Dr Umer Chaudhry

Background Ivermectin is an endectocide against many parasites. Though being a macrocyclic lactone, its activity against bacteria has been less known, possibly due to the fact that micromolar concentrations at tissue levels are required to achieve a therapeutic effect. Among pathogenic bacteria of major medical significance, Staphylococcus aureus cause a number of diseases in a wide variety of hosts including humans and animals. It has been attributed as one of the most pathogenic organisms. The emergence of methicillin resistance has made the treatment of S. aureus even more difficult as it is now resistant to most of the available antibiotics. Thus, search for alternate anti-staphylococcal agents requires immediate attention. Methods Twenty-one clinical isolates of S. aureus were isolated from bovine milk collected from Lahore and Faisalabad Pakistan. Different anthelmintics including levamisole, albendazole and ivermectin were tested against S. aureus to determine their minimum inhibitory concentrations. This was followed-up by growth curve analysis, spot assay and time-kill kinetics. Results The results showed that ivermectin but not levamisole or albendazole exhibited a potent anti-staphylococcal activity at the concentrations of 6.25 and 12.5 μg/ml against two isolates. Interestingly, one of the isolate was sensitive while the other was resistant to methicillin/cefoxitin. Conclusions Our novel findings indicate that ivermectin has an anti-bacterial effect against certain S. aureus isolates. However, to comprehend why ivermectin did not inhibit the growth of all Staphylococci needs further investigation. Nevertheless, we have extended the broad range of known pharmacological effects of ivermectin. As pharmacology and toxicology of ivermectin are well known, its further development as an anti-staphylococcal agent is potentially appealing.

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 parasite’s emergence and spread, based on Illumina MiSeq deep sequencing of meta barcoded amplicons of a fragment of the cytochrome c oxidase subunit I (mt-COX-1) locus. Our results show high levels of genetic diversity in 32 C. daubneyi populations derived from finished prime cattle consigned to slaughter from northern United Kingdom. The results are consistent with a single introduction of C. daubneyi infection to some of the farms where the cattle had been grazed during their lifetime and multiple introductions to most. The 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. 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.

Parasitic infestations are continuously posing enormous threats to food security and poverty alleviation. While different drugs are proposed to minimize the impact of parasite, these come with established genetic resistance. Understanding markers of genetic resistance will streamline the process of use of alternative medication as well as assessing the affect of currently applied anthelmintic. This review briefly outlines the roles of parasites in diseases and proposes the use of different genetic markers to mitigate the emerging resistance in parasite.

Development of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples. The online version contains supplementary material available at 10.1007/s12639-021-01414-w.

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 may implicate a novel mechanism that allows resistant worms to survive treatment. 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 response to treatment in parasitic helminths.

More than 23 Trichuroidea species have been identified in ruminants in different parts of the world. Most are pathogenic, causing trichurosis. Trichuris adults of most species within this family have a predilection for the ceca, where they may cause damage to the epithelial wall. In the present study, Trichuris spp. from large intestine of goats were analysed based on morphological and molecular characteristics. Fifty adult worms (male = 25 and female = 25) were selected for morphometric and molecular analysis. Male Trichuris were distinguished by their longer spicules, typical spicule sheaths, and small spicules that were always completely covered by the spicule sheath. The presence of an uneverted vulva in the vagina distinguished female worms. We have performed the molecular characterisation of adult warms to identify as Trichuris skrjabini. Genetic comparison of T. skrjabini rDNA ITS2 sequences with those from other Trichuris spp. was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for T. skrjabini identification. This work provides the first report of this parasite species from Pakistan and validated species-specific marker of T. skrjabini that reduces the production potential of goats in the country.

Haemonchus contortus is arguably one of the most economically important and ubiquitous parasites of livestock globally and commonly involved in cases of anthelmintic resistance. Here, we performed reciprocal genetic crosses using susceptible (MHco3(ISE)) and multiple anthelmintic resistant (MHco18(UGA2004)) H. contortus isolates. Resultant admixed populations were designated MHco3/18 or MHco18/3, where the lead isolate reflects the origin of the females. Three independent filial generations were generated for each cross, which were subjected to bioassays, molecular approaches and population genetic analyses to investigate the phenotypic and genotypic inheritance of benzimidazole (BZ) resistance at each stage. A panel of microsatellite markers confirmed the success of the genetic cross as markers from both parents were seen in the F1 crosses. Egg hatch tests revealed a stark difference between the two F1 crosses with ED50 estimates for MHco18/3 being 9 times greater than those for MHco3/18. Resistance factors based on ED50 estimates ranged from 6 to 57 fold in the filial progeny compared to MHco3(ISE) parents. Molecular analysis of the F167Y and F200Y SNP markers associated with BZ resistance were analysed by pyrosequencing and MiSeq deep amplicon sequencing, which showed that MHco3/18.F1 and MHco18/3.F1 both had similar frequencies of the F200Y resistant allele (45.3% and 44.3%, respectively), whereas for F167Y, MHco18/3.F1 had a two-fold greater frequency of the resistant-allele compared to MHco3/18.F1 (18.2% and 8.8%, respectively). Comparison between pyrosequencing and MiSeq amplicon sequencing revealed that the allele frequencies derived from both methods were concordant at codon 200 (rc = 0.97), but were less comparable for codon 167 (rc = 0.55). The use of controlled reciprocal genetic crosses have revealed a potential difference in BZ resistance phenotype dependent on whether the resistant allele is paternally or maternally inherited. These findings provide new insight and prompt further investigation into the inheritance of BZ resistance in H. contortus. [Display omitted] •Reciprocal cross used to investigate benzimidazole (BZ) resistance.•Phenotypic and genotypic tools combined for analysis.•Inheritance of BZ resistance influenced by maternal &/or cytoplasmic mechanisms.•Double homozygous resistant genotypes at F167Y and F200Y detected on β−tubulin gene.

Background The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. Methods We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. Results We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. Conclusion The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.

Control of tropical theileriosis, caused by the apicomplexan Theileria annulata, 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 T. 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 12 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 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. [Display omitted] •First link between genetic mutations and buparvaquone drug resistance in T. annulata.•The data will help to design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis.

It is important to understand how anthelmintic drug resistance mutations arise and spread in order to determine appropriate mitigation strategies. We hypothesised that a molecular genetic study of Haemonchus contortus in southern India, a region where resistance may be less advanced than in western Europe and North America, might provide some important insights into the origin and spread of anthelmintic resistance. The F200Y (TAC) isotype-1 β-tubulin benzimidazole resistance mutation is common in H. contortus throughout the world and the F167Y (TAC) and E198A (GCA) mutations, although less common, have been reported in a number of different countries. We have investigated the haplotypic diversity and phylogenetic relationship of isotype-1 β-tubulin benzimidazole resistance alleles for 23 H. contortus populations from small ruminants across southern India. The F200Y (TAC) mutation was most common, being detected in 18/23 populations at frequencies between 9% and 84% and the E198A (GCA) mutation was also detected in 8/23 populations at frequencies between 8% and 18%. The F167Y (TAC) mutation was not detected in any of the 23 populations. Phylogenetic haplotype network analysis suggested that the F200Y (TAC) mutation has arisen multiple independent times in the region with at least three independent origins of resistance alleles across the populations surveyed. In contrast, the E198A (GCA) mutation was present on a single haplotype which, given the high level of haplotypic diversity of the susceptible alleles in the region, suggests this particular mutation has spread from a single origin, likely by anthropogenic animal movement. Population genetic analysis of 12 of the H. contortus populations, using a panel of eight microsatellite markers, revealed extremely low genetic differentiation between populations, consistent with the hypothesis of high gene flow among sites. Additionally, there was no significant genetic differentiation between H. contortus taken from sheep and goats which is consistent with H. contortus populations being freely shared between these two different hosts. Overall, we believe these results provide the first clear genetic evidence for the spread of an anthelmintic resistance mutation to multiple different locations from a single origin.

The development of antibiotic resistance in bacteria is a major public health threat. Infection rates of resistant pathogens continue to rise against nearly all antimicrobials, which has led to development of different strategies to combat the antimicrobial resistance. In this review, we discuss how the newly popular CRISPR-cas system has been applied to combat antibiotic resistance in both extracellular and intracellular pathogens. We also review a recently developed method in which nano-size CRISPR complex was used without any phage to target the mecA gene. However, there is still challenge to practice these methods in field against emerging antimicrobial resistant pathogens.

