International Journal of Fisheries and Aquaculture Sciences. ISSN 2248-9975 Volume 2, Number 2 (2012), pp.

141-156 International Research Publication House http://www.irphouse.com

Phenotypic and Genotypic Methods for Identifications of Aeromonas hydrophila Strains from Carp Labeo rohita and their Virulence Study
1*

I. Sahu, B.K. Das, 1N.P. Marhual, J. Pradhan, 1D.R. Sahoo, 2 B. Behra and 4B.K. Mishra Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India 2 Department of Bio Science & Biotechnology, Fakir Mohan University, Balasore, India *Corresponding Author E-mail: basantadas@yahoo.com
1

Abstract In the present study two strains of A. hydrophila were characterized by using various phenotypic as well as genotypic methods and their virulent properties were examined through in vitro and in vivo pathogenicity tests. The strains were isolated from the diseased food fish i.e. Labeo rohita obtained from the ponds of Central Institute of Freshwater Aquaculture. Identification of bacterial strains was carried out through phenotypic methods that included biochemical and extracellular enzymatic activity tests. Genotypic characterization was conducted through analysis of 16S ribosomal RNA sequence which proved to be a useful, rapid and reliable method for identification of A. hydrophila. The 16S rRNA profiles of all the strains tested, consisted of band at 1.5 Kbp. Also a phylogenetic tree was constructed with the 16S rRNA sequence of present strains and other sequence of Aeromonas sp. that has been already published. Dot ELISA test was also performed by employing antisera raised against the outer membrane proteins of A. hydrophila which ensured the confirmatory identification of this bacterial pathogen. Further, the pathogenicity tests of the present strains confirmed them as highly virulent strains. Keywords: Aeromonas hydrophila; Phenotypic; 16S ribosomal RNA; Phylogenetic tree; Pathogenicity

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Introduction
Fish and Fishery products gained a tremendous importance due to their high nutritional value, clear health benefits and whole some properties (Darlington et al., 2001). Nowadays the aquaculture industry is facing a lot of problems due to microbial infection and mass mortalities in fishes. Aeromonas hydrophila is considered as an opportunistic and zoonotically important bacterial pathogen of both aquatic and terrestrial organisms, including humans (Figueras, 2005; Rahman et al., 2009). It acts as a significant constraint to aquaculture production and trade resulting in severe economic losses to this industry. It has also gained the status of food borne pathogen of emerging importance (Daskalov, 2006). In fishes, it is mainly responsible for causing fatal hemorrhagic septicemia, epizootic ulcerative syndrome, fin and tail rot diseases which lead to huge mortality among wild and cultured fish (Nayak et al., 1999; Vivekanandhan et al., 2005; Rahman et al., 2007). The virulent factors which contribute to their pathogenicity mostly include extracellular enzymes (Pemberton et al., 1997), various cytotoxic, hemolytic, enterotoxic and outer membrane proteins (Yu et al., 2007; Gochhayat, 2010; Sahu et al., 2011). Before adopting any preventive measure accurate identification of causative organisms is pre-requisite. Epidemiological investigation has been hampered due to lack of availability of rapid and precise diagnostic tools. Hence, specific, sensitive and rapid methods for detecting and identifying pathogenic microorganisms are needed to control microbial infection in intensive fish farming practice. Although the traditional culture techniques for direct isolation and identification of pathogens are available but they are usually time taking and laborious (Rathore et al., 2005). Recently, studying the diversity of microflora through sequencing of 16S ribosomal RNA gene proved to be a stable and specific marker for bacterial identification (Martinez-Murcia et al., 2008; Chan et al., 2011) and in the investigation of the phylogenetic tree. Since the gene contains highly variable regions flanked by conserved regions it allows amplification using universal primers and also help in the phylogenetic analysis and taxonomic identifications (Fox et al., 1977; Tringe et al., 2005). Besides, dot ELISA technique act as another efficient (specific and sensitive), simple to use and rapid diagnosis technique for the detection of aetiological agent of diseases (Swain et al., 2001). In the present study, the pathogens isolated from infected rohu (Labeo rohita) were identified using both phenotypic and genotypic methods, based on biochemical studies, enzymatic assay and phylogenetic analysis of 16S rRNA sequences. Also the polyclonal antibody raised against the outer membrane protein of single strain of A. hydrophila was used in the detection of pathogen through dot ELISA technique. Further, the pathogenicity of the isolated strains was studied under in vitro and in vivo conditions. Moreover, the aim of the above study was to give a platform for accurate identification of A. hydrophila so that, suitable control measures could be adopted in orders to put a check point in the outbreak of diseases.

