| Mycoplasma suis (M.suis) is a kind of bacteria that attach to the surface of host erythrocytes. This pathogen can cause high mortality and mild chronic anemia, illthrift and infertility on acute and chronic cases, respectively.The disease caused by M.suis widely spread in the world, leading to serious economic loss in the swine industry. To date, few studies are focusing on the sera diagnostic methods and the assembly of ELISA Kit. So, it is significant to study the kit of ELISA for epidemiology investigation. The surface-localised protein MSG1, an important protein with good immunogenicity, can be chosed as a good candidate to establish ELISA assay.Bartonella henselae (B.henselae) is one of the most known pathogenic species in Bartonella species. It is the etiologic agent of cat scratch disease (SCD).17-kDa protein is one of few characterized proteins of B.henselae that induce antibody responses in hosts.In this study, the main contents of the research were as followings:1. Preparation and quality detection of each cotents of ELISA KitBasen on the blocking ELISA for Mycoplasma suis established by our laboratory, each cotents in ELISA Kit were prepared and quality controlled in this study. The concentration of three batches of protein were more than0.5mg/mL and SDS-PAGE and western blot showed that purified recombinant MSG1is highly purified and antigenicity is excellent. The antigen was fit with the quality standards and can be used for the kit. ELISA Plates, positive and negative control sera, HRP-1A7, TMB, washing solution, sample diluents and stop diluents were fit with characteristic traits, sterility and titer.2. Assembly and preservation of ELISA KitThe ELISA kit was assembled with key components treated by Protectant. The storage time of ELISA kit was primarily assessed by keeping the components and the ELISA kit at37℃for7days. Acceleration destruction test at37℃for7days showed that there was little variation in OD450value or PI value. The real-time and real-temperature test showd that the ELISA kit could be stable at4℃for at least six months.The above results showed that the stability of kit is good, which will laid an important data foundation for the development of the final ELISA kit.3. Seroepidemiology investigation on Mycoplasma suis infectionTotal of fourteen hundred and ninety two sera samples from7areas were detected by using blocking ELISA and three distribution in epidemiological survey was analyzed. The results showed that the overall positive rate of sera was17.36%and the highest and lowest positive rates were appeared in Shanghai (57.01%) and Guangxi (4.55%), respectively. The positive rate of Ahui、 Zhejiang、Jiangsu、Jiangxi and Sichuan were28.40%,20.25%,13.93%,7.14%and5.49%, respectively. The infection time began from June and July and amount to the highest (33.94%) in September, and then decreased slowly. The positive rate of sow and piglet were65.48%and4.8%, respectively. According to our results, M.suis infection was widespread in swine group and increase year by year.4.Development of an indirect ELISA of17-kDa protein of Bartonella henselaeThe17-kDa protein gene of Bartonella henselae was cloned into a prokaryotic expression vector (pET-28a) and constructed a recombinant plasmid pET-28a-17kDa. After, the construct was transformed into competent E.coli strain (BL21). The17-kDa protein was expressed with IPTG induction and purified with nickel-agarose by affinity chromatography. Moreover, an indirect ELISA assay was developed to detect anti-B. henselae antibody using the purified protein as coating antigen. The optimized reaction conditions were as follows:antigen protein was coated at37℃for2h with0.4μg/ml concentration and then overnight at4℃; the plates were blocked with1%BSA at37℃for3h; the serum sample was incubated at37℃for lh with1:100dilution and secondary antibody was incubated at37℃for0.5h with1:15000dilution. The cutoff values of positive and negative results were OD450nm≥0.392and OD45onm≤0.3326, respectively. The specificity results showed that the protein could not react with positive sera of other six diseases (PRRSV, PCV2, CSFV, PRV, FMDV, M.suis) and the repeatability of intra-and inter-assay were excellent. Furthermore, total of379clinical sera samples obtained from swine farms were detected and the positive rate was51.72%. In conclusion, the results showed that the indirect ELISA could be used for epidemiological surveys and diagnosis on Bartonella henselae.In summary, this study has developed an ELISA kit for the detection of M.suis antibodies. The ELISA kit could be stable at2-8℃for at least six months. It laid an important material foundation for the industrialization of ELISA kit. An indirect ELISA method to detect anti-B. henselae antibody was developed successfully, which helpful for epidemiological survey of B. henselae in swine group. Based on the epidemiological investigation on M.suis and B.henselae, we should pay more attention to the prevention and control of two diseases. |
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