Antoinette E. Marsh

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae).

Neospora hughesi n. sp. was isolated from the central nervous system tissue of an adult equine (Equus caballus) from California. The tachyzoites are crescent-shaped, approximately 2 x 5 microm (1.8-3.0 x 4.0-7.0 microm), with characteristic apical complex structures consisting of an anterior polar ring, conoid, numerous rhoptries filled with a uniform electron-dense material, and 22 microtubules extending posteriorly from the polar ring. Comparison of N. hughesi to canine and bovine Neospora caninum isolates showed phenotypic differences in immunoreactive proteins. Molecular analysis of the small subunit ribosomal RNA gene revealed no differences in the nucleotide sequence between N. hughesi and N. caninum isolates examined. However, the internal transcribed spacer I region revealed 7 nucleotide base differences between N. hughesi and N. caninum isolates (CN1 and BPA1) analyzed in this study. The existence of nucleotide base differences in the internal transcribed spacer regions suggests that this region may be a genetic marker for discriminating species within the genus Neospora. The ultrastructural, antigenic, and molecular data support distinction of N. hughesi as a new species, separate from N. caninum, the only recognized species in this genus.

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.

Meningoencephalitis due to a Sarcocystis neurona-like protozoan in Pacific harbor seals (Phoca vitulina richardsi)

Seven Pacific harbor seals with meningoencephalitis associated with Sarcocystis neurona-like protozoa are described. Six of the 7 seals were free-ranging and were found stranded over an 80-km stretch of central California coastline; the other was captive. All had marked to severe nonsuppurative meningoencephalitis, most severe in the cerebellar cortex. Immunohistochemistry for S. neurona antigens was positive on brain tissue in all cases, revealing numerous merozoites as well as developing and mature schizonts, including rosette forms. Electron microscopy performed on 3 animals revealed merozoites and schizonts consistent with Sarcocystis sp., with the absence of rhoptries in merozoites, lack of a parasitophorous vacuole around schizonts, and division by endopolygeny. Serology using western blotting revealed the presence of anti-S. neurona immunoglobulins in the sera of 4 of 5 seals tested. Four animals also had a concurrent mild to moderate nonsuppurative myocarditis; in 1 seal, rare sarcocysts of undetermined species were present within cardiomyocytes.

Comparison of the Internal Transcribed Spacer, ITS-1, from Sarcocystis falcatula Isolates and Sarcocystis neurona

The genetic diversity among 6 Sarcocystis falcatula isolates derived from geographically distinct regions in the U.S.A. was detected using the first internal transcribed spacer region 1 (ITS-1) of the rRNA gene. These sequences were then compared to the full sequence from a Sarcocystis neurona isolate obtained from a California horse diagnosed with equine protozoal myeloencephalitis. No nucleotide differences were detected over partial sequence analysis of 2 additional S. neurona isolates: however, the complete nucleotide sequence for the ITS-1 region was not compared. Twelve nucleotide differences were consistently detected when aligned sequences of S. neurona were compared to those of the S. falcatula isolates. Additional nucleotide base changes were detected among the S. falcatula isolates, but these changes were not consistent in all the S. falcatula isolates. These results indicate that S. falcatula may be comprised of a heterogeneous population and that the ITS-1 region can be used to distinguish S. neurona from S. falcatala used in this study.

Differentiation of Neospora hughesi from Neospora caninum based on their immunodominant surface antigen, SAG1 and SRS2.

Neospora hughesi is a newly recognised parasite that is closely related to Neospora caninum, and is a cause of equine protozoal myeloencephalitis. We have characterised two N. hughesi immunodominant tachyzoite antigens which exhibit antigenic and molecular differences from the homologous tachyzoite antigens on N. caninum. These antigens on N. hughesi are referred to as NhSAG1 and NhSRS2, using the same mnemonics as used for the N. caninum antigens (NcSAG1 and NcSRS2), and are homologous to Toxoplasma gondii surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2). The NcSAG1 and NcSRS2 were antigenically conserved in six different N. caninum isolates from cattle and dogs. The two equine-derived Neospora isolates, one designated as N. hughesi, were similar to each other but different from N. caninum. There was 6% difference in amino acid identity between NcSAG1 and NhSAG1, whereas there was a 9% difference when NcSRS2 and NhSRS2 were compared. The polymorphism of these genes and their corresponding proteins provide additional markers which can be used to distinguish N. caninum from N. hughesi.

