Objective To clone Rhipicephalus linnaei Boophilin-H2 gene, construct the recombinant expression vector, express the Boophilin-H2 recombinant protein in Escherichia coli, and assess its anticoagulant activity. Methods Specific primers were designed to amplify the Boophilin-H2 gene fragment using cDNA, synthesized from engorged Rhipicephalus linnaei tick RNA through reverse transcription, as a template. The gene fragment was cloned and connected to plasmid pSmart-I, and the recombinant expression vector pSmart-I/Boophilin-H2 was constructed. The recombinant expression vector was verified by double restriction enzyme digestion with BamHⅠand XhoⅠ, transferred into the competent state of Escherichia coli BL21(DE3, and expressed under low-temperature induction with IPTG. The recombinant protein was purified by Ni-NTA Resin, and its expression and purification were detected by 12.5% SDS-PAGE. The femoral venous blood of New Zealand white rabbits was collected by 3.8% sodium citrate blood collection tube, and the upper plasma was centrifugally separated to measure the anticoagulant activity of the recombinant protein using four test plates of in vitro coagulation. Results A 387 bp gene fragment of Boophilin-H2 of Rhipicephalus linnaei was successfully amplified and cloned; the prokaryotic expression vector pSmart-I/Boophilin-H2 was constructed and verified by double enzyme digestion. Following induction with 0.8 mmol/L IPTG for 16 hours in Escherichia coli, SDS-PAGE showed that the recombinant protein was expressed in the supernatant primarily in a soluble form, with the Boophilin-H2 recombinant protein approximately 35 000 in size. The anticoagulant activity assays of the purified recombinant protein Boophilin-H2 showed that the recombinant protein significantly prolongs the activated partial thromboplastin time (APTT) in a concentration-dependent manner, while its effects on thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB) levels were not significant. Conclusions The expression vector for Boophilin-H2 was successfully constructed, and its product exhibited potent APTT anticoagulant activity, implying its role in the intrinsic coagulation pathway, possibly acting upon intrinsic coagulation factors VIII, XI, and XII to inhibit blood coagulation. This study provides a reference and theoretical foundation for further research and development of tick control vaccines and anticoagulant drugs.

Abstract: Objective To understand the morphological characteristics and ultrastructure of the dominant species of Rhipicephalus sanguineus in Hainan at different developmental stages, and provide theoretical basis for the identification of the lineage and control of Rhipicephalus sanguineus. Methods The external morphology of different developmental stages of the dominant species of Rhipicephalus sanguineus, including larva, nymph and adult tick in Hainan were observed by scanning electron microscope. Results The division between each segment of larva pedipalps was not obvious, and setae was serrated; dental formula type 2 | 2; 3 pairs of podomere; a pair of setae on the anal valve; none of anal groove, spiracular plate, porous area and genital aperture. There was a clear boundary at the beginning of each segment of nymph pedipalps; dental formula type 2 | 2; 4 pairs of podomere; 3 pairs of setae on the anal valve; anal groove; none of porous area and genital aperture. The male adult tick's trichotheca are covered by the pedipalps, and the whole bristles are conical; dental formula type 3 | 3; 4 pairs of podomere; anal groove and paraprocts; 7 setae on the anal valve; genital aperture was oval. The female of adult tick can be distinguished by dental formula 3 | 3; pairs of podomere; porous areas with 3 short setae; anal groove; 4 pairs of setae and 2 pores on the anal valve; genital pore was broadly U-shaped. In addition, the male adult's scutum occupies almost the entire dorsal surface, the basis capituli of larva, nymph and adult tick all were hexagonal, and the existence of Haller's organ was found on the first pair of legs. Conclusions Scanning electron microscopy observation of the different developmental stages of R.sanguineus revealed clear morphological features, preliminarily suggesting that R.sanguineus in Hainan Province may belong to the tropical lineage, which provide a certain experimental basis for the identification of the tick and the comprehensive prevention and control of local tick-borne diseases.

