Development of a Real-Time Recombinase Polymerase Amplification Assay for the Rapid Detection of African Swine Fever Virus Genotype I and II.

African swine fever (ASF) is a contagious viral disease in pigs and wild boars which poses a major threat to the pig industry. Rapid and accurate diagnosis is necessary to control ASF. Hence, we developed a rapid diagnostic method using a recombinase polymerase amplification (RPA) assay targeting the conserved sequences of CP204L (p30) thatcan rapidly detect ASF virus (ASFV) genotype strains I and II. The lower detection limit of the real-time RPA assay was 5 × 101 copies per reaction. The real-time RPA assay effectively detected ASFV isolates and clinical specimens belonging to ASFV genotypes I and II. The sensitivity and specificity of the assay were 96.8% (95% confidence interval (CI): 83.3−99.9) and 100% (95% CI: 88.4−100.0), respectively. The agreement between the real-time RPA assay and a reference commercial real-time quantitative polymerase chain reaction (qPCR) was 100%. The real-time RPA assay had a detection time of 6.0 min (95% CI: 5.7−6.2), which was significantly shorter than that of qPCR (49 min; 95% CI: 47.4−50.6; p < 0.001). Thus, the developed real-time RPA assay is a rapid and accurate diagnostic tool for detecting ASFV genotypes I and II.

Glutamate mediated metabolic neutralization mitigates propionate toxicity in intracellular Mycobacterium tuberculosis.

Metabolic networks in biological systems are interconnected, such that malfunctioning parts can be corrected by other parts within the network, a process termed adaptive metabolism. Unlike Bacillus Calmette-Guérin (BCG), Mycobacterium tuberculosis (Mtb) better manages its intracellular lifestyle by executing adaptive metabolism. Here, we used metabolomics and identified glutamate synthase (GltB/D) that converts glutamine to glutamate (Q → E) as a metabolic effort used to neutralize cytoplasmic pH that is acidified while consuming host propionate carbon through the methylcitrate cycle (MCC). Methylisocitrate lyase, the last step of the MCC, is intrinsically downregulated in BCG, leading to obstruction of carbon flux toward central carbon metabolism, accumulation of MCC intermediates, and interference with GltB/D mediated neutralizing activity against propionate toxicity. Indeed, vitamin B12 mediated bypass MCC and additional supplement of glutamate led to selectively correct the phenotypic attenuation in BCG and restore the adaptive capacity of BCG to the similar level of Mtb phenotype. Collectively, a defective crosstalk between MCC and Q → E contributes to attenuation of intracellular BCG. Furthermore, GltB/D inhibition enhances the level of propionate toxicity in Mtb. Thus, these findings revealed a new adaptive metabolism and propose GltB/D as a synergistic target to improve the antimicrobial outcomes of MCC inhibition in Mtb.

Molecular and genomic features of Mycobacterium bovis strain 1595 isolated from Korean cattle.

The aim of this study was to investigate the molecular characteristics and to conduct a comparative genomic analysis of Mycobacterium (M.) bovis strain 1595 isolated from a native Korean cow. Molecular typing showed that M. bovis 1595 has spoligotype SB0140 with mycobacterial interspersed repetitive units-variable number of tandem repeats typing of 4-2-5-3-2-7-5-5-4-3-4-3-4-3, representing the most common type of M. bovis in Korea. The complete genome sequence of strain 1595 was determined by single-molecule real-time technology, which showed a genome of 4351712 bp in size with a 65.64% G + C content and 4358 protein-coding genes. Comparative genomic analysis with the genomes of Mycobacterium tuberculosis complex strains revealed that all genomes are similar in size and G + C content. Phylogenetic analysis revealed all strains were within a 0.1% average nucleotide identity value, and MUMmer analysis illustrated that all genomes showed positive collinearity with strain 1595. A sequence comparison based on BLASTP analysis showed that M. bovis AF2122/97 was the strain with the greatest number of completely matched proteins to M. bovis 1595. This genome sequence analysis will serve as a valuable reference for improving understanding of the virulence and epidemiologic traits among M. bovis isolates in Korea.

Seroprevalence of tuberculosis in domesticated elk (Cervus canadensis) in Korea.

