scholarly journals Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array

2017 ◽  
Vol 24 (7) ◽  
Author(s):  
John P. Bannantine ◽  
Joseph J. Campo ◽  
Lingling Li ◽  
Arlo Randall ◽  
Jozelyn Pablo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
The United States ◽  
Johne's Disease ◽  
Protein Array ◽  
Johne’S Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
A Genome ◽  
Elisa Testing ◽  
Close Phylogenetic Relationship

ABSTRACT Johne's disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis, is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. avium subsp. paratuberculosis and the human pathogen Mycobacterium tuberculosis, here, we applied a whole-proteome M. tuberculosis protein array to identify seroreactive and diagnostic M. avium subsp. paratuberculosis antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. avium subsp. paratuberculosis and M. tuberculosis showed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the M. tuberculosis protein array probed with sera from M. avium subsp. paratuberculosis-infected cattle showed antibody binding to 729 M. tuberculosis proteins, with 58% of them having ≥70% identity to M. avium subsp. paratuberculosis orthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosis antigens were orthologs of previously reported M. avium subsp. paratuberculosis antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. avium subsp. paratuberculosis recombinant proteins, representing reactive and nonreactive M. tuberculosis orthologs, further confirmed that the M. tuberculosis array has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. avium subsp. paratuberculosis proteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays.

scholarly journals A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne’s Disease

2020 ◽  
Vol 58 (12) ◽  
Author(s):  
Satoko Kawaji ◽  
Reiko Nagata ◽  
Yasutaka Minegishi ◽  
Yumi Saruyama ◽  
Akiko Mita ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pcr Assay ◽  
Serological Tests ◽  
Fecal Samples ◽  
Content Type ◽  
Cattle Herds

ABSTRACT Johne’s disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis. As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.

scholarly journals MAP1272c Encodes an NlpC/P60 Protein, an Antigen Detected in Cattle with Johne's Disease

2012 ◽  
Vol 19 (7) ◽  
pp. 1083-1092 ◽  
Author(s):  
John P. Bannantine ◽  
Cari K. Lingle ◽  
Judith R. Stabel ◽  
Kasra X. Ramyar ◽  
Brandon L. Garcia ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Recombinant Proteins ◽  
Binding Assay ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Integral Membrane Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mycobacterial Species ◽  
Content Type

ABSTRACTThe protein encoded by MAP1272c has been shown to be an antigen ofMycobacterium aviumsubsp.paratuberculosisthat contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen ofM. aviumsubsp.paratuberculosis.

scholarly journals Genome Sequencing Identifies Two Nearly Unchanged Strains of Persistent Listeria monocytogenes Isolated at Two Different Fish Processing Plants Sampled 6 Years Apart

2013 ◽  
Vol 79 (9) ◽  
pp. 2944-2951 ◽  
Author(s):  
Anne Holch ◽  
Kristen Webb ◽  
Oksana Lukjancenko ◽  
David Ussery ◽  
Benjamin M. Rosenthal ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Random Amplified Polymorphic Dna ◽  
The United States ◽  
Genome Comparison ◽  
Physiological Factors ◽  
Content Type ◽  
Human Pathogenic Bacterium ◽  
A Genome ◽  
Genome Analyses

ABSTRACTListeria monocytogenesis a food-borne human-pathogenic bacterium that can cause infections with a high mortality rate. It has a remarkable ability to persist in food processing facilities. Here we report the genome sequences for twoL. monocytogenesstrains (N53-1 and La111) that were isolated 6 years apart from two different Danish fish processers. Both strains are of serotype 1/2a and belong to a highly persistent DNA subtype (random amplified polymorphic DNA [RAPD] type 9). We demonstrate usingin silicoanalyses that both strains belong to the multilocus sequence typing (MLST) type ST121 that has been isolated as a persistent subtype in several European countries. The purpose of this study was to use genome analyses to identify genes or proteins that could contribute to persistence. In a genome comparison, the two persistent strains were extremely similar and collectively differed from the reference lineage II strain, EGD-e. Also, they differed markedly from a lineage I strain (F2365). On the proteome level, the two strains were almost identical, with a predicted protein homology of 99.94%, differing at only 2 proteins. No single-nucleotide polymorphism (SNP) differences were seen between the two strains; in contrast, N53-1 and La111 differed from the EGD-e reference strain by 3,942 and 3,471 SNPs, respectively. We included a persistentL. monocytogenesstrain from the United States (F6854) in our comparisons. Compared to nonpersistent strains, all three persistent strains were distinguished by two genome deletions: one, of 2,472 bp, typically contains the gene forinlF, and the other, of 3,017 bp, includes three genes potentially related to bacteriocin production and transport (lmo2774,lmo2775, and the 3′-terminal part oflmo2776). Further studies of highly persistent strains are required to determine if the absence of these genes promotes persistence. While the genome comparison did not point to a clear physiological explanation of the persistent phenotype, the remarkable similarity between the two strains indicates that subtypes with specific traits are selected for in the food processing environment and that particular genetic and physiological factors are responsible for the persistent phenotype.

