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Keywords:

  • horse;
  • parasites;
  • cyathostomins;
  • anthelmintic resistance;
  • research

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Research areas
  5. Conclusions
  6. Conflicts of interest
  7. Acknowledgements
  8. References

Horses worldwide are exposed to a complex mixture of intestinal parasitic helminths. When burdens are high, these parasites can seriously compromise health and welfare. Some helminth species have an extremely high prevalence and are difficult to control, not least because there is a limited understanding of their most basic biology. Furthermore, levels of resistance to some of the commonly used anthelmintics are widespread and increasing. The cyathostomins are the most common nematode species affecting equids worldwide. Within this group of parasites are more than 50 different species. Until recent research activities, little was known about the contribution that individual species make to clinical disease, parasite epidemiology and anthelmintic resistance. This review describes some of the recent research advances in the understanding of cyathostomins in these areas. As part of the research effort, molecular tools were developed to facilitate identification of the non-parasitic stages of cyathostomins. These tools have proved invaluable in the investigation of the relative contributions that individual species make to the pathology and epidemiology of mixed infections. At the more applied level, research has also progressed in the development of a diagnostic test that will allow numbers of cyathostomin encysted larvae to be estimated. This test utilises cyathostomin-specific serum antibody responses as markers of infection. As anthelmintic resistance will be the major constraint on parasite control in future, researchers are actively investigating mechanisms of drug resistance and how to improve the detection of resistance in the field. Recent developments in these areas are also outlined.


Glossary
Anthelmintics

Drugs that expel parasitic worms from the body, generally by paralysing or starving them

Antigen

A molecule (or part of a molecule) that is recognised by components of the host immune system

Cestodes

Parasitic flatworms, commonly called tapeworms, which usually live in the digestive tract of vertebrates as adults and in the bodies of various intermediate hosts as juvenile stages

Codon

3-nucleotide sequences that encode a specific single amino acid

Colitis

Inflammation of the colon: often used to describe an inflammation of the large intestine

Egg reappearance period

The time taken (usually expressed in weeks) for eggs to reappear in faeces after anthelmintic treatment. Usually this is described for drug-sensitive worm populations at the time of product licensing

ELISA (enzyme-linked immunosorbent assay)

A technique used primarily in immunology to detect the presence of an antibody or an Antigen in a sample. Basically, an unknown amount of antigen is bound to the surface of a plastic well, then a specific antibody is added and if specific will bind the antigen. This antibody is linked to an enzyme or is detected by incubation with a second antibody that is linked to an enzyme. In the final step a substance is added that the enzyme can convert to some detectable signal (usually a colour change which is detectable by a spectrophotometer)

Epidemiology

The study of factors affecting the health of populations and often how diseases are transmitted

Faecal egg count reduction test

A test that measures the effect on faecal egg output of anthelmintic treatment. Generally, efficacy is assessed by comparing FECs obtained on the day of treatment with those obtained 14 days after treatment. This is an important tool in detecting anthelmintic resistance in the field

Genome

An organism's entire hereditary information, encoded either in DNA or, for some types of virus, in RNA. The genome includes the genes that code for proteins and noncoding sequences of the DNA

Genotype

The inherited instructions organisms carry in their genetic code

Helminths

A group of eukaryotic parasites that live inside their host. They are worm-like and live and feed off animals

Heterozygous

A genotype consisting 2 different alleles at a given locus

Homozygous

A genotype consisting 2 identical alleles at a given locus

Larvae

Juvenile forms that many animals undergo before the mature adult stage. Larvae are frequently adapted to environments different to those adult stages live in

Larval cyathostominosis

The clinical syndrome that results from mass emergence of cyathostomin larval stages from the gut wall. It has a high fatality rate despite treatment

Lumen

The inner space of a tubular structure, such as the intestine

Mutations

Alterations in DNA sequence in a genome that spontaneously occur during meiosis or DNA replication or are caused by factors such as by radiation, viruses or chemicals. Mutations can have no effect, alter the product of a gene, or prevent the gene from functioning properly if at all

Nematodes

Roundworms, one of the most diverse phyla of all animals

Pathogenicity

The ability of a pathogen to produce signs of disease in an organism

PCR (polymerase chain reaction)

A technique to amplify a single or a few copies of a piece of DNA by several orders of magnitude generating thousands to millions of copies of a particular sequence. Polymerase chain reaction relies on cycles of repeated heating and cooling for DNA melting and enzymatic replication of DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, DNA generated is used as a template for replication, setting in motion a chain reaction in which the template is exponentially amplified

