Nematodes Of Horses Research Articles

Patterns of Equine Small Strongyle Species Infection after Ivermectin Intervention in Thailand: Egg Reappearance Period and Nemabiome Metabarcoding Approach.

The indiscriminate use of anthelmintics to control parasitic nematodes in horses has led to the emergence of anthelmintic resistance worldwide. However, there are no data available on using ivermectin for treating strongyle infections within domesticated horses in Thailand. Therefore, this study aimed to use the fecal egg count reduction (FECR) test to determine the strongylid egg reappearance period (ERP). Additionally, the nemabiome metabarcoding approach is incorporated to study patterns of strongyle species infection following ivermectin treatment. The study results indicate that, although ivermectin effectively eliminated adult strongyle parasites within two weeks post-treatment, the ERP was shortened to 6 weeks post-treatment with a mean FECR of 70.4% (95% CI 46.1-84.0). This potentially indicates a recent change in drug performance. In addition, nemabiome metabarcoding revealed that strongyle species have different levels of susceptibility in response to anthelmintic drugs. The reduction in ERP was associated with the early reappearance of specific species, dominated by Cylicostephanus longibursatus and Cylicocyclus nassatus, indicating the lower susceptibility of these species. In contrast, Poteriostomum imparidentatum, Triodontophorus nipponicus, and Triodontophorus serratus were not found post-treatment, indicating the high level of susceptibility of these species. This information is vital for comprehending the factors contributing to the emergence of resistance and for devising strategies to manage and control strongyle infections in horses.

Understanding temporal and spatial distribution of intestinal nematodes of horses using faecal egg counts and DNA metabarcoding

This study reports the spatial and temporal distribution of ascarid and strongylid nematodes in Thoroughbred horses by age category across different climatic zones in Australia over an 18-month period. Faecal samples (n = 2046) from individual horses were analysed using the modified McMaster technique for faecal egg counts (FECs). Strongylids were identified using PCR-directed next-generation sequencing of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA. Yearlings had the highest prevalence (82%) of strongyle eggs followed by weanlings (79%), foals (58%), wet mares (49%) and dry mares (46%). For Parascaris spp., foals had the highest prevalence (35%) followed by weanlings (21%) and yearlings (10%). The highest mean FECs for Parascaris spp. were observed in foals (525 eggs per gram [EPG] of faeces) while those for strongyles were in yearlings (962 EPG). Among horses that were classified as adults at the time of sampling, 77% (860 of 1119) of mares were low (i.e., <250 EPG) strongyle egg-shedders. Mean strongyle FEC counts were highest in the Mediterranean (818 EPG) followed by summer (599 EPG), winter (442 EPG), and non-seasonal (413 EPG) rainfall zones. Twenty-six nematode species were detected, with Cylicostephanus longibursatus (26.5%), Cylicocyclus nassatus (23.7%) and Coronocyclus coronatus (20.5%) being the most frequently detected species. Their richness and relative abundance varied with horse age, season and climatic zone. In addition, Strongylus equinus and Triodontophorus spp. (T. brevicauda and T. serratus) were also detected. This comprehensive study elucidates spatial (climatic zone) and temporal (i.e., seasonal) trends in prevalence and burdens of intestinal nematodes in Australian horses using non-invasive conventional and molecular methods. The information presented in this study is crucial for developing integrated management strategies to control horse parasites in farmed horses.

Prevalence and diversity of ascarid and strongylid nematodes in Australian Thoroughbred horses using next-generation sequencing and bioinformatic tools

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n = 1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10–14%) and 72% (70–74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251–500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses.

Evidence of resistance to ivermectin in the gastrointestinal nematodes of horses from Mexican southeast.

