Molecular typing of Leptospira interrogans strains isolated from Rattus tanezumi in Guizhou Province, Southwest of China.

OBJECTIVE: To identify and type three leptospires isolated from Rattus tanezumi in Guizhou Province by using three molecular techniques (PFGE, MLVA, and MLST), reveal the molecular characteristic of causative agents of local leptospirosis and evaluate these three molecular methods based on their detection resolution and efficiency. METHODS: Three Leptospira strains were isolated from the kidney of Rattus tanezumi and cultured with EMJH medium. PFGE, MLVA, and MLST assays were applied to type the three strains isolated from Rattus tanezumi in Guizhou Province. RESULTS: PFGE, MLVA, and MLST typing showed that the three leptospiral isolates matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai. The findings of the genotyping methods were consistent. MLVA and MLST defined genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes, and the findings of molecular typing were also consistent with those of traditional techniques. CONCLUSION: Three leptospiral isolates from Guizhou Province matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai, and PFGE, MLVA, and MLST, as reliable molecular techniques for identifying and typing of Leptospira interrogans, would contribute to the active surveillance, outbreak investigation and source tracking for leptospirosis in Guizhou Province.

[Analysis of the genetic differences in the nucleoprotein between rabies virus and its vaccine strains in Guizhou province from year 2005 to 2010].

OBJECTIVE: This study was to explore the differences in the nucleoprotein gene between rabies virus (RABV) and its vaccine strains in Guizhou province from year 2005 to 2010. METHODS: Samples from 4 rabies patients and cerebral tissue samples of 28 rabies infected dogs were collected from different districts in Guizhou province between year 2005 and 2010. Direct Immunofluorescence Assay (DFA) and RT-nested PCR assay were applied to detect the overall length of N gene sequence. Meanwhile, based on the comparison between the homology and phylogenetic tree, the differences in N gene sequence between the prevalent RABV and the RABV vaccine strains collected from NCBI database in these years. RESULTS: According to DFA and RT-nested PCR assay, the antigen and nucleic acid of the 21 dogs and 4 human samples were both confirmed positive; whose full length of N gene sequences were both 1353 bp. The homological analysis showed that the 25 strains of RABV virus and the RABV type I virus stored by GenBank database shared a high homology in N gene nucleotide and amino acid sequences, which were 89%-100% and 98%-100%, respectively. Besides, the homology between the 25 strains of RABV virus and its vaccines in nucleotide and amino acid sequences were separately 86%-95% and 96%-100%. The N gene of vaccines for livestock shared the highest homology with HEP-Flury strain in the nucleotide and amino acid, which were 88%-89% and 98%-99%, respectively. The vaccines for human use showed its greatest homology with the CTN strain in nucleotide (86%-100%) and amino acid (96%-100%). The phylogenetic tree analysis indicated that the 25 strains of RABV virus, RABV type I virus and the CTN vaccine strains constituted one individual cluster, which was least different from the CTN vaccine for human use. CONCLUSION: The prevalent RABV virus, the vaccine HEP-Flury for livestock and the vaccine CTN for human use were found to be highly similar in N gene expression in Guizhou province from 2005 to 2010.

Molecular Detection of Tick-borne Pathogens in Ticks Collected from Hainan Island, China.

Pathogens like bacteria and protozoa, which affect human and animal health worldwide, can be transmitted by vectors like ticks. To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province, China, 285 adult hard ticks belonging to two species [ Rhipicephalus sanguineus ( sensu lato): 183, 64.21% and Rhipicephalus microplus: 102, 35.79%] from dogs, cattle, and goats were collected. Microbial families were identified in these ticks by amplifying the 18S rRNA, 16S rRNA ( rrs), citrate synthase ( gltA), and heat shock protein ( groEL) genes. Our data revealed the presence of four recognized species and two Candidatus spp. of Anaplasmataceae and Coxiellaceae. In sum, these data reveal an extensive diversity of Anaplasmataceae bacteria, Coxiellaceae bacteria, Babesiidae, and Hepatozoidae in ticks from Hainan Island, highlighting the need to understand the tick-borne pathogen infection in local animals and humans.

