Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Primary Aspergillus and Fusarium keratitis in a Holstein cow

A 5-year-old female Holstein cow was examined for ocular discharge, periorbital swelling, an area of full-thickness corneal cellular infiltrate, fibrin, hypopyon, diffuse corneal edema, and miosis. The patient was diagnosed with a corneal stromal abscess and secondary anterior uveitis. Histopathology, mycotic culture, and polymerase chain reaction positive for Aspergillus and Fusarium DNA confirmed the presence of fungal infection. Response to therapy was adequate, and follow-up with the patient 1 year after diagnosis revealed a focal area of corneal fibrosis.     

Comparison of various doses of carbon 13-labeled aminopyrine for a carbon 13-labeled aminopyrine demethylation blood test in healthy dogs

OBJECTIVE: To determine an optimal dose of carbon 13 ((13)C)-labeled aminopyrine for use in a (13)C-aminopyrine demethylation blood test in healthy dogs. ANIMALS: 9 adult dogs. PROCEDURES: Food was withheld from each dog for 12 hours. A 2-mL baseline blood sample was obtained from each dog and placed into an evacuated tube containing sodium heparin. Carbon 13-labeled aminopyrine was administered IV at doses of 1, 2, 5, or 10 mg/kg. Additional blood samples (2 mL) were obtained and placed into evacuated tubes containing sodium heparin 30, 45, 60, and 75 minutes after (13)C-aminopyrine administration. Hydrochloric acid was used to extract CO(2) from blood samples. The extracted gas was analyzed by fractional mass spectrometry to determine the percentage dose of (13)C administered as (13)C-aminopyrine and recovered in extracted gas (PCD). RESULTS: Gross evidence of clinical adverse effects was not detected in any dog after administration of (13)C-aminopyrine. The mean coefficient of variation (CV) for PCD was significantly lower than the mean CV for the summation of PCD values up to a given sampling time (CUMPCD). Mean PCD values among the 4 doses for each sample time were not significantly different. Administration of (13)C-aminopyrine at a dose of 2 mg/kg resulted in the lowest interindividual variability. CONCLUSIONS AND CLINICAL RELEVANCE: The PCD is superior to CUMPCD for the quantification of aminopyrine demethylation. Administration of (13)C-(13)C-aminopyrine at a dose of 2 mg/kg is appropriate for use in the (13)C-aminopyrine demethylation blood test in healthy dogs.     

Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro

OBJECTIVE: To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. SAMPLE POPULATION: Mononuclear cells from 18 horses and 3 dogs. PROCEDURES: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. RESULTS: Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPS-binding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosan-induced procoagulant activity of canine cells. CONCLUSION AND CLINICAL RELEVANCE: Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPS and zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.     

Posterior capsular opacification in diabetic and nondiabetic canine patients following cataract surgery

Posterior capsular opacification (PCO) is the most common postoperative complication of contemporary cataract surgery. Limited information is available regarding PCO formation and factors that influence PCO development in the dog. Two hundred sixty-five eyes (144 from diabetic dogs and 121 from dogs with breed-related cataracts) were prospectively evaluated for PCO formation for up to 12 months postoperatively. The mean age of all dogs in the study was 7.77 years and diabetic dogs were significantly older than dogs with breed-related cataracts. There were 73 males (61 neutered, 12 intact) and 74 females (70 neutered, 4 intact) in the study. Statistical analysis was performed based on age, breed/size, gender, stage of cataract at the time of surgery, PCO score at each time point, breed-related vs. diabetic cataract, right eyes compared to left eyes, and presence/absence of uveitis. Age and gender did not significantly influence PCO formation. Small and medium-sized breeds developed significantly more PCO in comparison to the large/giant breeds at 2 weeks and 2-4 months postoperatively, but the differences were not significant at later time points. There was an overall significant increase in PCO formation in eyes with early immature cataracts when compared to other stages of cataract up to 4 months postoperatively but not at later time points. There were no statistical differences in PCO score at 6 months or at 1 year postoperatively in eyes with breed-related and diabetic cataracts. Right eyes did not differ from left eyes in PCO score. PCO score significantly increased over time in breed-related and diabetic groups and in the overall population. No difference was found in the degree of PCO formation in eyes with inflammation prior to or after surgery compared with those without inflammation. In summary, age, gender, presence of inflammation, and cause of cataract (breed-related vs. diabetes mellitus) do not influence the development of PCO in canine cataract dogs. Small and medium-sized breeds develop significant PCO earlier than larger breeds. It is important to note that all eyes from all dogs in this study developed PCO in a time dependent manner.     

