Sarcocystis Cruzi Research Articles

Prevalence of Sarcocystis spp. cysts in Japanese and imported beef (Loin: Musculus longissimus)

A survey was carried out to investigate the occurrence of Sarcocystis infection in the loin ( Musculus longissimus) of Japanese and imported beef. In all, the muscle tissue of 482 samples were examined by histological method. The prevalence of Sarcocystis unspecified species cysts was lower in Japanese beef (total 6.31%: 0% in Holstein castrated, 12.96% in Holstein milk cow, 3.33% in Japanese shorthorn and 11.58% in Japanese black cattle) than in beef imported from America (36.78%) or Australia (29.49%). The infection density of imported beef, especially in American, was higher than in Japanese beef. All detected cysts except one were identified as Sarcocystis cruzi. One thick walled cyst was found in Australian beef but it could not be distinguished as either Sarcocystis hirsuta or Sarcocystis hominis.

Blood clotting disorders during experimental sarcocystiosis in calves

The effects of experimental infection of calves with Sarcocystis cruzi on the blood coagulation cascade were investigated. Calves were inoculated orally with 1×10 5 sporocysts (group S1, n = 6) or with 5×10 5 sporocysts of S. cruzi (group S2, n = 3). A group of eight calves served as non-infected controls (group C). The animals were bled once during the first 4 weeks of infection and twice a week thereafter until day 40 p.i. The following parameters were measured: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, thrombin coagulase time, factors XII, XI, X, IX, VIII:C, VII, V, prekallikrein, fibrinogen, α2-antiplasmin, antithrombin III, α1-antitrypsin, α2-macroglobulin, haematocrit, haemoglobin, numbers of erythrocytes and thrombocytes. The infected calves developed acute sarcocystiosis from 25 (S1) or 29 (S2) days p.i. onwards. During the acute disease, antiproteases tended to elevated values and thrombocyte counts were generally reduced. In group S1 prolonged prothrombin time and reduced activities of factors VII or V were found. In group S2 accelerated prothrombin time and activated partial thromboplastin time, as well as elevated factor X activities, were recorded even before the onset of clinical disease at 19 days p.i. While prothrombin time returned to normal levels thereafter, activated partial thromboplastin time remained short. Activities of factor V, factor VII and factor X were significantly reduced in group S2 at the onset of acute sarcocystiosis, and one of the three calves died at 29 days p.i. The other parameters were not significantly affected by either dose of infection. No evidence for a classical disseminated intravascular coagulation syndrome could be found; however, it was demonstrated that S. cruzi alters plasma coagulation in a dose-dependent way.

Characterization of Bovine Sarcocystis Species by Analysis of Their 18S Ribosomal DNA Sequences

Two Sarcocystis hirsuta isolates from 2 cattle (Bos taurus), 2 Sarcocystis hominis-like isolates from 2 cattle, 1 each Sarcocystis hominis-like isolate from a zoo-born dwarf zebu (B. taurus) and from a zoo-born bison (Bison bison), and 1 each Sarcocystis cruzi isolate from the dwarf zebu and the bison have been characterized by comparing the directly sequenced polymerase chain reaction products of their 18S rRNA genes. Sequences of 2 different isolates from the same species always showed a very high, nearly complete identity to each other both within all or only the conserved overlapping nucleotides. Thus, the 18S rDNA sequences of both S. hirsuta isolates proved to belong to a single species. The same is true for all 3 S. cruzi sequences. The 4 S. hominis-like isolates could not be placed within a single species using these criteria. They formed 2 separate groups (1 cattle isolate and the bison isolate, and the other cattle isolate and the dwarf zebu isolate). The results suggest that zoo-born large mammals do serve as intermediate hosts for Sarcocystis species known from indigenous herbivores, and that there might be another species found in cattle that is morphologically very similar to S. hominis.

In vitro isolation and characterisation of a bovine Neospora species in Japan

Eleven aborted bovine fetuses and five calves suspected as having neosporosis were necropsied and tissues from these animals were inoculated into bovine cardiopulmonary aortic endothelial cells and monkey kidney cells and maintained at 37°C with 5 per cent CO 2. Neospora tachyzoites were observed in one cell 49 days after inoculation. The isolated parasite ( JPA1) was morphologically identical to the previously reported bovine Neospora species ( BPA1) and confirmed by its strong antigenic reactivity with bovine control antisera to Neospora species and its lack of reactivity with Toxoplasma gondii and Sarcocystis cruzi antisera. This is the first bovine Neospora species isolate in Asia and further studies with this isolate are now expected.

