Cytokine responses to various larval stages of equine strongyles and modulatory effects of the adjuvant G3 in vitro.

AIMS: To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites. METHODS AND RESULTS: EqPBMC were exposed to S. vulgaris larvae (L3, exsheated L3 and L4) and cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL-4, IL-5 and IL-13 was induced by both S. vulgaris and cyathostomin L3. Moulting of S. vulgaris from L3 to L4 stage was accompanied by a shift to high expression of IL-5 and IL-9 (exsheated L3 and L4) and IFN-γ (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL-4, IL-5 and IL-10 while concomitantly enhancing the expression of IFN-γ. CONCLUSION: The L4 stage of S. vulgaris generated a cytokine profile different from that induced by the earlier L3 stage of S. vulgaris and cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.

Eficacy of ivermectin, moxidectin and febendazole in equine in Brazil.

The objectives of the present study were to evaluate the efficacy of three anthelmintic drugs, i.e. fenbendazole, ivermectin and moxidectin; to determine the genera and species of the most abundant strongyles; and to investigate parasite control measures used in herds of horses in the western region of the state of São Paulo, Brazil. This study was onducted between February and December 2013 on 10 farms in this region. Coprological evaluations were conducted for counting the numbers of eggs per gram of feces (EPG) and coprocultures were made in order to identify third-stage larvae (L3) of strongyles. Parasite control measures were investigated by surveying each farm's management and characteristics. A fecal egg count reduction test (FECRT) was used to determine the anthelmintic efficacy based on EPG count results before and after treatment. The FECRT showed that anthelmintic resistance to fenbendazole was present and that ivermectin and moxidectin had high effectiveness in the western region of the state of São Paulo. Identification of L3 revealed that there was high prevalence of cyathostomins among strongyle larvae. The highest prevalence of Strongylus vulgaris occurred on farms where the intervals between deworming were long. The questionnaire applied showed that ivermectin was the drug most used among these farmers.

The in vitro effect of Ferula asafoetida and Allium sativum extracts on Strongylus spp.

The high incidence of equine gastrointestinal worms and their increased resistance against anthelmintics has encouraged research into the effectiveness of rational phytotherapy. This study investigates the in vitro anti-parasitic effects of extracts of Ferula asafoetida and Allium sativum, two native plants that are widespread in Iran on Strongylus spp. larvae. Faecal samples were collected from horses, examined by routine parasitology methods and positive samples were used for future examination. After incubation, the third-stage larvae were harvested by the Baermann technique. A hydroalcoholic extract from the plants was used for the antiparasitic study, while tap water was used for controls. Trials for each concentration and control group were performed in three replicates. The results showed that that during the first day of exposure, the hydroalcoholic extract of F. asafoetida at concentration of 10, 50 and 100 mg/ml killed over the 90% of the larvae, and A. sativum extract at concentration of 50 and 100 mg/ml killed over the 95% of larvae (p<0.05). The results obtained from the bioassay showed that two plant extracts have a larvicidal effect on the Strongylus spp. larval stages compared with the control group.

Strongyle egg reappearance period after moxidectin treatment and its relationship with management factors in UK equine populations.

Parasitic nematodes, particularly cyathostomins, are ubiquitous in grazing horses world-wide. Considerable burdens of cyathostomin larvae can encyst in the large intestinal wall. The most recommended treatment against these pathogenic stages is moxidectin. Information is required on how effective moxidectin is against cyathostomin populations in different regions. The objectives here were to determine the efficacy of moxidectin treatment and estimate the strongyle egg reappearance period (ERP) after treatment in several equine populations, to confirm the type of strongyle nematodes present and to identify other (i.e. management) factors associated with shortened ERP. Eight yards were recruited and moxidectin in combination with praziquantel administered to all horses (n=261). Faecal egg count (FEC) analysis was performed at weeks 0, 2, 6, 10 and 12 after treatment to determine efficacy and ERP. The ERP was estimated using two previously published methods. Morphological identification of cultured third stage larvae from the sample population was compared to a Strongylus vulgaris-specific end-point PCR to examine the presence of S. vulgaris in samples before and after treatment. Strongyle egg shedding patterns were also compared to worm management practices at each site. At 2 weeks post-treatment, moxidectin was highly effective (faecal egg count reduction range, 99.9-100%). The strongyle ERP ranged from 6 weeks to >12 weeks depending on the calculation method applied. Only cyathostomin larvae were detected by morphological identification. The results from the coprocultures and PCR showed that S. vulgaris was absent before and after treatment. Analysis revealed that regular faecal removal from pasture was associated with lower average FEC and lower prevalence of egg shedding.

