The gamma interferon assay is used to identifyMycobacterium aviumsubsp.paratuberculosis-infected animals. It has been suggested that regulatory mechanisms could influence the sensitivity of the test when it is performed with cells from cattle and that the neutralization of interleukin-10 (IL-10) in vitro would increase the gamma interferon responses. To investigate the regulatory mechanisms affecting the gamma interferon assay with cells from goats, blood was collected fromM. aviumsubsp.paratuberculosis-infected,M. aviumsubsp.paratuberculosis-exposed, and noninfected goats. Neutralization of IL-10 by a monoclonal antibody resulted in increased levels of gamma interferon production inM. aviumsubsp.paratuberculosispurified protein derivative (PPDj)-stimulated samples from both infected and exposed goats. However, the levels of gamma interferon release were also increased in unstimulated cells and in PPDj-stimulated cells from some noninfected animals following neutralization. Depletion of putative regulatory CD25^highT cells had no clear effect on the number of gamma-interferon-producing cells. The IL-10-producing cells were identified to be mainly CD14⁺major histocompatibility complex class II-positive monocytes in both PPDj-stimulated and control cultures and not regulatory T cells. However, possible regulatory CD4⁺CD25⁺T cells produced IL-10 in response to concanavalin A stimulation. The numbers of CD4⁺, CD8⁺, and CD8⁺γδT-cell receptor-positive cells producing gamma interferon increased following IL-10 neutralization. These results provide insight into the source and the role of IL-10 in gamma interferon assays with cells from goats and suggest that IL-10 from monocytes can regulate both innate and adaptive gamma interferon production from several cell types. Although IL-10 neutralization increased the sensitivity of the gamma interferon assay, the specificity of the test could be compromised.

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

The Maedi-visna virus (MVV) causes a persistent infection in small ruminants, and its high genetic heterogeneity affects the performance of diagnostic tests when used in different populations. Therefore, the aim of this study was to develop a bead-based multiplex immunoassay tailored to detect antibodies against a Norwegian MVV strain. We used tissue samples from 14 PCR-positive sheep from a recent MVV outbreak in Norway to sequence the viral strain and produced recombinant antigens based on sequences from one animal. The assay included commercial TM-A and recombinant Norwegian p25, p16–25 and SU5 antigens. Cut-off values for each antigen were determined using receiver operating characteristic curves on 40 ELISA-negative and 67 ELISA-positive samples from the outbreak. The intraplate and interplate repeatability were investigated by testing a quadruplicate of five samples over three days, while the analytical sensitivity (aSe) and specificity (aSp) were measured in comparison to a commercial ELISA. The repeatability showed a coefficient of variation below 15% for most positive samples. The aSe was equal or higher for the multiplex assay than the ELISA, and the aSp of each antigen was 91.7, 93.3, 95.0 and 93.3% for p25, p16–25, SU5 and TM-A, respectively. The assay shows promising results; however, further evaluations of diagnostic characteristics are necessary before implementation in the Norwegian surveillance programme.

Actinobacillus pleuropneumoniae (APP) remains a major cause of respiratory disease in swine and accurate serological tools are essential for surveillance, particularly in sub-clinical herds and low prevalence settings. We evaluated the diagnostic performance of a microsphere-based multiplex fluorescent immunoassay (MFIA) and two commercial ELISAs (1–12 ELISA and ApxIV ELISA) using a Bayesian latent class model and field samples from Norwegian swine herds. Although both ELISAs and the MFIA showed high specificity, the ApxIV ELISA demonstrated lower sensitivity (89.4%) than the other assays. These findings align with previously reported variability in the sensitivity of the ApxIV-based ELISA. The MFIA showed high sensitivity, demonstrating the potential of multiplex immunoassays for APP serology. We also explored optimization of the ApxIV ELISA cut-off, which indicated that lowering the threshold could improve the sensitivity of the test without compromising the specificity for our samples. Our estimates can be influenced by several factors, including the serotype diversity in the population and assumptions related to latent class modelling. Despite these constraints, our findings support the use of MFIA and the ELISAs as accurate tools for APP surveillance, while highlighting that the ApxIV ELISA may be less suitable as a stand-alone screening test. These results underscore the importance of validating diagnostic assays in populations where they will be applied to ensure that they are fit-for-purpose.