Next-generation phylogeography resolves post-glacial colonization patterns in a widespread carnivore, the red fox (Vulpes vulpes), in Europe.

Carnivores tend to exhibit a lack of (or less pronounced) genetic structure at continental scales in both a geographic and temporal sense and this can confound the identification of post-glacial colonization patterns in this group. In this study we used genome-wide data (using genotyping by sequencing [GBS]) to reconstruct the phylogeographic history of a widespread carnivore, the red fox (Vulpes vulpes), by investigating broad-scale patterns of genomic variation, differentiation and admixture amongst contemporary populations in Europe. Using 15,003 single nucleotide polymorphisms (SNPs) from 524 individuals allowed us to identify the importance of refugial regions for the red fox in terms of endemism (e.g., Iberia). In addition, we tested multiple post-glacial recolonization scenarios of previously glaciated regions during the Last Glacial Maximum using an Approximate Bayesian Computation (ABC) approach that were unresolved from previous studies. This allowed us to identify the role of admixture from multiple source population post-Younger Dryas in the case of Scandinavia and ancient land-bridges in the colonization of the British Isles. A natural colonization of Ireland was deemed more likely than an ancient human-mediated introduction as has previously been proposed and potentially points to a larger mammalian community on the island in the early post-glacial period. Using genome-wide data has allowed us to tease apart broad-scale patterns of structure and diversity in a widespread carnivore in Europe that was not evident from using more limited marker sets and provides a foundation for next-generation phylogeographic studies in other non-model species.

Lack of efficacy of triclabendazole against Fasciola hepatica is present on sheep farms in three regions of England, and Wales.

The liver fluke Fasciola hepatica is a parasitic trematode that has a major impact on livestock production and human health. Control of F hepatica is difficult and relies on anthelmintics, particularly triclabendazole, due to its efficacy against both adult and juvenile stages of the parasite. Emergence of triclabendazole-resistant F hepatica populations has been reported in a number of countries, including the UK, but the overall prevalence and distribution of triclabendazole resistance is unknown. In this study, the authors established the presence of reduced efficacy of triclabendazole in sheep flocks in England and Wales, using a validated composite faecal egg count reduction test. Seventy-four sheep farms were sampled from Wales, southwest, northwest and northeast England between Autumn 2013 and Spring 2015. F hepatica eggs were detected in samples from 42/74 farms. Evidence of a lack of efficacy of triclabendazole was detected on 21/26 farms on which the faecal egg count reduction test was completed, with faecal egg count reductions ranging from 89 per cent to 0per cent. Regression analysis suggested that both prevalence of F hepatica and lack of efficacy of triclabendazole were spatially correlated, with higher faecal egg counts and lower percentage reductions on farms located in the northwest of England, and Wales. Overall, the results show that reduced efficacy of triclabendazole is present across England and Wales, with a complete lack of therapeutic efficacy observed on 9/26 farms.

An observational study of ewe treatments at lambing on early infection in lambs on UK sheep farms.

Anthelmintic treatments to ewes around lambing time, often with long acting products, have become common practice on UK sheep farms, but these treatments have also been shown to be highly selective for anthelmintic resistance in New Zealand and Australia, with field data supported by modelling results. The purpose of this study was to determine (1) the effect of treating or withholding anthelmintic treatments and (2) the effect of treatment of ewes with a persistent or non-persistent anthelmintic, on early infection in lambs in the UK. Faecal egg count data for 10-16 weeks old lambs collected over a three year period (2012-2014) was analysed. Samples were grouped according to whether the ewes on the farm had or had not been treated with an anthelmintic at lambing. For both analyses, data for early infection were analysed by generalised linear mixed model. There was no effect of withholding or treating ewes on subsequent early infection in lambs. In addition, there was no effect of region, farm type or management type on the faecal egg counts. There was, however an effect of year, with lambs having lower counts in 2014 than in 2012 and an interaction between year and ewe treatment, with data suggesting lower infection levels over time for those farms withholding anthelmintic treatments altogether. There was no effect of drug type on early infection in lambs nor region, farm type or treatment on the lamb faecal egg counts. However, there was an effect of year and an interaction between year and drug type with lower egg count over time with the short acting drugs. Our study supports data generated by other researchers suggesting that the practice of treating ewes at lambing to reduce contamination on pasture and minimise subsequent disease may not in fact always result in lower levels of infection in lambs. The study also demonstrated no significant benefit in early infection in lambs when ewes were treated with long acting compared to short acting anthelmintics. This provides further evidence to support the potential benefits of a more targeted approach to anthelmintic treatment on sheep farms.

