Infection with Brucella abortus causes contagious zoonosis, brucellosis, and leads to abortion in animals and chronic illness in humans. Chitosan nanoparticles (CNs), biocompatible and nontoxic polymers, acts as a mucosal adjuvant. In our previous study, B. abortus malate dehydrogenase (Mdh) was loaded in CNs, and it induced high production of pro-inflammatory cytokines in THP-1 cells and systemic IgA in BALB/C mice. In this study, the time-series gene expression analysis of nasal-associated lymphoid tissue (NALT) was performed to identify the mechanism by which Mdh affect the target site of nasal immunization. We showed that intranasal immunization of CNs-Mdh reduced cell viability of epithelial cells and muscle cells at first 1 h, then induced cellular movement of immune cells such as granulocytes, neutrophils and lymphocytes at 6h, and activated IL-6 signaling pathway at 12h within NALT. These activation of immune cells also promoted signaling pathway for high-mobility group box 1 protein (HMGB1), followed by the maturation of DCs required for mucosal immunity. The CNs also triggered the response to other organism and inflammatory response, showing it is immune-enhancing adjuvant. The ELISA showed that significant production of specific IgA was detected in the fecal excretions and genital secretions from the CNs-Mdh-immunized group after 2 weeks-post immunization. Collectively, these results suggest that B. abortus Mdh-loaded CNs triggers activation of HMGB1, IL-6 and DCs maturation signaling within NALT and induce production of systemic IgG and IgA.

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.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), and it causes diarrhea and weakness in cattle. During a long subclinical stage, infected animals without clinical signs shed pathogens through feces. For this reason, the diagnosis of JD during the subclinical stage is very important. Circulating miRNAs are attracting attention as useful biomarkers in various veterinary diseases because of their expression changes depending on the state of the disease. Based on current knowledge, circulating miRNAs extracted from bovine serum were used to develop a diagnostic tool for JD. In this study, the animals were divided into 4 groups according to fecal shedding, the presence of antibodies, and clinical signs. Gene expression was analyzed by performing miRNA sequencing for each group, and it was identified that the miRNA expression changed more as the MAP infection progressed. The eight miRNAs that were differentially expressed in all infected groups were selected as biomarker candidates based on their significant differences compared to the control group. These biomarker candidates were validated by qRT-PCR. Considering the sequencing data, two upregulated miRNAs and two downregulated miRNAs showed the same trend in the validation results. Network analysis was also conducted and the results showed that mRNAs (IL-10, TGF-β1) associated with regulatory T cells were predicted to be activated in the subclinical stage. Taken together, our data suggest that two miRNAs (bta-miR-374b, bta-miR-2887) may play major roles in the immune response to MAP infection during the subclinical stage.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic emaciating disease of ruminants that causes enormous economic losses to the bovine industry, globally. However, there are still remaining clues to be solved in the pathogenesis and diagnosis of the disease. Therefore, an in vivo murine experimental model was tried to understand responses in early stage of MAP infection by oral and intraperitoneal (IP) routes. In the MAP infection size, and weight of spleen and liver were increased in the IP group compared with oral groups. Severe histopathological changes were also observed in the spleen and liver of IP infected mice at 12 weeks post-infection (PI). Acid-fast bacterial burden in the organs was closely related to histopathological lesions. In the cytokine production from splenocytes of MAP-infected mice, higher amounts of in TNF-α, IL-10, and IFN-γ were produced at early stage of IP-infected mice while IL-17 production was different at time and infected groups. This phenomenon may indicate the immune shift from Th1 to Th17 through the time course of MAP infection. Systemic and local responses in the MAP-infection were analyzed by using transcriptomic analysis in the spleens and mesenteric lymph nodes (MLN). Based on the analysis of biological processes at 6 weeks PI in spleen and MLN in each infection group, canonical pathways were analyzed with ingenuity pathway analysis in the immune responses and metabolism especially lipid metabolism. Infected host cells with MAP increased in the production of proinflammatory cytokines and reduced the availability of glucose at early stage of infection (p < 0.05). Also, host cells secreted cholesterol through cholesterol efflux to disturb energy source of MAP. These results reveal immunopathological and metabolic responses in the early stage of MAP infection through the development of a murine model.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease, which is a chronic and debilitating disease in ruminants. MAP is also considered to be a possible cause of Crohn’s disease in humans. However, few studies have focused on the interactions between MAP and human macrophages to elucidate the pathogenesis of Crohn’s disease. We sought to determine the initial responses of human THP-1 cells against MAP infection using single-cell RNA-seq analysis. Clustering analysis showed that THP-1 cells were divided into seven different clusters in response to phorbol-12-myristate-13-acetate (PMA) treatment. The characteristics of each cluster were investigated by identifying cluster-specific marker genes. From the results, we found that classically differentiated cells express CD14, CD36, and TLR2, and that this cell type showed the most active responses against MAP infection. The responses included the expression of proinflammatory cytokines and chemokines such as CCL4, CCL3, IL1B, IL8, and CCL20. In addition, the Mreg cell type, a novel cell type differentiated from THP-1 cells, was discovered. Thus, it is suggested that different cell types arise even when the same cell line is treated under the same conditions. Overall, analyzing gene expression patterns via scRNA-seq classification allows a more detailed observation of the response to infection by each cell type.