Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Metagenomic analysis of individual mosquitos reveals the ecology of insect viruses.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Using a meta-transcriptomic approach, we analysed the virome of 2,438 individual mosquitos (79 species), spanning ~4000 km along latitudes and longitudes in China. From these data we identified 393 core viral species associated with mosquitos, including seven (putative) arbovirus species. We identified potential species and geographic hotspots of viral richness and arbovirus occurrence, and demonstrated that host phylogeny had a strong impact on the composition of individual mosquito viromes. Our data revealed a large number of viruses shared among mosquito species or genera, expanding our knowledge of host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, possibly facilitated by long-distance mosquito migrations. Together, our results greatly expand the known mosquito virome, linked the viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the ecology of viruses of insect vectors.

Prevalence of Spotted Fever Group Rickettsia and Candidatus Lariskella in Multiple Tick Species from Guizhou Province, China.

Rickettsiales (Rickettsia spp., Ehrlichia spp., and Anaplasma spp., etc.) are generally recognized as potentially emerging tick-borne pathogens. However, some bacteria and areas in China remain uninvestigated. In this study, we collected 113 ticks from mammals in Guizhou Province, Southwest China, and screened for the Rickettsiales bacteria. Subsequently, two spotted fever group Rickettsia species and one Candidatus Lariskella sp. were detected and characterized. "Candidatus Rickettsia jingxinensis" was detected in Rhipicephalus microplus (1/1), Haemaphysalis flava (1/3, 33.33%), Haemaphysalis kitaokai (1/3), and Ixodes sinensis (4/101, 3.96%), whereas Rickettsia monacensis was positive in H. flava (1/3), H. kitaokai (2/3), and I. sinensis ticks (74/101, 73.27%). At least two variants/sub-genotypes were identified in the R. monacensis isolates, and the strikingly high prevalence of R. monacensis may suggest a risk of human infection. Unexpectedly, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected from Ixodes ovatus (1/4) and I. sinensis (10/101, 9.90%). The gltA and groEL gene sequences were successfully obtained, and they show the highest (74.63-74.89% and 73.31%) similarities to "Candidatus Midichloria mitochondrii", respectively. Herein, we name the species "Candidatus Lariskella guizhouensis". These may be the first recovered gltA and groEL sequences of the genus Candidatus Lariskella.

Diversity of Rickettsiales in Rhipicephalus microplus Ticks Collected in Domestic Ruminants in Guizhou Province, China.

Rhipicephalus microplus ticks are vectors for multiple pathogens infecting animals and humans. Although the medical importance of R. microplus has been well-recognized and studied in most areas of China, the occurrence of tick-borne Rickettsiales has seldom been investigated in Guizhou Province, Southwest China. In this study, we collected 276 R. microplus ticks from cattle (209 ticks) and goats (67 ticks) in three locations of Guizhou Province. The Rickettsia, Anaplasma, and Ehrlichia were detected by targeting the 16S rRNA gene and were further characterized by amplifying the key genes. One Rickettsia (Ca. Rickettsia jingxinensis), three Ehrlichia (E. canis, E. minasensis, Ehrlichia sp.), and four Anaplasma (A. capra, A. ovis, A. marginale, Ca. Anaplasma boleense) species were detected, and their gltA and groEL genes were recovered. Candidatus Rickettsia jingxinensis, a spotted fever group of Rickettsia, was detected in a high proportion of the tested ticks (88.89%, 100%, and 100% in ticks from the three locations, respectively), suggesting the possibility that animals may be exposed to this type of Rickettsia. All the 16S, gltA, groEL, and ompA sequences of these strains are 100% identical to strains reported in Ngawa, Sichuan Province. E. minasensis, A. marginale, and Candidatus Anaplasma boleense are known to infect livestock such as cattle. The potential effects on local husbandry should be considered. Notably, E. canis, A. ovis, and A. capra have been reported to infect humans. The relatively high positive rates in Qianxinan (20.99%, 9.88%, and 4.94%, respectively) may indicate the potential risk to local populations. Furthermore, the genetic analysis indicated that the E. minasensis strains in this study may represent a variant or recombinant. Our results indicated the extensive diversity of Rickettsiales in R. microplus ticks from Guizhou Province. The possible occurrence of rickettsiosis, ehrlichiosis, and anaplasmosis in humans and domestic animals in this area should be further considered and investigated.

