Objective-To investigate the effects of exercise on activation of mitogen-activated protein kinase (MAPK) signaling proteins in horses. Animals-6 young trained Standardbred geldings. Procedure-Horses performed a 20-minute bout of exercise on a treadmill at 80% of maximal heart rate. Muscle biopsy specimens were obtained from the vastus lateralis and pectoralis descendens muscles before and after exercise. Amount of expression and intracellular location of phosphospecific MAPK pathway intermediates were determined by use of western blotting and immunofluorescence staining. Results-Exercise resulted in a significant increase in phosphorylation of p38 pathway intermediates, c-Jun NH2 terminal kinase (JNK), and heat shock protein 27 (HSP27) in the vastus lateralis muscle, whereas no significant changes were found in phosphorylation of extracellular regulated kinase. In the pectoralis descendens muscle, phosphorylation of p38 and HSP27 was significantly increased after exercise. Immunohistochemical analysis revealed fiber-type-specific locations of phosphorylated JNK in type 2a/b intermediate and 2b fibers and phosphorylated p38 in type 1 fibers. Phosphorylated HSP27 was strongly increased after exercise in type 1 and 2a fibers. Conclusions and Clinical Relevance-The p38 pathway and JNK are activated in the vastus lateralis muscle after a single 20-minute bout of submaximal exercise in trained horses. Phosphorylation of HSP27 as detected in the study reported here is most likely induced through the p38 signaling pathway.

Objective-To determine whether inflammation of the jejunum of horses decreases the number of motilin receptors and amounts of motilin receptor mRNA and alters erythromycin lactobionate binding affinity to the motilin receptor in jejunal tissues. Sample Population-Jejunal segments in 6 adult horses. Procedure-Each horse was anesthetized, and a ventral median celiotomy was performed; 2 segments of jejunum underwent a sham operation, 2 segments underwent ischemic strangulation obstruction (ISO), and 2 segments underwent intraluminal distension (ILD). Treatments were maintained for 120 minutes. From each segment, full-thickness biopsy samples were collected and smooth-muscle homogenates were prepared. Affinity and distribution of motilin binding to these preparations were determined by use of iodine 125 (125I)-labeled synthetic porcine motilin. Via displacement experiments, competition between 125I-labeled motilin and erythromycin lactobionate for binding to motilin receptors in the different segments was investigated. A quantitative real-time PCR technique was used to assess motilin receptor mRNA content in the muscle preparations. Results-Compared with the ISO or ILD segments, the number of motilin receptors was significantly higher in the sham-operated segments; ILD segments contained the lowest number of motilin receptors. The expression of motilin receptor mRNA was significantly decreased in ILD segments but not in ISO segments. Erythromycin lactobionate displacement of 125I-labeled motilin from motilin receptors did not differ significantly among the jejunal segments. Conclusions and Clinical Relevance-Results suggest that downregulation and decreased production of motilin receptors in inflamed jejunal tissue contribute to the altered prokinetic response to erythromycin in horses with gastrointestinal disease.

Objective-To test horses for serologic evidence of an association between vesiviral antibodies and abortion. Sample Population-Sera from 141 horses. Procedures-2 experiments were conducted. Experiment 1 comprised sera obtained in 2001 and 2002 from 3 groups of horses (58 mares from farms with a history of abortion problems, 25 mares between 3 and 13 years of age with unknown reproductive histories that were sold at auction breeding-age control mares, and 29 mixed-age males and yearling females sold at auction negative control population). Experiment 2 comprised sera from 3 groups of pregnant mares (10 pregnant mares fed Eastern tent caterpillars ETCs, 9 pregnant mares fed ETC frass only, and 10 pregnant control mares). Sera were analyzed for antibodies against vesivirus by use of a validated recombinant vesivirus-specific peptide antigen in an indirect ELISA. Results-For experiment 1, 37 of 58 (63.8%) mares from farms with abortion problems were seropositive for vesivirus antibodies, whereas 10 of 25 (40%) breeding-age control mares were seropositive. All 29 mixed-age males and yearling females were seronegative for vesivirus antibodies. For experiment 2, 17 of 29 mares aborted (some from each group). Seropositive status for vesivirus antibodies increased from 47.1% (8/17) to 88.2% (15/17) for the pregnant mares that aborted during the experiment. Conclusion and Clinical Relevance-Significant association was detected between seropositive status for vesivirus and abortion in mares; consequently, vesivirus appears to be a pathogenic virus associated with abortion in mares. These data support adding vesivirus antibody testing into diagnostic screening to determine the cause for abortion in mares.

