Metabolomic profiling of permethrin-treated Drosophila melanogaster identifies a role for tryptophan catabolism in insecticide survival.

Insecticides and associated synergists are rapidly losing efficacy in target insect pest populations making the discovery of alternatives a priority. To discover novel targets for permethrin synergists, metabolomics was performed on permethrin-treated Drosophila melanogaster. Changes were observed in several metabolic pathways including those for amino acids, glycogen, glycolysis, energy, nitrogen, NAD(+), purine, pyrimidine, lipids and carnitine. Markers for acidosis, ammonia stress, oxidative stress and detoxification responses were also observed. Many of these changes had not been previously characterized after permethrin exposure. From the altered pathways, tryptophan catabolism was selected for further investigation. The knockdown of some tryptophan catabolism genes (vermilion, cinnabar and CG6950) in the whole fly and in specific tissues including fat body, midgut and Malpighian tubules using targeted RNAi resulted in altered survival phenotypes against acute topical permethrin exposure. The knockdown of vermilion, cinnabar and CG6950 in the whole fly also altered survival phenotypes against chronic oral permethrin, fenvalerate, DDT, chlorpyriphos and hydramethylnon exposure. Thus tryptophan catabolism has a previously uncharacterized role in defence against insecticides, and shows that metabolomics is a powerful tool for target identification in pesticide research.

Development of an in vitro screen for compound bioaccumulation in Haemonchus contortus.

The objective of the current study was to establish an in vitro screen and a highly sensitive analytical assay to delineate key physicochemical properties that favor compound bioaccumulation in the L3 life stage of a Haemonchus contortus isolate. Time-dependent studies revealed that absorption and elimination kinetics during the first 6 hr of exposure were sufficient to achieve maximum bioaccumulation for the majority of compounds tested. In subsequent studies, the larvae were incubated for 6 hr in a medium containing 146 compounds (5 µM initial concentration), including both human and veterinary medicines, characterized by a broad range of physicochemical properties. Bioaccumulation of the compounds by the nematodes was determined, and multiple physicochemical descriptors were selected for correlation. Data analysis using Bayes classification model and partial least-square regression revealed that clogD7.4, rotatable bond, E-state, and hydrogen bond donor each correlated with compound bioaccumulation in H. contortus L3. The finding that lipophilicity was critical for transcuticle compound permeation was consistent with previous studies in other parasitic species and in adult H. contortus . The finding of additional physicochemical properties that contribute to compound conformational flexibility, polarity, and electrotopological state shed light on the mechanisms governing transcuticle permeation. The relatively poor correlation between transcuticle and transmembrane permeation indicated the distinct mechanisms of compound permeation, likely due to the different constituents, and their contributions to overall transport function, of the lipid membranes and the porous collagen barrier of the nematode cuticle. Our study, for the first time, establishes a high-throughput screen for compound bioaccumulation in a parasitic nematode and further elucidates physicochemical factors governing transcuticular permeation of compounds. Application of this methodology will help explain the basis for discrepancies observed in receptor binding and whole organism potency assays and facilitate incorporation of drug delivery principles in the design of candidate anthelmintics.

Confirmation of the efficacy of a novel fipronil spot-on for the treatment and control of fleas, ticks and chewing lice on dogs.

A novel spot-on formulation containing fipronil (Eliminall(®)/Exproline vet™) Spot-on Solution for Dogs, Pfizer Animal Health, registered and manufactured by Krka, d.d, Novo mesto) was evaluated in three laboratory studies to confirm efficacy against fleas, ticks and chewing lice on dogs for at least one month. Control of two laboratory strains of cat flea (Ctenocephalides felis), two tick species (Rhipicephalus sanguineus and Dermacentor reticulatus) and the chewing louse (Trichodectes canis) was evaluated. In all studies, dogs were randomly allocated to treatment groups and compared with untreated dogs. The studies also included a commercial, comparator product containing fipronil (Frontline(®) spot-on, Merial Limited). All treatments were applied to the skin at one spot between the scapulae on Day 0. In the studies, dogs were infested with fleas and/or ticks prior to treatment and then reinfested at weekly intervals for up to 8 weeks after treatment and evaluated for efficacy at 2 days (48 h) after treatment and each reinfestation. These studies confirmed that treatment with the novel fipronil spot-on at the proposed commercial dose rate rapidly reduced existing infestations of fleas, ticks and chewing lice on dogs. Treatment provided control of reinfesting fleas for up to 8 weeks, up to 4 weeks control of ticks, and control of chewing lice.

