Quantitative analyses of lymphoid tissue in the spleen, lymph nodes and Peyer's patches in cynomolgus monkeys

To clarify the morphological characteristics of the cynomolgus monkey immune system, we analyzed quantitative data on their lymphoid organs. Spleens, major lymph nodes and Peyer's patches were sampled from cynomolgus monkeys, and the lymphoid follicle and germinal center areas and percentages of CD3- and CD20-positive areas were calculated. All the organs analyzed showed large interindividual variations in the sizes of lymphoid follicles and germinal centers. Lymphoid follicle in the spleen, submandibular lymph nodes and Peyer's patches showed no marked difference in size. Germinal center size in the mesenteric lymph nodes and Peyer's patches were significantly smaller than those in the spleen. Areas containing T cells were largest in the lymph nodes, while those containing B cells were largest in the spleen and Peyer's patches. The mean size of the splenic lymphoid follicle in cynomolgus monkeys is larger than that in rats and similar to that in humans. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in standard toxicity studies.     

A new epitope on sheep CD45R molecule detected by a monoclonal antibody

This paper describes the production and characterization of a monoclonal antibody (mAb), Co-46D5, which recognizes a new epitope on the isoform of the homologous sheep leukocyte common antigen (LCA) or CD45. This nmAb was submitted to the 3rd workshop on ruminant leukocyte antigens and was assigned to a cluster reactive with B- and T-cells subsets. Co-46D recognizes a 220 kDa molecule on peripheral blood mononuclear cells (PBMC) and spleen cells but not on thymocytes. Flow cytometry (FCM) analysis shows that Co-46D5 reacted with 30% of PBMC and 50% of spleen cells and more than 95% of cells freshly isolated from lymphoid follicles of the ileal Peyer's patches (IPP) of young lambs. By immunohistochemistry, the antigen was detected mainly on B-cell areas of lymph nodes and spleen. It was also found on a subpopulation of medullar thymocytes. Based on these results, we assume that Co-46D5 recognizes a new epitope on the largest isoform of the sheep CD45 receptor, probably on the homologous to the human CD45RA isoform.     

Precursor B-1 B cell lymphoma in a newborn calf.

A newborn Holstein female calf had neoplastic lesions in the skin and within the thoracic and abdominal cavities but not in the bone marrow, spleen, thymus, or most lymph nodes. Because the tumor cells were positive for CD79a (B cell marker), CD5 (B-1 cell marker) and terminal deoxynucleotidyl transferase (marker for immature lymphoid precursors), a diagnosis of precursor B-1 B cell lymphoma was made. The diagnosis was strongly supported by the fact that B-1 cells can develop in the fetus, unlike B-2 cells, which are produced after birth. The lymphoma was distinct from the typical calf form of lymphoma of B-2 cell origin, which does not express CD5 and is characterized by generalized lymphadenopathy and involvement of the bone marrow, blood and spleen.     

Lymphatic organ development in dogs: major lymphocyte subsets and activity

In the present study, we have characterized lymphocyte subsets and activity in peripheral blood, spleen, mesenteric and popliteal lymph nodes in pups from birth till the age of one month and compared the results with the situation in the group of three adult dogs. In neonatal pups, lower numbers of CD3(+) T-cells were detected in both the spleen and peripheral blood than in lymph nodes. In contrast to the other compartments, CD21(+) B-cells prevailed in the spleen, which resulted in low values (<1) of the CD3(+)/CD21(+) ratio. Low numbers of CD8(+) lymphocytes were characteristic in all compartments immediately after birth; consequently a high CD4(+)/CD8(+) ratio has been calculated. Postnatal development was characterized by an increasing frequency of CD8(+) lymphocytes in all organs studied. Another typical feature of the early period of life was a relative decrease of B-cell numbers, which was compensated by an increasing proportion of T-lymphocytes, particularly in the peripheral blood and spleen. DNA synthesis in newborn pups' cells as measured by in vitro thymidine incorporation was surprisingly high in non-stimulated control samples, notably in the spleen. Further development of lymphocyte activity was characterized by the decline in spontaneous activity in all organs. Stimulation indices upon mitogen-induced proliferation increased proportionally to the decrease in spontaneous activity. Based on our experimental data, we have concluded that pups are born with a relatively competent immune system the structure of which, however, markedly develops during a few postnatal weeks.     

