RESUMO
Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.
Assuntos
Doenças dos Peixes , Oncorhynchus mykiss , Yersiniose , Animais , Yersinia ruckeri/genética , Oncorhynchus mykiss/microbiologia , Yersiniose/epidemiologia , Yersiniose/veterinária , Sorogrupo , Peru/epidemiologia , Doenças dos Peixes/microbiologiaRESUMO
Yersinia enterocolitica is a foodborne pathogen and pigs are the main reservoir of it in their tonsils. As Brazil is a large producer and exporter of pork meat and information regarding this pathogen is still quite scarce, this study aimed at evaluating the direct detection of Y. enterocolitica followed by pathogenic Y. enterocolitica (PYE) determination in tonsils of slaughtered pigs. For this purpose, 400 pig tonsils were collected from 15 farms in four federally certified slaughterhouses in Southern Brazil. Initially, samples were screened using conventional PCR targeting of the 16sRNA gene, followed by multiplex PCR (mPCR) in order to detect three virulence genes (ail, yadA, and virF) and quantitative real-time PCR (qPCR) for the detection of the ail gene. One hundred and one (25.2%) of the samples tested positive for the 16sRNA gene. However, a PYE was detected in one out of the 101 Y. enterocolitica positive samples. The three virulence genes were determined by mPCR and confirmed by partial DNA sequencing. Thus, a significant occurrence of Y. enterocolitica was observed in pig tonsils from federally inspected slaughterhouses in Brazil, although the presence of pathogenic strains was quite low.
Assuntos
Tonsila Palatina/microbiologia , Carne Vermelha/microbiologia , Doenças dos Suínos/microbiologia , Suínos/microbiologia , Yersiniose/veterinária , Yersinia enterocolitica , Matadouros , Animais , Brasil/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real , Doenças dos Suínos/epidemiologia , Virulência , Yersiniose/epidemiologiaAssuntos
Surtos de Doenças/veterinária , Doenças dos Peixes/epidemiologia , Oncorhynchus kisutch , Yersiniose/veterinária , Yersinia ruckeri/isolamento & purificação , Animais , Chile/epidemiologia , Doenças dos Peixes/microbiologia , Sorogrupo , Yersiniose/epidemiologia , Yersiniose/microbiologia , Yersinia ruckeri/genéticaRESUMO
OBJECTIVE: To apply a multiplex real-time polymerase chain reaction (PCR) technique to detect Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica as a diagnostic support tool for the surveillance of foodborne disease outbreaks. MATERIALS AND METHODS: Molecular methodology was applied on clinical samples taken from individuals who were associated with foodborne disease outbreaks in two departments of Colombia. The results were compared with the data obtained by conventional culture methodology. In addition, the clonal relation of the isolations was evaluated using the Pulsed Field Gel Electrophoresis (PFGE) technique. RESULTS: 123 cases of foodborne disease were determined, of which 45 biological samples were confirmed by laboratory and 88 by epidemiological link. The molecular methodology detected 35/45 positive samples versus 17/45 positive samples detected by conventional methodology. PFGE demonstrated a clonal relation during each outbreak. CONCLUSION: The results of the study demonstrate the applicability of the molecular technique as a useful diagnostic support tool to characterize foodborne disease outbreaks, allowing a timely and reliable response.
OBJETIVO: Aplicar una técnica de reacción en cadena de la polimerasa (PCR) múltiple en tiempo real para la detección de Salmonella spp., Listeria monocytogenes y Yersinia enterocolitica, como herramienta de apoyo diagnóstico en la vigilancia de brotes de enfermedad transmitida por alimentos. MATERIALES Y MÉTODOS: Se aplicó la metodología molecular en muestras clínicas provenientes de individuos que estaban asociados a brotes de enfermedad transmitida por alimentos de dos departamentos de Colombia. Los resultados se compararon con los datos arrojados por la metodología convencional de cultivo. Adicionalmente a los aislamientos obtenidos se les evaluó relación clonal mediante la técnica de electroforesis de campo pulsado (PFGE). RESULTADOS: Se determinó un total de 123 casos de enfermedad transmitida por alimentos de los cuales 45 muestras biológicas fueron confirmadas por laboratorio y 88 mediante nexo epidemiológico. La metodología molecular detectó 35/45 muestras positivas frente a 17/45 muestras positivas detectadas mediante la metodología convencional. La PFGE demostró relación clonal en cada brote. CONCLUSIÓN: Los resultados del estudio demuestran la aplicabilidad de la técnica molecular como herramienta útil de apoyo diagnóstico en la caracterización de brotes de enfermedad transmitida por alimentos, permitiendo una respuesta oportuna y confiable.
