Refugia, climatic conditions and farm management factors as drivers of adaptation in Nematodirus battus populations.

Parasites are known for their ability to rapidly adapt to changing conditions. For parasitic helminths, changes in climate, along with farming and management practices associated with the intensification of livestock farming, provide novel challenges which can impact on their epidemiology and control. The sustainability of livestock production partially relies on effective control of helminth infection. Therefore, understanding changes in parasite behaviour, and what drives these, is of great importance. Nematodirus battus is an economically important helminth in the UK and temperate regions. Its infective larvae typically overwinter in eggs on pasture and hatch synchronously in spring, causing acute disease in lambs. Attempts to control disease typically rely on whole-flock benzimidazole (BZ) treatments. In recent years, the emergence of BZ-resistance, alongside the hatching of eggs without the classical over-winter 'chill stimulus', have made N. battus more difficult to control. In three previous studies, after collecting a large number of N. battus populations alongside farm management data from commercial farms, we explored the prevalence of genetic mutations associated with BZ-resistance (n = 253 farms), the ability of eggs to hatch with and without a chill stimulus (n = 90 farms) and how farm management practices varied throughout the UK (n = 187 farms). In the present study, we identify factors which may be acting as drivers, or barriers, to either the development of resistance or the variable hatching behaviour of N. battus eggs. Generalised linear mixed effect models were applied to regress experimental hatching and genotyping data on farm management and additional environmental data. Both variable hatching and resistance development appeared associated with the maintenance of parasite refugia as well as grazing management, particularly reseeding of pasture routinely grazed by young lambs each spring and the practice of set-stocked grazing. Effective quarantine measures were identified as the main protective factor for the development of BZ-resistance whereas set stocked grazing and population bottlenecks, resulting from reseeding heavily contaminated pastures, were risk factors. Spring maximum temperature and other climatic factors were associated with 'typical' hatching of eggs following a chill stimulus whilst several management factors were linked with hatching without prior chilling. For example, practices which reduce parasite numbers on pasture (e.g. re-seeding) or restrict availability of hosts (e.g. resting fields), were found to increase the odds of non-chill hatching. Retention of the timing of lambing and infection level of the host within the fitted model indicated that requirement for a chill stimulus prior to hatching may be plastic, perhaps subject to change throughout the grazing season, in response to immune development or parasite density-dependence within the host. Further investigation of the influence of the factors retained within the fitted models, particularly the theme of parasite refugia which was highlighted in relation to both the presence of BZ-resistance alleles and alternative hatching, is required to establish robust, sustainable parasite control and farm management strategies.

Gastrointestinal parasites of domestic sheep from Patagonia throughout historical times: A paleoparasitological approach.

Sheep husbandry in Patagonia, Argentina, started at the beginning of the 20th century from European settlers. Sanitary management is minimal, which has serious implications for the sheep health. Sheep can be infested by diverse parasites, with well over 150 species of internal and external parasites reported worldwide. Gastrointestinal parasitism is one of the most common and important infections in sheep concerning the health status, and is the cause of significant morbidity and mortality, which generates considerable production losses. The present work is the first paleoparasitological study of sheep coprolites from Patagonia throughout historical times. Fifty-seven coprolites from the 'Cueva Peligro' archaeological site (Patagonia, Argentina) were analyzed using the Lutz spontaneous sedimentation technique and the modified Faust flotation technique. Ancient DNA (aDNA) study was carried out in order to identify the zoological origin of coprolites. The results obtained from Cytb analysis confirmed the identity of the host as Ovis aries (domestic sheep). A total of 39 coprolites examined were positive for parasites by at least one of the studied methods. Thirty eight samples were positive by spontaneous sedimentation and 10 samples were positive by the modified Faust technique. The parasitic fauna found was Trichuris sp. (Trichinellida: Trichuridae), Nematodirus sp., Nematodirus spathiger (Strongylida, Trichostrongyloidea), two unidentified Strongylida-type egg morphotypes, Fasciola hepatica (Trematoda: Digenea) and coccidia oocysts of Eimeria spp. (Apicomplexa: Eimeriidae). The modified Faust technique provided satisfactory results in terms of sensitivity for the detection of Eimeria spp. The use of this methodology as a routine procedure enables the processing of ancient samples, in order to increase recoveries. These results show the importance of integrating different diagnostic approaches in order to optimize parasitic findings. The recorded parasite diversity appears to have not changed over the last 120 years. The study displayed the presence of different parasitic species which suggests potential exposure to parasitic diseases through the historical times, both for sheep and for other domestic and wild mammals from Patagonia. Also, suggests the presence of fasciolosis, a zoonotic disease that implies a potential risk for Patagonian populations in the past.

