Biology subjects

Bartley, D. J.

Publications and source records attributed to Bartley, D. J..

7 recordsLinked to original sources

bioRxiv2026

A comparative analysis of liver tissue and novel primary organoid cultures from ruminants reveals species-specific immune architecture and metabolic specialization

The liver is widely considered to be one of the most conserved organs amongst vertebrates, with it being involved in blood detoxification, bile production and the metabolism of xenobiotic compounds. Liver organoids have previously been derived from several species and used as models of drug metabolism, toxicity, and fundamental tissue biology. To date, however, these models have not been developed from ruminant species, specifically cattle and sheep. Here we present the first report of the development and comprehensive characterisation of bovine and ovine liver organoids derived from primary liver tissue. When initially established, organoids from both species were comprised of KRT19- and KRT18-positive cholangiocytes. The capacity for organoids to differentiate into hepatocyte-enriched cultures was evaluated and it was noted that there was an increase in hepatocyte markers in bovine cultures. A comparative analysis of the liver tissue and organoids of both species revealed species-specific differences in gene expression, which were conserved within organoid cultures. Most notably, bovine liver tissue and organoids had enriched expression of genes associated with fatty acid uptake and storage whereas ovine samples had higher expression of genes associated with fatty acid conversion, highlighting fundamental differences between these two ruminant species. Differences in expression of cytochrome P450 family genes were identified alongside those associated with an inflammatory response specifically in bovine samples, whereas ovine samples had higher expression of genes associated with a protective immune response. Despite this, transcriptomic analysis of organoids from both species, cultured in both growth and differentiation media, revealed preserved expression of genes associated with key liver functions, including gluconeogenesis and xenobiotic metabolism. Transcripts associated with the flavin-containing monooxygenases (FMO) family were expressed in both organoid growth media and organoid development media (OGM and ODM respectively), and both species could metabolise triclabendazole into its primary metabolite triclabendazole sulfoxide, therefore validating the potential of the organoids to be applied as in vitro models of metabolism and/or toxicity. Overall, this study provides novel insights into differences in liver composition and function between ruminant species, as well as providing novel experimental models of the liver for both cattle and sheep.

cell biology↗

bioRxiv2022

Allele specific PCR for a major marker of levamisole resistance in Haemonchus contortus

Haemonchus contortus is a haematophagous parasitic nematode that infects small ruminants and causes significant animal health concerns and economic losses within the livestock industry on a global scale. Treatment primarily depends on broad-spectrum anthelmintics, however, resistance is established or rapidly emerging against all major drug classes. Levamisole (LEV) remains an important treatment option for parasite control, as resistance to LEV is less prevalent than to members of other major classes of anthelmintics. LEV is an acetylcholine receptor (AChR) agonist that, when bound, results in paralysis of the worm. Numerous studies implicated the AChR sub-unit, ACR-8, in LEV sensitivity and in particular, the presence of a truncated acr-8 transcript or a deletion in the acr-8 locus in some resistant isolates. Recently, a single non-synonymous SNP in acr-8 conferring a serine-to-threonine substitution (S168T) was identified that was strongly associated with LEV resistance. Here, we investigate the role of genetic variation at the acr-8 locus in a controlled genetic cross between the LEV susceptible MHco3(ISE) and LEV resistant MHco18(UGA 2004) isolates of H. contortus. Using single worm PCR assays, we found that the presence of S168T was strongly associated with LEV resistance in the parental isolates and F3 progeny of the genetic cross surviving LEV treatment. We developed and optimised an allele-specific PCR assay for the detection of S168T and validated the assay using laboratory isolates and field samples that were phenotyped for LEV resistance. In the LEV-resistant field population, a high proportion (>75%) of L3 encoded the S168T variant, whereas the variant was absent in the susceptible isolates studied. These data further support the potential role of acr-8 S168T in LEV resistance, with the allele-specific PCR providing an important step towards establishing a sensitive molecular diagnostic test for LEV resistance.

microbiology↗

bioRxiv2021

Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and recovery in a parasitic nematode

