Section: Aquatic Bacteria

Aeromonas hydrophila Infections in Freshwater Fish: Pathogenesis and Treatment Options

Introduction

Aeromonas hydrophila is a Gram-negative, facultatively anaerobic, rod-shaped bacterium belonging to the family Aeromonadaceae. It is a ubiquitous inhabitant of aquatic environments, including freshwater lakes, rivers, and aquaculture systems. Under conditions of host stress, immunosuppression, or environmental degradation, A. hydrophila transitions from a commensal organism to a primary or opportunistic pathogen capable of causing significant morbidity and mortality in a wide range of freshwater fish species. The disease manifestations, collectively termed motile aeromonad septicemia (MAS) or hemorrhagic septicemia, represent a major constraint to global freshwater aquaculture productivity [1, 2]. This article provides a comprehensive review of the pathogenesis, clinical presentation, diagnostic isolation, antimicrobial susceptibility, and treatment strategies for A. hydrophila infections in freshwater fish, with a focus on biosecurity measures relevant to aquaculture settings.

Etiology and Taxonomy

Aeromonas hydrophila is a member of the genus Aeromonas, which is divided into two major groups: the psychrophilic, non-motile species (e.g., Aeromonas salmonicida) and the mesophilic, motile species (e.g., A. hydrophila, A. veronii, A. caviae). A. hydrophila is characterized by its polar flagellation, which confers motility, and its ability to grow at a wide range of temperatures (optimum 22 degrees Celsius to 28 degrees Celsius) and pH values (5.5 to 9.0) [3]. The species is further subdivided into multiple genomospecies and serogroups, with O-antigen serotyping used for epidemiological tracking. The genetic heterogeneity of A. hydrophila strains contributes to variability in virulence potential and antimicrobial resistance profiles [4].

Pathogenesis and Virulence Factors

The pathogenesis of A. hydrophila infection is multifactorial, involving a complex interplay of bacterial virulence determinants and host susceptibility factors. The bacterium adheres to and colonizes the host epithelium, primarily the skin, gills, and gastrointestinal tract, before invading deeper tissues and the bloodstream.

Adhesion and Invasion

Initial attachment is mediated by flagella, type IV pili, and outer membrane proteins (OMPs). Flagella provide motility and chemotaxis toward host mucus, while pili facilitate intimate contact with epithelial cells [5]. Once adherent, A. hydrophila secretes a range of hydrolytic enzymes, including proteases, lipases, and hemolysins, which degrade host tissues and facilitate bacterial dissemination.

Exotoxins and Secreted Factors

The most extensively characterized virulence factors are the aerolysin/hemolysin family of pore-forming toxins. Aerolysin (AerA) and hemolysin (HlyA) are secreted via the type II secretion system and insert into host cell membranes, forming hexameric pores that cause osmotic lysis of erythrocytes, leukocytes, and epithelial cells [6, 7]. These toxins are directly responsible for the characteristic hemorrhagic lesions observed in infected fish.

Additional secreted factors include:

  • Proteases: Serine protease and metalloprotease degrade host connective tissue and immune proteins such as complement and immunoglobulins [8].
  • Lipases: Phospholipase C and glycerophospholipid-cholesterol acyltransferase (GCAT) disrupt cell membrane integrity and contribute to tissue necrosis [9].
  • Siderophores: The amonabactin siderophore system chelates iron from host transferrin and ferritin, an essential nutrient for bacterial growth within the host [10].

Biofilm Formation

A. hydrophila readily forms biofilms on both biotic and abiotic surfaces, including fish skin, gill tissue, and aquaculture equipment. Biofilm formation is regulated by quorum sensing systems, particularly the N-acyl homoserine lactone (AHL) mediated LuxR/LuxI homologs. Biofilm embedded bacteria exhibit increased resistance to antimicrobial agents and host immune defenses, complicating treatment and facilitating persistent infections [11, 12].

