Seroepidemiological Investigation of Lyme Disease and Human Granulocytic Anaplasmosis among People Living in Forest Areas of Eight Provinces in China

Objective Lyme disease and Human granulocytic anaplasmosis are tick-borne diseases caused by Borrelia burgdorferi and Anoplasrna phagocytophilum respectively. We have investigated infection and co-infection of the two diseases in the population of forest areas of eight provinces in China by measuring seroprevalence of antibodies against B. burgdorferi and A. phagocytophilum. Methods Forest areas in 8 provinces were chosen for investigation using whole sampling and questionnaire survey methods. 3 669 serum samples from people in the forest areas were tested for the presence of antibodies by indirect immunofluorescent assay （IFA）. Results Seroprevalence against B. burgdorferi was 3% to 15% and against A. phagocytophilum was 2% to 18% in the study sites in the 8 provinces in China. We also found co-infection of B. burgdorferi and A. phagocytophilum in 7 of the 8 provinces （the exception being the Miyun area in Beijing）. The seroprevalence for both B. burgdorferi and A. phagocytophilum was significantly higher among people exposed to ticks than among people who were not exposed to ticks. Conclusion We conclude that both pathogens are endemic in the forest areas in the eight provinces, but the prevalence of B. burgdorferi and A. phagocytophilum differs between the provinces.

MicroRNA-135a in ABCA1-labeled Exosome is a Serum Biomarker Candidate for Alzheimer’s Disease

Objective In the present study,the ABCA1 was used as a label to capture specific exosomes,the level of ABCA1-labeled exosomal microRNA-135 a(miR-135 a)was evaluated for the diagnosis of Alzheimer’s disease(AD),especially in patients with early stages of AD.Methods This is a preliminary research focused on the levels of ABCA1 in WBCs,RBCs,HT-22 cells,and neuron cells.The diagnostic value of ABCA1-labeled exosomal miR-135 a was examined using the CSF and serum of APP/PS1 double transgenic mice,and 152 patients with SCD,131 patients with MCI,198 patients with DAT,and 30 control subjects.Results The level of ABCA1 exosomes harvested from HT-22 cells and neuron culture medium was significantly higher compared to that of RBCs and WBCs(P<0.05).The levels of ABCA1-labeled exosomal miR-135 a increased in the CSF of MCI and DAT group compared to those of control group(P<0.05),slightly increased(P>0.05)in the serum of SCD patient group,and significantly increased in MCI and DAT patient groups compared to those of the control group(P<0.05).Conclusion This study outlines a method to capture specific exosomes and detect them using immunological methods,which is more efficient for early diagnosis of AD.

Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR

Objective This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase(DGC)in Vibrio cholerae and how its transcription is regulated by Fur and Hap R.Methods The roles of VCA0560 was investigated by utilizing various phenotypic assays,including colony morphological characterization,crystal violet staining,Cyclic di-GMP(c-di-GMP)quantification,and swimming motility assay.The regulation of the VCA0560 gene by Fur and Hap R was analyzed by luminescence assay,electrophoretic mobility shift assay,and DNase I footprinting.Results VCA0560 gene mutation did not affect biofilm formation,motility,and c-di-GMP synthesis in V.cholerae,and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity.The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator Hap R.Conclusion Overexpressed VCA0560 functions as an active DGC in V.cholerae,and its transcription is repressed by Fur and Hap R.

Species-level Microbiota of Biting Midges and Ticks from Poyang Lake

Objective The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area,namely,Qunlu Practice Base,Peach Blossom Garden,and Huangtong Animal Husbandry,and whether vectors carry any bacterial pathogens that may cause diseases to humans,to provide scientific basis for prospective pathogen discovery and disease prevention and control.Methods Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit(OPU)analysis,we characterized the species-level microbial community structure of two important vector species,biting midges and ticks,including 33 arthropod samples comprising 3,885 individuals,collected around Poyang Lake.Results A total of 662 OPUs were classified in biting midges,including 195 known species and 373 potentially new species,and 618 OPUs were classified in ticks,including 217 known species and 326 potentially new species.Surprisingly,OPUs with potentially pathogenicity were detected in both arthropod vectors,with 66 known species of biting midges reported to carry potential pathogens,including Asaia lannensis and Rickettsia bellii,compared to 50 in ticks,such as Acinetobacter lwoffii and Staphylococcus sciuri.We found that Proteobacteria was the most dominant group in both midges and ticks.Furthermore,the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria.Pantoea sp7 was predominant in biting midges,while Coxiella sp1 was enriched in ticks.Meanwhile,Coxiella spp.,which may be essential for the survival of Haemaphysalis longicornis Neumann,were detected in all tick samples.The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors.Conclusion Biting midges and ticks carry large numbers of known and potentially novel bacteria,and carry a wide range of potentially pathogenic bacteria,which may pose a risk of infection to humans and animals.The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Amino acid deletions at positions 893 and 894 of cytotoxinassociated gene A protein affect Helicobacter pylori gastric epithelial cell interactions

