Int. J. Aquat. Biol. (2015) 3(1): 52-59 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2015 Iranian Society of Ichthyology Original Article Simultaneous detection of Aeromonas hydrophila, and Escherichia coli in Rainbow trout (Oncorhynchus mykiss) by Duplex PCR Fatemeh Fattahi1, Alireza Mirvaghefi*1, Hamid Farahmand1, Gholamreza Rafiee1, Alireza Abdollahi2 1Department of Fisheries, Faculty of Natural Resource, University of Tehran, Karaj, Iran. 2Department of Pathology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Article history: Received 14 October 2014 Accepted 6 January 2015 Available online 2 5 February 2015 Keywords: Duplex PCR Rainbow trout Simultaneous detection 16SrRNA gene Abstract: Rapid and accurate identification of microorganisms have a significant impact on strategies and fish health management programs. Hence, in this study a duplex PCR assay based on the 16s rRNA gene for simultaneous detection of Aeromonas hydrophila RTICC 1032 and Escherichia coli RTICC 2325 from pure cultures, and challenged fish tissues was performed and their results compared with the results of single PCR assays for each bacterium. For this purpose, an experiment with three treatments including artificially infected with A. hydrophila, E. coli and a mixture of them with a control group was designed. Fish were injected intraperitoneally with 1 ml of sterile physiological saline containing 106 CFU/ml of the corresponding bacteria. Samples were collected from liver, kidney and spleen 48 hrs post-injection. A duplex PCR based 16S rRNA genes was developed for the simultaneous detection of A. hydrophila and E. coli. The PCR reaction conditions were optimized to permit detection of organisms from agar plates and fish tissues in less than 8 hrs. Each of the two pairs of oligonucleotide primers exclusively targeted 16S rRNA gene of the specific microorganism. When duplex PCR assay was used to simultaneous detection of the pathogens in asymptomatic fish, spleen and liver were negative for A. hydrophila, whereas kidney was positive for two bacteria. Samples of control group with negative results of duplex PCR were also negative by the culture method. On the whole, the duplex PCR has advantages in terms of its accuracy, sensitivity, ease of use, time of length analysis and cost-effectiveness compared to the single PCR and traditional method. Introduction Fishes are often exposed to various microorganisms such as Aeromonas hydrophila that is naturally present in the fresh water environments (Belanco, 2000). This bacterium does not cause disease under optimal conditions, whereas under unfavorable environmental conditions, physiological stress or infection by other pathogens can cause motile aeromonad septicemia (MAS), epizootic ulcerative syndrome (EUS) and ulcerous dermatitis, which are led to economic losses in aquaculture industry (Plumb et al., 1976; Fang et al., 2000; Laptera et al., 2010). In addition, the presence of Escherichia coli in fishes is considered an indicator of the polluted or * Corresponding author: Alireza Mirvaghefi E-mail address: avaghefi@ut.ac.ir stressful environments where fish inhabit (DHSS, 1991; Gelderich et al., 1966; Sinderman, 1988). Escherichia coli enters aquatic ecosystems via animal excreta, agricultural runoff and human consumed wastes (Ferreira da silva et al., 2007; Berier et al., 2008). Therefore, significant numbers of E. coli on the skin and gut of the fishes can be led health risk to human (Janssen 1974; Ishii et al., 2007). Rapid and accurate identification of the microorganisms, especially pathogen bacteria have a significant impact on fish health management programs (Adams et al., 2006). Traditionally, the diagnosis of the pathogenic bacteria is performed by 53 Fattahi et al./ Duplex PCR method detection of A. hydrophila, and E. coli in Rainbow trout culturing bacteria on agar plates followed by phenotypic and serological properties (Altinok et al., 2008). However, detections of some bacteria are difficult due to their morphological variations and unusual biochemical reactions. Therefore, molecular diagnosis methods such as reverse transcription PCR, quantitative PCR, real-time PCR, AFLP, RFLP and RAPD have been developed to detect specific nucleic acids without culture and isolation of the pathogens (Tang et al., 2006; Adams et al., 2008). In this regard, the individual PCR assay is an effective method for identification of the fish pathogens; however, a large number of individual PCR assays are necessary when a single primer set is used on a large number of the clinical samples, which can be a relatively costly and time-consuming process. Hence, the simultaneous detection of several pathogens with a multiplex PCR (m PCR) approach would be a relatively rapid and cost effective method (Mata et al., 2004). In this method, we seek to diagnose all possible pathogens, which can be occurred in each disease (Belak, 2007). The multiplex PCR assay for the simultaneous detection of fish pathogenic bacteria has been recently described (del Cerro et al., 2002; Mata et al., 2004; Altinok et al., 2008). Hence, in this work, a duplex PCR assay based on the 16s rRNA genes for the simultaneous detection of A. hydrophila and E. coli from pure cultures, and the challenged rainbow trout tissues (Oncorhynchus mykiss) including liver, kidney and spleen were performed and the results compared with single PCR assays for each bacterium. Materials and methods Fish: Fifty-two rainbow trout with a mean weight of 246 ± 20. 91 g (Mean ± SD) and mean length of 27.04 ± 0.90 cm (Mean ± SD) were obtained from a commercial fish farm in Karaj (Alborz Province, Iran). They were introduced at a rate of 13 fish per l000-liter to four tanks with proper aeration. Fish were acclimatized to the laboratory conditions for 2 weeks prior to experiment. The water temperature during acclimatization period and experiment was 14.5°C (± 1.5). Bacteria: Bacterial isolates (A. hydrophila RTICC 1032 and E. coli RTICC 2325) were obtained from the Razi Vaccine and Serum Research Institute (Table 1). For the challenges, each bacterium strain was cultured on TSA (Himedia-M 290) at 37°C for 24 hrs and harvested in a sterile physiological saline to 10-9 and diluted to an optical density of 1.0 at a wavelength of 640 nm. This corresponds to a bacterial concentration of 1 x 106 CFU/ml. Challenge with A. hydrophila and E. coli: For bacterial challenge, 9 fish (three from every tank) were randomly selected, anaesthetized in 100 mg/ml of Tricaine Methane Sulphonate (TMS) and injected intraperitoneally with 1 ml of the bacterial suspension of A. hydrophila, E. coli and mixture of A. hydrophila and E. coli, respectively. Then, injected fish were returned to treatment tanks and allowed to recover from the anesthetic. One of tanks was considered as controls group without injection. Fish sampling: Forty-eight hours after injection, three fish from each treatment were sampled and their body surface were swabbed using 70% ethyl alcohol after killing by overdosing using TMS to prevent contamination from the rearing environment and normal external bacterial flora. To obtain similar size samples, 1 mm cubes of the liver, kidney and spleen were aseptically removed and put in the microcenterfuge tubes for detection of microorganism from fish tissue. Then, the samples of liver, kidney and spleen were streaked on tryptic soy agar. Following incubation, one typical colony was selected from each isolate and sub-cultured on Bacteria species Strain number Donor Escherichia coli RTICC 2325 Razi Vaccine and Serum Research Institute Aeromonas hydrophila RTICC 1032 Razi Vaccine and Serum Research Institute Table 1. Bacteria strains used in the experiment. 