Johne’s disease is a chronic wasting disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), resulting in inflammation of intestines and persistent diarrhea. The initial host response against MAP infections is mainly regulated by the Th1 response, which is characterized by the production of IFN-γ. With the progression of disease, MAP can survive in the host through the evasion of the host’s immune response by manipulating the host immune response. However, the host response during subclinical phases has not been fully understood. Immune regulatory genes, including Th17-derived cytokines, interferon regulatory factors, and calcium signaling-associated genes, are hypothesized to play an important role during subclinical phases of Johne’s disease. Therefore, the present study was conducted to analyze the expression profiles of immune regulatory genes during MAP infection in whole blood. Different expression patterns of genes were identified depending on the infection stages. Downregulation of IL-17A, IL-17F, IL-22, IL-26, HMGB1, and IRF4 and upregulation of PIP5K1C indicate suppression of the Th1 response due to MAP infection and loss of granuloma integrity. In addition, increased expression of IRF5 and IRF7 suggest activation of IFN-α/β signaling during subclinical stages, which induced indoleamine 2,3-dioxygenase mediated depletion of tryptophan metabolism. Increased expression of CORO1A indicate modulation of calcium signaling, which enhanced the survival of MAP. Taken together, distinct host gene expression induced by MAP infection indicates enhanced survival of MAP during subclinical stages.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic debilitating disease affecting ruminants worldwide. In the present study, we aimed to determine the major gene networks and pathways underlying the immune response to MAP infection using whole-blood cells, as well as provide the potential transcriptional markers for identifying the status of MAP infection. We analyzed the transcriptional profiles of whole-blood cells of cattle identified and grouped according to the presence of MAP-specific antibodies and the MAP shed by them. The grouping was based on the results obtained by ELISA and PCR analyses as follows: i) Test1 group: MAP-negative results obtained by ELISA and positive results obtained by PCR; ii) Test2 group: MAP-positive results obtained by ELISA and negative results obtained by PCR; iii) Test3 group: MAP-positive results obtained by ELISA and positive results obtained by PCR; iv) uninfected control: MAP-negative results obtained both by ELISA and PCR analysis. The results showed down-regulated production and metabolism of reactive oxygen species in the Test1 group, activation of pathways related to the host-defense response against MAP (LXR/RXR activation and complement system) in the Test2 and Test3 groups, and anti-inflammatory response (activation of IL-10 signaling pathway) only in the Test3 group. Our data indicate a balanced response that serves the immune-limiting mechanism while the host-defense responses are progressing.

This study was conducted to analyze the gene expression of prognostic potential biomarker candidates using the whole blood of cattle naturally infected with ITALIC! Mycobacterium aviumsubsp. ITALIC! paratuberculosis(MAP). We conducted real-time PCR to evaluate 23 potential biomarker candidates. Experimental animals were divided into four groups based on fecal MAP PCR and serum ELISA. Seven ( ITALIC! KLRB1, ITALIC! HGF, ITALIC! MPO, ITALIC! LTF, ITALIC! SERPINE1, ITALIC! S100A8and ITALIC! S100A9) genes were up-regulated in fecal MAP-positive cattle and three ( ITALIC! KLRB1, ITALIC! MPOand ITALIC! S100A9) were up-regulated in MAP-seropositive cattle relative to uninfected cattle. In subclinically infected animals, 17 genes ( ITALIC! TFRC, ITALIC! S100A8, ITALIC! S100A9, ITALIC! MPO, ITALIC! GBP6, ITALIC! LTF, ITALIC! KLRB1, ITALIC! SERPINE1, ITALIC! PIGR, ITALIC! IL-10, ITALIC! CXCR3, ITALIC! CD14, ITALIC! MMP9, ITALIC! ELANE, ITALIC! CHI3L1, ITALIC! HPand ITALIC! HGF) were up-regulated compared with the control group. Moreover, six genes ( ITALIC! CXCR3, ITALIC! HP, ITALIC! HGF, ITALIC! LTF, ITALIC! TFRCand ITALIC! GBP6) showed significant differences between experimental groups. Taken together, our data suggest that six genes ( ITALIC! LTF, ITALIC! HGF, ITALIC! HP, ITALIC! CXCR3, ITALIC! GBP6and ITALIC! TFRC) played essential roles in the immune response to MAP during the subclinical stage and therefore might be useful as prognostic biomarkers.

The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltosebinding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-α, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-γ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-γ, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

Non-tuberculous mycobacteria (NTM) are ubiquitous microorganisms that have the potential to cause disease in both humans and animals. Recently, NTM infections have rapidly increased in South Korea, especially in urbanized areas. However, the distribution of species and the antibiotic resistance profile of NTM in environmental sources have not yet been investigated. Therefore, we analyzed the distribution of species and the antibiotic resistance profile of NTM in soil within urban areas of South Korea. A total of 132 isolates of NTM were isolated from soil samples from 1 municipal animal shelter and 4 urban area parks. Among the 132 isolates, 105 isolates were identified as slowly growing mycobacteria (SGM) and 27 isolates as rapidly growing mycobacteria (RGM) based on the sequences of the rpoB and hsp65 genes. The antibiotic resistance patterns of NTM isolates differed from species to species. Additionally, a mutation in the rrs gene found in this study was not associated with aminoglycoside resistance. In conclusion, our results showed that NTM isolates from South Korean soil exhibit multidrug resistance to streptomycin, amikacin, azithromycin, ethambutol, isoniazid, and imipenem. These results suggest that NTM may pose a public threat.

