Transcriptomic analyses implicate neuronal plasticity and chloride homeostasis in ivermectin resistance and response to treatment in a parasitic nematode.

The antiparasitic drug ivermectin plays an essential role in human and animal health globally. However, ivermectin resistance is widespread in veterinary helminths and there are growing concerns of sub-optimal responses to treatment in related helminths of humans. Despite decades of research, the genetic mechanisms underlying ivermectin resistance are poorly understood in parasitic helminths. This reflects significant uncertainty regarding the mode of action of ivermectin in parasitic helminths, and the genetic complexity of these organisms; parasitic helminths have large, rapidly evolving genomes and differences in evolutionary history and genetic background can confound comparisons between resistant and susceptible populations. We undertook a controlled genetic cross of a multi-drug resistant and a susceptible reference isolate of Haemonchus contortus, an economically important gastrointestinal nematode of sheep, and ivermectin-selected the F2 population for comparison with an untreated F2 control. RNA-seq analyses of male and female adults of all populations identified high transcriptomic differentiation between parental isolates, which was significantly reduced in the F2, allowing differences associated specifically with ivermectin resistance to be identified. In all resistant populations, there was constitutive upregulation of a single gene, HCON_00155390:cky-1, a putative pharyngeal-expressed transcription factor, in a narrow locus on chromosome V previously shown to be under ivermectin selection. In addition, we detected sex-specific differences in gene expression between resistant and susceptible populations, including constitutive upregulation of a P-glycoprotein, HCON_00162780:pgp-11, in resistant males only. After ivermectin selection, we identified differential expression of genes with roles in neuronal function and chloride homeostasis, which is consistent with an adaptive response to ivermectin-induced hyperpolarisation of neuromuscular cells. Overall, we show the utility of a genetic cross to identify differences in gene expression that are specific to ivermectin selection and provide a framework to better understand ivermectin resistance and response to treatment in parasitic helminths.

Nemabiome metabarcoding shows a high prevalence of Haemonchus contortus and predominance of Camelostrongylus mentulatus in alpaca herds in the northern UK.

Gastrointestinal nematodes (GINs) are a common threat faced by pastoral livestock. Since their major introduction to the UK in the early 1990s, South American camelids have been cograzed with sheep, horses, and other livestock, allowing exposure to a range of GIN species. However, there have been no molecular-based studies to investigate the GIN populations present in these camelids. In the current study, we sampled nine alpaca herds from northern England and southern Scotland and used high-throughput metabarcoded sequencing to describe their GIN species composition. A total of 71 amplicon sequence variants (ASVs) were identified representing eight known GIN species. Haemonchus contortus was the most prevalent species found in almost all herds in significant proportions. The identification of H. contortus in other livestock species is unusual in the northern UK, implying that alpacas may be suitable hosts and potential reservoirs for infection in other hosts. In addition, the camelid-adapted GIN species Camelostrongylus mentulatus was identified predominantly in herds with higher faecal egg counts. These findings highlight the value of applying advanced molecular methods, such as nemabiome metabarcoding to describe the dynamics of gastrointestinal nematode infections in novel situations. The results provide a strong base for further studies involving cograzing animals to confirm the potential role of alpacas in transmitting GIN species between hosts.

Mapping restricted introgression across the genomes of admixed indigenous African cattle breeds.

BACKGROUND: The genomes of indigenous African cattle are composed of components with Middle Eastern (taurine) and South Asian (indicine) origins, providing a valuable model to study hybridization and to identify genetic barriers to gene flow. In this study, we analysed indigenous African cattle breeds as models of hybrid zones, considering taurine and indicine samples as ancestors. In a genomic cline analysis of whole-genome sequence data, we considered over 8 million variants from 144 animals, which allows for fine-mapping of potential genomic incompatibilities at high resolution across the genome. RESULTS: We identified several thousand variants that had significantly steep clines ('SCV') across the whole genome, indicating restricted introgression. Some of the SCV were clustered into extended regions, with the longest on chromosome 7, spanning 725 kb and including 27 genes. We found that variants with a high phenotypic impact (e.g. indels, intra-genic and missense variants) likely represent greater genetic barriers to gene flow. Furthermore, our findings provide evidence that a large proportion of breed differentiation in African cattle could be linked to genomic incompatibilities and reproductive isolation. Functional evaluation of genes with SCV suggest that mitonuclear incompatibilities and genes associated with fitness (e.g. resistance to paratuberculosis) could account for restricted gene flow in indigenous African cattle. CONCLUSIONS: To our knowledge, this is the first time genomic cline analysis has been applied to identify restricted introgression in the genomes of indigenous African cattle and the results provide extended insights into mechanisms (e.g. genomic incompatibilities) contributing to hybrid differentiation. These results have important implications for our understanding of genetic incompatibilities and reproductive isolation and provide important insights into the impact of cross-breeding cattle with the aim of producing offspring that are both hardy and productive.

Comparative clinico-pathological assessment of velogenic (sub-genotype VIIi) and mesogenic (sub-genotype VIm) Avian avulavirus 1 in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 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.

Population genetics of benzimidazole-resistant Haemonchus contortus and Haemonchus placei from buffalo and cattle: implications for the emergence and spread of resistance mutations.

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.

Molecular characterization of infectious bursal disease viruses from Pakistan.

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.

Near-infrared fluorescent cholangiography facilitates identification of biliary anatomy during laparoscopic cholecystectomy.