54 Int. J. Aquat. Biol. (2015) 3(1): 52-59 MacConkey (Merck-5465), EMB (OXOID-CM69) and Blood Agar media to check purity of the isolates. All isolates were stored in a broth culture supplemented with 15% glycerol at -70°C. The isolates were classified as A. hydrophila or E. coli according to their reactions in the following conventional tests including catalase, motility, indole, voges-proskaues, urea, triple sugar iron (TSI), glucose, methyl red, H2S production and citrate utilization tests based on Bergey’s manual of determinative bacteriology (Holt, 2000). Every substrate was incubated at 37°C and reactions read after 24 and 48 hrs. DNA extraction: For DNA extraction of the isolates (pure cultures), a boiled method was used based on Sambrook et al. (2001) by phenol-chloroform– isoamyl alcohol. DNA concentrations of samples were evaluated using a spectrophotometer. In addition, DNA was extracted form liver, kidney and spleen of artificially infected fish based on Altinok et al. (2008). The extracted DNA quality was evaluated using electrophorese on a 0.8% agarose gel. Primers and PCR conditions: The used primers in this study were based on Nilsen et al. (2001) and Sabat et al. (1991) to validate the duplex PCR assay for the simultaneous detection of A. hydrophila and E. coli in asymptomatic carrier fish. The PCR protocol was optimized by amplification reaction in a thermal cycler (Astec, Japan) using the ready–to– go PCR beads (Cinagene, Iran). Reaction mixtures had 1 µL of each primer, 1 µL of the DNA template, 17.5 µL of sterile distilled water, 1 µL of Mgcl2, 0.5 µL of dNTP, 2.5 µL of 10 x PCR buffer. PCR conditions consisted of an initial denaturation step at 94°C for 7 min followed by 30 cycles of amplification (denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min) and a final 10 min elongation period at 72°C. Controls received the PCR mixture containing (1) No template, (2) DNA from control fish and (3) DNA from E. coli and A. hydrophyla (Positive control). After the PCR, the products were transferred to a 1.5% agarose gel, electrophoresed, and DNA was visualized by ethidium bromide staining. Table 2 shows the sequences of the two primer pairs used in this study. Duplex PCR assay: A duplex PCR assay was developed for the simultaneous detection of A. hydrophila and E. coli in which rainbow trout were experimentally challenged with both bacteria. The specificity and sensitivity of this assay was evaluated by performing the duplex PCR to the detection of healthy carriers. Samples were collected from kidney, liver and spleen and analyzed for the presence of these two pathogens by duplex PCR. To avoid contamination, each of the following steps were performed in a separate room: autopsy, DNA extraction, PCR master mix preparation, DNA quantification, addition to the PCR mixture, PCR reaction and electrophoresis. New disposable razor blades, forceps, and gloves were used for each fish to reduce potential contamination between fish. Sequencing method: To verify that the specific primer-pair amplified A. hydrophila and E. coli DNA, the PCR product was purified with a PCR purification Kit (Qiagen) and directly sequenced with an ABI 3130 genetic analyzer (Applied Biosystems Instrument) in Avicenna Research Institute. The results of the sequencing were used for homology searches by the BLAST program available at the NCBI (National Center for Biotechnology Information) website (http://www. ncbi.nlm.nih.gov). Name Gene Sequence (5-3) Described Pathogen Size (bp) FES RES 16s rRNA F:GAAAGGTTGATGCCTAATACGA R:CGTGCTGGCAACAAAGGACAG Nielsen et al., 2006 A. hydrophila 700 FES RES 16s rRNA F:GGAAGAAGCTTGCTTCTTTGCTG R:AGCCCGGGGATTTCACATCTGA Sabat et al., 1999 E. coli 544 Table 1. Primers used in this study. 