Among helminthes, the trichostrongyloidea consists of most significant parasites of ruminants, which are posing significant threats to livestock productivity, health and well-being. This brief commentary provides an overview of this important group of parasite to showcase the crucial roles these play in hosts.

Haemonchus contortus is a globally distributed and economically important gastrointestinal pathogen of small ruminants and has become a key nematode model for studying anthelmintic resistance and other parasite-specific traits among a wider group of parasites including major human pathogens. Here, 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 for the MHco3(ISE).N1 isolate. We show a remarkable pattern of conservation of chromosome content with Caenorhabditis elegans, but almost no conservation of gene order. Short and long-read transcriptome sequencing allowed us to define coordinated transcriptional regulation throughout the parasite’s life cycle and refine our understanding of cis- and trans-splicing. Finally, we provide a comprehensive picture of chromosome-wide genetic diversity both within a single isolate and globally. These data provide a high-quality comparison for understanding the evolution and genomics of Caenorhabditis and other nematodes and extend the experimental tractability of this model parasitic nematode in understanding helminth biology, drug discovery and vaccine development, as well as important adaptive traits such as drug resistance.

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 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. The different mock pools demonstrate the accurate detection ability and to show the proportions of each of the species being present. 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. The deep amplicon sequencing showed that P. vivax was present in 69.8%, P. falciparum in 29.5% and mixed infection in 0.7% patients examined. The immunochromatographic assay showed that P. vivax was present in 65.6%, P. falciparum in 27.4%, mixed infection 0.7% patients and 6.32% malaria-positive cases were negative in immunochromatographic assay, but positive in the deep amplicon sequencing. Overall, metabarcoded DNA sequencing demonstrates 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. •We reported for the first time the development of Haemoprotobiome technology to quantify the P. falciparum and P. vivax.•P. falciparum and P. vivax mock pools demonstrate the accurate detection ability and to show the proportions of each of the species.•Haemoprotobiome demonstrates better diagnostic performance than immunochromatographic assay.

Purpose Dicrocoeliosis can be an important cause of production loss in ruminants due to the cost of liver condemnation at slaughter. The aim of the present study was to determine the prevalence of Dicrocoelium infection and to predict the ecological niches and climatic variables that support dicrocoeliosis in the Himalayan ranges of Pakistan. Methods and Results Dicrocoelium was detected in 33 of 381 liver samples and 238 of 6060 blood samples taken from sheep and goat herds in the area. The prevalence of dicrocoeliosis was higher in sheep than in goats and highest in females aged more than 3 years. An environmental risk map was created to predict active zones of transmission and showed the highest probability values in central parts of the Chitral district in the northwest of Pakistan. Climatic variables of the mean monthly diurnal temperature range (Bio2), annual precipitation (Bio12), and normalised difference vegetation index (NDVI) were found to be significantly (p 

Our current understanding of differences in the epidemiology of gastrointestinal nematode (GIN) species in co-grazed sheep and goats is inadequate with reference to the development of sustainable control strategies. The next-generation metabarcoding sequencing method referred to as the ‘nemabiome’ allows some of these differences to be explored to describe the intensity of co-infecting GIN species. We applied this platform to study sheep and goats that were co-grazed on Guinea grass pasture in northeastern Brazil. Co-grazed goats and sheep were treated with a monepantel anthelmintic, then exposed to the same gastrointestinal nematode species. Overall, there were differences in the prevalence of GIN species identified in the sheep and goats; Trichostrongylus colubriformis and Teladorsagia circumcincta predominated in goat kids, while Haemonchus contortus predominated in adult does, ewes and lambs once burdens became re-established after anthelmintic treatment. Description of the pattern of re-infection following anthelmintic treatment was prevented by the unpredicted poor efficacy of 2.5 mg/kg and 5 mg/kg, respectively, of monepantel against O. columbianum and T. circumcincta in lambs, and T. circumcincta adult does. Differences in drug efficacy between host age and species groups may be important when considering sustainable GIN control strategies for co-grazed animals. The aggregated FECs of the adult does and goat kids representing re-established GIN burdens, were higher than those of the co-grazed adult ewes and lambs. This implies that there are inherent differences in GIN species adaptation to the two naïve small ruminant host species, and shows the need for better understanding of the factors giving rise to this situation associated with exposure to infective larvae and host responses. At the start of the study, the adult does were co-infected with several GIN species, with the highest intensity of T. circumcincta, contrasting with the situation in the adult ewes, in which H. contortus predominated. However, once burdens became re-established after treatment, H. contortus predominated in both adult does and ewes. This demonstrates the potential for host burdens of H. contortus to establish and predominate after anthelmintic treatment when burdens of co-infecting GIN species are low.

Ivermectin (IVM) is a versatile drug used against many microorganisms. Staphylococcus aureus is one of the most devastating microorganisms. IVM sensitive and resistant S. aureus strains were recently reported. However, the underlying molecular mechanisms of resistance are unknown. Clinical isolates of S. aureus were used for determination of the sensitivities against IVM by growth curve analysis and time-kill kinetics. Then, proteomic, and biochemical approaches were applied to investigate the possible mechanisms of resistance. Proteomic results showed a total of 1849 proteins in the dataset for both strains, 425 unique proteins in strain O9 (IVM sensitive), and 354 unique proteins in strain O20 (IVM resistant). Eight proteins with transport functions were differentially expressed in the IVM resistant strain. Among them, three efflux pumps (mepA, emrB, and swrC) were confirmed by qPCR. The IVM resistant S. aureus may overexpress these proteins as a key resistance determinant. Further experiments are required to confirm the exact mechanistic relationship. Nevertheless, the possibility of blocking these transporters to reverse or delay the onset of resistance and reduce selection pressure is potentially appealing.

Fasciola gigantica is considered to be a major pathogen causing fasciolosis in the Indian subcontinent, resulting in production losses of millions of dollars in the livestock industry. Understading the dispersal origin and the patterns of spread of F. gigantica is important. A total of 53 Fasciola flukes collected from buffaloes and goats in Punjab, Pakistan between 2017 and 2018 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 buffaloes and goats was indicated by the genetic analyses of mitochondrial markers, NADH dehydrogenase subunit 1 (nad1) and cyto-chrome 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 identified in Pakistan 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, probably from neighbouring Middle East countries. However, cautious interpretation is required due to the very limited samples size of this study. Our study provides a proof of concept for a method that could be used to investigate the epidemiology of F. gigantica.

More than 70 species of the family Paramphistomatidae, have been identified in ruminants in different parts of the world. Most are pathogenic, causing amphistomosis. Adult flukes of this Family have a predilection for the rumen, liver or bile duct of ruminants where they may cause damage to the epithelium. Identification of adult paramphistomes to the species level based on morphology alone requires specialized knowledge, whereas, molecular genetic marker analysis is more precise and transferable. In the present study, we performed both morphological and molecular characterization of fifteen adult flukes collected from the liver of domesticated buffalo in the Punjab province of Pakistan. The morphology of five of these flukes was examined in detail and on this basis these were identified as either Explanatum explanatum or Explanatum bathycotyle. PCR and sequencing of the ITS-2 rDNA region from these 5 flukes, plus 10 others, revealed a single haplotype in all cases. This differed by just a single nucleotide polymorphism from a previously described E. explanatum ITS-2 rDNA sequence. Phylogenetic comparison of these E. explanatum ITS2-rDNA sequences with those from other Paramphistomatidae, Fasciola and Dicrocoelium species was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for E. explanatum. This work provides a validated species-specific marker of E. explanatum and the first report of this parasite species from Pakistan.

There is a need for improved methods for the study of the impacts of climatic and livestock management change on the epidemiology of production-limiting helminth parasitic diseases. In this study we report the application of molecular methods to describe the natural history of the small lancet fluke, Dicrocoelium dendriticum on Machair pastures on the Inner Hebridean Isle of Coll. Our results build upon those of the only previous historic field study of D. dendriticum in the British Isles that had been undertaken on the same study site. We demonstrate the value of combining conventional parasitological methods with PCR amplification of a mitochondrial DNA fragment for the detection of D. dendriticum in ants and snails, and PCR amplification of ITS2 and 28S ribosomal DNA fragments to support the species identity of the intermediate hosts, to improving understanding of the epidemiology of D. dendriticum. We report the presence of D. dendriticum infection in cattle, sheep and rabbits grazing on Machair pastures. D. dendriticum infection was identified in a high percentage of the snails, identified as Cochlicella acuta and Cernuella virgata, and in a high percentage of Formica fusca and Myrmica ruginoides ants that were collected from, or clinging to, the tops of flowers. We have identified the involvement of different intermediate host species and higher prevalences of snail and ant infection than previously reported, in part reflecting differences between the sensitivity and specificity of morphological and molecular speciation methods. Overall, our results highlight the complex life history of dicrocoeliosis and illustrate the parasite’s generalist host strategy that confers potential to exploit new niches created by climatic change or grazing management for habitat conservation.

