In vitro cytopathic effects of a cysteine protease of Tritrichomonas foetus on cultured bovine uterine epithelial cells

OBJECTIVE: To evaluate the cytopathic effects of Tritrichomonas foetus and a purified cysteine protease (ie, CP30) of T foetus on cultured bovine uterine epithelial cells (BUECs) in vitro. SAMPLE POPULATION: 10 reproductive tracts were obtained from late-term bovine fetuses at a commercial abattoir. PROCEDURE: An in vitro culture system of BUECs was developed to study the cytopathic effects of T foetus and purified CP30 of T foetus on host cells. Cytotoxicity of T foetus or CP30 on exposed BUECs was determined. Fluorescence microscopy and flow cytometry analyses were used to detect apoptosis. A fluorometric assay was used to detect BUEC caspase 3 activation. The CP inhibitor E-64 and a caspase inhibitor were used to inhibit apoptosis. RESULTS: Cytopathic effects were observed in BUECs treated with parasites or CP30 and were concentration and time dependent. The BUECs underwent apoptosis in the presence of parasites or CP30. The specific CP inhibitor E-64 abolished the induction of apoptosis in BUECs by CP30. The caspase inhibitor reduced the amount of apoptosis in BUECs. CONCLUSIONS AND CLINICAL RELEVANCE: T foetus and its CP30 induce apoptosis in cultured BUECs in vitro. Induction of apoptosis by CP30 is correlated with protease activity. Endometrial cell death as a result of a T foetus infection is likely to be more important in mediating infertility than a direct effect on the conceptus. Provoking an apoptotic reaction in the host may mitigate an inflammatory reaction or immune response and therefore favor survival of the parasite in a chronic infection.     

In vitro effects and in vivo efficacy of a novel cyclooxygenase-2 inhibitor in cats with lipopolysaccharide-induced pyrexia

OBJECTIVE: To determine cyclooxygenase (COX)-2 selectivity, pharmacokinetic properties, and in vivo efficacy of firocoxib (ML-1,785,713) in cats. ANIMALS: 5 healthy male and 14 healthy female domestic shorthair cats. PROCEDURE: Selectivity of firocoxib for inhibiting COX-2 was determined by comparing the potency for inhibiting COX-1 with that of COX-2 in feline blood. Pharmacokinetic properties were determined after i.v. (2 mg/kg) and oral (3 mg/kg) administration in male cats. In vivo efficacy was evaluated in female cats with lipopolysaccharide (LPS)-induced pyrexia with administration of firocoxib 1 or 14 hours before LPS challenge. RESULTS: Blood concentrations resulting in 50% inhibition of COX-1 and COX-2 activity in vitro were 75 +/- 2 microM and 0.13 +/- 0.03 microM, respectively, and selectivity for inhibiting COX-2 relative to COX-1 was 58. Firocoxib had moderate to high oral bioavailability (54% to 70%), low plasma clearance (4.7 to 5.8 mL/min/kg), and an elimination half-life of 8.7 to 12.2 hours. Firocoxib at doses from 0.75 to 3 mg/kg was efficacious in attenuating fever when administered to cats 1 or 14 hours before LPS challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Firocoxib is a potent COX-2 inhibitor and is the only selective COX-2 inhibitor described for use in cats to date. It is effective in attenuating febrile responses in cats when administered 14 hours before LPS challenge, suggesting it would be suitable for once-a-day dosing. Because selective COX-2 inhibitors have an improved therapeutic index relative to nonselective nonsteroidal anti-inflammatory drugs in humans, firocoxib has the potential to be a safe, effective anti-inflammatory agent for cats.     

Distribution of power across the hind limb joints in Labrador Retrievers and Greyhounds

OBJECTIVE: To quantify angular excursions; net joint moments; and powers across the stifle, tarsal, and metatarsophalangeal (MTP) joints in Labrador Retrievers and Greyhounds and investigate differences in joint mechanics between these 2 breeds of dogs. ANIMALS: 12 clinically normal dogs (6 Greyhounds and 6 Labrador Retrievers) with no history of hind limb lameness. PROCEDURE: Small retroreflective markers were applied to the skin over the pelvic limb joints, and a 4-camera kinematic system captured data at 200 Hz in tandem with force platform data while the dogs trotted on a runway. Breed-specific morphometric data were combined with kinematic and force data in an inverse-dynamics solution for stance-phase net joint moments and powers at the stifle, tarsal, and MTP joints. RESULTS: There were gross differences in kinematic patterns between Greyhounds and Labradors. At the stifle and tarsal joints, moment and power patterns were similar in shape, but amplitudes were larger for the Greyhounds. The MTP joint was a net absorber of energy, and this was greater in the Greyhounds. Greyhounds had a positive phase across the stifle, tarsal, and MTP joints at the end of stance for an active push-off, whereas for the Labrador Retrievers, the only positive phase was across the tarsus, and this was small, compared with values for the Greyhounds. CONCLUSIONS AND CLINICAL RELEVANCE: Gross differences in pelvic limb mechanics are evident between Greyhounds and Labrador Retrievers. Joint kinetics in specific dogs should be compared against breed-specific patterns.     

