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Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.

Free access
in American Journal of Veterinary Research
in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association


To evaluate alterations in lymphocyte subpopulations, CBC results, and clinical signs in neonatal calves inoculated with 3 commercially available proprietary multiple-antigen vaccines containing known quantities of endotoxin.


Prospective, randomized controlled field trial.


36 healthy Holstein heifer calves between 3 and 31 days old.


Vaccines were administered to 18 calves according to label instructions, except for the recommended age of administration. The 18 other calves served as unvaccinated controls. Two weeks after entry into the study, calves were given secondary doses of the same vaccines. Calves in both groups were examined and blood samples were collected for determination of lymphocyte subpopulations and hematological parameters once daily for 5 days beginning on the day that both the primary and the secondary vaccinations were given. Lymphocyte subpopulations, including BoCD2*, BoCD4+, BoCD8+, B cells, and γ/δ T cells, were determined by use of flow cytometry, using monoclonal antibodies as markers.


Vaccinated calves did not develop clinical signs of illness. There were no significant differences in absolute numbers of lymphocyte subpopulations between vaccinated and unvaccinated calves. Vaccinated calves had significantly higher rectal temperatures, total WBC counts, and absolute neutrophil counts than did control calves. These differences persisted for 3 to 4 days after vaccination.

Clinical Implications

Findings confirm empirical observations that vaccination with multiple products at the same time may induce evidence of an inflammatory response in most calves. Additional research is indicated to further evaluate the safety of using multiple vaccines simultaneously. (J Am Vet Med Assoc 1996;209:638-642)

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in Journal of the American Veterinary Medical Association