Eprinomectin 5% w/v extended-release injection for the treatment of larval and/or adult Bunostomum phlebotomum, Haemonchus contortus, H. placei, Nematodirus helvetianus, Oesophagostomum radiatum, and Trichostrongylus colubriformis infections in cattle.

In an effort to generate data for regulatory purposes on the therapeutic efficacy of eprinomectin 5% w/v extended-release injection (Eprinomectin ERI) (LongRange®, Boehringer Ingelheim) against infections of immature and adult stages of some nematode species (or stages) which are generally less common or predominantly seen in younger cattle, nine studies (minimum two per parasite and stage) were conducted in the USA and Germany. A total of 218 young cattle were included in seven experimentally induced infection studies (180 animals) and two studies with naturally acquired nematode infections (38 animals), which were compliant with WAAVP and VICH guidelines. In each study, cattle were randomly assigned into groups which received 1 mL per 50 kg body weight of either saline (controls) or Eprinomectin ERI (equivalent to 1 mg eprinomectin per kg body weight) via subcutaneous injection when the parasites were developing fourth-stage larval (L4) or adult nematodes. Following necropsy and parasite recovery, percentage efficacy was calculated based on the comparison of geometric mean nematode counts of the Eprinomectin ERI- vs. the saline-treated animals. Eprinomectin ERI-treated cattle had significantly (p < 0.05) lower counts of each species and stage of nematodes than the controls. Eprinomectin ERI treatment was demonstrated to be efficacious (> 90%) against L4 and adult Bunostomum phlebotomum and Nematodirus helvetianus; against L4 Haemonchus placei, Oesophagostomum radiatum, and Trichostrongylus colubriformis; and against adult H. contortus. Percentage efficacy against L4 H. contortus was variable (69.6 to 100%). All treatments were well accepted, and no treatment-related health problems were observed in any study.

Descriptive analysis of nematode management practices and Nematodirus battus control strategies on UK sheep farms.

BACKGROUND: Farm management practices have a major impact on nematode population dynamics. The presented study aimed to understand current nematode management practices on UK sheep farms; with a particular focus on Nematodirus battus because of the changing epidemiology, and emerging anthelmintic resistance observed in this species. METHODS: A 42 question online survey covering grazing management, farm demographics and parasite control strategies was developed and distributed to the farming community in 2016. Analysis of the 187 completed questionnaires explored regional variations in practices. RESULTS: Uptake of recommendations was variable, particularly quarantine practices and monitoring tools. Results also highlighted variation in the epidemiology of N. battus; respondents in the north (Scotland, north-west and north-east England) typically reported N. battus in spring with a perception of more severe clinical symptoms than those from the south (Midlands, Wales, south-east and south-west England; p = 0.03). Farms in the south observed greater changes in the timing of disease (p = 0.006) with N. battus being reported throughout the year on some holdings and more frequent use of faecal egg count monitoring (p = 0.006). CONCLUSIONS: Control of N. battus infection is challenging and 'one-size-fits-all' advice is not applicable; however, the information gathered will enable the development of effective, adaptable control strategies.

Comparing two predictive risk models for nematodirosis in Great Britain.

BACKGROUND: Nematodirus battus infection is a major health concern in lambs. Development and hatch of infective larvae on pastures is temperature dependent, making model-based risk forecasting a useful tool for disease control. METHODS: Air and 30 cm soil temperature-based risk models were used to predict hatch dates using meteorological data from 2019 and compared to infection dates, estimated from the first appearance of N. battus eggs, on 18 sheep farms distributed across Great Britain. RESULTS: The air temperature model was more accurate in its predictions than the soil temperature model on 12 of the 18 farms, but tended to predict late hatch dates in the early part of the season. CONCLUSION: Overall, the air temperature model appears the more appropriate choice for predicting N. battus peak hatch in the UK in terms of accuracy and practicality, but some adjustment might be needed to account for microclimatic variations at the soil-air interface.