5 5 F at ta h i et a l. / D u p le x P C R m et h o d d et ec ti o n o f A . h y d ro p h il a, a n d E . co li i n R ai n b o w t ro u t C h a r a c te r is ti c o f is o la te s id e n ti fi e d a s E . c o li a n d A .h y d ro p h y la b y b o th P C R a n d b io c h e m ic a l te st s B io c h e m ic a l te st s L it e r a tu r e ( M il le r sh ip , 1 9 9 6 ) - A . h y d ro p h il a L it e r a tu r e ( F o r b e s e t a l. , 1 9 9 8 ) - E . c o li M o le c u la r d ia g n o si s C it T S I G a s/ g lu c o se U C a t M R - V P N it S IM G a s/ g lu c o se U C a t M R - V P N it S I M C it T S I G a s/ g lu c o se U C a t M R - V P N it S I M C it T S I A h -1 + A/ A + - + + + + + + + + - + + + + + + + + A/ A - 1 0 % + - + - + + + + - - A/ A A/ K 7 0 0 -b p E -1 - A/ A - - + - + + + + - G a s/ g lu c o se U C a t M R - V P N it S I M C it T S I 5 4 4 b p T y p e s tr a in R T IC C 1 0 3 2 - A/ A + - + + + + + + + - 1 0 % + - + - + + + + - - A/ A A/ K 7 0 0 -b p T y p e s tr a in R T IC C 2 3 2 5 - A/ A - - + - + + + + - G a s/ g lu c o se U C a t M R - V P N it S I M C it T S I 5 4 4 b p U re a = U , C a ta la se = C a t , C it ra te = C it , G a s fr o m g lu c o se = g a s/ g , N it ra te = N it , S u lf id e , In d o le , M o ti li ty = S IM , M R -V P = M e th y l re d , V o g e s- p ro sk a u ; T S I= T ri p le S u g a r Ir o n T ab le 3 . Id e n ti fi ca ti o n a n d c h ar ac te ri za ti o n o f E . co li a n d A . h y d ro p h y la . F ig u re 1 . F is h i n je ct e d w it h A . h y d ro p h y la ( n o te p et ec h ia l at t h e fi n b as ed a n d t ai l ro t) . F ig u re 2 . F is h i n je ct ed w it h E . co li ( sh o w s n o c li n ic al s ig n s o f d is ea se ). 56 Int. J. Aquat. Biol. (2015) 3(1): 52-59 Results Isolation and identification of A. hydrophyla and E. coli: Pure cultures were obtained from all tissue samples and biochemical analysis were carried out on A. hydrophyla and E. coli isolates. All tissue samples were positive for microbiological and PCR identification (Table 3). Variation in citrate reaction was observed within the group identified as A. hydrophyla when compared to the type strain RTICC 1032. Occurrence of A. hydrophila and disease signs: Mortality was observed two days post-injection in the group injected with A. hydrophila. Aeromonas hydrophila clinical signs were observed in five days post-injection including anorexia, exophtalmus, petechiae and reddening due to haemorrhage of the skin, erosion of the tail and fins and swimming at the surface of the tank (Fig. 1), and similar clinical signs were observed in the groups injected with both bacteria, while mortalities or clinical signs of disease were not observed in fish injected with E. coli (Fig. 2). PCR identification of A. hydrophyla and E. coli: Three annealing temperatures (58, 60 and 62°C) and two Mgcl2 concentrations (1.5 and 2 mM) were examined for the optimal sensitivity of the duplex PCR assay. A good intensity of the amplicons for each target DNA, as well as the absence of unspecific bands, was considered in selecting the optimal duplex PCR conditions. Thus, the best results were obtained with an annealing temperature of 58°C and 2 mM Mgcl2. Each of the two pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganisms. Positive PCR amplification of DNA templates from A. hydrophila and E. coli were produced a single fragment of the expected, for each pathogen (700 bp and 544 bp, respectively) (Fig. 3). The two bacterial pathogens were simultaneously amplified with relatively equal DNA band intensities (Fig. 3). Escherichia coli and A. hydrophila were detected from cultures on agar plates and fish tissues (Fig. 4). Detection of the two bacterial pathogens within DNA templates derived from liver, kidney and spleen were possible as early as 48 hrs after challenge in dead fish (Fig. 4A). The two bacterial pathogens were simultaneously amplified in kidney (Fig. 3); whereas, only E. coli was amplified in the liver and spleen tissues of fish injected with two bacteria. The size of PCR products from colonies were the same as tissues. Representative examples of the product formation from each source are shown in Figure 4. All tissue samples were positive for