Molecular mechanisms for anthelmintic resistance in strongyle nematode parasites of veterinary importance.

Veterinarians rely on a relatively limited spectrum of anthelmintic agents to control nematode parasites in domestic animals. Unfortunately, anthelmintic resistance has been an emerging problem in veterinary medicine. In particular, resistance has emerged among the strongyles, a group of gastrointestinal nematodes that infect a variety of hosts that range from large herbivores to small companion animals. Over the last several decades, a great deal of research effort has been directed toward developing an understanding of the mechanisms conferring resistance against the three major groups of anthelmintics: macrocyclic lactones, benzimidazoles, and nicotinic agonists. Our understanding of anthelmintic resistance has been largely formed by determining the mechanism of action for each drug class and then evaluating drug-resistant nematode isolates for mutations or differences in expression of target genes. More recently, drug efflux pumps have been recognized for their potential contribution to anthelmintic resistance. In this mini-review, we summarize the evidence for mechanisms of resistance in strongyle nematodes.

Serum Strongylus vulgaris-specific antibody responses to anthelmintic treatment in naturally infected horses.

Strongylus vulgaris is the most pathogenic helminth parasite of horses, causing verminous endarteritis with thromboembolism and infarction. A serum enzyme-linked immunosorbent assay (ELISA) has been validated for detection of antibodies to an antigen produced by migrating larvae of this parasite. The aim was to evaluate ELISA responses to anthelmintic treatment in cohorts of naturally infected horses. Fifteen healthy horses harboring patent S. vulgaris infections were turned out for communal grazing in May 2013 (day 0). On day 55, horses were ranked according to ELISA titers and randomly allocated to the following three groups: no treatment followed by placebo pellets daily; ivermectin on day 60 followed by placebo pellets daily; or ivermectin on day 60 followed by daily pyrantel tartrate. Fecal and serum samples were collected at ∼28-day intervals until study termination on day 231. Increased ELISA values were observed for the first 53 days following ivermectin treatment. Titers were significantly reduced 80 days after ivermectin treatment. Horses receiving daily pyrantel tartrate maintained lower ELISA values from 137 days post ivermectin treatment until trial termination. These results illustrate that a positive ELISA result is indicative of either current or prior exposure to larval S. vulgaris infection within the previous 5 months.

Strongyle infections and parasitic control strategies in German horses - a risk assessment.

BACKGROUND: As a consequence of the increasing levels of anthelmintic resistance in cyathostomes, new strategies for equine parasite control are being implemented. To assess the potential risks of these, the occurrence of strongyles was evaluated in a group of 1887 horses. The distribution of fecal egg counts (FECs), the frequency of anthelmintic drug use, and the deworming intervals were also analyzed. Between June 2012 and May 2013, 1887 fecal samples from either selectively or strategically dewormed horses were collected at 195 horse farms all over Germany and analyzed quantitatively with a modified McMaster technique. All samples with FEC ≥20 eggs per gram (EPG) were subjected to coproculture to generate third-stage larvae (LIII) for species differentiation. RESULTS: Egg counts were below the limit of detection (20 EPG) in 1046 (55.4%) samples and above it in 841 (44.6%) samples. Strongylus vulgaris larvae were identified in two of the 841 positive samples. Infections with cyathostomes were found on every farm. The most frequently applied anthelmintic was ivermectin (788/50.8%), followed by pyrantel (336/21.6%). The mean time since last treatment was 6.3 months. High-egg-shedding (>500 EPG) strategically dewormed horses (183/1357) were treated, on average, three times/year. The planned treatment date was already exceeded by 72.5% of the high egg-shedders and by 58.1% of the moderate (200-500 EPG) and low egg-shedders (20-199 EPG). CONCLUSIONS: S. vulgaris seems to be rare in Germany and no difference in its frequency has yet been found between selectively treated horses and horses receiving treatment in strategic intervals. However, inconsistent parasite control has been observed. Therefore, to minimize the risks for disease, consistent and efficient parasite control should be implemented.

