Jacqueline B. Matthews

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.

An update on cyathostomins: Anthelmintic resistance and worm control

Summary Intestinal nematodes are an important cause of equine disease. Of these parasites, the Cyathostominae are the most important group, both in terms of their prevalence and their pathogenicity. Cyathostomin infections are complex and control is further complicated by ever-increasing levels of resistance to some of the commonly used anthelmintics. There are no new equine anthelmintics under development, so it is imperative that the efficacy of any currently-effective drug classes be maintained for as long as possible. It is believed that the proportion of refugia (i.e. the percentage of parasites not exposed to a drug at each treatment) is one of the most crucial factors in determining the rate at which anthelmintic resistance develops. It is important, therefore, that levels of refugia be taken into account when designing nematode control programmes for horses. This can be assisted by knowledge of the local epidemiology of the infection, supplemented by faecal egg count analysis to identify those animals that are making the major contribution to pasture contamination. This type of rational nematode control requires equine veterinary surgeons to get involved in designing and implementing deworming programmes. The advice given must be based on a combination of knowledge of cyathostomin biology and epidemiology as well as an awareness of the parasite populations current drug sensitivity and a sound history of husbandry at the establishment. As anthelmintic resistance will be the major constraint on the future control of cyathostomins, researchers are now actively investigating this area. Studies are underway to develop tests that will enable earlier detection of anthelmintic resistance and an assay that will help identify those horses that require anthelmintic treatments targeted at intestinal wall larvae.

Proteomic analysis of excretory/secretory products released by Teladorsagia circumcincta larvae early post-infection

Teladorsagia circumcincta is an important parasitic nematode of domestic small ruminants. Drug resistance in this species is common so alternative methods of control are required. As animals develop immunity to T. circumcincta, vaccination is a valid option. Little is known about the antigens that play a role in stimulating immunity at this host/parasite interface. As responses generated between 1 and 5 dpi are known to affect development of these nematodes in their gastric niche, we focused on proteins released during the early stages of infection. To identify molecules potentially involved in immunity, we undertook a proteomics analysis of proteins released from larvae harvested at 1‐, 3‐ and 5‐days post‐infection (dpi). This analysis produced peptide sequence data that was used to search information available in T. circumcincta expressed sequence tag (EST) databases and enabled identification of a number of excretory/secretory (ES) proteins. Immunoblots were performed to assess the relative molecular weight of ES antigens that were targets of local IgA responses in mucus from sheep rendered immune to infection. ELISA was performed to assess antigen‐specific mucus IgA levels in individual sheep. These experiments provided preliminary evidence that the proteins identified in the larval secretome were subject to these antibody responses.

Anthelmintic resistance in equine nematodes

Graphical abstract

Anthelmintic efficacy on UK Thoroughbred stud farms.

Anthelmintic drugs have been applied indiscriminately to control horse nematodes for over 40 years. We undertook a comprehensive study to investigate efficacy of the four available broad-spectrum anthelmintic drugs on 16 Thoroughbred stud farms using the faecal egg count reduction test. Efficacy against strongyles was determined by calculating the percentage of reduction in faecal egg count between the group mean at Day 0 and Days 14-17 post-treatment and the 95% lower confidence intervals estimated by non-parametric bootstrapping. Individual strongyle faecal egg count reduction tests (n=429) were performed in which 179, 131, 89 and 30 horses were administered ivermectin, moxidectin, pyrantel and fenbendazole, respectively. Moxidectin was efficacious in all tests (faecal egg count reduction range: 99.8-100%; 95% lower confidence intervals range: 96.8-100%) and reduced efficacy of ivermectin (faecal egg count reduction range: 85.7-100%; 95% lower confidence intervals range: 65-100%) was observed in one group of yearlings. Reduced pyrantel efficacy was observed in five groups of yearlings (faecal egg count reduction range: 0-73%; 95% lower confidence intervals range: 0-59.5%), but pyrantel was found to be efficacious when administered to mares (faecal egg count reduction range: 98-99.4%; 95% lower confidence intervals range: 91.8-99.3%). Low efficacy of fenbendazole was always observed (faecal egg count reduction range: 0.4-41%; 95% lower confidence intervals not calculable). Two further methods for estimating efficacy were applied and outputs obtained using all methodologies were in agreement. Efficacy against Parascaris equorum was assessed on four farms: fenbendazole had acceptable efficacy (faecal egg count reduction range: 97.5-99.9%; 95% lower confidence intervals range: 96.3-99.1%), but reduced efficacy of ivermectin was observed (faecal egg count reduction range: 25.5-91.2%; 95% lower confidence intervals range: 6.7-82.4%). Strongyle faecal egg count were analysed at approximately 2 week intervals for up to 12 weeks after anthelmintic drug administration to determine the egg reappearance period for moxidectin, ivermectin and pyrantel. The egg reappearance period for all three anthelmintic drugs was shorter than previously observed. Overall, our results indicate that ivermectin and moxidectin administration provided acceptable efficacy at 14 days; however, egg reappearance period results suggest that these products are working less effectively than measured previously. As shortened egg reappearance period is believed to be an early indicator of resistance, this highlights the issue of impending multi-drug resistance in strongyles on stud farms.

