Development and field evaluation of a species-specific mt-COI targeted SYBR-Green Real Time PCR for detection and quantification of Haemonchus contortus in cattle in Turkey

Abstract

Haemonchus contortus is one of the most important gastrointestinal nematodes (GINs) infecting sheep, goats, and cattle worldwide. We developed a SYBR Green real-time PCR (qPCR) assay for detection and quantification of H. contortus by using specific primers based on a conserved region of the mitochondrial cytochrome oxidase subunit I (mt-COI) gene, and evaluated this technique in the detection of H. contortus infections in cattle in Central Anatolia Region of Turkey. The newly developed qPCR assay successfully discriminated H. contortus from other GIN species infecting cattle in the specificity evaluations, with a specific melt peak of 77.5 °C. Our results revealed the efficient amplification of the proposed target COI region within the range of plasmid copies, from 2 × 106 to 2 × 101 per μl, with 96.9 % efficiency, R² value of 0.999, and a slope of -3.398. Among the 920 cattle fecal samples from the field, 58 samples (6.3 %) were positive with qPCR assay, whereas 45 samples (4.9 %) were positive, as determined by larval culture, suggesting the utility of SYBR Green qPCR. Phylogenetic characterization of the partial COI gene of H. contortus isolates was also evaluated for 100 eggs and third stage larvae recovered from positive cattle faecal samples, which were verified with the qPCR assay prior to analyses. COI sequences were classified into three haplotypes (THC1 to THC3) with intraspecific nucleotide differences of 0.50 to 0.76 %. Phylogenetic analyses revealed that the haplotypes grouped with H. contortus isolates from several countries in a monophyletic cluster, with evidence of at least two main haplogroups. Overall, the SYBR Green qPCR assay was highly specific and sensitive, suggesting that it can be used for screening of H. contortus infections in livestock populations in epidemiological studies and the control of this important parasite.