Repertoire of P-glycoprotein drug transporters in the zoonotic nematode Toxocara canis.

Toxocara canis has a complex lifecycle including larval stages in the somatic tissue of dogs that tolerate macrocyclic lactones. In this study, we investigated T. canis permeability glycoproteins (P-gps, ABCB1) with a putative role in drug tolerance. Motility experiments demonstrated that while ivermectin failed to abrogate larval movement, the combination of ivermectin and the P-gp inhibitor verapamil induced larval paralysis. Whole organism assays revealed functional P-gp activity in larvae which were capable of effluxing the P-gp substrate Hoechst 33342 (H33342). Further investigation of H33342 efflux demonstrated a unique rank order of potency for known mammalian P-gp inhibitors, suggesting that one or more of the T. canis transporters has nematode-specific pharmacological properties. Analysis of the T. canis draft genome resulted in the identification of 13 annotated P-gp genes, enabling revision of predicted gene names and identification of putative paralogs. Quantitative PCR was used to measure P-gp mRNA expression in adult worms, hatched larvae, and somatic larvae. At least 10 of the predicted genes were expressed in adults and hatched larvae, and at least 8 were expressed in somatic larvae. However, treatment of larvae with macrocyclic lactones failed to significantly increase P-gp expression as measured by qPCR. Further studies are needed to understand the role of individual P-gps with possible contributions to macrocyclic lactone tolerance in T. canis.

Shortened egg reappearance periods of equine cyathostomins following ivermectin or moxidectin treatment: morphological and molecular investigation of efficacy and species composition.

Macrocyclic lactones have been the most widely used drugs for equine parasite control during the past four decades. Unlike ivermectin, moxidectin exhibits efficacy against encysted cyathostomin larvae, and is reported to have persistent efficacy with substantially longer egg reappearance periods. However, shortened egg reappearance periods have been reported recently for both macrocyclic lactones, and these findings have raised several questions: (i) are egg reappearance period patterns different after ivermectin or moxidectin treatment? (ii) Are shortened egg reappearance periods associated with certain cyathostomin species or stages? (iii) How does moxidectin's larvicidal efficacy affect egg reappearance period? To address these questions, 36 horses at pasture, aged 2-5 years old, were randomly allocated to three treatment groups: 1, moxidectin; 2, ivermectin; and 3, untreated control. Strongylid fecal egg counts were measured on a weekly basis, and the egg reappearance period was 5 weeks for both compounds. Strongylid worm counts were determined for all horses: 18 were necropsied at 2 weeks post-treatment (PT), and the remaining 18 at 5 weeks PT. Worms were identified to species morphologically and by internal transcribed spacer-2 (ITS-2) rDNA metabarcoding. Moxidectin and ivermectin were 99.9% and 99.7% efficacious against adults at 2 weeks post treatment, whereas the respective efficacies against luminal L4s were 84.3% and 69.7%. At 5 weeks PT, adulticidal efficacy was 88.3% and 57.6% for moxidectin and ivermectin, respectively, while the efficacy against luminal L4s was 0% for both drugs. Moxidectin reduced early L3 counts by 18.1% and 8.0% at 2 or 5 weeks, while the efficacies against late L3s and mucosal L4s were 60.4% and 21.2% at the same intervals, respectively. The luminal L4s surviving ivermectin treatment were predominantly Cylicocyclus (Cyc.) insigne. The ITS-2 rDNA metabarcoding was in good agreement with morphologic species estimates but suggested differential activity between moxidectin and ivermectin for several species, most notably Cyc. insigne and Cylicocyclus nassatus. This study was a comprehensive investigation of current macrocyclic lactone efficacy patterns and provided important insight into potential mechanisms behind shortened egg reappearance periods.

Margin of safety of extended-duration transdermal buprenorphine solution following multiple-dose administrations to cats.

