Bernadette C. Taylor

Alteration in lymphocyte subpopulations in bovine leukosis virus-infected cattle

Alterations in peripheral blood lymphocyte subpopulations were examined in bovine leukosis virus (BLV)-infected cattle using antibodies specific for differentiation antigens in conjunction with analytical flow cytometry. Animals considered to be aleukemic and lymphocytotic were included in the study. Significantly fewer numbers of circulating B-lymphocytes (surface Ig-positive) and T-helper lymphocytes (BoCD4-positive) were identified in BLV-infected aleukemic cattle compared to non-infected controls while no significant differences were established for T-cytotoxic/suppressor lymphocytes (BoCD8-positive). In contrast, BLV-infected animals with persistent lymphocytosis had elevated numbers of circulating B-lymphocytes with no significant perturbation in circulating T-lymphocyte subsets identified when compared as a group with the negative control cattle. Application of regression analysis to data from individual lymphocytotic cattle demonstrated a significant correlation between absolute numbers of B- and T-lymphocytes. Increased numbers of B-lymphocytes were correlated with increased numbers of T-helper and T-cytotoxic/suppressor lymphocytes.

Response of the regional lymph node to bluetongue virus infection in calves.

Bluetongue virus (BTV) infection of cattle is characterized by prolonged cell-associated viremia. In an effort to further evaluate the antiviral response of BTV-infected cattle, the role of the regional lymph node (LN) in the immune response of calves to BTV was characterized. Calves were inoculated with BTV in the skin of the neck in an area drained by the superficial cervical LN. Calves were euthanized at regular intervals after inoculation and both BTV-challenged and contralateral (control) superficial cervical LNs were harvested. In addition, some calves had cannulation of the superficial cervical efferent lymphatics prior to inoculation. Lymphocyte subpopulation analysis was done on LN cell suspensions and lymph cells using a panel of cell-specific monoclonal antibodies. There was a significant increase in the proportion of B cells in the challenged LN after inoculation as compared with the control LN. In addition, BTV-specific antibodies were detected in efferent lymph plasma from the challenged LN in one cannulated calf by 9 days after inoculation (DAI), as determined by competitive enzyme-linked immunosorbent assay, whereas BTV-specific antibodies were not detected in serum from this calf through 12 DAI. Analysis of lymph cells revealed a sustained increase in cell output from the challenged LN due to an increase in lymphoblasts and CD8+ T cells. In contrast, the cell output from the control LN dropped markedly by 8 DAI and there was no significant increase in any specific cell population. Double label analysis characterized lymphoblasts as activated CD8+ cells, as determined by expression of MHC Class II antigens (CD8+ MHC II+). These cells were only transiently present as CD8+ MHC II+ cells were not identified in lymph from the challenged LN at 14 DAI. Few CD8+ MHC II+ cells were identified at any time in lymph from the control LN or in lymph from a mock infected calf. The data indicate that B cell proliferation in the challenged LN and release of activated CD8+ cells from this LN were specific responses to BTV infection. The rapid expansion of activated CD8+ T cells indicates that these cells may limit early viral spread. It is concluded that the regional LN draining inoculated skin is critical to the immune response of calves to BTV infection.

Measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion.

Killer whales and sea otters maintained in captivity are the subjects of routine health monitoring programs, and interest in immunologic studies in sea otters has been rising recently in response to potential impacts from infectious disease and environmental pollution on the threatened southern sea otter population. Development of species-specific reagents for immunologic studies in these two marine mammals is currently in its infancy. In this study, killer whale and sea otter immunoglobulin-specific polyclonal antibodies were generated, and used to develop tests for serum Ig concentration in the killer whale (Orcinus orca) and the southern (Enhydra lutris nereis) and northern sea otter (Enhydra lutris lutris). Killer whale serum IgG was purified using caprylic acid/ammonium sulfate precipitation. Sea otter plasma IgG was purified using protein-A-agarose. Polyclonal anti-Ig antisera were produced in rabbits, and specificity confirmed by immunoelectrophoresis. Radial immunodiffusion was used to measure Ig concentration in serum or plasma samples derived from 21 captive killer whales, 18 wild and 4 captive southern sea otters and 15 wild and 4 captive northern sea otters grouped by age. Mean killer whale serum Ig concentration (+/-95% confidence interval) ranged from 15.04 +/- 3.97 g/l for animals aged 0-5 years to 26.65 +/- 9.8 g/l for animals aged >10 years. Mean sea otter serum Ig concentration (+/-95% confidence interval) ranged from 28.39 +/- 11.00 g/l for southern sub-adults to 32.76 +/- 11.58 g/l for southern adults. No significant difference in serum Ig concentration was found between southern and northern sea otters. Serum Ig concentrations in two northern sea otter pups were low compared to those of adult sea otters. The two serum Ig quantitation assays produced were highly specific and reproducible and will be useful additions to the limited number of tests available for immune function in these marine mammal species.

Memory and CD8+ are the predominant bovine bronchoalveolar lymphocyte phenotypes.

Bovine lymphocytes obtained by bronchoalveolar lavage (BAL) of healthy calves were simultaneously analyzed and compared to peripheral blood lymphocytes using monoclonal antibodies specific for bovine leukocyte differentiation antigens. Phenotypic differences were observed between bronchoalveolar and peripheral blood T-lymphocyte subpopulations, demonstrating selective lymphocyte migration to the bovine lung. The bronchoalveolar and peripheral blood T-lymphocyte populations, defined by expression of CD2, were similar, but bronchoalveolar T lymphocytes were predominately CD8+ while peripheral blood T cells were predominately CD4+. In addition, memory lymphocytes, characterized by low expression of CD45R and activated lymphocytes (CD25+), were found in significantly higher proportions in the bronchoalveolar compartment. The proportion of gammadelta T lymphocytes was, however, significantly higher in peripheral blood. B cells were observed in similar proportions in the bronchoalveolar compartment and peripheral blood.

PRIMARY AND ANAMNESTIC RESPONSES OF BOVINE BRONCHOALVEOLAR AND PERIPHERAL BLOOD LYMPHOCYTE SUBSETS TO AEROSOLIZED PASTEURELLA HAEMOLYTICA A1

Site-specific responses of bronchoalveolar and peripheral blood lymphocyte subsets were compared during primary and anamnestic immune responses against live Pasteurella haemolytica A1 (Ph1). Eight 1-year old calves were sequentially exposed intrabronchially with aerosolized Ph1 on days 0, 14, and 21, and two calves were sham exposed. Bronchoalveolar and peripheral blood lymphocytes were analyzed before each Ph1 exposure, and on days 3 and 7 post exposure using single and two-color flow cytometry to identify CD2+, CD4+, CD8+, CD21+, CD45R+, CD25+ and gammadelta lymphocyte subsets. Significant differences (p < 0.05) in bronchoalveolar and peripheral blood lymphocyte subsets were observed before Ph1 exposure. Subsequent aerosol exposures, resulted in significant (p < 0.05) changes in bronchoalveolar lymphocyte subsets and the CD4:CD8 bronchoalveolar lymphocyte ratio, but concomitant changes were not observed in peripheral blood lymphocytes. Expression of CD2, CD4 and CD8 lymphocyte differentiation antigens was consistently lower and more heterogeneous on bronchoalveolar lymphocytes. Differential analysis of bronchoalveolar leukocytes revealed a significant increase in bronchoalveolar lymphocytes and neutrophils during anamnestic responses.