Molecular epidemiology of Pseudomonas aeruginosa isolated from lower respiratory tract of ICU patients / Epidemiologia molecular de Pseudomonas aeruginosa isolada em pacientes com infecções do trato respiratório inferior admitidos em UTI

Lower respiratory tract infections (LRTIs) caused by Pseudomonas aeruginosa are the most common infection among hospitalized patients, associated with increased levels of morbidity, mortality and attributable health care costs. Increased resistant Pseudomonas worldwide has been quite meaningful to patients, especially in intensive care unit (ICUs). Different species of Pseudomonas exhibit different genetic profile and varied drug resistance. The present study determines the molecular epidemiology through DNA fingerprinting method and drug resistance of P. aeruginosa isolated from patients with LTRIs admitted in ICU. A total of 79 P. aeruginosa isolated from patients with LRTIs admitted in ICU were characterized by Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Repetitive Extrapalindromic PCR (REP-PCR). Antibiotic resistance was determined by minimum inhibitory concentration (MIC) assay while MDR genes, viz, blaTEM, blaOXA, blaVIM, blaCTX-M-15 were detected by polymerase chain reaction (PCR). Of the 137 Pseudomonas sp isolated from ICU patients, 57.7% of the isolates were reported to be P. aeruginosa. The overall prevalence of P. aeruginosa among the all included patients was 34.5%. The RAPD analysis yielded 45 different patterns with 72 clusters with 57% to 100% similarity level. The RFLP analysis yielded 8 different patterns with 14 clusters with 76% to 100% similarity level. The REP PCR analysis yielded 37 different patterns with 65 clusters with 56% to 100% similarity level. There was no correlation among the different DNA patterns observed between the three different methods. Predominant of the isolates (46.8%) were resistant to amikacin. Of the 79 isolates, 60.8% were positive for blaTEM gene and 39.2% were positive for blaOXA gene. P. aeruginosa was predominantly isolated from patients with LRTIs admitted in ICU. The difference in the similarity level observed between the three DNA fingerprinting methods indicates that there is high inter-strain variability. The high genetic variability and resistance patterns indicates that we should continuously monitor the trend in the prevalence and antibiotic resistance of P. aeruginosa especially in patients with LRTIs admitted in ICU.

Infecções do trato respiratório inferior (ITRIs) são as infecções mais comuns entre pacientes internados em unidade de terapia intensiva (UTI). Pseudomonas aeruginosa é a causa mais comum de ITRIs e está associada ao aumento da mortalidade. Diferentes espécies de Pseudomonas exibem diferentes perfis genéticos e resistência variada as drogas. O presente estudo determina a epidemiologia molecular através do método de fingerprinting de DNA e resistência as drogas de P. aeruginosa isoladas de pacientes com LTRIs internados em UTI. Um total de 79 P. aeruginosa isoladas de pacientes com ITRIs internados em UTI foram caracterizados por Polimorfismo de Comprimento de Fragmentos de Restrição (RFLP), DNA Polimórfico Amplificado ao Acaso (RAPD) e PCR Extrapalindrômico Repetitivo (REP-PCR). A resistência aos antibióticos foram determinadas pelos ensaios de concentrações inibitória mínima (MIC), enquanto os genes MDR, blaTEM, blaOXA, blaVIM, blaCTX-M-15 foram detectados pela reação em cadeia da polimerase (PCR). Das 137 Pseudomonas sp isoladas de pacientes de UTI, 57,7% dos isolados foram relatados como P. aeruginosa. A prevalência geral de P. aeruginosa entre os pacientes incluídos foram de 34,5%. A análise RAPD renderam 45 padrões diferentes com 72 clusters com nível de similaridade de 57% a 100%. A análise RFLP renderam 8 padrões diferentes com 14 clusters com 76% a 100% de similaridade. A análise de PCR do REP produziram 37 padrões diferentes com 65 clusters com nível de similaridade de 56% a 100%. Não houveram correlações entre os diferentes padrões de DNA observados entre os três diferentes métodos. Predominantes dos isolados (46,8%) eram resistentes à amicacina. Dos 79 isolados, 60,8% foram positivos para o gene blaTEM e 39,2% foram positivos para o gene blaOXA. P. aeruginosa foi predominantemente isolado de pacientes com ITRIs internados em UTI. A diferença no nível de similaridade observado entre os três métodos de fingerprinting do DNA indica que há alta variabilidade inter-strain. A alta variabilidade genética e os padrões de resistência indicam que devemos monitorar continuamente a tendência na prevalência e resistência a antibióticos de P. aeruginosa, especialmente em pacientes com ITRIs internados em UTI.

