Molecular Epidemiology Of 9 Critical Bacterial Pathogens In Patients With Undefined Febrile Illness In Hainan And The Expression Of The Major Immune Response Antigens Of Orientia Tshtsugamushi

Objective:(1)To provide scientific bases for the diagnosis、treatment and the control of 9 clinical important bacteria,the epidemiology of these bacteria was evaluated in patients with undefined febrile illness in Hainan.(2)To test the feasibility of the major immune response antigens,47 k Da and 56 k Da of Orientia tshtsugamushi,as diagnostic target proteins for ELISA,and as candidates for protein subunit vaccine,the corresponding genes are cloned,expressed and tested.Methods:(1)Blood samples from patients with undefined febrile illness were collected in Hainan during 2018-2021(N=503)and screened for 9 important bacterial pathogens by real-time PCR method.The pathogens and target genes included Rickettsia sibirica(17 k Da gene),Orientia tshutshugamushi(47 k Da gene),Anaplasma phagocytophilum(msp2 gene),Coxiella burnetii(23S r RNA gene),Ehrlictsia chaffeensis(dsb gene),Chlamydia psittaci(Ch 23 S gene),Burkholderia pseudomallei(orf 2 gene),Borrelia burgdorferi(rec 2 gene)and Brucella spp(bcsp31 gene).Positive samples were further verified by Nest PCR and phylogenetic analyses of positive samples were performed based on Sanger sequencing of the Nest PCR product.(2)Truncated 56 k Da was amplified from scrub typhus patient blood DNA sample by high fidelity PCR and the truncated 47 k Da gene was synthesized.The fragments were cloned into prokaryotic expression vector PET-28a(+)and transformed into BL21(DE3)p Lys S,and then induced by IPTG and purified.Rabbits were immunized with the purified 47 k Da protein to generate anti-serum,96-well plates were then coated by the anti-serum to establish ELISA and WB method.Result:(1)Orientia tshutshugamushi and Anaplasma phagocytophilum were detected in the patients with undefined febrile in Hainan.The positive rate of scrub typhus was 14.51%(73 / 503).Scrub typhus was detected in 12 cities among which Qiongzhong County has the highest number of cases(N=25),and Changjiang county showed the highest detection rate(28.57%).The peak of the cases was found in August to November,and the highest incidence population was between 50 and 70 years old and the genotypes included Karp and Gillam.The positive rate of anaplasmosis was 5.57%(28 / 503).Anaplasmosis was detected in 6 cities among which Haikou city has the highest number of cases(N=9),and Changjiang County showed the highest detection rate(14.29%).The peak of the cases was found from July to September.Co-infection of Scrub typhus and anaplasmosis was first detected from patients in Hainan,accounting for 1.59%(8 / 503).(2)Recombinant plasmids for 47 k Da and 56 k Da protein gene were successfully constructed and the proteins were expressed.However,only the 47 k Da protein can be purified for rabbit immunization.Rabbit anti-46 k Da serum was harvested with a high titer up to1:640,000,indicating the high immunogenicity of the expressed 47 k Da protein.Unfortunately,the 46 k Da protein can not be recognized by antibodies in the serum of patients from Hainan and the 56 k Da protein failed to be largely purified.Conclusions:(1)The results showed that Orientia tshutshugamushi is the dominant pathogen in the 9 important bacterial pathogens with undefined febrile illness in Hainan.Clinical infection of Orientia tsutsugamushi in Hainan showed genotypic diversity.The second is Anaplasma phagocytophilum.Scrub typhus and phagocytic anaplasmosis are widely distributed in Hainan.The populations with the highest prevalence are farmers and students,health testing for these populations should be strengthened.Similar peak time of scrub typhus and anaplasmosis,specially the discovery of co-infection cases implied a shared vector for the diseases.Unlike previous reports with high positive rate,rickettsiae was not detected in this study,which indicates the shift in pathogenic species of rickettsia diseases in Hainan.(2)In this study,the 47 k Da and 56 k Da protein of Orientia tshtsugamushi can be expressed in BL21(DE3)p Lys S.However,56 k Da protein was not largely purified due to the low expression.The 47 k Da recombinant protein possesses a high immunogenicity in rabbits.However,47 k Da is not sufficient as target protein for the diagnosis of scrub typhus.