Helminth egg excretion with regard to age, gender and management practices on UK Thoroughbred studs.

Few studies have described the combined effect of age, gender, management and control programmes on helminth prevalence and egg shedding in grazing equines. Here, fecal samples collected from 1221 Thoroughbred horses, residing at 22 studs in the UK, were analysed. The distribution of strongyle eggs amongst individuals in relation to age, gender and management practices was investigated. Fecal worm egg counts (FWECs), described as the number of eggs per gramme (epg) of feces, were determined using a modification of the salt flotation method. The FWEC prevalence (mean%) of strongyles, Parascaris equorum, tapeworm spp. and Strongyloides westeri was 56, 9, 4 and 8%, respectively. Strongyle, P. equorum, tapeworm spp. and S. westeri infections were detected on 22 (100%), 11 (50%), 9 (41%) and 8 (36%) of studs, respectively. Within all age and gender categories, strongyle FWECs were highly over-dispersed (arithmetic mean = 95 epg, aggregation parameter k=0·111) amongst horses. Animal age, last anthelmintic type administered and management practices (for example, group rotation on grazing) most strongly influenced strongyle prevalence and level of egg shedding (P < 0·05). Overall, 11% of equines (range: 234-2565 epg) were responsible for excreting 80% of the strongyle eggs detected on FWEC analysis. The results confirm that the judicious application of targeted treatments has potential to control equine strongyle populations by protecting individual horses from high burdens, whilst promoting refugia for anthelmintic susceptible genotypes.

The first report of macrocyclic lactone resistant cyathostomins in the UK.

In recent years, resistance to the benzimidazole (BZ) and tetrahydropyrimidine (PYR) anthelmintics in global cyathostomin populations, has led to reliance on the macrocyclic lactone drugs (ML-of which ivermectin and moxidectin are licensed in horses) to control these parasites. Recently, the first confirmed case of resistance to both ivermectin (IVM) and moxidectin (MOX) was reported in the USA in yearlings imported from Ireland. This suggests that ML resistance in cyathostomins has emerged, and raises the possibility that regular movement of horses may result in rapid spread of ML resistant cyathostomins. Resistance may go undetected due to a lack of surveillance for ML efficacy. Here, we report anthelmintic efficacies in cyathostomins infecting UK Thoroughbreds on four studs. Faecal egg count reduction tests (FECRT) were performed to define resistance (resistance = FECR <95% lower credible interval (LCI) < 90%). Stud A yearlings had FECRs of 36.4-78.6% (CI:15.7-86.3) after three IVM treatments, 72.6% (CI: 50.8-85.2) after MOX, and 80.8% (CI: 61.9-90.0) after PYR. Mares on stud A had a FECR of 97.8% (CI: 93.3-99.9) and 98% (95.1-99.4) after IVM and MOX treatment, respectively. Resistance to MLs was not found in yearlings or mares on studs B, C or D with FECR after MOX OR IVM treatment ranging from 99.8 to 99.9% (95.4-100); although yearlings on studs B, C and D all had an egg reappearance period (ERP) of six weeks for MOX and stud C had a four-week ERP for IVM. This study describes the first confirmed case of resistance to both licensed ML drugs on a UK Thoroughbred stud and highlights the urgent need for a) increased awareness of the threat of ML resistant parasites infecting horses, and b) extensive surveillance of ML efficacy against cyathostomin populations in the UK, to gauge the extent of the problem.

New insights into sequence variation in the IGS region of 21 cyathostomin species and the implication for molecular identification.

Cyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, additional IGS DNA sequences were generated from individual worms of 8 species already studied. Comparative analysis of these sequences revealed a greater range of intra-specific variation than previously reported (up to 23%); whilst the level of inter-specific variation (3-62%) was similar to that identified in earlier studies. The reverse line blot (RLB) method has been used to exploit the cyathostomin IGS DNA region for species identification. Here, we report validation of novel and existing DNA probes for identification of cyathostomins using this method and highlight their application in differentiating life-cycle stages such as third-stage larvae that cannot be identified to species by morphological means.

Fasciola hepatica in UK horses.