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Materials and Methods

Experimental fish Moribund fish (L. rohita, 40 in numbers) weight ranging from 100 to 150 g, having disease symptoms like hemorrhages, tail rot and fin rot were collected from the carp culture ponds of Central Institute of Freshwater Aquaculture, Bhubaneswar, India for the detection of causal pathogenic agents. Healthy advance fingerlings of rohu, L. rohita weighing 20-30 g, brought from fish farm were acclimatized in fiberglass reinforced plastic (FRP) tanks (500 L) for two weeks prior to the start of experiments. Fish were fed with commercial pellet at 2 % of their body weight during the experimental period. Bacterial isolation and identification For bacterial isolation, swabs were taken aseptically from the infected organs like tail, fin, kidney, liver, intestine, gill and from skin lesions in nutrient broth (Himedia Ltd., Mumbai, India) and incubated at 37oC for 24 h. For the presumptive isolation and identification of A. hydrophila the broth cultures were streaked on to the selective RSmedia (Himedia Ltd., Mumbai, India) and incubated at 37oC for 24 h. Phenotypic characterization Phenotypic analyses of the isolates were carried out through Gram staining, cellular morphology (by compound microscope), colonial characteristics on RS-media and biochemical studies. Conventional biochemical studies were carried out as per standard protocol (MacFaddin, 1980) for identification purpose and mostly it includes motility, urease, indole, MethylRed,Voges Proskauer, citrate utilization, arginine utilization, lysine decarboxylase and ornithinie decarboxylase tests. The presence of catalase was tested with hydrogen peroxide, and cytochrome oxidase test was performed by using oxidase discs (Himedia Ltd., Mumbai, India). Carbohydrate usage was determined by using various sugar discs in phenol red broth base (MacFaddin, 1980). Extracellular enzymatic activity Extracellular enzymatic activity e.g. amylase, caseinase, gelatinase, lipase and DNase were studied as per the method given by Pemberton et al. (1997) with certain modification. Starch hydrolysis test This test was performed by inoculating the bacterial culture onto starch agar plates containing nutrient agar with 1 % soluble starch followed by addition of iodine solution after incubation at 37oC for 24 h. Clear zone formed around colonies indicated hydrolysis of starch. Gelatin hydrolysis test This test was carried out by inoculating the bacterial culture onto gelatin agar plates containing nutrient agar with 2 % gelatin and were flooded with acidic mercury

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chloride solution after incubation at 37oC for 24-48 h. Appearance of clear zone around the colonies indicated the hydrolysis of gelatin by the enzyme gelatinase. Lipase test (Tween-20 degradation) Tween-20 degradation was studied by supplementing basal medium with 1 % (v/v) Tween 20. Clear zone around the colonies flooded with 1 % CaCl2 solution after incubation for a period of 24 h at 37oC indicated positive result. Lecithinase test The organisms were spot inoculated onto the nutrient agar plates containing 1 % egg yolk and then incubated at 37oC for 24-48 h. Phospholipase production was indicated by turbidity in the medium surrounding each colony. DNase test This test was performed with basal medium supplemented with 0.2 % of fresh DNA of any organism. The plates were examined for pink halo zones around the colonies after flooding with 0.001 % toluidine blue. Phylogenetic analysis of the isolates based on 16S rRNA sequences Extraction of genomic DNA The genomic DNA was extracted using MB505 HiPurATM Bacterial and yeast genomic DNA purification kit (Himedia Ltd., Mumbai, India) as per the manufacturers instruction. Bacterial culture (1.5 ml) was taken from 24 h broth culture in an eppendrof tube and centrifuged at 10,000 rpm for 5 min. The pellet collected were resuspended in180 l of Lysis solution AL and 20 l of RNase A followed by incubation of 2 min at room temperature. Again Proteinase K (20 mg/ml) of 20 l was added to it and incubated for 30 min at 55oC. Then 200 l of Lysis solution C1 was added, mixed properly and incubated at 55oC for 10 min. It was followed by addition of 200 l 95-100 % ethanol. The entire contents of the tube were loaded onto binding column and centrifuged at 10,000 rpm for 1 min. The column was then placed in a new 2 ml collection tube. To it 500 l of prewash solution-PWB was added and centrifuged at 10,000 rpm for 1 min. After that 500 l of wash solution was added to the column and centrifuged at 10,000 rpm for 1 min. Then the column was placed in another new 2 ml collection tube followed by addition of 200 l of Elution buffer-ET. It was incubated at room temperature for 2 min and centrifuged at 10,000 rpm for 1 min to elute DNA. Finally, the genomic DNA obtained was stored at -20oC. Amplification and sequencing of 16S r RNA The universal bacterial primer: forward primer (5AAGAGTTTGATCCTGGCTCAG-3) and reverse primer(5GGTTACCTTGTTACGACTT- 3) obtained from Bangalore Genei ( Bangalore, India) were used to amplify the 16S rRNA sequences of both the strains as well as reference strain ATCC 49140 (Martinez-Murcia et al., 1992). The PCR was carried out in the thermal cycler (Minicycler, MJResearch) under the following conditions:

