Long-Term Survival of Mycobacterium avium subsp. paratuberculosis in Fecal Samples Obtained from Naturally Infected Cows and Stored at -18°C and -70°C.
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ABSTRACT:
SUBMITTER: Raizman EA
PROVIDER: S-EPMC3157033 | biostudies-other | 2011
REPOSITORIES: biostudies-other
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Project description:Mycobacterium avium subsp. paratuberculosis causes an enteric infection in cattle, with a great impact on the dairy industry in the United States and worldwide. Characterizing the gene expression profile of M. avium subsp. paratuberculosis exposed to different stress conditions, or shed in cow feces, could improve our understanding of the pathogenesis of M. avium subsp. paratuberculosis. In this report, the stress response of M. avium subsp. paratuberculosis on a genome-wide level (stressome) was defined for the first time using DNA microarrays. Expression data analysis revealed unique gene groups of M. avium subsp. paratuberculosis that were regulated under in vitro stressors while additional groups were regulated in the cow samples. Interestingly, acidic pH induced the regulation of a large number of genes (n=597), suggesting the high sensitivity of M. avium subsp. paratuberculosis to acidic environments. Generally, responses to heat shock, acidity, and oxidative stress were similar in M. avium subsp. paratuberculosis and Mycobacterium tuberculosis, suggesting common pathways for mycobacterial defense against stressors. Several sigma factors (e.g., sigH and sigE) were differentially coregulated with a large number of genes depending on the type of each stressor. Subsequently, we analyzed the virulence of six M. avium subsp. paratuberculosis mutants with inactivation of differentially regulated genes using a murine model of paratuberculosis. Both bacterial and histopathological examinations indicated the attenuation of all gene mutants, especially those selected based on their expression in the cow samples (e.g., lipN). Overall, the employed approach profiled mycobacterial genetic networks triggered by variable stressors and identified a novel set of putative virulence genes. A similar approach could be applied to analyze other intracellular pathogens.
| S-EPMC2168719 | biostudies-literature
Project description:In this study, samples of 16 dairy cows from a MAP infected farm were used. Serum, milk and fecal samples were collected. Categorizing these cows into two groups based on their MAP infection status different standard methods for detection MAP were applied. Healthy controls showed no positive results in enzyme-linked immunosorbent assay (ELISA) with serum and milk samples (cattletype MAP Ab, Qiagen, Hilden, Germany; In-direct, IDVet, Grabels, France) and after cultivation of fecal samples on commercial Her-rold´s Egg Yolk Agars (HEYM agar, Becton Dickinson, Heidelberg, Germany) for 12 weeks. Cows with positive results were grouped into MAP infected cows. Specifically, for mass spectrometry analysis serum of seven MAP infected cows and seven healthy controls were used. All animals were from the same farm and were kept under the same environmental conditions. For additional mass spectrometry analysis with a further control group sam-ples of 21 dairy cows from an uninfected farm were examined. All cattle from this farm showed negative results in ELISA with serum and milk samples. Additionally, there was never a positive result in regularly tested fecal samples and sock swab samples of this farm. For verification of differential CTSS expression in Western blot analysis five dairy cows from another infected farm were consultedincluded. MAP status of these cows was analyzed by cultivation of fecal samples on HEYM agar for 12 weeks and ELISA with se-rum samples. In detail, two cattle were categorized into healthy controls and three cattle into MAP infected cows. Withdrawal of bovine venous whole blood and experi-mental protocols were approved by the local authority, Government of Upper Bavaria, permit no. ROB-55.2-2532.Vet_03-17-106.
2024-06-22 | PXD048042 | Pride
Project description:Johne's disease (JD) is a chronic, intestinal infection of cattle, caused by Mycobacterium avium subsp. paratuberculosis (MAP). It results in granulomatous inflammation of the intestinal lining, leading to malabsorption, diarrhea, and weight loss. Crohn's disease (CD), a chronic, inflammatory gastrointestinal disease of humans, has many clinical and pathologic similarities to JD. Dysbiosis of the enteric microbiota has been demonstrated in CD patients. It is speculated that this dysbiosis may contribute to the intestinal inflammation observed in those patients. The purpose of this study was to investigate the diversity patterns of fecal bacterial populations in cattle infected with MAP, compared to those of uninfected control cattle, using phylogenomic analysis. Fecal samples were selected to include samples from 20 MAP-positive cows; 25 MAP-negative herdmates; and 25 MAP-negative cows from a MAP-free herd. The genomic DNA was extracted; PCR amplified sequenced on a 454 Roche platform, and analyzed using QIIME. Approximately 199,077 reads were analyzed from 70 bacterial communities (average of 2,843 reads/sample). The composition of bacterial communities differed between the 3 treatment groups (P < 0.001; Permanova test). Taxonomic assignment of the operational taxonomic units (OTUs) identified 17 bacterial phyla across all samples. Bacteroidetes and Firmicutes constituted more than 95% of the bacterial population in the negative and exposed groups. In the positive group, lineages of Actinobacteria and Proteobacteria increased and those of Bacteroidetes and Firmicutes decreased (P < 0.001). Actinobacteria was highly abundant (30% of the total bacteria) in the positive group compared to exposed and negative groups (0.1-0.2%). Notably, the genus Arthrobacter was found to predominate Actinobacteria in the positive group. This study indicates that MAP-infected cattle have a different composition of their fecal microbiota than MAP-negative cattle.
| S-EPMC4975387 | biostudies-literature
Project description:The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO(+) CD25(+) T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.
| S-EPMC3675962 | biostudies-literature
Project description:Johne's disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and provides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection.
| S-EPMC4836751 | biostudies-literature
Project description:A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (10(1) to 10(6)) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortutitum, M. scrofalaceum, M. intracellular, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.
| S-EPMC2801105 | biostudies-literature
Project description:Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSP(P)15. We obtained a transposon (Tn) mutant with a disruption in the LSP(P)15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSP(P)15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSP(P)15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.
| S-EPMC4810606 | biostudies-literature
Project description:Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures.
| S-EPMC88986 | biostudies-literature
Project description:The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.
| S-EPMC140397 | biostudies-literature
Project description:BackgroundMycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study.ResultsTo characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical.ConclusionsWe present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.
| S-EPMC156888 | biostudies-literature