BACKGROUND: Fasciola hepatica (liver fluke) affects grazing animals including horses but the extent to which it affects UK horses is unknown. OBJECTIVES: To define how liver fluke affects the UK horse population. STUDY DESIGN: Descriptive, cross-sectional, observational study. METHODS: An F. hepatica excretory-secretory antibody detection ELISA with a diagnostic sensitivity of 71% and specificity of 97% was validated and used to analyse serum samples. An abattoir study was performed to determine prevalence. A case-control study of 269 horses compared fluke exposure between horses with liver disease and controls. Data on clinical signs and blood test results were collected for sero-positive horses. Genotyping of adult fluke was used to produce a multilocus genotype for each parasite. RESULTS: Four (2.2%) of 183 horses registered in the UK, sampled in the abattoir, had adult flukes in the liver, and the sero-prevalence of F. hepatica was estimated as 8.7%. In the case-control study, horses showing signs consistent with liver disease had significantly higher odds of testing positive for F. hepatica on ELISA than control horses. In 23 sero-positive horses, a range of non-specific clinical signs and blood test abnormalities was reported, with a third of the horses showing no signs. Genotypic analysis of liver flukes from horses provided evidence that these came from the same population as flukes from sheep and cattle. MAIN LIMITATIONS: Bias could have arisen in the prevalence and case-control studies due to convenience sampling methods, in particular the geographic origin of the horses. Only a small number of horses tested positive so the data on clinical signs are limited. CONCLUSIONS: Exposure to liver fluke occurs frequently in horses and may be an under-recognised cause of liver disease. Flukes isolated from horses are from the same population as those found in ruminants. When designing and implementing parasite control plans, fluke should be considered, and horses should be tested if appropriate.

The role of polymorphisms at beta tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins.

Cyathostomins are recognised as the primary parasitic pathogens of horses. Despite the use of benzimidazole (BZ) anthelmintics in horses for more than 40 years and widespread drug resistance in the field, the mechanisms of resistance to this drug class in cyathostomins are not fully understood. The results presented here constitute a detailed comparison of beta tubulin gene mutations and mRNA transcript levels in populations of BZ-susceptible (BZ-S) and -resistant (BZ-R) cyathostomins. Full-length cDNA sequences were generated from individual parasites of four (n=24) and two (n=19) cyathostomin species for isotypes 1 and 2, respectively. Levels of intra- and inter-specific nucleotide sequence variation were comparable with previous findings and single amino acid substitutions were observed at several locations. On comparison of BZ-S and BZ-R parasites, differences were consistently observed at only two sites, codons 167 and 200 of the beta tubulin isotype 1 gene. Four populations of parasites were genotyped at these two loci by pyrosequencing; one that was fenbendazole (FBZ)-sensitive (FBZ-rS), two that were FBZ-resistant (FBZ-R1 and -R2) and one that was oxibendazole-resistant (OBZ-R), as previously assessed by faecal egg count reduction tests. This analysis revealed statistically significant differences between FBZ-rS and FBZ-R populations at both loci and this was highly significant for codon 167. For the OBZ-R population, the only significant difference compared with the FBZ-rS population was observed at codon 200. These observations suggest that mutations at codons 167 and 200 are important in BZ resistance and raise the possibility that selection at different loci may occur in FBZ- and OBZ-resistant parasites. Multiple parasites (n=158) were genotyped for both codons 167 and 200, the majority of which showed homozygous 'resistant' mutations at one locus only and none showed homozygous 'resistant' genotypes at both loci. No significant differences in mRNA levels of beta tubulin isotypes 1 and 2 were observed between the FBZ-rS and FBZ-R1 populations.

The relationships between faecal egg counts and gut microbial composition in UK Thoroughbreds infected by cyathostomins.

