Comparative structural and immunological analysis of outer membrane proteins and dermonecrotic toxin in Bordetella bronchiseptica canine isolate.

Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals' welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components.

Emergence and genomic chion of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs.

Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-ß-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a "one health approach".

Serological investigation of seven zoonotic pathogens in companion dogs in South Korea, 2018-2021.

Based on the current situation of Korean culture and society, the population of companion animals in South Korea is growing rapidly along with zoonotic risks. The current data regarding zoonotic infections in companion dogs reported in Korea is sparse. This study aims to investigate the seroprevalence of seven potential zoonotic pathogens in companion dogs in South Korea: Anaplasma phagocytophilum, Borrelia burgdoferi, Ehrlichia canis, Coxiella burnetii, Brucella canis, Leptospira spp. and canine influenza A virus. A total of 284 serum samples were collected from 2018 to 2021, and the immunoglobulin G (IgG) antibodies against 7 zoonotic pathogens were detected using enzyme-linked immunosorbent assays. Samples were divided into five groups and analysed based on age. IgG antibodies against six of the seven pathogens were detected. The highest seropositivity rate was detected for canine influenza A virus exposure (59.1%) for which the rates were the highest in dogs under 1 year old and declined with age. Positivity rates of the other pathogens were relatively low: 1.76% for Leptospira spp., 1.40% for A. phagocytophilum and E. canis, 1.06% for B. canis and 0.35% for B. burgdoferi. No antibodies against C. burnetii were detected in this study. The exposure of dogs in South Korea to six zoonotic pathogens was serologically confirmed, highlighting a potential risk for human infection. The zoonotic risk of companion dogs cannot be neglected, and implementation of One Health approach should be advocated to establish effective preventive measures.

Comparison of blood parameters according to fecal detection of Mycobacterium avium subspecies paratuberculosis in subclinically infected Holstein cattle.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic and progressive granulomatous enteritis and economic losses in dairy cattle in subclinical stages. Subclinical infection in cattle can be detected using serum MAP antibody enzyme-linked immunosorbent assay (ELISA) and fecal polymerase chain reaction (PCR) tests. OBJECTIVES: To investigate the differences in blood parameters, according to the detection of MAP using serum antibody ELISA and fecal PCR tests. METHODS: We divided 33 subclinically infected adult cattle into three groups: seronegative and fecal-positive (SNFP, n = 5), seropositive and fecal-negative (SPFN, n = 10), and seropositive and fecal-positive (SPFP, n = 18). Hematological and serum biochemical analyses were performed. RESULTS: Although the cows were clinically healthy without any manifestations, the SNFP and SPFP groups had higher platelet counts, mean platelet volumes, plateletcrit, lactate dehydrogenase levels, lactate levels, and calcium levels but lower mean corpuscular volume concentration than the SPFN group (p < 0.017). The red blood cell count, hematocrit, monocyte count, glucose level, and calprotectin level were different according to the detection method (p < 0.05). The SNFP and SPFP groups had higher red blood cell counts, hematocrit and calprotectin levels, but lower monocyte counts and glucose levels than the SPFN group, although there were no significant differences (p > 0.017). CONCLUSIONS: The cows with fecal-positive MAP status had different blood parameters from those with fecal-negative MAP status, although they were subclinically infected. These findings provide new insights into understanding the mechanism of MAP infection in subclinically infected cattle.

Whole genome structure and resistance genes in carbapenemase-producing multidrug resistant ST378 Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CPKP) is one of the most dangerous multidrug-resistant (MDR) pathogens in human health due to its widespread circulation in the nosocomial environment. CPKP carried by companion dogs, which are close to human beings, should be considered a common threat to public health. However, CPKP dissemination through companion animals is still under consideration of major diagnosis and surveillance systems. METHODS: Two CPKP isolates which were genotyped to harbor bla NDM-5-encoding IncX3 plasmids, were subjected to the whole-genome study. Whole bacterial DNA was isolated, sequenced, and assembled with Oxford Nanopore long reads and corrected with short reads from the Illumina NovaSeq 6000 platform. The whole-genome structure and positions of antimicrobial resistance (AMR) genes were identified and visualized using CGView. Worldwide datasets were downloaded from the NCBI GenBank database for whole-genome comparative analysis. The whole-genome phylogenetic analysis was constructed using the identified whole-chromosome SNP sites from K. pneumoniae HS11286. RESULTS: As a result of the whole-genome identification, 4 heterogenous plasmids and a single chromosome were identified, each carrying various AMR genes. Multiple novel structures were identified from the AMR genes, coupled with mobile gene elements (MGE). The comparative whole-genome epidemiology revealed that ST378 K. pneumoniae is a novel type of CPKP, carrying a higher prevalence of AMR genes. CONCLUSIONS: The characterized whole-genome analysis of this study shows the emergence of a novel type of CPKP strain carrying various AMR genes with variated genomic structures. The presented data in this study show the necessity to develop additional surveillance programs and control measures for a novel type of CPKP strain.

