Anthelmintic resistance in strongylids (Nematoda: Strongylidae) parasitizing wild and domestic equids in the Askania Nova Biosphere Reserve, Ukraine

Resistance of strongylids in domestic horses to benzimidazole anthelmintics (BZ) has been detected worldwide; however, information on the presence of BZ-resistance in wild equids has not been published to date. The purpose of this study was to analyze the manifestations of the BZ resistance in strongylids in domestic and wild equids kept in the Askania Nova Biosphere Reserve, Ukraine. Four species of equids: domestic horses and Shetland ponies (Equus caballus), donkeys (E. asinus), plains zebras (E. burchelli) and Grévy's zebras (E. grevyi) kept under semi-free conditions were examined using the Fecal Egg Count Reduction Test (FECRT) in order to detect the presence of resistance to the BZ anthelmintics. Analysis of long-term data (2009­2019) revealed a decrease in the efficacy of BZ drugs against strongylids in these four species of equids from 97.6% in 2009 to <75% in 2019. The efficacy of anthelmintic treatments was low in all species of equids: in plains zebras ­ 69.4%, Grévy's zebras ­ 72.7%, horses ­ 66.4%, ponies ­ 61.1% and donkeys ­ 45.2%. Ten species of cyathostomins (Cyathostomum catinatum, Cylicocyclus nassatus, C. ashworthi, C. leptostomus, Cylicostephanus calicatus, C. goldi, C. longibursatus, C. minutus, Coronocyclus labiatus, C. labratus) were found after horse deworming with albendazole. Our results are the first detection of BZ-resistance in strongylids of wild equids kept under semi-free conditions in the Reserve.

New record of Steinernema arenarium (Artyukhovsky) (Rhabditida: Steinernematidae) from Ukraine and a note on its distribution.

During a survey of the biodiversity of entomopathogenic nematodes in Ukraine, a population of Steinernema arenarium, strain Ch, was recovered in the sensitive Chornobyl Exclusion Zone. In the present work, this strain was morphologically and molecularly characterised using light microscopy and the sequences of the ITS and D2-D3 region of the 28S rDNA. In addition, we sequenced the ITS and D2-D3 regions of four populations of S. arenarium from a laboratory collection. Phylogenetic analyses were performed and the phylogenetic structure and geographic distribution of S. arenarium are discussed.

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.

Morphology and diagnosis of the fourth-stage larva of Coronocyclus labratus (Looss, 1900) (Nematoda: Strongyloidea) parasitising equids.

The fourth larval stage (L4) of Coronocyclus labratus (Looss, 1900) Hartwich, 1986, one of the common species of the Cyathostominae found in equids, is identified and described. The larvae found were identified as C. labratus by finding moulting forms possessing characters of both larval and adult stages. The larvae are similar to those of Cylicocyclus leptostomum (Kotlán, 1920) Foster, 1936, Cyathostomum catinatum Looss, 1900 and Cylicostephanus goldi (Boulenger, 1917) Lichtenfels, 1975. The buccal capsule (BC) wall of the L4 of Cylicocyclus leptostomum is thinner than that of Coronocyclus labratus, and the ring of the oesophageal funnel is comparatively less well developed. In C. labratus the dorsal tooth protrudes only slightly into the buccal cavity, and this larva possesses a characteristically elongate, pyriform oesophagus. In Cyathostomum catinatum, the BC walls are thicker than in Coronocyclus labratus, and the dorsal tooth is more bluntly pointed. Sublateral teeth, if present, are bluntly pointed in the former species, but usually absent or indistinct in C. labratus. Larvae of Cylicostephanus goldi differ from those of Coronocyclus labratus in the larger size of the BC.

Illustrated identification keys to strongylid parasites (Strongylidae: Nematoda) of horses, zebras and asses (Equidae).

