Publikationsdatenbank

Identification of β-tubulin isotypes and development of pyrosequencing assays for Benzimidazole resistance in Heterakis gallinarum and Ascaridia galli (2020)

Art

Hochschulschrift

Autor

Ameen, Vahel J. (WE 13)

Quelle

Berlin: Mensch und Buch Verlag Berlin, 2020 — IX, 115 Seiten

ISBN: 978-3-96729-042-4

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/27666

Kontakt

Institut für Parasitologie und Tropenveterinärmedizin

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 62310
parasitologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Poultry is considered to be one of the best sources of high quality protein for humans. Following recent changes in husbandry systems for poultry in Europe from cage to other, alternative husbandry systems such as free-range systems, increased prevalence of helminth infections, predominantly Ascaridia galli and Heterakis gallinarum, was observed. The basic principles for the effective control of helminth infections in poultry farms are a combination of preventive measures such as biosecurity and disinfection with broad-spectrum anthelmintic therapy. The benzimidazoles (BZs) flubendazole and fenbendazole have been certified for treatment of chicken nematodes within the European Union and are widely used as save broad-spectrum anthelmintics in poultry. Benzimidazole (BZ)-resistance is widespread and a significant problem in several parasitic nematodes of ruminants and horses and was correlated with the presence of three single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of strongyle nematodes. However, BZ-resistance has so far not been reported in the chicken parasitic nematodes but the risk for resistance development has to be considered as existant and will increase with parasite infection intensity and treatment frequencies. Therefore, it is necessary to establish sensitive and reliable diagnostic tests for detection of BZ-resistance at least for the most important nematodes of chicken. Single nucleotide polymorphisms in the β-tubulin gene, particularly at the three specific codons F200Y, E198A and F200Y, are used as markers for the molecular detection of BZ-resistance in-several strongyle nematodes in veterinary medicine. Among the molecular tests, pyrosequencing is considered an excellent technique for quantification of BZ-resistance associated β-tubulin alleles even when the allele frequency is still quite low. Here, partial cDNAs representing a single β-tubulin isotype were obtained using degenerated primers. The obtained nine A. galli sequences were 100% identical to a previously published sequence while the 40 new H. gallinarum sequences were 93-99% identical to each other and 88% identical to the A. galli sequence. The 1497 bp full-length β-tubulin isotype 1 cDNA sequence with an 1353 bp open reading frame of poultry the nematode H. gallinarum was identified using 3'/5' RACE protocols. Phylogenetic maximum-likelihood analysis revealed that both A. galli and H. gallinarum β-tubulin cDNAs cluster together with high statistical support values and share a close phylogenetic relationship with the other ascarid nematode β-tubulins. Overall, there is a clear separation between β-tubulins from clade V and clade III nematodes. The latter form a highly supported group. This together with the fact that in both clusters several β-tubulin paralogs are found per species prevents to identify clear one-to-one orthologs between the ascarid β-tubulins and those of Caenorhabditis elegans and strongyles. Even worse, such one-to-one orthologs cannot even be identified in comparison to the pig roundworm A. suum since the sister group in the phylogenetic tree to the A. galli and H. gallinarum group contains A. suum tbb-1 and tbb-2 cDNAs. Pyrosequencing assays for the quantitative analysis of potentially BZ-resistance associated SNPs in the β-tubulin isotype 1 genes of A. galli and H. gallinarum were successfully developed. Plasmids with artificially synthesised inserts carrying combinations of SNPs were combined in defined ratios to evaluate sensitivity and linearity of the assays. The correlation of calculated and observed frequencies for the SNP determination at codons F167Y, E198A and F200Y was very high. Pyrosequencing assays carried out for A. galli collected from naturally infected chicken on Swedish farms showed no evidence for increased frequencies of potentially BZ-resistance associated SNPs. The developed assays offer the tools to screen for the presence and to quantify the relative frequency of BZresistance associated SNPs in populations of important poultry parasites. The assays have the potential of detecting the development of resistance in an early phase before it becomes clinically apparent. This offers the chance to implement measures to counteract further selection before resistance becomes widespread.