Background The impact of drug selection pressure on the overall genetic diversity of parasitic nematode populations in the field is poorly understood. In this study, we address this issue for the small ruminant parasite Haemonchus contortus in the Punjab, Pakistan. This region provides an opportunity to compare H. contortus populations that have been subjected to a prolonged period of frequent benzimidazole drug treatments on government farms with parasite populations that have been exposed to little or no drug treatment in neighbouring pastoral herds. Methods Adult H. contortus worms were collected from the abomasa of small ruminants from three government farms frequently using benzimidazole drugs, and closed to animal movement, for over 30 years and also from from eighteen pastoral herds subject to minimal drug selection. The frequency of three known benzimidazole resistance associated mutations was determined in each parasite population. For the seven parasite populations in which resistance mutations were found, the diversity, geographical distribution and phylogenetic relationships of isotype-1 β-tubulin benzimidazole resistance haplotypes were determined. In addition, the genetic diversity of the parasite populations on the three government farms were compared with those from four pastoral herds. Results The F200Y (TAC) resistance mutation was present at a very high frequency in H. contortus populations from government herds, but not from pastoral herds, consistent with their respective drug selection histories. Population genetic analysis, using a panel of microsatellite markers, revealed that there was little genetic differentiation among the parasite populations with no significant difference in the overall genetic diversity between government and pastoral herds. In addition, sequence analysis of the isotype-1 β-tubulin locus revealed multiple F200Y (TAC) haplotypes demonstrating soft selective sweeps even in government herds with little or no contemporary parasite migration. Conclusions The results suggest that, although the frequent drug treatment used on government farms has selected for a high frequency of benzimidazole resistance mutations, there has been little or no reduction in the overall genetic diversity of the selected parasite populations.

Since its first report in 1942, peste-des-petits-ruminants virus (PPRV) has caused several epidemics in a wide range of susceptible hosts around the world. In the last 30 years, the evidence of natural and experimental infections and virus isolation were reported from novel but unusual hosts such as camel, cattle, buffalo, dogs, Asiatic lion and pigs. In addition, PPRV in a potential vector, biting midges (Culicoides imicola), has been reported. Either presented as clinical and/or subclinical infections, the presence of the virus in an extended range of susceptible hosts highlights the cross-species transmission and supports the hypothesis of an endemic circulation of PPRV among susceptible hosts. However, the potential role of large ruminants, camels and unusual hosts for PPRV epidemiology is still obscure. Therefore, there is a need for molecular and epidemiological investigations of the disease among usual and unusual hosts to achieve the goals of disease control and eradication programmes initiated by national and international organisations, such as the FAO and OIE. This review is the first to summarise the scattered data on PPR in large ruminants, camels and unusual hosts to obtain the global scientific communities' attention for further research on epidemiological aspects, not only in its native hosts, but also in large ruminants, camels and other unusual hosts.

Foot and mouth disease (FMD) is a viral disease that affects predominantly cloven-footed animal species within the order Artiodactyla. The potential of the virus to transmit, maintain and circulate itself across a wide range of susceptible hosts, including both domestic and wild ungulates, remains a single major obstacle in an effective eradication of disease worldwide, particularly in disease-endemic settings. Hence, a better understanding of virus transmission dynamics is very much crucial for an efficient control of the disease, particularly at places or regions where wildlife and livestock rearing co-exists. Both OIE and FAO have jointly launched the FMD-control program as FMD-Progressive Control Pathway (PCP) in various disease-endemic developing countries. Nevertheless, the propensity of virus to inter- and intra-species transmission may be a possible constraint in disease control and, hence, its subsequent eradication in such countries. Other than this, cross-species transmission, among domestic and wild ungulates living in close proximities, can undermine the conservation efforts for endangered species. We reviewed and summarized the so-far available information about inter- and intra-species disease transmission, and its impact on wildlife populations to better comprehend disease epidemiology and substantiate efforts for eventual disease eradication across the globe, particularly in settings where the disease is endemic.

•F200Y (TAC) benzimidazole resistance mutation in H. placei was found on a single haplotype in multiple populations across Punjab province.•F200Y (TAC) mutations was found on multiple divergent haplotypes in H. contortus.•The P167 (TAC) and E198A (GCA) mutations were not detected in any H. contortus and H. placei populations. Benzimidazoles have been intensively (for over 40 years) used in the livestock sector, particularly in small ruminants. This has been led to the widespread emergence of resistance in a number of small ruminant parasite species, especially Haemonchus contortus. In many countries benzimidazole resistance has severely compromised the control of H. contortus in small ruminants; but there is a little information on benzimidazole resistance in H. contortus infecting buffalo and cattle. Resistance to benzimidazoles have also been reported in the large ruminant parasite, Haemonchus placei, but again there is relatively little information on its prevalence. Hence it is very important to understand how resistance-conferring mutations emerge and spread in both parasites in buffalo and cattle hosts in order to develop approaches for the recognition of the problem at an early stage of its development. The present study suggests that the F200Y (TAC) mutation is common in H. contortus, being detected in 5/7 populations at frequencies between 7 and 57%. Furthermore, 6/10 H. placei populations contained the F200Y (TAC) mutation, albeit at low frequencies of between 0.4 and 5%. The phylogenetic analysis suggests that the F200Y (TAC) mutation in H. contortus has emerged on multiple occasions in the region, with at least three independent emergences across the populations. In contrast, the F200Y (TAC) resistance-conferring mutation in H. placei is only seen on a single haplotype. A high level frequency of the resistance haplotypes in the region, suggests that the unique resistance conferring-mutation has spread from a single emergence; likely by anthropogenic animal movement. Overall, these results provide the first clear genetic evidence for the spread of benzimidazole resistance-conferring mutations to multiple different locations from a single emergence in H. placei; while being consistent with previous small ruminant-based observations of multiple emergence of resistance mutations in H. contortus.

•58R and 173L single mutants and 58R/117N double mutants are present on a single lineage; suggesting a single origin of these mutations.•117N mutant is present on two separate lineages suggesting that there are multiple origins of this mutation.•Single mutation of 117N and double mutations of 58R/117N arise commonly, whereas the single mutation of 173L are less common. 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.

Toxoplasma gondii is a major foodborne pathogen capable of infecting all warm-blooded animals, including humans. Although oocyst-associated toxoplasmosis outbreaks have been documented, the relevance of the environmental transmission route remains poorly investigated. Thus, we carried out an extensive systematic review on T. gondii oocyst contamination of soil, water, fresh produce, and mollusk bivalves, following the PRISMA guidelines. Studies published up to the end of 2020 were searched for in public databases and screened. The reference sections of the selected articles were examined to identify additional studies. A total of 102 out of 3201 articles were selected: 34 articles focused on soil, 40 focused on water, 23 focused on fresh produce (vegetables/fruits), and 21 focused on bivalve mollusks. Toxoplasma gondii oocysts were found in all matrices worldwide, with detection rates ranging from 0.09% (1/1109) to 100% (8/8) using bioassay or PCR-based detection methods. There was a high heterogeneity (I-2 = 98.9%), which was influenced by both the sampling strategy (e.g., sampling site and sample type, sample composition, sample origin, season, number of samples, cat presence) and methodology (recovery and detection methods). Harmonized approaches are needed for the detection of T. gondii in different environmental matrices in order to obtain robust and comparable results.

