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Caseous lymphadenitis (CLA) is a chronic and subclinical bacterial disease of ruminants caused by Corynebacterium pseudotuberculosis ( C. pseudotuberculosis ) infection. Until 2014, there were no reports of CLA outbreaks in South Korea; however, the prevalence of CLA cases has steadily increased. In this study, we used recently obtained field isolates to develop the first inactivated CLA vaccine in South Korea and evaluated it in various animal models. The inactivated vaccine was evaluated for virulence and effectiveness. Mice were tested for virulence and immunization challenges, and guinea pigs and Korean Native Black Goats (KNBGs) evaluated various vaccine concentrations to determine the optimal dose and effectiveness. In the case of KNBGs, clinical symptoms were not observed after vaccination. In addition, CLA-specific IgG was detected at a significantly ( p < 0.05) high level and was maintained. In histopathological evaluations, inflammation was predominantly observed in the prefemoral lymph nodes in the non-vaccinated+CHAL group. The genetic diversity of C. pseudotuberculosis , which has become widespread in South Korea, is less than 0.5% our vaccine is expected to prevent infection by a wide range of strains effectively. In summary, our CLA vaccine can potentially prevent CLA and foster the growth of South Korea's domestic KNBG industry.

Porcine respiratory coronavirus (PRCV) is a member of the species Alphacoronavirus 1 within the genus Alphacoronavirus of the family Coronaviridae . A few studies have been conducted on the prevalence of PRCV since its first identification in 1997, but there have been no recent studies on the prevalence and genetic characterization of the virus in Korea. In this study, the seroprevalence of PRCV was determined in Korean pig farms using a commercially available TGEV/PRCV differential enzyme-linked immunosorbent assay kit. The farm-level seroprevalence of PRCV was determined to be 68.6% (48/70), similar to previous reports in Korea, suggesting that PRCV is still circulating in Korean pig herds nationwide. Among the 20 PRCV-seropositive farms tested in this study, PRCV RNAs were detected in 17 oral fluid samples (28.3%) from nine farms (45.0%), while TGEV RNAs were not detected in any sample. To investigate the genetic characteristics of Korean PRCV strains, genetic and phylogenetic analyses were conducted on PRCV spike gene sequences obtained in this study. The three Korean PRCV strains (KPRCV2401, KPRCV2402, and KPRCV2403) shared 98.5-100% homology with each other and 96.2-96.6% and 91.6-94.5% homology with European and American strains, respectively. A 224-amino acid deletion was found in the S gene of both Korean and European PRCVs but not in that of American PRCVs, suggesting a European origin for Korean PRCVs. Phylogenetic analysis showed that Korean PRCVs are more closely related to European PRCVs than American PRCVs but clustered apart from both, suggesting that Korean PRCV has evolved independently since its emergence in Korean PRCVs. The results of this study will help expand knowledge on the epidemiology and molecular biology of PRCV currently circulating in Korea.

All pigs in the Republic of Korea are given the foot-and-mouth disease virus (FMDV) vaccine intramuscularly (IM) as part of the country's vaccination policy. However, the IM administration of the FMDV vaccine to pig results in residual vaccine components in the muscle and undesirable changes in muscle and soft tissues, causing economic losses in swine production. In this study, we evaluated whether intradermal (ID) vaccination could be proposed as an alternative to IM administration. ID vaccination (0.2 mL on each side of the neck muscle) and IM vaccination (2 mL on each side of the neck muscle) were performed twice, separated by 14 days, using a commercial FMD vaccine in specific-pathogen-free pigs. We observed growth performance, gross and microscopic lesions at the inoculation site, FMDV-specific antibodies, and neutralizing antibodies for 35 days after vaccination. Side effects on the skin grossly appeared following ID administration, but most were reduced within two weeks. All ID-vaccinated pigs showed inflammatory lesions limited to the dermis, but IM-vaccinated pigs had abnormal undesirable changes and pus in the muscle. ID-vaccinated pigs performed comparably to IM-vaccinated pigs in terms of growth, FMD virus-specific antibodies, protection capability against FMDV, and T-cell induction. This study demonstrated that the ID inoculation of the inactivated FMD vaccine induced immune responses comparable to an IM injection at 1/10 of the inoculation dose and that the inoculation lesion was limited to the dermis, effectively protecting against the formation of abnormal undesirable changes in muscle and soft tissues.

Novel swine orthopneumovirus (SOV) infections have been identified in pigs in the USA and some European countries but not in Asian countries, including South Korea, to date. The current study reports the first SOV infections in four domestic pig farms located in four provinces across South Korea. The detection rate of SOV in oral fluid samples using qRT-PCR was 4.4% (14/389), indicating the presence of the virus in pigs at commercial farms in Korea. Two complete genome sequences and one glycoprotein (G) gene sequence were obtained from SOV-positive samples. The complete genome analysis of KSOV-2201 and KSOV-2202 strains showed 98.2 and 95.4% homologies with a previously reported SOV, and the phylogenetic tree exhibited a high correlation with a previously reported SOV strain from the US and a canine pneumovirus (CPnV) strain from China. Based on the genetic analysis of the viral G gene, the murine pneumonia virus (MPV)-like orthopneumoviruses (MLOVs) were divided into two genogroups (G1 and G2). Seventeen CPnVs and two feline pneumoviruses were grouped into G1, while the Korean SOV strains identified in this study were grouped into G2 along with one SOV and two CPnVs. These results will contribute to expanding our understanding of the geographical distribution and genetic characteristics of the novel SOV in the global pig population.

For rapid and reliable detection of porcine epidemic diarrhea virus (PEDV) from pig clinical samples, a multiplex, real-time, reverse transcription loop-mediated isothermal amplification (mqRT-LAMP) was developed using two sets of primers and assimilating probes specific to the PEDV N gene and the Sus scrofa β-actin gene, which was used as an endogenous internal positive control (EIPC) to avoid false-negative results. The assay specifically amplified both target genes of PEDV and EIPC in a single reaction without any interference but did not amplify other porcine viral nucleic acids. The limit of detection was 10 copies/μL, 100-fold lower than that of a reverse transcription-polymerase chain reaction (RT-PCR) and equivalent to that of quantitative/real-time RT-PCR (qRT-PCR). This assay has high repeatability and reproducibility with coefficients of variation < 4.0%. The positive signal of the mqRT-LAMP assay was generated within 25 min, demonstrating advantages in rapid detection of PEDV over RT-PCR or qRT-PCR assay, which require at least 2 h turnaround times. In clinical evaluation, the detection rate of PEDV by mqRT-LAMP assay (77.3%) was higher than that of RT-PCR assay (69.7%), and comparable to qRT-PCR (76.8%) with almost 100% concordance (kappa value 0.98). The developed mqRT-LAMP assay can serve as an advanced alternative method for PEDV diagnosis because it has high sensitivity and specificity, rapidity, and reliability even in resource-limited laboratories.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection severely affects the swine industry each year. Although the host mechanisms against PRRSV infection have been identified in key target tissues through whole transcriptome sequencing, specific molecular regulators have not been elucidated. Long non-coding RNA (lncRNA) expression is highly specific and could thus be used to effectively identify PRRSV-specific candidates. Here, we identified novel lncRNAs in lungs, bronchial lymph nodes, and tonsils after PRRSV infection and constructed phenotype-based integrative co-expression networks using time-series differentially expressed (DE) lncRNAs and mRNAs. After the analyses, a total of 309 lncRNA-mRNA interactions were identified. During early host innate signalling, interferon-inducible and interferon genes were positively regulated by specific lncRNA. Moreover, T-cell receptor genes in lung adaptive immune signalling were negatively regulated by specific lncRNA. Collectively, our findings provide insights into the genome-wide lncRNA-mRNA interactions and dynamic regulation of lncRNA-mediated mechanisms against PRRSV infection., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: The antiviral activity of different mutagens against single-stranded RNA viruses is well documented; however, their activity on the replication of double-stranded RNA viruses remains unexplored. This study aims to investigate the effect of different antivirals on the replication of a chicken embryo fibroblast-adapted Infectious Bursal Disease virus, FVSKG2. This study further explores the antiviral mechanism utilized by the most effective anti-IBDV agent., Methods: The cytotoxicity and anti-FVSKG2 activity of different antiviral agents (ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride) were evaluated. The virus was serially passaged in chicken embryo fibroblasts 11 times at sub-cytotoxic concentrations of ribavirin, 5-fluorouracil or amiloride. Further, the possible mutagenic and non-mutagenic mechanisms utilized by the most effective anti-FVSKG2 agent were explored., Results and Discussion: Ribavirin was the least cytotoxic on chicken embryo fibroblasts, followed by 5-fluorouracil, amiloride and 5-azacytidine. Ribavirin inhibited the replication of FVSKG2 in chicken embryo fibroblasts significantly at concentrations as low as 0.05 mM. The extinction of FVSKG2 was achieved during serial passage of the virus in chicken embryo fibroblasts at ≥0.05 mM ribavirin; however, the emergence of a mutagen-resistant virus was not observed until the eleventh passage. Further, no mutation was observed in 1,898 nucleotides of the FVSKG2 following its five passages in chicken embryo fibroblasts in the presence of 0.025 mM ribavirin. Ribavarin inhibited the FVSKG2 replication in chicken embryo fibroblasts primarily through IMPDH-mediated depletion of the Guanosine Triphosphate pool of cells. However, other mechanisms like ribavirin-mediated cytokine induction or possible inhibition of viral RNA-dependent RNA polymerase through its interaction with the enzyme's active sites enhance the anti-IBDV effect. Ribavirin inhibits ds- RNA viruses, likely through IMPDH inhibition and not mutagenesis. The inhibitory effect may, however, be augmented by other non-mutagenic mechanisms, like induction of antiviral cytokines in chicken embryo fibroblasts or interaction of ribavirin with the active sites of RNA-dependent RNA polymerase of the virus., Competing Interests: NC is employed by Daskdan Innovations Pvt. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Akram, Gul, Parveez Zia, Hassan, Khatun, Shah, Ahmad, Ganai, Chikan, Kim and Shabir.)