microbiological and PCR identification (Table 3). The total procedure was accomplished in less than 8 hrs. No amplification products were obtained from control group (Fig. 3). To confirm the positive PCR results, we sequenced the amplified DNA products Figure 3. Representative PCR products from dead fish tissues, agar plate and positive control using the Duplex PCR and single PCR assay under optimized condition. Lane M, molecular size marker; lane 1, A. hydrophila RTICC 1032 (700bp); lane 2, E. coli RTICC 2325(544 bp); lane 3, mixture of the two bacteria; lanes 4 to 6, bacteria isolated from agar plates; lane 7, kidney tissue from fish injected with A. hydrophila; lane 8, kidney tissue from fish injected with E. coli; lane 9, kidney tissue from fish injected with two bacteria (asymptomatic fish); lane 10, liver tissue from fish injected with E. coli; lanes 11 to 12, liver and spleen tissue from fish were injected with two bacteria (asymptomatic fish), respectively; lanes 13 to15, negative control containing Duplex PCR from fish tissues (kidney, liver and spleen, respectively); lane16, no DNA. 57 Fattahi et al./ Duplex PCR method detection of A. hydrophila, and E. coli in Rainbow trout from different PCR reactions and DNA extractions. The BLAST analysis from sequencing of four randomly amplicons showed similar results, and found that the amplified fragment exactly matched the sequence of two bacteria. These findings suggest the high specificity of the primers to detect E. coli and A. hydrophila. Discussion In aquaculture industry, diagnosis and treatment of the microbial diseases is crucial both from economic and sanitary point of views (Blanco et al., 2000; Stevenson, 1999). In recent years, there has been much interest in the development of multiplex PCR assay for the simultaneous detection of bacterial fish pathogens (del Cerro et al., 2002; Mata et al., 2004; Altinok et al., 2008). Mortality was observed in 48 hrs post-injection in injected fish with A. hydrophila, showing a hemorrhagic septicemia with hemorrhagic in internal organs and a red tinged ascetic fluid. The kidney, liver and spleen of challenged fish were processed for both microbiological and single PCR analysis. In the literature, A. hydrophila is usually reported to be citrate positive (Millership, 1996). However, Neil and Nair (2004) reported that A. hydrophila to be citrate variable, which was confirmed in this study. This could be due to many biochemical identification schemes based on the analysis of human clinical isolates and that fish isolates may differ in several biochemical characters. Different reaction patterns may be influenced by physical parameters, such as pH, temperature, and growth substrate concentrations (Haenninen et al., 1994; Janda et al., 2002; Sautour et al., 2003). In this study, the characterization by biochemical identification methods was supported by the use of molecular technique. Escherichia coli was detected 48 hrs after injection in the kidney and liver of the injected fish with this bacterium. Buras et al. (1987) reported that the peaks in the concentration of fecal coliforms in water can be detected after 2 weeks in the kidney and liver. These fish showed no clinical signs of disease. All tissue samples were positive for microbiological and PCR identification. The results demonstrated that A. hydrophila and E. coli can be simultaneously detected in kidney from asymptomatic carrier fish and agar plates. In the duplex PCR assay, the amplification products corresponding to A. hydrophila (700 bp) and E. coli (544 bp) were obtained, which was supported by the sequencing results. The duplex PCR did not produce any non-specific amplification products. Aeromonas hydrophila was not isolated from liver and spleen. The high specificity of this assay was verified by the absence of amplified A. hydrophila DNA fragment in these samples. Figure 4. Specificity PCR products from