Efforts to develop live attenuated vaccines against Mycobacterium avium subspecies paratuberculosis (Map), using indirect methods to screen Map deletion mutants for potential efficacy, have not been successful. A reduction in the capacity to survive in macrophages has not predicted the ability of mutants to survive in vivo. Previous studies for screening of three deletion mutants in cattle and goats revealed one mutant, with a deletion in relA (ΔMap/relA), could not establish a persistent infection. Further studies, using antigen presenting cells (APC), blood dendritic cells and monocyte derived DC, pulsed with ΔMap/relA or a 35 kDa Map membrane protein (MMP) revealed a component of the response to ΔMap/relA was directed towards MMP. As reported herein, we developed a bacterium viability assay and cell culture assays for analysis and evaluation of cytotoxic T cells generated against ΔMap/relA or MMP. Analysis of the effector activity of responding cells revealed the reason ΔMap/relA could not establish a persistent infection was that vaccination elicited development of cytotoxic CD8 T cells (CTL) with the capacity to kill intracellular bacteria. We demonstrated the same CTL response could be elicited with two rounds of antigenic stimulation of APC pulsed with ΔMap/relA or MMP ex vivo. Cytotoxicity was mediated through the perforin granzyme B pathway. Finally, cognate recognition of peptides presented in context of MHC I and II molecules to CD4 and CD8 T cells is required for development of CTL.

Objective—To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms. Sample Population—Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results. Procedures—Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry. Results—Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect. Conclusions and Clinical Relevance—Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.

Raising chinchilla (Chinchilla laniger) for commercial purpose has increased significantly; however, hematological and serum biochemical reference values have not yet been determined for chinchillas raised in south Brazil. Establishing blood cells and serum biochemistry reference values might be helpful to evaluate health status of chinchillas and might be used as a tool by clinicians. The purpose of this study was to determine the reference values for blood cells and serum biochemistry of Chinchilla laniger. Blood samples were collected by cardiac puncture from 16 adult males, at the time they were killed to remove the fur coat, and from 8 adult males anesthetized with ketamine and xylazine. Blood cell counts and serum biochemistry analysis were performed using standard techniques and the results were expressed as mean ± SEM. Analysis of blood parameters from post-mortem cardiac punctured and from anesthetized chinchillas indicated that blood samples from anesthetized chinchillas had higher PCV, Hemoglobin, MCHC and WBC (P < .05); in contrast, had lower levels of monocytes, basophils and eosinophils (P < .05). Serum biochemical parameters were less affected by sampling method: anesthetized chinchillas had lower levels of urea, glucose and triglycerids (P < .05). The data obtained might be useful as a parameter to monitor the health status of chinchillas raised in south Brazil.

Analysis of the primary and recall responses to a membrane molecule (MMP), encoded by MAP2121c demonstrated that tri-directional signaling between the antigen-presenting cell (APC), CD4 and CD8 is essential for eliciting a CD8 cytotoxic T cell (CTL) response against Mycobacterium avium subsp. paratuberculosis. As reported here, RNA-sequencing was used to initiate the characterization of the signaling pathways involved in eliciting the development of CD8 CTL, starting with the characterization of the activation status of genes in monocyte-derived macrophages (MoMΦ) following uptake and processing MMP for the presentation of antigenic epitopes to CD4 and CD8 T cells. Activation status was compared with the uptake and processing of LPS, a nonspecific stimulator of macrophages. 1609 genes were identified that were upregulated, and 1277 were downregulated three hours after uptake and processing MMP. No significant difference was observed in the cytokine genes selected for analysis of the signaling that must occur between APC, CD4, and CD8 for the development of CTL. The initial observations indicate screening of the transcriptome should include genes involved in signaling between APC and CD4, and CD8 regardless of their activation status. Four genes of interest in this study, IL12A, IL12B, IL15, and IL23A, were not significantly different from control values. The initial studies also indicate MoMΦ can be included with dendritic cells and monocyte-derived dendritic cells for further analysis of the tri-directional signaling required for the development of CTL. A análise das respostas primárias e de recall a uma molécula de membrana (MMP), codificada por MAP2121c demonstrou que a sinalização tridirecional entre a célula apresentadora de antígeno (APC), CD4 e CD8 é essencial para provocar uma resposta de células T citotóxicas CD8 (CTL) contra Mycobacterium avium subsp. paratuberculose. Conforme relatado aqui, o sequenciamento de RNA foi usado para iniciar a caracterização das vias de sinalização envolvidas na indução do desenvolvimento de CTL CD8, começando com a caracterização do status de ativação de genes em macrófagos derivados de monócitos (MoMΦ) após captação e processamento de MMP para a apresentação de epítopos antigênicos às células T CD4 e CD8. O status de ativação foi comparado com a captação e processamento de LPS, um estimulador inespecífico de macrófagos. Foram identificados 1.609 genes que foram regulados positivamente e 1.277 foram regulados negativamente três horas após a captação e processamento de MMP. Nenhuma diferença significativa foi observada nos genes de citocinas selecionados para análise da sinalização que deve ocorrer entre APC, CD4 e CD8 para o desenvolvimento de CTL. As observações iniciais indicam que o rastreio do transcriptoma deve incluir genes envolvidos na sinalização entre APC e CD4 e CD8, independentemente do seu estado de activação. Quatro genes de interesse neste estudo, IL12A, IL12B, IL15 e IL23A, não foram significativamente diferentes dos valores de controle. Os estudos iniciais também indicam que o MoMΦ pode ser incluído com células dendríticas e células dendríticas derivadas de monócitos para análise adicional da sinalização tridirecional necessária para o desenvolvimento de CTL.