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

Abstract

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...

Pathologic changes in cultured Nile tilapia (Oreochromis niloticus) associated with an outbreak of Edwardsiella anguillarum

“…In conjunction with E. piscicida , E. anguillarum is emerging as a fish pathogen with an expanding host range (Buján et al, ; Dubey et al, ; Griffin et al, ). Information concerning impacts of E. anguillarum infection and its associated histopathologic changes is limited, although several investigations indicate variation in pathogenicity among isolates, as well as differences in virulence among diverse fish species (Abayneh et al, ; Reichley, Ware, Greenway, Wise, & Griffin, ; Reichley et al, ). The severity of lesions associated with these particular isolates suggests they are highly virulent in Nile tilapia.…”

Histologic and molecular characterization of Edwardsiella piscicida infection in largemouth bass (Micropterus salmoides)

“…Alternatively, E. piscicida isolate NFAVS-1 possessed a unique gyrB sequence compared with other published gyrB sequences available in GenBank, being most similar (1,891/1,894 bp; 99.8%) to E. piscicida isolate S07-1019 (GenBank JX867002) from a blue catfish (Ictalurus furcatus) in Mississippi. This identification was further supported using a validated end-point and real-time PCR targeting the E. piscicida fimbrial subunit, 23 which identified all 4 isolates as E. piscicida. Genetic fingerprints using the BOX and ERIC (Enteric Repetitive Intergenic Consensus) primer sets, 26,27 following protocols previously outlined, 10 demonstrated these isolates were largely similar to E. piscicida isolate S11-285, which was cultured from channel catfish in Mississippi 10 ( Fig.…”

Identification and Detection of the Pathogenic Bacteria Responsible for Swollen Abdomen Disease in Cultured Turbot, Scophthalmus maximus, and Flounder, Paralichthys olivaceus

“…tarda or V . alginolyticus (Cai et al ; Castro et al ; Zhao et al ; Reichley et al ). However, very few methods were designed for the detection of multiple pathogenic bacterial species.…”