Molecular typing and antibiotic resistance of Shigella isolated from 2011 to 2014 in Shandong Province / 中华疾病控制杂志

Objective To study the characteristics and epidemic trend of Shigella in Shandong province through the analysis of serotype, virulence genes, molecular typing and drug sensitivity. Methods The serotype was classified using the method of slide agglutination. Polymerase chain reaction (PCR) was used to amplify the related virulence genes. The molecular typing was carried out by pulsed field gel electrophoresis (PFGE), and the antibiotic sensitivity of the strains was determined by micro-broth dilution method. Results The main serogroups of 44 Shigella strains were Shigella flexneri (54.55%) and Shigella sonnei (43.18%). The carrying rates of ipaH, Set1, Sen and ial were 100%, 43.18%, 56.82% and 50.00%, respectively. By PFGE typing, the strains of Shigella flexneri were divided into 18 patterns with a low similarity. The strains of Shigella sonnei were divided into 14 patterns, and the similarity of 89.47% of the strains was more than 90%. 44 strains of Shigella had different levels of resistance to 14 of the 15 antibiotics. 93.18% of the strains were multidrug resistant. Conclusion The Shigella in Shandong province is dominated by serogroups of Shigella flexneri and Shigella sonnei, with high virulence gene carrying rate, clustering distribution and severe antibiotic resistance. It is necessary to strengthen the monitoring on serotype, traceability and antibiotic resistance of Shigella in Shandong province.

Clinical and epidemiological characteristics of laboratory confirmed cases of severe fever with thrombocytopenia syndrome infection in Shandong Province, China / 中国人兽共患病学报

We investigated the clinical and epidemiological characteristics of laboratory confirmed cases of severe fever with thrombocytopenia syndrome (SFTS) in Shandong Province,China.A descriptive epidemiological method combined with case investigation was used in this study.Cases information were collected by standard questionnaire and analyzed by Epidata3.1,SPSS 17.0 and ArcGIS10.0 software.Results showed that a total of 154 cases were analyzed and the case fatality rate was 7.1％.Epidemic peak was from May to October,the high incidence areas were located in the middle and east hilly areas of Shandong Province.The characteristic of SFTS cases were farmers (93.5％),and the age was over 40 years.Most of them were living in the hilly areas (85.7 ％),and had outdoor activities within the previous 2 weeks prior to fever onset (83.8 ％).The 16.8％ of them had tick bites history.Tick carrying rates of sheep,cattle,dogs and cats were 66.7％,40％,34.3％ and 12.5％,respectively.Directly contact with bloody secretion of SFTS death cases can be infected with the disease.Major symptoms include high fever (98.1％),anorexia (90.9 ％),fatigue (53.3％),thrombocytopenia (73.4 ％) and leukocytopenia (60.4 ％).The 35.7 ％ cases need to go through more than three referrals for treatment,the interval time between onset and diagnosis was 5 days(3-15),only hospitals above county level can make the correct diagnosis of the disease.Compared with survival patients,the death cases were elderly patients (t =2.03,P=0.044) and with bleeding performance (x2 =13.09,P＜0.01).In conclusion,SFTS is a severe disease with high mortality.Living hilly environment,doing agricultural labor,feeding animals,tick carrying rates of animals and direct contacting with bloody secretion of deaths maybe possible risk factors.To reduce morbidity and mortality of SFTS,measures should be carried out to propagandize the basic knowledge for SFTS prevention and control and to improve the medical treatment skills of doctors in the epidemic foci.

The status of awareness, treatment and control of hypertension in adult population in Shandong province in 2011 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To identify the status of awareness, treatment and control of hypertension in adult population in Shandong province in China.</p><p><b>METHODS</b>A total of 15 350 representative subjects aged 18 to 69 in Shandong province were selected with multistage stratified and clustered sampling design. Questionnaire investigation and physical examination including measurement of blood pressure, height and weight, were taken for all of them. The prevalence was estimated by weighted SURVEYFREQ model.</p><p><b>RESULTS</b>In Shandong province, 34.5% of the hypertensive patients were aware of their high blood pressure (31.1% in male, 38.5% in female), 27.5% of them were taking antihypertensive medications (24.1% in male, 31.7% in female), and 14.9% of them (13.7% in male, 16.4% in female) were under control for their blood pressure (<140/90 mm Hg).</p><p><b>CONCLUSION</b>The rates of awareness, treatment and control of hypertension in adult hypertensive population in Shandong province, China were low, and it is urgently needed to take steps for intervention and control for hypertension prevention, particularly in rural areas.</p>