Background: The porcine epidemic diarrhea virus (PEDV) represents a major health issue for piglets worldwide and does significant damage to the pork industry. Thus, new therapeutic approaches are urgently needed to manage PEDV infections. Due to the current lack of a reliable remedy, this present study aims to identify novel compounds that inhibit the 3CL protease of the virus involved in replication and pathogenesis., Results: To identify potent antiviral compounds against the 3CL protease, a virtual screening of natural compounds (n = 97,999) was conducted. The top 10 compounds were selected based on the lowest binding energy and the protein-ligand interaction analyzed. Further, the top five compounds that demonstrated a strong binding affinity were subjected to drug-likeness analysis using the ADMET prediction, which was followed by molecular dynamics simulations (500 ns), free energy landscape, and binding free energy calculations using the MM-PBSA method. Based on these parameters, four putative lead (ZINC38167083, ZINC09517223, ZINC04339983, and ZINC09517238) compounds were identified that represent potentially effective inhibitors of the 3CL protease., Conclusion: Therefore, these can be utilized for the development of novel antiviral drugs against PEDV. However, this requires further validation through in vitro and in vivo studies., (© 2023. The Author(s).)

Recently, intercalation pseudocapacitance has received significant interest as an abnormal charge storage mechanism owing to the battery-like intercalation energy storage into the bulk electrodes and the fast charge storage kinetics of electrochemical capacitors. However, intercalation pseudocapacitance of molybdenum-based polyoxometalates (POMs) for high-performance Zn ion battery (ZIB) cathodes is yet to be exploited. Herein, we demonstrate the fast and reversible intercalation pseudocapacitance of vanadium-substituted Keggin-type molybdenum-based POMs (XPMoV), where H of HPMoV is replaced by X cations (X = Li, Na, K, or Rb). This cation exchange allows cation-exchanged XPMoV to exhibit the morphological evolution into an anisotropic rodlike structure and to achieve a pillar effect on the improved chemical and structural integrity. Despite the micron-size rod morphology and the contracted lattice of (100) plane, the intercalation pseudocapacitance kinetics of XPMoV was dominated by the fast surface-confined electrochemistry and became highly reversible after the 1st cycle activation process by co-intercalation of Li + and Zn 2+ ions. Therefore, the ZIB with the KPMoV cathode delivered a high rate capability of 74.0 mAh g -1 at 20,000 mA g -1 and 87% capacity retention over 2000 cycles at 1000 mA g -1 , far exceeding HPMoV and other Mo-based cathodes. This study paves the way to design the fast and reversible intercalation pseudocapacitance of POMs and the cation exchange chemistry into the improved (electro)chemical and structural integrity.

Background: Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment., Objectives: Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses., Methods: Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis , and porcine reproductive and respiratory syndrome virus (PRRSV)., Results: The frequency of IFN-γ + CD3 + T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ + CD4 - CD8 + cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples., Conclusions: We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Korean Society of Veterinary Science.)

Porcine reproductive and respiratory syndrome (PRRS) is a disease that has inflicted economic losses in the swine industry. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), is known to have a high genetic diversity which leads to heterogeneous pathogenicity. To date, the impact of PRRS outbreaks on swine production and the economy of the swine industry in South Korea has been rarely reported. In this study, we compare the reproductive performance in the breeding-farrowing phase and growth performance in the nursery phase, in two 27-week periods, one before and one after a PRRSV1 outbreak on a 650-sow farrow-to-nursery farm caused by a Korean PRRSV1 isolate which was genetically distinct from vaccine strains or other global strains. The reproductive performance of sows and the growth performance of nursery pigs were compared using row data consisting of 1907 mating records, 1648 farrowing records, and 17,129 weaning records from 32 breeding batches. The following variables were significantly different between the pre-PRRS outbreak period and the post-PRRS outbreak period: the farrowing rate (−7.1%, p < 0.0001), the abortion rate (+3.9%, p < 0.0001), the return rate (+2.9%, p = 0.0250), weaning to estrus interval days (+1.9 days, p < 0.0001), total piglets born (−1.2 pigs/litter, p < 0.0001), piglets born alive (−2.2 pigs/litter, p < 0.0001), weaned piglets (−2.7 pigs/litter, p < 0.0001), pre-weaning mortality (+7.4%, p < 0.0001), weaning weight (−0.9 kg/pig, p = 0.0015), the mortality rate (+2.8%, p < 0.0001), average daily gain (−69.8 g/d, p < 0.0001), and the feed conversion ratio (+0.26, p = 0.0036). Economic losses for a period of 27 weeks after a PRRS outbreak were calculated at KRW 99,378 (USD 82.8) per mated female for the breeding-farrowing phase, KRW 8,968 (USD 7.5) per pig for the nursery growth phase, and KRW 245,174 (USD 204.3) per sow in the post-outbreak period. In conclusion, the farrow-to-nursery farm in our study suffered extensive production and economic losses as a result of a PRRSV1 outbreak.

Porcine respirovirus 1 (PRV1) is a recently emerging porcine respiratory virus that belongs to the genus Respirovirus of the Paramyxoviridae family. Since its first detection in Hong Kong, China in 2009, PRV1 has been subsequently identified in several American and European countries, suggesting that the emerging virus may have been globally distributed. However, in Asia, the virus has been reported only in China. Here, we report that PRV1 was first detected in pigs from 16 farms located in seven provinces across Korea, with a prevalence of 71.4% based on the tested oral fluid samples, suggesting that the virus is already widespread in Korean pig herds. For further genetic characterization of the Korean PRV1 strains, a complete genome and two F gene sequences were obtained from PRV1-positive samples collected from three different pig farms. Phylogenetic analysis based on the complete genome and F gene sequences showed that all three Korean PRV1 strains were grouped into European lineage 1 and were closely related to strains from Hong Kong (China), Germany and Poland. We could not obtain evidence for the origin of Korean PRV1 because of the limited availability of PRV1 sequences. In conclusion, PRV1 was first identified in Korean pig herds and genetically characterized in the present study. These results contribute to a better understanding of the global geographical distribution and genetic characteristics of PRV1., (© 2022 Wiley-VCH GmbH.)