Phenotype

Any observable characteristic or trait of an organism: such as its morphology, development, biochemical or physiological properties, or behaviour. Phenotypes result from the expression of an organism's genes as well as the influence of environmental factors and possible interactions between the 2

Pyrosequencing

A method of DNA sequencing. It differs from conventional sequencing by relying on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Research areas
  5. Conclusions
  6. Conflicts of interest
  7. Acknowledgements
  8. References

Horses play host to many species of internal parasite; for example, unicellular protozoan species and multicellular nematodes and cestodes. The nematodes are the most important parasitic pathogens and these primarily affect the gastrointestinal (GI) tract. Virtually all horses that graze experience some level of GI nematode parasitism and, in some cases, infection is continuous throughout an animal's life. Often, the presence of parasites does not lead to overt clinical disease; however, in the low number of animals that do experience illness, this can be manifest as colic, severe weight loss and a life-threatening colitis with diarrhoea. It is a general rule that the higher the level of infection, the more likely it is that an animal will develop clinical signs. Therefore, it is important to identify animals that carry higher burdens of parasitic nematodes and to combine this with appropriate control practices that ensure horses do not develop substantial levels of infection.

The parasites that primarily cause GI symptoms fall into the broad categories of the nematodes (cyathostomins and large strongyles) and cestodes (or tapeworms). Anoplocephala perfoliata is the cestode species most associated with the presence of clinical disease in horses (Proudman and Trees 1999). In addition to these parasites, Parascaris equorum is a large nematode that can cause respiratory and GI signs in foals. Of the parasites listed above, the cyathostomins are the most important because of their high prevalence and pathogenicity (Matthews 2008). This complex group of roundworms are found worldwide and their control relies heavily on anthelmintics. The general life cycle of cyathostomins is depicted in Figure 1. The life cycle is direct and infection is via ingestion of infective third stage larvae (L3). Ingested L3 enter the mucosa and submucosa of the large intestine and develop through a number of encysted stages: early third L3 (EL3), late L3 and developing fourth stage larvae (DL4). The L4 emerge into the lumen and develop to fifth stage larvae, which mature to male and female adult worms. Eggs are excreted in faeces, hatch and develop to L3 in a temperature dependent manner (Lyons et al. 1999). The prepatent period is considered to be between 6 and 12 weeks; however, this is extended by up to many months when encysted larvae undergo inhibiteddevelopment in the large intestinal wall (Eysker and Mirck 1986; Love and Duncan 1992). In northern temperate areas, up to 90% of the total cyathostomin burden has been recorded as encysted larvae, with levels of immature stage worms in the order of several million (Dowdall et al. 2002). The larvae can subsequently reactivate in large numbers and in so doing can cause a clinical syndrome known as larval cyathostominosis. The factors associated with this re-emergence are unknown but could include changes in host immune response, environmental effects on L3 prior to ingestion, recent treatments with adulticidal anthelmintics and the population density of parasites in the large intestine (Matthews 2010). Larval cyathostominosis often occurs in winter or early spring and is more common in animals less than 5-years-old. Clinical signs include sudden onset weight loss, subcutaneous oedema and diarrhoea (Giles et al. 1985). In some animals, the syndrome presents as various forms of colic. Larval cyathostominosis is fatal in up to 50% of cases.

image

Figure 1. Life cycle of cyathostomins: L1: first stage larvae; L2: second stage larvae; L3: third stage larvae; EL3: early L3; DL: developing fourth stage larvae (comprising late L3 and developing L4).

Download figure to PowerPoint

Currently, 3 classes of anthelmintic are licensed for use against cyathostomins; these are the macrocyclic lactones (MLs), tetrahydropyrimidines (THPs) and benzimidazoles (BZs). Only moxidectin (MOX), a ML and fenbendazole (FBZ), administered as a 5 day course, have licensed efficacy against encysted cyathostomin larvae. There are now numerous reports of resistance to BZ and THP anthelmintics in cyathostomin populations (Kaplan 2002) and, recently, single cyathostomin populations have been identified that exhibit resistance to all 3 drug classes (Molento et al. 2008). The increasing threat of anthelmintic resistance in cyathostomins is reducing the long-term options for the control of these important parasitic nematodes: it is essential that the risk factors for the development of resistance be identified. Much of the recent research in cyathostomins has focused on developing tools to allow this research to happen.