The first study of molecular prevalence and species characterization of Cryptosporidium in free-range chicken (Gallus gallus domesticus) from Brazil / Primeiro estudo de prevalência molecular e caracterização de espécies de Cryptosporidium em galinhas (Gallus gallus domesticus) colonial/caipiras do Brasil

Rearing free-range chicken is based on grazing feeding patterns, and these animals could be potential environmental contaminants of Cryptosporidium oocysts for humans and other animals. Therefore, the present study aimed to evaluate the molecular prevalence of Cryptosporidium spp. in free-range chickens from Brazil. A total of 351 fecal samples from chickens were examined from 20 farms. For detection of Cryptosporidium spp., 18S rRNA gene fragments were amplified using a nested PCR reaction. Positive samples were sent for sequencing. The overall prevalence of Cryptosporidium was 25.6% (95% CI = 21.2% - 30.6%). Sequencing of the amplified fragments allowed for the identification of three species: C. meleagridis in 57 (62.6%), C. baileyi in 15 (16.4%), C. parvum in 3 (3.2%) samples, and a new Cryptosporidium genotype (C. genotype BrPR1) in 3 (3.2%) samples. Cryptosporidium genotype BrPR1 has not yet been classified as a species, and its host spectrum is not known. Cryptosporidium, including zoonotic species, exists at a high prevalence in free-range chickens within the region studied.(AU)

A criação de galinhas no estilo colonial/caipira é baseada em padrões de alimentação de pastagem, o que as torna potenciais contaminantes ambientais de oocistos de Cryptosporidium para humanos e outros animais. Portanto, o presente estudo teve como objetivo avaliar a prevalência molecular de Cryptosporidium spp. em galinhas criadas em sistema colonial/caipira. Um total de 351 amostras de fezes de frangos foram examinadas em 20 fazendas. Para a detecção de Cryptosporidium spp., os fragmentos do gene rRNA 18S foram amplificados utilizando-se a reação de nested-PCR. A prevalência global de Cryposporidium foi de 25,6% (IC 95% = 21,2% - 30,6%). O sequenciamento dos fragmentos amplificados permitiu a identificação de três espécies que infectam aves: C. meleagridis em 57 (62,6%), C. baileyi em 15 (16,4%), C. parvum em 3 (3,2%) amostras, bem como, um novo genótipo de Cryptosporidium (C. genótipo BrPR1) foi identificado em 3 (3,2%) amostras. Cryptosporidium genotipo BrPR1 não foi ainda classificado como uma espécie, e seu espectro de hospedeiros é desconhecido. O presente trabalho permitiu concluir que Cryptosporidium, incluindo espécies zoonóticas, existe com alta prevalência em galinhas criadas em sistema colonial/caipira na região estudada.(AU)

Molecular epidemiology of Anaplasma platys, Ehrlichia canis and Babesia vogeli in stray dogs in Paraná, Brazil / Epidemiologia molecular de Anaplasma platys, Ehrlichia canis e Babesia vogeli em cães não domiciliados do Paraná

