Molecular Characterization of Porcine Reproductive and Respiratory Syndrome Virus in Korea from 2018 to 2022.

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease in the Republic of Korea. Surveillance of PRRS virus (PRRSV) types is critical to tailor control measures. This study collected 5062 serum and tissue samples between 2018 and 2022. Open reading frame 5 (ORF5) sequences suggest that subgroup A (42%) was predominant, followed by lineage 1 (21%), lineage 5 (14%), lineage Korea C (LKC) (9%), lineage Korea B (LKB) (6%), and subtype 1C (5%). Highly virulent lineages 1 (NADC30/34/MN184) and 8 were also detected. These viruses typically mutate or recombine with other viruses. ORF5 and non-structural protein 2 (NSP2) deletion patterns were less variable in the PRRSV-1. Several strains belonging to PRRSV-2 showed differences in NSP2 deletion and ORF5 sequences. Similar vaccine-like isolates to the PRRSV-1 subtype 1C and PRRSV-2 lineage 5 were also found. The virus is evolving independently in the field and has eluded vaccine protection. The current vaccine that is used in Korea offers only modest or limited heterologous protection. Ongoing surveillance to identify the current virus strain in circulation is necessary to design a vaccine. A systemic immunization program with region-specific vaccinations and stringent biosecurity measures is required to reduce PRRSV infections in the Republic of Korea.

Evaluation and Determination of a Suitable Passage Number of Codon Pair Deoptimized PRRSV-1 Vaccine Candidate in Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is major economic problem given its effects on swine health and productivity. Therefore, we evaluated the genetic stability of a codon pair de-optimized (CPD) PRRSV, E38-ORF7 CPD, as well as the master seed passage threshold that elicited an effective immune response in pigs against heterologous virus challenge. The genetic stability and immune response of every 10th passage (out of 40) of E38-ORF7 CPD was analyzed through whole genome sequencing and inoculation in 3-week-old pigs. E38-ORF7 CPD passages were limited to 20 based on the full-length mutation analysis and animal test results. After 20 passages, the virus could not induce antibodies to provide effective immunity and mutations accumulated in the gene, which differed from the CPD gene, presenting a reason for low infectivity. Conclusively, the optimal passage number of E38-ORF7 CPD is 20. As a vaccine, this may help overcome the highly diverse PRRSV infection with substantially enhanced genetic stability.

Codon Pair Deoptimization (CPD)-Attenuated PRRSV-1 Vaccination Exhibit Immunity to Virulent PRRSV Challenge in Pigs.

Commercially used porcine respiratory and reproductive syndrome (PRRS) modified live virus (MLV) vaccines provide limited protection with heterologous viruses, can revert back to a virulent form and they tend to recombine with circulating wild-type strains. Codon pair deoptimization (CPD) is an advanced method to attenuate a virus that overcomes the disadvantages of MLV vaccines and is effective in various virus vaccine models. The CPD vaccine against PRRSV-2 was successfully tested in our previous study. The co-existence of PRRSV-1 and -2 in the same herd demands protective immunity against both viruses. In this study, live attenuated PRRSV-1 was constructed by recoding 22 base pairs in the ORF7 gene of the E38 strain. The efficacy and safety of the CPD live attenuated vaccine E38-ORF7 CPD to protect against virulent PRRSV-1 were evaluated. Viral load, and respiratory and lung lesion scores were significantly reduced in animals vaccinated with E38-ORF7 CPD. Vaccinated animals were seropositive by 14 days post-vaccination with an increased level of interferon-γ secreting cells. In conclusion, the codon-pair-deoptimized vaccine was easily attenuated and displayed protective immunity against virulent heterologous PRRSV-1.