The COVID-19 pandemic was caused by the zoonotic SARS-CoV-2. A variety of animals involved in human life worldwide have been investigated for infection. As the degree of infection increased, extensive monitoring in animals became necessary to determine the degree of infection in animals. The study was conducted on a sample of dogs and cats, which were randomly sampled according to the number of confirmed cases in the region. Animals from both COVID-19-confirmed households and generally disease-negative families and animal shelters were included. Tests included real-time qPCR tests for SARS-CoV-2 antigens, ELISA for antibodies, and plaque reduction neutralization tests (PRNT) for neutralizing antibodies. As a result, SARS-CoV-2 viral RNA was detected in 2 cats out of 1018 pets (672 dogs and 346 cats). A total of 16 dogs (2.38%) and 18 cats (5.20%) tested positive using ELISA, and 14 dogs (2.08%) and 17 cats (4.91%) tested positive using PRNT. Antigens of- and/or antibodies to SARS-CoV-2 were detected in the animals regardless of whether the companion family was infected; this was the case even in animal shelters, which have been regarded as relatively safe from transmission. In conclusion, continuous viral circulation between humans and animals is inevitable; therefore, continuous monitoring in animals is required.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen that causes porcine circovirus-associated diseases (PCVADs). The objective of this study was to evaluate the use of specific pathogen-free Yucatan miniature pigs (YMPs) as an experimental model for PCV2d challenge and vaccine assessment because PCV2-negative pigs are extremely rare in conventional swine herds in Korea. In the first experiment, every three pigs were subjected to PCV2d field isolate or mock challenge. During three weeks of experiments, the PCV2d infection group exhibited clinical outcomes of PCVAD with high viral loads, lymphoid depletion, and detection of PCV2d antigens in lymphoid organs by immunohistochemistry. In the second experiment, three groups of pigs were challenged with PCV2d after immunization for three weeks: a nonvaccinated group (three pigs), a PCV2b-Vac group vaccinated with a commercial PCV2b-based inactivated vaccine SuiShot ® Circo-ONE (five pigs), and a PCV2d-Vac group vaccinated with an experimental PCV2d-based inactivated vaccine (five pigs). During the three weeks of the challenge period, nonvaccinated pigs showed similar clinical outcomes to those observed in the PCV2d infection group from the first experiment. In contrast, both the PCV2b and PCV2d vaccinations produced good levels of protection against PCV2d challenge, as evidenced by reduced viral loads, improved growth performance, high virus-neutralizing antibody titers, and less development of PCV2-associated pathological lesions. Taken together, these data suggest that YMPs could be an alternative model for PCV2 challenge experiments, and these animals displayed typical clinical and pathological features and characteristics of protective immunity induced by the vaccines that were consistent with those resulting from PCV2 infections in conventional pigs.

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is a macrophage-tropic arterivirus with extremely high genetic and pathogenic heterogeneity that causes significant economic losses in the swine industry worldwide. PRRSV can be divided into two species [PRRSV1 (European) and PRRSV2 (North American)] and is usually diagnosed and genetically differentiated into several lineages based on the ORF5 gene, which constitutes only 5% of the whole genome. This study was conducted to achieve nonselective amplification and whole-genome sequencing (WGS) based on a simplified sequence-independent, single-primer amplification (SISPA) technique with next-generation sequencing (NGS), and to genetically characterize Korean PRRSV field isolates at the whole genome level., Methods: The SISPA-NGS method coupled with a bioinformatics pipeline was utilized to retrieve full length PRRSV genomes of 19 representative Korean PRRSV strains by de novo assembly. Phylogenetic analysis, analysis of the insertion and deletion (INDEL) pattern of nonstructural protein 2 (NSP2), and recombination analysis were conducted., Results: Nineteen complete PRRSV genomes were obtained with a high depth of coverage by the SISPA-NGS method. Korean PRRSV1 belonged to the Korean-specific subtype 1A and vaccine-related subtype 1C lineages, showing no evidence of recombination and divergent genetic heterogeneity with conserved NSP2 deletion patterns. Among Korean PRRSV2 isolates, modified live vaccine (MLV)-related lineage 5 viruses, lineage 1 viruses, and nation-specific Korean lineages (KOR A, B and C) could be identified. The NSP2 deletion pattern of the Korean lineages was consistent with that of the MN-184 strain (lineage 1), which indicates the common ancestor and independent evolution of Korean lineages. Multiple recombination signals were detected from Korean-lineage strains isolated in the 2010s, suggesting natural interlineage recombination between circulating KOR C and MLV strains. Interestingly, the Korean strain GGYC45 was identified as a recombinant KOR C and MLV strain harboring the KOR B ORF5 gene and might be the ancestor of currently circulating KOR B strains. Additionally, two novel lineage 1 recombinants of NADC30-like and NADC34-like viruses were detected., Conclusion: Genome-wide analysis of Korean PRRSV isolates retrieved by the SISPA-NGS method and de novo assembly, revealed complex evolution and recombination in the field. Therefore, continuous surveillance of PRRSV at the whole genome level should be conducted, and new vaccine strategies for more efficient control of the virus are needed., (© 2022. The Author(s).)

Background: Classical porcine parvovirus (PPV1) and novel porcine parvoviruses designated porcine parvovirus 2 through 7 (PPV2-PPV7) are widespread in pig populations. The objective of this study was to investigate the prevalence rates of PPV1-PPV7 in Korea by detecting PPVs in serum, lung and fecal samples and to elucidate the association of PPVs with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory virus (PRRSV), major pathogens involved in porcine respiratory disease complex (PRDC). A total of 286 serum, 481 lung, and 281 fecal samples collected from 2018 to 2020 were analyzed., Results: The results showed that PPVs are widespread in Korea; the highest detection rates were found in lung samples and ranged from 7.9% (PPV1) to 32.6% (PPV2). Regarding age groups, fattening pigs had the highest detection rates of PPVs, ranging from 6.4% (PPV1) to 36.5% (PPV6); this finding suggests the chronic nature of PPV infections and the continual circulation of these viruses. When compared with PCV2- and PRRSV-negative lung samples, PCV2-positive samples with or without PRRSV positivity had significantly higher detection levels of PPV1 and PPV6. In contrast, the prevalence of PPV2 and PPV7 was significantly higher in PRRSV-infected lung samples regardless of PCV2 detection. PPV5 was detected significantly more frequently in samples  with both PCV2 and PRRSV positivity., Conclusions: This study could offer a better understanding of the role of PPVs in PCV2 and/or PRRSV infection though further studies are needed to experimentally assess the impact of PPVs in coinfections., (© 2022. The Author(s).)

Immortalized cell lines are valuable resources to expand the molecular characterization of major histocompatibility complex genes and their presented antigens. We generated a panel of immortalized cell lines by transfecting human telomerase reverse transcriptase ( hTERT ) into primary fibroblast cells prepared from ear, fetal, and lung tissues of 10 pigs from five breeds and successfully cultured them for 30-45 passages. The cell growth characteristic of the immortalized fibroblasts was similar to that of primary fibroblast, which was unable to form colonies on soft agar. The genotypes of major swine leukocyte antigen (SLA) genes, including three classical class I ( SLA-1, -2, and -3 ) and three class II genes ( DQB1 , DRB1 , and DQA ), were determined using high-resolution typing. A total of 58 alleles, including a novel allele for SLA-2 , were identified. Each cell line was unique. A cell line derived from a National Institutes of Health miniature pig was homozygous across the six major SLA genes. The expression levels of SLA classical class I genes varied among the cell lines and were slightly upregulated in the immortalized compared to the primary cells based on semiquantitative reverse transcription polymerase chain reaction. The immortalized porcine fibroblast cell lines with diverse SLA haplotypes that were developed in this study have potential to be applied in studies regarding the molecular characteristics and genetic structure of SLA genes and epitope-major histocompatibility complex interactions in pigs., Competing Interests: Author C-SH was employed by the company Gift of Hope Organ & Tissue Donor Network. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Le, Youk, Choi, Jeon, Kim, Ho and Park.)

