Diagnosis of Bovine Paratuberculosis by a Novel Enzyme-Linked Immunosorbent Assay Based on Early Secreted Antigens of Mycobacterium avium subsp. paratuberculosis

Shin, Sung Jae,Cho, Donghee,Collins, Michael T. American Society for Microbiology 2008 CLINICAL AND VACCINE IMMUNOLOGY Vol.15 No.8

<B>ABSTRACT</B><P>We previously reported that protein antigens of serodiagnostic potential were more abundant in culture filtrates than cellular extracts from liquid cultures of <I>Mycobacterium avium</I> subsp. <I>paratuberculosis</I> (D. Cho and M. T. Collins, Clin. Vaccine Immunol. 13:1155-1161, 2006). Based on this observation, a novel enzyme-linked immunosorbent assay (ELISA) using antigens secreted by young (early- to mid-log-phase) cultures of <I>M. avium</I> subsp. <I>paratuberculosis</I> JTC303 (a low-passage isolate originating from the ileum of a Holstein bull) in mycobactin-supplemented Watson-Reid medium (pH 6.0) was developed and evaluated using a previously described panel of bovine sera (M. T. Collins et al., Clin. Diagn. Lab. Immunol. 12:685-692, 2005) that included 444 paratuberculosis cases and 412 controls. The new assay, called JTC-ELISA, had a significantly higher diagnostic sensitivity and an equivalent specificity compared to those of five commercial paratuberculosis ELISA kits. By receiver-operating characteristic analysis, the JTC-ELISA had the highest area under the curve of the six assays evaluated. The JTC-ELISA was particularly sensitive at detecting low-level fecal shedders of <I>Mavium</I> subsp. <I>paratuberculosis</I> (40%; the sensitivity of the commercial kits was 20%). The JTC-ELISA works effectively on both serum and milk samples for the detection of cattle with subclinical <I>M. avium</I> subsp. <I>paratuberculosis</I> infections, providing a cost-effective diagnostic tool to support paratuberculosis control programs in cattle herds.</P>

Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

Cho, Donghee,Collins, Michael T. AMERICAN SOCIETY FOR MICROBIOLOGY 2006 CLINICAL AND VACCINE IMMUNOLOGY Vol.13 No.10

<B>ABSTRACT</B><P>The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of <I>Mycobacterium paratuberculosis</I> were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with <I>M. paratuberculosis</I>. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (<I>P</I> < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from <I>M. paratuberculosis</I>-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all <I>M. paratuberculosis</I>-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.</P>

Thiopurine Drugs Azathioprine and 6-Mercaptopurine Inhibit Mycobacterium paratuberculosis Growth In Vitro

Shin, Sung Jae,Collins, Michael T. American Society for Microbiology 2008 Antimicrobial agents and chemotherapy Vol.52 No.2

<B>ABSTRACT</B><P>The in vitro susceptibility of human- and bovine-origin <I>Mycobacterium paratuberculosis</I> to the thioupurine drugs 6-mercaptopurine (6-MP) and azathioprine (AZA) was established using conventional plate counting methods and the MGIT 960 ParaTB culture system. Both 6-MP and AZA had antibacterial activity against <I>M. paratuberculosis</I>; isolates from Crohn's disease patients tended to be more susceptible than were bovine-origin isolates. Isolates of <I>Mycobacterium avium</I>, used as controls, were generally resistant to both AZA and 6-MP, even at high concentrations (≥64.0 μg/ml). Among rapidly growing mycobacteria, <I>Mycobacterium phlei</I> was susceptible to 6-MP and AZA whereas <I>Mycobacterium smegmatis</I> strains were not. AZA and 6-MP limited the growth of, but did not kill, <I>M. paratuberculosis</I> in a dose-dependent manner. Anti-inflammatory drugs in the sulfonamide family (sulfapyridine, sulfasalazine, and 5-aminosalycilic acid [mesalamine]) had little or no antibacterial activity against <I>M. paratuberculosis</I>. The conventional antibiotics azithromycin and ciprofloxacin, used as control drugs, were bactericidal for <I>M. paratuberculosis</I>, exerting their killing effects on the organism relatively quickly. Simultaneous exposure of <I>M. paratuberculosis</I> to 6-MP and ciprofloxacin resulted in significantly higher CFU than use of ciprofloxacin alone. These data may partially explain the paradoxical response of Crohn's disease patients infected with <I>M. paratuberculosis</I> to treatment with immunosuppressive thiopurine drugs, i.e., they do not worsen with anti-inflammatory treatment as would be expected with a microbiological etiologic pathogen. These findings also should influence the design of therapeutic trials to evaluate antibiotic treatments of Crohn's disease: AZA drugs may confound interpretation of data on therapeutic responses for both antibiotic-treated and control groups.</P>

Identification of proteins of potential diagnostic value for bovine paratuberculosis

Cho, Donghee,Sung, Nackmoon,Collins, Michael T. WILEY-VCH Verlag 2006 Proteomics Vol.6 No.21

