Evidence from two independent backcross experiments supports genetic linkage of microsatellite Hcms8a20, but not other candidate loci, to a major ivermectin resistance locus in Haemonchus contortus

Haemonchus contortus is the leading parasitic nematode species used to study anthelmintic drug resistance. A variety of candidate loci have been implicated as being associated with ivermectin resistance in this parasite but definitive evidence of their importance is still lacking. We have previously performed two independent serial backcross experiments to introgress ivermectin resistance loci from two H. contortus ivermectin-resistant strains – MHco4(WRS) and MHco10(CAVR) – into the genetic background of the ivermectin-susceptible genome reference strain MHco3(ISE). We have interrogated a number of candidate ivermectin resistance loci in the resulting backcross populations and assessed the evidence for their genetic linkage to an ivermectin resistance locus. These include the microsatellite marker Hcms8a20 and six candidate genes Hco-glc-5, Hco-avr-14, Hco-lgc-37 (previously designated Hco-hg-1), Hco-pgp-9 (previously designated Hco-pgp-1), Hco-pgp-2 and Hco-dyf-7. We have sampled the haplotype diversity of amplicon markers within, or adjacent to, each of these loci in the parental strains and fourth generation backcross populations to assess the evidence for haplotype introgression from the resistant parental strain into the genomic background of the susceptible parental strain in each backcross. The microsatellite Hcms8a20 locus showed strong evidence of such introgression in both independent backcrosses, suggesting it is linked to an important ivermectin resistance mutation in both the MHco4(WRS) and MHco10(CAVR) strains. In contrast, Hco-glc-5, Hco-avr-14, Hco-pgp-9 and Hco-dyf-7 showed no evidence of introgression in either backcross. Hco-lgc-37 and Hco-pgp-2 showed only weak evidence of introgression in the MHco3/4 backcross but not in the MHco3/10 backcross. Overall, these results suggest that microsatellite marker Hcms8a20, but not the other candidate genes tested, is linked to a major ivermectin resistance locus in the MHco4(WRS) and MHco10(CAVR) strains. This work also emphasises the need for genome-wide approaches to identify mutations responsible for the ivermectin resistance in this parasite

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta, Canada

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America (NA) and that European-type (EU) haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) of western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the EU-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite

Increased expression of a microRNA correlates with anthelmintic resistance in parasitic nematodes

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3′ UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3′ UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species

The confounding effects of high genetic diversity on the determination and interpretation of differential gene expression analysis in the parasitic nematode Haemonchus contortus

Differential expression analysis between parasitic nematode strains is commonly used to implicate candidate genes in anthelmintic resistance or other biological functions. We have tested the hypothesis that the high genetic diversity of an organism such as Haemonchus contortus could complicate such analyses. First, we investigated the extent to which sequence polymorphism affects the reliability of differential expression analysis between the genetically divergent H. contortus strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR). Using triplicates of 20 adult female worms from each population isolated under parallel experimental conditions, we found that high rates of sequence polymorphism in RNAseq reads were associated with lower efficiency read mapping to gene models under default TopHat2 parameters, leading to biased estimates of inter-strain differential expression. We then showed it is possible to largely compensate for this bias by optimising the read mapping single nucleotide polymorphism (SNP) allowance and filtering out genes with particularly high single nucleotide polymorphism rates. Once the sequence polymorphism biases were removed, we then assessed the genuine transcriptional diversity between the strains, finding ≥824 differentially expressed genes across all three pairwise strain comparisons. This high level of inter-strain transcriptional diversity not only suggests substantive inter-strain phenotypic variation but also highlights the difficulty in reliably associating differential expression of specific genes with phenotypic differences. To provide a practical example, we analysed two gene families of potential relevance to ivermectin drug resistance; the ABC transporters and the ligand-gated ion channels (LGICs). Over half of genes identified as differentially expressed using default TopHat2 parameters were shown to be an artifact of sequence polymorphism differences. This work illustrates the need to account for sequence polymorphism in differential expression analysis. It also demonstrates that a large number of genuine transcriptional differences can occur between H. contortus strains and these must be considered before associating the differential expression of specific genes with phenotypic differences between strains

Resurrection and redescription of Varestrongylus alces (Nematoda; Protostrongylidae), a lungworm of the Eurasian moose (Alces alces), with report on associated pathology

