Biology subjects

Maitland, K.

Publications and source records attributed to Maitland, K..

13 recordsLinked to original sources

bioRxiv2024

Optimisation of single-nuclei isolation and RNA sequencing of parasitic nematodes

Single-cell/nuclei transcriptomics has revolutionised our understanding of cell, tissue, and organismal biology during health and disease of humans and model organisms. However, for non-model species, reassessment and refinement of conventional methods to address novel technical and biological challenges are needed. Here, we evaluate three established nuclei isolation protocols and, building on experiences, optimised single nuclei isolation for parasitic nematodes (SNIP). We test the versatility of SNIP across diverse species and life stages, validating it with snRNA-seq to reveal sex, stage, and tissue-specific gene expression. Optimised protocols will advance the understanding of parasitic nematodes, from fundamental biology to infection and disease.

molecular biology↗

bioRxiv2024

Chromosomal genome assembly resolves drug resistance loci in the parasitic nematode Teladorsagia circumcincta

The parasitic nematode Teladorsagia circumcincta is one of the most important pathogens of sheep and goat farming in temperate climates worldwide and can rapidly evolve resistance to drugs used to control it. To understand the genetics of drug resistance, we have generated a highly contiguous genome assembly for the UK T. circumcincta isolate, MTci2. Assembly using PacBio long-reads and Hi-C long-molecule scaffolding together with manual curation resulted in a 573 Mb assembly (N50 = 84 Mb, n = 1,286) with five autosomal and one sex-linked chromosomal-scale scaffolds consistent with its karyotype. The genome resource was further improved via annotation of 22,948 genes, with manual curation of over 3,200 of these, resulting in a robust and near complete resource (96.3% complete protein BUSCOs) to support basic and applied research on this important veterinary pathogen. Genome-wide analyses of drug resistance, combining evidence from three distinct experiments, identified selection around known candidate genes for benzimidazole, levamisole and ivermectin resistance, as well as novel regions associated with ivermectin and moxidectin resistance. These insights into contemporary and historic genetic selection further emphasise the importance of contiguous genome assemblies in interpreting genome-wide genetic variation associated with drug resistance and identified key loci to prioritise in developing diagnostic markers of anthelmintic resistance to support parasite control. AUTHOR SUMMARYUnderstanding the genetics of anthelmintic resistance is a critical part of the sustainable control of parasitic worms. Here, we have generated a chromosome-scale genome assembly for the gastrointestinal nematode, Teladorsagia circumcincta, one of the most important pathogens of sheep in temperate climates worldwide. This genome and its annotation offers a substantial improvement over existing genetic resources, and will enable new insight into the biology for this parasite and new opportunities to characterise therapeutic targets such as drug and vaccine candidates. We used this resource to map genetic variation associated with resistance to multiple anthelmintic drug classes used as the primary means of parasite control, confirming known candidate genes and variants (i.e., beta-tubulin isotypes 1 and 2 associated with benzimidazole resistance, and acr-8 associated with levamisole resistance, and pgp-9 copy number variation, of which overexpression is associated broadly with anthelmintic resistance) and revealing new regions of the genome associated with drug treatment responses (i.e. 34-38 Mb on chromosome 5 associated with ivermectin resistance). Our results highlight the importance of a contiguous genome assembly and the use of complementary experimental approaches to reveal the impact of drug-mediated selection, both historically and directly in response to treatment, on genome-wide genetic variation.

genomics↗

bioRxiv2024

Modelling the gut microbiota of children with malnutrition: in vitro models reveal differences in fermentability of widely consumed carbohydrates

