Genotyping and traceability analysis of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau by MLVA / 中华地方病学杂志

Objective:To observe multiple locus variable-number tandem repeat analysis (MLVA) typing of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province, and to explore the relationship between the strains and strains previous isolated from Qinghai Province. Methods:Blood samples of Himalayan marmot were collected in Qinghai-Tibet Plateau of Qinghai Province from March 2019 to October 2020. Pathogens were isolated and cultured from Brucella antibody positive samples identified by using the rose bengal test (RBT). Conventional biological methods and molecular biological methods (BCSP31-PCR and AMOS-PCR) were used for strain identification. At the same time, MLVA method was used to genotype the isolated strains, and cluster analysis was used to analyze the genetic relationships between the strains based on the genotype of 70 Brucella isolated from different hosts in Qinghai Province. Results:A total of 1 466 blood samples of Himalayan marmot were collected from Qinghai-Tibet Plateau. Two strains of Brucella were isolated and cultured from 64 RBT-positive samples, named QH2013054 and QH2013062, respectively. They were identified as Brucella ovis biotype Ⅲ by conventional and molecular biological methods. The MLVA genotyping results showed that QH2013054 and QH2013062 were different at the Bru16 locus, indicating different MLVA genotypes. Cluster analysis showed that strain QH2013054 had the same MLVA genotype as 7 strains, among which 6 strains were from 3 farmers and 3 sheep from the same family in Gonghe County, and 1 strain was from a farmer in Menyuan Hui Autonomous County. The strain QH2013062 had the same MLVA genotype as 4 strains, including 3 strains from 3 farmers in Menyuan Hui Autonomous County and 1 strain from a farmer in Tu Autonomous County of Huzhu. Conclusions:The strains of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province have the same MLVA genotype as some strains of Brucella isolated from humans and sheep in Qinghai Province. It is speculated that the host humans, sheep and Himalayan marmot in Qinghai-Tibet Plateau may have a common source of infection.

Genotyping of Listeria monocytogenes isolates by high-resolution melting curve (HRM) analysis of tandem repeat locus

Abstract Listeria monocytogenes is responsible for causing listeriosis, a type of food poisoning with high mortality. This bacterium is mainly transmitted to humans through the consumption of contaminated foods. Detection of L. monocytogenes through molecular methods is crucial for food safety and clinical diagnosis. Present techniques are characterized by low discrimination power and high cost, as well as being time-consuming and taking several days to give the final result. In our study, MLVA-HRM (Multiple-Locus Variable-number tandem repeats Analysis ‒ High-Resolution Melting) was investigated as an alternative method for a fast and precise method for the genotyping of L. monocytogenes isolates. Forty-eight isolates of L. monocytogenes obtained from the microbial bank of Department of Microbiology, Iran University of Medical Sciences, were typed by MLVA-HRM analysis using five Variable Numbers of Tandem Repeat (VNTR) loci. A total of 43 different types were obtained. This research demonstrated the usefulness of the MLVA-HRMA method and its ability to discriminate L. monocytogenes isolates. Since this method is easier and more efficient than existing methods, it can be widely used in food processing plants and diagnostic laboratories as a fast and accurate method.

Determination of investigation of the link between human and animal Brucella isolates in Iran using multiple-locus variable number tandem repeat method comprising 16 loci (MLVA-16)

