Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp.

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 µm (10 µg mL-1) and IVM at 1.1 µm (1 µg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Dealing with double trouble: Combination deworming against double-drug resistant cyathostomins.

An alternative control regimen for drug-resistant parasites is combination deworming, where two drugs with different modes of action are administered simultaneously to target the same parasite. Few studies have investigated this in equine cyathostomins. We previously reported that an oxibendazole (OBZ) and pyrantel pamoate (PYR) combination was not sustainable against a cyathostomin population with high levels of OBZ and PYR resistance. This study consisted of a field study and two computer simulations to evaluate the efficacy of a moxidectin-oxibendazole (MOX-OBZ) combination against the same cyathostomin population. In the field study, anthelmintic treatments occurred when ten horses exceeded 100 eggs per gram. Fecal egg counts and efficacy evaluations were performed every two weeks. The two simulations utilized weather data as well as equine and parasite population parameters from the field study. The first simulation repeated the treatment schedule used in the field study over a 40 year period. The second evaluated efficacies of combination treatments using selective therapy over 40 years. In the field study, efficacies of MOX and both combination treatments were 100%. The egg reappearance period for MOX was 16 weeks, and the two combination treatments were 12 and 18 weeks. The first (46.7%) and last (40.1%) OBZ efficacies were not significantly different from each other. In the simulation study, the combination treatment delayed MOX resistance development compared to when MOX was used as a single active. This occurred despite the low efficacy of OBZ. The second set of simulations identified combination treatments used with selective therapy to be the most effective at delaying MOX resistance. Overall, this study supports the use of combination treatment against drug-resistant cyathostomins, when one of the actives exhibits high efficacy, and demonstrates benefits of this approach despite substantially lowered efficacy of the other active ingredient.

Long live the worms: methods for maintaining and assessing the viability of intestinal stages of Parascaris spp. in vitro.

In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms obtained from naturally infected foals at necropsy were immediately placed in culture flasks containing 200 mL of culture media. A variety of media types, nutrient supplementation and environmental conditions were examined. A motility-based scoring system was used to assess worm viability. Worms maintained in Roswell Park Memorial Institute-1640 had significantly better viability than any other media (P < 0.0001) and all media types supplemented with any of the nutrients examined (P < 0.0001). The use of a platform rocker also significantly improved viability (P = 0.0305). This is the first study to examine the requirements for maintaining Parascaris spp. intestinal stages in vitro and to evaluate their viability based on movement using an objective scoring system.

Effects of homogenizing methods on accuracy and precision of equine strongylid egg counts.

Recommendations for control of equine strongylid parasites are based on regular determination of fecal egg counts to identify high strongylid shedders and to evaluate treatment efficacy. The McMaster technique has long been used as the standard egg counting technique in equine veterinary practice in most parts of the world, but recent work has found the Mini-FLOTAC technique to perform with significantly better accuracy and precision. The Mini-FLOTAC system comes with a homogenizing device, termed the Fill-FLOTAC, and it has been hypothesized that this device might have a significant impact on accuracy and precision. The aim of the present study was to investigate the impact of the Fill-FLOTAC homogenizer in comparison with the classical McMaster approach, where samples are suspended in flotation medium by stirring with tongue depressor in a plastic cup. The study compared the McMaster and Mini-FLOTAC techniques, but also included cross-over versions where the Fill-FLOTAC was used with the McMaster chamber, and the tongue depressor and plastic cup homogenizing method was used with the Mini-FLOTAC counting disc. Fecal samples were collected from horses naturally infected with mixed strongylid species. Five samples were included from each of the following egg count levels: 0-500, 501-1000, and >1000 eggs per gram (EPG). Each sample was then analyzed with all four set-ups with three subsamples collected from the same suspension, and three repeated counts determined on each subsample. Both the Fill-FLOTAC homogenizer (p = 0.0098) and the McMaster counting chamber (p = 0.0298) were significantly associated with higher strongylid egg counts, whereas the Mini-FLOTAC chamber was associated with a lower coefficient of variation (p < 0.0001). Precision, however, was not associated with homogenization method (p = 0.9341). Taken together, this study suggests that while the homogenizing method has a positive effect on egg count accuracy, the counting chamber appears to primarily affect precision.