Objective:To evaluate the effectiveness of HIV/syphilis joint self-testing in promoting syphilis testing among men who have sex with men (MSM).Methods:In July 2019, the research participants were recruited through the Danlan website (https://www.danlan.org). Participants who met the selection criteria, and were randomly assigned into one of the three study groups (1∶1∶1) including HIV/syphilis joint self-testing group and lottery incentive self-testing group and control group. Self-test reagents were mailed to HIV/syphilis joint self-testing group and lottery incentive self-testing group, and the subjects in control group were encouraged to go to offline locations for testing. One month later, follow-up was conducted to evaluate the differences in the testing rates of syphilis among the three groups.Results:A total of 145 subjects were included in this study, including 48 in control group, 49 in HIV/syphilis joint self-testing group and 48 in lottery incentive self-testing group. During the follow-up period, the self-testing rate of syphilis was 74.4% (32/43) in HIV/syphilis joint self-testing group, 70.0% (28/40) in lottery incentive self-testing group and 36.4% (16/44) in control group. Multivariate logistic analysis revealed that the proportions of syphilis testing in HIV/syphilis joint self-testing group and lottery incentive self-testing group were 5.38 (95% CI: 2.06-14.04) times and 4.54 (95% CI: 1.75-11.74) times higher than that in control group during the follow-up period. Conclusions:HIV/syphilis joint self-testing and lottery-incentives-prompted self-testing significantly increased the testing rate of syphilis in MSM, respectively. HIV/syphilis joint self-testing is feasible for promotion.

Objective:To explore the spatiotemporal distribution and macro-related factors of congenital syphilis in Guangdong province and provide suggestions and recommendations for prevention.Methods:Yearly reported cases of syphilis and some influencing factor data of Guangdong province were collected from 2005 to 2017. The spatiotemporal distribution of congenital syphilis was described. Meanwhile, the spatial panel data model was constructed to analyze the relationship between the incidence rates of congenital syphilis and related factors.Results:From 2005 to 2017, 13 361 cases of congenital syphilis were reported in Guangdong province. The number of congenital syphilis cases rose to its highest point during 2005-2011. A slow downward trend followed. The peaks of incidence were observed from August to December. The incidence of the non-Pearl River Delta region has experienced a process of rising first and then decreasing. The spatial panel data model results showed that congenital syphilis had significant positive spatial autocorrelation ( P<0.001). The incidence of primary and secondary syphilis in women ( β=0.822, P<0.001), gross domestic product per capita ( β=3.511, P<0.001), net migrate rate ( β=0.215, P=0.047) and maternal system management rate ?(β=0.017, P=0.021) were all positively correlated with the incidence rates of congenital syphilis. Registered population density ( β=-1.167, P<0.001) and prenatal examination rate ( β=-0.038, P=0.031) was negatively correlated with congenital syphilis. Conclusions:The incidence of congenital syphilis was spatially aggregated in Guangdong province from 2005 to 2017. The intensity of prevention might be strengthened in cities with developed economies and high net migration rates, which have high risks of congenital syphilis. Controlling the incidence of primary and secondary syphilis in women and increasing the prenatal examination rate for pregnant women appears effective prevention measures of congenital syphilis.

Objective To analyze the operation of water improvement projects in drinking water-borne fluorosis areas of Qinghai Province and the trend of dental fluorosis in children aged 8 to 12 years, and to evaluate the effects of prevention and control measures comprehensively, so as to provide basis for improving prevention and control strategies. Methods In 2009 - 2017, according to the "Qinghai Drinking Water Endemic Fluorosis Surveillance Program", four project counties of Huangyuan, Minhe, Hualong and Guide were selected in Qinghai Province. Three villages in each project county were selected according to their mild, moderate and severe conditions, and monitored the operation of water improvement projects, water fluoride content and dental fluorosis among children aged 8 to 12-year-old in each diseased village. Results In 2009-2017, the normal operation rate of water improvement projects increased from 2/8 to 7/8; the batch operation rate of the projects decreased from 5/8 to 1/8; the number of abandoned projects decreased from 1 to 0; the number of people covered by qualified water fluoride increased from 9962 to 80760 people; there was no significant difference in water fluoride content between different years (F = 0.758, P > 0.05). The total detection rate of children's dental fluorosis was 33.01％(1812/5489) from 2009 to 2017, the index of dental fluorosis was 0.65, and the epidemic intensity was slightly prevalent;the detection rate of children's dental fluorosis was 30.02％(951/3168) in villages with normal operation of water improvement projects and qualified water fluoride content, the index of dental fluorosis was 0.58, and the epidemic intensity was marginal;the detection rate of children's dental fluorosis was 32.72％(583/1782) in villages with abnormal operation of water improvement projects or excessive fluoride, the index of dental fluorosis was 0.66, and the epidemic intensity was slight; the detection rate of children's dental fluorosis in villages without changing the water was 51.58％ (278/539), and the index of dental fluorosis was 1.04, belonging to the moderate epidemic intensity; the detection rate of children's dental fluorosis in villages without changing the water was significantly higher than that in villages with normal operation of water improvement projects and qualified water fluoride content (χ2 = 102.30, P < 0.01). Conclusions The water improvement project in drinking water-borne fluorosis areas is running well and the operation rate is increasing year by year. The detection rate of dental fluorosis in children aged 8-12 years old in villages with normal operation of water improvement projects and qualified water fluoride content was significantly lower than that in villages without changing the water. The monitoring of fluorosis and the maintenance of water-renovation projects should be strengthened, and the problem of drinking water for residents in villages without changing the water should be resolved as soon as possible.

