Detecção e caracterização molecular de piroplasmas em cães naturalmente infectados no Sudeste do Brasil

Rangelia vitalii é um protozoário que infecta cães e foi descrito nas regiões Sul e Sudeste do Brasil. R. vitalii é filogeneticamente próxima à Babesia spp., mas dados deste misterioso parasito ainda são escassos. O objetivo deste trabalho foi detectar a presença de piroplasmas em cães naturalmente infectados no estado do Rio de Janeiro, através da amplificação do gene 18S rRNA pela PCR, clivagem com enzimas de restrição (RFLP) e caracterização genética através do sequenciamento. De 103 cães, sete (6,8%) foram positivos para Babesia spp. pela PCR. Os produtos amplificados foram digeridos por enzimas de restrição para a diferenciação das espécies de Babesia e uma amostra foi identificada como Babesia vogeli. O padrão de amplificação observado nas outras seis amostras não correspondeu ao padrão descrito para babesias que infectam cães. O sequenciamento das seis amostras confirmou ser uma espécie geneticamente idêntica a R. vitalii apresentando grande homologia (99-100%) com a sequência do sul do Brasil. Este estudo confirma a presença de Babesia vogeli e Rangelia vitalii infectando cães em Teresópolis, Rio de Janeiro, Brasil.Rangelia vitalii is a protozoon described from dogs in the south and southeast regions of Brazil. It is phylogenetically related to Babesia spp. that infects dogs, but data on this enigmatic parasite is still limited. The aim of this work was to detect piroplasm species in dogs in the state of Rio de Janeiro, Brazil, by 18S rRNA gene-based PCR assay, restriction fragment length polymorphism (RFLP) and sequence analyses. Of 103 dogs examined, seven (6.8%) were positive for Babesia spp. by PCR. The amplified products were digested by restriction enzymes to differentiate the Babesia species, and one sample was identified as Babesia vogeli. The pattern observed for the other six amplification products did not match with pattern described for large Babesia infecting dogs. Sequencing analysis confirmed these six samples as R. vitalii, with high homologies (99-100%) with a sequence from south Brazil. This study confirms the presence of Babesia vogeli and Rangelia vitalii circulate in domestic dogs in Teresópolis, Rio de Janeiro, Brazil

Shiga toxin-producing Escherichia coli (STEC): prevalence in animal reservoir, virulence markers and analysis of clonal structure

Dentre as diversas categorias diarreiagenicas de Escherichia coli, as amostras produtoras de toxina Shiga (STEC) destacam-se pela potencial letalidade, principalmente em criancas, decorrente de graves sequelas extra-intestinais e pelo importante papel de animais, especialmente bovinos, como reservatorios destas bacterias para o ser humano. Apesar da relativa baixa ocorrencia de STEC como agente de diarreia humana em nosso meio, dados previos revelaram a predominancia desta categoria em produtos carneos comercializados no Rio de Janeiro. O presente estudo buscou avaliar em relacao a STEC a sua ocorrencia em bovinos no Estado do Rio de Janeiro, caracterizar a presenca de mercadores de virulencia em amostras previamente isoladas de alimentos e analisar a possivel estrutura clonal de amostras isoladas de diferentes origens em nosso meio. Uma elevada ocorrencia de STEC foi caracterizada pela amplificacao por PCR de sequencias geneticas de stx em 71 por cento das 197 amostras fecais de bovinos analisadas, sendo maior em animais de regime leiteiro (82 por cento) quando comparada a animais de corte (53 por cento). Constatou-se ainda, apos ensaios de hibridizacao com sondas geneticas, a predominancia da presenca simultanea das toxinas Stx1 e Stx2 (58 por cento) nas amostras amplificadas. Atraves da utilizacao de meio seletivo (TC-SMAC), foi possivel o isolamento de 3 amostras STEC pertencentes ao sorotipo 0157:H7 provenientes de 3 animais distintos, sendo este o primeiro relato do isolamento de amostras deste sorotipo no Brasil. Doze amostras STEC adicionais foram obtidas a partir de amostras fecais positivas ufilizando-se um protocolo de isolamento baseado em PCR. A predominancia do genotipo stxl/stx2 foi confinada entre as amostras isoladas. A expressao da citotoxina foi caracterizada em todas as amostras a excecao de 2 pertencentes ao sorotipo 0157:H7. A caracterizacao de mercadores de virulencia foi analisada em 21 amostras STEC de origem alimentar acrescidas de 8 amostras de origem bovina (5 isoladas de animais diarreicos e as 3 amostras 0157:H7 isoladas neste estudo) e 4 amostras isoladas de criancas diarreicas. De modo geral as amostras de origem humana e animal apresentaram um perfil de virulencia compativel com aquele descrito para o subgrupo mais virulento das STEC, constituido pelas amostras enterohemorragicas (EHEC), principalmente pela presenca do gene eae, da producao de enterohemolisina e por uma interacao eficiente e caracteristica ...(au)BV UNIFESP: Teses e dissertaçõe