Septicaemia secondary to infection by Corynebacterium macginleyi in an Indian python (Python molurus)

A seven-year-old female Indian python (Python molurus) weighing about 35kg was euthanased after several clinical episodes of stomatitis, pneumonia, ophthalmitis and dystocia over a period of four years. The animal had been maintained in a terrarium in a circus truck at an adequate temperature. During shows, however, the snake was considered to be exposed to stressful conditions for several hours at a time at low temperatures and with noise and bright lights. A post-mortem examination indicated ulcerative stomatitis, osteomyelitis, severe pneumonia and numerous granulomata and multifocal necrosis in stomach and spleen. Corynebacterium macginleyi was isolated in pure culture from the ulcerative stomatitis, and mixed with Stenotrophomonas maltophilia from the lungs and spleen. The findings indicated that the snake had died from a septicaemic process caused by C. macginleyi, probably originating from the stomatitis. The role of S. maltophilia as a secondary agent is discussed. The stress of the circus show and poor husbandry may have predisposed the animal to infection and septicaemia. This is the first report of C. macginleyi causing disease in a snake.     

Non-invasive assessment of growth, gender and time of day related changes of respiratory pattern in healthy cats by use of barometric whole body plethysmography

The objective of this study was to establish a reference base for respiratory variables (respiratory rate [R(R)], inspiratory and expiratory time [T(i) and T(e)], peak inspiratory and expiratory pseudoflow [PIF and PEF], tidal volume [V(T)], minute ventilation [V(E)] and enhanced pause [Penh]) of healthy cats by use of barometric whole body plethysmography (BWBP). Eighteen healthy European cats (10 male, 8 female) were studied from the age of 3 to 13 months in order to assess growth- and gender-related changes of BWBP variables. Chest radiographs and bronchoalveolar lavage cytology were performed to confirm pulmonary health status. Diurnal changes were investigated every 2 h over a period of 24 h when the cats were adult. V(T), V(E), PIF and PEF significantly increased during somatic growth and were higher in males than in females, whereas R(R), T(i), T(e), T(e)/T(i) ratio, PEF/PIF ratio and Penh remained unchanged and were not affected by gender. When measured over 24 h, Penh, T(e) and T(i) were significantly increased in the early morning hours (04:00 h), whereas R(R), PIF and PEF were decreased at that time. This study provides reference values of BWBP variables for healthy male and female cats and indicates when circadian changes might be observed.     

Comparison of biopsy samples obtained using standard endoscopic instruments and the harmonic scalpel during laparoscopic and laparoscopic-assisted surgery in normal dogs

Objectives— To compare tissue specimens of canine abdominal organs collected by standard techniques (ST) and harmonic scalpel (HS) and to determine the effect of each technique on wound healing.
Study Design— Experimental.
Animals— Twelve dogs.
Methods— Paired biopsy samples (ST, HS) were collected from liver, spleen, adrenal gland, pancreas, stomach, jejunum, and bladder using laparoscopic or laparoscopic-assisted methods. Hemorrhage at biopsy sites was assessed (present or absent). Specimens were evaluated for diagnostic quality and histologic changes. Dogs were necropsied at 4 or 14 days postoperatively.
Results— HS incision caused less frequent hemorrhage of all organs except spleen. HS specimens had more coagulation necrosis than ST specimens, but both techniques yielded specimens that were sized similarly and were of diagnostic quality. At necropsy, HS biopsy sites of the stomach, jejunum, bladder, adrenal gland, and pancreas were more hyperemic than ST sites. There were more adhesions at jejunal and pancreatic ST biopsy sites. HS biopsy sites had more coagulation necrosis at days 4 and 14 postoperatively. Differences in fibrin deposition, inflammation, and fibrosis were present at biopsy sites of some organs at days 4 or 14 and in comparisons between days 4 and 14.
Conclusions— Diagnostic quality biopsy specimens were obtained with HS and ST. Although HS-induced gross and histologic changes during the first 2 postoperative weeks, no clinical complications were observed.
Clinical Relevance— Both HS and ST can yield specimens with minimal hemorrhage and HS resulted in no apparent postoperative problems in normal dogs. Although HS caused more inflammation and adhesions at biopsy sites of the pancreas, adrenal gland, and jejunum, no clinical complications occurred.