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and recovery in parasitic helminths. Author SummaryParasitic helminths (worms) infect people and animals throughout the world and are largely controlled with mass administration of anthelmintic drugs. There are a very limited number of anthelmintics available and parasitic helminths can rapidly develop resistance to these drugs. Ivermectin is a widely used anthelmintic in both humans and animals, but resistance is now widespread in the veterinary field. We crossed ivermectin resistant and ivermectin susceptible parasitic helminths and treated them with ivermectin or left them as untreated controls. This provided resistant and susceptible populations with a similar genetic background with which to study differences in gene expression associated with ivermectin resistance. We identified upregulation of a gene with no previous association with drug resistance (HCON_00155390:cky-1) in male and female worms in all resistant populations. This gene is thought to be expressed in the helminth pharynx (mouthpart) and, in mammals, plays a role in controlling nerve function and protecting nerves from damage. This is consistent with the known effects of ivermectin in inhibiting helminth feeding through pharyngeal paralysis and implicates a novel mechanism that allows resistant worms to survive treatment.

genomics↗

bioRxiv2019

Development of high throughput methods for the analysis of anthelmintic resistance allele frequencies in field populations of gastrointestinal nematodes.

Drug resistant helminths have become a major cause of poor health and production in sheep and goats, and there is a need for diagnostic markers and tools to determine the frequency of resistance alleles in field parasite populations. Gastrointestinal nematode resistance to benzimidazole drugs is caused by a mutation in one of three positions on the isotype 1 {beta}-tubulin locus, and in the absence of markers for resistance to other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field gastrointestinal nematode populations can be impractical using conventional molecular methods, which may be error prone or lack sensitivity at low levels of resistance. Here, we report the development of a novel method based on an Illumina Mi-seq deep amplicon sequencing platform; to sequence the isotype 1 {beta}-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias in the isotype 1 {beta}-tubulin locus, comparing the results of Illumina Mi-seq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible L3. Finally, we applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic drug resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

molecular biology↗

bioRxiv2018

A major locus for ivermectin resistance in a parasitic nematode

BackgroundInfections with helminths cause an enormous disease burden in billions of animals and plants worldwide. Large scale use of anthelmintics has driven the evolution of resistance in a number of species that infect livestock and companion animals, and there are growing concerns regarding the reduced efficacy in some human-infective helminths. Understanding the mechanisms by which resistance evolves is the focus of increasing interest; robust genetic analysis of helminths is challenging, and although many candidate genes have been proposed, the genetic basis of resistance remains poorly resolved. ResultsHere, we present a genome-wide analysis of two genetic crosses between ivermectin resistant and sensitive isolates of the parasitic nematode Haemonchus contortus, an economically important gastrointestinal parasite of small ruminants and a model for anthelmintic research. Whole genome sequencing of parental populations, and key stages throughout the crosses, identified extensive genomic diversity that differentiates populations, but after backcrossing and selection, a single genomic quantitative trait locus (QTL) localised on chromosome V was revealed to be associated with ivermectin resistance. This QTL was common between the two geographically and genetically divergent resistant populations and did not include any leading candidate genes, suggestive of a previously uncharacterised mechanism and/or driver of resistance. Despite limited resolution due to low recombination in this region, population genetic analyses and novel evolutionary models supported strong selection at this Q.TL, driven by at least partial dominance of the resistant allele, and that large resistance-associated haplotype blocks were enriched in response to selection. ConclusionsWe have described the genetic architecture and mode of ivermectin selection, revealing a major genomic locus associated with ivermectin resistance, the most conclusive evidence to date in any parasitic nematode. This study highlights a novel genome-wide approach to the analysis of a genetic cross in non-model organisms with extreme genetic diversity, and the importance of a high quality reference genome in interpreting the signals of selection so identified.

genomics↗

bioRxiv2018

Effect of Infection with, and Treatment of, Sensitive and Resistant Strains of Teladorsagia Circumcincta on the Ovine Intestinal Microbiota

