David E. Granstrom

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.

Equine Protozoal Myeloencephalitis: Antigen Analysis of Cultured Sarcocystis Neurona Merozoites

Antigens of cultured Sarcocystis neurona merozoites were examined using immunoblot analysis. Blotted proteins were probed with S. cruzi, S. muris, and S. neurona antisera produced in rabbits, S. fayeri (pre- and post-infection) and S. neurona (pre- and post-inoculation) sera produced in horses, immune sera from 7 histologically confirmed cases of equine protozoal myeloencephalitis (EPM), and pre-suckle serum from a newborn foal. Eight proteins, 70, 24, 23.5, 22.5, 13, 11, 10.5, and 10 Kd, were detected only by S. neurona antiserum and/or immune serum from EPM-affected horses. Equine sera were titered by the indirect immunofluorescent antibody (IFA) method using air-dried, cultured S. neurona merozoites. Anti-Sarcocystis IFA titers were found in horses with or without EPM. Serum titers did not correspond to the number of specific bands recognized on immunoblots.

Equine Protozoal Myelitis in Panamanian Horses and Isolation of Sarcocystis neurona

Schizonts of Sarcocystis neurona were identified microscopically in hematoxylin-eosin-stained spinal cord sections from 2 native Panamanian horses that exhibited clinical signs of equine protozoal myelitis (EPM). Spinal cord homogenate from a third Panamanian horse with EPM was inoculated onto monolayers of cultured bovine monocytes (M617). Intracytoplasmic schizonts containing merozoites arranged in rosette forms surrounding a central residual body first were observed 13 wk postinoculation. Parasites divided by endopolygeny and lacked rhoptries. Schizonts from each horse reacted with Sarcocystis cruzi antiserum in an immunohistochemical test.

Transmission of some species of internal parasites in horses born in 1993, 1994, and 1995 on the same pasture on a farm in central Kentucky

Data are presented on the last 3 years of a 7-year study (1989-1995) on transmission of natural infections of internal parasites in horse foals (n = 27) born in 1993, 1994, and 1995 on the same pasture on a farm in central Kentucky. The foals were in a closed breeding herd of horses. Research on the first 4 years (1989-1992) of the study was published earlier (Lyons et al., 1991, 1994). Thirty-five species of endoparasites were identified, including 24 species of small strongyles. Monthly, seasonal, and host-age transmission patterns were elucidated for the parasites. Comparison of data between the first 4 years and last 3 years of the study indicates similarities, but also differences, including an increase in prevalence and numbers of Thelazia lacrymalis and Anoplocephala perfoliata.

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.

Critical and controlled tests of activity of moxidectin (CL 301, 423) against natural infections of internal parasites of equids

The activity of moxidectin was evaluated in 1988 and 1989 against natural infections of internal parasites in 20 critical tests (n = 20 equids) and three controlled tests (n = 20 equids). Two formulations, injectable administered intramuscularly (i.m.) or intraorally (i.o.) and gel i.o., were given at dose rates of 0.2, 0.3 or 0.4 mg kg-1 body weight. For the critical tests (all three dose rates evaluated), removals of second instar Gasterophilus intestinalis were 93-100%, except (89%) for the injectable formulation (i.m.) at 0.2 mg kg-1. Removals of third instar G. intestinalis were 88-100% for the injectable formulation given i.m. or i.o. and 93-100% for the gel formulation, except (53%) for one batch (0.4 mg kg-1). Activity was 100% for third instar Gasterophilus nasalis, Parascaris equorum, Strongylus vulgaris and Strongylus edentatus. For Oxyuris equi, removals were 91-100%, except (27%) for one batch of the injectable formulation given i.o. at 0.3 mg kg-1. There was apparent activity against migrating S. vulgaris and S. edentatus at various dose rates and routes of administration for both formulations. At necropsy, there were local reactions observed at the injection site of three equids. In the controlled tests, dose rates were 0.2 or 0.4 mg kg-1. Removal of third instar G. intestinalis was highest for the injectable formulation given i.m. All formulations and dose rates were highly effective against S. vulgaris and S. edentatus, but variable and incomplete against O. equi. Removal was excellent on Habronema muscae and on migrating S. vulgaris and S. edentatus, although incomplete on S. vulgaris. Gasterophilus nasalis third instars and P. equorum were present in low numbers in some non-treated equids, but none were recovered from treated equids. Toxicosis was not evident.

