Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the β tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay.

Vb-AphaS-CL131 is a novel cyanosiphovirus that infects harmful Aphanizomenon flos-aquae. This cyanophage has an isometric head, 97 nm in diameter and a long, flexible non-contractile tail, 361 nm long. With a genome size of ~120 kb, it is the second largest cyanosiphovirus isolated to date. The latent period was estimated to be ~36 h and a single infected cell produces, on average, 218 infectious cyanophages. Cyanophage infection significantly suppresses host biomass production and alters population phenotype.

The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr- 8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing “resistant” allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous “resistant” genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the “resistant” allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.

Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform–based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites’ ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.Electronic supplementary materialThe online version of this article (10.1007/s00436-019-06414-7) contains supplementary material, which is available to authorized users.

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