Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae

Abstract

Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (P<0.05). However, some measurements overlapped. For this reason, a discriminate function did not allow the correct identification of 13 H. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/ or H. contortus in a population of domestic ruminants.Keywords: Diagnosis, Trichostrongyloidea, molecular biology, ruminants, epidemiology. ResumoMétodos moleculares e morfológicos foram avaliados para a identificação de Haemonchus contortus e Haemonchus placei. No total, 141 H. contortus e 89 H. placei machos adultos, obtidos de cordeiros artificialmente infectados, foram identificados individualmente por PCR com o emprego de um par de "primers" espécie-específico. Esses resultados da análise por PCR foram considerados como padrão para a identificação das espécies de Haemonchus. Haemonchus placei apresentou valores médios de espículos e ganchos superiores aos de H. contortus (P<0,05). Entretanto, houve sobreposição de alguns valores. Por essa razão, a função discriminante não permitiu a identificação correta de 13 exemplares de H. contortus e de um, de H. placei. Foi medida a cauda da bainha de larvas infectantes (L3), que compreende a distância entre a ponta da cauda da larva e a ponta da cauda da bainha. Apenas três das 485 L3 de H. placei (0,619%) apresentaram a cauda da bainha com medida inferior a 85 µm e somente em quatro das 500 L3 de H. contortus (0,8%) essa medida foi superior a 85 µm. Os resultados demonstraram que 6,09% dos machos adultos seriam identificados erroneamente com base na função discriminante, enquanto a identificação incorreta de L3 seria de apenas 0,71%. Portanto, a identificação de L3 pode ser utilizada como método inicial para indicar a presença de H. placei e/ou H. contortus em uma população de ruminantes domésticos.

Overview of anthelmintic resistance of gastrointestinal nematodes of small ruminants in Brazil

“…In Brazil, Nunes et al (2013) evaluated goat, sheep and buffalo herds with respect to the presence of polymorphism which confers resistance to benzimidazole, and stated that molecular diagnosis is the guide of choice for anthelmintic drugs before they are used on the herd. Studies of molecular tests on AHR in Brazil have also been evaluated, involving testing of beta-tubulin mutations or genetic evaluations of nematodes, corroborating researches into AHR (SILVA et al, 2015;SANTOS et al, 2014;AMARANTE et al, 2014;BRASIL et al, 2012).…”

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).…”

“…In the second year (2016), we collected 127 Haemonchus adult male worms from 12 lambs. The spicule measurement and a DNA sample were obtained from each specimen according to the protocol described by Santos et al (2014).…”

“…The use of molecular tools is particularly important in Haemonchus spp. 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).…”

A panel of microsatellite markers to discriminate and study interactions between Haemonchus contortus and Haemonchus placei

“…Attempts to use morphology and morphometrics to distinguish Haemonchus species, have encountered considerable issues with overlapping measurement ranges (Santos et al, 2014). Molecular techniques offer advantages over morphology for the identification of parasites, their genetic characterization, the isolation and characterization of expressed genes, and the detection of drug resistance and mutations (Gasser, 1999;Gasser et al, 2016).…”