A growing body of evidence, particularly in humans and rodents, supports the existence of a complex network of interactions occurring between gastrointestinal (GI) helminth parasites and the gut commensal bacteria, with substantial effects on both host immunity and metabolic potential. However, little is known of the fundamental biology of such interactions in other animal species; nonetheless, given the considerable economic losses associated with GI parasites, particularly in livestock and equines, as well as the global threat of emerging anthelmintic resistance, further explorations of the complexities of host-helminth-microbiota interactions in these species are needed. This study characterises the composition of the equine gut commensal flora associated with the presence, in faecal samples, of low (Clow) and high (Chigh) numbers of eggs of an important group of GI parasites (i.e. the cyathostomins), prior to and following anthelmintic treatment. High-throughput sequencing of bacterial 16S rRNA amplicons and associated bioinformatics and statistical analyses of sequence data revealed strong clustering according to faecal egg counts (P = 0.003). A trend towards increased populations of Methanomicrobia (class) and Dehalobacterium (genus) was observed in Clow in comparison with Chigh. Anthelmintic treatment in Chigh was associated with a significant reduction of the bacterial Phylum TM7 14 days post-ivermectin administration, as well as a transient expansion of Adlercreutzia spp. at 2 days post-treatment. This study provides a first known insight into the discovery of the intimate mechanisms governing host-parasite-microbiota interactions in equines, and sets a basis for the development of novel, biology-based intervention strategies against equine GI helminths based on the manipulation of the commensal gut flora.

Fasciola and fasciolosis in ruminants in Europe: Identifying research needs.

Fasciola hepatica is a trematode parasite with a global distribution, which is responsible for considerable disease and production losses in a range of food producing species. It is also identified by WHO as a re-emerging neglected tropical disease associated with endemic and epidemic outbreaks of disease in human populations. In Europe, F. hepatica is mostly associated with disease in sheep, cattle and goats. This study reviews the most recent advances in our understanding of the transmission, diagnosis, epidemiology and the economic impact of fasciolosis. We also focus on the impact of the spread of resistance to anthelmintics used to control F. hepatica and consider how vaccines might be developed and applied in the context of the immune-modulation driven by the parasite. Several major research gaps are identified which, when addressed, will contribute to providing focussed and where possible, bespoke, advice for farmers on how to integrate stock management and diagnosis with vaccination and/or targeted treatment to more effectively control the parasite in the face of increasing the prevalence of infection and spread of anthelmintic resistance that are likely to be exacerbated by climate change.

P-glycoproteins play a role in ivermectin resistance in cyathostomins.

Anthelmintic resistance is a global problem that threatens sustainable control of the equine gastrointestinal cyathostomins (Phylum Nematoda; Superfamily Strongyloidea). Of the three novel anthelmintic classes that have reached the veterinary market in the last decade, none are currently licenced in horses, hence current control regimens focus on prolonging the useful lifespan of licenced anthelmintics. This approach would be facilitated by knowledge of the resistance mechanisms to the most widely used anthelmintics, the macrocyclic lactones (ML). There are no data regarding resistance mechanisms to MLs in cyathostomins, although in other parasitic nematodes, the ABC transporters, P-glycoproteins (P-gps), have been implicated in playing an important role. Here, we tested the hypothesis that P-gps are, at least in part, responsible for reduced sensitivity to the ML ivermectin (IVM) in cyathostomins; first, by measuring transcript levels of pgp-9 in IVM resistant versus IVM sensitive third stage larvae (L3) pre-and post-IVM exposure in vitro. We then tested the effect of a range of P-gp inhibitors on the effect of IVM against the same populations of L3 using the in vitro larval development test (LDT) and larval migration inhibition test (LMIT). We demonstrated that, not only was pgp-9 transcription significantly increased in IVM resistant compared to IVM sensitive L3 after anthelmintic exposure (p < 0.001), but inhibition of P-gp activity significantly increased sensitivity of the larvae to IVM in vitro, an effect only observed in the IVM resistant larvae in the LMIT. These data strongly implicate a role for P-gps in IVM resistance in cyathostomins. Importantly, this raises the possibility that P-gp inhibitor-IVM combination treatments might be used in vivo to increase the effectiveness of IVM against cyathostomins in Equidae.

Molecular diagnosis and equine parasitology.

The future implementation of improved and sustainable control strategies for the major equine parasites will be dependent on a greater insight into their basic biology, pathogenicity and epidemiology together with an enhanced ability for accurate diagnosis. This paper will provide a review of the current molecular methods under development for the detection of equine parasites and their application to current scientific questions. In particular, the strongyles are recognised as important pathogens of horses and recent advances made in the study of this parasitic group at the single species level will be addressed. The ribosomal (r)DNA region of the parasite genome has been employed to distinguish between closely related species. Molecular probes designed to this target region were used in combination with PCR technology to allow the identification of individual species within mixed infections. They have been applied to all parasite stages to look at the role of individual species in natural infection, disease and drug resistance. Similar techniques have been developed to detect other equine parasites and these will also be discussed. Further opportunities for employing existing techniques and the need for new diagnostic tools will be highlighted.