Acute Sarcocystis Falcatula-Like Infection in a Carmine Bee-Eater (Merops Nubicus) and Immunohistochemical Cross Reactivity Between Sarcocystis Falcatula and Sarcocystis Neurona

An unidentified Sarcocystis falcatula-like infection was diagnosed in a captive bee-eater (Merops nubicus) in a zoo in Florida. The bird died suddenly, probably due to protozoa-associated pneumonia. Protozoal schizonts were found in lungs and heart, and immature sarcocysts were seen in skeletal muscles. Ultrastructurally, schizonts were located in capillary endothelium and merozoites lacked rhoptries, consistent with the structure of Sarcocystis species. Sarcocysts were immature, microscopic, and contained only metrocytes. The sarcocyst wall had finger-like villar protrusions that were up to 0.7 µm long and up to 0.2 µm wide. The villar protrusions lacked microtubules, characteristically seen in sarcocysts of S. falcatula. Antigenically, parasites in lungs and muscles of the bee-eater reacted with a varying intensity with polyclonal rabbit antisera to S. falcatula and Sarcocystis neurona. Results indicated that sarcocysts in the bee-eater were morphologically different from the reported structure for sarcocysts of other S. falcatula infections.

Fatal hepatic sarcocystosis in two polar bears (Ursus maritimus).

Fatal hepatic sarcocystosis was diagnosed in 2 polar bears from a zoo in Anchorage, Alaska. Gross lesions were icterus and systemic petechiae. Marked microscopic lesions were detected only in the liver and included severe random necrotizing hepatitis with hemorrhage. Only asexual stages of an apicomplexan parasite were detected within hepatocytes, and rare extracellular zoites were seen in foci of necrosis. The parasite divided by endopolygeny, and occasionally merozoites formed rosettes around a central residual body. Ultrastructural features of the merozoites included a conoid and low numbers of micronemes at the apical pole, centrally located nuclei, and absence of rhoptries. The parasites failed to react with anti-Neospora sp., anti-Toxoplasma gondii, or anti-Sarcocystis neurona sera. The microscopic and ultrastructural morphology of the parasite are most compatible with an apicomplexan protozoan of the genus Sarcocystis. The life cycle of this parasite in bears is not known.

Molecular characterisation of a major 29 kDa surface antigen of Sarcocystis neurona.

A gene encoding a major 29 kDa surface antigen from Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), was cloned, sequenced, and expressed as a recombinant protein. A cDNA library was prepared in the expression vector lambda ZAP from polyA+mRNA isolated from S. neurona merozoites cultivated in vitro. Random sequencing of 96 clones identified a clone of an abundant transcript having a translated amino acid sequence with 30% identity to the 31-kDa surface antigen of Sarcocystis muris cyst merozoites. Southern blot analysis indicated that the corresponding gene exists in low copy number within the S. neurona genome, but RNA blot analysis and other data indicated that the gene transcript is highly abundant. The sequence of the cDNA clone encoded an open reading frame specifying a polypeptide of 276 amino acids with a predicted size of 28.7 kDa. The deduced amino acid sequence displayed a hypothetical N-terminal signal peptide sequence followed by a polypeptide containing 12 cysteines. The coding region of the cDNA insert was subcloned into the expression vector pET14b, and a fusion protein expressed. The recombinant polypeptide was recognised by mAb 2A7 and mAb 1631, directed against a 29 kDa native protein found on the surface of cultured merozoites. Antibodies in serum and cerebrospinal fluid from a horse with EPM recognised a 29 kDa native protein of S. neurona merozoites and the 29 kDa recombinant protein. This S. neurona surface antigen is named SnSAG1.

Characterization of monoclonal antibodies developed against Sarcocystis neurona

Abstract. Equine protozoal myeloencephalitis (EPM), caused by a protozoal parasite infection of the central nervous system, is the most commonly diagnosed neurologic disease of horses in North America. In specific regions of the United States approximately 50% of the horse population is seropositive to Sarcocystis neurona. However, not all seropositive horses develop clinical signs. Detailed clinical examination, along with cerebrospinal fluid antibody evaluation are often used to diagnose EPM. Postmortem evaluation of the brain stem and spinal cord for histopathologic lesions compatible with nonsuppurative meningoencephalomyelitis is used for reaching a diagnosis since organisms are difficult to detect by routine staining methods. Immunohistochemical staining aids detection of organisms; however, the polyclonal antibodies that react with S. neurona may react with merozoites of other closely related Sarcocystis species. In this study, two different monoclonal antibodies, mAb 2A7–18 and mAb 2G5–2, were developed against the merozoite stage of S. neurona UCD-SN1 strain. The antibodies were evaluated by immunoblot, immunofluorescence, immuno-electron microscopy and immunohistochemistry for their ability to react with S. neurona. MAb 2G5–2 reacted with antigenically distinct S. neurona isolates whereas mAb 2A7–18 appeared to be limited in its ability to recognize different isolates. These two monoclonal antibodies recognize protein epitopes of two different immunodominant proteins of S. neurona.