Gastrointestinal nematodes (GIN) are a major impediment to breeding and exploitation of horses. Traditional control of GIN has generated resistance to main anthelmintics, including ivermectin. An analysis of five ranches with a history of IVM use was done to determine the efficacy and resistance of GIN to IVM treatment in horses from the Mexican southeast. Predesigned questionnaires were applied to collect information on previous treatment protocols. The fecal egg count reduction test (FECRT) was applied to determine resistance. Before IVM application, a McMaster test was used to diagnose GIN infection in horses, and feces cultures were done to identify L3 larvae for Strongylida eggs. Pre-treatment samples showed that 72.7% (80/110) of horses were GIN positive, with cyathostomins being the most frequent (91.8%), followed by Oxyuris equi (7.0%), Parascaris equorum (1.0%), and Strongylus vulgaris (0.2%). Based on the results, the horses at each ranch were divided in control (CG) and experimental (EG) groups with similar eggs per gram of feces (EPG). The EG (40/80) was dewormed with IVM (0.2mg/kg orally) and the CG (40/80) remained untreated. After 14days, EPG were measured, and feces cultures were done again to identify L3 larvae. After treatment of EG, 40% (16/40) of horses were positive, the most frequently identified GIN were cyathostomins (98.6%), followed by P. equorum (1.0%) and S. vulgaris (0.2%). Three of the five ranches were classified as resistant, according to the FECRT, with a percentage of reduction from 53 to 68%, all of which used IVM ≥4 times annually. This is the first evidence of resistance in cyathostomins to IVM treatment in horses from the Mexican southeast, adding to the current problem of anthelmintic resistance in equine GIN.

Biological control of gastrointestinal nematodes in horses fed with grass in association with nematophagus fungi Duddingtonia flagrans and Pochonia chlamydosporia

Biological control with nematophagous fungi can be used to reduce populations of intestinal parasites, since these are their natural antagonists, minimizing the use of chemical anthelmintics. Therefore, the objective of the present work was to evaluate the combined effect of two nematophagous fungi Duddingtonia flagrans and Pochonia chlamydosporia in the biological control of gastrointestinal helminthoses in horses kept on pasture. Thirty six mares were divided into three groups, one control and two trated, for twelve months. As for the group of animals treated with fungi, inwhich animals in which the animals received a dose of 1 g of formulation containing 105 chlamydospores of D. flagrans and 105 chlamydospores of P. chlamydosporia per gram of the commercial product for each 10 kg of body weight, per day. The group Abamectin, with the animals being treated every 3 months with the anthelmintic 1% abamectin, at a dose of 0.2 mg/kg, administered orally. The proceeding of all treatments was evaluated based on the count of eggs per gram of faeces (EPG) with samples of faeces collected every 28 days and preparation of fecal cultures to identify the different genera of infective larvae, count of infective larvae retrieved from the pasture and microbiological analysis of the faeces. The data were submitted for analysis of variance and means were compared by Tukey's test (5%). The overall average of EPG in the control group was 635 (±61.7); as for the fungi group, 385 (±60.4) and in the abamectin group 313 (±67.3), showing a statistically significant difference in the group treated with fungi and abamectin in comparison to the control group, with the control group having the highest averages, while the two other treated groups were overall similar. In the count of infective larvae retrieved from the pasture, the general averages presented in the control group was 2267 (±789.4), the group treated with fungi 1100 (±384.8) and the group abamectin 3115 (±1038.4), writing down that the three groups differed significantly from each other, given that the group being treated with fungi presented the lowest overall average. The fecal cultures showed a predominance of small strongyles in the three groups. It is concluded that the use of Duddingtonia flagrans and Pochonia chlamydosporia fungi is efficient in the control of gastrointestinal nematodes in horses kept on pasture, promoting a reduction in pasture infestation by infective larvae and so the degree of helminthoses.

A review of the use of Duddingtonia flagrans as a biological controller of strongylid nematodes in horses.

In horses, the nematodes of the Strongylidae family are the most important due to their prevalence and pathogenicity. Sanitary plans include parasite control based on chemical anthelmintics. Among these, the benzimidazole compounds have been used since the 1960s to control the nematode Strongylus vulgaris. Its inappropriate use resulted in the development of resistance in parasites with a shorter biological cycle, such as the small strongyles. Currently, the genera that make up this group show widespread resistance to all chemical treatments available in veterinary medicine, except for macrocyclic lactones, where less effective action has been detected. The need to find alternative routes for its control is recognized. International organizations and markets are increasingly restrictive in the allowed levels of drugs in products of animal origin, so one of the drawbacks is the permanence of chemical compounds in tissues. Therefore, other tools not chemically based are proposed, such as the biological control of gastrointestinal nematodes. Various research groups around the world have carried out tests on the control capacity of the nematophagous fungus Duddingtonia flagrans against this group of parasites. The objective of this review is to compile the different tests that are available on biological control in this species, in in vivo and in vitro tests, and the possible incorporation of this tool as an alternative method of antiparasitic control in an integrated control scheme of parasites.

