Local and systemic inflammatory and immunologic reactions to cyathostomin larvicidal therapy in horses.

Encysted cyathostomin larvae are ubiquitous in grazing horses. Arrested development occurs in this population and can lead to an accumulation of encysted larvae. Large numbers of tissue larvae place the horse at risk for developing larval cyathostominosis. This disease complex is caused by mass emergence of these larvae and is characterized by a generalized acute typhlocolitis and manifests itself as a profuse protein-losing watery diarrhea with a reported case-fatality rate of about 50%. Two anthelmintic formulations have a label claim for larvicidal therapy of these encysted stages; moxidectin and a five-day regimen of fenbendazole. There is limited knowledge about inflammatory and immunologic reactions to larvicidal therapy. This study was designed to evaluate blood acute phase reactants as well as gene expression of pro-inflammatory cytokines, both locally in the large intestinal walls and systemically. Further, mucosal tissue samples were evaluated histopathologically as well as analyzed for gene expression of pro- and anti-inflammatory cytokines, cluster of differentiation (CD) cell surface proteins, and select transcription factors. Eighteen juvenile horses with naturally acquired cyathostomin infections were randomly assigned to three treatment groups; one group served as untreated controls (Group 1), one received a five-day regimen of fenbendazole (10mg/kg) (Group 2), and one group received moxidectin (0.4mg/kg) (Group 3). Horses were treated on day 0 and euthanatized on days 18-20. Serum and whole blood samples were collected on days 0, 5, and 18. All horses underwent necropsy with collection of tissue samples from the ventral colon and cecum. Acute phase reactants measured included serum amyloid A, iron and fibrinogen, and the cytokines evaluated included interferon γ, tumor necrosis factor α, transforming growth factor (TGF)-ß, and interleukins 1ß, 4, 5, 6, and 10. Transcription factors evaluated were FoxP3, GATA3 and tBet, and CD markers included CD163, CD3z, CD4, CD40, and CD8b. Histopathology revealed an inflammatory reaction with higher levels of lymphocytes, T cells, B cells, eosinophils and fibrous tissue in the moxidectin-treated group compared to controls or horses treated with fenbendazole. No apparent systemic reactions were observed. Expression of IL-5 and TGF-ß in intestinal tissues was significantly lower in Group 3 compared to Group 1. This study revealed a subtle inflammatory reaction to moxidectin, which is unlikely to cause clinical issues.

Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis.

The link between the fermentation of carbohydrate in the equine large intestine and the development of acute laminitis is poorly understood. Absorption of endotoxin (lipopolysaccharide; LPS) into the plasma has been observed in one experimental model of laminitis, but does not cause laminitis when administered alone. Thus, the potential role of endotoxin is unclear. Platelet activation has previously been demonstrated in the developmental stage of laminitis. Equine platelets are more sensitive than leukocytes to activation by endotoxin, and can be activated directly by LPS in the low pg/ml range, activating p38 MAP kinase and releasing serotonin (5-HT) and thromboxane. The objectives of this study were firstly to determine whether endotoxin and platelet activation could be measured in the plasma of horses in the developmental phase of laminitis induced with oligofructose. Secondly, the time course of events involving platelet activation and platelet-derived vasoactive mediator production was investigated. Laminitis was induced in six Standardbred horses by the administration of 10 g/kg bwt of oligofructose. Plasma samples were obtained every 4h, and platelet pellets were obtained by centrifugation. LPS was measured using a kinetic limulus amebocyte lysate assay, and platelet activation was assessed by Western blotting for the phosphorylated form of p38 MAP kinase. Plasma 5-HT was assayed by HPLC with electrochemical detection and thromboxane B(2) was measured by radioimmunoassay. Clinical signs of laminitis and histopathologic changes were observed in lamellar sections from five of the six horses. Onset of lameness was between 20 and 30 h after the administration of oligofructose. LPS increased above the limit of detection (0.6 pg/ml) to reach a peak of 2.4+/-1.0 pg/ml at 8 h. TNFalpha was also detectable in the plasma from 12 to 24 h. There was a time-dependent increase in platelet p38 MAPK phosphorylation, which peaked at approximately 12 h (3.8+/-1.3 fold increase); plasma 5-HT and thromboxane increased steadily after this time (2.9+/-0.6 and 11.3+/-5.0 fold increases, respectively). These data indicate that small quantities of endotoxin may move into the circulation from the large intestine after the sharp decrease in pH that occurs as a result of carbohydrate fermentation. Correlating these findings with in vitro studies suggests that LPS may primarily activate platelets, leading indirectly to the activation of leukocytes. Therefore, endotoxin may contribute in the initiation of the early inflammatory changes observed in experimental models of acute laminitis.

Evaluation of the efficacy of emodepside+praziquantel topical solution against cestode (Dipylidium caninum, Taenia taeniaeformis, and Echinococcus multilocularis) infections in cats.