Immune Evasion

The bacterium employs several strategies to subvert the host immune response. The O-antigen of lipopolysaccharide (LPS) provides resistance to complement mediated killing. Additionally, secreted proteases degrade host antibodies and complement proteins, while the aerolysin toxin induces apoptosis in macrophages and neutrophils [13, 14].

Clinical Signs and Pathological Findings

The clinical presentation of A. hydrophila infection in freshwater fish is highly variable, ranging from peracute mortality with few premonitory signs to chronic, low-grade morbidity. The classic syndrome is hemorrhagic septicemia.

External Clinical Signs

  • Hemorrhages: Petechial and ecchymotic hemorrhages on the skin, fins, opercula, and around the vent. These lesions are most prominent on the ventral surface and flanks.
  • Ulceration: Progressive dermal ulceration, often with a necrotic center and erythematous border. Deep ulcers may expose underlying muscle.
  • Exophthalmos: Unilateral or bilateral ocular protrusion due to retrobulbar edema or hemorrhage.
  • Ascites: Abdominal distension due to accumulation of serosanguinous fluid in the peritoneal cavity.
  • Gill Pallor: Anemia and gill necrosis, leading to respiratory distress.
  • Behavioral Changes: Lethargy, anorexia, erratic swimming, and surface gasping.

Internal Pathological Findings

  • Splenomegaly and Renomegaly: Enlargement and congestion of the spleen and kidney.
  • Hepatic Necrosis: Focal to diffuse hepatic necrosis, often with a mottled appearance.
  • Enteritis: Hemorrhagic inflammation of the intestinal mucosa, with lumenal fluid accumulation.
  • Pericarditis and Myocarditis: In severe cases, inflammation of the pericardium and cardiac muscle.

Histopathological examination reveals extensive necrosis of hematopoietic tissue in the kidney and spleen, hepatocellular degeneration, and severe branchitis with lamellar fusion and epithelial sloughing [15, 16].

Host Susceptibility and Environmental Risk Factors

Disease outbreaks are strongly correlated with environmental stressors that compromise fish immune function. Key predisposing factors include:

  • Elevated Water Temperature: Optimal bacterial growth and toxin production occur at temperatures above 20 degrees Celsius. Outbreaks are most common during summer months.
  • Poor Water Quality: High ammonia, nitrite, and organic load, combined with low dissolved oxygen, suppress the fish immune system and enhance bacterial proliferation.
  • Overcrowding: High stocking densities facilitate horizontal transmission and increase stress.
  • Coinfections: Concurrent infections with parasites (e.g., Ichthyophthirius multifiliis, see Ichthyophthirius multifiliis (Ich) in Freshwater Aquaculture: Rapid Detection and Integrated Control) or viruses (e.g., Canine Coronavirus variants, though not directly relevant, illustrate the principle of viral-bacterial synergy) can predispose fish to secondary bacterial infections.
  • Nutritional Deficiencies: Deficiencies in vitamins C and E, and essential fatty acids impair immune function [17, 18].

Diagnostic Isolation and Identification

Accurate diagnosis of A. hydrophila infection requires a combination of clinical observation, microbiological culture, and molecular confirmation.

Sample Collection

Samples should be collected from moribund or freshly dead fish. Target tissues include kidney, spleen, liver, and external lesions. Aseptic technique is critical to avoid contamination from the aquatic environment.

Culture and Isolation

  1. Media: A. hydrophila grows readily on standard bacteriological media such as tryptic soy agar (TSA) or brain heart infusion agar (BHIA). Selective media, such as Rimler-Shotts agar or Aeromonas selective agar (ASA) containing ampicillin, are used to suppress competing flora [19].
  2. Incubation: Plates are incubated aerobically at 25 degrees Celsius to 28 degrees Celsius for 24 to 48 hours.
  3. Colony Morphology: Colonies are typically 2 to 4 mm in diameter, smooth, convex, and translucent to opaque. On blood agar, a clear zone of beta-hemolysis is often observed.