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initial denaturation at 95C for 5 min followed by 35 cycles at 94C for 1 min (denaturing temperature), 53C for 1 min (annealing temperature) and at 72C for 2 min (extension temperature) followed by a final extension of 10 min at 72C. The PCR amplified products, which had an expected size of 1.5 kb were analyzed through 1.2 % agarose gel containing ethidium bromide (0.1 g/ml), by using standard molecular weight marker of 1 kbp DNA ladder (Banglore Genei, India). The gel was visualized under gel documentation system. The amplified PCR products were subjected for sequencing. Sequence analysis and construction of phylogenetic tree After sequencing, database searches were conducted with the BLAST (Basic Local Alignment Sequencing Tool) algorithm provided by NCBI (National Center for Biotechnology Information). Phylogenetic analysis was carried out using MEGA-4 software (Tamura et al., 2007). For phylogenetic analysis a total 17 numbers of 16S rRNA sequences, including the isolated strains, reference strain and other related taxa, obtained from Gene bank database were manually aligned using multiple-sequence alignment software CLUSTAL W (Thompson et al., 1994). The phylogenetic tree was inferred using Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of taxa analyzed and the percentage of replicate trees in which the associated taxa were clustered together are shown next to the branches. The evolutionary distances were computed using the Kimura 2-parameter methods (Kimura, 1980). Extraction of Outer membrane protein antigen Outer membrane protein (OMP) of the bacterial cell was obtained as per the method given by Austin and Rodgers (1981). Nutrient broth culture (24 h old) of 500 ml was centrifuged at 8000 rpm for 45 min. The pellet obtained was washed in tris buffer (20 mM, pH-7.2) and resuspended in the tris buffer containing EDTA (10 mM). This bacterial cell suspension was sonicated at 50 Hz in a sonicator (Artek Sonic Dismembrator, Model-150, Artek, New York, USA). After sonication the unbroken cells were sedimented by centrifugation at 8000 rpm and the supernatant was collected which was subjected to ultracentrifugation at a speed of 27,000 rpm in a ultracentrifuge (HITACHI, Model number-CP 100x). The supernatant was discarded and the sediment was resuspended in Tris buffer containing 0.5 % sarcosyl. It was further centrifuge at 27,000 rpm for about 45 min and finally the pellets collected after ultracentrifugation was OMP. Raising of polyclonal antibodies in rabbit Protein concentration of OMP extracted from CAHH14 strain of A. hydrophila was determined by using Bradford method (Bradford, 1976). OMP of 150 l i.e. about 100 g of protein along with Freunds complete adjuvant (FCA) was injected intramuscularly into the hind leg of rabbit. The animal was given a booster dose on 14th and 28th days of immunization with same dose of emulsion containing antigen and Freunds incomplete adjuvant (FIA) instead of FCA.

