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.

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.

Investigating hill sheep farmers and crofters' experiences of blackloss in the Highlands and Islands of Scotland.

Hill sheep farming is an important component of Scottish agriculture and comprises a significant land use in much of the Highlands and Islands. However it faces significant challenges due to the natural constraints of the landscape. Hill sheep farming uses hardy traditional breeds, such as the Scottish blackface and North Country Cheviot to graze extensive areas, where the sheep are not housed and tend to lamb on the open hill. Flocks are gathered several times a year for stock checks, husbandry, and health treatments. Between these handling events, stock will disappear and be unaccounted for. These unexplained losses are known as blackloss in the Highlands and Islands. Previously reported figures for annual lamb blackloss give an average of 18.6%. These losses are in addition to the known losses of lambs and represent a significant welfare and sustainability issue. High parasite burdens, predation, a photosensitisation disease known as plochteach or yellowses, and poor nutrition are often given as presumed reasons for blackloss. A questionnaire was developed to assess the experiences, impacts and understanding flock managers have of blackloss. Typology analysis using partitioning around medoids was used to cluster respondents into three distinct groups: 1- very large extensive farms and Sheep Stock Clubs, 2- medium sized farms, and 3- small-scale crofts. The responses of these groups were subsequently analysed to see if their experiences and perceptions of blackloss differed with relation to lamb health challenges and predation impacts. The groups reported similar health challenges, apart from Group 1 which had a significantly higher plochteach challenge. In terms of predators, Group 1 also perceived white-tailed eagles (Haliaeetus albicilla) as a much higher threat to their lambs than the other groups. It was observed that many of the respondents believed blackloss is inevitable and that predators pose a large threat to lambs. However, most agreed that reducing these losses is important and that understanding the causes would enable them to do so.

Investigating the perceived versus actual gastrointestinal nematode challenge on extensive sheep farms.

Extensive farming systems form an integral part of sheep production systems across Europe. However, with innate production handicaps, declining sheep numbers and narrow economic margins, production is becoming increasingly challenging threatening the future sustainability of the industry. Gastrointestinal nematodes (GINs) are a significant cause of production losses to the global sheep industry, with well-established resistance to the major anthelmintic groups. Traditionally, extensive farming systems are not thought to have a significant parasite challenge compared with intensive farms, but there is a need to identify the scale and importance of GINs on extensive farms to inform the need for sustainable control strategies. In this study, a questionnaire of extensive farmers (n=34) was conducted and parasitological data were collected from nine study farms to investigate the perceived versus actual GIN and anthelmintic resistance challenge faced by extensive farms. The results showed a production-limiting challenge on most farms, with a higher GIN challenge observed on improved pastures. Furthermore, over half of the extensive farmers perceived anthelmintic resistance to be a greater problem for intensive farmers, with only 20% of respondents reporting known anthelmintic resistance. However, all study farms had evidence of resistance to at least one group of anthelmintics. Consequently, this study has demonstrated that despite the traditional perception of parasitism on extensive farms, there is a need to increasingly consider its impact and take a proactive approach to sustainable control, with solutions tailored to their unique management.

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.

The Immune Response in the Uteri and Placentae of Chlamydia abortus-Infected Ewes and Its Association with Pregnancy Outcomes.

The enzootic abortion of ewes, caused by the bacterium Chlamydia abortus (C. abortus), is one of the main causes of abortion in sheep. There are multiple contributory factors, including chlamydial growth, host immune response, and hormonal balance, that result in different pregnancy outcomes, such as abortion, the birth of weak lambs that may die, or healthy lambs. This study aimed to determine the relationship between phenotypical patterns of immune cell infiltration and different pregnancy outcomes in twin-bearing sheep (both lambs born dead; one alive and one dead; both alive) when experimentally infected with C. abortus. Both the sheep uteri and placentae were collected after parturition. All samples were analysed for specific immune cell features, including cell surface antigens and the T-regulatory (Treg) cell-associated transcription factor and cytokines, by immunohistochemistry and in situ hybridisation. Some of these immunological antigens were evaluated in ovine reproductive tissues for the first time. Differential patterns of T helper/Treg cells revealed significant group effects in the placentae. It suggests the potential role that the balance of lymphocyte subsets may play in affecting different pregnancy outcomes in C. abortus-infected sheep. The present study provides novel detailed information about the immune responses observed at the maternofoetal interface in sheep at the time of pre-term abortion or lambing.

