Heidi Mikkelsen

Association between milk antibody and interferon-gamma responses in cattle from Mycobacterium avium subsp. paratuberculosis infected herds.

Paratuberculosis is a chronic infection of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is possible to detect infection with paratuberculosis at different stages of disease by means of various diagnostic test strategies. The objective of the present study was to evaluate if early cell-mediated immunity could predict the antibody results of milk samples in cattle with different faecal culture (FC) status. A group of 975 cows from 18 Danish MAP infected dairy herds was studied during a 3-year period. Cell-mediated immunity was measured in blood samples from heifers by use of an IL-12 potentiated IFN-gamma protocol. Following calving, milk samples were collected and analysed for MAP specific antibodies by ELISA and faecal samples were cultured. The relationship between the variables IFN-gamma and FC and the outcome of ELISA was assessed using generalised additive models. The results of the study showed that a significant association exists between early IFN-gamma and later FC status with occurrence of antibodies. In addition, the early IFN-gamma and FC status affect the antibody ELISA result at different stages post calving. We observed that only some IFN-gamma positive animals developed a positive antibody response against MAP, which indicate that cell-mediated immune responses can control or eradicate MAP in many animals.

Review of Mycobacterium avium subsp. paratuberculosis antigen candidates with diagnostic potential

Mycobacterium avium subsp. paratuberculosis (MAP) is a slow growing bacterium that can infect ruminants and remain latent for years without development of any clinical signs or disease. Diagnosis is often based on detection of MAP antibodies in milk or serum samples or culture of bacteria from faeces; however, these diagnostic tools are often not applicable until years after infection. Detection of MAP specific cell-mediated immune (CMI) responses can serve as an alternative and be implemented in a diagnostic tool. CMI responses can be measured at an early stage of infection, prior to development of antibodies and shedding of detectable amounts of MAP. At present, available diagnostic assays are limited by the lack of MAP specific antigens included in these assays resulting in poor specificity. The objective of this review is to provide a systematic overview of diagnostic MAP antigen candidates described to date with special emphasis on antigen candidates tested for CMI responses. Relevant information on 115 different MAP antigens was systematically extracted from literature and summarized in 6 tables of CMI antigens, secreted antigens, cell wall and membrane antigens, lipoprotein antigens, heat shock antigens and hypothetical antigens. Strategies for evaluation of novel antigen candidates are discussed critically. Relatively few of the described antigens were evaluated for their use in CMI based diagnostic assays and so far, no obvious candidate has been identified for this application. Most of the novel diagnostic candidates were evaluated in few animals and it is recommended that an appropriate sample size is included for evaluation of antigen candidates in future studies.

Use of the johnin PPD interferon-gamma assay in control of bovine paratuberculosis.

Although the interferon-gamma (IFN-γ) assay for measurements of cell-mediated immune (CMI) responses to paratuberculosis PPD (johnin) has been available for close to 20 years, the assay has not yet emerged as the long desired test to identify infected animals at an early time point. Among other issues, this relates to problematic interpretation of the test results and maybe an over-expectation of what can be deducted from this kind of test given the chronic nature and slow development of infection of paratuberculosis. Over a number of years a modified IFN-γ assay with addition of recombinant bovine IL-12 to the PPDj stimulation of blood samples from the heifer group in more than 20 Danish dairy herds which also perform surveillance of MAP antibodies in milk have been performed. The results indicate that IFN-γ assay results are specific for paratuberculosis, but the IFN-γ assay result of an individual animal cannot establish whether the animal is infected or predict the future progression of disease in this animal. The IFN-γ assay should thus be used on a group of animals to test the level of exposure to paratuberculosis bacteria the animals have experienced, and thereby assist in maintaining rational in-herd management procedures and in the establishment of paratuberculosis status of a given herd. Indeed, for any diagnostic test applied in paratuberculosis, both the diagnostic target condition and the purpose of the diagnostic testing must be considered before any meaningful estimates of sensitivity or specificity can be given.

Novel antigens for detection of cell mediated immune responses to Mycobacterium avium subsp. paratuberculosis infection in cattle.