There is increasing evidence in children suffering from Severe Acute Malnutrition (SAM) that there is disruption of the gut microbiome and low gut microbiota diversity, which may be contributing factors to poor outcomes during nutritional treatment and recovery. The gut microbiome of children with SAM has been demonstrated to have a lower production of beneficial short chain fatty acids, which may contribute to impaired gut barrier function. Recently, several microbiota-directed therapies have been tested in clinical trials in children with SAM. Among them we hypothesized that feeds containing fermentable carbohydrates from various sources (legumes, chicory, milk oligosaccharides) would be fermented to produce beneficial microbial metabolites by the microbiota of children with SAM. In this study we used an in vitro model system inoculated with stool from children with SAM to investigate the fermentability of four substrates; inulin (a chicory-derived fructan), two milk powders (one supplemented with a human milk oligosaccharide) and a chickpea enriched feed. We demonstrated that while the milk powders and chickpea feed were fermented to produce short chain fatty acids, inulin was only fermented to a very limited degree. Through 16S rRNA sequencing we demonstrated that the samples inoculated with inulin had low microbial diversity and linked this to the limited ability to metabolise inulin. Through revealing the fermentability of different complementary feeds, the findings of this study will be of use for the design of future therapeutic feeds for treatment of SAM. ImportanceMalnutrition is a major contributor to childhood mortality globally and is a major public health problem primarily affecting Lower- and Middle-Income Countries. Despite the development of nutritional recovery therapies, for those with the severe and complicated form of malnutrition (SAM), mortality and relapse rates remain high. Emerging evidence suggests a role for the gut microbiome in these poor outcomes, which is known to be significantly altered in children in SAM, compared to healthy age matched controls. To aid in recovery from SAM, nutritional interventions should be designed to support the gut microbiome, using a range of ingredients targeted for colonic fermentation. It is important to understand the fermentation capacity of the gut microbiome of children with SAM, to design future nutritional interventions. In this work, we demonstrate that inulin, a widely used chicory-derived prebiotic, is not a suitable fermentation substrate for the gut microbiome of SAM children, while legume-based formulations and milk oligosaccharides result in increased production of beneficial metabolites.

microbiology↗

bioRxiv2022

Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria

Blood group O is associated with protection against severe malaria and reduced size and stability of P. falciparum-host red blood cell (RBC) rosettes compared to non-O blood groups. Whether the non-O blood groups encoded by the specific ABO genotypes AO, BO, AA, BB and AB differ in their associations with severe malaria and rosetting is unknown. The A and B antigens are host RBC receptors for rosetting, hence we hypothesized that the higher levels of A and/or B antigen on RBCs from AA, BB and AB genotypes compared to AO/BO genotypes could lead to larger rosettes, increased microvascular obstruction and higher risk of malaria pathology. We used a case-control study of Kenyan children and in vitro adhesion assays to test the hypothesis that "double dose" non-O genotypes (AA, BB, AB) are associated with increased risk of severe malaria and larger rosettes than "single dose" heterozygotes (AO, BO). In the case-control study, compared to OO, the double dose genotypes consistently had higher odds ratios (OR) for severe malaria than single dose genotypes, with AB (OR 1.93) and AO (OR 1.27) showing most marked difference (P=0.02, Wald test). In vitro experiments with blood group A-preferring P. falciparum parasites showed that significantly larger rosettes were formed with AA and AB host RBCs compared to OO, whereas AO genotype rosettes were indistinguishable from OO. Overall, the data show that ABO genotype influences P. falciparum rosetting and support the hypothesis that double dose non-O genotypes confer a greater risk of severe malaria than AO/BO heterozygosity.

genetics↗

bioRxiv2021

Genomic landscape of drug response reveals novel mediators of anthelmintic resistance

Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to improving the efficacy and sustainability of parasite control. Here, we use a genetic cross in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies novel alleles for resistance to benzimidazoles and levamisole and implicates the transcription factor, cky-1, in ivermectin resistance. This gene is within a locus under selection in ivermectin resistant populations worldwide; functional validation using knockout experiments supports a role for cky-1 overexpression in ivermectin resistance. Our work demonstrates the feasibility of high-resolution forward genetics in a parasitic nematode, and identifies variants for the development of molecular diagnostics to combat drug resistance in the field. One-Sentence SummaryGenetic mapping of known and novel anthelmintic resistance-associated alleles in a multi-drug resistant parasitic nematode

genomics↗

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↗

bioRxiv2021

Improving statistical power in severe malaria genetic association studies by augmenting phenotypic precision