ABSTRACT Background: Epidemiological studies are important tools to assess the diversity of Brucella isolates and to estimate their epidemiological relationship among isolates from different geographical origins. In this study the MLVA16 (multiple-locus variable number tandem repeat analysis based on 16 loci) was employed to investigate the diversity of Brucella spp. Isolated from humans and animals for epidemiological purposes and to determine the most common Brucella genotypes in Iran. Methods: We designed a molecular-based study to evaluate the potential reservoirs of human brucellosis. After isolation and identification of 54 Brucella spp human and animal specimens from three regions of Iran, bacterial genomic DNA was extracted MLVA with three panel was used for the genotyping of isolates. The size of PCR products were analyzed and converted to repeat unit numbers using a published allele numbering system and data set was imported into Bionumerics. Results: Three isolates (5.55%) were identified as Brucella abortus and 51 (94.44%) as Brucella melitensis. Two isolates of Brucella abortus were from humans and one from an animal. Thirty-four Brucella melitensis isolates were from humans and 17 from animals. Using MLVA16-genotyping, 54 isolates with genetic similarity coefficient of 80% were divided into 46 genotypes and 22 genotypes were represented by a single isolate, while 4, 2, 1 and 2 genotypes were represented by 2, 3, 4 and 7 isolates, respectively. The most prevalent genotype was represented by 14 isolates. There were two other frequent genotypes each represented by seven isolates, among which only one was restricted to a geographic region. Discriminatory power for each locus was determined in this study and panel 2B shows the high discretionary power [Bruce04 (0.837), Bruce30 (0.806), Bruce 09 (0.787), Bruce 07 (0.772), Bruce16 (0.766)]. Conclusion: MLVA16 analysis of 54 Brucella isolates showed high level polymorphism in their genotypes. Only two genotypes, each observed in seven isolates, were related to one another and only one of these genotypes were found in to two separate regions.

Progress of molecular epidemiology of mycoplasma pneumoniae in children / 国际儿科学杂志

Mycoplasma pneumoniae(MP)is the main pathogen causing community-acquired pneumonia in children, usually treated with macrolide drugs.The type of MP genes is mainly based on PCR-RFLP(P1-restriction fragment length polymorphism analysis)and MLVA(multiple locus variable number tandem repeat analysis)typing methods.During epidemics, MP subtypes will undergo certain transformation.Studying and mastering the prevalence characteristics and transformation laws of MP subtypes can deeper understand the distribution area, prevalence year and clinical relevance of each subtype of MP.Due to the extensive use of antibiotics, the resistance of mycoplasma pneumoniae to macrolides has increased , which has become a global public health concern.Studies have shown that MP resistance is related to mutations in the V region of 23S rRNA gene domain.The improvement of typing technology also guides significance for the rational selection of antibiotics.It is imperative to carry out systematic and comprehensive molecular epidemiological studies of MP genotypes and its resistance mutations, and reveal the distribution characteristics, epidemic trends and resistance mutations of MP subtypes at the molecular level.This paper reviews the research progress of molecular epidemiology of mycoplasma pneumoniae in children, and provides ideas for the monitoring, prevention and clinical treatment of MP infection.

Genetic diversity of Mycoplasma hyopneumoniae in Mendoza province / Diversidad genética de Mycoplasma hyopneumoniae en la provincia de Mendoza

En Argentina, la neumonía enzoótica porcina (NEP) es altamente prevalente y se han identificado diferentes tipos genéticos de Mycoplasma hyopneumoniae. Sin embargo, se carece de información acerca de la prevalencia de NEP y de otros aspectos epidemiológicos de esta entidad en la provincia de Mendoza. En esta investigación se usó un análisis multilocus de regiones repetidas en tándem (MLVA) de los loci P97 R1, P97 R1A y P146 R3 para evaluar la diversidad genética de M. hyopneumoniae a partir de muestras clínicas de cerdos de cinco granjas localizadas en diferentes distritos de la provincia de Mendoza. M. hyopneumoniae pudo ser tipificado a partir de 27 muestras de lavado broncoalveolar (LBA) y se identificaron 8 diferentes MLVA-tipos. Este es el primer informe acerca de la diversidad genética de M. hyopneumoniae en Mendoza. Los resultados obtenidos permiten describir de manera más acabada la diversidad genética de este agente en nuestro país.

