Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis

Oral infection of goats with Mycobacterium avium subsp. hominissuis (MAH) resulted in a large variety of granulomas in organized gut-associated lymphatic tissues and intestinal lymph nodes. To characterize the cellular composition of granulomas, CD4⁺, CD8⁺, γδ, B lymphocytes and plasma, CD25⁺, CD68⁺, MHC-II⁺, Ki67⁺ and endothelial cells were labeled in consecutive frozen sections by immunohistochemistry and acid fast bacilli (AFB) by Kinyoun stain. Granulomas with extensive necrosis, little mineralization and variable numbers of AFB surrounded by many CD4⁺ T cells, but only few epitheloid macrophages were observed in severely sick goats at 2–3 mpi. They were interpreted as exuberant immune reaction. Organized granulomas with very few AFB were seen in clinically healthy goats at 13 mpi. The necrotic cores were surrounded by a zone of granulomatous infiltrate with many epitheloid macrophages and few lymphocytes. This zone was initially wide and highly vascularized and became progressively smaller. It was enclosed by an increasing layer of connective tissue. All organized granulomas were surrounded by compartimentalized tertiary lymphoid tissue. The granulomas in experimental infection of goats with MAH reflect the heterogeneity of lesions seen in mycobacterial infections of humans and ruminants and are therefore valuable for comparative research.     

Characterization of an intravenous lipopolysaccharide inflammation model in calves with respect to the acute-phase response

Our objective was to develop a lipopolysaccharide (LPS) inflammation model in calves to evaluate the acute-phase response with respect to the release of pro-inflammatory cytokines and acute-phase proteins, fever development and sickness behaviour. Fourteen 4-week-old male Holstein Friesian calves were included and randomly assigned to a negative control group (n = 3) and an LPS-challenged group (n = 11). The latter received an intravenous bolus injection of 0.5 μg of LPS/kg body weight. Blood collection and clinical scoring were performed at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 18, 24, 28, 32, 48, 54 and 72 h post LPS administration (p.a.). In the LPS group, the following clinical signs were observed successively: tachypnoea (on average 18 min p.a.), decubitus (29 min p.a.), general depression (1.75 h p.a.), fever (5 h p.a.) and tachycardia (5 h p.a.). Subsequent to the recovery from respiratory distress, general depression was prominent, which deteriorated when fever increased. One animal did not survive LPS administration, whereas the other animals recovered on average within 6.1 h p.a. Moreover, the challenge significantly increased plasma concentrations of tumour necrosis factor-α, interleukin 6, serum amyloid A and haptoglobin, with peaking levels at 1, 3.5, 24 and 18 h p.a., respectively. The present LPS model was practical and reproducible, caused obvious clinical signs related to endotoxemia and a marked change in the studied inflammatory mediators, making it a suitable model to study the immunomodulatory properties of drugs in future research.     

Use of Fourier-transform infrared spectroscopy to quantify immunoglobulin G concentration and an analysis of the effect of signalment on levels in canine serum

Deficiency in immunoglobulin G (IgG) is associated with an increased susceptibility to infections in humans and animals, and changes in IgG levels occur in many disease states. In companion animals, failure of transfer of passive immunity is uncommonly diagnosed but mortality rates in puppies are high and more than 30% of these deaths are secondary to septicemia. Currently, radial immunodiffusion (RID) and enzyme-linked immunosorbent assays are the most commonly used methods for quantitative measurement of IgG in dogs. In this study, a Fourier-transform infrared spectroscopy (FTIR) assay for canine serum IgG was developed and compared to the RID assay as the reference standard. Basic signalment data and health status of the dogs were also analyzed to determine if they correlated with serum IgG concentrations based on RID results.Serum samples were collected from 207 dogs during routine hematological evaluation, and IgG concentrations determined by RID. The FTIR assay was developed using partial least squares regression analysis and its performance evaluated using RID assay as the reference test. The concordance correlation coefficient was 0.91 for the calibration model data set and 0.85 for the prediction set. A Bland–Altman plot showed a mean difference of −89 mg/dL and no systematic bias. The modified mean coefficient of variation (CV) for RID was 6.67%, and for FTIR was 18.76%.The mean serum IgG concentration using RID was 1943 ± 880 mg/dL based on the 193 dogs with complete signalment and health data. When age class, gender, breed size and disease status were analyzed by multivariable ANOVA, dogs <2 years of age (p = 0.0004) and those classified as diseased (p = 0.03) were found to have significantly lower IgG concentrations than older and healthy dogs, respectively.     

Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis

Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs.     

The γδ cells as marker of non-seroconverted cattle naturally infected with Mycobacterium avium subspecies paratuberculosis

Early diagnosis of MAP infection is a pressing need to enable efficient intervention with the spread of MAP infection in herds. Hence, study of lymphocyte subsets and their expressed adhesion molecules could contribute in defining a distinct diagnostic marker (or markers) at the subclinical period of the infection that could in turn facilitate the development of effective diagnostic approach. In accordance with this objective, milk and blood samples were collected from two groups of cattle naturally infected with MAP and their corresponding negative controls. Group (C) comprised 3-4 year-old ELISA negative/PCR positive-cattle that were considered as subclinical seronegative low shedder group (early stage). Group (A) included 6-8 year-old ELISA positive-cattle, which were considered as a clinical seropositive group (late stage). Flow cytometry of B cells, CD8⁺, CD4⁺ and γδ cells and the adhesion molecules CD44⁺, CD62L, LFA-1 and LPAM-1 indicated increase in CD4⁺ and B cells levels, with higher levels in blood than milk of group A, and significant expression of CD44⁺ in blood and milk and LPAM-1 in blood only. The CD8⁺ cells count in milk was higher than blood in the late stage. The peculiar feature of the early stage (group C) was the high level of γδ cells in the blood and milk, with tendency to express high level of CD62L. Compelling evidence could support the assumption that the dominant γδ cells at early stage of MAP infection could be of CD8CD2⁻WC+1⁺ phenotype. γδ cells appear as promising markers in defining early changes of MAP infection due to their important role in priming innate and cell mediated immunity. Possible utilization of these peculiar changes in the γδ cells level in the early diagnosis of MAP infection should be the subject of further research.     

Changes in Lymphocyte Subsets in the Bronchus‐associated Lymphoid Tissue of Goats Naturally Infected with Different Mycoplasma Species

The distribution of cells containing lysozyme, S‐100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus‐associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3–5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1–6), M. mycoides ssp. capri (goats no. 7–12) and M. capricolum ssp. capricolum (goats no. 13–18). Microscopically, infected animals showed a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3⁺ T lymphocytes, and the ratio of CD4⁺:CD8⁺ cells was >2. The BALT showed large germinal centres mainly composed of IgG⁺ B lymphocytes, with numerous S‐100⁺ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG⁺ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.     

Postnatal Development of Lymphocyte Subpopulations in the Intestinal Lymph Nodes in Goats

The incidence and location of CD2⁺, CD4⁺, CD8⁺ and γ/δ T lymphocytes and IgM⁺ B lymphocytes were studied in the intestinal lymph nodes in 1-week, 1-month, 3-month and 7-month-old goats, using monoclonal antibodies and immuno-histochemical methods. The cortical area of the intestinal lymph nodes in 1-week-old animals contains only primary follicles occupied by IgM⁺ B lymphocytes and some CD2⁺CD4⁺ T lymphocytes. In goats older than 1 month, secondary follicles, that increased in number and size with age, were observed; the light zone of the germinal centre was occupied by IgM⁺ lymphocytes and some CD2⁺ and CD4⁺ T lymphocytes. In the other compartments of the lymph nodes, B lymphocytes were scarce, their number increasing with age in the medulla and diminishing in the paracortex. The numerous CD2⁺ T lymphocytes in the interfollicular area increased in number in the paracortical area of the 7-month-old goats, simultaneously with an increase in the MHC II⁺ dendritic cells and the CD4/CD8 ratio, which was greater than 1. The γ/δ T lymphocytes represented a minor subpopulation scattered through the lymph nodes.     

Local assessment of WC1+ γδ T lymphocyte subset in the different types of lesions associated with bovine paratuberculosis

The local expression of WC1⁺ γδ T lymphocytes subset has been evaluated by immunohistochemical methods at the different types of lesions present in cows naturally infected with Mycobacterium avium subsp. paratuberculosis (Map) and in non-infected control animals. Infected cattle were either in the latent/subclinical (focal lesions) or clinical (diffuse paucibacillary and multibacillary forms) stage of paratuberculosis. To assess the cell distribution, a differential cell count was carried out at the lamina propria, gut-associated lymphoid tissue and submucosa. A significant increase in the number of WC1⁺ γδ T cells was observed in all the infected animals, regardless of the type of lesion. Cows with focal lesions showed higher number of labeled cells than those with diffuse forms, where no differences were found between the two types. This increase in the number of positively immunolabelled lymphocytes in infected animals was seen in the lamina propria, with higher values in those with focal lesions. While in the lymphoid tissue no differences in the numbers were observed, in animals with focal lesions, WC1⁺ γδ T cells tended to be located at the periphery of the granulomas. These findings suggest a proinflammatory action of WC1⁺ γδ T lymphocytes in bovine paratuberculosis, which might play an important role in the containment of the Map-infection in the focal granulomas located in the lymphoid tissue, helping to prevent the progression toward diffuse forms responsible for the clinical signs.     

