Studies On Epidemiological Characteristics Of Chlamydiosis In Yak(Bos Grunniens)in Qinghai-Tibet Plateau And Subunit Vaccine Of Chlamydia Abortus

Chlamydia spp.is a large group of obligate intracellular,gram-negative bacterium with biphasic development cycle of elementary body and initial body.Chlamydia is widely distributed all over the world,with strong infectivity and pathogenicity,and is an important zoonotic infectious disease pathogen.In recent years,Chlamydia abortus has caused large-scale abortions in yaks in some areas of the Qinghai-Tibet Plateau,causing great losses to the local livestock industry.Most of the research on yak chlamydiosis is limited to serology,and there are very few data on its molecular epidemiology.Chlamydia abortus mainly infects placenta and causes inflammation of placenta,resulting in abortion.Vaccination is the key to prevent and control Chlamydia in yak.At present,inactivated Chlamydia abortus vaccines have been successfully developed locally and internationally,but the pathogen needs to be cultured in the yolk sac membrane of chicken embryos,and the extraction of the yolk sac membrane of chicken embryos has biosafety risks,and the vaccine production process is relatively backward,which is not suitable for modern vaccine production enterprises.So far there is no commercialized inactivated Chlamydia abortus vaccine.Therefore,the development of a safe and efficient Chlamydia abortus subunit vaccine has become a top priority.In the development of subunit vaccines,proteins with important biological functions and good immunogenicity are often selected as candidate antigens.OmcB,an adhesion molecule of Chlamydia trachomatis and Chlamydia pneumoniae,plays a key role in the infection of host cells.However,its role in Chlamydia abortus infection of host cells and whether it can be used as a candidate antigen for the preparation of subunit vaccines is unclear.This study was designed to first explore the epidemiological characteristics of chlamydiosis in yak in Qinghai-Tibet Plateau and subsequently,the role of OmcB in the process of Chlamydia abortus infection of host cells was clarified,and OmcB was further combined with MOMP,CPAF and inc A proteins with important biological functions in different developmental cycles to prepare subunit vaccines.Finally,the immune effect of the prepared subunit vaccine was evaluated by animal experiments.It is expected that the prepared subunit vaccine can provide materials for the prevention and control of yak chlamydiosis.The results are as follows.1.A total of 3352 samples from yaks were collected from Tibet,Qinghai,Gansu and Sichuan provinces（regions）of China,including 2724 rectal swabs and 628 vaginal swabs.Of these,836 rectal and 398 vaginal swabs were collected from Tibet,1086 rectal and 189vaginal swabs were from Qinghai,524 rectal and 70 vaginal swabs were from Gansu,and208 rectal and 41 vaginal swabs were collected from Sichuan.The results of fluorescence quantitative PCR showed that the total positive rate of Chlamydia infection in yaks in Qinghai-Tibet Plateau was 26.8%（899/3352）,of which the positive rate of Chlamydia infection in rectal swabs was 24.3%（644/2654）.The positive rate of Chlamydia in vaginal swabs was 36.5%（255/698）.The positive rate of Chlamydia varies greatly in different regions,among them,the highest positive rate was 44.9%（554/1234）in Tibet,19.8%（253/1275）in Qinghai,12.0%（71/594）in Gansu,and the lowest was recorded in Sichuan8.4%（21/249）.Chlamydia abortus,Chlamydia pecorum and Chlamydia psittaci were the main endemic species,with positive rates of 33.6%（302/899）,61.7%（555/899）and 17.0%（153/899）,respectively.Analysis of omp A gene showed that the prevalent strains of Chlamydia abortus were very conservative,while the prevalent strains of Chlamydia pecorum showed some genetic diversity.938 yak serum samples were collected from Tibet and tested by ELISA.The results showed that the positive rate of Chlamydia abortus antibody was 11.1%（104/938）.2.The expression vectors pET-30a-MOMP,pET-30a-OmcB,pET-30a-CPAF,pET-30a-inc A were successfully constructed,and 4 recombinant proteins with His label were