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Safety and Immunogenicity of Salmonella enterica Serovar Typhimurium llaB in Mice

  • CHO SUN-A;LEE IN-SOO;PARK JONG-HWAN;SEOK SEUNG-HYEOK;LEE HUI-YOUNG;KIM DONG-JAE;BACK MIN-WON;LEE SEOK-HO;HUR SOOK-JIN;BAN SANG-JA;LEE YOO-KYOUNG;PARK JAE-HAK
    • Journal of Microbiology and Biotechnology
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    • v.15 no.3
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    • pp.609-615
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    • 2005
  • The safety and immunogenicity of an attenuated recombinant Salmonella vaccine strain, Salmonella enterica serovar Typhimurium llaB, was assessed. This vaccine strain could survive in low pH condition, and its ability of intracellular survival did not differ from that of S. enterica serovar Typhimurium UK1, which is the wild-type of the vaccine strain. The mortality of the mice orally administered with the vaccine strain was $50\%$ at the dose of $10^7$ CFU. All mice administered with $10^5\;or\;10^3$ CFU of the vaccine strain survived for 3 days postinoculation (pi). However, all mice administered with more than $10^3$ CFU of the vaccine strain died within 3 days pi. To examine the protective effect of the vaccine strain, mice were orally immunized with $10^4\;and\;10^6$ CFU of the bacteria. Control mice were given with 0.5 ml of phosphate buffered saline (PBS). After 8 days, the mice were challenged with $10^9$ CFU of S. enterica serovar Typhimurium UK1, and mortality was examined for 5 days. The survival rates of the mice immunized with $10^4\;and\;10^6$ CFU of the vaccine strain were $60\%\;and\;80\%$, respectively, whereas all control mice died within 2 days after challenging. To investigate the immunogenicity of S. enterica serovar Typhimurium llaB, mice were orally immunized with $10^5\;or\;10^6$ CFU ml of the vaccine strain. Five mice of each group were sacrificed at 5 and 12 days after immunization, and results showed that immunization of the vaccine strain led to increases of IgG1, IgG2, and IgM titers against S. enterica serovar Typhimurium UK1 in mouse sera, cytokine expressions such as IL-2, IL-4, IL-6, and IL-10 in spleen, and the lymphocyte proliferation response to mitogens (concanavalin A or LPS) stimulation.

Comparison of tissue tropism of Newcastle disease vaccine viruses by Immunohistochemistry techniques (면역조직화학기법을 이용한 뉴캣슬병 백신바이러스의 조직친화성 비교)

  • Kim, Min-Jeong;Kwon, Yong-Kuk;Seong, Hwan-Woo;Kang, Shien-Young;Mo, In-Pil
    • Korean Journal of Veterinary Research
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    • v.44 no.4
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    • pp.539-549
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    • 2004
  • Mean death time of inoculated embryonated egg is one of the methods to determine the virulence of the Newcastle disease viruses (NDV). Evaluation of tissue tropism of NDV in the host has been proposed as an another way to determine the pathogenicity of NDV based on the principal site of viral replication. To evaluate the tissue tropism among NDV, an immunohistochemistry(IHC) technique using monoclonal antibody was applied in one-day-old SPF chickens inoculated with different ND vaccine strains such as Ulster 2C, VG/GA and B1 viruses by eye drop instillation. The tissues used for this comparison were trachea, intestine, Harderian gland and cecal tonsil, which were collected at 0.5, 1, 2, 3, 5, 8, 10, 14 days post inoculation. Among test groups, chickens inoculated with B1 viurs, which is known to replicate in the respiratory system, have IHC positive staining mainly in the trachea and those inoculated with Ulster 2C have IHC positive staining mainly in the intestine. However, chickens inoculated with VG/GA strain have IHC positive staining in both the trachea and intestine. Therefore, a differences in tissue tropism among ND vaccine strains has been proved by the IHC technique. Based on this results, the IHC staining technique could be used to classify the NDV or to study the pathogenesis of NDV in chickens.

