Resistance to infection by respiratory disease viruses involves several aspects of the immune response. Serum virus-neutralizing antibodies are important and are the component of immunity that is most often measured as an indication of resistance to infection by respiratory disease viruses. Other components of the immune response that may contribute to protection include IgA in mucus of the nasal passage, which can prevent virus attachment to epithelial cells; T-helper-1 cells, which can secrete IFN-γ and other cytokines; cytotoxic T cells, which can kill virus-infected cells; and γδ T cells, which are believed to help control viral infection of epithelial cells. Serum virus-neutralizing antibodies may be sufficient to protect healthy nonstressed animals from lowlevel challenge during experimental conditions. However, animals in field conditions exposed to various stressors and to varying amounts of multiple infectious agents may be better protected if all of the relevant aspects of acquired immunity have been effectively stimulated by vaccination.
Several modified-live and killed-virus vaccines containing the 5 major viruses that contribute to the bovine respiratory disease complex (BHV-1, BRSV, PI-3, and BVDV types 1 and 2) are licensed by the USDA and are commercially available.1 These vaccines may contain various strains and concentrations of viruses and various adjuvants, and they may induce various levels and types of immune responses.
The purpose of the study reported here was to determine the efficacy of a new modified-live virus vaccinea containing all 5 major bovine respiratory disease viruses to induce antigen-specific T-helper cells, cytotoxic T cells, and γδ T cells to heterologous strains of BHV-1, BRSV, and BVDV types 1 and 2 for as long as 6 months after vaccination. Flow cytometry was used to detect upregulation of CD25 (α chain of IL-2 receptor) and IFN-γ expression on CD4+, CD8+, and γδ TCR+ T-cell subsets. Induction of SVN antibody titers to all 5 viruses after vaccination was evaluated. Protection from BHV-1 challenge 6 months after vaccination was also evaluated as well as the induction of anamnestic antibody and T-cell responses after BHV-1 challenge.
Flow cytometry has been used in our laboratory to test for responsiveness of bovine T-cell subsets to specific antigens. Initially, 2-color flow cytometry2–8 was used to detect a single T-cell marker and an activation marker CD25 on each cell. This method was improved to 4-color flow cytometry,9 which can detect all major T-cell subsets and CD25 simultaneously. In our study, we introduced the use of 5-color flow cytometry to also simultaneously detect intracellular IFN-γ from the same cells. Five-color flow cytometry diminishes well-to-well variation inherent in 2-color flow cytometry, and the percentage of each T-cell subset can be studied more accurately. T-cell subsets with double positive T-cell markers can be identified with no overlapping results with single T-cell marker cells. We did not evaluate the CMI response to the PI-3 virus because that assay had not been validated at the time of the study.
Bovine herpes virus 1
Bovine respiratory syncytial virus
Parainfluenza virus 3
Bovine viral diarrhea virus
Serum virus neutralization
Peripheral blood mononuclear cell
Fetal bovine serum
PBS solution with 0.5% FBS and 0.1% sodium azide
Net increases in the percentage of IFN-γ-positive cells
Vista 5 SQ, Intervet Inc, Millsboro, Del.
Center for Veterinary Biologics, Ames, Iowa.
Titanium 5, Diamond Animal Health Inc, Des Moines, Iowa.
Sigma Chemical Co, St Louis, Mo.
BOVI-SHIELD IBR, Pfizer Animal Health, Exton, Pa.
BOVI-SHIELD BRSV, Pfizer Animal Health, Exton, Pa.
Courtesy of Dr. Julia Ridpath, National Animal Disease Center, Ames, Iowa.
VMRD Inc, Pullman, Wash.
Alexa Fluor 488, Molecular Probes Inc, Eugene, Ore.
Southern Biotech, Birmingham, Ala.
Alexa Fluor 647, Molecular Probes Inc, Eugene, Ore.
Caltag Laboratories, Burlingame, Calif.
Serotec, Raleigh, NC.
DyeMer 488/615, Molecular Probes Inc, Eugene, Ore.
BD Perm/Wash, BD Biosciences Pharmingen, San Diego, Calif.
GolgiStop, BD Biosciences Pharmingen, San Diego, Calif.
Cytofix/Cytoperm solution, BD Biosciences Pharmingen, San Diego, Calif.
Polyscience, Warrington, Pa.
BD, Franklin Lakes, NJ.
Epic Altra, Beckman Coulter Inc, Fullerton, Calif.
FlowJo, Tree Star Inc, San Carlos, Calif.
Invitrogen Corp, Carlsbad, Calif.
MEM, Mediatech Inc, Herndon, Va.
JMP 5.1, SAS Institute Inc, Cary, NC.
Inglis S, Stahle D, Schwartz JL. Compendium of veterinary products. 7th ed. Port Huron, Mich: North American Compendiums Inc, 2003;302–303.
Isaacson JA, Flaming KP, Roth JA. Increased MHC class II and CD25 expression on lymphocytes in the absence of persistent lymphocytosis in cattle experimentally infected with bovine leukemia virus. Vet Immunol Immunopathol 1998;4:235–248.
Quade MJ, Roth JA. Antigen-specific in vitro activation of Tlymphocyte subsets of cattle immunized with a modified live bovine herpesvirus 1 vaccine. Viral Immunol 1999;12:9–21.
Quade MJ, Roth JA. Dual-color flow cytometric analysis of phenotype, activation marker expression, and proliferation of mitogen-stimulated bovine lymphocyte subsets. Vet Immunol Immunopathol 1999;67:33–45.
Endsley JJ, Quade MJ & Terhaar B, et al. Bovine viral diarrhea virus type 1- and type 2-specific bovine T lymphocyte-subset responses following modified-live virus vaccination. Vet Ther 2002;3:364–372.
Endsley JJ, Roth JA & Ridpath JF, et al. Maternal antibody blocks humoral but not T cell responses to BVDV. Biologicals 2003;31:123–125.
Ridpath JE, Neill JD & Endsley J, et al. Effect of passive immunity on the development of a protective immune response against bovine viral diarrhea virus in calves. Am J Vet Res 2003;64:65–69.
Endsley JJ, Ridpath JF & Neill JD, et al. Induction of T lymphocytes specific for BVDV in calves with maternal antibody. Viral Immunol 2004;17:13–23.
Sandbulte MR, Platt R, Roth JA. T cells from a high proportion of apparently naïve cattle can be activated by modified vaccinia virus Ankara (MVA). Viral Immunol 2004;17:39–49.
Solerte SB, Cravello L & Ferrari E, et al. Overproduction of IFN-gamma and TNF-alpha from natural killer (NK) cells is associated with abnormal NK reactivity and cognitive derangement in Alzheimer's disease. Ann N Y Acad Sci 2000;917:331–340.
He XS, Draghi M & Mahmood K, et al. T cell-dependent production of IFN-gamma by NK cells in response to influenza A virus. J Clin Invest 2004;114:1812–1819.