Novel ELISA for detection of Neospora-specific antibodies in cattle

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Neospora species in cattle was developed. Whole formalin-fixed Neospora caninum (NC-Liverpool) tachyzoites were used as antigen and a monoclonal antibody to...

Characterization of a Swedish bovine isolate of Neospora caninum

The brain of a stillborn calf, seropositive to Neospora caninum and born to a seropositive cow, was homogenized and cultured on Vero cells, where growth of Neospora-like tachyzoites was detected after 8 weeks. The ultrastructural features of the new isolate (Nc-SweB1) corresponded to those of previously published Neospora isolates. In indirect immunofluorescence tests, antigens on Nc-SweB1 tachyzoites were recognized by antibodies raised to a canine N. caninum isolate (Nc-1) but not by antibodies to Toxoplasma gondii, Sarcocystis cruzi, S. tenella, Eimeria alabamensis, Babesia divergens, or B. motasi. Immunoblot analyses revealed no major antigenic difference between Nc-SweB1 and Nc-1, whereas several differences were seen between Nc-SweB1 and protozoa related to N. caninum. The sequences of 16S-like rRNA and the internal transcribed spacer 1 of Nc-SweB1 revealed complete homology with corresponding sequences of two canine N. caninum isolates. Thus, no dissimilarity between Nc-SweB1 and the canine isolates was found, confirming that Nc-SweB1 is N. caninum and suggesting that Neospora-like organisms isolated from cattle are indeed N. caninum.

On the diagnostics of Sarcocystis in cattle: sarcocysts of a species unusual for Bos taurus in a dwarf zebu

Sarcocysts from the musculature of a dwarf zebu born in a German zoo are described by light and transmission electron microscopy. In addition to the three named bovine Sarcocystis species ( Sarcocystis cruzi, S. hominis, and S. hirsuta) a fourth species was found which was not previously reported from cattle. It mostly resembles Sarcocystis gracilis Rátz, 1909 from roe deer.

Studies on pathogenesis, tissue infection and congenital transmission in cows experimentally infected with Sarcocystis cruzi by various routes

A group of nine cows, naturally infected with Sarcocystis, were challenged with Sarcocystis cruzi: three intrarumenally with sporocysts, two intrarumenally with water (controls), two intravenously with merozoites grown in vitro and two intravenously with saline solution (controls). The animals intrarumenally challenged with sporocysts developed acute sarcocystiosis and produced stillborn calves, whereas those intravenously challenged with merozoites suffered from subclinical sarcocystiosis with premature births. No parasites were found in calves from cows challenged with sporocysts; however, a meront of Sarcocystis was found in calves from cows the cerebrospinal fluid of a calf from a cow intravenously inoculated with merozoites of S. cruzi. This is the first time that merozoites of S. cruzi grown in vitro have been demonstrated to retain the ability to infect their natural intermediate host and complete their life cycle.

Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.

Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta.

Use of genomic DNA probes for the diagnosis of acute sarcocystosis in experimentally infected cattle

Two clones of 1.4 and 4.33 kilobase pairs (kbp) DNA inserts, were selected from a Sarcocystis cruzi sporozoite genomic library constructed in bacteriophage lambda gt10. These clones strongly hybridized with sporozoite and merozoite DNA and were evaluated as probes for detection of merozoite DNA in clinical samples. Of five calves in the experiment, four were each orally dosed with approximately 200 000 S. cruzi sporocysts; one calf served as non-infected control. Subsequently, blood was collected from the calves twice weekly for 3.5 months and fractionated into buffy coats, polymorphonuclear cells, and plasma. Total cellular DNA extracted from these fractions was dot blotted on nylon membranes and hybridized with the probes radiolabeled with [ α- 32P]dATP. The probes detected merozoites on Day 22 post infection in the buffy coats and intermittently from Day 25–39 in the granulocyte fraction. Parasitemia (ie. merozoites in blood) was also detected by indirect fluorescent antibody technique (IFAT) and direct microscopy. Diagnosis of sarcocystosis in cattle using genomic DNA probes by dot blot hybridization provides an alternative method of detecting parasitemia that is more rigorous than the other two tests (IFAT, direct microscopy) which rely on morphology of the merozoite and visualization by the examiner. As probes detected merozoite DNA in the granulocyte fraction, polymorphonuclear cells may be involved in the pathogenesis of S. cruzi; however this hypothesis requires further study.