Efficacy of Brucella abortus strain RB51 vaccine in Korean mongrel dogs against virulent strains of B. abortus biotype 1 and B. canis

  • Hur, Jin;Baek, Byeong-Kirl
    • Korean Journal of Veterinary Service
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    • v.33 no.1
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    • pp.29-35
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    • 2010
  • This study was performed to test the hypothesis that Brucella abortus strain RB51 (SRB51) might protect Korean indigenous mongrel dog against challenge with either virulent B. abortus biotype 1 or B. canis. A total of 12 Korean mongrel dogs were divided into four groups (Group A, B, C and D). Dogs belonging to Group A and C were inoculated subcutaneously with $1{\times}10^9$ CFU of SRB51 in 1ml of sterile phosphate buffered saline (PBS). Dogs of Group B and D were inoculated subcutaneously with 1ml of sterile PBS as control. At 12 weeks post vaccination, dogs of Group A and B were challenged by oral inoculation of virulent strain of B. canis ($5.0{\times}10^9$ CFU) and dogs of Group C and D were challenged by oral inoculation of virulent strain of B. abortus biotype 1 ($4.4{\times}10^{10}$ CFU). The serum antibodies titers in all dogs were monitored at regular interval for eight weeks after challenge (AC) by standard tube agglutination test, plate agglutination test, rose bengal test, 2-mercaptoethanol rapid slide agglutination test and 2-mercaptoethanol tube agglutination test. No antibody titers in Group A and C was detected. Also, the challenge strains were not found from blood of all dogs of Group A and C from 1 week AC till the end of the experiment by culture and modified AMOS-PCR, whereas B. canis and B. abortus challenge strains were detected from blood of Group B and D, respectively. In addition, neither of two challenge bacteria was recovered from liver, spleen, kidneys, lymph nodes and reproductive tracts of Group A and C dogs after postmortem. However, B. canis and B. abortus challenge strains were isolated from these tissues of Group B and D, respectively. These data suggest that SRB51 could be a promising vaccine candidate for immunizing dogs to control canine brucellosis caused by B. canis or B. abortus.

Studies on the brucellosis in Kyongbuk area (경북지역의 부루셀라병에 관한 연구)

  • 박노찬;김상윤;조광현;도재철;김영환;신상희;조민희;오강희;김우현
    • 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.

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Sequence analysis of the hypervariable region in VP2 gene of infectious bursal disease vaccine strains (Infectious bursal disease 백신주의 VP2 gene의 hypervariable region 분석)

  • Park, Yoo-jin;Kim, Soo-joung;Kwon, Hyuk-moo
    • Korean Journal of Veterinary Research
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    • v.41 no.3
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    • pp.333-342
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    • 2001
  • To detect the genetic variations among infectious bursal disease (IBD) vaccine strains, the hypervariable region of VP2 gene of seven IBDV vaccine strains were amplified using reverse transcriptase/polymerase chain reation(RT/PCR). Ampllified PCR products of IBDV were cloned, sequenced, and compared with published sequences for IBDV. Vaccine strains (JOONG, HAN, B7, IB, BU2, G2, CIL) used in Korea and Korean field isolates (SH/92, K1, 310) had 81%(310 and HAN) ~ 98%(SH/92 and CIL) amino acid sequence similarity. Vaccine strains had 80%(HAN and IB) ~ 99%(JOONG and BU2) amino acid sequence similartiy. Intermediate plus vaccine strain, CIL was not substituted at positions 279(D $\rightarrow$ N) and 284(A $\rightarrow$ T), and conserved in serine-rich heptapeptide. At the two hydrophilic region, JOONG, IB and Bu2 strains had identical amino acid sequence comparing with STC strain. By phylogenetic analysis, JOONG and DAE strains were categorized in same group with BU2. The CIL and STC strains closely related but seperated from G2, HAN, B7 and IB strains.

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Brucella melitensis omp31 Mutant Is Attenuated and Confers Protection Against Virulent Brucella melitensis Challenge in BALB/c Mice

  • Verdiguel-Fernandez, L;Oropeza-Navarro, R;Ortiz, Adolfo;Robles-Pesina, MG;Ramirez-Lezama, J;Castaneda-Ramirez, A;Verdugo-Rodriguez, A
    • Journal of Microbiology and Biotechnology
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    • v.30 no.4
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    • pp.497-504
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    • 2020
  • For control of brucellosis in small ruminants, attenuated B. melitensis Rev1 is used but it can be virulent for animals and human. Based on these aspects, it is essential to identify potential immunogens to avoid these problems in prevention of brucellosis. The majority of OMPs in the Omp25/31 family have been studied because these proteins are relevant in maintaining the integrity of the outer membrane but their implication in the virulence of the different species of this genus is not clearly described. Therefore, in this work we studied the role of Omp31 on virulence by determining the residual virulence and detecting lesions in spleen and testis of mice inoculated with the B. melitensis LVM31 mutant strain. In addition, we evaluated the conferred protection in mice immunized with the mutant strain against the challenge with the B. melitensis Bm133 virulent strain. Our results showed that the mutation of omp31 caused a decrease in splenic colonization without generating apparent lesions or histopathological changes apparent in both organs in comparison with the control strains and that the mutant strain conferred similar protection as the B. melitensis Rev1 vaccine strain against the challenge with B. melitensis Bm133 virulent strain. These results allow us to conclude that Omp31 plays an important role on the virulence of B. melitensis in the murine model, and due to the attenuation shown by the strain, it could be considered a vaccine candidate for the prevention of goat brucellosis.