Transdermal buprenorphine solution (TBS) is approved for the control of postoperative pain in cats where a single preoperative dose provides 4 days of analgesia. It is administered as a unit dose of 8 mg to cats weighing 1.2-3 kg and 20 mg to cats weighing to >3-7.5 kg, which is equivalent to a dosage on a bodyweight basis of 2.7-6.7 mg/kg. In this safety study, the 1X dose was defined as 6.7 mg/kg. Thirty-two cats (16 males and 16 females) were randomly allocated to placebo, 1, 2, and 3X TBS administered topically to the dorsal cervical skin every 4 days for 3 doses. Clinical observations, behavioral scores, mydriasis score (yes/no), and physiological variables were assessed or measured prior to each dose administration (0 h) and at 1, 2, 4, 8, 12, 24, 36, 48, and 72 h following each treatment and prior to euthanasia on Day 12 or 13. Blood samples for clinical pathology were collected on Days - 1, 4, 8, and prior to euthanasia. There was little evidence of respiratory, cardiovascular, or gastrointestinal effects. Respiratory rates were above the reference range in all groups and lower by 10 breaths/min in the 3X group during the third dosing interval compared to placebo. There were no differences in heart rates. Constipation was transiently observed in approximately equal numbers in placebo- and TBS-treated cats. Behavioral scores showed sedation or euphoria was transient in the first dosing interval but became more prolonged with each dosing interval. Mydriasis was prolonged in the first dosing interval and diminished by the third dosing interval consistent with accommodation. Mean body temperatures in TBS-treated cats were up to 0.6°C (1.8°F) greater than placebo-treated cats. There were no clinically relevant changes to serum chemistry, hematology, or urinalysis outcomes nor gross or microscopic observations attributable to TBS. These data demonstrate that TBS is safe and well-tolerated when administered to 16-week-old cats at multiples of the approved dose and duration and supports clinical safety in the event of delayed buprenorphine metabolism, medication errors, or alterations in the dosing regimen.

Single-dose pharmacokinetics and bioavailability of a novel extended duration transdermal buprenorphine solution in cats.

A novel transdermal buprenorphine solution (TBS) was developed for evaluation in order to make available an extended duration opioid analgesic for cats. Healthy adult cats were administered a single TBS dose of 10 mg (1.57-4.35 mg/kg), 30 mg (4.72-13.0 mg/kg), or 50 mg (7.87-21.7 mg/kg) (4 cats per group) applied topically to the unclipped dorsal cervical skin and plasma buprenorphine concentrations were evaluated through 7 days. To determine the absolute bioavailability of TBS, healthy cats were administered single TBS dose of 20 mg (3.33-4.76 mg/kg) or 0.05 mg (0.008-0.011 mg/kg) IV buprenorphine (6 cats per group). The mean ± standard deviation maximum plasma buprenorphine concentrations (Cmax ) were 10.5 ± 6.28, 18.6 ± 8.68, and 22.5 ± 4.47 ng/ml following 10, 30, and 50 mg doses, respectively, with the time of Cmax occurrence (tmax ) typically occurring at 2-12 h post-dosing. Mean plasma buprenorphine terminal half-lives ranged between 78.3 and 91.2 h. Increasing the dose threefold and fivefold from the 10 mg dose increased the exposure by 2.8- and 3.6-fold, respectively, indicating that plasma buprenorphine exposure increased in a less than proportional manner at doses >30 mg. Transient sedation, mydriasis, and euphoria were observed within 4 h post-dosing. Mean rectal temperatures were increased 0.6-0.9°C greater than baseline (37.4-37.8°C) through 168 h post-dosing. The absolute bioavailability was 16.0% (90% CI: [11.8%-21.7%]). Flip-flop pharmacokinetics were observed with a terminal elimination half-life of 0.82 ± 0.13 and 64.9 ± 15.0 h for IV buprenorphine and 20 mg of TBS, respectively. A single administration of TBS over a range of doses resulted in extended plasma buprenorphine concentrations and opioid physiological and behavioral effects.

World association for the advancement of veterinary parasitology (WAAVP): Third edition of guideline for evaluating the efficacy of equine anthelmintics.

This guideline have been developed to assist in the design, execution, and interpretation of studies to assess the efficacy of anthelmintic drugs against internal parasites of equines, including nematodes, cestodes, and larval instars of Gasterophilus spp. The design and execution of critical and controlled studies are outlined, and their advantages and disadvantages are discussed. Unique considerations for specific target parasites are included. Information is also provided on selection of animals, procedures for randomization, housing, feeding, dosage titration, dosage confirmation and field studies, record keeping and necropsy procedures. Finally, this document includes guidance for group size determination and statistical analysis of study results. This guideline should assist investigators in the evaluation of anthelmintic drugs in horses by using comparable and standardized procedures in studies with appropriate numbers of animals.

Effectiveness of a novel orally administered combination drug product containing milbemycin oxime and lotilaner (Credelio® Plus) for the treatment of larval and immature adult stages of Ancylostoma caninum in experimentally infected dogs.