Susceptibility to Acaricides and the Frequencies of Point Mutations in Etoxazole- and Pyridaben-Resistant Strains and Field Populations of the Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

The two-spotted spider mite Tetranychus urticae Koch is a major agricultural pest worldwide and is known to rapidly develop resistance to pesticides. In the present study, we explored a field strain that was collected in 2000 and 2003 and has been exhibiting resistance to etoxazole and pyridaben over the last 16 years. The resistance ratios of the etoxazole- and pyridaben-resistant strains (ER and PR) to etoxazole or pyridaben were more than 5,000,000- and 4109.6-fold higher than that of the susceptible strain, respectively. All field-collected populations showed resistance to etoxazole and pyridaben. The ER and PR strains showed cross-resistance to several acaricides. Both I1017F and H92R point mutations were detected in 7 out of 8 field groups. Spirodiclofen and spiromesifen resulted in more than 77.5% mortality in the 8 field groups. In addition, the genotype frequency of the I1017F point mutation was 100.0% in the ER strain, and that of the H92R point mutation was 97.0% in the PR strain. All of the field populations were found to have a high frequency of I1017F. These results suggest that the observation of resistance patterns will help in designing a sustainable IPM program for T. urticae.

Current status of pharmacovigilance regulatory structures, processes, and outcomes in the Asia-Pacific region: Survey results from 15 countries.

PURPOSE: Regulatory discrepancies may exist in pharmacovigilance (PV) structure, process, and outcome status worldwide. Our study's objective was to survey the current status of PV in each regulatory body in the Asia-Pacific Economic Cooperation (APEC) region. METHODS: A modified questionnaire was sent to the PV team heads of 21 PV agencies based in the APEC countries, between June 28 and September 12, 2017, to gather information on the structure, process, and outcome of PV status in these countries. RESULTS: Of the 21 APEC countries, 15 responded. We found harmonized laws and regulations for general PV and risk management systems. However, variations were found in PV structure: for example, 11 out of 15 countries had national regulatory representatives responsible for PV in pharmaceutical companies, while four did not. For PV process, discrepancies were also found in the source type of adverse drug reaction (ADR) reports and reporting of medication errors and therapeutic ineffectiveness in cumulative ADR reports. With respect to PV outcomes, among countries that performed active surveillance, the United States of America was more active, with hundreds of projects including additional pharmacoepidemiological studies etc. Among the nine countries that responded, Japan had the greatest number of product label changes followed by Taiwan, Malaysia, and Korea. CONCLUSION: We have identified substantial variations in the structures, processes, and outcomes of PV status among the countries of the APEC region. Therefore, efforts to reduce variations in the PV administration and regulation are warranted for harmonization of PV within the APEC region.

Evidence for recent acquisition and successful transmission of bla(CTX-M-15) in Salmonella enterica in South Korea.

We identified two distinct bla(CTX-M)-bearing and five distinct bla(CMY-2)-bearing genetic structures located on plasmids from Salmonella enterica and Escherichia coli isolates (n = 35) collected from chickens in South Korea. All Salmonella plasmids shared a common replicon, bla(CTX-M-15) transposon, and core resistance phenotype, while E. coli bla(CTX-M-15) plasmids included four distinct replicons.

Comparative proteome and transcriptome analyses of wild-type and live vaccine strains of Salmonella enterica serovar Gallinarum.

Salmonella enterica serovar Gallinarum causes fowl typhoid in chickens and is of economic importance to the chicken industry. A serovar Gallinarum live vaccine strain 9R (SG 9R) has been used to control fowl typhoid in many areas where the disease is endemic. Because the attenuation mechanism of SG 9R was not defined, there has been continued concern about reversion to virulence. In this study, we examined the molecular characteristics, which might provide better insight into attenuation of SG 9R, by comparing its proteome and transcriptome with those of two wild-type strains (287/91 and 06Q110). Proteins present in wild-type strains but absent in SG 9R were identified by two-dimensional gel electrophoresis and MALDI-TOF MS. Genes up- or down-regulated in SG 9R compared to wild-type strains were identified using an expression array. The proteome analysis identified nine proteins absent in SG 9R of which one protein had relevance to virulence. The transcriptome analysis revealed 24 up-regulated and 97 down-regulated genes in SG 9R. Approximately one-half of down-regulated genes (42 genes) were associated with virulence mechanisms. This finding suggests that attenuation of SG 9R may be associated with a combination of impaired virulence factors and thus reversion to virulence would not be caused by any single mutation event.