<P>Previously we showed that Mycobacterium paratuberculosis culture filtrates (CFs) contain more antigens that react with sera from infected cattle than do cellular extracts of the organism. The goal of the present study was to identify proteins of potential diagnostic value among these CF proteins. Proteins of potential interest were first separated by 2-DE. Roughly 240 CF protein spots were detected on CBB-stained gels using Phoretix 2D software. Of these, 83% reacted with serum from M. paratuberculosis-infected cattle in immunoblots. When bovine serum was absorbed with M. phlei antigens, however, only 37 of these antigenic protein spots were reactive. Twenty-four of these spots were selected for identification based on their immunoblot staining intensity and differences in pI and mass. A total of 14 proteins were ultimately identified by MS and BLAST searches as ModD, PepA, ArgJ, CobT, Antigen 85C, and nine hypothetical proteins. N-terminal peptide analysis of PepA, Antigen 85C, ModD, MAP1693c, MAP2168c, and MAP1022c showed that each protein has 27–39 amino acids that may function as a signal sequence suggesting they are secreted through a Sec-dependent pathway. These 14 proteins from M. paratuberculosis CF are strong candidates for use as antigens for improved serodiagnostic tests for bovine paratuberculosis.</P>

B-cell epitope specificity of carboxy terminus of Mycobacterium paratuberculosis ModD.

Cho, Donghee,Shin, Sung Jae,Collins, Michael T Marcel Dekker 2010 Journal of immunoassay & immunochemistry Vol.31 No.3

<P>Epitope mapping of ModD of Mycobacterium paratuberculosis was performed using overlapping peptides. In total, 80 overlapping peptides, covering the entire mature ModD, were commercially synthesized. Each peptide spanned 14 amino acids with an offset of 4 amino acids, i.e., with an overlap of 10 amino acids. Synthetic peptide antigenicity was evaluated by enzyme-linked immunosorbent assay (ELISA) using rabbit antisera to culture filtrate (CF) of M. avium or M. paratuberculosis or recombinant ModD (rModD). The peptides o f ModD reacting most strongly (ELISA OD > 1.0) were clustered near the N- and C-terminal ends. The peptides around the C-terminal end only showed the greatest specificity for M. paratuberculosis, yielding high ELISA OD values with rabbit anti-M. paratuberculosis CF serum and low ELISA OD values with rabbit anti-M. avium CF serum. Sera from naturally M. paratuberculosis-infected cattle, however, bound poorly to the short, 14-amino-acid peptides. Thus, two longer peptides covering amino acids 100 to 125 and 328 to 353 were synthesized based on their broad reactivity to rabbit serum against M. paratuberculosis CF. The peptide covering amino acids 328 to 353 showed the highest level of specific bovine antibody binding.</P>

Mycobacterium avium subsp. paratuberculosis Fibronectin Attachment Protein Activates Dendritic Cells and Induces a Th1 Polarization

Lee, Jun Sik,Shin, Sung Jae,Collins, Michael T.,Jung, In Duk,Jeong, Young-Il,Lee, Chang-Min,Shin, Yong Kyoo,Kim, Daejin,Park, Yeong-Min American Society for Microbiology 2009 Infection and immunity Vol.77 No.7

<B>ABSTRACT</B><P>Paratuberculosis is a chronic infectious disorder and a major problem in farmed ruminants. This disease is caused by <I>Mycobacterium avium</I> subsp. <I>paratuberculosis</I>. <I>M. avium</I> subsp. <I>paratuberculosis</I> is an important pathogen that causes Johne's disease in animals and also has been implicated as a possible cause of Crohn's disease in humans, but little is known about the protective immune responses to this microorganism. Fibronectin attachment protein (FAP) is a member of a family of fibronectin-binding proteins produced by several species of mycobacteria which is important in the pathogenesis of <I>M. avium</I>. Addition of recombinant FAP to human respiratory tract organ cultures inhibits <I>M. avium</I> binding to areas where there is epithelial damage. We characterized the role of FAP in promoting adaptive and innate immune responses. FAP functionally activated dendritic cells by augmenting the expression of CD80, CD86, major histocompatibility complex class I, and major histocompatibility complex class II. Moreover, FAP induced the allogeneic immunostimulatory capacity of dendritic cells by stimulating dendritic cell production of Th1-promoting interleukin-12. FAP also increased the production of gamma interferon by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in dendritic cells was mediated by both mitogen-activated protein kinases and NF-κB pathways. These results show that FAP modulates the adaptive immune responses to <I>M. avium</I> subsp. <I>paratuberculosis</I> by inducing maturation and activation of dendritic cells, which drives Th1 polarization.</P>

Identification of seroreactive proteins in the culture filtrate antigen of<i>Mycobacterium avium</i>ssp.<i>paratuberculosis</i>human isolates to sera from Crohn's disease patients

Shin, A-Rum,Kim, Hwa-Jung,Cho, Sang Nae,Collins, Michael T.,Manning, Elizabeth J.B.,Naser, Saleh A.,Shin, Sung Jae Oxford University Press 2010 FEMS immunology and medical microbiology Vol.58 No.1

Production of and Applications for a Polyclonal IgY Diagnostic Reagent Specific for Mycobacterium avium subsp. paratuberculosis

신성재,Seung-Sub Lee,Elizabeth J. B. Manning,Michael T. Collins 한국미생물학회 2009 The journal of microbiology Vol.47 No.5

Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-labeled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immuno- magnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.