Prevalence of trichostrongylus in sheep in the district Zhob, Balochistan, Pakistan / [Prevalência de trichostrongylus em ovelhas no distrito Zhob, Balochistão, Paquistão]

Trichostrongylid em ovelhas foi estudado no Distrito Zhob, Balochistan. Foram coletados 120 tratos gastrointestinais (GIT) de ovelhas do matadouro do distrito. Estas amostras foram processadas para isolamento e identificação de nematódeos tricostrônquicos no Laboratório do Hospital Veterinário do Distrito Zhob. A taxa de prevalência geral foi de 39,1% em machos e 60,8% em fêmeas (p=0,001). A taxa de prevalência em duas raças viz Balochi e Rakhshani foi de 58,3% e 41,6%, respectivamente (p=0,01). A prevalência da espécie observada com Trichostrongylus foi 19,1%, Haemonchus foi 20,8%, Cooperia foi 29,1% e Nematodirus foi 30,8% (p=0,087). Quanto a quantidade de espécies infestadas pelas ovelhas, um único tipo de parasita estava em 32,5% de animais, dois tipos de espécies parasitárias em 36,3% dos animais e três tipos de espécies parasitárias em 30,8% dos animais (p=0,366).(AU)

Variation in hatching responses of Nematodirus battus eggs to temperature experiences.

BACKGROUND: Nematodirus battus, unlike most other gastrointestinal nematodes, undergoes maturation to an infective larva within the egg. Historically, eggs were considered to require a period of chilling over winter followed by a period of temperature above 10 °C for synchronous hatching to occur (generally in spring). Anecdotal reports of Nematodirus infection out-with spring in veterinary journals and the farming press suggest that the concentrated pasture abundance of N. battus infective larvae may be changing. In order for control practices to be adapted, and unexpected disease outbreaks to be avoided, it is important to quantify how parasite epidemiology is changing and research the drivers behind it. METHOD: The present study investigated the in vitro hatching response to temperature experiences (with and without a period of chilling) for egg samples of 90 N. battus populations obtained from 73 commercial sheep farms. Six aliquots of larvated eggs were prepared per population, three aliquots were placed at 4 °C for 6  weeks to provide a chill stimulus then incubated at the optimal hatching temperature for the species. The remaining three aliquots of eggs were incubated at the hatching temperature without a prior chill stimulus and the number of hatched larvae was compared between treatments. RESULTS: Median hatch rate across all populations with chilling was 45% (95% CI: 42-48%) and without chilling was 4% (95% CI: 2-6%). Inter-population variation in hatching ranged from 0 to 87% of eggs able to hatch in the absence of a chill stimulus, mean non-chill hatching was 13 ± 2% of eggs (mean ± SE). Non-chill hatching rates were greater than chilled hatching rates in seven of the 90 populations tested. CONCLUSIONS: Clearly, the variation in hatching responses to temperature experience is very large and therefore the seasonality of the parasite may vary not only between regions but also at farm level. In contrast to what previous work has suggested, there was a geographical trend towards higher non-chill hatching in the Northern parts of the UK.

Large scale screening for benzimidazole resistance mutations in Nematodirus battus, using both pyrosequence genotyping and deep amplicon sequencing, indicates the early emergence of resistance on UK sheep farms.

