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Amplification of the IS900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis, which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/ P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 39 end, and it does not bind all copies of IS900 due to 59 deletions at some IS900 loci. These IS900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/ 921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. [ABSTRACT FROM AUTHOR]

Macrophages are important host defense cells in ruminant paratuberculosis (Johne's Disease; JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). Classical macrophage functions of pathogen trafficking, degradation, and antigen presentation are interrupted in mycobacterial infection. Immunologic stimulation by 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D3 on macrophage phenotype and endosomal trafficking of MAP in monocyte-derived macrophages (MDMs) cultured from JD-, JD+ subclinical, and JD+ clinically infected cattle. MDMs were pre-treated 100 ng/ml 25(OH) D3 or 4 ng/ml 1,25(OH)2D3 and incubated 24 hrs with MAP at 10:1 multiplicity of infection (MOI). In vitro MAP infection upregulated pro-inflammatory (M1) CD80 and downregulated resolution/repair (M2) CD163. Vitamin D3 generally decreased CD80 and increased CD163 expression. Furthermore, early endosomal marker Rab5 was upregulated 140x across all stages of paratuberculosis infection following in vitro MAP infection; however, Rab5 was reduced in MAP-activated MDMs from JD+ subclinical and JD+ clinical cows compared to healthy controls. Rab7 expression decreased in control and clinical cows following MDM infection with MAP. Both forms of vitamin D3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH)2D3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro. Vitamin D3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D3 analog. Results from this study show exogenous vitamin D3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture. [ABSTRACT FROM AUTHOR]

Mycobacterium avium subspecies paratuberculosis (MAP) is an environmentally hardy pathogen of ruminants that plagues the dairy industry. Hallmark clinical symptoms include granulomatous enteritis, watery diarrhea, and significant loss of body condition. Transition from subclinical to clinical infection is a dynamic process led by MAP which resides in host macrophages. Clinical stage disease is accompanied by dysfunctional immune responses and a reduction in circulating vitamin D3. The immunomodulatory role of vitamin D3 in infectious disease has been well established in humans, particularly in Mycobacterium tuberculosis infection. However, significant species differences exist between the immune system of humans and bovines, including effects induced by vitamin D3. This fact highlights the need for continued study of the relationship between vitamin D3 and bovine immunity, especially during different stages of paratuberculosis. [ABSTRACT FROM AUTHOR]

Little is known of protein expression in Mycobacterium avium subsp, paratuberculosis and how this contributes to pathogenesis. In the present study, proteins from both membranes and cytosol were prepared from two strains of M. avium subsp, paratuberculosis, i.e., laboratory-adapted strain K-10 and a recent isolate, strain 187, obtained from a cow exhibiting clinical signs of Johne's disease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosol and membrane proteins from K-10 and 187 showed marked differences in protein expression. Relative levels of protein expression from both M. avium subsp, paratuberculosis strains were measured by using amine-reactive isobaric tagging reagents (iTRAQ) and tandem mass spectroscopy. Protein identification and relative expression data were obtained for 874 membrane and cytosolic proteins from the M. avium subsp, paratuberculosis proteome. These data showed a number of significant differences in protein expression between strain K-10 and clinical isolate 187. Examples of proteins expressed at higher levels in clinical isolate 187 compared to strain K-10 are AtpC, RpoA, and several proteins involved in fatty acid biosynthesis. In contrast, proteins such as AhpC and several proteins involved in nitrogen metabolism were expressed at higher levels in strain K-10 compared to strain 187. These data may provide insights into the proteins whose expression is important in natural infection but are modified once M. avium subsp, paratuberculosis is adapted to laboratory cultivation. Results from these studies will provide tools for developing a better understanding of M. avium subsp, paratuberculosis infection in the host and offer potential as diagnostic reagents and vaccine candidates.

