Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR

BMC Microbiology - Tập 14 - Trang 1-10 - 2014
Nicole Strepparava1,2, Thomas Wahli2, Helmut Segner2, Orlando Petrini3
1Laboratory of Applied Microbiology, University of Applied Sciences and Arts of Southern Switzerland, Bellinzona, Switzerland
2Centre for Fish and Wildlife Health, University of Bern, Bern, Switzerland
3POLE Pharma Consulting, Breganzona, Switzerland

Tóm tắt

Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. We describe a qPCR technique based on the single copy gene β’ DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak.

Tài liệu tham khảo

Baker GC, Gaffar S, Cowan DA, Suharto AR: Bacterial community analysis of Indonesian hot springs. FEMS Microbiol Lett. 2001, 200 (1): 103-109. 10.1111/j.1574-6968.2001.tb10700.x.

Eiler A, Bertilsson S: Flavobacteria blooms in four eutrophic lakes: linking population dynamics of freshwater bacterioplankton to resource availability. Appl Environ Microbiol. 2007, 73 (11): 3511-3518. 10.1128/AEM.02534-06.

Peeters K, Willems A: The gyrB gene is a useful phylogenetic marker for exploring the diversity of Flavobacterium strains isolated from terrestrial and aquatic habitats in Antarctica. FEMS Microbiol Lett. 2011, 321 (2): 130-140. 10.1111/j.1574-6968.2011.02326.x.

Barnes ME, Brown ML: A review of Flavobacterium psychrophilum biology, clinical signs, and Bacterial Cold Water Disease prevention and treatment. Open Fish Sci J. 2011, 4: 1-9.

Bernardet JF, Kerouault B: Phenotypic and genomic studies of “Cytophaga psychrophila” isolated from diseased rainbow trout (Oncorhynchus mykiss) in France. Appl Environ Microbiol. 1989, 55 (7): 1796-1800.

Hesami S, Allen KJ, Metcalf D, Ostland VE, MacInnes JI, Lumsden JS: Phenotypic and genotypic analysis of Flavobacterium psychrophilum isolates from Ontario salmonids with bacterial coldwater disease. Can J Microbiol. 2008, 54 (8): 619-629. 10.1139/W08-058.

Madetoja J, Dalsgaard I, Wiklund T: Occurrence of Flavobacterium psychrophilum in fish-farming environments. Dis Aquat Organ. 2002, 52 (2): 109-118.

Valdebenito S, Avendano-Herrera R: Phenotypic, serological and genetic characterization of Flavobacterium psychrophilum strains isolated from salmonids in Chile. J Fish Dis. 2009, 32 (4): 321-333. 10.1111/j.1365-2761.2008.00996.x.

Wakabayashi H, Huh GJ, Kimura N: Flavobacterium branchiophila sp. nov., a causative agent of Bacterial Gill Disease of freshwater fishes. Int J Syst Bacteriol. 1989, 39 (3): 213-216. 10.1099/00207713-39-3-213.

Nematollahi A, Decostere A, Pasmans F, Haesebrouck F: Flavobacterium psychrophilum infections in salmonid fish. J Fish Dis. 2003, 26 (10): 563-574. 10.1046/j.1365-2761.2003.00488.x.

Anderson JI, Conroy DA: The pathogenic myxobacteria with special reference to fish diseases. J appl Bact. 1969, 32: 30-39. 10.1111/j.1365-2672.1969.tb02186.x.

Carlson RV, Pacha RE: Procedure for the isolation and enumeration of myxobacteria from aquatic habitats. Appl Microbiol. 1968, 16 (5): 795-796.

Lehmann J, Mock D, Stürenberg FJ, Bernardet JF: First isolation of Cytophaga psychrophila from a systemic disease in eel and cyprinids. Dis Aquat Organ. 1991, 10: 217-220.

Pacha RE: Characteristics of Cytophaga psychrophila (Borg) isolated during outbreaks of bacterial cold-water disease. Appl Microbiol. 1968, 16 (1): 97-101.

Rangdale RE, Richards RE, Alderman DJ: Isolation of Cytophaga psychrophila, causal agent of rainbow trout fry syndrome (RTFS) from reproductive fluids and egg surfaces of rainbow trout (Oncorhynchus mykiss). Bull Eur Ass Fish Pathol. 1996, 16 (2): 63-

Strepparava N, Wahli T, Segner H, Polli B, Petrini O: Fluorescent in situ Hybridization: a new tool for the direct identification and detection of F. psychrophilum. PLoS One. 2012, In Press

Langendijk PS, Schut F, Jansen GJ, Raangs GC, Kamphuis GR, Wilkinson MH, Welling GW: Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Appl Environ Microbiol. 1995, 61 (8): 3069-3075.