Benzimidazoles (BZ) have been the anthelmintic of choice for controlling Nematodirus battus infections since their release in the 1950s. Despite heavy reliance on this single anthelmintic drug class, resistance was not identified in this nematode until 2010 (Mitchell et al., 2011). The study aimed to explore the prevalence of BZ-resistance mutations in N. battus from UK sheep flocks using deep amplicon sequencing and pyrosequencing platforms. Based on evidence from other gastrointestinal nematodes, resistance in N. battus is likely to be conferred by single nucleotide polymorphisms (SNP) within the ß-tubulin isotype 1 locus at codons 167, 198 and 200. Pyrosequencing and deep amplicon sequencing assays were designed to identify the F167Y (TTC to TAC), E198A (GAA to GCA) and F200Y (TTC to TAC) SNPs. Nematodirus battus populations from 253 independent farms were analysed by pyrosequencing; 174 farm populations were included in deep amplicon sequencing and 170 were analysed using both technologies. F200Y was the most prevalent SNP identified throughout the UK, in 12-27% of the populations tested depending on assay, at a low overall individual frequency of 2.2 ±â€¯0.6% (mean ±â€¯SEM, based on pyrosequencing results). Four out of the five populations with high frequencies (>20%) of the F200Y mutation were located in NW England. The F167Y SNP was identified, for the first time in this species, in four of the populations tested at a low frequency (1.2% ±â€¯0.01), indicating the early emergence of the mutation. E198A or E198L were not identified in any of the isolates. Results obtained were comparable between both techniques for F200Y (Lins' CCC, rc = 0.96) with discrepancies being limited to populations with low frequencies. The recent emergence of resistance in this species will provide a unique opportunity to study the early stages of anthelmintic resistance within a natural setting and track its progress in the future.

Gastrointestinal parasites in reindeer (Rangifer tarandus tarandus) calves from Fennoscandia: An epidemiological study.

Reindeer (Rangifer tarandus tarandus) host numerous parasites. Although there is a general knowledge about parasite diversity in reindeer, detailed baseline information about parasitic infections is limited. Detailed knowledge of parasite prevalence and diversity provide a pathway for more targeted parasite control, an increasing need expected in the future. The main aim of our cross-sectional study was to estimate the prevalence of gastrointestinal parasites in semidomesticated reindeer calves. The 480 reindeer calves included in our study were aged 6-7 months, originated from 9 reindeer herding cooperatives in Finland and 1 in Norway, and were slaughtered during September-November 2015 in 10 reindeer slaughterhouses. All the reindeer calves passed meat inspection, and the detected parasitic infections were subclinical. As the reindeer included in this study were young animals intended for slaughter, they had never been administrated any antiparasitic treatment. Assessments of gastrointestinal parasitism among these reindeer calves were based on fecal examination and morphological identification of coccidian oocysts or helminth eggs. Individual fecal samples collected from the rectum of each of the reindeer were examined using a modified McMaster method. Most (78.3%) of the reindeer calves had eggs or oocysts of at least one parasite species in their feces, and more than half (53.5%) had a mixed infection. Strongylid eggs were detected in 75.6%, Eimeria sp. oocysts in 50.6%, Moniezia sp. eggs in 28.1%, Nematodirus sp. eggs in 22.1%, Capillaria sp. eggs in 9.4%, and Trichuris sp. eggs in 0.6% of the samples. The prevalence of gastrointestinal parasites was similar or higher relative to previous estimates from the region; the proportion of reindeer calves shedding strongylid eggs and the proportion of reindeer calves shedding Moniezia sp. eggs had increased. Prevalence varied by geographical region, which may reflect different herding practices or environmental parameters. Higher reindeer density was a risk factor for testing positive for Eimeria sp. oocysts, and the odds of testing positive for Nematodirus sp. eggs were higher if a peroral route was used for antiparasitic treatment in the reindeer herding cooperative. The mean proportion of reindeer estimated to receive antiparasitic treatment in Finland was 86% in 2004-2005 and 91% in 2014-2015. During the historical time frames of current management practices, this routine annual antiparasitic treatment of breeding reindeer has not decreased the prevalence of gastrointestinal parasites in reindeer calves, which can be seen as sentinels or indicators of the infection pressure.

Apex Predators, Rockshelters, and Zoonoses in the Patagonian Holocene.

Carnivorous mammals are a trophic guild with an important role in the dissemination of parasite infective stages (larvae, eggs, cysts, and oocysts). In the present study, new samples of coprolites attributed to carnivorous mammals, obtained from 2 archaeological caves, were analyzed for the presence of parasites with the aim to increase the knowledge about parasites in rockshelters that could have spread to humans and other mammals. To this purpose, fragments of 3 coprolites from Cerro Casa de Piedra, cave 5 and cave 7, were examined. Coprolites were rehydrated in aqueous trisodium phosphate and processed by spontaneous sedimentation. High parasite richness was observed and new parasite species for archaeological contexts were found. The parasitological findings in Puma concolor coprolites associated with caves suggest the importance of these carnivores in the dissemination of parasites in areas with high re-use of space and steady conditions of temperature, humidity, and radiation.