The problem of anthelminthic resistance in horse breeding

The purpose of the research is to outline the growing problem of anthelmintic resistance of horse nematodes in the world, to describe the methods currently used to detect it, and the proposed ways to overcome it.Materials and methods. A review of the world literature on the resistance of horse nematodes to anthelmintic drugs is carried out.Results and discussion. Currently, veterinary parasitology is faced with the growing problem of the emergence of resistant races of helminths, against which previously tested anthelmintic drugs in recommended doses are ineffective. This phenomenon has been noted in many animal species and manifests itself in relation to many drugs of the main groups of anthelmintics. This is evidenced by numerous reports. In horse breeding, benzimidazoles have been used for over 40 years, leading to widespread resistance to them in intestinal nematodes. There is a loss or weakening of the effectiveness of treatment of nematodes of horses with anthelmintic drugs: thiabendazole, pyrantel pamoat, drugs from the benzimidazole groups and macrocyclic lactones. There is no doubt that a similar situation of the spread of resistant races of helminths is also typical for Russia, since here the same anthelmintic drugs are used for treatment as abroad. However, this problem in our country remains practically unexplored. In this situation, the uncontrolled use of anthelmintics, which does not take into account the possibility of resistance to them, inevitably leads (and possibly in some cases has already led) to the emergence and spread of resistant populations of helminths, against which existing drugs will be ineffective.

Cyathostomin resistance to moxidectin and combinations of anthelmintics in Australian horses

BackgroundCyathostomins are the most important and common parasitic nematodes of horses, with > 50 species known to occur worldwide. The frequent and indiscriminate use of anthelmintics has resulted in the development of anthelmintic resistance (AR) in horse nematodes. In this study we assessed the efficacy of commonly used anthelmintics against cyathostomins in Australian thoroughbred horses.MethodsTwo drug efficacy trials per farm were conducted on two thoroughbred horse farms in the state of Victoria, Australia. In the first trial, the horses on Farm A were treated with single and combinations of anthelmintics, including oxfendazole (OFZ), abamectin (ABM), abamectin and morantel (ABM + MOR), moxidectin (MOX) and oxfendazole and pyrantel (OFZ + PYR), at the recommended doses, whereas the horses on Farm B only received MOX, at the recommended dose. The faecal egg count reduction test (FECRT) was used to determine the efficacy and egg reappearance period (ERP) of anthelmintics. Based on the results of the first trial, the efficacies of MOX and a combination of ABM + MOR were reassessed to confirm their activities against cyathostomins.ResultsOf the five anthelmintic products tested on Farm A, resistance against OFZ, ABM and OFZ + PYR was found, with efficacies of − 41% (− 195% lower confidence limit [LCL]), 73% (60% LCL) and 82% (66% LCL) at 2 weeks post-treatment, respectively. The FECRT showed high efficacies of MOX and ABM + MOR (100%) at 2 week post-treatment and shortened ERPs for these anthelmintics (ABM + MOR: 4 weeks; MOX: 5 weeks). Resistance to MOX was found on Farm B, with a reduced efficacy of 90% (70% LCL) and 89% (82% LCL) at 2 weeks post-treatment in trials one and two, respectively.ConclusionsThis study provides the first evidence of MOX- and multidrug-resistant (ABM and combinations of anthelmintics) cyathostomins in Australia and indicates the need for continuous surveillance of the efficacy of currently effective anthelmintics and large-scale investigations to assess the ERP for various anthelmintics.Graphical

Anthelmintic resistance in Creole horses in the South of Rio Grande do Sul, Brazil

