Biology subjects

Chaudhry, U.

Publications and source records attributed to Chaudhry, U..

At least 19 recordsLinked to original sources

bioRxiv2026

Development and validation of a multilocus sequence typing scheme for Fasciola hepatica using next-generation deep amplicon sequencing

Fasciolosis caused by Fasciola hepatica is an economically important disease in sheep and cattle. Knowledge of the population genetic structure of F. hepatica is important for understanding gene flow and informing disease control. In the present study, we designed, developed, and validated a multilocus sequence typing (MLST) scheme based on six markers. These markers were selected by aligning newly sequenced whole-genome sequence (WGS) data with available reference genomes and selecting variable regions with five or more single-nucleotide polymorphisms SNPs from different scaffolds of the F. hepatica reference genome Fasciola 10x pilon (GCA_900302435.1). Twenty markers were initially identified, of which 12 were multiplexed for deep amplicon sequencing after validation on worm and faecal eggs DNA; six markers were ultimately retained for downstream population genetics analysis. These markers were used to investigate population genetic structure in 15 cattle- and 27 sheep-derived F. hepatica populations in UK. A total of 53 unique alleles from six MLST markers were identified from 30 faecal (cattle = 13, sheep = 17) and 12 adult worm (cattle = 2, sheep = 10) populations. Shared alleles were observed in sheep- and cattle-derived populations. The highest allelic variation was observed in the Scottish Borders, Southern Scotland, and South-West England, and the lowest in North-West England. Minimal genetic differentiation was observed between cattle- and sheep-derived populations, with most genetic structuring within rather than between populations. Five markers showed high allelic polymorphism, whereas one marker showed low levels of allelic polymorphism, highlighting the importance of multilocus approaches. Overall, this six MLST-marker panel provides a tool for population genetic studies, revealing high gene flow and clonal expansion of F. hepatica across hosts and regions in the UK.

genetics↗

bioRxiv2026

Limited genetic structure and high gene flow in Fasciola hepatica populations infecting ruminants in different geographic areas in the UK

The liver fluke, Fasciola hepatica, is a major parasitic threat to ruminant health and productivity worldwide, with important implications for food security, animal welfare, and zoonotic risk. This study developed and validated a multiplex deep amplicon sequencing assay targeting the mitochondrial NADH dehydrogenase 1 (mt-ND1) and cytochrome c oxidase subunit 1 (mt-COX1) loci for high-throughput genotyping of F. hepatica. DNA was extracted from eggs sedimented from sheep and cattle faeces (n = 78) received from farms and from adult worm pools (n = 12) isolated at abattoirs from diverse regions across the UK. Following high-throughput sequencing, bioinformatics analysis was performed to demultiplex Illumina sequence reads and extract amplicon sequence variants (ASVs). A total of 11 ASVs were identified at each locus (mt-ND1: 264-279 bp; mt-COX1: 312-319 bp), with two or three predominant ASVs per locus, along with rare variants. Network and PCA analyses revealed two distinct clusters at the mt-ND1 locus: one primarily associated with sheep and another shared between sheep and cattle. In contrast, mt-COX1 sequence reads formed a single dominant cluster. Population analyses revealed extensive ASV sharing across regions, indicating high gene flow, likely facilitated by livestock movement and parasite adaptation.

evolutionary biology↗

bioRxiv2025

Scalable Assembly of Ascaris Mitogenomes from Whole-Genome Data Reveals a Novel Clade

The genus Ascaris is an important group of giant parasitic roundworms, infecting over 700 million people globally, with substantial economic losses in domestic pigs. Whilst species of Ascaris are morphologically indistinguishable, analysis of mitochondrial and selected nuclear DNA loci has revealed three clades (A, B, C), which may inflate with the addition of genomic data. Here, we present a bioinformatic pipeline for de novo assembly of complete mitochondrial genomes (mtDNA) from low-coverage whole-genome data through host-read depletion or mtDNA read enrichment, followed by mtDNA-specific assembly. Our approach yielded 149 high-quality Ascaris mtDNA assemblies, enabling the study of population-level diversity, including the identification of a novel clade (Clade D, designated here) associated with human samples from Ethiopia. Our analysis further revealed Clade C to comprise of pig-derived samples from Europe based on characterisation of worms isolated in Germany. Furthermore, genetic diversity across all mitochondrial loci was high (concatenated dataset: S = 1261; h = 94; {pi} = 0.0224), indicating substantial intraspecies variation. Our methods described here provide a scalable framework for mtDNA genome reconstruction with insights into roundworm population-genomic and phylogenetic studies.