An investigation into the effects of maternal supplementation with excess iodine on the mechanisms and impacts of reduced IgG absorption in the lamb postpartum.

An experiment was conducted to determine: (1) the effect of excess maternal I supplementation on the thyroid hormone status of the ewe and her progeny; (2) potential mechanisms underpinning the failure of passive transfer associated with excess I and (3) the growing lambs' response to natural gastrointestinal infection. Twin-bearing ewes received one of two treatments (n 32/treatment group): basal diet (C) or C plus 26·6 mg of iodine/ewe per d (I), supplied as calcium iodate. Ewes were individually fed from day 119 of gestation to parturition. Progeny of I ewes had lower (P<0·01) serum IgG concentrations from 24 h to 28 d postpartum but higher serum IgG concentrations at day 70 postpartum (P<0·05). I supplementation increased the relative expression of Fc receptor, IgA, IgM high affinity and polymeric Ig receptor in the ileum of the lamb at 24 h postpartum; however, thyroid hormone receptor-ß (THRB) and ß-2-microglobulin (B2M) expression declined (P<0·05). Progeny of I ewes had higher growth rates to weaning (P<0·05) and lower faecal egg count (FEC) for Nematodirus battus (P<0·05) between weeks 6 and 10 postpartum. In conclusion, excess maternal I supplementation negatively affected the thyroid hormone status, serum IgG concentration, ileal morphology and the gene expression of THRB and B2M in the ileum and ras-related protein (RAB) RAB25 and the mucin gene (MUC) MUC1 in the duodenum of the lamb postpartum. These effects were followed by an enhancement of average daily gain and lower N. battus FEC in the pre-weaning period of I-supplemented lambs.

Chilling requirements for hatching of a New Zealand isolate of Nematodirus filicollis.

The eggs of some species of the parasitic nematode Nematodirus require a period of chilling before they can hatch; N. filicollis is one such species. This study investigated this requirement for chilling in a New Zealand strain of this species. Eggs of N. filicollis were extracted from lamb's faeces and incubated at 20°C to allow development to the third stage larvae within the egg. These eggs were then placed into tissue culture plates and incubated at: 2.7°C (±0.99), 3.6°C (±0.90), 4.7°C (±0.35), 6.4°C (±0.37), 8.0°C (±1.54) or 9.9°C (±0.14) for up to 224 days. At 14day intervals until day 84, then every 28 days, one plate was removed from each temperature and placed at 13.1°C (±0.44) for 14 days. Eggs were then assessed for hatching. From this data, chill units were calculated by subtracting the culture temperature from a constant threshold of 11°C and multiplying by the number of days for which the sample was cultured; then the Gompertz model fitted. Even though hatching overall was low, a greater proportion of eggs hatched with chill accumulation. Maximum hatching of eggs required 800-1000 chill units. Consequently in the field, more than one season of chilling would be required before hatching. As such a generation time could take more than one year to complete. This is different to the hatching dynamics of N. spathiger, the other main species found in New Zealand sheep, which does not display this requirement for chilling and hatches immediately once the third stage larvae are developed.

Pooling sheep faecal samples for the assessment of anthelmintic drug efficacy using McMaster and Mini-FLOTAC in gastrointestinal strongyle and Nematodirus infection.