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.

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.

Genomic landscape of drug response reveals mediators of anthelmintic resistance.

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.

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.

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.

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.

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.

Practices employed by veterinary practitioners for controlling canine gastrointestinal helminths and ectoparasites.

The present study attempted to evaluate the practical experience and methods employed by Brazilian veterinary practitioners for control of parasites. Twenty-one questions were asked of 403 veterinary practitioners based in different climatic zones with reference to parasite epidemiology from the country. Administration of a combination of drugs at three-month intervals was the most common regime recommended for prophylaxis against gastrointestinal helminths, with a single treatment repeated after 15 days. Routine prophylaxis against dog ectoparasites was recommended by 82.4% veterinary practitioners, and 46.6% changed the drug compound used. Monthly prophylaxic treatments for ectoparasites, using systemic, topical and/or collar-impregnated drugs, was recommended by 21.5% veterinary practitioners. Side-effects of ectoparasiticide-impregnated collars were suspected by 58% of the veterinary practitioners. Isoxazolines were the most frequently used chemical group to treat ectoparasites in dogs. Poor efficacy of fipronil in controlling ticks was suspected by 79.5% of the veterinary practitioners. The isoxazolines and combination of anthelmintic compounds are the most common drugs to prevent or treat ectoparasites and gastrointestinal nematodes, respectively. The suspect of the inefficacy of antiparasitic drugs is shared among the veterinary practitioners from part of Brazil. Guidelines are needed, specifically for the control of gastrointestinal helminths and ectoparasites in Brazilian dogs.

Livestock vaccination programme participation among smallholder farmers on the outskirts of National Parks and Tiger Reserves in the Indian states of Madhya Pradesh and Assam.

Effective livestock vaccination has the potential to raise prosperity and food security for the rural poor in low and middle income countries. To understand factors affecting access to vaccination services, and guide future policy, smallholder farmers in three locations in India were questioned about vaccination of their cattle and buffalo, with particular reference to foot and mouth disease (FMD), haemorrhagic septicaemia (HS) and blackquarter (BQ). In the three regions 51%, 50%, and 31% of respondents reported vaccinating their livestock; well below any threshold for effective population level disease control. However, within the third region, 65% of respondents in villages immediately surrounding the Kaziranga National Park reported vaccinating their cattle. The majority of respondents in all three regions were aware of FMD and HS, awareness of BQ was high in the Kanha and Bandhavgarh regions, but much lower in the Kaziranga region. The majority of respondents had positive attitudes to vaccination; understood vaccination protected their animals from specific diseases; and wished to immunise their livestock. There was no significant association between the age or gender of respondent and the immunisation of their livestock. Common barriers to immunisation were: negative attitudes to vaccination; lack of awareness of date and time of vaccination events; and difficulty presenting animals. Poor access to vaccination services was significantly associated with not vaccinating livestock. Fear of adverse reactions to vaccines was not significantly associated with not vaccinating livestock. Respondents who reported that vets or animal health workers (AHWs) were their main source of animal health knowledge were significantly more likely to have immunised their livestock in the last twelve months. Participants cited poor communication from vaccinators as problematic, both in publicising immunisation programmes, and explaining the purpose of vaccination. Where vaccinations were provided free of charge, farmers commonly displayed passive attitudes to accessing vaccination services, awaiting organised "immunisation drives" rather than seeking vaccination themselves. Based on these findings the following recommendations are made to improve participation and effectiveness of immunisation programmes. Programmes should be planned to integrate with annual cycles of: disease risk, agricultural activity, seasonal climate, social calendar of villages; and maximise efficiency for vaccinators. Dates and times of immunisation in each village must be well publicised, as respondents frequently reported missing the vaccinators. Relevant farmer education should precede immunisation programmes to mitigate against poor knowledge or negative attitudes. Immunisation drives must properly engage beneficiaries, particularly ensuring that services are accessible to female livestock keepers, and sharing some responsibilities with local farmers. Payment of a small monetary contribution by animal keepers could be considered to encourage responsibility for disease prevention, making vaccination an active process by farmers.

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.