Shigatoxigenic Escherichia coli serogroups O157, O111 and O113 in feces, water and milk samples from dairy farms

Este trabalho teve como objetivo determinar a prevalência de Escherichia coli Shigatoxigênicas dos sorogrupos O157, O111 e O113 em bovinos leiteiros, água e leite de propriedades rurais do Município de Jaboticabal/SP. Para isso, foram colhidas 454 amostras de fezes, 54 amostras de água e 30 amostras de leite e pesquisada a presença de seqüências stx1, stx2 e eae pela reação em cadeia da polimerase (PCR). Todas as amostras stx e/ eae positivas foram submetidas a uma nova reação de PCR para detecção das seqüências rfb O157, rfb O111 e rfb O113. Uma alta ocorrência (59,9%) de stx foi detectada nas fezes dos bovinos. Os coeficientes de prevalência das seqüências rfb O157, O111 e O113 nas fezes dos bovinos foram, respectivamente, 18,9%, 3,3% e 30,4%. Todas as seqüências foram encontradas com maior freqüência em bezerros e novilhas. As seqüências eae e stx2 foram as mais detectadas entre as amostras de fezes. Foram detectados 1,9% e 3,3% de seqüências stx na água e no leite, respectivamente. Nas amostras de água observou-se uma baixa prevalência de O113 e não foram detectadas O157 e O111. Nenhum dos sorogrupos pesquisados foi encontrado nas amostras de leite. STEC foram identificadas em todos os rebanhos (100%) e os sorogrupos O157, O111 e O113 foram observados em 40,0%, 50,0% e 90,0% dos rebanhos. Conclui-se que a prevalência de STEC em fezes de bovino, no Município de Jaboticabal/SP é alta, caracterizando-a como importante via de contaminação ambiental para esses microrganismos.The objective of this study was to determine the prevalence of Shigatoxigenic Escherichia coli (STEC) and STEC serogroups O157, O111 and O113 in feces, water and milk sampled in dairy farms in Jaboticabal, SP, Brazil. Feces (n=454), water (n=54) and milk samples (n=30) were collected from 10 herds and assessed for the presence of the virulence genes stx1, stx2 and eae by polymerase chain reaction (PCR). All stx and eae positive samples were submitted to a second PCR reaction targeting the sequences rfb O157, rfb O111 and rfb O113. High prevalence of stx was detected (59.9%) in fecal samples, whereas the prevalence of sequences rfb O157, rfb O111 and rfb O113 was 18.9%, 3.3% and 30.4%, respectively. All sequences were detected more frequently in calves and heifers. Sequences stx2 and eae were prevalent in the fecal samples. stx sequences were detected in 1.9% and 3.3% of water and milk samples, respectively. Low prevalence of E. coli O113 was observed in water samples, whereas no E. coli O157 or E. coli O111 was detected. Furthermore, none of the serogroups were identified in milk samples. STEC was identified in all herds (100%), and serogroups O157, O111 and O113 were observed in 40%, 50% and 90% of the herds, respectively. In conclusion, the high STEC prevalence detected in dairy herds evidences that bovine feces might play an important role as a contamination source in the region of Jaboticabal, Brazil.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Microbiological Findings in Tracheal Wash From Mule Foals With and Without Clinical Evidence of Respiratory Disease

