Unravelling the genetic diversity and relatedness of Echinococcus multilocularis isolates in Eurasia using the EmsB microsatellite nuclear marker.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe helminthic zoonotic disease distributed in the Northern Hemisphere. The lifecycle of the parasite is mainly sylvatic, involving canid and rodent hosts. The absence of genetic data from most eastern European countries is a major knowledge gap, affecting the study of associations with parasite populations in Western Europe. In this study, EmsB microsatellite genotyping of E. multilocularis was performed to describe the genetic diversity and relatedness of 785 E. multilocularis isolates from four western and nine eastern European countries, as well as from Armenia and the Asian parts of Russia and Turkey. The presence of the same E. multilocularis populations in the Benelux resulting from expansion from the historical Alpine focus can be deduced from the main profiles shared between these countries. All 33 EmsB profiles obtained from 528 samples from the nine eastern European countries belonged to the European clade, except one Asian profile form Ryazan Oblast, Russia. The expansion of E. multilocularis seems to have progressed from the historical Alpine focus through Hungary, Slovakia, the Czech Republic and southern Poland towards Latvia and Estonia. Most of the samples from Asia belong to the Asian clade, with one EmsB profile shared between Armenia and Turkey, and two between Turkey and Russia. However, two European profiles were described from two foxes in Turkey, including one harboring worms from both European and Asian clades. Three EmsB profiles from three Russian samples were associated with the Arctic clade. Two E. multilocularis profiles from rodents from Lake Baikal belonged to the Mongolian clade, described for the first time here using EmsB. Further worldwide studies on the genetic diversity of E. multilocularis using both mitochondrial sequencing and EmsB genotyping are needed to understand the distribution and expansion of the various clades.

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.

Characterisation of serum IgG(T) responses to potential diagnostic antigens for equine cyathostominosis.

Cyathostomins are ubiquitous parasitic nematodes of horses. These worms spend substantial periods as intestinal wall stage encysted larvae, which can comprise up to 90% of the total burden. Several million larvae have been reported in individuals. Emergence of these larvae from the gut wall can lead to life-threatening colitis. Faecal egg count tests, increasingly used by horse owners to inform anthelmintic treatments, do not correlate with the intra-host burden of cyathostomins; this represents a key gap in the diagnostic toolbox. Previously, a cyathostomin Gut Associated Larval Antigen was identified as a promising marker for the intra-host stages of infection. Here, cyathostomin Gut Associated Larval Antigen and an additional protein, Cyathostomin Immuno-diagnostic antigen, were investigated to examine their value in providing information on cyathostomin burden. ELISA analyses examined serum IgG(T) responses to recombinant proteins derived from individual cyathostomin species. Receiver Operator Characteristic curve analysis was performed on the ELISA data; proteins with the highest Area Under the Curve values were selected to test protein combinations to investigate which were the most informative in identifying the infection status of individuals. Three cocktail combinations were tested, comprising: (a) Cy-GALA proteins from two species and a Cy-CID protein from a third species (CT3), (b) Cy-GALA proteins from five species (CT5), and (c) all CT5 components, plus a Cy-CID protein from an additional species (CT6). The best predictive values for infection were obtained using CT3 and CT6, with similar values achieved for both. Proteins in CT3 are derived from the most commonly reported species, Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus longibursatus. This combination was selected for future development since it represents a more commercially viable format for a diagnostic test.

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.

Development of a recombinant protein-based ELISA for diagnosis of larval cyathostomin infection.

Cyathostomins are ubiquitous nematodes of horses. Once ingested, they can spend a substantial time as encysted larvae in the intestinal wall. The larvae can comprise up to 90% of the total burden, with up to several million worms reported in individuals. These stages can emerge in large numbers to cause life-threatening colitis. Direct methods for detection of encysted larval burdens in live horses do not exist. Previously, two antigen complexes were identified as promising markers for infection. A component of these, cyathostomin gut associated larval antigen-1 (Cy-GALA-1), was identified following immunoscreening of a complementary DNA library. Serum immunoglobulin G(T) (IgG(T)) responses to Cy-GALA-1 were shown to inform on larval infection. Sequence analysis of polymerase chain reaction products amplified from individual worms indicated that Cy-GALA-1 was derived from Cyathostomum pateratum. As cyathostomin infections always comprise multiple species, a diagnostic test must account for this. Here, segments of the Cy-gala gene were isolated from four common species, Cyathostomum catinatum, Cylicocyclus ashworthi, Cylicostephanus goldi and Cylicostephanus longibursatus, and the associated proteins expressed in recombinant form. The specificity and immunogenicity of each protein was confirmed. Each protein was assessed by enzyme linked immuno sorbent assay (ELISA) for its ability for informing on the presence of encysted larval infection and the level of burden.

Analysis of study comprehensiveness for nematode fauna of hydrophilic birds in Ukrainian Polissya.

The fauna of helminths of hydrophilic birds was subject of many works in Ukraine. But the most of investigators dealt with helminth fauna of waterfowls in general. And only few works were dedicated to the parasitic nematodes. There are registered 81 species of helminths on Ukraine and 36 species of nematode of hydrophilic bird for Polissya (23 species for Belarus. 15 for Poland and 18 for Ukraine). As well it was compared lists of parts of Polissya between itself. The comprehension of the parasitic nematodes from different part of Polissya showed that 6 species of nematodes are common for whole Polissya: Eucoleus contortus, Amidostomum anseris, Echinuria uncinata, Porrocaecum crassum, Porrocaecum semiteres and Tetrameres fissispina. Further studies are necessary to increase our knowledge on biodiversity of the parasitic nematodes in waterfowls and clear up their taxonomic status.

Human dirofilariasis due to Dirofilaria repens in Ukraine, an emergent zoonosis: epidemiological report of 1465 cases.

The filarial nematode Dirofilaria repens is currently considered to be one of the most extensively spreading human and animal parasites in Europe. In Ukraine, reporting cases of dirofilariasis has been mandatory since 1975, and the disease was included in the national surveillance system for notifiable diseases. Up until December 31st 2012, a total of 1533 cases have been registered, with 1465 cases occurring within the previous 16 years. Most of the cases of dirofilariasis were registered in 6 regions: Kyiv, and the Donetsk, Zaporizhzhya, Dnipropetrovsk, Kherson and Chernihiv oblasts. In the years 1997-2002 the highest incidence rate was noted in the Kherson oblast in the south of the country (9.79 per 100 000 people), and the lowest in western Ukraine (0.07-1.68 per 100 000 people). D. repens infections were registered in all oblasts. Parasitic lesions were most often located in the head, the subconjunctival tissue and around the eyes. D. repens lesions were also found in the limbs, torso, male sexual organs, and female mammary glands. Dirofilariasis was diagnosed in persons aged from 11 months to 90 years old, most often among people between 21-40 years of age. Most patients had only one parasitic skin lesion; the majority of isolated nematodes were female. The results of our analysis point to a constant increase in D. repens dirofilariasis incidence in humans in Ukraine. Despite educational efforts, infections have become more frequent and the territory in which the disease occurs has enlarged to encompass the whole of Ukraine. Nevertheless, the Ukrainian sanitary-epidemiological services managed to achieve some measure of success, e.g. by creating a registration system for D. repens infections and establishing proper diagnostics for the disease.

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.

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.

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.

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.

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.