Ad P. Koets

Carvacrol Induces Heat Shock Protein 60 and Inhibits Synthesis of Flagellin in Escherichia coli O157:H7

ABSTRACT The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7.

Subpopulations of bovine WC1+ γδ T cells rather than CD4+CD25highFoxp3+ T cells act as immune regulatory cells ex vivo

Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4+CD25high T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4+CD25high T cells and γδ T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4+CD25high T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4+CD25low T cells, WC1+, WC1.1+ and WC1.2+ γδ T cells, NK cells, CD8+ T cells and CD14+ monocytes. Only the WC1.1+ and WC1.2+ γδ T cells and CD14+ monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4+CD25highFoxp3+ and CD4+CD25low T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the γδ T cell population, more precisely in the WC1.1+ and the WC1.2+ subpopulation, major populations present in blood of cattle in contrast to non-ruminant species.

Differential Changes in Heat Shock Protein-, Lipoarabinomannan-, and Purified Protein Derivative-Specific Immunoglobulin G1 and G2 Isotype Responses during Bovine Mycobacterium avium subsp. paratuberculosis Infection

ABSTRACT Bovine paratuberculosis is caused by infection of young calves withMycobacterium avium subsp. paratuberculosis. In some of the chronically infected cows the long asymptomatic stage (2 to 4 years) is followed by a rapid progression to a clinical stage due to protein-losing enteropathy, which will ultimately be fatal. The current dogma is that in early stages of disease the cell-mediated responses predominate, whereas in the clinical stage of the disease the humoral responses prevail, possibly signaling a switch in immune reactivity related to disease progression. We developed immunoglobulin M (IgM)-, IgA-, and IgG1- and IgG2-isotype-specific enzyme-linked immunosorbent assays for M. avium subsp.paratuberculosis-derived antigens (heat shock proteins of 70 kDa [Hsp70] and 65 kDa [Hsp65], lipoarabinomannan [LAM], andM. avium subsp. paratuberculosis purified protein derivative PPD [PPDP]). The serological responses of cows in different stages of paratuberculosis were used to evaluate the putative shift in immune responsiveness. In the clinical stage the PPDP-specific IgG1 responses were increased compared to those in the asymptomatic stage. However, total IgG1 and IgG2 and the Hsp70-, Hsp65-, and LAM-specific isotype responses were decreased in the clinical stage were decreased compared to those in the asymptomatic stage of disease. Thus, the classical pattern was found only for PPDP antigens and the IgG1 isotype. For other antigens and isotypes and the total IgG levels, the response pattern is different and indicates that there is no uniform association with increased antibody responses during the progression from the asymptomatic stage to the clinical stage of bovine paratuberculosis.

Immunopotentiating heat shock proteins: negotiators between innate danger and control of autoimmunity.

Heat shock proteins (hsps) are known to be immunodominant antigens of bacteria. Hsps are evolutionarily strongly conserved proteins present in all eukaryotic and prokaryotic cellular organisms and upregulated by several forms of stress. Despite (the paradigm of) self-tolerance, hsp-epitopes homologous to endogenous host hsp sequences have been implicated as T cell epitopes to endow crossreactive, hsp-specific T cells with the capacity to regulate inflammation, such as in experimentally induced autoimmune diseases. Such T cells were found to produce regulatory cytokines like IL10, in contrast to T cells induced with other conserved microbial proteins that are not upregulated by stress. Hsps have been implicated in immune regulation not only as upregulated targets of adaptive immunity during inflammatory stress, but recently also as triggering factors for innate immunity through activation via Toll-like receptors (TLRs).

Progressive bovine paratuberculosis is associated with local loss of CD4(+) T cells, increased frequency of gamma delta T cells, and related changes in T-cell function

ABSTRACT Bovine paratuberculosis is caused by the infection of young calves with Mycobacterium avium subsp. paratuberculosis, resulting in a chronic granulomatous infection of predominantly the ileum. After an incubation period of 2 to 5 years, the disease becomes progressive in some of the chronically infected, but asymptomatic cows. This results in a protein-losing enteropathy that will ultimately be fatal. A loss of cell-mediated immune responses in symptomatic animals has been described, but no information is available concerning immune reactivity in the intestine. We sought to investigate putative disease status-associated lymphocyte subset distributions and antigen-specific functional characteristics of mononuclear cells isolated from blood, gut-associated lymphoid tissue, and the intestinal walls of 22 cows in different stages of disease and in control animals. The results demonstrated a significant decrease in CD4+ T-cell frequency and a significant increase in TcR1-N12+ γδ T-cell frequency in ileum lamina propria lymphocytes of symptomatic animals compared to the asymptomatic shedders. Immunohistology revealed that there was also an absolute decrease in the number of CD4+ T cells in sections of the lesional ileum. Our findings also indicated that both peripheral and intestinal cell-mediated responses are decreased in symptomatic animals compared to asymptomatic animals. We conclude that the decrease in cell-mediated responses is likely related to a loss of antigen-specific CD4+ T cells, which is most prominent in the lesional ileum from symptomatic animals, thus contributing to the progressive nature of bovine paratuberculosis.

The Bovine CD1 Family Contains Group 1 CD1 Proteins, but No Functional CD1d

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-γ and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, α-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.

Paratuberculosis (Johne's Disease) in Cattle and Other Susceptible Species

Paratuberculosis (Johnes disease) is a widespread and costly disease. This consensus statement will summarize recommendations regarding diagnosis, control, and treatment of Johnes disease in cattle and other species. Each section of recommendations is followed by a statement that subjectively characterizes the strength of the supporting evidence. The role played by Mycobacterium avium subsp. paratuberculosis (MAP) in the pathogenesis has been a matter of controversy for many years. This statement concludes with an assessment of the evidence in favor of MAP as a potential zoonotic pathogen.

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.

Detection of spatial and temporal spread of Mycobacterium avium subsp. paratuberculosis in the environment of a cattle farm through bio-aerosols.

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.

Low predictive value of seroprevalence of Toxoplasma gondii in cattle for detection of parasite DNA

The role of beef in human infections with Toxoplasma gondii is not clear. To get a better understanding of the value of seroprevalence as an indication of the role of beef in human infections with T. gondii we studied the seroprevalence of T. gondii in Dutch cattle and analysed the correlation between detection of antibodies and parasitic DNA. An indirect ELISA was developed and used to test a sample of the Dutch cattle population. Since validation of the ELISA was hampered by a lack of sufficient bovine reference sera, the results were analysed in two different ways: using a cut-off value that was based on the course of the OD in 27 calves followed from birth until 16 months of age, and by fitting a mixture of two normal distributions (binormal mixture model) to the log-transformed ODs observed for the different groups of cattle in the study population. Using the cut-off value, the seroprevalence was estimated at 0.5% for white veal, 6.4% for rosé veal and 25.0% for cattle. However, using the frequency distributions the prevalences were higher: 1.9% for white veal, 15.6% for rosé veal and 54.5% for cattle. Next, for 100 cattle the results with two different serological assays (ELISA and Toxo-Screen DA) were compared with detection of parasites by our recently developed sensitive magnetic capture PCR. Toxoplasma gondii DNA was detected in only two seronegative cattle. This discordance demonstrates that seroprevalence cannot be used as an indicator of the number of cattle carrying infectious parasites. Demonstrating parasitic DNA in seronegative cattle and not in seropositive cattle suggests that only recent infections are detectable. Whether beef from these PCR-positive cattle is infectious to humans remains to be studied.