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Blood from the rabbit was collected by ear vein puncture on 42nd days of post immunization. Serum was collected by centrifugation (5000 rpm) of the clotted blood for 30 min at room temperature and then stored at 20oC until further use. Specificity test through Dot-ELISA Dot-ELISA test was performed as per the method given by Swain et al. (2001) with slight modifications. Nitrocellulose paper (NCP) strip was coated separately with 2 l of OMP antigen of CAHH14, CAHH15 and ATCC 491040 strain. The NCP was also coated with OMP of other bacteria V. alginolyticus ATCC 17749, Edwardsiella tarda ATCC 15947, taken as negative controls. The coated NCP strip was dried at room temperature for 2 h. Then, the strips were blocked in PBS-T (phosphate buffer saline and Tween-20) containing 5 % skimmed milk powder at 37 oC for 30 min followed by washing with PBS-T and incubated with primary antibody (1: 400 dilutions) for 1 h at 37 oC. Then it was washed several times in PBS-T and incubated further with secondary antibody i.e. anti rabbit horse radish peroxidase conjugate (Vector Lab, USA) for 30 min. The NCP was again incubated with Anti-Bovine-Complex (ABC) reagent for 30 min. Lastly, NCP was transferred into a new plate and then incubated with 3-amino-9 ethyl carbazole (AEC) reagent, for 10 min in dark. The NCP strip was washed properly with distilled water, air dried at room temperature and blots were detected. Pathogenicity study In vitro virulence study In vitro virulence study was carried out by hemolytic (Radu et al., 2003) and congo red binding assay (Illanchezin et al., 2010). The strains were tested for hemolytic activity by inoculation on to nutrient agar plates containing 5 % sheep defibrinated red blood cells and incubated at 37oC for 24 h. The patterns of hemolysis around the colonies were categorized as alpha (), beta () and gamma. Congo red binding assay was performed by inoculation of bacterial strains on to nutrient agar plates supplemented with 0.03 % (w/v) congo red and incubated at 37oC for 48 h The colonies used were then examined for congo red uptake. Deep red, raised colony and high slime production indicated virulence nature of strain. In vivo virulence study Prior to challenges, the acclimatized healthy advanced fingerlings of rohu were taken in FRP tanks containing 20 fish. Based on the LD50 values of A. hydrophila (Sahu et al., 2011) fish were intraperitoneally injected with 0.1 ml of bacterial inoculums at a concentration of 1.7 x 105 cfu/ ml. In parallel, control group were injected intraperitoneally with PBS. Fish mortality was recorded daily for an observation period of 5 days. Kochs postulates were verified from the moribund fish.

Results
Phenotypic characterization On the basis of colony morphology over RS plates presumptively 50 isolates of A.

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hydrophila were isolated from the skin lesions, kidney, liver, gill and intestine of moribund fish. They appeared to be small, rounded and yellowish in color over RS plates incubated for 24 h at 37C. Under microscope Gram negative, small rods were observed. Although the biochemical study was time taking but it proved to be another method for typing and differentiation of bacterial fish pathogen. From the biochemical studies the isolates were identified as A. hydrophila due to typical reactions of majority of biochemical tests were similar to the reference strain of A. hydrophila ATCC 49140. All the isolates were found to be motile and showed positive reactions towards oxidase, catalase, Methyl Red, urease, citrate utilization test and negative reactions towards ornithine decarboxylase test, Vogus Proskauer test where as variable results were found in indole test and acid production from different sugars. Based on these results two different strains of A. hydrophila i.e. CAHH14 and CAHH15 were obtained. Extracellular enzymatic activity The enzymatic activity of both the strain was measured as the ratio between the diameter of zone of activity and diameter of the smear. The amylase and gelatinase activity was found to be highest in CAHH14 strain whereas the gelatinase activity was found to be lowest in ATCC strain. The lipase activity was found to be highest in CAHH15 strain and lowest in CAHH14 strain. lecithinase activity was found to be lowest in CAHH15 strain where as DNase activity was found to be highest in ATCC strain (Fig. 1).

Figure 1: Enzyme activity of CAHH14, CAHH15 and ATCC 49140 strains of A. hydrophila

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Phylogenetic analysis of t isolates based on 16S rRNA sequences the For confirmatory identific cation, the phylogenetic analysis of the isolate strains based ed on 16S rRNA sequence st trongly suggested to be A. hydrophila. In bot the strains as th well as in reference strain ATCC 49140 the 16S rRNA amplified at 1.5 kbp region n (Fig. 2). The accession nu umbers of the 16S rRNA gene sequences d deposited to the gene bank of both the stra are as follows: A. hydrophila CAHH14: JF330411 and ains A. hydrophila CAHH15: J JN621033. The phylogenetic tree constructed from 16S rRNA sequences of the isolated e strains and 15 other taxa of Aeromonas sp using Neighbor-Joining m method (Fig. 3) showed the bacterial spec with identical DNA sequences in a giv region were cies ven present on a single dendr rogram. The degree of sequence similarity (or number of sequence differences) bet tween any pair of bacteria could also be determined by calculating the sum of ho orizontal distances between each pair of bact terial species as measured on the dendrog gram. In general the tree confirms the dista relationship ant between the isolated A. hy ydrophila CAHH14, CAHH15 strain with oth strains of A. her hydrophila and other iden ntified Aeromonas sp like A. punctata, A. e enteropelogenes etc.