BACKGROUND: The hookworm, Ancylostoma caninum, is a common and important zoonotic intestinal nematode parasite that infects dogs globally. Both the immature and adult stages of A. caninum ingest large volumes of blood during the feeding process and can cause severe anemia and death in young dogs, even before patent infections can be diagnosed using routine faecal examination methods. Thus, effective treatment of any pre-patent stages of immature hookworms can reduce or eliminate the risk of clinical disease in infected dogs and additionally reduce environmental contamination of eggs and infective larvae. Two randomized, blinded, GCP-compliant, pivotal laboratory dose confirmation studies were conducted to evaluate the effectiveness and safety of a new novel combination of lotilaner and milbemycin oxime tablets (Credelio Plus®) administered orally to dogs experimentally infected with immature (L4 and immature adult [L5]) stages of A. caninum. METHODS: Treatments using the intended global commercial tablet formulation of Credelio Plus were administered in a time frame relative to inoculation with infective larvae so that effectiveness could be assessed against each specific immature stage of A. caninum. In each study, dogs were randomized to one of six (study 1) or four (study 2) treatment groups. Each treatment group contained 8 (study 1) or 10 (study 2) dogs that had been experimentally inoculated with infective A. caninum larvae on day 0 and were dosed once on day 7 or day 11. Enrolled subjects were administered placebo tablets, Credelio Plus tablets, or lotilaner mono tablets to provide minimum dosages of 0.75 mg/kg of milbemycin oxime and 20 mg/kg of lotilaner. All dogs were necropsied 5 days after their respective treatment. All nematodes recovered from the gastrointestinal tract at necropsy were counted by species and stage. RESULTS: For both dose confirmation studies and based on geometric mean worm counts, efficacy of Credelio Plus was ≥ 97.3% against L4 larval stage of A. caninum and ≥ 98.7% against immature adult (L5) A. caninum. CONCLUSIONS: These studies demonstrated that the orally administered Credelio Plus combination tablet was highly efficacious in treating immature (L4 and immature adult [L5]) stages of A. caninum in experimentally infected dogs.

Effectiveness of Credelio® Plus, a novel chewable tablet containing milbemycin oxime and lotilaner for the treatment of larval and immature adult stages of Toxocara canis in experimentally infected dogs.

BACKGROUND: The ascarid, Toxocara canis, is a common and important zoonotic intestinal nematode parasite that infects dogs globally. An effective treatment that kills any pre-patent stages of immature T. canis could additionally reduce or eliminate the development of patent infections that can result in clinical disease in infected dogs and would further reduce environmental contamination of eggs. Two randomized, blinded, GCP-compliant, pivotal laboratory dose confirmation studies were conducted to assess the effectiveness and safety of a new novel combination of lotilaner and milbemycin oxime tablets (Credelio Plus) administered orally to dogs that were experimentally infected with immature (L4 or immature adult [L5]) stages of T. canis. METHODS: The commercial tablet formulation of Credelio Plus® was administered in a time frame relative to inoculation with infective eggs. This allowed for effectiveness to be assessed against each specific immature stage of T. canis. In each study, dogs were randomized and allocated to one of four treatment groups. Each treatment group contained ten dogs that had been experimentally inoculated on Day 0 with infective T. canis eggs and then were dosed once on Day 14 or Day 24 using either placebo tablets or Credelio Plus tablets (IP) to provide minimum dosages of 0.75 mg/kg of milbemycin oxime and 20 mg/kg of lotilaner. All dogs were necropsied 5 or 6 days after their respective treatment. At necropsy, all nematodes recovered from the gastrointestinal tract were counted by species and stage. RESULTS: In both dose confirmation studies using geometric mean worm counts, effectiveness of Credelio Plus was ≥ 98.6% and ≥ 96.8% against L4 larval stage T. canis and immature adult [L5] T. canis in both studies, respectively. CONCLUSIONS: These studies demonstrated that the Credelio Plus combination tablet administered orally to dogs was highly efficacious against experimental infections with L4 and immature adult [L5] stages of T. canis.

Efficacy of a novel topical combination of esafoxolaner, eprinomectin and praziquantel in cats against Toxocara cati and Dipylidium caninum.