Genomic molecular epidemiology of carbapenemase-producing Escherichia coli ST410 isolates by complete genome analysis.

The circulation of carbapenemase-producing Escherichia coli (CPEC) in our society is a serious concern for vulnerable patients in nosocomial environments. However, the genomic epidemiology of the circulation of CPEC bacteria among companion animals remains largely unknown. In this study, epidemiological analysis was conducted using complete genome identification of CPEC ST410 isolates obtained from companion animals. To estimate the genomic distance and relatedness of the isolates, a total of 37 whole-genome datasets of E. coli ST410 strains were downloaded and comparatively analysed. As a result of the analysis, the genomic structure of the chromosomes and plasmids was identified, revealing the genomic positions of multiple resistance and virulence genes. The isolates in this study were grouped into the subclade H24/RxC, with fimH24, and substituted quinolone resistance-determining regions (QRDRs) and multiple beta-lactamases, including extended-spectrum ß-lactamase (ESBL) and carbapenemase. In addition, the in silico comparison of the whole-genome datasets revealed unidentified ST410 H24/Rx subgroups, including either high pathogenicity islands (HPIs) or H21 serotypes. Considering the genetic variations and resistance gene dissemination of the isolates carried by companion animals, future approaches for preventive measurement must include the "One Health" perspective for public health in our society.

Effects of synthetic peptide RP557 and its origin, LL-37, on carbapenem-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common bacterium in nosocomial infection. The biofilm-forming ability and antimicrobial resistance make P. aeruginosa biofilm infection refractory to patients requiring hospitalization, especially patients in the intensive care unit. Therefore, many alternative compounds have been developed. A newly synthesized peptide, RP557, derived from human cathelicidin LL-37, was evaluated for its antimicrobial and antibiofilm effect toward carbapenem-resistant P. aeruginosa (CRPA). The results showed that regardless of the resistance to carbapenems, the minimal inhibition concentrations of RP557 and LL-37 against P. aeruginosa were 32 µg/mL and 256 µg/mL, respectively. Both RP557 and LL-37 significantly reduced the P. aeruginosa biofilm mass at subMICs, while subMICs of carbapenems induced biofilm formation. RP557 could also remove approximately 50% of the mature biofilm at a concentration of 64 µg/mL, while 256 µg/mL LL-37 was needed to remove it. A quarter MIC of RP557 and LL-37 was used together with carbapenems (ertapenem, imipenem, and meropenem). The results show that both RP-557 and LL-37 might increase the susceptibility to CRPA by 4-16 times. Significant gene expression level changes were observed in RP557- or LL-37-treated CRPA. Confocal images showed that biofilm structures and biofilm cell viability were significantly reduced in the LL-37- or RP557-treated groups. Therefore, RP557 and its structural origin, LL-37, could be potential treatments for carbapenem-resistant P. aeruginosa infection, especially for chronic biofilm infection. IMPORTANCE Pseudomonas aeruginosa is one of the major pathogens of nosocomial infection. Combined its biofilm-forming ability with carbapenem-resistance, it is hard to handle P. aeruginosa infection, especially for patients requiring hospitalization. Antimicrobial peptide is a type of potential compound for bacterial infection treatment. Among these, RP557 was found effective in inhibiting biofilm previously. By assessing its effect on both carbapenem-resistant P. aeruginosa planktonic cells and biofilm, our results offered a potential treatment for carbapenem-resistant P. aeruginosa infection. It could be helpful to treat severe nosocomial infection related to carbapenem-resistant bacteria and increase the patients' survival rate.