Epidemic status of brucellosis and MLVA genotyping characteristics of Brucella isolated from Hebei Province in 2016 / 中华地方病学杂志

Objective To analyze the brucellosis epidemic and genotyping characteristics of Brucella strains isolated from Hebei Province,and to provide scientific basis for brucellosis control and prevention.Methods Descriptive epidemiological analysis (including region,population,and time distribution characteristics) was performed using individual data of human brucellosis in Hebei Province from the National Notifiable Infectious Disease Reporting Information System of China Information System for Disease Control and Prevention in 2016,Brucella strains were isolated by phase culture bottles in Brucellosis Lab from Hebei Provincial Center for Disease Control and Prevention.Multiple-locus variable-number tandem-repeat analysis (MLVA) was used to identify the genotyping characteristics of the isolates.Results There were 3 774 reported cases in 2016,and 11 cities in the province had reported the cases.The cases were found mainly in Zhangjiakou,Chengde and Tangshan.And 95.98％ (167/174) of all counties were reported brucellosis cases.Vocational high risk population was farmers and herdsmen engaged in livestock and processing of animal products,accounting for 90.22％ (3 405/3 774).Male had the highest incidence,accounting for 74.38％ (2 807/3 774).The onset time was concentrated in January-August,accounting for 83.67％ (3 161/3 774).Twenty-one strains of Brucella were isolated,twenty isolates were Brucella melitensis biovar 3,and one isolate was Brucella abortus biovar 3.Panel 1 (MLVA-8) identified three genotypes 42,43,and 114,with genotype 42 representing 85.00％ (17/20);Panel 1 + Panel 2A (MLVA-11) identified three genotypes 116,122,291,with genotype 116 representing 85.00％ (17/20),which were identified as belonging to the East Mediterranean group.Panel 1 (MLVA-8) identified the genotype 36,Panel 1 + Panel 2A (MLVA-11) identified the genotype 72 of one Brucella abortus strain.It was identified as belonging to abortus C.Conclusions The brucellosis epidemic in Hebei is relatively severe,the pathogenic bacteria is mainly Brucella melitensis.The MLVA assay is a very practical and important molecular genotyping tool,it provides a basis for tracing the source of brucellosis infection.

Relationship between macrolide resistance mutations in Mycoplasma pneumoniae and its genotype / 中华微生物学和免疫学杂志

Objective To analyze the relationship between macrolide resistance mutations in My-coplasma pneumoniae (Mp) and its genotype by multiple-locus variable-number tandem-repeat analysis (MLVA). Methods One hundred and forty-three Mp-positive specimens were collected in Beijing(54 col-lected at the Affiliated Children′s Hospital of the Capital Institute of Pediatrics),the United States(59 col-lected at four different geographical locations:Kansas City,Missouri;Seattle,Washington;New York,New York;Chicago,Illinois) and Australia(30 provided by the diagnostic laboratory at the Centre for Infectious Diseases and Microbiology Laboratory Services,Institute of Clinical Pathology and Medical Research,West-mead Hospital,Sydney). Nested PCR was used to detect mutations in 23S rRNA. A capillary electrophore-sis-based single tube multiplex PCR (mPCR-CE) was used to analyze the MLVA types of Mp in those sam-ples. Results A2063G mutation was identified in 57 specimens including 49 from Beijing,seven from the United States and one from Australia. The 143 Mp-positive specimens were typed into 10 distinct MLVA types. Fifty-four specimens collected in Beijing belonged to four MLVA types, which were M4-5-7-2 (44/54,81.5%),M3-5-6-2 (7/54,13.0%), M4-5-6-2 (2/54,3.70%) and M4-5-5-2 (1/54,1.85%). Fifty-nine specimens collected in the United States belonged to six MLVA types including M4-5-7-2(27/59, 45.8%),M3-5-6-2 (18/59,30.5%),M3-6-6-2 (11/59,18.6%),M3-5-6-1 (1/59,1.69%),M4-5-7-3 (1/59,1.69%) and M5-5-7-2 (1/59,1.69%). Thirty specimens of Mp from Australia were grouped to five types with M3-5-6-2 (12/30, 40.0%) and M4-5-7-2 (10/30, 33.3%) and M3-5-7-2 (5/30, 16.7%) being the predominant types. Macrolide resistance mutations were detected in 57 out of 143 speci-mens (49 from Beijing,seven from the United States and one from Sydney) and 50 of them were MLVA type of M4-5-7-2. Conclusion The MLVA type of M4-5-7-2 is associated with macrolide resistance in Mp.