Immunohistochemical characterization of Kisselev nodules (ectopic lymphoid follicles) in wild boar (Sus scrofa L.)

This paper describes the histopathological features and cellular distribution of T lymphocytes (CD3), B cells (CD79), follicular dendritic cells (FDC) and macrophages (alpha-1-antitrypsin, lysozyme) in lymphoid aggregates (Kisselev nodules) found in the lung, kidney and liver of wild boar (Sus scrofa L.). The distribution of immunoreactive cells, tested for antibodies, was similar to that found in the cortex of lymph nodes: lymphoid follicles with germinal centers mainly consisting of CD79⁺ B cells with sparse interfollicular tissue (CD3⁺ T lymphocytes). This finding and the association of these structures with helminthic infections suggests that local humoral immunity is central to the organism’s response to parasitic challenge. The presence of follicular dendritic cells confirms the high degree of organization of these lymphoid-like structures. The role of other pathogenic factors and the induction of chronic inflammatory reaction in these ectopic lymphoid sites is also discussed.     

Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep

A method to assess the expansion of antigen-specific intracellular IFN-γ positive T cell subsets during the infection will be helpful for a better understanding of mycoplasmal infections physiopathology in the sheep. We analysed the percentage of antigen-specific lymphocytes positive for intracellular IFN-γ during the infection of sheep with Mycoplasma agalactiae by culturing peripheral blood mononuclear cells of infected or uninfected animals with irradiated M. agalactiae. The expansion of antigen-specific IFN-γ positive lymphocytes in infected sheep was initially sustained by CD4⁺ T cells at day 15 after infection, when antigen specific IgG start to be detectable, followed by CD8/IFN-γ double positive cells. γδ T-cells were not expanded at any time point analysed. IFNγ⁺ T cells disappear 60 days after infection, suggesting that antigen specific IFNγ⁺ T cells, mainly detected in the early phase of the disease, could be useful to understand the role of cell-mediated immunity during M. agalactiae infection.     

Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep

A method to assess the expansion of antigen-specific intracellular IFN-γ positive T cell subsets during the infection will be helpful for a better understanding of mycoplasmal infections physiopathology in the sheep. We analysed the percentage of antigen-specific lymphocytes positive for intracellular IFN-γ during the infection of sheep with Mycoplasma agalactiae by culturing peripheral blood mononuclear cells of infected or uninfected animals with irradiated M. agalactiae. The expansion of antigen-specific IFN-γ positive lymphocytes in infected sheep was initially sustained by CD4⁺ T cells at day 15 after infection, when antigen specific IgG start to be detectable, followed by CD8/IFN-γ double positive cells. γδ T-cells were not expanded at any time point analysed. IFNγ⁺ T cells disappear 60 days after infection, suggesting that antigen specific IFNγ⁺ T cells, mainly detected in the early phase of the disease, could be useful to understand the role of cell-mediated immunity during M. agalactiae infection.     

Engraftment of canine peripheral blood lymphocytes into nonobese diabetic-severe combined immune deficient IL-2R common gamma chain null mice

To study the canine immune system we generated a mouse model engrafted with canine lymphocytes using NOD SCID IL2R common gamma chain ?/? (NSG) mice as recipients (Ca-PBL-SCID). Engraftment of canine peripheral blood lymphocytes (PBLs) was determined post-injection with 10⁷ peripheral blood mononuclear cells (PBMCs) into irradiated NSG mice using flow cytometry and fluorescently labeled antibodies specific to canine helper T cells (CD45⁺ CD4⁺), cytotoxic lymphocytes (CD45⁺ CD8⁺), regulatory T cells (CD45⁺ CD4⁺ Foxp3⁺), and B cells (CD45⁺ Ig⁺ CD21_lo). Canine CD45⁺ lymphocytes were detectable as early as day 1 in the peritoneal cavity, and beginning at 9 days in the blood, bone marrow, and spleen. CD4⁺ T cells, of which Foxp-3⁺ CD25_hi cells constituted a minor percentage, were the predominant lymphocyte population at 9 days post engraftment contrasting with increasing proportions of CD8⁺ CTL's and Ig⁺ B cells beginning at 16 days. Canine immunoglobulin was initially detected in the serum of Ca-PBL-SCID mice at 9 days post-engraftment and peaked in concentration at day 36. From day 28 to 52 post-engraftment 30% of the Ca-PBL-SCID mice became markedly anemic and thrombocytopenic, yet gross and histopathologic examination of bone marrow, kidneys, spleen, liver, and intestine revealed no obvious lesions. Blood smear evaluation revealed agglutination of mature red blood cells, reticulocytes and a regenerative anemia. These findings demonstrate that NSG mice are capable of engraftment of canine PBLs yet develop graft versus host disease similar to Hu-PBL-SCID mice.     