Safety and Protective Efficacy of a Candidate Vector-Based Vaccine for Bovine Tuberculosis.

This study presents the results of a survey of the safety and protective efficacy of a candidate vector-based vaccine for bovine tuberculosis, using an influenza vector with the NS1 mutation and expressing M. bovis protective antigens ESAT-6 and TB10.4. We vaccinated Balb/c outbred mice two times at 21 days apart. Our experimental design includes mice immunised with the candidate vaccine with or without adjuvant 15% Montanide Gel. The candidate vaccine's safety was determined by biometric analysis, and protective efficacy was assessed by bacteriological and histological experiments following a virulent M. bovis-8 strain challenge. Our data indicated that the adjuvant-free version of the vaccine ensured complete protection from the M. bovis-8 infection in mice.

Current Status of Q Fever and the Challenge of Outbreak Preparedness in Korea: One Health Approach to Zoonoses.

Human Q fever, a zoonosis caused by Coxiella burnetii, presents with diverse clinical manifestations ranging from mild self-limited febrile illnesses to life-threatening complications such as endocarditis or vascular infection. Although acute Q fever is a benign illness with a low mortality rate, a large-scale outbreak of Q fever in the Netherlands led to concerns about the possibility of blood transfusion-related transmission or obstetric complications in pregnant women. Furthermore, a small minority (< 5%) of patients with asymptomatic or symptomatic infection progress to chronic Q fever. Chronic Q fever is fatal in 5-50% of patients if left untreated. In South Korea, Q fever in humans was designated as a notifiable infectious disease in 2006, and the number of Q fever cases has increased sharply since 2015. Nonetheless, it is still considered a neglected and under-recognized infectious disease. In this review, recent trends of human and animal Q fever in South Korea, and public health concerns regarding Q fever outbreaks are reviewed, and we consider how a One Health approach could be applied as a preventive measure to prepare for zoonotic Q fever outbreaks.

Lumpy skin disease as an emerging infectious disease.

Lumpy skin disease (LSD) is one of the most important emerging transboundary diseases. Recently, LSD has emerged in many countries in the northern hemisphere. The LSD virus has a huge genome and is highly resistant to environmental conditions. The virus is also host-specific and large ruminants, such as cattle and domestic water buffalo, are particularly susceptible. In addition, wild ruminants can serve as potential reservoirs for spreading the LSD virus. The emergence might be related to climate change in various regions because LSD is an arthropod-borne infectious disease. This disease causes enormous economic losses, such as leather damage, decreased milk production, abortion, and death in infected ruminants. The economic importance of LSD in the bovine industry has forced countries to develop and implement control strategies against the disease. With the recent global spread and the economic impact, LSD will be discussed intensively. In addition, effective preventive measures are suggested based on the presence or absence of LSD outbreaks.

Gene expression of Toll-like receptors, cytokines and a nuclear factor and cytokine secretion in DH82 canine macrophage cells infected with Brucella canis.

Canine brucellosis caused by Brucella canis infection occurs mainly in dogs, and is a zoonotic disease that also has the possibility of infection in humans. Many studies have been conducted to understand the immunopathological mechanism of B. canis infection. However, the precise immune mechanism remains to be elucidated because compared to other Brucella spp., B. canis has different immune evasion mechanisms. In this study, gene expression levels of Toll-like receptors (TLRs) and TLR-associated molecules and cytokine production were analyzed to figure out the roles of immune-related host factors in B. canis infection. Time-dependent gene expression of TLRs (1-10) and TLR-related molecules (TNF-α, IL-5, IL-23, CCL4, CD40 and NFκ-B) and release of Th1, Th2 and Th17-related cytokines (IFN-γ, IL-1ß, IL-4, IL-6, IL-10 and IL-17A) were investigated in DH82 canine macrophages infected with B. canis. Time-dependent induction of TLRs 3, 7 and 8 was observed, and TLR 7 had the highest expression level (p <0.05). The expression levels of all TLR-related genes were significantly increased after infection. In particular, the expression of the CCL4 and IL-23 genes was highly induced. The amounts of IL-1ß, IL-6 and IL-10 were significantly increased by B. canis infection, but the amounts of IL-4 and IL-17A were not. The production of IL-1ß and IL-6 was the highest at 24 hr after B. canis infection (p <0.05). This study demonstrates that TLRs 3, 7 and 8 are prominent sites of to immune response induction with the production of related cytokines and a nuclear factor in DH82 cells infected with B. canis. These results suggest a sequential immune mechanism of B. canis infection, involving TLRs, cytokines and their associated factors.

