Establishment Of Colloidal Gold Strip Test Method For Clostridium Septicum And Preparation And Application Of Toxoid Vaccine

Braxy is an acute,lethal infectious disease caused by Clostridium septicum.C.septicum is a Gram-positive bacterium that infects humans and livestock and causes malignant edema,so it was once known as malignant edema.Braxy is characterized by acute onset,rapid death,and no obvious symptoms at the initial stage of infection,which causes serious harm to human health and animal husbandry production.Therefore,it is urgent to establish a rapid diagnostic method for C.septicum diseases such as braxy,so that it can be used in grass-roots production,so as to achieve timely and effective control.At the same time,vaccine immunization is an effective preventive measure against C.septicum diseases.It is very important to develop a more effective vaccine for the prevention and control of this disease.Therefore,according to the growth and propagation characteristics of Clostridium putrefaciens,a new culture method of C.septicum was invented.Based on this,polyclonal antibody against C.septicum was prepared,colloidal gold test strip detection method was established and C.septicum toxoid vaccine was prepared,which provided technical support for diagnosis and control of braxy.The research content can be divided into the following three parts:1.Establishment of a new culture method for C.septicumAccording to the needs of growth and culture of C.septicum,the culture medium was designed,the reference strain of C.septicum?ATCC 12464?was inoculated,and the culture was carried out in different gas environment.The OD_600nm00nm determination of the proliferation of C.septicum at different times was performed.Finally,the composition and proportion of the medium were determined,and it was found that the OD_600nm00nm value of the culture of C.septicum reached the highest when the pH was 7.8 under the condition of a certain mixture of gases.This study showed that the OD_600nm00nm of C.septicum in the self-made medium could reach 1.981,but it was only 1.055 in the commonly used anaerobic broth medium,and the effect of increasing bacteria is obviously improved.In addition,this study explores that C.septicum has a better ability to produce toxins at a pH of 7.8.2.Establishment of detection method for C.septicum colloidal gold stripAfter resuscitation and enrichment culture of the reference strain of C.septicum?ATCC12464?,0.4%formaldehyde was used to inactivate the concentrated bacterial cells to obtain the antigen.The protein was analyzed by Sodium dodecyl silfate-Polyacrylamide gel electrophoresis?SDS-PAGE?.The antigen was mixed with Freund's adjuvant,and Japanese white rabbits were immunized 4 times to obtain a polyclonal antibody.The antibody titer can reach 1:1024000,showing good specificity.The optimal labeling amount of the purified antibody was 10?g/mL,the optimal labeling pH was determined to be 8.0,and the optimal working concentration of the colloidal gold-antibody conjugate stock solution was 1:4.The detection of the C.septicum colloidal gold test strip showed that the bacterial solution was still positive after being diluted 6 times.The sera of C.septicum infection in guinea pigs were positive,while the sera of Clostridium novyi infection in guinea pigs were negative,showing good sensitivity and specificity.3.Preparation and application of C.septicum toxin vaccineThe reference strain?ATCC 12464?of C.septicum was used to recover the bacteria,enrichment,and produce toxins.The exotoxin was crudely extracted and inactivated to form a toxoid,and then thoroughly mixed with a white oil adjuvant to prepare a C.septicum toxoid vaccine.After 21 days of immunizing mice with this vaccine,the antibody titer of C.septicum toxin in the serum reaches 1:6400,and they were able to resist the challenge of 1 mouse MLD.The sheep were immunized with different doses,and the results showed that the immune effect of the 3.5mL group was the best,and the antibody titer of C.septicum toxin reached the highest in the serum on the 21st day after immunization,and maintained to the 6th week,which indicated that the first immunization lasted for a long time.After 21 days of immunization with this dose,the neutralization titer of serum mice reached 1:600.In this study,a new culture method of C.septicum was established,and based on this,a colloidal gold detection method for C.septicum and a toxin vaccine for C.septicum were established.After detection,the colloidal gold detection method of C.septicum has good specificity and sensitivity,which can provide a basis for the clinical diagnosis of braxy.The C.septicum toxoid vaccine has a high effective antigen content,ideal immune effect,high antibody titer and long-lasting time.This study provides a new technical method for the cultivation of C.septicum,and provides a theoretical basis and technical support for the diagnosis,prevention and control of braxy.