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PCR-Based Assays for the Fish Pathogen Aeromonas Salmonicida

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PCR-Based Assays for the Fish Pathogen Aeromonas Salmonicida DISEASES OF AQUATIC ORGANISMS Vol. 49: 129–138, 2002 Published May 10 Dis Aquat Org PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification Helen K. Byers, Nicholas Gudkovs, Mark St. J. Crane* Australian Animal Health Laboratory, CSIRO Livestock Industries, Private Bag 24, Geelong, Victoria 3220, Australia ABSTRACT: In an effort to develop a rapid diagnostic test for the fish pathogen Aeromonas salmoni- cida, the performance of 2 polymerase chain reaction (PCR) primer sets (AP and PAAS) targeting the fish pathogen A. salmonicida and 1 PCR primer set (MIY) targeting A. salmonicida subsp. salmoni- cida were evaluated. Initially, the PCR assays were used to screen purified DNA extracted from 308 A. salmonicida isolates. The AP and PAAS PCR tests were demonstrated to be 100% specific for the species A. salmonicida and did not cross-react with any of the non-target organisms (bacterial species other than A. salmonicida) used in this study. The combined sensitivity of the AP and PAAS tests was 99.4% and offered the best coverage in terms of identifying the target organism. The MIY PCR appeared to be 100% sensitive and specific for A. salmonicida subsp. salmonicida. Studies with tis- sues, spiked with known quantities of bacteria, were conducted to determine the lower detection limit of the PCR tests, and then the ability of these PCR tests to detect A. salmonicida in experimen- tally infected salmonids was assessed. KEY WORDS: Aeromonas salmonicida · Furunculosis · Detection · Identification · Experimental infections · Polymerase chain reaction Resale or republication not permitted without written consent of the publisher INTRODUCTION mary location within the carrier host remains un- known. All of these factors lead to the possibility that Aeromonas salmonicida is a complex group of bacte- A. salmonicida subsp. salmonicida could enter Aus- rial pathogens, many strains of which cause a range of tralia undetected, establish covert infections in fish and diseases in a wide variety of fish species (Austin & become widely disseminated before the first clinical Austin 1999). A. salmonicida subsp. salmonicida, also case of furunculosis is reported. known as ‘typical A. salmonicida’, is the causative Some atypical strains of Aeromonas salmonicida are agent of furunculosis, an often devastating disease of already considered enzootic to Australia (Whittington salmonids (Hiney & Olivier 1999). et al. 1995, Trust et al. 1996). The incidence of disease Furunculosis poses a very serious exotic disease outbreaks caused by atypical strains, not only in Aus- threat to the growing salmonid aquaculture industries tralia but also world-wide, appears to be increasing in Australia. This threat is further complicated by the and could potentially affect both aquaculture and nat- fact that the bacterium can be present as clinically ural fisheries over a wide geographic range within inapparent or ‘covert’ infections in ‘carrier’ fish (Hiney southern Australia. It is essential that the technology to et al. 1997). Carriers can be extremely difficult to differentiate between those strains exotic to Australia detect, due to the fact that Aeromonas salmonicida is (particularly typical A. salmonicida) and enzootic strains likely to be present only in low numbers, and its pri- be developed. Identification of Aeromonas salmonicida, based on *Corresponding author. E-mail: [email protected] traditional methods including classical biochemical © Inter-Research 2002 · www.int-res.com 130 Dis Aquat Org 49: 129–138, 2002 testing, can be time consuming and problematic. Phe- cytochrome oxidase; motility; growth on sheep blood notypic variation displayed by this group of bacteria agar (SBA) at 37°C; indole; methyl red; Voges- can make assigning new isolates to the existing taxon- Proskauer; arginine dihydrolase (Moeller); nitrate omy, based on phenotype, a difficult procedure (Austin reduction; aesculin hydrolysis; gas production (1% et al. 1998). Polymerase chain reaction (PCR) technol- carbohydrate in oxidation-fermentation [OF] basal ogy may be able to provide a rapid means of identify- medium) from D-glucose; and acid production from L- ing and differentiating bacterial isolates that display arabinose, D-galactose, maltose, D-mannitol, sucrose, phenotypic properties that would otherwise hinder their and trehalose. Tests were conducted according to timely identification. Our objective was to develop Balows et al. (1993) except that cultures were incu- improved procedures for the detection and identifica- bated at 22°C. Tests were examined at 1, 2, 3, 5, 7 and tion of A. salmonicida species and subspecies, thus en- 10 d