| Swine Dysentery is a more serious intestinal infectious disease, caused by Brachyspira hyodysenteriae. It causes a severe mucohaemorrhagic diarrhea, most of the pathogenic sites are in the colon,in some severe cases could cause the death of pig. Brachyspira hyodysenteriae with short spirochetes serpentine shape in both end, about6～8.5μm length and a diameter of320-380nm, have4-6sparse spiro curves. It is a gram-negative and anaerobic bacteria, has a harsh growth condition, so it is not easy to separate. This bacterria is one of important pathogen of pig gut syndrome, is widespread especially in the developed coutrieds. It mainly cause slow growth, low feed utilization and a high cost, causing serious economic loss. Compared to other coutrties, our domestic researth is little, so it is necessary to conduct epidemiological investigation and related characteristics. The following three aspects is a brief summary of this study.1TaqMan-MGB probe real time fluorescence quantitative PCR for rapid detection of brachyspira hyodysenteriaeIn order to increase the positive detection rate of brachyspira hyodysenteriae, a pair of primers and a probe were designed according for conserved sequence nox. After optimization the methods conditions, a TaqMan-MGB probe real time fluorescence quantitative PCR method was established. This method had specificity, sensitivity and good reproducibility. Using the recombinant plasmid to establish a standard curve, the linear correlation coefficient was0.99. This method had a good linear relationship, which could detect a minimum of2.17copis, as well as with no cross reactivity compared to other bacteria.107samples DNA had been detected using TaqMan-MGB probe real time fluorescence quantitative PCR, the results showed that this method better than the ordinary PCR. Furthmore, as compared to the ordinary method, this method also costed much less time and could get a accurate quantification, which can be used for detecting brachyspira hyodysenteriae of pig farms and epidemiological investigation. 2The infection survey of Swine dysentery in Shanghai and surrounding areaIn order to know the infection and prevalence of this disease in different farms. During octber2010to December2012, collecting624samples were detected. These samples were the colon, stool and rectal swab form Shanghai animal disease prevetion and control center clinic, large-scale farms of Shanghai, Zhejiang and Henna province, also with five slaughterhouses in Shanghai. These samples were detected by TaqMan-MGB probe real time fluorescence quantitative PCR. The results showed that different symptoms, different week-old samples, the positive rate had different. The average of positive rate was8.49%.The positive rate of diarrhea was15%and none diarrhea was1.93%. Among2-5weeks of age,6-9weeks of age,10-13weeks of age,14-17weeks of age and18-19weeks of age, positive rates were respectively14%,22%,32%,13%and8.2%, ortherwise the slaughterhouses’s detection was low. Piglet and finisher in herds have a higher infection. Through anaerobic culture, a Brachyspira hyodysenteriae(SH-BH-1) was successfully isolated. Swine dysentery infection investigation in Shanghai and surrouding areas, which could be helpful to prevent and control this disease in the future.3Expression, purification and characterization of BIpA with immunogenicity analysisThough analyzing the sequence of outer membrane protien BlpA of brachyspira hyodysenteriae in NCBI, the primers were desighed. Using genetic engneering technique, the target fragment was cloned into expression plasmid vector pET-28a(+), then recombinant plasmids was identified by PCR identification, restriction endonuclease and sequencing. The recombinant was transformed into BL21(ED3)plysS and induced by IPTG. After SDS-PAGE identification get a29.6kDa expectable protein. The purified protein immunized the mices for6weeks. Though elisa detection, it shows that the recombinant protein can induce high lever of specific IgG antibodies, may be used as a potential vaccine. |
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