Effects of daily pyrantel tartrate on strongylid population dynamics and performance parameters of young horses repeatedly infected with cyathostomins and Strongylus vulgaris.

Strongylid infections are ubiquitous in grazing horse populations. Infections with cyathostomin (small strongyle) and strongylin (large strongyle) nematodes have long been associated with clinical disease in horses, but little is known about their subclinical impact. A masked, randomized, controlled study was conducted to evaluate the effects of daily administration of pyrantel tartrate on body condition scores, weight gain, fecal egg counts, and total worm counts of young horses repeatedly inoculated with strongylid larvae. Twenty eight immature horses were treated with larvicidal anthelmintic regimens and randomly allocated to two groups. Group 1 horses were given a pelleted placebo product once daily, and those in Group 2 received pyrantel tartrate once daily at ∼ 2.64 mg/kg body weight. On five days during each week, ∼ 5000 infective cyathostomin larvae were administered to each horse. In addition, horses received ∼ 25 infective Strongylus vulgaris larvae once weekly. Horses were maintained on pasture for 154 days and had ad libitum access to grass hay throughout. At approximate, 14-day intervals, body weights were measured, body condition scores were assigned, fecal samples were collected for egg counts, and blood samples were collected for measurement of S. vulgaris antibodies and various physiologic parameters. After 22 weeks at pasture and 14-17 days in confinement, horses were euthanatized and necropsied. Nematodes were recovered and counted from aliquots of organ contents, representative samples of large intestinal mucosa, and the root of the cranial mesenteric artery. Daily treatment with pyrantel tartrate at the recommended dosage significantly reduced numbers of adult cyathostomins in the gut lumen and early third-stage larvae in the cecal mucosa, increased the proportions of fourth-stage larvae in the gut contents, and was accompanied by significant improvements in body condition scores. Fecal egg counts of horses receiving daily pyrantel tartrate were significantly reduced, with percentages of efficacy ranging from 84.4% to 98.9%, but egg counts of both groups increased significantly over the course of the study. Treatment also significantly reduced the numbers of S. vulgaris larvae in the cranial mesenteric artery by 99.2%. Serum antibodies to S. vulgaris apparently persisted from pre-enrollment infections, but ELISA values gradually declined over the course of the study. This study has provided useful insights into the effects of daily pyrantel tartrate on the dynamics of cyathostomin infection, and into some subclinical effects of strongylid parasitism in horses.

[The resistance status of gastrointestinal strongyles against anthelmintics in three Estonian sheep flocks]. / Die Resistenzlage von Magen-darm- Strongyliden gegenüber Anthelminthika in drei estnischen Schafherden.