Efficacy of a novel fipronil spot-on for the treatment and control of induced infestations of adult cat fleas (Ctenocephalides felis) and castor bean ticks (Ixodes ricinus) on cats.

Cat fleas (Ctenocephalides felis) and the castor bean tick (Ixodes ricinus) cause discomfort and health effects due to bites and ingestion of blood and they serve as vectors for several animal and human pathogens. Effectiveness of a novel 10 % w/v fipronil spot-on (Eliminall®/Exproline vet™, marketed by Pfizer Animal Health and registered and manufactured by Krka, d.d., Novo mesto) was confirmed against these parasites on experimentally infested cats. Two parallel, unicentre and masked controlled studies were conducted with European mixed breed and mixed sex cats. Cats were allocated randomly to one of two treatment groups based on either pre-treatment flea counts (study 1) or pre-treatment tick counts (study 2). In each of the study, eight animals served as control, while another eight animals were treated once topically with the unit label dose of 50 mg fipronil per cat (10.6-23.8 mg/kg). At each reinfestation, animals were infested with approximately 100 fleas or 60 ticks to achieve adequate infestation rates. Parasites were removed and counted on days 2, 9, 16, 23, 30 and 37, 48 h after the treatment or experimental infestation. Excellent effectiveness was demonstrated on day 2 (100 and 94 % efficacy against fleas and ticks, respectively) and lasted for up to 5 weeks (efficacy ≥96 %) against fleas and up to 4 weeks against ticks (efficacy ≥94 %). The product was well tolerated and no adverse reactions were observed.

Efficacy of pyrantel pamoate and ivermectin paste formulations against naturally acquired Oxyuris equi infections in horses.

In recent years, numerous veterinary practitioners have reported anecdotal episodes in which anthelmintic treatment did not appear to deliver the expected efficacy against equine pinworms (Oxyuris equi). Anthelmintic resistance has not been demonstrated formally in equine pinworms, so a clinical study was designed to evaluate the efficacy of paste formulations of pyrantel pamoate or ivermectin against naturally acquired infections with O. equi. Twenty-one horses (>4 months to 15 years of age) with patent, naturally acquired pinworm infections were blocked by source of origin and allocated randomly to one of three treatment groups: horses (n=7) assigned to Group 1 were treated orally with pyrantel pamoate paste at a dosage of 13.2 mg/kg (2x label dosage), Group 2 horses (n=7) were untreated controls, and horses (n=7) assigned to Group 3 were treated orally with ivermectin paste at a dosage of 200 microg/kg. Fourteen days after treatment, horses were euthanatized, necropsied, and large intestinal contents were processed for recovery of adult pinworms. In addition, duplicate 1% aliquots of intestinal contents from the cecum, ventral colon, dorsal colon, and small colon were collected, preserved, and examined for recovery and enumeration of fourth-stage larval O. equi. Anthelmintic efficacy against pinworms was evaluated by comparing the post-treatment worm counts of Groups 1 and 3 to those of control animals. Mean numbers of O. equi adults recovered postmortem were significantly decreased by both pyrantel pamoate (P=0.0366) and ivermectin (P=0.0137) treatment, with respective efficacies of 91.2% and 96.0%. In addition, both products demonstrated >99% efficacy against fourth-stage O. equi larvae. The current study demonstrated acceptable adulticidal and larvicidal efficacy of both pyrantel pamoate and ivermectin paste formulations against O. equi and did not support the existence of macrocyclic lactone or pyrimidine resistance in the pinworm populations evaluated.

Efficacy of pyrantel pamoate against a macrocyclic lactone-resistant isolate of Parascaris equorum in horses.