To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A novel porcine circovirus 4 has been recently identified in China and Korea. A sensitive and specific diagnostic method is urgently required to detect the virus in field samples. We developed a loop-mediated isothermal amplification (LAMP) the assay for the visual detection of PCV4 and evaluated its sensitivity, specificity, and applicability in clinical samples. This assay's results can be directly visualized by the naked eye using hydroxynaphthol blue after incubation for 40 min at 64 °C. The assay specifically amplified PCV4 DNA and no other viral nucleic acids. The sensitivity of the assay was <50 DNA copies/reaction, which was 10 times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). Clinical evaluation revealed that the PCV4 detection rate in individual pig samples and at the farm level was 39.3 % (57/145) and 45.7 % (32/70), respectively, which were higher than cPCR (46 samples, 24 farms) and qPCR (52 samples, 29 farms) results. Cumulatively, owing to the advantages of high sensitivity and specificity, direct visual monitoring of the results, no possibility for cross-contamination, and being a low-cost equipment, the developed LAMP assay will be a valuable tool for the detection of the novel PCV4 in clinical samples, even in resource-limited laboratories., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This study aimed to determine the prevalence of several pathovirotypes and evaluate the association of haemolysis with the virotypes of pathogenic E. coli isolated from post-weaning piglets in South Korea from 2015 to 2019. We isolated 890 E. coli and tested for O-serogroups, virulence genes, haemolysis, and multilocus sequence typing. The predominant virotypes were STb:EAST1:AIDA-I, F18b:Stx2e:AIDA-I, F18:STa:STb:Stx2e, and eae:Paa in enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), ETEC/STEC, and enteropathogenic E. coli (EPEC), respectively. Regarding serogroups, O139, O149, O141, and O121 were mostly detected in F18:Stx2e:AIDA-I, F4:LT:STb:EAST1, F18:STa:STb, and F18:Stx2e:EAST1, respectively. There was a significant change in the frequency of the O141:F18ac:STa:STb (an increase from 1.6% to 10.1%) and O139:F18ab:Stx2e:AIDA-I (a decrease from 13.0% to 5.3%) virotypes in ETEC and STEC, respectively, from 2015 to 2019. The O141:F18ac:STa:STb virotype was mostly detected in the central area and was spreading to the southern area. The odds ratios between haemolysis and virotypes were 11.0, 6.25, and 8.57 in F18:STa:STb, F18:Stx2e:AIDA-I, and F4:LT:STb:EAST1, respectively. Our findings provide insights regarding the recent prevalence of pathogenic E. coli in South Korea and could be used for the development of vaccines for E. coli responsible for PWD and ED in post-weaning piglets.

Despite the routine use of porcine reproductive and respiratory syndrome (PRRS)-modified live vaccines, serious concerns are currently being raised due to their quick reversion to virulence and limited cross-protection against divergent PRRS virus (PRRSV) strains circulating in the field. Therefore, a PRRS chimeric vaccine (JB1) was produced using a DNA-launched infectious clone by replacing open reading frames (ORFs) 3-6 with those from a mixture of two genetically different PRRSV2 strains (K07-2273 and K08-1054) and ORF1a with that from a mutation-resistant PRRSV strain (RVRp22) exhibiting an attenuated phenotype. To evaluate the safety and cross-protective efficacy of JB1 in a reproductive model, eight PRRS-negative pregnant sows were purchased and divided into four groups. Four sows in two of the groups were vaccinated with JB1, and the other 4 sows were untreated at gestational day 60. At gestational day 93, one vaccinated group and one nonvaccinated group each were challenged with either K07-2273 or K08-1054. All of the sows aborted or delivered until gestation day 115 (24 days post challenge), and the newborn piglets were observed up to the 28th day after birth, which was the end of the experiment. Overall, pregnant sows of the JB1-vaccinated groups showed no meaningful viremia after vaccination and significant reductions in viremia with K07-2273 and K08-1054, exhibiting significantly higher levels of serum virus-neutralizing antibodies than non-vaccinated sows. Moreover, the JB1-vaccinated groups did not exhibit any abortion due to vaccination and showed improved piglet viability and birth weight. The piglets from JB1-vaccinated sows displayed lower viral concentrations in serum and fewer lung lesions compared with those of the piglets from the nonvaccinated sows. Therefore, JB1 is a safe and effective vaccine candidate that confers simultaneous protection against two genetically different PRRSV strains.

Vaccines against porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are routinely used by intramuscular injection. However, since intramuscular vaccination causes stress and increases the risk of cross-contamination among pigs, research on intradermal vaccination is currently being actively conducted. This study was designed to evaluate the efficacy of intradermally administered inactivated vaccines against PCV2 and Mhp in pigs. Three-week-old specific pathogen-free pigs were divided into three groups (5 pigs per group). Pigs in the two groups were intradermally vaccinated with the PCV2 or Mhp vaccine using a needle-free injector. Pigs in the third group were kept as nonvaccinated controls. At 21 days post-vaccination, pigs in one of these vaccinated groups and the nonvaccinated group were intranasally challenged with PCV2b and Mhp, while the other vaccinated group pigs were maintained as vaccine controls. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes, and humoral or cellular immune responses. As a result, vaccinated pigs revealed significantly higher body weight gain, with lower clinical scores. Vaccinated pigs also showed higher antibody responses but lower PCV2b or Mhp loads in sera, nasal swabs, or lungs than nonvaccinated pigs. Intriguingly, vaccinated pigs upregulated cytotoxic T cells (CTLs), helper T type 1 cells (Th1 cells), and helper T type 17 cells (Th17 cells) after immunization and showed significantly higher levels of CTLs, Th1 and Th17 cells at 14 days post-challenge than nonvaccinated and challenged pigs. This study demonstrated that protective immune responses against PCV2 and Mhp could be efficiently induced in pigs using a relatively small volume of intradermal vaccines, probably due to effective antigen delivery to antigen-presenting cells in the dermis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the Korean swine industry. Despite efforts including improved biosecurity and vaccination protocols, the virus continues to circulate and evolve. Based on phylogenetic analysis of open reading frame 5 (ORF5), Korean PRRSVs are known to form not only globally circulating lineages but also country-specific lineages (Lin Kor A, B, and C). To understand the recent epidemiological status of PRRSV in Korea, a total of 1349 ORF5 sequences of Korean PRRSV isolates from 2014 to 2019 were analyzed. Phylogenetic analysis was conducted using the maximum-likelihood method, and temporal changes in the relative prevalence of lineages were investigated. The analysis showed that PRRSV1 and PRRSV2 were both highly prevalent throughout the years examined. Among the PRRSV1 isolates, subgroup A (90.1%) and vaccine-like subgroup C (9.0%) composed most of the population. For PRRSV2 isolates, vaccine-like lineage 5 (36.3%) was dominant, followed by Lin Kor B (25.9%), Kor C (16.6%), lineage 1 (11.6%), and Kor A (9.1%). The PRRSV2 lineage 1 population increased from 2014 (1.8%) to 2019 (29.6%) in Korea due to the continual spread of sublineage 1.8 (NADC30-like) and introduction of sublineage 1.6 into the country. Additional genetic analysis, including analysis of non synonymous and synonymous mutations, revealed evidence of diversification and positive selection in immunologically important regions of the genome, suggesting that current vaccination is failing and promoting immune-mediated selection. Overall, these findings provide insights into the epidemiological and evolutionary dynamics of cocirculating viral lineages, and constant surveillance of PRRSV occurrence is needed., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

We aimed to comprehensively understand the functional mechanisms of immunity, especially of the CD8 +/- subsets of gamma delta (γδ) T cells, using an RNA-sequencing analysis. Herein, γδ T cells were obtained from bronchial lymph node tissues of 38-day-old (after weaning 10-day: D10) and 56-day-old (after weaning 28-day: D28) weaned pigs and sorted into CD8 + and CD8 - groups. Differentially expressed genes (DEGs) were identified based on the CD8 groups at D10 and D28 time points. We confirmed 1699 DEGs between D10 CD8 + versus D10 CD8 - groups and 1784 DEGs between D28 CD8 + versus D28 CD8 - groups; 646 upregulated and 561 downregulated DEGs were common. The common upregulated DEGs were enriched in the cytokine-cytokine receptor interaction and T cell receptor (TCR) signaling pathway, and the common downregulated DEGs were enriched in the B cell receptor signaling pathway. Further, chemokine-related genes, interferon gamma, and CD40 ligand were involved in the cytokine-cytokine receptor interaction and TCR signaling pathway, which are associated with inter-regulation in immunity. We expect our results to form the basic data required for understanding the mechanisms of γδ T cells in pigs; however, further studies are required in order to reveal the dynamic changes in γδ T cells under pathogenic infections, such as those by viruses.