Genetic analysis of group A streptococcus isolates recovered during acute glomerulonephritis outbreaks in Guizhou Province of China.

In this study, 68 group A streptococcus (GAS) isolates associated with two outbreaks of acute glomerulonephritis (AGN) in China were analyzed by emm typing. A total of 11 different emm types were identified. Analysis of emm type distribution suggested that AGN outbreaks in two counties were caused by emm60.1- and emm63.0-type GAS. These two types were further characterized by pulsed-field gel electrophoresis, multilocus sequence typing, sof sequence typing, and PCR-based identification of streptococcal pyrogenic exotoxin A, B, and C (speA, speB, and speC) genes. In antimicrobial susceptibility tests, all outbreak strains were resistant to erythromycin and tetracycline, and the rates of resistance of nonoutbreak strains to the two antibiotics were 63.6% and 90.9%. This study is also the first to report a nephritogenic M63 GAS strain.

Serological Survey of Zika Virus in Humans and Animals in Dejiang Prefecture, Guizhou Province, China.

OBJECTIVE: The current outbreak of Zika virus (ZIKV) poses a severe threat to human health. Two ZIKV strains were isolated from mosquitoes collected from the Dejiang prefecture in China in 2016, which was the first isolation of ZIKV in nature in China. METHODS: In this study, serum samples were collected from 366 healthy individuals and 104 animals from Dejiang prefecture in 2017, and the plaque reduction neutralization test (PRNT) was used to evaluate the seroprevalence of ZIKV. RESULTS: None of the 366 residents from whom the samples were collected were seropositive for ZIKV. None of the 11 pigs from whom the samples were collected were seropositive for ZIKV, while 1 of 63 (1.59%) chickens and 2 of 30 (6.67%) sheep were seropositive for ZIKV. CONCLUSION: The extremely low seropositivity rate of ZIKV antibodies in animals in the Dejiang prefecture, Guizhou province in this study indicates that ZIKV can infect animals; however, there is a low risk of ZIKV circulating in the local population.

Novel genospecies of Borrelia burgdorferi sensu lato from rodents and ticks in southwestern China.

By using multilocus sequence analysis, five Borrelia valaisiana-related strains isolated from rodents and ticks in southwestern China were eventually classified as a new genospecies of B. burgdorferi sensu lato rather than B. valaisiana. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-related strains from eastern Asia.

Feasibility study of enforcing immunization certificate check before primary school or kindergarten enrollment in Guizhou Province, China.

OBJECTIVE: To study the feasibility of enforcing immunization certificate check before children enroll in primary schools or kindergartens in Guizhou Province. METHODS: Quantitative and qualitative studies were conducted. The multi-stage and cluster sampling approach was adopted for the quantitative part of the study. A questionnaire was designed and 996 children and their keepers were interviewed. Principals, doctors or teachers of the primary schools, directors and child care nurses of kindergarten, and staff of immunization agencies were invited to take part in 12 focus group discussions; meanwhile, face-to-face individual in-depth interviews with 16 officials of the Health, Education and Governmental Departments at various levels were conducted. RESULTS: The total number of subjects was 996. 16.7% of the children in the study completed all the procedures of the National Immunization Programme. 34.3% of them had immunization certificates while the remainder 44.7% registered in immunization agencies. Factors, including the migrant children, doubt about vaccine efficiency, mother's occupation and educational background, knowledge of the National Immunization Programme on targeted vaccines, played an important role in obtaining or not immunization certificates. 95% of the keepers interviewed thought the immunization certificates were useful; 94.8% of them considered the check was critical while only 3.6% of them thought it unnecessary. The first reason from those who found it unnecessary was that they feared that repeated immunization might affect their children's health. The second reason was the cost of immunization, which some of them could not afford to pay. However, the Health Department expressed a favorable attitude to the checking scheme. Though the Education Department agreed that the scheme was essential, they worried that it would affect the enrollment rate. CONCLUSION: In spite of the difficulty in administering immunization certificate check, the effort would be rewarding for raising the immunization coverage rate among the children in Guizhou Province.