Severe falciparum malaria has substantially affected human evolution. Genetic association studies of patients with clinically defined severe malaria and matched population controls have helped characterise human genetic susceptibility to severe malaria, but phenotypic imprecision compromises discovered associations. In areas of high malaria transmission the diagnosis of severe malaria in young children and, in particular, the distinction from bacterial sepsis, is imprecise. We developed a probabilistic diagnostic model of severe malaria using platelet and white count data. Under this model we re-analysed clinical and genetic data from 2,220 Kenyan children with clinically defined severe malaria and 3,940 population controls, adjusting for phenotype mis-labelling. Our model, validated by the distribution of sickle trait, estimated that approximately one third of cases did not have severe malaria. We propose a data-tilting approach for case-control studies with phenotype mis-labelling and show that this reduces false discovery rates and improves statistical power in genome-wide association studies.

genetics↗

bioRxiv2020

Extensive genomic and transcriptomic variation defines the chromosome-scale assembly of Haemonchus contortus, a model gastrointestinal worm

BackgroundHaemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants, and has become the key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Two draft genome assemblies for H. contortus were reported in 2013, however, both were highly fragmented, incomplete, and differed from one another in important respects. While the introduction of long-read sequencing has significantly increased the rate of production and contiguity of de novo genome assemblies broadly, achieving high quality genome assemblies for small, genetically diverse, outcrossing eukaryotic organisms such as H. contortus remains a significant challenge. ResultsHere, we report using PacBio long read and OpGen and 10X Genomics long-molecule methods to generate a highly contiguous 283.4 Mbp chromosome-scale genome assembly including a resolved sex chromosome. We show a remarkable pattern of almost complete conservation of chromosome content (synteny) with Caenorhabditis elegans, but almost no conservation of gene order. Long-read transcriptome sequence data has allowed us to define coordinated transcriptional regulation throughout the life cycle of the parasite, and refine our understanding of cis- and trans-splicing relative to that observed in C. elegans. Finally, we use this assembly to give a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally. ConclusionsThe H. contortus MHco3(ISE).N1 genome assembly presented here represents the most contiguous and resolved nematode assembly outside of the Caenorhabditis genus to date, together with one of the highest-quality set of predicted gene features. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes, and extends the experimental tractability of this model parasitic nematode in understanding pathogen biology, drug discovery and vaccine development, and important adaptive traits such as drug resistance.

genomics↗

bioRxiv2019

Evaluation of DNA extraction methods on individual helminth egg and larval stages for whole genome sequencing

Whole genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative, however, these samples are typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole genome sequencing of individual immature helminth samples. These approaches do not use whole genome amplification, a common but costly approach to increase the yield of low input samples. We first tested individual parasites from two species spotted onto FTA cards - egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni - before further testing on an additional six species - Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Dracunculus medinensis, Strongyloides stercoralis, and Trichuris muris - with an optimal protocol. Whole genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation between species, and within species but between life stages, described. These results demonstrate the feasibility of whole genome sequencing of individual parasites, and highlight a new avenue towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

genomics↗

bioRxiv2018

The impact of 10-valent Pneumococcal Conjugate Vaccine on the incidence of radiologically-confirmed pneumonia and clinically-defined pneumonia in Kenyan children.

BackgroundPneumococcal conjugate vaccines (PCV) are highly protective against invasive pneumococcal disease caused by vaccine serotypes but the burden of pneumococcal disease in developing countries is dominated by pneumonia, most of which is non-bacteraemic. We examined the impact of PCV on pneumonia incidence.\n\nMethodsWe linked prospective hospital surveillance for clinically-defined WHO severe or very-severe pneumonia at Kilifi County Hospital from 2002-2015 to population surveillance at Kilifi Health and Demographic Surveillance System, comprising 45,000 children aged <5 years. Chest radiographs were read according to a WHO standard. A 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PCV10) was introduced in Kenya in January 2011. In Kilifi, there was a catch-up campaign for children aged <5 years. We estimated the impact of PCV10 on pneumonia incidence through interrupted time series analysis accounting for seasonal and temporal trends.\n\nFindingsThe incidence of admission with clinically-defined pneumonia in 2002/3 was 21{middle dot}7/1000/year in children aged 2-59 months. This declined progressively over 13 years. By the end of March 2011, 61{middle dot}1% of children aged 2-11 months received [≥]2 doses and 62{middle dot}3% of children aged 12-59 months received [≥]1 dose of PCV10. Adjusted incidence rate ratios for admissions with radiologically-confirmed pneumonia, clinically-defined pneumonia, and diarrhoea (control condition), associated with PCV10 introduction, were 0{middle dot}52 (95% CI 0{middle dot}32-0{middle dot}86), 0{middle dot}73 (95% CI 0{middle dot}54-0{middle dot}97) and 0{middle dot}63, (95% CI 0{middle dot}31-1{middle dot}26), respectively. The annual incidence of clinically-defined pneumonia in December 2010 was 12{middle dot}2/1000; this was reduced by 3{middle dot}3/1000 with PCV10 introduction.\n\nInterpretationOver 13 years, hospitalisations for clinically-defined pneumonia declined progressively at Kilifi County Hospital but fell abruptly by 27% in association with PCV10 introduction. The incidence of radiologically-confirmed pneumonia fell by 48%. The burden of childhood pneumonia in Kilifi, Kenya, has been reduced substantially by PCV10.\n\nFundingGavi, Wellcome Trust