ABSTRACT The objective of the study was to evaluate the antiparasitic resistance against horse nematodes in the South of Rio Grande do Sul, Brazil. The results concerning the tests of anthelmintic efficacy on horses, stored in the database of the Parasitic Diseases Study Group (GEEP) - Veterinary Faculty, at the Federal University of Pelotas (UFPel), were carried out in the laboratory from 2018 to 2019. Stool samples were received from farms with breeding of adult female and male Criollo horses naturally infected, located in municipalities in the country’s southern region. The antiparasitic agents tested were Triclorfon + Fenbendazole, Closantel + Albendazole, Ivermectin + Praziquantel, Fenbendazole, Ivermectin, Doramectin, Mebendazole and Moxidectin. Techniques such as Gordon and Whitlock, Coproculture and Fecal Egg Count Reduction Test were performed. Of all the antiparasitic drugs tested, it was observed that only treatments with Ivermectin 2% showed desired values. The observed results indicate that resistance to macrocyclic lactones is usual in equine parasites in this Brazilian region, despite the results with isolated Ivermectin.

Prevalence and degree of gastrointestinal nematode infection of horses (Equus caballus) used as public transport in Mataram city, Indonesia

Horses of Equus caballus species which are used as public transportation in the form of Cidomo (traditional horse cart) in Mataram City need to be considered about their welfare and health. A common problem in horses is gastrointestinal nematode worms that can induce negative health effects on horses. Furthermore, the infective larvae of nematodes can survive in the environment under optimum conditions of temperature and humidity. The purpose of this study was to determine the prevalence and degree of infection of gastrointestinal nematodes in horses used as public transportation (Cidomo) in Mataram City. The cross-sectional study was conducted from July to August 2020 to collect feces of Cidomo horses at 8 traditional markets in Mataram City. A total of 131 feces of horses as Cidomo were taken directly from the rectum of Cidomo horses by the purposive method. The Morphology, prevalence of nematode egg, and Egg per gram (EPG) for a degree of infection were used examined using the floating McMaster technique. Morphological data and the prevalence of gastrointestinal nematodes in horses (Equus caballus) as Cidomo were presented descriptively, and the EPG value was analyzed by the Anova test, while the degree of infection has been categorized as low, medium, and high levels. The eggs of gastrointestinal nematodes (Trichosstrongylus spp, Strongylus spp, and Capillaria spp) have been found in feces of horses as Cidomo at 8 traditional markets in Mataram City with a prevalence of 74.04% (97/131). The EPG value of the gastrointestinal nematode of feces of horses as Cidomo between 8 traditional markets in Mataram City showed no significant difference with p > 0.05 (p-value = 0.227). Overall the degree of infection of gastrointestinal nematodes in horses (Equus caballus) as Cidomo in Mataram City was a medium level with a mean value of EPG = 547.628 ± 82.059 The results showed that the gastrointestinal nematodes have been infected horses as Cidomo in 8 traditional markets in Mataram City with a medium level of infection degree.

Anthelmintic resistance of horse strongyle nematodes to ivermectin and pyrantel in Lithuania

BackgroundWith intensive use of anthelmintic drugs in recent decades, anthelmintic resistance (AR) in horse nematodes is becoming a growing issue in many countries. However, there is little available information about the parasites, treatment practices or AR in the horse population in Lithuania. The aim of this study was to assess the current situation of AR on horse farms in Lithuania. The study was conducted in 25 stables on horses with a strongyle faecal egg count (FEC) of ≥ 200 eggs per gram. A faecal egg count reduction test (FECRT) was performed on each farm after administration of ivermectin (IVM) or pyrantel (PYR).ResultsThe efficacy of IVM was comparatively high, with 98.8% of 250 horses having a zero egg count 14 days after treatment. Two conditions were used to interpret the FECRT results for PYR: firstly, resistance was determined when FECR was < 90% and the lower 95% confidence interval (LCL) was < 80%, and secondly when in addition the upper confidence level (UCL) was < 95%. Under the first condition, resistance against PYR was found in five stables (25% of all tested herds), while when considering the UCL as well, resistance was only detected in two stables (8%). The FEC showed a significant (P < 0.01) difference between the treatment and control groups. Only cyathostomin larvae were detected in larval cultures derived from strongyle-positive faecal samples collected 14 days after treatment of a test group with PYR.ConclusionsThis in vivo study showed that PYR resistance is prevalent on horse farms in Lithuania, while the efficacy of IVM still appears to be unaffected. However, further studies of ivermectin resistance are needed. These findings should guide the implementation of more sustainable management of strongyle infections in horses in Lithuania.