Poor efficacy of anthelmintic drugs in sheep due to infections with resistant gastrointestinal strongyles is reported worldwide. The aim of this pilot study was to gain an insight into the current situation of anthelmintic efficacy in Estonian sheep flocks. From September to November 2012, faecal egg count reduction tests (FECRT) were carried out in three Estonian sheep farms, evaluating the efficacy of albendazole and ivermectin. Individual faecal samples were taken at the day of treatment and 10 to 14 days later and examined by a modified McMaster technique. Anthelmintic treatment was carried out with ivermectin (Bimectin 10 mg/ml, Bimeda Chemicals Export) subcutaneously with a dose rate of 0.2 mg/kg of body weight in the IVM group (n = 20 animals/farms 1 and 2; n = 5 for farm 3) or albendazol (Endospec 10%, Bimeda Chemicals Export) orally in the dose of 5 mg/kg of body weight in the ALB group (n = 20 animals/ farm); animals in the control group (n = 20 animals for farms 1 and 3, n = 18 for farm 2) were left untreated. The FECRT was carried out according to the WAAVP guidelines. On farm 1 the efficacy of albendazole and ivermectin was reduced with 66% and 65% FECR, respectively. With a FECR of 26% the results of farm 2 showed a pronounced albendazole resistance while ivermectin was still sufficiently efficient (99% reduction). Farm 3 showed nearly 100% efficacy of albendazole and ivermectin, but earlier problems with high endoparasite burden and mortality may indicate a possible developing albendazole resistance which could not be detected with the FECRT yet. The results of this study show that in Estonia resistance against benzimidazoles and macrocyclic lactones does occur, indicating that close countrywide monitoring is advisable.

Strongylus vulgaris associated with usage of selective therapy on Danish horse farms-is it reemerging?

Nematodes belonging to the order Strongylida are ubiquitous in grazing horses, and the large strongyle Strongylus vulgaris is considered the most pathogenic. This parasite was originally described widely prevalent in equine establishments, but decades of frequent anthelmintic treatment appears to have reduced the prevalence dramatically. Increasing levels of anthelmintic resistance in cyathostomin parasites have led to implementation of selective therapy to reduce further development of resistance. It has been hypothesized that S. vulgaris could reoccur under these less intensive treatment circumstances. The aim with the present study was to evaluate the occurrence of S. vulgaris and the possible association with usage of selective therapy. A total of 42 horse farms in Denmark were evaluated for the presence of S. vulgaris using individual larval cultures. Farms were either using a selective therapy principle based on regular fecal egg counts from all horses, or they treated strategically without using fecal egg counts. A total of 662 horses were included in the study. Covariate information at the farm and horse level was collected using a questionnaire. The overall prevalence of S. vulgaris was 12.2% at the individual level and 64.3% at the farm level. Farms using selective therapy had horse and farm prevalences of 15.4% and 83.3%, respectively, while the corresponding results for farms not using selective therapy were 7.7% and 38.9%. These findings were found statistically significant at both the horse and the farm level. Stud farms using selective therapy were especially at risk, and occurrence of S. vulgaris was significantly associated with the most recent deworming occurring more than six months prior. The results suggest that a strict interpretation of the selective therapy regimen can be associated with an increased prevalence of S. vulgaris. This suggests that modifications of the parasite control programs could be considered on the studied farms, but it remains unknown to which extent this can be associated with increased health risks for infected horses.

Field tests demonstrating reduced activity of ivermectin and moxidectin against small strongyles in horses on 14 farms in Central Kentucky in 2007-2009.

Efficacy of ivermectin (IVM) and moxidectin (MOX) against small strongyles was evaluated in horses (n=363) in field tests on 14 farms in Central Kentucky between 2007 and 2009. Most of the horses were yearlings but a few were weanlings and mares. The number of horses treated with IVM was 255 and those treated with MOX was 108. Horses on six farms were allotted into two groups. One group was treated with each of the two drugs, whereas horses on the other eight farms were treated with only one of the two drugs--IVM on six farms and MOX on two farms. Strongyle eggs per gram of feces (EPGs) compared to initial use of IVM and MOX returned almost twice as quickly after treatment of horses on all of the farms. IVM has been used much more extensively in this geographical area than MOX. Reduced activity of MOX was evident even on farms with rare or no apparent previous use of MOX but with probable extensive use of IVM.

Determination of ivermectin efficacy against cyathostomins and Parascaris equorum on horse farms using selective therapy.

