The characterisation of the 5’ end of the glc-3 of Cyathostomins: Gaining insight into the variation of glc-3 in these species

Abstract

Background: A widespread resistance of cyathostomins against macrocyclic lactones has been reported. Changes in the glutamate-gated chloride (GluCl) channels of cyathostomins are suspected to contribute to this development of anthelmintic resistance. To monitor the development of anthelmintic resistance it is important to know the genetic basis of the proteins involved. This research builds further on research where cDNA variations were found in in vitro resistant L3 stadia. The variation lies in the 5’end of the glc-3 gene coding for the GluCl channel protein. Cwiklinski et al (2013) mapped with the use of DNA sequencing the biggest part of the genome of Cylicostephanus goldi, but did not succeed to sequence the 5’end of the glc-3 gene. Objectives: To map the DNA-sequence of the glc-3 gene in cyathostomins and gain insight into the variation of this gene within individual cyathostomins. Methods: Steps were taken to optimise the current lysis and polymerase chain reaction (PCR) protocol, especially for cyathostomins which have been stored in ethanol. Moderations were made in lysis type, Proteinase K amounts, inactivation temperature and purification manner. With PCR, different variations of the glc-3 gene in individual cyathostomins were detected. PCR, multi displacement amplification, restriction enzymes, ligation and precipitation were used to map the unknown part of the glc-3 gene. Results: No satisfactory PCR protocol has been found for adult cyathostomins which have been fixated in alcohol. The results of detecting different variations of the glc-3 in individual cyathostomins were inconclusive. No previously unknown part of the glc-3 gene could be mapped. Conclusions; A different research method must be developed to map the DNA sequence of the glc-3 gene in cyathostomins, as the used method proved to be inadequate.