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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.

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.)

Environmental samples were collected to investigate the spatial and temporal spread of Mycobacterium avium subsp. paratuberculosis (MAP) in a dairy cattle barn before and after the introduction of two groups of MAP-shedding animals. Samples collected off the floor of the barn reflected the moment of sampling whereas samples collected by microfiber wipes at a minimal of 3m height contained the accumulated settled dust over a 3-week period. Samples were analysed by IS900 qPCR for the presence of MAP DNA and by culture for viable MAP bacteria. MAP DNA was detected in a large number of sites both before and after introduction cattle. MAP DNA was detected inside the barn in floor and dust samples from cubicles and slatted floors and in settled dust samples located above the slatted floors and in the ventilation ridge opening. Outside the barn MAP DNA was detected by PCR in samples reflecting the walking path of the farmer despite hygiene measures. No viable MAP was detected before the introduction of shedder cattle. Three weeks later viable MAP was found inside the barn at 7/49 locations but not outside. Fifteen weeks later viable MAP was also detected in environmental samples outside the barn. In conclusion, introduction of MAP shedding cattle lead to widespread contamination of the internal and external environment of a dairy barn, including the presence of viable MAP in settled dust particles suggesting potential transmission of MAP infection through bio-aerosols., ((c) 2009 Elsevier B.V. All rights reserved.)

Paratuberculosis is a chronic intestinal infection in ruminants, caused by Mycobacterium avium subspecies paratuberculosis (Map). To study the role of host genetics in disease susceptibility, the Toll-like receptor 2 (TLR2) gene, selected based on its potential role in immunity to mycobacterial infections, was analyzed for single nucleotide polymorphisms (SNP) and their potential association with disease. For SNP discovery and to study SNP association with disease, a case-control study including 24 cows from farms with paratuberculosis was conducted. Sequence analysis of the TLR2 genes from 12 paratuberculosis-infected animals and 12 age-matched healthy herd mates revealed 21 different SNP. The TLR2-1903 T/C SNP was significantly associated with resistance to Map. This and four additional TLR2 SNP were studied in a subsequent observational field study with 553 cows from farms with paratuberculosis. The allelic distribution of the TLR2-1903 T/C SNP was confirmed to be significantly different between the infected and non-infected animals. For the TLR2-1903 T/C SNP the odds ratio was calculated, and similar to the dominance model in the association study, the CT and CC genotypes were compared to the TT genotype. Cows with the TLR2-1903 T/C mutation (i.e., the CT and CC genotypes) were at 1.7 (95% CI: 1.2, 2.8) times the odds of being Map-infected compared to cows with the TT genotype. In in vitro functional assays, monocyte-derived macrophages from animals with a TLR2-1903 TT genotype produced more IL12p40 and IL1beta when stimulated with Map compared to cells derived from TLR2-1903 CT and CC genotypes. Also, T cell proliferative responses to mycobacterial antigens were higher in animals with a TLR2-1903 TT genotype. In conclusion, we have found a significant association between SNP TLR2-1903 T/C in the bovine TLR2 gene and bovine paratuberculosis infection. This SNP and other genetic markers could be useful in marker-assisted breeding strategies as an additional tool in paratuberculosis control strategies. In addition, the functional studies suggest that genetic polymorphisms in bovine TLR2 which result in higher macrophage activity may contribute to enhanced T cell activation and a lower susceptibility to paratuberculosis in cattle.