Extensive diversity of coronaviruses in bats from China.

To help reveal the diversity and evolution of bat coronaviruses we collected 1067 bats from 21 species in China. A total of 73 coronaviruses (32 alphacoronaviruses and 41 betacoronaviruses) were identified in these bats, with an overall prevalence of 6.84%. All newly-identified betacoronaviruses were SARS-related Rhinolophus bat coronaviruses (SARSr-Rh-BatCoV). Importantly, with the exception of the S gene, the genome sequences of the SARSr-Rh-BatCoVs sampled in Guizhou province were closely related to SARS-related human coronavirus. Additionally, the newly-identified alphacoronaviruses exhibited high genetic diversity and some may represent novel species. Our phylogenetic analyses also provided insights into the transmission of these viruses among bat species, revealing a general clustering by geographic location rather than by bat species. Inter-species transmission among bats from the same genus was also commonplace in both the alphacoronaviruses and betacoronaviruses. Overall, these data suggest that high contact rates among specific bat species enable the acquisition and spread of coronaviruses.

[Analysis on molecular epidemiological features of enterovirus type 71 in Guizhou Province, 2008-2011].

To study the genotype and molecular epidemiological features of enterovirus type 71 (EV71) in Guizhou Province. The hand-foot-mouth disease(HFMD) patients since 2008 in Guizhou Province were surveyed. EV71 RNA positive samples, including mild cases, death cases and cases of serious conditions, were used for subsequent cell culture and sequencing of VP1 complete gene. Genotypes were compared between domestic and foreign epidemic strains. The results showed that the main pathogenwas EV71 subtype C4a in 2008, 2009 and 2011. The nucleotide homology was 95. 3 - 99. 7% among 109 sequenced strains isolated in Guizhou Province. Guizhou strains were more similar to the representative strains from neighbor Province or City, including Shandong, Shanghai, Nanjing, Jilin and Ningbo. There was no distinct sequence difference among the isolates from either of death, mild case or case of serious condition at the nucleotide and amino acid levels. The diversity of nucleotide sequence of isolates was less within same region and year than those trans-regional strains or strains in different year.

[Identification and genetic characteristics on the bacteria isolate from a case of human Brucellosis in Guizhou province].

OBJECTIVE: A suspected Brucella (B.) strain(GZZA), isolated from a case of anti-Brucella antibody positive patient was identified and its' genetic characteristics was analyzed, to provide etiologic basis for the confirmation of patient in Guizhou province. METHODS: Conventional methods and polymerase chain reaction(PCR)were used to identify the bacteria strain, with genetic characteristics analyzed by MLVA-16. RESULTS: The bacteria strain was identified as B. melitensis biovar 3 under the conventional and PCR methods. Results from the MLVA-16 analysis indicated that the bacteria strain was closely clustered with B. melitensis biovar 3, and differences of repeated numbers at VNTR loci bruce42, bruce04, bruce09 and bruce16 were also displayed. CONCLUSION: Both traditional and molecular methods to identify one bacteria strain isolated from the human patient as B. melitensis biovar 3 and the genetic characteristics of the strain was closely related to that of B. melitensis biovar 3. Differences of repeated numbers at part of VNTR loci were also showed. The results of this study provided etiologic evidences for the confirmation of Brucella infection of the patient, also providing scientific basis for the control and prevention of Brucellosis in Guizhou province.

[Fever monitoring program in areas with high incidence of typhoid and paratyphoid fever in Guizhou province].