Objective-To evaluate the effect of dietary supplementation with the probiotic strain Lactobacillus acidophilus DSM13241 in healthy adult cats. Animals-15 adult cats. Procedures-Cats were fed a nutritionally complete dry food for 5 weeks. Fecal character was assessed daily, and a single fecal sample and 3-mL blood sample were collected for bacterial enumeration and hematologic analysis, respectively. Cats were then fed the same diet supplemented with L acidophilus DSM13241 (2 X 10(8) CFU/d) for 4.5 weeks. Repeat fecal and hematologic measurements were taken prior to the return to control diet for a 4-week period. Results-The probiotic species was recovered from feces, demonstrating survival through the feline gastrointestinal tract. Probiotic supplementation was associated with increased numbers of beneficial Lactobacillus and L acidophilus groups in feces and decreased numbers of Clostridium spp and Enterococcus faecalis, indicating an altered bacterial balance in the gastrointestinal tract microflora. Fecal pH was also decreased suggesting a colonic environment selective for the beneficial lactic acid bacterial population. Systemic and immunomodulatory effects were associated with administration of L acidophilus DSM13241 including altered cell numbers within WBC subsets and enhanced phagocytic capacity in the peripheral granulocyte population. In addition, plasma endotoxin concentrations were decreased during probiotic feeding, and RBCs had a decreased susceptibility to osmotic pressure. Conclusions and Clinical Relevance-Probiotic strain L acidophilus DSM13241 fed at 2 X 10(8) CFU/d can alter the balance of gastrointestinal microflora in healthy cats. Furthermore, administration of this probiotic results in beneficial systemic and immunomodulatory effects in cats.

Objective-To determine the maximum tolerated dose and characterize the pharmacokinetic disposition of an orally administered combination of docetaxel and cyclosporin A (CSA) in dogs with tumors. Animals-16 client-owned dogs with metastatic or advanced-stage refractory tumors. Procedures-An open-label, dose-escalation, single-dose, phase I study of docetaxel administered in combination with a fixed dose of CSA was conducted. Docetaxel (at doses of 1.5, 1.625, or 1.75 mg/kg) and CSA (5 mg/kg) were administered concurrently via gavage twice during a 3-week period. Plasma docetaxel concentrations were quantified by use of high-performance liquid chromatography, and pharmacokinetic disposition was characterized by use of noncompartmental analysis. Dogs' clinical signs and results of hematologic and biochemical analyses were monitored for evidence of toxicosis. Results-No acute hypersensitivity reactions were observed after oral administration of docetaxel. Disposition of docetaxel was dose independent over the range evaluated, and pharmacokinetic variables were similar to those reported in previous studies involving healthy dogs, with the exception that values for clearance were significantly higher in the dogs reported here. The maximum tolerated dose of docetaxel was 1.625 mg/kg. Gastrointestinal signs of toxicosis were dose limiting. Conclusions and Clinical Relevance-The absence of myelosuppression suggested that the docetaxel-CSA combination may be administered more frequently than the schedule used. Further studies are warranted to evaluate combination treatment administered on a biweekly schedule in dogs with epithelial tumors.

Objective-To study the pharmacokinetics of difloxacin (5 mg/kg) following IV, IM, and intragastric (IG) administration to healthy horses. Animals-6 healthy mature horses. Procedures-A crossover study design with 3 phases was used (15-day washout periods between treatments). An injectable formulation of difloxacin (5%) was administered IV and IM in single doses (5 mg/kg); for IG administration, an oral solution was prepared and administered via nasogastric tube. Blood samples were collected before and at intervals after each administration. A high-performance liquid chromatography assay with fluorescence detection was used to determine plasma difloxacin concentrations. Pharmacokinetic parameters of difloxacin were analyzed. Plasma creatine kinase activity was monitored to assess tissue damage. Results-Difloxacin plasma concentration versus time data after IV administration were best described by a 2-compartment open model. The disposition of difloxacin following IM or IG administration was best described by a 1-compartment model. Mean half-life for difloxacin administered IV, IM, and IG was 2.66, 5.72, and 10.75 hours, respectively. Clearance after IV administration was 0.28 L/kg.h. After IM administration, the absolute mean +/- SD bioavailability was 95.81 +/- 3.11% and maximum plasma concentration (C(max)) was 1.48 +/- 0.12 mg/L. After IG administration, the absolute bioavailability was 68.62 +/- 10.60% and C(max) was 0.732 +/- 0.05 mg/L. At 12 hours after IM administration, plasma creatine kinase activity had increased 7-fold, compared with the preinjection value. Conclusions and Clinical Relevance-Data suggest that difloxacin is likely to be effective for treating susceptible bacterial infections in horses.

