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Little is known about the role that B cells play in immune responses to infection with the intracellular pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). Traditionally, the role of B cells has been constrained to their function as antibody-producing cells, however, antibodies are not thought to play a protective role in mycobacterial infections. The present study was designed to characterize B cell subpopulations as well as activation/maturation states in cattle with paratuberculosis. Peripheral blood mononuclear cells (PBMCs) were isolated from noninfected control cows (n = 8); as well cattle naturally infected with MAP in the subclinical (n = 8) and clinical (n = 7) stage of infection and stimulated with MAP antigen for 6 days. MAP infection resulted in greater numbers of total B cells for clinical cows compared to control noninfected cows. The major subpopulation in freshly isolated PBMCs in clinical cows was B-1a B cells, but this shifted to a composite of both B-1a and B-2 B cells upon stimulation of PBMCs with either MAP antigen or pokeweed mitogen, with higher numbers of B-2 B cells. Early B cells were observed to predominate the population of B cells in PBMCs, with lesser populations of germinal B cells, memory B cells and plasma cells. These subpopulations were elevated in clinical cows upon stimulation of PBMCs with MAP antigen, except for plasma cells which were lower compared to control noninfected cows. Increased numbers of B cells in clinical cows aligned with higher expression of B cell markers such as MAPK1/3, BTG1, Bcl2, CD79A and SWAP70, depending upon in vitro stimulation with either mitogen or antigen. This would indicate that the B cells were capable of activation but were anti-apoptotic in nature. The shift to B-2 B cells in the periphery of clinical cows seems to be indicative of an expansion of memory B cells, rather than plasma cells. This may be a last attempt by the host to control the rampant inflammatory state associated with advanced clinical disease. [ABSTRACT FROM AUTHOR]

The immune system is not always successful in recognizing and destroying pathogens it may encounter. Host immunity to mycobacteria is characterized by a very complex series of events, designed to clear the infection. The first line of defense is uptake and processing of the pathogen by macrophages, followed by the initiation of cell-mediated immunity. The secretion of pro-inflammatory cytokines such as IFN-?? is credited with containment of mycobacterial infections. Yet it is clear that activated T-cells may contain but fail to clear the infection in some hosts. Further, it is recognized that if infection progresses to a more clinical state, the production of pro-inflammatory cytokines is suppressed and expression of anti-inflammatory cytokines is increased. It is unclear what defines a host that can successfully contain the infection versus one that succumbs to severe immunopathologic disease. This review will address some of the key elements in host immunity to mycobacterial pathogens, with an emphasis on Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), in an attempt to understand the dialogue between immune cells and their mediators during infection and what causes this discourse to go awry. [ABSTRACT FROM PUBLISHER]

Our objective was to evaluate the effects of time and temperature on whole blood used in the gamma interferon enzyme-linked immunosorbent assay (IFN- γ ELISA) for paratuberculosis along with evaluating four potential positive controls, and four different mycobacterial antigens for the ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium ssp. paratuberculosis were used in a randomized complete block design. Forty-nine blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6 and 4.4°C for 0, 4, 8, 12, 18, 24, 32, 48 and 72 h. Each blood tube was tested with four mycobacterial antigens (two johnin PPDs, an avain PPD and a whole cell sonicate) and four potential positive controls [concanavalin A (conA), phytohaemagglutinin A (PHA), pokeweed mitogen (PWM) and Staphylococcus enterotoxin A (SEA)]. After incubation for 24 h, the plasma was assayed with a commercial IFN- γ ELISA. Blood stored at 21.1 and 15.6°C maintained the highest ELISA optical densities (OD) over time with severe reduction in OD values at or above 37.8°C. None of the potential positive controls exactly mimicked the antigen response. SEA and PWM were able to elicit a response after the whole blood quit responding to the antigen and conA underestimated the responsiveness. Phytohemagglutinin A was similar to the antigens on an average, but there was significant disagreement among samples. The PPDs were more potent at stimulating IFN- γ production than the whole cell sonicate. In conclusion, whole blood should be stored/transported at ambient room temperature and stimulated within 12 h of collection. [ABSTRACT FROM AUTHOR]

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is estimated to infect more than 22% of US dairy herds. Periods of immunosuppression, typically seen at parturition, may contribute to the transition from subclinical, or asymptomatic, to clinical stage of infection. Understanding the effects of stressors disease may provide information that will help manage JD. The objective of this study was to characterize cytokine gene expression in periparturient dairy cows naturally infected with MAP. Twenty-two multiparous Holstein cows were placed into 3 groups consisting of 5 noninfected healthy cows, 12 subclinical cows, and 5 clinical cows. Blood was collected from the jugular vein 3 wks preand 4 wks post-calving. Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat fractions of blood and cultured for 24 h with and without concanavalin A. At 24 h, RNA was extracted from all cells and converted to first-strand cDNA. Real-time PCR was performed on each sample to evaluate the expression of the following genes: IFN-γ, TNF-α, IL-12, IL-10, TGF-β, IL-4 and β-actin gene. All reactions were performed in triplicate and RT-PCR data was analyzed using delta-Ct values and Proc Mix procedure of SAS. Across the periparturient period, expression of IFN-γ, TNF-α, and TGF-β mRNA did not differ between nil- and stimulated PBMCs isolated from infected and control cows. ConA-stimulated PBMCs showed greater IL-12 mRNA expression for both subclinical (P <0.03) and clinical (P <0.04) cows compared to controls. Expression of IL-4 mRNA from nil- (P <0.03) and stimulated (P <0.01) PBMCs was greater in infected compared to healthy control cows. Expression of IL-10 mRNA from nil- (P <0.08) and stimulated (P <0.05) PBMCs was greater for infected cows compared to control cows. The data indicate an ability of parturition to regulate IL-12 and IL-4 gene expression, but not IFN-γ, TNF-α, TGF-β, or IL-10. Expression for the Th1 cytokine IL-12 and the Th2 cytokines IL-4 and IL-10 from infected cows were significantly elevated compared to healthy controls. [ABSTRACT FROM AUTHOR]