Wiebren J. Santema

Susceptibility to paratuberculosis infection in cattle is associated with single nucleotide polymorphisms in Toll-like receptor 2 which modulate immune responses against Mycobacterium avium subspecies paratuberculosis.

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.

Heat shock protein 70 subunit vaccination against bovine paratuberculosis does not interfere with current immunodiagnostic assays for bovine tuberculosis

Paratuberculosis or Johnes 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.

The mycobacterial glycolipid glucose monomycolate induces a memory T cell response comparable to a model protein antigen and no B cell response upon experimental vaccination of cattle

Glycolipids are presented to T cells by human group 1 CD1 proteins, but are not used as subunit vaccines yet. Experimental immunizations with pure mycobacterial glucose monomycolate (GMM) and keyhole limpet haemocyanin (KLH) in cattle, a species which, unlike mice, expresses group 1 CD1, showed that GMM was equally efficient as KLH in generating T cell responses in blood, but not in the draining lymph node. Also, KLH induced strong antibody responses whereas GMM did not. These data suggest that non-overlapping T cell populations are targeted and demonstrate the potential of glycolipids as a special class of subunit vaccine candidates.

γδ T Cell Homing to Skin and Migration to Skin-Draining Lymph Nodes Is CCR7 Independent

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.

Different Mycobacterium avium subsp. paratuberculosis MIRU-VNTR patterns coexist within cattle herds

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.

Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins.

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.

The bovine CD1D gene has an unusual gene structure and is expressed but cannot present α-galactosylceramide with a C26 fatty acid.

Although CD1d and NKT cells have been proposed to have highly conserved functions in mammals, data on functions of CD1d and NKT cells in species other than humans and rodents are lacking. Upon stimulation with the CD1d-presented synthetic antigen α-galactosylceramide, human and rodent type I invariant NKT cells release large amounts of cytokines. The two bovine CD1D (boCD1D) genes have structural features that suggest that they cannot be translated into functional proteins expressed on the cell surface. Here we provide evidence that despite an intron-exon structure and signal peptide that are different from all other known CD1 genes, boCD1D can be translated into a protein that is expressed on the cell surface. However, in vivo treatment of cattle (Bos taurus) with 0.1, 1, or 10 µg kg⁻¹ of the most commonly used α-galactosylceramide, which has a C26 fatty acid, did not lead to an increase in body temperature and serum cytokine levels of the animals. This lack of reactivity is not due to a complete inability of boCD1d to present glycosphingolipids because α-galactosylceramide variants with shorter fatty acids could be presented by boCD1d to human NKT cells in vitro. This suggests that the natural ligands of boCD1d are smaller lipids.

Early infection dynamics after experimental challenge with Mycobacterium avium subspecies paratuberculosis in calves reveal limited calf-to-calf transmission and no impact of Hsp70 vaccination

Efficient control of bovine paratuberculosis is hampered by lack of a vaccine. The purpose of this study was to evaluate efficacy of a candidate vaccine, consisting of recombinant Mycobacterium avium subspecies paratuberculosis (MAP) Hsp70 with DDA adjuvant, in calves experimentally infected with MAP. Four groups of 14 animals each were used. Animals in group 1 and 2 were all vaccinated with Hsp70/DDA at day 0, 84, 168 and 357, and those in group 3 and 4 were non-vaccinated controls. In each group half (n=7) of the animals were challenged and the remaining half served as contacts. Blood and fecal samples were collected at three week intervals until day 588, and subsequently all animals were subjected to necropsy. The primary outcomes assessed were fecal culture (FC) of MAP, tissue colonization of MAP, and transmission of infection to contact animals. The kinetics of MAP shedding in feces of challenged animals showed a peak around 130 days post-challenge, irrespective of vaccination status. At necropsy no differences in the level of tissue colonization between vaccinated animals and controls were observed in the challenged groups. Only one contact animal (non-vaccinated) was positive at necropsy, indicating limited to no transmission within groups. These findings indicate that Hsp70/DDA vaccination does not influence early infection dynamics after experimental infection. However, early shedding of MAP in calves did not result in efficient transmission of infection to contact animals. The latter implies that introduction of an infected calf in a cohort of susceptibles has limited consequences for spread of infection.

Hsp70 vaccination-induced antibodies recognize B cell epitopes in the cell wall of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic intestinal infection of ruminants and has been associated with the etiology of human Crohns 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.

Postexposure Subunit Vaccination against Chronic Enteric Mycobacterial Infection in a Natural Host

ABSTRACT 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.