Myocardial concentrations of fatty acids in dogs with dilated cardiomyopathy

OBJECTIVE: To compare myocardial concentrations of fatty acids in dogs with dilated cardiomyopathy (DCM) with concentrations in control dogs. SAMPLE POPULATION: Myocardial tissues from 7 dogs with DCM and 16 control dogs. PROCEDURE: Myocardial tissues were homogenized, and total fatty acids were extracted and converted to methyl esters. Myocardial concentrations of fatty acids were analyzed by use of gas chromatography and reported as corrected percentages. RESULTS: The amount of docosatetraenoic acid (C22:4 n-6) was significantly higher in myocardial samples from dogs with DCM (range, 0.223% to 0.774%; median, 0.451%), compared with the amount in samples obtained from control dogs (range, 0.166% to 0.621%; median, 0.280%). There were no significant differences between DCM and control dogs for concentrations of any other myocardial fatty acids. CONCLUSIONS AND CLINICAL RELEVANCE: Although concentrations of most myocardial fatty acids did not differ significantly between dogs with DCM and control dogs, the concentration of docosatetraenoic acid was significantly higher in dogs with DCM. Additional investigation in a larger population is warranted to determine whether this is a primary or secondary effect of the underlying disease and whether alterations in fatty acids may be a target for intervention in dogs with DCM.     

Contractile activity and motility responses of the dog heartworm Dirofilaria immitis to classical anthelmintics and other compounds

A variety of compounds including classical anthelmintics and avermectin analogs were screened for their effects on movements of adult heartworms (HW) (Dirofilaria immitis). Contractile activity was measured by tension recording of spontaneous movements of intact HW coil preparations (6 min compound exposure) and motility was evaluated by observation of spontaneous, free movements in culture (3 and 7 days compound exposure). Results for female HW indicated that some compounds caused spastic paralysis of contractile activity and inhibition of motility in culture (bephenium, DL-tetramisole, and pyrantel); some caused only spastic paralysis of contractile activity (methyridine and disophenol); and some caused only inhibition of motility in culture (chlorpromazine, dithiazanine, 1-ethoxycarbonylmethyl-1-methylpyrrolidinium, and 4-methyltropolone). Effects on motility in culture appeared to be lethal. The following compounds lacked effects: amprolium, 2-amino-2-thiazoline, bithionol, bitoscanate, bitriben, hexachlorophene, ivermectin, and 10 H-phenothiazine. A group of avermectin analogs was screened for effects only on motility in culture of both adult female and male HW. Several of the analogs affected motility, but the effects appeared to be non-lethal. Microfilaria release into the culture media was suppressed by two of the analogs (an aglycone and avermectin B2). The HW maintenance system used in the present study facilitated screening of compounds for effects on this parasite.     

Failure to protect goats following vaccination with soluble proteins of Sarcoptes scabiei: evidence for a role for IgE antibody in protection

Developing an anti-scabies vaccine is thought to be a feasible alternative to chemical control, since animals which have recovered from sarcoptic mange become resistant against mite reinfestation. The purpose of this study was to evaluate the protective value of immune responses developed in animals after immunisation with soluble mite proteins. Soluble proteins from Sarcoptes scabiei were extracted then subjected to ion exchange chromatography, and proteins from the column were eluted step-wise with 0%, 10%, 25% and 50% of 1 M solution of NaCl in a Tris buffer. Each protein fraction was concentrated and dialysed against PBS. To evaluate the immunogenicity of the fractions, 36 goats were allocated into six groups, group1 goats were unvaccinated, group 2 were vaccinated with intact soluble mite proteins, and groups 3-6 were vaccinated respectively with the fractionated proteins. Vaccinations were conducted four times with 1 mg protein/dose and 4-week intervals between vaccinations. One week after the last vaccination, all goats were challenged with approximately 2000 live mites on the auricles and infestations were allowed to progress for 6 weeks. The severity of lesions caused by the infestation was assessed throughout the study. The challenge caused mange or encrustation dermatitis in all animals and no differences in severity of lesions were observed between vaccinated and unvaccinated control goats. Vaccination with each fraction of the mite proteins invoked high levels of scabies-specific IgG in the serum of all animals but failed to induce specific IgE as determined by Elisa. In contrast, goats challenged experimentally with a primary or repeated mite challenge developed strong serum IgE and IgG antibody responses to Sarcoptes antigens. The latter animals were shown in a previous study to be resistant to reinfestation. The lack of immune protection in the vaccinated animals may be attributed to the absence of protective levels of IgE antibody, and the present findings indicate that allergens and IgE antibody is important in immunity to S. scabiei infection.     

A case study of capture-recapture methodology using scrapie surveillance data in Great Britain

We applied capture–recapture methodology (CRC) to data from three surveillance sources (statutory notification, abattoir survey (AS) and fallen stock (FS) survey) to estimate the number of holdings infected with scrapie in Great Britain and to assess the sensitivity of the surveillance network. Between January 1, 2002 and March 31, 2003, 144 holdings were identified by the three sources. Using CRC modelling techniques, we estimated a minimum lower bound for the total number of holdings infected as 642. A biologically plausible positive dependence between the statutory reporting and the fallen stock survey was found statistically significant. The sensitivity of the three sources combined was very low. The integration of the three overlapping sources provided a better understanding of the interactions within the surveillance network. However, the scarcity of the data and reduced overlapping among sources only allowed for very cautious inferences to be drawn about the true proportion of scrapie affected holdings in the national population. Future surveys and surveillance activities should be planned such that the resulting data can be used more effectively as part of CRC modelling approaches.