BACKGROUND Helicobacter pylori(H.pylori)persistently colonizes the human gastric mucosa in more than 50%of the global population,leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma.Cytotoxin-associated gene A(CagA)protein,an important oncoprotein,has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus,which play crucial roles in pathogenesis.Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer.AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function.METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894.The cagA gene was amplified and mutated into cagA-NT and cagA-NE(sequence characteristics of strains from nongastric cancer patients),cloned and inserted into pAdtrack-CMV,and then transfected into AGS cells.The expression of cagA and its mutants was examined using realtime polymerase chain reaction and Western blotting,cell elongation via cell counting,F-actin cytoskeleton visualization using fluorescence staining,and interleukin-8(IL-8)secretion via enzyme-linked immunosorbent assay.RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE(40.88±3.10 vs 32.50±3.17,P<0.001 and 40.88±3.10 vs 32.17±3.00,P<0.001)at 12 hours after transfection.At 24 hours,pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT(46.02±2.12 vs 53.90±2.10,P<0.001 and 46.02±2.12 vs 51.15±3.74,P<0.001).The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE(27.54±17.37 vs 41.51±11.90,P<0.001 and 27.54±17.37 vs 41.39±14.22,P<0.001)at 12 hours after transfection.Additionally,pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection.CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity,which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H.pylori.

A Study of the Technique of Western Blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China

Objective To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. Methods FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and irnmunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples （159 patients with Lyme disease and 292 controls）, all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Results Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection

AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

Dynamics of Rodent and Rodent-borne Disease during Construction of the Three Gorges Reservoir from 1997 to 2012

Objective To investigate the impact of impoundment and active public health interventions on rodent populations and rodent-borne diseases in the Three Gorges reservoir region from 1997 to 2012. Methods Surveillance data from 1997 to 2012 were extracted from the Public Health Surveillance System of The Three Gorges established in 1997. Temporal changes in the incidences of hemorrhagic fever with renal syndrome （HFRS） and leptospirosis, rodent density, pathogen-carrying rates, and their correlations were analyzed. Results The average indoor and outdoor rodent densities decreased overall from 1997 to 2012. The average densities decreased by 47.72% （from 4.38% to 2.29%） and 39.68% （from 4.41% to 2.66%）, respectively, after impoundment （2003-2012） compared with before impoundment （1997-2002）. The average annual incidence rates of HFRS and leptospirosis were 0.29/100,000 and 0.52/100,000, respectively, and decreased by 85.74% （from 0.68/100,000 to 0.10/100,000） and 95.73% （from 1.47/100,000 to 0.06S/100,000）, respectively, after impoundment compared with before impoundment. Incidences of HFRS and leptospirosis appear to be positively correlated with rodent density in the reservoir area. Conclusion This study demonstrated that rodent density and incidences of rodent-borne diseases decreased and were maintained at low levels during construction of the Three Gorges dam. Measures that reduce rodent population densities could be effective in controlling rodent-borne diseases during large-scale hydraulic engineering construction.

Rapid and Sensitive Chemiluminescent Enzyme Immunoassay for the Determination of Neomycin Residues in Milk

Immunoassays greatly contribute to veterinary drug residue analysis. However, there are few reports on detecting neomycin residues by immunoassay. Here, a rapid and sensitive chemiluminescent enzyme immunoassay （CLIEA） was successfully developed for neomycin residue analysis. CLIEA demonstrated good cross-reactivity for neomycin, and the IC50 value was 2.4 ng/mL in buffer.

Screening Pathogenic Microorganism Standard Strains for Disinfection Efficacy Evaluation

Infectious diseases would always threaten human health,especially emerging infectious diseases(EIDs),such as the coronavirus disease-2019(COVID-19).In 2019,COVID-19 spread rapidly worldwide.On January 30,2020,World Health Organization(WHO)declared its outbreak a public health emergency of international concern.