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.

Reduced efficacy of ivermectin and moxidectin against Parascaris spp. in foals from Argentina

Macrocyclic lactones are the most widely used drugs for the control of gastrointestinal nematodes of horses in Argentina. Ivermectin and moxidectin are used as broad spectrum anthelmintics and although there are several international reports on the resistance of Parascaris spp., the resistance status of the local nematode population is largely unknow. This report informs a case of suboptimal efficacy to both drugs to control Parascaris spp in foals in central Argentina. In February 2018, routine fecal parasite egg counts showed a moderate-high number of Parascaris spp eggs (mean=680 eggs per gram of feces) in foals treated approximately one month before with moxidectin. Upon suspicion of resistance of this parasite to the macrocyclic lactones, 24 of these animals were selected for a fecal egg count reduction test (FECRT). Twelve foals were treated with ivermectin and the remaining 12 animals with moxidectin. Two weeks after treatment, the FECRT was 48.1% and 34.8% for moxidectin and ivermectin respectively (25% of the animals increased the number of eggs in feces after treatment). Five days later, the administration of fenbendazole resulted in a FECRT=100%. The monitoring of the status of susceptibility or resistance in each establishment is critical for the design of control programs based on rational and sustainable use of anthelmintics.

Anthelmintic Resistance and Associated Management Practices in Local Horses in Sokoto Metropolis, Nigeria

Abstract This study was carried out to assess the management practices used in the control of gastrointestinal (GI) nematodes of horses and to determine the efficacy of three anthelmintics commonly used in Sokoto metropolis. A questionnaire was administered on management practices, while an anthelmintic efficacy test was carried out using 15 horses. The 15 horses were divided into three groups (A, B and C) comprising of 5 each and treated with albendazole, ivermectin and fenbendazole, respectively. The faecal egg count reduction test (FECRT) was used to determine the efficacy and faecal culture was used to determine the parasite species. Majority of the respondents (80%) claimed to have worm control strategies, but only 32.5% used anthelmintics for the control of GI parasites. 62.5% of respondents designed their deworming plan, while only 25% relied on veterinarians. Most of the treatments were done by the horse owners and/or handlers and they largely depended on visual judgement in dosage determination. Their selection of anthelmintics was based on familiarity and 52.5% of the respondents dewormed their horses six times a year using a particular class of anthelmintic or herbal remedies. Resistance against albendazole as well as suspected resistance against fenbendazole by the GI nematodes identified was observed, while ivermectin demonstrated high efficacy against all nematodes isolated. In conclusion, a single dose of subcutaneous injection of ivermectin was highly effective against gastrointestinal parasites in horses, while the worm control strategies employed by respondents enhanced the selection of nematode resistance to albendazole and fenbendazole.

Screening of Model Animals for Experimental Infection with Equine Cyathostomes

Abstract Various laboratory animals – mice (Mus musculus) of six strains, rabbits (Oryctolagus cuniculus), guinea pigs (Cavia porcellus), rats (Rattus norvegicus), and Mongolian gerbils (Meriones unguiculatus) were experimentally infected with larvae of small strongyles (Cyathostominae), obtained from horse faeces and cultured to the infective larval stage L3. The attempt to transfer cyathostome larvae was aimed at developing a model for the investigation of different aspects of the life cycle and biology of these nematodes in the laboratory. Some animals were immunized (hydrocortisone) for the duration of the study. The laboratory animals were orally infected with 2–10 thousand sheathed or ex-sheathed L3 larvae of mixed cyathostome species. All attempts to inoculate any animal failed; there was no larval development in the experimental rodents and it can be stated that none of the investigated animals may serve as a suitable model host for horse nematodes of the subfamily Cyathostominae.