scholarly journals Meningococcal Antigen Typing System (MATS)-Based Neisseria meningitidis Serogroup B Coverage Prediction for the MenB-4C Vaccine in the United States

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Gowrisankar Rajam ◽  
Maria Stella ◽  
Ellie Kim ◽  
Simon Paulos ◽  
Giuseppe Boccadifuoco ◽  
...  
Keyword(s):  
United States ◽  
Vaccine Coverage ◽  
Binding Protein ◽  
The United States ◽  
Factor H ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immune Reactivity ◽  
Content Type ◽  
Efficacy Trials ◽  
Typing System

ABSTRACT The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA)-based system that assesses the levels of expression and immune reactivity of the three recombinant MenB-4C antigens and, in conjunction with PorA variable 2 (VR2) sequencing, provides an estimate of the susceptibility of NmB isolates to killing by MenB-4C-induced antibodies. MATS assays or similar antigen phenotype analyses assume importance under conditions in which analyses of vaccine coverage predictions are not feasible with existing strategies, including large efficacy trials or functional antibody screening of an exhaustive strain panel. MATS screening of a panel of NmB U.S. isolates (n = 442) predicts high MenB-4C vaccine coverage in the United States. Neisseria meningitidis is the most common cause of bacterial meningitis in children and young adults worldwide. A 4-component vaccine against N. meningitidis serogroup B (MenB) disease (MenB-4C [Bexsero]; GSK) combining factor H binding protein (fHBP), neisserial heparin binding protein (NHBA), neisserial adhesin A (NadA), and PorA-containing outer membrane vesicles was recently approved for use in the United States and other countries worldwide. Because the public health impact of MenB-4C in the United States is unclear, we used the meningococcal antigen typing system (MATS) to assess the strain coverage in a panel of strains representative of serogroup B (NmB) disease in the United States. MATS data correlate with killing in the human complement serum bactericidal assay (hSBA) and predict the susceptibility of NmB strains to killing in the hSBA, the accepted correlate of protection for MenB-4C vaccine. A panel of 442 NmB United States clinical isolates (collected in 2000 to 2008) whose data were down weighted with respect to the Oregon outbreak was selected from the Active Bacterial Core Surveillance (ABCs; CDC, Atlanta, GA) laboratory. MATS results examined to determine strain coverage were linked to multilocus sequence typing and antigen sequence data. MATS predicted that 91% (95% confidence interval [CI95], 72% to 96%) of the NmB strains causing disease in the United States would be covered by the MenB-4C vaccine, with the estimated coverage ranging from 88% to 97% by year with no detectable temporal trend. More than half of the covered strains could be targeted by two or more antigens. NHBA conferred coverage to 83% (CI95, 45% to 93%) of the strains, followed by factor H-binding protein (fHbp), which conferred coverage to 53% (CI95, 46% to 57%); PorA, which conferred coverage to 5.9%; and NadA, which conferred coverage to 2.5% (CI95, 1.1% to 5.2%). Two major clonal complexes (CC32 and CC41/44) had 99% strain coverage. The most frequent MATS phenotypes (39%) were fHbp and NHBA double positives. MATS predicts over 90% MenB-4C strain coverage in the United States, and the prediction is stable in time and consistent among bacterial genotypes. IMPORTANCE The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA)-based system that assesses the levels of expression and immune reactivity of the three recombinant MenB-4C antigens and, in conjunction with PorA variable 2 (VR2) sequencing, provides an estimate of the susceptibility of NmB isolates to killing by MenB-4C-induced antibodies. MATS assays or similar antigen phenotype analyses assume importance under conditions in which analyses of vaccine coverage predictions are not feasible with existing strategies, including large efficacy trials or functional antibody screening of an exhaustive strain panel. MATS screening of a panel of NmB U.S. isolates (n = 442) predicts high MenB-4C vaccine coverage in the United States.

scholarly journals Metabolic Modeling of Streptococcus mutans Reveals Complex Nutrient Requirements of an Oral Pathogen

mSystems ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Kenan Jijakli ◽  
Paul A. Jensen
Keyword(s):  
Streptococcus Mutans ◽  
Single Gene ◽  
Metabolic Model ◽  
The United States ◽  
Defined Medium ◽  
Oral Microbiome ◽  
Tooth Decay ◽  
Oral Pathogen ◽  
Content Type ◽  
A Genome