Resistance delaying strategies on UK sheep farms: A cost benefit analysis.

UK guidelines for the sustainable control of parasites in sheep (SCOPS) were formulated with the primary aim of delaying development of anthelmintic resistance (AR) on UK sheep farms. Promoting their use requires the engagement and commitment of stakeholders. An important driver for behavioural change in sheep farmers is evidence of economic benefits. A recent evaluation of SCOPS guidance in practice demonstrated a significant reduction in anthelmintic use, suggesting economic benefits through a direct reduction in product and labour costs. However, in order to maintain production, a range of alternative control strategies are advised, resulting in additional costs to farmers and so a full cost benefit analysis of best practice management was undertaken. We allocated financial values to the management recommendations described in the SCOPS technical manual. Benefits were calculated using data for production variables and anthelmintic use measured during studies to evaluate the effect of SCOPS recommendations on 16 UK sheep farms and from other published work. As SCOPS control is not prescriptive and a range of different diagnostics are available, best and worst case scenarios were presented, comparing the cheapest methods (e.g. egg counts without larval culture) and management situations (e.g closed flocks not requiring quarantine treatments) with the most laborious and expensive. Simulations were run for farms with a small, medium or large flock (300; 1000; 1900 ewes) as well as comparing scenarios with and without potential production benefits from using effective wormers. Analysis demonstrated a moderate cost for all farms under both scenarios when production benefits were not included. A cost benefit was demonstrated for medium and large farms when production benefits were included and the benefit could be perceived as significant in the case of the large farms for the best case scenario (>£5000 per annum). Despite a significant potential reduction in anthelmintic use by farmers employing SCOPS guidance, the very low price of the older anthelmintic classes meant that the benefit did not always outweigh the additional management/diagnostic costs unless an increase in production was also achieved. This is an important finding. Focussing research on key innovations that will improve the cost effectiveness of diagnostic assays in a diagnostic driven control strategy, as well as designing treatment options that can improve production outcomes, and presenting them in a clear and transparent way, must be high priority goals. Coupling targeted research with improvements in the delivery of messages to the end user is important in the light of increasing global concerns over drug resistance.

Simulating control of a focal wildlife outbreak of Echinococcus multilocularis.

The parasitic tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a serious zoonotic infection present in Europe that can be fatal. The United Kingdom currently has E. multilocularis free status but the possibility of introduction exists, most likely via an imported or returning dog or other deliberately introduced animal that has not had anthelmintic treatment. We have developed a model to predict the probability of successfully eliminating a focal outbreak of E. multilocularis using a programme of anthelmintic bait distribution. We investigated three different potential control programmes, each with 36 monthly campaigns commencing five, ten or 15 years after disease introduction over an area of 2827km2. We assumed equilibrium disease prevalence of 30%, 40% and 55% based on the range of values reported across Europe. However, for all of these scenarios, equilibrium had not been reached at five to 15 years after introduction and simulated local prevalence values were between 0.5% and 28%. We found that it is possible to eliminate the disease with a 38%-86% success rate if control is started five years after introduction, dropping to 0% to 56% if control is delayed until 15 years after introduction, depending upon the prevalence equilibrium. We have also estimated the costs involved in these programmes to be from €7 to €12 million (2013 prices).

Serological and molecular epidemiology of canine adenovirus type 1 in red foxes (Vulpes vulpes) in the United Kingdom.