genomics↗

bioRxiv2025

Inactivating conditions of therapeutic mycobacteriophages

There is a need for new therapies to treat drug resistant nontuberculous mycobacteria (NTM) disease. Bacteriophages (phages), which are viruses that infect and kill bacteria, are actively being explored as an alternative approach for treating mycobacterial diseases. Several compassionate-use cases of phage therapy for drug resistant NTM infections exhibit favorable outcomes. To further the development of phage therapy it is important to recognize and avoid conditions that negatively impact phage activity during phage production, storage, formulation, or treatment. Conversely, there is a need to inactivate free phages in certain preclinical phage therapy experiments. In this study, we investigated three mycobacteriophages BPs{Delta}33HTH-HRM10, Muddy, and ZoeJ{Delta}45 from compassionate-use NTM treatment cases for their sensitivity to a variety of conditions that included temperature, acid pH, detergents, mucus, and phage inactivating buffers. Several conditions resulted in dramatic and rapid reductions in the level of active phage while others had no effect. We also observed different sensitivities between the phages. The results provide valuable information to support further investigation and development of these phages as therapeutics.

microbiology↗

bioRxiv2025

Development of a qPCR assay and tremabiome deep amplicon sequencing method for differentiation of fluke species in livestock

BackgroundTrematode parasites, or flukes, are a significant economic threat to ruminant production worldwide. Traditional diagnostic methods rely on egg sedimentation from faeces, a time-consuming methodology lacking sensitivity and specificity. This study aimed to develop and validate two diagnostic methods: firstly, qPCR for accurate identification of Fasciola spp, and secondly, tremabiome, deep amplicon sequencing technique for identifying fluke species using faecal egg DNA. MethodologyTo detect fluke infection primers targeting mitochondrial DNA were repurposed to develop a SYBR Green qPCR diagnostic. For the identification of fluke species, a tremabiome approach was developed. A reference sequence library and taxonomy file were generated for 21 fluke species, enabling species sequence read separation and extracting amplicon sequence variants (ASVs). To validate the qPCR and tremabiome approach, 402 faecal samples were collected from cattle and sheep across the UK. Fluke eggs were isolated by sedimentation, detected by microscopy and qPCR, and tremabiome used to identify fluke eggs to species level. ResultsqPCR demonstrated high analytical sensitivity, detecting Fasciola hepatica DNA down to 19.2fg and F. gigantica down to 6.4fg, with no cross-amplification of other flukes. Tremabiome was able to detect as few as five F. hepatica and Calicophoron daubneyi eggs and identify mixed infections. High levels of co-infection (14.4%) of F. hepatica and C. daubneyi were observed in faecal samples, followed by single infections with C. daubneyi (12.6%) and F. hepatica (3.2%). Notably, tremabiome detected F. hepatica in 20 samples missed by qPCR. Data analysis identified 55 and 32 ASVs for F. hepatica and C. daubneyi, respectively, with phylogenetic clustering within their respective clades. ConclusionThis study developed qPCR assay for Fasciola detection and validated a tremabiome deep amplicon sequencing for fluke species differentiation. These approaches have improved capacity to identify fluke species compared to microscopy and are valuable tools for enhancing fasciolosis surveillance and control. Author SummaryFlukes are flatworm parasites that cause disease domestic and wild animals and humans. The main species infecting cattle and sheep globally are the liver flukes F. hepatica and F. gigantica, with other species including the rumen fluke Calicophoron daubneyi locally important or emerging. Infections result in serious economic losses. The traditional method of diagnosing fluke infection involves observation of eggs in faecal samples under the microscope, but this can be time-consuming and error prone, since the eggs of different species often look similar. In this study, we developed and validated two methods to improve detection: qPCR, a sensitive DNA-based test to identify Fasciola infections, and tremabiome, a DNA sequencing technique that can accurately differentiate between different fluke species. We tested these methods using faecal samples collected from cattle and sheep across the UK. The qPCR could detect small amounts of Fasciola DNA, while tremabiome was more sensitive, identifying different fluke species from as few as five eggs. Our study found that co-infections of F. hepatica and C. daubneyi are common in the UK. The approaches we have developed could be valuable tools for to improve fluke diagnosis and enable better control of this important parasitic disease.