epidemiology↗

bioRxiv2018

Population Turnover in Remote Oceania Shortly After Initial Settlement

Ancient DNA analysis of three individuals dated to ~3000 years before present (BP) from Vanuatu and one ~2600 BP individual from Tonga has revealed that the first inhabitants of Remote Oceania (\"First Remote Oceanians\") were almost entirely of East Asian ancestry, and thus their ancestors passed New Guinea, the Bismarck Archipelago, and the Solomon Islands with minimal admixture with the Papuan groups they encountered [1]. However, all present-day populations in Near and Remote Oceania harbor 25-100% Papuan ancestry, implying that there must have been at least one later stream of migration eastward from Near Oceania. We generated genome-wide data for 14 ancient individuals from Efate and Epi Islands in Vanuatu ranging from 3,000-150 BP, along with 185 present-day Vanuatu individuals from 18 islands. We show that people of almost entirely Papuan ancestry had arrived in Vanuatu by 2400 BP, an event that coincided with the end of the Lapita cultural period, changes in skeletal morphology, and the cessation of long-distance trade between Near and Remote Oceania [2]. First Remote Oceanian ancestry subsequently increased via admixture but remains at 10-20% in most islands. Through a fine-grained comparison of ancestry profiles in Vanuatu and Polynesia with diverse groups in Near Oceania, we find that Papuan ancestry in Vanuatu is consistent with deriving from the Bismarck Archipelago instead of the geographically closer Solomon Islands. Papuan ancestry in Polynesia also shows connections to the ancestry profiles present in the Bismarck Archipelago but is more similar to Tolai from New Britain and Tutuba from Vanuatu than to the ancient Vanuatu individuals and the great majority of present-day Vanuatu populations. This suggests a third eastward stream of migration from Near to Remote Oceania bringing a different type of Papuan ancestry.

genetics↗

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↗

bioRxiv2017

Genomic And Transcriptomic Profiling Of Nematode Parasites Surviving After Vaccine Exposure

Some nematode species are economically important parasites of livestock, while others are important human pathogens causing some of the most important neglected tropical diseases. In both humans and animals, anthelmintic drug administration is the main control strategy, but the emergence of drug-resistant worms has stimulated the development of alternative control approaches. Among these, vaccination is considered to be a sustainable and cost effective strategy. Currently, Barbervax(R) for the ruminant strongylid Haemonchus contortus is the only registered subunit vaccine for a nematode parasite, although a vaccine for the human hookworm Necator americanus is undergoing clinical trials (HOOKVAC consortium). As both these vaccines comprise a limited number of proteins there is potential for selection of nematodes with altered sequence or expression of the vaccine antigens. Here we compared the transcriptome of H. contortus populations from sheep vaccinated with Barbervax(R) with worms from control animals. Barbervax(R) antigens are native integral membrane proteins isolated from the brush border of the intestinal cells of the adult parasite and many of them are proteases. Our findings provide no evidence for changes in expression of genes encoding Barbervax(R) antigens in the surviving parasite populations. However, surviving parasites from vaccinated animals showed increased expression of other proteases and regulators of lysosome trafficking, and displayed up-regulated lipid storage and defecation abilities that may have circumvented the vaccine effect. Implications for other potential vaccines for human and veterinary nematodes are discussed.

genomics↗