Influence of advanced age on Mycobacterium bovis BCG vaccination in guinea pigs aerogenically infected with Mycobacterium tuberculosis.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only tuberculosis (TB) vaccine currently available, but its efficacy against adult pulmonary TB remains controversial. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against TB. TB remains a major public health problem, especially among the elderly, yet the efficacy of BCG in the elderly is unknown. We investigated the ability of BCG vaccination to prevent TB in young (6-week-old), middle-aged (18-month-old), and old (60-month-old) guinea pigs. BCG-Tokyo vaccination reduced the growth of Mycobacterium tuberculosis H37Rv in all three groups. By use of an enzyme-linked immunospot (ELISPOT) assay, antigen-specific gamma interferon (IFN-γ)-producing cells were detected in the 60-month-old guinea pigs after a booster vaccination with BCG-Tokyo. Our findings suggest that BCG-Tokyo has a protective effect against tuberculosis infection regardless of age.

Novel two-parameter flow cytometry (MIL4/SSC followed by MIL4/CT7) allows for identification of five fractions of guinea pig leukocytes in peripheral blood and lymphoid organs.

Though the guinea pig has been an extremely useful animal model for a variety of diseases, the tools necessary to undertake a full-scale immunological analysis of the guinea pig have been lacking. For instance, traditional two-parameter forward/side scatter (FSC/SSC) flow cytometry, though effective in human and other animal models, is unable to adequately identify the distinct fractions of guinea pig peripheral blood leukocytes (PBL). We introduce here a new flow cytometric technique (MIL4/SSC followed by MIL4/CT7) which redresses this lack by identifying and characterizing five distinct fractions of PBL: neutrophils, lymphocytes, monocytes, eosinophils plus basophils, and the novel MIL4(-)SSC(large)CT7(high) population. The MIL4(-)SSC(large)CT7(high) cells possess cytoplasmic inclusion bodies of variable size that were positive for periodic acid Schiff (PAS). Their cell surface stained positive for the helper/inducer lymphocyte markers, T cell markers, CD45, Thy-1, asialo GM1 and FcR, but negative for B cell markers, such as membrane-type IgM, CD8 and MHC class II. The novel flow cytometric technique also allowed us to establish that the five leukocyte fractions were found in PBL, splenocytes, thymocytes and lymph node cells. Cells which were positive for inclusion bodies comprised 16.6% of splenocytes, 9.9% of PBL and 4.3% of liver cells, but were comparatively rare in lymph node cells, thymocytes, and BM cells. The novel flow cytometric technique introduced here will allow a better understanding of the response of each type of guinea pig leukocyte and thereby shed light on the diseases with which they are associated.

The normalization of guinea pig leukocyte fractions and lymphocyte subsets in blood and lymphoid tissues using a flow cytometric procedure.

Many hematological and immunological parameters remain unclear in the study of the guinea pig. In this study, we established the mean values of blood counts, the percentage of leukocyte fractions and lymphocyte subsets in blood and various lymphoid tissues of the guinea pig with a flow cytometric procedure using MIL4/SSC. The mean counts of WBC and RBC in the blood were lower, and MCV and MCH were higher than those of other rodents, resembling those of humans. Furthermore, the mean percentages of blood lymphocytes were smaller and that of granulocyte was larger than those of other rodents, resembling those of humans. We further established a flow cytometric procedure for lymphocyte subsets and clarified the mean percentages of T- and B-cells, CD4(+)-, CD8(+)- and MHC Class II(+)- T-cells, and CD4(-)CD8 (-) T-cells. The latter were morphologically larger in cell size and cytoplasm than CD4(+)- plus CD8(+) T-cells, and this subset had a significantly higher percentage in newborn animals. Furthermore, the appearance of the MHC Class II(+) T-cell subset was suggested to be a marker of hyper-activation of T-cells in BCG-immunized animals. Thus, both the novel flow cytometric procedure for leukocyte fractions and lymphocyte subsets, and the established normal values will be useful tools in studying guinea pigs as models of various diseases and biological phenomena.