Ehrlichia, Coxiella and Bartonella infections in rodents from Guizhou Province, Southwest China.

Rodents are generally recognized to be the reservoir hosts of a great many zoonotic pathogens. In some areas of China, rodent-borne pathogens, as well as the role of rodents in the natural cycle of these pathogens, are still poorly investigated. To increase our knowledge on the distribution and epidemiology of rodent-borne bacterial pathogens, 81 rodent liver samples were collected in three locations of Guizhou province located in Southwest China, and screened for the presence of Ehrlichia, Coxiella, and Bartonella in them. A putative novel Ehrlichia species was identified in 5 Berylmys bowersi samples (100%, 5/5). Its 16S rRNA, gltA, and groEL genes have highest 99.84%, 89.11%, and 98.02% identities to those from known Ehrlichia species, and form distinct clades in the phylogenetic trees. Herein we name it "Candidatus Ehrlichia zunyiensis". Bartonella was tested positive in 8 A. agrarius (striped field mouse), 2 A. chevrieri (Chevrier's field mouse), 1 R. norvegicus (Norway rat), 1 N. confucianus, and 1 N. lotipes, with a total positive rate of 16.05% (13/81). Sequence analysis indicated high genetic diversity in these Bartonella strains. Unexpectedly, two Coxiella strains were identified from the rodents (1 Niviventer confucianus and 1 Mus pahari). Genetic and phylogenetic analysis indicated that both of them are closely related to the Coxiella endosymbiont of ticks. This result supported previous conjectures that vertebrate hosts such as rodents may play a role in the preservation and transmission of Coxiella endosymbiont of ticks.

Epidemiological Investigation and Etiological Analysis of a Cutaneous Anthrax Epidemic Caused by Butchering Sick Cattle in Guizhou, China.

A suspected human cutaneous anthrax epidemic caused by butchering sick cattle occurred in Zhijin County of Guizhou Province, Southwest of China, in 2016. Epidemiological investigation and etiological analysis were performed to provide a scientific basis for the source tracking of the epidemic. The epidemic was epidemiologically investigated, and skin blister samples collected from patients and soil samples collected from the butchering spots were used for Bacillus anthracis isolation. The suspicious B. anthracis isolates were identified using conventional methods and PCR, followed by genotyping using multiple-locus variable-number tandem repeats (VNTRs) analysis (MLVA-15) and canonical single-nucleotide polymorphism (canSNP). The genetic relationship of epidemic strains and isolates collected from other regions was analyzed. Epidemiological investigation results showed that the patients may be infected by B. anthracis during butchering sick cattle. Two suspected B. anthracis strains were isolated from blood samples and blister fluids, respectively. Conventional methods identified the two suspected isolates as B. anthracis, while PCR results showed that anti-protective antigen (PA) and capsule (CAP) gene were positive in the two isolates. MLVA-15 showed that the MLVA profiles of the two isolates were 9-20-12-53-16-2-8-8-8-4-4-4-4-10-4, which is different from the MLVA profiles of representative strains from other regions. CanSNP analysis showed that the two strains belonged to cluster A.Br.001/002. Clustering analysis and minimum spanning tree (MST) demonstrated that the two isolates were clustered with strains previously isolated from Guizhou Province. The results indicated that B. anthracis was the pathogen for this epidemic, and the patients were infected during butchering the sick. The genetic characteristics and the relationship of the B. anthracis isolates to strains from other regions indicated that the epidemic was a local occurrence.

Capacity assessment of the health laboratory system in two resource-limited provinces in China.

