Epizootic of equine protozoal myeloencephalitis on a farm.

OBJECTIVE: To determine the clinical findings, course of treatment, and long-term outcome of horses on a farm in central Kentucky during an epizootic of equine protozoal myeloencephalitis (EPM). DESIGN: Cohort study. ANIMALS: 21 horses on a farm in central Kentucky, 12 of which developed clinical signs of EPM. PROCEDURE: Horses on the farm were serially examined for signs of neurologic disease and serum and CSF antibodies to Sarcocystis neurona. Horses were considered to have EPM if they had neurologic signs and positive test results for antibodies to S neurona in CSF. Blood values were monitored for evidence of abnormalities resulting from long-term pyrimethamine and trimethoprim-sulfamethoxazole administration Physical, neurologic, and fetal necropsy examinations were performed as needed. Horses were treated for EPM until they had negative test results for CSF antibodies to S neurona. RESULTS: Of 21 horses on the farm, 12 had EPM over the course of 6 months. The duration of treatment ranged from 45 to 211 days, excluding 1 horse that persistently had CSF antibodies to S neurona. Adverse effects from pyrimethamine and trimethoprim-sulfamethoxazole administration included transient fever, anorexia, and depression (n = 2); acute worsening of ataxia (2); mild anemia (4); and abortions (3). CLINICAL IMPLICATIONS: EPM may develop as an epizootic. In the horses of this report subtle clinical signs that were originally considered unimportant ultimately progressed to obvious neurologic signs. Adverse effects associated with EPM treatment included worsening of neurologic signs, anemia, abortion, and leukopenic and febrile episodes.

Experimental induction of equine protozoal myeloencephalitis in horses using Sarcocystis sp. sporocysts from the opossum (Didelphis virginiana).

Sarcocystis sp. sporocysts isolated from eight feral opossums (Didelphis virginiana) were pooled and fed to 18 commercially reared budgerigars (Melopsittacus undulatus), 14 wild-caught sparrows (Passer domesticus), one wild-caught slate-colored Junco (Junco hyemalis) and five weanling horses (Equus caballus). All budgerigars died within 5 weeks post inoculation (wpi). Histologic examination revealed meronts within the pulmonary epithelia and typical Sarcocystis falcatula sarcocysts developing in the leg muscles. Sparrows were euthanized 13 and 17 wpi and their carcasses were fed to four laboratory raised opossums. Sporocysts were detected in the feces of two opossums on 15 days post inoculation (dpi) and in a third opossum on 40 dpi. Fecal samples from the fourth opossum remained negative; however, sporocysts were found in intestinal digests from all four opossums. Sporocysts were not found in feces or intestinal digest of an additional opossum that was fed three uninoculated sparrows. Five foals were fed sporocysts (Foals 2, 3, 4, 5, and 7) and two foals were maintained as uninoculated controls (Foals 1 and 6). Sporocysts from two additional feral opossums also were fed to foals. Foal 5 was given 0.05 mg kg-1 dexamethasone sodium phosphate daily beginning 2 days before inoculation for a total of 2 weeks. Horse sera were tested three times per week, and cerebrospinal fluid (CSF) samples were tested biweekly for anti-Sarcocystis neurona antibodies by Western blot analysis. No foals had any S. neurona-specific antibodies by Western blot analysis prior to sporocysts ingestion. Seroconversion occurred in Foals 3, 5, and 7 by 24 dpi, followed by positive CSF tests on 28 dpi. Foals 2 and 4 seroconverted by 40 dpi. Cerebrospinal fluid from Foal 2 tested positive by 42 dpi, but Foal 4 remained seronegative throughout the study. Sera and CSF from control Foals 1 and 6 remained seronegative. All foals with positive CSF developed neurologic clinical signs. Neurologic disease was evident in Foals 2 and 3 by 42 dpi and in Foal 7 by 28 dpi. The severity of clinical signs progressed to marked spasticity, hypermetria and ataxia in Foal 7 by the end of the trial. Necropsy examination of inoculated foals did not reveal gross lesions; however, microscopic lesions consistent with equine protozoal myeloencephalitis (EPM) were found in Foals 2, 3, and 7. Protozoa were not observed in the tissue sections. Microscopic lesions consistent with EPM were not found in Foals 4 and 5 or in uninoculated control Foals 1 and 6. Foal 5 had unilateral non-inflammatory lesions in the cervical and thoracic spinal cord consistent with cord compression. These data indicate that the opossum is a definitive host of S. neurona.