Figure 2: PCR amplificat tion of 16S rRNA gene (1.5 Kbp) of A. hydr rophila. Lane 1: CAHH14; 2: CAHH15; 3: ATCC 49140; M: 1 kbp DNA ladder. :

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Figure 3: The phylogenetic tree, based on 16S rRNA sequences, generated by using the neighbor-joining method. The bootstrap values (%) were shown besides the clades, accession numbers were written besides the name of type strains and scale bars represented distant values. Specificity test through Dot-ELISA In dot ELISA brown colouration was developed on the NCP strips coated with OMP of CAHH14, CAHH15 and ATCC 49140 strains due to strong reactions of antigen with homologous antiserum but no colour development was seen with OMP of V. alginolyticus ATCC 17749 and E. tarda ATCC 15947 which suggest the test to be specific and rapid for identification of A. hydrophila.

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Pathogenicity study In vitro virulence study Both the strains as well as the reference strain showed positive reactions towards the congo red binding assay and developed deep red coloration with slime production after incubation for a period of 24 h. The isolated strains and the reference strain were found to be - hemolytic as they developed clear zones around the smear which demonstrated the virulent nature of present strains (Fig. 4.).

Figure 4: CAHH14 A. hydrophila strain showing -hemolysis on blood agar plates.

In vivo virulence study Fish mortality began to occur from day 2 onwards of challenge with subsequent losses in the following 72 h. They showed reduced appetite and become prostrate in the swimming the day before of their mortality. Externally skin lesions and internally haemorrhagic patches were found in liver, intestine and gill. Pure cultures of the inoculated strains were recovered from the internal organ of moribund fish. No mortality or visible changes were observed in the control group.

Discussion
Among the motile aeromonads, the most frequently encountered bacterial agent associated with fish diseases are A. hydrophila (Janda, 1991; Otta et al., 2003). It is found to be distributed widely in the aquatic environment and responsible for causing several diseases in cold-blooded animals besides fish like reptiles, amphibians and in warm blooded animals like mammals and birds (Gossling, 1996). In our study, primary isolation of A. hydrophila was carried out mostly from the infected organs like tail, fin, kidney, liver, intestine, gill and skin lesions region of moribund fish collected from carp culture ponds of Central Institute of Freshwater Aquaculture. Presumptive identification was carried out through RS-media which

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posses 94 % accuracy to facilitate diagnosis of A. hydrophila infection in both animals and human (Shotts and Rimmler, 1973). Hus et al. (1981) identified 127 isolates of A. hydrophila producing yellow colonies over RS media. Further, identifications and strain a differentiation of A. hydrophila was carried out from their cellular morphology as well as through biochemical study (Joseph and Carnahan, 1994; Sharma et al., 2009). Since a single difference in character among isolate is consider to be different strain so from all the isolates two strains were obtained and both the strain differ from each other in many biochemical test like indole test and acid production from different sugars. Based on biochemical properties Thankappan et al. (2008) reported twelve strains of A. hydrophila from the fresh water fishes of eastern India. Similarly Abulhamd (2009) characterized ten strains of A. hydrophila isolated from water samples based on biochemical study. Another character common to all strains of A. hydrophila is the production of different extracellular enzymes that may be virulent or possibly virulent determinants (Pemberton et al., 1997; Gonzalez-Serrano et al., 2002). Secreted enzymes produced by them are thought to contribute to their wide distribution and great adaptatibility to environmental change. In our study both the strains exhibit positive enzymatic activity towards amylases, proteases (casein and gelatin hydrolysis), lipases and DNases with only minor variation in their pattern of enzymatic activity. This could be due to the difference in physiological properties of the isolates and the media used along with the incubation time as reported by Nayak et al. (1999). Since the classical identification methods based on colony morphology study and biochemical study are not adequate for the confirmation of A. hydrophila strains, 16S rRNA gene sequence analysis was carried out (Marchandin et al., 2003). In the modern taxonomy of bacteria the sequence analysis of 16S rRNA is used as a stable and confirmatory marker for the identification of bacteria as it is ubiquitous among all the cellular organisms and posses highly conserved sequence (Woese et al., 1987; Martinez-Murica et al., 2005). Both the strains isolated were amplified at 1.5 kbp region. The sequence analysis of these amplified product help in the establishment of phylogentic relationship with other Aeromonas sp. In the phylogenetic tree, out of the two isolated strains A. hydrophila CAHH15 strain remained in a separate cluster which may be due to partial sequence of 16S rRNA. But the A. hydrophila CAHH14 strain remained in a single cluster together with other A. hydrophila strains with 64 % of bootstrap values. However, due to slight sequence disimilarities with other strains it was little distance from the root of the cluster, in comparison to others. At present, fish disease diagnosis using dot ELISA technique is widely accepted (Swain et al., 2001). It seems to be rapid and confirmatory test for the identification of causative organisms (Sachan et al., 2002). A number of monoclonal antibodies were raised against A. hydrophila to report this pathogen as the causative agent of wide range of diseases in freshwater fishes (Longyant et al., 2010). However, in the present study we have raised polyclonal antibody against the outer membrane protein of this pathogen for its detection. We have focused OMP because they have been described as one of the very important antigen in vaccine formulation against the gram negative bacteria (Xu et al., 2005) and might simply be helpful for the positive result through dot ELISA. Both the strains and the reference strain were found to be positive but it