Madetoja J, Wiklund T: Detection of the fish pathogen Flavobacterium psychrophilum in water from fish farms. Syst Appl Microbiol. 2002, 25 (2): 259-266. 10.1078/0723-2020-00105.

Wiklund T, Madsen L, Bruun MS, Dalsgaard I: Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification. J Appl Microbiol. 2000, 88 (2): 299-307. 10.1046/j.1365-2672.2000.00959.x.

Altinok I, Capkin E, Kayis S: Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens. Vet Microbiol. 2008, 131 (3–4): 332-338.

Del Cerro A, Marquez I, Guijarro JA: Simultaneous detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, three major fish pathogens, by multiplex PCR. Appl Environ Microbiol. 2002, 68 (10): 5177-5180. 10.1128/AEM.68.10.5177-5180.2002.

Carelli G, Decaro N, Lorusso A, Elia G, Lorusso E, Mari V, Ceci L, Buonavoglia C: Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR. Vet Microbiol. 2007, 124 (1–2): 107-114.

Lewin SR, Vesanen M, Kostrikis L, Hurley A, Duran M, Zhang L, Ho DD, Markowitz M: Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy. J Virol. 1999, 73 (7): 6099-6103.

Trombley AR, Wachter L, Garrison J, Buckley-Beason VA, Jahrling J, Hensley LE, Schoepp RJ, Norwood DA, Goba A, Fair JN, Kulesh DA: Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses. Am J Trop Med Hyg. 2010, 82 (5): 954-960. 10.4269/ajtmh.2010.09-0636.

Marancik DP, Wiens GD: A real-time polymerase chain reaction assay for identification and quantification of Flavobacterium psychrophilum and application to disease resistance studies in selectively bred rainbow trout Oncorhynchus mykiss. FEMS Microbiol Lett. 2013, 339 (2): 122-129. 10.1111/1574-6968.12061.

Orieux N, Bourdineaud JP, Douet DG, Daniel P, Le Henaff M: Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR. J Fish Dis. 2011, 34 (11): 811-821. 10.1111/j.1365-2761.2011.01296.x.

Chelo IM, Ze-Ze L, Tenreiro R: Congruence of evolutionary relationships inside the Leuconostoc-Oenococcus-Weissella clade assessed by phylogenetic analysis of the 16S rRNA gene, dnaA, gyrB, rpoC and dnaK. Int J Syst Evol Microbiol. 2007, 57 (Pt 2): 276-286.

Mittenhuber G: Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins). J Mol Microbiol Biotechnol. 2001, 3 (4): 585-600.

Morse R, Collins MD, O’Hanlon K, Wallbanks S, Richardson PT: Analysis of the beta’ subunit of DNA-dependent RNA polymerase does not support the hypothesis inferred from 16S rRNA analysis that Oenococcus oeni (formerly Leuconostoc oenos) is a tachytelic (fast-evolving) bacterium. Int J Syst Bacteriol. 1996, 46 (4): 1004-1009. 10.1099/00207713-46-4-1004.

Duchaud E, Boussaha M, Loux V, Bernardet JF, Michel C, Kerouault B, Mondot S, Nicolas P, Bossy R, Caron C, Bessieres P, Gibrat JF, Claverol S, Dumetz F, Le Henaff M, Benmansour A: Complete genome sequence of the fish pathogen Flavobacterium psychrophilum. Nat Biotechnol. 2007, 25 (7): 763-769. 10.1038/nbt1313.

Morillo JM, Lau L, Sanz M, Herrera D, Silva A: Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. J Periodontal Res. 2003, 38 (5): 518-524. 10.1034/j.1600-0765.2003.00684.x.

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009, 55 (4): 611-622. 10.1373/clinchem.2008.112797.

Barbier P, Houel A, Loux V, Poulain J, Bernardet JF, Touchon M, Duchaud E: Complete genome sequence of Flavobacterium indicum GPSTA100-9T, isolated from warm spring water. J Bacteriol. 2012, 194 (11): 3024-3025. 10.1128/JB.00420-12.

McBride MJ, Xie G, Martens EC, Lapidus A, Henrissat B, Rhodes RG, Goltsman E, Wang W, Xu J, Hunnicutt DW, Staroscik AM, Hoover TR, Cheng YO, Stein JL: Novel features of the polysaccharide-digesting gliding bacterium Flavobacterium johnsoniae as revealed by genome sequence analysis. Appl Environ Microbiol. 2009, 75 (21): 6864-6875. 10.1128/AEM.01495-09.

Tekedar HC, Karsi A, Gillaspy AF, Dyer DW, Benton NR, Zaitshik J, Vamenta S, Banes MM, Gulsoy N, Aboko-Cole M, Waldbieser GC, Lawrence ML: Genome sequence of the fish pathogen Flavobacterium columnare ATCC 49512. J Bacteriol. 2012, 194 (10): 2763-2764. 10.1128/JB.00281-12.