Canine adenovirus type 1 (CAV-1) causes infectious canine hepatitis (ICH), a frequently fatal disease which primarily affects canids. In this study, serology (ELISA) and molecular techniques (PCR/qPCR) were utilised to investigate the exposure of free-ranging red foxes (Vulpes vulpes) to CAV-1 in the United Kingdom (UK) and to examine their role as a wildlife reservoir of infection for susceptible species. The role of canine adenovirus type 2 (CAV-2), primarily a respiratory pathogen, was also explored. In foxes with no evidence of ICH on post-mortem examination, 29 of 154 (18.8%) red foxes had inapparent infections with CAV-1, as detected by a nested PCR, in a range of samples, including liver, kidney, spleen, brain, and lung. CAV-1 was detected in the urine of three red foxes with inapparent infections. It was estimated that 302 of 469 (64.4%) red foxes were seropositive for canine adenovirus (CAV) by ELISA. CAV-2 was not detected by PCR in any red foxes examined. Additional sequence data were obtained from CAV-1 positive samples, revealing regional variations in CAV-1 sequences. It is concluded that CAV-1 is endemic in free-ranging red foxes in the UK and that many foxes have inapparent infections in a range of tissues.

The development of anthelmintic resistance with best practice control of nematodes on commercial sheep farms in the UK.

Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections. The widespread development of anthelmintic resistance is a major global issue affecting the effective control of parasite diseases in grazing livestock production. Sustainable control strategies that reduce dependence on antimicrobials have the potential to slow the further development of resistance but there is little data on the effect of control strategies on resistance development in the field. This report documents a study undertaken to measure the temporal effect of the UK sustainable control of parasites in sheep (SCOPS) guidelines on the development of anthelmintic resistance. Farms carrying out SCOPS or traditional worm control (TRADITIONAL) were tested for resistance to the benzimidazole and imidazothiazole anthelmintics in vitro using a discriminating dose (dd) larval development test (LDT) in year 1 and then 7 years later. In years 5 and 7, resistance was also measured using a dose-response LDT assay. There was a significant increase in Teladorsagia survivors at the benzimidazole dd assay between year 1 and year 7 for both treatment groups, but the increase in survivors was greater for the farms carrying out their traditional worm control compared to the SCOPS farms. There was also a significant difference between benzimidazole dd results generated across years for Trichostrongylus, but the year and treatment interaction was not significant. Only one of the farm Teladorsagia populations had survivors in the imidazothiazole dd assay in years 1 and 7 and none of the Trichostrongylus populations survived in year 1 compared to isolates from three of the farms in year 7. Dose-response data showed a significant effect for time for both the benzimidazole and imidazothiazole anthelmintics and the increase was again significantly higher for the Teladorsagia populations in the TRADITIONAL group compared to the SCOPS group. This data suggests an increased sensitivity both to detect and to measure changes in response to anthelmintics with the dose-response assay compared to the dd and this is important particularly when allele frequencies are low as might be the case when novel compounds are released to the market. Anthelmintic use across years 5-7 was significantly lower for the farms in the SCOPS group compared to the TRADITIONAL group and farmers in the SCOPS group had selected products from the benzimidazole group less often than farmers in the TRADITIONAL group. Both groups had made minimal use of the imidazothiazole anthelmintic classes and the majority of ewe treatments were selected from the macrocyclic lactone class. Further research is required to determine the effect of these anthelmintic choices on the development of resistance to the macrocyclic lactones.

Three-year evaluation of best practice guidelines for nematode control on commercial sheep farms in the UK.