Hemoparasitic infections are tick-borne diseases, which affect animals and humans. Considering the importance of canine hemoparasitic infections in veterinary clinics, this study aimed to determine the occurrence of Anaplasma platys, Ehrlichia canis and Babesia vogeli in blood samples from 182 dogs not domiciled in the city of Pato Branco, southwestern region of Paraná State, Brazil, using polymerase chain reaction (PCR). The prevalence of A. platys and B. vogeli was 32.9% and 10.9% respectively, and A. platys infection prevailed (p<0.001). The number of dogs positive for A. platys was larger in Winter (p<0.05). All blood samples were negative for E. canis. In the dogs, infestation by Amblyomma cajennense predominated over that by Rhipicephalus sanguineus (p<0.001); but there was no significant association between PCR and the variables presence of ticks, sex and age. Dogs infected by A. platys and B. vogeli showed thrombocytopenia, lymphopenia and leukocytosis; but there was no correlation between such hematological changes and infection by hemoparasites. This appears to be the first molecular study that demonstrates the existence of A. platys and B. vogeli in dogs from the southwestern region of Paraná.(AU)

As hemoparasitoses são enfermidades transmitidas por carrapatos que afetam os animais e os humanos. Considerando a importância das hemoparasitoses caninas na clínica médica veterinária, este estudo objetivou determinar a ocorrência de Anaplasma platys, Ehrlichia canis e Babesia vogeli em amostras de sangue de 182 cães não domiciliados do município de Pato Branco, região sudoeste paranaense, Brasil, utilizando a reação em cadeia da polimerase (PCR). A prevalência de A. platys e B. vogeli foi de 32,9% e 10.9%, respectivamente, predominando a infecção por A. platys (p<0,001). Constatou-se um maior número de cães positivos para A. platys no período do inverno (p<0.05). Todas as amostras de sangue foram negativas para E. canis. Nos cães, a infestação por Amblyomma cajennense prevaleceu sobre a infestação por Rhipicephalus sanguineus (p<0,001), mas não foi observada associação significativa entre a PCR e as variáveis presença de carrapatos, sexo e idade. Cães infectados por A. platys e B. vogeli apresentaram trombocitopenia, linfopenia e leucocitose, porém não houve correlação destas alterações hematológicas com a infecção pelos hemoparasitas. Este é o primeiro estudo molecular que demonstra a existência de A. platys e de B. vogeli em cães da região sudoeste paranaense.(AU)

Molecular epidemiology of Anaplasma platys, Ehrlichia canis and Babesia vogeli in stray dogs in Paraná, Brazil / Epidemiologia molecular de Anaplasma platys, Ehrlichia canis e Babesia vogeli em cães não domiciliados do Paraná

Hemoparasitic infections are tick-borne diseases, which affect animals and humans. Considering the importance of canine hemoparasitic infections in veterinary clinics, this study aimed to determine the occurrence of Anaplasma platys, Ehrlichia canis and Babesia vogeli in blood samples from 182 dogs not domiciled in the city of Pato Branco, southwestern region of Paraná State, Brazil, using polymerase chain reaction (PCR). The prevalence of A. platys and B. vogeli was 32.9% and 10.9% respectively, and A. platys infection prevailed (p < 0.001). The number of dogs positive for A. platys was larger in Winter (p < 0.05). All blood samples were negative for E. canis. In the dogs, infestation by Amblyomma cajennense predominated over that by Rhipicephalus sanguineus (p < 0.001); but there was no significant association between PCR and the variables presence of ticks, sex and age. Dogs infected by A. platys and B. vogeli showed thrombocytopenia, lymphopenia and leukocytosis; but there was no correlation between such hematological changes and infection by hemoparasites. This appears to be the first molecular study that demonstrates the existence of A. platys and B. vogeli in dogs from the southwestern region of Paraná.(AU)