NexGard® Combo, a novel topical antiparasitic product for cats, combines the insecticide/acaricide esafoxolaner with the nematocide eprinomectin and cestodicide praziquantel. The efficacy of this combination product was evaluated against two common endoparasites of global occurrence in cats, the nematode Toxocara cati and the cestode Dipylidium caninum, in five controlled studies using naturally or experimentally infected cats with parasites of North American, South African or European origin. Cats evaluated in these studies harbored patent infection of the target parasite confirmed through a pre-treatment fecal examination. In each study, cats were allocated randomly to two groups of equal size (8 or 10 cats per group per study), one group treated with a placebo (mineral oil) and the other with NexGard® Combo. Both treatments were administered once as a spot-on at 0.12 mL per kg body weight to deliver the minimum label dosage (1.44 mg/kg esafoxolaner, 0.48 mg/kg eprinomectin, and 10.0 mg/kg praziquantel) to the NexGard® Combo-treated cats. To determine efficacy, geometric mean parasite counts seven to 12 days after treatment of placebo-treated (control) cats and NexGard® Combo-treated cats were compared. The efficacy of NexGard® Combo was 98.8% and 100% against adult T. cati in two studies; and 98.0%, 98.3% and 93.2% against D. caninum in three studies. No adverse events related to treatment were observed throughout the studies. These studies demonstrate high efficacy against these major feline endoparasites and excellent acceptability of the novel topical antiparasitic combination of esafoxolaner, eprinomectin and praziquantel.

TITLE: Efficacité d'une nouvelle association topique d'esafoxolaner, d'éprinomectine et de praziquantel chez le chat contre Toxocara cati et Dipylidium caninum. ABSTRACT: NexGard® Combo, un nouveau produit antiparasitaire topique pour chats combine l'insecticide/acaricide esafoxolaner avec le nématocide éprinomectine et le cestodicide praziquantel. L'efficacité de ce produit d'association a été évaluée contre deux endoparasites communs d'occurrence mondiale chez le chat, le nématode Toxocara cati et le cestode Dipylidium caninum, dans cinq études contrôlées utilisant des chats naturellement ou expérimentalement infectés par des parasites d'origine nord-américaine, sud-africaine ou européenne. Les chats évalués dans ces études présentaient une infection patente du parasite cible confirmée par un examen fécal avant le traitement. Dans chaque étude, les chats ont été répartis au hasard en deux groupes de taille égale (8 ou 10 chats par groupe et par étude), un groupe traité avec un placebo (huile minérale) et l'autre avec NexGard® Combo. Les deux traitements ont été administrés une fois par spot-on à 0,12 mL par kg de poids corporel pour délivrer la dose minimale indiquée sur l'étiquette (1,44 mg/kg d'esafoxolaner, 0,48 mg/kg d'éprinomectine et 10,0 mg/kg de praziquantel) pour les chats du groupe traité par NexGard® Combo. Pour déterminer l'efficacité, les nombres moyens géométriques de parasites sept à 12 jours après le traitement des chats traités par placebo (témoins) et des chats traités par NexGard® Combo ont été comparés. L'efficacité de NexGard® Combo était de 98,8 % et de 100 % contre T. cati adulte dans deux études, et de 98,0 %, 98,3 % et 93,2 % contre D. caninum dans trois études. Aucun événement indésirable lié au traitement n'a été observé tout au long des études. Ces études démontrent la grande efficacité contre ces principaux endoparasites félins et l'excellente acceptabilité de la nouvelle combinaison antiparasitaire topique d'esafoxolaner, d'éprinomectine et de praziquantel.

Efficacy of a new oral chewable tablet containing sarolaner, moxidectin and pyrantel (Simparica Trio™) against induced ascarid infections in dogs.

BACKGROUND: Ascarid infections are among the most prevalent intestinal parasitic infections occurring in dogs around the world, with Toxocara canis and Toxascaris leonina commonly observed. Toxocara canis can cause considerable disease in dogs and humans, and year-round prophylactic treatment and control in dogs is recommended. Elimination of immature stages of these parasites before egg-laying will reduce environmental contamination and the risk of infection for both dogs and humans. Studies were conducted to evaluate the efficacy of a novel, oral chewable tablet containing sarolaner, moxidectin and pyrantel (Simparica Trio™) against induced immature adult (L5) and adult T. canis, and adult T. leonina infections in dogs. METHODS: Six negative-controlled, masked, randomized laboratory studies were conducted. Two studies each evaluated efficacy against immature adult (L5) T. canis, adult T. canis, and adult T. leonina. Sixteen to 40 dogs were included in each study. Dogs experimentally infected with the target parasite were dosed once on Day 0 with either placebo tablets or Simparica Trio™ tablets to provide minimum dosages of 1.2 mg/kg sarolaner, 24 µg/kg moxidectin and 5.0 mg/kg pyrantel (as pamoate salt). Efficacy was based on the number of worms recovered at necropsy 7-10 days after treatment compared to placebo control. RESULTS: Based on geometric mean worm counts, efficacy of the sarolaner + moxidectin + pyrantel combination was ≥ 95.2% against immature adult T. canis, ≥ 97.3% against adult T. canis, and ≥ 89.7% against adult T. leonina. There were no treatment-related adverse events in any study. CONCLUSIONS: These studies confirm the efficacy of a single dose of a new oral chewable tablet containing sarolaner, moxidectin and pyrantel (Simparica Trio™) against immature adult and adult T. canis, and adult T. leonina infections in dogs.