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did not show any cross reaction with other group of bacteria like V. alginolyticus and E. tarda. Uptake of congo red dyes as well as slime production was considered to be highly significant to the pathogenesis as the slime inhibits the neutrophils, chemotoxin, phagocytosis and anti-microbial drugs (Merino et al., 1995). Sontosh et al. (1988) reported binding of congo red dyes to be characteristics of virulent A. hydrophila. In our study both CAHH14 and CAHH15 strains were found to be highly virulent based on red colorations and high production of slime on congo red bindng assay plate. Similarly, Illianchezian et al. (2010) characterized 73 isolates of A. hydrophila out of which 76.9 % were found to be positive for slime production as well as uptake of dyes and considered as virulent strains. The production of hemolytic toxins has been regarded as strong evidences of pathogenic potential in A. hydrophila and is responsible for showing hemolytic activities (Brenden and Janda, 1987; Radu et al., 2003). In our study both the strains of A. hydrophila and the reference strain showed - hemolysis on blood agar plates. To illustrate the in vivo pathogenecity, the advanced rohu fingerlings challenged with CAHH14 strains, demonstrated that the main organ infected were skin, gill, liver, kidney and intestine because of typical signs of hemorrhages and lesions found in the above organ of moribund challenged fish. Similarly, Thune et al. (1986) and Khalil and Mansour (1997) studied effect the viable cells of A. hydrophila in channel catfish and tilapia respectively which were responsible for huge mortality. Hence, in order to overcome the mortalities caused by this harmful microbial pathogen among the fishes, development of molecular and serological techniques for the detection and screening of pathogens and subsequent treatment is highly important. In this regard, the study has immense importance for successful aquaculture production and trade. Besides this it is also advisable to set up proper management procedures in intensive fish culture systems, including the reduction of overcrowding and the prompt removal of moribund fish along with adoption of suitable preventive measures.

Acknowledgements
We express our heartiest gratitude to The Director, Central Institute of Freshwater Aquaculture Bhubaneswar, India, for providing all the possible assistance and sincere co-operation for conducting this investigation.

References
[1] Abulhamd AT (2009). Characterization of Aeromonas hydrophila isolated from aquatic environments using phenotypic and genotyping methods. Res J Agr Biol. Sci. 5: 923-931. [2] Austin B and Rodgers CJ (1981). Evaluation of Aeromonas salmonicida vaccines. Dev. Biol. Stand. 49: 387393.