Porcine parvoviruses (PPVs) are small, nonenveloped DNA viruses that are widespread in the global pig population. PPV type 1 (PPV1) is a major causative agent of reproductive failure and has been recognized since the 1960s. In recent decades, novel PPVs have been identified and designated as PPVs 2 through 7 (PPV2~PPV7). Although the epidemiological impacts of these newly recognized parvoviruses on pigs are largely unknown, continuous surveillance of these PPVs is needed. The aim of this study was to develop an improved and efficient detection tool for these PPVs and to assess the developed method with field samples. Using 7 sets of newly designed primers, a multiplex polymerase chain reaction (mPCR) protocol was developed for the simultaneous detection of the seven genotypes of PPV (PPV1~PPV7). The sensitivity of the mPCR assay was analyzed, and the detection limit was determined to be 3×103 viral copies. The assay was highly specific in detecting one or more of the viruses in various combinations in specimens. The mPCR method was evaluated with 80 serum samples, 40 lung or lymph node samples and 40 intestine or fecal samples. When applied to these samples, the mPCR method could detect the 7 viruses simultaneously, providing rapid results regarding infection and coinfection status. In conclusion, the developed mPCR assay can be utilized as an effective and accurate diagnostic tool for rapid differential detection and epidemiological surveillance of various PPVs in numerous types of field samples., Competing Interests: The authors have declared that no competing interests exist.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is the most important viral disease causing severe economic losses in the swine industry. However, mechanisms underlying gene expression control in immunity-responsible tissues at different time points during PRRSV infection are poorly understood. We constructed an integrated gene co-expression network and identified tissue- and time-dependent biological mechanisms of PRRSV infection through bioinformatics analysis using three tissues (lungs, bronchial lymph nodes [BLNs], and tonsils) via RNA-Seq. Three groups with specific expression patterns (i.e., the 3-dpi, lung, and BLN groups) were discovered. The 3 dpi-specific group showed antiviral and innate-immune signalling similar to the case for influenza A infection. Moreover, we observed adaptive immune responses in the lung-specific group based on various cytokines, while the BLN-specific group showed down-regulated AMPK signalling related to viral replication. Our study may provide comprehensive insights into PRRSV infection, as well as useful information for vaccine development.

This study examined the presence of Treponema in lesions using conventional PCR detection methods and investigated the microbiome by performing high-throughput DNA sequencing. Twenty-nine bovine digital dermatitis (BDD) lesions were collected from 25 dairy farms in South Korea that were tested by PCR amplification using sets of one universal, one genus-specific, and three species specific Treponema PCR primers. Three BDD samples were randomly selected and normal tissue samples were submitted for 16S rRNA sequencing using the Illumina MiSeq platform. The dominant phylum present in all tested BDD lesions was Spirochaetes with a mean relative abundance of 46.9 %, and Treponema was the most abundant genus. Spirochaetes abundance was followed by the phyla Tenericutes and Bacteroidetes with 14.1 % and 11.8 % mean abundances, respectively. Co-infecting bacteria from phyla Tenericutes and Bacteroidetes may be involved in the progression of BDD. Bovine digital dermatitis infection is polymicrobial in nature, but Treponema spp. are the main etiologic agents of the disease. In the microbiome results, Treponema pedis had the highest mean relative abundance (20.9 %) in the BDD lesions in this study followed by T. denticola, T. medium, T. lecithinolyricum, Spirochaeta africana, and Sediminispirochaeta bajacalifoniensis. All 29 samples were positive in the genus-specific Treponema PCR results. The species-specific PCR resulted in 75.9 %, 86.2 %, and 69.0 % of samples in groups T. medium/T. vincentii-like, T. phagedenis-like, and T. pedis, respectively. Understanding how these microorganisms mutually interact in the host during certain stages of infection may help in the development of better practices for controlling BDD., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicting interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) from infected pigs is currently poorly understood. To better understand the dynamics of host-pathogen interactions, seventy-five of 100 pigs infected with PRRSV-JA142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). Blood, lung, bronchoalveolar lavage (BAL) and bronchial lymph node (BLN) samples were collected to evaluate the cellular immune responses. The humoral responses were evaluated by measuring the levels of anti-PRRSV IgG and serum virus-neutralizing (SVN) antibodies. Consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. At peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived DC/macrophage and conventional DC frequencies were increased, and these effects coincided with the early induction of local T-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, BAL, and BLN as early as 10 dpc. Conversely, the systemic T-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. Taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining PRRSV through the early induction of T-cell responses at the sites of virus replication.

Background: Multifocal spherical nonstaining cavities and gram-positive, rod-shaped, and endospore-forming bacteria were found in the liver of a sow that died suddenly. Clostridium novyi type B was identified and isolated from the sudden death case, and the isolate was characterized by molecular analyses and bioassays in the current study., Results: C. novyi was isolated from the liver of a sow that died suddenly and was confirmed as C. novyi type B by differential PCR. The C. novyi isolate fermented glucose and maltose and demonstrated lecithinase activity, and the cell-free culture supernatant of the C. novyi isolate exhibited cytotoxicity toward Vero cells, demonstrating that the isolate produces toxins. In addition, whole-genome sequencing of the C. novyi isolate was performed, and the complete sequences of the chromosome (2.29 Mbp) and two plasmids (134 and 68 kbp) were identified for the first time. Based on genome annotation, 7 genes were identified as glycosyltransferases, which are known as alpha toxins; 23 genes were found to be related to sporulation; 12 genes were found to be related to germination; and 20 genes were found to be related to chemotaxis., Conclusion: C. novyi type B was isolated from a sow in a sudden death case and confirmed by biochemical and molecular characterization. Various virulence-associated genes were identified for the first time based on whole-genome sequencing.

This study evaluated a combined method for the detection of Listeria monocytogenes in mushrooms, involving enrichment and quantitative real-time polymerase chain reaction (qPCR), to improve sensitivity and reduce detection time. The growth of L. monocytogenes was evaluated in Listeria enrichment broth (LEB) with modified carbon and nitrogen sources, increasing sodium concentrations, and added micronutrients. Primers targeting the L. monocytogenes iap ( iap 1 and iap 2), hlyA ( hlyA 1- hlyA 6), and prfA ( prfA 1- prfA 4) genes were developed and their sensitivity and specificity were evaluated. The greatest increase in L. monocytogenes cell count was observed after 6-h incubation at 30°C in LEB+2 × FAC (LEB plus 20 mL/L ferric ammonium citrate), where cell count increased by 1.4 log CFU (colony-forming unit)/mL, compared with 0.9 log CFU/mL in LEB ( p  < 0.05). iap 2 primers targeting the iap gene showed high specificity and were the most sensitive among those tested, with a detection limit of 2 log CFU/mL in LEB medium, 3.1 log CFU/g in golden needle mushroom, and 3.5 log CFU/g in large oyster mushroom. When applied to detection in golden needle mushrooms, a combination of 3-h incubation in LEB+2 × FAC medium and qPCR analysis with iap 2 primers permitted detection of L. monocytogenes , even at an inoculum of 1 log CFU/g. Similarly, in large oyster mushrooms, 10-h enrichment in LEB+2 × FAC medium resulted in a cell count of 3.7 log CFU/g. These results indicate that a combined detection method, using LEB+2 × FAC medium for enrichment followed by qPCR with iap 2 primer pair, can reduce enrichment time and improve the sensitivity and specificity of L. monocytogenes detection in mushrooms.