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring specific cell mediated immune responses, using the whole blood interferon-γ (IFN-γ) assay. Available IFN-γ assays use purified protein derivative of MAP (PPDj) which are complex antigen mixtures with low specificity. The objectives of this study were to evaluate immunogenicity and specificity of 14 novel recombinant antigens for use in the IFN-γ assay and to assess the consistency of IFN-γ responses. The study included blood samples from 26 heifers from a MAP infected herd, collected three times with four to five-week intervals, and blood samples from 60 heifers of a non-infected herd collected once. Heifers of the non-infected herd were used to establish cut-off values for each antigen. The case definition was an animal with ≥ 2 positive tests for ≥ 4 antigens, resulting in 13 cases and 13 non-cases. IFN-γ levels of cases were higher compared to IFN-γ levels of non-cases (P<0.05). The results of the IFN-γ assay using PPDj did not correlate well with the results using the novel antigens. PPDj produced elevated IFN-γ responses of samples from both the non-infected and the MAP infected herd, indicating unspecific IFN-γ responses and showed low consistency. Three latency proteins, LATP-1, LATP-2 and LATP-3 gave positive IFN-γ tests that correlated very well with the case definition suggesting high immunogenicity. Three tested antigens, LATP-2, MAP-1 and MAP-2 have no homologue in the M. avium subsp. avium or M. bovis genome and could be promising diagnostic antigens, especially LATP-2 correlated highly with the case definition.

Correlation of antigen-specific IFN-γ responses of fresh blood samples from Mycobacterium avium subsp. paratuberculosis infected heifers with responses of day-old samples co-cultured with IL-12 or anti-IL-10 antibodies

Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10-21 months of age from a MAP infected herd with addition of either recombinant bovine IL-12 or anti-bovine IL-10 antibody with IFN-γ production in sample day samples. IFN-γ responses of sample day samples showed high correlation with responses to some antigens in day-old samples with addition of IL-12 or anti-IL-10 antibodies to cultures, indicating that day-old protocols can be applied as an alternative to the conventional IFN-γ protocol. Immunogenicity of the novel antigens was generally low for day-old samples. The most promising antigen using the day-old protocol with addition of IL-12 was latency protein LATP-2 as correlations, immunogenicity and diagnostic specificity collectively was high. The latency protein LATP-1 was the most promising antigen in the day-old protocol with addition of anti-IL-10 antibodies.

Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals

The quantitative detection of viable pathogen load is an important tool in determining the degree of infection in animals and contamination of foodstuffs. Current conventional culture methods are limited in their ability to determine these levels in Mycobacterium avium subspecies paratuberculosis (MAP) due to slow growth, clumping and low recoverability issues. The principle goal of this study was to evaluate a novel culturing process (TiKa) with unique ability to stimulate MAP growth from low sample loads and dilutions. We demonstrate it was able to stimulate a mean 29-fold increase in recoverability and an improved sensitivity of up to three logs when compared with conventional culture. Using TiKa culture, MAP clumping was minimal and produced visible colonies in half the time required by standard culture methods. Parallel quantitative evaluation of the TiKa culture approach and qPCR on MAP loads in tissue and gut mucosal samples from a MAP vaccine-challenge study, showed good correlations between colony counts (cfu) and qPCR derived genome equivalents (Geq) over a large range of loads with a 30% greater sensitivity for TiKa culture approach at low loads (two logs). Furthermore, the relative fold changes in Geq and cfu from the TiKa culture approach suggests that non-mucosal tissue loads from MAP infected animals contained a reduced proportion of non-viable MAP (mean 19-fold) which was reduced significantly further (mean 190-fold) in vaccinated “reactor” calves. This study shows TiKa culture equates well with qPCR and provides important evidence that accuracy in estimating viable MAP load using DNA tests alone may vary significantly between samples of mucosal and lymphatic origin.

Targeting the Mincle and TLR3 receptor using the dual agonist cationic adjuvant formulation 9 (CAF09) induces humoral and polyfunctional memory T cell responses in calves

There is a need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium avium subsp. paratuberculosis in a number of species. One of the main challenges for vaccine development is the lack of safe adjuvants that induce protective immune responses. Cationic Adjuvant Formulation 01 (CAF01)—an adjuvant based on trehalose dibehenate (TDB) and targeting the Mincle receptor—has entered human trials based on promising pre-clinical results in a number of species. However, in cattle CAF01 only induces weak systemic immune responses. In this study, we tested the ability of three pattern recognition receptors, either alone or in combination, to activate bovine monocytes and macrophages. We found that addition of the TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)) to either one of the Mincle receptor agonists, TDB or monomycoloyl glycerol (MMG), enhanced monocyte activation, and calves vaccinated with CAF09 containing MMG and Poly(I:C) had increased cell-mediated and humoral immune response compared to CAF01 vaccinated animals. In contrast to the highly reactogenic Montanide ISA 61 VG, CAF09-primed T cells maintained a higher frequency of polyfunctional CD4+ T cells (IFN-γ+ TNF-α+ IL-2+). In conclusion, CAF09 supports the development of antibodies along with a high-quality cell-mediated immune response and is a promising alternative to oil-in-water adjuvant in cattle and other ruminants.