molecular biology↗

bioRxiv2022

Genetic characterisation of the Theileria annulata cytochrome b locus and its impact on buparvaquone resistance in ruminants

Control of tropical theileriosis depends on the use of a single drug, buparvaquone, the efficacy of which is compromised by the emergence of resistance. The present study was undertaken to improve understanding of the role of mutations conferring buparvaquone resistance in Theileria annulata, and the effects of selection pressures on their emergence and spread. First, we investigated genetic characteristics of the cytochrome b locus associated with buparvaquone resistance in 10 susceptible and 7 resistant T. annulata isolates. The 129G (GGC) mutation was found in the Q01 binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q02 binding pocket. Next, we examined field isolates and identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 cattle-derived T. annulata isolates, providing evidence of positive selection pressure. Both hard and soft selective sweeps were identified, with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high frequency, implying the emergence of resistance by a single mutation. Two buffalo-derived and 11 cattle-derived isolates contained equally high frequencies of 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes, implying the emergence of resistance by pre-existing and or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting a single origin. We propose that animal migration between farms is an important factor in the spread of buparvaquone resistance in endemic regions of Pakistan. The overall outcomes will be useful in understanding how drug resistance emerges and spreads, and this information will help design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis.

microbiology↗

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↗

bioRxiv2020

Genetic models suggest single and multiple origins of dihydrofolate reductase mutations in Plasmodium vivax

Pyrimethamine was first introduced for the treatment of malaria in Asia and Africa during the early 1980s, replacing chloroquine, and has become the first line of drugs in many countries. In recent years, development of pyrimethamine resistance in Plasmodium vivax has become a barrier to effective malaria control strategies. Here, we describe the use of meta-barcoded deep amplicon sequencing technology to assess the evolutionary origin of pyrimethamine resistance by analysing the flanking region of dihydrofolate reductase (dhfr) locus. The genetic modelling suggests that 58R and 173L single mutants and 58R/117N double mutants are present on a single lineage; suggesting a single origin of these mutations. The triple mutants (57L/58R/117N, 58R/61M/117N and 58R/117N/173L) share the lineage of 58R/117N, suggesting a common origin. In contrast, the 117N mutant is present on two separate lineages suggesting that there are multiple origins of this mutation. We characterised the allele frequency of the P. vivax dhfr locus. Our results support the view that the single mutation of 117N and double mutations of 58R/117N arise commonly, whereas the single mutation of 173L and triple mutations of 57L/58R/117N, 58R/61M/117N and 58R/117N/173L are less common. Our work will help to inform mitigation strategies for pyrimethamine resistance in P. vivax.

genetics↗

bioRxiv2020

Molecular characterization of Fasciola gigantica in Punjab, Pakistan to infer the dispersal route among the neighbouring countries of the Indian subcontinent

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in millions of dollars production losses to the livestock industry. To understand the dispersal origin and the spread patterns of F. gigantica is important for preventing the disease. A total of 53 Fasciola flukes collected from buffalo and goat in the Punjab province of Pakistan, were identified as F. gigantica based on the multiplex PCR for the phosphoenolpyruvate carboxykinase (pepck) and the PCR-restriction fragment length polymorphism (RFLP) for DNA polymerase delta (pold). A significant genetic difference between F. gigantica from buffalo and goats in Pakistan was indicated by the genetic analysis of two distinct mitochondrial markers [NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1)]. Phylogenetic analysis of the seventeen nad1 haplotypes of F. gigantica from Pakistan with those in neighbouring countries of the Indian subcontinent revealed that all the haplotypes were clustered in haplogroup A. Fasciola gigantica with the eight haplotypes might be expanded in Pakistan from Indian origin, along with the migration of the domestic animals, since they were related to Indian haplotypes. In contrast, the remaining nine haplotypes were not shared with any neighbouring countries, suggesting independent origin, or possibly come from neighbouring Middle East countries. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica regarding the development of sustainable parasite control strategies.