Local circulating clones of Staphylococcus aureus in Ecuador

ABSTRACT The spread of pandemic Staphylococcus aureus clones, mainly methicillin-resistant S. aureus (MRSA), must be kept under surveillance to assemble an accurate, local epidemiological analysis. In Ecuador, the prevalence of the USA300 Latin American variant clone (USA300-LV) is well known; however, there is little information about other circulating clones. The aim of this work was to identify the sequence types (ST) using a Multiple-Locus Variable number tandem repeat Analysis 14-locus genotyping approach. We analyzed 132 S. aureus strains that were recovered from 2005 to 2013 and isolated in several clinical settings in Quito, Ecuador. MRSA isolates composed 46.97% (62/132) of the study population. Within MRSA, 37 isolates were related to the USA300-LV clone (ST8-MRSA-IV, Panton-Valentine Leukocidin [PVL] +) and 10 were related to the Brazilian clone (ST239-MRSA-III, PVL−). Additionally, two isolates (ST5-MRSA-II, PVL−) were related to the New York/Japan clone. One isolate was related to the Pediatric clone (ST5-MRSA-IV, PVL−), one isolate (ST45-MRSA-II, PVL−) was related to the USA600 clone, and one (ST22-MRSA-IV, PVL−) was related to the epidemic UK-EMRSA-15 clone. Moreover, the most prevalent MSSA sequence types were ST8 (11 isolates), ST45 (8 isolates), ST30 (8 isolates), ST5 (7 isolates) and ST22 (6 isolates). Additionally, we found one isolate that was related to the livestock associated S. aureus clone ST398. We conclude that in addition to the high prevalence of clone LV-ST8-MRSA-IV, other epidemic clones are circulating in Quito, such as the Brazilian, Pediatric and New York/Japan clones. The USA600 and UK-EMRSA-15 clones, which were not previously described in Ecuador, were also found. Moreover, we found evidence of the presence of the livestock associated clone ST398 in a hospital environment.

Association of number of tandem repeats in two important adhesins in Mycoplasma hyopneumoniae / Associação entre os números de repetições em tandem em duas importantes adesinas de Mycoplasma hyopneumoniae

Diversidade genética de Mycoplasma hyopneumoniae tem sido relatada em análise múltipla de repetições em tandem em número variável (MLVA). O objetivo deste estudo foi descrever a distribuição espacial e a heterogeneidade genética de tipos de M. hyopneumoniae no Brasil, bem como investigar a correlação entre regiões de repetição 1 (RR1) e 3 (RR3) de duas adesinas importantes (P97 e P146). Foram identificados 39 tipos de MLVA baseados no número de repetições em tandem em P97 RR1 e RR3 P146. A correlação negativa significativa (Spearman's rho = -0,26; P = 0,022) entre P97 RR1 e RR3 P146 foi observada, o que sugere um possível mecanismo compensatório que permitiria a bactéria manter a sua capacidade de adesão. Os resultados contribuem para compreender a epidemiologia das M. hyopneumoniae no quarto maior país produtor de suínos do mundo.(AU)

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Isolation of Leptospira spp. from a man living in a rural area of the Municipality of Cruz Alta, RS, Brazil / Isolamento de Leptospira spp. em um morador da zona rural do Município de Cruz Alta, RS, Brasil

The aim of this study was to describe the isolation of a pathogenic strain of Leptospira interrogans from the urine sample of a male human living in the rural area of the County of Cruz Alta, Rio Grande do Sul. An aliquot of each urine sample was sown in a Fletcher and Ellinghausen - McCullough - Johnson - Harris (EMJH) media. Samples in which there was growth of spirochetes were sent to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were typified by the Multiple Locus of Variable Number Tandem Repeat technique (MLVA). Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 was isolated, and this is a very important finding that serves as a warning to characterize risk situation of leptospirosis epidemic by a pathogenic strain. Health professionals need to be more committed to the primary health care in Brazil and routinely apply actions of preventive medicine in rural communities in order to get success in the control of leptospirosis and other important zoonoses.

