Molecular approaches to studying benzimidazole resistance in trichostrongylid nematode parasites of small ruminants.

Molecular techniques are of growing importance in the study of anthelmintic resistance in trichostrongylid worm populations. A knowledge of the genetic determinants of benzimidazole (BZ) resistance has made it possible to construct a molecular tool for genotyping individual worms, in respect of mutation of the beta-tubulin gene responsible for BZ resistance. This tool offers new possibilities in the diagnosis of BZ resistance, and also in the study of anthelmintic use and other breeding management factors that can affect the selection of BZ-resistant alleles in worm populations. New molecular methods have also made it possible to study the origin and diversity of BZ-resistant alleles in trichostrongylid populations. The results demonstrate the value of a multidisciplinary approach to the study of anthelmintic resistance, combining molecular, ecological and epidemiological techniques.

Importance of the mutation of amino acid 200 of the isotype 1 beta-tubulin gene in the benzimidazole resistance of the small-ruminant parasite Teladorsagia circumcincta.

In this work we demonstrated that the acquisition of benzimidazole (BZ) resistance in the small-ruminant parasite Teladorsagia circumcincta is linked to the selection of individuals that are characterized by a tyrosine (Tyr) at amino acid 200 of their isotype 1 beta-tubulin gene. This mutation appears to be recessive, since only homozygous mutant (Tyr/Tyr) individuals survived after BZ treatment of two resistant populations in which the three genotypes (rr, rs, ss) were initially present. In comparison with natural BZ-susceptible populations, a decrease in the restriction polymorphism (RFLP) of the isotype 1 beta-tubulin gene was observed in natural resistant populations. It seems that this decrease in beta-tubulin polymorphism results from the selection of homozygous mutant individuals.

PCR diagnosis of benzimidazole-susceptibility or -resistance in natural populations of the small ruminant parasite, Teladorsagia circumcincta.

We have developed a new molecular tool for the diagnosis of the benzimidazole (BZ)-susceptibility or resistance in Teladorsagia circumcincta a nematode parasite of small ruminants. This tool is based on the use of the PCR and allows the genotyping of resistant (rr) or susceptible (rS and SS) adult worms or larvae. By using four primers in the same reaction mixture, worms can be genotyped in regard to the mutation on the residue 200 (phenylalanine to tyrosine) of the beta-tubulin which is implicated in BZ resistance. A very high proportion of homozygous SS (Phe/Phe) individuals characterized the BZ susceptible populations, whereas a variable proportion of homozygous rr (Tyr/Tyr) individuals characterized the BZ resistant populations. A positive correlation was observed between the LD50 estimated by egg hatch assay, and the proportion of mutant homozygous individuals rr (Tyr/Tyr). Our PCR method allows the rapid genotyping of numerous worms and permits the detection of the first resistant individuals in a worm population.

Fitness of benzimidazole-resistant and -susceptible worms of Teladorsagia circumcincta, a nematode parasite of small ruminants.

We compared, some fitness-related traits of benzimidazole resistant (rr) and susceptible (rS, SS) worms of Teladorsagia circumcincta, a gastrointestinal parasite of the small ruminants, under laboratory conditions. PCR was used to determine the genotypes (rr, SS, rS) and the fitness of each was compared within the same strain. There was no significant difference in egg production, development rate from egg to infective larvae stage, establishment of these larvae in the host or the survival of adult worms and infective larvae for the 3 genotypes. The same results were obtained for the establishment rate of larvae in the host and the production of infective larvae under conditions of strong competition between resistant and susceptible worms. The fact that there were no differences in fitness suggests that the installation of benzimidazole resistance in a worm population is irreversible. This agrees with field observations.

Study by ribosomal DNA ITS 2 sequencing and RAPD analysis on the systematics of four Metastrongylus species (Nematoda:Metastrongyloidea).

Four species of lungworms of the Metastrongylus genus are found sympatrically in the lungs of the wild boars (Sus scrofa L.) on the Chambord game reserve (France): Metastrongylus asymmetricus, M. confusus, M. pudendotectus, and M. salmi. These species are difficult to identify using morphological characters alone, and epidemiological evidence raises doubt about the existence of 4 distinct species. Two molecular approaches (sequencing of ribosomal DNA Internal Transcribed Spacer 2 [ITS 2] and Random Amplified Polymorphic DNA assay [RAPD]) were used to evaluate the genetic similarities between the 4 taxa. The ITS 2 sequences of M. salmi and M. confusus were identical, whereas the sequences of the other species were distinct. On the other hand, RAPD analysis indicated unambiguously that the 4 species are genetically different. These results reveal the limitation of using only 1 molecular approach for taxonomic studies and indicate the need to examine different regions (ribosomal, mitochondrial DNA) in the genome of a species in complement with morphological and epidemiological data.

Using molecular tools for diagnosis in veterinary parasitology.

The development of molecular biology has made available tools that identify parasites which are important in veterinary parasitology. PCR (polymerase chain reaction) is the most frequently used tool. Target sequences are ribosomal DNA and RNA as well as fragments derived from RAPD (random amplified polymorphic DNA). The most studied protozoan genera have been: Eimeria, Babesia, Theileria, Trypanosoma and Cryptosporidium. Trichinella species and ruminant Trichostrongylid parasites have been the most investigated helminths. The use of molecular tools for identification in veterinary parasitology, however, remains a research technique rather than one that is currently used in the field for diagnostic purposes.