A model for the dynamics of the parasitic stages of equine cyathostomins.

A model was developed to reproduce the dynamics of the parasitic stages of equine cyathostomins. Based on a detailed review of published literature, a deterministic simulation model was constructed using the escalator boxcar-train approach, which allows for fully-overlapping cohorts of worms and approximately normally distributed variations in age/size classes. Key biological features include a declining establishment of ingested infective stage larvae as horses age. Development rates are constant for all the parasitic stages except the encysted early third stage larvae, for which development rates are variable to reflect the sometimes extended arrestment of this stage. For these, development is slowed in the presence of adult worms in the intestinal lumen, and when ingestion of infective larvae on herbage is high or extended. In the absence of anthelmintic treatments, the life span of adult worms is approximately 12 months, and the presence of an established adult worm burden largely blocks the transition of luminal fourth stage larvae to the adult stage, resulting in mortality of the larvae. This inhibition is removed by effective anthelmintic treatment allowing the rapid replacement of adult worms from the pool of mucosal stages. Within the model, the rate and seasonality at which infective stage larvae are ingested strongly influences the dynamics of the pre-adult stages. While the adult worm burden remains relatively stable within a year, due to the negative feedback they have on developing stages, the numbers and proportions of larval stages relative to the total worm burden increase with the numbers of infective larvae ingested. Further, within the model, the seasonal rise and fall of encysted stages is largely driven by the seasonal pattern of infective larvae on pasture. Because of this, the model reproduces the contrasting seasonal patterns of mucosal larvae, typical of temperate and tropical environments, using only the appropriate seasonality of larvae on pasture. Thus, the model reproduces output typical of different climatic regions and suggests that observed patterns of arrested development may simply reflect the numbers and seasonality of free-living stages on pasture as determined by different management practices and weather patterns.

Anthelmintic therapy of equine cyathostomin nematodes - larvicidal efficacy, egg reappearance period, and drug resistance.

Cyathostomins are ubiquitous in grazing horses across the world, and anthelmintic resistance has been reported with increasing levels over past decades. The aims of the present study were (i) to investigate the efficacy against encysted larval stages of moxidectin (0.4 mg/kg) and fenbendazole (10 mg/kg daily for five consecutive days) and compare these regimens at 2 and 5 weeks post-treatment, (ii) to investigate individual cyathostomin species associated with shortened egg reappearance periods, and (iii) to document species exhibiting decreased susceptibility to the evaluated compounds. Thirty-six ponies were allocated to treatment groups with half euthanatized 2 weeks post-treatment, and the remainder necropsied after 5 weeks. Luminal and mucosal worm counts were conducted and strongyle egg counts were determined at weekly intervals. At 2 weeks, mean reductions of early L3s were 50.4% and 73.8% for fenbendazole and moxidectin, respectively. At 5 weeks, the respective efficacies were 51.3% and 71.8%. Two week efficacies against late L3s and L4s (LL3s/L4s) were 70.8% and 74.6% for fenbendazole and moxidectin, respectively, whereas very low numbers were found in all three groups at 5 weeks. None of the mucosal counts were significantly different between treatment groups. Fenbendazole and moxidectin reduced luminal worm counts by 93.2% and 98.3% at 2 weeks following administration, with moxidectin group adult counts being significantly lower than the other two groups (P < 0.0001). Both treatment groups had increased counts 3 weeks later (P = 0.0415). A moxidectin ERP of 4 weeks was associated with surviving luminal L4s, and adult species contributing to this were Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicocyclus ashworthi and Cylicocyclus nassatus. This study documented (i) larvicidal efficacy of fenbendazole much lower than historical standards, (ii) survival of luminal immatures (L4) following moxidectin administration, and (iii) new information about cyathostomin species associated with these phenomena.

Efficacy of a new spot-on formulation of selamectin plus sarolaner in the treatment of Otodectes cynotis in cats.