Emodepside+praziquantel topical solution was developed to provide broad-spectrum anthelmintic activity against gastrointestinal parasites in cats. Eight controlled studies were conducted to evaluate the efficacy of a topical solution of emodepside (3 mg/kg) and praziquantel (12 mg/kg) (Profender, BayerAG, Leverkusen, Germany) against feline infections with three species of cestodes. Studies featured naturally acquired infections of Dipylidium caninum or Taenia taeniaeformis, or experimental infections with Echinococcus multilocularis that were placebo-controlled, randomized and blinded. Cats were euthanatized and necropsied between 2 and 11 days after treatment, depending on the target parasite. The efficacy of emodepside+praziquantel topical solution was 100% against D. caninum and T. taeniaeformis, and 98.5- 100% against E. multilocularis. No significant systemic or local adverse reactions to treatment were noted in cats that received the combination. Topical treatment of cats with emodepside+praziquantel topical solution was safe and highly effective against cestode infections.

Evaluation of the efficacy of emodepside plus praziquantel topical solution against ascarid infections (Toxocara cati or Toxascaris leonina) in cats.

Eleven controlled studies were conducted in the United States and Europe to evaluate the efficacy of a topical solution of emodepside (3 mg/kg)+praziquantel (12 mg/kg) (Profender, Bayer AG, Leverkusen, Germany) against infection with various stages of the ascarid nematodes Toxocara cati and Toxascaris leonina. Infections were induced by administration of larvated ascarid eggs, and stage-specific efficacy was evaluated by treating cats at scheduled intervals post-inoculation. All studies featured random allocation to treatment groups, placebo-treated control animals and assessment of outcome measures by masked personnel. The product (emodepside+praziquantel topical solution) was 100% effective against mature adults and immature adult T. cati. In addition, it was 96.8% effective against third stage larvae and at least 99.4% effective against fourth stage larvae of T. cati, respectively. Efficacy against mature, immature adult and L4 stages of T. leonina exceeded 93.4%, but regulatory "adequacy of infection" criteria were not met in some studies. No adverse reactions to treatment were noted in cats treated with the emodepside+praziquantel topical solution.

Comparison of endoscopic, necropsy and histology scoring of equine gastric ulcers.

Equine gastric ulcer syndrome (EGUS) represents a major health problem in performance horses. Much debate exists regarding endoscopic gastric ulcer scoring systems and their ability accurately to predict severity or depth of gastric ulcers. The purpose of this study was to evaluate the ability of an endoscopist to count gastric ulcers and predict gastric ulcer severity or depth using 2 endoscopic scoring systems and compare them to the same gastric ulcers see on necropsy and histopathology. Endoscopic examination of the stomach was performed under general anaesthesia on 23 mixed breed yearling horses, after feed was withheld for 24 h. Gastric ulcers were scored using 2 systems, number/severity-scoring (N/S) and practitioner simplified (PS) systems. After endoscopy, the horses were subjected to euthanasia and the stomach mucosa examined blindly and scored again at necropsy using above scoring systems. Representative gastric ulcers were then placed in 10% formalin and processed routinely for histopathology. The gastric ulcers were scored using a histopathology system (HSS) based on ulcer depth. Number scores in the N/S scoring system and PS on endoscopic and necropsy examinations were compared using Friedman 2 way analysis of variance. Where significant differences between variables were found a post hoc analysis was conducted using a Tukey's Studentised range (HSD) test. Severity scores using the N/S (ENGS) and PS scores recorded for the stomach via endoscopy and scores from HSS were evaluated for significant association using a Mantel-Haenszel Chi-square and Pearson moment correlation coefficient analysis. Significance was P < 0.05. All horses had gastric ulcers in the nonglandular mucosa via endoscopic examination and at necropsy examination. Mean nonglandular ulcer number (ENGN) score was significantly (P = 0.0024) lower on endoscopic examination compared to the score at necropsy (NNGN); whereas PS scores were not significantly different on endoscopy when compared to necropsy examination. A significant but weak association was found between ENGS and HSS (3.89, P = 0.048; r = 0.453, P = 0.045) and no correlation was found between PS and HSS (1.2, P = 0.272; r = 0.117; P = 0.622). Only 1/23 horses had glandular ulcers observed via endoscopic examination whereas, 6/23 horses had glandular ulcers at necropsy and on histopathology. The prevalence of EGUS is high in stalled yearling horses. The endoscopist may underestimate the number of gastric ulcers and may not be able accurately to predict the severity or depth of those ulcers present in the nonglandular equine stomach. Furthermore, the endoscopist may miss glandular gastric ulcers.

Efficacy of moxidectin equine oral gel against endoscopically-confirmed Gasterophilus nasalis and Gasterophilus intestinalis (Diptera: Oestridae) infections in horses.