Changes in macrophage and lymphocyte subpopulations of lymphoid tissues from pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV)

We used an immunohistochemical method to investigate changes in macrophage and lymphocyte subpopulations in various lymphoid tissues of pigs in the acute phase of porcine reproductive and respiratory syndrome virus (PRRSV) infection. The numbers of CD8+ cells and B-cells varied among lymphoid tissues after PRRSV infection. In the infected pigs, numbers of CD8+ cells increased in systemic lymphoid tissues whereas numbers of B-cells increased in mucosa-associated lymphoid tissues. There was no difference in the distribution of virus-infected cells and macrophages between lymphoid tissues of the infected pigs. These changes may be associated with the establishment of virus persistence or the emergence of concurrent infection in mucosal organs.     

Active immunity and T-cell populations in pigs intraperitoneally inoculated with baculovirus-expressed transmissible gastroenteritis virus structural proteins

The intraperitoneal inoculation of pigs with baculovirus-expressed transmissible gastroenteritis virus (TGEV) structural proteins (S, N, M) in conjunction with thermolabile Escherichia coli mutant toxin (LT-R192G) in incomplete Freund’s adjuvant (IFA) was tested in an attempt to elicit active immunity to TGEV in gut-associated lymphoid tissues (GALT). Four groups of 63 (1–5-week-old) suckling, TGEV-seronegative pigs were used to assess the efficacy of the recombinant protein vaccine (group 3) in comparison with sham (group 1), commercial vaccine (group 2), and virulent TGEV Miller-strain-inoculated pigs (group 4). The TGEV-specific mucosal and systemic immune responses were measured after in vivo and in vitro stimulation with TGEV-antigens. The major T-cell subset distribution was analyzed in vivo and in vitro after stimulation of mononuclear cells with TGEV (from mesenteric lymph nodes of group 3 inoculated with TGEV-recombinant proteins). Induction of active immunity was assessed by challenge of pigs with virulent TGEV at 27 days of age. Baculovirus-expressed TGEV proteins coadministered with LT-R192G in IFA induced mesenteric lymph node immune responses associated with IgA-antibodies to TGEV and partial protection against TGEV-challenge. The high titers of serum IgG- and virus-neutralizing-antibodies to TGEV in group 3 pigs most likely reflected the dose of TGEV S-protein administered. At the day of TGEV-challenge, the in vitro stimulation of mononuclear cells from the mesenteric lymph nodes of group 3 pigs with inactivated TGEV resulted in an increase in double positive (CD4+CD8+), natural killer (CD2+CD4−CD8+dim) and cytotoxic (CD2+CD4−CD8+bright) T-cell phenotypes, accompanied by increased expression of interleukin-2 receptor and a decrease of the null (CD2−CD4−CD8−/SW6+) cell phenotype.     

Tacrolimus-FK506 induced immunosuppression in gnotobiotic piglets

Tacrolimus (FK506), an inhibitor of calcineurin, is an immunosuppressive agent used in clinical trials of transplant patients. Although FK506 targets Ca(2+)-mediated T-cell signaling, phenotype(s) of the specific target cells and the corresponding cytokine pathways are not well known. In this study, the impact of FK506 on number and characteristic of T-cells in selected lymphoid tissues of gnotobiotic (GB) piglets was determined. FK506-treated GB piglets were compared with untreated GB and conventional piglets. The T-helper, cytotoxic, natural killer, double-positive, and activated T-cell populations were analyzed in suspensions of mononuclear cells isolated from thymus, mesenteric lymph nodes and peripheral blood. In vitro secretion of interleukin-8 and interferon-gamma in concanavalin A-stimulated lymphoid cell-cultures was measured by ELISA. Daily intramuscular treatment of GB piglets with 1mg/kg of FK506 from birth for 4 weeks resulted in lowered (P<0.05) in vitro secretion of interferon-gamma and interleukin-8. Moreover, depletions of MNC in systemic and mucosa-associated lymphoid tissues were observed in piglets treated with FK506. The depletions of mononuclear cells and low levels of interferon-gamma and interleukin-8 in piglets treated with FK506 were accompanied by lower proportion of CD3+, CD2+CD4+ and CD2+CD8+ T-cell phenotypes in peripheral blood but not in thymus and mesenteric lymph nodes. These results indicate that FK506-treatment causes immunosuppression in GB piglet, and this effect could be exploited further to study opportunistic pathogens in pig model.     