A study (1977–1992) of population dynamics of endoparasites featuring benzimidazole-resistant small strongyles (Population S) in Shetland ponies

Critical tests (91) were done between 1977 and 1992 in Shetland ponies to evaluate drug susceptibility and population dynamics (present paper) of endoparasites. The test ponies, most less than 1 year old, were from a herd where older animals were treated every 8 weeks initially with cambendazole (CBZ) (1974-1978) and then with oxibendazole (OBZ) (1978-1992). Previous field test data (1974-1992) on older ponies in the breeding herd indicated the presence of benzimidazole (BZ) resistant small strongyles. Data on population dynamics from the present critical tests indicated that 28 species of small strongyles persisted over the study period in spite of initial susceptibility and later refractiveness of six species to both CBZ and OBZ. Changes in intensities and other aspects were observed for the six BZ-resistant species (Cyathostomum catinatium, Cyathostomum coronatum, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus goldi, and Cylicostephanus longibursatus). Variabilities, some striking, were found in prevalence and intensity in bots, stomach worms, ascarids, eyeworms, large strongyles, pinworms and tapeworms.

Critical test evaluation (1977–1992) of drug efficacy against endoparasites featuring benzimidazole-resistant small strongyles (Population S) in Shetland ponies

Several compounds (n = 13 single or combinations; most at therapeutic dosages) were evaluated between 1977 and 1992 in critical tests (n = 91) against benzimidazole (BZ) resistant small strongyles (Population S) and several other species of internal parasites in Shetland ponies, mostly under 1 year old. The closed breeding herd, from which the test ponies were selected, had been treated every 8 weeks with cambendazole (CBZ) for 4 years (1974-1978) and oxibendazole (OBZ) for 14 years (1978-1992). Published field test data (1974-1992) on older ponies in the herd showed BZ resistance of small strongyles. Average efficacies in the present critical tests against small strongyles for OBZ (n = 59 animals) were high in early years (95% or higher), but gradually declined to a low of 1% in 1991. Side-resistance of small strongyles was evident in critical tests (n = 1-6/single drug or combination) for several other BZs and a pro-BZ; ivermectin and piperazine were highly active, but pyrantel pamoate exhibited weak activity. BZ resistance was evident for six small strongyle species (Cyathostomum catinatum, Cyathostomum coronatum, Cylicocylus nassatus, Cylicostephanus calicatus, Cylicostephanus goldi, and Cylicostephanus longibursatus). Activity on bots, ascarids, large strongyles, and pinworms was essentially as expected, indicating no drug resistance.

Natural infections of Strongyloides westeri: prevalence in horse foals on several farms in central Kentucky in 1992.

During the period 28 February-1 July 1992, fecal samples were collected and examined for eggs of Strongyloides westeri once from each of 382 horse foals (364 Thoroughbreds, 16 Standardbreds, one Lippizaner, and one draft-type horse) in central Kentucky. Ages of the foals at the time of sampling ranged from 7 to 63 days (mean 22 days). The nine farms in the study were considered to have overall excellent deworming programs. None of the foals had been treated with an antiparasitic compound before the study. Eggs of S. westeri were found in 6% (22 of 382) of the foals on 78% (7 of 9) of the farms. Current prevalence of this parasite in foals is discussed relative to studies before the availability and usage of effective drugs.

Immunohistochemical Diagnosis of Protozoan Parasites in Lesions of Equine Protozoal Myeloencephalitis

asite also differed structurally from Caryospora sp. because gamonts were not found and schizonts and merozoites were much larger than those of Caryospora sp. In structure, the parasite in the present case resembles the Sarcocystis parasite that causes fatal encephalomyelitis in horses, cattle, and sheep. Although the central nervous system in the infected dog was not examined, its litter-mate died of protozoan encephalomyelitis. 4 Acknowledgements. We thank John Jenkins and Eva Kovats for technical assistance. Montana State University, Agricultural Experiment Station Journal series No. 525847.