Touchon M, Barbier P, Bernardet JF, Loux V, Vacherie B, Barbe V, Rocha EP, Duchaud E: Complete genome sequence of the fish pathogen Flavobacterium branchiophilum. Appl Environ Microbiol. 2011, 77 (21): 7656-7662. 10.1128/AEM.05625-11.

Nematollahi A, Decostere A, Pasmans F, Ducatelle R, Haesebrouck F: Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow trout Oncorhynchus mykiss. Dis Aquat Organ. 2003, 55 (2): 101-107.

Brown LL, Cox WT, Levine PL: Evidence that the causal agent of bacterial cold-water disease Flavobacterium psychrophilum is transmittes within salmonid eggs. Dis Aquat Organ. 1997, 29: 213-218.

McCormick A, Loeffler J, Ebel F: Aspergillus fumigatus: contours of an opportunistic human pathogen. Cell Microbiol. 2010, 12 (11): 1535-1543. 10.1111/j.1462-5822.2010.01517.x.

Miceli MH, Diaz JA, Lee SA: Emerging opportunistic yeast infections. Lancet Infect Dis. 2011, 11 (2): 142-151. 10.1016/S1473-3099(10)70218-8.

Morgan AL, Thompson KD, Auchinachie NA, Migaud H: The effect of seasonality on normal haematological and innate immune parameters of rainbow trout Oncorhynchus mykiss L. Fish Shellfish Immunol. 2008, 25 (6): 791-799. 10.1016/j.fsi.2008.05.011.

Banks JL: Raceway density and water flow as factors affecting spring chinook salmon (Oncorhynchus tshawytscha) during rearing and after release. Aquaculture. 1994, 119 (2–3): 201-217.

Karvonen A, Rintamaki P, Jokela J, Valtonen ET: Increasing water temperature and disease risks in aquatic systems: climate change increases the risk of some, but not all, diseases. Int J Parasitol. 2010, 40 (13): 1483-1488. 10.1016/j.ijpara.2010.04.015.

Pulkkinen K, Suomalainen LR, Read AF, Ebert D, Rintamaki P, Valtonen ET: Intensive fish farming and the evolution of pathogen virulence: the case of columnaris disease in Finland. Proc Biol Sci. 2010, 277 (1681): 593-600. 10.1098/rspb.2009.1659.

Suomalainen LR, Tiirola MA, Valtonen ET: Influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum). J Fish Dis. 2005, 28 (5): 271-277. 10.1111/j.1365-2761.2005.00631.x.

Lorenzen E, Olesen NJ: Characterization of isolates of Flavobacterium psychrophilum associated with coldwater disease or rainbow trout fry syndrome II: serological studies. Dis Aquat Organ. 1997, 31: 209-220.

Green DM, Gregory A, Munro LA: Small- and large-scale network structure of live fish movements in Scotland. Prev Vet Med. 2009, 91 (2–4): 261-269.

Tonolla M, Peduzzi S, Hahn D, Peduzzi R: Spatio-temporal distribution of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland). FEMS Microbiol Ecol. 2003, 43 (1): 89-98. 10.1111/j.1574-6941.2003.tb01048.x.

Griffiths E, Gupta RS: Signature sequences in diverse proteins provide evidence for the late divergence of the Order Aquificales. Int Microbiol. 2004, 7 (1): 41-52.

Yamamoto S, Harayama S: PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol. 1995, 61 (10): 3768-

Tamura K, Dudley J, Nei M, Kumar S: MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007, 24 (8): 1596-1599. 10.1093/molbev/msm092.

Bikandi J, San Millan R, Rementeria A, Garaizar J: In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction. Bioinformatics. 2004, 20 (5): 798-799. 10.1093/bioinformatics/btg491.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215 (3): 403-410. 10.1016/S0022-2836(05)80360-2.

Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J: qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007, 8 (2): R19-10.1186/gb-2007-8-2-r19.

Mackay IM: Real-time PCR in the microbiology laboratory. Clin Microbiol Infect. 2004, 10 (3): 190-212. 10.1111/j.1198-743X.2004.00722.x.

Yun JJ, Heisler LE, Hwang II, Wilkins O, Lau SK, Hyrcza M, Jayabalasingham B, Jin J, McLaurin J, Tsao MS, Der SD: Genomic DNA functions as a universal external standard in quantitative real-time PCR. Nucleic Acids Res. 2006, 34 (12): e85-10.1093/nar/gkl400.

Joly P, Falconnet PA, Andre J, Weill N, Reyrolle M, Vandenesch F, Maurin M, Etienne J, Jarraud S: Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl Environ Microbiol. 2006, 72 (4): 2801-2808. 10.1128/AEM.72.4.2801-2808.2006.

Thông tin
Thông tin xuất bản

BMC Microbiology

Tập 14

1-10

Thông tin tác giả