Drug resistance in parasitic helminths of veterinary importance in Chile: Status review and research needs

Abstract: The increasing development of anthelmintic resistance (AR) in parasites of livestock is threatening animal health and production worldwide. In Chile, studies evaluating the field efficacy of anthelmintics and the detection of AR have been performed since the 2000s, but until now, no previous attempt has tried to systematise the available information. This article reviews general concepts about AR in helminths of veterinary importance, methods for diagnosis of AR and summarises the published reports of AR in Chile. Anthelmintic resistance in Chile has been reported in gastrointestinal nematodes of horses (benzimidazole resistance) and ruminants (sheep and cattle, macrocyclic lactone and benzimidazole resistance). However, these cases involved a limited number of selected farms and no further conclusions can be made of the status of parasite drug resistance at a regional or national level. No published cases of AR in Fasciola hepatica have been reported in Chilean livestock, but human infections with triclabendazole-resistant F. hepatica have been described in patients with previous consumption of watercress or untreated water from marshes grazed by livestock. Given the zoonotic potential and endemic nature of F. hepatica in Chile, it is urgent to determine the extent of liver fluke resistance. Current research gaps of the situation of AR in Chile and suggestions for the performance of laboratory and field studies are further discussed.

In vitro evaluation of ivermectin, moxidectin, albendazole and pyrantel against cyathostomins of horses.

Cyathostomins are the most prevalent nematodes of horses, and multidrug resistance has been reported worldwide. There is a need to implement alternative drug monitoring analytical tests. The objective of this study was to determine the consistency (5 repetitions) of the larval migration on agar test (LMAT) using ivermectin, moxidectin, pyrantel or albendazole against cyathostomin infective-stage larvae in eight different concentrations. LMAT showed a strong coefficient of determination (R2 > 0.91), between the test repetitions (n=5). The average 50% effective concentration (EC50) for ivermectin, moxidectin, pyrantel and albendazole were 0.0404, 0.0558, 0.0864 and 0.0988 nMol, respectively. The results of the EC50 for albendazole showed the greatest range of concentration. Ivermectin and moxidectin had the lowest in between-test variation. In the future, internationally certified susceptible isolates could be used for screening new drug candidates, or to follow up the pattern of drug efficacy from field populations.

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.

Anthelmintic resistance in horse nematodes: Where does it come from?

Anthelmintic resistance in horse nematodes: Where does it come from?

Semi-quantitative differentiation of cyathostomin larval cultures by reverse line blot

Cyathostomins are the most prevalent horse nematodes worldwide and over 50 species are described. The eggs and the infective larvae (L3) can easily be obtained or cultured from infected horses, but cannot be differentiated morphologically at species level. A reverse line blot (RLB) method based on the hybridization of a PCR fragment with a species specific probe, has previously been developed for the differentiation of individual eggs and/or L3s, but is too labor intensive for large scale studies. In the present study a RLB method on multiple pooled L3s for the semi-quantitative differentiation of cyathostomin larval cultures was developed and validated. First, the probability of the presence of a certain species within a pool was calculated as function of the frequency and the number of L3s within a pool. Ten L3s per pool were found to be optimal. Next, the probability, the chance of occurrence was calculated when 4 pools per culture were used. The probability distributions for 0, 1, 2, 3 or 4 positive pools were transformed into the corresponding median frequency of the cumulative probability: 0.014, 0.04, 0.08, 0.16 and 0.59, respectively. Based on these calculated probabilities, RLB on 10 L3s per pool and 4 pools per sample was validated by estimating the cross-hybridization, precision and accuracy in 3 groups of horses. First, absence of cross-hybridization was confirmed by differentiation of the same L3s (160 L3s from the 4 horses from group 1) in the RLB on individual as well as on pooled L3s. Cross-hybridization was excluded for 9 of the most common cyathostomins. Next, the precision and accuracy were determined by the differentiation of 10 replicates of 3 cultures from 3 horses from group 2 (1200 L3s). The coefficient of variation (CV) was between 0 and 0.90 and the accuracy was between 0.42 and 1.73. A Monte Carlo simulation based on the observed scores and associated probability distributions gave similar results as the use of a fixed median frequency. The LPGs obtained from 276 larval culture counts from a larger cohort (23 horses, group 3) were not significantly different from the LPGs obtained from summation of the LPG per species found by RLB on pooled L3s. The RLB on pooled L3s was found therefore an useful semi-quantitative method for the differentiation of the most common cyathostomin L3, with a workload of approximately one tenth of that of the RLB on individual L3s.