Ivermectin resistance has recently been described in Parascaris equorum and there have been reports from several countries of a shortened egg reappearance period (ERP) following ivermectin treatment for cyathostomins. This study was aimed at determining the efficacy of ivermectin in treating cyathostomins and P. equorum in Danish horses. A total of 196 animals were selected from 52 farms, all of which were using a selective anthelmintic treatment strategy. ERP was investigated with weekly samples from 96 horses from nine farms. Horses were treated with ivermectin oral paste by their owners at an estimated dose rate of 0.2mg/kg. Overall, faecal egg counts were reduced 10-14 days after treatment by 96.9% and 100% for P. equorum and cyathostomins, respectively. Mean faecal egg count reductions at 4 and 6 weeks post treatment were 99.5% and 96.9%, respectively. No signs of developing ivermectin resistance were found in either cyathostomins or P. equorum in the studied horses.

Anthelmintic efficacies of a tablet formula of ivermectin-praziquantel on horses experimentally infected with three Strongylus species.

In this blinded randomized and controlled study, the anthelmintic efficacy of a tablet formula of ivermectin-praziquantel was evaluated in horses experimentally infected with three species of Strongylus larvae. Eighteen previously dewormed horses were inoculated on study day 0 with third-stage larvae of Strongylus vulgaris, Strongylus equinus, and Strongylus edentatus. The horses were randomly allocated to three groups (n = 6): test-drug (tablet formula), positive-control (reference gel), and negative-control (placebo tablet). On day 56, the horses were treated once with the respective drugs. On day 95, the horses were sacrificed, and necropsy examinations were performed to assess the status of the parasite burden (L4 and immature L5) and pathological lesions on selected organs and tissues. By the criteria of worm counts, the test-drug and positive-control showed, respectively, 100% and 97.3% anthelmintic efficacies on S. vulgaris, 100% and 81.4% on S. equinus, and equally 100% on S. edentatus. However, the efficacies on S. equinus and S. edentatus should be taken only as face values considering their respective low worm counts in the placebo group. The S. vulgaris-induced arterial lesions were also reduced in the test-drug and positive-control groups with efficacies of 73.9% and 62.9%, respectively. No adverse reactions were observed with either of the drugs. Our data demonstrate that the Equimax tablet formula was as safe and efficacious as the gel formula anthelmintic on large strongyles in horses.

Effects of previous control programmes on the proportion of horses shedding small numbers of strongyle-type eggs.

Between 30 and 70 per cent of horses at 18 stables had their anthelmintic treatments withdrawn for a year. Three of the stables (group 1) had used no anthelmintics for many years, 10 (group 2) had treated their horses with anthelmintics approximately twice a year, and five (group 3) had used anthelmintics five to seven times a year. A total of 1068 samples of faeces were collected from the 267 untreated horses and faecal egg counts were carried out four times during the year. Overall 50.2 per cent of the samples contained less than 200 epg. When the four counts from individual horses were collated, 35.6 per cent of the horses were low egg-shedders (LES). However, the previous control programme had a marked effect on both the mean log(10) epg and the proportion of horses in the stables that were LES. The proportions of LES among the horses in the stables in groups 1 and 2 were similar, although the log(10) epg was significantly lower in group 2. There was a markedly higher proportion of LES and a lower mean log(10) epg in the untreated horses at the stables in group 3 than in either of the other groups. The horses at the stables in groups 2 and 3 that remained on the anthelmintic programme previously used at the stables had lower egg counts than the untreated horses at the same stables, but the difference was usually not statistically significant. At the stables in group 2 the proportions of horses that were LES in the treated and untreated groups were not significantly different. At the stables in group 3 the proportion of LES was significantly lower in the treated horses than in the untreated horses.

Prevalence of anthelmintic resistance on sheep and goat farms in the southeastern United States.