Altered lymphocyte homeostasis after oral prion infection in mouse

Transmissible spongiform encephalopathies (TSEs) or prion diseases develop as central nervous system (CNS) disorders characterized by extremely long incubation periods. Although TSEs do not go along with inflammatory infiltrates and/or antibody production against the prion protein (PrP(Sc)), the immune system plays an important role in pathogenesis as long as different lymphoid organs (Peyer's patches, lymph nodes and spleen) may facilitate the accumulation and further spread of prions after peripheral exposure. In this work we investigated the changes in lymphoid and dendritic cell (DC) populations as well as the implications of different cytokines during disease progression after experimental oral inoculation of prions in a transgenic mouse model. At different days post-inoculation (dpi), T and B lymphocytes and DC populations from lymphoid organs, blood and brain were analyzed by flow cytometry and immunohistochemistry. Besides time related variations in lymphoid cell numbers due to the aging of the animals significant changes related with the infection were found in mesenteric lymph nodes, peripheral blood leukocytes (PBLs) as well as in spleen, affecting the CD4/CD8 ratio. In contrast, little or no variation was detected in Peyer's Patches or in thymus either associated with aging or the infection status. At individual time points significant differences between infected and control mice were seen in the CD8, CD4 and DC populations, with less evidence of differences in the B cell compartment. Finally, a pro-inflammatory phenotype occurred at early times in the spleen, where the levels of lymphotoxin-beta mRNA were found augmented with respect to controls. Altogether, these results suggest that normal regulation of lymphocyte populations becomes altered along the progression of a prion infection.     

Barley-derived β-glucans increases gut permeability, ex vivo epithelial cell binding to E. coli, and naive T-cell proportions in weanling pigs

Weaning in young animals is associated with an increased incidence of gastrointestinal infections. β-glucans exert numerous physiological effects, including altering immune function. The objective of this study was to determine the effects of feeding barley (Hordeum vulgare L.)-derived β-glucans on immune and intestinal function in weanling pigs (Sus scrofa). Thirty-one individually-housed Dutch Landrace pigs (21 d; initial BW, 6,298 ± 755 g) were weaned and fed a wheat-based diet (control) or a low (Lo-BG), medium (Med-BG), or high β-glucan-containing barley-based diet (Hi-BG) for 2 wk with 7 or 8 pigs/treatment. Intestinal segments were analyzed for permeability using Ussing chambers and K88 Escherichia coli adhesion to enterocytes was assessed ex vivo. Immune cells from mesenteric lymph nodes, peripheral blood, and Peyer's patches were analyzed for lymphocyte subsets by indirect immunofluorescence and the ability to respond ex vivo to mitogens by (3)H-thymidine incorporation. Hematology and neutrophil function were determined by flow cytometry. Neutrophil burst, size, and granularity, lymphocyte proliferation, and B-cell distribution in peripheral blood lymphocytes, Peyer's patches, and mesenteric lymph nodes were not affected by β-glucans content of the diet. The β-glucans content of the diet altered blood concentrations of erythrocytes and leukocytes, CD4, CD45RA, and CD8 blood cells (P < 0.05). In addition, feeding β-glucan resulted in increased (P < 0.05) percentage CD45RA positive cells in peripheral blood lymphocytes, Peyer's patches, and mesenteric lymph nodes. Mannitol permeability and tissue conductance were increased (P < 0.05) in Hi-BG fed pigs compared with control pigs. Percentage maximum K88-E.coli binding was increased in proportion to the β-glucan content of the diet (P < 0.05). Although β-glucan feeding during the weaning period increased blood lymphocytes and the proportion of na?ve T-cells, it also increased E. coli-enterocyte binding and intestinal permeability. β-glucan may alter immune and intestinal function of weaning pigs.     

Estudio Histoquímico e Immunohistoquímico del Tejido Linfoide Porcino: Ganglio Linfático, Bazo y Timo