Objective To understand the prevalence state of drinking-tea type fluorosis in Hainan Prefecture,Qinghai Province,and provide scientific data for its prevention and control.Methods In 2016,a cross-sectional survey method was used to select five counties of Guide,Guinan,Xinghai,Gonghe and Tongde in Hainan according to "National Investigation Measure of Prevalence of Drinking-tea Type Fluorosis".In each county,5 villages (towns) were selected according to the directions of east,west,north,south and center positions,and 1 administrative village was randomly selected in each village (town) as the survey site.All the children aged 8 to 12 years old at the survey site and 60 adults aged 16 years old and older at each survey site were selected as the survey subjects.We investigated the volume of brick-tea water consumed per day among objective population with questionnaire,and collected water,brick-tea water and urine samples (if each region has one water source,we collected one water sample,if this place have different water sources,we collected all the water samples from each water source;brick-tea water samples were collected based on different locations;more than 12 urine samples were collected in each survey site).The fluoride concentration in water,urine and tea water were detected with fluoride ion-selective electrode,the dental fluorosis of children aged 8-12 years were diagnosed by "Diagnosis of Dental Fluorosis" (WS/T 208-2011),and clinic skeletal fluorosis of population was diagnosed based on the "Diagnostic Criteria for Endemic Skeletal Fluorosis" (WS 192-2008).Results The medians of fluoride in drinking water,drinking-tea water were 0.29 and 1.43 mg/L,respectively.The mean of daily fluoride intake of each person was 1.82 mg,and the geometric mean of urinary fluoride was 0.83 mg/L;the rate of dental fluorosis among children was 14.06％ (559/3 975),its index was 0.24,the prevalence rate of clinic skeletal fluorosis among adult was 8.01％ (119/1 484).The rate of dental fluorosis among children in Tongde County was the highest,about 15.96％ (83/520),while the rate of dental fluorosis among children in Guinan County was the lowest,about 10.76％ (111/1 032).The prevalence rate of clinic skeletal fluorosis among adult in Xinghai County was the highest,about 10.87％ (30/276),while Tongde County was the lowest,about 5.75％ (15/261).Conclusions The prevalence of drinking-tea type fluorosis is relative low.However,the local population are still under the risk of brick-tea with high fluoride,so it is necessary to strengthen the health education propaganda to keep the population healthy.

Objective To understand the epidemiological prevalence of drinking-tea type fluorosis in Haidong Region,Qinghai Province,and provide scientific data for its prevention and control.Methods According to the investigation plan of drinking-tea type fluorosis in China,cross sectional survey was adopted to select 6 counties in Haidong Prefecture from May to July in 2016.Five townships were selected according to the eastern,western,north and south directions,1 administrative village was randomly selected in each township (town) as the survey site.Water and brick tea samples were collected to test fluoride ion concentration;dental fluorosis in children aged 8 to 12 years and adult skeletal fluorosis were examined.Fluorine ion selective electrode was used to determine fluoridated fluoride (WS/T 106-1999),fluorine in urine (WS/T 30-1996) and fluorine content in brick tea (GB 19965-2005).Dental fluorosis was examined by Deans method.The diagnosis of skeletal fluorosis was referred to the "Diagnostic Criteria for Endemic skeletal Fluorosis" (WS 192-2008).Results Fifty-seven drinking-water and 56 brick-tea samples were determined,the means of fluoride in drinking water and brick-tea were 0.25 mg/L and 724.71 mg/kg [which was significantly higher than the national standard (300 mg / kg)],respectively.Totally 1 207 urinary samples of children and 1 428 urinary samples of adults were determined,the geometric means of urinary fluoride of children and adults were 0.57 and 0.62 mg/L,respectively;2 677 children and 1 968 adults were examined,the rate of dental fluorosis among children was 15.09％ (404/2 677),its index was 0.26,the prevalence rate of clinic skeletal fluorosis among adults was 2.44％ (48/1 968).Conclusion The fluoride content of brick tea in 6 counties of Haidong Prefecture of Qinghai is higher than that of the national standard.Local residents are still exposed to high fluoride brick tea,and health education and publicity should continue to be strengthened.