As hemoparasitoses são enfermidades transmitidas por carrapatos que afetam os animais e os humanos. Considerando a importância das hemoparasitoses caninas na clínica médica veterinária, este estudo objetivou determinar a ocorrência de Anaplasma platys, Ehrlichia canis e Babesia vogeli em amostras de sangue de 182 cães não domiciliados do município de Pato Branco, região sudoeste paranaense, Brasil, utilizando a reação em cadeia da polimerase (PCR). A prevalência de A. platys e B. vogeli foi de 32,9% e 10.9%, respectivamente, predominando a infecção por A. platys (p < 0,001). Constatou-se um maior número de cães positivos para A. platys no período do inverno (p < 0.05). Todas as amostras de sangue foram negativas para E. canis. Nos cães, a infestação por Amblyomma cajennense prevaleceu sobre a infestação por Rhipicephalus sanguineus (p < 0,001), mas não foi observada associação significativa entre a PCR e as variáveis presença de carrapatos, sexo e idade. Cães infectados por A. platys e B. vogeli apresentaram trombocitopenia, linfopenia e leucocitose, porém não houve correlação destas alterações hematológicas com a infecção pelos hemoparasitas. Este é o primeiro estudo molecular que demonstra a existência de A. platys e de B. vogeli em cães da região sudoeste paranaense.(AU)

Antimicrobial resistance in Neisseria gonorrhoeae: history, molecular mechanisms and epidemiological aspects of an emerging global threat

ABSTRACT Neisseria gonorrhoeae is the agent of gonorrhea, a sexually transmitted infection with an estimate from The World Health Organization of 78 million new cases in people aged 15-49 worldwide during 2012. If left untreated, complications may include pelvic inflammatory disease and infertility. Antimicrobial treatment is usually effective; however, resistance has emerged successively through various molecular mechanisms for all the regularly used therapeutic agents throughout decades. Detection of antimicrobial susceptibility is currently the most critical aspect for N. gonorrhoeae surveillance, however poorly structured health systems pose difficulties. In this review, we compiled data from worldwide reports regarding epidemiology and antimicrobial resistance in N. gonorrhoeae, and highlight the relevance of the implementation of surveillance networks to establish policies for gonorrhea treatment.(AU)

Molecular diagnosis of Hepatozoon canis in symptomatic dogs in the city of Goiania, Goiás, Brazil / Diagnóstico molecular de Hepatozoon canis em cães sintomáticos do município de Goiânia, Goiás, Brasil

More than 300 species have been described in the genus Hepatozoon, occurring in different vertebrates. Among these, only Hepatozoon canis and Hepatozoon americanum are seen in dogs. Different methods may be used for laboratory diagnosis. The most common of these is direct parasitological examination of parasite stages in blood smears. The aim of this investigation was to conduct a phylogenetic study on Hepatozoon isolates from symptomatic dogs in the city of Goiânia, Goiás, Brazil. Blood samples were obtained from 40 symptomatic dogs that had been referred to the Veterinary Hospital of the Federal University of Goiás. Among these, only two samples were positive for Hepatozoon spp. using the direct parasitological method. These samples were then subjected to a DNA extraction process and amplification of a fragment of the 18S rRNA by means of PCR. Subsequently, the PCR products from each sample were purified and sequenced. The sequences obtained were then analyzed using the BLASTn algorithm, which identified both sequences of this study as Hepatozoon canis. By applying the Mega4 software, it was confirmed that these isolates of H. canis from dogs in Goiânia are similar to other reference isolates of the same species from other regions of Brazil and worldwide.(AU)

São descritas mais de 300 espécies do gênero Hepatozoon que acometem diferentes vertebrados. Entre estas, apenas Hepatozoon canis e Hepatozoon americanum são descritas em cães. Diferentes métodos podem ser utilizados para o diagnóstico laboratorial. O mais empregado é o exame parasitológico direto do parasito em esfregaços sanguíneos. O objetivo deste trabalho foi realizar um estudo filogenético em Hepatozoon isolados de cães sintomáticos de Goiânia, Goiás. As amostras de sangue foram obtidas de 40 cães sintomáticos encaminhados ao Hospital Veterinário da Universidade Federal de Goiás. Entre essas, duas únicas amostras foram positivas para Hepatozoon spp. pelo método parasitológico direto. Estas amostras foram, então, submetidas ao processo de extração de DNA e de amplificação de um fragmento de 18S rRNA por PCR. Ambas as amostras foram positivas na PCR. Posteriormente, os produtos de PCR de cada amostra foram purificados e sequenciados. As sequências obtidas foram analisadas pelo algoritmo BLASTn, sendo identificadas como Hepatozoon canis. Por meio do software Mega4 foi confirmado que estes isolados de H. canis de cães de Goiânia são semelhantes a outros isolados de referência da mesma espécie de outras regiões do Brasil e do mundo.(AU)

