Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Abstract

A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 × 10 2 and 2.5 × 10 5 cells g -1 of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the timeconsuming traditional bacteriological culture techniques. KEY WORDS: Multiplex-PCR · Fish · Edwardsiella · Flavobacterium · AeromonasResale or republication not permitted without written consent of the publisher Dis Aquat Org 74: 199-208, 2007 most important bacterial diseases affecting the aquaculture industry in the USA (United States Department of Agriculture 2003). The prevalence and impact of septicemic disease in fish caused by Aeromonas hydrophila (referred to as motile aeromonad septicemia, MSA), is less known. However, A. hydrophila is ubiquitous in aquatic ecosystems (Holmes et al. 1996) and recent studies have shown that catfish latently infected with A. hydrophila (carriers) could shed the organism when co-infected with Edwardsiella ictaluri (Nusbaum & Morrison 2002). Since all 3 species of bacteria (Flavobacterium columnare, E. ictaluri, and A. hydrophila) are ubiquitous in the aquatic environment and fish are reared in extensive pond acreages with high stocking densities (20 000 to 30 000 fish ha -1 ), the occurrence of coinfections or multiple infections in the same host should not be overlooked (Jack et al. 1992). Traditional methods of diagnosing bacterial infections using culture techniques require several days to arrive at a definitive diagnosis, resulting in delayed implementation of control measures and increased potential for spreading of disease. Because PCR can target unique genetic sequences of microorganisms, PCR-based nucleic acid amplification techniques have gained recognition as rapid, sensitive, and specific methods for detection of disease-causing pathogens in various aquaculture species (Vantarakis et al. 2000, Del Cero e...

Comparison and evaluation of Renibacterium salmoninarum quantitative PCR diagnostic assays using field samples of Chinook and coho salmon

“…Newer molecular techniques for the detection and quantification of pathogenic bacteria in fish are being developed, including DNA microarrays coupled with PCR (Call et al 2003, Warsen et al 2004, multiplex qPCR (at present, none have included Renibacterium salmoninarum in their panels; see Ji et al 2004, Panangala et al 2007, and the newer technique of loopmediated isothermal amplification (Gahlawat et al 2009). Eventually, a newly developed assay will surpass those currently available.…”

Concurrent infections of Flavobacterium columnare and Edwardsiella ictaluri in striped catfish, Pangasianodon hypophthalmus in Thailand

“…Aeromonas hydrophila (Crumlish et al, 2010;Panangala et al, 2007;Shetty et al, 2014). Among bacterial pathogens, E. ictaluri and F. columnare are recognized as the most highly pathogenic bacteria that cause enteric septicemia of catfish (ESC) and columnaris disease in freshwater fish respectively (Declercq et al, 2013;Hawke et al, 1981).…”

Virulence of Aeromonas hydrophila to channel catfish Ictaluras punctatus fingerlings in the presence and absence of bacterial extracellular products

“…The AL98-C1B isolate of A. hydrophila was isolated from diseased channel catfish in July 1998 and stored in tryptic soy broth containing 5% glycerol at -80°C. All 4 isolates were cultured on tryptic soy agar (TSA) plates according to published procedures (Panangala et al 2007) and re-isolated from channel catfish after 3 passages. The isolates were previously confirmed as A. hydrophila (Pridgeon & Klesius 2011).…”