Research areas

  1. Top of page
  2. Summary
  3. Introduction
  4. Research areas
  5. Conclusions
  6. Conflicts of interest
  7. Acknowledgements
  8. References

The need to identify cyathostomins to species

The cyathostomin group comprises over 50 species. The species can be differentiated on the basis of the morphological characteristics of the adult worm head and tail. In almost all cases, the accessible life cycle stages (i.e. eggs and larvae) cannot be distinguished to species. This inability to identify these stages has provided a major barrier to the study of even the most basic aspects of cyathostomin biology. For example, until relatively recently, virtually nothing was known of the individual species' contributions to the epidemiology of infection or if all species could acquire anthelmintic resistance. Over the last 15 years, concerted efforts have been made to develop DNA-based tools that enable the non-invasive identification of cyathostomin species (Matthews et al. 2004). In so doing, subsequent experiments became feasible that allowed assessment of species' contributions to observations that were made in the laboratory and in the field. For instance, there is a possibility that the anthelmintic resistance phenotype is associated with the presence of particular species; this hypothesis could not be tested non-invasively without tools that allowed eggs and larvae to be unequivocally identified. DNA tools were developed that enabled species identification at all stages of the life cycle: the ribosomal intergenic spacer (IGS) region of the cyathostomin genome was chosen as the target for molecular identification. Evidence in other, related organisms indicated that there was a sufficient balance of sequence similarity/variability to allow clear-cut species discrimination (Kaye et al. 1998; Hodgkinson et al. 2001). A number of IGS sequences were obtained using DNA derived from single, identified adult cyathostomins and a species-specific polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) protocol was developed using these sequences. First, the assay was used to investigate the relative pathogenicity of species for which DNA probes were available and was applied to identify fourth stage larvae (L4) obtained from the diarrhoeic faeces of horses that had displayed clinical signs of larval cyathostominosis (Hodgkinson et al. 2003). In these studies, species proportions were assessed in samples derived from 17 horses using DNA probes designed for the following species: Cylicostephanus longibursatus, Cylicocyclus nassatus, Cylicocyclus ashworthi, Cylicostephanus goldi, Cyathostomum catinatum and Cylicocyclus insigne. The data indicated that larval cyathostominosis is predominantly caused by mixed-species infections and does not appear to be related to the presence of a single species.

In further studies to investigate the possible impact of particular species on the development of anthelmintic resistance, eggs were isolated from horse faeces before and after treatment with the 2 commonly used anthelmintic drugs, pyrantel (PYR) and FBZ (Hodgkinson et al. 2005). Pyrantel was administered to horses to eliminate any FBZ-resistant adult worms that were present. Fenbendazole was administered 7 days later as a 5-day dose in an attempt to reduce the numbers of gut wall encysted larvae. The horses were stabled to prevent further cyathostomin infections and, thereafter, faecal samples were obtained weekly and egg counts performed and eggs harvested for species identification. Eggs harvested from faeces from 7 weeks post treatment were subjected to species composition analysis. The resultant data showed that, although PYR treatment was efficacious in reducing faecal egg counts (FECs) by >95% at 7 and 14 days post treatment, a return to FEC-positive samples occurred within 28 days of treatment suggesting that the combined therapy did not clear FBZ-resistant worms. This was a significant observation as, at the time, it had been speculated that 5-day FBZ treatments could eliminate cyathostomin populations that were resistant to a single dose of FBZ. The IGS PCR-ELISA species composition data indicated that the occurrence of FBZ resistance, in this instance, was not associated with the presence of a particular species.

The IGS sequence generated in the aforementioned studies were deposited in the public DNA database at GenBank (http://www.ncbi.nlm.nih.gov/GenBank/) and have been used subsequently by a number of research groups worldwide to develop DNA detection techniques to analyse cyathostomin species compositions (Ionita et al. 2010; Traversa et al. 2010). Currently, the IGS DNA sequences are being used to develop Pyrosequencing technology for the identification 17 different cyathostomin species (K. Cwiklinski, J.B. Matthews and J.E. Hodgkinson, unpublished data). This is a faster and more cost effective method than the PCR-ELISA and is better suited to processing large numbers of DNA samples. It is hoped that these further studies will provide insights into cyathostomin biology that will be translatable to improvements in control strategies in practice.