Establishment of minireplicon system for severe fever with thrombocytopenia syndrome bunyavirus / 病毒学报

Severe fever with thrombocytopenia syndrome bunyavirus is a newly emerging virus in China, enveloped with a tripartite, single-stranded RNA genome of negative polarity. The regulatory elements for viral transcription and replication, as well as encapsidation and packaging signals, are thought to be located within these noncoding regions (NCRs). The terminal nucleotides are genus specific and highly conserved. The function of the remaining nucleotides of the NCRs is still not well understood. In this study, we developed the plasmid-driven RNA polymerase I minireplicon system for SFTSV firstly, using reporter genes GFP and luciferase. The function of the noncoding regions of the three Bunyaviridae RNA segments (L, M, S) in transcription was analyzed. Reporter genes are successfully expressed in SFTSV minireplicon system. Our results suggest that the NCRs of SFTSV from all three segments contain the necessary signals to initiate transcription. Quantitative detection of the luciferase expression level shows that promoter activity in the three segments is different.

Isolation, identification and characterization of SFTS bunyavirus from ticks collected on the surface of domestic animals / 病毒学报

To understand the maintenance and transmission of SFTS virus, the potential vector ticks were collected from sheep, cattle and dogs in the endemic areas of SFTSV in Shandong Province. Among the collected ticks, the dominant species was H. longicornis ticks. Real-time PCR for RNA detection, virus isolation and characterization, genomic sequencing, phylogenetic and antigenic analysis were performed in this investigation. The results showed that the SFTS viral RNA was detected in 2.14% H. longicornis, and a SFTS virus was isolated from one of viral RNA positive ticks collected from sheep. Whole genome analysis of the SFTSV isolates with 11 human-origin SFTS virus revealed a highly pairwise similarity, and the growth curve analysis showed nearly identical in virus yield and the dynamic of virus reproduction compared to human derived viral isolates. Immunofluorescence and neutralization test showed identical serological reaction character of the two different origin viral strains. In this study, the characters of a SFTSV isolate was firstly described, which suggested that the tick species H. longicornis acting important vector role in the transmission of SFTS virus.

Study of molecular subtypes of biotype 1A Yersinia enterocolitica in Shandong province from 2008 to 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the molecular subtypes of 73 strains of Yersinia enterocolitica biotype 1A isolated in Shandong province by PFGE, and thereby to analyze the relationship between PFGE typing and biological characteristics.</p><p><b>METHODS</b>Seventy-three strains of Yersinia enterocolitica biotype 1A were isolated from animal feces and meat products in Gaomi city and Wulian county in Shandong province from 2008 to 2009. Motility test, serum agglutination and virulent genes detection by PCR were used to learn the biological characteristics of the isolated strains. The molecular subtypes were determined by PFGE, whose relationships with motility, serotypes and virulent genotypes were also analyzed.</p><p><b>RESULTS</b>Out of the 73 strains of Yersinia enterocolitica, 5 showed medium-active motility while the other 68 showed well-active motility. The dominated serotypes were O:5(17/73) and O:8(14/73), followed by O:9(5/73) and O:7, 8(1/73), and there was no O:3 serotype found. Meanwhile, 36 strains couldn't be serotyped. All the strains were negative with the gene ail, ystA, yadA and virF, yet the positive rate of ystB gene was 72.6% (53/73). The 73 strains of Yersinia enterocolitica isolated could be subtyped into 54 PFGE patterns (K6GN11SD0001-K6GN11SD0054), most of which only had 1 or 2 isolated strains, and no pattern was dominant. The strains in the same or similar cluster were from different hosts; each serotype and toxic genotype scattered in the clustering trees, without specific correlation with PFGE subtypes. 4 out of 5 strains, which showed medium-active motility, belonged to one branch, with the similarity coefficient at 80.9% - 100.0%; while all the toxic genotype belonged to type B.</p><p><b>CONCLUSION</b>Biotype 1A Yersinia enterocolitica has many clones, whose PFGE types had relations with motility, but no relations with virulent genotype and host.</p>