dead fish tissues, agar plate and positive control using FAS-RAS primer set for detection of A. hydrophila (A). Lane M, molecular size marker; lane 1, A. hydrophila RTICC 1032 (700bp); lane 2, kidney; lane 3, spleen; lane 4, bacteria isolated from agar plate; lane 5, negative control (no DNA). (B) Specificity PCR products from fish tissues, agar plate and positive control using FES-RES primer set for detection of E. coli. Lane M, molecular size marker; lane 1, E. coli RTICC 2325(544 bp); lane 2, kidney; lane 3, liver; lane 4, bacteria isolated from agar plate; lane 5, negative control (no DNA). 58 Int. J. Aquat. Biol. (2015) 3(1): 52-59 The duplex PCR method described in the present work provides improved capabilities to detect A. hydrophila and E. coli in healthy carriers and also decreases the time required to amplify the 16SrRNA gene. Our results, based on the PCR suggested that the first target of A. hydrophila colonization is kidney of the rainbow trout showing the pathogen prior to clinical symptoms. Furthermore, our findings suggest that the combination of sampling method and duplex PCR is suitable for a rapid detection and discrimination between apparently healthy and asymptomatic infected and uninfected fish. Uninfected fish used as controls did not produce any amplification products with either method. Our study has confirmed that the usage of an individual PCR reaction for each pathogen and laboratory culture based methods is costly, tedious and time consuming. But the duplex PCR method could identify two bacterial pathogens in less than 8 hrs. In conclusion, the duplex PCR presented in the current work is suitable tool for the detection of fish pathogen and at the same time, allows for rapid identification of bacterial causative agents of human diseases. These results, together with those obtained in dead fish and asymptomatic fish experiments, indicate that the kidney is suitable for epidemiological sampling. On the whole, the duplex PCR has advantages in terms of its accuracy, sensitivity, ease of use, time of length analysis and cost-effectiveness compared to the single PCR and traditional method. Acknowledgement The authors acknowledge the aquatic animal laboratory and aquatic biotechnology laboratory staff of University of Tehran for their assistance. References Adams A., Thompson K.D. (2006). Bio technology offers revolution to fish health management. Trends in Biotechnology, 24: 201-205. Adams A., Thompson K.D. (2008). Recent applications of biotechnology to novel diagnostics for aquatic animals. Revue Scientifique et Technique, 27(1): 197- 209. Altinok I., Capkin E., Kayis S. (2008). Development of multiples PCR assay for simultaneous detection of fire bacterial fish pathogens. Veterinary Microbiology, 131: 332-338. Blanco M., Gibello A., Fernández-Garayzábal J.F. (2000). Influence of fish health management: Bases, Procedures and economic implications. VisaVet, 51: 45-49. Bela'k S. (2007). Molecular diagnosis of viral diseases, present trends and future aspects a view from the OIE collaborating center for the application of polymerase chain reaction methods for Diagnosis of Viral Diseases in Veterinary Medicine. Vaccine, 25: 5444- 5452. Beier R.C., Duke S.E., Ziprin, R.L., Harvey R.B., Hume M.E. (2008). Antibiotic and disinfectant susceptibility profiles of vancomycin-resistant Enterococcus faecium (VRE) isolated from community wastewater in Texas. Bulletin of Environmental Contamination and Toxicology, 80: 188-194. Buras N., Duek L., Niv S., Hepher B., Sandbank E. (1987). Microbiological aspects of fish grown in treated waste water. Water Research, 21: 1-10. del Cerro A., Marquez I., Guijarro J.A. (2002). Simultaneous detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, three major fish pathogens, by multiplex PCR. Applied and Environmental Microbiology, 68: 5177- 5180. DH S.S. (1991). The bacteriological examination of drinking water