A fresh look at the SarcoFluor antibody test for the detection of specific antibodies to Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis.

Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, including the use of serum:CSF antibody ratios, the SarcoFluor antibody test for Sarcocystis neurona requires revalidation. The SarcoFluor, a previously validated immunofluorescent antibody test (IFAT) for the detection of antibodies specific to S. neurona in serum and cerebrospinal fluid (CSF) of naturally infected horses was analyzed using recent data and considering a serum:CSF antibody ratio threshold. Utilization of serum and CSF phosphorylated neurofilament heavy protein (pNfH) concentrations in support of an EPM diagnosis was also evaluated. 172 horses were divided into three groups: EPM-positive horses (EPM+, n=42), neurological non-EPM horses (n=74) confirmed with non-EPM neurological diseases (cervical vertebral compressive myelopathy, equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy), and control horses (control, n=56) without neurological signs and neurological abnormalities on histology. Logistic regression was used to compare EPM diagnostic regimens. Specifically, EPM+ horses were compared with neurological non-EPM horses showing neurological signs. To consider diagnostic utility, post-test probabilities were calculated by titer. When differentiating between EPM and other neurological diseases, the combination of serum and CSF SarcoFluor testing added more information to the model accuracy than either test alone. Using serum and CSF for pNfH in support of an EPM diagnosis did not identify cutoffs with statistically significant odds ratios but increased the overall model accuracy when used with the IFAT. Utilization of IFAT titers against S. neurona in serum and CSF result in a high post-test probability of detecting EPM+ horses in a clinical setting.

Histologic characterization of eosinophilic encephalitis in horses in Florida.

Eosinophils within the central nervous system are abnormal and are usually associated with fungal or parasitic infections in horses. Causative agents include Halicephalobus gingivalis, Sarcocystis neurona, and Neospora hughesi. Confirmation of these organisms via specific testing is typically not performed, and final diagnoses are often presumptive. With molecular technology, many of these organisms can now be confirmed. This is important for emerging and zoonotic pathogens, including Angiostrongylus cantonensis, an emerging parasite of interest in the southeastern United States. We retrospectively analyzed eosinophilic encephalitides in Floridian horses for H. gingivalis, S. neurona, and A. cantonensis, applied descriptors to equine eosinophilic encephalitides, and determined if a relationship existed between these descriptions and specific etiologic agents. In a database search for horses with eosinophilic and other encephalitides submitted to the University of Florida, College of Veterinary Medicine, Anatomic Pathology Service, we identified 27 horses with encephalitis, and performed DNA isolation and rtPCR on formalin-fixed, paraffin-embedded blocks from these cases. Real-time PCR identified 6 horses positive for S. neurona and 4 horses positive for H. gingivalis; all horses were negative for A. cantonensis. All 25 control horses were negative for H. gingivalis, S. neurona, and A. cantonensis. Pattern analysis and eosinophil enumeration were not useful in differentiating among causes of eosinophilic encephalitides in horses in our study.

Review of triazine antiprotozoal drugs used in veterinary medicine.

Triazines are relatively new antiprotozoal drugs that have successfully controlled coccidiosis and equine protozoal myeloencephalitis. These drugs have favorably treated other protozoal diseases such as neosporosis and toxoplasmosis. In this article, we discuss the pharmacological characteristics of five triazines, toltrazuril, ponazuril, clazuril, diclazuril, and nitromezuril which are used in veterinary medicine to control protozoal diseases which include coccidiosis, equine protozoal myeloencephalitis, neosporosis, and toxoplasmosis.

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development.

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.

Immunohistochemical study to demonstrate Sarcocystis neurona in equine protozoal myeloencephalitis.

A 5-year (1985-1989) retrospective immunohistochemical study was conducted using an avidin-biotin complex (ABC) immunoperoxidase method to demonstrate Sarcocystis neurona in histologically suspect cases of equine protozoal myeloencephalitis (EPM). Primary antibodies against S. neurona and S. cruzi were utilized for the ABC technique. The findings were compared with those from cases in which the organisms were detected by examination of hematoxylin and eosin (HE)-stained neuronal sections. HE-stained sections detected the presence of the organisms in 20% of the suspect cases; whereas the ABC technique confirmed the presence of S. neurona in 51% and 67% of the cases by S. neurona and S. cruzi antibodies, respectively. A review of clinical case histories showed that 21/47 (45%) of the EPM horses with parasites in the tissue sections had prior treatment with antiprotozoal drugs and/or steroids. Using the test results of S. neurona and S. cruzi as a standard reference, HE test sensitivity based on examination of up to 30 neuronal sections per case was only 25%, and test specificity was 91%.

Equine protozoal myeloencephalitis in a pony.

A 10-year-old pony died 5 days after the onset of a nervous disorder. Necropsy revealed a yellowish area of discoloration (1.5 by 1 cm) in the medulla oblongata. Microscopically, necrosis and nonsuppurative myeloencephalitis were found in the medulla oblongata. Immature and mature meronts (25 by 10 microns) were seen in neural tissue and in capillaries of the brain stem. Organisms were similar structurally to those seen in equine protozoal myeloencephalitis of horses.

Identification of a new Venezuelan equine encephalitis virus from Brazil.

Two strains of recently isolated Venezuelan equine encephalitis (VEE) complex virus from southern Brazil, avirulent for 6- to 8-week-old mice and short-haired guinea pigs, were characterized by biologic, serologic, and biochemical means. They were shown serologically to represent a single, newly recognized variant of subtype I. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of ribonuclease T1 digests of viral ribonucleic acid showed considerable homology between the genomes of the new variant prototype and variant IA. Three structural proteins were visualized by discontinuous sodium dodecyl sulfate-PAGE (SDS-PAGE). Although the smallest protein of both recent isolates migrates with the capsid proteins of other subtype I viruses, the larger structural proteins of the new variants differ in molecular weight from the E1 and E2 envelope glycoproteins of the other subtype I variants. The new isolates produced peptide fragment patterns that were identical to each other, but different from the patterns of other subtype I viruses, following SDS-PaGE of dissociated virions digested with Staphylococcus aureus V8 protease. Since these two isolates were from Culex (Melanoconion) species mosquitoes and from a bat (Carollia perspicillata), were postulated that this is an enzootic VEE virus variant for which the classification IF is suggested.