• Journal of Microbiology and Biotechnology
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• v.32 no.3
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• pp.278-286
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• 2022

Live bacterial vector vaccines are one of the most promising vaccine types and have the advantages of low cost, flexibility, and good safety. Meanwhile, protein secretion systems have been reported as useful tools to facilitate the release of heterologous antigen proteins from bacterial vectors. The twin-arginine translocation (Tat) system is an important protein export system that transports fully folded proteins in a signal peptide-dependent manner. In this study, we constructed a live vector vaccine using an engineered commensal Escherichia coli strain in which amiA and amiC genes were deleted, resulting in a leaky outer membrane that allows the release of periplasmic proteins to the extracellular environment. The protective antigen proteins SLY, enolase, and Sbp against Streptococcus suis were targeted to the Tat pathway by fusing a Tat signal peptide. Our results showed that by exploiting the Tat pathway and the outer membrane-defective E. coli strain, the antigen proteins were successfully secreted. The strains secreting the antigen proteins were used to vaccinate mice. After S. suis challenge, the vaccinated group showed significantly higher survival and milder clinical symptoms compared with the vector group. Further analysis showed that the mice in the vaccinated group had lower burdens of bacteria load and slighter pathological changes. Our study reports a novel live bacterial vector vaccine that uses the Tat system and provides a new alternative for developing S. suis vaccine.

• Journal of Practical Agriculture & Fisheries Research
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• v.20 no.1
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• pp.89-96
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• 2018

The object of this study was to evaluate the change of antibody titers on virus strains after inoculation with commercial killed equine influenza (EI) vaccines in horses. Serum antibodies of 20 Thoroughbred yearlings were detected using hemagglutination inhibition test for 41 weeks. Second vaccination is inoculated 4 weeks after the initial vaccination. Most of antibody titers were not increased until 4 weeks after first vaccination. The highest titers were detected 6-10 weeks after vaccination. The titers were decreased slowly and maintained for 16 weeks after inoculation. We could barely detect the antibody 41 weeks after vaccination in most cases. Vaccine anergia were appeared in 3 horses (15%) but it depended on virus strains. A/Equine/La Plata/93(H3N8) strain that induce high and durable antibody responses was the most effective among three strains. This study presents the first comprehensive data on the endurance of antibody titers against EI. Our data also suggests that yearlings should be inoculated three times in order to maintaining optimal antibody titers against EI. We speculate the causes of anergia were vaccine break down or individual specificity. Further research is needed to investigate immunological unresponsiveness. This was the first study on strain of equine vaccine in Korea.

• Korean Journal of Veterinary Service
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• v.21 no.4
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• pp.451-465
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• 1998

The present study was carried out to investigate the prevalence of brucellosis in Kyungbuk area for the 3 years from 1966 to 1998. Collective milk samples were routinely screened to detect positive farms by using the milk ring test(MRT), and serum agglutination test was performed to detect sero-positive individuals in the MRT positive farms. Attempt were made to isolate the causative organismas from slaughtered sero-positive reactors and some biochemical and polymerase chain reation characters of the isolates were also made to identify the organisms. Seroprevalence to brucellosis in peoples who are close contact with infected dairy herds was also investigated. Brucellosis of dairy cattle was rare before 1997, but has been broken more frequently since early 1998. By the MRT for dairy herds, positive rate was gradually increased every year : 0.6% in 1996, 1.5% in 1997, 3.9% in 1998. Among 262 MRT-positive herds, only 21 herds(8.0%) showed positive brucellosis in serological test. The isolation rates of Brucella sp from tested materials were 51.2% in supramammary glands, 39.5% in milks, and 50.0% in pulmonary Iymphnode, respectively. Isolated strain and biotype were Brucella(B) arbortus biotype 1 in 26 heads, and were B suis biotype 1 in 2 heads. Isolated strain and vaccine strain were very similar in their colony morphology and staining. In drug susceptibility, isolated stains(B abortus) and vaccine strain(B abortus RB-51) were sensitive to ampicillin, gentamycin, kanamycin, neomycin, penicillin, streptomycin, and to tetracycline, but resistant to erythromycin. In the PCR, field strains reacted to BA and IS711 primers, and vaccine strain reacted to BA, IS711, and RB5l primers. In the plate agglutination test of 96 sera of human contacted with animals, serum antibody titer detected 1 : 100 in one person, 1 : 200 in one, and below 1 : 25 in the others.

• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.167-179
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• 2023

The rifampicin-resistant strain E9-302 of Edwardsiella ictaluri strain 669 (WT) was generated by continuous passage on BHI agar plates containing increasing concentrations of rifampicin. E9-302 was attenuated significantly by 119 times to zebrafish Danio rerio compared to WT in terms of the 50% lethal dose (LD₅₀). Zebrafish vaccinated with E9-302 via intraperitoneal (IP) injection at a dose of 1 × 10³ CFU/fish had relative percentage survival (RPS) rates of 85.7% when challenged with wild-type E. ictaluri via IP 14 days post-vaccination (dpv). After 14 days of primary vaccination with E9-302 via immersion (IM) at a dose of 4 × 10⁷ CFU/ml, a booster IM vaccination with E9-302 at a dose of 2 × 10⁷ CFU/ml exhibited 65.2% RPS against challenge with wild-type E. ictaluri via IP 7 days later. These results indicated that the rifampicin-resistant attenuated strain E9-302 had potential as a live vaccine against E. ictaluri infection. A previously unreported amino acid site change at position 142 of the RNA polymerase (RNAP) β subunit encoded by the gene rpoB associated with rifampicin resistance was identified. Analysis of the whole-genome sequencing results revealed multiple missense mutations in the virulence-related genes esrB and sspH2 in E9-302 compared with WT, and a 189 bp mismatch in one gene, whose coding product was highly homologous to glycosyltransferase family 39 protein. This study preliminarily explored the molecular mechanism underlying the virulence attenuation of rifampicin-resistant strain E9-302 and provided a new target for the subsequent study of the pathogenic mechanism of E. ictaluri.

• Korean Journal of Veterinary Research
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• v.40 no.3
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• pp.533-541
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• 2000

Biochemical and genetic analysis were carried out to investigate the potential recovery of pathogenecity or related mutations of Brucella abortus RB51 vaccine strains. RB51 strains were recovered from commercial vaccines, including related seed stocks from private companies in Republic of Korea, strain from USA, a reference strain from C university and a field isolate (Daehungjin) from aborted dairy cow after RB51 vaccination were compared with two identified virulent wild strains (S2308 and a field strain isolated from dairy cow in Korea) at the same conditions. All the strains examined, except identified pathogenic strains, revealed the identical characteristics to the original RB51 in biochemical properties, antigen and bacteriophage typing. Outer membrane protein (OMP) profiles from strains of RB51 showed the same patterns with standard RB51 in SDS-PAGE. In addition, Western blotting with the brucella specific monoclonal antibody also indicated that all the vaccine strains were completely deficient in their LPS compared to the pathogenic Br abortus strains. The differences in DNA structures among strains were also possible to detect after PCR. All vaccine strains, except S19, S1119-3, S1075, S544 and Br suis, were amplified a 178bp DNA fragment of eri-gene, and 364bp of IS711 elements. In contrast, 498bp DNA product was only found with Br abortus. Overall evidences in the present study confirmed that the RB51 strains for vaccine production in Korea did not originated from the phenomena of possible recovery of pathogenicity or related to any potential mutation event at all.

• Korean Journal of Veterinary Research
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• v.8 no.2
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• pp.125-146
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• 1968

Throughout the studies the following experimental results were summarized. 1. It was impossible to infect and kill the mice, weighing 10 to 12 gm, by inoculating 0.2ml of virulent Cl. chauvoei, diluted 1 to 10 with physiological saline, via subcutaneous, intramuscular, intraperitoneal or intraveonus, route. 2. The mice which were inoculated in brain with 0.03ml of Cl. chauvoei diluted 1 : 5120 with physiological saline were resulted in all death after infection, but not in case of attenuated strain even in dilution of one to five. 3. Virulent Cl. chauvoei were diluted with each of those of whole blood, erythrocytes and serum of horse, calf, swine, sheep, rabbit, guinea pig, chicken and duck, human plasma and 2% CaCl solution, and inoculated subcutaneously 0.25 to 0.5ml in mice, weighing 12 to 15gm. It was resulted in significant increase in virulence as comparing with the case of physiological saline solution except when horse and pig sera were used. Such a phenomena were not seen in attenuated strain. 4. Virulence of virulent Cl. Chauvoei could be increased significantly in rat, as the procedures used in mice, by suspending in whole blood, erythrocytes, serum, or plasma of various animals, or 2% $CaCl_2$ solution and by inoculating subcutaneously 0.5 to 10ml in rat, weighing 30 to 60 gm, as compared with those of control group which used physiological saline solutionos diluent. 5. Mice resisted 100 and 80 percent against challenge of $10^3$ and $10^4$ M.L.D.. respectively, 24 hours after inoculation of 0.5ml black leg antiserum. 6. Immune response to the black leg living vaccine in mice could be obtained more favorably in the group of respected vaccination rather than those of single inoculation and the most profitable inoculm size of the vacine was 0.5 to 1.0ml. 7. Challenge for the immunized mice could be carried out effectively 3 weeks after first vaccination. 8. Satisfactory results could be obtained by inoculating subcutaneously for the immunization and intracerebrally or subcutaneously for the challenge. 9. Mice which were inoculated with 0.5ml of black leg living vaccine via subtaneucously two times at seven days interval and 21 days after first inoculation and challenged with 5 and 10 M.L.D. of virulent strain, resited 100 and 70 to 80 percent respectively. Same results were obtainable in black leg killed vaccine as the procedures used in living vaccine. 10. There were significantly different resistances against the definite challenge does between the mice groups which were immnuized with the living vaccine diluted five or 10 times and the undiluted. 11. For the biological assay of black leg living vaccine and antiserum, satisfactory results could be obtained using mice.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.318-324
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• 2006