Anthelmintic resistance

As mentioned above, anthelmintic resistance is widespread in equine nematode populations. Reversion to drug sensitivity does not appear to occur; even when nematode populations have not been exposed to a specific drug class for many years. Furthermore, no new anthelmintic classes are anywhere near commercial market for use in horses, so the efficacy of the currently effective drugs needs to be preserved. The manner in which many horses are managed is not conducive to this: they are often dewormed far too frequently, with little thought put into the type of helminth that may be present or, importantly, the level of parasite burden. Horses vary markedly in their susceptibility to cyathostomins; most animals regulate the nematodes well and have low infection levels. In general, few animals in a population have sizeable nematode burdens, which may or may not be identifiable externally as a moderate to high FEC. This over-dispersed pattern in GI nematode burdens in equine populations normally persists throughout life and means that not all horses require anthelmintic at every treatment and, also, that the same animals tend to have the higher FECs when monitored over time. Equine parasite control programmes need to be designed to take this type of distribution pattern into consideration so that only the horses that contribute the bulk of contamination are treated regularly, whilst those with negative or low burdens receive specific treatments to eliminate pathogenic worms and/or those worm stages not detectable by routine FECs (e.g. cyathostomin encysted larvae). Such targeted approaches will help maintain drug efficacy because they help increase the proportion of a nematode population not under constant selection pressure by anthelmintics. This concept is critical in determining the rate at which resistance develops, but is often ignored in equine management systems. An over-reliance on anthelmintic treatments often goes hand-in-hand with an ignorance of the nematode population's drug sensitivity with the obvious risk that the drugs being administered have no useful effect.

To promote targeted treatment approaches, awareness of the prevalence of nematode species and, importantly, on how sensitive populations are to the anthelmintics available is required. Only when such information is available can rational, effective programmes be put in place for parasite control. The principle aims of research in this field should be to:

  • (i) 
    obtain information on the type of helminths that are prevalent and their drug sensitivity;
  • (ii) 
    develop sensitive and robust tests to examine anthelmintic efficacy in field populations.

To serve these purposes, it is essential that tools be developed that enable timely detection of resistance. Such tools can have a phenotypic or molecular basis. Generation of the latter requires detailed analyses of the gene or genes that encode the target proteins of the relevant drug, or of the genes that encode proteins involved in removal of the anthelmintic from the worms.

A molecular test for BZ resistance

The frequent use of BZs in horses since the 1970s has led to widespread resistance to this drug class in cyathostomin populations. The acquisition of BZ resistance in nematodes is thought to be associated with structural changes in the drug target molecule (beta-tubulin). This alteration results in reduced binding of BZ thereby allowing the parasites to survive in anthelmintic concentrations that would normally kill them (Lubega and Prichard 1990). Research on nematodes of sheep had previously revealed that specific point mutations (a change from TTC to TAC) at codons 167 (Silvestre and Cabaret 2002) and 200 (Kwa et al. 1994) of the beta-tubulin (isotype 1) gene are sufficient to cause resistance to BZs in these parasites (Kwa et al. 1995). In cyathostomins, the beta-tubulin codon 200 mutation was studied initially: it was concluded that a mutation at this site was not the only molecular mechanism of BZ resistance in these worms (Pape et al. 2003). Subsequent work led to the isolation of additional beta-tubulin gene sequences that allowed investigation of their relative contribution to BZ resistance (Clark et al. 2005). As is often the case with the cyathostomin group, the analysis was complicated by the existence of many species that exhibited inherent differences in their DNA sequences. For this reason, detailed groundwork had to be undertaken to ensure that any tests designed to detect DNA mutations would take into account the variation in sequences encoded amongst the species. The generation of these sequences (Clark et al. 2005) provided essential information on which laboratory tests were developed for the detection of BZ resistance-conferring mutations in cyathostomins. For example, beta-tubulin gene sequences were investigated in adult parasites of known BZ sensitivity to identify which polymorphisms are associated with resistance phenotype. This work showed that mutations at codons 167 and 200 both have a role to play in BZ resistance in cyathostomins (Hodgkinson et al. 2008). To increase sample throughput, Pyrosequencing has been developed for analysing DNA polymorphisms at these sites. Analysis using this technique has revealed statistically significant differences in sequence between FBZ-sensitive and resistant cyathostomin populations at codon 167 (Hodgkinson et al. 2008). A cyathostomin population resistant to the BZ compound, oxibendazole (OBZ), was shown to have significant differences at codon 200. This data indicated that mutations at codons 167 and 200 are both relevant to BZ resistance in cyathostomins and that differential selection at the two sites may occur in FBZ- vs. OBZ-resistant populations. Homozygous-resistant mutations occurring concurrently at both codons are probably lethal to cyathostomins, as these were not detected in the large number of parasites analysed. More recent research to examine the genomic structure of beta-tubulin genes in cyathostomins (Lake et al. 2009) has revealed a structure for the isotype 1 gene different to that published previously by other groups (Pape et al. 1999). This has important implications for diagnostic assay design because the later studies demonstrated that the 2 resistance-conferring mutations (167 and 200) are further apart on the genomic DNA template than was formerly presumed. This consequence of this is that 2 distinct assays need to be designed to analyse polymorphisms at each of these sites when studying the genomic DNA template. A multi-species, codon 167-based assay has now been developed to directly genotype field derived L3 of unknown species and BZ sensitivity (Lake et al. 2009). Preliminary results using this genotyping assay have revealed that just a single FBZ treatment can result in a substantial reduction in heterozygous (drug sensitive) genotypes with a consequent an increase in homozygous (resistant) genotypes. This is the first time that this effect had been quantified in cyathostomin L3 at the molecular level. Such an assay will become a valuable asset in future should cyathostomin resistance to the other classes of anthelmintics (the MLs and THPs) spread.

