDa protein isolated from the cysts induced diarrhea and lethal toxicity in rabbits, and the protein produced enterotoxicity in the ileal loop test as did the extracts of the horsemeat containing the cysts. The partial amino acid sequence of the 15-kDa protein was homologous to the actin-depolymerizing factor of Toxoplasma gondii and Eimeria tenella. These findings indicate that the 15-kDa protein of S. fayeri is a toxin that causes food poisoning after consumption of parasitized horsemeat.

Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites.

An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.

Cloning, sequencing and recombinant expression of the open reading frame encoding a novel member of the Sarcocystis muris (Apicomplexa) microneme lectin family.

Micronemes are characteristic secretory organelles located within the apical cell region of apicomplexan parasites. The protein contents are exocytosed during an early phase of host cell invasion and contribute to parasite motility and the invasion of target cells. We report here on the cloning and heterologous expression of a novel member of the Sarcocystis muris microneme lectin family. The deduced amino acid sequence is in total agreement with that obtained after sequencing the native protein and is characterized by two copies of the apple domain motif. The recombinant polypeptide is expressed in a biologically active conformation as demonstrated by its galactose binding properties.

Characterization and crystallization of a novel Sarcocystis muris lectin, SML-2.

A novel lectin (SML-2) consisting of 138 amino acids was isolated from cyst merozoites of Sarcocystis muris and sequenced by Edman degradation and mass spectrometry. All 12 cysteinyl residues are involved in disulfide bridges, four of which are attributed to a characteristic pattern of cysteines as found in the so-called PAN-module superfamily. Crystals of SML-2 diffracting to 2.1 A resolution at a synchrotron were grown by the hanging-drop vapour-diffusion technique. They belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 53.6, b = 128.8, c = 158.2 A and eight molecules in the asymmetric unit. SML-2 cocrystallized with Au galactose results in two different crystal forms. The first form is isomorphous with the native crystals and the second form adopts space group C222(1), with unit-cell parameters a = 74.7, b = 82.0, c = 131.0 A, and diffracts to 2.4 A at a rotating-anode X-ray generator.

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona.

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis, Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species; however, life cycles of these species have not been determined. Following feeding of armadillo muscles containing sarcocysts to the Virginia opossum (Didelphis virginiana), the opossums shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0x7.5 microm and each contained four sporozoites and a residual body. Sporocysts were identified as Sarcocystis neurona using PCR and DNA sequencing. A 2-month-old foal that was negative for S. neurona antibodies in the CSF was orally inoculated with 5x10(5) sporocysts. At 4 weeks post-infection, the foal had a 'low positive' result by immunoblot for CSF antibodies to S. neurona and by week 6 had a 'strong positive' CSF result and developed an abnormal gait with proprioceptive deficits and ataxia in all four limbs. Based on the results of this study, the nine-banded armadillo is an intermediate host of S. neurona.

Expression, purification, and biochemical characterization of a recombinant lectin of Sarcocystis muris (Apicomplexa) cyst merozoites.

The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia colias a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.

A comparison of sporozoite and cyst merozoite surface proteins of Sarcocystis.

Surface labeling of Sarcocystis muris and S. suicanis sporozoites with N-hydroxysuccinimide biotin led to the detection of major membrane proteins with relative molecular weights of 29 and 30 kDa, respectively. Immunoblots of Sarcocystis sporozoite proteins probed with sera from infected hosts or with polyclonal monospecific antibodies generated against membrane antigens of cyst merozoites (noncorresponding stages) showed cross-reactivity between the two developmental stages (cyst merozoites and sporozoites) as well as between the species S. muris and S. suicanis. Two-dimensional gel electrophoresis resulted in the identification of isoforms of the sporozoite membrane antigens, with isoelectric points ranging from pH 4.7 to pH 6.4 for S. muris and from pH 4.7 to pH 5.2 for S. suicanis. The molecular masses, the charge heterogeneity, and the immunological reactivity of the surface proteins of Sarcocystis sporozoites were similar to those of cyst merozoites of both species.