BACKGROUND: Strong laboratory capacity is essential for detecting and responding to emerging and re-emerging global health threats. We conducted a quantitative laboratory assessment during 2014-2015 in two resource-limited provinces in southern China, Guangxi and Guizhou in order to guide strategies for strengthening core capacities as required by the International Health Regulations (IHR 2005). METHODS: We selected 28 public health and clinical laboratories from the provincial, prefecture and county levels through a quasi-random sampling approach. The 11-module World Health Organization (WHO) laboratory assessment tool was adapted to the local context in China. At each laboratory, modules were scored 0-100% through a combination of paper surveys, in-person interviews, and visual inspections. We defined module scores as strong (> = 85%), good (70-84%), weak (50-69%), and very weak (< 50%). We estimated overall capacity and compared module scores across the provincial, prefecture, and county levels. RESULTS: Overall, laboratories in both provinces received strong or good scores for 10 of the 11 modules. These findings were primarily driven by strong and good scores from the two provincial level laboratories; prefecture and county laboratories were strong or good for only 8 and 6 modules, respectively. County laboratories received weak scores in 4 modules. The module, 'Public Health Functions' (e.g., surveillance and reporting practices) lagged far behind all other modules (mean score = 46%) across all three administrative levels. Findings across the two provinces were similar. CONCLUSIONS: Laboratories in Guangxi and Guizhou are generally performing well in laboratory capacity as required by IHR. However, we recommend targeted interventions particularly for county-level laboratories, where we identified a number of gaps. Given the importance of surveillance and reporting, addressing gaps in public health functions is likely to have the greatest positive impact for IHR requirements. The quantitative WHO laboratory assessment tool was useful in identifying both comparative strengths and weaknesses. However, prior to future assessments, the tool may need to be aligned with the new WHO IHR monitoring and evaluation framework.

Correction to: Genetic characterization of Bacillus anthracis in Guizhou Province, Southwest of China.

ERRATUM: Upon publication of the original article (1) it was highlighted by the authors that a grant awarded to support the research work of the study was missed in the acknowledgements. It should also be acknowledged that the grant titled "Genotyping and Molecular Epidemiological Characteristic of Bacillus anthracis in Guizhou Province" awarded by the Program of Natural Science Foundation of Guizhou Province (Grant No. Qian Ke He J Word [2015] 2084)also contributed to the resources for this research. This has since been formally noted in this correction article.

Leptospira interrogans infection leads to IL-1ß and IL-18 secretion from a human macrophage cell line through reactive oxygen species and cathepsin B mediated-NLRP3 inflammasome activation.

Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Although there is a large diversity of clinical signs and symptoms, a severe inflammatory response is common to all leptospirosis patients. The mechanism of IL-1ß secretion during Leptospira infection has been previously studied in mouse macrophages. However, the outcome of Leptospira infection is very different in human and murine macrophages, and the mechanisms responsible for IL-1ß secretion in human macrophages had not been investigated. This study therefore examines the effects of Leptospira interrogans infection on inflammasome activation and proinflammatory cytokine expression in human macrophages. Increased mRNA and protein expression of NLRP3 was observed by real time RT-PCR and flow cytometry at 1 h after co-cultivation. Enzyme-linked immunosorbent assay (ELISA) determination showed that IL-1ß and IL-18 are released in the culture supernatants at 1 h after cultivation. The inhibition assay showed that glybenclamide (a K+ efflux inhibitor that blocks NLRP3 inflammasome activation) and N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) and NLRP3 depletion with siRNAs reduced the levels of IL-1ß and IL-18 release. Moreover, the levels of IL-1ß and IL-18 production decreased in CA-074 (a cathepsin B inhibitor) and NAC (an anti-oxidant) pretreated human macrophages, compared to untreated controls. This study suggests that L. interrogans infection leads to reactive oxygen species (ROS)- and cathepsin B-dependent NLRP3 inflammasome activation, which subsequently mediates caspase-1 activation and IL-1ß and IL-18 release.

The role of reactive oxygen intermediates in the intracellular fate of Leptospira interrogans in the macrophages of different hosts.

