The Pentezug Wildhorse Reserve, located in the Hortobágy National Park, Hungary, has one of the biggest ex situ populations of Przewalski's horses and aims to preserve its landscape and to study this subspecies. Between September and November 2018, 79 faecal samples were collected from Przewalski's horses. The McMaster, Willis flotation, natural sedimentation and coproculture methods were applied to all the samples. Results showed an average level of 1287 eggs per gram (EPG), which is a high faecal egg-shedding level. All the samples were positive for strongyle-type eggs (100%). There were no statistical differences regarding the EPG values between different harems of the population. The same happened when considering sexes, ages, lactating status or when bachelors are compared with harem members. Cyathostominae were dominant, when compared to Strongylinae and Tricostrongylidae, and 15 different morphological infective third-stage larvae types and/or species belonging to the order Strongylida were identified. The subfamily Cyathostominae was prevalent in 100% of the horses. Strongylus vulgaris was the most prevalent strongylin (40.5%). Additionally, 27.8% were positive for Parascaris sp. and 2.5% showed Oxyuris equi in their faeces. This study revealed that there is a higher prevalence of Triodontophorus serratus and Poteriostomum spp. in juveniles. Horses with S. vulgaris showed lower levels of EPG. This was the first study involving this population, showing 100% prevalence of intestinal parasites.

To study the prevalence of the polymorphism in position 200 of the beta-tubulin gene in the mechanism of benzimidazole (BZ) resistance in cyathostomes of horses, an allele-specific PCR was used to detect the genotype of individuals of BZ-susceptible and BZ-resistant populations. The molecular analysis of 100 adults recovered from an anthelmintic-naïve horse revealed 80% homozygous TTC/TTC individuals, 17% heterozygous TTC/TAC and 3% homozygous TAC/TAC. A naturally infected horse was treated with increasing fenbendazole (FBZ) dosages to select a BZ-resistant population of cyathostomes. The PCR based analysis of 3rd-stage larvae (L3) during the experiment revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals. After treatment 42·3% of the adults (n=104) were homozygous TTC/TTC, 55·8% were heterozygous TTC/TAC and only 1·9% showed the homozygous genotype TAC/TAC. The results of the molecular analysis lead to the proposal that polymorphism within codon 200 is not the only reason for the development of BZ resistance in small strongyles.

Three nucleotide data sets, one nuclear (ITS-2) and two mitochondrial (COI and l-rRNA), have been investigated in order to determine relationships among species of Strongylinae and Cyathostominae, intestinal parasites of the horse.Nucleotide sequence data reported in this paper are available in the NCBI, GenBank, accession numbers: AF263472 to AF263472 and AF268479. The data exhibited a strong mutational bias towards A and T and in the COI gene, silent sites appeared to saturate rapidly partly due to this substitution bias. Thus, the COI gene was found to be less phylogenetically informative than the l-rRNA and ITS-2 genes. Combined analysis of the l-rRNA and ITS-2 genes supported a monophyletic clade of the cyathostomes with Tridentoinfundibulum gobi, which had previously been classified as a nematode of ‘uncertain origin'. The Strongylinae grouped consistently outside the clade containing the cyathostomes and T. gobi. Molecular analysis failed to provide strong evidence for the separation of cyathostomes into classical genera, as previously defined by morphological classification.