Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND: Guidelines published by the Food and Drug Administration and Center for Human Medicinal Products describe the need to assess immunotoxic effects in nonclinical studies that evaluate drug toxicity, including the use of immunophenotyping to measure immunotoxicity. We are not aware of previous studies, however, that have validated methods for immunophenotyping peripheral blood lymphocyte subsets in whole blood samples from cynomolgus monkeys. OBJECTIVE: The purpose of this study was to optimize and validate a flow cytometric assay for immunophenotyping lymphocytes in the peripheral blood of cynomolgus monkeys. METHODS: A series of prevalidation experiments were done to determine optimal reagents, volumes, timing, and other procedural details of the flow cytometric assay. Using the optimized method, we then determined precision, interindividual variation, laboratory-to-laboratory variability, and sample stability. Stabilized human blood was used as a positive control for staining, processing, and analysis. The percentage and number of pan-T cells (CD3+), T-helper cells (CD3+4+), T cytotoxic/suppressor cells (CD3+8+), natural killer cells (CD3-16+), and B-cells (CD3-20+) were determined in 146 male and 140 female, clinically healthy monkeys and reference intervals were calculated. RESULTS: By doing 4-color staining with a lyse-wash method, intra- and interassay precision were <5% for all lymphocyte subsets. Variability between technicians and laboratories was minimal (CVs<3%). Samples were stable for up to 24 hours after staining and fixing. CONCLUSIONS: The validated method is extremely robust and can be performed under good laboratory practice conditions to support nonclinical studies. Reference intervals for lymphocyte subsets were similar to those previously reported.     

Primary central nervous system B-cell lymphoma in a young dog

This report describes a primary central nervous system B-cell lymphoma in a 3-year-old intact female Maltese dog. Canine primary central nervous system lymphomas constitute about 4% of all intracranial primary neoplasms, but comprehensive histopathologic classifications have rarely been carried out. This is the first report of this disease in a young adult dog.     

Adherence to various host cell lines of Mycoplasma bovis strains differing in pathogenic and cultural features

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified.     

The effect of immunosuppression on protective immunity of turkey poults against infection with turkey coronavirus

The objective of the present study was to evaluate the protective effect of humoral and cellular immunities on turkeys infected with turkey coronavirus (TCV). Two trials were conducted with two separate hatches of turkey poults. Turkey's were experimentally immunosuppressed with cyclosporin A (CsA) or cyclophosphamide (CY) and infected with TCV. Prior to infection, treatment with CsA selectively suppressed T cell activity as revealed by 2-3 fold decreased (p < 0.1) lymphocyte proliferation responses to a T cell mitogen, concanavalin A (Con A). Treatment with CY mainly induced B cell deficiency as indicated by significant reductions (p < 0.05) in antibody responses to sheep erythrocytes 7 days after injection. Body weight gain of turkeys treated with CY was significantly lower (p < 0.05) than that of untreated turkeys at 9 days post-infection (PI). Turkeys treated with CY had 1-2 fold higher immunofluorescent antibody assay (IFA) scores for TCV antigens (p < 0.05) in the intestine than untreated turkeys at 9 or 14 days PI. These results suggested that humoral immunity against TCV infection may be important in turkeys.     

Neutrophil myeloperoxidase measurements in plasma, laminar tissue, and skin of horses given black walnut extract

OBJECTIVE: To compare measurements of myeloperoxidase (MPO) in plasma, laminar tissues, and skin obtained from control horses and horses given black walnut heartwood extract (BWHE). ANIMALS: 22 healthy 5- to 15-year-old horses. PROCEDURES: Horses were randomly assigned to 4 groups as follows: a control group given water (n = 5) and 3 experimental groups given BWHE (17) via nasogastric intubation. Experimental groups consisted of 5, 6, and 6 horses that received BWHE and were euthanatized at 1.5, 3, and 12 hours after intubation, respectively. Control horses were euthanatized at 12 hours after intubation. Plasma samples were obtained hourly for all horses. Laminar tissue and skin from the middle region of the neck were harvested at the time of euthanasia. Plasma and tissue MPO concentrations were determined via an ELISA; tissue MPO activity was measured by use of specific immunologic extraction followed by enzymatic detection. RESULTS: Tissues and plasma of horses receiving BWHE contained significantly higher concentrations of MPO beginning at hour 3. Laminar tissue and skin from horses in experimental groups contained significantly higher MPO activity than tissues from control horses. Concentrations and activities of MPO in skin and laminar tissues were similar over time. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, BWHE administration causes increases in MPO concentration and activity in laminar tissue and skin and the time of increased MPO concentration correlates with emigration of WBCs from the vasculature. These findings support the hypothesis that activation of peripheral WBCs is an early step in the pathogenesis of acute laminitis.     

SONOGRAPHIC EVALUATION OF EXPERIMENTAL BILE DUCT LIGATION IN THE DOG

The common bile duct was surgically ligated in five normal adult dogs. Ultrasonographic examinations of the gallbladder and biliary system were performed after duct ligation at intervals of 24 hours. The sequence of biliary system dilation was from the common duct to the peripheral intrahepatic ducts. Common duct enlargement was evident in 24–48 hours, while peripheral biliary duct dilation was recognized by five to seven days after obstruction. When compared with hepatic and portal veins, dilated biliary ducts were more tortuous and had irregular branching patterns. Gross pathologic changes were correlated with ultrasonographic findings at seven, 14, 15, 18 and 21 days after obstruction.