Molecular diagnosis of Hepatozoon canis in symptomatic dogs in the city of Goiania, Goiás, Brazil / Diagnóstico molecular de Hepatozoon canis em cães sintomáticos do município de Goiânia, Goiás, Brasil

More than 300 species have been described in the genus Hepatozoon, occurring in different vertebrates. Among these, only Hepatozoon canis and Hepatozoon americanum are seen in dogs. Different methods may be used for laboratory diagnosis. The most common of these is direct parasitological examination of parasite stages in blood smears. The aim of this investigation was to conduct a phylogenetic study on Hepatozoon isolates from symptomatic dogs in the city of Goiânia, Goiás, Brazil. Blood samples were obtained from 40 symptomatic dogs that had been referred to the Veterinary Hospital of the Federal University of Goiás. Among these, only two samples were positive for Hepatozoon spp. using the direct parasitological method. These samples were then subjected to a DNA extraction process and amplification of a fragment of the 18S rRNA by means of PCR. Subsequently, the PCR products from each sample were purified and sequenced. The sequences obtained were then analyzed using the BLASTn algorithm, which identified both sequences of this study as Hepatozoon canis. By applying the Mega4 software, it was confirmed that these isolates of H. canis from dogs in Goiânia are similar to other reference isolates of the same species from other regions of Brazil and worldwide.(AU)

São descritas mais de 300 espécies do gênero Hepatozoon que acometem diferentes vertebrados. Entre estas, apenas Hepatozoon canis e Hepatozoon americanum são descritas em cães. Diferentes métodos podem ser utilizados para o diagnóstico laboratorial. O mais empregado é o exame parasitológico direto do parasito em esfregaços sanguíneos. O objetivo deste trabalho foi realizar um estudo filogenético em Hepatozoon isolados de cães sintomáticos de Goiânia, Goiás. As amostras de sangue foram obtidas de 40 cães sintomáticos encaminhados ao Hospital Veterinário da Universidade Federal de Goiás. Entre essas, duas únicas amostras foram positivas para Hepatozoon spp. pelo método parasitológico direto. Estas amostras foram, então, submetidas ao processo de extração de DNA e de amplificação de um fragmento de 18S rRNA por PCR. Ambas as amostras foram positivas na PCR. Posteriormente, os produtos de PCR de cada amostra foram purificados e sequenciados. As sequências obtidas foram analisadas pelo algoritmo BLASTn, sendo identificadas como Hepatozoon canis. Por meio do software Mega4 foi confirmado que estes isolados de H. canis de cães de Goiânia são semelhantes a outros isolados de referência da mesma espécie de outras regiões do Brasil e do mundo.(AU)

Phylogenetic analysis: basic concepts and its use as a tool for virology and molecular epidemiology / Análise filogenética: conceitos básicos e suas utilizações como ferramenta para virologia e epidemiologia molecular

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology. Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. [...](AU)

Phylogenetic analysis: basic concepts and its use as a tool for virology and molecular epidemiology / Análise filogenética: conceitos básicos e suas utilizações como ferramenta para virologia e epidemiologia molecular

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology. Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. [...]

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.(AU)

Prevalence of Helicobacter pylori in children in eastern Turkey and molecular typing of isolates

The objectives of the present study were to determine Helicobacter pylori via culture, polymerase chain reaction and histopathological diagnosis in 101 children ranging in age from 4 to 18 years, to identify the association among restriction fragment length polymorphism types and clinical disease and to investigate the relationships among different isolates of H. pylori in different age groups. We observed a high prevalence of H. pylori infections in children between the ages of 13 and 18 (75.8%), while children aged 4 to 6 years had the lowest prevalence of infection (40%). H. pylori was detected in 30.7% (31 of 101), 66.3% (67 of 101) and 63.2% (60 of 95) of children as determined by culture methods, PCR and histological examination, respectively. H. pylori isolates with RFLP types I and III were the most common among children with antral nodularity, whereas RFLP types II and IV were the least detected types. Interestingly, all isolates from peptic ulcer patients were type III. Although our results show a high prevalence of H. pylori infections in the pediatric population in eastern Turkey, no association was identified between H. pylori infection with antral nodularity and recurring abdominal pain. In addition, we found low genetic variation among H. pylori isolates from children and no association between RFLP types and antral nodularity (p > 0.05). Additionally, we found that H. pylori isolates with specific RFLP types were predominant in different age groups.(AU)