Successful immunization against a parasitic nematode by vaccination with recombinant proteins

Infection of humans and livestock with parasitic nematodes can have devastating effects on health and production, affecting food security in both developed and developing regions. Despite decades of research, the development of recombinant sub-unit vaccines against these pathogens has been largely unsuccessful. We have developed a strategy to identify protective antigens from Teladorsagia circumcincta, the major pathogen causing parasitic gastroenteritis in small ruminants in temperate regions, by studying IgA responses directed at proteins specific to post-infective larvae. Antigens were also selected on the basis of their potential immunomodulatory role at the host/parasite interface. Recombinant versions of eight molecules identified by immunoproteomics, homology with vaccine candidates in other nematodes and/or with potential immunoregulatory activities, were therefore administered to sheep in a single vaccine formulation. The vaccine was administered three times with Quil A adjuvant and the animals subsequently subjected to a repeated challenge infection designed to mimic field conditions. Levels of protection in the vaccinates were compared to those obtained in sheep administered with Quil A alone. The trial was performed on two occasions. In both trials, vaccinates had significantly lower mean fecal worm egg counts (FWECs) over the sampling period, with a mean reduction in egg output of 70% (Trial 1) and 58% (Trial 2). During the period of peak worm egg shedding, vaccinates shed 92% and 73% fewer eggs than did controls in Trials 1 and 2, respectively. At post mortem, vaccinates had 75% (Trial 1) and 56% (Trial 2) lower adult nematode burdens than the controls. These levels of protection are the highest observed in any system using a nematode recombinant sub-unit vaccine in the definitive ruminant host and indicate that control of parasitic helminths via vaccination with recombinant subunit vaccine cocktails is indeed an alternative option in the face of multi-drug resistance.

Parasite loss and inhibited development of Teladorsagia circumcincta in relation to the kinetics of the local IgA response in sheep

Groups of yearling sheep, which had been trickle infected with Teladorsagia circumcincta for 8 weeks and then drenched, were challenged with 50 000 T. circumcincta larvae together with groups of worm‐free controls. Fewer parasites and a greater proportion of early fourth stage larvae were recovered from previously infected sheep compared to controls. Worm loss and arrested development were evident by 5 days after challenge whereas growth retardation of developing worms was observed by day 10. In the previously infected sheep a secondary IgA response was observed in the efferent gastric lymph from 5 days post‐infection. Western blot analysis showed the lymph IgA to be predominantly dimeric and nonsecretory in nature and that the somatic antigens recognized were predominantly in the 100–250 kDa range. The concentration of IgA in lymph was always higher than in blood and in the previously infected sheep increased fivefold 8 days post‐challenge in contrast to blood where IgA levels were unchanged. The timing of the response suggested that it occurred too late to have been the cause of worm loss or arrested development, though it may have retarded the growth of developing parasites.

A case control study to examine the pharmacological factors underlying ventricular septal defects in the North of England

Background: Amphetamine exposure is associated with congenital cardiac abnormalities in animals. We previously reported an association between recreational use of 2,4-methylenedioxymethamphetamine (ecstasy, MDMA) and ventricular septal defect in babies born to users. We have carried out a case control study to investigate risks in the occurrence of ventricular septal defect in a cohort of babies born in the North East of England. Methods: Cases were identified from paediatric cardiology units in Newcastle upon Tyne and Leeds, and controls were recruited from the mothers of babies born in the same hospital as the index case. Research nurses carried out interviews using a structured questionnaire. Results: A total of 296 case control pairs were studied. There was insufficient exposure to ecstasy to test the primary hypothesis. Increased risk of ventricular septal defect was found to be associated with consumption of cough and cold remedies [pre-conception OR 2.2, 95% CI 1.41, 3.51; pregnancy OR 5.1, 95% CI 2.56, 11.27; exposure in either OR 2.83, 95% CI 1.85, 4.45; P<0.005] and in the case of non-steroidals for exposures in pregnancy (OR 4.2, 95% CI 1.54, 14.26; P<0.005). Conclusions: These findings suggest that ventricular septal defect is associated with consuming the medications identified. They are also compatible with the hypothesis that sympathomimetics (pseudoephedrine, phenylephrine and phenylpropanolamine) present in cough mixtures cause the increased risk, and with our original hypothesis that sympathomimetics and amphetamines are potentially cardiotoxic in utero.

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freunds incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.

Identification and characterisation of an immunodiagnostic marker for cyathostomin developing stage larvae.

Parasitic nematodes of the group Cyathostominae are an important cause of disease in horses. This group consists of approximately 50 species, all of which have similar life cycles that involve encystment of larval stages in the large intestinal wall. Encysted larvae can persist for months to years and, occasionally, large numbers can accumulate and emerge synchronously to cause severe pathology, resulting in diarrhoea, weight loss, colic and/or oedema. This syndrome, known as larval cyathostominosis, can be fatal in up to 50% of cases, despite treatment. There is no diagnostic method that enables detection of the encysted larval burden. Previously, we identified two native antigen complexes that showed utility as diagnostic markers for the estimation of cyathostomin encysted larval burdens. This paper reports the identification of a likely protein component of one of these antigen complexes. The protein, designated cyathostomin gut-associated larval antigen-1 (Cy-GALA-1), was isolated by immunoscreening a cyathostomin mixed-species, larval complementary DNA library using sera from experimentally-infected horses. The resultant recombinant protein, rCy-GALA-1, was expressed in Escherichia coli and shown to be a target of serum IgG(T) responses in experimentally- and naturally-infected horse populations. Transcription of Cy-gala-1 was restricted to cyathostomin encysted larvae and the presence of native protein was limited to developing larval stages. Importantly, rCy-GALA-1 exhibited no reactivity to serum from horses mono-specifically infected with other helminth species, nor did antisera, raised to the recombinant protein, bind to adult stage extracts of heterologous species. Immunohistochemical experiments located Cy-GALA-1 to the nematode gut. A region of the gene encoding orthologous GALA sequences was isolated from 10 separate cyathostomin species, indicating the ubiquity of the protein in this nematode group.