BACKGROUND: Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease. Our previous work showed that leptospires survive and replicate in human macrophages but are killed in murine macrophages. However, the mechanism responsible for the different intracellular fates of leptospires within the macrophages of different hosts remains unclear. RESULTS: The present study demonstrates that infection with Leptospira interrogans caused significant up-regulation of reactive oxygen species (ROS) and superoxide in J774A.1 cells but did so to a lesser extent in THP-1 cells. The up-regulation of ROS and superoxide was significantly inhibited by the NADPH oxidase inhibitor apocynin. The damaged leptospires and remnants of leptospires within membrane-bound vacuoles were significantly inhibited by apocynin in J774A.1 cells but were less inhibited in THP-1 cells. In addition, apocynin significantly prevented damage to leptospires and the co-localization of L. interrogans with lysosomes in J774A.1 cells but did so to a lesser extent in THP-1 cells. Furthermore, the relative fluorescence intensity levels of intracellular leptospires and the viability of the intracellular leptospires increased in apocynin pretreated J774A.1 and THP-1 cells after 2 h of infection. CONCLUSIONS: The present study, based on our previous findings, further demonstrated that ROS contributed substantially to the bactericidal ability of mouse macrophages to kill intracellular leptospires. However, ROS did not contribute as much in human macrophages, which partially explains the different intracellular fates of L. interrogans in human and mouse macrophages.

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.

Mechanism study on a plague outbreak driven by the construction of a large reservoir in southwest china (surveillance from 2000-2015).

BACKGROUND: Plague, a Yersinia pestis infection, is a fatal disease with tremendous transmission capacity. However, the mechanism of how the pathogen stays in a reservoir, circulates and then re-emerges is an enigma. METHODOLOGY/PRINCIPAL FINDINGS: We studied a plague outbreak caused by the construction of a large reservoir in southwest China followed 16-years' surveillance. CONCLUSIONS/SIGNIFICANCE: The results show the prevalence of plague within the natural plague focus is closely related to the stability of local ecology. Before and during the decade of construction the reservoir on the Nanpan River, no confirmed plague has ever emerged. With the impoundment of reservoir and destruction of drowned farmland and vegetation, the infected rodent population previously dispersed was concentrated together in a flood-free area and turned a rest focus alive. Human plague broke out after the enzootic plague via the flea bite. With the construction completed and ecology gradually of human residential environment, animal population and type of vegetation settling down to a new balance, the natural plague foci returned to a rest period. With the rodent density decreased as some of them died, the flea density increased as the rodents lived near or in local farm houses where had more domestic animals, and human has a more concentrated population. In contrast, in the Himalayan marmot foci of the Qinghai-Tibet Plateau in the Qilian Mountains. There are few human inhabitants and the local ecology is relatively stable; plague is prevalence, showing no rest period. Thus the plague can be significantly affected by ecological shifts.

Simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique.

Many viruses can cause respiratory diseases in humans. Although great advances have been achieved in methods of diagnosis, it remains challenging to identify pathogens in unexplained pneumonia (UP) cases. In this study, we applied next-generation sequencing (NGS) technology and a metagenomic approach to detect and characterize respiratory viruses in UP cases from Guizhou Province, China. A total of 33 oropharyngeal swabs were obtained from hospitalized UP patients and subjected to NGS. An unbiased metagenomic analysis pipeline identified 13 virus species in 16 samples. Human rhinovirus C was the virus most frequently detected and was identified in seven samples. Human measles virus, adenovirus B 55 and coxsackievirus A10 were also identified. Metagenomic sequencing also provided virus genomic sequences, which enabled genotype characterization and phylogenetic analysis. For cases of multiple infection, metagenomic sequencing afforded information regarding the quantity of each virus in the sample, which could be used to evaluate each viruses' role in the disease. Our study highlights the potential of metagenomic sequencing for pathogen identification in UP cases.

Molecular Characterization of Shigella sonnei: An Increasingly Prevalent Etiologic Agent of Shigellosis in Guizhou Province, Southwest of China.

