Detection of SNPs and benzimidazole resistance in strongyle nematode eggs of horses by allele-specific PCR.

This study was conducted to determine single nucleotide polymorphisms (SNPs) and the benzimidazole (BZ) resistance in strongyle nematode egg populations in horses using molecular techniques. A total of 200 fecal samples were collected from horses in 26 farms in two provinces (Kayseri and Nevsehir) of the Central Anatolia Region of Türkiye between May and August 2022. The flotation method was used to detect strongyle nematode eggs in the fecal samples of the horses. Afterward, strongyle nematode eggs were collected, and the allele-specific polymerase chain reaction (AS-PCR) technique was used to detect the BZ resistance. BZ-susceptible and BZ-resistant PCR products were sequenced to determine single nucleotide polymorphisms (SNPs) in the ß-tubulin isotype 1 gene. The strongyle nematode eggs were determined in 85 (42.5%) out of 200 fecal samples. AS-PCR detected 50.58% (43/85) BZ-resistant (homozygous resistant) and 36.4% (31/85) BZ-susceptible (homozygous susceptible) genes in the strongyle eggs. Both BZ-resistant and BZ-susceptible genes (heterozygous) were determined in 11 samples. BZ-resistant and BZ-susceptible allele frequencies were determined as 57.0% (48.5/85) and 43.0% (36.5/85), respectively. SNPs were detected only in codon 200 of the ß-tubulin isotype 1 gene in four sequenced isolates of the two resistant and two susceptible isolates. This study is the first molecular report on BZ resistance in strongyle nematode eggs in horses in Türkiye. The widespread prevalence of BZ-resistant alleles in equine strongyle nematodes shows the requirement for the immediate usage of other anthelmintics instead of the BZ group drugs for the effective management and control of equine strongyle nematodes.

Strongylids in domestic horses: Influence of horse age, breed and deworming programs on the strongyle parasite community.

An extensive analysis of the relationships between strongylid egg shedding in domestic horses and the strongylid community structure in regard to the age of the horses, their breeds and different strategies of horse management, particularly with anthelmintic treatment programs was performed. Domestic horses (n=197) of different ages (5 months to 22 years) and of various breeds from 15 farms with different types of deworming programs were included in this study. Strongylids (totally, 82,767 specimens) were collected in vivo after deworming of the horses with the macrocyclic lactone anthelmintic ("Univerm", 0.2% aversectin C), and identified to the species level. Models of multiple regressions with dummy variables were used to estimate the effects of age, breed, type of farm and deworming programs on number of eggs shed per gram of feces (EPG value) and the strongylid community. Totally, 33 strongylid species were collected (8 species of Strongylinae and 25 - of Cyathostominae); a significant correlation (r=0.67; p<0.001) between the prevalence and proportion of species in the strongylid community was observed. The highest number of species (32) was found in young horses (1.5-4 years old); the lowest (17) - in old horses (>16years). Foals (<1year old) had significantly higher EPG value than older horses. The linear regression models of the strongyle egg counts (EPG) with three predictors: horse age (AGE), number of strongylids (SN), and type of farm (FARM) revealed significant effects of SN and FARM, but an effect of AGE was near the limit of significance. Horses from farms with rare or no anthelmintic treatments (type A) shed significantly more strongyle eggs than horses from farms with regular treatments; frequency of dewormings - 1-2 (type B) or 3-4 and more times per year (type C) did not have a significant impact on the EPG value. Thoroughbreds, Ukrainian Saddlers and Russian Racers had much higher EPG values comparing to non-breed horses. Analysis of the relation of age of the horses and structure of the strongylid communities revealed that foals (<1year old) and old horses (>16years old) were significantly less infected by large strongyles as compared to other horses. Species from the genus Triodontophorus and Strongylus vulgaris infected foals and young horses; whereas S. edentatus and S. equinus were registered in horses >1.5years old. Differences in proportions of separate strongylid species in the community related to age were insignificant (p>0.05). Frequency of anthelmintic treatments was found to affect horses infections with strongylids and strongylid community structure much more than intrinsic factors such as age and breed.

Estimation of genetic parameters for resistance to gastro-intestinal nematodes in pure blood Arabian horses.

Equine internal parasites, mostly cyathostomins, affect both horse welfare and performance. The appearance of anthelmintic-resistant parasites creates a pressing need for optimising drenching schemes. This optimization may be achieved by identifying genetic markers associated with host susceptibility to infection and then to drench carriers of these markers. The aim of our study was to characterise the genetics of horse resistance to strongyle infection by estimating heritability of this trait in an Arabian pure blood population. A population of 789 Arabian pure blood horses from the Michalów stud farm, Poland were measured for strongyle egg excretion twice a year, over 8 years. Low repeatability values were found for faecal egg counts. Our analyses showed that less than 10% of the observed variation for strongyle faecal egg counts in this population had a genetic origin. However, additional analyses highlighted an age-dependent increase in heritability which was 0.04 (±0.02) in young horses (up to 3 years of age) but 0.21 (±0.04) in older ones. These results suggest that a significant part of the inter-individual variation has a genetic origin. This paves the way to a genomic dissection of horse-nematode interactions which might provide predictive markers of susceptibility, allowing individualised drenching schemes.

Lower shedding of strongylid eggs by Warmblood horses with recurrent airway obstruction compared to unrelated healthy horses.

An association between equine recurrent airway obstruction (RAO) and increased resistance to intestinal parasites has been demonstrated in descendants of an RAO-affected stallion. It was hypothesised that members of another high-incidence RAO family (F) and unrelated RAO-affected Warmblood horses (UA) would shed fewer strongylid eggs than unrelated RAO-unaffected pasture mates (PM) under the same environmental conditions. Faecal worm egg counts were performed on faecal samples (63 F, 86 UA, 149 PM) and classified into three categories: 0, 1-100 and >100 eggs per gram. While results for F did not differ from PM, UA were 2.5-times less likely to shed strongylid eggs than PM. RAO-affected Warmblood horses may be more resistant to strongylid nematodes than unrelated unaffected pasture mates and a family history of RAO does not necessarily confer protection against helminth infections.

Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs.

In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation in the ITS-2 for the Strongylus species examined, whereas the levels of interspecific differences (13-29%) were significantly greater. Interspecific differences in the ITS-2 sequences allowed unequivocal species identification of single worms and eggs using PCR-linked restriction fragment length polymorphism. These results demonstrate the potential of the ribosomal spacers as genetic markers for species identification of single strongyle eggs from horse faeces.