Objective To detect urinary bio-markers of hydroxyproline (HYP) and c-terminal telopeptide of collagen type Ⅱ (CTX-Ⅱ) among population from Kashin-Beck disease (KBD) regions in Qinghai Province,and to provide the scientific data for prevention and control of adult KBD.Methods According to the "Diagnosis of Kashin-Beck Disease" (WS/T 207-2010),using case-control study,120 KBD patients (males 55,females 65) and 89 healthy controls (males 41,females 48) in Qinghai KBD regions were divided into case group and control group.Morning urine samples were collected.HYP and CTX-Ⅱ contents were analyzed by enzyme-linked immunosorbent assay (ELISA),then these results were corrected with creatinine.All the data were analyzed by SPSS 17.0 software.Results There was no significant difference in the age of male and female between case group and control group (t =1.813,1.131,P ＞ 0.05).The medians of urinary HYP and CTX-Ⅱ contents among male patients were 74.91 μg/μmol Cr and 630.77 ng/μmol Cr,respectively,which were higher than those of control groups (51.38 μg/μ mol Cr,401.32 ng/μmol Cr,Z =3.068,3.246,P ＜ 0.01).The medians of urinary HYP and CTX-Ⅱ contents among female patients were 91.07 μg/μmol Cr and 637.17 ng/μmol Cr,respectively,compared with those of control groups (88.37μg/μmol Cr,546.47 ng/μmol Cr),there was no significant difference in HYP content (Z =0.273,P ＞ 0.05),however,the difference in CTX-Ⅱ content was statistically significant (Z =2.002,P ＜ 0.05).Conclusion The urinary HYP contents of male patients with KBD change significantly,while the degradation of type Ⅱ collagen in male and female patients increases,and CTX-Ⅱ could reflect the metabolic changes of collagen in KBD.

Objective To investigate the prevalence of genital Chlamydia trachomatis (GCT) infection and associated risk factors in male clients attending sexually transmitted disease (STD) clinics in Guangdong and provide integrated intervention strategy for this group.Methods Convenient sampling was used to recruit participants from April to June in 2015 in Guangdong province.The information about their socio-demographic characteristics and sexual behaviors were collected by using a questionnaire,and blood samples were taken from them to test the antibodies against HIV,syphilis and HCV.First pass urine was taken to test GCT and gonorrhea.Results A total of 1 749 participants with the average age of 39.53 years were recruited.The majority of them were married (73.87％,1 292/1 749),residents of Guangdong (92.28％,1 614/1 749) and in Han ethnic group (99.49％,1 740/1 749).The positive rates for GCT,HIV,syphilis,HCV,Neisseria gonorrhea,and WBC in urinalysis were 6.06％ (106/1 749),0.46％ (8/1 749),3.43％ (60/1 749),0.45％ (7/1 550),2.74％ (48/1 749),7.89％ (138/1 749) respectively.Multivariate analysis showed that risk factors for GCT infection include IDUs (OR=13.98,95％CI:3.35-58.38),anal sex with men (OR=3.11,95％ CI:1.45-6.71),Neisseria gonorrhea positive (OR =9.64,95％ CI:5.09-18.24),and WBC positive (OR =1.96,95％ CI:1.08-3.55).Conclusions This study demonstrated the high prevalence of GCT infection in male clients attending STD clinics in Guangdong.Therefore precision intervention should target this population at high-risk.