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Title: DIFFERENTIATION OF GASTROINTESTINAL NEMATODE SPECIES INFECTING BOVINE BY MULTIPLEX PCR

Author
item Zarlenga, Dante
item Chute, M
item Gasbarre, Louis
item Boyd, Patricia

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/12/2001
Publication Date: 5/12/2001
Citation: N/A

Interpretive Summary: The goal of effective nematode control is to protect cattle from production losses by reducing parasite transmission and the rate of parasite establishment in the host. Accurate and rapid identification of species infecting the host is essential to achieving these goals. Presently, no rapid, sensitive test exists that will diagnose gastrointestinal (GI) nematode eggs in feces that contribute to propagation of infection from pastures. We identified PCR primer sequences within 5 species of common GI nematodes that when mixed together in a single reaction can differentiate a species simultaneously and allow a qualitative assessment of the relative e burden from each egg species. This test will allow more selective drug treatment of animals, reduce the potential of drug resistance developing in parasites, reduce the potential for harmful drug residues to collect in animals and reduce costs to the producer groups by identifying and treating gonly those animals harboring the pathogenic parasite.

Technical Abstract: A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal nematodes that commonly infect cattle, and that can discriminate at the species and genera levels, genomic material derived from parasite eggs acquired from the feces of infected animals. Sequence data from internal (ITS) and external (ETS) transcribed spacers of fthe ribosomal DNA (rDNA) repeats as well as the 3'-end of the small subuni rDNA and 5'-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern characterized by a single DNA fragment for Ostertagia ostertagi (257 bp), Haemonchus placei (176 bp), Oesophagostomum radiatum (329 bp), Trichostrongylus colubriformis (243 bp) and Cooperia oncophora (151 bp). In a similar manner, the chosen primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogeneity in the ETS that is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Though not designed as a quantitative technique, relative PCR signal intensities corresponded with relative egg burdens within the DNA samples from mixed species of eggs.