Immunohistological (S-100, cIg) and enzyme histochemical (ANAE/ANBE, beta-glu, ATPase, AcP) investigations were carried out to identify lymphocyte and reticular cell subpopulations "in situ", in pig lymphoid tissue (lymph node, spleen, and thymus) of a 6 months old group, and a 6-9 days old one. By means of immunohistological techniques, in the 6 month old pigs we could detect S-100 protein (PAP), chiefly in T-areas lymphocytes, but we also found some S-100 positive lymphocytes in spleen follicles. Also S-100 protein were detected at Follicular Dendritic Cells (FDC) in lymph node and spleen; and Reticular Fibroblastic Cells (RFC) only in the first one. Finally, S-100 were noted in Hassall corpuscles (thymus), nervous fibres, and endothelial cells too. Using PAP (IgG, IgM) and IPI (IgA) techniques we could detect lymphocyte cytoplasmatic surface immunoglobulins (cIg) in lymphocytes, lymphoblastoid and plasmacytoid cells in nearly all tissue compartments. By means of histochemical techniques we could identify T-area lymphocytes ANAE/ANBE and beta-glu positives (cytoplasmatic spots) and B-area lymphocytes ATPase positive; macrophages, and polymorphonuclear eosinophiles PHNE being ANAE/ANBE and beta-glu positives (diffuse cytoplasmic stain); and Hassall corpuscles ANAE/ANBE and AcP positives. Concerning to reticular cells, we found FDC and RFC in lymphoid follicles, and Interdigitating Reticular Cells (IDC) in lymphoid diffuse tissue, with enzyme activity (all the enzymes studied) in nearly all the cases. In piglets, the immunohistological and histochemical pattern was nearly the same.     

Comparative studies on the distribution and population of immunocompetent cells in bovine hemal node, lymph node and spleen

The distribution and population of immunocompetent cells in bovine hemal node, mesenteric lymph node and spleen were analyzed comparatively by immunohistochemistry and flow cytometry. Many CD8(+) cells, CD172a(+) cells and γδ T cells were found in the lymphatic cord along the sinus of the hemal node and the splenic red pulp. A few CD8(+) cells and γδ T cells were distributed diffusely in the paracortex and medullary cord of the mesenteric lymph node. Many germinal centers were recognized in the lymphatic regions such as the cortex and white pulp of these lymphoid organs. The populations of CD8(+) cells and γδ T cells in the hemal node and the spleen were higher than those of the mesenteric lymph node. In addition, the populations of CD21(+) cells and MHC class II(+) cells in the hemal node and the mesenteric lymph node were higher than those of the spleen. The results suggest that the hemal node has an important role in both cellular and humoral immunity as well as the lymph node and the spleen in cattle.     

Lymphocyte populations and adhesion molecule expression in bovine tonsils

Bovine tonsils are mucosal-associated lymphoid tissue (MALT) located at the entry of the pharynx where both inhaled and ingested antigens can induce an immune response. This study was conducted to determine the lymphocyte populations and adhesion molecule expression in the palatine tonsil (PT) and pharyngeal tonsil (PhT) of adult cattle and compare them with typical MALT (discrete Peyer's patches, PP) and a peripheral lymph node (parotid lymph node, PLN). The distribution of various lymphocyte subsets was determined in situ by immunofluorescence, and their proportions were determined by multicolor flow cytometry. The tonsils were similar to PP in the proportions of B- and T-cells (25-32% T-cells, 39-45% B-cells), and T cell subpopulations (CD4, CD8, and gammadelta). The PP contained the highest proportion of memory T-helper cells with beta7 integrin (30.3%+/-5.4), the tonsils intermediate (PT: 19.8%+/-4.4 and PhT: 19.7%+/-4.9), and the PLN had the lowest proportion (15.4%+/-3.1). The opposite relationship was observed with CD62L on na?ve T- helper cells as PP had the lowest proportion (14.2%+/-6.4), the tonsils intermediate (PT: 17.4%+/-2.5 and PhT: 24.3%+/-7.3), and the PLN the highest proportion (45.3%+/-6.5). MAdCAM-1 was highly expressed in the high endothelial venules (HEV) of PP, with variable and weak expression in the tonsils and PLN. PNAd, on the other hand, was highly expressed in HEV of tonsils and PLN, and weakly expressed in the PP. These results indicate that the bovine tonsils share characteristics with both PP and PLN. The alpha4beta7/MadCAM-land CD62L/PNAd interaction may be involved in lymphocyte migration to the tonsils, but it is likely that other adhesion molecules participate as well. Similarities between the human and bovine tonsils suggest that cattle may provide a good model to study the role of the tonsil in the respiratory immune response.     