Bovine paratuberculosis (PTB) is a chronic enteric inflammatory disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) that causes large economic losses in the dairy industry. Spread of PTB is mainly provoked by a long subclinical stage during which MAP is shed into the environment with feces; accordingly, detection of subclinical animals is very important to its control. However, current diagnostic methods are not suitable for detection of subclinical animals. Therefore, the current study was conducted to develop a diagnostic method for analysis of the expression of genes of prognostic potential biomarker candidates in the whole blood of cattle naturally infected with MAP. Real-time PCR with nine potential biomarker candidates was developed for the diagnosis of MAP subclinical infection. Animals were divided into four groups based on fecal MAP PCR and serum ELISA. Eight genes (Timp1, Hp, Serpine1, Tfrc, Mmp9, Defb1, Defb10, and S100a8) were up-regulated in MAP-infected cattle (p <0.05). Moreover, ROC analysis revealed that eight genes (Timp1, Hp, Serpine1, Tfrc, Mmp9, Defb1, Defb10, and S100a8) showed fair diagnostic performance (AUC≥0.8). Four biomarkers (Timp1, S100a8, Defb1, and Defb10) showed the highest diagnostic accuracy in the PCR positive and ELISA negative group (PN group) and three biomarkers (Tfrc, Hp, and Serpine1) showed the highest diagnostic accuracy in the PCR negative and ELISA positive group (NP group). Moreover, three biomarkers (S100a8, Hp, and Defb10) were considered the most reliable for the PCR positive and ELISA positive group (PP group). Taken together, our data suggest that real-time PCR based on eight biomarkers (Timp1, Hp, Serpine1, Tfrc, Mmp9, Defb1, Defb10, and S100a8) might be useful for diagnosis of JD, including subclinical stage cases.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease, which is a chronic granulomatous enteropathy in ruminants. Determining the genetic diversity of MAP is necessary to understand the epidemiology and biology of MAP, as well as establishing disease control strategies. In the present study, whole genome-based alignment and comparative analysis were performed using 40 publicly available MAP genomes, including newly sequenced Korean isolates. First, whole genome-based alignment was employed to identify new genomic structures in MAP genomes. Second, the genomic diversity of the MAP population was described by pangenome analysis. A phylogenetic tree based on the core genome and pangenome showed that the MAP was differentiated into two major types (C- and S-type), which was in keeping with the findings of previous studies. However, B-type strains were discriminated from C-type strains. Finally, functional analysis of the pangenome was performed using three virulence factor databases (i.e., PATRIC, VFDB, and Victors) to predict the phenotypic diversity of MAP in terms of pathogenicity. Based on the results of the pangenome analysis, we developed a real-time PCR technique to distinguish among S-, B- and C-type strains. In conclusion, the results of our study suggest that the phenotypic differences between MAP strains can be explained by their genetic polymorphisms. These results may help to elucidate the diversity of MAP, extending from genomic features to phenotypic traits.

The aim of this study was to describe the genetic diversity of Mycobacterium avium subsp. paratuberculosis (MAP) obtained from individual cows in Korea. Twelve MAP-positive fecal DNA samples and 19 MAP isolates were obtained from 10 cattle herds located in 5 provinces in Korea. In addition, 5 MAP isolates obtained from the Czech Republic and Slovakia and 3 isolates from Australia were genotyped for comparison with the domestic isolates. The most prevalent strains in Korea were of the “bison-type” genotype (23 of 31 fecal DNA/isolates) and were distributed nationwide. The remaining MAP isolates (8) and all of the foreign isolates were identified as “cattle-type”. The bison-type strains which were discriminated only as INMV 68 in variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing. Multilocus short sequence repeat (MLSSR) typing differentiated the bison-type strains into 3 different subtypes. The cattle-type strains were divided into 3 subtypes by MIRU-VNTR and 8 subtypes by MLSSR. The allelic diversities in the MIRU-VNTR and MLSSR results were calculated as 0.567 and 0.866, respectively. These results suggest that MIRU-VNTR typing cannot provide a sufficient description of the epidemiological situation of MAP. Therefore, an alternative method, such as MLSSR, is needed for typing of MAP strains to elucidate the molecular epidemiology of MAP infections. Overall, this study is the first epidemiological survey report in Korea using both MIRU-VNTR and MLSSR typing methods, and it has provided basic data necessary to elucidate the characteristics of MAP infections in Korea.