Análise da presença do marcador molecular de resistência ao princípio ativo do antihelmíntico Levamisol em espécimes de Haemonchus contortus
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Data
2021-11
Autores
Ciconi, Talita Caroline Teli
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Universidade Estadual Paulista (Unesp)
Resumo
Haemonchus contortus (que parasita, preferencialmente, ovinos) apresenta resistência a todas as classes de antihelmínticos resultando em perdas econômicas significativas em todo mundo. Também há resistência anti-helmíntica em Haemonchus placei (parasita preferencial de bovinos) embora ocorra mais raramente. Porém, infecções mistas têm sido observadas quando ovinos e bovinos compartilham pastagens em sistemas extensivos de produção e já foram identificados híbridos tanto em hospedeiros infectados naturalmente, quanto em infecções artificiais, havendo evidência de fluxo gênico entre essas espécies. A identificação das diferentes espécies de Haemonchus é realizada, preferencialmente, através de análise morfológica, mas sobreposições entre medidas têm sido observadas, o que dificulta a identificação espécie-específica. Embora os métodos moleculares sejam considerados laboriosos e mais caros, têm maior sensibilidade e especificidade. A resistência anti-helmíntica tem sido frequentemente descrita como sendo poligênica, porém, os mecanismos genéticos envolvidos não são totalmente conhecidos. Oligonucleotídeos iniciadores (primers) foram desenvolvidos por Barrère et al. (2014) para avaliar a inserção/deleção (indel) de 63 pares de base (pb) no gene acr-8 em H. contortus. Esta deleção promove a formação da proteína truncada do gene ACR-8b, que está relacionada com a resistência ao Levamisol. Santos et al. (2014) analisaram morfologica e molecularmente os espécimes de H. contortus empregando o isolado denominado de RsHco1, obtido em ovinos criados no estado do Rio Grande do Sul. Este isolado é suscetível a todas as classes de antihelmínticos, destoando das amostras de H. contortus obtidas a campo no estado de São Paulo, pois as mesmas tem se mostrado resistentes aos princípios ativos analisados até o momento. Porém, Santos et al. (2014) tiveram problemas para identificar os espécimes de Haemonchus. Assim sendo, os objetivos deste projeto são: identificar molecularmente, com o emprego da Reação em Cadeia da Polimerase (PCR convencional) os mesmos espécimes de H. contortus com o emprego do par de primers espécie-específico descrito por Amarante et al. (2017), bem como testar o par de primers descrito por Barrère et al. (2014) para a verificação de resistência/susceptibilidade ao antihelmíntico Levamisol. As análises moleculares dos 152 espécimes machos de H. contortus foram repetidas com o par de primers HcBotuF1/R2 (Amarante et al., 2017), visando dirimir os problemas de identificação. Os produtos de PCR foram submetidos à eletroforese em gel de agarose a 2% em tampão TAE e fotografados sob luz UV. Na avaliação dos géis, foi observada uma banda de amplificação de 260 pb, que resultou na identificação molecular das 152 amostras de DNA como H. contortus. Essas mesmas amostras também foram submetidas à PCR convencional para a identificação da resistência/susceptibilidade ao Levamisol, o que resultou em: 96 amostras (63,2%) que apresentaram somente bandas de 256 pb, ou seja, os respectivos espécimes de H. contortus foram considerados resistentes ao levamisol; 44 amostras (28,9%), somente bandas de 319 pb, cujos espécimes foram considerados susceptíveis a este antihelmíntico e 12 amostras amostras (7,9%) apresentaram ambas as bandas de amplificação, sendo considerados molecularmente heterozigotas quanto ao levamisol. Uma vez que os espécimes de H. contortus utilizados neste estudo eram provenientes do isolado RsHco1 (suscetível a todas as classes de anti helmínticos) conclui-se que o par de primers descrito por Barrère et al (2014) é capaz de detectar um segmento de DNA relacionado à susceptibilidade/resistência ao Levamisol, mas não pode ser empregado como um marcador molecular confiável, em amostras de campo, para a verificação da susceptibilidade e/ou resistência a este antihelmíntico.
Haemonchus contortus primarily infects sheep and has shown resistance to all major anthelmintic drug classes and causes significant economic losses in the livestock industry worldwide. There also have been reports of anthelmintic resistance in H. placei, although the level of resistance is not so extensive. In addition, mixed infections have been reported in livestock production systems in which cattle and sheep share the same pasture and hybrids have been found in naturally and artificial infected animals and there is evidence of gene flow. Morphological identification of Haemonchus species is usually performed but overlap between measurements among specimens can occur, casting doubt upon the correct species identification. Although molecular techniques are considered more expensive and laborious they show higher sensitivity and specificity. Anthelmintic resistance has been described to be polygenic, but all genetic mechanisms involved have not been determined. Oligonucleotide primers were designed by Barrère et al. (2014) to evaluate an insertion/deletion (indel) of 63 bp of the acr-8 gene in H. contortus. The truncated isoform of the ACR-8b protein arises due to the 63bp deletion that is associated with the phenotypic resistance to levamisol. Santos et al. (2014) used molecular and morphological methods in order to identify H. contortus species. They analysed specimens from the laboratory isolate RsHco1 which is susceptible to all chemical classes of anthelmintics. The opposite situation has been observed in São Paulo state, Brazil where anthelmintic resistance is widespread. Once Santos et al. (2014) were not able to identify all H. contortus samples, the goals of this study were: to identify the same H. contortus specimens using the species-specific primer pair (conventional PCR) described by Amarante et. al (2017) and to verify resistance/susceptibility to levamisol using the primer described by Barrère et al. (2014). Molecular analysis of 152 male specimens of H. contortus employed primers HcBotuF1/R2 (Amarante et al., 2017) in order to solve identification problems. PCR products were electrophoresed on 2% agarose gels in 1% Tris–acetate–EDTA (TAE) buffer. A 260 bp amplification band was observed and allowed the identification of 152 DNA samples as H. contortus. The same DNA samples were also submitted to conventional PCR to identify resistance/susceptibility to levamisol. And 96 samples presented a 256 bp band, 44 samples presented a 319 bp one and 12 samples (7.9%) showed both bands, which means 63.2% of the H. contortus specimens were resistant to levamisol, 28.9% were susceptible and 7.9% of them presented genetic heterozygosity, respectively. Considering H. contortus specimens were obtained from the laboratory isolate RsHco1, which is susceptible to all chemical classes of anthelmintics, it can be concluded that Barrère et al (2014) described primers that are able to detect one DNA segment correlated with the levamisol resistance/susceptibility. But that can’t be used as a reliable molecular marker to verify susceptibility or resistance to levamisol in field populations.
Haemonchus contortus primarily infects sheep and has shown resistance to all major anthelmintic drug classes and causes significant economic losses in the livestock industry worldwide. There also have been reports of anthelmintic resistance in H. placei, although the level of resistance is not so extensive. In addition, mixed infections have been reported in livestock production systems in which cattle and sheep share the same pasture and hybrids have been found in naturally and artificial infected animals and there is evidence of gene flow. Morphological identification of Haemonchus species is usually performed but overlap between measurements among specimens can occur, casting doubt upon the correct species identification. Although molecular techniques are considered more expensive and laborious they show higher sensitivity and specificity. Anthelmintic resistance has been described to be polygenic, but all genetic mechanisms involved have not been determined. Oligonucleotide primers were designed by Barrère et al. (2014) to evaluate an insertion/deletion (indel) of 63 bp of the acr-8 gene in H. contortus. The truncated isoform of the ACR-8b protein arises due to the 63bp deletion that is associated with the phenotypic resistance to levamisol. Santos et al. (2014) used molecular and morphological methods in order to identify H. contortus species. They analysed specimens from the laboratory isolate RsHco1 which is susceptible to all chemical classes of anthelmintics. The opposite situation has been observed in São Paulo state, Brazil where anthelmintic resistance is widespread. Once Santos et al. (2014) were not able to identify all H. contortus samples, the goals of this study were: to identify the same H. contortus specimens using the species-specific primer pair (conventional PCR) described by Amarante et. al (2017) and to verify resistance/susceptibility to levamisol using the primer described by Barrère et al. (2014). Molecular analysis of 152 male specimens of H. contortus employed primers HcBotuF1/R2 (Amarante et al., 2017) in order to solve identification problems. PCR products were electrophoresed on 2% agarose gels in 1% Tris–acetate–EDTA (TAE) buffer. A 260 bp amplification band was observed and allowed the identification of 152 DNA samples as H. contortus. The same DNA samples were also submitted to conventional PCR to identify resistance/susceptibility to levamisol. And 96 samples presented a 256 bp band, 44 samples presented a 319 bp one and 12 samples (7.9%) showed both bands, which means 63.2% of the H. contortus specimens were resistant to levamisol, 28.9% were susceptible and 7.9% of them presented genetic heterozygosity, respectively. Considering H. contortus specimens were obtained from the laboratory isolate RsHco1, which is susceptible to all chemical classes of anthelmintics, it can be concluded that Barrère et al (2014) described primers that are able to detect one DNA segment correlated with the levamisol resistance/susceptibility. But that can’t be used as a reliable molecular marker to verify susceptibility or resistance to levamisol in field populations.
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Palavras-chave
Haemonchus contortus, Levamisol, PCR, Resistência, Suscetibilidade, Resistance, Susceptibility