Distribution of lymphocyte subsets in the small intestine lymphoid tissue of 1-month-old lambs

Distribution of lymphocyte subpopulations along the small intestine lymphoid tissue has been examined in 1-month-old lambs using flow cytometric and immunohistochemical techniques. Monoclonal antibodies against CD4, CD8, gamma delta, CD45R and B receptors have been employed in samples from continuous ileal Peyer's patch (IPP), discrete jejunal Peyer's patches (JPP), ileocaecal valve lymphoid tissue (ICVPP), mesenteric lymph node (MLN) and intra-epithelial (IEL) and lamina propria (LPL) lymphocytes. Histological studies were also done. Differences in the lymphocyte distribution have been observed between some of the regions examined, especially between IPP and JPP for most of the markers. A remarkable feature was the existence of morphological and lymphocyte distribution differences between ICVPP and IPP, locations that had been traditionally considered as similar. The antibody against CD45R receptor used in this study, that was supposed to mark B cells and some T cells, detected cell populations located in the dome of the follicles in all the samples, whereas the centre was negative. Lymphocytes positive to the B marker employed were located mainly in the centre, suggesting that both antibodies would mark B cells in different maturation status.     

Immune dysfunction following infection with chicken anemia agent and infectious bursal disease virus. I. Kinetic alterations of avian lymphocyte subpopulations.

The potential effect of chicken anemia agent (CAA) alone or in combination with infectious bursal disease virus (IBDV) on the immune system of young chickens was determined by measuring alterations in hematocrit values, lymphoid organ-to-body weight ratios and lymphoid cell concentrations at 4, 7, 10, 14, 17, 21, 28 and 42 days post-inoculation (PI). Lymphocyte subpopulations were identified and counted by flow cytometry using cell suspensions stained with monoclonal antibodies (Mabs) for panlymphocytes (K55), cytotoxic T-cells (CTLA3), T-helper cells (CT3), Ia-expressing cells (P2M11) and macrophages (P7). Chicken anemia agent induced a substantial but transient decrease in hematocrit value, thymus-to-body weight ratio and bursa-to-body weight ratio between 7 and 21 days PI corresponding to a generalized lymphocytopenia in the thymus, bursa and spleen. However, cytotoxic T-cell, T-helper cell and Ia-expressing cell concentrations increased in the bone marrow of birds inoculated with CAA alone or in combination with IBDV during the same time period. T-helper-to-cytotoxic T-cell ratios increased in the thymus and spleen during severe lymphocytopenia, indicating a selective decrease in cytotoxic T-cells. T-helper-to-cytotoxic T-cells ratios increased in the bone marrow, indicating a selective increase in T-helper cell concentrations. The increase in Ia-expressing cells in the bone marrow may be a reflection of increased number of activated T-cells which express Ia antigen. Infectious bursal disease virus alone induced a persistent depression of Ia-expressing cells in the bursa and the spleen and no measurable change in the bone marrow lymphocyte subpopulations. Chickens inoculated simultaneously with CAA and IBDV experienced clinical signs observed in chickens inoculated with each virus separately with a prolonged acute phase prior to recovery or mortality.     

Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens.

Postnatal development of various T-lymphocyte subpopulations expressing CD3, CD8, CD4, and antigen-specific TCR heterodimers alpha beta (TCR2) or gamma delta (TCR1) was investigated in two different inbred chicken strains, SC and TK. The ratios of jejunum T-cells expressing TCR1 to TCR2 in the intraepithelium of SC and TK strains gradually increased after hatching and were 3.40 and 4.28 by 12 weeks in TK and SC chickens respectively. The ratios of TCR1+ to TCR2(+)-cells in intraepithelium and the lamina propria in SC chickens were 0.96 and 1.23 at 8 weeks and 4.29 and 2.15 at 12 weeks, respectively. Jejunum intraepithelial lymphocytes expressing the CD8 antigen increased gradually until 4-6 weeks of age and subsequently declined as chickens aged. CD4(+)-cells represented a minor subpopulation among the intestinal lymphocyte subpopulations. Therefore, the composition of various T-cell subpopulations in the intestine depended upon host age, the regions of the gut examined and host genetic background. These results suggest that changes in T-cell subpopulations in the intestine may reflect age-related maturation of the gut-associated lymphoid tissues.     