Assuntos
Surtos de Doenças , Doenças Transmitidas por Alimentos/diagnóstico , Listeriose/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Vigilância em Saúde Pública/métodos , Infecções por Salmonella/diagnóstico , Yersiniose/diagnóstico , Adolescente , Adulto , Idoso , Técnicas de Tipagem Bacteriana/métodos , Criança , Pré-Escolar , Colômbia/epidemiologia , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado , Feminino , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Listeriose/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Salmonella/epidemiologia , Yersiniose/epidemiologia , Yersinia enterocolitica/isolamento & purificação , Adulto JovemRESUMO
INTRODUCTION: Yersinia enterocolitica is a well-known foodborne pathogen widely distributed in nature with high public health relevance, especially in Europe. METHODOLOGY: This study aimed to analyze the pathogenic potential of Y. enterocolitica isolated strains from human, animal, food, and environmental sources and from different regions of Brazil by detecting virulence genes inv, ail, ystA, and virF through polymerase chain reaction (PCR), phenotypic tests, and antimicrobial susceptibility analysis. Pulsed-field gel electrophoresis (PFGE) was used for the assessment of phylogenetic diversity. RESULTS: All virulence genes were detected in 11/60 (18%) strains of serotype O:3, biotype 4 isolated from human and animal sources. Ten human strains (4/O:3) presented three chromosomal virulence genes, and nine strains of biotype 1A presented the inv gene. Six (10%) strains were resistant to sulfamethoxazole-trimethoprim, seven (12%) to tetracycline, and one (2%) to amikacin, all of which are used to treat yersiniosis. AMP-CEF-SXT was the predominant resistance profile. PFGE analysis revealed 36 unique pulsotypes, grouped into nine clusters (A to I) with similarity ≥ 85%, generating a diversity discriminatory index of 0.957. Cluster A comprised all bio-serotype 4/O:3 strains isolated from animal and humans sources. CONCLUSIONS: This study shows the existence of strains with the same genotypic profiles, bearing all virulence genes, from human and animal sources, circulating among several Brazilian states. This supports the hypothesis that swine is likely to serve as a main element in Y. enterocolitica transmission to humans in Brazil, and it could become a potential threat to public health as in Europe.
Assuntos
Yersinia enterocolitica/classificação , Yersinia enterocolitica/isolamento & purificação , Animais , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Brasil/epidemiologia , Análise por Conglomerados , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Microbiologia Ambiental , Microbiologia de Alimentos , Genes Bacterianos , Variação Genética , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Sorogrupo , Suínos , Fatores de Virulência/genética , Yersiniose/epidemiologia , Yersiniose/microbiologia , Yersiniose/veterinária , Yersinia enterocolitica/genética , Yersinia enterocolitica/fisiologia , Zoonoses/epidemiologia , Zoonoses/microbiologiaRESUMO
A polyphasic analysis was carried out on Yersinia ruckeri strains isolated from recently outbreaks in vaccinated fish using a combination of different phenotypic and molecular typing methods in order to study their variability and epidemiological relationships. Eighty strains were subjected to biotyping with conventional tests and API 20E system, serotyping, outer membrane protein (OMP) and lipopolysaccharide (LPS) profiling, and genetic fingerprinting by ERIC-PCR and REP-PCR techniques. The strains showed a high diversity, as evidenced by the formation of different phenotypic groups mainly related to the serotypes, LPS and OMP profiles. The diversity among all isolates, calculated as Simpson's diversity index (Di), varied between 0.35 (REP-PCR) and 0.70 (OMP). The most discriminative values (Di value ≥0.86) were obtained from any combination of three methods including biotype, serotype, API 20E profile, LPS or OMP. With the combination of all typing methods used a Di value of 0.90 was obtained. Association between different groups to the host species was evidenced. Furthermore, it seems that strains with similar characteristics are associated with recent outbreaks occurred in vaccinated fish in certain geographical areas. Our results emphasize the usefulness of using a combination of several different typing methods for epidemiological and bacterial diversity studies.
Assuntos
Surtos de Doenças , Doenças dos Peixes/microbiologia , Salmonidae , Yersiniose/veterinária , Yersinia ruckeri/classificação , Animais , Impressões Digitais de DNA , Europa (Continente)/epidemiologia , Doenças dos Peixes/epidemiologia , Lipopolissacarídeos/metabolismo , Reação em Cadeia da Polimerase , Sorotipagem , América do Sul/epidemiologia , Estados Unidos/epidemiologia , Yersiniose/epidemiologia , Yersiniose/microbiologia , Yersinia ruckeri/genética , Yersinia ruckeri/isolamento & purificação , Yersinia ruckeri/metabolismoRESUMO
We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.