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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]

Johne's disease affects ruminants causing an economic burden to dairy, meat and wool industries. Vaccination against Mycobacterium avium subspecies paratuberculosis (Map), which causes Johne's disease, is a primary intervention for disease control in livestock. Previously, a comprehensive, multi-institutional vaccine trial for Johne's disease was conducted to test the efficacy of live attenuated Map strains. Here, we report the humoral and cell-mediated immune responses from kid goats enrolled in that trial. Both vaccinated and unvaccinated animals showed IFN-γ stimulation and proliferation of T cell subpopulations on challenge with Map. CD4+, CD25+ and γδ cells from cultured PBMCs in the vaccinated goats showed significantly greater proliferation responses on stimulation with Map antigens. The increase in CD44+ and decrease in CD62L+ cells suggest that vaccine administration reduced the inflammatory responses associated with Map infection. Overall, a stronger antibody response was observed in the infected goats as compared to vaccinated goats. Two independent experimental approaches were used to identify differences in the antibody responses of vaccinated and unvaccinated goats. The first approach involved screening a phage expression library with pooled serum from infected goats, identifying previously reported Map antigens, including MAP_1272c and MAP_1569. However, three specific antigens detected only by vaccinated goats were also identified in the library screens. A second approach using dot blot analysis identified two additional differentially reacting proteins in the vaccinated goats (MAP_4106 and MAP_4141). These immunological results, combined with the microbiological and pathological findings obtained previously, provide a more complete picture of Johne's disease control in goats vaccinated against Map. [ABSTRACT FROM AUTHOR]

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]

Antigens extracted using ethanol (EtOH) and incorporated in the EtOH vortex ELISA (EVELISA) test have previously shown high specificity and sensitivity for detecting Mycobacterium avium subspecies paratuberculosis (Map) and M. bovis infections in cattle. The objective of this study is to define the components present in the EtOH extract. We show that this extract is composed of lipid, carbohydrate, and proteins on the surface of the bacilli, and that EtOH removes the outer layer structure of Map which comprise these elements. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a Map genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits, but this protein is not strongly immunogenic in cattle with Johne's disease. A panel of monoclonal antibodies was used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that a well-known antigen, termed MPB83, is present in M. bovis EtOH extracts and a fatty acid desaturase (MAP_2698c) is present in Map EtOH extracts, while lipoarabinomannan was common to both. The lipid and carbohydrate components of the extract were analyzed using thin layer chromatography and lectin binding, respectively. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle. [ABSTRACT FROM AUTHOR]

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in midileal tissue sections was higher for clinically affected cows, followed by subclinically affected cows and then uninfected control cows. Macrophages were present throughout the tissue sections in clinical cows, including the tunica muscularis, submucosa, and the lamina propria around the crypts and in the villous tips, with progressively fewer macrophages in subclinically affected and control cows. Clinically affected cows also demonstrated significantly higher numbers of MAP and higher numbers of macrophages with intracellular MAP compared to subclinically affected cows. MAP IF labeling was present within the submucosa and lamina propria around the crypts, progressing into the villous tips in some clinically affected cows. Our findings indicate that number of macrophages increases with progression of infection, but a significant number of the macrophages present in the midileum are not associated with MAP. [ABSTRACT FROM AUTHOR]

A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis ( MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases. [ABSTRACT FROM AUTHOR]

Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne's disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne's disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development. [ABSTRACT FROM AUTHOR]

Abstract: In the first step of a comprehensive large-scale antigen discovery project, 651 genes of Mycobacterium avium subspecies paratuberculosis were expressed in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS–PAGE gels. Collectively, these purified recombinant proteins represent 14.9% of the total M. avium subsp. paratuberculosis proteome. This volume of protein expression and purification has yielded unique observations that may be missed in smaller scale expression and purification projects. For example, the 252 putative membrane proteins predicted by PSORTb analysis, resulted in lower average expression yields (3.51mg/l culture) than the 176 predicted cytoplasmic proteins (7.27mg/l culture). A few proteins (MAP0107c, MAP3169c and MAP3640) appear to promote lysis of E. coli since there was a drop in optical density of the growth culture minutes after the inducing agent was added. Certain M. avium subsp. paratuberculosis proteins, when expressed in E. coli changed the color of the column resin or appearance of harvested cell pellets. Finally, 19 proteins showed an absorbance maximum at 260nm rather than 280nm that was attributed to binding of nucleic acid during purification. This extensive recombinant protein repository provides a powerful tool for proteome- and genome-scale research of this organism. [Copyright &y& Elsevier]