O objetivo do presente estudo foi descrever um caso de isolamento de espécie patogênica de Leptospira interrogans em amostra de urina de um humano morador da zona rural do Município de Cruz Alta, Estado do Rio Grande do Sul. De cada amostra de urina, uma alíquota foi semeada nos meios Fletcher e Ellinghausen - McCullough - Johnson - Harris (EMJH). As amostras, nas quais houve crescimento de espiroquetas, foram encaminhadas para o Laboratório de Leptospirose do Instituto de Patobiologia do Instituto Nacional de Tecnologia Agropecuária, Buenos Aires, Argentina e foram tipificadas pela técnica Multiple Locus of Variable Number Tandem Repeat (MLVA). De um residente do sexo masculino da área rural do município de Cruz Alta, foi isolada Leptospira interrogans sorovariedade Copenhageni cepa Fiocruz L1-130, uma descoberta muito importante e que serve como um alerta por caracterizar uma situação de risco de epidemia de leptospirose por uma cepa patogênica. Os profissionais de saúde precisam ser mais comprometidos com a atenção primária à saúde no Brasil e rotineiramente aplicar ações de medicina preventiva nas comunidades rurais, a fim de obter sucesso no controle da leptospirose e de outras importantes zoonoses.

MLVA based Mycobacterium tuberculosis clinical isolates genotyping analysis f rom HIV co-infection patients in Dali area,China / 中国人兽共患病学报

A new method based on the multiple locus variable number tandem repeat analysis (MLVA) was applied for the genotyping of combined HIV Mycobacterium tuberculosis in Dali to investigate the genotyping and distribution pattern of Myco‐bacterium tuberculosis clinical isolates with MLVA .Mycobacterium tuberculosis clinical isolates were selected from Dali area , and the polymorphism of VNTR locus was tested with PCR .The clustering of genotype was analyzed by BioNumerics (6 .6) . Result showed that 15 VNTR loci of 61 combined HIV Mycobacterium tuberculosis clinical isolates were analyzed respectively . There were obvious polymorphisms of VNTRs .The discrimination power of these loci appeared different from each other ,with the biggest Hunter‐Gaston index (0 .839) loci was MIRU26 ,and the smallest one (0 .341) loci was MIRU4 .The clustering of genotype showed that these strains could be categorized into 5 gene clusters and 61 genotype ,the proportions of cluster Ⅰ was the biggest one ,51 .6% were cluster Ⅰ which including 32 strains .The standard strain H37Rv was belongs to cluster Ⅱ .Its indicated that there are obvious polymorphisms of VNTRs of combined HIV Mycobacterium tuberculosis clinical isolates in Da‐li .The main genotype was Beijing family genotype .

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter pittii and Development of an Optimized Multiple-Locus VNTR Analysis Typing Scheme / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Acinetobacter pittii typing.</p><p><b>METHODS</b>Polymorphic VNTRs were searched by Tandem Repeats Finder. The distribution and polymorphism of each VNTR locus were analyzed in all the A. pittii genomes deposited in the NCBI genome database by BLAST and were evaluated with a collection of 20 well-characterized clinical A. pittii strains and one reference strain. The MLVA assay was compared with pulsed-field gel electrophoresis (PFGE) for discriminating A. pittii isolates.</p><p><b>RESULTS</b>Ten VNTR loci were identified upon bioinformatic screening of A. pittii genomes, but only five of them showed full amplifiability and good polymorphism. Therefore, an MLVA assay composed of five VNTR loci was developed. The typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. Compared with PFGE, the new optimized MLVA typing scheme provided the same and even greater discrimination.</p><p><b>CONCLUSION</b>Compared with PFGE, MLVA typing is a faster and more standardized alternative for studying the genetic relatedness of A. pittii isolates in disease surveillance and outbreak investigation.</p>