BACKGROUND: Shigellosis is a serious problem in Guizhou and Shigella sonnei is an increasingly prevalent etiologic agent of local shigellosis cases. No data, however, are available about the molecular characterization of the local isolates of S. sonnei. We have conducted this study to molecularly characterize the clinical isolates of S. sonnei in Guizhou Province. RESULTS: 76 S. sonnei isolates, including four isolates from 1974-1982 and 72 isolates from 2008-2010, were used for analysis in this study. Pulsed-field gel electrophoresis (PFGE) based on XbaI digestion divided the 76 isolates into 38 PFGE patterns (PT) and 15 PTs were represented by more than one isolates with PT31 (N = 8) containing the most number of isolates, followed with PT2 (N = 6). Multiple-Locus Variable number tandem repeat (VNTR) Analysis (MLVA) based on seven VNTR loci discriminated them into 19 different MLVA types (MTs), and four MTs were represented by more than one isolate with MT4 (N = 39) containing the most number of isolates, followed with MT12 (N = 18). 15 Multilocus sequence typing (MLST) base on 15 loci differentiated the isolates into six sequence types (STs), among which four STs were novel. The most common STs are ST76 (N = 43) and ST116 (N = 25), accounting for 92.1%. Correlation between genetic relationships and geographical origins or isolation years was observed among the isolates studied. Majority of isolates were clustered in accordance with the origin of isolation years based on the genetic data, which were also from similar geographical origins. CONCLUSIONS: Our results revealed the molecular characteristics including the specific genotypes such as four novel STs, clonal relationship, and genetic changes of local isolates from different years, which enhances our understanding of molecular characteristics of S. sonnei and contributes to the prevention and control of shigellosis in Guizhou Province.

[Investigation on the causes of increased hepatitis E cases reported in Guizhou province].

OBJECTIVE: To analyze causes of growing hepatitis E (HE) cases reported in Guizhou province, and probe into existing problems faced by medical institutions in diagnosis of clinical and laboratory-confirmed cases, for the purpose of improving the quality of HE surveillance system. METHODS: Six hospitals reporting greater HE cases from 2007 to 2011 were pinpointed, whose reported cases rose suddenly in 2011. Such cases were investigated by means of impatient medical record review, results of laboratory test and clinician interview. RESULTS: 136 of the 354 reported HE cases investigated were found compliant with the diagnostic criteria of HE with an accordance rate of 38.42%. Difference of the HE diagnostic accordance rate among individual years, hospitals and reporting departments was statistically significant. Such rate of hospital reports was found to be the lowest in 2011, ranging from 0 to 18.18% respectively; HE cases reported by non-infectious departments accounted for 61.30% of total cases reported, with its accordance rate considerably below the infectious departments (8.29%). HE positive cases and HE positive rate in 2011 were significantly higher than that of preceding years. CONCLUSION: Such increase of reported HE cases in 2011 in the province was mostly attributable to more HE laboratory tests made by the hospitals, yet the accordance rates were lower than satisfactory. In this regard, the medical institutions in question were advised to enhance their competency training for HE diagnosis and case report quality.

A returning migrant worker with avian influenza A (H7N9) virus infection in Guizhou, China: a case report.

INTRODUCTION: Human infection with avian influenza A (H7N9) virus was first reported on March, 2013 in the Yangtze River Delta region of China. The majority of human cases were detected in mainland China; other regions out of mainland China reported imported human cases, including Hong Kong SAR, Taiwan (the Republic of China) and Malaysia, due to human transportation. Here, we report the first human case of H7N9 infection imported into Guizhou Province during the Spring Festival travel season in January 2014. CASE PRESENTATION: In early January 2014, a 38-year-old healthy Chinese man, a migrant worker returning from previously H7N9-affected Zhejiang Province, was identified as the first human case of infection with avian influenza A(H7N9) virus in Guizhou Province. He developed fever in Zhejiang at the beginning of January 2014, and returned to Guizhou for the Chinese New Year the next day. He went to seek medical care, but deteriorated rapidly and died on day 8 after his illness onset. The influenza virus A/Guizhou/01502/2014 isolated from the patient had 99% identity with viruses circulating in the Yangtze River Delta region. Selected amino acids substitutions, well-known to be associated with mammalian adaptation, viral replication and drug resistance were similar to other H7N9 viruses circulating in humans. CONCLUSIONS: Epidemiology investigation and laboratory results confirmed it was the first imported case of H7N9 infection in Guizhou Province. This finding further indicated that more human H7N9 cases may be detected in other regions due to frequent travel both domestically and internationally.