Respiratory diseases are common in horses; however, there is a lack of information in the literature on respiratory disease affecting mule foals. The objective of this study was to investigate the presence of aerobic bacteria in tracheal wash samples from 20 mule foals up to 6 months of age, with and without clinical evidence of respiratory disease. Samples were collected via endoscopy in two separate occasions and sent for cytology, microbial culture, and PCR for detection of Rhodococcus equi. Based on clinical evidence, 32.5% (13/40) of the samples were obtained from mule foals displaying signs of a respiratory condition, whereas 50% (20/40) of the samples showed cytologic evidence of respiratory tract infection. One hundred percent of samples provided positive cultures with Escherichia coli (45%), Enterococcus (37.5%), and coagulase-negative Staphylococcus (30%) being the most common bacterial genera isolated. R. equi was not identified in any of the samples. The correlation between isolated bacterial agents and the presence of respiratory infection was not statistically significant. The microorganisms found in the samples may be naturally present in the soil, feces, and environment in which the animals live, presenting a risk of opportunistic respiratory infection

Characterization of epidemic clones of Listeria monocytogenes serotype 4b isolated from humans and meat products in Brazil

Submitted by sandra infurna ([email protected]) on 2016-03-08T15:06:05Z No. of bitstreams: 1 andre_barbosa_etal_IOC_2015.pdf: 771307 bytes, checksum: f6af03ce204ee1aef411a720590d5e7c (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-03-08T15:15:59Z (GMT) No. of bitstreams: 1 andre_barbosa_etal_IOC_2015.pdf: 771307 bytes, checksum: f6af03ce204ee1aef411a720590d5e7c (MD5)Made available in DSpace on 2016-03-08T15:15:59Z (GMT). No. of bitstreams: 1 andre_barbosa_etal_IOC_2015.pdf: 771307 bytes, checksum: f6af03ce204ee1aef411a720590d5e7c (MD5) Previous issue date: 2015Universidade Federal Fluminense. Departamento de Microbiologia e Parasitologia. Laboratório de Enteropatógenos, Microbiologia Veterinária e de Alimentos. Niterói, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense. Departamento de Microbiologia e Parasitologia. Laboratório de Enteropatógenos, Microbiologia Veterinária e de Alimentos. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Departamento de Microbiologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Introduction: Listeria monocytogenes is an important foodborne pathogen and the 4b serotype is responsible for many cases of human listeriosis reported in Brazil. Several listeriosis outbreaks worldwide have involved a small number of well-defined clonal groups, designated as epidemic clones (ECs). Methodology: We studied 71 strains of serotype 4b, including 25 isolates from human cases of listeriosis and 46 from meat-based foods, collected in Brazil between 1977 and 2010. The presence of ECs (I and II) markers and virulence genes (inlA, inlB, ilnC, inlJ and actA) were evaluated by PCR assay. The genetic relationship of ECs-positive strains was assessed by pulsed field gel electrophoresis. Results: ECI and ECII markers were found both in human and food strains, with 19.7% positive for the ECI marker and 40.8% for ECII. Most strains (97.2%) were positive for the virulence genes that were studied. Nevertheless, the actA gene amplicons showed two distinct sizes, with all ECI positive strains exhibiting a 105bp deletion. Pulsed field gel electrophoresis (PFGE) analysis allowed the recognition of highly related strains, particularly from two outbreaks of neonatal listeriosis in São Paulo State occurred in 1992 and 1997, both ECIIpositive; and two ECI strains from a human case (1982) and from bovine meat (2009). Conclusions: The presence of ECs among clinical samples and beef isolates of serotype 4b from some regions of Brazil highlights the need for rigorous control of production procedures. Furthermore, the association of ECII with two nosocomial outbreaks suggests its ability to spread in these settings

Detection and molecular characterization of piroplasms species from naturally infected dogs in southeast Brazil Detecção e caracterização molecular de piroplasmas em cães naturalmente infectados no Sudeste do Brasil

