Trypsin enhances SARS-CoV-2 infection by facilitating viral entry.

Coronaviruses infect cells by cytoplasmic or endosomal membrane fusion, driven by the spike (S) protein, which must be primed by proteolytic cleavage at the S1/S2 furin cleavage site (FCS) and the S2' site by cellular proteases. Exogenous trypsin as a medium additive facilitates isolation and propagation of several coronaviruses in vitro. Here, we show that trypsin enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in cultured cells and that SARS-CoV-2 enters cells via either a non-endosomal or an endosomal fusion pathway, depending on the presence of trypsin. Interestingly, trypsin enabled viral entry at the cell surface and led to more efficient infection than trypsin-independent endosomal entry, suggesting that trypsin production in the target organs may trigger a high level of replication of SARS-CoV-2 and cause severe tissue injury. Extensive syncytium formation and enhanced growth kinetics were observed only in the presence of exogenous trypsin when cell-adapted SARS-CoV-2 strains were tested. During 50 serial passages without the addition of trypsin, a specific R685S mutation occurred in the S1/S2 FCS (681PRRAR685) that was completely conserved but accompanied by several mutations in the S2 fusion subunit in the presence of trypsin. These findings demonstrate that the S1/S2 FCS is essential for proteolytic priming of the S protein and fusion activity for SARS-CoV-2 entry but not for viral replication. Our data can potentially contribute to the improvement of SARS-CoV-2 production for the development of vaccines or antivirals and motivate further investigations into the explicit functions of cell-adaptation-related genetic drift in SARS-CoV-2 pathogenesis.

Molecular detection and phylogenetic analysis of tick-borne pathogens in wild Korean water deer and farmed elk in Gyeongbuk and Gangwon Provinces of Korea.

The purpose of this study was to assess tick-borne pathogenic infections in 42 wild Korean water deer (KWD) and 26 farmed elk in the Gyeongbuk and Gangwon Provinces of Korea. Among the 42 wild KWD tested, the eighteen (42.9%) and five (11.9%) samples tested positive for Anaplasma phagocytophilum and A. bovis, respectively, by PCR and DNA sequencing. All positive samples were only from wild KWD. All samples were negative for other tick-borne pathogens tested. Detected 16S rRNA sequences of A. phagocytophilum and A. bovis showed 98.6-99.8% and 94.4-100% identity to those of sequences in GenBank, respectively. Because few studies have examined tick-borne pathogens in wild animals, appropriate control programs and studies are needed to prevent pathogen transmission.

Mycobacterium tuberculosis Infection in a Domesticated Korean Wild Boar ( Sus scrofa coreanus).

Tuberculosis, a chronic progressive disease, has been reported in bovine, swine, and primate species. Here, we report the first case of Mycobacterium tuberculosis infection in a Korean wild boar ( Sus scrofa coreanus). The owners this domesticated boar brought it to the Gyeongbuk Veterinary Service Laboratory in Korea after it was found dead and severely emaciated. Demarcated yellowish white nodules were found around the larynx and retropharyngeal lymph node during necropsy. The lungs had diffuse fibrinous pleuritis, severe congestion, and scattered nodules. More nodules were found in the spleen. Tuberculosis is characterized by massive macrophage infiltration and central caseous necrosis; both characteristics were found in the lungs. Histopathologic examination revealed that the alveolar lumen had marked fibrosis and exudates. Examination of the fluid revealed extensive macrophage permeation. To confirm a Mycobacterium infection, PCR was performed using two primer sets specific to the rpoB gene of Mycobacterium; Mycobacterium was detected in the lungs and spleen. To identify the species of Mycobacterium, immunohistochemical evaluation was performed using antibodies against Mycobacterium tuberculosis and Mycobacterium bovis . The results revealed immunoreactivity against M. tuberculosis but not against M. bovis . The consumption of undercooked or raw meat from game animals may expose humans and other animals to sylvatic infection. Consequently, Koreans who ingest wild boar may be at risk of a tuberculosis infection. To reduce the risk of foodborne infection and maintain public health, continuous monitoring and control strategies are required.

Detection of Antibodies against Toxoplasma gondii in Cattle Raised in Gyeongbuk Province, Korea.

