| dc.description.abstract | Background: Gastrointestinal nematodes of the cyathostomin species pose a threat to equine health worldwide. Even more so, since cyathostomin species are developing resistance against all groups of veterinary anthelmintics. Macrocyclic lactones, such as ivermectin and moxidectin, are still the most effective anthelmintic drugs registered for use in horses. However, recent research shows a reduced egg reappearance period and a reduced efficacy, suggesting a developing resistance. Glutamate gated chloride channels are the receptor targets for macrocyclic lactones. It was discovered that the glutamate gated chloride channel-α4 is a ivermectin sensitive glutamate gated channel and so the gene encoding for this channel (glc-3) is strongly suspected to play a role in developing resistance. Especially the 5’-end of this gene, which has proven to comprise several variations on mRNA level. So far, however, the genomic sequence of the 5’-end of glc-3 has not been mapped. This research focusses on the characterisation of the 5’-end of glc-3 and the possible relation with developing resistance.
Objectives: To characterize the 5’-end of the glc-3 gene by a genome walking method using DNA from the nematode species Cylicostephanus goldi.
Methods: Different lysis methods have been researched such as worm lysis buffer in combination with proteinase K and an alkaline lysis buffer from the REPLI-g Mini kit or REPLI-g Single Cell kit. PCR was used to visualise efficacy of lysis methods on different life stages of cyathostomins. Polymerase chain reaction was also used to examine successfulness of the multiple displacement amplification. A genome walking method was performed using rolling circle amplification of the products of the multiple displacement amplification with primers turned outwards. This way large quantities of linear templates are generated suitable for inverse PCR. This allows for sequencing and identification of unknown genomic sequences flanking known regions.
Results: Lysis of eggs, L1s and L3s with worm lysis buffer and proteinase K was successful most of the time. Lysis with the alkaline lysis buffer showed very inconsistent results, with only a small success rate. DreamTaq PCR with primer pair CY1-CY18 has proven to be a sufficient method of visualising the efficacy of the lysis methods as well as visualising downstream obtained products. Multiple displacement amplification was successful for two lysed eggs and genome walking was performed on the product. Genome walking was partially completed for both eggs and resulted in products ready for sequencing. Sequencing of the genome walking product revealed the template to be glc-3.
Conclusion: cyathostomin eggs have proven to be promising substitutes for adult cyathostomins in performing downstream molecular work such as polymerase chain reaction, multiple displacement amplification and genome walking. Genome walking on lysed eggs has led to amplification of glc-3, which is a promising step towards the characterization of the 5’-end of this gene. | |