Inhibitory effects of SKF96365 on the activities of K+ channels in mouse small intestinal smooth muscle cells

In order to investigate the effects of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (SKF), which is a non-selective cationic channel blocker, on K⁺ channel currents, we recorded currents through ATP sensitive K⁺ (I_KATP), voltage-gated K⁺ (I_Kv) and Ca²⁺ activated K⁺ channels (I_BK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished I_KATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K⁺ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC₅₀) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC₅₀ value in I_KATP suppression) reversibly inhibited the I_Kv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient I_BK and caffeine-induced I_BK. These results indicated that SKF inhibited ATP sensitive K⁺ channels and voltage-gated K⁺ channels, with the ATP sensitive K⁺ channels being more sensitive than the voltage-gated K⁺ channels. These inhibitory effects on K⁺ channels should be considered when SKF is used as a cationic channel blocker.     

Comparison of cytokine mRNA expression in peripheral CD4+, CD8+ and γδ T cells between healthy Holstein and Japanese Black calves

Japanese Black (JB) calves are more susceptible to infectious diseases compared to Holstein (Hol) calves. To clarify the immunological differences between JB and Hol calves, expression of cytokine messenger RNA (mRNA) was examined using peripheral CD4⁺, CD8⁺ and γδ T cells. Healthy calves, 24 from each species, were examined. Blood samples were obtained from calves at 1 week, 1 month and 3 months old, eight calves for each age of each species. Peripheral blood mononuclear cells were stimulated with phytohemagglutin (PHA), and T cell subsets were isolated by positive selection using magnetic cell sorting (MACS). Levels of interlekin (IL)‐2, IL‐4, IL‐10 and interferon (IFN)‐γ mRNA in three T cell subsets were analyzed. WC1‐N1⁺ γδ T cell percentages were significantly lower in JB calves at 1 week and 1 month of age compared to Hol calves. In addition JB calves had significantly lower IL‐2, IL‐10 and IFN‐γ mRNA in WC1‐N1⁺ γδ T cells at 1 and 3 months of age, whereas there were no significant differences in cytokine mRNA of CD4⁺ and CD8⁺ cells between the two groups. Decreased cytokine mRNA and cell number of peripheral γδ T cells may affect negatively on the immune system of JB calves.     

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3⁺, CD4⁺ and γδ T-cells; whereas CD8⁺ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.     

Induction of inducible CD4+CD25+Foxp3+ regulatory T lymphocytes by porcine reproductive and respiratory syndrome virus (PRRSV)

Increases in numbers or activities of regulatory T lymphocytes (Tregs) have been linked to the establishments of several persistent infections. It has been previously shown that porcine reproductive and respiratory syndrome virus (PRRSV) can negatively modulate the host immune responses, resulting in persistent infection and secondary immunodeficiency. Recently, the existence of porcine CD4⁺CD25⁺ Tregs has been demonstrated. We investigated the effect of PRRSV on the CD4⁺CD25⁺ Tregs. The CD4⁺CD25⁺Foxp3⁺ T lymphocytes in the peripheral blood mononuclear cells (PBMCs) were identified, using the anti-human anti-Foxp3 monoclonal antibody. In vitro culture of porcine PBMC in the presence of PRRSV, but not classical swine fever virus, significantly increased the numbers of Foxp3⁺ lymphocytes, particularly in the CD4⁺CD25^high subpopulation. The time-course study revealed that PRRSV significantly increased the numbers of viral-specific CD4⁺CD25^highFoxp3⁺ subpopulation in the culture starting from 12 h through the end of the observation period. Consistent to the results obtained by flow cytometry, enhanced Foxp3 gene expression was observed in the PBMC cultured with PRRSV in a time-course manner. The presence of monocyte-derived DC in the co-culture significantly enhanced the induction of CD4⁺CD25⁺ Foxp3⁺ T lymphocytes. The PRRSV-induced CD4⁺CD25^high T lymphocytes exhibited suppressive activity when co-cultured with PHA-activated, autologous peripheral blood leukocytes, indicating the suppressive activity of the PRRSV-specific Tregs. In addition, PRRSV exposure significantly increased the numbers of PRRSV-specific CD4⁺CD25⁺Foxp3⁺ subpopulation in the PBMC of infected pigs at 10 days post-infection. In summary, the results indicated that PRRSV could increase the numbers of viral-specific, inducible regulatory T lymphocytes in the porcine PBMC, both in vitro and in vivo. The findings suggested the novel immunomodulatory mechanism induced by PRRSV.     

Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ⁺CD8⁺IFN-γ⁺ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ⁺CD4⁺ T cells were also found to increase in the infected mice, however later than CD8⁺ T cells. Interestingly, splenic and MLN CD4⁺CD25⁺ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ⁺CD8⁺ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.     

Study of the local immune response to Fasciola hepatica in the liver and hepatic lymph nodes of goats immunised with a peptide of the Sm14 antigen

The nature of the local immune response was assessed studying the distribution of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes, IgM⁺ B cells, IL-4⁺ and IFN-γ⁺ cells in the liver and hepatic lymph nodes (HLN) of goats immunised with a synthetic peptide of the Sm14 antigen from Schistosoma mansoni and challenged with Fasciola hepatica. A morphometric study of HLN was also carried out in order to evaluate the hyperplasia of lymphoid follicles. Despite the decrease in fluke burdens found in the immunised group (45.9%) respect to the infected control group, this difference was not statistically significant due to the high individual variability. In liver, a significant increase of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes was found in the infected groups respect to the uninfected control and in the infected control respect to the immunised group. HLN showed a significant enlargement due to the hyperplasia of lymphoid follicles and infiltration of CD2⁺, CD4⁺, CD8⁺, γδ⁺ T lymphocytes in both infected groups respect to the uninfected control, with no significant differences between the infected control and immunised group. IFN-γ⁺ lymphoid cells was absent or very occasional in HLN where the number of IL-4⁺ cells was higher than that of IFN-γ, suggesting a polarized Th2 response in immunised and in infected control group.     

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4⁺CD25⁺Foxp3⁺ T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3⁺CD4⁺ cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4⁺ cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4⁺ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4⁺ and CD25⁺ T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4⁺ cells in the CD4⁺ T cell subpopulation was positively correlated with TGF-β mRNA expression, suggesting that CD4⁺CTLA-4⁺ T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.     

Investigation of antigen-specific T-cell responses and subcutaneous granuloma development during experimental sensitization of calves with Mycobacterium avium subsp paratuberculosis

OBJECTIVE: To characterize the early cellular immune response to Mycobacterium avium subsp paratuberculosis (MAP) infection and evaluate the development of granulomatous inflammation at the SC injection site in experimentally inoculated calves. ANIMALS: Forty-eight 4-week-old calves. PROCEDURE: Calves received an SC injection of MAP strain 19698 (n = 25), sterile saline (0.9% NaCl) solution (20), or a commercial paratuberculosis vaccine (3); the inoculation site tissue and associated draining lymph node were excised at postinoculation day (PID) 0 (n = 36), 7 (14), 14 (6), 21 (8), and 60 (32). Sections of inoculation site tissues were evaluated immunohistochemically for T-cell subsets; lymph node mononuclear cells (LNMCs) were assessed for T-cell surface markers and for intracellular interferon-gamma via flow cytometry. RESULTS: At MAP inoculation sites, calves developed mild, focal granulomatous inflammation by PID 7; by PID 60, areas of inflammation contained macrophages with numerous lymphocytes. Compared with control calves, there was increased antigen-specific LNMC proliferation in MAP- and vaccine-inoculated calves at PID 60, although proliferation among lymphocyte subsets was not significantly different between MAP-inoculated and control calves; in vaccine-inoculated calves, CD4+ T-cells predominated. In MAP-inoculated and control calves, antigen-specific interferon-gamma production by LNMCs did not differ significantly; vaccine-inoculated calves had marked interferon-gamma expression by CD4+ T-cells. CONCLUSIONS AND CLINICAL RELEVANCE: In calves, SC administration of MAP resulted in granulomatous inflammation at inoculation sites and an antigen-specific T-cell proliferative response. Results suggest that this experimental system can be used to reproducibly generate antigen-specific T-cells during MAP infection for functional analysis.