Benzimidazole resistance allele haplotype diversity in United Kingdom isolates of Teladorsagia circumcincta supports a hypothesis of multiple origins of resistance by recurrent mutation

Polymorphisms in the isotype I β-tubulin gene are important genetic determinants of benzimidazole (BZ) resistance in a number of parasitic nematode species including Teladorsagia circumcincta, a major gastrointestinal nematode of sheep. This study investigates the genetic diversity at this locus in a BZ-resistant isolate of T. circumcincta (MTci5) derived from a sheep farm in the United Kingdom (UK) that was open to animal, and therefore parasite, migration. Pyrosequencing was used to determine the frequency of single nucleotide polymorphisms (SNPs) known to be associated with BZ resistance. This was followed by a combination of single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing to sample allelic diversity in a 276 bp fragment immediately surrounding the isotype I β-tubulin F200Y mutation. The genetic diversity at this locus was extremely high, with seven different haplotypes found to contain the resistant F200Y polymorphism in this single resistant isolate. Genotyping by SSCP interfaced with pyrosequencing demonstrated that the P200^Y mutation is also present on multiple haplotypes in two other BZ-resistant T. circumcincta isolates from the UK. This contrasts with much lower levels of haplotype diversity in BZ-resistant alleles present in T. circumcincta isolates from French goat farms that are closed to parasite migration. Taken together with our knowledge of T. circumcincta population genetic structure, these results are most consistent with multiple independent origins of resistance and mixing of alleles due to the large amount of livestock movement in the UK.     

Genetic Variants and Increased Expression of Parascaris equorum P-glycoprotein-11 in Populations with Decreased Ivermectin Susceptibility

Macrocyclic lactones (MLs) represent the major drug class for control of parasitic infections in humans and animals. However, recently reports of treatment failures became more frequent. In addition to human and ruminant parasitic nematodes this also is the case for the horse-nematode Parascaris equorum. Nevertheless, to date the molecular basis of ML resistance is still not understood. Unspecific resistance mechanisms involving transporters such as P-glycoproteins (Pgps) are expected to contribute to ML resistance in nematodes. Here, complete sequences of two P. equorum Pgps were cloned and identified as orthologs of Caenorhabditis elegans Ppg-11 and an unnamed Caenorhabditis briggsae Pgp designated as Pgp-16 using phylogenetic analysis. Quantitative real-time PCR was used to compare expression between tissues. Significantly higher PeqPgp-11 expression was found in the gut for both genders, whereas for PeqPgp-16 the body wall was identified as predominant expression site. Furthermore, Pgps were analyzed regarding their participation in resistance development. Using SeqDoC analyses, Pgp-sequences of P. equorum populations with different ML susceptibility were compared. This approach revealed three single nucleotide polymorphisms (SNPs) causing missense mutations in the PeqPgp-11 sequence which correlated with decreased ML susceptibility. However, no resistance associated differences in mRNA expression levels were detected between embryonated eggs of these populations. In contrast, comparison of two pre-adult groups with different ivermectin (IVM) susceptibility revealed the presence of the three SNPs and in addition statistically significant PeqPgp-11 overexpression in the group of worms with reduced susceptibility. These results indicate that Pgp-11 might be involved in IVM resistance in P. equorum as it shows increased expression in an IVM exposed life-cycle stage of an IVM resistant population as well as occurrence of putatively resistance associated SNPs in populations with reduced IVM susceptibility. These SNPs are promising diagnostic candidates for detection of ML resistance with potential also for other parasitic nematode species.     

Macrocyclic Lactones Differ in Interaction with Recombinant P-Glycoprotein 9 of the Parasitic Nematode Cylicocylus elongatus and Ketoconazole in a Yeast Growth Assay

Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the presence of ivermectin, moxidectin, daunorubicin and ketoconazole but not selamectin. These results demonstrate direct effects of MLs on a recombinant nematode Pgp in an ML-specific manner.     

The Emergence of Resistance to the Benzimidazole Anthlemintics in Parasitic Nematodes of Livestock Is Characterised by Multiple Independent Hard and Soft Selective Sweeps

Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the “genetic signature” of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs.     

A tandemly repetitive DNA sequence is present at diverse locations in the genome of Ostertagia circumcincta

A novel repetitive DNA sequence in the sheep parasitic nematode Ostertagia circumcincta was cloned and sequenced. This 1.2-kb sequence (Oc1B) was not found in the closely related cattle parasite Ostertagia ostertagi, nor in the more distantly related sheep parasites Haemonchus contortus or Trichostrongylus colubriformis. Sequences similar to Oc1B were found at various genomic locations and contained a pair of 33-bp direct repeats. Oc1B also contained a single copy of a 218-bp sequence (designated OcREP) which was present in 100 to 200 copies in the O. circumcincta genome and mostly organized in distinctive tandem arrays. The dual organizational pattern of OcREP as both a satellite-like sequence as well as interspersed as single copies amongst dissimilar sequences adds to the growing evidence for the fluidity of the parasitic nematode genome, and of eukaryotic genomes in general.     

Isolation and characterization of a naturally occurring multidrug-resistant strain of the canine hookworm,Ancylostoma caninum

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500?million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50?mg/kg for 3?days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the β-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous β-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.     

Comparison of Gene Expression Profiles Between Pansensitive and Multidrug-Resistant Strains of Mycobacterium tuberculosis

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.     