Immunopathological mechanisms in the early stage of Mycobacterium avium subsp. paratuberculosis infection via different administration routes in a murine model.

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.

A Machine Learning Approach Reveals a Microbiota Signature for Infection with Mycobacterium avium subsp. paratuberculosis in Cattle.

Although Mycobacterium avium subsp. paratuberculosis (MAP) has threatened public health and the livestock industry, the current diagnostic tools (e.g., fecal PCR and enzyme-linked immunosorbent assay [ELISA]) for MAP infection have some limitations, such as inconsistent results due to intermittent bacterial shedding or low sensitivity during the early stage of infection. Therefore, this study aimed to develop a novel biomarker focusing on elucidating the gut microbial signature of MAP-positive ruminants, since the clinical signs of MAP infection are closely related to dysbiosis. 16S rRNA-based gut microbial community analysis revealed both a decrease in microbial diversity and the emergence of several distinct taxa following MAP infection. To determine the discriminant taxa diagnostic of MAP infection, machine learning-based feature selection and predictive model construction were applied to taxon abundance data or their transformed derivatives. The selected taxa, such as Clostridioides (formerly Clostridium) difficile, were used to build models using a support vector machine, linear support vector classification, k-nearest neighbor, and random forest with 10-fold cross-validation. The receiver operating characteristic-area under the curve (ROC-AUC) analysis of the models revealed their high accuracy, up to approximately 96%. Collectively, taxonomic signatures of cattle gut microbiotas according to MAP infection status could be identified by feature selection tools and applied to establish a predictive model for the infection state. IMPORTANCE Due to the limitations, such as intermittent bacterial shedding or poor sensitivity, of the current diagnostic tools for Johne's disease, novel biomarkers are urgently needed to aid control of the disease. Here, we explored the fecal microbiota of Johne's disease-affected cattle and tried to discover distinct microbial characteristics which have the potential to be novel noninvasive biomarkers. Through 16S rRNA sequencing and machine learning approaches, a dozen taxa were selected as taxonomic signatures to discriminate the disease state. In addition, when constructing predictive models using relative abundance data of the corresponding taxa, the models showed high accuracy for classification, even including animals with subclinical infection. Thus, our study suggested novel noninvasive microbiological biomarkers that are robustly expressed regardless of subclinical infection and the applicability of machine learning for diagnosis of Johne's disease.

Delineating transcriptional crosstalk between Mycobacterium avium subsp. paratuberculosis and human THP-1 cells at the early stage of infection via dual RNA-seq analysis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic debilitating disease in ruminants. To control this disease, it is crucial to understand immune evasion and the mechanism of persistence by analyzing the early phase interplays of the intracellular pathogens and their hosts. In the present study, host-pathogen interactions at the transcriptomic level were investigated in an in vitro macrophage infection model. When differentiated human THP-1 cells were infected with MAP, the expression of various genes associated with stress responses and metabolism was altered in both host and MAP at 3 h post-infection. MAP upregulates stress-responsive global gene regulators, such as two-component systems and sigma factors, in response to oxidative and cell wall stress. Downstream genes involved in type VII secretion systems, cell wall synthesis (polyketide biosynthesis proteins), and iron uptake were changed in response to the intracellular environment of macrophages. On the host side, upregulation of inflammatory cytokine genes was observed along with pattern recognition receptor genes. Notably, alterations in gene sets involved in arginine metabolism were observed in both the host and MAP, along with significant downregulation of NOS2 expression. These observations suggest that the utilization of metabolites such as arginine by intracellular MAP might affect host NO production. Our dual RNA-seq data can provide novel insights by capturing the global transcriptome with higher resolution, especially in MAP, thus enabling a more systematic understanding of host-pathogen interactions.

Fetuin as a potential serum biomarker to detect subclinical shedder of bovine paratuberculosis.

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods.

Mycobacterium intracellulare induces a Th17 immune response via M1-like macrophage polarization in canine peripheral blood mononuclear cells.

