Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.

Proteome Analysis of Alkylhydroxide Peroxidase-Deficient Isogenic Mutant of Helicobacter pylori 26695

In order to investigate the antioxidant effect of alkylhydroxide peroxidase (ahpC) of Helicobacter pylori (H. pylori) 26695, an ahpC-deficient mutant (H. pylori 26695 ahpC::cat) was generated. ahpC-deficient mutant was grown slowly at lower pressure of oxygen (5% oxygen) compared to the H. pylori 26695. Whole cell proteins isolated form H. pylori 26695 and H. pylori 26695 ahpC::cat were analyzed by MALDI-TOF and tandem-MS. The expression of 15 proteins, including Ppa, HypB, GrpE, Elp, RecA, GroES, Mda66, RibE, NapA, GlnA, BioB, TrxB, Tsf, FumC and Icd, was more than doubled in H. pylori 26695 ahpC::cat. Production of 10 proteins such as UreG, FabE, Adk, Pnp, OorC, AtpA, AtpD, Nqq3, Pfr, and TagD decreased below 50% in H. pylori 26695 ahpC::cat compared to the H. pylori 26695. In microarray analysis, 9 genes including sul1, amiE, frxA, fecA, hyuA, and katA increased in transcription level in H. pylori 26695 ahpC::cat compared to H. pylori 26695. A total of 24 genes, including flaB, protein kinase C inhibitor, cag16, pabC, and sabA, reduced in transcription. 27 genes, including HP0889, showed common expression changes in ahpC, katA, and sodB-deficient mutations. As a result of this study, there were not many genes whose expression was commonly changed by the deletion of each of the three major antioxidant enzymes of H. pylori. These results showed the functions and regulation of the three antioxidant enzymes were different in H. pylori.

Gene Expression Profiles of Th1-type Chemokines in Whole Blood of Mycobacterium avium subsp. paratuberculosis-Infected Cattle

Johne's disease (JD) is a chronic, debilitating disease of ruminants including cows, and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is not only important in animal husbandry, but also in public health as it is associated with the onset of Crohn's disease, a chronic inflammatory bowel disease in humans. JD, like other mycobacterial diseases including tuberculosis, is classified into different stages based on the progression of infection. In addition, development of diagnostic assays that can distinguish between subclinical and clinical stages of JD is essential to control mycobacterial infection by providing an effective treatment. For the development of novel diagnostic methods of JD, it is important to investigate and understand the mRNA expression of the various immune markers in individuals at each stage of infection. In this study, we measured the levels of Th1-type chemokines, CXCR3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11 in MAP-infected bovine blood by interferon (IFN)-γ release assay (IGRA) using IFN-γ as an alternative biomarker. The association of mRNA expression patterns of these chemokines with the MAP infection stages was analyzed and IFN-γ, CCL5, and CXCL10 were found to be significantly upregulated compared to IFN-γ, the biomarker used in IGRA. Our results further indicate that IFN-γ levels significantly increased in individuals with MAP-specific antibody, and CCL5 and CXCL10 levels significantly increased in those with MAP DNA. In particular, CCL5 was significantly upregulated in individuals, in which both MAP-specific antibody and MAP DNA were detected, but the expression of CXCL10 was specifically elevated in MAP DNA-detected individuals without MAP-specific antibody.

Different invasion efficiencies of Brucella abortus wild-type and mutantsin RAW 264.7 and THP-1 phagocytic cells and HeLa non-phagocytic cells

Brucellosis is one of the common zoonoses caused by Brucella abortus (B. abortus). However, little has been reported on factors affecting invasion of B. abortus into host cells. To investigate cell-type dependent invasion of B. abortus, phagocytic RAW 264.7 and THP-1 cells and non-phagocytic HeLa cells were infected with wild-type and mutant B. abortus, and their invasion efficiencies were compared. The invasion efficiencies of the strains were cell-type dependent. Wild-type B. abortus invasion efficiency was greater in phagocytic cells than in epithelial cells. The results also indicated that there are different factors involved in the invasion of B. abortus into phagocytic cells.

Different invasion efficiencies of Brucella abortus wild-type and mutantsin RAW 264.7 and THP-1 phagocytic cells and HeLa non-phagocytic cells / 대한수의학회지

Brucellosis is one of the common zoonoses caused by Brucella abortus (B. abortus). However, little has been reported on factors affecting invasion of B. abortus into host cells. To investigate cell-type dependent invasion of B. abortus, phagocytic RAW 264.7 and THP-1 cells and non-phagocytic HeLa cells were infected with wild-type and mutant B. abortus, and their invasion efficiencies were compared. The invasion efficiencies of the strains were cell-type dependent. Wild-type B. abortus invasion efficiency was greater in phagocytic cells than in epithelial cells. The results also indicated that there are different factors involved in the invasion of B. abortus into phagocytic cells.

Potential biomarkers as an indicator of vertical transmission of Johne's disease in a Korean native cattle farm

Paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is one of the most widespread and economically important diseases in cattle. After birth, calves are raised with natural breast feeding without separation from their mothers in most Korean native cattle (Hanwoo breed) farms. Vertical transmission of PTB has been reported, but the exact PTB infection route has not been revealed in Hanwoo farms. Calves of MAP seropositive dams were tested for MAP presence and MAP antibodies in feces and tissues. MAP was detected in calf tissues by using polymerase chain reaction. Expressions of genes reported to be prognostic biomarkers of MAP infection changed in both calves and cows (p < 0.05). Expression of two genes (HGF and SERPINE1) were significantly decreased in MAP-infected cattle and their offspring (p < 0.01). The results suggest that biomarker gene expression profiles can be useful in detecting early stage MAP infection. Based on the results, complete eradication of MAP may be possible if accurate diagnostic methods to detect infected calves are added to the current PTB eradication strategy, which, because infected individuals are likely to develop into fecal MAP shedders at any time, includes isolation of new born calves and feeding sterilized colostrum.