Rangelia vitalii is a protozoon described from dogs in the south and southeast regions of Brazil. It is phylogenetically related to Babesia spp. that infects dogs, but data on this enigmatic parasite is still limited. The aim of this work was to detect piroplasm species in dogs in the state of Rio de Janeiro, Brazil, by 18S rRNA gene-based PCR assay, restriction fragment length polymorphism (RFLP) and sequence analyses. Of 103 dogs examined, seven (6.8%) were positive for Babesia spp. by PCR. The amplified products were digested by restriction enzymes to differentiate the Babesia species, and one sample was identified as Babesia vogeli. The pattern observed for the other six amplification products did not match with pattern described for large Babesia infecting dogs. Sequencing analysis confirmed these six samples as R. vitalii, with high homologies (99-100%) with a sequence from south Brazil. This study confirms the presence of Babesia vogeli and Rangelia vitalii circulate in domestic dogs in Teresópolis, Rio de Janeiro, Brazil.<br>Rangelia vitalii é um protozoário que infecta cães e foi descrito nas regiões Sul e Sudeste do Brasil. R. vitalii é filogeneticamente próxima à Babesia spp., mas dados deste misterioso parasito ainda são escassos. O objetivo deste trabalho foi detectar a presença de piroplasmas em cães naturalmente infectados no estado do Rio de Janeiro, através da amplificação do gene 18S rRNA pela PCR, clivagem com enzimas de restrição (RFLP) e caracterização genética através do sequenciamento. De 103 cães, sete (6,8%) foram positivos para Babesia spp. pela PCR. Os produtos amplificados foram digeridos por enzimas de restrição para a diferenciação das espécies de Babesia e uma amostra foi identificada como Babesia vogeli. O padrão de amplificação observado nas outras seis amostras não correspondeu ao padrão descrito para babesias que infectam cães. O sequenciamento das seis amostras confirmou ser uma espécie geneticamente idêntica a R. vitalii apresentando grande homologia (99-100%) com a sequência do sul do Brasil. Este estudo confirma a presença de Babesia vogeli e Rangelia vitalii infectando cães em Teresópolis, Rio de Janeiro, Brasil

Genetic diversity of Ehrlichia canisstrains from naturally infected dogs in Rio de Janeiro, Brazil

The aim of this study was to characterize Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. In addition, all the clinical and hematological findings observed in these dogs were reported. PCR targeting the 16S rRNA gene was used for diagnostic purposes, and the TRP19 and TRP36 genes were sequenced to evaluate the genetic diversity. Fifteen samples were positive for E. canis. The polymerase chain reaction for the TRP19 gene resulted in 11 amplicons (11/15), which were cloned into the pGEM-T easy vector for sequencing. The complete sequence of TRP19 gene was compared to those in the GenBank, revealing high identicalness. Phylogenetic analysis on the TRP36 gene sequences demonstrated two distinct strains from two dogs, named 56C and 70C. The 56C strain was grouped with the strain Cuiaba 16, which is a hybrid strain formed by Brazilian and US genogroups; and the 70C strain was grouped with other strains of the US genogroup, thus suggesting that there are at least two genogroups of E. canis in Rio de Janeiro (US and Brazilian). Those animals, in which the 70C and 56C strains were isolated, showed distinct clinical and hematological manifestations of 1the disease. The appearance of different genotypes may express new phenotypes, thus resulting in different forms of presentation of the disease and making its diagnosis more complex

Cytauxzoon felis and 'Candidatus Mycoplasma haemominutum' coinfection in a Brazilian domestic cat (Felis catus)

This article describes the first detection of Cytauxzoon felis, using molecular techniques, in a naturally infected domestic cat from Brazil, South America. Coinfection with 'CandidatusMycoplasma haemominutum' was also found. The molecular identification of the piroplasmid species was performed by Polymerase Chain Reaction (PCR) and sequencing analysis. A 284 pb fragment of the gene encoding the 18S ribosomal RNA region was amplified and showed 99% identity with other C. felis strains from North America. In addition, PCR-RFLP (restriction fragment length polymorphism) analysis, which amplifies a 595 bp fragment of the gene encoding 16S ribosomal RNA of some bacterial species, identified the co-infecting species as 'Candidatus M. haemominutum'