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.

Salmonella enterica outbreaks in sprouts originate from contaminated seeds; conventional prevention technologies have been reported from many research institutes. In this study, we applied a biological control approach to inhibit S. enterica growth using the seed-dwelling non-antagonistic bacteria. We isolated non-antibacterial seed-dwelling bacteria from vegetable sprouts. A total of 206 bacteria exhibiting non-antibacterial activity against S. enterica were subjected to alfalfa sprout development tests. Eight isolates exhibiting no deleterious effect on the growth of alfalfa sprouts were tested for S. enterica growth inhibition on alfalfa seeds and sprouts, and an isolate EUS78 was finally selected for further investigation. Based on 16S rRNA, gyrB, and rpoB gene sequence analyses, strain EUS78 was identified as Erwinia persicina. In population competition, the S. enterica population increased by >3 log CFU/g after 6 days of alfalfa sprout growth, whereas S. enterica growth was significantly inhibited by treatment with EUS78 (P < .05). This effect of S. enterica growth inhibition by EUS78 was sustained until the end of the alfalfa sprout harvest. Overall, bacterial strain EUS78 significantly reduced S. enterica growth on alfalfa sprouts in a manner consistent with competitive exclusion. These findings led us to monitor EUS78 behavior on seeds during early sprout development using fluorescence and scanning electron microscopy. Strain EUS78 initially colonized alfalfa sprout seed coat edges, cotyledons, and finally root surfaces during early sprout germination. As alfalfa sprouts grew, EUS78 bacterial cells established colonies on newly emerged plant tissues such as root tips. The results of this study suggest that strain EUS78 has potential as a biological control agent to inhibit S. enterica contamination in the sprout food industry., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Pasteurella multocida is responsible for significant economic losses in pigs worldwide. In clinically diseased pigs, most P. multocida isolates are characterised as subspecies multocida, biovar 2 or 3 and capsular type A or D; however, there is little information regarding subspecies, biovars, and other capsular types of P. multocida isolates in Korea. Here, we provided information covering an extended time period regarding P. multocida in pigs with pneumonia in Korea using phenotypic and genotypic characterisations and data associated with the minimum inhibitory concentrations., Results: The overall prevalence of P. multocida between 2008 and 2016 was 16.8% (240/1430), with 85% of the P. multocida isolates (204/240) coinfected with other respiratory pathogens. Of the 240 isolates, 166 were included in this study; all of these P. multocida isolates were characterised as subspecies multocida and the most prevalent phenotypes were represented by biovar 3 (68.7%; n = 114) and capsular type A (69.9%; n = 116). Additionally, three capsular type F isolates were identified, with this representing the first report of such isolates in Korea. All biovar 1 and 2 isolates were capsular types F and A, respectively. The virulence-associated gene distribution was variable; all capsular type A and D isolates harboured pmHAS and hsf-1, respectively (P < 0.001), with type F (biovar 1) significantly correlated with hsf-1 (P < 0.05) and pfhA (P < 0.01), biovar 2 highly associated with pfhA and pmHAS, and biovar 3 significantly correlated with hsf-1, pmHAS, and hgbB (P < 0.001), whereas biovar 13 was related only to hgbB (P < 0.05). The highest resistance rate was found to be to oxytetracycline (63.3%), followed by florfenicol (16.3%)., Conclusions: P. multocida subspecies multocida, biovar 3, and capsular type A was the most prevalent isolate in this study, and our findings indicated the emergence of capsular type F in Korea. Moreover, prudent use of oxytetracycline and florfenicol is required because of the identified high resistance rates. Further studies are required for continuous monitoring of the antimicrobial resistance, prevalence, and epidemiological characterisation of P. multocida, and experimental infection models are needed to define the pathogenicity of capsular type F.

DiNap [( E )-1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(naphthalen-1-yl)prop-2-en-1-one], an analog of a natural product (the chalcone flavokawain), was synthesized and characterized in this study. Porcine reproductive and respiratory syndrome virus (PRRSV) is the most challenging threat to the swine industry worldwide. Currently, commercially available vaccines are ineffective for controlling porcine reproductive and respiratory syndrome (PRRS) in pigs. Therefore, a pharmacological intervention may represent an alternative control measure for PRRSV infection. Hence, the present study evaluated the effects of DiNap on the replication of VR2332 (a prototype strain of type 2 PRRSV). Initially, in vitro antiviral assays against VR2332 were performed in MARC-145 cells and porcine alveolar macrophages (PAMs). Following this, a pilot study was conducted in a pig model to demonstrate the effects of DiNap following VR2332 infection. DiNap inhibited VR2332 replication in both cell lines in a dose-dependent manner, and viral growth was completely suppressed at concentrations ≥0.06 mM, without significant cytotoxicity. Consistent with these findings, in the pig study, DiNap also reduced viral loads in the serum and lungs and enhanced the weight gain of pigs following VR2332 infection, as indicated by comparison of the DiNap-treated groups to the untreated control (NC) group. In addition, DiNap-treated pigs had fewer gross and microscopic lesions in their lungs than NC pigs. Notably, virus transmission was also delayed by approximately 1 week in uninfected contact pigs within the same group after treatment with DiNap. Taken together, these results suggest that DiNap has potential anti-PRRSV activity and could be useful as a prophylactic or post-exposure treatment drug to control PRRSV infection in pigs.

Background: Currently, an in vitro immunogenicity screening system for the immunological assessment of potential porcine reproductive and respiratory syndrome virus (PRRSV) vaccine candidates is highly desired. Thus, in the present study, two genetically divergent PRRSVs were characterized in vitro and in vivo to identify an in vitro system and immunological markers that predict the host immune response. Porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) collected from PRRSV-negative pigs were used for in vitro immunological evaluation, and the response of these cells to VR2332c or JA142c were compared with those elicited in pigs challenged with the same viruses., Results: Compared with VR2332c or mock infection, JA142c induced increased levels of type I interferons and pro-inflammatory cytokines (TNF-α, IL-1α/β, IL-6, IL-8, and IL-12) in PAMs, and these elevated levels were comparable to the cytokine induction observed in PRRSV-challenged pigs. Furthermore, significantly greater numbers of activated CD4 + T cells, type I helper T cells, cytotoxic T cells and total IFN-γ + cells were observed in JA142c-challenged pigs than in VR2332c- or mock-challenged pigs., Conclusions: Based on these results, the innate immune response patterns (particularly IFN-α, TNF-α and IL-12) to specific PRRSV strains in PAMs might reflect those elicited by the same viruses in pigs.

O-2- 18 F-fluoroethyl-l-tyrosine ([ 18 F]FET) has been widely used for glioblastomas (GBM) in clinical practice, although evaluation of its applicability in non-clinical research is still lacking. The objective of this study was to examine the value of [ 18 F]FET for treatment evaluation and prognosis prediction of anti-angiogenic drug in an orthotopic mouse model of GBM. Human U87MG cells were implanted into nude mice and then bevacizumab, a representative anti-angiogenic drug, was administered. We monitored the effect of anti-angiogenic agents using multiple imaging modalities, including bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET/CT). Among these imaging methods analyzed, only [ 18 F]FET uptake showed a statistically significant decrease in the treatment group compared to the control group ( P =0.02 and P =0.03 at 5 and 20 mg/kg, respectively). This indicates that [ 18 F]FET PET is a sensitive method to monitor the response of GBM bearing mice to anti-angiogenic drug. Moreover, [ 18 F]FET uptake was confirmed to be a significant parameter for predicting the prognosis of anti-angiogenic drug ( P =0.041 and P =0.007, on Days 7 and 12, respectively, on Pearson's correlation; P =0.048 and P =0.030, on Days 7 and 12, respectively, on Cox regression analysis). However, results of BLI or MRI were not significantly associated with survival time. In conclusion, this study suggests that [ 18 F]FET PET imaging is a pertinent imaging modality for sensitive monitoring and accurate prediction of treatment response to anti-angiogenic agents in an orthotopic model of GBM., Competing Interests: Conflict of interests: The authors declare that there is no financial conflict of interests to publish these results.