genetics↗

bioRxiv2020

Genetic analysis confirms the presence of Dicrocoelium dendriticum in the Himalaya ranges of Pakistan.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to address a lack of knowledge of lancet flukes in the Himalaya ranges of Pakistan by characterising Dicrocoelium species collected from the Chitral valley. The morphology of 48 flukes belonging to eight host populations was examined in detail and according to published keys, they were identified as either D. dendriticum or Dicrocoelium chinensis. PCR and sequencing of fragments of ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) and NADH dehydrogenase-1 (ND-1) mitochondrial DNA from 34, 14 and 3 flukes revealed 10, 4 and 1 unique haplotypes, respectively. Single nucleotide polymorphisms in these haplotypes were used to differentiate between D. chinensis and D. dendriticum, and confirm the molecular species identity of each of the lancet flukes as D. dendriticum. Phylogenetic comparison of the D. dendriticum rDNA, COX-1 and ND-1 sequences with those from D. chinensis, Fasciola hepatica and Fasciola gigantica species was performed to assess within and between species variation and validate the use of species-specific markers for D. dendriticum. Genetic variations between D. dendriticum populations derived from different locations in the Himalaya ranges of Pakistan illustrate the potential impact of animal movements on gene flow. This work provides a proof of concept for the validation of species-specific D. dendriticum markers and is the first molecular confirmation of this parasite species from the Himalaya ranges of Pakistan. The characterisation of this parasite will allow research questions to be addressed on its ecology, biological diversity, and epidemiology.

genetics↗

bioRxiv2020

Contrasting population genetics of cattle- and buffalo- derived Theileria annulata causing tropical theileriosis

The present study was designed to improve understanding of Theileria annulata in sympatric water buffalo and cattle in the Punjab province of Pakistan. The prevalence of tropical theileriosis is high, buparvaquone resistance is widespread, and vaccine protection is poor in the field. Better understanding is, therefore, needed of the factors that influence the genetics of T. annulata populations both within its hosts and in its overall populations. Here we utilise a panel of six satellites and a mitochondrial cytochrome b marker to explore the multiplicity of T. annulata infection and patterns of emergence and spread of different parasite genotypes. Parasite materials were collected from infected animals in defined regions, where water buffalo and cattle are kept together. Our results show that T. annulata is genetically more diverse in cattle- than in water buffalo-derived populations (the mean numbers of unique satellite alleles were 13.3 and 1.8 and numbers of unique cytochrome b locus alleles were 65 and 27 in cattle- and water buffalo-derived populations, respectively). The data show a high level of genetic diversity among the individual host-derived populations (the overall heterozygosity (He) indices were 0.912 and 0.931 in cattle, and 0.874 and 0.861 in buffalo, based on satellite and cytochrome b loci, respectively). When considered in the context of high parasite transmission rates and frequent animal movements between different regions, the predominance of multiple T. annulata genotypes, with multiple introductions of infection in the hosts from which the parasite populations were derived, may have practical implications for the spread of parasite genetic adaptations; such as those conferring vaccine cross-protection against different strains affecting cattle and buffalo, or resistance to antiprotozoal drugs.

genetics↗

bioRxiv2019

Novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

Various PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have been recently developed for the first time a novel high throughput haemoprotobiome metabarcoded DNA sequencing method and applied it for the quantification of haemoprotozoan parasites (Theleria and Babesia) of livestock. Here, we describe a novel, high throughput method using an Illumina MiSeq platform to demonstrate the proportions of Plasmodium species in metabarcoded DNA samples derived from human malaria patients. Plasmodium falciparum and Plasmodium vivax positive control gDNA was used to prepare mock DNA pools of parasites to evaluate the detection threshold of the assay for each of the two species and to assess the accuracy of proportional quantification. We then applied the assay to malaria-positive human samples to show the species composition of Plasmodium communities in the Punjab province of Pakistan and in the Afghanistan-Pakistan tribal areas. The diagnostic performance of the deep amplicon sequencing method was compared to an immunochromatographic assay that is widely used in the region. Metabarcoded DNA sequencing showed better diagnostic performance, greatly increasing the estimated prevalence of Plasmodium infection. The next-generation sequencing method using metabarcoded DNA has potential applications in the diagnosis, surveillance, treatment, and control of Plasmodium infections, as well as to study the parasite biology.

genetics↗

bioRxiv2019

Genetic diversity and multiplicity of infection in Fasciola gigantica isolates of Pakistani livestock

