PCR primers for straightforward differentiation ofHaemonchus contortus, Haemonchus placeiand their hybrids

Abstract

Haemonchus contortus and Haemonchus placei are among the major parasites of small ruminants and cattle. Although infection with these nematodes is host-specific, with H. placei predominating in cattle and H. contortus in sheep, cross-infections are observed in areas where both parasites are sympatric, and hybrid offspring can occur. Therefore, a fast and precise method is required for differentiating the parasites. Identification based on spicule morphometry is the most common technique for differentiating Haemonchus species. However, because these measurements overlap between species, morphological analysis is insufficient for differentiating between helminth species. In this work, we present a reliable, conventional polymerase chain reaction (PCR)-based method that uses two species-specific primer pairs to differentiate between H. contortus and H. placei specimens and their hybrids. Each primer pair produces one single and distinct amplification band for each species, which enables the detection of hybrid specimens. These primer pairs were validated by testing eight different populations of H. contortus, H. placei and hybrids.

Helminth infections and hybridization between Haemonchus contortus and Haemonchus placei in sheep from Santana do Livramento, Brazil

“…This would be a major concern given the high prevalence of anthelmintic resistance in H. contortus. Molecular tools are very useful for elucidating the dynamics of H. contortus and H. placei infections when the two species are sympatric, because identification based on morphological analysis has limitations for differentiating species and detecting hybrids within a population (SANTOS et al, 2014;AMARANTE et al, 2017).…”

“…The primer pair HcBotuF1/R2 amplified an approximately 260 base pair (260 bp) band from H. contortus samples and HpBotuF/R amplified a PCR product of approximately 459 bp from H. placei samples. The PCR conditions were previously described by Amarante et al (2017).…”

“…diagnosis because morphological measurements of spicules may overlap, raising issues about the precise differentiation among species (SANTOS et al, 2014(SANTOS et al, , 2017. In addition, co-infection with H. contortus and H. placei can result in hybrids (HUSSAIN et al, 2014;AMARANTE et al, 2017). For this reason, the accurate identification of both species and hybrids is imperative in epidemiological studies where the species are sympatric, especially when small ruminants and cattle share the same pastures (AMARANTE, 2011).…”

Establishment of co-infection and hybridization of Haemonchus contortus and Haemonchus placei in sheep

“…The identification of the Haemonchus species and hybrids was performed by PCR (the gold standard analysis) using the species-specific primer pairs HcBotuF1/R2 and HpBotuF/R, designed from sequences proximal to the 3′- and 5′- ends of the large and small rDNA, which span the ETS sequence (Amarante et al , 2017). Each primer pair produces one single and distinct amplification band for each species.…”

“…Negative (no-DNA) and known positive ( H. contortus or H. placei ) controls were included in each set of PCRs. The PCR conditions have been described by Amarante et al (2017).…”