Document Type : Original Article


1 South of Iran Aquaculture Research Institute, Iranian Fisheries Science Research Institute, Agricultural Research Education and Extension ‎Organization (AREEO), Ahvaz, Iran‎

2 Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran

3 Department of Aquatic Animal Health, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran‎

4 Department of Fish diseases, Iranian Fisheries Research Science Institute (IFSRI), Agriculture Research, Education and Extension Organization (AREEO), Tehran, Iran

5 Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

6 Centre for Sustainable Aquatic Ecosystems, Harry Butler Institute, Murdoch University, Perth, Australia


Aeromonas hydrophila is a bacterium associated with many diseases and disorders such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. It bears several virulence factors including type III secretion system (T3SS), aerolysin, cytolytic enterotoxin and enzymes (e.g. hemolysins, lipase) that seem to play an important role in its pathogenesis. Detection of virulence markers by polymerase chain reaction (PCR) is a key procedure in defining the pathogenic ability of pathogenic bacteria and preparing a vaccine for its treatment. In this sense, this study was aimed to determine the frequency of virulence genes in isolates obtained from infected cultured carps in Khuzestan province. Out of 200 moribund carps with septicemic symptoms, 125 isolates were belonged to the motile aeromonads and 59 isolates were identified as A. hydrophila by biochemical methods. Finally, using PCR analysis, 31 isolates were identified as A. hydrophila. Five virulence genes were detected in these isolates including hemolysin, aerolysin, cytolytic enterotoxin and T3SS (aopB and ascV) by specific primers. Results showed that 23 (74.19%), 18 (58.06%), 16 (51.61%), 13 (41.63%) and 10 (32.25%) isolates possessed cytolytic enterotoxin, hemolysin, aerolysin, and T3SS genes, respectively. The results of the present study showed that among 31 isolates, only five isolates had all of dominant virulence genes. Thirteen other isolates had genotypes including hlyA+, aerA+, and act+. The remaining isolates had at least one virulence gene. This study showed that determination of the virulence genes by PCR can be a reliable method to identify a potential pathogenic Aeromonad strain.



