Detection Of Klebsiella Pneumoniae And Streptococcus Suis Infections And Evaluation Of The Immune Efficacy Of The Combined Vaccine

Klebsiella pneumoniae(KP)is a conditional pathogen that often causes diseases such as pneumonia,bacteremia,and meningitis in humans.In the veterinary field,KP can cause diseases such as pneumonia,sepsis,and mastitis in pigs.In recent years,KP has attracted increasing attention due to its increased drug resistance and virulence.In veterinary clinical research,the main focus is on the high isolation rate and drug resistance of the bacteria in aquaculture environments.However,there are still few reports on the prevalence of the bacteria in pig populations,and there is currently no vaccine available to prevent KP infection in pigs.Streptococcus suis(SS)is an important zoonotic pathogen that can cause acute sepsis,meningitis,arthritis,pneumonia,and other diseases in pigs.Some strains can cause infection in humans,causing bacterial encephalitis or toxic shock syndrome,posing a serious threat to the aquaculture industry and public health safety.Although there are already vaccines to prevent SS related diseases,their effectiveness is limited.Currently,there are many cases of mixed bacterial infections in veterinary clinical practice,and both KP and SS infections are common in pig herds.Therefore,developing a dual vaccine for KP and SS is a better option.In order to understand the prevalence of KP in pig herds,the study established an indirect ELISA method for the detection of KP antibodies using recombinant khe protein as the coating protein,and established a nested PCR method for the detection of KP antibodies targeting the relatively conservative khe gene of KP.920 serum samples from pigs from Shandong and Inner Mongolia that were not immunized with KP vaccine were tested.The positive rate of ELISA was 27.28%(251/920),and the positive rate of nested PCR was 19.13%(176/920),indicating a high prevalence of KP in pig populations.The antibody positive rate is higher than the PCR positive rate,indicating that some pigs have been cleared of bacteria and DNA after infection.Subsequently,a correlation analysis was conducted between the prevalence of KP and human activities in the blood sample collection area,and it was found that the prevalence of KP in pig herds was positively correlated with local population density,GDP(gross domestic product),and the number of domestic tourists,indicating that human activities affected the flow of KP in pig herds.Nested PCR was then used to detect the co-infection of KP and SS,and it was found that the co-infection rate of the two bacteria was about 9.24%(85/920).In response to the high co-infection rate of the two bacteria,we extracted the capsular polysaccharide(CPS)from KP,tested its immune regulatory function on spleen lymphocytes and macrophages,and then mixed it with inactivated SS to prepare a KP-SS combined vaccine.In vitro studies have found that CPS of KP can promote the proliferation of mouse spleen cells in vitro,while at low concentrations it can promote the proliferation of spleen B cells and T cells,while at high concentrations it exhibits an inhibitory effect;After stimulation of mouse mononuclear macrophages(RAW)with CPS,the phagocytic ability of RAW to fluorescent microspheres and SS was enhanced;After adding CPS to the SS inactivated vaccine,compared to using the SS inactivated vaccine alone,the proportion of CD4~+T cells significantly increased,while the proportion of CD8~+T cells had no significant change.The immune mouse test confirmed that the CPS of KP has the potential of adjuvant,which can improve the antibody level induced by SS inactivated vaccine to a certain extent;CPS can also act as an antigen to induce the body to produce immune protective antibodies against KP.The KP-SS combined vaccine constructed using KP’s CPS and inactivated SS has a certain protective effect on both bacteria.Subsequently,it is necessary to optimize the antigen ratio and adjuvant selection.To sum up,this study clarified the prevalence of KP in pig herds and the co infection of KP and SS.A dual vaccine was prepared using a mixture of KP’s CPS and inactivated SS,and the antibody production level and immune protection effect of the dual vaccine were tested,providing a theoretical basis for the adjuvant application of KP CPS and the development of KP-SS combined vaccine.