Relatively little is known about regulatory T (Treg) cells and their functional responses in dogs. We have used the cross-reactive anti-mouse/rat Foxp3 antibody clone FJK-16s to identify a population of canine CD4⁺ FOXP3^high T cells in both the peripheral blood (PB) and popliteal lymph node (LN). FOXP3⁺ cells in both PB and LN yielded positive staining with the newly developed anti-murine/human Helios antibody clone 22F6, consistent with the notion that they were naturally occurring Treg cells. Stimulation of mononuclear cells of LN origin with concanavalin A (Con A) in vitro yielded increased proportions and median fluorescence intensity of FOXP3 expression by both CD4⁺ and CD8⁺ T cells. Removal of the Con A and continued culture disclosed a CD4⁺ FOXP3^high population, distinct from the CD4⁺ FOXP3^intermediate T cells; very few CD8⁺ FOXP3^high T cells were observed, though CD8⁺ FOXP3^intermediate cells were present in equal abundance to CD4⁺ FOXP3^intermediate cells. The CD4⁺ FOXP3^high T cells were thought to represent activated Treg cells, in contrast to the FOXP3^intermediate cells, which were thought to be a more heterogeneous population comprising predominantly activated conventional T cells. Co-staining with interferon-γ (IFN-γ) supported this notion, because the FOXP3^high T cells were almost exclusively IFN-γ⁻, whereas the FOXP3^intermediate cells expressed a more heterogeneous IFN-γ phenotype. Following activation of mononuclear cells with Con A and interleukin-2, the 5% of CD4⁺ T cells showing the highest CD25 expression (CD4⁺ CD25^high) were enriched in cells expressing FOXP3. These cells were anergic in vitro, in contrast to the 20% of CD4⁺ T cells with the lowest CD25 expression (CD4⁺ CD25⁻), which proliferated readily. The CD4⁺ CD25^high FOXP3^high T cells were able to suppress the proliferation of responder CD4⁺ T cells in vitro, in contrast to the CD4⁺ CD25⁻ cells, which showed no regulatory properties.

Background: Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is widespread in sub-Saharan Africa. The current live vaccine T1/44 has limited efficacy and occasionally leads to severe side effects in the animals. A better understanding of the immune responses triggered by Mycoplasma mycoides subsp. mycoides and their role in disease progression will help to facilitate the design of a rational vaccine. Currently, knowledge of cytokines involved in immunity and immunopathology in CBPP is rather limited. The aim of this study was to characterize the in vivo plasma concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10 and the overall role of CD4⁺ T cells in the development of cytokine levels during a primary infection. Plasma cytokine concentrations in two groups of cattle (CD4⁺ T cell-depleted and non-depleted cattle) experimentally infected with Mycoplasma mycoides subsp. mycoides were measured and their relationship to the clinical outcomes was investigated. Results: Plasma cytokine concentrations varied between animals in each group. Depletion of CD4⁺ T cells did not induce significant changes in plasma levels of TNF-α, IL-4, and IL-10, suggesting a minor role of CD4⁺ T cells in regulation or production of the three cytokines during the time window of depletion (1-2 weeks post depletion). Unexpectedly, the IFN-γ concentrations were slightly, but statistically significantly higher in the depleted group (p < 0.05) between week three and four post infection. Three CD4⁺ T cell-depleted animals that experienced severe disease, had high levels of TNF-α and IFN-γ. Only one severely diseased non-depleted animal showed a high serum concentration of IL-4 post infection. Conclusions: Comparison of most severely diseased animals, which had to be euthanized prior to the expected date, versus less severe diseased animals, irrespective of the depletion status, suggested that high TNF-α levels are correlated with more severe pathology in concomitance with high IFN-γ levels.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4⁺ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known. We investigated the role of CD4⁺ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4⁺ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4⁺ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4⁺ T cells in control of CBPP.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae is a severe disease widespread in Africa and Asia. Limited knowledge is available on the pathogenesis of this organism, mainly due to the lack of a robust in vivo challenge model and the means to do site-directed mutagenesis. This work describes the establishment of a novel caprine challenge model for CCPP that resulted in 100% morbidity using a combination of repeated intranasal spray infection followed by a single transtracheal infection employing the recent Kenyan outbreak strain ILRI181. Diseased animals displayed CCPP-related pathology and the bacteria could subsequently be isolated from pleural exudates and lung tissues in concentrations of up to 109 bacteria per mL as well as in the trachea using immunohistochemistry. Reannotation of the genome sequence of ILRI181 and F38T revealed the existence of genes encoding the complete glycerol uptake and metabolic pathways involved in hydrogen peroxide (H2O2) production in the phylogenetically related pathogen M. mycoides subsp. mycoides. Furthermore, the expression of L-α-glycerophosphate oxidase (GlpO) in vivo was confirmed. In addition, the function of the glycerol metabolism was verified by measurement of production of H2O2 in medium containing physiological serum concentrations of glycerol. Peroxide production could be inhibited with serum from convalescent animals. These results will pave the way for a better understanding of host–pathogen interactions during CCPP and subsequent vaccine development.

Capsular polysaccharides have been confirmed to be an important virulence trait in many gram-positive and gram-negative bacteria. Similarly, they are proposed to be virulence traits in minimal Mycoplasma that cause disease in humans and animals. In the current study, goats were infected with the caprine pathogen Mycoplasma mycoides subsp. capri or an engineered mutant lacking the capsular polysaccharide, galactofuranose. Goats infected with the mutant strain showed only transient fever. In contrast, 5 of 8 goats infected with the parental strain reached end-point criteria after infection. These findings confirm that galactofuranose is a virulence factor in M. mycoides.