Search

ABSTRACTCD40 and CD40 ligand (CD40L) have costimulatory effects as part of a complex series of events in host immunity. In this study, the expression of CD40 and CD40L on peripheral blood mononuclear cells (PBMCs) isolated from cattle with Johne's disease were measured on freshly isolated PBMCs and on cells cultured for 8, 24, and 72 h in the presence or absence of live Mycobacterium aviumsubsp. paratuberculosisand exogenous gamma interferon, interleukin 10, and transforming growth factor ß. Results demonstrated greater CD40 and CD40L expression on fresh PBMCs obtained from animals in the clinical stage of disease (symptomatic) than those from healthy control animals or cows in the subclinical stage of disease (asymptomatic). A similar expression profile with greater magnitude was noted for cultured PBMCs, with increased CD40 expression after 8 and 24 h of culture and increased CD40L expression between 24 and 72 h on PBMCs obtained from clinically infected animals. The addition of live M. aviumsubsp. paratuberculosisto cell cultures resulted in downregulation of CD40L expression in naturally infected cows, regardless of the disease stage. In contrast, the addition of live M. aviumsubsp. paratuberculosisto cultures resulted in upregulation of CD40 expression on cells obtained from clinically infected animals, while a decrease in expression was noted for healthy and subclinically infected cows. No effects of exogenous cytokines on CD40 or CD40L expression were observed. These results clearly point for the first time to a disparity in the expression of these costimulatory molecules on immune cells from cattle in different stages of Johne's disease and suggest further investigation into their roles in paratuberculosis pathogenesis.

ABSTRACTThe cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium aviumsubsp. paratuberculosisrecombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. aviumsubsp. paratuberculosisantigens were compared in calves inoculated with live M. aviumsubsp. paratuberculosis, M. aviumsubsp. avium(M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-?) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. aviumsubsp. paratuberculosisproteins also elicited nonspecific IFN-? responses in inoculated calves, with the exception of calves infected with M. bovis. M. aviumsubsp. paratuberculosisproteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (?d) T, and B cells for the calves that were inoculated with either M. aviumsubsp. paratuberculosisor M. aviumafter antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO+CD25+T cells from calves inoculated with M. aviumsubsp. paratuberculosisand M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. aviumsubsp. paratuberculosis, the differences in immune responses to M. aviumsubsp. paratuberculosisantigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. aviumsubsp. paratuberculosisand those inoculated with M. aviumthat were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.

ABSTRACTA major drawback of current whole-cell vaccines for Mycobacterium aviumsubsp. paratuberculosisis the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n= 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n= 5). The results from this study demonstrated a rapid initiation of M. aviumsubsp. paratuberculosis-specific gamma interferon (IFN-?) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovispurified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovisrecombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. aviumsubsp. paratuberculosisantibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovisantigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis.

ABSTRACTAs a consequence of continued spillover of Mycobacterium bovisinto cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovisantigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at ~90 to 100 days after experimental M. bovischallenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacteriumspp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing.

ABSTRACTThe objective of this study was to determine if experimental infection of neonatal calves with Mycobacterium aviumsubsp. paratuberculosiswould invoke changes in the percentages of total B cells in the peripheral blood mononuclear cell population and of subpopulations of B cells as determined by CD5, CD25, and CD45RO markers during a 12-month period. Experimental infection groups included control (noninfected), oral (infected with M. aviumsubsp. paratuberculosisstrain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), i.p. (intraperitoneal inoculation), and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. Over the course of the study, the percentages of total B cells in nonstimulated and antigen-stimulated cell cultures increased for oral and i.p. group calves, with the highest percentages noted at 3 and 6 months. Oral/M group calves had increased percentages of activated B cells, as determined by CD5dimand CD5brightmarkers, at 9 and 12 months. Experimental infection by all methods resulted in increased expression of CD25+and CD45RO+B cells early in the study, but the most significant results were observed at 12 months for oral/DXM and oral/M group calves. Immunoblot analyses with a whole-cell sonicate of M. aviumsubsp. paratuberculosisdemonstrated the most reactivity with sera from i.p. group calves and the least reactivity with sera from oral group calves. Further evidence of M. aviumsubsp. paratuberculosis-specific antibody responses in the i.p. group calves was demonstrated using the ethanol vortex enzyme-linked immunosorbent assay (EvELISA) method. In summary, an induction of B cell responses was noted after experimental infection with M. aviumsubsp. paratuberculosis, with differences in responses noted according to the method of experimental inoculation.

ABSTRACTThe objective of this study was to observe early markers of cell-mediated immunity in nai¨ve calves infected with Mycobacterium aviumsubsp. paratuberculosisand how expression of these markers evolved over the 12-month period of infection. Groups for experimental infection included control (noninfected), oral (infected orally with M. aviumsubsp. paratuberculosisstrain K-10), oral/DXM (pretreatment with dexamethasone before oral inoculation), intraperitoneal (i.p.) inoculation, and oral/M (oral inoculation with mucosal scrapings from a cow with clinical disease) groups. One of the earliest markers to emerge was antigen-specific gamma interferon (IFN-?). Only i.p. inoculated calves had detectable antigen-specific IFN-? responses at 7 days, with responses of the other infection groups becoming detectable at 90 and 120 days. All infection groups maintained robust IFN-? responses for the remainder of the study. At 1 month, calves in the oral and oral/M groups had higher antigen-stimulated interleukin-10 (IL-10) levels than calves in the other treatment groups, but IL-10 secretion declined by 12 months for all calves. T-cell activation markers such as CD25, CD26, CD45RO, and CD5 were significantly upregulated in infected calves compared to noninfected controls. Oral inoculation of calves resulted in significantly increased antigen-specific lymphocyte proliferation at 9 and 12 months, as well as inducible nitric oxide synthase (iNOS) secretion at 6 and 12 months. These results demonstrate that infection of nai¨ve calves with M. aviumsubsp. paratuberculosisinvoked early immunologic responses characterized by robust antigen-specific IFN-? responses and induction of CD25 and CD45RO expression on T-cell subsets. These were followed by antigen-specific lymphocyte proliferation, iNOS secretion, and expression of CD26 and CD5brightmarkers in the latter part of the 12-month study.

ABSTRACTImmunological diagnosis of Mycobacterium bovisinfection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-?)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. aviumexposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. aviumor experimentally challenged with Mycobacterium aviumsubsp. aviumor Mycobacterium aviumsubsp. paratuberculosiswere compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-a), IFN-?, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P< 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. aviumsubsp. paratuberculosisinduced significant (P< 0.05) IFN-? and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-? responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P< 0.05) the corresponding responses of noninfected, M. avium-infected, and M. aviumsubsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. aviumand M. aviumsubsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. aviumand/or M. aviumsubsp. paratuberculosis.