ABSTRACT Streptococcus mutans is a Gram-positive bacterium that thrives under acidic conditions and is a primary cause of tooth decay (dental caries). To better understand the metabolism of S. mutans on a systematic level, we manually constructed a genome-scale metabolic model of the S. mutans type strain UA159. The model, called iSMU, contains 675 reactions involving 429 metabolites and the products of 493 genes. We validated iSMU by comparing simulations with growth experiments in defined medium. The model simulations matched experimental results for 17 of 18 carbon source utilization assays and 47 of 49 nutrient depletion assays. We also simulated the effects of single gene deletions. The model’s predictions agreed with 78.1% and 84.4% of the gene essentiality predictions from two experimental data sets. Our manually curated model is more accurate than S. mutans models generated from automated reconstruction pipelines and more complete than other manually curated models. We used iSMU to generate hypotheses about the S. mutans metabolic network. Subsequent genetic experiments confirmed that (i) S. mutans catabolizes sorbitol via a sorbitol-6-phosphate 2-dehydrogenase (SMU_308) and (ii) the Leloir pathway is required for growth on complex carbohydrates such as raffinose. We believe the iSMU model is an important resource for understanding the metabolism of S. mutans and guiding future experiments. IMPORTANCE Tooth decay is the most prevalent chronic disease in the United States. Decay is caused by the bacterium Streptococcus mutans, an oral pathogen that ferments sugars into tooth-destroying lactic acid. We constructed a complete metabolic model of S. mutans to systematically investigate how the bacterium grows. The model provides a valuable resource for understanding and targeting S. mutans’ ability to outcompete other species in the oral microbiome.

scholarly journals The collection of lymphatic fluid from the bovine udder and its use for the detection of Mycobacterium avium subsp. paratuberculosis in the cow

2011 ◽  
Vol 24 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Johannes L. Khol ◽  
Pablo J. Pinedo ◽  
Claus D. Buergelt ◽  
Laura M. Neumann ◽  
Walter Baumgartner ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Fluid Collection ◽  
Johne's Disease ◽  
Johne’S Disease ◽  
Mycobacterium Avium Subsp ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lymph Fluid ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Lymphatic Fluid ◽  
Elisa Result

The objective of the current study was to evaluate the feasibility of lymph collection from the bovine udder and to investigate if the lymphatic fluid might be of diagnostic value in cows infected with Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis. Lymph fluid collection was attempted from 58 cows, and the reactions of the cows as well as the level of difficulty of the procedure were recorded in 56 animals. Lymph samples (51 in total) were tested for the presence of MAP by nested polymerase chain reaction. Collection of the lymphatic fluid caused no or mild signs of discomfort in 94.6% of the cows; in 51.8% of cows, lymphatic fluid was attained on the first attempt, while sample collection was unsuccessful in 12.1%. Mycobacterium avium subsp. paratuberculosis was detected in 43.1% of all lymph samples. The bacterium was present in 66.7% of cows with clinical Johne’s disease, in 42.8% of asymptomatic cows with a positive or suspicious enzyme-linked immunosorbent assay (ELISA) result in blood, and in 38.7% of cows with a negative ELISA result in blood. The present study shows that the procedure was well tolerated by most cows and can easily be performed on farm. The current report of the isolation of MAP from lymph fluid suggests that the present approach could be used for the early detection of Johne’s disease in cattle.

scholarly journals Key Role for the Alternative Sigma Factor, SigH, in the Intracellular Life of Mycobacterium avium subsp. paratuberculosis during Macrophage Stress

2013 ◽  
Vol 81 (6) ◽  
pp. 2242-2257 ◽  
Author(s):  
Pallab Ghosh ◽  
Chia-wei Wu ◽  
Adel M. Talaat
Keyword(s):  
Oxidative Stress ◽  
Mycobacterium Avium ◽  
Sigma Factor ◽  
Johne's Disease ◽  
Factor H ◽  
Johne’S Disease ◽  
Activated Macrophages ◽  
Phagosome Maturation ◽  
Content Type ◽  
Ifn Γ

ABSTRACTMycobacterium aviumsubsp.paratuberculosiscauses Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell,M. aviumsubsp.paratuberculosisis known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding ofM. aviumsubsp.paratuberculosispathogenesis, we examined phagosome maturation associated with transcriptional responses ofM. aviumsubsp.paratuberculosisduring macrophage infection. Monitoring cellular markers, only liveM. aviumsubsp.paratuberculosisbacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300M. aviumsubsp.paratuberculosisgenes were significantly and differentially regulated in both naive and IFN-γ-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important forM. aviumsubsp.paratuberculosissurvival inside gamma interferon (IFN-γ)-activated bovine macrophages. Interestingly, ansigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to thesigHregulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in thesigHregulon with a putative consensus sequence for SigH binding that was recognized in a subset of these genes (n= 30), suggesting direct regulation by SigH. Finally, mice infections showed a significant attenuation of the ΔsigHmutant compared to its parental strain, suggesting a role forsigHinM. aviumsubsp.paratuberculosisvirulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne's disease.