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153

[3] Bradford MM (1976). A rapid method for the quantification of microgram quantities protein utilizing the principle of protein dyes binding. Anal. Biochem. 72: 248-254. [4] Brenden R and Janda JM (1987). Detection, quantification and stability of the beta-haemolysin of Aeromonas species. J. Med. Microbiol. 24: 247-251. [5] Chan ER, Hester J, Kalady M, Xiao H, Li X and Serre D (2011). A novel method for determining microflora composition using dynamic phylogenetic analysis of 16S ribosomal RNA deep sequencing data. Genomics. doi: 10.1016/j.ygeno.2011.04.002. [6] Darlington LG and Stone TW (2001). Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders. Br J Nutr. 85: 251269. [7] Daskalov H (2006). The importance of Aeromonas hydrophila in food safety. Food Cont. 17: 474-483. [8] Felsenstein J (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 39: 783-791. [9] Figueras MJ (2005) Clinical relevance of Aeromonas. Rev Med Microbiol. 16: 145-153. [10] Fox GE, Magrum LJ, Balch WE, Wolfe RS and Woese CR (1977). Classification of methanogenic bacteria by 16S ribosomal RNA characterization. Proc Natl Acad Sci, U.S.A. 74: 45374541. [11] Gochhayat AA (2010). Evaluation of outer membrane protein from Aeromonas hydrophila. M.Sc. Thesis. SOA University, Bhubaneswar, Odisha. [12] Gonzalez-Serrrano CJ, Santos JA, Garcia-Lopez ML and Otero A (2002). Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates obtained from fresh water fish and from a diarrhoea case. J. Appl. Microbiol. 93: 414-419. [13] Gosling PJ (1996). Aeromonas species in disease of animals. In Austin B, Altwegg M, Gosling PJ and Joseph S (Eds.) The Genus Aeromonas. pp. 175196. J. Wiley and Sons, Ltd. [14] Hus TS, Waltman WP and Shotts EB (1981). Correlation of extracellular enzymatic activity and biochemical characteristics with regard to virulence of Aeromonas hydrophila. Dev Biol Stand. 49: 101-111. [15] Illanchezian S, Jayaraman SK, Manoharan MS and Valsalam S (2010). Virulence and cytotoxicity of seafood borne Aeromonas hydrophila. Braz J Microbiol. 41: 978-983. [16] Jhanda JM (1991) Recent advances in the study of taxonomy, pathogenicity and infectious syndrome associated with the genus Aeromonas. Clin. Microbiol. Rev. 4: 397-410. [17] Joseph SW and Carnahan A (1994). The isolation and identification and systematic of motile Aeromonas species. Annu Rev Fish Dis. 4: 315-343. [18] Khalil A.H and Mansour EH (1997). Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapia nilotica. Lett Appl Microbiol. 25: 269-273.

154

I. Sahu et al

[19] Kimura M (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120. [20] Longyant S, Chaiyasittrakul K, Rukpratanporn S, Chaivisuthangkura P and Sithigorngul P (2010). Simple and direct detection of Aeromonas hydrophila infection in the gold fish, Carassius auratus (L.), by dot blotting using specific monoclonal antibodies. J Fish Diseases. 33: 973-984. [21] MacFaddin JE (1980). Biochemical Tests for Identification of Medical Bacteria. William Wilkinson, Baltimore, MD, pp.1-523. [22] Marchandin H, Teyssier C, Simeon De Buochberg M, Jean-Pierre H, Carriere C and Jumas-Bilak E (2003). Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy. Curr. Microbiol. 149: 1493-1501. [23] Martnez-Murcia AJ, Monera A, Alperi A, Figueras MJ and Saavedra MJ (2008). Phylogenetic evidence suggests that strains of Aeromonas hydrophila subsp. dhakensis Huys et al. 2002 belong to the species Aeromonas aquariorum sp. nov. Martnez-Murcia et al. 2008. Curr. Microbiol. 58:76-80. [24] Martnez-Murcia AJ, Benlloch S and Collins MD (1992). Phylogenetic interrelationship of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridization. Int J Syst Bacteriol. 42: 412-421. [25] Martnez-Murcia AJ, Soler L, Saavedra MJ, Chacn MR, Guarro J, Stackebrandt E and Figueras MJ (2005). Phenotypic, genotypic, and phylogenetic discrepancies to differentiate Aeromonas salmonicida from Aeromonas bestiarum. Int Microbiol. 8: 259-269. [26] Merino S, Rubires X, Knochel S and Tomas JM (1995). Emerging pathogens: Aeromonas spp. Int J Food Microbiol. 28: 157-168. [27] Nayak KK, Mukherjee SC and Das BK (1999). Observation on different strains of Aeromonas hydrophila from various diseased fishes. Indian J Fish. 46: 245250. [28] Otta SK, Karunasagar I and Karunasagar I (2003). Disease problems affecting fish in tropical environments. J.Appl. Aquacult. 13: 231-249. [29] Pembertona JM, Kidd SP, Schmidt R (1997). Secreted enzymes of Aeromonas. FEMS Microbiol. Lett. 152: 1-10. [30] Radu S, Ahmad N, Ling FH and Reezal A (2003). Prevalence and resistance to antibiotics for Aeromonas species from retail fish in Malaysia. Int J Food Microbiol. 81: 261-266. [31] Rahman M, Huys G, Kuhn I, Rahman M and Mollby R (2009). Prevalence and transmission of antimicrobial resistance among Aeromonas populations from a duckweed aquaculture based hospital sewage water recycling system in Bangladesh. Antonie Van Leeuwenhoek. 96: 313321. [32] Rahman, M.H., Huys, G., John, A.M., Kuhn, I., Mollby, R., 2007. Persistence, transmission, and virulence characteristics of Aeromonas strains in a duckweed aquaculture-based hospital sewage water recycling plant in Bangladesh. Appl Environ Microbiol. 73: 1444-1451.