Abstract: An international committee of Johne''s disease (JD) researchers was convened to develop guidelines for JD challenge studies in multiple animal species. The intent was to develop and propose international standard guidelines for models based on animal species that would gain acceptance worldwide. Parameters essential for the development of long-term and short-term infection models were outlined and harmonized to provide a “best fit” JD challenge model for cattle, goats, sheep, cervids, and mice. These models will be useful to study host–pathogen interactions, host immunity at the local and systemic level, and for evaluating vaccine candidates and therapeutics. The consensus guidelines herein list by animal species strains of Mycobacterium avium subsp. paratuberculosis used, challenge dose, dose frequency, age of challenge, route of challenge, preparation of inoculum, experimental animal selection, quality control, minimal experimental endpoints and other parameters. [Copyright &y& Elsevier]

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, γδ+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or γδ+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation. [Copyright &y& Elsevier]

Monoclonal antibodies against Mycobacterium avium subspecies paratuberculosis(Map) proteins are important tools in Johne's disease research and diagnostics. Johne's disease is a chronic inflammatory intestinal disease of cattle, sheep, and other ruminant animals. We have previously generated multiple sets of monoclonal antibodies (mAbs) in different studies; however, because many were generated and screened against a whole-cell extract of Map, the antigens that bind to these antibodies remained unknown. In this study, we used three different approaches to identify the corresponding Map antigens for 14 mAbs that could not be identified previously. In the first approach, a new Map-lambda phage expression library was screened to identify corresponding antigens for 11 mAbs. This approach revealed that mAbs 7C8, 9H3, 12E4, 3G5, and 11B8 all detect MAP_3404 encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, while mAbs 7A6, 11F8, and 10C12 detect the GroEL2 chaperonin (MAP_3936), 6C9 detects electron transfer flavoprotein (MAP_3060c), and 14G11 detects MAP_3976, a lipoprotein anchoring transpeptidase. The epitopes to a selection of these mAbs were also defined. In a second approach, MAP_2698c bound monoclonal antibody (mAb) 14D4 as determined using protein arrays. When both of these approaches failed to identify the antigen for mAb 12C9, immunoprecipitation, mass spectrometry analysis, and codon optimization was used to identify the membrane protein, MAP_4145, as the reacting antigen. Characterized antibodies were used to quickly interrogate mycobacterial proteomic preps. We conclude by providing a complete catalog of available mAbs to Map proteins, along with their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes. [ABSTRACT FROM AUTHOR]

Abstract: Infection of ruminants with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) leads to a chronic and often fatal granulomatous enteritis known as Johne''s disease. Most infections with M. paratuberculosis occur during the first 6 months of life, and there is some evidence for transmission in utero. Once established, infections typically exist in a subclinical state for several years. Recent gene-expression profiling studies suggested the hypothesis that inherent gene-expression profiles in peripheral blood mononuclear cells (PBMCs) from M. paratuberculosis-infected cattle may be different than expression profiles in PBMCs from uninfected controls. If true, this would suggest that it is possible to identify an M. paratuberculosis infection “signature” through transcriptional profiling of peripheral immune cells. In addition, identification of groups or classes of genes showing inherently different expression in PBMCs from M. paratuberculosis-infected cattle relative to PBMCs from uninfected controls might highlight important interactions between this pathogen and the host immune system. In this report, we describe studies aimed at testing this hypothesis. Our novel results indicate that, indeed expression profiles of at least 42 genes are inherently different in freshly isolated PBMCs from M. paratuberculosis-infected cattle when compared to similar cells from uninfected controls. Gene-expression differences observed following microarray analysis were verified and expanded upon by quantitative real-time PCR (Q-RT-PCR). Our results indicate that T cells within PBMCs from M. paratuberculosis-infected cows have adopted a predominant Th 2-like phenotype (enhanced expression of IL-5, GATA 3, and possibly IL-4 mRNA), that cells within infected cow PBMCs may exhibit tissue remodeling deficiencies through higher expression of tissue inhibitor of matrix metalloproteinase (TIMP) 1 and TIMP2 RNA and lower expression of matrix metalloproteinase (MMP) 14 RNA than similar cells from healthy controls, and that cells within the PBMC population of M. paratuberculosis-infected cows are likely poised for rapid apoptosis (upregulation of CIDE-A, Bad, TNFRI, and Fas). [Copyright &y& Elsevier]