BACKGROUND: Intraoperative cholangiography (IOC) is the current gold standard for biliary imaging during laparoscopic cholecystectomy (LC). However, utilization of IOC remains low. Near-infrared fluorescence cholangiography (NIRF-C) is a novel, noninvasive method for real-time, intraoperative biliary mapping. Our aims were to assess the safety and efficacy of NIRF-C for identification of biliary anatomy during LC. METHODS: Patients were administered indocyanine green (ICG) prior to surgery. NIRF-C was used to identify extrahepatic biliary structures before and after partial and complete dissection of Calot's triangle. Routine IOC was performed in each case. Identification of biliary structures using NIRF-C and IOC, and time required to complete each procedure were collected. RESULTS: Eighty-two patients underwent elective LC with NIRF-C and IOC. Mean age and body mass index (BMI) were 42.6 ± 13.7 years and 31.5 ± 8.2 kg/m(2), respectively. ICG was administered 73.8 ± 26.4 min prior to incision. NIRF-C was significantly faster than IOC (1.9 ± 1.7 vs. 11.8 ± 5.3 min, p < 0.001). IOC was unobtainable in 20 (24.4 %) patients while NIRF-C did not visualize biliary structures in 4 (4.9 %) patients. After complete dissection, the rates of visualization of the cystic duct, common bile duct, and common hepatic duct using NIRF-C were 95.1, 76.8, and 69.5 %, respectively, compared to 72.0, 75.6, and 74.3 % for IOC. In 20 patients where IOC could not be obtained, NIRF-C successfully identified biliary structures in 80 % of the cases. Higher BMI was not a deterrent to visualization of anatomy with NIRF-C. No adverse events were observed with NIRF-C. CONCLUSIONS: NIRF-C is a safe and effective alternative to IOC for imaging extrahepatic biliary structures during LC. This technique should be evaluated further under a variety of acute and chronic gallbladder inflammatory conditions to determine its usefulness in biliary ductal identification.

Validation of deep amplicon sequencing of Dicrocoelium in small ruminants from Northern regions of Pakistan.

Dicrocoelium lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple Dicrocoelium species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe Dicrocoelium species in sheep and goats. First, rDNA ITS-2 sequences of four Dicrocoelium species (Dicrocoelium dendriticum, Dicrocoelium hospes, Dicrocoelium orientalis, and Dicrocoelium chinensis) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of Dicrocoelium species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the Dicrocoelium genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as D. dendriticum and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the Dicrocoelium species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from D. orientalis and D. chinensis, which sit very close to each other in a single large clade whereas D. hospes and D. dendriticum are separated into their own clade. In conclusion each sample was identified as D. dendriticum based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of Dicrocoelium in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.

Worldwide absence of canonical benzimidazole resistance-associated mutations within ß-tubulin genes from Ascaris.

BACKGROUND: The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with ß-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the ß-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the ß-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. METHODS: To further determine the putative absence of key ß-tubulin polymorphisms, we screened two ß-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris ß-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts. RESULTS: In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of ß-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for ß-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of ß-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts. CONCLUSIONS: Our work suggests an absence of canonical ß-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.

Obstructive jaundice expands intrahepatic regulatory T cells, which impair liver T lymphocyte function but modulate liver cholestasis and fibrosis.

Although obstructive jaundice has been associated with a predisposition toward infections, the effects of bile duct ligation (BDL) on bulk intrahepatic T cells have not been clearly defined. The aim of this study was to determine the consequences of BDL on liver T cell phenotype and function. After BDL in mice, we found that bulk liver T cells were less responsive to allogeneic or syngeneic Ag-loaded dendritic cells. Spleen T cell function was not affected, and the viability of liver T cells was preserved. BDL expanded the number of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), which were anergic to direct CD3 stimulation and mediated T cell suppression in vitro. Adoptively transferred CD4(+)CD25(-) T cells were converted into Treg within the liver after BDL. In vivo depletion of Treg after BDL restored bulk liver T cell function but exacerbated the degrees of inflammatory cytokine production, cholestasis, and hepatic fibrosis. Thus, BDL expands liver Treg, which reduce the function of bulk intrahepatic T cells yet limit liver injury.

Histopathology and antibody responses describe the seasonal pattern of dicrocoeliosis in small ruminants in the Himalayan ranges of Pakistan.

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.

Spatial Distribution of Dicrocoelium in the Himalayan Ranges: Potential Impacts of Ecological Niches and Climatic Variables.

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 < 0.05) associated with the presence of Dicrocoelium infection. CONCLUSION: Together, the findings of this study demonstrate the most suitable ecological niches and climatic variables influencing the risk of dicrocoeliosis in the Himalayan ranges of Pakistan. The methods and results could be used as a reference to inform the control of dicrocoeliosis in the region.

A new record of the occurrence of Trichuris skrjabini Baskakov, 1924 in goats of Pakistan.

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.

Proteomic Comparison of Ivermectin Sensitive and Resistant Staphylococcus aureus Clinical Isolates Reveals Key Efflux Pumps as Possible Resistance Determinants.

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.

A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 ß-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA.

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 ß-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 ß-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 ß-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 ß-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. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-021-01414-w.

Current methods for the detection of Plasmodium parasite species infecting humans.

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.

A novel metabarcoded deep amplicon sequencing tool for disease surveillance and determining the species composition of Trypanosoma in cattle and other farm animals.

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.

Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd.

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.

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

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.

Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations.

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited 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 selection 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 populations: 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 signature of selection was detected on chromosome I, centred on the isotype-1 ß-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 annotated genes, including the isotype-2 ß-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 ß-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.