Neurotrophin receptor-like proteins in the bovine (Bos taurus) lymphoid organs, with special reference to thymus and spleen

Increasing evidence suggests that neurotrophins could regulate immune functions acting directly or indirectly on immunocompetent cells. The indirect pathway involves stromal cells of the primary and secondary lymphoid organs. In the present study the occurrence of Trk proteins (TrkA, TrkB and TrkC), regarded as the high-affinity signal-transducing receptors for neurotrophins, was investigated in cow lymphoid organs using immunohistochemistry. The thymus and spleen of both fetal and adult animals, and the palatine tonsils, lymph nodes and Peyer's patches of adult animals, were analysed. Unidentified cells displaying TrkA-like immunoreactivity were found in the fetal thymus, whereas those expressing this protein in the adult gland were identified as epithelial cells. In the spleen, immunoreactive TrkA was observed in cells of the white pulp. TrkB immunoreactivity in both fetal and adult thymus and spleen was localized in monocyte/macrophage cells. As a rule, TrkC was absent from the thymus and the spleen independent of the animal's age. Different types of stromal cells, but never the lymphocytes themselves, displayed TrkA, TrkB, or TrkC immunoreactivity in the other lymphoid organs analysed. As in other vertebrate species, Trk proteins in the lymphoid organs of the cow were localized in the stromal, non-lymphoid cells, thus suggesting that neurotrophins might regulate the immune function acting indirectly on lymphocytes.     

Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues

The frequency and the distribution of apoptotic cells were investigated in formalin-fixed paraffin-embedded lymphoid tissues from healthy conventional pigs at four different ages (6 days, 2 months, 3.5 months and 5 months). Samples of tonsil, mesenteric lymph node, spleen, thymus and Peyer's patches were histologically processed and apoptosis evaluated with the TUNEL reaction and cleaved caspase-3 immunohistochemistry. In each technique, quantification of positive labelling was done for each particular lymphoid tissue area. The labelling pattern and distribution were similar for TUNEL and cleaved caspase-3. TUNEL stained mainly apoptotic bodies inside macrophages, but signal was also seen in free apoptotic bodies and in the nuclei of lymphocyte-like cells. The anti-cleaved caspase-3 antibody labelled mainly nuclei of lymphocyte-like cells. All tissues presented a similar distribution pattern of apoptosis, except for the 6-day-old group. In this group, a scattered distribution of positive cells was detected in tonsil, lymph node and spleen. In the tonsil and mesenteric lymph nodes from the older pigs, follicular areas presented higher amounts of positive cells than interfollicular areas. Moreover, the splenic white pulp showed more positive reaction than the red pulp, especially when they included germinal centres. In all groups, the follicular areas of ileal Peyer's patches presented more labelled cells than the dome and the lamina propria. In the thymus, the higher apoptotic rates were found in the cortex. In general, TUNEL yielded higher rates of positive cells than cleaved caspase-3 immunolabelling. A good correlation between the two techniques was found for thymus, tonsil and mesenteric lymph node, but not for Peyer's patches and spleen. This study describes a detailed histochemical characterization of apoptosis in pig lymphoid tissues using TUNEL and a cleaved caspase-3 immunolabelling at different ages. Moreover, our results indicate that TUNEL and cleaved caspase-3 techniques can be equivalent only when tissues have a high or low levels of apoptosis, since a considerable discrepancy was found in intermediate situations. Data from this study should be useful for future comparative studies under disease conditions.     

Differences in lymphocyte subpopulations from peripheral blood and lymphoid organs in natural caprine tuberculosis infection

Although the cell-mediated immune response is known to be a critical factor in host defence against intracellular mycobacterial infection, the different components of the T-cell response are unclear, particularly in caprine infection. In this study we examine the differences in the lymphocyte population of peripheral blood, spleen and mediastinal and superficial lymph nodes in 11 naturally infected goats showing positive reactions in the comparative tuberculine intradermal test. According to the different types of lesion showing, the goats were classified into proliferative or exudative tuberculosis. The results obtained by fflow cytometry analysis indicated that the main differences in peripheral blood were in the CD4 T-cell population, which decreased markedly in goats with exudative tuberculosis, while the CD8 and B cells increased in number. The gamma/delta T cells did not show significant differences in either type of tuberculosis, while interleukin-2 receptor cells decreased slightly in the exudative tuberculosis. The CD4:CD8 ratio was higher than 1 in goats with proliferative tuberculosis and lower than 1 in goats with exudative tuberculosis. In general, the lymphoid organs of the goats with exudative tuberculosis showed a significant increase in the number of CD8 T cells (CD4:CD8 ratio of less than 1) whereas no significant differences were observed in the CD4 T population between either type of tuberculosis.     

Characterization and Distribution of B Cells in the Lymphoid Organs of Goats

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.