The expanding prevalence of Parascaris equorum populations that are resistant to macrocyclic lactone (ML) anthelmintics makes it desirable to identify dewormers which remain effective. The objective was to evaluate the efficacy of pyrantel pamoate in 14 suckling foals that had been infected orally with approximately 600 larvated eggs of a P. equorum isolate selected for ML resistance (ML-R). Seventy days after inoculation, foals were weaned, housed individually, and fecal samples were examined frequently to detect the onset of patency. Between 73 and 80 days post-inoculation, all 14 foals developed P. equorum egg counts>or=150 eggs per gram (EPG). An initial cohort of eight foals was treated orally with ivermectin paste (200 microg/kg) 84-91 days post-inoculation. Egg counts were reduced by only 47% at 2 weeks after ivermectin treatment, confirming the ML-R status of the isolate. A second cohort of six foals was not treated with ivermectin. Within each cohort, eligible foals were allocated randomly to treated (pyrantel pamoate; n=7) or untreated control (n=7) groups. Treated foals were dosed orally on Day 0 with a paste formulation of pyrantel pamoate at 13.2mg/kg. Mean ascarid egg counts of treated foals were reduced by 96.0% and 98.8% at 1 and 2 weeks post-treatment, respectively. On Day 14, foals were euthanatized and specimens of P. equorum were recovered from the gut contents, preserved in 10% formalin, and counted. Mean numbers of P. equorum adults recovered postmortem were significantly lower (P=0.0031) in foals treated with pyrantel pamoate (X=1.7; range 0-16) compared to control foals (X=63.0; range 0-320). A paste formulation of pyrantel pamoate, at a dosage of 13.2 mg/kg, was 97.3% effective against a ML-R isolate of P. equorum.

Future of the animal health industry at a time of food crisis.

It is popular in some quarters to say that there is no food crisis; that there is food aplenty; and that the problem is one of distribution or other over-arching technical difficulty. To the starving, however, there is a food crisis; and it neither speaks well nor bodes well for humanity if we dismiss their plight so glibly. The United Nations has called for a large and rapid increase in food production. Veterinary parasitologists and industry leaders can contribute to the production of healthier livestock and the expansion of aquaculture, but enhanced production and better delivery of plant foods may provide faster relief. Although livestock farming is not the most energy-efficient way of producing food, meat will remain a significant component of the global diet for the foreseeable future. New measures for parasite control will be needed, and we must improve our methods of inventing them. They need not act directly against the parasite. In the distant future lie other threats to the inhabitants of planet Earth, and here we must acknowledge the cogency of the no-food-crisis argument. In the long term, the production of animal foods and animal feeds will be revamped in ways that depend on how (or whether) we solve the energy crisis, the environmental crisis, the increasingly dire regional population crises, and the current world financial crisis. Throughout the 20th century, the animal health industry had to adapt to industrialization and expansive agribusiness. It will have to adapt to even greater changes in the 21st century and beyond.

Dose-confirmation studies of the cestocidal activity of pyrantel pamoate paste in horses.

Dose confirmation studies of the cestocidal activity of pyrantel pamoate paste were conducted at two sites in North America during 2001. Horses with naturally-acquired cestode infections were identified by detection of typical Anoplocephala spp. eggs in feces collected between 7 and 92 days prior to treatment. Twenty and 22 horses were enrolled at Site 1 (Urbana, IL) and Site 2 (Knoxville, TN), respectively. Candidate horses were acclimated to study conditions for 14 days, ranked by length of interval since coprologic confirmation, and allocated randomly to one of two treatment groups: (T1) pyrantel pamoate paste 13.2mg pyrantel base per kilogram body weight administered orally, and (T2) untreated controls. Individual doses of pyrantel pamoate paste were prepared on the basis of contemporaneous body weights and administered to Group T1 horses on Day 0. Trained personnel monitored the animals at regular intervals after treatment to detect potential adverse reactions. Horses were euthanatized and necropsied 10-12 days after treatment. The contents of the large and small intestines were collected, and the walls of each organ were rinsed with water and inspected. Attached cestodes were recovered and preserved in 10% formalin. The intestinal contents and rinsed ingesta were washed over a #10-mesh (2mm aperture) sieve and tapeworms were extracted and preserved. Recovered cestodes were counted and examined at 1-4x magnification for identification to genus and species. At Site 1, specimens of Anoplocephala perfoliata were recovered from seven of 10 control horses, and from one of 10 horses treated with pyrantel pamoate. Mean cestode numbers were 4.52 in the control group and 0.07 for treated horses. At Site 2, cestodes were found in 10 of 11 controls (mean 26.2) and in five of 11 horses (mean 1.2) treated with pyrantel pamoate. In both studies, Group T1 means were significantly lower than the control group (P<0.005). The calculated efficacies were 98.4 and 95.5% at Sites 1 and 2, respectively. In two dose-confirmation studies, a single, oral treatment of pyrantel pamoate paste (19.13% w/w pyrantel base) at 13.2mg/kg was >or=95.5% effective against A. perfoliata in naturally-infected horses.