Genotipos de cepas de Leptospira spp. aisladas de perros en Buenos Aires, Argentina / Genotypes of Leptospira spp. strains isolated from dogs in Buenos Aires, Argentina

La leptospirosis es una enfermedad infecciosa de amplia distribución global; endémica en Argentina. El objetivo de este estudio fue obtener los perfiles genéticos de las cepas de Leptospira spp. aisladas de casos clínicos de perros provenientes de la provincia de Buenos Aires, empleando el análisis de repeticiones en tándem de número variable en múltiples locus [multiple-locus variable-number tandem repeats analysis (MLVA)]. Fueron genotipificadas por MLVA ocho cepas aisladas de perros. Se obtuvo un perfil idéntico al de Leptospira interrogans serovar Canicola Hond Utrecht IV en las cepas denominadas Dogy y Mayo. Las cepas denominadas Bel, Sarmiento, La Plata 4581 y La Plata 5478 mostraron un perfil idéntico al genotipo de L. interrogans serovar Portlandvere MY 1039. La cepa denominada Avellaneda presentó un perfil idéntico al genotipo L. interrogans serovar Icterohaemorrhagiae RGA, y la cepa denominada SB mostró un perfil idéntico al genotipo de L. interrogans serovar Pomona Baires y similar al serovar Pomona Pomona. Sería de gran utilidad incluir un mayor número de cepas provenientes de distintas poblaciones caninas de diversas provincias de Argentina a fin de conocer los perfiles de las cepas circulantes en el país. La información así obtenida contribuirá al control de la leptospirosis en la población canina

Leptospirosis is an infectious disease of wide global distribution, which is endemic in Argentina. The objective of this study was to obtain the genetic profiles of Leptospira spp. strains isolated from clinical cases of dogs in the province of Buenos Aires by the multiple-locus variable-number tandem repeat analysis (MLVA). Eight isolated canine strains were genotyped by MLVA, obtaining the identical profile of Leptospira interrogans serovar Canicola Hond Utrecht IV in the strains named Dogy and Mayo. The strains named Bel, Sarmiento, La Plata 4581 and La Plata 5478 were identical to the profile of the genotype of L. interrogans serovar Portlandvere MY 1039.The strain named Avellaneda was identical to the genotype profile of L. interrogans serovar Icterohaemorrhagiae RGA and the strain named SB had the same profile as the L. interrogans serovar Pomona Baires genotype and was similar to the profile of serovar Pomona Pomona genotype. It would be useful to include a larger number of isolates from different dog populations in various provinces of Argentina and to characterize the genetic profiles of the strains circulating in the country. The information obtained will be useful for the control of leptospirosis in the dog population

Application and evaluation of PFGE and MLVA subtyping methods on Brucella genotype in Guangdong Province,China / 中国人兽共患病学报

To compare and evaluate the discriminatory ability and potential value of pulsed field gel electrophoresis (PF-GE) and multiple locus VNTRs analysis (MLVA) on the genotyping of Brucella ,a total of 60 strains of Brucella and three standards (16M ,544A ,1330S) were genotyped simultaneously by PFGE and MLVA .The result indicated that the similarity coefficient among the 63 isolates was from 72 .1-100 .0% by PFGE ,and could distinguish three species of B .melitensis ,B .su-is and B .abortus at the similarity level of 94 .4% .There were 14 clusters and 29 PFGE types identified by PFGE with discrim-inatory index (DI) of 0 .957 5 at the similarity level of 100% ;the similarity coefficient among the 63 isolates was from 16 .9-100 .0% by MLVA ,and could distinguish three species of Brucella at the similarity level of 52 .3% .There were 8 clusters and 47 MLVA types identified by MLVA with discriminatory index (DI) of 0 .985 2 at the similarity level of 100% .It's suggested that PFGE and MLVA could be used to distinguish three species of Brucella in the similarity coefficient of certain ,but could not effectively distinguish the type in the same species .Both of these two methods could be used for Brucella molecular typing , but MLVA is better than PFGE for its relatively higher discriminating ability .