Epidemiological Characteristics of Notifiable Infectious Diseases among Foreign Cases in China,2004–2017

Objective We aimed to assess the features of notifiable infectious diseases found commonly in foreign nationals in China between 2004 and 2017 to improve public health policy and responses for infectious diseases.Methods We performed a descriptive study of notifiable infectious diseases among foreigners reported from 2004 to 2017 in China using data from the Chinese National Notifiable Infectious Disease Reporting System(NNIDRIS). Demographic, temporal-spatial distribution were described and analyzed.Results A total of 67,939 cases of 33 different infectious diseases were reported among foreigners.These diseases were seen in 31 provinces of China and originated from 146 countries of the world. The infectious diseases with the highest incidence number were human immunodeficiency virus(HIV) of18,713 cases, hepatitis B(6,461 cases), hand, foot, and mouth disease(6,327 cases). Yunnan province had the highest number of notifiable infectious diseases in foreigners. There were different trends of the major infectious diseases among foreign cases seen in China and varied among provinces.Conclusions This is the first description of the epidemiological characteristic of notifiable infectious diseases among foreigners in China from 2004 to 2017. These data can be used to better inform policymakers about national health priorities for future research and control strategies.

Establishment and Comparison of Pulsed-field Gel Electrophoresis,Multiple-locus Variable Number Tandem Repeat Analysis and Automated Ribotyping Methods for Subtyping of Citrobacter Strains

Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration （turbidity of 2.5 to 3.5）, SDS addition （no SDS needed） and lysis time （1 h）, and selected the appropriate restriction enzyme （Xbal） and the electrophoretic parameters （1.0 s-20.0 s for 19 h） of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources （with the same species from the same place at the same time identified as the same source） and divided strains from different sources （from different years, places and hosts） into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.

Establishment of Multiple Locus Variable-number Tandem Repeat Analysis Assay for Genotyping of Borrelia burgdorferi sensu lato Detected in China

Objective Human Lyme Borreliosis （LB）, which is caused by Borrefia burgdorferi sensu lato （B. burgdorferi）, has been identified as a major arthropod-borne infectious disease in China. We aimed to develop a multiple locus variable-number tandem repeat （VNTR） analysis （MLVA） assay for the genotyping of Borrelia burgdorJ：eri strains detected in China. Methods B. garinii PBi complete 904.246 kb chromosome and two plasmids （cp26 and Ip54） were screened by using Tandem Repeats Finder program for getting potential VNTR loci, the potential VNTR loci were analyzed and identified with PCR and the VNTR loci data were analyzed and MLVA clustering tree were constrcted by using the categorical coefficient and the unweighted pair-group method with arithmetic means （UPGMA）. Results We identified 5 new VNTR loci through analyzing 47 potential VNTR loci. We used the MLVA protocol to analyse 101 B. burgdorferi strains detected in China and finally identified 51 unique genotypes in 4 major clusters including B. burgdorferi sensu stricto （B.b.s.s）, B. garinii, B. a[zelii, and B. valaisiana, consistent with the current MLSA phylogeny studies. The allele numbers of VNTR-1, VNTR-2, VNTR-3, VNTR-4, and VNTR-5 were 7, 3, 9, 7, and 6. The Hunter-Gaston index （HGI） of five VNTR loci were 0.79, 0.22, 0.77, 0.71, and 0.67, respectively. The combined HGI of five VNTR loci was 0.96. Clustering of the strains of Xinjiang, Inner Mongolia and Heilongjiang was confirmed, and this situation was consistent with the close geographical distribution of those provinces. Conclusion The MLVA protocol esytablished in this study is easy and can show strains＇ phylogenetic relationships to distinguish the strains of Borrelia species. It is useful for further phylogenetic and epidemiological analyses of Borrelia strains.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Evaluation of Four Candidate VNTR Loci for Genotyping 225 Chinese Clinical Mycobacterium Tuberculosis Complex Strains

Objective To evaluate four candidate variable number tandem repeat （VNTR） loci for genotyping Mycobacterium tuberculosis complex strains. Methods Genomic sequences for two M. tuberculosis strains （CCDC5079 and CCDC5180） were generated, and using published sequence data, four candidate VNTR loci were identified. The VNTRs were used to genotype 225 Chinese clinical M. tuberculosis complex strains. The discriminatory power of the VNTRs was evaluated using BioNumerics 5.0 software. Results The Hunter-Gaston Index （HGI） for BJ1, BJ2, BJ3, and BJ4 loci was 0.634, 0.917, 0.697, and 0.910, respectively. Combining all four loci gave an HGI value of 0.995, thus confirming that the genotyping had good discriminatory power. The HGI values for BJ1, BJ2, BJ3, and BJ4, obtained from Beijing family strain genotyping, were 0.447, 0.878, 0.315, and 0.850, respectively. Combining all four loci produced an HGI value of 0.988 for genotyping the Beijing family strains. We observed unique patterns for M. boris and M. africanum strains from the four loci. Conclusion We have shown that the four VNTR loci can be successfully used for genotyping M. tuberculosis complex strains. Notably, these new loci may provide additional information about Chinese M. tuberculosis isolates than that currently afforded by established VNTR loci typing.