The Equidae (the horse, Equus caballus, the ass, Equus asinus, zebras and their hybrids) are hosts to a great variety of nematode parasites, some of which can cause significant morbidity or mortality if individual hosts are untreated. Worldwide the nematode parasites of horses belong to 7 suborders, 12 families, 29 genera and 83 species. The great majority (19 of 29 genera and 64 of 83 species) are members of the family Strongylidae, which includes the most common and pathogenic nematode parasites of horses. Only the Strongylidae are included in this treatise. The Strongylidae (common name strongylids) of horses--nematodes with a well-developed buccal capsule, a mouth collar with two leaf-crowns, and a strongyloid (common name of superfamily Strongyloidea) copulatory bursa--can be separated into two subfamilies: Strongylinae (common name strongylins), usually large or medium-sized with a globular or funnel-shaped buccal capsule; and Cyathostominae (common name cyathostomins), usually small to medium-sized with a cylindrical buccal capsule. The increased attention to strongylid nematode parasites of horses has resulted in the need for updated diagnostic keys to these parasites using readily recognizable characters and the most recent literature on their systematics. Because the cyathostomins have been historically difficult to identify, and because they have emerged as the most significant nematode pathogens of horses, we provide a brief nomenclatural and taxonomic history and an introduction to the morphology of this group. This treatise is intended to serve as a basic working tool--providing easy identifications to genus and species of adult strongylid nematodes of equids. All strongylid nematodes normally parasitic in horses, the ass (and their hybrids), and zebras are included. The keys are illustrated with line drawings and halftone photomicrographs of each species. A short discussion of the systematics of the genus and species is provided for each genus following the species descriptions. Species diagnoses and a synonymy of each species is provided. Geographic distribution, prevalence, and location in host are also given for each species.

Haplotypic variability within the mitochondrial gene encoding for the cytochrome c oxidase 1 (cox1) of Cylicocyclus nassatus (Nematoda, Strongylida): evidence for an affiliation between parasitic populations and domestic and wild equid hosts.

This study investigated the genetic variability within Cylicocyclus nassatus (Nematoda, Strongylida, Cyathostominae) collected from different domestic and wild hosts (i.e. horse, donkey, Przewalskii horse, tarpan and Turkmen kulan) and localities in Europe and/or USA. The ribosomal Internal Transcribed Spacer 2 (ITS2) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were PCR-amplified and sequences characterized from seventy individual parasitic specimens. While ITS2 displayed 0-0.6% variation rate among all individual adult specimens of C. nassatus examined, 22 different sequence variants (haplotypes) of cox1 were detected. Nucleotide variation was detected at 75 of the total 689 positions (overall 10.8% rate of intraspecific nucletidic difference) in the cox1, with the absence of invariable positions among specimens collected from each equid species or country. Conversely, two haplotypes were detected in horses from USA and in donkeys of Italy and Ukraine, respectively. The absence of haplotypes shared by the equid species suggests an affiliation of C. nassatus populations to their specific host. The results of the present study demonstrated that the characterization of mitochondrial regions may have important implications for studying the genetic structure and biology of equine cyathostomes, and to exploit taxonomic issues and practical implications related to the spread of anthelmintic resistance.

New method for simultaneous species-specific identification of equine strongyles (nematoda, strongylida) by reverse line blot hybridization.

The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species.

A redescription of Cylicocyclus adersi Boulenger, 1920 (Nematoda: Strongyloidea: Cyathostominae) parasitic in equids.

As part of a revision of the systematics of the Cyathostominea of equids, we determined that the single type-specimen of Cylicocyclus adersi Boulenger, 1920 is missing and a modern description of this rare species parasitic in donkeys Equus asinus L. and zebras E. burchelli (Gray) and E. zebra L. is not available. Systematists at a workshop on the systematics of the Cyathostominea of horses, convened on August 11, 1997 in Sun City, South Africa, listed C. adersi as a species inquirenda . Herein, we provide a redescription of C. adersi and propose a neotype for the species. The distinctive shape of the buccal capsule of C. adersi , with a relatively tiny 'hoop-like thickening' at its base, a relatively small oesophageal funnel with a thick cuticular lining, and a short, but distinct, dorsal gutter readily distinguishes this species from other large species of Cylicocyclus Ihle, 1922.