OBJECTIVE: To determine prevalence of anthelmintic resistance on sheep and goat farms in the southeastern United States. DESIGN: Cross-sectional study. ANIMALS: Sheep and goats from 46 farms in 8 southern states, Puerto Rico, and St Croix in the US Virgin Islands. PROCEDURES: Parasite eggs were isolated from fecal samples, and susceptibility to benzimidazole, imidathiazole, and avermectin-milbemycin anthelmintics was evaluated with a commercial larval development assay. RESULTS: Haemonchus contortus was the most common parasite on 44 of 46 farms; Trichostrongylus colubriformis was the second most commonly identified parasite. Haemonchus contortus from 45 (98%), 25 (54%), 35 (76%), and 11 (24%) farms were resistant to benzimidazole, levamisole, ivermectin, and moxidectin, respectively. Resistance to all 3 classes of anthelmintics was detected on 22 (48%) farms, and resistance to all 3 classes plus moxidectin was detected on 8 farms (17%). CONCLUSIONS AND CLINICAL RELEVANCE: Findings provided strong evidence that anthelmintic resistance is a serious problem on small ruminant farms throughout the southeastern United States. Owing to the frequent movement of animals among regions, the prevalence of resistance in other regions of the United States is likely to also be high. Consequently, testing of parasite eggs for anthelmintic resistance should be a routine part of parasite management on small ruminant farms.

Field studies indicating reduced activity of ivermectin on small strongyles in horses on a farm in Central Kentucky.

Field studies (n=6) were completed on evaluation of activity of ivermectin (200 microg/kg) paste formulation against small strongyles in horses (foals, yearlings, and older animals) on a farm (Farm MC) in Central Kentucky in late 2006 and during 2007. A girth tape was used to estimate body weights which were then used to calculate the proper dose rate of ivermectin. The foals, yearlings, and some of the older horses were born and raised on the farm. However, most of the older horses which were not raised on the farm had been there for several years. The horse herd was given ivermectin exclusively, usually four times a year, since 1990. An exception was that during the foal's period of life fenbendazole, pyrantel pamoate, and oxibendazole were given occasionally besides ivermectin. Efficacy of drug activity was determined by pretreatment and posttreatment counts of strongyle eggs per gram of feces (EPGs). Culture of strongyle eggs in feces from some of the horses showed that only small strongyle larvae were present. The research included two studies (A and B) in foals (n=24) and four studies (C, D, E, and F) in yearlings (n=13) alone or with older horses (n=10). For each of the studies (B through F), there was a treated and a nontreated group. These groups were switched for each treatment, i.e., the treated group in one study was the nontreated group in the next study and vice versa. Eggs per gram of feces counts were determined at 1- or 2-week posttreatment intervals for 4 weeks for study A and 6 weeks for studies B through F. Also, for studies B, E, and F, counts of EPGs were done either two or three times during the third week posttreatment. The studies showed a similar posttreatment pattern of strongyle EPG counts beginning to return at about 4 weeks and increasing at 5 and 6 weeks posttreatment. Two horses in study E and one in study F had low EPG values toward the end of the third week posttreatment. The results of this ivermectin investigation showed that the strongyle EPG counts started returning about twice as quickly post-ivermectin-treatment of horses than when the drug was first marketed in the early 1980s.

A field study on the efficacy of doramectin against strongyles and its egg reappearance period in horses.

The aim of the present study was to determine the efficacy and the so-called "egg reappearance period" (ERP) of doramectin in horses naturally infected with strongyles during a period of 34 weeks. A group of yearlings of 10 animals was treated intramuscularly with doramectin at a dose rate of 0.2 mg/kg bodyweight (BW) at the begin of the grazing season. To obtain comparable data, another group of yearlings (n = 10) was treated orally with ivermectin at a dose rate of 0.2 mg/kg BW. Individual faecal samples were examined for strongyle egg counts per gram of faeces (EPG) in two-week intervals. Twelve weeks later, a second treatment was given in both groups with the respective anthelmintic followed by a third treatment when the group mean egg count reached > or = 200 EPG. The efficacy of doramectin was > or = 96 % and that of ivermectin 100%, based on the mean egg counts two weeks post treatments (wpt). The highest and the lowest extensity of the efficacy (average values) for doramectin were 90% and 41% two and ten wpt, respectively, whereas these values for ivermectin differed from 100% (two wpt) to 24.3% (eight wpt). The ERP was found to be 10 and 8 weeks for doramectin and ivermectin, respectively.