Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2003
Publication Date: 10/25/2003
Citation: Hodgkinson, J.E., Lichtenfels, J.R., Mair, T.S., Cripps, P., Freeman, K.L., Ramsey, Y.H., Love, S., Matthews, J.B. 2003. A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis. International Journal for Parasitology. 33:1427-1435.
Interpretive Summary: Strongyloid nematodes are a major cause of morbidity and mortality in equines in the United States. Resistance to antiparasitic drugs (currently the only means of controlling the nematodes) is common and alternative control methods for these parasites are needed. The 40 species in horses worldwide can be identified by a few authorities using comparative anatomy of adult stages, but larval stages are exceptionally difficult to identify and eggs are impossible to identify to even subfamily level. The development of diagnostic DNA probes that can be used to identify all stages of the nematodes including eggs in the feces is a high priority need. This report describes the use of six oligoprobes designed from intergenic spacer (IGS) region sequences to identify fourth stage larvae (L4) of the tribe, Cyathostominea associated with cases of larval cyathostominosis. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocylus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed to identify members of the entire subfamily. The results will be used by researchers working to control these economically important nematodes of horses, especially in the pharmaceutical industry, the Food and Drug Administration, and scientists evaluating biological control agents and antiparasitic drugs for larval cyathostominosis, an emerging disease of horses worldwide.
Technical Abstract: Here, we report the use of six oligoprobes designed from intergenic spacer (IGS) region sequences to identify fourth stage larvae (L4) of the tribe, Cyathostominea. Oligoprobes were designed for identification of the following speices: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocylus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed to identify members of the Strongylinae. DNA was extracted from 546 fourth stage larvae (L4) isolated from the diarrhoea of 17 clinical cases of larval cyathostominosis. The IGS region was amplified by PCR using conserved primers CY1/CY18. Three probes were used initially in Southern blotting and developed further using a PCR-ELISA, to allow high throughput identification to species. These sequences and another four probes were fully validated for sensitivity and specificity using the PCR-ELISA. A total of 522 L4 were subject to Southern blotting and PCR-ELISA techniques, with 79% of the L4 being identified to species, the remaining 21% were designated as other, unidentified species. Five of the species tested were identified; 28.54% of the 522 larvae identified were Cylicostephanus longibursatus, 25.67% C. nassatus, 15.90% C. ashworthi, 7.28% C. goldi, 1.72% C. catinatum and 0% C. insigne. C. longibursatus, C. nassatus and C. goldi species were found in proportions which reflect previous studies where adult cyathostomins were identified. No case comprised exclusively one species, with two species the lowest number identified in any one individual. This study suggests mixed species infections are associated with larval cyathostominosis.