In small ruminants, faecal egg counts (FECs) and reduction in FECs (FECR) are the most common methods for the assessment of intensity of gastrointestinal (GI) nematodes infections and anthelmintic drug efficacy, respectively. The main limitation of these methods is the time and cost to conduct FECs on a representative number of individual animals. A cost-saving alternative would be to examine pooled faecal samples, however little is known regarding whether pooling can give representative results. In the present study, we compared the FECR results obtained by both an individual and a pooled examination strategy across different pool sizes and analytical sensitivity of the FEC techniques. A survey was conducted on 5 sheep farms in Scotland, where anthelmintic resistance is known to be widespread. Lambs were treated with fenbendazole (4 groups), levamisole (3 groups), ivermectin (3 groups) or moxidectin (1 group). For each group, individual faecal samples were collected from 20 animals, at baseline (D0) and 14 days after (D14) anthelmintic administration. Faecal samples were analyzed as pools of 3-5, 6-10, and 14-20 individual samples. Both individual and pooled samples were screened for GI strongyle and Nematodirus eggs using two FEC techniques with three different levels of analytical sensitivity, including Mini-FLOTAC (analytical sensitivity of 10 eggs per gram of faeces (EPG)) and McMaster (analytical sensitivity of 15 or 50 EPG).For both Mini-FLOTAC and McMaster (analytical sensitivity of 15 EPG), there was a perfect agreement in classifying the efficacy of the anthelmintic as 'normal', 'doubtful' or 'reduced' regardless of pool size. When using the McMaster method (analytical sensitivity of 50 EPG) anthelmintic efficacy was often falsely classified as 'normal' or assessment was not possible due to zero FECs at D0, and this became more pronounced when the pool size increased. In conclusion, pooling ovine faecal samples holds promise as a cost-saving and efficient strategy for assessing GI nematode FECR. However, for the assessment FECR one will need to consider the baseline FEC, pool size and analytical sensitivity of the method.

Benzimidazole resistance in Nematodirus spathiger and N. filicollis in New Zealand.

AIM: To determine the prevalence of benzimidazole resistance in Nematodirus spathiger and N. filicollis from a sample of New Zealand farms. METHODS: The efficacy of albendazole (ABZ) against Nematodirus spp. was assessed by faecal nematode egg count reduction (FECR) tests undertaken in lambs aged 3-8 months old on 27 sheep farms throughout New Zealand. On each farm, groups of 10-16 lambs were either treated with ABZ (4.75 mg/kg) or remained as untreated controls. Faecal samples were collected from all animals at the time of treatment and 7-10 days later. Faecal nematode egg counts (FEC) were performed using a modified McMaster technique. Larvae were cultured from pooled faecal samples, collected 7-10 days after treatment from each group, by incubation at 20°C for 6 weeks, 4°C for 26 weeks then 13°C for 2 weeks. The resulting third stage larvae were identified to species using a multiplex PCR assay, that identified species-specific sequences in the second internal transcribed spacer region of ribosomal DNA. The efficacy of ABZ for N. spathiger and N. filicollis was calculated from the proportion of the two species in culture and the group mean FEC before and after treatment. Only farms with a mean of 10 epg for each species in untreated samples were included for analysis. Resistance was defined as an efficacy <95%. RESULTS: On farms that met the threshold of 10 epg in faecal samples, benzimidazole resistance was found on 20/21 (95%) farms for N. spathiger compared with 4/10 (40%) farms for N. filicollis (p<0.05). In samples collected following treatment, a mean of 83 (min 46, max 100)% of Nematodirus spp. larvae recovered from the untreated groups were N. spathiger, compared with 94 (min 45, max 100)% in the ABZ treated groups (p=0.03). This change in percentage was not influenced by the overall efficacy of treatment based on the FECR test (p=0.324). CONCLUSION: The results confirm the high level of resistance in N. spathiger in New Zealand and that benzimidazole resistance was more common in N. spathiger than N. filicollis. While resistance to benzimidazole anthelmintics has been reported previously in New Zealand, this is the first report of N. filicollis being resistant to benzimidazole anthelmintics.

Finding new ways to prevent disease in food-producing animals.

Increasing concern about antimicrobial resistance and moves to restrict the use of antibiotics in food-producing animals mean that farmers will need new ways of preventing and controlling disease in their animals. With its focus on addressing the needs of the farming industry, the Moredun Research Institute sees this as an opportunity to be at the forefront of developing new solutions. Kristy Ebanks reports from an event organised to showcase some of the institute's latest research.

Asynchrony in host and parasite phenology may decrease disease risk in livestock under climate warming: Nematodirus battus in lambs as a case study.