Detection of equine infectious anemia viral RNA in plasma samples from recently infected and long-term inapparent carrier animals by PCR.

Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detected in 63 of 63 horses with EIAV antibody test-positive histories on approved serologic tests, demonstrating that RT-nPCR was probably directed against highly conserved sequences in the viral genome. The RT-nPCR assay, agar gel immunodiffusion test, and conventional virus isolation were compared for detection of early infection in 12 experimentally infected ponies. Viral gag sequences were detected in all 12 animals by 3 days postinfection (p.i.) by RT-nPCR, whereas virus could not be routinely isolated on cell culture until 9 to 13 days p.i. and EIAV antibodies could not be detected by agar gel immunodiffusion until 20 to 23 days p.i. Finally, specificity of the RT-nPCR assay was examined by testing plasma from 43 horses with serologic test-negative histories and no known contact with EIAV-infected animals. Viral gag sequences were not detectable in this control group. These data suggest that the EIAV RT-nPCR assay effectively detects EIAV and is more sensitive than current standard methods for detection of early stages of infection.

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.

Phylogenetic relationship of Sarcocystis neurona to other members of the family Sarcocystidae based on small subunit ribosomal RNA gene sequence.

Sarcocystis neurona is a coccidial parasite that causes a neurologic disease of horses in North and South America. The natural host species are not known and classification is based on ultrastructural analysis. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was amplified using polymerase chain reaction techniques and sequenced by Sanger sequencing reactions. The sequence was compared with partial sequences of S. muris, S. gigantea, S. tenella, S. cruzi, S. arieticanis, S. capracanis, Toxoplasma gondii, Eimeria tenella, and Cryptosporidium parvum. Alignments of available sites for all 10 species and alignments of the entire SSURNA sequence of S. neurona, S. muris, S. cruzi, T. gondii, and C. parvum were performed. Alignments were analyzed using maximum parsimony and maximum likelihood methods to determine relative phylogeny of these organisms. These analyses confirmed placement of S. neurona in the genus Sarcocystis and suggested a close relationship to S. muris, S. gigantea, and T. gondii. Molecular phylogeny suggests that Sarcocystis spp., which utilize the dog (Canis familiaris) as the definitive host, evolved from a common ancestor, whereas those species (including T. gondii) that utilize the cat (Felis domesticus) as the definitive host evolved from another common ancestor. This suggests a possible definitive host for S. neurona.

Application of cycle dideoxy fingerprinting to screening heterogeneous populations of the equine infectious anemia virus.

Nucleotide sequence heterogeneity in a population of the equine infectious anemia virus (EIAV) was investigated using a modification of the dideoxy fingerprinting (ddF) technique. PCR-amplified regions of the gag gene from EIAV isolates were ligated into plasmid vectors and used to transform bacteria. The single dideoxynucleotide sequencing step was performed using plasmid DNA prepared from individual bacterial colonies using an 35S end-labeled primer and Taq DNA polymerase. Analysis of the products of this reaction was conducted using non-denaturing polyacrylamide gel electrophoresis. Polymorphism within this gene was suggested by the presence of several distinct electrophoretic profiles. Significantly, each profile could be correlated with variations in nucleotide sequence, which demonstrates that cycle ddF (CddF) offers a rapid and sensitive approach to identify polymorphism in PCR-amplified products.