•We reported for the first time the development of haemoprotobiome technology to quantify the trypanosoma species.•Results are the proof of concept for the use of this method in disease surveillance programmes in an endemic regions. The World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have developed strategies to control trypanosomiasis in humans and livestock in endemic areas. These require a better understanding of the distribution of different Trypanosoma species and improved predictions of where they might appear in the future, based on accurate diagnosis and robust surveillance systems. Here, we describe a metabarcoding deep amplicon sequencing method to identify and determine the Trypanosoma species in co-infecting communities. First, four morphological verified Trypanosoma species (T. brucei, T. congolense, T. vivax and T. theileri) were used to prepare test DNA pools derived from different numbers of parasites to evaluate the method's detection threshold for each of the four species and to assess the accuracy of their proportional quantification. Having demonstrated the accurate determination of species composition in Trypanosoma communities, the method was applied to determine its detection threshold using blood samples collected from cattle with confirmed Trypanosoma infections based on a PCR assay. Each sample showed a different Trypanosoma species composition based on the proportion of MiSeq reads. Finally, we applied the assay to field samples to develop new insight into the species composition of Trypanosoma communities in cattle, camels, buffalo, horses, sheep, and goat in endemically infected regions of Pakistan. We confirmed that Trypanosoma evansi is the major species in Pakistan and for the first time showed the presence of Trypanosoma theileri. The metabarcoding deep amplicon sequencing method and bioinformatics pathway have several potential applications in animal and human research, including evaluation of drug treatment responses, understanding of the emergence and spread of drug resistance, and description of species interactions during co-infections and determination of host and geographic distribution of trypanosomiasis in humans and livestock.

Understanding the composition of gastrointestinal nematode communities may help to mitigate or exploit parasite adaptations within their host. We have used nemabiome deep amplicon sequencing of internal transcribed spacer-2 (ITS-2) ribosomal DNA to describe the temporal and host species composition of gastrointestinal nematode communities following sampling of six Scottish ponies across 57 months. In the absence of parasite control, each horse showed seasonal trends of increases and decreases in faecal egg counts, consistent with the epidemiology of equine strongylid parasites, however, the composition of parasites within individuals changed over time. Sixteen presumptive strongylid species were identified in each of the horses, 13 of which were distributed in a complex clade together with small numbers of amplicon sequences which could not be classified beyond the Cyathostominae subfamily level. Egg shedding of seven trichostrongylid species, which had previously been identified in co-grazed Soay sheep, was identified during the early spring. Faecal egg counts and the percentage of amplicon sequences assigned to each gastrointestinal nematode species were combined to describe their relative abundance across both host and time. Significant differences in species diversity between horses and between months were observed, being greatest from March to May and least from October to December. The magnitude of the individual horse effect varied between months and, conversely, the magnitude of the seasonal effect varied between individual horses. The most abundant gastrointestinal nematode in each of the horses was Cylicostephanus longibursatus (46.6% overall), while the abundance of the other strongylid species varied between horses and relative to each other. Patent C. longibursatus infections over the winter months might represent a genetic adaptation towards longer adult worm survival, or a lower rate of developmental arrest in the autumn. This study provides insight into highly complex phylogenetic relationships between closely related cyathostomin species; and describes the dynamics of egg shedding and pasture contamination of co-infecting equine gastrointestinal nematode communities. The results could be applied to determine how climatic and management factors affect the equilibrium between hosts and their parasites, and to inform the development of sustainable gastrointestinal nematode control strategies for different host species.

Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds, especially those from Africa for which genomic resources are particularly limited, despite their disproportionate importance to the continent’s economies. To mitigate this issue, we have generated assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into a graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1 Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research. Cattle reference genomes are valuable resources but are currently heavily biased towards European breeds. Here the authors integrate assemblies for African breeds into a more representative cattle graph genome capturing global breed diversity.

Canine parvoviruses (CPV) exist as antigenic variants with varying frequencies and genetic variabilities across the globe. Given the endemicity and high prevalence in Pakistan, we characterized the CPVs originating from dogs-population to elucidate viral diversity and evolution. Fecal samples from clinically diseased pups (n = 17) of different breeds and age (2-6 months) were processed for hemagglutination assay (HA), and later for partial amplification of VP2 gene sequence and amino acid analysis. A total of 11 samples (64.71%) were found positive both in hemagglutination and PCR assays. Phylogenetic and evolutionary analysis demonstrated higher genetic heterogeneity in studied strains and constituted seven clusters within the CPV-2a group, however, they shared high level of identity with Chinese strains. Further studies are necessary to elucidate genetic analysis and epidemiology of CPV variants across a wide geographical area of the country.

Since the first report of infectious bursal disease in Pakistan in 1987, outbreaks have been common even in vaccinated flocks. Despite appropriate administration of vaccines, concerns arise if the circulating strains are different from the ones used in the vaccine. Here, we sequenced the hypervariable region (HVR) of the VP2 gene of circulating strains of infectious bursal disease virus (IBDV) originating from outbreaks (n = 4) in broiler flocks in Pakistan. Nucleotide sequencing followed by phylogeny and deduced amino acid sequence analysis showed the circulating strains to be very virulent (vv) and identified characteristic residues at position 222 (A), 242 (I), 256 (I), 294 (I) and 299 (S). In addition, a substitution at positions 221 (Q→H) was found to be exclusive to Pakistani strains in our analysis, although a larger dataset is required to confirm this finding. Compared to vaccine strains that are commonly used in Pakistan, substitution mutations were found at key amino acid positions in VP2 that may be responsible for potential changes in neutralization epitopes and vaccine failure.

The large stomach worm, Haemonchus, commonly known as the barber’s pole worm, is a blood sucking nematode found in the abomasa of small and large ruminants. Allele-specific amplification of the rDNA internal transcribed spacer-2 (ITS-2) sequences was performed from the total of 78 individual adult worms to screen Haemonchus placei at species level, which is the significant diagnostic tool to identify this major economically important species. Further full sequences analysis of the ITS-2 region revealed that there are 4 sites shows intraspecific variations at position 65, 111, 125 and 148. For instance this study is the first documented report of intraspecific genetic variations in the rDNA ITS-2 sequences of H. placei from cattle in Pakistan and the results shows that H. placei is genetically different from the isolates studied previously. However, detailed and large size samples strategy will be required to identify the co-infection and interspecies hybridization between H. placei and Haemonchus contortus in cattle.

Fascioliasis is an important disease affecting livestock, with great costs to producers worldwide. It has also become a serious issue for human populations in some endemic areas as an emerging zoonotic infection. There are two Fasciola species of liver fluke responsible for this disease, which occur worldwide, Fasciola hepatica and Fasciola gigantica. Identifying these two species on the basis of adult or egg morphology requires specialist knowledge due to the similarity of characters, and may misidentify putative intermediate or hybrid forms. In this study we sequenced the internal transcribed spacer 2 (ITS-2) rDNA of liver flukes collected from multiple species of hosts from seven localities in the Punjab and Baluchistan provinces of Pakistan, to determine the distribution of these two species. All 46 flukes processed in this study, collected from seven sites, showed the rDNA ITS-2 genotype corresponding to F. gigantica, contradicting previous reports, based on adult and egg morphology, that both species are present in Pakistan, with F. hepatica being the more common.

Haemonchus populations were collected from cattle from mid-western and eastern Southern US (four and six populations, respectively) to determine the relative prevalence of Haemonchus contortus and Haemonchus placei and the frequency of the three isotype-1 β-tubulin polymorphisms associated with benzimidazole resistance. A minimum of 32 individual adult worms were genotyped at position 24 of the rDNA ITS-2 for each population to determine species identity (296 worms in total). One population from Georgia was identified as 100% H. contortus with the remaining nine populations identified as 100% H. placei. For the H. contortus population, 29 out of 32 worms carried the P200Y (TAC) isotype-1 β-tubulin and 2 out of 32 worms carried the P167Y (TAC) benzimidazole resistance associated polymorphisms respectively. For H. placei, six out of the nine populations contained the P200Y (TAC) isotype-1 β-tubulin benzimidazole resistance associated polymorphism at low frequency (between 1.6% and 9.4%) with no resistance associated polymorphisms being identified at the P198 and P167 codons. This is the first report of the P200Y (TAC) isotype-1 β-tubulin benzimidazole resistance associated polymorphism in H. placei. The presence of this mutation in multiple independent H. placei populations indicates the risk of resistance emerging in this parasite should benzimidazoles be intensively used for parasite control in US cattle.

Toxoplasma gondii is an intracellular parasite, which infects human and animals by ingestion of tissue cyst, raw or undercooked meat or oocyst from soil, vegetables, fruits, water, soil and food contaminated by cat faeces or by transmission through the placenta, milk and blood transfusion. Seropositivity levels vary widely among different regions of the globe and according to sociocultural habits, geographic factors; climate and transmission routes and typically rise with age. In view of the worldwide importance of T. gondii, a study was conducted to determine the prevalence of T. gondii antibody in camels by using Toxoplasma Latex Test Kit. The overall prevalence of T. gondii infection in camels was recorded as 10%. Two camels were found seropositive at 1:16 dilution showing residual or nonspecific immunity, five camels were found seropositive at 1:128 showing acquired or evolving immunity, whereas three camels were positive at antibody titre of 1:256 giving an evidence of present infection. It was also noted that seropositivity of T. gondii in camels was higher in age group from 6–10 years; infection was higher in female camels having abortion history.