Mismatch in the phenology of trophically linked species as a result of climate warming has been shown to have far-reaching effects on animal communities, but implications for disease have so far received limited attention. This paper presents evidence suggestive of phenological asynchrony in a host-parasite system arising from climate change, with impacts on transmission. Diagnostic laboratory data on outbreaks of infection with the pathogenic nematode Nematodirus battus in sheep flocks in the UK were used to validate region-specific models of the effect of spring temperature on parasite transmission. The hatching of parasite eggs to produce infective larvae is driven by temperature, while the availability of susceptible hosts depends on lambing date, which is relatively insensitive to inter-annual variation in spring temperature. In southern areas and in warmer years, earlier emergence of infective larvae in spring was predicted, with decline through mortality before peak availability of susceptible lambs. Data confirmed model predictions, with fewer outbreaks recorded in those years and regions. Overlap between larval peaks and lamb availability was not reduced in northern areas, which experienced no decreases in the number of reported outbreaks. Results suggest that phenological asynchrony arising from climate warming may affect parasite transmission, with non-linear but predictable impacts on disease burden. Improved understanding of complex responses of host-parasite systems to climate change can contribute to effective adaptation of parasite control strategies.

Molecular approaches to differentiate three species of Nematodirus in sheep and goats from China based on internal transcribed spacer rDNA sequences.

Internal transcribed spacer (ITS) rDNA sequences of three Nematodirus species from naturally infected goats or sheep in two endemic provinces of China were analysed to establish an effective molecular approach to differentiate Nematodirus species in small ruminants. The respective intra-specific genetic variations in ITS1 and ITS2 rDNA regions were 0.3-1.8% and 0-0.4% in N. spathiger, 0-6.5% and 0-5.4% in N. helvetianus, and 0-4.4% and 0-6.1% in N. oiratianus from China. The respective intra-specific variations of ITS1 and ITS2 were 1.8-4.4% and 1.6-6.1% between N. oiratianus isolates from China and Iran, 5.7-7.1% and 6.3-8.3% between N. helvetianus samples from China and America. For N. spathiger, compared with samples from China, sequence differences in ITS1 rDNA were 0.3-2.4% in isolates from America, 0.3-2.9% in New Zealand and 2.1-2.4% in Australia. Genetic variations in ITS2 rDNA of N. spathiger were 0-0.4% between samples from China and America, and 0-0.8% between samples from China and New Zealand. Using mutation sites, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and specific PCR techniques were developed to differentiate these three Nematodirus species. The specific PCR assay allowed the accurate identification of N. oiratianus from other common nematodes with a sensitivity of 0.69 pg and further examination of Nematodirus samples demonstrated the reliability of these two molecular methods.

Phenotypic and genotypic analysis of benzimidazole resistance in the ovine parasite Nematodirus battus.

Benzimidazole resistance is common amongst many ovine trichostrongylid nematodes species globally. Although anthelmintics have been used for over half a century in some areas of the world for the control of Nematodirus battus, resistance has never been detected. Veterinary investigations conducted in 2010 demonstrated reduced efficacy in a flock that had been treated previously with fenbendazole (FBZ), suggesting probable resistance in N. battus. Infective larvae (L3; designated MNba2) were generated from the original material to conduct a controlled efficacy test (CET). Faecal egg counts showed an average of 37% reduction in the FBZ treated group 7 days post treatment compared to the untreated lambs. Average worm burden results showed no reduction after FBZ treatment compared to the untreated group (3850 and 3850 worms respectively). A molecular assay to assess the frequency of the commonly associated single nucleotide polymorphisms (SNP) in the ß-tubulin isotype 1 gene, F200Y and E198A, was developed. Larval genotypes were predominantly homozygous resistant at codon 200 SNP, ranging from 56%-83% and remained stable at 70% for adult worm populations taken from treated and control lambs in the CET. Only susceptible genotypes were found at codon 198. The allele frequency for F200Y ranged between 80-83% in adult worms taken from the CET from treated and control lambs. The results confirmed initial findings and demonstrated the first report of FBZ resistance in N. battus whilst providing evidence that the P200 point mutation in the ß-tubulin isotype 1 gene is a potential mechanism of resistance in the species.