Differentiating Strongylus vulgaris-associated, non-strangulating intestinal infarctions from idiopathic peritonitis and acute colitis in horses.

BACKGROUND: Horses with non-strangulating intestinal infarction (NSII) are often misdiagnosed with idiopathic peritonitis or acute colitis. Early diagnosis is essential to ensure early surgical intervention and improve survival. METHODS: Clinical and laboratory data from horses admitted to the University of Copenhagen Large Animal Teaching Hospital with NSII, idiopathic peritonitis or acute colitis between 2009 and 2018 were used for univariate comparisons and a multivariable logistic regression model for prediction of NSII. RESULTS: Two hundred and thirty-one horses were included. A multivariable model for the prediction of NSII included gastric reflux (more than 5 L) (odds ratio [OR] 8.7; 95% confidence interval [CI] 2.1-36.2), abnormal findings palpated per rectum (intestinal dilatations/impactions [OR 4.43; 95% CI 1.43-13.38], colon displacements [OR 23.16; 95% CI 5.26-101.97] or intestinal mass [OR 179.7; 95% CI 23.5-1375.5]), white blood cell count (OR 1.2; 95% CI 1.1-1.4), packed cell volume (OR 0.9; 95% CI 0.8-0.9), age (OR 0.9; 95% CI 0.8-1.0) and heart rate (OR 1.1; 95% CI 1.0-1.1). The model had a low false positive rate (5%), but a high false negative rate (50%). LIMITATIONS: Due to the retrospective nature of the study, sample collection was inconsistent, resulting in missing values. CONCLUSION: The model had some capability in predicting NSII. However, the high risk of false negatives means that exploratory laparotomy should be considered in horses with peritonitis of unknown aetiology in areas where Strongylus vulgaris is prevalent and occurrence of idiopathic peritonitis is low.

SvSXP: a Strongylus vulgaris antigen with potential for prepatent diagnosis.

BACKGROUND: 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. 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. METHODS: Immunoscreening 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. RESULTS: The 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. CONCLUSION: IgG(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.

Examination of equine glandular stomach lesions for bacteria, including Helicobacter spp by fluorescence in situ hybridisation.

BACKGROUND: The equine glandular stomach is commonly affected by erosion and ulceration. The aim of this study was to assess whether bacteria, including Helicobacter, could be involved in the aetiology of gastric glandular lesions seen in horses. RESULTS: Stomach lesions, as well as normal appearing mucosa were obtained from horses slaughtered for human consumption. All samples were tested for urease activity using the Pyloritek assay, while mucosal bacterial content was evaluated using Fluorescence In Situ Hybridisation. In selected sub samples, bacteria characterisation was pursued further by cloning and sequencing. Mucosal lesions were found in 36/63 stomachs and included hyperplastic rugae, polypoid structures and focal erosions. None of the samples were tested positive for urease activity or for FISH using the Helicobacter genus specific probe. In samples of lesions, as well as normal samples, clones with 99% similarities to Lactobacillus salivarius and Sarcina ventriculi were found. Escherichia like bacterium clones and Enterococcus clones were demonstrated in one focal erosion. Based on a phylogenetic tree these clones had 100% similarity to Escherichia fergusonii and Enterococcus faecium. The Enterococcus were found colonising the mucosal surface, while E. fergusonii organisms were also demonstrated intraepithelial. CONCLUSION: Gastric Helicobacter spp. could not be verified as being involved in lesions of the glandular stomach of the horse. Since E. fergusonii has been described as an emerging pathogen in both humans and animals, the finding of this bacterium in gastric erosion warrants further clarification to whether gastric infection with this type bacterium is important for horses.

Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR.

Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can reliably and semi-quantitatively detect minute quantities of S. vulgaris eggs in faecal samples.

Climatic influences on development and survival of free-living stages of equine strongyles: implications for worm control strategies and managing anthelmintic resistance.

Development of resistance to anthelmintic drugs by horse strongyles constitutes a growing threat to equine health because it is unknown when new drug classes can be expected on the market. Consequently, parasite control strategies should attempt to maintain drug efficacy for as long as possible. The proportion of a parasite population that is not exposed to anthelmintic treatment is described as being "in refugia" and although many factors affect the rate at which resistance develops, levels of refugia are considered the most important as these parasites are not selected by treatment and so provide a pool of sensitive genes in the population. Accordingly, treatment should be avoided when pasture refugia are small because such treatments will place significant selection pressure for resistance on worm populations. Given this new paradigm for parasite control, it has become important to identify seasons and circumstances wherein refugia are diminished. Free-living stages of equine strongyles are highly dependent on climatic influences, and this review summarises studies of strongyle development and survival under laboratory and field conditions in Northern (cool) temperate, Southern (warm) temperate and subtropical/tropical climates. In Northern temperate climates, refugia are smallest during the winter. In contrast, refugia are lowest during the summer in warm temperate and subtropical/tropical climates. Although adverse seasonal changes clearly have significant effects on the ability of free living stages of strongyle nematode parasites to survive and develop, available data suggest that climatic influences cannot effectively "clean" pastures from one grazing season to the next.