The ancient wisdom found in iron gall ink guides this work to a simple but advanced solution to the molecular engineering of fluidic interfaces. The Fe(II)-tannin coordination complex, a precursor of the iron gall ink, transforms into interface-active Fe(III)-tannin species, by oxygen molecules, which form a self-assembled layer at the fluidic interface spontaneously but still controllably. Kinetic studies show that the oxidation rate is directed by the counteranion of Fe(II) precursor salts, and FeCl 2 is found to be more effective than FeSO 4 -an ingredient of iron gall ink-in the interfacial-film fabrication. The optimized protocol leads to the formation of micrometer-thick, free-standing films at the air-water interface by continuously generating Fe(III)-tannic acid complexes in situ. The durable films formed are transferable, self-healable, pliable, and postfunctionalizable, and are hardened further by transfer to the basic buffer. This O 2 -instructed film formation can be applied to other fluidic interfaces that have high O 2 level, demonstrated by emulsion stabilization and concurrent capsule formation at the oil-water interface with no aid of surfactants. The system, inspired by the iron gall ink, provides new vistas on interface engineering and related materials science., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One-step assembly of iron(III)-tannic acid (Fe 3+ -TA) complex forms nanothin (∼10 nm) films on various substrates within minutes. In this deposition scheme, however, the film does not grow continuously over time even though Fe 3+ -TA complex is still abundant in the coating solution. In this paper, we report that the salt addition dramatically changes the one-off coating characteristic to continuous one, and each salt has its optimum concentration ( C MFT ) that produces maximum film thickness. For detailed investigation of the salt effects, we employed various salts, including LiCl, NaCl, KCl, CaCl 2 , SrCl 2 , BaCl 2 , NaBr, and NaNO 3 , and found that only cations played an important role in the continuous deposition of the Fe 3+ -TA complex, with smaller C MFT values for the cations of higher valency and larger size. On the basis of the results, we suggested that the positively charged cations screened the negative surface charges of Fe 3+ -TA complex particles, leading to coagulation and continuous deposition, further supported by the ζ-potential measurement and time-resolved dynamic light-scattering analysis.

An adult male Eurasian river otter ( Lutra lutra) was diagnosed with systemic infection. Microbiologic findings identified Streptococcus suis serotype 2, clonal complex 28, and sequence type 629. Genetic analysis strongly suggested the transmission of S. suis isolate from pigs to wild animals through environmental contamination.

Background: Porcine circovirus-associated diseases (PCVAD), caused by porcine circovirus type 2 (PCV2), threaten the pig industry worldwide. Five genotypes of PCV2 were recently identified: PCV2a, PCV2b, PCV2c, PCV2d and PCV2e. In addition, a novel porcine circovirus from a case of a sow with dermatitis, nephropathy syndrome and reproductive failure has been identified based on metagenomic analysis and classified as porcine circovirus type 3 (PCV3). Therefore, the current study was conducted to determine the prevalence and genetic characteristics of PCV2 and PCV3 in clinical samples., Results: A total of 471 samples (161 tissue samples of lungs and lymph nodes from 34 farms and 310 serum samples from 47 farms) were tested for PCV2. Among them, 171 samples from 59 farms that had been positive for PCV2 were genotyped. Another 690 samples (296 tissue samples of lungs and lymph nodes from 91 farms, 108 samples of aborted foetuses from 26 farms, and 286 serum samples from 47 farms) were tested for PCV3. Based on PCV2 genotyping results, PCV2d was the most prevalent genotype (107 of 171 samples), and co-infections with combinations of PCV2a, 2b and 2d were identified in 48 samples from 17 farms. A total of 14 samples from 11 farms were also positive for both PCV2 and PCV3. For PCV3, 57 samples (9.8%) from 32 farms (23.2%) were positive. Among the 108 aborted foetuses from 26 farms, only 2 samples were positive for PCV3. Based on sequence comparisons, PCV2d shares 89.6-91.0% and 93.2-94.3% homology with PCV2a and PCV2b, respectively; 98.6-100% homology is shared among PCV2d strains. The PCV3 strains identified in this study share 98.0-99.5% homology., Conclusions: Our study concludes that PCV2d has become the most predominant genotype in Korea. PCV3 was also identified in clinical samples, though no significant association with clinical symptoms was observed in PCV3-positive cases.

Primary porcine alveolar macrophages (PAM) are useful for studying viral infections and immune response in pigs; however, long-term use of these cells is limited by the cells' short lifespan. We immortalized primary PAMs by transfecting them with both hTERT and SV40LT and established two immortalized cell lines (iPAMs) actively proliferating even after 35 passages. These cells possessed the characteristics of primary PAMs, including strong expression of swine leukocyte antigen (SLA) class II genes and the inability to grow anchorage-independently. We characterized their SLA genes and subsequently performed peptide-SLA binding assays using a peptide from porcine circovirus type 2 open reading frame 2 to experimentally measure the binding affinity of the peptide to SLA class II. The number of peptides bound to cells measured by fluorescence was very low for PK15 cells (7.0% ± 1.5), which are not antigen-presenting cells, unlike iPAM61 (33.7% ± 3.4; SLA-DQA*0201/0303, DQB1*0201/0901, DRB1*0201/1301) and iPAM303 (73.3% ± 5.4; SLA DQA*0106/0201, DQB1*0202/0701, DRB1*0402/0602). The difference in peptide binding between the two iPAMs was likely due to the allelic differences between the SLA class II molecules that were expressed. The development of an immortal PAM cell panel harboring diverse SLA haplotypes and the use of an established method in this study can become a valuable tool for evaluating the interaction between antigenic peptides and SLA molecules and is important for many applications in veterinary medicine including vaccine development.

An enzyme-instructed method is developed for material-independent, cytocompatible coating of phenolic amines, inspired by melanogenesis found in nature. Tyrosinase-based film formation proceeds smoothly in an aqueous solution at neutral pH, and can use various phenolic amines including catecholamines, such as tyrosine, tyramine, dopamine, norepinephrine, and DOPA, as a coating precursor. Compared with polydopamine coating, the method is fast and efficient, and forms uniform films. Its high cytocompatibility is advantageously applied to cell-surface engineering, where chemically labile Jurkat cells are coated individually without any noticeable decrease in viability. Considering the huge potential of polyphenolic-based coatings, the strategy developed herein will provide an advanced tool for manipulating biological entities, including living cells, in biomedical and medicinal applications.

This study examined the effects of alfalfa seed germination on growth of Salmonella enterica . We investigated the population changes of S. enterica during early sprout development. We found that the population density of S. enterica , which was inoculated on alfalfa seeds was increased during sprout development under all experimental temperatures, whereas a significant reduction was observed when S. enterica was inoculated on fully germinated sprouts. To establish a model for predicting S. enterica growth during alfalfa sprout development, the kinetic growth data under isothermal conditions were collected and evaluated based on Baranyi model as a primary model for growth data. To elucidate the influence of temperature on S. enterica growth rates, three secondary models were compared and found that the Arrhenius-type model was more suitable than others. We believe that our model can be utilized to predict S. enterica behavior in alfalfa sprout and to conduct microbial risk assessments.

The broad-spectrum lytic capability of Salmonella bacteriophages against various Salmonella species was evaluated to determine their potential as an alternative for antibiotics, and the safety and preventive effects of the bacteriophages were assessed on mice and pigs. Four bacteriophage cocktails were prepared using 13 bacteriophages, and the lytic capability of the four bacteriophage cocktails was tested using Salmonella reference strains and field isolates. Bacteriophage cocktail C (SEP-1, SGP-1, STP-1, SS3eP-1, STP-2, SChP-1, SAP-1, SAP-2; ≥10 9 pfu/ml) showed the best lytic activity against the Salmonella reference strains (100% of 34) and field isolates (92.5% of 107). Fifty mice were then orally inoculated with bacteriophage cocktail C to determine the distribution of bacteriophages in various organs, blood and feces. The effects of bacteriophages on Salmonella infection in weaned pigs (n=15) were also evaluated through an experimental challenge with Salmonella Typhimurium after treatment with bacteriophage cocktail C. All mice exhibited distribution of the bacteriophages in all organs, blood and feces until 15 days post infection (dpi). After 35 dpi, bacteriophages were not detected in any of these specimens. As demonstrated in a pig challenge study, treatment with bacteriophage cocktail C reduced the level of Salmonella shedding in feces. The metagenomic analyses of these pig feces also revealed that bacteriophage treatment decreased the number of species of the Enterobacteriaceae family without significant disturbance to the normal fecal flora. This study showed that bacteriophages effectively controlled Salmonella in a pig challenge model and could be a good alternative for antibiotics to control Salmonella infection.

