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Bovine paratuberculosis is a highly prevalent chronic infection of the small intestine in cattle, caused by Mycobacterium avium subspecies paratuberculosis (MAP). In earlier studies we showed the protective effect of Hsp70/DDA subunit vaccination against paratuberculosis. In the current study we set out to measure primary immune responses generated at the site of Hsp70 vaccination. Lymph vessel cannulation was performed to obtain efferent lymph from the prescapular lymph node draining the neck area where the vaccine was applied. Hsp70 vaccination induced a significant increase of CD21(+) B cells in efferent lymph, accounting for up to 40% of efferent cells post-vaccination. Proliferation (Ki67(+)) within the CD21(+) B cell and CD4(+) T cell populations peaked between day 3 and day 5 post-vaccination. From day 7, Hsp70-specific antibody secreting cells (ASCs) could be detected in efferent lymph. Hsp70-specific antibodies, mainly of the IgG1 isotype, were also detected from this time point onwards. However, post-vaccination IFN-γ production in efferent lymph was non-sustained. In conclusion, Hsp70-vaccination induces only limited Th1 type immune responsiveness as reflected in efferent lymph draining the vaccination site. This is in line with our previous observations in peripheral blood. The main primary immunological outcome of the Hsp70/DDA subunit vaccination is B cell activation and abundant Hsp70-specific IgG1 production. This warrants the question whether Hsp70-specific antibodies contribute to the observed protective effect of Hsp70 vaccination in calves., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The control of chronic bacterial diseases with high prevalence in areas of endemicity would strongly benefit from availability of postexposure vaccines. The development of these vaccines against mycobacterial infections, such as (para)tuberculosis, is hampered by lack of experience in natural hosts. Paratuberculosis in cattle is both a mycobacterial disease of worldwide importance and a natural host model for mycobacterial infections in general. The present study showed beneficial effects of therapeutic heat shock protein 70 (Hsp70) vaccination in cattle with naturally acquired chronic infection with Mycobacterium avium subsp. paratuberculosis. Vaccination-induced protection was associated with antibody responses, rather than with induction of specific T helper 1 cells. Targeted therapeutic postexposure vaccination complementary to selective use of antibiotics could be an effective approach for control of chronic mycobacterial infections.

In most species, γδ T cells preferentially reside in epithelial tissues like the skin. Lymph duct cannulation experiments in cattle revealed that bovine dermal γδ T cells are able to migrate from the skin to the draining lymph nodes via the afferent lymph. For αβ T cells, it is generally accepted that epithelial and mucosal tissue egress is regulated by expression of the CCR7 chemokine receptor. In this study, we tracked the migratory route of bovine lymph-derived γδ T cells and examined their CCR7 cell surface expression in several compartments along this route. Total lymph cells from afferent and efferent origin were labeled with PKH fluorescent dyes and injected into the bloodstream. PKH(+) cells already reappeared in the afferent lymph after 4 h. The vast majority of the PKH(+) cells retrieved from the afferent lymph were of the WC1(+) γδ T cell phenotype, proving that this PKH(+) γδ T cell subset is able to home to and subsequently exit the skin. PKH(+) γδ T cells from afferent and efferent lymph lack CCR7 surface expression and display high levels of CD62L compared with CD4 T cells, which do express CCR7. Skin homing receptors CCR4 and CCR10 in contrast were transcribed by both CD4 and γδ T cells. Our findings suggest that γδ T cell skin egress and migration into the peripheral lymphatics is CCR7-independent and possibly mediated by CD62L expression.

Bovine paratuberculosis is a highly prevalent chronic infection of the small intestine in cattle, caused by Mycobacterium avium subspecies paratuberculosis. Current control strategies based on test-and-cull and biosecurity measures do not suffice in lowering the prevalence of paratuberculosis in an adequate manner. Therefore, control programmes are in need of an effective vaccine, but at the moment no vaccine is registered for use in cattle in the European Union. This review provides a brief overview of the microbiology, epidemiology and immunology of bovine paratuberculosis, and focuses on recent advances in the development of vaccines against paratuberculosis.