Phenotypic analysis of host-parasite interactions in lambs infected with Teladorsagia circumcincta

Female Blackface lambs expected to exhibit genetic variability for resistance to gastrointestinal nematodes, were either exposed to continuous experimental infections of Teladorsagia circumcincta or were sham-dosed to monitor phenotypic responses to infection. As a measure of parasitism and host response, worm-eggs in faeces (faecal egg count, FEC) were counted over a 3-month period and worm burdens were ascertained at post-mortem. The host response to the infection was also measured by differential counts of white blood cells, anti-T. circumcincta IgA antibody levels and body weight. Results suggest that nematode abundance (mean number of parasites per host) and prevalence (proportion of infected animals) were maximal shortly after the beginning of infection (21 days p.i.) when virtually all the infected animals were shedding worm eggs. Increasing anti-T. circumcincta IgA antibody and eosinophil concentrations were associated with a reduction in total numbers of adult worms and an increase in the frequency of early L4s. The data also suggest that genetic selection for an enhanced anti-T. circumcincta IgA response might complement selection based on a reduced FEC as a strategy to select for resistance to gastrointestinal nematodes.     

Association of BMPR-1B and GDF9 genes polymorphisms and secondary protein structure changes with reproduction traits in Mehraban ewes

BMPR-1B and GDF9 genes are well known due to their important effects on litter size and mechanisms controlling ovulation rate in sheep. In the present study, polymorphisms of BMPR-1B gene exon 8 and GDF9 gene exon 1 were detected by single strand conformational polymorphism (SSCP) analysis and DNA sequencing methods in 100 Mehraban ewes. The PCR reaction forced to amplify 140 and 380-bp fragments of BMPR-1B and GDF9 genes, respectively. Two single nucleotide polymorphisms (SNP_S) were identified in two different SSCP patterns of BMPR-1B gene (CC and CA genotypes) that deduced one amino acid exchange. Also, two SNP_S were identified in three different SSCP patterns of GDF9 gene (AA, AG and GG genotypes) that deduced one amino acid exchanges. Two different secondary structures of protein were predicted for BMPR-1B exon 8, but the secondary protein structures predicted for GDF9 exon 1 were similar together. The evaluation of the associations between the SSCP patterns and the protein structure changes with reproduction traits showed that BMPR-1B exon 8 genotypes have significant effects on some of reproduction traits but the GDF9 genotypes did not have any significant effect. The CA genotype of BMPR-1B exon 8 had a significant positive effect on reproduction performance and could be considered as an important and new mutation, affecting the ewes reproduction performance. Marker assisted selection using BMPR-IB gene could be noticed to improve the reproduction traits in Mehraban sheep.     

Circadian control of permethrin-resistance in the mosquito Aedes aegypti

Daily fluctuation of permethrin-resistance was found in adult mosquito Aedes aegypti, the major vector of dengue viruses in Taiwan. We hypothesized there is a relationship between resistance and the circadian clock. To test our hypothesis we correlated changes in the knock-down time (KT₅₀) response to permethrin with the expression of the pyrethroid-resistant gene CYP9M9 and the clock gene period (per) during a 12:12 h photoperiodic cycle. Rhythmic expression of per peaked at early scotophase of the light-dark cycle and at early subjective night in constant darkness. The values of KT₅₀ and the expression of CYP9M9 also exhibited circadian rhythms in both susceptible and permethrin-resistant mosquito strains, from which we inferred a link to the circadian clock. The KT₅₀ was significantly longer in the light than in the dark phase, and the level of CYP9M9 mRNA was maximal in early scotophase, dropped to a minimum in the midnight and then slowly increased through the photophase. Existence of a clock control over mosquito sensitivity to permethrin was further indicated by reduced expression of CYP9M9 and reduced mosquito resistance to permethrin after temporal silencing of the per gene. These data provide the first evidence on the circadian control of insect resistance to permethrin.     

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

Serial passage in resistant sheep drives the infectivity and fitness of Teladorsagia circumcincta in susceptible lambs: Experimental evidence

Gastrointestinal nematodes (GIN) of small ruminants have adapted their life history strategies to thrive in diverse and fluctuating environments. Environments which alter their expression of life traits may also drive changes in the infection or transmission dynamics, particularly if transferred to a foreign setting. This study aimed to explore how repeated exposure to a resistant sheep host environment would alter the life history traits and infection dynamics of Teladorsagia circumcincta when consequently infected in susceptible lambs. Following just three generations of passage in resistant sheep, T. circumcincta significantly increased their infectivity and fitness in susceptible lambs compared to a control population. This is the first evidence to indicate the resistant host environment can drive such rapid changes in the expression of GIN life traits, with potentially undesirable epidemiological outcomes.     

Selection for cold resistance alters gene transcript levels in Drosophila melanogaster

Microarrays have been used to examine changes in gene expression underlying responses to selection for increased stress resistance in Drosophila melanogaster, but changes in expression patterns associated with increased resistance to cold stress have not been previously reported. Here we describe such changes in basal expression levels in replicate lines following selection for increased resistance to chill coma stress. We found significant up- or down-regulation of expression in 94 genes on the Affymetrix Genome 2.0 array. Quantitative RT-PCR was used to confirm changes in expression of six genes. Some of the identified genes had previously been associated with stress resistance but no previously identified candidate genes for cold resistance showed altered patterns of expression. Seven differentially expressed genes that form a tight chromosomal cluster and an unlinked gene AnnX may be potentially important for cold adaptation in natural populations. Artificial selection for chill coma resistance therefore altered basal patterns of gene expression, but we failed to link these changes to plastic changes in expression under cold stress or to previously identified candidate genes for components of cold resistance.