supplies 1982. Public Health Laboratory Service, London. HMSO. Fang H.M., Ling K.C., Sin G.Y.M. (2000). Enhancement of protective immunity in blue gourami, Trichogaster trichopterus (Pallas), against Aeromonas hydrophila and Vibrio anguillarum by A. hydrophila major adhesin. Journal of Fish Diseases, 23: 137-145. Ferreira da Silva M., Vaz-Moreira I., Gonzalez-Pajuelo M., Nunes O.C., Manaia C.M. (2007). Antimicrobial resistance patterns in Enterobacteriaceae isolated from an urban wastewater treatment plant. FEMS Microbiology Ecology, 60: 166-176. Forbes B., Sahm D. (1998). Weissfeld, A diagnostic Microbiology, 10th edition, Mosby, Inc: 509-526. Geldreich E.E., Clarke N.A. (1966). Bacterial pollution indicators in the intestinal tract of freshwater fish. Applied Microbiology, 14: 429-437. 59 Fattahi et al./ Duplex PCR method detection of A. hydrophila, and E. coli in Rainbow trout Haenninen M.L. (1994). Phenotypic characteristics of the three hybridization groups of Aeromonas hydrophila complex isolated from different sources. Journal of Applied Bacteriology, 76: 455-462. Holt J.G. (2000). Bergey’s Manual of Determinative Bacteriology (7th edition), Lippincott Willions and Wilkins, Philadelphia, USA. Ishii S., Hansen D.L., Hicks R.E., Sadowsky M.J. (2007). Beach sand and sediments are temporal sinks and sources of Escherichia coli in Lake Superior. Environmental Science and Technology, 41: 2203- 2209. Janda J.M., Abbott S.L. (2002). Bacterial identification for publication: when is enough? Journal of Clinical Microbiology, 40: 1887-1891. Janssen W.A. (1970). Fish as potential vectors of human bacterial diseases of fishes and shellfishes. American Fisheries Society Special Publication, 5: 90-284. Lapatera S.E., Plant K.P., Alcorn S., Ostland V., Winton J. (2010). An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout, Oncorhynchus mykiss (Walbaum). Fish Diseases, 33: 143-151. Mata A.I., Gibello A., Casamayor A., Blanco M.M., Domı´nguez L., Fernandez-Garayzabal J.F. (2004). Multiplex PCR assay for detection of bacterial pathogens associated with warm-water streptococcosis in fish. Applied and Environmental Microbiology, 70: 3183-3187. Millership S.E. (1996). Identification. In: B. Austin, M. Altwegg, P.J. Gosling, S Joseph. (eds.) The genus Aeromonas. John Wiley and Sons, Chichester, 85- 107. Neil G., Nair M. (2004). Fish and shell fish bacteriology manual techniques and procedures. A Blackwell Publishing. America, 1-258. Nielsen M.E., Hqi L., Schmidt A.S., Qian D., Shimada T., Shen J., Larsen J.L. (2001). Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease out breaks in aquaculture production in the Zhejinag Province of China?. Diseases of Aquatic Organisms 46: 23-29. Plumb J.A., Grizzle J.M., Defigueiredo J. (1976). Necrosis and bacterial infection in channel catfish (Ictalurus punctatus) following hypoxia. Journal of Wildlife Diseases, 12: 247-253. Sabat, G., Rose, P., Hickey, W., Harkin, J.M. 1999. Selective and sensitive method for PCR amplification of Escherichia coli 16Sr RNA genes in soil. Applied and Environmental Microbiology, 66: 844-849. Sambrook J., Russell D.W. (2001). Molecular cloning, 3th edition, New York, Cold Press, 800 p. Sautour M., Mary P., Chihib N.E. Hornez J.P. (2003). The effects of temperature, water activity and pH on the growth of Aeromonas hydrophila and on its subsequent survival in microcosm water. Journal of Applied Microbiology, 95: 807-813. Sindermann C.J. (1988). Epizotic ulcerative syndrome in costal/Estuarine Fish. U.S. Department of Commerce. National Marine Fish service NOAA Tech Memo. NMFS-F/NEC-54. Stevenson R.M.W. (1999). Bacterial diseases in cold- water aquaculture: Tracking Pathogens. Proceedings of the 8th International Symposium on Microbial Ecology; 1999. 1-6; Halifax, Canada. Microbial Interactions in Aquaculture. Tang Y.W., Stratton C. (2006). Advanced techniques in diagnostic microbiology. USA, Springer, 1-551.