Genetic variability of the β-Tubulin gene in gastrointestinal nematodes

Abstract

A series of DNA extractions and polymerase chain reactions (PCR) was carried out to investigate anthelmintic resistance in different life stages (egg, L₃ and adult) of various nematode species Trichostrongylus colubrifonnis (two strains and three duplicates of each) Ostertagia circumcincta, Haemonchus contortus and Cooperia oncophora. The results of this study indicated that the PCR may be of potential as a method of detection for anthelmintic resistance. Successful amplifications of genomic DNA from O.circumcincta and T.colubrifonnis infective larvae were obtained using primers from C.elegans sequence data. This confirms the homology of the β-tubulin gene, within these three species. The amplifications indicated some subtle differences between O.circumcincta and T.colubriformis. Several high molecular weight fragments (800-1050bp) were present in the T.colubriformis products but absent from O.circumcincta. A double band of 600-700bp was present in the PCR products of both species.The amplification of the two strains of T.colubrifonnis susceptible and resistant L₃ DNA indicated some differences between the two. The most consistently appearing fragments in the products of the resistant strain has a molecular weight of 550-650bp, smaller than that of the double band of the susceptible strains. One the resistant L₃ duplicates produced one fragment with a molecular weight of a similar size (960bp) to that of the susceptible strain (lOOO-1050bp).There was successful extraction and amplification of adult DNA from O.circumcincta. The PCR products revealed a pattern of fragments similar (600-800bp) to that of the L₃s but with two fragments at a higher molecular weight, at 900-1000bp. There was no amplification of the DNA from the eggs but this is not indicative that PCR will not amplify nematode egg DNA, but that other unknown preventative factors were in operation. In conclusion this preliminary study has allowed some insight into the possible utilisation of the PCR as a method to resistance detection and as a result has found some variability within the β-tubulin gene of some important nematode species. However, a lot more research needs to be undertaken in this area in order to determine the exact molecular nature of anthelmintic resistance in gastrointestinal nematodes.