OBJECTIVE: To understand the incidence rates of both typhoid fever and paratyphoid fever in the high prevalent areas of Guizhou province so as to provide evidence for the development of programs on comprehensive intervention and effectiveness evaluation. METHODS: Six townships in Pingba county were selected as intervention areas while six townships in Kaiyang county were taken as control. All hospitals and clinics were classified into A, B and C types according to its level and the capacity of the blood culture. Surveillance on typhoid and paratyphoid fever was conducted based on all population and all hospitals, clinics and county CDCs among the patients with unknown fever. RESULTS: In the surveillance area in those two counties, there were 12 944 blood samples from patients with unknown fever which have been tested and cultured. Among them, 200 strains of Salmonella including 16 typhoid strains, 184 paratyphoid A strains were identified, with the total positive rate as 1.55%. The positive rate before the intervention program was higher than the after. The detection rate was 1.91% in the type A hospitals. 39 strains of Salmonella have been cultured from 2039 samples which accounting for 19.50% (39/200) of the total strains. 4315 blood samples were cultured at the 'Class B' sites which isolated 82 strains of Salmonella, accounting for 41.00% (82/200), with a detection rate as 1.90%. 6590 samples were cultured at the 'Class C' sites, which identified 79 strains of Salmonella, accounting for 39.50% (79/200), with a detection rate as 1.20%. The detection rate was much higher before the use of antibiotics than after using them (P < 0.05). The annual peak time of positive detection was in spring and fall. The outbreaks or epidemics often appeared in the same places, with farmers, students as the high-risk populations. Symptoms of both typhoid and paratyphoid fever were not typical. CONCLUSION: Typhoid and paratyphoid monitoring programs which covered primary health care institutions in the high incidence area seemed to be effective in reflecting the pictures as well as the burden of both typhoid and paratyphoid.

A foodborne outbreak of Aeromonas hydrophila in a college, Xingyi City, Guizhou, China, 2012.

BACKGROUND: On 12 May 2012, over 200 college students with acute diarrhoea were reported to the Guizhou Center for Disease Control and Prevention. We conducted an investigation to identify the agent and mode of transmission and to recommend control measures. METHODS: A suspected case was a person at the college with onset of ≥ two of the following symptoms: diarrhoea (more than three loose stools in 24 hours), abdominal pain, vomiting or fever (> 37.5C) between 6 and 15 May 2012. A confirmed case also had a positive Aeromonas hydrophila culture from a stool sample. A retrospective-cohort study of 902 students compared attack rates (AR) by dining place, meals and food history. We reviewed the implicated premise, its processes and preparation of implicated food. RESULTS: We identified 349 suspected cases (AR = 14%) and isolated Aeromonas hydrophila from three stools of 15 cases. Students who ate in cafeteria A were more likely to be ill compared to those eating in other places (relative risk [RR]: 3.1, 95% confidence interval [CI]: 2.0-4.8). The cohort study implicated cold cucumber (RR: 2.6, 95% CI: 2.0-3.3) and houttuynia dishes (RR: 1.8, 95% CI: 1.4-2.3). Environmental investigation showed that vegetables were washed in polluted water from a tank close to the sewage ditch, then left at 30 °C for two hours before serving. The Escherichia coli count of the tank was well above the standard for drinking-water. CONCLUSION: This outbreak of Aeromonas hydrophila was most probably caused by salad ingredients washed in contaminated tank water. We recommended enhancing training of foodhandlers, ensuring tanks and sewerage systems comply with appropriate standards and adequate monitoring of drinking-water sources.

[Analysis on nucleoprotein gene sequence of 25 rabies virus isolates in Guizhou Province, China].