Identification of Opossums (Didelphis virginiana) as the Putative Definitive Host of Sarcocystis neurona

Sarcocystis neurona is an apicomplexan that causes equine protozoal myeloencephalitis (EPM) in North and South America. Horses appear to be an aberrant host, because the merozoites continually divide in the central nervous system, without encysting. The natural host species has not previously been identified. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was compared to those of Sarcocystis muris, Sarcocystis cruzi, Toxoplasma gondii, and Cryptosporidium parvum to identify a unique region suitable for a species-specific amplification primer. The S. neurona SSURNA primer was used in a polymerase chain reaction (PCR) assay for the purpose of identifying this organism in feces and intestinal digest of wildlife specimens. Sporocysts were isolated from 4 raccoons (Procyon lotor), 2 opossums (Didelphis virginiana), 7 skunks (Mephitis mephitis), 6 cats (Felis catus), 1 hawk (Accipiter sp.), and 1 coyote (Canis latrans). The S. neurona SSURNA PCR assay and a control PCR assay using protist-specific primers were applied to all sporocyst DNA samples. All sporocyst DNA samples tested positive on the control assay. The SSURNA PCR assay yielded a 484-bp product only when applied to opossum samples. The SSURNA gene of both opossum sporocyst samples was sequenced to determine its relationship to the S. neurona SSURNA gene. The sequence had 99.89% similarity with S. neurona. This suggests that opossums are the definitive host of S. neurona.

Serological evidence implicating Neospora species as a cause of abortion in British cattle

By means of an immunofluorescence antibody test (IFAT), using in vitro cultured parasites as antigen, antibodies to Neospora species at titres > or = 1/1280 were found in 11 of 120 Scottish cattle that had recently aborted but in only one of 97 cattle from herds in which there had been no recent abortions (P < 0.01). The specificity of the antibodies was confirmed by the lack of cross reactivity between samples with high titres to Neospora and toxoplasma antigen in a direct agglutination test, and by the absence of reactivity at > or = 1/640 in the IFAT of convalescent sera from cattle infected experimentally with Toxoplasma gondii, Sarcocystis cruzi, Eimeria bovis, E alabamensis, Cryptosporidium parvum and Babesia divergens. These results demonstrate that Neospora species infection occurs commonly in aborting cattle in Britain, and that the IFAT may be a useful tool for investigating the infection.

Survey of Sarcocystis infection in cattle in east Hokkaido, Japan.

The prevalence of Sarcocystis infection was examined in older cows and imported cattle slaughtered in East Hokkaido. Samples of myocardial tissues were examined for Sarcocystis microscopically. Sarcocystis cysts were detected in 15.7% of 83 older cows, 48.4% of 91 imported cattle which were kept in East Hokkaido prior to slaughter and 51.1% of 94 imported cattle slaughtered immediately after quarantine check. Based on the morphology of the cyst wall and the establishment of infection in experimentally inoculated dog, the Sarcocystis species was identified as Sarcocystis cruzi.

Preparation and applicability of Sarcocystis cruzi antigens and their anti-S. cruzi rabbit sera for serodiagnosis of bovine sarcocystosis.

Sarcocystis cruzi antigens and their antisera were prepared for the seroimmunological diagnosis of bovine sarcocystosis. Fresh S. cruzi cysts directly removed from cardial muscle of slaughtered cattle were digested with trypsin to release bradyzoites. By twelve cycles of freezing at-22 degrees C and thawing at 37 degrees C, bradyzoites were let to leach out soluble material. The soluble antigens were inoculated four times to rabbits at a dose of 343 micrograms protein and anti-S. cruzi sera were prepared with blood of the rabbits. Gel immunodiffusion test showed no cross-reaction between the present antigens and any of anti-Toxoplasma gondii, -Hammondia hammondi and -Besnoitia wallacei rabbit sera. Avidin-biotin complex immunoperoxidase (ABC) technique with the present anti-S. curzi rabbit sera showed clear positive reaction against S. cruzi cysts in the muscular sections of infected cattle.

