Occurrence of Strongylid Nematode Parasites on Horse Farms in Berlin and Brandenburg, Germany, With High Seroprevalence of Strongylus vulgaris Infection

Abstract

The infection of horses with strongylid nematodes is highly prevalent, with multi-species infections being the rule. Strongylus spp. and in particular Strongylus vulgaris are amongst the most pathogenic strongyle equine parasites. Presumably due to regular strategic anthelmintic treatments in combination with long prepatencies, prevalence of these worms was severely reduced in past decades. In this study, 484 horses from 48 farms in Berlin/Brandenburg, Germany were sampled between May 2017 and January 2018. Mini-FLOTAC and combined sedimentation/flotation were used to analyse faecal samples and larval cultures were carried out from individual strongyle infected horses for molecular testing for Strongylus spp. infection. Additionally, for Strongylus vulgaris, antibodies against a recombinant larval antigen were quantified in an ELISA. Strongyle type eggs were detected in 66.7% of the individual faecal samples. Nematode DNA was amplifiable from 311 samples and S. vulgaris and Strongylus edentatus were detected in four (1.3%) and 10 (6.3%) of these, respectively, the latter using a novel high-resolution-melt PCR targeting S. edentatus, Strongylus equinus, and Strongylus asini. On the farm level, prevalence for Strongylus spp. by PCR was 12.5%. Applying a conservative cut-off (sensitivity 0.43, specificity 0.96), 21.2% of all serum samples were positive for antibodies against S. vulgaris larvae (83.3% prevalence on farm level). Newly developed pyrosequencing assays to analyse putatively benzimidazole resistance associated polymorphisms in codons 167, 198, and 200 of the isotype 1 β-tubulin gene of S. vulgaris did not detect such polymorphisms in the four positive samples. Low age and increasing access to pasture were risk factors for egg shedding and seropositivity for S. vulgaris. Time since last treatment increased whereas use of moxidectin and ivermectin for the last treatment decreased the risk for strongyle egg shedding. Noteworthy, horses under selective treatment had significantly higher odds to be seropositive for anti-S. vulgaris antibodies than horses treated four times per year (odds ratio 4.4). The serological findings suggest that exposure to S. vulgaris is considerably higher than expected from direct diagnostic approaches. One potential explanation is the contamination of the environment by a few infected horses, leading to the infection of many horses with larvae that never reach maturity due to regular anthelmintic treatments.

Keywords: ELISA; Strongylus spp.; equine parasites; large strongyles; nematodes.

Figure 1

Comparison of faecal analyses methods, referring to counting results of strongyle type eggs of 484 faecal samples. Results of the combined sedimentation/flotation methods are plotted as classes (0: no eggs found; 1: 1–10 eggs per slide; 2: 11–40 eggs per slide; 3: ≥ 41 eggs per slide) vs. eggs per gram faeces (epg) as determined using Mini-FLOTAC.

Figure 2

Amplification plots for the Strongylus vulgaris real-time PCR. Plasmid DNAs with 500, 50, and 5 copies were used as positive control (green). The single negative control is shown in yellow but mostly hidden by the black curves. Field samples considered to be negative are shown in black and samples scored as positive are in blue. All positive samples, including the one with a very high Cq value were repeatedly tested positive and identity of all PCR products was confirmed by Sanger sequencing.

Figure 3

Establishment of a high-resolution melt PCR to distinguish Strongylus edentatus, Strongylus equinus, and Strongylus asini. PCR. The amplification plots show that presence of 10,000–5 copies (S. edentatus, S. equinus) or 1,000–5 copies (S. asini) ITS-2 plasmid resulted exponential amplification while there was no exponential increase of relative fluorescence units (RFU) in the presence of 10,000 to 5 copies of S. vulgaris ITS-2 DNA or in the absence of any template (NC). (A). Using the data from (A), the quantification cycles (Cq) were plotted against the log₁₀ transformed copy numbers and linear regressions were calculated (B). There were no significant differences between slopes (p = 0.760). Raw melting curves (C) and their first derivative dRFU/dT (D) were recorded between 65°C and 98°C. Normalisation of the melting curves between 73.1 and 79.9°C resulted in clear separation of the melting curves (E), which was also visible in the difference plot (F), in which the mean normalised fluorescence for S. edentatus was subtracted from all individual plots.

Figure 4

High-resolution melting curves for DNA from four field samples compared to standards containing 500 copies of the ITS-2 of S. edentatus, S. equinus, and S. asini. All positive samples were assigned to S. edentatus, which was confirmed by Sanger sequencing of the PCR products.

Figure 5

Regression analysis of pyrosequencing assays for S. vulgaris isotype 1 β-tubulin for polymorphisms F167Y (A), E198A (B) and F200Y (C). Artificial mixtures of plasmids were prepared and analysed using pyrosequencing. For each mixture, four to six replicates were used and included in the analyses. Regression plots with 95% confidence bands as calculated by Pearson regression are shown. Raw data are shown as circles (dots) ± SEM.

Figure 6

Risk factor analysis for the odds of horses in the study population to be positive for strongyle eggs. Risk factors for the odds to be positive or negative for strongyle eggs were determined using logistic regression analysis. Anoplocephala_Serum, positive in the Anoplocephala serum ELISA; Drugs refer to the drug used for the last treatment; Time since treatment represents the time since the last anthelmintic treatment in weeks [w]. The age refers to the age of the animals in years [y]. The age of horses was rounded into whole years. For foals and yearlings, the age was converted from months to years by dividing the month measurement by the conversion ratio 12. ***, p < 0.001; **, p < 0.01; *, p < 0.05.

Figure 7

Risk factor analysis for the odds of horses in the study population to be positive for antibodies against the Strongylus vulgaris recombinant SvSXP larval antigen. Risk factors for the odds to be seropositive or negative for antibodies against S. vulgaris using logistic regression analysis. The treatment scheme refers to whether horses were selectively dewormed based on previous diagnosis (selective) or regularly dewormed 1–2.5 times (<3 times), 3–3.5 times (<4 times), or 4 times per year on average. The age of the horses was rounded into whole years [y]. For foals and yearlings, the age was converted from months to years by dividing the month measurement by the conversion ratio 12. **, p < 0.01; *, p < 0.05.