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Toxicity detection of heat-labile enterotoxin in non-toxic mutant of Escherichia coli and investigation on its adjuvant effect of E.coli heat-labile enterotoxin

Xuefei BAI ; Jingyu GUO ; Wanjun LEI ; Guangcai DUAN

Chinese Journal of Zoonoses.2010;(1):69-71. doi:10.3969/j.issn.1002-2694.2010.01.017

In the present study, the expression, purification, toxicity detection and the adjuvant effect of the heat-labile enterotoxin in non-toxic mutant mLT63 of Escherichia coli were investigated, in which the inductive expression was performed under optimal condition for inductive expression and the toxicity of the products obtained from inductive expression were tested for toxicity after being purified and concentrated with affinity chromatography. BALB/c mice were immunized orally with the mutant mLT63 associated with Helicobacter pylori (Hp) subunit vaccine UreB, Omp11. After immunization, the specific antibody levels in serum, extract from gastric tissues and fecal extract were determind by means of ELISA assay and the results were subjected to statistical analysis. It was demonstrated that the mutant mLT63 of heat-labile entrotoxin of E.coli constructed in our laboratory devoided of any toxic effect as revealed by the rabbit ileal loop assay, but its adjuvant effect could be demonstrated in the associated immunization of mice with Hp subunit vaccine UreB and Omp11.

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Genetic analysis on the main virulence genes in the Fujian strain of Streptococcus suis type 2 from slaughtered pigs

Qinmei FANG ; Shaoqian HUANG ; Fusong YU ; Jichang ZHU ; Tianlong LIN

Chinese Journal of Zoonoses.2010;(1):65-68. doi:10.3969/j.issn.1002-2694.2010.01.016

To investigate the distribution of virulence associated factors of 5 strains of Streptococcus suis type 2 from slaughtered pigs isolated in Fujian province , 7 virulence genes were detected through PCR amplification. They were the glutamate dehydrogenase gene(gdh), the capsular polysaccharides gene(cps), the extracellular factor gene(ef), the muramidate-released protein gene(mrp), the suilysin gene(sly), the fibronectin-binding protein gene(fbps) and the virulence-associated sequence or f2. Among these 5 strains isolated, the virulence genotypes of 4 strains S.suis type 2 and strain SS2PFJ07 was gdh+/cps2J+/mrp+/ef-/sly-/fbps+/orf2+ and the other one strain isolated from slaughtered pigs was gdh+/cps2J+/mrp+/ef+/sly+/fbps+ /orf2+ respectively. This results showed that there are at least 2 virulence genotypes in Streptococcus suis type 2 from slaughtered pigs isolated in Fujian province.

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Sequence analyses of the Cps2J gene in 9 strains of Streptococcus suis serotype2 isolated in Zhejiang province

Pingping YAO ; Fusu WANG ; Hanping ZHU ; Lingling MEI ; Juyian YE ; Jin LUO ; Zheng ZHANG ; Chenhui YAO

Chinese Journal of Zoonoses.2010;(1):62-64. doi:10.3969/j.issn.1002-2694.2010.01.015

To explore the molecular characteristics of Streptococcus suis serotype 2(ss2) isolated in Zhejiang province by deciding the variation loci and its variation frequency of Cps2J gene.The total DNA of 9 strains of ss2 isolated in Zhejiang province were extracted and amplifed by PCR. Then,the Cps2J fragments were cloned into plasmid carrier and completely sequenced after purification.Finally,the sequence results of all 9 ss2 isolates were compared with those obtained by other studies around the world.It was found that the open reading fragments of Cps2J in 9 SS2 isolates encoding 333 amino acids were 999 bp in length.Comparisons of this region among ss2 isolates revealed a similarity of between 98.8% and 99.9%, while the homology to ss1 strains varied between 56.8% and 57.0%.Our study shows the sequences of complete Cps2J segment are fairly stable and all these 9 ss2 strains of different sources possibly have the same evolutionary origin.

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Effect of Toxoplasma gondii infection on the placental apoptosis-related protein of BALB/c mice during the second trimester of pregnancy

Huaxiang RAO ; Yuying HOU ; Huiping YUE ; Yanxia HE ; Yunhe ZHAO ; Junfeng YANG ; Hongye CHANG ; Hui LIU ; Jie ZHANG ; Liping HOU

Chinese Journal of Zoonoses.2010;(1):57-61. doi:10.3969/j.issn.1002-2694.2010.01.014