Establishment of multiple locus variable-number tandem repeat analysis assay for genotyping of Borrelia burgdorferi sensu lato detected in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Human Lyme Borreliosis (LB), which is caused by Borrelia 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 burgdorferi strains detected in China.</p><p><b>METHODS</b>B. garinii PBi complete 904.246 kb chromosome and two plasmids (cp26 and lp54) 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).</p><p><b>RESULTS</b>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. afzelii, 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.</p><p><b>CONCLUSION</b>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.</p>

Detección de un complejo clonal con el genotipo de Brucella abortus biovariedad 2 como fundador en aislamientos de B. abortus de Argentina / Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina

Brucella abortus es el agente causal de la brucelosis bovina, enfermedad zoonótica que se encuentra ampliamente distribuida en el mundo. Actualmente existen ocho biovariedades de B. abortus. En Argentina se encuentra con mayor frecuencia la biovariedad 1, pero también se suele aislar la biovariedad 2, que es más patogénica que la anterior. Resulta necesario contar con métodos de tipificación que tengan la resolución suficiente para permitir el seguimiento epidemiológico de los brotes de brucelosis y de los programas de control de la enfermedad. Debido a la gran homogeneidad genética que existe entre las distintas especies del género Brucella, ha sido dificultoso el desarrollo de herramientas moleculares para realizar el análisis epidemiológico de los aislamientos. La publicación del genoma de varias especies de Brucella facilitó el diseño de estas herramientas. El objetivo del presente trabajo fue emplear un esquema de análisis multilocus de VNTR en aislamientos de Argentina obtenidos en nuestro laboratorio. De los 56 aislamientos analizados se obtuvieron 47 perfiles genotípicos diferentes. El empleo de este esquema permitió asignarles a dichos aislamientos la biovariedad correspondiente. A través del análisis goeBURST se pudo relacionar a todos los genotipos entre sí, y además, proponer al genotipo de la biovariedad 2 como fundador.

Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder.

Study on polymorphism of Brucella melitensis biovar 3 strains isolated from Guangdong province / 中华微生物学和免疫学杂志

Objective To investigate the polymorphism of Brucella melitensis biovar 3 ( B.melitensis biovar 3) strains isolated from Guangdong province .Methods PCR assays followed by agar-ose gel electrophoresis and capillary electrophoresis based on the multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) were performed to analyze 43 clinic isolates of B.melitensis biovar 3 strains isolated from clinical patients in Guangdong province .Results MLVA typing showed that the simi-larities of the analyzed locus among 43 strains of Brucella ranged from 68.2%to 100%.32 genotypes identi-fied among the isolates were identical (100%similarity).27 out of 43 strains (62.8%) were single geno-types, while the other 16 strains (37.2%) belonged to 5 other genotypes with 2 to 5 strains in each of them . Conclusion B.melitensis biovar 3 isolates showed polymorphism distribution in Guangdong province as in-dicated by MLVA typing analysis .Single-genotype isolates accounted for 62.8% of all studied strains.No predominant genotype was found among all isolates .

Isolation of Leptospira borgpetersenii in synanthropic Didelphis albiventris in Jaboticabal, São Paulo, Brazil / Isolamento de Leptospira borgpetersenii em Didelphis albiventris sinantrópicos em Jaboticabal, São Paulo, Brasil

