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.     

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.     

Association between heterophil/lymphocyte ratio, a marker of 'resistance' to stress, and some production and fitness traits in chickens

1. A stock of cross-bred CD hens was divided into 'resistant' (R) and 'sensitive' (S) to stress on the basis of the heterophil/lymphocyte (H/L) ratio using the lower 99% confidence limit method for selection. A first generation was produced from these R and S females mated with similarly selected R and S local Barred males. The association between 'resistance' and some production traits was investigated. 2. The proportions of R:S among CD females and Barred males was 51.2:48.8 and 52:48, respectively. Mean H/L ratios for Barred males and females, respectively, were 0.87 +/- 0.03 and 0.83 +/- 0.03 (P > 0.05). 3. Compared with the S group in CD females, the R group had greater egg production (59% vs 51%), egg weight (65.5 g vs 62.6 g) and body weight at 39 weeks (115 g heavier). 4. The superiority of R in terms of proportion of fertile eggs, hatchability and the proportion hatched from those set amounted to 4.8, 6.1 and 5.4%, respectively. 5. The proportion of dead embryos in the R group (5.4%) was lower than in the S group (9.3%). 6. With the F1 generation the R chicks were heavier at hatching. 7. Percentage mortality of male F1 chicks, from hatching to 4 weeks and from 4 to 8 weeks, was lower in the R group. A similar, but non-significant, trend was noted for R females. 8. The progeny of the R group was heavier than the S progeny at 4 and 8 weeks and also the percentage dressed weight of males was greater at 8 weeks. The R group was heavier than the S group at 4, 8 and 12 weeks. 9. These results suggest that selection for general 'resistance', on the basis of the H/L ratio using the 99% lower confidence limit method of selection, is positively correlated with several important production and reproduction traits. Establishing resistant lines with high production rates seems feasible.     

Primary erythrocytosis in a Japanese black calf: a case report

An 8-month-old Japanese Black heifer with severe erythropoietic symptoms was subjected to clinical, histological and cytological examinations. During the 1 month clinical observation period, severe increases in RBC count, packed cell volume and haemoglobin concentration were observed. The plasma erythropoietin (Epo) concentration of the heifer (20.7 mIU/ml) was similar to that observed in normal control heifers. Blood gas examinations of the arterial and venous blood revealed low levels of partial pressure O(2) (PaO(2)), partial pressure CO(2) (PaCO(2)) and O(2) saturation (SaO(2)), while the blood pH was within the normal range. Gross lesions could not be detected. However, microscopic observation revealed severe proliferation of erythroblasts in the bone marrow and in the spleen without evidence of neoplastic changes. Based on these clinical and pathological examinations, we diagnosed the heifer as being the first case of primary erythrocytosis in Japanese Black cattle.     

Severe weight loss in lambs infected with Giardia duodenalis assemblage B

An outbreak of giardiasis was observed in a sheep farm in Central Italy. Infected lambs (30-90 days of age) showed a malabsorption syndrome, decreased weight gain and impairment in feed efficiency. The most relevant clinical sign was the excretion of malodorous and poorly formed faeces, whereas diarrhoea was rarely observed in the flock. Laboratory investigations revealed the presence of Giardia in affected animals, while no other significant viral, bacterial or parasitic pathogens were identified in faeces or tissue samples. A mild to severe infiltrative enteritis with eosinophils, lymphocytes and plasma cells was detected in histological sections of the gut. Giardia parasites collected from duodenal aspirates were typed as Giardia duodenalis Assemblage B, by PCR amplification and sequencing of the TPI gene. Treatment with fenbendazole at a dose of 10mg/kg for 3 consecutive days, successfully cleared the infection. These results show that G. duodenalis can cause significant economic losses in sheep farming.     

Prospective analysis for community participation in trypanosomosis control in The Gambia

The shift towards community participation in the eradication of trypanosomosis calls for investigation the underlying incentive structure for individuals in the community to cooperate in the provision of various control methods. Survey data were used to assess patterns of the community’s demand for insecticide pour-ons and trypanocidal drugs and factors affecting individual} demand in The Gambia. The results show that insecticide pour-on is strongly preferred. Similarly, farmers revealed a preference for community-based provision scheme. Factors affecting an individual farmer’s decision to invest in either pour-on or trypanocidal drugs were highlighted. While there are many factors associated with farmer’s decisions to invest in trypanosomosis control methods and to participate in collective actions, the results indicate that farmers} are ready to anticipate complete privatization of veterinary services through community-based schemes.     

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.