Clinical field efficacy and safety of pyrantel pamoate paste (19.13% w/w pyrantel base) against Anoplocephala spp. in naturally infected horses.

Clinical field trials were conducted at five geographical locations in the USA (Oklahoma, Wisconsin, Tennessee, Virginia and Idaho) to evaluate the efficacy and safety of pyrantel pamoate paste (19.13%, w/w, pyrantel base) administered at the recommended dosage of 13.2 mg pyrantel base/kg (6.0 mg pyrantel base/lb) body weight (b.w.) against tapeworm infections of Anoplocephala spp. in naturally infected horses. Horses at each study site were allocated by restricted randomization based on the cestode status (positive or negative) of pre-treatment fecal egg counts to complete sets of four animals each or incomplete sets of fewer than four animals. Within sets comprising of two to four horses, one animal was randomly allocated to receive placebo vehicle paste and the remaining horse(s) received pyrantel pamoate paste administered orally at a minimum dosage of 13.2 mg pyrantel base/kg b.w. on Test Day (TD) 0. Single animal sets received pyrantel pamoate paste. Fecal samples of horses were collected and examined for equine tapeworm (Anoplocephala spp.) eggs a minimum of four times (once or thrice between TD -28 and -14, twice between TD -14 and -7, and once on TD 0) prior to treatment on TD 0. Fecal samples of horses that were positive for cestode infection pre-treatment were examined for cestode eggs on TD 7, 8, 9, 14, 15 and 16. Cestode-negative pre-treatment horses were not sampled again after treatment. A total of 241 horses (141 mares, 16 stallions and 84 geldings; 6 months-30 yrs of age; 173-646 kg; 13 recognized breeds and various crossbreds) were evaluated. The prevalence of Anoplocephala spp. determined by pre-treatment fecal examination ranged from 38.3% in Idaho to 68.1% in Tennessee with an overall prevalence of 52.3%. Ninety cestode-positive and 88 cestode-negative horses were treated with pyrantel pamoate paste, 36 cestode-positive and 27 cestode-negative horses were treated with placebo vehicle paste. Overall, 178 horses were treated with pyrantel pamoate paste, and 63 horses were treated with placebo paste. Of the 178 horses treated with pyrantel pamoate paste, no drug related, adverse clinical or neurological health events were observed. No doses of pyrantel pamoate paste were refused or lost during dosing. At each post-treatment time sampling interval, significantly fewer cestode eggs (P < 0.0115) were passed by cestode-positive horses treated with pyrantel pamoate paste compared to cestode-positive horses that received placebo paste. Efficacy of the pyrantel pamoate paste treatment ranged from 92 to 96% from TD 7 to TD 16 with an overall efficacy of 95%. The results of these trials demonstrated that pyrantel pamoate paste (19.13%, w/w, pyrantel base) administered orally at a dosage of 13.2 mg pyrantel base/kg b.w. is highly efficacious (95%) against Anoplocephala spp. and safe for use in horses with no adverse clinical or neurological health events observed under field use conditions.

Target animal safety and tolerance study of pyrantel pamoate paste (19.13% w/w pyrantel base) administered orally to horses.

Pyrantel pamoate paste (19.13% w/w pyrantel base) for the treatment of tapeworm, Anoplocephala spp was evaluated for target animal safety and tolerance in horses treated orally at 0, 1, 3, 5, and 10 times the clinical dose of 13.2 mg pyrantel base/kg body weight administered daily for six consecutive days. Parameters evaluated included clinical signs, food and water consumption, body weights, physical examinations, clinical pathology (hematology, coagulation, serum chemistry, urinalyses, and fecal examinations), complete necropsy, organ weights, and histopathology. No adverse events or test article-related effects were observed in any treatment group during daily clinical observations of the test animals. Statistically significant changes (P < .05) lacked a dose- and/or time-dependent trend and were considered incidental. Administration of pyrantel pamoate paste did not produce any macroscopic or microscopic tissue effects in any dose group of either sex. The no-observed-effect-level (NOEL) for pyrantel pamoate paste, when administered orally to horses once daily for 6 consecutive days, was determined to be 132 mg/kg/day. Pyrantel pamoate paste (19.13% w/w pyrantel base) can be safely administered orally to horses at 13.2 mg of pyrantel base/kg for the treatment of Anoplocephala infestations.