Molecular typing of Mycobacterium bovis isolates: a review

Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.

Prior oropharyngeal colonization and ventilator-associated pneumonia

This study evaluated the relationship between previous colonization of the oropharynx and development of ventilator-associated pneumonia through the classification of genomic fingerprint pattern by pulsed-field gel electrophoresis of both oxacillin-resistant and oxacillin-susceptible Staphylococcus aureus isolates obtained from hospitalized patients in an intensive care unit.

Drug resistance profiles and clonality of sporadic Shigella sonnei isolates in Ankara, Turkey

The aims of this study were to investigate drug resistance rates, types of extended spectrum beta lactamases (ESBLs), and molecular epidemiological characteristics of 43 Shigella sonnei isolates. Ampicillin-sulbactam, amoxicillin-clavulanate, chloramphenicol, and ciprofloxacin were the most active antibiotics. Five isolates harbored blaSHV-12, blaTEM-1 and blaCTX-M-15. More than 90% of the isolates had an indistinguishable pulsotype.

Epidemiologia molecular de surto de raiva bovina na região central do Rio Grande do Sul, 2012 / Molecular epidemiology of an outbreak of bovine rabies in central Rio Grande do Sul, Brazil, 2012

A raiva é uma doença infecciosa do sistema nervoso central de mamíferos causada pelo vírus da raiva (RabV), geralmente, transmitido pela mordedura de animais infectados. No Brasil, os morcegos hematófagos Desmodus rotundus são as principais fontes de infecção do RabV para bovinos e equinos. Este artigo descreve uma investigação epidemiológica e molecular de surtos de raiva ocorridos na região central do Rio Grande do Sul, entre maio e agosto de 2012. Nesse período, 45 casos suspeitos de raiva foram relatados em 22 pequenos rebanhos, localizados dentro de um raio de 4,7km, no município de Pinhal Grande. Desses, 32 amostras foram submetidas para diagnóstico da raiva, sendo que o RabV e/ou antígenos virais foram identificados em 27 amostras. Em um segundo momento, 11 amostras foram submetidas à transcrição reversa/reação em cadeia da polimerase (RT-PCR) para o gene da nucleoproteína (N) do RabV, seguido de sequenciamento nucleotídico e análise filogenética. Sete das 11 amostras apresentaram sequências nucleotídicas idênticas e uma apresentou mutação sinônima, não-codificante, indicando uma provável origem comum dos vírus. Por outro lado, três amostras apresentaram mutações que resultaram em alterações de aminoácidos, sugerindo uma origem diferente do vírus. Esses resultados sugerem que RabV de diferentes origens/linhagens co-circulam na região e foram envolvidos nos surtos descritos. Investigações sobre a circulação de ambos os genótipos em morcegos na região estão em andamento.(AU)

Rabies is an infectious disease of the central nervous system of mammals caused by rabies virus (RabV), generally transmitted by the bite of rabid animals. In Brazil, vampire bats Desmodus rotundus are the main reservoirs of RabV for livestock. The present study describes a molecular and epidemiological investigation of outbreaks of bovine rabies occurring in the central region of Rio Grande do Sul state, Brazil, between May and August 2012. In this period, 45 cases suspected of rabies were reported in 22 small herds, located within a 4.7km range, in the county of Pinhal Grande. From these, 32 samples were submitted to rabies diagnosis and RabV and/or viral antigens were identified in 27 samples. Subsequently, 11 brain samples were submitted to reverse transcription/polymerase chain reaction (RT-PCR) for the nucleoprotein gene (N) followed by nucleotide sequencing and phylogenetic analysis. Seven out of 11 samples yielded identical sequences; one presented a synonymous, non-coding mutation, indicating a likely common origin of the virus. However, three other samples presented nucleotide mutations which resulted in amino acid changes, suggesting a different origin of the virus. In summary, these results suggest that RabV strains of different origin/lineages co-circulate in the region and were involved in the outbreaks. Investigations on the circulation of both genotypes in bats in the region are currently underway.(AU)