    1. Zorrilla I, Chabrillón M, Arijo S, et al. Bacteria recovered from diseased cultured gilthead sea bream (Sparus aurata) in southwestern Spain. Aquaculture 2003; 218: 11-20.
    2. Al-Fatlawy HNK ,Al-Ammar MH. Molecular study of Aeromonas hydrophila isolated from stool samples in Najaf (Iraq). Int J Microbiol Res 2013; 5(1): 362-365
    3. Inglis V, Roberts RJ, Bromage NR. Bacterial diseases of fish. New York, USA: Halsted Press 1993; 312.
    4. Yogananth N, Bhakyaraj R, Chanthuru A, et al. Detection of virulence gene in Aeromonas hydrophila isolated from fish samples using PCR technique. J Biotechnol Biochem 2009; 4(1): 51-53.
    5. Janda JM, Abbott SL, The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 2010; 23(1): 35-73.
    6. Albert MJ, Ansaruzzaman M, Talukder KA, et al. Prevalence of enterotoxin genes in Aeromonas isolated from children with diarrhea, healthy controls, and the environment. J Clin Microbiol 2000; 38(10): 3785-3790.
    7. Heuzenroeder MW, Wong CY, Flower RL. Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas: correlation with virulence in a suckling mouse model. FEMS Microbiol Lett 1999; 174(1): 131-136.
    8. Kingombe CI, Huys G, Tonolla M, et al. PCR detection, characterization, and distribution of virulence genes in Aeromonas Appl Environ Microbiol 1999; 65(12): 5293-5302.
    9. Wong CY, Mayrhofer G, Heuzenroeder MW, et al. Measurement of virulence of aeromonads using a suckling mouse model of infection. FEMS Immunol Med Microbiol 1996; 15(4): 233-241.
    10. Burr SE, Stuber K, Wahli T, et al. Evidence for a type III secretion system in Aeromonas salmonicida salmonicida. J Bacteriol 2002; 184(21): 5966-5970.
    11. Francis MS, Wolf-Watz H, Forsberg A. Regulation of type III secretion systems. Curr Opin Microbiol 2002; 5(2): 166-172.
    12. Dean P. Functional domains and motifs of bacterial type III effector proteins and their roles in infection. FEMS Microbiol Rev 2011; 35(6): 1100-1125.
    13. Chacón MR, Soler L, Groisman EA, et al. Type III secretion system genes in clinical Aeromonas J Clin Microbiol 2004; 42(3): 1285-1287.
    14. Sen K. Development of a rapid identification method for Aeromonas species by multiplex-PCR. Can J Microbiol 2005; 51(11): 957-966.
    15. Chang Y-C, Wang J-Y, Selvam A, et al. Multiplex PCR detection of enterotoxin genes in Aeromonas from suspect food samples in northern Taiwan. J Food Prot 2008; 71(10): 2094-2099.
    16. Nielsen ME, Høi L, Schmidt AS, et al. Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China? Dis Aquat Organ 2001; 46(1): 23-29.
    17. Buller NB, Bacteria and fungi from fish and other aquatic animals: a practical identification manual. 2nd Oxford, UK: CABI 2014; 137-277.
    18. Ottaviani D, Parlani C, Citterio B, et al. Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: a comparative study. Int J Food Microbiol 2011; 144(3): 538-545.
    19. Porteen K, Agarwal RK, Bhilegaonkar KN. PCR-based detection of Aeromonas from milk samples. J Food Technol 2006; 4(2): 111-115.
    20. Dorsch M, Ashbolt NJ, Cox PT, et al. Rapid identification of Aeromonas species using 16S rDNA targeted oligonucleotide primers: a molecular approach based on screening of environmental isolates. J Appl Bacteriol 1994; 77(6): 722-726.
    21. Cascón A, Anguita J, Hernanz C, et al. Identification of Aeromonas hydrophila hybridization group 1 by PCR Appl Environ Microbiol 1996; 62(4): 1167-1170.
    22. Aslani MM, Seyyed Hamzeh H. Characterization and distribution of virulence factors in Aeromonas hydrophila strains isolated from fecal samples of diarrheal and asymptomatic healthy persons, in Ilam, Iran. Iran Biomed J 2004; 8(4): 199-203.
    23. Granum PE, O'Sullivan K, Tomás JM, et al. Possible virulence factors of Aeromonas from food and water. FEMS Immunol Med Microbiol 1998; 21(2): 131-137.
    24. Shirali T. Effect of para coumaric acid on expression of some virulence genes of Aeromonas hydrophila and its protective effects in Comman Carp (Cyprinus carpio). PhD Thesis. Shahid Chamran University of Ahvaz. Ahvaz, Iran: 2015.
    25. Lee S, Kim S, Oh Y, et al. Characterization of Aeromonas hydrophila isolated from rainbow trouts in Korea. J Microbiol 2000; 38(1): 1-7.
    26. Wong CYF, Heuzenroeder MW, Flower RLP. Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model. Microbiology (Reading) 1998; 144(Pt 2): 291-298.
    27. Uma A, Rebecca G, Meena S, et al. PCR detection of putative aerolysin and hemolysin genes in an Aeromonas hydrophila isolate from infected Koi carp (Cyprinus carpio). Tamil Nadu J Vet Anim Sci 2010; 6(1): 31-33.
    28. Yousr AH, Napis S, Rusul GRA, et al. Detection of aerolysin and hemolysin genes in Aeromonas isolated from environmental and shellfish sources by polymerase chain reaction. ASEAN Food J 2007; 14(2): 115-122.
    29. Ye YW, Fan TF, Li H, et al. Characterization of Aeromonas hydrophila from hemorrhagic diseased freshwater fishes in Anhui Province, China. Int Food Res J 2013; 20(3): 1449-1452.
    30. Castro-Escarpulli G, Figueras MJ, Aguilera-Arreola G, et al. Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico. Int J Food Microbiol 2003; 84(1): 41-49.
    31. Tomás JM, The main Aeromonas pathogenic factors. ISRN Microbiology 2012; 2012: 256261. doi: 10.5402/ 2012/256261.
    32. Yu HB, Rao PSS, Lee HC, et al. A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis. Infect Immun 2004; 72(3): 1248-1256.
    33. Vance RE, Rietsch A, Mekalanos JJ. Role of the type III secreted exoenzymes S, T, and Y in systemic spread of Pseudomonas aeruginosa PAO1 in vivo. Infect Immun 2005; 73(3): 1706-1713.
    34. Schulert GS, Feltman H, Rabin SDP, et al. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J Infect Dis 2003; 188(11): 1695-1706.
    35. Merino S, Aguilar A, Rubires X, et al. Mesophilic Aeromonas strains from different serogroups: the influence of growth temperature and osmolarity on lipopolysaccharide and virulence. Res Microbiol 1998; 149(6): 407-416.