Surveillance of tick-borne bacteria infection in ticks and forestry populations in Inner Mongolia, China.

Ticks are one of the most important vectors that can transmit pathogens to animals and human beings. This study investigated the dominant tick-borne bacteria carried by ticks and tick-borne infections in forestry populations in Arxan, Inner Mongolia, China. Ticks were collected by flagging from May 2020 to May 2021, and blood samples were collected from individuals at high risk of acquiring tick-borne diseases from March 2022 to August 2023. The pooled DNA samples of ticks were analyzed to reveal the presence of tick-borne bacteria using high-throughput sequencing of the 16S rDNA V3-V4 region, and species-specific polymerase chain reaction (PCR) related to sequencing was performed to confirm the presence of pathogenic bacteria in individual ticks and human blood samples. All sera samples were examined for anti-SFGR using ELISA and anti-B. burgdorferi using IFA and WB. A total of 295 ticks (282 Ixodes persulcatus and 13 Dermacentor silvarum) and 245 human blood samples were collected. Rickettsia, Anaplasma, Borrelia miyamotoi, and Coxiella endosymbiont were identified in I. persulcatus by high-throughput sequencing, while Candidatus R. tarasevichiae (89.00%, 89/100), B. garinii (17.00%, 17/100), B. afzelii (7.00%, 7/100), and B. miyamotoi (7.00%, 7/100) were detected in I. persulcatus, as well the dual co-infection with Candidatus R. tarasevichiae and B. garinii were detected in 13.00% (13/100) of I. persulcatus. Of the 245 individuals, B. garinii (4.90%, 12/245), R. slovaca (0.82%, 2/245), and C. burnetii (0.41%, 1/245) were detected by PCR, and the sequences of the target genes of B. garinii detected in humans were identical to those detected in I. persulcatus. The seroprevalence of anti-SFGR and anti-B. burgdorferi was 5.71% and 13.47%, respectively. This study demonstrated that Candidatus R. tarasevichiae and B. garinii were the dominant tick-borne bacteria in I. persulcatus from Arxan, and that dual co-infection with Candidatus R. tarasevichiae and B. garinii was frequent. This is the first time that B. miyamotoi has been identified in ticks from Arxan and R. solvaca has been detected in humans from Inner Mongolia. More importantly, this study demonstrated the transmission of B. garinii from ticks to humans in Arxan, suggesting that long-term monitoring of tick-borne pathogens in ticks and humans is important for the prevention and control of tick-borne diseases.

Prevalence and prediction of Lyme disease in Hainan province.

Lyme disease (LD) is one of the most important vector-borne diseases worldwide. However, there is limited information on the prevalence and risk analysis using correlated factors in the tropical areas. A total of 1583 serum samples, collected from five hospitals of Hainan Province, were tested by immunofluorescence assay (IFA) and western blot (WB) analyses using anti-Borrelia burgdorferi antibodies. Then, we mapped the distribution of positive rate (by IFA) and the spread of confirmed Lyme patients (by WB). Using ArcGIS, we compiled host-vector-human interactions and correlated data as risk factor layers to predict LD risk in Hainan Province. There are three LD hotspots, designated hotspot I, which is located in central Hainan, hotspot II, which contains Sanya district, and hotspot III, which lies in the Haikou-Qiongshan area. The positive rate (16.67% by IFA) of LD in Qiongzhong, located in hotspot I, was higher than that in four other areas. Of confirmed cases of LD, 80.77% of patients (42/52) whose results had been confirmed by WB were in hotspots I and III. Hotspot II, with unknowed prevalence of LD, need to be paid more attention considering human-vector interaction. Wuzhi and Limu mountains might be the most important areas for the prevalence of LD, as the severe host-vector and human-vector interactions lead to a potential origin site for LD. Qiongzhong is the riskiest area and is located to the east of Wuzhi Mountain. In the Sanya and Haikou-Qiongshan area, intervening in the human-vector interaction would help control the prevalence of LD.

Case report: A patient coinfected by Borrelia burgdorferi sensu lato and spotted fever group Rickettsiae in Urumqi, China.

RATIONALE: Both Borrelia burgdorferi sensu lato and spotted fever group Rickettsiae (SFGR) are pathogens carried by ticks. There is a possibility of co-infection with these tick-borne diseases. PATIENT CONCERNS: Male patient, 63 years-of-age, admitted to hospital with skin rash presenting for 1 week and fever with cough and expectoration for 3 days before admission. DIAGNOSES: We diagnosed that the patient was co-infected by B burgdorferi sl and SFGR using laboratory test results and the patient's clinical manifestations. INTERVENTIONS: The patient started therapy with oral minocycline, then levofloxacin by intravenous injection for SFGR. Meanwhile, he was treated with penicillin G sodium, cefoperazone sulbactam sodium and ceftriaxone by intravenous injection for B burgdorferi sl. OUTCOMES: After the patient was in stable condition, he was discharged from hospital. LESSONS: This case report highlights the possibility of co-infection by 2 tick-borne diseases in Urumqi, Xinjiang Uygur Autonomous Region, China. The antibiotic therapy should be based on the detection of pathogenic bacteria, and the different susceptibilities of co-infecting bacteria should be considered.

