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.

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.

Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes.

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

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.

The Prevalence and Control of Lungworms of Pastoral Ruminants in Iran.

Lungworms of the genera Dictyocaulus, Muellerius, Protostrongylus, and Cystocaulus are common helminths of domestic and wild ruminants with substantial veterinary and economic importance. Several studies have assessed the presence and prevalence of lungworm infections in ruminants in Iran. This report compiles the available scientific information about the occurrence of lungworms in domestic and wild ruminants in Iran between 1931 and June 2022 to give an insight into their epidemiology, and where possible to describe drug treatment efficacy. For this purpose, national and international scientific databases were searched. Overall, 54 publications comprising 33 articles in peer-reviewed journals, 8 conference papers, and 13 dissertations were evaluated regarding prevalence data; and an additional 4 peer-reviewed articles were evaluated regarding drug efficacy. Seven species of lungworms, namely Dictyocaulus filaria, Dictyocaulus viviparus, Dictyocaulus eckerti, Protostrongylus rufescens, Protostrongylus raillietti, Muellerius capillaris, and Cystocaulus ocreatus have been recorded from different ruminant hosts in Iran. Thirty-three studies conducted on small ruminant (sheep and goat) lungworms reported prevalences of lungworm infection of 11.6%, 45.81% and 66.29% using abattoir meat inspection, Baermann technique and fecal flotation, respectively. Eight studies conducted on large ruminants (cattle and water buffalo) reported prevalences of infection of 14.83%, 13.98% and 5% using abattoir meat inspection, the Baermann technique and fecal flotation, respectively. The prevalence of infection in wild ruminants was variable across examined species; 38% in urial, 37% in wild goats, 5% in goitered gazelles and 67% in red deer, in addition to a single case report in roe deer. There are few contemporary studies assessing the efficacy of currently available broad-spectrum anthelmintic compounds against lungworms in Iran. The high prevalence of multiple lungworm species in Iran, combined with a lack of information about drug efficacy, supports the need to improve the understanding of these important nematode parasites and inform the development of sustainable control strategies. The aim of this review and meta-analysis is to provide a baseline for future conventional parasitology and next generation molecular epidemiological studies of lungworm infection in pastoral ruminants in Iran.

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.

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.

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.

Determining the influence of socio-psychological factors on the adoption of individual 'best practice' parasite control behaviours from Scottish sheep farmers.

Since 2003, the Sustainable Control Of Parasites in Sheep (SCOPS) group have provided the UK sheep farming industry with guidance on ways to mitigate the development and dissemination of anthelmintic resistance (AR). However our empirical understanding of sheep farmers' influences towards such 'best practice' parasite control approaches is limited, and therefore requires further assessment and evaluation to identify the potential factors influencing their implementation. In 2015, a telephone questionnaire was conducted in order to elicit Scottish sheep farmers' attitudes and behaviours regarding the SCOPS recommended practices, as well as gauging farmers' general attitudes to gastrointestinal nematodes (GIN; term roundworm used in questionnaire) control. A quantitative structural equation modelling (SEM) approach was employed to determine the influences of socio-psychological factors and the uptake of individual anthelmintic resistance mitigating practices including: the implementation of a quarantine strategy for parasite control and the use of parasite diagnostic testing for monitoring faecal egg counts (FEC) and detecting AR. The proposed models established a good fit with the observed data and explained 61%, 54% and 27% of the variance in the adoption of AR testing, FEC monitoring, and quarantine behaviours respectively. The results presented highlight a number of consistent and distinct factors significantly influencing the implementation of selected SCOPS recommended practices. The negative influences of topography and farmer experience was frequently demonstrated in relation to multiple GIN control practices, as well as the positive influences of social norms, worm control knowledge, AR risk perception and positive attitudes to the services provided by the veterinary profession. Factors that were shown to have the greatest relative effects on individual parasite control practices included: the perceived expectation of others (i.e. Social norms) for implementing a quarantine strategy, farmer's suspicions to the presence of AR on the holding for instigating AR testing and the confirmation of AR for adopting FEC monitoring. Determining the influences of behaviour-specific factors on farmers' decision making processes will help to identify and address positive and negative influences concerning implementation of AR mitigating practices, as well as contribute to the development of more evidence based intervention strategies in the future.

High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan, but only Fasciola gigantica in the Punjab province of Pakistan.

Fasciola gigantica and Fasciola hepatica are digenetic trematodes causing fasciolosis in ruminants. The host and geographical distribution of both Fasciola species are influenced by environmental and climatic conditions favouring survival and development of free-living stages and intermediate hosts, and livestock management practices. The aim of the present study was to describe the host distribution of the two Fasciola species in buffalo, cattle, goats, and sheep in the Balochistan and Punjab provinces of Pakistan. 359 flukes were collected from a total of 32 livers from the four livestock species. Deep amplicon sequencing of the internal transcribed spacer region 2 of ribosomal DNA (rDNA ITS-2) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND-1) loci confirmed co-infection of F. hepatica and F. gigantica in Balochistan and single species F. gigantica infection in Punjab. In Balochistan, co-infections and hybrids of both Fasciola species were identified in cattle, with more F. hepatica detected than F. gigantica. However, F. hepatica was the only species identified in goats, and F. gigantica was the only species identified in buffalo. In Punjab, all flukes were confirmed as F. gigantica in each of the four livestock species. Overall, the results indicate differences in the host and geographical distribution of F. gigantica and F. hepatica, and provide useful knowledge for the development of control strategies for livestock and humans.