Antimicrobial Susceptibility Testing and Molecular Characterization of Mycobacterium fortuitum Isolates in China

We performed molecular identification of clinical isolates of Mycobacterium fortuitum（M. fortuitum） and conducted drug susceptibility testing to analyze the in vitro susceptibility of clinical M. fortuitum isolates and potential molecular mechanism conferring resistance to fluoroquinolone and macrolide drugs. The results showed that moxifloxacin had the highest in vitro activity against M. fortuitum, and most M. fortuitum isolates were resistant to clarithromycin and linezolid in China. The loss of genetic mutation in clarithromycin-and amikacin-resistant isolates indicates that some other intrinsic mechanism conferring clarithromycin and amikacin resistance plays an essential role in M. fortuitum infection.

T4SP: A Novel Tool and Database for Type IV Secretion Systems in Bacterial Genomes

Secretion systems, macromolecules to pass which can mediate the across cellular membranes, are essential for virulent and genetic material exchange among bacterial species[1]. Type IV secretion system （T4SS） is one of the secretion systems and it usually consists of 12 genes： VirB1, VirB2 ...VirB11, and VirD4[2]. The structure and molecular mechanisms of these genes have been well analyzed in Gram-negative strains[3] and Gram-positive strains were once believed to be lack of T4SS. However, some recent studies revealed that one or more virB/D genes also exist in some kinds of Gram-positive bacteria and play similar role, and form a T4SS-like system[3]. The VirBl-like, VirB4, VirB6, and VirD4 genes were identified in the chromosome of Gram-positive bacterium Streptococcus suis in our previous studies and their role as important mobile elements for horizontal transfer to recipients in an 89 K pathogenicity island （PAl） was demonstrated[45]. However, their structure and molecular mechanisms in other strains, especially in Gram-positive strains, are remained unclear.

The Role of Standards for Communicable Disease Prevention and Control in Protecting People’s Health and Safety During COVID-19

World Standards Day was established by the International Organization for Standardization(ISO)on October 14 to enhance awareness on the significance of international standardization to respond to the needs of businesses,industries,governments and consumers worldwide.Since its rejoining the ISO in 1978,China held various presentations,symposiums,and commemorative events for World Standards Day in major and medium-sized cities nationwide to publicize the role of standardization in societal development and enhance the awareness of standardization among the people.

A Core Genome Multilocus Sequence Typing Scheme for Proteus mirabilis

Objective A core genome multilocus sequence typing(cgMLST)scheme to genotype and identify potential risk clonal groups(CGs)in Proteus mirabilis.Methods In this work,we propose a publicly available cgMLST scheme for P.mirabilis using chew BBACA.In total 72 complete P.mirabilis genomes,representing the diversity of this species,were used to set up a cgMLST scheme targeting 1,842 genes,635 unfinished(contig,chromosome,and scaffold)genomes were used for its validation.Results We identified a total of 205 CGs from 695 P.mirabilis strains with regional distribution characteristics.Of these,159 unique CGs were distributed in 16 countries.CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes.Nine virulence genes(papC,papD,papE,papF,papG,papH,papI,papJ,and papK)related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20.These CGs require attention due to potential risks.Conclusion This research innovatively performs high-resolution molecular typing of P.mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline(chewBBACA).We found that the CGs of P.mirabilis showed regional distribution differences.We expect that our research will contribute to the establishment of cgMLST for P.mirabilis.

A Campylobacteriosis Outbreak Caused by One Asymptomatic Food Handler Carrier

In August 2021,three students with diarrhea from the same school visited a local hospital in the S district of Beijing.An epidemic investigation showed that there were more students with diarrhea in the same school and they had one meal together.Campylobacter jejuni was isolated from both patients with diarrhea and asymptomatic food handlers;however,the latter also carried Campylobacter coli.Phylogenomic analysis showed that there was a campylobacteriosis outbreak among the students,and the asymptomatic food handler may have been the source of the infection.Routine inspection and surveillance for Campylobacter is needed for the food producing staff,particularly those cooking in the cafeteria in schools or other public food services.