The efficacy of a new spot-on formulation of selamectin/sarolaner was evaluated against induced Otodectes cynotis infestations in cats in two randomized, blinded studies. Fourteen and 16 cats were randomly assigned to treatment groups in Studies 1 and 2, respectively. On Day 0, animals were either treated with placebo or with the spot-on formulation at the minimal dose of 6.0mg selamectin and 1.0mg sarolaner per kg bodyweight. Treatments were administered topically at the base of the neck. Presence of live mites was evaluated 14days after treatment administration by otoscopic examination and total live mite counts (adults plus immature) were conducted on Day 30 by ear lavage. Efficacy was calculated based on the reduction of mean total live mite counts on Day 30 in the selamectin/sarolaner-treated group versus the placebo-treated group. There were no treatment-related adverse reactions during the studies, apart from one cat in each treatment group with alopecia at the administration site. In both studies combined, live mites were present on Day 14, in 14 out of 15 cats in the placebo-treated groups and in 2 out of 15 cats in the selamectin/sarolaner-treated groups. On Day 30, the arithmetic mean live mite counts were 576.9 and 875.8 in the placebo-treated groups and 5.8 and 4.7 in the selamectin/sarolaner-treated groups, in Studies 1 and 2, respectively. The live mite counts were significantly (P≤0.0021) lower in the selamectin/sarolaner-treated groups compared to the placebo-treated groups with efficacies of 99.2% and 99.3%, in Studies 1 and 2 respectively. A single administration of a new spot-on formulation of selamectin/sarolaner at the minimum dose was safe and highly efficacious in the treatment of ear mite infestations in cats.

Efficacy of oral afoxolaner plus milbemycin oxime chewables against induced gastrointestinal nematode infections in dogs.

The efficacy of oral afoxolaner plus milbemycin oxime combination chewables against induced gastrointestinal nematode infections in dogs was evaluated in six separate studies. Two studies were performed to evaluate the efficacy of the product against Toxocara canis, two studies evaluated the efficacy against Toxascaris leonina, one study evaluated the efficacy against Ancylostoma braziliense, and one study evaluated the efficacy against Ancylostoma caninum. In the A. caninum study, the efficacy of milbemycin oxime alone and afoxolaner alone was also evaluated. Dogs in all studies were inoculated with infective eggs or larvae and confirmed to have patent infections based on a fecal examination prior to allocation to study group and treatment. Each study utilized a randomized block design with blocks based on pre-treatment body weight. All dogs were assigned to blocks based on body weight, and then each dog within a block was randomly assigned to treatment group. There were two groups of 10 dogs each in the T. canis, T. leonina, and A. braziliense studies: 1) an untreated (control) group and 2) a group treated with afoxolaner plus milbemycin oxime chewables (NexGard Spectra(®), Merial). This group was treated at a dose as close as possible to the minimum effective dose of afoxolaner and milbemycin oxime (2.5mg+0.5mg per kg body weight, respectively) once on Day 0 using whole chews. There were four groups of 10 dogs each in the A. caninum study: 1) untreated (control), 2) NexGard Spectra(®) as described above, 3) milbemycin oxime alone (dose of at least 0.5mg per kg of body weight) and 4) afoxalaner alone (dose of at least 2.5mg per kg body weight). For parasite recovery and counts, dogs were euthanized humanely and necropsied seven days after treatment. The efficacy of the afoxolaner plus milbemycin oxime combination was ≥98% against T. canis, ≥95.8% against T. leonina, and 90.2% against A. braziliense. Efficacy of the combination against A. caninum was 99.7%, while the efficacy of milbemycin oxime alone was 99.6% and the efficacy of afoxolaner alone was 2.1%. Dogs treated with afoxolaner plus milbemycin oxime chewables had significantly (p≤0.0002) fewer nematodes than the untreated controls in all studies. There were no adverse events or other health problems that were related to treatment with Nexgard Spectra(®) in these studies. The results of these controlled studies demonstrate the high efficacy of the afoxolaner plus milbemycin oxime chewables against a broad range of canine intestinal nematode infections.

Changes in Serum Strongylus Vulgaris-Specific Antibody Concentrations in Response to Anthelmintic Treatment of Experimentally Infected Foals.