Anthelmintics are commonly used on the majority of UK commercial sheep farms to reduce major economic losses associated with parasitic diseases. With increasing anthelmintic resistance worldwide, several countries have produced evidence-based, best practice guidelines with an example being the UK's Sustainable Control of Parasites in Sheep (SCOPS) initiative. In 2012, a pilot study demonstrated that SCOPS-managed farms used fewer anthelmintic treatments than traditionally managed farms, with no impact on lamb productivity and worm burden. Building on these results, we collected data for three consecutive years (2012-2014) with the following aims: (1) To compare the effects of traditional and SCOPS-based parasite management on lamb productivity and worm burden; (2) To evaluate the effect of region and farm type on lamb productivity and worm burden; (3) To compare the frequency and patterns of use of anthelmintic treatment on traditional and SCOPS-managed farms. The study was carried out on 16 farms located in the North east and the South west of England and Wales. Lamb productivity was assessed by quantifying birth, mid-season and finish weights and calculating daily live-weight gains and time to finish in a cohort of 40-50 lambs on each farm. Five annual faecal egg counts were carried out on each farm to assess worm burden. No differences in lamb productivity and worm burdens were found between farms that adopted SCOPS guidelines and traditional farms across the three years. However, mean infection levels increased for both the SCOPS and the traditional groups. Lamb production was not significantly different for farm type and region but the effect of region on infection was significant. For both ewes and lambs, SCOPS farms carried out significantly fewer anthelmintic treatments per year, and used fewer anthelmintic doses/animal than traditional farms. The data suggest a trend to increasing use of anthelmintics in ewes on traditional but not on the SCOPS farms and a decreasing use of anthelmintics in lambs on both SCOPS and traditional farms. Across time, an increasing number of SCOPS farmers left their ewes and lambs untreated and the reverse was true for traditional farmers. Overall, farms implementing SCOPS guidelines used less anthelmintic treatments and less frequently than traditionally managed farms, without loss of animal performance or increased worm burden. Implementing SCOPS guidelines might have economic benefits for farmers, help reduce development of anthelmintic resistance on farms and decrease any environmental impact of anthelmintics. Furthermore, these data suggest some important epidemiological trends that should be investigated in long-term studies.

First report of Trichinella pseudospiralis in a red fox in mainland Britain.

Active surveillance of red foxes for Trichinella has been undertaken in mainland Britain since 1999. Post-mortems are carried out, followed by a magnetic stirrer method for sample digestion based on European Commission (EC) Regulation 216/2014 (which amends 2075/2005). Initially samples are tested in batches of 20 foxes and in December 2013, for the first time under the surveillance programme, a batch tested positive for Trichinella at the Animal and Plant Health Agency, York. Further individual tests identified one infected fox, from the Bristol area. The larvae were identified as Trichinella pseudospiralis. This is the first report of T. pseudospiralis in Great Britain and suggests the possibility of a cycle of infection existing in wildlife.

Evaluation of 'best practice' (SCOPS) guidelines for nematode control on commercial sheep farms in England and Wales.

Parasitic diseases are a major constraint to optimum livestock production and are the major cause of economic loss in UK sheep flocks, with farmers remaining dependant on anthelmintics for control. In the UK, research and evidence based, "best practice" guidelines for sustainable control of parasites in sheep (SCOPS) were first produced in 2004 and have been regularly updated since. This study was designed to evaluate the effect of these best practice guidelines for worm control on lamb production and infection levels, compared with more traditional management. Sixteen farms were selected based on a 2 cube factorial design with 3 factors known to affect worm epidemiology: control regimen; farm type; and climatic region. A formalised plan for worm control using 7 potential resistance-delaying practices was prepared for each of the 8 best practice (SCOPS) farms, in conjunction with the farms veterinarians. The 8 farms in the traditional management group (CONTROL farms) were selected based on ongoing evidence of them using worm control strategies deemed to be "higher-risk". A cohort of 40-50 study lambs at each farm was monitored from birth to finishing, allowing evaluation of lamb productivity, worm infection levels and for comparison of numbers of anthelmintic treatments. Birth and mid-season weights were used to calculate daily live-weight gain. Birth and finish dates were used to calculate time to finish and finish weights were also compared. Faecal egg counts, larval culture and species differentiation were undertaken throughout the year to assess the impact of the control strategies on worm burdens. There was no significant difference in results for any of the 3 production responses when comparing predicted means accounting for the differences in birth weight. In fact SCOPS farms had, on average, a higher daily weight gain and finish weight than CONTROL farms when comparing observed means. Statistical analysis of infection levels clearly showed no significant effect according to farm type (p=0.71) or treatment (p=0.81). In contrast the effect of region (p=0.08), although not significant, had a much larger effect size (standardised mean difference) with lower parasite burdens based on faecal egg counts on Northern farms compared to Southern farms. For both ewes and lambs, significantly fewer treatments were carried out on the SCOPS farms. The data collected from this study suggests that farms implementing SCOPS principles use less anthelmintic than other farms, without loss of animal performance or increased worm burden.