A 3 m, video gastroscope was used to screen 47 horses suspected of being naturally infected with equine bot larvae. 17 of 47 (36.2%) candidate horses harbored Gasterophilus nasalis larvae in the proximal duodenum and 46 of 47 (97.9%) had G. intestinalis larvae in the stomach. All horses infected with G. nasalis had concurrent infections with G. intestinalis. 14 horses with dual infections were allocated randomly to two treatment groups. Seven horses in Group 1 received 2% moxidectin oral gel once at a dosage of 0.4 mg/kg bodyweight (BW), and seven horses in Group 2 were untreated controls. 14 days after treatment, all horses were necropsied and the stomach and proximal duodenum harvested from each. Bot larvae were recovered, identified to species and instar, and counted. At the label dosage, moxidectin oral gel was 100 and 97.6% effective (P < 0.05) against third-instar G. nasalis and G. intestinalis, respectively. In addition to demonstrating the boticidal efficacy of moxidectin, this trial illustrated that gastroscopy/duodenoscopy is a feasible method for confirming infections with different species of bot larvae in the horse.

Small strongyles. Recent advances.

The recent increased interest in cyathostomes can be traced to simplification of their taxonomy, improved knowledge of pathogenicity, and failures of practical control due to anthelmintic resistance. Cyathostome ova develop to infective third-stage larvae (L3) at a rate that is directly proportional to environmental temperature. Equine feces serve as a reservoir for L3, which are liberated by moderate amounts of rainfall. Third-stage larvae persist for longer periods at low temperatures, easily surviving over-winter on pastures to provide a source of infection during the following grazing season. Third-stage larvae exsheath within the host and enter the mucosa and submucosa of the cecum and large colon. Larvae develop within mucosal cysts, molt to the fourth stage, and may persist within the tissues for up to 2 1/2 years. Larvae ultimately emerge from the mucosa to become adults in the lumen. Adult populations are replenished by recently ingested larvae and by immature worms newly emerged from arrested development. The magnitude of larval and adult populations within the host displays seasonal variations, with peak numbers occurring in early spring and autumn in the United States. In typical natural infections, a small number of species comprise the majority of the cyathostome populations. Cyathostome infection may result in anorexia, weight loss, diarrhea, colic, and death. Cyathostome ova are easily detected in feces, but ova may not be present during larval cyathostomiasis. Increased concentrations of beta-globulins, hypoalbuminemia, anemia, and leukocytosis occur inconsistently. Two major problems in the treatment of cyathostome infections are anthelmintic resistance and the insusceptibility of encysted larvae to recommended dosages of most anthelmintics. The major goal of cyathostome control is prevention of environmental contamination with nematode ova. Host resistance appears to protect against cyathostome disease rather than cyathostome infection, and one manifestation of this resistance appears to be prolongation of the prepatent period.

Anatomic distribution of encysted cyathostome larvae in the horse.

The large intestines of 6 horses were divided by length into 12 segments, and each segment was washed and weighed. At least 5% by weight of each segment was examined by mural transillumination, and encysted cyathostome larvae were counted. Total numbers of larvae in each segment were calculated. Encysted larvae (98%) were present in the proximal 7 segments of the large intestine (cecum and proximal 75% of the ventral colon), and 2% were present in the distal 25% of the ventral colon and entire dorsal colon. Encysted larvae (6%) were located in the dorsal colon of 1 heavily infected horse. Larval density was greatest in the cecum, which harbored 57% of encysted worms yet contributed only 27% to the total weight of the large intestine. Larvae dissected from tissue cysts consisted of species that were highly prevalent as adult worms. However, there were few Cylicostephanus longibursatus, probably because of the small size and fragility of its 4th-stage larvae.

Comparison of two techniques for quantitation of encysted cyathostome larvae in the horse.

Haustral portions of intestine of 6 horses were isolated by excising the taeniae coli from the cecum and the ventral colon. Uniform 5-cm X 5-cm sections were cut from the haustra and were illuminated from the serosal side with a strong light source (mural transillumination). Cyathostome larvae encysted in the mucosa and submucosa were observed at 15 X magnification and counted. Two separate counts of the larvae in 80 replicates of tissue by the mural transillumination technique (MTT) revealed no significant (P less than 0.05) difference between sample means. Larvae in tissue sections were counted in situ by MTT, and the mucosal scrapings of the tissue sections were digested in pepsin and HCl to determine larval yields for comparison with the MTT counts. Numbers of larvae recovered by pepsin and HCl digestion for 3 and 6 hours were significantly (P less than 0.01) lower than were numbers originally determined by MTT. Larvae recovered by tissue digestion for 3 or 6 hours were examined individually and given objective scores for morphologic damage. Distribution of scores was time-dependent; increased damage to larvae was associated with a longer time of digestion. Individual 4th-stage cyathostome larvae were dissected from cysts in the large intestinal wall and were incubated in water, 0.9% saline solution, 1.1% HCl, or pepsin (7,000 U of activity/ml). Significantly fewer (P less than 0.05) larvae were recovered from all solutions after 3 and 6 hours. The proportion of dissected larvae that were given high scores after exposure to pepsin was significantly (P less than 0.01) greater than were those held in HCl, saline solution, or water for both periods.