Coronaviruses (CoVs) are enveloped, positive sense, single-stranded RNA viruses. The viruses have adapted to infect a large number of animal species, ranging from bats to camels. At present, seven CoVs infect humans, of which Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for causing the Coronavirus Disease 2019 (COVID-19) in humans. Since its emergence in late 2019, SARS-CoV-2 has spread rapidly across the globe. Healthcare systems around the globe have been stretched beyond their limits posing new challenges to emergency healthcare services and critical care. The outbreak continues to jeopardize human health, social life and economy. All known human CoVs have zoonotic origins. Recent detection of SARS-CoV-2 in pet, zoo and certain farm animals has highlighted its potential for reverse zoonosis. This scenario is particularly alarming, since these animals could be potential reservoirs for secondary zoonotic infections. In this article, we highlight interspecies SARS-CoV-2 infections and focus on the reverse zoonotic potential of this virus. We also emphasize the importance of potential secondary zoonotic events and the One-Health and One-World approach to tackle such future pandemics.

Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can seriously impact on the health of livestock and production. Piroplasms of multiple species can be present in a single host, but reliable molecular diagnostic tools are needed in order to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction with co-infecting species, but are similar in that infected 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 DNA pools with random numbers of the parasites amplified by four different numbers of PCR cycles to assess sequence representation for 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.

Anthelmintic resistance is a threat to global food security. In order to alleviate the selection pressure for resistance and maintain drug efficacy, management strategies increasingly aim to preserve a proportion of the parasite population in ‘refugia’, unexposed to treatment. While persuasive in its logic, and widely advocated as best practice, evidence for the ability of refugia-based approaches to slow the development of drug resistance in parasitic helminths is currently limited. Moreover, the conditions needed for refugia to work, or how transferable those are between parasite-host systems, are not known. This review, born of an international workshop, seeks to deconstruct the concept of refugia and examine its assumptions and applicability in different situations. We conclude that factors potentially important to refugia, such as the fitness cost of drug resistance, the degree of mixing between parasite sub-populations selected through treatment or not, and the impact of parasite life-history, genetics and environment on the population dynamics of resistance, vary widely between systems. The success of attempts to generate refugia to limit anthelmintic drug resistance are therefore likely to be highly dependent on the system in hand. Additional research is needed on the concept of refugia and the underlying principles for its application across systems, as well as empirical studies within systems that prove and optimise its usefulness. [Display omitted] •Refugia is currently recommended for strategic control of AR in GI nematodes of livestock.•Conditions required for refugia-based control may differ between parasite systems.•An improved understanding of the parameters influencing refugia-based control is required.

Gastrointestinal nematodes (GIN) are a serious concern for sheep producers worldwide. However, there is a paucity of evidence describing the epidemiology of GIN on modern UK sheep farms. The aim of this paper was to understand whether expected seasonal variations of infection are still found in ewes and lambs under varying management strategies in temperate climates. Faecal egg counts (FEC) were conducted on freshly voided samples collected from groups of ewes and lambs every third week for twelve months on three farms in southeast Scotland. The patterns of egg output have been described here in relation to management practices undertaken on the farms. Despite changes in farming practice and climatic conditions, the findings complement historical studies detailing the epidemiology of GIN. Findings include a periparturient rise in ewe FEC on two of the farms, while lambing time treatment appeared to suppress this on the third farm. On the same two farms lamb FEC increased during the summer, reaching a peak in the autumn. The work also highlights how the ad hoc use of anthelmintics does little to impact these patterns.

Newcastle disease (ND), caused by virulent 1 (AAvV 1), affects a wide range of avian species worldwide. Recently, several AAvVs of diverse genotypes have emerged with varying genomic and residue substitutions, and subsequent clinical impact on susceptible avian species. We assessed the clinico-pathological influence of two different AAvV 1 pathotypes [wild bird originated-velogenic strain (sub-genotype VIIi, MF437287) and feral pigeon originated-mesogenic strain (sub-genotype VIm, KU885949)] in commercial broiler chickens and pigeons. The velogenic strain caused 100% mortality in both avian species while the mesogenic strain caused 0% and 30% mortality in chickens and pigeons, respectively. Both strains showed tissue tropism for multiple tissues including visceral organs; however, minor variances were observed according to host and pathotype. The observed gross and microscopic lesions were typical of AAvV 1 infection. Utilizing oropharyngeal and cloacal swabs, a comparable pattern of viral shedding was observed for both strains from each of the infected individuals of both avian species. The study concludes a varying susceptibility of chickens and pigeons to different wild bird-originated AAvV 1 pathotypes and, therefore, suggests continuous monitoring and surveillance of currently prevailing strains for effective control of the disease worldwide, particularly in disease-endemic countries.

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 confirm the species identity of Dicrocoeliid flukes collected from the Chitral valley in the Himalayan ranges of Pakistan. The morphology of 48 flukes belonging to eight host populations was examined; but overlapping traits prevented accurate species designation. Phylogenetic comparison of published D. dendriticum ribosomal cistron DNA, and cytochrome oxidase-1 (COX-1) mitochondrial DNA sequences with those from D. chinensis was performed to assess within and between species variation and re-affirm the use of species-specific single nucleotide polymorphism markers. PCR and sequencing of 34 corresponding fragments of ribosomal DNA and 14 corresponding fragments of mitochondrial DNA from the Chitral valley flukes, revealed 10 and 4 unique haplotypes, respectively. These confirmed for the first time the molecular species identity of Pakistani lancet liver flukes as D. dendriticum. This work provides a preliminary illustration of a phylogenetic approach that could be developed to study the ecology, biological diversity, and epidemiology of Dicrocoeliid lancet flukes when they are identified in new settings. •First molecular confirmation of Dicrocoelium dendriticum in Himalayan Pakistan.•Use of ribosomal and mitochondrial DNA phylogenetic markers.•Demonstration of the complementary value of morphological and molecular speciation methods for Dicrocoeliid flukes.

A study was designed to improve understanding of the genetics of Theileria annulata populations in sympatric cattle and Asian buffalo (Bubalus bubalus). The study was undertaken in the Punjab province of Pakistan, where the prevalence of tropical theileriosis is high. Parasite materials were collected from infected animals in defined regions, where cattle and Asian buffalo are kept together. Six satellite DNA markers and a mitochondrial cytochrome b marker were used to explore the multiplicity of T. annulata infection and patterns of emergence and spread of different parasite genotypes. The results show differences in the numbers of unique satellite locus alleles, suggesting that T. annulata is genetically more diverse in cattle- than in buffalo-derived populations. Heterozygosity (He) indices based on satellite and cytochrome b loci data show high levels of genetic diversity among the cattle- and buffalo-derived T. annulata populations. When considered in the context of high parasite transmission rates and frequent animal movements between different regions, the predominance of multiple T. annulata genotypes and multiple introductions of infection may have practical implications for the spread of parasite genetic adaptations; such as those conferring vaccine cross-protection against different strains affecting cattle and Asian buffalo, or resistance to antiprotozoal drugs.

Fasciola spp. are responsible for over 3 billion US dollars of production loss annually in livestock and cause widespread zoonotic disease. Nevertheless, 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 favouring the completion of the parasite's 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. [Display omitted] •To confirm the species identity of recovered Fasciola spp.•To identify the presence of single or multiple genotypes per infection (multiplicity of infection)•Demonstrate the spread of F. gigantica mt-ND-1 haplotypes

A recurrent mite infestation affecting a room used to inspect fabric in a UK textile mill was investigated to allay concerns of any potential health risks to factory staff, and to inform the unknown risk of downgrading of the product. The approach integrated conventional morphological examination of adult female mites by referring to published identification keys, with molecular speciation based on amplification of a 16S ribosomal DNA fragment. The methods enabled the mites to be unambiguously identified as Dermanyssus gallinae ‘special lineage L1’. Subsequent investigations showed the source of infestation to be pigeons nesting in the air ducts, with the gamasid mites moving into the room once the young birds had fledged. This is the first report of D. gallinae ‘special lineage L1’ in northern Europe. Previous reports of nosocominal gamasoidosis caused by D. gallinae ‘special lineage L1’ originating from feral pigeon populations have been from southern Europe. Confirmation of the mite identity was important in allowing the mill to take remedial and preventive action. In this clinical communication, we provide images of the key morphological features used to identify D. gallinae and describe a molecular protocol to confirm ‘special lineage L1’. •Investigation of mite infestation in a textile factory•Morphological keys to identify poultry red mites•Molecular speciation of Dermanyssus gallinae and confirmation of ‘special lineage L1’•First report of D. gallinae ‘special lineage L1’ in northern Europe•Risk of zoonotic gamasoidosis from pigeons nesting in air ducts

Internal parasites are a major concern in livestock production because they can impact the health and well-being of animals clinically and subclinically, and ultimately cause significant production loss. Among these internal parasites are nematodes, tapeworms, flukes, and coccidian protozoans. This review focuses on the diagnostic tests that are routinely performed by veterinarians and diagnostic laboratories, but also highlights recently developed tools that may improve diagnostic capabilities, including molecular and immunodiagnostic tests. Overall, diagnostic tests for parasites of livestock are an integral part of health management practices, and for assessing individual animal and herd health.