induced to be purified.The purified recombinant proteins were mixed with adjuvant in a ratio of 1:1（v/v）and fully emulsified to obtain the desired subunit vaccine.The anti-OmcB polyclonal antibody was prepared by immunizing the New Zealand white rabbits with recombinant OmcB.Western blotting showed that the polyclonal antibody could specifically bind to Chlamydia abortus OmcB,indicating that the OmcB had immunogenicity.Chlamydia abortus OmcB is a surface exposed protein with adhesive effects by adhesion and infection inhibition assays.The OmcB XBBXBX motif deletion mutants OmcB_{1-310Δmotif1},OmcB_{1-310Δmotif2}and OmcB_{1-310Δmotif1,2} were successfully constructed by site-directed mutation.The first XBBXBX motif at the N-terminus of Chlamydia abortus OmcB protein was essential for adhesion to host cells.3.The mice were randomly divided into 6 groups,including PBS group（negative control group）,chicken embryo yolk sac membrane formaldehyde inactivated vaccine group（positive control group）,MOMP+OmcB group,CPAF group,inc A group and Mixture group（Mixture group of 4 recombinant proteins）.After immunization,the mice showed good appetite and mental state,and no adverse reactions were found at the inoculation site.All recombinant proteins could induce high level of specific antibodies in mice.Inactivated Chlamydia abortus may significantly stimulate the proliferation of splenocytes in the Mixture group（P<0.05）,and induce splenocytes to secrete Th1cytokines IFN-γand IL-2.Chlamydia abortus could be detected in the tissues and organs of mice in each group after challenge,mainly distributed in the uterus and placenta.Except for the inc A group,the load of Chlamydia abortus in the uterus and placenta of the other immunization groups was significantly lower than that in the PBS group（P<0.05）;histopathological examination results showed that a large number of inflammatory cells infiltrated in the uterus of mice,the placental tissue showed decidual cell necrosis,trophoblast strip necrosis,and multifocal necrosis of labyrinth layer in PBS group and inc A group.A small amount of inflammatory cell infiltration in the uterus of mice in CPAF group and MOMP+OmcB group,but no obvious pathological changes in the Mixture group.Birth rate of mice in Mixture group was 73%,and was higher than that other groups.4.According to the results of mice assay,the protective effect of 4 recombinant proteins（Mixture group）with the best protective effect was evaluated for the protective effect of sheep.There were 13 sheep in the immune challenge group,10 sheep in the non-immune challenge group and 11 sheep in the blank control group.The results showed that all pregnant sheep in immune challenge group had higher level of specific antibody;the body temperature of both the immune challenge group and the non-immune challenge group increased（≥40°C）on day 2 after challenge,and the average body temperature of the immune challenge group was significantly lower than that of the non-immune challenge group on day 4 after challenge（P<0.05）;the Chlamydia abortus load in vaginal swabs of sheep in the immunized challenge group was significantly lower than that in the non-immunized challenge group（P<0.01）;compared with non-immune challenge group,the pathological changes of placental tissue in immune challenge group were lighter;the abortion rate was 38.5%in the immune challenge group and 100%in the non-immune challenge group.This study confirmed that yaks were mainly infected with Chlamydia abortus,Chlamydia pecorum and Chlamydia psittaci in the Qinghai-Tibet Plateau of China.It was found that OmcB plays an important role in the infection of host cells by Chlamydia abortus,and the first XBBXBX motif at the N-terminus of OmcB is essential for adhesion to host cells.Subunit vaccines with different protein combinations were prepared.Among them,the subunit vaccine containing 4 recombinant proteins had better immune protection effect on pregnant mice than other groups,and the vaccine also had better immune protection effect on pregnant sheep.The results of this study lay a foundation for the prevention and control of yak chlamydiosis in the future.