Antibiotic resistance and molecular typing of Listeria monocytogenes from foods in Shandong province from 2009 to 2010 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To know the antibiotic resistance and molecular characteristics of Listeria monocytogenes in Shandong province and to study the relationship between antibiotic resistance phenotypes and genome types.</p><p><b>METHODS</b>From 2009 to 2010, a total of 80 Listeria monocytogenes isolates were collected from raw meat, cooked meat, aquatic products and other foods in 6 cities of Shandong province. The antibiotic susceptibility was measured by broth microdilution method, PFGE was performed for molecular typing and the relationship between antimicrobial resistance and PFGE patterns was analyzed.</p><p><b>RESULTS</b>16.25% (13/80) of the isolates were drug-resistant. Imipenem resistance was the most prevalent (12.50%, 10/80), followed by tetracycline and doxycycline (3.75%, 3/80 and 2.50%, 2/80). A total of the 80 isolates were subtyped into 9 antibiotic resistance patterns and 34 PFGE types which were largely dominated by the type 17 and 29. Antibiotic resistance pattern A corresponded to 79.41% (27/34) of PFGE types.</p><p><b>CONCLUSION</b>The antibiotic resistance of Listeria monocytogenes in Shandong province is serious from 2009 to 2010 and there is no correlation between PFGE types and antibiotic resistance patterns.</p>

Etiological and molecular characteristics of pandemic influenza A (H1N1) virus during 2009-2010 in Shandong Province / 病毒学报

To isolate and identify the influenza virus in Shandong Province in 2009-2010 and analyze the genetic characteristics of hemagglutinin and neuraminidase gene, further study the variation of gene. A total of 17 126 nasopharyngeal swabs from fever patients were collected and detected by real time quantitative RT-PCR method. The results showed 4004 samples were pandemic influenza A (H1N1) virus positive, with an overall positive rate as 23.38%. The positive samples were incubated and cultured in MDCK cells. The HA and NA genes of isolated pandemic influenza A(H1N1) virus were sequenced, the homology analysis of the HA and NA genes showed an average of 96.9%-99.3% and 99.1%-99.6% sequence identity, respectively, compared with WHO-recommended vaccine strain. The genetic evolution and amino acid substitutions were performed with Mega 4.0 Software. Twenty one amino acids were changed in HA protein, of which 11 were located in the antigenic site; Sixteen amino acids were changed in NA protein, which didn't lead to the changes of enzyme sites. Furthermore, one glycosylation site of HA protein and NA protein were changed respectively. No H275Y mutation in NA protein was found. The results showed that the HA and NA genes of the epidemic strains were highly homologous, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process. All the strains were sensitive to oseltamivir.

Analysis of etiology of four pandemic influenza A (H1N1) virus outbreaks in Shandong province, in 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To isolate and identify the influenza virus that caused four influenza-like-illness outbreaks in Jining city of Shandong Province in 2009 and analyze the genetic characteristics of hemagglutinin (HA) and neuraminidase (NA) gene, the variation of these genes were studied.</p><p><b>METHODS</b>34 nasopharyngeal swabs from fever patients of four influenza-like-illness outbreaks were collected and diagnosed by real time quantitative RT-PCR method. The positive samples were incubated and cultured for virus. HA and NA genes of isolated pandemic influenza A (H1N1) virus were sequenced, the homology analysis was done with DNAStar software and the genetic evolution and amino acid substitutions were performed with Mega 4.0 software. The sequences were compared with WHO recommended vaccine virus, native reference virus.</p><p><b>RESULTS</b>Seventeen of 34 nasopharyngeal swabs were positive, 11 pandemic influenza A (H1N1) viruses were isolated and HA and NA genes of 7 strains were sequenced. Phylogenetic analysis for hemagglutinin and neuraminidase gene of Shandong outbreak strains showed that there were 98.4% - 99.6% and 99.2% - 100.0% sequence identity. Compared with WHO-recommended vaccine strain, the reference virus in mainland China strain, eleven amino acids were changed for HA protein, including position 38, 40, 56, 90, 100, 145, 172, 173, 220, 303 and 338, and 38, 40, 303 of HA protein were located in the antigenic determination C cluster, 172, 173 in the D cluster, 56 in the E cluster, site 40 of HA protein were glycosylated. In NA protein, seven amino acids were changed, including position 80, 106, 241, 248, 351, 369 and 386, site 40 of NA protein were glycosylated. No mutations of 275 in NA protein were found.</p><p><b>CONCLUSION</b>The HA and NA genes of the epidemic strains showed high homology, some mutations in the HA and NA proteins were found, the antigenic site and glycosylation site of some strains were changed during the epidemic process.</p>

