This study aimed to investigate whether bacterial ghosts (BGs), empty cell envelopes of a gram-negative bacterium, delivering envelope protein domain III (EDIII) of dengue virus (DENV) serotype 2 could induce protective immune responses against dengue infection. In this study, we constructed Salmonella Typhimurium BGs expressing and delivering EDIII (BG-EDIII) and evaluated these ghosts for their immunogenicity studies in C57BL/6 mice. Our results demonstrated that the mice vaccinated once orally with BG-EDIII followed by an intramuscular boosting with a recombinant EDIII protein elicited significantly higher humoral and cell-mediated immune responses compared to the BGs alone vaccinated group (p<0.001). Upon challenge with DENV2, significantly lower viral load and liver damage was observed in BG-EDIII vaccinated group than BGs alone control group (p<0.05). The outcomes of this study revealed the ability of BG- EDIII to stimulate immune response with no observable damage to the vital organs.

Fowl adenovirus serotype-4 (FAdV-4) infection, also known as hydropericardium-hepatitis syndrome (HHS), is one of the most prevalent diseases in the Korean poultry industry. Therefore, an effective vaccination against FAdV-4 should be developed for prevention of HHS infection. However, a standard animal model with significant pathologic characteristics has not been established for evaluation of HHS vaccine efficacy. In this study, we comparatively evaluated FAdV-4 pathogenicity from specific- pathogen-free (SPF) chickens using FAdV-4 strain (ADL091024) isolated in laying chickens in Korea according to chicken age, passage number and virus titers. In brief, FAdV-4 passaged four times in yolk sac of SPF eggs with the highest viral titers could induce the most severe clinical signs in 1-day-old chickens, similar to natural HHS outbreaks. Furthermore, the efficacy of a newly developed attenuated live FAdV-4 vaccine candidate was successfully determined using the established 1-day-old SPF chickens. There was no significant pathogenicity based on the criteria such as mortality, body weight, gross lesions, histological lesions and virus detection rates in the vaccination and non-challenge control groups. However, the challenge group without vaccination showed significant pathogenicity including hepatic necrosis, histological lesion scores in the liver and heart, virus replication and decreased body weight gain. In conclusion, the chicken challenge model established in this report was proposed for consistent and reliable evaluation of FAdV- 4 vaccine efficacy.

Avian pathogenic Escherichia coli (APEC) is an agent associated with colibacillosis and an important primary pathogen with responsible for significant economic losses in chickens. This study investigated the molecular characteristics including virulence and antimicrobial resistance of serotype O78 APEC isolates, the predominant serotype, in Korea. Among 16 O78 APEC isolates, 13 isolates carried the genes conferring resistance to ß-lactam (blaTEM), aminoglycoside [aac(3)-II], plasmid-mediated quinolone (qnrA), tetracycline (tetA and tetB), sulfonamide (sul1 and sul2), or chloramphenicol (catA1). Three isolates showed resistance to gentamicin and carried aminoglycoside-modifying enzyme gene, aac(3)-II, simultaneously. Ten O78 APEC isolates showed resistance to nalidixic acid, but only qnrA gene among plasmid-mediated quinolone resistance genes was detected in one isolate. The tetA and tetB genes were also detected in nine and two isolates, respectively. In distribution of phylogenetic groups, four O78 APEC isolates only belonged to group D. But all isolates carried three to five essential virulence genes regardless of phylogenetic groups. Virulence factors and antimicrobial resistance of the most predominant serotype, O78, in chickens tested in this study can be significant role in persistence of APEC in Korea.

Bacillus thuringiensis (Bt) strain K4 was isolated from fallen leaves which had been collected at a forest stand in Mungyeong city, Republic of Korea. The flagellated vegetative cells of Bt K4 were agglutinated with the H3 reference antiserum among 55 reference H-antisera. In a further test to identify subfactors, 3b and 3d monospecific antisera were reactive to the cells, followed up with introducing a novel serogroup of 3a3b3d, designated as serovar mogi. The strain K4 had mosquitocidal activity against Dipteran larvae, Anopheles sinensis and Culex pipiens pallens, with no Lepidopteran toxicity observed. The SDS-PAGE profile of K4 crystal protein, ovoidal-shaped, included several bands ranging from 30-75 kDa. Four putative peptides, Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected from the bands by a nano-LC-ESI-IT MS analysis. Through a thermal asymmetric interlaced PCR, cry19Ba, cry40ORF2 and cry27Aa genes were partially cloned from K4 strain. Three cry genes were further found in the strain by a 454 pyrosequencing, ending up to showing 58%, 39% and 84% homology in amino acids with Cry56Aa, Cry8Ba and Cry39ORF2 toxins, respectively. This novel 3a3b3d type strain, B. thuringiensis subsp. mogi, can be used as a good resource for studying unknown mosquitocidal cry genes.

The present study was conducted to investigate serotype distribution and biofilm formation of Actinobacillus (A.) pleuropneumoniae isolated from pneumonic lungs of pigs. A total of 37 A. pleuropneumoniae were isolated between January 2009 and June 2010. Serotypes of A. pleuropneumoniae isolates were determined using two different PCRs. The majority of isolates belonged to serotype 5 (n=31, 83.8%), and the others belonged to serotype 1 (n=4, 10.8%), and 2 (n=2, 5.4%), respectively. The ability of biofilm formation of the isolates was also determined by quantitative microtiter plate assay. Biofilm formation was observed in both 23 (62.2%) of the 37 field isolates and seven (43.8%) of the 16 reference strains. On the other hand, biofilm formation was various according to the serotypes: 20 (64.5%) of serotype 5, and three (75.0%) of serotype 1. However, two isolates of serotype 2 did not produce the biofilm in this study. Consequently, A. pleuropneumoniae serotype 5 was the most frequently detected (83.8% of the isolates), and 23 (62.2%) of 37 isolates exhibited biofilm positive phenotype in this study.

The Bacillus thuringiensis strain K4 was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. The flagellated vegetative cells of B. thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific antisera but non-reactive for 3c and 3e factor sera. These results create a new serogroup with flagellar antigenic structure of 3a3b3d, designated serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced a single ovoidal-shaped parasporal crystal whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected. In contrast to the plasmid profile of B. thuringiensis H3 serotype strains, the strain K4 contained only a large plasmid (~100 kb) and we cloned partial cry27Aa, cry19Ba and cry40ORF2 genes from it by thermal asymmetric interlaced PCR. Sequencing analysis showed 87%, 88% and 88% homologous with known cry27Aa, cry19Ba and cry40ORF2 genes, respectively. The new type strain, B. thuringiensis subsp. mogi (H3a3b3d) will be a good resource for new mosquitocidal cry genes.