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

Abstract

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.

Introduction

Members of the nematode group Cyathostominae infect almost all grazing horses. Most animals have burdens to the order of tens of thousands of nematodes and usually do not exhibit clinical disease. In some animals, however, cyathostomin infection leads to severe colitis (Giles et al., 1985). This occurs following the accumulation of large numbers of larvae that encyst and undergo development as early and late L3s and developing L4s, in the large intestinal wall. When vast numbers of encysted larvae (EL) emerge simultaneously from the large intestinal mucosa, an inflammatory enteropathy ensues. This has been termed larval cyathostominosis, the principal effect of which is weight loss; however, horses can exhibit other clinical signs including diarrhoea, colic, s.c. oedema and/or pyrexia (Love et al., 1999). Unfortunately, up to 50% of animals with larval cyathostominosis die (Giles et al., 1985). This disease most commonly occurs in younger horses but animals have a lifelong susceptibility to infection and the disease can occur at any age (Giles et al., 1985, Reid et al., 1995). Encysted larvae can persist for prolonged periods and it has been proposed that their existence is favoured by negative feedback from mature nematodes in the large intestinal lumen, large larval challenges or ‘trickle’ infections (Murphy and Love, 1997).

Drug resistance in cyathostomins is common, particularly with regard to benzimidazole and pyrantel compounds (Kaplan, 2002). Moxidectin (MOX) is the only commercially-available anthelmintic for which there is high efficacy against all encysted larval stages, but for which drug resistance is not yet widespread (Matthews, 2008). Nevertheless, reports of MOX resistance have recently started to emerge (Molento et al., 2008). To slow the rate at which MOX resistance develops or spreads, it is important to maintain elements of nematode refugia by utilising targeted treatment control programmes (Proudman and Matthews, 2000). For cyathostomins, this can be achieved by treating only those horses that have moderate to high nematode burdens at the appropriate time of year. Current protocols for targeted treatments in horses are based on faecal egg count analysis; however, this has no value in estimating burdens of encysted larvae. Indeed, horses with large EL burdens often have low or negative faecal egg counts (Paul, 1998). Previously, we identified two antigen complexes (observed to migrate at 20 and 25 kDa by one-dimensional SDS–PAGE), derived from encysted larvae, that showed potential as markers for estimating burdens of these stages (Dowdall et al., 2002, Dowdall et al., 2003, Dowdall et al., 2004). Significant increases in serum IgG(T) levels to the complexes were observed 6 weeks p.i. in experimentally-infected ponies (Dowdall et al., 2003) and, when serum IgG(T) levels were analysed in naturally- and experimentally-infected horses, significant correlations were observed between anti-25 kDa complex serum IgG(T) levels and the total encysted larval burden (Dowdall et al., 2004). Furthermore, serum IgG(T) levels specific to either complex were significantly higher in larval cyathostominosis cases than in helminth-naïve ponies and parasite-negative horses (Dowdall et al., 2004). When cyathostomin developmental stages were compared, antigens in both complexes appeared specific for EL stages, indicating their utility as markers for prepatent infections (Dowdall et al., 2003). An immunoassay based on antigens present within these complexes could ultimately be used to differentially diagnose larval cyathostominosis cases or could be used to assist in delivering targeted anthelmintic treatments to horses with high EL burdens. As somatic antigen preparations of cyathostomin EL are extremely labour intensive to prepare and rely on a continuous source of infected equine tissue for their production, we have developed a strategy to identify genes encoding proteins present in the larval antigen complexes and to test the associated recombinant proteins’ utility for diagnostic immunoassay. The data described here detail the identification, cloning, characterisation and recombinant protein expression of one component that shows promise as an immunodiagnostic marker for developing larval stage cyathostomins.