To observe the influence of the placental apoptosis on the expression of Bax,Bcl-2, Fas, FasLand TNF-α during the second trimester of pregnancy, mice of experimental group were intraperitoneal injected with 100 purified Toxoplasma gondii tachyzoites added in 0.2mL of PBS, while those of the control group were injected with 0.2 mL of sterile PBS (0.01 mol/L, pH 7.4) in the 8-th day of pregnancy. During the 12, 14, 16 and 18-th days of pregnancy, 5 mice both in experimental and control group were randomly killed and the expression levels of the apoptosis-related proteins Bax, Bcl-2, Fas, FasL and TNF-α in the placental tissues were determined by means of immunohistochemical methods. It was showed that the apoptosis-related protein expressed both in villus and decidua of the placenta, most of which were expressed in syneytiotrophoblast (ST). The positive cells with expression of Bax, Fas, FasL and TNF-α increased along with the increase of the pregnant days in both the experimental group and the control group, and the positive cells with expression of Bcl-2 decreased along with the increase of the pregnant days. It was also demonstrated that the positive cells with expression of Bax, Fas, FasL and TNF-α of the experimental group showed a higher percentage of expression than that of the control group on the same pregnant days, but the positive cells with Bcl-2 expression of the experimental group were fewer than that of the control group. It is concluded that the expression of apoptosis-related protein Bax, Bcl-2, Fas, FasL and TNF-α in the placenta were altered when the pregnant mice were infected with Toxoplasma gondii during the second trimester, which may induce the apoptosis through the endogenic and ectogenic pathway.

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Application of interferon-γ testing and the comparative cervical skin test in herds infected with Mycobacterium bovis

Xiyue ZHANG ; Xidan HU ; Jingwei YANG ; Rui CHAO ; Baoxu HUANG ; Weixing FAN

Chinese Journal of Zoonoses.2010;(1):53-56. doi:10.3969/j.issn.1002-2694.2010.01.013

The interferon-γ testing and the comparative cervical skin test were compared with the simple cervical hypersensitivity test routinely used in our country in herds infected with Mycobacterium bovis, in which 167 infected cattle from 5 herds were tested with interferon-γ testing and the comparative cervical test (CCT) simultaneously and other 106 cattle were tested with other hypersensitivity tests used as control for comparison. In these 167 cattle tested with both tests, 89 cattle were proved to be positive both in interferon-γ testing and CCT using the bovine PPD of Netherland with a coincidence rate of 92.7% (89/96). In 106 cattle the coincidence rate of positive reactions detected by γ-interferon test and CCT was 93.41% (78/83.5), that detected by the routine skin of foreign country was 62.26%(66/106); and that between the hypersensitivity test of China and foreign country was 92.19% (59/64). It is obvious that the interferon-γ testing and CCT seem to have higher coincidence rate and the specificity of the skin tests used in our country was rather low. Consequently, the use of the comparative cervical skin test should be considered to replace the routinely used shin tests.

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Identification and susceptibility test of bacteria isolated from miniature pigs in China

Zhengqin GAO ; Qiang ZHANG ; Zhengming HE ; Bingfei YUE ; Qiang YE

Chinese Journal of Zoonoses.2010;(1):46-52. doi:10.3969/j.issn.1002-2694.2010.01.012

To investigate the prevalence and the antibiotic resistance of bacteria isolated from 25 miniature pigs. 45 bacterial strains were isolated, which were identified by biochemical assays, amplification of 16S rRNA genes by PCR and sequence analysis, and were evaluated for resistance to 30 antibiotics. The identification results showed that these bacteria belonged to Campylobacter (Campylobacter jejuni), Helicobacterium (Helicobacter pylori), Klebsiella (Klebsiella pneumoniae), Escherichia (Escherichia coli, Escherichia fergusonii), Pseudomonas (Pseudomonas aeruginosa), Stenotrophomonas (Stenotrophomonas maltophilia), Staphylococcus (Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus simulans), Streptococcus (Streptococcus pneumoniae, Streptococcus suis, Streptococcus vestibularis, Streptococcus mitis, Gemella measles, Aerococcus viridans) and Bacillus (Bacillus subtilis, Bacillus licheniformis, Bacillus alvei, Bacterium megaterium). These bacteria were all susceptible to aztreonam and cephalothin. However, the resistence to furazolidone was found. Microbial population carried by miniature pigs in China had characters of diversity. Results of this study provided scientifical accordance for the microorganism monitoring of miniature pigs in China.

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A preliminary proteome analysis on the fluconazole-resistant strains of Penicillium marneffei

Donghua LIU ; Ming LIANG ; Hong LUO ; Shengshun TAN

Chinese Journal of Zoonoses.2010;(1):36-40. doi:10.3969/j.issn.1002-2694.2010.01.010

The correlated proteome of Penicillium marneffei resistant to fluconazole was investigated in the present study, in which 11 strains of P. marneffei of both the mycelial and yeast forms sensitive to fluconazole were cultivated in Sabouraud's liquid medium containing 8 g fluconazole and the MICs of fluoconazile to P.marneffei before and after inducing cultures were tested by E-test method. The strains of the mycelial and yeast forms showing the most significant increase in MIC value were selected and the protein clip CM10 was used to detect the proteome differences before and after inducing cultures. It was found that 11 strains of the mycelial forms and 2 strains of the yeast forms could tolerate the action of fluconazole and could grow at a drug concentration of 8 μg/mL. Furthermore, the MICs of fluoconazole to the mycelial forms were significantly increased after 7 days of inducing culture and the geometric means of MICs were increased from 1.22 μg /mL to 55.56 μg/mL. After the mycelial forms had been induced to be resistant, 16 proteins were highly expressed with specific expression of the 4581.1 Da and 6109.7 Da proteins; whereas 22 resistant yeast form were highly expressed with specific expression of the 3575.2 Da, 8507.0 Da and 8563.3 Da proteins. These results suggest that the mycelial form of P.narneffei seems to be more tolerant to the action of fluconazole than the yeast form. The resistance of these organisms to fluconazole may be associated to some specific proteins.