Papaya latex supernatant has a potent effect on the free-living stages of equid cyathostomins in vitro.

The control of equid gastrointestinal nematodes in developed countries, in particular the cyathostomins, is threatened by high levels of anthelmintic resistance. In recent years, there has been increasing interest in the evaluation of traditional 'ethnoveterinary' medicines as alternatives to chemical anthelmintics. The cysteine proteinases (CPs), a group of enzymes derived from fruits such as papaya (Carica papaya), pineapple (Ananas comosus) and figs (Ficus spp.), have shown good efficacy against adult stages of a range of parasitic nematodes, in vitro and in vivo. The efficacy of CPs against cyathostomins remains to be explored. In this study, the efficacy of a crude preparation of CPs, papaya latex supernatant (PLS), against the free-living stages of cyathostomins was evaluated using two in vitro tests, the egg hatch test (EHT) and the larval migration inhibition test (LMIT). It was demonstrated that PLS had a potent effect in the EHT, with EC-50 values in the range of 0.12-0.22µM. At concentrations above 6.25µM the eggs did not develop, below this concentration the L1 developed but they lost integrity of the cuticle upon hatching. These effects were inhibited by pre-incubation of PLS with the CP inhibitor L-trans-epoxysuccinyl-l-leucylamido-(4-guanidino butane) (E64), indicating that CPs were responsible for the anti-parasitic activity. A dose-dependent inhibition of migration of third stage larvae (L3) in the LMIT was demonstrated at higher concentrations of PLS, with EC-50 values in the range of 67.35-106.31µM. Incubation of PLS with E64 prior to use in the LMIT did not reverse the anti-migratory effect, suggesting that CPs were not responsible for the reduced migration of cyathostomin L3 and that PLS also contains an additional active compound. This is the first report of PLS and/or CPs showing activity against the free-living stages of a parasitic helminth. In addition, it suggests that cyathostomins are highly sensitive to the effects of CPs and further evaluation of their efficacy against parasitic stages and in vivo are strongly indicated.

Expression of a human homeobox-containing gene is regulated by 1,25(OH)2D3 in bone cells.

The murine msh-like homeobox-containing gene Msx-2 (Hox-8) has a multiphasic pattern of embryonic expression which includes the developing skull bones and teeth. In order to determine whether this gene might be expressed in adult mineralizing tissues, an adult human bone cell (osteoblastic) cDNA library was screened with a murine Msx-2 probe. One of the positive clones obtained was sequenced in full and shown to be highly homologous to the murine Msx-2 gene. Expression of this gene, MSX2, was studied in human bone-derived cells, where a low level of expression was detected which was greatly induced by stimulation with 1,25(OH)2D3. Little or no increase in expression was detected with retinoic acid or TPA stimulation. In cultures of skin fibroblasts no stimulation of expression with 1,25(OH)2D3 was detected. MSX2 is the first homeobox-containing gene to be shown to be regulated by 1,25(OH)2D3 and to be expressed in adult human bone cells. These results suggest a possible role for MSX2 in bone cell differentiation and/or calcium homeostasis.

Isolation and characterisation of a beta tubulin isotype 2 gene from two species of cyathostomin.

This study describes the isolation and characterisation of beta tubulin isotype 2 cDNA sequences from two common species of cyathostomin, Cylicocyclus nassatus and Cyathostomum catinatum. The full-length cDNAs for these species were 1709 and 1753 bp in length, respectively, including 1350 bp of sequence inferred to encode 450 amino acids of peptide sequence. They had greatest identity with previously characterised isotype 2 sequences from Trichostrongylus colubriformis, Cooperia oncophora and Haemonchus contortus (96% for C. nassatus and 95% for C. catinatum), and grouped together with these sequences in phylogenetic analysis. Both cyathostomin beta tubulin isotype 2 sequences contained the isotype-specific carboxyl terminal region described previously in other nematode species. Alignment with beta tubulin isotype 1 proteins from other trichostrongyloids, indicated 95 and 94% identity for the isotype 2 sequences of C. nassatus and C. catinatum, respectively. This comparison revealed 14 isotype-specific amino acid substitutions. Also, 2605 bp of beta tubulin isotype 2 genomic DNA sequence were isolated from C. nassatus. Comparison with the previously published isotype 1 gene of C. nassatus indicated differences in genomic organisation between the two isotypes. Reverse transcriptase (RT)-polymerase chain reaction analysis revealed constitutive temporal expression of beta tubulin isotype 1, whilst isotype 2 appeared to be developmentally expressed, with transcripts detected only in RNA derived from adult parasites.