scholarly journals Mycobacterium avium subsp. paratuberculosis Inhibits Gamma Interferon-Induced Signaling in Bovine Monocytes: Insights into the Cellular Mechanisms of Johne's Disease

2012 ◽  
Vol 80 (9) ◽  
pp. 3039-3048 ◽  
Author(s):  
Ryan J. Arsenault ◽  
Yue Li ◽  
Kelli Bell ◽  
Kimberley Doig ◽  
Andrew Potter ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Rational Design ◽  
Cell Function ◽  
Johne's Disease ◽  
Gamma Interferon ◽  
Johne’S Disease ◽  
Cellular Mechanisms ◽  
Content Type ◽  
Ifn Γ ◽  
Stimulation Of

ABSTRACTMycobacterium aviumsubsp.paratuberculosisis the causative agent of Johne's disease in cattle and may have implications for human health. Establishment of chronic infection byM. aviumsubsp.paratuberculosisdepends on its subversion of host immune responses. This includes blocking the ability of infected macrophages to be activated by gamma interferon (IFN-γ) for clearance of this intracellular pathogen. To define the mechanism by whichM. aviumsubsp.paratuberculosissubverts this critical host cell function, patterns of signal transduction to IFN-γ stimulation of uninfected andM. aviumsubsp.paratuberculosis-infected bovine monocytes were determined through bovine-specific peptide arrays for kinome analysis. Pathway analysis of the kinome data indicated activation of the JAK-STAT pathway, a hallmark of IFN-γ signaling, in uninfected monocytes. In contrast, IFN-γ stimulation ofM. aviumsubsp.paratuberculosis-infected monocytes failed to induce patterns of peptide phosphorylation consistent with JAK-STAT activation. The inability of IFN-γ to induce differential phosphorylation of peptides corresponding to early JAK-STAT intermediates in infected monocytes indicates thatM. aviumsubsp.paratuberculosisblocks responsiveness at, or near, the IFN-γ receptor. Consistent with this hypothesis, increased expression of negative regulators of the IFN-γ receptors SOCS1 and SOCS3 as well as decreased expression of IFN-γ receptor chains 1 and 2 is observed inM. aviumsubsp.paratuberculosis-infected monocytes. These patterns of expression are functionally consistent with the kinome data and offer a mechanistic explanation for this criticalM. aviumsubsp.paratuberculosisbehavior. Understanding this mechanism may contribute to the rational design of more effective vaccines and/or therapeutics for Johne's disease.

scholarly journals Antibodies to Borrelia burgdorferi OspA, OspC, OspF, and C6 Antigens as Markers for Early and Late Infection in Dogs

2012 ◽  
Vol 19 (4) ◽  
pp. 527-535 ◽  
Author(s):  
Bettina Wagner ◽  
Heather Freer ◽  
Alicia Rollins ◽  
David Garcia-Tapia ◽  
Hollis N. Erb ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
The United States ◽  
Sensu Stricto ◽  
Antibody Responses ◽  
Surface Proteins ◽  
Early Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Antibody Levels

ABSTRACTLyme disease in the United States is caused byBorrelia burgdorferisensu stricto, which is transmitted to mammals by infected ticks.Borreliaspirochetes differentially express immunogenic outer surface proteins (Osp). Our aim was to evaluate antibody responses to Osp antigens to aid the diagnosis of early infection and the management of Lyme disease. We analyzed antibody responses during the first 3 months after the experimental infection of dogs using a novel multiplex assay. Results were compared to those obtained with two commercial assays detecting C6 antigen. Multiplex analysis identified antibodies to OspC and C6 as early as 3 weeks postinfection (p.i.) and those to OspF by 5 weeks p.i. Antibodies to C6 and OspF increased throughout the study, while antibodies to OspC peaked between 7 and 11 weeks p.i. and declined thereafter. A short-term antibody response to OspA was observed in 3/8 experimentally infected dogs on day 21 p.i. Quant C6 enzyme-linked immunosorbent assay (ELISA) results matched multiplex results during the first 7 weeks p.i.; however, antibody levels subsequently declined by up to 29%. Immune responses then were analyzed in sera from 125 client-owned dogs and revealed high agreement between antibodies to OspF and C6 as robust markers for infection. Results from canine patient sera supported that OspC is an early infection marker and antibodies to OspC decline over time. The onset and decline of antibody responses toB. burgdorferiOsp antigens and C6 reflect their differential expression during infection. They provide valuable tools to determine the stage of infection, treatment outcomes, and vaccination status in dogs.

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