Improving methods for the detection of ML resistance

The MLs are by far the most commonly administered anthelmintics in horses (Lind et al. 2007). Members of this class, ivermectin (IVM) and MOX, play a central role in the management of cyathostomin (and other nematode) infections in horses and other equids worldwide. MOX, in particular, is popular, in part due to its recorded high efficacy against most stages of cyathostomins including the encysted larval stages (Bairden et al. 2001). Indeed, because of the extent of BZ resistance, MOX is realistically the only drug that is now effective against all stages of cyathostomins and is therefore an important part of armoury in controlling these infections. For this reason, the preservation of MOX effectiveness should be an essential component of all equine health programmes. To maintain efficacy, it is important that animals do not receive unnecessary treatments and that they are administered with the appropriate dose of drug at each treatment. It is also essential to monitor nematode population drug sensitivity, so that any reduction in efficacy is detected at as early stage as is possible. The faecal egg count reduction test (FECRT) can be used for analysing anthelmintic efficacy in the field; however, this test has a number of drawbacks, the major one being that the currently recommended methods of analysis do not take into account the distribution and level of variability of FECs in horses. This greatly impacts on the accuracy of inference on efficacy.

The FECRT and its improvement

Precise standards for FECRT study design and data analysis are lacking and there are few clear benchmarks for diagnosing anthelmintic resistance in horses. Data interpretation is often complicated by small group sizes, high proportions of zero FECs and substantial variation in FEC between animals. These variables are exacerbated by the fact that, even in drug-sensitive populations, efficacy levels differ amongst the various anthelmintic classes. Statistical modelling methods have been developed to overcome these drawbacks (Vidyashankar et al. 2007) and are currently being investigated to analyse FECRT data to test if these have sufficient power to detect differences between presumed and true ML efficacy. This work will be published in the near future and its applicability to field situations assessed.

The larval migration assay (LMA)

Studies in other nematode species have indicated that once a lack of efficacy of a specific anthelmintic is obvious (as demonstrated by a failed FECRT), it is too late to affect the preponderance of drug resistance within a given population (Martin et al. 1989). Hence, the containment of ML resistance should be a priority in equine parasite control programmes. It is important that assays more sensitive than the FECRT be developed for anthelmintic resistance detection. Laboratory assays such as the larval migration assay (LMA) may provide a possible means by which to discriminate anthelmintic resistance in a more sensitive and less time-consuming manner than the standard FECRT. In the LMA, L3 are cultured from equine faecal samples and then subjected to migration through a small filter in the presence of increasing concentrations of anthelmintic such as a ML, which paralyses sensitive worms (Matthews et al. in press). Assessment of the percentage migration of different cyathostomin populations in incremental concentrations of ML allows the generation of a dose-response curve, from which can be derived an EC50 value. The latter is the concentration of anthelmintic at which 50% of the L3 do not migrate and enables a comparative quantifiable analysis amongst populations or within a single population over time. A robust LMA has recently been developed for the examination of the ML sensitivity of cyathostomin populations in the laboratory (Matthews et al. in press). This assay is currently being used to compare ML sensitivity of L3 derived from various UK Thoroughbred stud farms where lack of efficacy of IVM or MOX has been detected using the FECRT. The LMA data derived from these populations is being compared with data obtained using well characterised ML-sensitive and ML-resistant cyathostomin isolates. Statistical comparison of the resistance indices derived from these various populations will confirm the value of this test as a pre-FECRT screen for ML sensitivity in large populations of horses.