Serologic Diagnosis of Encephalomyocarditis Virus Infection in Swine by the Microtiter Serum Neutralization Test

ophilic cytoplasmic and sometimes intranuclear inclusions characteristic of canine distemper infection. No viral inclusions were seen in the brain and lungs. However, in lung there was moderately severe patchy interstitial alveolar thickening. Other incidental findings included moderate numbers of Sarcocystis in striated muscles (heart, tongue, diaphragm, esophagus, masseter, and extraorbital muscles), a few Capillaria sp. in the mucosa of the esophagus and urinary bladder, large numbers of Cryptosporidium sp. in the intestines, and a few unidentified helminthic eosinophilic granulomas in the lung and mesenteric lymph nodes. The eosinophilic meningitis observed in the present case appears to be an incidental finding not related to the protozoa1 infection. Based on these findings, the primary disease in the raccoon was distemper. The neurologic signs were attributed to the extensive multifocal lesions produced by the unidentified protozoa. Based on morphologic and antigenic differences, the organism was neither T. gondii nor N. caninum. Both N. caninum and T. gondii divide into two progeny by endodyogeny, whereas the present organism divided into numerous progeny by schizogony. The low T. gondii antibody titer (10) in the raccoon serum is considered nondiagnostic in the agglutination test. The organism causing encephalitis in the raccoon is morphologically similar to the organism associated with equine protozoa1 myeloencephalitis (EPM). The organism in the raccoon and that causing EPM resemble Sarcocystis in structure, react with Sarcocystis immunologically, are located free in the host cell cytoplasm, divide by schizogony, have no rhoptries, multiply in neurons, and cause malacia of the central nervous system. Acknowledgements. This research was supported in part by grants from the Pennsylvania Department of Agriculture. The authors thank Eva Kovacs, M. Coicou, S. Hindman, B. Lehmann, and J. Dieter for technical assistance, Charlie Brown for transmission electron microscopy, and Dr. D. E. GranStrom for providing Sarcocystis cruzi serum.

Immunofluorescent Localization of Sarcocystis Cruzi Antigens, IgG and IgE, in Lesions of Eosinophilic Myositis in Cattle

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Elevated IgG Antibody to Sarcocystis Cruzi Associated with Eosinophilic Myositis in Cattle

Blood sera, skeletal muscle, and cardiac muscle from 24 bovine carcasses condemned for eosinophilic myositis by US Department of Agriculture meat inspection veterinarians were compared with similar specimens from 35 random carcasses passed for human consumption. Fluorescence values determined by using a fluorometric immunoassay system were used to measure the specific antibodies to Sarcocystis cruzi. The values were significantly elevated in carcasses condemned for eosinophilic myositis as compared to carcasses passed for human consumption. The elevated fluorescence values appeared to be more than coincidental, suggesting that S. cruzi may be a causative agent of eosinophilic myositis. Microscopic examination of affected muscle revealed lesions typical of eosinophilic myositis. Lesions were characterized by extensive multifocal areas of myofiber hyaline degeneration, necrosis with sarcoplasmic fragmentation, mineralization of occasional myofibers, and atrophy of fibers with varied stages of fibrosis. Inflammatory cell exudates were predominantly eosinophils, with some macrophages and lymphocytes, and extravasated erythrocytes within, and adjacent to, affected myofibers. Affected muscles contained more S. cruzi than unaffected muscle from passed carcasses. However, a distinct cause and effect relationship could not be determined between the parasite and the presence of eosinophilic myositis.

Identification of Antigens of Sarcocystis cruzi Sporozoites, Merozoites, and Bradyzoites with Monoclonal Antibodies

Sporozoites and culture-derived merozoites of Sarcocystis cruzi were used to elicit monoclonal antibodies (MAb's) in mice. Some of these antibodies reacted with the surface of live sporozoites and merozoites as determined by immunofluorescence. An array of similar antigens was identified in Western blots of sporozoites by both anti-merozoite MAb's and an anti-sporozoite MAb. At least 1 antigen in blots of bradyzoites was identified by anti-merozoite MAb's and a cluster of antigens was identified by an anti-sporozoite antibody. These results indicate that several surface epitopes of sporozoites and merozoites are shared with molecules of bradyzoites and that antigen patterns of molecules bearing these epitopes in 3 stages of Sarcocystis may be either distinct or similar.

The prevalence and identity of Sarcocystis in beef cattle in New Zealand

Muscle tissue from the oesophagus and diaphragm of 500 beef cattle slaughtered in New Zealand was examined for Sarcocystis infection by microscopic examination of cysts isolated from muscle samples. All cattle were infected with Sarcocystis; based on light microscopy of cysts, 98% had thin-walled Sarcocystis cruzi cysts and 79.8% had thick-walled ( Sarcocystis hirsuta/Sarcocystis hominis) cysts. Cysts were also collected for electron microcopy and transmission experiments. Thick-walled cysts could not be distinguished as S. hirsuta or S. hominis by light or electron microscopy. Thick-walled cysts were fed to three cats and one human volunteer; one cat shed sporocysts but not the human volunteer. Electron microscopy of the cysts revealed many features that have not been described previously.