Strong anti glycolipid IgG responses can occur in humans and animals, but contrary to anti protein responses and anti glycoprotein responses, the exact mechanism of induction is unknown. We have previously shown that experimental immunization with the glycolipid glucose monomycolate (GMM) causes the development of specific T cell responses, but not of anti GMM antibodies. However, cattle naturally infected with Mycobacterium avium ssp. paratuberculosis produce high levels of anti GMM IgG. In the present study, we tested whether vaccination with GMM conjugated to a protein mimics natural infection in its capacity to induce the production of antibodies against GMM. Cattle were immunized (n=5 per group) with GMM conjugated to a protein, or GMM and protein non-conjugated and administered at contralateral locations, or carrier only. Although immunization with the GMM-protein conjugate vaccine and the non-conjugated vaccine induced protein specific antibody responses, GMM specific antibodies were not detected in either of the groups. In conclusion, the generation of isotype-switched anti lipid antibodies appears to require more than providing peptide epitopes for T helper cells to support glycolipid specific B cells in antibody production., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A better understanding of the biodiversity of Mycobacterium avium subsp. paratuberculosis (MAP) offers more insight in the epidemiology of paratuberculosis and therefore may contribute to the control of the disease. The aim of this study was to investigate the genetic diversity in bovine MAP isolates using PCR-based methods detecting genetic elements called Variable-Number Tandem Repeats (VNTRs) and Mycobacterial Interspersed Repetitive Units (MIRUs) to determine if multiple MAP strains can coexist on farms with endemic MAP infection. For 52 temporal isolates originating from infected cattle from 32 commercial dairy herds with known trading history, MIRU-VNTR analysis was applied at 10 loci of which six showed variation. Within the group of 52 isolates, 17 different MIRU-VNTR patterns were detected. One MIRU-VNTR pattern was found in 29 isolates, one pattern in four isolates, one pattern in three isolates, two times one MIRU-VNTR pattern was found occurring in two isolates, and 12 patterns were found only once. Eleven herds provided multiple isolates. In five herds a single MIRU-VNTR pattern was detected among multiple isolates whereas in six herds more than one pattern was found. This study confirms that between dairy farms as well as within dairy farms, infected animals shed MAP with different MIRU-VNTR patterns. Analysis of trading history and age within herds indicated that cows born within the same birth cohort can be infected with MAP strains exhibiting variations in the number of MIRU-VNTR repeats. These data indicate that such multiple genotypes of MAP can coexist within one herd., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic intestinal infection of ruminants and has been associated with the etiology of human Crohn's disease. A MAP Hsp70/DDA subunit vaccine previously showed a significant reduction in fecal shedding of MAP in cattle, concomitant with pronounced antibody production against MAP Hsp70, rather than T cell reactivity. Our hypothesis is that if Hsp70-specific antibodies are able to confer protection, the first requisite would be that the Hsp70 molecule is accessible for antibodies in intact MAP bacteria. In the current study monoclonal antibodies identified MAP Hsp70 B cell epitopes. Two linear epitopes were also recognized by antibodies of vaccinated calves and goats. These epitopes showed to be accessible by antibodies in the bacterial cell wall and in intestinal lesional tissue during natural infection. These results indicate that vaccination-induced antibodies can bind intact bacteria and have the potential to contribute to the protective effect of Hsp70/DDA subunit vaccination against bovine paratuberculosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens. One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in Escherichia coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested. In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N=20) were significantly higher (p<0.01) than in control animals (N=20), despite the conserved nature of this protein. In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases.

Paratuberculosis or Johne's disease in ruminants is an infectious disease of the small intestine caused by Mycobacterium avium spp. paratuberculosis, and a global problem of the livestock industry. No therapy is available and the use of a whole bacterin vaccine is limited due to interference with tuberculosis diagnostics. In earlier studies we showed the protective effect of Hsp70/DDA subunit vaccination against paratuberculosis. In the current study, potential interference of this vaccination with immunodiagnostic procedures to detect bovine tuberculosis and paratuberculosis was studied. Vaccination of cattle with an Hsp70/DDA subunit vaccine has little side-effects, and does not interfere with current tuberculosis immunodiagnostic assays. Serological assays for paratuberculosis diagnostics can be adapted by inclusion of an Hsp70 pre-absorption step that enables differentiation between vaccinated and infected animals. In conclusion, the Hsp70/DDA subunit vaccine may contribute to paratuberculosis control strategies, without compromising diagnosis of bovine tuberculosis or paratuberculosis.

Paratuberculosis is a chronic granulomatous inflammation of the small intestine of cattle and other ruminants, caused by infection with Mycobacterium avium ssp. paratuberculosis (MAP). The disease can be found in ruminant herds worldwide, causing substantial economic losses at farm level due to premature culling and production losses. In previous studies, it has been shown that immune responses to recombinant MAP Hsp70 proteins were predominantly cell mediated. As protective immunity to the intracellular mycobacterial pathogens is thought to be cell-mediated in origin, we have studied the use of a recombinant MAP Hsp70 as a subunit vaccine in cattle experimentally infected with MAP. The results of the current study demonstrate that recombinant MAP Hsp70 can be successfully used as a subunit vaccine against bovine paratuberculosis, significantly reducing shedding of bacteria in feces during the first 2 years following experimental infection.