Nematodes are one of the main impactors on health, welfare and productivity of farmed animals. Teladorsagia circumcincta is arguably one of the most globally important nematode species in sheep. Control of these nematode infections is essential and heavily reliant on chemotherapy (anthelmintics), but this has been complicated by the development of anthelmintic resistance. In mammals the composition of the intestinal microbiota has been shown to have a significant effect on overall health. The interactions between host, microbiota and pathogens are complex and influenced by numerous factors. In this study, the interactions between T. circumcincta infections and microbial composition and abundance were investigated. In a preliminary study the intra-and inter-individual diversity and composition of the microbiota of grazing sheep was assessed in two distinct regions of the ovine intestinal tract, the terminal ileum (TI) and rectal contents. Additionally, the effects of experimental infection of sheep with two strains of T. circumcincta (anthelmintic resistant or sensitive) on the microbiota were assessed with and without anthelmintic (monepantel) administration. The inter-animal variability was greater in the TI compared to the rectal samples. However, the alpha-diversity (species richness) was significantly lower in the TI samples. In the experimental study, clear differences were observed between successfully treated animals and those sheep that were left untreated and/or those carrying resistant nematodes. Differences in microbiota between the four different experimental conditions were observed and potential predictive biomarkers were identified. In particular, a restoration of potentially beneficial Bifidobacteria sp. in successfully-treated animals was observed.

microbiology↗

bioRxiv2017

A genome resequencing-based genetic map reveals the recombination landscape of an outbred parasitic nematode in the presence of polyploidy and polyandry

The parasitic nematode Haemonchus contortus is an economically and clinically important pathogen of small ruminants, and a model system for understanding the mechanisms and evolution of traits such as anthelmintic resistance. Anthelmintic resistance is widespread and is a major threat to the sustainability of livestock agriculture globally; however, little is known about the genome architecture and parameters such as recombination that will ultimately influence the rate at which resistance may evolve and spread. Here we performed a genetic cross between two divergent strains of H. contortus, and subsequently used whole-genome re-sequencing of a female worm and her brood to identify the distribution of genome-wide variation that characterises these strains. Using a novel bioinformatic approach to identify variants that segregate as expected in a pseudo-testcross, we characterised linkage groups and estimated genetic distances between markers to generate a chromosome-scale F1 genetic map composed of 1,618 SNPs. We exploited this map to reveal the recombination landscape, the first for any parasitic helminth species, demonstrating extensive variation in recombination rate within and between chromosomes. Analyses of these data also revealed the extent of polyandry, whereby at least eight males were found to have contributed to the genetic variation of the progeny analysed. Triploid offspring were also identified, which we hypothesise are the result of nondisjunction during female meiosis or polyspermy. These results expand our knowledge of the genetics of parasitic helminths and the unusual life-history of H. contortus, and will enable more precise characterisation of the evolution and inheritance of genetic traits such as anthelmintic resistance. This study also demonstrates the feasibility of whole-genome resequencing data to directly construct a genetic map in a single generation cross from a non-inbred non-model organism with a complex lifecycle.\n\nAuthor summaryRecombination is a key genetic process, responsible for the generation of novel genotypes and subsequent phenotypic variation as a result of crossing over between homologous chromosomes. Populations of strongylid nematodes, such as the gastrointestinal parasites that infect livestock and humans, are genetically very diverse, but little is known about patterns of recombination across the genome and how this may contribute to the genetics and evolution of these pathogens. In this study, we performed a genetic cross to quantify recombination in the barbers pole worm, Haemonchus contortus, an important parasite of sheep and goats. The reproductive traits of this worm make standard genetic crosses challenging, but by generating whole-genome sequence data from a female worm and her offspring, we identified genetic variants that act as though they come from a single mating cross, allowing the use of standard statistical approaches to build a genetic map and explore the distribution and rates of recombination throughout the genome. A number of genetic signatures associated with H. contortus life history traits were revealed in this analysis: we extend our understanding of multiple paternity (polyandry) in this species, and provide evidence and explanation for sporadic increases in chromosome complements (polyploidy) among the progeny. The resulting genetic map will aid in population genomic studies in general and enhance ongoing efforts to understand the genetic basis of resistance to the drugs used to control these worms, as well as for related species that infect humans throughout the world.

genomics↗