Tuberculosis is a contagious disease in animals, primarily cattle, although it also affects wild animals and humans. There are few data on the state of tuberculosis in domesticated elk (Cervus canadensis) in Korea. In order to investigate tuberculosis in elk, the effectiveness of an enzyme linked immunosorbent assay (ELISA) using MPB70 and MPB83 antigens was compared with the tuberculin skin test (TST), and seroprevalence was measured with this assay using serum samples collected from domesticated elk herds in Korea. The respective sensitivities of the MPB70 and MPB83 ELISAs were 51.9% (95% CI 42.0-61.6) and 49.1% (95% CI 39.3-58.9), and their specificities were 100.0% (95% CI 92.6-100.0) and 97.9% (95% CI 88.9-100.0), respectively, in comparison with the TST. The herd prevalence ranged from 50 to 80% and the mean herd seropositive rate was 67.7% (21 of 31). Of 819 serum samples, 163 (19.9%) were seropositive, and the within-region prevalence ranged from 18.5-58.0%. In conclusion, the ELISA using the MPB70 and MPB83 antigens showed moderate sensitivity and high specificity compared to TST in elk, and tuberculosis was assumed to be fairly prevalent in domesticated elk in Korea.

Complete Genome Sequence of Mycobacterium bovis Clinical Strain 1595, Isolated from the Laryngopharyngeal Lymph Node of South Korean Cattle.

Mycobacterium bovis strain 1595 was isolated from the lymph node of South Korean native cattle. The complete genome sequence of strain 1595 was determined in 2 contigs and was found to be 4,351,712 bp in size, with a 65.64% G+C content and 4,358 predicted protein-coding genes.

Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals.

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.

Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells.

BACKGROUND: All mucosal epithelia, including those of the tubotympanium, are secreting a variety of antimicrobial innate immune molecules (AIIMs). In our previous study, we showed the bactericidal/bacteriostatic functions of AIIMs against various otitis media pathogens. Among the AIIMs, human beta-defensin 2 is the most potent molecule and is inducible by exposure to inflammatory stimuli such as bacterial components or proinflammatory cytokines. Even though the beta-defensin 2 is an important AIIM, the induction mechanism of this molecule has not been clearly established. We believe that this report is the first attempt to elucidate NTHi induced beta-defensin expression in airway mucosa, which includes the middle ear. METHODS: Monoclonal antibody blocking method was employed in monitoring the TLR-dependent NTHi response. Two gene knock down methods - dominant negative (DN) plasmid and small interfering RNA (siRNA) - were employed to detect and confirm the involvement of several key genes in the signaling cascade resulting from the NTHi stimulated beta-defensin 2 expression in human middle ear epithelial cell (HMEEC-1). The student's t-test was used for the statistical analysis of the data. RESULTS: The experimental results showed that the major NTHi-specific receptor in HMEEC-1 is the Toll-like receptor 2 (TLR2). Furthermore, recognition of NTHi component(s)/ligand(s) by TLR2, activated the Toll/IL-1 receptor (TIR)-MyD88-IRAK1-TRAF6-MKK3/6-p38 MAPK signal transduction pathway, ultimately leading to the induction of beta-defensin 2. CONCLUSION: This study found that the induction of beta-defensin 2 is highest in whole cell lysate (WCL) preparations of NTHi, suggesting that the ligand(s) responsible for this up-regulation may be soluble macromolecule(s). We also found that this induction takes place through the TLR2 dependent MyD88-IRAK1-TRAF6-p38 MAPK pathway, with the primary response occurring within the first hour of stimulation. In combination with our previous studies showing that IL-1alpha-induced beta-defensin 2 expression takes place through a MyD88-independent Raf-MEK1/2-ERK MAPK pathway, we found that both signaling cascades act synergistically to up-regulate beta-defensin 2 levels. We propose that this confers an essential evolutionary advantage to the cells in coping with infections and may serve to amplify the innate immune response through paracrine signaling.

Identification of the Coxiella sp. detected from Haemaphysalis longicornis ticks in Korea.

Two Haemaphysalis longicornis ticks were found positive in PCR assay of com-1 gene to detect Coxiella burnetii DNA from 100 ticks. The nucleotide sequences of com-1 and 16S rRNA gene were determined from 2 ticks and compared with those of other C. burnetii strains. The results suggest that H. longicornis harbor Coxiella sp. bacteria in Korea. Furthermore, icd, cbhE', and cbbE' genes are C. burnetii specific genes whereas com-1 gene is Coxiella genus specific gene. This study gives the first documentation to prove the existence of Coxiella sp. in tick collected in Korea.

Identification of the spotted fever group rickettsiae detected from Haemaphysalis longicornis in Korea.

Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea.