Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish

Abayneh, Takele; Colquhoun, Duncan J.; Sørum, Henning

doi:10.1016/j.vetmic.2012.03.006

Cited by 50 publications

(79 citation statements)

References 32 publications

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“…To determine whether the isolates described by Griffin et al (2013) and Abayneh et al (2012Abayneh et al ( , 2013 are conspecific, sequencing of the gyrB gene was performed on the 18 Edwardsiella isolates genetically characterized by Griffin et al (2013) (Table 2). As the gyrB fragments determined by Abayneh et al (2012) and Griffin et al (2013) did not overlap, new sequencing primers were designed to specifically encompass the gyrB fragments sequenced in these previous studies so they could be compared (Table 3).…”

Section: Gyrb Sequencing For Species Identificationmentioning

confidence: 99%

“…As the gyrB fragments determined by Abayneh et al (2012) and Griffin et al (2013) did not overlap, new sequencing primers were designed to specifically encompass the gyrB fragments sequenced in these previous studies so they could be compared (Table 3). Primer locations were based on both complete and incomplete genome sequences available via the National Center for Biotechnology Information's GenBank (Table 4).…”

Section: Gyrb Sequencing For Species Identificationmentioning

confidence: 99%

“…Recent phenotypic and genetic studies have revealed that Edwardsiella tarda isolates from fish fell into different genetic groups, suggesting the existence of multiple distinct taxa within the group of organisms classified as E. tarda (Abayneh et al 2012, Yang et al 2012). The result of these studies has been the adoption of a fourth member of the Edwardsiella, E. piscicida, which was characterized from fish in Europe and Asia (Abayneh et al 2013).…”

Section: Introductionmentioning

confidence: 99%

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Edwardsiella piscicida identified in the southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR

Griffin¹,

Ware²,

Quiniou³

et al. 2014

Dis. Aquat. Org.

125

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A new Edwardsiella taxon was recently described from fishes of Europe and Asia. Phenotypically similar to E. tarda, extensive genetic and phenotypic characterization determined this new strain does not belong to any established Edwardsiella taxa, leading to the adoption of a new taxon, E. piscicida. Concurrent research in the USA also identified 2 genetically distinct taxa within the group of organisms traditionally classified as E. tarda. Comparisons of gyrB sequences between US isolates and E. piscicida from Europe and Asia identified several US isolates with > 99.6% similarity to the gyrB sequence of the E. piscicida type strain (ET883) but < 87% similarity to the E. tarda type strain (ATCC #15947). A discriminatory PCR was developed for the identification of E. tarda and 2 genetic variants of E. piscicida (E. piscicida and E. piscicida-like species). Using these PCR assays, a survey was conducted of 44 archived bacterial specimens from disease case submissions to the Aquatic Research and Diagnostic Laboratory (Stoneville, MS, USA) between 2007 and 2012. All 44 isolates, originally identified phenotypically and biochemically as E. tarda, were identified as E. piscicida by PCR. Repetitive sequence-mediated PCR (rep-PCR) analysis of these archived specimens suggests they are largely homogenous, similar to what has been observed for E. ictaluri. The gyrB sequence data, coupled with the E. piscicida specific-PCR and rep-PCR data, confirms that E. piscicida has been isolated from fish disease cases in the southeastern USA. Moreover, our survey data suggests E. piscicida may be more prevalent in catfish aquaculture than E. tarda.

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Section: Gyrb Sequencing For Species Identificationmentioning

confidence: 99%

Section: Gyrb Sequencing For Species Identificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Edwardsiella piscicida identified in the southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR

Griffin¹,

Ware²,

Quiniou³

et al. 2014

Dis. Aquat. Org.

125

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“…2013, based on comparative phylogenomics, 30 multilocus sequence analysis 1 and DNA-DNA hybridization experiments, the taxon E. piscicida was adopted. 2 A 2014 survey of Edwardsiella isolates from diseased catfish in Mississippi demonstrated that E. piscicida was more commonly associated with disease case submissions of farmraised catfish than E. tarda or E. piscicida-like sp.…”

Section: Research-article2014mentioning

confidence: 99%

Real-time polymerase chain reaction assays for the detection and quantification of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella piscicida–like species in catfish tissues and pond water

et al. 2015

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Abstract.Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida-like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published primers, for E. tarda, E. piscicida, and E. piscicida-like sp. to provide rapid quantitative confirmatory tests for these phenotypically ambiguous bacteria. The qPCR assays were shown to be repeatable and reproducible, with high degrees of sensitivity and specificity. Each assay showed a linear dynamic range covering 8 orders of magnitude and a sensitivity limit of 5 copies of target DNA in a 15-µL reaction. In addition, each assay was found specific to their respective targets with no observed amplification from nontarget organisms, including the closely related E. ictaluri and E. hoshinae. Under the conditions used in this study, the 3 assays had a quantifiable limit ranging from 10 3 (E. piscicida) to 10 2 (E. piscicida-like and E. tarda) colony forming units in kidney tissue biopsies (approximately 25 mg), pond water samples (35 mL), and broth culture (20 µL). In experimental challenges, the assays were able to detect their respective targets in both clinically and subclinically infected channel catfish (Ictalurus punctatus) fingerlings. In addition to quantifying target bacteria from various substrates, the assays provide rapid identification, differentiation, and confirmation of the phenotypically indistinguishable E. tarda, E. piscicida, and E. piscicida-like sp., a valuable tool for diagnostic assessments. 13 Speciesspecific polymerase chain reaction (PCR) assays targeting the fimbrial gene cluster were developed for each individual taxa and were demonstrated specific to their respective target organisms. 13,26 In the current study, real-time PCR (qPCR) assays were developed using these established primer sets and were validated for the detection and quantification of E. tarda, E. piscicida, and E. piscicida-like sp. from catfish kidney tissues, pond water, and broth culture. Materials and methods Bacterial cultures and isolation of genomic DNAThe Edwardsiella strains used in the validation of the assays in the current study were characterized as part of an earlier study 13 and identified by gyrB sequencing and speciesspecific PCR (Table 1). In addition, an Edwardsiella hoshinae strain (ATCC 35051), an Escherichia coli strain (ATCC 25922), 2 Flavobacterium columnare strains (94-081 and ATCC 49512), and 2 Aeromonas hydrophila strains (ML 09-119 and TN 97-08), including a highly virulent strain (ML 09-119) attributed to disease outbreaks in farm-raised catfish 18 were also included in the validation process. Design of primer and probe setsThe development of the qPCR assays specific to E. tarda, E. piscicida, and E. piscicida-like sp. was based on previously published PCR primers. 13,26 Oligonucleotide probes corresponding to each primer set were designed using primer design software 25 and synthesized...

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“…HK genes are broadly distributed around the chromosome and encode conserved proteins that are not considered to be under diversifying selection (32). Phylogenies based on these loci have been useful in differentiating bacterial pathogens (33)(34)(35), including bacteria that are highly recombinogenic and undergo frequent lateral gene transfer (36,37).…”

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confidence: 99%

Multilocus Sequence Analysis Provides Insights into Molecular Epidemiology of Chlamydia pecorum Infections in Australian Sheep, Cattle, and Koalas

et al. 2013

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Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts.

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Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish

Cited by 50 publications

References 32 publications

Edwardsiella piscicida identified in the southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR

Edwardsiella piscicida identified in the southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR

Real-time polymerase chain reaction assays for the detection and quantification of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella piscicida–like species in catfish tissues and pond water

Multilocus Sequence Analysis Provides Insights into Molecular Epidemiology of Chlamydia pecorum Infections in Australian Sheep, Cattle, and Koalas

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