Development of TaqMan real-time PCR in detection of Aeromonas hydrophila / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a TaqMan real-time PCR for the detection of Aeromonas hydrophila.</p><p><b>METHODS</b>The conserved region of major adhesion gene of Aeromonas hydrophila (aha) was used to design primers and TaqMan probe. A total of six concentration gradients for forward and reverse primers ranging from 200 -700 nmol/L were chosen, and four concentration gradients for probe ranging from 100 - 400 nmol/L were chosen. And then the concentration of primers and probe were optimized by ANOVA of completely randomized design respectively. The specificity of the established method was evaluated by using bacteria as contrast, including 45 strains Vibrio cholerae, 20 strains Vibrio parahaemolyticus, 10 strains Vibrio fluvialis, 4 strains Vibrio mimicus, 5 strains Vibrio vulnificus, 1 strain Vibrio alginolyticus, 1 strain Vibrio furnissii, 5 strains Salmonella, 10 strains Shigella and 2 strains Plesiomonas shigelloides. The sensitivity, bacterial sensitivity and DNA sensitivity included,were evaluated. The stool of healthy people was contaminated by Aeromonas hydrophila artificially, and the ability of the established TaqMan real-time PCR system for detection of Aeromonas hydrophila was also evaluated.</p><p><b>RESULTS</b>The cycle threshold (Ct) value deserved from 6 groups of primers concentration gradient was (x +/- s):20.69 +/- 0.33, 20.72 +/- 0.21, 20.81 +/- 0.12, 20.74 +/- 0.12, 20.51 +/- 0.16 and 20.69 +/- 0.11, respectively, and the concentration of forward primer and reverse primer was determined to be 200 nmol/L (F = 1.33, P = 0.28). The Ct value deserved from 4 groups of probe concentration gradient was (x +/- s):20.56 +/- 0.08, 20.82 +/- 0.05, 20.82 +/- 0.11 and 20.93 +/- 0.09, respectively, and the concentration of probe was determined to be 100 nmol/L (F = 5.26, P = 0.01). The bacterial sensitivity and DNA sensitivity were 80 CFU/ml and 100 fg/microl respectively, and the sensitivity to detect Aeromonas hydrophila from stool was 8 x 10(3) CFU/ml, which might be 8 CFU/ml after 8 hours' enrichment. No amplification was observed in the templates of other bacterial.</p><p><b>CONCLUSION</b>The TaqMan real-time PCR method targeting the aha gene of Aeromonas hydrophila had a high sensitivity and specificity and might be used to detect Aeromonas hydrophila from pure bacterial and stool rapidly.</p>

Cloning and sequence analysis of envelope glycoprotein G2 gene of hantavirus in Shandong province / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the cloning vector of glycoprotein G2 gene of hantavirus (HV), to analyze the sequence of G2 gene by the phylogenetic tree, and to study the differences among glycoprotein G2 genes from the world around.</p><p><b>METHODS</b>Envelope glycoprotein G2 gene was amplified from four specimens of Shandong province by RT-PCR, and the product recombined into the PMD-18T vector. The clones that carry the G2 gene were identified. After sequencing, the gene sequence was handled with the software DNASTAR, compared with 24 strains worldwide and the phylogenetic tree was drawn.</p><p><b>RESULTS</b>HV G2 gene was amplified by RT-PCR from 4 specimens, named GM04-38.G2, ZB8.G2, JUN5-14.G2, RCH5.G2, respectively. The map of the phylogenetic tree showed that all the 4 strains belonged to SEO-type hantavirus. The analysis of the sequence showed that all the four HV strains had the highest rates of homology with Z37 strain. The sequence homology of SEO-type HV strains was from 82.3% to 99.8%.</p><p><b>CONCLUSION</b>The four cloning vectors containing the glycoprotein G2 genes were successfully constructed. Envelope glycoprotein G2 gene of four specimens from Shandong province had high homology rates.</p>