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.

A novel isothermal detection for recombinase polymerase amplification with lateral flow (LF-RPA) was established for Borrelia burgdorferi (B. burgdorferi) detection in this study. This assay with high sensitivity and specificity can get a visible result without any additional equipment in 30 min. We designed a pair of primers according to recA gene of B. burgdorferi strains and a methodology evaluation was performed. The results showed that the RPA assay based on the recA gene was successfully applied in B. burgdorferi detection, and its specific amplification was only achieved from the genomic DNA of B. burgdorferi. The detection limit of the new assay was about 25 copies of the B. burgdorferi genomic DNA. Twenty Lyme borreliosis patients' serum samples were detected by LF-RPA assay, real-time qPCR and nested-PCR. Results showed the LF-RPA assay is more effective than nested-PCR for its shorter reaction time and considerably higher detection rate. This method is of great value in clinical rapid detection for Lyme borreliosis. Using the RPA assay might be a megatrend for DNA detection in clinics and endemic regions.

Genomic Characteristics of Chinese Borrelia burgdorferi Isolates.

In China, B. burgdorferi, B.garinii, B. afzelii and B. yangtze sp. nov have been reported; B.garinii and B. afzelii are the main pathogenic genotypes. But until now only one Chinese strain was reported with whole genome sequence. In order to further understand the genomic characteristics and diversity of Chinese Borrelia strains, 5 isolates from China were sequenced and compared with the whole genome sequences of strains in other areas. The results showed a high degree of conservation within the linear chromosome of Chinese strains, whereas plasmid showed a much larger diversity according to the majority genomic information of plasmids. The genome sequences of the five Chinese strains were compared with the corresponding reference strains, respectively, according to the genospecies. Pairwise analysis demonstrates that there are only 70 SNPs between the genomes of CS4 and B31. However, there are many more SNPs between the genomes of QX-S13 and VS116, PD91 and PBi, FP1 and PKo, R9 and Pko, respectively. Gene comparison showed some important different genes. OspA was one of the important different genes. Comparative genomic studies have found that OspA gene sequences of PD91 and R9 had great differences compared with the sequence of B31. OspA gene sequence of R9 had a 96bp deletion; OspA gene of PD91 had two deletions: 9bp and 10 bp. To conclude, we showed the genomic characteristics of four genotype Chinese B. burgdorferi strains. The genomic sequence of B. yangtze sp. nov and differences from B. valaisiana were first reported. Comparative analysis of Chinese strains with the different Borrelia species from other areas will help us to understand evolution and pathogenesis of Chinese Borrelia burgdorferi strains.

[Prediction of potential geographic distribution of Lyme disease in Qinghai province with Maximum Entropy model].

OBJECTIVE: To predict the potential geographic distribution of Lyme disease in Qinghai by using Maximum Entropy model (MaxEnt). METHODS: The sero-diagnosis data of Lyme disease in 6 counties (Huzhu, Zeku, Tongde, Datong, Qilian and Xunhua) and the environmental and anthropogenic data including altitude, human footprint, normalized difference vegetation index (NDVI) and temperature in Qinghai province since 1990 were collected. By using the data of Huzhu Zeku and Tongde, the prediction of potential distribution of Lyme disease in Qinghai was conducted with MaxEnt. The prediction results were compared with the human sero-prevalence of Lyme disease in Datong, Qilian and Xunhua counties in Qinghai. RESULTS: Three hot spots of Lyme disease were predicted in Qinghai, which were all in the east forest areas. Furthermore, the NDVI showed the most important role in the model prediction, followed by human footprint. Datong, Qilian and Xunhua counties were all in eastern Qinghai. Xunhua was in hot spot areaⅡ, Datong was close to the north of hot spot area Ⅲ, while Qilian with lowest sero-prevalence of Lyme disease was not in the hot spot areas. The data were well modeled in MaxEnt (Area Under Curve=0.980). CONCLUSIONS: The actual distribution of Lyme disease in Qinghai was in consistent with the results of the model prediction. MaxEnt could be used in predicting the potential distribution patterns of Lyme disease. The distribution of vegetation and the range and intensity of human activity might be related with Lyme disease distribution.

Polymorphism of 41 kD Flagellin Gene and Its Human B-Cell Epitope in Borrelia burgdorferi Strains of China.

