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.

Nocturnal Gamma-Hydroxybutyrate Reduces Cortisol-Awakening Response and Morning Kynurenine Pathway Metabolites in Healthy Volunteers.

BACKGROUND: Gamma-hydroxybutyrate (GHB; or sodium oxybate) is an endogenous GHB-/gamma-aminobutyric acid B receptor agonist. It is approved for application in narcolepsy and has been proposed for the potential treatment of Alzheimer's disease, Parkinson's disease, fibromyalgia, and depression, all of which involve neuro-immunological processes. Tryptophan catabolites (TRYCATs), the cortisol-awakening response (CAR), and brain-derived neurotrophic factor (BDNF) have been suggested as peripheral biomarkers of neuropsychiatric disorders. GHB has been shown to induce a delayed reduction of T helper and natural killer cell counts and alter basal cortisol levels, but GHB's effects on TRYCATs, CAR, and BDNF are unknown. METHODS: Therefore, TRYCAT and BDNF serum levels, as well as CAR and the affective state (Positive and Negative Affect Schedule [PANAS]) were measured in the morning after a single nocturnal dose of GHB (50 mg/kg body weight) in 20 healthy male volunteers in a placebo-controlled, balanced, randomized, double-blind, cross-over design. RESULTS: In the morning after nocturnal GHB administration, the TRYCATs indolelactic acid, kynurenine, kynurenic acid, 3-hydroxykynurenine, and quinolinic acid; the 3-hydroxykynurenine to kynurenic acid ratio; and the CAR were significantly reduced (P < 0.05-0.001, Benjamini-Hochberg corrected). The quinolinic acid to kynurenic acid ratio was reduced by trend. Serotonin, tryptophan, and BDNF levels, as well as PANAS scores in the morning, remained unchanged after a nocturnal GHB challenge. CONCLUSIONS: GHB has post-acute effects on peripheral biomarkers of neuropsychiatric disorders, which might be a model to explain some of its therapeutic effects in disorders involving neuro-immunological pathologies. This study was registered at ClinicalTrials.gov as NCT02342366.

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.

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.

Evaluation of the mucosal inflammatory responses to equine cyathostomins in response to anthelmintic treatment.

Members of Cyathostominae are pervasive parasites of equids that can cause larval cyathostominosis, a potentially life-threatening disease that occurs when a multitude of encysted larvae synchronously excyst from the wall of the large intestine. Moxidectin and fenbendazole are the two current labeled drugs that target the encysted larval stages; however, there is limited knowledge of the local inflammatory response to the larvae and to the two treatments in clinically healthy horses. This study is the first to evaluate the local inflammatory response to cyathostomin larvae and to larvicidal treatment at 2 and 5 weeks post treatment. Thirty-six ponies with naturally acquired cyathostomin infections were randomly allocated into 3 groups: Group 1, fenbendazole at 10 mg/kg for 5 days, Group 2, a single dose of moxidectin at 0.4 mg/kg, and Group 3, untreated controls. Tissue samples from the cecum and dorsal and ventral colons were used for histopathological and immunohistochemical evaluation. Tissues were stained with routine hematoxylin and eosin (H&E) for light microscopy and immunohistochemically for MAC387, CD20, and CD3 for differentiation of activated macrophages, B cells, and T cells, respectively. Semiquantitative scores were assigned for all inflammatory cell types and fibrous connective tissue. Larvae observed by light microscopy were enumerated and classified by stage. Mucosal ulcerations and submucosal granulomas were also enumerated. Mean macrophage scores were higher in the moxidectin group than the fenbendazole group (p = 0.0185) and the control group had a higher activated macrophage score than both treatment groups (p = 0.0104, p = 0.0004). T lymphocyte scores were higher in the moxidectin group when compared to the control group (p = 0.0069). Goblet cell hyperplasia scores were elevated at 5 weeks post treatment compared to 2 weeks post treatment (p = 0.0047) and were elevated in the ventral colon compared to the dorsal colon (p = 0.0301). Eosinophil scores were elevated surrounding degenerative larvae when compared to intact larvae (p = 0.0001). Mucosal ulcerations were found only in the control group at 2 weeks post treatment. This study found subtle inflammatory differences between treatment groups but provided new information about goblet cells and eosinophils in relation to encysted cyathostomin larvae.

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.

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.