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. This study showed that a gene encoding the M. paratuberculosis 35 kDa major membrane protein (MMP) is expressed at a higher level in low-oxygen and high-osmolarity conditions that are similar to the environment of the intestine. In addition, cattle with Johne's disease produced antibodies against MMP, suggesting that the protein is present during infection. The gene encoding MMP was cloned and expressed as a fusion protein with the maltose-binding protein (MBP-MMP) in Escherichia coli. Rabbit antisera were raised against a M. paratuberculosis whole-cell sonicate and MMP-specific antibodies were purified from these sera by affinity chromatography. MMP was localized to the surface of M. paratuberculosis by immunoelectron microscopy and by immunoblot analysis of fractionated protein lysates. Both anti-MMP antibodies and MBP-MMP protein inhibited M. paratuberculosis invasion of cultured Madin-Darby bovine kidney cells by 30 %. In similar invasion experiments with M. paratuberculosis incubated in low oxygen tension, these antibodies and protein decreased invasion by 60%. Collectively, these data show that the 35 kDa MMP is a surface exposed protein that plays a role in invasion of epithelial cells. The authors suggest that the MMP is a virulence factor of M. paratuberculosis that may be important in the initiation of infection in vivo.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of ruminant enteritis, targets intestinal macrophages. During infection, macrophages contribute to mucosal inflammation and development of granulomas in the small intestine which worsens as disease progression occurs. Vitamin D3 is an immunomodulatory steroid hormone with beneficial roles in host-pathogen interactions. Few studies have investigated immunologic roles of 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in cattle, particularly cattle infected with MAP. This study examined the effects of exogenous vitamin D3 on immune responses of monocyte derived macrophages (MDMs) isolated from dairy cattle naturally infected with MAP. MDMs were pre-treated with ± 100 ng/ml 25(OH)D3 or ± 4 ng/ml 1,25(OH)2D3, then incubated 24 hrs with live MAP in the presence of their respective pre-treatment concentrations. Following treatment with either vitamin D3 analog, phagocytosis of MAP by MDMs was significantly greater in clinically infected animals, with a greater amount of live and dead bacteria. Clinical cows had significantly less CD40 surface expression on MDMs compared to subclinical cows and noninfected controls. 1,25(OH)2D3 also significantly increased nitrite production in MAP infected cows. 1,25(OH)2D3 treatment played a key role in upregulating secretion of pro-inflammatory cytokines IL-1β and IL-12 while downregulating IL-10, IL-6, and IFN-γ. 1,25(OH)2D3 also negatively regulated transcripts of CYP24A1 , CYP27B1 , DEFB7 , NOS2 , and IL10. Results from this study demonstrate that vitamin D3 compounds, but mainly 1,25(OH)2D3, modulate both pro- and anti-inflammatory immune responses in dairy cattle infected with MAP, impacting the bacterial viability within the macrophage. [ABSTRACT FROM AUTHOR]

Abstract Background Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection. Results Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease. Conclusion Collectively these results demonstrate that M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens.

Abstract Background The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed. Results MAP0261c is conserved in mycobacteria, showing a 95% amino acid identity in M. avium subspecies avium, 84% in M. intracellulare and 76% in M. bovis and M. tuberculosis. MAP0261c was cloned, expressed, and purified as a fusion protein with the maltose-binding protein (MBP-19 kDa) in Escherichia coli. IFN-γ production was measured from 21 naturally infected and 9 control cattle after peripheral blood mononuclear cells (PBMCs) were stimulated with a whole cell lysate (WCL) of M. avium subsp. paratuberculosis or the recombinant MBP-19 kDa. Overall, the mean response to MBP-19 kDa was not as strong as the mean response to the WCL. By comparison, cells from control, non-infected cattle did not produce IFN-γ after stimulation with either WCL or MBP-19 kDa. To assess the humoral immune response to the 19-kDa protein, sera from cattle with clinical Johne's disease were used in immunoblot analysis. Reactivity to MBP-19 kDa protein, but not MBP alone, was observed in 9 of 14 infected cattle. Antibodies to the 19-kDa protein were not observed in 8 of 9 control cows. Conclusions Collectively, these results demonstrate that while the 19-kDa protein from M. avium subsp. paratuberculosis stimulates a humoral immune response and weak IFN-γ production in infected cattle, the elicited responses are not strong enough to be used in a sensitive diagnostic assay.

Abstract Background Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host. Results Serial plating of M. paratuberculosis infected macrophage lysates on Herold's egg yolk medium showed that mycobacterial replication takes place between 0 and 24 hours post-infection. This initial growth phase was followed by a steady decline in viability over the next six days. Antibodies against M. paratuberculosis were affinity purified and used in conjunction with transmission electron microscopy to track the development of intracellular bacilli. Immunogold labeling of infected macrophages with antibody against M. paratuberculosis showed degraded intracellular mycobacteria that were unrecognizable by morphology alone. Conversely, when macrophages were heavily infected with M. paratuberculosis, no degraded forms were observed and macrophages were killed. Conclusions We present a general description of M. paratuberculosis survival within cultured macrophages using transmission electron microscopy and viability counts. The results of this study provides further insight surrounding M. paratuberculosis-macrophage infections and have implications in the pathogenesis of M. paratuberculosis, a pathogen known to persist inside cattle for many years.