Epidemiologia molecular de vírus da raiva isolados de herbívoros e suínos procedentes da Amazônia Brasileira / Molecular epidemiology of rabies virus isolated herbivorous and swine from Brazilian Amazon

A raiva é uma antropozoonose com alta letalidade, acomete todos os animais de sangue quente, essa doença causa enormes prejuízos econômicos ao rebanho pecuário nacional, é impossível estimar os custos reais com controle dessa doença, em decorrência do grande número de subnotificações. O uso de ferramentas moleculares permite identificar as linhagens virais circulantes, facilitando desse modo à compreensão da epidemiologia da raiva. A técnica de RT-PCR para amplificação parcial dos genes N e G, foi aplicada em 60 amostras positivas para o vírus da raiva pelas provas de imunofluorescência direta e prova biológica, procedentes dos Estados do Pará, Tocantins, Rondônia e Acre das espécies bovina, equídea, bubalina e suína. As sequências nucleotídicas obtidas para os genes N (41 amostras) e G (17 amostras) foram analisadas pelo algoritmo Neighbor-Joining e modelo evolutivo Kimura 2-parâmetros. Essa análise permitiu identificar distintas linhagens circulantes nas regiões estudadas, foi evidenciado ainda, um padrão de distribuição geográfico dessas linhagens. Além disso, este estudo possibilitou identificar marcadores moleculares para diferentes regiões geográficas, promovendo um melhor entendimento da epidemiologia molecular da raiva das linhagens circulantes da região em estudo

Rabies is an anthropozoonosis with high mortality, affects all warm-blooded animals, this disease causes major economic losses to livestock. It is difficult to estimate the actual costs of disease control, due to sub notification of cases. Molecular techniques allow identification of genetic viral strains circulating, improving rabies epidemiology comprehension. Sixty rabies virus isolates from bovines, equines, swine and buffaloes coming from the states of Pará, Tocantins, Rondônia and Acre were analyzed in the present study. All samples were previously submitted to direct immunofluorescence test and inoculation in mice, afterwards were submitted to RT-PCR for amplification of partial Nucleoprotein and Glycoprotein genes. Nucleotide sequences from nucleoprotein (41 samples) and glycoprotein G (17 samples) genes were analyzed by Neighbor-joining algorithm and Kimura two-parameter model. This analysis allowed to identify different genetic strains circulating and its geographic distribution patterns. Moreover this study revealed molecular markers for different geographic regions, promoting a better understanding of rabies molecular epidemiology of circulating strains of study area

Epidemiologia molecular de vírus da raiva isolados de herbívoros e suínos procedentes da Amazônia Brasileira / Molecular epidemiology of rabies virus isolated herbivorous and swine from Brazilian Amazon

A raiva é uma antropozoonose com alta letalidade, acomete todos os animais de sangue quente, essa doença causa enormes prejuízos econômicos ao rebanho pecuário nacional, é impossível estimar os custos reais com controle dessa doença, em decorrência do grande número de subnotificações. O uso de ferramentas moleculares permite identificar as linhagens virais circulantes, facilitando desse modo à compreensão da epidemiologia da raiva. A técnica de RT-PCR para amplificação parcial dos genes N e G, foi aplicada em 60 amostras positivas para o vírus da raiva pelas provas de imunofluorescência direta e prova biológica, procedentes dos Estados do Pará, Tocantins, Rondônia e Acre das espécies bovina, equídea, bubalina e suína. As sequências nucleotídicas obtidas para os genes N (41 amostras) e G (17 amostras) foram analisadas pelo algoritmo Neighbor-Joining e modelo evolutivo Kimura 2-parâmetros. Essa análise permitiu identificar distintas linhagens circulantes nas regiões estudadas, foi evidenciado ainda, um padrão de distribuição geográfico dessas linhagens. Além disso, este estudo possibilitou identificar marcadores moleculares para diferentes regiões geográficas, promovendo um melhor entendimento da epidemiologia molecular da raiva das linhagens circulantes da região em estudo (AU)

