Identification and semi-quantitation of Ostertagia ostertagi eggs by enzymatic amplification of ITS-1 sequences

• Published in: Veterinary parasitology 1998-06, Vol.77 (4), p.245-257
• Main Authors: Zarlenga, D.S, Gasbarre, L.C, Boyd, P, Leighton, E, Lichtenfels, J.R
• Format: Article
• Language: English
• Subjects: ADN; Animals; Base Sequence; Cattle; Cattle Diseases - diagnosis; Cloning, Molecular; DIAGNOSIS; DIAGNOSTIC; DIAGNOSTICO; DNA; DNA Primers - chemistry; DNA, Helminth - chemistry; DNA, Ribosomal - chemistry; Electrophoresis, Agar Gel - veterinary; Haemonchus - genetics; Internal transcribed spacer; Molecular Sequence Data; Oesophagostomum - genetics; Ostertagia; Ostertagia - genetics; Ostertagia - isolation & purification; OSTERTAGIA OSTERTAGI; Ostertagiasis - diagnosis; Ostertagiasis - veterinary; Parasite Egg Count - veterinary; PCR; Polymerase Chain Reaction - veterinary; Quantitation; Ribosomal DNA; Sequence Analysis, DNA; Strongyle; Tandem Repeat Sequences - genetics; Trichostrongyle; Trichostrongyloidea - genetics
• ISSN: 0304-4017; 1873-2550
• DOI: 10.1016/S0304-4017(98)00114-9

A region within the first internal transcribed spacer (ITS-1) of the ribosomal DNA repeat of Ostertagia ostertagi has been identified that is 408 base pairs (bp) in length and is comprised of a 2×204 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 bp (approximate) 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 percentage of O. ostertagi DNA within a mixture of parasite DNAs. A similar high correlation was obtained between the percentage of O. ostertagi DNA and percent O. ostertagi eggs in feces containing eggs of other nematode genera. This resulted in the generation of a protocol that can determine the percentage of O. ostertagi eggs within a mixed population of gastrointestinal nematode eggs. Results indicate a detection equivalent to 0.05 eggs.