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes devastating disease characterized by reproductive failure and respiratory problems in the swine industry. To understand the recent prevalence and genetic diversity of field PRRSVs in the Republic of Korea, open reading frames (ORFs) 5 and 7 of PRRSV field isolates from 631 PRRS-affected swine farms nationwide in 2013-2016 were analyzed along with 200 Korean field viruses isolated in 2003-2010, and 113 foreign field and vaccine strains., Results: Korean swine farms were widely infected with PRRSVs of a single type (38.4 and 37.4% for Type 1 and Type 2 PRRSV, respectively) or both types (24.2%) with up to approximately 83% nucleotide sequence similarity to prototype PRRSVs (Lelystad or VR2332). Phylogenetic analysis based on the ORF5 nucleotide sequence revealed that Korean Type 1 field isolates were classified as subgroups A, B, and C under subtype 1, while Korean Type 2 field isolates were classified as lineages 1 and 5 as well as three Korean lineages (kor A, B, and C) with the highest infection prevalence in subgroup A (50.5%) and lineage 5 (15.3%) for Type 1 and Type 2 PRRSV, respectively, among ORF5-positive farms. In particular, the lineages kor B and C were identified as novel lineages in this study, and lineage kor B comprised only the field viruses isolated from Gyeongnam Province in 2014-2015, establishing regionally unique genetic characteristics. It has also recently been confirmed that commercialized vaccine-like viruses (subgroup C) of Type 1 PRRSV and NADC30-like viruses of Type 2 PRRSV (lineage 1) are spreading rapidly in Korean swine farms. The Korean field viruses were also expected to be antigenically variable as shown in the high diversity of neutralizing epitopes and N-glycosylation sites., Conclusions: This up-to-date information regarding recent field PRRSVs should be taken into consideration when creating strategies for the application of PRRS control measures, including vaccination in the field.

Main Conclusion: We demonstrated successful overexpression of porcine reproductive and respiratory syndrome virus (PRRSV)-derived GP4D and GP5D antigenic proteins in Arabidopsis. Pigs immunized with transgenic plants expressing GP4D and GP5D proteins generated both humoral and cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, the most economically significant disease affecting the swine industry worldwide. However, current commercial PRRSV vaccines (killed virus or modified live vaccines) show poor efficacy and safety due to concerns such as reversion of virus to wild type and lack of cross protection. To overcome these problems, plants are considered a promising alternative to conventional platforms and as a vehicle for large-scale production of recombinant proteins. Here, we demonstrate successful production of recombinant protein vaccine by expressing codon-optimized and transmembrane-deleted recombinant glycoproteins (GP4D and GP5D) from PRRSV in planta. We generated transgenic Arabidopsis plants expressing GP4D and GP5D proteins as candidate antigens. To examine immunogenicity, pigs were fed transgenic Arabidopsis leaves expressing the GP4D and GP5D antigens (three times at 2-week intervals) and then challenged with PRRSV at 6-week post-initial treatment. Immunized pigs showed significantly lower lung lesion scores and reduced viremia and viral loads in the lung than pigs fed Arabidopsis leaves expressing mYFP (control). Immunized pigs also had higher titers of PRRSV-specific antibodies and significantly higher levels of pro-inflammatory cytokines (TNF-α and IL-12). Furthermore, the numbers of IFN-γ + -producing cells were higher, and those of regulatory T cells were lower, in GP4D and GP5D immunized pigs than in control pigs. Thus, plant-derived GP4D and GP5D proteins provide an alternative platform for producing an effective subunit vaccine against PRRSV.

This study was conducted to develop a screening method using Colilert-18 and a device for the detection of E. coli from agri-food production environments and fresh vegetables. The specificity and sensitivity of Colilert-18 by temperature (37°C and 44°C) were evaluated with 38 E. coli and 78 non- E. coli strains. The false-positive rate was 3.8% (3/78) and 0% (0/78) at 37°C and 44°C, respectively. The detection limit of E. coli at 37°C at <1.0 log CFU/250 ml was lower than that at 44°C. The efficiency of the developed device, which comprised an incubator equipped with a UV lamp to detect E. coli in the field, was evaluated by measuring the temperature and UV lamp brightness. The difference between the set temperature and actual temperature of the developed device was about 1.0°C. When applying the developed method and device to various samples, including utensils, gloves, irrigation water, seeds, and vegetables, there were no differences in detection rates of E. coli compared with the Korean Food Code method. For sanitary disposal of culture samples after experiments, the sterilization effect of sodium dichloroisocyanurate (NaDCC) tablets was assessed for use as a substitute for an autoclave. The addition of one tablet of NaDCC per 50 ml was sufficient to kill E. coli cultured in Colilert-18. These results show that the developed protocol and device can efficiently detect E. coli from agri-food production environments and vegetables.

An injectable adhesive hydrogel composed of carboxymethyl cellulose (CMC) and pullulan is developed and evaluated as a postoperative anti-adhesion barrier. CMC was modified with tyramine to introduce crosslinking site via an EDC-NHS reaction. The in situ hydrogel was prepared by an enzyme-mediated reaction of tyramine-immobilized CMC with horseradish peroxidase (HRP) and hydrogen peroxide (H 2 O 2 ). Pullulan was added to the hydrogel solution to improve adhesiveness to the wound area and accelerate biodegradation. The modified CMC was confirmed by ATR-FTIR spectroscopy. The gelation time, storage modulus (G'), and weight loss of the hydrogels were measured as functions of the amounts of HRP and H 2 O 2 . The hydrogel group showed negligible cell proliferation and cytotoxicity, compared to that shown by the control group. The in vivo animal test demonstrated that significant decrease of postoperative tissue adhesion by applying the hydrogels. The CMC-pullulan hydrogel could be a useful treatment as an injectable in situ anti-adhesive agent., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Although campylobacteriosis caused by Campylobacter jejuni and Campylobacter coli has been increasingly reported worldwide owing to the consumption of contaminated poultry and fresh produce, the current detection protocols are not selective enough to inhibit unspecific microbes other than these pathogens. Five antibiotics were separately added to Bolton broth, and the survival rates of 18 Campylobacter spp. and 79 non-Campylobacter spp. were evaluated. The survival rate of the non-Campylobacter spp. was the lowest in Bolton broth with rifampin (6.3%), followed by cefsulodin (12.7%), novobiocin (16.5%), and potassium tellurite and sulfamethozaxole (both 17.7%). Also the most effective concentration of rifampin was found to be 12.5 mg/L, which markedly inhibited non-Campylobacter strains while not affecting the survival of Campylobacter strains. After the Campylobacter spp. were enriched in Bolton broth supplemented with 12.5 mg/L rifampin (R-Bolton broth), CampyFood Agar (CFA) was found to be better in selectively isolating the pathogens in the enrichment broth than the International Organization for Standardization method of using modified charcoal cefoperazone deoxycholate agar (mCCDA) for this step. When applied to natural food samples-here, romaine lettuce, pepper, cherry tomato, Korean leek, and chicken-the R-Bolton broth-CFA combination decreased the number of false-positive results by 50.0, 4.2, 20.8, 50.0, and 94.4%, respectively, compared with the International Organization for Standardization method (Bolton broth-mCCDA combination). These results demonstrate that the combination of R-Bolton broth and CFA is more efficient in detecting C. jejuni and C. coli in poultry and fresh produce and thus should replace the Bolton broth-mCCDA combination., (Copyright ©, International Association for Food Protection.)