Rabies is an anthropozoonosis with high mortality, affects all warm-blooded animals, this disease causes major economic losses to livestock. It is difficult to estimate the actual costs of disease control, due to sub notification of cases. Molecular techniques allow identification of genetic viral strains circulating, improving rabies epidemiology comprehension. Sixty rabies virus isolates from bovines, equines, swine and buffaloes coming from the states of Pará, Tocantins, Rondônia and Acre were analyzed in the present study. All samples were previously submitted to direct immunofluorescence test and inoculation in mice, afterwards were submitted to RT-PCR for amplification of partial Nucleoprotein and Glycoprotein genes. Nucleotide sequences from nucleoprotein (41 samples) and glycoprotein G (17 samples) genes were analyzed by Neighbor-joining algorithm and Kimura two-parameter model. This analysis allowed to identify different genetic strains circulating and its geographic distribution patterns. Moreover this study revealed molecular markers for different geographic regions, promoting a better understanding of rabies molecular epidemiology of circulating strains of study area (AU)

Aplicação da técnica de REP PCR no rastreamento de Staphylococcus aureus em sala de ordenha, para o monitoramento da qualidade do leite / Using the REP-PCR technic in the tracking of Staphylococcus aureus in milking room, for milk quality production

A identificação e classificação bacteriana são de suma importância no ambiente, na indústria, na medicina veterinária, na microbiologia e na ecologia microbiana. Um número de diferentes métodos genotípicos e fenotípicos estão sendo empregados para identificação e classificação microbiana. A técnica de REP-PCR é baseada no uso de primers sintetizados a partir de sequências repetidas de DNA, chamadas depalindrômicas extragênicas repetidas (REP), e têm sido descrita como um método o qual gera impressões digitais (fingerprints) de DNA que podem diferenciar bactérias entre gêneros e espécies. Neste estudo, o método de fingerprint foi usado para Staphylococcus aureus com o objetivo de avaliar a higiene de ordenha em duas fazendas leiteiras.Foram obtidos vários fingerprints de todos os isolados coletados das diferentes fontes estudadas (mãos de ordenhadores, tetos das vacas,leite e ordenhadeira), e foram obtidos comportamentos muito similares das bandas indicando que os isolados podem ser relatados como clones epidemiológicos. Em nosso estudo, a técnica mostrou ser eficiente para a análise da similaridade entre indivíduos da mesma espécie, no caso, o Staphylococcus aureus, mostrando ser uma ferramenta útil para investigação de falhas no manejo e, em um controle mais eficiente para evitar e/ou diminuir a disseminação de microrganismos causadores de sérias enfermidades em humanos e em animais, que podem ser transmitidas através de produtos como o leite e seus derivados.(AU)

The identification and classification of bacteria are of crucial importance in environmental, industrial, veterinary, microbiology and microbial ecology. A number of different phenotypic and genotypic methods are presently being employed for microbial identification and classification. Repetitive-element PCR (Rep-PCR) with primers basedon repetitive extragenic palindromic (REP) repeated DNA sequencesis a recently described method which generates DNA fingerprints that descriminate between bacterial species and strains. In this study, this method was used for genomic fingerprinting of Staphylococcus aureus in control of hygiene in milk line production on two farms. Complex fingerprinting patterns were obtained for all isolates from different sources (milking handlers, cows teats, milk and milk machine) were very similar, and the data indicated that the isolated were closely related.In our study, this technic shows to be usefull for investigation in fails on milk line production and for more efficient control for patogenic microrganisms that cause serious illness in humans and animals.(AU)