Varestrongylus alces, a lungworm in Eurasian moose from Europe has been considered a junior synonym of Varestrongylus capreoli, in European roe deer, due to a poorly detailed morphological description and the absence of a type-series. Methods Specimens used in the redescription were collected from lesions in the lungs of Eurasian moose, from Vestby, Norway. Specimens were described based on comparative morphology and integrated approaches. Molecular identification was based on PCR, cloning and sequencing of the ITS-2 region of the nuclear ribosomal DNA. Phylogenetic analysis compared V. alces ITS-2 sequences to these of other Varestrongylus species and other protostrongylids. Results Varestrongylus alces is resurrected for protostrongylid nematodes of Eurasian moose from Europe. Varestrongylus alces causes firm nodular lesions that are clearly differentiated from the adjacent lung tissue. Histologically, lesions are restricted to the parenchyma with adult, egg and larval parasites surrounded by multinucleated giant cells, macrophages, eosinophilic granulocytes, lymphocytes. The species is valid and distinct from others referred to Varestrongylus, and should be separated from V. capreoli. Morphologically, V. alces can be distinguished from other species by characters in the males that include a distally bifurcated gubernaculum, arched denticulate crura, spicules that are equal in length and relatively short, and a dorsal ray that is elongate and bifurcated. Females have a well-developed provagina, and are very similar to those of V. capreoli. Morphometrics of first-stage larvae largely overlap with those of other Varestrongylus. Sequences of the ITS-2 region strongly support mutual independence of V. alces, V. cf. capreoli, and the yet undescribed species of Varestrongylus from North American ungulates. These three taxa form a well-supported crown-clade as the putative sister of V. alpenae. The association of V. alces and Alces or its ancestors is discussed in light of host and parasite phylogeny and host historical biogeography. Varestrongylus alces is a valid species, and should be considered distinct from V. capreoli. Phylogenetic relationships among Varestrongylus spp. from Eurasia and North America are complex and consistent with faunal assembly involving recurrent events of geographic expansion, host switching and subsequent speciation. Cervidae, Cryptic species, Historical biogeography, ITS-2, Metastrongyloidea, Parasite biodiversity, Varestrongylinae, Varestrongylus capreoli, Verminous pneumoniapublishedVersio

Microsattilite genotypes for all individuals at all 16 loci

This file contains the microsattilite data that was attained for this study. It is in genepop format. This file contains all loci that were genotyped including those that were removed from the analysis. The 16 numbers at the top of the file represent loci names. Each entry under a "POP" header represents an individual of that population, where A = Alaska, B = Brannen Lake, E = East Coast, M = Mayliewan (our Alberta population), and N = Norway. Each of the 16 sets of 4 digits represent diploid genotypes at each of the 16 loci for that individual. For example, 0414 would represent a heterozygous call for allele 4 and allele 14 for that individual for whatever locus it is under. The first column of 4 digits represents locus 51 and the last represents locus 222 (the rest are ordered accordingly). 00s represent missing data

Data from: Toward the genetic origins of a potentially non-native population of threespine stickleback (Gasterosteus aculeatus) in Alberta

Disentangling the origin of putatively introduced/invasive species is of increasing priority in conservation biology. The presence of a previously undocumented species may be due to an undetected recent population or range expansion associated with environmental change, or due to an introduction by humans. We used molecular tools to address the origin of a population of threespine stickleback (Gasterosteus aculeatus) first identified in central Alberta, Canada in 1980 from a single lake. We characterized this inland, high elevation Alberta population in comparison to samples from five representative geographic regions worldwide, using mtDNA and nine microsatellite loci to elucidate genetic structure and estimate divergence times. We found significantly lower levels of genetic variation in the Alberta population, which could reflect either a recent colonization or periodic bottlenecks associated with winterkills. While we did find that the Alberta samples were most closely related to the North American West Coast populations, we did not uncover a putative source population. Alberta samples formed a clade in phylogenetic analyses, with divergence time estimates between the Alberta and British Columbia samples ca. 250–750 kya. The hypothesis that the Alberta population represents a natural colonization during North America’s last glacial recession could not be rejected. Collectively, these data suggest that the genetic signature of colonizing populations following introductions may be similar to populations at their range limit, contributing to difficulties establishing population origins

Resurrection and Redescription of \u3ci\u3eVarestrongylus alces\u3c/i\u3e (Nematoda: Protostrongylidae), a Lungworm of the Eurasian Moose (\u3ci\u3eAlces alces\u3c/i\u3e), with Report on Associated Pathology