Isolation and epidemiological characterization of heat-labile enterotoxin-producing Escherichia fergusonii from healthy chickens.

Escherichia fergusonii has been associated with a wide variety of intestinal and extraintestinal infections in both humans and animals. The aim of this study was to demonstrate the presence of heat-labile enterotoxin (LT)-producing E. fergusonii in healthy chickens and its plasmid-mediated LT toxin gene transfer to other Enterobacteriaceae. We tested faecal samples from 184 chicken flocks (consisting of 109 broilers and 75 layers) of 78 commercial chicken farms for the presence of the LT gene using a polymerase chain reaction-based screening and identified samples from 43 flocks (23.4%) as positive for the LT gene. We subsequently isolated and identified E. fergusonii harboring the LT gene from all LT-positive samples and observed 21 various biochemical types. The plasmids encoding LT in 16 (37.2%) of the 43 isolates were conjugally transferred to the recipient strain Escherichia coli J53. Southern hybridization showed that all plasmids from the transconjugants carried the eltAB gene (Ent plasmid) and belonged to the narrow-host-range IncF type. In addition, all the E. fergusonii strains identified were classified into 17 pulsed-field gel electrophoresis (PFGE) types, and it is likely that there was an association between the PFGE types and geographical location or breed of flocks. In conclusion, this is the first study to demonstrate that LT-producing E. fergusonii strains were present in the faeces of healthy chickens and that plasmid-mediated virulence genes can be transferred to E. coli and may have a great potential to cause human disease.

Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R.

Salmonella enterica serovar Gallinarum biovar Gallinarum causes fowl typhoid in chickens and has been of economic importance to the chicken industry in many countries. The biovar Gallinarum live vaccine strain 9R (SG 9R) has been used to control fowl typhoid in many areas where the disease is endemic. Therefore, a definitive diagnosis of this disease may require differentiation of wild-type field isolates of biovar Gallinarum from the live vaccine strain SG 9R. Here, we report the development of a triplex polymerase chain reaction (PCR) assay to differentially identify serovar Gallinarum biovars Gallinarum and Pullorum and SG 9R. Sequences specific to SG 9R, which are absent or highly divergent in the fully sequenced biovar Gallinarum strain 287/91, were identified by constructing the suppression subtractive hybridization (SSH) library. A total of 565 nonredundant inserts were obtained from successfully sequenced SSH clones (718 clones). Sequences of 14 inserts were unique to SG 9R, but single nucleotide polymorphisms (SNPs) found in another insert (9R22C9) were more useful for strain discrimination. A new PCR primer set was designed to target SNP regions of the insert and was integrated into a duplex PCR assay developed previously (Kang et al., 2011). Boiled lysates of 138 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the triplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) and SG 9R (n=7) tested were differentially identified, whereas the other strains (n=57) were PCR negative. This triplex PCR assay will be very useful for rapid differential diagnoses of fowl typhoid and pullorum disease in veterinary laboratories.

Direct and quantitative analysis of Salmonella enterica serovar Typhimurium using real-time PCR from artificially contaminated chicken meat.

For quantitative PCR assay of Salmonella enterica serovar Typhimurium in food samples, a real-time PCR method was developed, based on DNA genome equivalent. Specific primers and probe designed based on the STM4497 gene of S. Typhimurium LT2 showed the specificity to S. Typhimurium. Threshold cycle (Ct) values of real-time PCR were obtained from a quantitative standard curve with genomic DNA of Salmonella Typhimurium. In addition, the recovery of S. Typhimurium inoculated artificially to chicken samples with 4.5 x 10(5) to 4.5 CFU/ml was evaluated by using real-time PCR and plate-count methods. Result showed that the number of cells calculated from the real-time PCR method had good correlation with that of the plate-count method. This real-time PCR method could be applicable to the detection and quantification of S. Typhimurium in food samples.