Strong antibody responses to Mycobacterium bovis infection in domestic pigs and potential for reliable serodiagnostics.

Mycobacterium bovis (M. bovis), the main cause of animal tuberculosis (TB), can infect a wide variety of domestic and wild animal species, including suids. Suids may serve as reservoir hosts or disease sentinels in different scenarios. Accurate detection of M. bovis infection in pigs is important for TB control programs. Although previous studies have shown the value of serological assays for screening animal populations, the diagnostic accuracy was considered suboptimal. In this study, we used Dual Path Platform (DPP) technology and multi-antigen print immunoassay (MAPIA) to characterize antigen recognition profiles and temporal antibody responses. Four M. bovis experimentally infected pigs developed an early antibody response to antigen MPB83, with a peak in IgG levels starting around 4-6 weeks post-inoculation, although none of the pigs developed antibodies to fusion protein CFP10/ESAT6 within 16 weeks of the experiment. Three of four experimentally infected pigs developed antibody responses before detectable antigen-specific interferon gamma responses. Naturally infected pigs with gross lesions containing viable M. bovis showed IgM (19/40 infected animals) and IgG (39/40) antibody responses to both MPB70/MPB83 (39/40) and CFP10/ESAT6 (34/40). Using MPB70/MPB83 antigen alone to measure IgG antibody levels by DPP assay, an estimated test sensitivity was 97.5 % (95 % CI: 85.3-99.9 %). None of the 57 negative control samples had detectable IgM or IgG antibodies to either of the two test antigens in DPP assay, suggesting an estimated specificity of 100 % (95 % CI: 92.1-100.0 %) in pigs. MAPIA showed robust IgG reactivity to multiple protein antigens of M. bovis in the naturally infected pigs. The results demonstrate that serological assays which detect IgG antibodies to MPB83 have high sensitivity and specificity for accurate detection of M. bovis infection in pigs. Further investigations should be done to validate anti-MPB70/MPB83 antibodies as a reliable serodiagnostic biomarker for TB diagnosis in pigs.

Antibody responses in European bison (Bison bonasus) naturally infected with Mycobacterium caprae.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, infects humans and animals causing lesions and disease like that of Mycobacterium bovis. The aim of this study was to evaluate antibody responses in European Bison (EB, Bison bonasus; a vulnerable species) naturally infected with M. caprae using dual path platform (DPP) BovidTB test and multi-antigen print immunoassay (MAPIA). Study cohorts consisted of naturally M. caprae-infected EB (n = 4), M. caprae-exposed but uninfected (n = 3), EB infected with non-tuberculous mycobacteria or other respiratory pathogens (n = 3), and negative controls (n = 19). M. caprae-infected EB were seropositive by both DPP and MAPIA; 3/4 were seropositive by DPP; and 4/4 were seropositive by MAPIA. One M. caprae-infected animal that developed generalized disease with most advanced gross lesions in the group produced the most robust antibody response. All 25 EB with no culture-confirmed M. caprae infection, including three animals exposed to M. caprae and three other animals infected with non-tuberculous pathogens, were seronegative on both tests. Antibody responses to M. caprae infection included IgM antibodies against MPB70/MPB83 and IgG antibodies to both MPB70/MPB83 and CFP10/ESAT-6. This study demonstrates the potential for use of serological assays in the ante-mortem diagnosis of M. caprae infection in EB.

Biomarkers of cell-mediated immunity to bovine tuberculosis.

Whole blood based assays, particularly interferon gamma (IFN-γ) release assays (IGRAs), are used for the diagnosis of both bovine and human tuberculosis (TB). The aim of the current study was to evaluate a panel of cytokines and chemokines for potential use as diagnostic readouts indicative of Mycobacterium bovis (M. bovis) infection in cattle. A gene expression assay was used to determine the kinetics of the response to M. bovis purified protein derivative and a fusion protein consisting of ESAT-6, CFP10, and Rv3615c upon aerosol infection with ∼104 cfu of M. bovis. The panel of biomarkers included: IFN-γ, CXCL9, CXCL10, CCL2, CCL3, TNF-α, IL-1α, IL-1ß, IL-1Ra, IL-22, IL-21 and IL-13. Protein levels of IFN-γ, CXCL9, and CXCL10 were determined by ELISA. Findings suggest that CXCL9, CXCL10, IL-21, IL-13, and several acute phase cytokines may be worth pursuing as diagnostic biomarkers of M. bovis infection in cattle.

Evaluation of granulysin and perforin as candidate biomarkers for protection following vaccination with Mycobacterium bovis BCG or M. bovisDeltaRD1.

The development of improved vaccines against tuberculosis (TB) is directly linked to the investigation of new and better correlates of protection after vaccination against TB. Cloning and characterization of bovine homologues of the antimicrobial protein granulysin (Bo-lysin) and perforin by our group could be used as potential biomarkers for TB vaccination efficacy. In the present study, we examined the kinetics of granulysin, perforin, IFNgamma and Fas-L responses to Mycobacterium bovis purified protein derivative (PPD) stimulation by peripheral blood mononuclear cells from M. bovisDeltaRD1-, BCG- and non-vaccinated cattle. Gene expression profiles following PPD stimulation showed significant increases in transcripts for granulysin and IFNgamma in both CD4(+) and CD8(+) T cells in BCG-vaccinated as compared with non-vaccinated animals. Perforin and IFNgamma examined by flow cytometry, showed a difference of 1-2% more PPD-specific cells in BCG-vaccinated than non-vaccinated animals. In the vaccine trial, granulysin and perforin were significantly increased in both vaccine groups as compared with control after vaccination and challenge. IFNgamma expression was increased only after vaccination and secretion was higher in the control, non-protected group as compared with both vaccine groups demonstrating no correlation with protection upon vaccination. In summary, results shown here provide evidence that granulysin and perforin are prospective candidates as biomarkers of protection after vaccination against TB.