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Title: IDENTIFICATION AND SEMI-QUANTITATION OF OSTERTAGIA EGGS BY ENZYMATIC AMPLIFICATION OF ITS-1 SEQUENCES

Author
item Zarlenga, Dante
item Gasbarre, Louis
item Boyd, Patricia
item LEIGHTON, E. - C-BAR GROUP, VA.
item LICHTENFELS, J. R - USDA BIOSYSTEMATICS, MD

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/4/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Ostertagia ostertagi is the most pathogenic of gastrointestinal nematodes infecting cattle in the Unites States. Presently, no rapid, sensitive test exists that will both diagnose and quantitate the level of eggs in feces that contribute to propagation of infection from pastures. We identified a region of DNA within O. ostertagi that can differentiate it from other common gastrointestinal nematodes and permit the semiquantitation of egg numbers. A simple PCR test was developed to differentiate O. ostertagi from related parasites. 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 enabling them to identify and treat only those animals harboring the pathogenic O. ostertagi parasite.

Technical Abstract: A region within the first internal transcribed spacer (ITS-1) of the ribosomal DNA repeat of O. ostertagi has been identified that is 408 base pairs (bp) in length and is comprised of a 2 X 204 bp repeat. Universal polymerase chain reaction (PCR) primers which span this region as well as a portion of the 5.8S rDNA generate a 1011 bp fragment using genomic DNA from O. ostertagi. However, these same primers generate only a 600 (approximate) bp fragment using DNA from Haemonchus contortus, Cooperia oncophora and Oesophagostomum radiatum as well as other species within the genus Ostertagia. When DNA samples derived from adult parasites of the different genera were mixed and simultaneously amplified, the O. ostertagi component could be identified within the mixed DNA populations. Furthermore, a correlation was observed between relative fluorescence intensities of the 1011 bp and the 600 bp PCR fragments and the % O. ostertagi DNA within a mixture of parasite DNAs. A similar high correlation was obtained between % O. ostertagi DNA and % O. ostertagi eggs in feces containing eggs of other parasite genera. This resulted in the generation of a protocol that can determine the % O. ostertagi eggs within a mixed population of gastrointestinal nematode eggs. Results indicate a detection equivalent to 0.05 eggs.