The early interaction of Lawsonia intracellularis with host cells was examined with the use of porcine ileum models. Two conventional swine were anesthetized, and ligated ileum loops were prepared during abdominal surgery. The loops were inoculated with 10⁸ L. intracellularis or saline. After 60 min, samples of each loop were processed for routine histologic and electron microscopic study. Histologic and ultrathin sections of all the loops appeared normal, with no apposition of bacteria and host cells or bacterial entry events in any loop. Portions of ileum from a single gnotobiotic piglet were introduced as xenografts into the subcutis of each flank of 5 weaned mice with severe combined immunodeficiency disease. After 4 wk, 10⁸ L. intracellularis were inoculated into each of 4 viable xenografts with a sterile needle; the other 3 viable xenografts received saline. Histologic and ultrathin sections of all the xenografts 3 wk after inoculation showed relatively normal porcine intestinal architecture, with normal crypts, crypt cell differentiation, and low villous structures; the xenografts treated with the bacteria also showed intracytoplasmic L. intracellularis within crypt and villous epithelial cells. Thus, entry of L. intracellularis into target epithelial cells and multiplication may not be sufficient alone to directly cause cell proliferation. A proliferative response may require active division of crypt cells and differentiation in conjunction with L. intracellularis growth.

Objective-To determine whether a novel third-generation chelating agent (8mM disodium EDTA dehydrate and 20mM 2-amino-2-hydroxymethyl-1, 3-propanediol) would act as an antimicrobial potentiator to enhance in vitro activity of antifungal medications against fungal isolates obtained from horses with mycotic keratitis. Sample Population-Fungal isolates (3 Aspergillus isolates, 5 Fusarium isolates, 1 Penicillium isolate, 1 Cladosporium isolate, and 1 Curvularia isolate) obtained from horses with mycotic keratitis and 2 quality-control strains obtained from the American Type Culture Collection (ATCC; Candida albicans ATCC 90028 and Paecilomyces variotii ATCC 36257). Procedure-Minimum inhibitory concentrations (MICs) against fungal isolates for 4 antifungal drugs (miconazole, ketoconazole, itraconazole, and natamycin) were compared with MICs against fungal isolates for the combinations of each of the 4 antifungal drugs and the chelating agent. The Clinical and Laboratory Standards Institute microdilution assay method was performed by use of reference-grade antifungal powders against the fungal isolates and quality-control strains of fungi. Results-Values for the MIC at which the antifungal drugs decreased the growth of an organism by 50% (MIC50) and 90% (MIC90) were decreased for the control strains and ophthalmic fungal isolates by 50% to 100% when the drugs were used in combination with the chelating agent at a concentration of up to 540 microgram/mL. Conclusions and Clinical Relevance-The chelating agent increased in vitro activity of antifungal drugs against common fungal pathogens isolated from eyes of horses with mycotic keratitis.

Objective-To compare 4 assay procedures for prediction of passive transfer status in lambs. Animals-Thirty-one 1-day-old Sardinian lambs. Procedure-Serum IgG concentration was determined by use of single radial immunodiffusion. The following were determined: serum total protein concentration as measured by refractometry (ie, refractometry serum total protein concentration), serum total protein concentration as determined by the biuret method (ie, biuret method serum total protein concentration), serum gamma-globulin concentration as determined by serum protein electrophoresis, and serum gamma-glutamyltransferase (GGT) activity as measured by spectrophotometry. Accuracy of these assays for estimation of serum IgG concentration in 1-day-old lambs was established by use of linear regression analysis. Results-Refractometry serum total protein concentration, biuret method serum total protein concentration, and serum gamma-globulin concentration were closely and linearly correlated with serum IgG concentration. The natural logarithm (ln) of serum GGT activity was closely and linearly correlated with serum IgG concentration (ln). Refractometry serum total protein concentration, biuret method serum total protein concentration, and gamma-globulin concentration accounted for approximately 85%, 91%, and 95% of the variation in serum IgG concentration, respectively. Serum GGT activity (ln) accounted for approximately 92% of the variation in serum IgG concentration (ln). Conclusions and Clinical Relevance-For prediction of passive transfer status in 1-day-old lambs, serum GGT activity or biuret method serum total protein concentration determination will allow for passive transfer monitoring program development. Immediate refractometry serum total protein concentration determination is beneficial in making timely management and treatment decisions. Serum gamma-globulin concentration determination can be used as a confirmatory test.

Objective-To determine gene expression of selected molecular markers (tumor necrosis factor TNF-alpha, interleukin IL-1beta, IL-6, IL-8, IL-10, procalcitonin PCT, and transforming growth factor TGF-beta) in the blood of healthy and sick foals. Animals-28 sick foals without sepsis, 21 foals with sepsis, and 21 healthy foals. Procedures-Total RNA was extracted from blood samples and converted into complementary DNA (cDNA). Gene expression was measured for the molecular markers by use of real-time PCR assay, and final quantitation was performed with the comparative threshold cycle method. Results-Samples from all foals yielded transcription for all markers. Expression of TNF-alpha and TGF-beta was significantly lower and that of IL-8 significantly greater in the sick-nonseptic and septic groups, compared with the healthy group. No significant difference in expression of IL-1beta, IL-6, and PCT was found between the healthy group and the 2 sick groups. Expression of IL-10 was significantly greater in nonsurvivors, compared with survivors. Conclusions and Clinical Relevance-The cytokine profile in foals with sepsis may suggest an immunosuppressive state. Expression of IL-10 may be a marker for identification of foals with a guarded prognosis.