The 41 kD flagellin of Borrelia burgdorferi (B. burgdorferi) is a major component of periplasmic flagellar filament core and a good candidate for serodiagnosis in early stage of Lyme disease. Here, we chose 89 B. burgdorferi strains in China, amplified the gene encoding the 41 kD flagellin, and compared the sequences. The results showed that genetic diversity presented in the 41 kD flagellin genes of all 89 strains among the four genotypes of B. burgdorferi, especially in the genotype of B. garinii. Some specific mutation sites for each genotype of the 41 kD flagellin genes were found, which could be used for genotyping B. burgdorferi strains in China. Human B-cell epitope analysis showed that thirteen of 15 nonsynonymous mutations occurred in the epitope region of 41 kD flagellin and thirty of 42 B-cell epitopes were altered due to all 13 nonsynonymous mutations in the epitope region, which may affect the function of the antigen. Nonsynonymous mutations and changed human B-cell epitopes exist in 41 kD flagellin of B. burgdorferi sensu lato strains; these changes should be considered in serodiagnosis of Lyme disease.

Test of 259 serums from patients with arthritis or neurological symptoms confirmed existence of Lyme disease in Hainan province, China.

Indirect Fluorescent-Antibody Test (IFA), Western Blot (WB) and Nested-PCR were applied to identify the Borrelia burgdorferi in human serum samples in Hainan province. A total of 259 serum samples were collected from Sanya Peoples' Hospital, Hainan province. These samples were examined for the presence of B. burgdorferi serologically and etiologically by the two tier tests (IFA and WB) and Nested-PCR. 43 in total of 259 serum samples were tested positive by IFA assay, the positive rate was 16.6%. Among 43 IFA-positive samples, 6 were identified positive by WB. Nested-PCR were also used to test B. burgdorferi DNA in 259 serum samples at the same time, 27 samples were tested positive with positive rate of 10.42%. It is the first time to confirm that there are Lyme patients in Hainan province of China. The study suggested that Lyme disease should be commonly considered by clinicians with the patients who had correlated symptoms with lyme disease in Hainan.

Prevalence of Borrelia burgdorferi sensu lato in rodents from Jiangxi, southeastern China region.

In order to investigate the prevalence of B.burgdorferi sensu lato in rodents from Jiangxi province of southeastern China. Isolation of B.burgdoferi strains and PCR-based studies were carried out in 204 mice collected from six counties of Jiangxi province in May of 2011 and 2012. The results showed the prevalence of Lyme spirochetal infection among seven species of wild and peridomestic rodents in Jiangxi. 3 strains isolated from 204 mice were all belonged to Borrelia yangze sp.nov. The study firstly showed the role of rodents in maintaining the pathogen of Lyme disease in the environment from Jiangxi province and there existed at least one genotype of Lyme spirochetes in Jiangxi.

Distribution of Borrelia burgdorferi sensu lato in China.

We genotyped 102 Borrelia burgdorferi sensu lato strains isolated from ticks, animals, and patients in 11 provinces in China by PCR-restriction fragment length polymorphism (PCR-RFLP) amplification of 5S (rrf)-23S (rrl) rRNA gene spacer amplicons and multilocus sequence analysis (MLSA). The results showed that Borrelia garinii was the main genotype in China (65/102) and that it was distributed mainly in northern China. Borrelia afzelii was the second most frequently found species (22/102), and it was distributed in both northern and southern China. All Borrelia valaisiana strains were isolated from Guizhou Province. Additionally, one B. burgdorferi strain was isolated from Hunan Province. Our results show the diversity and wide distribution of B. burgdorferi sensu lato in China.

Interpretation criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China.

OBJECTIVE: Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for its performance and interpretation have been established in China. The present study was designed to determine the criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China, in which WB was produced with strain PD91 as the representative strain attributed to predominant genospecies Borrelia garinii of Borrelia burgdorferi sensu lato. METHODS: Approximately 13 bands between 14 and 100 kD were differentiated for strain PD91 by using Gel-Pro analysis software. In a study with 631 serum samples (taken from 127 patients with Lyme borreliosis and 504 controls), all observed bands were documented. To establish criteria for a positive WB result for strain PD91, receiver operating characteristic (ROC) curves were used. RESULTS: The following interpretation criteria were recommended: for IgG, at least one band of P83/100, P58, P39, P30, OspC, P17, P66, and OspA; for IgM, at least one band of P83/100, P58, OspA, P30, OspC, P17 or P41. In addition, syphilis, leptospirosis and other related diseases should be excluded when the positive band is P41 in IgM. For IgG criteria, the sensitivity is 73.2%, the specificity is 99.4% and Youden index is 0.726; for IgM criteria, the sensitivity is 50.6%, the specificity is 93.1% and Youden index is 0.437. CONCLUSION: Standardization of WB assays is necessary for comparison of results from different laboratories. Moreover, the criteria of other genospecies of Borrelia burgdorferi sensu lato should be determined in the future to complete the criteria of WB for the diagnosis of the Lyme disease in China.