Quantifying larval burden to avoid disease and facilitate targeted anthelmintic treatments

As previously mentioned, the clinical syndrome of larval cyathostominosis occurs as a result of the mass emergence of larval stages from the gut wall and has a high fatality rate despite treatment (Corning 2009). Management measures to prevent this syndrome currently rely heavily on the repeated use of anthelmintics, particularly those with larvicidal activities. There is no specific method by which to detect encysted cyathostomin larvae noninvasively: horses with high burdens often have low or negative FECs and animals that develop disease often have no specific clinical features that help discriminate the disease in practice (Dowdall et al. 2002). A diagnostic test for encysted cyathostomin larvae would aid the diagnosis of larval cyathostominosis and would allow the identification of those horses that require larvicidal anthelmintic treatments. The latter capability would facilitate targeted treatment strategies and hence help preserve MOX efficacy. Previous research studies led to the identification of 2 antigen complexes that have diagnostic potential for estimating cyathostomin encysted larval burdens (Dowdall et al. 2002, 2004). Antibody responses to these antigens in horse sera were shown to be good indicators of the levels of encysted larvae that were present. These antibody responses were also shown to be specific to the cyathostomin group, with no or limited crossreactivity to the other helminth species that affect equines. Furthermore, antigen-specific antibody levels were significantly higher in clinical larval cyathostominosis cases than in cyathostomin-naïve or cyathostomin-negative animals. As the antigens are extremely time consuming to purify and prepare and rely on a continuous source of tissue from infected horses, the genes encoding them were isolated and the antigens produced synthetically using Escherichia coli (McWilliam et al. 2010). So far, most work has been undertaken on the protein called the cyathostomin gut associated larval antigen (Cy-GALA). This protein, or its transcript, has been detected specifically in encysted larval stages: early L3, LL3 and DL4. Recombinant versions representative of this protein have been generated for a number of common cyathostomin species and these are currently being assessed for their utility in a diagnostic ELISA. The proteins have been shown to exhibit no reactivity to serum obtained from horses specifically infected with non-cyathostomin helminth species, nor does antisera, raised to Cy-GALA, bind to extracts from other helminth species. The receiver operating characteristic (ROC) curve, a graphical plot of the sensitivity (or true positive rate vs. false positive rate) of a diagnostic test, has been used to assess the value of an ELISA using these proteins. This analysis has indicated that the GALA proteins have great potential as the basis of a diagnostic assay for cyathostomin encysted larval stages. Currently, this area of research is directed at increasing the level of cyathostomin species coverage for the GALA proteins and for other proteins that have been identified as having diagnostic potential. It is anticipated that a diagnostic assay based on a cocktail combination of these proteins will be developed for commercial use in the short to medium term.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Research areas
  5. Conclusions
  6. Conflicts of interest
  7. Acknowledgements
  8. References

Our knowledge of the cyathostomins has expanded considerably over the last 10–15 years. State-of-the-art technologies in molecular biology and protein chemistry have been utilised in a number of ways. First, to allow the identification of some of the molecular mechanisms underlying anthelmintic resistance; however, work remains to be done in this area to address the imminent spread of ML resistance. Progress towards the development of a diagnostic assay for encysted larval cyathostomin stages provides real potential for a test that can be used to help target larvicidal treatments on the basis of burden instead of the current practice of blanket treatments, which are a substantial risk factor for the development of resistance. More traditional methodologies in parasitology also have a part to play in future control strategies; accurate FECRTs should be made more accessible to veterinary surgeons in practice so that they have confidence in assessing anthelmintic efficacy. This will require considerable investment in knowledge transfer and continuing professional development activities. For those dealing with large groups of horses, laboratory-based assays such as the LMA may provide a preliminary screening mechanism by which to assess the relative sensitivity of specific cyathostomin populations to ML anthelmintics. Taken together, the basic and more advanced tests, along with a philosophy of treating for infection type and level present, rather than random blanket treatments, will help maintain effective anthelmintics for longer. Ongoing studies on patterns of parasite epidemiology and risk factors for anthelmintic resistance will also inform on how parasite control strategies can be refined to help meet this objective.

Conflicts of interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Research areas
  5. Conclusions
  6. Conflicts of interest
  7. Acknowledgements
  8. References

HBLB's Veterinary Advisory Committee commissioned and sponsored this article as part of a series summarising progress made in areas relating to their priorities for research funding. The author and other workers at her institute hold current and previous research grants funded by the HBLB.

