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.

Clinical profile of Asian and African strains of Zika virus in immunocompetent mice

The mosquito-borne pathogen Zika virus may result in neurological disorders such as Guillain–Barré syndrome and microcephaly. The virus is classified as a member of the Flaviviridae family and its wide spread in multiple continents is a significant threat to public health. So has created a need to develop animal models to examine the pathogenesis of the disease and develop vaccines. To examine the clinical profile during Zika virus infection, we infected neonatal and adult wild-type mice (C57BL/6 and Balb/c) and compared the clinical signs of African-lineage strain (MR 766) and Asian-lineage strain (PRVABC59, MEX2-81) of Zika virus. Consistent with previous reports, eight-week-old female Balb/c mice infected with these viral strains showed no changes in body weight, survival rate, and no neurologic signs, but demonstrated increases in the weights of spleens and hearts. However, one-day-old neonates showed significantly lower survival rate and body weight with the African-lineage strain than the Asian-lineage strain. These results confirmed the pathogenic differences between Zika virus strains. We also evaluated the clinical responses in neonatal and adult mice of different strains. Our findings suggest that these are useful mouse models for characterization of Zika virus for vaccine development.

Clinical profile of Asian and African strains of Zika virus in immunocompetent mice

The mosquito-borne pathogen Zika virus may result in neurological disorders such as Guillain–Barré syndrome and microcephaly. The virus is classified as a member of the Flaviviridae family and its wide spread in multiple continents is a significant threat to public health. So has created a need to develop animal models to examine the pathogenesis of the disease and develop vaccines. To examine the clinical profile during Zika virus infection, we infected neonatal and adult wild-type mice (C57BL/6 and Balb/c) and compared the clinical signs of African-lineage strain (MR 766) and Asian-lineage strain (PRVABC59, MEX2-81) of Zika virus. Consistent with previous reports, eight-week-old female Balb/c mice infected with these viral strains showed no changes in body weight, survival rate, and no neurologic signs, but demonstrated increases in the weights of spleens and hearts. However, one-day-old neonates showed significantly lower survival rate and body weight with the African-lineage strain than the Asian-lineage strain. These results confirmed the pathogenic differences between Zika virus strains. We also evaluated the clinical responses in neonatal and adult mice of different strains. Our findings suggest that these are useful mouse models for characterization of Zika virus 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.

Generation of a cold-adapted PRRSV with a nucleotide substitution in the ORF5 and numerous mutations in the hypervariable region of NSP2

A cold-adapted porcine reproductive and respiratory syndrome virus (CA-VR2332) was generated from the modified live virus strain VR2332. CA-VR2332 showed impaired growth when cultured at 37°C with numerous mutations ( S731F , E819D , G975E , and D1014N) in the hypervariable region of the NSP2, in which the mutation S731F might play a vital role in viral replication at 30°C. Conserved amino acid sequences of the GP5 protein suggests that CAVR2332 is a promising candidate for producing an effective vaccine against PRRSV infection.Further studies on replication and immunogenicity in vivo are required to evaluate the properties of CA-VR2332.

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.

Molecular epidemiology of sequence type 33 of Shiga toxin-producing Escherichia coli O91:H14 isolates from human patients and retail meats in Korea

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.

Pathogenic and phylogenetic characteristics of non-O157 Shiga toxin-producing Escherichia coli isolates from retail meats in South Korea

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx₁, stx(2c), stx(2e), or stx₁ + stx(2b)) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

Genetic variations in open reading frame 2 gene of porcine circovirus type 2 isolated in Korea during 2016–2017 / 대한수의학회지

The capsid protein of porcine circovirus type 2 (PCV2) encoded by open reading frame 2 (ORF2) is important for neutralizing activity against PCV2 infection. This study investigated the heterogeneity of the ORF2 gene of PCV2 isolated in Korea during 2016–2017. The results revealed that PCV2d is currently the predominant genotype. Moreover, comparison of ORF2 from 17 PCV2 isolates revealed 88.3–100% homology at the nucleotide (deduced amino acid 86.3–100%) level. Interestingly, 61.5% (8/13) of the PCV2d isolates had glycine at position 210. These data provide a useful information for PCV2 epidemiology in Korea.

Genetic variations in open reading frame 2 gene of porcine circovirus type 2 isolated in Korea during 2016–2017

The capsid protein of porcine circovirus type 2 (PCV2) encoded by open reading frame 2 (ORF2) is important for neutralizing activity against PCV2 infection. This study investigated the heterogeneity of the ORF2 gene of PCV2 isolated in Korea during 2016–2017. The results revealed that PCV2d is currently the predominant genotype. Moreover, comparison of ORF2 from 17 PCV2 isolates revealed 88.3–100% homology at the nucleotide (deduced amino acid 86.3–100%) level. Interestingly, 61.5% (8/13) of the PCV2d isolates had glycine at position 210. These data provide a useful information for PCV2 epidemiology in Korea.

Evaluation of safety and immunogenicity of a new octavalent inactivated vaccine containing porcine parvovirus, erysipelas, and leptospira

Porcine parvovirus, Erysipelothrix (E.) rhusiopathiae, and Leptospira (L.) interrogans are considered major etiologic agents of reproductive failure in pigs, causing economic loss in the swine industry. In this study, the safety and immunogenicity of a new octavalent inactivated vaccine were evaluated. The vaccine contained inactivated porcine parvovirus, E. rhusiopathiae, and six L. interrogans serovars (Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona). Safety test results showed no notable side effects or clinical signs after vaccination in mice, guinea pigs, and sows. In addition, we assessed immunogenicity of the vaccine in 25 sows under field conditions. The vaccinated group (n = 20) had a significantly higher antibody level than the non-vaccinated group (n = 5). Moreover, the stillbirth rate decreased in piglets born from vaccinated sows, resulting in an increased fertility rate. The results of this study demonstrate that the new octavalent inactivated vaccine can be applied safely and effectively to improve reproductive performance in sows.

Establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines

With the increase in international human and material exchanges, contagious and infectious epidemics are occurring. One of the effective methods of epidemic inhibition is the rapid development and supply of vaccines. Considering the safety of the brain during vaccine development is very important. However, manuals for brain safety assays for new vaccines are not uniform or effective globally. Therefore, the aim of this study is to establish a positive-control protocol for an effective brain safety test to enhance rapid vaccine development. The blood-brain barrier's tight junctions provide selective defense of the brain; however, it is possible to destroy these important microstructures by administering lipopolysaccharides (LPSs), thereby artificially increasing the permeability of brain parenchyma. In this study, test conditions are established so that the degree of brain penetration or brain destruction of newly developed vaccines can be quantitatively identified. The most effective conditions were suggested by measuring time-dependent expressions of tight junction biomarkers (zonula occludens-1 [ZO-1] and occludin) in two types of mice (C57BL/6 and ICR) following exposure to two types of LPS (Salmonella and Escherichia). In the future, we hope that use of the developed positive-control protocol will help speed up the determination of brain safety of novel vaccines.

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

Application of Ecklonia cava Kjellman by-product as a feed additive: enhancing weight gain, immunity and protection from Salmonella infection in chickens

The Ecklonia cava Kjellman by-product (ECBP) as a feed additive was evaluated in improvement of productivity and immune enhancement against Salmonella Gallinarum (SG). Lohmann Brown chickens proved SG-free were randomly divided into 3 groups of 8 chickens each. Chickens were fed with the experimental diet treatment: T0, Non treatment-commercial feed; T1, commercial feed with 0.5% ECBP; T2, commercial feed with 0.1% Lactobacillus plantarum. In this study, we evaluated the effect of T1 and T2 groups on the body weight and protective efficacy against SG in chickens. The results demonstrated that treatment of T1 group as a feed additive affected significantly body weight gaining in chickens. In addition, T1 group showed a significant different colonization of SG when compared to T2 and T0 groups. We also studied that serum IgG and interferon-γ levels were significantly different compared with other treatment groups. Therefore, we suggest that ECBP can be used as a good candidate of feed additives in chicken industry.

Establishment of hydrochloric acid/lipopolysaccharide-induced pelvic inflammatory disease model

Pelvic inflammatory disease (PID), which is one of the most problematic complications experienced by women with sexually transmitted diseases, frequently causes secondary infections after reproductive abnormalities in veterinary animals. Although the uterus is self-protective, it becomes fragile during periods or pregnancy. To investigate PID, bacteria or lipopolysaccharide (LPS) extracted from gram negative bacteria has been used to induce the disease in several animal models. However, when LPS is applied to the peritoneum, it often causes systemic sepsis leading to death and the PID was not consistently demonstrated. Hydrochloric acid (HCl) has been used to induce inflammation in the lungs and stomach but not tested for reproductive organs. In this study, we developed a PID model in mice by HCl and LPS sequential intracervical (i.c.) administration. The proinflammatory cytokines, interleukin (IL)-1β, IL-6 and tumor necrosis factor-α, were detected in the mouse uterus by western blot analysis and cytokine enzyme-linked immunosorbent assay after HCl (25 mg/kg) administration i.c. followed by four LPS (50 mg/kg) treatments. Moreover, mice exhibited increased infiltration of neutrophils in the endometrium and epithelial layer. These results suggest that ic co-administration of HCl and LPS induces PID in mice. This new model may provide a consistent and reproducible PID model for future research.

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.

In vitro antibiotic susceptibility of field isolates of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis from Korea

The present study was conducted to determine the antibiotic susceptibilities of local Mycoplasma hyopneumoniae (Mhp) and Mycoplasma hyorhinis (Mhr) filed isolates. Minimum inhibitory concentrations (MICs) of Mhp and Mhr field isolates (twelve each) obtained from enzootic pneumonia-like lung lesions during 2009-2011 from Korea were determined using the broth microdilution method. Tylvalosin showed the highest activity against Mhp and Mhr field isolates, with MIC90 values of 0.06 µg/mL and 0.12 µg/mL, respectively. Therefore, Korean Mhp and Mhr isolates are highly susceptible to tylvalosin.

Efficacy of orally administered ginseng stem and leaf in chickens / 대한수의학회지

Ginseng has been widely used in Korea as a natural medicine due to its saponin contents. Although the total amount of ginseng stem and leaf saponins (GSLS) is 4~5 times higher than that of saponin in the root, the root is mainly used. This is due to two reasons: nervous system-stimulant activity of GSLS and pesticide residues in GSLS. In this study, residual agricultural pesticides were removed from GSLS using two types of bacterial treatments. Two GSLS treatment groups of chickens (GSLS-1 and GSLS-2) were established. The chickens were fed 0.4% GSLS-1 or GSLS-2 mixed with crop. We then evaluated the effects of GSLS on bodyweight and several immune parameters. At the end of the experiments, chickens fed GSLS-1 and red ginseng saponin had significantly higher growth rates (16.6% and 8.0%, respectively) compared to the vaccine control group treated with Noblis Salenvac-T. The group fed GSLS-1 also had the highest IgG titer that was significantly different at the end of experiments compared to the other groups. These findings imply that GSLS-1 is a good candidate feed additive for the chicken industry.

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.