Anthelmintic efficacy against equine strongyles in the United States.

Equine strongyle parasites are ubiquitous in grazing equids across the world. Anthelmintic resistance is widely developed in cyathostomin populations, but very few surveys have evaluated anthelmintic efficacy in equine populations in the United States, and most of these are over 15 years old. The present study was carried out as part of the National Animal Health Monitoring Systems (NAHMS) Equine 2015-2016 study. The aims were to investigate anthelmintic treatment efficacy by means of the fecal egg count reduction test (FECRT) and identify parameters associated with decreased efficacy. Data were collected from equine operations in 28 states via questionnaires and fecal samples submitted for fecal egg count analysis. Participants were instructed to collect samples from six equids at the day of anthelmintic treatments and 14 days later, and they were asked to include an empty syringe with a legible label of the anthelmintic product used in the shipment. Overall, dewormer treatment was effective for 76.3% of operations (84.6% of animals). Macrocyclic lactone use was effective for 88.7% of operations (95.0% of animals) while pyrimidine/benzimidazole use was effective for 21.4% of operations (43.5% of animals). Univariate analysis revealed that overall, macrocyclic lactones exhibited significantly higher efficacy than the pyrimidine and benzimidazole drug classes (p < 0.0001). There were no statistically significant differences observed between geographic regions (West, South Central, North East, and Southeast). Body weight (p = 0.0355), amount of anthelmintic administered (p = 0.0119), and operation size (p = 0.0162) were statistically associated with anthelmintic efficacy, while anthelmintic treatment frequency in the previous 12 months was not (p = 0.7081). Multiple, mixed-effect logistic regression revealed that anthelmintic drug class (p < 0.0001) was the most impactful factor in predicting anthelmintic efficacy, after accounting for operation size, region and clustering of equids at the operation level. Pasture rotation (p = 0.0129) also demonstrated a significant effect using this model. These data document widespread occurrence of reduced anthelmintic efficacy of benzimidazole and pyrimidine products against strongyle infections in equids in the United States. Anthelmintic efficacy patterns were relatively uniform between the four studied regions, and some epidemiological factors were identified to be associated with anthelmintic efficacy against strongyle infections. This information can be useful in devising sustainable parasite control strategies in the future.

Risk factors associated with strongylid egg count prevalence and abundance in the United States equine population.

Equine strongyle parasites are considered ubiquitous in grazing equids across the world, and cyathostomin parasites are known pathogens causing well-described disease complexes in horses. Decades of intensive anthelmintic treatments have led to anthelmintic resistance in cyathostomins, and current recommendations are to lower treatment intensity and base control strategies on fecal egg count surveillance. Little is known about risk factors associated with strongyle parasite egg shedding patterns in the United States equine population, as the most recent national survey was conducted 20 years ago. The present study was carried out as part of the National Animal Health Monitoring Systems (NAHMS) Equine 2015-2016 study. The aims were to describe strongyle parasite egg shedding patterns in the United States equine population and identify risk factors associated with prevalence and egg count magnitude. Data were collected from equine operations in 28 states via questionnaires and fecal samples submitted to a parasitology research laboratory for fecal egg count analysis and the data gathered underwent comprehensive statistical analyses. Though region and season were related, overall, the summer months and the fall in the southeast tended to have the greatest odds of presence of strongyles eggs on a FEC. Generally, equids resident in the Western region (Arizona, California, Colorado, Montana, Oregon, and Wyoming) had significantly lower strongyle prevalence, no matter the season, as well as a markedly different distribution between strongyle egg shedding levels (p = 0.0005). Overall, egg counts were over-dispersed with about 27% of equids (95% Confidence Interval (CI): 20-34%) contributing 80% of the egg output. Pasture history was significantly associated with strongyle egg prevalence (p = 0.0003) and egg shedding levels (p = 0.0063) with daily access in the previous 30 days being associated with higher odds of presence and greater median egg count levels. Equid gender was significantly associated with strongylid presence (p = 0.0081) and egg count level (p = 0.0008), with male equids having significantly lower odds and median egg counts than female equids, and age was significantly negatively associated with strongylid prevalence (p < 0.0001). Time since last deworming was significantly positively associated with prevalence of strongyle eggs, and this was dependent on the class of dewormer used (p = 0.0086), with equids treated with macrocyclic lactone class of drugs having lower odds of strongyle egg presence at 120 days since the last deworming. These data provide useful insights into strongylid egg shedding patterns in the United States equine population, and they can help refine parasite control recommendations depending on region, pasture access, and age distribution.