An extended range of host susceptibility including camel has been evidenced for some of the important veterinary and public health pathogens, such as brucellosis, peste des petits ruminants (PPR) and bluetongue (BT). However, in disease endemic settings across many parts of the globe, most of the disease control interventions accounts for small and large ruminants, whereas unusual hosts and/or natural reservoirs, such as camels, remain neglected for disease control measures including routine vaccination. Such a policy drawback not only plays an important role in disease epizootiology particularly in settings where disease is endemic, but also serves an obstacle in disease control and subsequent eradication in future. With this background, using pre-validated ELISA and molecular assays [multiplex PCR, reverse transcriptase (RT)-PCR and real-time (rt)-PCR], we conducted a large-scale pathogen- and antibody-based surveillance for brucellosis, peste des petits ruminants and bluetongue in camel population (n = 992) originating from a wide geographical region in southern part of the Punjab province, Pakistan. Varying in each of the selected districts, the seroprevalence was found to be maximum for bluetongue [n = 697 (70.26%, 95% CI: 67.29–73.07)], followed by PPR [n = 193 (19.46%, 95% CI: 17.07–22.09)] and brucellosis [n = 66 (6.65%, 95% CI: 5.22–8.43)]. Odds of seroprevalence were more significantly associated with pregnancy status (non-pregnant, OR = 2.23, 95% CI: 1.86–5.63, p

•Summary of the methods currently available for the detection of antimalarial drug resistance within blood samples of human patients.•No single method is perfect for every application, many newly developed methods give promise for a more reliable and efficient characterisation of a Plasmodium resistance.•Deeper understanding of the evolutionary and spaciotemporal dynamics of this disease. Malaria is the world's deadliest parasitic disease. Great progress has been made in the fight against malaria over the past two decades, but this has recently begun to plateau, in part due to the global development of antimalarial drug resistance. The ability to track drug resistance is necessary to achieve progress in treatment, disease surveillance and epidemiology, which has prompted the development of advanced diagnostic methods. These new methods provide unprecedented access to information that can help to guide public health policies. Development of new technologies increases the potential for high throughput and reduced costs of diagnostic tests; improving the accessibility of tools to investigate the forces driving disease dynamics and, ultimately, clinical outcomes for malaria patients and public health. This literature review provides a summary of the methods currently available for the detection of antimalarial drug resistance from the examination of patients’ blood samples. While no single method is perfect for every application, many of the newly developed methods give promise for more reliable and efficient characterisation of Plasmodium resistance in a range of settings. By exploiting the strengths of the tools available, we can develop a deeper understanding of the evolutionary and spatiotemporal dynamics of this disease. This will translate into more effective disease control, better-informed policy, and more timely and successful treatment for malaria patients.

Toxoplasmosis is a globally distributed disease of warm-blooded animals. It is caused by the opportunistic parasite ( ). One-third of the global human population is believed to be infected with . Cats serve as final host of and are the main source of contamination of soil and water. This study aimed to detect genotypes of in cats. Fecal samples (n = 400) were collected from districts of South Punjab (Khanewal and Sahiwal), and were processed by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. The obtained oligonucleotide sequences ( ) were submitted to the GenBank database, and the evolutionary tree was constructed using MEGA-X software. Seven fecal samples (3.5%) from cats were positive. Five out of thirteen fecal samples (38.46%) found to be positive for with microscopy were confirmed by PCR. After phylogenetic analysis with 3 clonal types and atypical strains, isolates of in current study were more closely linked to a typical strain (AF249696). Besides genotyping from cats, seroprevalence from humans and ruminants is still considered to be the best and easiest way to identify the . Blood samples were collected from sheep and goats (n = 2000 each), and human blood samples (n = 400) were collected from the same vicinity. Seroprevalence was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit. In Khanewal, the blood samples of 292 goats (29.2%) and 265 sheep (26.5%), and 6 fecal samples from cats (3%) were positive. Out of 200 human blood samples, 52 were positive, with a seroprevalence of 26%. In the Sahiwal district, the blood samples from 49 humans, 235 sheep and 348 goats were positive, with seroprevalence of 24.5%, 23.5% and 34.8%, respectively. The present study revealed the current circulating genotype of from cats in the districts Khanewal and Sahiwal and the seroprevalence of the organism in small ruminants and humans living in the same vicinity. Further genotype analyses of the organism from ruminants and humans are needed.

Like other pathogens, parasitic helminths can rapidly evolve resistance to drug treatment. Understanding the genetic basis of anthelmintic drug resistance in parasitic nematodes is key to tracking its spread and improving the efficacy and sustainability of parasite control. Here, we use an in vivo genetic cross between drug-susceptible and multi-drug-resistant strains of Haemonchus contortus in a natural host-parasite system to simultaneously map resistance loci for the three major classes of anthelmintics. This approach identifies new 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; expression analyses and functional validation using knockdown experiments support that cky-1 is associated with ivermectin survival. 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. • High genomic diversity differentiates susceptible and multi-drug-resistant helminths • A forward genetic cross reveals QTLs that are specific to each of three drug classes • Novel causal variants are defined for benzimidazole and levamisole anthelmintics • cky-1 expression is correlated with ivermectin resistance in three nematode species Doyle et al. describe a forward genetic cross between susceptible and multi-drug-resistant strains of the globally distributed parasitic worm Haemonchus contortus . Whole-genome analysis reveals loci associated with resistance to each drug class, and novel resistance alleles are validated for two classes.

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, lim-ited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selec-tion in parasitic nematodes and on how best to detect them. This study addresses these knowledge gaps by using field populations of Haemonchus contortus with well-defined benzimidazole treatment histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 genomic markers to genotype 20 individual adult worms from each of four H. contortus popu-lations: two from closed sheep flocks with an approximate 20 year history of frequent benzimidazole treatment, and two populations with a history of little or no treatment. Sampling occurred in the same geographical region to limit genetic differentiation and maximise the detection sensitivity. A clear signa-ture of selection was detected on chromosome I, centred on the isotype-1 6-tubulin gene. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I spanning 3.75 Mbp and 259 annotated genes, and one on chromosome II spanning a region of 3.3 Mbp and 206 anno-tated genes, including the isotype-2 6-tubulin locus. We also assessed how sensitivity was impacted by sequencing depth, worm number, and pooled versus individual worm sequence data. This study provides the first known direct genome-wide evidence for any parasitic nematode, that the isotype-1 6-tubulin gene is quantitatively the single most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole resistance loci of secondary importance. This study provides an experimental framework to maximise the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes.(c) 2022 Published by Elsevier Ltd on behalf of Australian Society for Parasitology.

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 resequencing of a female worm and her brood to identify the distribution of genome-wide variation that characterizes these strains. Using a novel bioinformatic approach to identify variants that segregate as expected in a pseudotestcross, we characterized linkage groups and estimated genetic distances between markers to generate a chromosome-scale F1 genetic map. We exploited this map to reveal the recombination landscape, the first for any 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 analyzed. Triploid offspring were also identified, which we hypothesize 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 enhance ongoing efforts to understand the genetic basis of resistance to the drugs used to control these worms and for related species that infect livestock and humans throughout the world. This study also demonstrates the feasibility of using whole-genome resequencing data to directly construct a genetic map in a single generation cross from a noninbred nonmodel organism with a complex lifecycle.