Cytokine responses to Cyathostominae larvae in the equine large intestinal wall.

To investigate cytokine responses in cyathostomin infection, we quantified mucosal interleukin-4 (IL-4), interleukin-10 (IL-10), tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma by reverse transcriptase-competitive polymerase chain reaction. The analysis was performed on large intestinal wall samples obtained from six anatomical sites spanning the caecum and colon of 17 naturally exposed horses. The numbers of developing larvae (DL) and early third stage larvae (EL3) were ascertained using transmural illumination and pepsin digestion techniques, respectively. Levels of each cytokine transcript were correlated with local intestinal wall burdens of Cyathostominae larvae. IL-4 and IL-10 levels showed significant correlations with EL3 and DL burdens at several sites. No significant correlations were observed with IFNgamma. A pro-inflammatory response, typified by detection of TNFalpha transcript, was observed at a few sites in some horses with inflammatory enteropathy associated with emerging or emerged larvae. However, this cytokine was measured at an insufficient number of sites to enable statistical analysis. Levels of IL-4, IL-10 and IFNgamma transcript were compared between two groups: one group consisting of horses with low to high mucosal burdens (Group A) and the other, of horses with negative/negligible mucosal burdens (Group B). Significant differences in IL-4 (P<0.001) and IL-10 (P<0.001) transcript levels were observed between the groups, with higher levels observed in Group A. No significant differences in IFNgamma were observed. Taken together, these results indicate that Th2 responses predominate in mucosal Cyathostominae infection prior to larval reactivation.

An evidence-based approach to the evaluation of ethnoveterinary medicines against strongyle nematodes of equids.

Cyathostomins are the most important gastrointestinal nematode infecting equids. Their effective control is currently under threat due to widespread resistance to the broad spectrum anthelmintics licenced for use in equids. In response to similar resistance issues in other helminths, there has been increasing interest in alternative control strategies, such as bioactive plant compounds derived from traditional ethnoveterinary treatments. This study used an evidence-based approach to evaluate the potential use of plant extracts from the UK and Ethiopia to treat cyathostomins. Plants were shortlisted based on findings from a literature review and additionally, in Ethiopia, the results of a participatory rural appraisal (PRA) in the Oromia region of the country. Systematic selection criteria were applied to both groups to identify five Ethiopian and four UK plants for in vitro screening. These included Acacia nilotica (L.) Delile, Cucumis prophetarum L., Rumex abyssinicus Jacq., Vernonia amygdalina Delile. and Withania somnifera (L.) Dunal from Ethiopia and Allium sativum L. (garlic), Artemisia absinthium L., Chenopodium album L. and Zingiber officinale Roscoe. (ginger) from the UK. Plant material was collected, dried and milled prior to hydro-alcoholic extraction. Crude extracts were dissolved in distilled water (dH2O) and dimethyl sulfoxide (DMSO), serially diluted and screened for anthelmintic activity in the larval migration inhibition test (LMIT) and the egg hatch test (EHT). Repeated measures ANOVA was used to identify extracts that had a significant effect on larval migration and/or egg hatch, compared to non-treated controls. The median effective concentration (EC-50) for each extract was calculated using PROBIT analysis. Of the Ethiopian extracts A. nilotica, R. abyssinicus and C. prophetarum showed significant anthelmintic activity. Their lowest EC-50 values were 0.18 (confidence interval (CI): 0.1-0.3), 1.1 (CI 0.2-2.2) and 1.1 (CI 0.9-1.4)mg/ml, respectively. All four UK extracts, A. sativum, C. album, Z. officinale and A. absinthium, showed significant anthelmintic activity. Their lowest EC-50 values were 1.1 (CI 0.9-1.3), 2.3 (CI 1.9-2.7) and 0.3 (CI 0.2-0.4)mg/ml, respectively. Extract of A. absinthium had a relatively low efficacy and the data did not accurately fit a PROBIT model for the dose response relationship, thus an EC-50 value was not calculated. Differences in efficacy for each extract were noted, dependent on the assay and solvent used, highlighting the need for a systematic approach to the evaluation of bioactive plant compounds. This study has identified bioactive plant extracts from the UK and Ethiopia which have potential as anthelmintic forages or feed supplements in equids.

Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses.

Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-specific. The fifth probe recognised all 16 cyathostomin species but did not bind to members of the genus Strongylus. Furthermore, these probes were used to identify individual infective L3, eggs and L4 indicating that they will be invaluable to furthering the study of the epidemiology and pathogenesis of these important equine nematodes.

A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis.

We report the use of six oligoprobes designed from intergenic spacer region sequences to identify fourth-stage larvae (L4) of the tribe Cyathostominae. Oligoprobes were designed for identification of the following species: Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, and Cylicostephanus longibursatus. A seventh probe was designed as a positive control to identify all these members of the Cyathostominae. The intergenic spacer region was amplified by PCR using conserved primers. Initially, three oligoprobes were used in Southern blot analysis. To facilitate high-throughput identification, these and a further four oligoprobes were developed for use in a PCR-ELISA. All probes were validated for their ability to detect cyathostomin PCR products in the PCR-ELISA, using DNA from morphologically identified adult parasites. Initially, 712 L4 were isolated from the diarrhoeic faeces from horses (n=17) with clinical larval cyathostominosis. PCR products from 522 of these L4 were subjected to analysis, with 413 L4 being identified as one of the aforementioned species. With reference to individual species analysis, 28.5% of the 522 L4 were identified as C. longibursatus, 25.7% as C. nassatus, 15.9% as C. ashworthi, 7.3% as C. goldi and 1.7% as C. catinatum. No L4 were identified as being C. insigne species. When L4 within faeces from individual horses were compared, no sample was found to comprise parasites of one species. The least number of species identified in a single sample was two. This study suggests that clinical larval cyathostominosis is predominantly caused by mixed-species infections.

Seasonal development of Cyathostominae larvae on pasture in a northern temperate region of the United Kingdom.

A study following the development of Cyathostominae from egg to the infective larval third stage was conducted from April to December 2001 in west central Scotland. Duplicate samples (1 kg) of naturally infected faeces were placed on a 78 cm2 plot each week on a cyathostomin-free pasture. Subsamples of the grass surrounding the faecal plot were collected weekly on four occasions and the number of larvae obtained determined. Few larvae were recovered in the first week of development of individual plots, followed by a rise in the numbers of larvae in second, third and fourth weeks of development of each sample. The climatic conditions were seen to have an effect on the rate of development. Specifically, from multilevel, multivariable linear regression models it was evident that the factors associated with numbers of infective larvae recovered from pasture were the time since the faeces samples were laid down, the average temperature and rainfall during the previous week, as well as the interaction between temperature and rainfall. Conversely, from the model, the number of larvae recovered from pasture was associated with neither the number of eggs within the faeces samples placed on the plots nor with the viability of these eggs.

Anthelmintic efficacy against cyathostomins in horses in Southern England.

Cyathostomins are considered to be the most important group of helminths to affect equids due to their high prevalence, potential pathogenicity and ability to develop anthelmintic resistance. Their control relies almost exclusively on frequent anthelmintic use. Currently, fenbendazole (FBZ), pyrantel embonate (PYR), ivermectin (IVM) and moxidectin (MOX) are licensed for use in horses in the UK. With no new anthelmintics likely to be licensed in the near future, it is essential that investigations into the efficacy of current anthelmintics in different locations are performed to help inform control programmes. Here, efficacy of FBZ, PYR, IVM and MOX in horse populations in the South of England was investigated. Horses with a strongyle faecal egg count (FEC) of ≥50 eggs per gram (EPG) were enrolled onto a faecal egg count reduction test (FECRT) study. Efficacy was determined by calculating the percentage reduction in FEC between the group mean at Day 0 and 14 days post-treatment. Efficacy was indicated when a group arithmetic faecal egg count reduction (FECR) of ≥90% was recorded for FBZ and PYR, and ≥95% for IVM and MOX. Between March and December 2012, 404 FECRT were performed on 12 yards examining 101, 110, 93 and 100 equids for FBZ, PYR, IVM, and MOX, respectively. FBZ resistance was identified on all yards (mean FECR range 0-65.8%). On 10 of 12 yards, PYR efficacy was >90% (91.0-99.4%) and on two yards, PYR resistance was suspected (86.8-87.2%). IVM (96.4-100%) and MOX (99.9-100%) were >95% efficacious on all yards. As the prevalence of FBZ resistance was 100%, the future use of this anthelmintic for the control of strongyles should be questioned. PYR should be used strategically to reduce reliance on the macrocyclic lactone class products. Over-dispersion of FEC between horses was observed (average k=0.21) with 80% of the strongyle eggs counted measured in 15% of horses tested, strongly supporting the application of targeted helminth control programmes in this host species.