Combination deworming for the control of double-resistant cyathostomin parasites - short and long term consequences.

Equine cyathostomin are pervasive gastrointestinal parasites with wide-spread resistance to the benzimidazole and tetrahydropyrimidine drug classes worldwide. Combination deworming has been proposed as a more sustainable parasite control strategy. Simulation studies have found combination deworming to be effective in controlling drug resistant ovine trichostrongylid parasites. One equine study demonstrated an additive effect of a combination of oxibendazole and pyrantel pamoate against cyathostomins. However, this is the only equine study evaluating combination therapy, and the effects of repeated combination treatments administered over time remain unknown. The purpose of this study was to observe the efficacy of repeated oxibendazole/pyrantel pamoate combination therapy administered over one year against a cyathostomin population with resistance to benzimidazole and pyrantel products. Fecal egg counts were determined for the entire herd (N = 21) at the day of anthelmintic treatment and at two-week intervals for eight weeks post treatment. Starting efficacies of oxibendazole (OBZ, 10 mg/kg) and pyrantel pamoate (PYR, 6.6 mg base/kg) were 66.7% and 63.3%, respectively. Hereafter, the herd was treated four times with an oxibendazole/pyrantel pamoate combination, eight weeks apart, followed by repeating the single active treatments before concluding the study. While the first combination treatment exhibited an additive effect of the two active ingredients, this efficacy was not sustained over the course of the study. Mean fecal egg count reduction (FECR) was significantly greater for the first combination treatment (76.6%) than the second (42.6%, p = 0.0454), third (41.6%, p = 0.0318), and fourth (40.7%, p = 0.0372) combination treatments. The final single active mean FECRs were 42.3% for oxibendazole, and 42.7% for pyrantel pamoate. These efficacies were not significantly different from the initial single active efficacies (OBZ, p = 0.4421; PYR, p = 0.8361). These results suggest that combination therapy against double resistant equine cyathostomin populations is not sustainable, when using actives with markedly decreased starting efficacies.

Management practices associated with strongylid parasite prevalence on horse farms in rural counties of Kentucky.

Anthelmintic resistance among cyathostomin parasites is a wide-spread problem. The parasite control guidelines written by the American Association of Equine Practitioners (AAEP) encourages the preservation of anthelmintic efficacy by reducing treatment frequency, using targeted deworming, and implementing environmental management practices. While there is knowledge regarding parasite management practices of affluent horse farms in the United States, surveys rarely explore the rural and underserved regions. The purpose of this study was to observe the management practices of horse farms in rural regions Kentucky, including working Amish farms, and determine factors associated with strongyle prevalence. A total of 160 horses among 38 owners from 28 different farms were enrolled in this study. A questionnaire survey regarding equine information, farm management, and deworming history was performed with each owner. Fecal samples were collected to determine fecal egg counts, perform coprocultures for subsequent strongyle larvae identification, and Strongylus vulgaris specific PCR. Serum samples were collected for the S. vulgaris antibody specific ELISA. The mean number of deworming treatments given in the last year was 2.1 with a 95% confidence interval of 1.9-2.3 with ivermectin being the most common active used. Statistical analysis showed horses treated within the last three months with a macrocylic lactone (ML) drug had significantly lower egg counts than horses treated with a ML 7-9 months ago (p = .0005). Despite the AAEP recommendations to reduce the overall number of treatments by using a surveillance-based approach and to no longer rotate treatments, only 17 horses reportedly had a fecal sample submitted for a fecal egg count and 65 horses were dewormed in a rotational manner. Horses whose owners utilized an informative deworming source (i.e., veterinarian, internet, magazine, local feed store) also had significantly lower counts (p = .0026). All coprocultures were negative for S. vulgaris while five horses were PCR positive. Interestingly, 95 horses tested ELISA positive for S. vulgaris. The strongyle egg counts of the working Amish horses were not significantly different from the other horses in this study and deworming practices including the use of efficacious drugs and low treatment frequencies were in accordance with the AAEP guidelines. This study was the first to summarize deworming management practices of rural regions in Kentucky, including a working Amish community. Overall, horse owners employed deworming practices recommended by the AAEP, however rotational deworming is still commonly implemented and fecal egg counts are rarely used.