Profiling of antimicrobial resistance and plasmid replicon types in beta-lactamase producing Escherichia coli isolated from Korean beef cattle

In this study, 78 isolates of Escherichia coli isolated from Korean beef cattle farms were investigated for the production of extended-spectrum beta-lactamase (ESBL) and/or AmpC beta-lactamase. In the disc diffusion test with ampicillin, amoxicillin, cephalothin, ceftiofur, cefotaxime, ceftazidime, and cefoxitin, 38.5% of the isolates showed resistance to all of ampicillin, amoxicillin, and cephalothin. The double disc synergy method revealed that none of the isolates produced ESBL or AmpC beta-lactamases. DNA sequencing showed that all isolates encoded genes for TEM-1-type beta-lactamase. Moreover, 78.2% of the isolates transferred the TEM-1-type beta-lactamase gene via conjugation. In plasmid replicon typing of all donors, IncFIB and IncFIA were identified in 71.4% and 41.0% of plasmids, respectively. In transconjugants, IncFIB and IncFIA were the most frequent types detected (61.5% and 41.0%, respectively). Overall, the present study indicates that selection pressures of antimicrobials on beta-lactamases in beef cattle may be low relative to other livestock animals in Korea. Moreover, to reduce selection pressure and dissemination of beta-lactamase, the long-term surveillance of antimicrobial use in domestic beef cattle should be established.

Virulence factors, antimicrobial resistance patterns, and genetic characteristics of hydrogen sulfide-producing Escherichia coli isolated from swine / 대한수의학회지

Escherichia (E.) coli is commensal bacteria found in the intestine; however, some pathogenic strains cause diseases in animals and humans. Although E. coli does not typically produce hydrogen sulfide (H2S), H2S-producing strains of E. coli have been identified worldwide. The relationship between virulence and H2S production has not yet been determined. Therefore, characteristics of H2S-producing isolates obtained from swine feces were evaluated including antibiotic resistance patterns, virulence gene expression, and genetic relatedness. Rates of antibiotic resistance of the H2Sproducing E. coli varied according to antibiotic. Only the EAST1 gene was detected as a virulence gene in five H2S-producing E. coli strains. Genes conferring H2S production were not transmissible although the seeA gene encoding 3-mercaptopyruvate sulfurtransferase was detected in all H2S-producing E. coli strains. Sequences of the seeA gene motif CGSVTA around Cys238 were also identical in all H2S-producing E. coli strains. Diverse genetic relatedness among the isolates was observed by pulsed-field gel electrophoresis analysis. These results suggested that H2S-producing E. coli strains were not derived from a specific clone and H2S production in E. coli is not associated with virulence genes.

Dietary germanium biotite supplementation enhances the induction of antibody responses to foot-and-mouth disease virus vaccine in pigs

We evaluated the potential ability of germanium biotite (GB) to stimulate the production of antibodies specific for foot-and-mouth disease virus (FMDV). To this aim, we measured the total FMDV-specific antibody responses and IgM production after vaccination against FMD both experimentally and in the field. GB supplementation with FMDV vaccination stimulated the production of anti-FMDV antibodies, and effectively increased IFN-gamma and TNF-alpha levels. These results suggest that GB may be a novel alternative feed supplement that can serve as a boosting agent and an immunostimulator for increasing the efficacy of FMDV vaccination in pigs.

Application of SYBR Green real-time PCR assay for the specific detection of Salmonella spp / 대한수의학회지

The aim of this study was to applicate and evaluate a SYBR Green real-time PCR for the specific detection of Salmonella spp. Specificity of the PCR method was confirmed with 48 Salmonella spp. and 5 non-Salmonella strains using invA gene primer. The average threshold cycle (C(T)) of Salmonella spp. was 11.83 +/- 0.78 while non-Salmonella spp. was 30.86 +/- 1.19. Correlation coefficients of standard curves constructed using C(T) versus copy number of Salmonella Enteritidis ATCC 13076 showed good linearity (R2 = 0.993; slope = 3.563). Minimum level of detection with the method was > 10(2) colony forming units (CFU)/mL. These results suggested that the SYBR Green real-time PCR might be applicable for the specific detection of Salmonella spp. isolates.

The effects of Germanium biotite on the adsorptive and inhibition of growth abilities against E. coli and Salmonella spp. in vitro / 대한수의학회지

Germanium biotite, a natural mineral, has been used as a feed supplement to reinforce innate immune ability. The aim of the present study was to evaluate the effects of germanium biotite on the adsorptive and inhibition of growth abilities against Escherichia (E.) coli and Salmonella spp. in vitro. Two strains of enterotoxigenic E. coli and four strains of two Salmonella serotypes (Salmonella Derby and Salmonella Typhimurium), major bacterial diarrheal pathogens, were used for this experiment. The absorptive ability of germanium biotite against most Salmonella used in present experiment was observed weakly. The germanium biotite, however, showed significant effect of bacterial growth inhibition in most experiment bacteria. These results suggest that the use of the germanium biotite as feed supplement could alleviate diarrhea following inhibition of bacteria growth. It is also presumed that antibiotics usage for farm animals, considered as causes of antibiotic residue in meat and emerging antibiotic resistance, could be reduced through the use of germanium biotite as a feed supplement, in place of antibiotics used for the prevention of diarrhea.