Discrimination of Culicoides midge larvae using multiplex polymerase chain reaction assays based on DNA sequence variation at the mitochondrial cytochrome C oxidase I gene.

The recent spread of Bluetongue disease in northwestern Europe has indicated the ability of Palaearctic Culicoides species to vector the disease. Because the different midge species vary in their ability to harbor and transmit the Bluetongue virus, quick and reliable identification is necessary to resolve the species composition of midge communities, both adult and larval, at any place at any given time point. Given that morphological identification of Culicoides species is problematic, we developed three multiplex polymerase chain reaction (PCR) assays that facilitate high-throughput analysis of midge specimens. One assay distinguishes between species of the so-called Culicoides obsoletus s.l. complex (including C. dewulfi), whereas two assays facilitate differentiation of species of the Culicoides pulicaris s.l. complex. These assays yield two PCR products: one species-specific and one generic band. We show the application of the assays in the analysis of Culicoides larvae from three different farms in northeast Scotland.

Real-time PCR assays for monitoring benzimidazole resistance-associated mutations in Ancylostoma caninum.

Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infection has led to drug resistance that often renders whole populations of livestock unresponsive to treatment. Therefore, it is important to detect mutations associated with drug resistance before it becomes clinically manifest. To monitor developing drug resistance against benzimidazoles (BZ), we developed real-time PCR assays and applied them to analyse the beta-tubulin isotype-1 gene of the hookworm Ancylostoma caninum, an important parasite of dogs. Previously, we developed PCR assays to monitor codon positions 167 and 200. Here, we describe an assay which is able to detect resistance alleles in codon 198. These real-time PCR assays were subsequently applied to screen hookworm specimens recovered from dogs in Georgia. No elevated levels of polymorphisms at the investigated loci were found, suggesting that selection for resistance in the tested samples did not occur.

Characterization of beta-tubulin genes in hookworms and investigation of resistance-associated mutations using real-time PCR.

Human hookworms (Ancylostoma duodenale, Necator americanus) are a major cause of malnutrition and anemia, particularly in children, and high worm burdens can lead to stunted growth and mental retardation. Mass drug administration (MDA) with benzimidazole (BZ) anthelmintics has the potential to greatly reduce morbidity and infection prevalence. However, such treatment strategies may apply significant selection pressure on resistance alleles. In several Strongylid parasites of livestock, resistance to BZ drugs is associated with single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codons 167 and 200. As an initial investigation into the possible development of BZ resistance in hookworms, we have cloned and sequenced the beta-tubulin isotype-1 genes of the canine hookworm Ancylostoma caninum and the two human hookworm species A. duodenale and N. americanus. The genomic sequences are highly conserved as evidenced by a similar structure of exons and introns; the 10 exons are of the same length in all three species and code for the same amino acids. The genomic sequences were then used to develop a real-time PCR assay for detecting polymorphisms in codons 167 and 200 in all three species. Hookworm specimens previously obtained from Pemba Island school children who had demonstrated a reduced response to treatment with mebendazole were then examined using the real-time PCR assay. None of the samples revealed significant levels of polymorphisms at these loci. If BZ resistance is present in the hookworm populations examined, the results do not support the hypothesis that changes in codons 167 and 200 of beta-tubulin isotype-1 are responsible for any resistance.

Microsatellite analysis reveals genetic structure of Leishmania tropica.

The current rapid spread of leishmaniases caused by Leishmania tropica and the complexity of its clinical spectrum call for this parasite's epidemiological and evolutionary investigation. Evaluation of its population structure by isoenzyme electrophoresis and previous molecular biological analysis has proved difficult. In this study, we used 21 microsatellite loci to type 117 strains from different African and Asian locations. Eighty-one different genotypes were found. A genetic bottleneck supported by a gradient in the number of alleles and consistent with the geographical structure of the Middle East suggests an African origin of this species. A Bayesian approach identified 10 genetic clusters that correlated predominantly with geographical origin. The strains in the 'Asia' cluster form a very heterogeneous sub-population, with a varied but inter-related genotype that is geographically very widely dispersed and consistent with anthroponotic transmission of the parasite. The other nine clusters were more homogenous. The propagation of L. tropica appears to be predominantly clonal. In Africa and the Middle East, anthroponotic and zoonotic systems of distribution may contribute to the development of overlapping, genetically distinct populations of L. tropica.

Evolution and conservation of microsatellite markers for Leishmania tropica.

Sixteen polymorphic microsatellite markers were developed for phylogenetic analysis of Leishmania tropica. The phylogenetic tests done demonstrated that they do provide a powerful tool for epidemiological studies. They were also tested for their ability to differentiate strains of other species of Leishmania, confirming that microsatellite markers developed for one leishmanial species cannot generally be used for other leishmanial species. In addition to length variation, a high degree of allelic heterozygosity was seen among the strains investigated, suggestive of sexual recombination within the species L. tropica.

Molecular and biological diagnostic tests for monitoring benzimidazole resistance in human soil-transmitted helminths.

In endemic countries with soil-transmitted helminths mass drug administration with albendazole or mebendazole are being implemented as a control strategy. However, it is well known in veterinary helminths that the use of the same benzimidazole drugs can place selection on the ß-tubulin gene, leading to resistance. Given the concern that resistance could arise in human soil-transmitted helminths, there is an urgent need to develop accurate diagnostic tools for monitoring resistance. In this study, we developed molecular assays to detect putative resistance genetic changes in Ascaris lumbricoides, Trichuris trichiura, and hookworms, and we optimized an egg hatch assay for the canine hookworm Ancylostoma caninum and applied it to Necator americanus. Both assays were tested on field samples. The molecular assays demonstrated their reproducibility and capacity to detect the presence of worms carrying putative resistance-associated genetic changes. However, further investigations are needed to validate our molecular and biological tests on additional field isolates.