Multiplex nested-polymerase chain reaction for the simultaneous detection ofAeromonas hydrophila, Edwardsiella tarda, Photobacterium damselaeandStreptococcus iniae, four important fish pathogens in subtropical Asia

Abstract

A multiplex nested‐polymerase chain reaction (PCR)‐based (m‐nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae. The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non‐specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m‐nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila, 62 CFU for E. tarda, 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.

In vitrocomparisons of the inhibitory activity of florfenicol, copper sulphate and potassium permanganate towardsAeromonas hydrophilaandFlavobacterium columnare

“…Aeromonas hydrophila HSB‐B2 was provided by Dr Julie Bebak (Aquatic Animal Health Research Laboratory, Auburn, AL, USA) and F. columnare LSU 06‐66 was provided by Dr John Hawke (Louisiana State University, Baton Rouge, LA, USA). The identity of both bacteria was definitively confirmed by PCR (Darwish, Ismaiel, Newton & Tang ; Chang, Wu, Tsai & Lin ). To ensure culture purity, A. hydrophila HSB‐B2 was streaked on Mueller Hinton agar (pH 7.3) plates while F. columnare LSU 06‐66 was streaked on modified Shieh agar (pH 7.2–7.4) plates (Decostere, Ducatelle & Haesebrouck ).…”

Development of a multiplex polymerase chain reaction to detect five common Gram‐negative bacteria of aquatic animals

“…Detection of food-borne vibriosis caused by V. parahaemolyticus, V. cholerae and V. vulnificus was also carried out using an m-PCR (Bauer & Rorvik 2007). Moreover, multiplex nested PCR methods based on the amplification of 16S rRNA genes have been developed for the identification of bacterial pathogens in aquaculture, such as A. hydrophila, E. tarda, Photobacterium damselae and Streptococcus iniae (Chang et al 2009) and A. salmonicida, Y. ruckeri and Flavobacterium psychrophilum (Taylor & Winton 2002). As the prevalence of fish disease may be diverse and depend on the farmed species and culture area, it is difficult to assess disease outbreaks by means of a universal diagnostic measure or procedure especially when newly emerging diseases are possible.…”

Assessment of Aquaflor®, copper sulphate and potassium permanganate for control of Aeromonas hydrophila and Flavobacterium columnare infection in sunshine bass, Morone chrysops female × Morone saxatilis male

“…All bacterial isolations were from external lesions, liver or trunk kidney on either tryptic soy agar supplemented with 5% sheep blood (TSA‐5SB) (Edge Biologicals, Inc.) or on selective cytophaga agar (SCA) (Hawke & Thune 1992) at 28 °C for 48 h. Aeromonas hydrophila was isolated on TSA‐5SB, Gram‐stained and identified using API 20E biochemical testing at 30 °C (bioMerieux, Inc.) and multiplex nested polymerase chain reaction (Chang et al. 2009).…”