With extensive use of the anthelmintic in many tropical and parasite-endemic countries, the graveness of parasite resistance is exaggerating. The trend in the use of excessive medication in animal is also posing threats to public health. The governmental bodies are either lacking understanding or underestimating the regime of this emergingissue. This brief overview highlights the need, rationale and urgency of the matter to establish foundations for unifiedaction plans at the national levels as well as international levels in mitigating the development of resistance.

More than 70 species of the Superfamily Paramphistomoidea, have been identified in ruminants in different parts of the world. Most are pathogenic, causing amphistomosis. Adult flukes within this family have a predilection for the forestomach (rumen) or bile duct of the liver, where they may cause epithelial damage. Identification of adult Paramphistomum, Calicophoron, Gastrothylax and Fischoederius at the species level based on morphology requires specialised expertise, whereas molecular genetic marker analysis is more precise and transferable. In the present study, we performed molecular characterisation of twenty seven adult flukes collected from the forestomachs of buffalo, cattle and goats in the Punjab province of Pakistan. PCR and sequencing of the ITS-2 rDNA region revealed a single haplotype in all cases. Phylogenetic comparison of P. epiclitum ITS2-rDNA sequences with those from other Paramphistomum, Calicophoron, Gastrothylax and Fischoederius species was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for P. epiclitum identification. This work provides a validated species-specific marker of P. epiclitum and the first report of this parasite species from Pakistan. The results of this study also have implications for the diagnosis and control of rumen flukes in the region and the need for accurate species identification to understand parasite distribution and population genetics. [Display omitted] •Molecular confirmation of P. epiclitum using rDNA ITS-2 and the significance of its phylogenetic relationship to other fluke.•Differentiation between Paramphistomum, Gastrothylax and Fischoederius as an aid to their identification in the rumen.•The present study has important implications for the diagnosis and control of rumen flukes.

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the β-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.

Varestrongylus eleguneniensis (Nematoda; Protostrongylidae) is a recently described species of lungworm that infects caribou (Rangifer tarandus), muskoxen (Ovibos moschatus) and moose (Alces americanus) across northern North America. Herein we explore the geographic distribution of V. eleguneniensis through geographically extensive sampling and discuss the biogeography of this multi-host parasite. We analyzed fecal samples of three caribou subspecies (n = 1485), two muskox subspecies (n = 159), and two moose subspecies (n = 264) from across northern North America. Protostrongylid dorsal-spined larvae (DSL) were found in 23.8%, 73.6%, and 4.2% of these ungulates, respectively. A portion of recovered DSL were identified by genetic analyses of the ITS-2 region of the nuclear rDNA or the cytochrome oxidase c subunit I (COI) region of the mtDNA. We found V. eleguneniensis widely distributed among caribou and muskox populations across most of their geographic prange in North America but it was rare in moose. Parelaphostrongylus andersoni was present in caribou and moose and we provide new geographic records for this species. This study provides a substantial expansion of the knowledge defining the current distribution and biogeography of protostrongylid nematodes in northern ungulates. Insights about the host and geographic range of V. eleguneniensis can serve as a geographically extensive baseline for monitoring current distribution and in anticipating future biogeographic scenarios under a regime of accelerating climate and anthropogenic perturbation. [Display omitted] •Varestrongylus eleguneniensis is a lungworm whose primary host is the caribou.•The muscleworm, Parelaphostrongylus andersoni, co-infects caribou across its range.•We expand the knowledge on distribution of the caribou lungworm and the muscleworm.•Muskoxen sympatric with caribou are infected with the caribou lungworm.•We discuss the biogeography of V. eleguneniensis and Rangifer across North America.

The benzimidazoles are one of the most important broad-spectrum anthelmintic drug classes for parasitic nematode control in domestic animals and humans. They have been widely used in livestock, particularly in small ruminants for over 40 years. This has resulted in widespread resistance in small ruminant gastrointestinal nematode parasite species, especially Haemonchus contortus. Benzimidazole resistance mutations have also been reported in Haemonchus placei, but only at low frequencies, suggesting resistance is at a much earlier stage of emergence than is the case for H. contortus. Here, we investigate the haplotype diversity of isotype-1 β-tubulin benzimidazole resistance mutations and the population genetic structure of H. contortus and H. placei populations from sheep and cattle from the southern USA. Microsatellite genotyping revealed a low level of genetic differentiation in six H.placei and seven H. contortus populations examined. This is consistent with several previous studies from other regions, mainly in H. contortus, supporting a model of high gene flow between parasite populations. There was a single F200Y(TAC) haplotype present in all six H. placei populations across Georgia, Florida and Arkansas. In contrast, there were at least two different F200Y(TAC) haplotypes (up to four) and two different F167Y(TAC) haplotypes across the seven H. contortus populations studied. These results provide further evidence to support a model for benzimidazole resistance in Haemonchus spp, in which resistance mutations arise from a single, or the small number of locations, in a region during the early phases of emergence, and subsequently spread due to animal movement.

Malaria is the world’s fatal parasitic disease. The ability to quickly and accurately identify malaria infection in challenging environments is crucial to allow efficient administration of the best treatment regime for human patients. If those techniques are accessible and efficient, global detection of Plasmodium species will become more sensitive, allowing faster and more precise action to be taken for disease control strategies. Recent advances in technology have enhanced our ability to diagnose different species of Plasmodium parasites with greater sensitivity and specificity. This literature review provides a summary and discussion of the current methods for the diagnosis and identification of Plasmodium spp. in human blood samples. So far not a single method is precise, but advanced technologies give consistent identification of a Plasmodium infection in endemic regions. By using the power of the recent methods, we can provide a broader understanding of the multiplicity of infection and or transmission dynamics of Plasmodium spp. This will result in improved disease control strategies, better-informed policy, and effective treatment for malaria-positive patients. [Display omitted] •Summary of the methods currently available for the detection of Plasmodium spp. infecting humans.•No single method is perfect for every application to identify Plasmodium spp.•Newly developed methods give promise for more reliable characterisation of Plasmodium spp.

In some parts of the world, Dicrocoelium spp. lancet flukes cause significant production loss in pastoral livestock, and accurate diagnosis of infection is important. The aims of the present study were to describe the histopathology and to investigate the transmission patterns of Dicrocoelium amongst ten sheep and goat farms in Khyber Pakhtunkhwa and Gilgit Baltistan, Pakistan. The liver histology and indirect enzyme-linked immunosorbent assay (ELISA) analyses followed standard procedures. The liver histopathology showed intensive tissue destruction and biliary hyperplasia associated with presence of adult flukes, severe inflammatory cell infiltration, congestion of blood vessels, damaged hepatocytes, and sinusoids in the infected areas. The time of onset of infection was investigated by ELISA detection of antibodies in sheep (n = 164) and goats (n = 152). Colostral transfer of Dicrocoelium antibodies from seropositive mothers was detected in sheep and goats up to 16 weeks of age. In both sheep and goats, the estimated time of infection differed between farms and years. Infection was seen in both sheep flocks and goat herds, with high variation between flocks and herds, and the highest infection rate in lambs. Dicrocoelium infection was most prevalent in sheep and goats in September (n = 84) and August (n = 63) respectively. This study concluded Dicrocoelium causes severe inflammation and necrosis of liver tissues in sheep and goats. Colostral transfer of antibodies can be detected up to about ten weeks of age. Higher infection rates are observed during August and September in sheep than in goats, putatively due to effects of different grazing and browsing behaviors on the ingestion of ants. The results will aid in the development of effective disease control strategies to ensure optimal growth and productivity of sheep and goats. •First to study onset of infection and transmission pattern of Dicrocoelium in Asia.•ELISA detection of antibodies in sheep (n = 164) and goats (n = 152).•Dicrocoelium causes severe inflammation and necrosis of liver tissues.•Colostral transfer of antibodies can be detected up to about ten weeks of age.•Higher infection rates are observed during August and September in sheep than in goats.