Genetic characterization of Bacillus anthracis in Guizhou Province, Southwest of China.

BACKGROUND: Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Guizhou Province is an old foci of anthrax in the southwest of China. Human anthrax has also been frequently reported in Guizhou in recent year. However, there is limited information on the genetic background of local B. anthracis isolates in Guizhou Province. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial genetics. In the present study, we employed Multiple Locus Variable Number Tandem Repeats (VNTR) Analysis (MLVA) assay to analyze the genetic characteristics of B. anthracis strains isolated in Guizhou Province and their relationships to worldwide distributed isolates. RESULTS: A total of 32 isolates of B. anthracis from soil, human, cattle, dog and water of different anthrax epidemics in Guizhou Province from 2006 to 2011 were confirmed with phage lysis test, penicillin inhibition test and PCR. MLVA-8 discriminated them into 28 unique MLVA types (MT G1 - G28), which were novel MTs compared with the previous reports. Cluster tree based on 32 isolates from Guizhou Province and 76 worldwide distributed isolates (30 MTs) showed they were divided into three clusters, designated A, B and C. All the 32 isolates were distributed in cluster A, which were further grouped into A1, A2, A3 and A4 sub-clusters. 32 isolates from Guizhou Province were closely grouped in each of the sub-clusters, respectively. Minimum Spanning Tree (MST) based on the MLVA data showed that the 28 MLVA profiles of isolates from Guizhou Province and 30 MLVA profiles of worldwide distributed isolates formed three clonal complexes (CCs) and ten singletons. CONCLUSIONS: 28 novel MTs of B. anthracis from Guizhou were revealed and their relationships to worldwide isolates were showed. The results will provide important information for prevention of anthrax and also enhances our understanding of genetic characteristics of B. anthracis in China.

Genetic characterization of Shigella flexneri isolates in Guizhou Province, China.

Shigella flexneri is one of the major etiologic causes of shigellosis in Guizhou Province, China. However, the genetic characteristics of circulating isolates are unknown. Phenotypic and molecular profiles of 60 S. flexneri isolates recovered in Guizhou between 1972 to 1982 and 2008 to 2010 were determined. Nine serotypes (1a, 2a, 3a, 1b, 2b, X, Y, 4av and Yv) were identified. Multi-locus sequence typing differentiated the isolates into 20 sequence types (STs); 18 were novel. Four STs, ST 129, ST 100, ST 126 and ST 18, were most abundant, accounting for 65% of the isolates. Thirty-nine NotI-pulsed field gel electrophoresis patterns (pulsotypes, PTs) were observed; eight PTs were represented by more than one isolate with six isolates sharing the PT 13 profile. Multi-locus variable-nucleotide tandem-repeat analysis recognized 44 different types (MTs); seven MTs were represented by more than one isolate and MT 1 was most commonly encountered. Correlation between genetic relationships and serotypes was observed among the isolates studied; the majority of isolates belonging to the same serotype from different years clustered together based on the molecular data. These clustered isolates were also from similar geographical origins. These results enhance our understanding of genetic relationships between S. flexneri in Guizhou Province and can be used to help understand the changing etiology of shigellosis in China.

[Molecular typing of 12 Brucella strains isolated in Guizhou province in 2010-2013].

OBJECTIVE: To identify and characterize the Brucella strains from Guizhou province in 2010-2013. METHODS: A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE). RESULTS: Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I. CONCLUSION: The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.