[Isolation and identification of Borrelia burgdorferi sensu lato from ticks in six provinces in China].

OBJECTIVE: To understand the carrying status of Borrelia burgdorferi in ticks from the mountain areas from six representative provinces, including Jilin, Shanxi, Gansu, Qinghai, Guizhou and Hunan in China. METHODS: Flagging and trapping methods were used to collect ticks in forest area and culture medium was used to isolate the pathogen. Nested-PCR was used to detect the germ-carrying rate of ticks. RESULTS: More than 2200 ticks from six representative provinces were collected and 1000 ticks were used to isolate the pathogen. 13 Lyme disease spirochetes from ixodes persulcatus in Changbai, Jilin province and 9 Lyme disease spirochetes from ixodes granulatus in Daozhen, Guizhou province were identified. There were 1255 ticks used for PCR testing. Specific fragments of the Borrelia burgdorferi in ticks were found from the six representative provinces in China. The carrier rate was higher in Jilin (Changbai 27.08%, Tonghua 20.41%), Qinghai (Huzhu 25.06%, Huangnan 21.11%) and Guizhou (Daozhen 25.63%), than in Shanxi (Yuanqu 4.72%, Jiaocheng 3.64%). Result from the sequence analysis showed that the genotype belong to Borrelia garinii in Jilin, Qinghai, Gansu, Shanxi provinces but Borrelia valaisiana in Guizhou and Hunan provinces. CONCLUSION: Our data showed that there existed Lyme disease spirochetes in all the six representative provinces in China, but the carriying rates of ticks were different. Borrelia garinii was found in Shanxi province, and Borrelia valaisiana in Hunan province.

[Investigation on the vectors of Borrella burgdorferi and on the identification of the isolates along China-Russia border in Eastern Heilongjiang province, China].

OBJECTIVE: To explore the fact that the east border of Heilongjiang had been a lyme disease natural focus,we investigated the species and distribution of ticks and isolated bacteria from ticks and identified genomic species of Borrelia burdorferi sensu lato. This study provided evidence for prevention and control of lyme disease. METHODS: Ticks were caught by flagging method and Direct immunofluorescence method was used to detect the rate of bacteria borne by the tick. BSK UI culture medium was used to isolate the agent and Specific McAbs were used to identify the bacteria. SDS-PAGE protein profile and PCR-RFLP method were also used to identify the species of Spirochetes. RESULTS: Ticks, collected from China-Russia border of east Heilongiiang province were classified including Ixodes persulcatus Schulze, Dermacentor sivarum Olener, Haemaphysalis concinna Kock,and Haemaphysalis japonica Kock. We found that the distributon of ticks was different under different circumstances and the predominant species were also different in different ports. The rate of bacteria borne by Iodes persulaatus Schulze was 31.4% ,by Dermacentor sivarum Olener and Haemaphysalis concinna Kock were 2.2% and 3.8%, respectively. However,it was negative for Haenaphysalis japonica Kock. Spirochetes isolated from Ixodes persulcatus Schulze were collected from Dongning and Tongjiang while Genomic species of Spirochetes, isolated from ticks of the border belonged to B. garinii. CONCLUSION: All the results showed that the east border of Heilongjiang province was the natural focus of lyme disease.

[Cloning and expression of flagellin gene from a Chinese Borrelia burgdorferi PD91 strain].

OBJECTIVE: To study the cloning and expression of flagellin gene from Chinese Borrelia burgdorferi, PD91 strain and to evaluate the feasibility of using recombinant protein as diagnostic antigen when comparing the gene sequence with flagellin gene from North American Borrelia burgdorferi B31. METHODS: The piece of genes coding flagellin from Chinese Borrelia burgdorferi PD91 by polymerase chain reaction (PCR) method was obtained, and constructed recombinant plasmid, before transformed into E. coli BL21 strain, and induced. The recombinant plasmid was identified with enzyme cutoff and gene sequence comparison. Efficient expression strain was selected and the expression product was analyzed with sodium amplified polymorphic-polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blot method. RESULTS: The recombinant protein (r-flagellin) expressed in host bacteria was successful. By means of western-blot assay, the immunological response showed the same antigenicity between r-flagellin and PD91 flagellin. The piece of genes coding flagellin of PD91 was 1011 bp, but when comparing with that of North American Borrelia burgdorferi it showed 94.70% homology. Homology between the sequence of amino acid of the r-flagellin and that of B31 flagellin was 95.85%. CONCLUSION: Flagellin gene of Borrelia garinii of Chinese Lyme disease spirochete was successfully cloned and expressed for the first time. It was proved that the immunoreactivity of r-flagellin was the same as the natural flagellin.