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Expression of Mb2277 of M. bovis and initial research on its reactogenicity

Fang LIU ; Jianguo ZHU ; Xiuguo HUA ; Qian GAO ; Yijia YAN

Chinese Journal of Zoonoses.2010;(1):33-35. doi:10.3969/j.issn.1002-2694.2010.01.009

To obtain fusion protein of Mycobacterium bovis with high purity, the recombinant prokaryotic expression vector for Mb2277 gene was constructed and the immunogenicity of its products was initially investigated in the present study.A pair of primer was designed according the gene sequence Mb2277 from the genomic DNA of M.Bovis in GenBank. and was amplified by PCR using DNA of M.Bovis 93006 strain as template. The PCR product and pET-28a(+) was then digested by BamHⅠ and EcoR Ⅰdouble enzyme. To constructed a prokaryotic expression plasmid, the purified Mb2277 was cloned to pET28a(+). Then the recombinant plasmid was transformed into competent cell of E.coli BL21(DE3).The bacteria were induced by IPTG and its lysates were analyzed by SDS-PAGE and Western-blotting. In this way, the prokaryotic expression plasmid for M. bovis Mb 2277 protin was obtained, and a expression band with molecular of 25 ku could be found in SDS-PAGE analysis. As demonstrated by Western blotting this expression product showed excellent reactivity with rabbit immune sera against M. bovis.

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Construction and expression of the eukaryotic expression vector containing the p55 gene fragment of rat Pneumocystis

Liangheng YI ; Yongwei QIN ; Jinling CHEN ; Dandan ZHU ; Xingxin HE ; Yinong DUAN

Chinese Journal of Zoonoses.2010;(1):25-28. doi:10.3969/j.issn.1002-2694.2010.01.007

To construct the eukaryotic expression plasmid containing the p55 gene fragment of Pneumocystis and to investigate the efficient expression in COS-7 cells, the gene fragment conaining the whole length of p55 gene was used as template to amplify this fragment with PCR and the amplified fragment was then cloned to vector pGEM-T. After enzyme digestion, p55 gene was cloned to the eukaryotic expression vector pcDNA3.1(+) to construct the plasmid pcDNA3.1(+)-582. This plasmid was then transfected to the eukaryotic expression cells COS-7 and PCR and SDS-PAGE assays were used to confirm the presence of target protein in these cells. In these ways, the eukaryotic expression vector for the p55 gene of Pneumocystis of rats was successfully constructed and expressed in COS-7 cells, thus providing the basis for further studies on the nucleic acid vaccine.

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Sequence determination and phylogenetic tree analysis of the E gene of dengue virus type 4 isolated from a patient in Shenzhen

Fan YANG ; Jianfan HE ; Huixia XIAN ; Yaqing HE ; Hailong ZHANG ; Xiangjie YAO ; Hong YANG

Chinese Journal of Zoonoses.2010;(1):17-20. doi:10.3969/j.issn.1002-2694.2010.01.005

To identify the genotype and analyze the molecular characteristics of dengue virus strain SZ0524 isolated from serum samples of patients with early stage of dengue fever in Shenzhen in 2005 so as to explore its possible origin. The C6/36 cell line was cultivated with virus strain SZ0524 and its suspension was harvested. The type of isolated virus strain was determined by RT-semi-nested PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of this dengue virus with the strains isolated from other areas were constructed. This SZ0524 strain was further identified by fluorescent PCR, and confirmed to be the type 4 virus after obtaining the 392bp band with type 4 specific primers. The homology of nucleotide sequence of E gene of SZ0524 strain with the standard type 4 dengue virus H241 strain were 99.7%, but the homology with the standard dengue virus 1,2,3 in the same fragment were 57.0%, 59.2% and 56.2% respectively. Analysis of the phylogenetic tree indicated that SZ0524 was more close to D4-73NIID and D4-61NIID strain, next to H241 strain, and they lied in the same branch of phylogenetic tree. The isolated dengue virus type 4 belonged to genotype Ⅰand the SZ0524 strain was proved to be dengue virus type 4 in the molecular level. Combined with epidemiology information, it is suggested that this case can be classified as an imported case and the SZ0524 strain may be transferred from the southeast asian region.

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