To analyze 25 nucleoprotein gene (N gene) sequences of rabies viruses circulating in Guizhou province during 2005-2010, China, and to explore the epidemic characteristics and the probable mutant of rabies in Guizhou Province. Rabies virus RNA in human brain tissues, human saliva, and domestic dog brain tissues derived from different prefectures of Guizhou Province were detected with RT-nested PCR, and the amplified products were then sequenced. Bioinformatics software was used to determine the genetic characteristics of these rabies viruses. The sequences of N gene of 25 Guizhou provincial isolates were identical with homogeny between 97.5% - 99.3% and 98.4% - 99.8% at nucleotide and deduced amino acid level, respectively, while the identities between them and isolated strains from other province of China were 88% - 99.1% and 88% - 99.7%. There were several amino acid substitutions in the nucleoprotein of 25 Guizhou isolates compared with the known genotype 1 isolates. The analysis of phylogenetic tree of 25 Guizhou isolates was demonstrated to be genetically divided into two groups, indicating that the virus presented a unique characteristics in geographic distribution and in a time dependent-manner. And phylogenetic tree of 25 Guizhou isolates and 7 genotype 1 strains isolated from other Province of China was also divided into two groups, which were further composed of several subgroups, respectively. From these observations, the rabies viruses derived from Guizhou province were still genotype 1. These isolates of rabies virus were diverged from the strains isolated from other provinces in both gene sequences and deduced amino acid sequences, and these divergences were characterized in geographic distribution and in a time-dependent manner.

Association of bone morphogenetic protein 4 gene polymorphisms with nonsyndromic cleft lip with or without cleft palate in Chinese children.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common congenital anomalies in humans. The pathogenesis of nsCL/P involves both genetic and environmental factors. On the basis of linkage data suggesting that 14q21-24 is one of the chromosomal regions that affects nsCL/P and data locating the BMP4 gene to 14q22-23, we performed a case-control study to evaluate whether BMP4 538T/C polymorphism, resulting in an amino acid change of Val/Ala (V152A) in the polypeptide, is associated with nsCL/P in a Chinese children population. Genotypes of 184 patients with nsCL/P and 205 controls were detected using a PCR-RFLP strategy. The results showed significant differences in the genotype and allele distribution of 538T/C polymorphisms of the BMP4 gene among the cases and controls. The 538C allele carriers were associated with a significantly increased risk of nsCL/P as compared with the noncarriers (odds ratio = 1.52; 95% confidence interval, 1.13-2.03; p = 0.005). Hence, our results support the hypothesis that this polymorphism contributes to risk of nsCL/P, which suggests that BMP4 538T/C polymorphisms could be used as genetic susceptibility markers of nsCL/P.

[Distribution of hepatitis B virus genotype among population of Dong, Miao minority and Han in Guizhou].

OBJECTIVE: To study the distribution of hepatitis B virus (HBV) genotype in population of Dong, Miao minority and Han in Guizhou. METHODS: S region nucleotides were compared in 127 strains whole sequence of HBV and three restriction enzymes which can be used for genotyping were found by DNA software analysis system. The partial gene fragment of HBV S region was amplified by nested polymerase chain reaction (nPCR). The products were digested with Mbo I, BstN I or BsmA I and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were analysed. The genotypes were determined by nPCR-RFLP in 166 asymptomatic HBV carriers (ASC), including 48 Dong minority, 52 Miao minority and 66 Han subjects. Some of the ASC were also analysed by direct sequencing of PCR products. RESULTS: The nPCR-RFLP method was simple and accurate. Of the 166 ASC, 138 (83.13%) were genotype B, and 28 (16.87%) were genotype C. Of the 48 Dong minority subjects, 47 (97.2%) were genotype B and 1 (2.08%) was type C. Of the 52 Miao minority subjects, 49 (94.23%) were genotype B and 3 (5.77%) were genotype C. Of the 66 Han subjects, 42 (63.64%) were genotype B and 24 (36.36%) were genotype C. There was a statistical significance in the distribution of genotype C between Dong, Miao minority and Han (7.85% vs 36.36% P less than 0.005). CONCLUSION: Genotype B and C exist in Guizhou and genotype B is the major genotype. Genotype C is found more frequently in Han than in Dong and Miao minority.