Development and validation of real-time PCR methods for diagnosis of Teladorsagia circumcincta and Haemonchus contortus in sheep.

Parasitic gastroenteritis, a disease caused by parasitic nematodes, is of major concern to the sheep industry and threatens sustainability. Traditional methods for diagnosis of the type and level of infection in a sheep flock require laborious laboratory extraction, culture and microscopic examination of eggs or larvae from faecal samples. Advances in molecular technology offer the potential for more efficient and reliable methods. This study aimed to develop and test a real-time PCR method for routine diagnosis of infection by Teladorsagia circumcincta and Haemonchus contortus in sheep. Primer/probe sets were designed around the ribosomal internal transcribed spacer 2 (ITS2) region as sequence data was available from other studies and so tests used published primer/probe sets, as well as those designed at the Food and Environment Research Agency (Fera). Different primer/probe combinations were tested for specificity against DNA extracted from T. circumcincta larvae or H. contortus DNA. All sets were tested for cross reactivity against four other closely related species, using real-time PCR technology. Reactions were optimised with the best primer/probe combination for each species and then tested for sensitivity against samples containing different T. circumcincta or H. contortus DNA concentrations. Faecal samples were collected from sheep infected with T. circumcincta or H. contortus alone and the eggs harvested, counted and DNA extracted. Serial dilutions were prepared to give a range of concentrations between approximately 3000 and 50 eggs per sample and real-time PCR reactions were carried out for each and mean cycle time (Ct) values were calculated. These Ct values were plotted against the sample egg concentration to produce a standard curve. Regression analysis was carried out using the generated data. Eggs were then harvested from faecal samples collected in the field from sheep carrying natural mixed infections, DNA extracted and Ct values recorded as before. Results were compared to the standard curve data in order to calculate the approximate number of T. circumcincta and H. contortus eggs in each field sample. These values were compared to the number of eggs determined using the traditional laboratory methods and Pearson product-moment correlation coefficients calculated. Results showed a strong correlation between numbers of eggs determined using the traditional and novel molecular methods suggesting that the developed molecular tools adequately predict egg numbers in this range. Further development and validation work should allow practical use of the method. This assay has significant advantages over the traditional methods currently used routinely at Fera and other parasitology laboratories and these are discussed.

Impact of an insecticide resistance strategy for house fly (Diptera: Muscidae) control in intensive animal units in the United Kingdom.

A strategy for house fly (Musca domestica L.) control in intensive animal units in the United Kingdom was proposed by the Pesticide Safety Directorate (PSD) of the Ministry of Agriculture, Fisheries and Food in 1993. An advice leaflet was circulated to farmers, and label recommendations for insecticides used to control house flies were altered to prevent their long-term and frequent use. A study was carried out between 1996 and 1998 to gather data on insecticide use and resistance in house fly populations and compared with results from a study carried out in 1990-1992 to assess the impact of the 1993 label recommendations. As in the 1990-1992 study, resistance to methomyl, azamethiphos and pyrethrins + piperonyl butoxide was assessed. Larvicide tests with cyromazine, which had recently been released in the United Kingdom, were also included in this study. Most of the farmers claimed to have received and read the PSD insecticide advice leaflet, and half claimed to have altered insecticide treatments as a result. Comparing results for insecticides used before and after 1993, the proportion of farmers claiming to have used each of the insecticides had decreased. However, there had been no amelioration in resistance to synergised pyrethrins, and the number of house fly populations with reduced response to the insecticide baits had increased between 1990-1992 and 1996-1998. All the house fly populations tested were fully susceptible to cyromazine. There is an urgent need, therefore, to devise new strategies and particularly to minimize the risk of selecting for resistance to cyromazine.