EVJ is delighted to publish HBLB's Advances in Equine Veterinary Science and Practice Review Series in recognition of the major contribution that HBLB research and educational funding has made to the health and welfare of the Thoroughbred.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Research areas
  5. Conclusions
  6. Conflicts of interest
  7. Acknowledgements
  8. References

The author would like to thank the Horserace Betting Levy Board, the Horse Trust, the BBSRC, the Donkey Sanctuary and the Thoroughbred Breeders' Association for generously funding some of the research cited in this review. The author would also like to thank Hamish McWilliam for the illustration used in Figure 1. The author also acknowledges Wikipedia for assistance with preparing the Glossary.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Research areas
  5. Conclusions
  6. Conflicts of interest
  7. Acknowledgements
  8. References
  • Bairden, K., Brown, S.R., McGoldrick, J., Parker, L.D. and Talty, P.J. (2001) Efficacy of moxidectin 2 per cent gel against naturally acquired strongyle infections in horses, with particular reference to larval cyathostomes. Vet. Rec. 148, 138-141.
  • Clark, H.J., Kaplan, R.M., Matthews, J.B. and Hodgkinson, J.E. (2005) Isolation and characterisation of a beta-tubulin isotype 2 gene from two species of cyathostomin. Int. J. Parasitol. 35, 349-358.
  • Corning, S. (2009) Equine cyathostomins: a review of biology, clinical significance and therapy. Parasit. Vectors 2, Suppl. 2, S1.
  • Dowdall, S.M.J., Matthews, J.B., Murphy, D., Love, S. and Proudman, C.J. (2002) Antigen-specific IgG(T) responses in natural and experimental cyathostomin infection. Vet. Parasitol. 106, 225-242.
  • Dowdall, S.M.J., Proudman, C.J., Klei, T.R., Mair, T.S. and Matthews, J.B. (2004) Antigen-specific IgGT responses as a marker for larval cyathostomin infection. Int. J. Parasitol. 34, 101-108.
  • Dowdall, S.M.J., Proudman, C.J., Love, S., Murphy, D., Klei, T.R. and Matthews, J.B. (2003) Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses. Res. vet. Sci. 75, 223-229.
  • Eysker, M. and Mirck, M.H. (1986) The distribution of inhibited early third stage Cyathostominae larvae in the large intestine of the horse. Parasitol. Res. 72, 815-820.
  • Giles, C.J., Urquhart, K.A. and Longstaffe, J.A. (1985) Larval cyathostomiasis (immature trichonema-induced enteropathy): a report of 15 clinical cases. Equine vet. J. 17, 196-201.
  • Hodgkinson, J.E., Clark, H.J., Kaplan, R.M., Lake, S.L. and Matthews, J.B. (2008) The role of polymorphisms at beta-tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. Int. J. Parasitol. 38, 1149-1160.
  • Hodgkinson, J.E., Love, S., Lichtenfels, J.R., Palfreman, S., Ramsey, Y.H. and Matthews, J.B. (2001) Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses. Int. J. Parasitol. 31, 197-204.
  • Hodgkinson, J.E., Freeman, K.L., Lichtenfels, J.R., Palfreman, S., Love, S. and Matthews, J.B. (2005) Identification of strongyle eggs from anthelmintic-treated horses using a PCR-ELISA based on intergenic DNA sequences. Parasitol. Res. 95, 287-292.
  • Hodgkinson, J.E., Lichtenfels, J.R., Mair, T.S., Cripps, P., Freeman, K.L., Ramsey, Y.H., Love, S. and Matthews, J.B. (2003) A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis. Int. J. Parasitol. 33, 1427-1435.
  • Ionita, M., Howe, D.K., Lyons, E.T., Tolliver, S.C., Kaplan, R.M., Mitrea, I.L. and Yeargan, M. (2010) Use of a reverse line blot assay to survey small strongyle (Strongylida: Cyathostominae) populations in horses before and after treatment with ivermectin. Vet. Parasitol. 168, 332-337.
  • Kaplan, R.M. (2002) Anthelmintic resistance in nematodes of horses. Vet. Res. 33, 491-507.
  • Kaye, J.N., Love, S., Lichtenfels, J.R. and McKeand, J.B. (1998) Comparative sequence analysis of the intergenic spacer region of cyathostome species. Int. J. Parasitol. 28, 831-836.
