S. Stamper

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.

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.

RECOMBINANT NhSAG1 ELISA: A SENSITIVE AND SPECIFIC ASSAY FOR DETECTING ANTIBODIES AGAINST NEOSPORA HUGHESI IN EQUINE SERUM

Neospora hughesi is a recently identified cause of equine protozoal myeloencephalitis. However, the significance of this parasite is poorly understood. An enzyme-linked immunosorbent assay (ELISA) with a recombinant form of the N. hughesi 29-kDa surface antigen (rNhSAG1) was developed for serodiagnosis of equine N. hughesi infections. Parallel ELISA analysis showed that animals immunized or infected with N. hughesi exhibited greater antibody reactivity with rNhSAG1 than with the Neospora caninum homolog, rNcSAG1. The rNhSAG1 ELISA showed 94.4% sensitivity and 95.0% specificity when compared with N. hughesi western blot results for 1,006 samples. The N. hughesi seroprevalence was 3.4% for the 1,917 samples tested by ELISA, which is less than earlier reports. Importantly, western blot analysis of ELISA-positive sera revealed only 18 true seropositive samples for an even lower seroprevalence of 0.9%. These results imply that Neospora spp. infections are uncommon in horses. The sensitivity and specificity exhibited by the rNhSAG1 ELISA suggest that it has a potential use for serodiagnosis of N. hughesi infection in equids. Furthermore, the high-throughput capability of the ELISA will allow for screening large sample sets, which should provide a better understanding of N. hughesi epidemiology.

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.

Control of cambendazole-resistant small strongyles (Population S) with oxibendazole in a pony band: an 8 year field test (1984–1992)

Studies in a band of ponies harboring Population S benzimidazole-resistant small strongyles were initiated in 1974 and have continued for 18 years. Treatment (bimonthly) was with cambendazole for the first 4 years and with oxibendazole (OBZ) for the next 14 years. Data on the first 10 years have been published. The present investigation includes the last 8 years (4 October 1984-11 September 1992), which are the seventh through fourteenth years, of treatment with OBZ. Pre- and posttreatment mean counts of strongyle eggs (epg) and larvae (lpg) per gram of feces were determined biweekly during the current study to monitor the efficacy of OBZ. The average annual percent reductions of epg counts effected by OBZ treatments were 51%, 53%, 38%, 38%, 39%, 28%, 40%, and 19% for the seventh through fourteenth years, respectively. Similar levels of reductions were observed for lpg counts. Although OBZ was initially highly effective on this population of small strongyles, epg and lpg counts gradually declined, but have remained more or less constant since the fifth year of research. However, reductions of the counts were the lowest for the last year of the study.

Activity of praziquantel (0.5 mg kg−1) against Anoplocephala perfoliata (Cestoda) in equids

Praziquantel injectable formulation was administered at 0.5 mg k-1 per os to 24 equids naturally infected with 1-183 (average 40) Anoplocephala perfoliata. Drug activity was evaluated by a modified critical test method with necropsy 24 h after treatment. There was variable efficacy of 0-100% (aggregate average 85%); for 18 equids, 93-100%, for three equids, 70-85%, and for three equids, 0-20%.

Survey for internal parasites in cattle in Kentucky (1993)

Fecal samples were examined between 17 September and 1 November 1993 from 1765 cattle (one bull, 533 cows, 474 heifers, 22 steers, and 735 calves) on pasture on 15 farms in 11 counties in Kentucky for eggs or larvae of internal parasites. All of the cattle were beef-type except for 22 which were dairy-type. In fecal samples from the bull, cows, heifers, steers, and calves, the types of helminth eggs present were trichostrongyles (excluding Nematodirus) in 0%, 25%, 31%, 86%, and 93%, Nematodirus in 0%, 0%, < 1%, 0%, and 34%, Strongyloides in 0%, 0%, < 1%, 0%, and 7%, Trichuris in 0%, 0%, 0%, 0%, and 2%, Capillaria in 0%, 0%, 0%, 0%, and < 1%, and Moniezia in 0%, 1%, 8%, 5%, and 21%, respectively. Dictyocaulus viviparus larvae were present in feces of calves (7%) on one farm and heifers (< 1%) on another farm.

Pheromone trap for the eastern tent caterpillar moth

Abstract The discovery that the eastern tent caterpillar Malacosoma americanum (F.) causes mare reproductive loss syndrome (MRLS), and thus has the potential to continue to result in major economic losses to the equine industry of Kentucky, has resulted in an intensive effort to identify practical means to monitor and control this defoliator, including these experiments to optimize a sex pheromone trap for this pest. A pheromone-baited delta trap with a large opening, such as InterceptST Delta, was more effective than other tested traps. Orange delta traps caught more moths than other tested colors. ETC males are caught at all tested heights within the tree canopy. For monitoring flights, setting traps at 1.5 m would allow easy counting of moths. A 9:1 blend of (E,Z)-5,7-dodecadienal (ETC-Ald) and (E,Z)-5,7-dodecadienol (ETC-OH) was most effective in capturing males. Increasing loading doses of a 3:1 blend (Ald:OH) resulted in the capture of increasing numbers of moths, but a 9:1 blend was more effective than 3:1 blend even at a nine-fold lower loading rate. Pheromone-impregnated white septa caught more moths than gray septa at the same loading dose. The advantages and limitations of using pheromone traps for monitoring M. americanum are discussed.