Background: Varestrongylus alces, a lungworm in Eurasian moose from Europe has been considered a junior synonym of Varestrongylus capreoli, in European roe deer, due to a poorly detailed morphological description and the absence of a type-series. Methods: Specimens used in the redescription were collected from lesions in the lungs of Eurasian moose, from Vestby, Norway. Specimens were described based on comparative morphology and integrated approaches. Molecular identification was based on PCR, cloning and sequencing of the ITS-2 region of the nuclear ribosomal DNA. Phylogenetic analysis compared V. alces ITS-2 sequences to these of other Varestrongylus species and other protostrongylids. Results: Varestrongylus alces is resurrected for protostrongylid nematodes of Eurasian moose from Europe. Varestrongylus alces causes firm nodular lesions that are clearly differentiated from the adjacent lung tissue. Histologically, lesions are restricted to the parenchyma with adult, egg and larval parasites surrounded by multinucleated giant cells, macrophages, eosinophilic granulocytes, lymphocytes. The species is valid and distinct from others referred to Varestrongylus, and should be separated from V. capreoli. Morphologically, V. alces can be distinguished from other species by characters in the males that include a distally bifurcated gubernaculum, arched denticulate crura, spicules that are equal in length and relatively short, and a dorsal ray that is elongate and bifurcated. Females have a well-developed provagina, and are very similar to those of V. capreoli. Morphometrics of first-stage larvae largely overlap with those of other Varestrongylus. Sequences of the ITS-2 region strongly support mutual independence of V. alces, V. cf. capreoli, and the yet undescribed species of Varestrongylus from North American ungulates. These three taxa form a well-supported crown-clade as the putative sister of V. alpenae. The association of V. alces and Alces or its ancestors is discussed in light of host and parasite phylogeny and host historical biogeography. Conclusions: Varestrongylus alces is a valid species, and should be considered distinct from V. capreoli. Phylogenetic relationships among Varestrongylus spp. from Eurasia and North America are complex and consistent with faunal assembly involving recurrent events of geographic expansion, host switching and subsequent speciation

Association of ligamentous laxity, male sex, chronicity, meniscal injury, and posterior tibial slope with a high-grade preoperative pivot shift: A post hoc analysis of the STABILITY study

Background: A spectrum of anterolateral rotatory laxity exists in anterior cruciate ligament (ACL)–injured knees. Understanding of the factors contributing to a high-grade pivot shift continues to be refined. Purpose: To investigate factors associated with a high-grade preoperative pivot shift and to evaluate the relationship between this condition and baseline patient-reported outcome measures (PROMs). Study Design: Cross-sectional study; Level of evidence, 3. Methods: A post hoc analysis was performed of 618 patients with ACL deficiency deemed high risk for reinjury. A binary logistic regression model was developed, with high-grade pivot shift as the dependent variable. Age, sex, Beighton score, chronicity of the ACL injury, posterior third medial or lateral meniscal injury, and tibial slope were selected as independent variables. The importance of knee hyperextension as a component of the Beighton score was assessed using receiver operator characteristic curves. Baseline PROMs were compared between patients with and without a high-grade pivot. Results: Six factors were associated with a high-grade pivot shift: Beighton score (each additional point; odds ratio [OR], 1.17; 95% CI, 1.06-1.30; P = .002), male sex (OR, 2.30; 95% CI, 1.28-4.13; P = .005), presence of a posterior third medial (OR, 2.55; 95% CI, 1.11-5.84; P = .03) or lateral (OR, 1.76; 95% CI, 1.01-3.08; P = .048) meniscal injury, tibial slope >9° (OR, 2.35; 95% CI, 1.09-5.07; P = .03), and chronicity >6 months (OR, 1.70; 95% CI, 1.00-2.88; P = .049). The presence of knee hyperextension improved the diagnostic utility of the Beighton score as a predictor of a high-grade pivot shift. Tibial slope <9° was associated with only a high-grade pivot in the presence of a posterior third medial meniscal injury. Patients with a high-grade pivot shift had higher baseline 4-Item Pain Intensity Measure scores than did those without a high-grade pivot shift (mean ± SD, 11 ± 13 vs 8 ± 14; P = .04); however, there was no difference between groups in baseline International Knee Documentation Committee, ACL Quality of Life, Knee injury and Osteoarthritis Outcome Score, or Knee injury and Osteoarthritis Outcome Score subscale scores. Conclusion: Ligamentous laxity, male sex, posterior third medial or lateral meniscal injury, increased posterior tibial slope, and chronicity were associated with a high-grade pivot shift in this population deemed high risk for repeat ACL injury. The effect of tibial slope may be accentuated by the presence of meniscal injury, supporting the need for meniscal preservation. Baseline PROMs were similar between patients with and without a high-grade pivot shift