Studies on the genetic population structure of Cooperia oncophora

Abstract

Cooperia oncophora is one of the most common intestinal parasitic nematodes of cattle in temperate climates worldwide contributing to serious production losses. It is considered as a mild pathogen which can be effectively controlled with anthelmintics. However, this control strategy is threatened by the development of anthelmintic resistance. Although resistance in cattle nematodes emerged just recently and is not yet common, it is a wide-spread phenomenon in nematode parasites of sheep. For the latter it has been proven that resistance can develop within 5–8 generations following the introduction of a drug. Molecular analysis of the most commonly encountered types of resistance, such as those to benzimidazoles, showed that a small resistant subpopulation was present within the susceptible population from the onset. This observation raises important questions like how much genetic variability is present in the starting population? And to what extent does the evolution to anthelmintic resistance result in changes in polymorphism within a population? As a consequence of the increased occurrence of anthelmintic resistance an alternative for controlling gastrointestinal nematodes could be the use of animals that are genetically resistant to worm infections. However, like the use of anthelmintic drugs, this creates a different environment in which the parasites have to live. It is generally accepted that parasites possess a variety of mechanisms for escaping or modulating the host immune response. Hence, it is of interest to know how a parasite population reacts if it encounters a host that is immune either through breeding or vaccination. To address these questions for C. oncophora populations, molecular markers were needed. Since C. oncophora infections are not a major threat to the livestock industry research addressing C. oncophora genetics has lagged behind. Therefore little sequence information was available and the first aim within the project was to characterize genetic markers. Therefore the majority of this thesis is dedicated to the search for markers within the genome of C. oncophora. An ideal marker must have sufficient variation, be reliable and be simple to generate and interpret. The amplified fragment length polymorphism (AFLP) analysis, revealed an enormous amount of genetic differences between individuals from the same population (Chapter 2). The within population variation disclosed by the AFLP was too high for simple interpretation and large population screening experiments. Although a different approach of the AFLP, using cDNA, demonstrated less polymorphism it failed when applied on individual worms. Since direct determination of possible genomic regions under selection was not feasible, neutral markers situated on the mitochondrial genome were assessed. Initially a PCR-RFLP experiment, performed on the partial mitochondrial encoded cytochrome oxidase subunit I gene (cox1), confirmed the existence of between and within population variation of C. oncophora (Chapter 3). It was demonstrated that variation in two restriction sites was present in two populations and that the distribution differed significantly among the two. The frequencies of the variant polymorphisms demonstrated a distinction between a laboratory maintained population and a field isolate of C. oncophora. Because the polymorphisms were identified in one of the most conserved genes of the mt genome, it indicated that the mt genome indeed contains sufficient variation for identification of genetic markers. Consequently the complete mt genome of C. oncophora was sequenced. The characteristics of the composition and the polymorphisms identified in the C. oncophora mt genome are described in Chapter 4. From 426 identified polymorphisms a selection was made which subsequently was used in a population study to address the distribution of the variation within the laboratory population. An additional population, derived from a passage experiment through immune hosts, was tested to establish whether this population had undergone a change in the level of diversity induced by the encountered immune pressure of the host. Although a phenotypic change was observed in the passaged population, genetic differences based on the mt polymorphisms between this population and the laboratory maintained population could not be demonstrated. However it cannot be concluded that no genetic changes had occurred. It remains possible that changes on the nuclear genome occurred, which did not affect the level of mt genome diversity of the population. Although the research described in this thesis could not demonstrate genetic changes in a parasite population under the influence of host immunity, it has contributed to describing the enormous genetic variability that exists within animal parasitic nematodes. In particular the delineation of the complete mt genome sequence, with an additional substantial number of its polymorphisms, will contribute to further studies on the genetic population structures of nematode species.