Postmortem examination (2016-2017) of weanling and older horses for the presence of select species of endoparasites: Gasterophilus spp., Anoplocephala spp. and Strongylus spp. in specific anatomical sites.

Parasite infections are more quantifiable postmortem than antemortem in horses. Thus a study was carried out examining dead horses for specific parasite species. Most of the weanling and older horses submitted to the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) for postmortem examination between November 22, 2016 and March 23, 2017 were examined for certain species of internal parasites. The stomach and duodenum from 69 horses were examined for bots (Gasterophilus spp.). Combined data for both Thoroughbred and non-Thoroughbred (16 other than Thoroughbred breeds/mixed breeds) horses revealed that the prevalence of Gasterophilus intestinalis was 19% (n=12) with 2nd instars (x̄ 8.5) and 39% (n=27) with 3rd instars (x̄ 90). The prevalence of Gasterophilus nasalis was 1.5% (n=1) for 2nd instars (x̄ 1) and 7% (n=5) for 3rd instars (x̄ 25). A few third instar G. intestinalis placed in 10% formalin showed slight movement at over two hundred hours later. The cecum and about 25cm of the terminal part of the ileum were examined from 139 horses for tapeworms (Anoplocephala spp.) and large strongyles (Strongylus spp.). The prevalence of A. perfoliata was 44% (n=62) and the average number of specimens per infected horse was 92.5. Strongylus vulgaris and Strongylus edentatus were not found in the gut of any horse.

Evaluation of accuracy and precision of a smartphone based automated parasite egg counting system in comparison to the McMaster and Mini-FLOTAC methods.

Fecal egg counts are emphasized for guiding equine helminth parasite control regimens due to the rise of anthelmintic resistance. This, however, poses further challenges, since egg counting results are prone to issues such as operator dependency, method variability, equipment requirements, and time commitment. The use of image analysis software for performing fecal egg counts is promoted in recent studies to reduce the operator dependency associated with manual counts. In an attempt to remove operator dependency associated with current methods, we developed a diagnostic system that utilizes a smartphone and employs image analysis to generate automated egg counts. The aims of this study were (1) to determine precision of the first smartphone prototype, the modified McMaster and ImageJ; (2) to determine precision, accuracy, sensitivity, and specificity of the second smartphone prototype, the modified McMaster, and Mini-FLOTAC techniques. Repeated counts on fecal samples naturally infected with equine strongyle eggs were performed using each technique to evaluate precision. Triplicate counts on 36 egg count negative samples and 36 samples spiked with strongyle eggs at 5, 50, 500, and 1000 eggs per gram were performed using a second smartphone system prototype, Mini-FLOTAC, and McMaster to determine technique accuracy. Precision across the techniques was evaluated using the coefficient of variation. In regards to the first aim of the study, the McMaster technique performed with significantly less variance than the first smartphone prototype and ImageJ (p<0.0001). The smartphone and ImageJ performed with equal variance. In regards to the second aim of the study, the second smartphone system prototype had significantly better precision than the McMaster (p<0.0001) and Mini-FLOTAC (p<0.0001) methods, and the Mini-FLOTAC was significantly more precise than the McMaster (p=0.0228). Mean accuracies for the Mini-FLOTAC, McMaster, and smartphone system were 64.51%, 21.67%, and 32.53%, respectively. The Mini-FLOTAC was significantly more accurate than the McMaster (p<0.0001) and the smartphone system (p<0.0001), while the smartphone and McMaster counts did not have statistically different accuracies. Overall, the smartphone system compared favorably to manual methods with regards to precision, and reasonably with regards to accuracy. With further refinement, this system could become useful in veterinary practice.