Abstract
BackgroundStrongyle parasites are ubiquitous in grazing horses. Strongylus vulgaris, the most pathogenic of the large strongyles, is known for its extensive migration in the mesenteric arterial system. The lifecycle of S. vulgaris is characterised by a long prepatent period where the migrating larvae are virtually undetectable as there currently is no test available for diagnosing prepatent S. vulgaris infection. Presence of S. vulgaris larvae in the arterial system causes endarteritis and thrombosis with a risk of non-strangulating intestinal infarctions. Emergence of anthelmintic resistance among cyathostomins has led to recommendations of reduced treatment intensity by targeting horses that exceed a predetermined strongyle faecal egg count threshold. One study suggests an apparent increase in prevalence of S. vulgaris on farms where reduced anthelmintic treatment intensity has been implemented. These issues highlight the need for an accurate and reliable assay for diagnosing prepatent S. vulgaris infection.MethodsImmunoscreening of a larval S. vulgaris cDNA library using hyperimmune serum raised against S. vulgaris excretory/secretory antigens was performed to identify potential diagnostic antigens. Immunoreactive clones were sequenced, one potential antigen was characterised, expressed as a recombinant protein, initially evaluated by western blot (WB) analysis, the diagnostic potential of the IgG subclasses was evaluated by ELISA, and the diagnostic accuracy evaluated using serum from 102 horses with known S. vulgaris infection status.ResultsThe clone expressing the potential antigen encoded a S. vulgaris SXP/RAL2 homologue. The recombinant protein, rSvSXP, was shown to be a potential diagnostic antigen by WB analysis, and a target of serum IgGa, IgG(T) and total IgG in naturally infected horses, with IgG(T) antibodies being the most reliable indicator of S. vulgaris infection in horses. Evaluation of diagnostic accuracy of the ELISA resulted in a sensitivity of 73.3%, a specificity of 81.0%, a diagnostic odds ratio of 11.69; a positive likelihood ratio (LR) of 3.85 and a negative LR was 0.33. The area under the ROC curve was 0.820.ConclusionIgG(T) antibodies to recombinant SvSXP show potential for use as an antigen for prepatent diagnosis of migrating stages of S. vulgaris with moderate to good diagnostic accuracy.
Highlights
Strongyle parasites are ubiquitous in grazing horses
Horse serum from naturally infected horses was initially used for immunoscreening of the cDNA library; strong levels of anti-E. coli antibodies in the horse serum resulted in high background staining that could not be controlled
Western blot analyses of IgG antibodies against rSvSXP in S. vulgaris-positive and negative horses The immunodiagnostic potential of antigen-specific IgG and IgG(T) antibodies against rSvSXP were evaluated by western blot (WB). These analyses revealed no IgG reactivity to rSvSXP in serum from two horses not infected with S. vulgaris and low level IgG(T) reactivity to rSvSXP in serum from eight horses not infected with S. vulgaris (Figure 4), as determined by post-mortem examination
Summary
Strongyle parasites are ubiquitous in grazing horses. Strongylus vulgaris, the most pathogenic of the large strongyles, is known for its extensive migration in the mesenteric arterial system. One study suggests an apparent increase in prevalence of S. vulgaris on farms where reduced anthelmintic treatment intensity has been implemented These issues highlight the need for an accurate and reliable assay for diagnosing prepatent S. vulgaris infection. Prior to the advent of modern paste-based dewormers, S. vulgaris was present in about 80–100% of horses [9,10], but frequent interval-dose anthelmintic regimens appear to have caused a dramatic reduction in prevalence [11,12] These frequent treatments have led to anthelmintic resistance in other parasite categories infecting horses; cyathostomins [13,14,15] and Parascaris equorum [16,17,18], and recommendations are given to reduce treatment intensity by basing control programs on systematic parasite surveillance [19,20,21]. Given the high pathogenicity of this parasite, there is a need for improved diagnostic assays to better diagnose and monitor this parasite while maintaining anthelmintic efficacy in equine populations
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