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. 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. 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.

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.

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.

A novel porcine circovirus 4 (PCV4) was recently emerged in Chinese and Korean pig herds, which provided epidemiological situation where three pathogenic PCVs, PCV2, PCV3, and newly emerged PCV4, could co-infect pig herds in these countries.In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) method was developed for the rapid and differential detection of these viruses.The assay specifically amplified each viral capsid gene, whereas no other porcine pathogenic genes were detected.The detection limit of the assay was below 10 copies/μL and the assay showed high repeatability and reproducibility.In the clinical evaluation using 1476 clinical samples from 198 Korean pig farms, the detection rates of PCV2, PCV3 and PCV4 by the tqPCR assay were 13.8%, 25.4%, and 3.8%, respectively, which were 100% agreement with those of previously reported monoplex qPCR assays for PCV2, PCV3, and PCV4, with a κ value (95% CI) of 1 (1.00∼1.00).The prevalence of PCV2, PCV3, and PCV4 at the farm levels were 46.5%, 63.6%, and 19.7%, respectively.The co-infection analysis for tested pig farms showed that single infection rates for PCV2, PCV3, and PCV4 were 28.8%, 44.4%, and 9.6%, respectively, the dual infection rates of PCV2 and PCV3, PCV2 and PCV4, and PCV3 and PCV4 were 12.6%, 3.5%, and 5.1%, respectively, and the triple infection rate for PCV2, PCV3, and PCV4 was 1.5%.These results demonstrate that three pathogenic PCVs are widely spread, and their co-infections are common in Korean pig herds, and the newly developed tqPCR assay will be useful for etiological and epidemiological studies of these pathogenic PCVs.

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.

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.

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.

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.

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.

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.

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.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) produces hemolytic cytotoxins, ApxI, ApxII, ApxIII, and ApxIV, causing a huge loss to the swine industry. We developed the PadLysis protocol for extracting genomic DNA (gDNA) for the LAMP—LeucoCrystal Violet (LCV) colorimetric detection. A paper-based well pad for gDNA extraction was fabricated using a wax printing method. Furthermore, the LAMP amplicons were detected in situ with colorimetric dye, LCV. Wax-printed well pads (WPWPs) have hydrophobic wax barriers by printing a well pattern that allows the establishment of simple and equipment-free PadLysis protocol consisting of the three steps: (1) 2 min lysis, (2) 2 min washing, and (3) 1 min drying. This proposed platform allowed direct LAMP amplification for A. pleuropneumoniae field isolates. We optimized LCV chemical components to enable the accurate LAMP signal readouts. The overall assay takes up to 45 min including 5 min PadLysis, 40 min LAMP amplification, and LCV signal observation with the naked eye. Our WPWPs–LAMP–LCV approach can be used as a rapid screening tool for A. pleuropneumoniae pleuropneumonia (APP) caused by ApxIA toxin in future outbreak areas.

Abstract The major concern for the pig industry is disease control and prevention, and porcine reproductive and respiratory syndrome (PRRS) is one of the main threats to it. Tonsil is a part of the immune system and its role is to recognize and reject foreign antigens to prevent them from invading the lungs. Interestingly, porcine tonsil can harbor PRRS viruses over 150 days post infection (dpi) with no clinical signs, causing PRRS re-break. This study was conducted to find out variation in levels of gene expression in tonsils and investigate functional roles of differentially expressed genes in tonsils depending on days post infection. We used porcine tonsils from weaned pigs following experimental infection with PRRSV-2 (JA142 strain) at 3, 10, 21, 28, 35 dpi. Based on RNA-seq analysis pipeline, differentially expressed genes (DEGs) were considered significant at False Discovery Rate (FDR) ≤ 0.05 level and above the two-fold change. Comparative analyses of 3 dpi vs. 10 dpi, 3 dpi vs. 21 dpi, 3 dpi vs. 28 dpi, and 3 dpi vs. 35 dpi produced 368, 315, 754, and 336 DEGs, respectively. Then we annotated the functions of each DEG set using DAVID tool. Significant GO terms of the MF, BP, and CC ontologies and KEGG pathways were selected within the limit of FDR < 0.05. Common overrepresented GO terms of all DEG sets were mainly negative regulation of viral genome replication, defense response to virus, negative regulation of type Ⅰ interferon production, and type Ⅰ interferon signaling pathway. Specific GO terms were found such as cellular response to interleukin-1, interferon-gamma-mediated signaling pathway, growth factor activity, and acute-phase response from each DEG set, respectively. It suggested tonsil responded to protect itself from PRRSV infection but further study is required to understand PRRSV persistence in tonsils.

BackgroundPasteurella 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.ResultsThe 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%).ConclusionsP. 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.

The study was performed to investigate the antimicrobial susceptibility of L. intracellularis field isolates from Korean pig farms. The novel four field isolates successfully obtained from clinical cases were prepared in IEC-18 cells to conduct the in vitro antimicrobial susceptibility testing (AST) by determining minimum inhibitory concentrations (MIC)s; either intracellular (InMIC) or extracellular MICs (ExMIC). The final MICs were assessed by counting the number of heavily infected cells (HICs;> 30 bacteria per cell) using an immunoperoxidase monolayer assay. Enrofloxacin (0.125 to 0.25 μg/mL: InMIC and 2 to 16 μg/mL; ExMIC) presented the most notable antimicrobial susceptibility, and marbofloxacin (0.25 to 0.5 μg/mL and 4 to 32 μg/mL) followed. Colistin (0.125 to 2 μg/mL and 2 to 4 μg/mL) presented a susceptibility followed by tylvalosin (0.5 to 1 μg/mL and 2 to 4 μg/mL). Florfenicol and lincomycin had the weakest susceptibility and amoxicillin, penicillin G, chlortetracycline, oxytetracycline, tiamulin, tilmicosin and tylosin displayed weak susceptibility. Dividing cells in culture and strict environmental conditions to isolate and cultivate L. intracellularis has limited the maintenance of this bacteria in vitro to only a few laboratories. Four isolates were successfully maintained in our laboratory, which enabled us to determine the antimicrobial susceptibility profile. In addition, the results could be one of the contributions in clinical fields.

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; ≥109 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.