ß-Tubulin genotypes in six species of cyathostomins from anthelmintic-naive Przewalski and benzimidazole-resistant brood horses in Ukraine.

Resistance to benzimidazoles (BZ) in the gastrointestinal nematodes of livestock is characterised by the presence of specific polymorphisms in the ß-tubulin isotype 1 protein, a component of microtubules. The most prevalent polymorphism associated with resistance in nematodes infecting cattle, sheep, and goats is found at codon 200, with minor occurrences of polymorphisms at codons 167 and 198. In the cyathostomins that infect horses, however, a polymorphism at codon 167 appears to be more common than the codon 200 polymorphism. In the present study, a focussed analysis of PCR-amplified ß-tubulin fragments incorporating the above-mentioned three polymorphic sites in isotype 1 and 2 genes in worms of six species of cyathostomins, Cylicocyclus nassatus, Cylicocyclus ashworthi, Cyathostomum catinatum, Cylicostephanus goldi, Cylicostephanus longibursatus, and Coronocyclus coronatus, was performed. Worms were collected from two distinct horse populations, i.e. they were collected from Przewalski horses of the Askania-Nova Biosphere Reserve that had never received any anthelmintic treatment and from brood horses of the Dubrovsky farm where benzimidazole resistance had become established. DNA was extracted and sequenced from three worms of each species and population as well as from pools of 50 male C. nassatus and C. catinatum from both populations. The vast majority of putatively BZ resistance-associated TAC alleles were found at codon 167, compared to codon 200. The former allele occurred in isotype 1 in all six species of the supposedly benzimidazole-resistant cyathostomins from Dubrovsky horses. None of the polymorphisms associated with resistance was found in the corresponding isotype 2 codons nor at codon 198 in any of the six species of cyathostomins (neither single nor pooled worm DNA) from either of the two populations. These findings further support the predominant association of ß-tubulin isotype 1 and therein codon 167 with BZ resistance in cyathostomins.

Morphology and diagnosis of some fourth-stage larvae of cyathostomines (Nematoda: Strongyloidea) in donkeys Equus asinus L. from Ethiopia.

Fourth-stage larvae of four species of the Cyathostominae Nicoll, 1927 parasitic in donkeys Equus asinus L. from Ethiopia were identified mainly using moulting specimens. They are Cylicocyclus asini Matthee, Krecek & Gibbons, 2001, C. auriculatus (Looss, 1900) Chaves, 1930, Cyathostomum tetracanthum (Mehlis, 1831) Molin, 1861 (sensu Looss, 1900) and Cylindropharynx brevicauda Leiper, 1911. The larva of Cylicocyclus asini is similar to those of C. nassatus (Looss, 1900) Chaves, 1930 and C. leptostomum Kotlán, 1920, but differs from the former by the shape of the dorsal tooth in the oesophageal funnel, which is notably smaller than in C. asini but similar to that of C. leptostomum. However, the larva of C. asini differs from that of C. leptostomum in the size of the buccal capsule, which is notably larger than in C. leptostomum but similar to that of C. nassatus. The larva of C. auriculatus is very similar to that of C. insigne (Boulenger, 1917) Chaves, 1930. The larva of Cyathostomum tetracanthum is similar to those of Cylicostephanus bidentatus (Ihle, 1925) Lichtenfels, 1975, C. goldi (Boulenger, 1917) Lichtenfels, 1975 and Cyathostomum catinatum Looss, 1900. The larva of Cylindropharynx brevicauda is similar to that of Petrovinema skrjabini (Ershov, 1930) Ershov, 1943 but smaller in size. Including the four cyathostomine species identified in the present study, 36 species belonging to 13 genera have so far been described and identified of a total of 64 recognised and confirmed species of equine strongyles.

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.

Anthelmintic resistance in cyathostomins of brood horses in Ukraine and influence of anthelmintic treatments on strongylid community structure.

In 2004-2006, 322 brood horses from 11 horse farms were examined using the faecal egg count reduction test (FECRT) to determine the presence and distribution of anthelmintic resistance in strongylids in Ukraine. The anthelmintic drugs "Albendazole-7.5" (7.5mg of albenazole, Ukraine) at a dose of 5mg per kg body weight and "Univerm" (0.2% aversectin C, Russia) at a dose of 0.5mg per kg body weight were used. Seventy-one horses from six farms were examined in vivo to investigate the influence of anthelmintic treatment on the gastrointestinal strongylid community structure. Horses were treated with anthelmintics; faecal sampling (200 g in each sample) for strongylid expulsion was performed 24, 36, 48 and 60 h after treatment; and all strongylids expelled (25,292 specimens) were collected and identified. Fourteen horses from the Dubrovsky horse farm were also examined to determine the benzimidazole-resistant cyathostomin species; 5208 specimens of benzimidazole-resistant cyathostomins were collected and identified. According to the FECRT data, benzimidazole resistance in strongylids was observed only at the Dubrovsky horse farm (FECRT=68.7%). No resistance to macrocyclic lactones in strongylids or in Parascaris equorum was observed. Twenty-nine strongylid species were found in horses from six horse farms. The number of species per horse ranged from 4-9 (5.8+/-1.5) to 10-20 (14.4+/-2.9) and depended on horse anthelmintic treatment strategies. From 4 to 13 strongylid species predominated (prevalence>66.7%) in the strongylid community. Eleven cyathostomin species (Cylicocyclus nassatus, C. ashworthi, C. leptostomum, Cyathostomum catinatum, C. pateratum, Cylicostephanus calicatus, C. longibursatus, C. goldi, C. minutus, Coronocyclus coronatus and C. labiatus) were found to be resistant to benzimidazoles at the Dubrovsky horse farm. Ten of these were the dominant species in the strongylid community; only C. labiatus was a rare species (prevalence 29.4%). Species richness and species diversity were significantly higher in horses from farms without treatment or with occasional treatments than from farms with regular treatments. The shape of the prevalence frequency distribution of strongylid species from farms with regular treatments was bimodal ("core" and "satellite" mode). This distribution was multimodal at farms without treatment or with occasional anthelmintic treatments. The results of the current study indicated the possibility of the further spread of anthelmintic resistance on horse farms in Ukraine and the necessity of monitoring the development of resistance in horse parasitic nematodes.