Leptospirosis is a waterborne disease and, therefore, stands out for the possibility of environmental contamination, the cross transmission between domestic and wild animals and humans. Opossum species are important reservoirs of this disease making them potential pathogen spreaders. Aiming to verify the presence of Leptospira spp. and the antibodies against Leptospira spp. in the Campus of São Paulo State University, in Jaboticabal, São Paulo, Brazil, freeliving wild life opossum (Didelphis albiventris) were captured for blood and urine sampling. Serological analysis was performed Microscopic Agglutination Test (MAT). Aliquots of urine were seeded in media Ellinghausen-McCullough- Johnson-Harris (EMJH) and Fletcher without antibiotics. The samples in which there was growth of leptospires were forwarded to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were genotyped using Multiple Locus Variable number tandem repeat Analysis (MLVA). Of the 15 analyzed animals, nine (60.0%) were reactant to Patoc serovar. The pathogenic specie Leptospira borgpetersenii was isolated and identified in three Didelphis albiventris. The isolation findings of pathogenic specie Leptopsira borgpetersenii in the urine culture of three Didelphis albiventris in a university campus are a major discovery in the area of preventive veterinary medicine and public health and open a discussion about the important role of free-living wild animals as reservoirs of this agent to domestic animals and humans, a condition that serves as a warning for the improvement of health practices...

A leptospirose é uma zoonose de veiculação hídrica e, portanto, se destaca pela possibilidade de contaminação ambiental, o que facilita a transmissão cruzada entre animais domésticos, selvagens e humanos. Espécies de gambás são importantes reservatórios dessa enfermidade, tornando-os potenciais disseminadores do agente. Com o objetivo de verificar a presença de Leptospira spp. e de anticorpos contra Leptospira spp. no Campus da Universidade Estadual Paulista, em Jaboticabal, foram capturados gambás (Didelphis albiventris) de vida livre para a colheita de amostras de sangue e de urina. As análises sorológicas foram efetuadas pela técnica de Soroaglutinação Microscópica (SAM). Alíquotas de urina foram semeadas nos meios Ellinghausen-McCullough-Johnson-Harris (EMJH) e Fletcher sem antibióticos. As amostras que apresentaram crescimento de espiroquetas foram levadas ao Laboratório de Leptospirose do Instituto de Patobiologia, no Instituto Nacional de Tecnologia Agropecuaria, Buenos Aires, Argentina e foram genotipadas com a técnica de Múltiplos Locus de Números Variáveis de Repetição em Tandem (MLVA). Dos 15 animais examinados pela SAM, nove (60,0%) foram reagentes à sorovariedade Patoc. Foi isolada e identificada a espécie patogênica Leptospira bosrpetersenii de três Didelphis albiventris. Os achados de isolamento da espécie patogênica Leptospira borgpetersenii na cultura de urina de três Didelphis albiventris são um grande descobrimento para as áreas da medicina veterinária preventiva e da saúde pública e reforçam a discussão sobre o importante papel dos animais selvagens de vida livre como reservatórios desse agente para animais domésticos e seres humanos, situação que serve de alerta para melhorias nas práticas sanitárias...

Genotype analysis of Mycoplasma pneumoniae during 2010-2012 in children from Beijing / 中华微生物学和免疫学杂志

Objective To inspect the source of an outbreak with Mycoplasma pneumoniae (Mp).Methods We carried out real-time PCR to analyze specimens collected from pediatric patients in Beijing during January 2010 to May 2012,diagnosed as pneumonia or a respiratory infection according to clinical symptoms.These positive samples were analyzed by the M-P typing system(M:multiple-locus variable-number tandem-repeat analysis,MLVA; P:P1-restriction fragment length polymorphism analysis,P1-RFLP).Results Sixty-nine specimens were tested positive to Mp by the real-time PCR in 446 specimens from pediatric patients.The infection rate was 11.69％,15.56％ and 20.00％ respectively in 2010,2011 and the first half of 2012.According to the M-P system,11 distinct genotypes were identified from 69 positive specimens,M43562P1 and M53562P1 were the two main genotypes that showed an increasing trend from 2010 to 2011,and M33562P1 and M63562P1 showed an increasing trend from 2011 to 2012 in China.Conclusion During this international Mp epidemic,the infection rate of Mp was also increase in Beijing in 2011,and M43562P1 and M53562P1 were the two main genotypes.Among them,M43562 were consistent with pop genotypes in Europe,and M53562 were consistent with pop genotype in Israel.The M-P system would be valuable to monitor the epidemic of Mp in different countries in the world.