In order to develop an oral vaccine to prevent H. pylori infection, we have expressed the hpaA gene of H. pylori K51 isolated from Korean patients, encoding 29-kDa HpaA that is known to be localized on the cell surface and flagella sheath, in a live delivery vector system, Lactococcus lactis. The hpaA gene, amplified by PCR using the genomic DNA of H. pylori K51, was cloned in the pGEX-2T vector, and the DNA sequence analysis revealed that the hpaA gene of H. pylori K51 had 99.7% and 94.8% identity with individual hpaA genes of the H. pylori 26695 strain (U.K) and the J99 strain (U.S.A). A polyclonal anti-HpaA antibody was raised in rats using GST-HpaA fusion protein as the antigen. The hpaA gene was inserted in an E. coli-L. lactis-shuttle vector (pMG36e) to express in L. lactis. Western blot analysis showed that the expression level of HpaA in the L. lactis transformant remained constant from the exponential phase to the stationary phase, without extracelluar secretion. These results indicate that the HpaA of H. pylori K51 was successfully expressed in L. lactis, and suggest that the recombinant L. lactis expressing HpaA may be applicable as an oral vaccine to induce a protective immune response against H. pylori.

• Korean Journal of Poultry Science
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• v.27 no.2
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• pp.109-114
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• 2000

The objective of these experiments was to develop an attenuated thermostable Newcastle disease virus(NDV), CBP-1 strain isolated from infected pheasants. Safety, pathogenicity and protective effects against velogenic NDV were also investigated to evaluate if the attenuated NDV, CBP-1 strain could be a candidate for a new NDV vaccine strain. CBP-1 strain was passaged up to the 173 times by nine days old embryonated eggs and chicken embryo fibroblast(CEF) cell cultures. Its sensitivitly to lipid solvents and low pH, thermostability, mean death time(MDT), intracerebral pathogenicity index(ICPI) of one day old chicks and intravenous pathogenicity index(IVPI) of four weeks old chicks were examined. Safety, boosting and protective effects were tested by chicks mortality. CBP-1 NDV strain had significant thermostability at 56$\^{C}$ for 30 minutes. by hemagglutinin activity and egg infectivity test, but was not resistant to lipid solvent. It showed possibility to use as a feed or water vaccine because of the resistance to low pH. MDT, ICPI and IVPI of CBP-1 were attenuated from 51.5, 1.96, 2.60 to 112.4, 1.12, 1.45. These results implied that the 173rd passages in embryonated egg and CEF cell cultures induced a substantial attenuation of the pathogenicity of the parent virus, changing the virulence from velogenic to intermediate between mesogenic and lentogenic. After vaccination with CBP-1 at one day old by drinking water mortality was 17.5%. However, spray vaccination with B1 at one day old, CBP-1 at two weeks ild and challenge with velogenic Kyojeongwon strain at four weeks old showed 93.5% survival rate. Mortality of chicks, vaccination with 173rd passaged CBP-1 strain at one day old, two weeks old and challenge with Kyokeongwon strain at four weeks old, was 20.0%. The results of these studies indicated that partial attenuated CBP-1 strain tended to be a low safety for ND of broiler chicks and would need to be more successive attenuation.

• Journal of Microbiology
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• v.45 no.2
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• pp.179-184
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• 2007

Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.

• Korean Journal of Veterinary Research
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• v.58 no.3
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• pp.137-141
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• 2018

The efficacy of the CA-2-MP120 vaccine, a cell culture-attenuated strain of virulent porcine reproductive and respiratory syndrome virus (PRRSV), was assessed in pigs. Despite the persistence of viremia in all vaccinated animals during the immunization period, the virus was not detected in vaccinated pigs following challenge. Furthermore, no pigs in the vaccinated group shed PRRSV nasally, orally or rectally throughout the experiment. Moreover, histopathological lung and lymph node lesions in the immunized group were much milder than those in the unimmunized and challenged group. These results indicated that CA-2-MP120 can provide effective protection against virulent wild-type PRRSV-2.