To study the prevalence of the polymorphism in position 200 of the beta-tubulin gene in the mechanism of benzimidazole (BZ) resistance in cyathostomes of horses, an allele-specific PCR was used to detect the genotype of individuals of BZ-susceptible and BZ-resistant populations. The molecular analysis of 100 adults recovered from an anthelmintic-naïve horse revealed 80% homozygous TTC/TTC individuals, 17% heterozygous TTC/TAC and 3% homozygous TAC/TAC. A naturally infected horse was treated with increasing fenbendazole (FBZ) dosages to select a BZ-resistant population of cyathostomes. The PCR based analysis of 3rd-stage larvae (L3) during the experiment revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals. After treatment 42.3% of the adults (n=104) were homozygous TTC/TTC, 55.8% were heterozygous TTC/TAC and only 1.9% showed the homozygous genotype TAC/TAC. The results of the molecular analysis lead to the proposal that polymorphism within codon 200 is not the only reason for the development of BZ resistance in small strongyles.
Several coding sequences of the benzimidazole (BZ) target beta-tubulin have been described for different parasitic nematodes. However, until recently no tubulin sequences from Cyathostome species were available, despite the importance of BZ resistance in horses in the field. Here, we describe several full-length beta-tubulin coding sequences of two major small strongyle species, namely Cylicocyclus nassatus and Cyathostomum coronatum. In the latter sequence, the putative BZ resistant mutation in codon 200 leading to a Phe to Tyr exchange is present. High nucleotide sequence similarities (>95%) were found among the tubulin sequences of the two different genera. This will be of advantage for the development of an allele-specific BZ resistance polymerase chain reaction (PCR) for multiple small strongyle species.
TaqMan minor groove binder probes were evaluated as to their suitability for the real-time allelic discrimination of the beta-tubulin codon 200 TTC/TAC single nucleotide polymorphism in cyathostomin species. Amplification of titrated cloned full-length beta-tubulin cDNA revealed that the TaqMan minor groove binder PCR is capable of specifically detecting as few as 10 copies. Testing of DNA from single adult and larval stages of several different species of cyathostomin allowed reproducible genotyping of individual worms. Using the real-time PCR approach, the throughput of samples was considerably increased compared with conventional post-PCR readout procedure. Only 7.8% homozygous TAC L3 were found among 102 L3 which were genotyped from phenotypically BZ-resistant small strongyle populations. The percentages of the homozygous TTC and heterozygous TTC/TAC were 41.3% and 50.9%, respectively. This resulted in a total TAC-allele percentage of only 33.3%. These findings correspond to data obtained by genotyping of an experimentally selected BZ-resistant cyathostomin population. It is concluded that the beta-tubulin codon 200 polymorphism is not the sole mechanism involved in the process of BZ resistance in cyathostomins.
The diversity of the beta-tubulin cDNAs of the cyathostominae and the occurrence of further isotypes were examined in adult worms isolated from an anthelmintic-naïve horse. cDNAs encoding beta-tubulin from Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus radiatus, Cylicocyclus elongatus, Cyathostomum coronatum, and Cyathostomum pateratum were characterized using specific primers developed from the cDNA sequence of Cc. nassatus. The cDNA sequences span 1,429 bp and show identities ranging from 95.6 to 100%. The deduced protein sequences span 448 amino acids and were 98-100% identical. The amino acid sequences of the 7 species varied within and between species at 10 positions. A 3' Rapid Amplification of cDNA ends using a degenerate forward primer was carried out with cDNA from Cy. pateratum, Cy. coronatum, Cy. catinatum, and Cc. nassatus to investigate the occurrence of further beta-tubulin isotypes. The expected polymerase chain reaction (PCR) product of 400 bp, including 306 bp of coding sequence, was amplified, as was an additional fragment of 600 nucleotides in the case of Cy. pateratum, Cy. coronatum, and Cy. catinatum. Sequencing of the PCR products revealed no evidence for the existence of a second beta-tubulin isotype in cyathostomes. The variation in size was caused by a length polymorphism within the 3' untranslated region, and 2 functional mRNAs seem to be transcribed from the same gene.
It has been shown that benzimidazole (BZ) resistance in sheep gastrointestinal nematodes is linked with an increase in beta-tubulin codon 200 tyrosine-expressing alleles in the resistant parasite populations. Here, an allele-specific PCR has been developed for the discrimination of the TAC/TTC polymorphism in the beta-tubulin 200 codon of small strongyles. One reverse primer was used in 2 separate amplifications with 1 of 2 forward primers that differed only in their final 3' nucleotide. The primers flank a facultative intron/exon. Therefore, the amplified fragments are either 251 or 308 bp in size, depending on the presence or absence of the intron in individual worms. Amplification of genomic DNA isolated from single adult small strongyles from a set of 7 species consistently generated allele-specific products. Three worms each of the following species were used: Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus elongatus, Cylicocyclus radiatus, Cyathostomum pateratum, Cyathostomum catinatum, and Cyathostomum coronatum. PCR with DNA isolated from single larvae also reproducibly generated specific fragments. This method might be applied for the future assessment of allele frequencies in susceptible and resistant populations to further investigate the mechanism of BZ-resistance in small strongyles.
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