Sang Gyun Kang

The potential of mannosylated chitosan microspheres to target macrophage mannose receptors in an adjuvant-delivery system for intranasal immunization

A vaccine delivery system based on mannosylated chitosan microspheres (MCMs) was studied in vitro and in vivo. Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) were loaded in MCMs or chitosan microspheres (CMs). Fluorescence confocal microscopy indicated that BBD-loaded MCMs (BBD-MCMs) bound with mannose receptors on murine macrophages (RAW264.7 cells). In vitro experiments using macrophages demonstrated that BBD-MCMs had more effective immune-stimulating activity than BBD-loaded CMs (BBD-CMs). Mice intranasally immunized with BBD-MCMs showed significantly higher BBD-specific IgA antibody responses in saliva and serum than mice immunized with BBD-CMs (p<0.05). After challenge with B. bronchiseptica via the nasal cavity, groups treated with BBD-MCMs or BBD-CMs showed similar patterns with a high survival rate even though there was no significant difference between those groups. These results suggested that mannose moieties in the MCMs enhanced immune-stimulating activities through mucosal delivery due to a specific interaction between mannose groups in the MCMs and mannose receptors on the macrophages.

Characterization of TEM-, SHV- and AmpC-type β-lactamases from cephalosporin-resistant Enterobacteriaceae isolated from swine

Extended-spectrum beta-lactamase (ESBL) and AmpC-producing Enterobacteriaceae are an increasing problem in human medicine and an emerging problem in the veterinary field. Our study, therefore, focused on assessing the prevalence of beta-lactamases isolated from swine. Sixty-six Salmonella enterica serovar Typhimurium (S. Typhimurium), 33 Salmonella enterica serovar Enteritidis (S. Enteritidis), 26 Klebsiella pneumonia (K. pneumoniae) and 130 Escherichia coli (E. coli) pig isolates collected from 1999-2006 were screened for beta-lactam resistance by the disk diffusion test (DDT) and micro-broth dilution. Among the isolates, five E. coli and five K. pneumoniae exhibited reduced susceptibility to the cephalosporins tested. PCR, plasmid profiling and Southern blot hybridization showed the presence of multiple beta-lactamases in these isolates of animal origin. Hybridization patterns of the DHA-1 specific probe indicated that dissemination of DHA-1 related beta-lactamases could be attributed to plasmids of one common size among the enteric microbes of animal origin. To the best of our knowledge, this study reports the first identification of SHV-28 and DHA-1 from microbes of animal origin.

Development of Multiplex Polymerase Chain Reaction Assays for Detecting Enterotoxigenic Escherichia Coli and their Application to Field Isolates from Piglets with Diarrhea

Fimbriae and enterotoxins are major virulence factors associated with enterotoxigenic Escherichia coli (ETEC). In this study, 3 sets of multiplex polymerase chain reaction (mPCR) assays targeting fimbriae, enterotoxins, and other adherence factors were developed for detecting ETEC. A total number of 188 E. coli field isolates were examined, and percentages of E. coli strains carrying each virulence factors were as follows: F4 (7.45%), F5 (29.79%), F6 (6.38%), F18 (15.43%), F41 (3.72%), STa (10.11%), STb (20.74%), LT (9.57%), Stx2e (2.13%), EAST1 (42.02%), F1 (67.55%), AIDA-I (2.66%), and pAA (7.45%). Of the 188 E. coli field isolates examined, 25.53% were found to be pathogenic ETEC, having both fimbriae and enterotoxins. However, the ratio increased to 44.68% when the presence of other adhesins was considered as criteria for virulence. Among the adherence factors, F1 was found to be the most prevalent. AIDA-I and pAA were also found with similar ratio as compared with other virulence factors. In addition, virulence patterns carrying these alternate adhesive genes with enterotoxins were detected with significant ratio. Therefore, it is desirable that alternate adhesins be considered as markers for diagnosis of ETEC.

Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.