  • Kwa, M.S., Veenstra, J.G. and Roos, M.H. (1994) Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in beta-tubulin isotype 1. Mol. Biochem. Parasitol. 63, 299-303.
  • Kwa, M.S., Veenstra, J.G., Van Dijk, M. and Roos, M.H. (1995) Beta-tubulin genes from the parasitic nematode Haemonchus contortus modulate drug resistance in Caenorhabditis elegans. J. Mol. Biol. 246, 500-510.
  • Lake, S.L., Matthews, J.B., Kaplan, R.M. and Hodgkinson, J.E. (2009) Determination of genomic DNA sequences for beta-tubulin isotype 1 from multiple species of cyathostomin and detection of resistance alleles in third-stage larvae from horses with naturally acquired infections. Parasit. Vectors 2, Suppl . 2, S6.
  • Lind, E.O., Rautalinko, E., Uggla, A., Waller, P.J., Morrison, D.A. and Hoglund, J. (2007) Parasite control practices on Swedish horse farms. Acta vet. Scand. 49, 25.
  • Love, S. and Duncan, J.L. (1992) The development of naturally acquired cyathostome infection in ponies. Vet. Parasitol. 44, 127-142.
  • Lubega, G.W. and Prichard, R.K. (1990) Specific interaction of benzimidazole anthelmintics with tubulin: high-affinity binding and benzimidazole resistance in Haemonchus contortus. Mol. Biochem. Parasitol. 38, 221-232.
  • Lyons, E.T., Tolliver, S.C. and Drudge, J.H. (1999) Historical perspective of cyathostomes: prevalence, treatment and control programs. Vet. Parasitol. 85, 97-112.
  • Martin, P.J., Anderson, N. and Jarrett, R.G. (1989) Detecting benzimidazole resistance with faecal egg count reduction tests and in vitro assays. Aust. vet. J. 66, 236-240.
  • Matthews, J.B. (2008) An update on cyathostomins: anthelmintic resistance and worm control. Equine vet. Educ. 20, 552-560.
  • Matthews, J.B. (2010) Clinical Forum: drug resistance in cyathostomins. UK. Vet. Companion. Anim. 15, 9-17.
  • Matthews, J.B., Hodgkinson, J.E., Dowdall, S.M. and Proudman, C.J. (2004) Recent developments in research into the Cyathostominae and Anoplocephala perfoliata. Vet. Res. 35, 371-381.
  • Matthews, J.B., McArthur, C., Robinson, A. and Jackson, F. (in press) Advances in the in vitro diagnosis of anthelmintic resistance in cyathostomins. Vet. Parasitol.
  • McWilliam, H.E., Nisbet, A.J., Dowdall, S.M., Hodgkinson, J.E. and Matthews, J.B. (2010) Identification and characterisation of an immunodiagnostic marker for cyathostomin developing stage larvae. Int. J. Parasitol. 40, 265-275.
  • Molento, M.B., Antunes, J., Bentes, R.N. and Coles, G.C. (2008) Anthelmintic resistant nematodes in Brazilian horses. Vet. Rec. 162, 384-385.
  • Pape, M., Posedi, J., Failing, K., Schnieder, T. and von Samson-Himmelstjerna, G. (2003) Analysis of the beta-tubulin codon 200 genotype distribution in a benzimidazole-susceptible and -resistant cyathostome population. Parasitology. 127, 53-59.
  • Pape, M., von Samson-Himmelstjerna, G. and Schnieder, T. (1999) Characterisation of the beta-tubulin gene of Cylicocyclus nassatus. Int. J. Parasitol. 29, 1941-1947.
  • Proudman, C.J. and Trees, A.J. (1999) Tapeworms as a cause of intestinal disease in horses. Parasitol. Today 15, 156-159.
  • Silvestre, A. and Cabaret, J. (2002) Mutation in position 167 of isotype 1 beta-tubulin gene of Trichostrongylid nematodes: role in benzimidazole resistance? Mol. Biochem. Parasitol. 120, 297-300.
  • Traversa, D., Milillo, P., Barnes, H., von Samson-Himmelstjerna, G., Schurmann, S., Demeler, J., Otranto, D., Lia, R.P., Perrucci, S., Frangipane di Regalbono, A., Beraldo, P., Amodie, D., Rohn, K., Cobb, R. and Boeckh, A. (2010) Distribution and species-specific occurrence of cyathostomins (Nematoda, Strongylida) in naturally infected horses from Italy, United Kingdom and Germany. Vet. Parasitol. 168, 84-92.
  • Vidyashankar, A.N., Kaplan, R.M. and Chan, S. (2007) Statistical approach to measure the efficacy of anthelmintic treatment on horse farms. Parasitology. 134, 2027-2039.