A questionnaire study on parasite control practices on UK breeding Thoroughbred studs.

REASONS FOR PERFORMING STUDY: Improved education of veterinarians and equine owners/managers is essential in implementing parasite control strategies that are less reliant on chemicals. METHODS: This questionnaire study, conducted on 61 UK Thoroughbred (TB) establishments during 2009 and 2010, was designed to obtain an understanding of current helminth control practices on studs. To our knowledge, this is the first occasion that statements obtained from TB studs via questionnaire have been supported by statistical analysis. RESULTS: Despite many respondents indicating high levels of concern regarding anthelmintic resistance, 56% of these establishments that received visiting equines co-grazed these animals with permanent stock and <74% administered anthelmintics prior to integration. In the 12 months preceding the study, most respondents administered frequent macrocyclic lactone (ML) treatments, with none appearing to leave any animals in groups untreated at each administration. Indiscriminate whole group treatments with MLs and movement of animals to 'clean grazing' post treatment (reported by >25% of respondents), indicates that many stud owners/managers are not aware of the strong risk factors for the development of anthelmintic resistance. Few studs had conducted faecal egg count (FEC) analysis in the past and only 22% indicated that they considered this form of analysis beneficial in determining anthelmintic choice. CONCLUSIONS AND POTENTIAL RELEVANCE: The challenge now is to convince stud owners/managers to deviate from their current practices to control strategies that are more likely to preserve anthelmintic efficacy. Veterinarians need to get more involved in implementing these control strategies, with better emphasis placed on the role of diagnostic tests in facilitating targeted treatments and in investigating anthelmintic sensitivity in the associated nematode populations.

The prevalence of pyrethroid resistance phenotype and genotype in Rhipicephalus (Boophilus) microplus in Yucatan, Mexico.

A field survey was conducted to evaluate susceptibility of Rhipicephalus (Boophilus) microplus to cypermethrin on 49 farms in three areas of Yucatan, Mexico. The modified larval packet test was used to evaluate larval mortality at different cypermethrin concentrations. Dose-mortality regressions, lethal concentrations (LC(50)-LC(99)), confidence intervals and slope were estimated by probit analysis. Phenotype was defined as susceptible, tolerant or resistant when the resistance factor (RF) derived from both LC(50) and LC(99) determinations were <3, 3-5 or >5, respectively. An allele specific PCR (AS-PCR) was used to determine the frequency of a sodium channel mutation (F1550I, Phe→Ile) associated with pyrethroid resistance. Overall, 26.5%, 40.8% and 32.6% of tick populations were susceptible, tolerant and resistant to cypermethrin, respectively. A substantial inter-population variation in the level of cypermethrin response was evident (resistance factors ranged from 0.3 to 2599 and from 0.7 to >5000 when were indicated by the LC(50) and LC(99), respectively). The F1550I mutation (R allele) in R. microplus was present in all studied areas. The increasing presence of the R allele correlated well with increased levels of response indicated by both the LC(50) (r(2)=0.659, p=0.001) and LC(99) (r(2)=0.688, p=0.001) to cypermethrin. These results indicated that the F1550I mutation is a major common mechanism responsible for pyrethroid resistance in field populations of R. microplus ticks in the Mexican tropics. Both bioassay and AS-PCR showed that the prevalence of cypermethrin-resistant/tolerant R. microplus is high in Yucatan, Mexico and the relationship between the RF and the frequency of the R allele supports the role of F1550I as one of the most important mechanisms conferring pyrethroid resistance in these R. microplus populations.