This study investigated the seroprevalence of Toxoplasma gondii in cattle in Gyeongbuk province, the largest producer of cattle in Korea. Of blood samples obtained from 568 animals, 0.5% (3 of 568) tested seropositive for T. gondii by using a commercial ELISA kit. Among young individuals (≤1 year old), adult individuals (2 years old), and old individuals (≥3 years old), 0 (0%) of 37, 3 (0.6%) of 474, and 0 (0%) of 57 were seropositive, respectively. Among male, female, and castrated individuals, 0 (0%) of 22, 0 (0%) of 74, and 3 (0.6%) of 472 were seropositive, respectively. Among individuals from east, south, and northwest regions of Gyeongbuk province, 0 (0%) of 155, 2 (0.7%) of 288, and 1 (0.8%) of 125 were seropositive, respectively. No statistical differences were observed among the groups. Although a low T. gondii seroprevalence was detected in cattle raised in Gyeongbuk province, toxoplasmosis is still a concern owing to the Korean habit of eating raw or undercooked meat and the consequent risk of Toxoplasma transmission to animals and humans.

The in vitro activity of 15 antimicrobial agents against bacterial isolates from dogs.

The in vitro activity of 15 antimicrobial agents against clinical isolates of Staphylococcus pseudintermedius, Staphylococcus aureus, Escherichia coli, Pasteurella spp. and Streptococcus canis from dogs was investigated. For Staphylococcus spp., the highest frequency of resistance was observed for penicillin, followed by ampicillin, tetracycline and chloramphenicol. The highest frequency of resistance in E. coli isolates was recorded for tetracycline and streptomycin. Pasteurella spp. and S. canis had the highest resistance rate for tetracycline and chloramphenicol. Most isolates showed full susceptibility to low-level resistance to colistin, florfenicol and fluoroquinolones. Further studies using larger number of isolates from both healthy and diseased dogs would provide a broader picture of antimicrobial resistance at a national level and promote prudent use of antimicrobial agents in companion animals.

Mutant prevention concentration and phenotypic and molecular basis of fluoroquinolone resistance in clinical isolates and in vitro-selected mutants of Escherichia coli from dogs.

The antibacterial activity, selection of Escherichia coli (E. coli) mutants and mechanisms of fluoroquinolone resistance were investigated by integrating the minimum inhibitory concentration (MIC), mutant prevention concentration (MPC) and in vitro dynamic model approaches. Difloxacin and orbifloxacin, for which the above information has been scarce, were used. A range of area under curve over a 24h interval (AUC(24h))/MIC ratios and selected E. coli strains were investigated using the dynamic models. Continuous incubation for three days in the presence of difloxacin or orbifloxacin resulted in losses in E. coli susceptibility. An AUC(24h)/MIC (AUC(24h)/MPC)-dependent fluoroquinolone activity and selection of E. coli mutants was confirmed. Maximum losses in susceptibility occurred at AUC(24h)/MIC ratios of 54 (orbifloxacin) and 57.3 (difloxacin). AUC(24h)/MIC ratios of 169.8 (orbifloxacin) and 199.5 (difloxacin) were estimated to be protective against the selection of E. coli mutants, and the corresponding ratios based on AUC(24h)/MPC predictions were 34 (orbifloxacin) and 36.3 (difloxacin). When integrating our in vitro data with pharmacokinetic data in dogs, the conventional clinical doses of both drugs were found to be inadequate to attain the above protective values for 90% of the mutant subpopulation (AUC(24h)/MPC(90)). Both target mutations, esp. at codon 83 (Ser to Leu) of gyrA, and overexpression of efflux pumps contributed to resistance development, with mutants also showing decreased susceptibility to enrofloxacin and marbofloxacin. Additional studies would determine the role of mutations found outside the QRDR, at codon 24 of gyrA, and at codon 116 of parC, and establish the significance of these observations in vivo.

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology.

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a 'one factor at a time' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.

Selection of Lactobacillus sp. PSC101 that produces active dietary enzymes such as amylase, lipase, phytase and protease in pigs.

This study was carried out to screen lactic acid bacteria that produce active dietary enzymes, such as amylase, lipase, phytase, and protease, using a two-step process in pigs. We isolated a total of 210 and 132 strains of bacteria, grown under aerobic and anaerobic conditions, respectively, in Man Rogosa Sharpe agar containing 0.13% bile after treatment of intestinal samples at pH 3 for 30 min. From fecal samples, a total of 134 aerobic and 111 anaerobic strains were isolated in the same manner. In the second screening test, we selected four strains that produced four dietary enzymes from isolates obtained in the first screening test. Each strain was characterized as lactobacilli based on the following criteria: rod shape, negative for catalase, Gram positive, and lack of acute oral toxicity in mice. Of these four strains, we finally selected Lactobacillus spp. PSC101, which was resistant to pH 3 for 8 h and grew in the presence of 1% bile. In summary, Lactobacillus sp. PSC101 may be a strong probiotic candidate in swine due to its resistance to both acid and bile, its production of dietary enzymes that promote animal growth, and its non-toxic nature in mice.