[Display omitted] •Metabarcoded sequencing was used to determined species composition (nemabiome) of gastrointestinal nematode (GIN) larvae.•Deep amplicon sequencing determined proportions of benzimidazole (BZ)-resistant SNPs in Teladorsagia circumcincta.•Nemabiomes varied with time and between ewes and lambs, possibly associated with weather.•The F200Y BZ resistance mutation was close to genetic fixation in T. circumcincta.•There was no genetic evidence of reversion to BZ anthelmintic susceptibility. Anthelmintic resistance threatens the sustainability of sheep production globally. Advice regarding strategies to reduce the development of anthelmintic resistance incorporates the outcomes of modelling exercises. Further understanding of gastrointestinal nematode species diversity, and population dynamics and genetics (which may vary between species) is required to refine these models; and field studies combining faecal egg outputs, species composition and resistance genetics are needed to calibrate them. In this study, faecal samples were taken from ewes and lambs on a commercial farm in south-eastern Scotland at approximately 3 t-4 week intervals between spring and autumn over a period of 4 years. Faecal egg counts were performed on these samples, and L3 were collected from pooled coprocultures. Deep amplicon sequencing was used to determine both the species composition of these L3 and the proportions of benzimidazole-resistant single nucleotide polymorphisms in the isotype-1 β-tubulin locus of the predominant species, Teladorsagia circumcincta L3. Despite consistent management throughout the study, the results show variation in gastrointestinal nematode species composition with time and between age groups, that was potentially associated with weather conditions. The F200Y benzimidazole resistance mutation is close to genetic fixation in the T. circumcincta population on this farm. There was no evidence of variation in isotype-1 β-tubulin single nucleotide polymorphisms frequency between age groups, and no genetic evidence of reversion to benzimidazole susceptibility, despite targeted benzimidazole usage. This study highlights the need to include speciation when investigating gastrointestinal nematode epidemiology and anthelmintic resistance, and serves as an example of how genetic data may be analysed alongside species diversity and faecal egg counts, when markers for other anthelmintic classes are identified.

The population genetics of nematode parasites are poorly understood with practical reference to the selection and spread of anthelmintic resistance mutations. Haemonchus species are important to study the nematode population genetics due to their clinical importance in ruminant livestock, and the availability of genomic resources. In the present study, it has been examined that Haemonchus contortus and Haemonchus placei populations from three buffalo and nine cattle hosts. Seventy-three individual adult worms of H. contortus and 148 of H. placei were analysed using a panel of seven microsatellite markers. The number of alleles per locus in H. contortus and H. placei indicated that all populations were polymorphic for the microsatellites used in the present study. Genetic diversity parameters included high levels of allelic richness and heterozygosity, indicating effective population sizes, high mutation rates and high transmission frequencies in the area. Genetic structure parameters revealed low genetic differentiation between and high levels of genetic variation within H. contortus and H. placei populations. Population dynamic analyses showed an absence of heterozygosity excess in both species, suggesting that there was no deviation from genetic drift equilibrium. Our results provide a proof of concept for better understanding of the consequences of specific control strategies, climatic change or management strategies on the population genetics of anthelmintic resistance alleles in Haemonchus spp. infecting co-managed buffalo and cattle.

Pyrimethamine resistance is a major concern for the control of human haemoprotozoa, especially Plasmodium species. Currently, there is little understanding of how pyrimethamine resistance developed in Plasmodium vivax in the natural field conditions. Here, we present for the first time evidence of positive selection pressure on a dihydrofolate reductase locus and its consequences on the emergence and the spread of pyrimethamine resistance in P. vivax in the Punjab province of Pakistan. First, we examined the dihydrofolate reductase locus in 38 P. vivax isolates to look for evidence of positive selection pressure in human patients. The S58R (AGA)/S117N (AAC) double mutation was most common, being detected in 10/38 isolates. Single mutation S117N (AAC), I173L (CTT) and S58R (AGA) SNPs were detected in 8/38, 2/38 and 1/38 isolates, respectively. The F57L/I (TTA/ATA) and T61M (ATG) SNPs were not detected in any isolates examined. Although both soft and hard selective sweeps have occurred with striking differences between isolates, there was a predominance of hard sweeps. A single resistance haplotype was present at high frequency in 9/14 isolates, providing a strong evidence for single emergence of resistance by the single mutation, characteristics of hard selective sweeps. In contrast, 5/14 isolates carried multiple resistance haplotypes at high frequencies, providing an evidence of the emergence of resistance by recurrent mutations, characteristics of soft selective sweeps. Our phylogenetic relationship analysis suggests that S58R (AGA)/S117N (AAC) and S117N (AAC) mutations arose multiple times from a single origin and spread to multiple different cities in the Punjab province through gene flow. Interestingly, the I173L (CTT) mutation was present on a single haplotype, suggesting that it arises rarely and has not spread between cities. Our work shows the need for responsible use of existing and new antimicrobial drugs and their combinations, control the movement of infected patients and mosquito vector control strategies. •Illumina MiSeq deep amplicon sequencing was used to identify pyrimethamine resistance associated SNPs in Plasmodium vivax.•Pyrimethamine resistance was identified in field populations.•Positive selection pressure for resistance in human patients was investigated.•Hard and soft selective sweep models were used for the emergence of resistance.•Both single and multiple recurrent resistance mutations were spread by migration.

Insular wildlife populations provide opportunities to examine biological questions in systems that are relatively closed and potentially tractable, striking examples being the long-term studies of ecology and evolution in the red deer and feral sheep populations on the Hebridean islands of Rum and St Kilda. In the case of parasitology, Understanding of parasitic infections insular wildlife populations in conjunction with knowledge of their origins has the potential to add a fresh perspective to disease control in humans and domestic animals. In the case of parasitology, understanding infections of insular wildlife populations, in conjunction with knowledge of their origins, has the potential to add a fresh perspective to disease control in humans and domestic animals. With this in mind, gross and molecular examination for the presence of cyclophyllidean tapeworms was performed on the viscera and rectal contents of 17 preserved specimens of Apodemus sylvaticus field mice and on the naturally voided faeces of a further four mice on the remote archipelago of St Kilda. Molecular speciation of hexacanth embryos extracted from the faeces of two mice, using nucleotide sequence analysis of the ribosomal cytochrome c-oxidase subunit-1, confirmed infection with Hymenolepis hibernia. Phylogenetic analysis showed that these were genetically distinct from Hymenolepis diminuta, previously reported in the insular A. sylvaticus mice, and from other published H. hibernia haplotypes. There was insufficient hymenolepidid tapeworm phylogeographic variation to resolve the origins of the co-evolved St Kilda mice, primarily due to a lack of published H. hibernia Cox-1 sequence data across the parasite's geographical range. Nevertheless, the Maximum Likelihood haplotype tree shows the potential for molecular parasitology to resolve a host-parasite relationship once more data become available. Morphological diagnostic features of zoonotic H. hibernia eggs are also described. [Display omitted] •First identification of Hymenolepis hibernia in Apodemus sylvaticus hirtensis mice.•Molecular speciation of hexocanth eggs.•Morphological description of Hymenolepis hibernia eggs.•Maximum likelihood hymenolepidid tapeworm haplotype tree.•Consideration of parasite origins and those of co-evolved hosts.

Genetic hybridisation between parasitic nematode species has potentially important consequences. It could lead to the introgression of genes between species including those involved in pathogenicity, host specificity, transmission and drug resistance. It could also complicate diagnosis and control. However, there are few compelling examples of its occurrence in parasites in the field. Haemonchus contortus and Haemonchus placei are two closely related parasitic nematode species that predominantly infect small ruminants and cattle, respectively. They are capable of experimental hybridisation when adult worms of each species are transplanted into the same individual host. Given that co-infection occurs in both small ruminants and cattle, there is potential for hybridisation in the field. However, this has not been definitively demonstrated and its extent is unknown. We investigated the occurrence of co-infection and interspecies hybridisation in H. contortus and H. placei in field populations from small ruminants from Pakistan and southern India using a number of independent genetic markers. Haemonchus contortus and H. placei co-infections were common in Pakistan but not in southern India where H. placei appeared to be absent in small ruminant hosts. In the former region, a number of worms were identified that were heterozygous for fixed, species-specific rDNA internal transcribed spacer 2 (ITS-2) single nucleotide polymorphisms. Genotyping of these ITS-2 heterozygotes with an additional four nuclear markers conclusively demonstrated them to be F1 interspecies hybrids. Mitochondrial NADH dehydrogenase subunit 4 haplotype analysis demonstrated that four of the hybrid worms had a H. placei maternal parent and one had a H. contortus maternal parent showing that hybridisation could occur in either direction. Interestingly, one of these hybrids contained an H. contortus isotype-1 β-tubulin benzimidazole resistance allele, suggesting there is a potential for interspecies introgression of drug resistance loci. We believe this is the first definitive genetic evidence of hybridisation between H. contortus and H. placei in the field and represents the most comprehensive genetic evidence of F1 hybrids between any human or livestock parasitic nematode species to date. Further, it suggests that interspecies transmission of anthelmintic resistance mutations warrants further investigation.