post-inoculation. Terminal end-product tests were hancing the diagnostic capability for this fish pathogen. evaluated 4 to 10 d post-inoculation. Genomic DNA extraction. Genomic DNA was extracted from all bacterial isolates using the DNAzol® MATERIALS AND METHODS reagent (Life Technologies™) or the Puregene® DNA Isolation Kit (Gentra Systems) as per the manufac- Aeromonas salmonicida isolates. In order to under- turer’s instructions. take extensive in vitro validation and determination of PCR protocols. PCRs were performed in 0.2 ml the specificity and sensitivity of the PCR tests, the Aus- thin-walled PCR tubes (Quantum Scientific) in a tralian Animal Health Laboratory (AAHL) Fish Dis- GeneAmp® 9600 thermal cycler (Perkin Elmer-Cetus). eases Laboratory (AFDL) sourced a wide variety of A. The DNA polymerase and buffering system used was salmonicida isolates (n = 308) from all regions of the either Taq DNA polymerase from Promega or Plat- world where this species has been isolated (Table 1). inum™ Taq from Life Technologies™. Unless other- The collection comprised type and reference cultures wise specified, 1 to 10 ng template DNA was added. from recognised culture collections, and clinical and laboratory strains originating from at least 38 teleost spe- Table 1. List of contributors to the Australian Animal Health Laboratory (AAHL) Fish Diseases Laboratory (AFDL) Aeromonas salmonicida collection cies. Approximately 85% of the iso- lates in the AFDL A. salmonicida col- lection are exotic to Australia. American Type Culture Collection, USA AAHL Fish Diseases Laboratory Cultures other than Aeromonas Commonwealth Scientific and Industrial Research Organisation (CSIRO) salmonicida. A variety of bacterial Livestock Industries, Australia cultures were employed to test the Australian Fish Health Reference Laboratory, Benalla, Australia specificity of the PCR assays. Isolates Dr. C. Rogers, Fish Diseases Laboratory, Centre for Environment, Fisheries and Aquaculture Science (CEFAS), UK included representatives of the Aero- Dr. C. Michel, Institut National de la Recherche Agronomique, France monas DNA hybridisation groups Dr. E. B. Shotts, Department of Microbiology, University of Georgia, USA (Huys & Swings 1999) and a range of Dr. H. Kalnins, Commonwealth Serum Laboratories, Australia common fish pathogens (Table 2). Dr. I. Dalsgaard, Danish Institute for Fisheries and Marine Research, Denmark Maintenance of bacterial strains. All Dr. J. Carson, Fish Health Unit, Mt. Pleasant Laboratory, Department of Primary Industries, Water and Environment (DPIWE), Australia bacterial isolates were stored in Dr. M. Hiney, Fish Disease Group, University College Galway, Ireland MicroBank™ vials (Pro-Lab) at –80°C Dr. O. Haenen, Institute for Animal Science and Health, The Netherlands as per the manufacturer’s instructions. Dr. P. Rintamaki, University of Oulu, Finland When required, isolates were grown Dr. R. Whittington, Elizabeth MacArthur Agricultural Institute, Australia Dr. R. B. Callinan, NSW Department of Fisheries, Australia on Columbia Blood Agar Base (Oxoid Dr. R. Cipriano, National Fish Health Research Laboratory, USA CM331), supplemented with 5% de- Dr. S. Høie, National Veterinary Institute, Norway fibrinated sheep blood, incubated aer- Dr. S. D. Miller, Institute of Aquaculture, University of Stirling, Scotland obically at 22°C for 2 to 4 d. Dr. T. P. T. Evelyn, Pacific Biological Station, Canada Biochemical characterisation. To Dr. T. Meyers, Department of Fish and Game, USA Dr. T. Wiklund, Institute of Parasitology, Abo Akademi University, Finland confirm their identity and to group the Dr. B. K. Gu7mundsdóttir, Institute for Experimental Pathology, isolates according to subspecies, a bio- University of Iceland chemical profile was obtained for 243 Mr. L D. Ashburner, Department of Conservation Forests and Lands, Australia of the 308 Aeromonas salmonicida iso- Mrs. J. Petrie, Fisheries Research Services, Scotland National Collection of Industrial and Marine Bacteria (NCIMB), Scotland lates. The profile consisted of the fol- Prof. B. Austin, Heriot Watt University, Scotland lowing tests: Gram stain; pigment Prof. T. Aoki, Tokyo University of Fisheries, Japan production on tryptic soy agar (TSA); Byers et al.: PCR-based assays for Aeromonas salmonicida. I 131 Table 2. Non-target bacterial isolates employed as specificity amplified for 28 cycles, with denaturation for 30 s at controls during this study. NCFB: National Collection of Food 94°C, annealing for 30 s at 49°C and elongation for Bacteria; other abbreviations in Table 1 1 min 15 s at 72°C. A final extension was performed at 72°C for 3 min. The expected PCR product size was Bacterial species Isolate number 1465 base pairs (bp). Yersinia ruckeri ATCC 29473 Amplification with the PAAS primers (O’Brien et al. Carnobacterium piscicola
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