Mycobacterium avium-intracellulare complex (MAC) is one of the most prevalent pathogenic nontuberculous mycobacteria that cause chronic pulmonary disease. The prevalence of MAC infection has been rising globally in a wide range of hosts, including companion animals. MAC infection has been reported in dogs; however, little is known about interaction between MAC and dogs, especially in immune response. In this study, we investigated the host immune response driven by M. intracellulare using the co-culture system of canine T helper cells and autologous monocyte-derived macrophages (MDMs). Transcriptomic analysis revealed that canine MDMs differentiated into M1-like macrophages after M. intracellulare infection and the macrophages secreted molecules that induced Th1/Th17 cell polarization. Furthermore, canine lymphocytes co-cultured with M. intracellulare-infected macrophages induced the adaptive Th17 responses after 5 days. Taken together, our results indicate that M. intracellulare elicits a Th17 response through macrophage activation in this system. Those findings might help the understanding of the canine immune response to MAC infection and diminishing the potential zoonotic risk in One Health aspect.

Efficacy of bivalent vaccines of porcine circovirus type 2 and Mycoplasma hyopneumoniae in specific pathogen-free pigs challenged with porcine circovirus type 2d.

BACKGROUND: Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MHP) are economically significant pathogens in the pig industry. The use of combined vaccines against PCV2 and MHP is one of the most effective ways of protecting pigs from both diseases, and it has become a part of general management. OBJECTIVES: This study evaluated the efficacy of two new bivalent vaccines of PCV2 and MHP (Myco-X and Myco-XD) in SPF pigs. Myco-X and Myco-XD are a combined vaccine of MHP with PCV2b and PCV2d, respectively. METHODS: Sixteen pigs were divided into four groups: Myco-X-vaccinated challenged, Myco-XD-vaccinated challenged, unvaccinated challenged, and unvaccinated unchallenged. Two milliliters of Myco-X were administered intramuscularly, and 0.5 mL of Myco-XD was injected intradermally at 3 wk of age. The pigs were challenged with virulent PCV2d via the intramuscular and intranasal route 4 wk post-vaccination. RESULTS: All vaccinated pigs showed effective reduction of the clinical signs, the PCV2d load in the blood and nasal swab samples, as well as lung and lymphoid tissue lesions in the challenge test. Compared to unvaccinated challenged animals, the vaccinated challenged animals showed significantly higher (p < 0.05) levels of anti-PCV2 IgG, PCV2d-specific interferon-γ (IFN-γ), and anti-MHP IgG. CONCLUSIONS: Based on clinical, microbiological, serological, and pathological assessments, this study confirmed that both combined vaccines could protect pigs against PCV2 infection caused by PCV2d. On the other hand, further research on the efficacy evaluation of these new vaccines against the MHP challenge and PCV2d/MHP co-challenge is needed.

Comparative genomic analysis of plasmids encoding metallo-ß-lactamase NDM-5 in Enterobacterales Korean isolates from companion dogs.

Carbapenems are broad-spectrum antibiotics widely used for the treatment of human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, emerging carbapenemase-producing Enterobacterales (CPE) are rising as a public threat to human and animal health. We screened clinical bacterial isolates from 241 dogs and 18 cats hospitalized at Veterinary Medical Teaching Hospital, Seoul National University, from 2018 to 2020 for carbapenemase production. In our study, 5 strains of metallo-ß-lactamase NDM-5-producing Escherichia coli and Klebsiella pneumoniae were isolated from 4 different dogs. Multilocus sequence typing (MLST) results showed that all E. coli strains were ST410 and all K. pneumoniae strains were ST378. Whole genome analysis of the plasmid showed that blaNDM-5 is carried on a IncX3 plasmid, showing a high concordance rate with plasmids detected worldwide in human and animal isolates. The blaNDM gene was associated with the bleMBL gene and the ISAba125 element, truncated with the IS5 element. The results of this study show that CPE has already become as a threat to both animals and humans in our society, posing the necessity to solve it in terms of "One Health". Therefore, preventive strategies should be developed to prevent the spread of CPE in animal and human societies.

MicroRNA profiling in bovine serum according to the stage of Mycobacterium avium subsp. paratuberculosis infection.

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-ß1) 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.