Fasciola gigantica liver flukes are responsible for over 3 billion US dollars of production loss annually in farmed livestock and cause widespread zoonotic disease. Nevertheless, the understating of the emergence and spread of the trematode species is poor. The multiplicity of F. gigantica infection and its spread is potentially influenced by multiple factors, including the abundance of suitable intermediate hosts, climatic conditions favoring the completion of the parasites lifecycle, and translocation of infected animals or free-living parasite stages between regions. Here we describe the development of a tremabiome metabarcoding sequencing method to explore the numbers of F. gigantica genotypes per infection and patterns of parasite spread, based on genetic characteristics of the mitochondrial NADH dehydrogenase 1 (mt-ND-1) locus. We collected F. gigantica from three abattoirs in the Punjab and Balochistan provinces of Pakistan, and our results show a high level of genetic diversity in 20 F. gigantica populations derived from small and large ruminants consigned to slaughter in both provinces. This implies that F. gigantica can reproduce in its definitive hosts through meiosis involving cross- and self-breeding, as described in the closely related species, Fasciola hepatica. The genetic diversity between the 20 populations derived from different locations also illustrates the impact of animal movements on gene flow. Our results demonstrate the predominance of single haplotypes, consistent with a single introduction of F. gigantica infection in 85% of the hosts from which the parasite populations were derived. This is consistent with clonal reproduction in the intermediate snail hosts.

genetics↗

bioRxiv2019

Evidence for the F200Y (TAC) mutation conferring benzimidazole resistance in a southern USA cattle population of Haemonchus placei spreading from a single emergence

The benzimidazoles are one of the most important broad-spectrum anthelmintic drug classes for the control of parasitic nematodes of domestic animals and humans. They have been widely used in the livestock sector, particularly in small ruminants for over 40 years. This has resulted in the development and wide spread of resistance in small ruminant gastrointestinal nematode parasite species, including Haemonchus contortus. Recently, resistance to benzimidazole drugs has been reported in Haemonchus placei, but there is relatively little information on its prevalence. It is important to develop a molecular tools to identify resistance mutations in H. placei early in their development in order to understand the emergence and spread. Our previous study demonstrated the F200Y (TAC) mutation at their early stage in 6/9 H. placei populations derived from southern USA, albeit at low frequencies between 2 and 10%. The present study analysis the phylogenetics of the isotype-1 {beta}-tubulin locus to suggest that F200Y (TAC) mutation has been spread from a single emergence in H. placei; likely by the anthroprogenic movement of ruminant livestock in southern USA. Population genetic data of H. placei using a panel of microsatellite markers revealed little genetic sub-structure, consistent with a high level of gene flow in this region. Overall, these results provide clear genetic evidence for the spread of F200Y (TAC) benzimidazoles resistance mutation to multiple different locations from a single emergence in H. placei.

molecular biology↗

bioRxiv2019

Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can cause serious impact on the health of livestock and production. Multiple piroplasm species can infect a single host, but reliable molecular diagnostic tools are needed with which to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction of co-infecting species, but are similar in the animals, become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a \"haemoprotobiome\". First, four phenotypically-verified species of Theileria and Babesia were used to prepare mock pools with random amounts of the parasites and amplified with four different numbers of PCR cycles to assess sequence representation of each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The \"haemoprotobiome\" concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.

microbiology↗

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↗

bioRxiv2019

A high throughput deep amplicon sequencing method to show the emergence and spread of Calicophoron daubneyi in United Kingdom cattle herds.

The prevalence of C. daubneyi infection in the United Kingdom has increased, but despite the potential for rumen flukes to cause production loss in ruminant livestock, understanding of their emergence and spread is poor. Here we describe the development of a method to explore the multiplicity of C. daubneyi infection and patterns of the parasites emergence and spread, based on Illumina MiSeq deep sequencing of meta barcoded amplicons of a fragment of the mt-COX-1 locus. Our results show high levels of genetic diversity per infection and between populations of 10 to 47 of adult C. daubneyi, each from a total of 32 finished prime cattle consigned to slaughter from northern United Kingdom; with 18 unique mt-COX-1 haplotypes. This has implications for the adaptability of environmental and intermediate host stages of the parasite to changing climatic and animal management conditions, or of parasitic stages to exposure to anthelmintic drugs; potentially allowing for greater pathogenicity, or the development of anthelmintic resistance, respectively. Our results illustrate the impact of high levels of animal movements in the United Kingdom, whereby multiple common mt-COX-1 haplotypes were identified in 26 populations in the absence of geographical clustering of clades.

evolutionary biology↗