Strongylus vulgaris is the most pathogenic nematode parasite of horses. Its extensive migration in the mesenteric blood vessels can lead to life-threatening intestinal infarctions. Recent work has shown that this parasite is still identified among managed horse populations. A serum enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of migrating larvae of S. vulgaris. Previous work has documented an increase in ELISA values following larvicidal treatment with ivermectin and suggested that the target parasite antigen is primarily produced by the later larval stages. The aim of this study was to experimentally inoculate cohorts of foals with S. vulgaris, and then compare ELISA responses to early or later ivermectin treatments. Fifteen foals were held in confinement and infected orally with ~25 S. vulgaris third-stage larvae on Days 0, 7, 14, and 21. Foals were weaned on Day 43 and turned out to a pasture not previously grazed by horses. Foals remained at pasture continuously until the study was terminated on Day 196. On Day 55, foals were randomly allocated to three treatment groups of five each. Group 1 received ivermectin on Day 56, Group 2 received ivermectin on Day 112, and Group 3 foals served as untreated controls. Serum and fecal samples were collected at 28-day intervals throughout the study. Serum samples were analyzed with the S. vulgaris-specific ELISA and fecal samples were processed for fecal egg counting. The ELISA values of Group 1 foals were significantly lower than Groups 2 or 3 on Days 140-196. Both treated groups exhibited increased ELISA values following ivermectin treatment. Results indicate that the target diagnostic antigen is produced throughout the course of arterial infection with S. vulgaris, but that an early ivermectin treatment can reduce the cumulative antigen produced over the course of an infection.

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.

Efficacy of milbemycin oxime in combination with spinosad in the treatment of larval and immature adult stages of Ancylostoma caninum and Toxocara canis in experimentally infected dogs.

Ancylostoma caninum and Toxocara canis are two important zoonotic parasites of dogs. The primary objective of these studies were to confirm the oral effectiveness of milbemycin oxime (MO) and spinosad in dogs experimentally infected with immature (L4 and immature adult) stages of T. canis or A. caninum. Both trials were conducted as randomized, blinded, placebo-controlled dose confirmation studies. Treatments using the intended European commercial tablet formulation of Trifexis were administered in a timeframe relative to inoculation so that effectiveness could be assessed against specific immature stages of A. caninum or T. canis. In each study on Day 0, each of 32, 3-4 month old dogs were inoculated with 250 infective eggs of T. canis or 300 infective L3 of the hookworm, A. caninum. All dogs were weighed before their scheduled treatment, randomized to 1 of the 4 treatment groups in each study (8 dogs/group). All dogs were fed just prior to dosing. For T. canis, dogs were treated orally with an MO/spinosad tablet on Day 14 or Day 24. For A. caninum, dogs were treated orally with an MO/spinosad tablet on Day 7 or Day 11. Corresponding control groups in each study received a placebo tablet. Dogs were necropsied 5 or 6 days after their respective treatments. The digestive tract was removed and processed to recover, count, and identify all stages. The GM worm count for the MO/spinosad tablet on Day 14 (L4 T. canis) was 0.0, with efficacy calculated as 100%; however, only 3 of 8 control dogs had adequate infections. The GM worm count for the MO/spinosad tablet on Day 24 (immature adult stage) was 0.30; efficacy calculated at 96.15%. This is based on 5 of the 8 control dogs with adequate infections. In the two A. caninum studies, GM worm counts for the MO/spinosad tablets on Day 7 (L4 efficacy) was 2.37 and 0.8 with efficacy calculated as 98.92% and 99.25%, respectively. The GM count for the group treated with the MO/spinosad combination on Day 11 (immature adult) was 6.19 and 1.4; efficacy calculated at 97.77% and 98.58%, respectively. A minimum MO oral dose of 0.75 mg/kg was highly effective for the treatment of immature stages of T. canis and A. caninum infections in dogs. The ability to kill immature stages of these two parasites before they become patent will benefit dogs, their owners and family members due to reduced exposure to these potentially zoonotic parasites.

Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against adult and larval stages of Toxocara cati in cats.

The efficacy of a novel topical combination of fipronil 8.3% (w/v), (S)-methoprene 10% (w/v), eprinomectin 0.4% (w/v), and praziquantel 8.3% (w/v) (BROADLINE(®), Merial) was evaluated against adult and larval Toxocara cati in four controlled studies. All studies included experimentally infected, purpose-bred, short-haired cats. In two studies, 22 or 20 cats harbouring patent infections as confirmed by pre-treatment faecal examination, were included. Within each study, cats were allocated to one of two groups: control or treated. In a further two studies, 30 cats were included in each; cats were allocated to one of three groups: control, treated when T. cati were expected to be either migrating third and/or fourth-stage larvae, or treated when T. cati were expected to be fourth-stage larvae. Cats allocated to the treated groups received a single topical application of the combination product at 0.12 mL/kg bodyweight (10mg fipronil+12 mg (S)-methoprene+0.5mg eprinomectin+10mg praziquantel per kg). For parasite recovery and count, cats were euthanized humanely at different intervals after treatment. In the studies targeting adult T. cati, ascarids were recovered from all controls (range 1-150) while only two worms were isolated from one treated cat. Thus, the efficacy of the novel combination was 99.4% and 100% against adult T. cati. For studies targeting larval T. cati, up to 21 worms were recovered from each of seven or eight of the control cats per study. No T. cati were recovered from the treated cats in two studies, corresponding to 100% efficacy against both, migrating third and/or fourth-stage larvae and luminal fourth-stage larvae. All cats accepted the treatment well and no adverse experiences or other health problems were observed throughout the studies.

