Porcine epidemic diarrhea viruses from Vietnam: isolation, characterization, and neutralizing activity

Porcine epidemic diarrhea (PED) is characterized by acute enteritis, watery diarrhea, weight loss, dehydration, and death, with high mortality in neonatal piglets. In this study, 3 virus isolates collected in Vietnam between 2016 and 2017 were propagated successfully in Vero cells at high virus titers. Sequence analysis of the full-length spike (S) gene showed that all 3 isolates belong to genogroup 2b, which is closely related to other prevalent Asian strains. A comparison of the amino acid sequence revealed a 98.19% to 99.13% homology with the Vietnam isolates circulating during 2013–2015, suggesting that field PED viruses (PEDVs) are evolving continuously. Experiments in animals showed that the antisera from guinea pigs immunized with the vaccine strain resulted in higher levels (5 log2) of neutralizing antibodies against the homologous strain and a relatively moderate level of neutralizing antibodies against the field isolates. This finding would be helpful in selecting a PEDV strain for vaccine development.

Isolation, characterization and neutralizing activity of porcine epidemic diarrhea viruses from Vietnam

Porcine epidemic diarrhea (PED) is characterized by acute enteritis, watery diarrhea, weight loss, dehydration, and death with high mortality in neonatal piglets. In this study, 3 virus isolates collected in Vietnam between 2016 and 2017 were successfully propagated in Vero cells at high virus titers. Sequence analysis of the full-length spike (S) gene revealed that all 3 isolates belong to genogroup 2a, which is closely related to other prevalent Asian strains. Amino acid sequence comparisons revealed 98.19% to 99.13% homology with the Vietnam isolates circulating during 2013–2015, suggesting that field PED viruses (PEDVs) evolve continuously. Experiments in animals demonstrated that antisera from guinea pigs immunized with the vaccine strain resulted in higher levels (5 log2) of neutralizing antibody against the homologous strain, and showed a relatively lower level of neutralizing antibody against the field isolates. This finding would be helpful in choosing a PEDV strain for vaccine development.

Construction and immunization with double mutant ΔapxIBD Δpnp forms of Oryctolagus cuniculus serotypes 1 and 5

Actinobacillus pleuropneumoniae (APP) causes a form of porcine pleuropneumonia that leads to significant economic losses in the swine industry worldwide. The apxIBD gene is responsible for the secretion of the ApxI and ApxII toxins and the pnp gene is responsible for the adaptation of bacteria to cold temperature and a virulence factor. The apxIBDand pnp genes were deleted successfully from APP serotype 1 and 5 by transconjugation and sucrose counter-selection. The APP1ΔapxIBD Δpnp and APP5ΔapxIBD Δpnp mutants lost hemolytic activity and could not secrete ApxI and ApxII toxins outside the bacteria because both mutants lost the ApxI- and ApxII-secreting proteins by deletion of the apxIBD gene.Besides, the growth of these mutants was defective at low temperatures resulting from the deletion of pnp. The APP1ΔapxIBD Δpnp and APP5ΔapxIBD Δpnp mutants were significantly attenuated compared with wild-type ones. However, mice vaccinated intraperitoneally with APP5ΔapxIBD Δpnpdid not provide any protection when challenged with a 10-times 50% lethal dose of virulent homologous (APP5) and heterologous (APP1) bacterial strains, while mice vaccinated with APP1ΔapxIBD Δpnp offered 75% protection against a homologous challenge.The ΔapxIBD Δpnp mutants were significantly attenuated and gave different protection rate against homologous virulent wild-type APP challenging.

Isolation and characterization of Brucella abortus isolates from wildlife species in South Korea

A total of 782 blood and 465 tissue samples from 1,039 wild animals and 127 dairy goats were collected from January 2011 to December 2013 in 10 provinces of South Korea and tested for the presence of brucellosis. The Rose Bengal test revealed that 8.0% (52/650) of the serum samples were seropositive, while 4.2% (33/782) of the serum samples were positive for Brucella antibodies by competitive enzyme-linked immunosorbent assay. Of the 650 sera examined, only 16 (2.5%) were positive by both serological tests. Direct polymerase chain reaction (PCR) assay using B4/B5 primers for Brucella abortus (BCSP31) revealed the prevalence of Brucella to be 26.5% (129/487) in blood samples and 21% (98/465) in tissue samples while, 16S rRNA PCR detected Brucella DNA in 6.8% (33/487) and 2.6% (12/465) in blood and tissue samples, respectively. Of PCR-positive samples, only 6.2% (30/487) of blood samples and 2.4% (11/465) of tissue samples were found to be positive by both BCSP31 and 16S rRNA PCRs. However, Brucella strains were isolated by blood culture from only two out of 487 blood samples (0.4%). This characterization and identification of pathogenic Brucella isolates is the first to clearly indicate that the organisms were Brucella abortus biovar 1.

Genetic diversity of nucleocapsid genes of recent porcine epidemic diarrhea viruses isolated in Korea

Porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea in piglets, resulting in large economic losses because of high mortality. In November 2013, PEDV reemerged in Korea, and these outbreaks have since continuously occurred. In the present study, we determined the full-length nucleocapsid (N) gene sequences of three Korean PEDV field isolates collected in 2014-2015. Sequence and phylogenetic analysis of N genes revealed that recent prevalent Korean PEDV isolates were very closely related to the US PEDV isolates in 2013. Interestingly, the phylogenetic tree based on the nucleotide sequencing of the PEDV N gene was similar to the tree topology of the PEDV complete genomes. Therefore, our data provide a better understanding of the genetic diversity and contribute to the accurate diagnosis and development of vaccines against PEDV.

The role of rpoS, hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H2O2, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGDelta3 (SGDeltarpoSDeltahmpDeltassrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGDelta3 mutant did not cause any mortality after inoculation with either 1 x 10(6) or 1 x 10(8) colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGDelta3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGDelta3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGDelta3 could be a promising candidate for a live Salmonella vaccine against FT.