Phenotypic and Genotypic Methods for Identifications

155

[33] Rathore G, Swaminathan TR, Abidi R, Mahanta PC and Kapoor D (2005). Isolation and Characterization of Motile Aeromonads from Aquatic Environment. Indian J. Fish. 52: 241248. [34] Sachan N and Agarwal RK (2002). A simple enzyme-linked immunosorbent assay for the detection of Aeromonas spp. Vet. Arhiv. 72: 327-334. [35] Sahu I, Das BK, Marhual N, Samanta M, Mishra BK and Ekanath, A.E (2011). Toxicity of crude extracellular product of Aeromonas hydrophila on Rohu, Labeo rohita (Ham.). Ind. J. Microbiol. 51: 515-520. [36] Saitou N and Nei M (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425. [37] Sharma I, Kumar A, Pramanik AK (2009). Review of Techniques on Isolation and Identification of Aeromonads from the Food of Animal and Fish Origin. A Review- Assam Univ. J. of Science and Technology: Biol and Env. Science. 4: 73-85. [38] Shotts EB and Rimler R (1973). Medium for the isolation Aeromonas hydrophila. J. Appl. Microbiol. 26: 550-553. [39] Sontos Y, Toranzo AE, Barja JL, Nieto TP and Villa TG (1988). Virulence properties and enterotoxin production of Aeromonas strains isolated from fish. Infect. Immun. 56: 3285-3293. [40] Swain P, Mukherjee SC, Sahoo PK, Das BK, Pattnaik P, Murjani G and Nayak SK (2001). Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for the diagnosis of Edwardsiella tarda infection in fish. Asian Fish Sci. 14: 8993. [41] Tamura K, Dudley J, Nei M and Kumar S (2007). MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599. [42] Thankappan A, Das BK, Barman HK and Samal SK (2008). Genetic Fingerprinting of Aeromonas hydrophila isolated from diseased fresh water fishes of Eastern India. e-planet. 6:1-6. [43] Thompson JD, Higgins DG and Gibson TJ (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res. 22: 4673-4680. [44] Thune RL, Johnson MC, Grahem TE and Amborski RL (1986). Aeromonas hydrophila beta haemolysin : purification and examination of its role in virulence in O group channel catfish, Ictalurus punctatus (Rafinesque ). J Fish Diseases. 9: 55-61. [45] Tringe SG, Von Mering C, Kobayashi A, Salamov AA, Chen K, Chang HW, Podar M, Short JM, Mathur EJ and Detter JC (2005). Comparative metagenomics of microbial communities, Science (New York). 308: 554557. [46] Vivekanandhan G, Hatha AAM and Lakhmanaperumalsamy P (2005). Prevalence of Aeromonas hydrophila in fish and prawns from the seafood market of Coimbatore, South India. Food Microbiol. 22: 133-137. [47] Woese CR (1987) Bacterial evolution. Microbiol Rev. 51: 221-271.

156 [48]

I. Sahu et al

Xu C, Wang S, Zhaoxia Z and Peng X (2005). Immunogenic cross-reaction among outer membrane proteins of Gram-negative bacteria. Int Immunopharmacol. 5: 1151-1163. [49] Yu HB, Kaur R, Lim S, Wang XH and Leung KY (2007). Characterization of extracellular proteins produced by Aeromonas hydrophila AH-1. Proteomics. 7: 436-449.