Studies on Inactivated Combined Vaccine of Bovine Anthrax and Blackleg I. Preparation of Vaccine and Its Evaluation in Guinea Pigs (소의 탄저기종저 불활화 혼합백신에 관한 연구 I. 백신 제조와 기니픽에서의 효과시험)

  • Jeon, Yun Seong
    • Korean Journal of Veterinary Research
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    • v.12 no.1
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    • pp.71-75
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    • 1972
  • Due to the fact that an inactivated anthrax vaccine may show no or lower immunogenicity and stability, a number of spore vaccines were exclusively used worldwide. In these studies non or less allergic strain of anthrax bacillus was selected and made a capsulated vegetative organisms. Anthrax organisms of a virulent strain were cultivated on sodium bicarbonate medium with or without adding I-alanine in which B. anthracis grew luxuriantly without forming spores. Inactivation of the organisims was carried out at $37^{\circ}C$ water bath for 3 days after the bacterial culture was mixed with formalin in a final concentration of two per cent. Aluminum hydroxide gel was added to the mixture of anthrax and blackleg bacterin. Guinea pigs were injected with the vaccine via subcutaneous or intramuscular route and challenged after three weeks, and the possibilities of protection was tested. Throughout the studies, the vaccines possibly protected the vaccinated guinea pigs more than 80 per cent compared to that of the controls. This experimental results strongly suggest that the vaccine may possibly applicable to the prevention of bovine anthrax and blackleg.

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Attenuation and Protective Effects of a Thermostable Newcastle Disease Virus Isolated from Korean Pheasants (한국산 꿩으로부터 분리한 열 안정성 뉴캣슬병 바이러스의 순화와 방어효과)

  • 한수철;곽길한;김태중;장경수;전무형;송희종
    • 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.

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Studies on the immunization against field strain after live Newcastle disease virus vaccination (뉴캣슬병 생독백신 접종 후 야외 분리 바이러스에 대한 면역성 조사)

  • 김순태;박인화;김성국;김영환;조광현;손재권
    • Korean Journal of Veterinary Service
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    • v.24 no.2
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    • pp.147-159
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    • 2001
  • This Study was conducted to determine vaccination programs for the control of Newcastle Disease(ND) in chickens and investigate protective effect against Newcastle disease virus (NDV) after live ND vaccination. Maternal HI antibody titer level of chickens according to day(age) 1, 7, 14, 21, 28 and 35 were decreased gradually as 7.10$\pm$0.74, 6.57$\pm$0.74, 3.71$\pm$1.25, 2.20$\pm$1.03, 1.20$\pm$1.23 and 0.50$\pm$0.71. As a result of HI test and ELISA, both chickens vaccinated with VG/GA strain live vaccine at 1-day-old and chickens not vaccinated do not have antibody titer for protection against NDV at 14-day-old. Except for LaSota strain vaccine, in case of vaccination with VG/GA spray and VG/GA, B1 and LaSota strain drinking water at 14-day-old, the protective effect was 100% in chickens inoculated NDV($10^{7.2}$ $EID_{50}$/50${\mu}\ell$, eye drop) at 21-day-old, but not 10~50% at 28-day-old. These data suggest that live NDV vaccination should be given at 10-day-old 20-25day-old for protect against NDV at periodic outbreaks of ND caused by velogenic viscerotropic NDV in the environment of a farm.

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Improved Purification Process for Cholera Toxin and its Application to the Quantification of Residual Toxin in Cholera Vaccines

  • Jang, Hyun;Kim, Hyo-Seung;Kim, Jeong-Ah;Seo, Jin-Ho;Carbis, Rodney
    • Journal of Microbiology and Biotechnology
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    • v.19 no.1
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    • pp.108-112
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    • 2009
  • A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-${\mu}m$ crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-${\mu}m$ permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH 7.0, containing 1.0 M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of $3.1\;EU/{\mu}g$ of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a $G_{M1}$ ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The $G_{M1}$ ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.