Phylogenetic analysis suggests single and multiple origins of dihydrofolate reductase mutations in Plasmodium vivax.

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

Nemabiome metabarcoding reveals differences between gastrointestinal nematode species infecting co-grazed sheep and goats.

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.

Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan.

Lancet liver flukes of the genus Dicrocoelium (Trematoda: Digenea) are recognised parasites of domestic and wild herbivores. The aim of the present study was to 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.

Contrasting population genetics of co-endemic cattle- and buffalo- derived Theileria annulata.

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.

Assessment of Knowledge, Attitudes and Practices Relating to Parasitic Diseases and Anthelmintic Resistance Among Livestock Farmers in Hamedan, Iran.

Iranian studies have shown a high prevalence of broad spectrum anthelmintic resistance (AR) in gastrointestinal helminths of ruminants. However, there is a lack of information about levels of knowledge, attitudes and practices among livestock farmers in Iran regarding the concept of parasite control and AR. This study aimed to evaluate the knowledge, attitudes and practices of livestock farmers of Hamedan, Iran, regarding parasitic diseases and AR by interviewing 150 farmers using a structured questionnaire. Most of farmers had some knowledge of the clinical signs associated with helminth parasitism, but more than half were unaware of the existence of zoonotic parasites. More than half of the participants had never heard about AR, but were interested to learn about it through their veterinarians. Those who were aware of the problem considered non-prescribed anthelmintic drugs to play a role in its emergence, while several of the participants believed that "more expensive" and "foreign-branded" drugs worked best. Almost all of the farmers reported that they frequently consulted with a veterinarian about anthelmintic treatments, but very few adhered to recognized principles of responsible and sustainable drug use. About half of the participating farmers treated their sheepdogs for helminth parasites, despite the common practice of regularly feeding likely infected livestock offal. Education had a significantly positive association with farmers' knowledge, attitudes, and best practice scores, while knowledge was significantly associated with both attitudes and practices. Based on these results, we recommend that regular country-wide classes should be held to educate farmers on the evidence-based principles of sustainable helminth control and prevention of zoonotic helminth diseases.

A novel metabarcoded 18S ribosomal DNA sequencing tool for the detection of Plasmodium species in malaria positive patients.

Various PCR based methods have been described for the diagnosis of malaria, but most depend on the use of Plasmodium species-specific probes and primers; hence only the tested species are identified and there is limited available data on the true circulating species diversity. Sensitive diagnostic tools and platforms for their use are needed to detect Plasmodium species in both clinical cases and asymptomatic infections that contribute to disease transmission. We have 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.

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

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.

The confounding effects of high genetic diversity on the determination and interpretation of differential gene expression analysis in the parasitic nematode Haemonchus contortus.

Differential expression analysis between parasitic nematode strains is commonly used to implicate candidate genes in anthelmintic resistance or other biological functions. We have tested the hypothesis that the high genetic diversity of an organism such as Haemonchus contortus could complicate such analyses. First, we investigated the extent to which sequence polymorphism affects the reliability of differential expression analysis between the genetically divergent H. contortus strains MHco3(ISE), MHco4(WRS) and MHco10(CAVR). Using triplicates of 20 adult female worms from each population isolated under parallel experimental conditions, we found that high rates of sequence polymorphism in RNAseq reads were associated with lower efficiency read mapping to gene models under default TopHat2 parameters, leading to biased estimates of inter-strain differential expression. We then showed it is possible to largely compensate for this bias by optimising the read mapping single nucleotide polymorphism (SNP) allowance and filtering out genes with particularly high single nucleotide polymorphism rates. Once the sequence polymorphism biases were removed, we then assessed the genuine transcriptional diversity between the strains, finding ≥824 differentially expressed genes across all three pairwise strain comparisons. This high level of inter-strain transcriptional diversity not only suggests substantive inter-strain phenotypic variation but also highlights the difficulty in reliably associating differential expression of specific genes with phenotypic differences. To provide a practical example, we analysed two gene families of potential relevance to ivermectin drug resistance; the ABC transporters and the ligand-gated ion channels (LGICs). Over half of genes identified as differentially expressed using default TopHat2 parameters were shown to be an artifact of sequence polymorphism differences. This work illustrates the need to account for sequence polymorphism in differential expression analysis. It also demonstrates that a large number of genuine transcriptional differences can occur between H. contortus strains and these must be considered before associating the differential expression of specific genes with phenotypic differences between strains.

Development of a deep amplicon sequencing method to determine the species composition of piroplasm haemoprotozoa.

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.

Development of amplicon sequencing for the analysis of benzimidazole resistance allele frequencies in field populations of gastrointestinal nematodes.

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