Illumina sequencing of the V4 hypervariable region 16S rRNA gene reveals extensive changes in bacterial communities in the cecum following carbohydrate oral infusion and development of early-stage acute laminitis in the horse.

In the equine carbohydrate overload model of acute laminitis, disease progression is associated with changes in bacteria found in the cecum. To date, research has focused on changes in specific Gram-positive bacteria in this portion of the intestinal tract. Metagenomic methods are now available making it possible to interrogate microbial communities using animal protocols that sufficiently power a study. In this study, the microbiota in cecal fluid collected from control, non-laminitic horses (n=8) and from horses with early-stage acute laminitis induced with either oligofructan (n=6) or cornstarch (n=6) were profiled. The microbiota were identified based on sequencing the V4 hypervariable region of the 16S rRNA gene. The results of the study show that the relative abundance of Lactobacillus sp. and Streptococcus sp. increased significantly (p<0.05) following OF and CS infusion. Other significant changes included an increase (p<0.05) in relative abundance of Veillonella sp. and Serratia sp., two potentially pathogenic, Gram-negative bacteria. Significant decreases in the relative abundance of presumptive normal flora were detected as well. Although changes in cecal microbiota described in this communication are from a pilot study, it is hypothesized that an overgrowth of pathogenic Gram-negative bacteria develops and contributes to enterocolitis, pyrexia and lameness in the carbohydrate overload model of acute laminitis.

SvSXP: a Strongylus vulgaris antigen with potential for prepatent diagnosis.

BACKGROUND: Strongyle parasites are ubiquitous in grazing horses. Strongylus vulgaris, the most pathogenic of the large strongyles, is known for its extensive migration in the mesenteric arterial system. The lifecycle of S. vulgaris is characterised by a long prepatent period where the migrating larvae are virtually undetectable as there currently is no test available for diagnosing prepatent S. vulgaris infection. Presence of S. vulgaris larvae in the arterial system causes endarteritis and thrombosis with a risk of non-strangulating intestinal infarctions. Emergence of anthelmintic resistance among cyathostomins has led to recommendations of reduced treatment intensity by targeting horses that exceed a predetermined strongyle faecal egg count threshold. One study suggests an apparent increase in prevalence of S. vulgaris on farms where reduced anthelmintic treatment intensity has been implemented. These issues highlight the need for an accurate and reliable assay for diagnosing prepatent S. vulgaris infection. METHODS: Immunoscreening of a larval S. vulgaris cDNA library using hyperimmune serum raised against S. vulgaris excretory/secretory antigens was performed to identify potential diagnostic antigens. Immunoreactive clones were sequenced, one potential antigen was characterised, expressed as a recombinant protein, initially evaluated by western blot (WB) analysis, the diagnostic potential of the IgG subclasses was evaluated by ELISA, and the diagnostic accuracy evaluated using serum from 102 horses with known S. vulgaris infection status. RESULTS: The clone expressing the potential antigen encoded a S. vulgaris SXP/RAL2 homologue. The recombinant protein, rSvSXP, was shown to be a potential diagnostic antigen by WB analysis, and a target of serum IgGa, IgG(T) and total IgG in naturally infected horses, with IgG(T) antibodies being the most reliable indicator of S. vulgaris infection in horses. Evaluation of diagnostic accuracy of the ELISA resulted in a sensitivity of 73.3%, a specificity of 81.0%, a diagnostic odds ratio of 11.69; a positive likelihood ratio (LR) of 3.85 and a negative LR was 0.33. The area under the ROC curve was 0.820. CONCLUSION: IgG(T) antibodies to recombinant SvSXP show potential for use as an antigen for prepatent diagnosis of migrating stages of S. vulgaris with moderate to good diagnostic accuracy.