Biochemical Identification

A. hydrophila is oxidase positive, catalase positive, and capable of fermenting glucose with acid and gas production. Key biochemical tests for species-level identification include:

| Test | A. hydrophila Reaction | | :-, | :-, | | Oxidase | Positive | | Catalase | Positive | | Motility | Positive (polar flagella) | | Indole Production | Positive | | Voges-Proskauer | Positive | | Citrate Utilization | Positive | | Lysine Decarboxylase | Positive | | Ornithine Decarboxylase | Negative | | Fermentation of Sucrose | Positive | | Fermentation of Mannitol | Positive | | Resistance to O/129 (vibriostatic agent) | Resistant |

Commercial biochemical test strips (e.g., API 20E, API 20NE) are widely used for rapid identification, though they may misidentify some environmental isolates [20].

Molecular Confirmation

Definitive identification is achieved through molecular methods targeting species-specific genes.

  • 16S rRNA Gene Sequencing: Universal primers amplify a conserved region of the 16S ribosomal RNA gene. Sequence comparison against databases (e.g., GenBank) provides genus and species identification [21].
  • Species-Specific PCR: Primers targeting the aerolysin gene (aerA) or the hemolysin gene (hlyA) provide rapid and specific detection of pathogenic A. hydrophila strains [22].
  • Multiplex PCR: Panels that simultaneously detect multiple Aeromonas species and virulence genes are used for epidemiological surveillance [23].
  • Whole Genome Sequencing (WGS): WGS provides the highest resolution for strain typing, antimicrobial resistance gene profiling, and virulence gene characterization. It is increasingly used in outbreak investigations [24].

Antimicrobial Susceptibility and Resistance

The treatment of A. hydrophila infections relies heavily on antimicrobial chemotherapy. However, the emergence and dissemination of antimicrobial resistance (AMR) genes in aquaculture environments pose a significant therapeutic challenge.

Commonly Used Antimicrobials

Historically, several classes of antimicrobials have been effective against A. hydrophila:

  • Tetracyclines: Oxytetracycline is a common first-line treatment administered via medicated feed.
  • Potentiated Sulfonamides: Trimethoprim-sulfamethoxazole (TMP-SMX) is widely used for systemic infections.
  • Fluoroquinolones: Enrofloxacin and ciprofloxacin are highly effective but are classified as critically important antimicrobials for human medicine, limiting their use in some jurisdictions.
  • Phenicols: Florfenicol is approved for use in several aquaculture species and has good activity against A. hydrophila.
  • Aminoglycosides: Gentamicin and kanamycin are used in bath treatments or injectable formulations for valuable broodstock.

Antimicrobial Resistance Mechanisms

Resistance in A. hydrophila is mediated by several mechanisms:

  • Enzymatic Inactivation: Beta-lactamases (e.g., TEM, SHV, CTX-M) hydrolyze beta-lactam antibiotics. Aminoglycoside modifying enzymes (e.g., acetyltransferases, phosphotransferases) inactivate aminoglycosides [25].
  • Efflux Pumps: Resistance-nodulation-division (RND) family efflux pumps, such as AdeABC homologs, actively export tetracyclines, fluoroquinolones, and chloramphenicol from the bacterial cell [26].
  • Target Site Modification: Mutations in DNA gyrase (gyrA) and topoisomerase IV (parC) confer resistance to fluoroquinolones. Methylation of the 16S rRNA target site confers resistance to aminoglycosides [27].
  • Acquired Resistance Genes: Mobile genetic elements, including plasmids, integrons, and transposons, facilitate the horizontal transfer of resistance genes between A. hydrophila and other Gram-negative bacteria in the aquatic microbiome [28].

Antibiogram Profiling

Given the high prevalence of multidrug resistant (MDR) strains, antimicrobial susceptibility testing (AST) is essential for guiding therapy. The disk diffusion method (Kirby-Bauer) and broth microdilution are standard techniques. Minimum inhibitory concentration (MIC) breakpoints should be interpreted using guidelines established for aquatic bacteria or, in their absence, human clinical breakpoints for Enterobacteriaceae [29].

A typical antibiogram for A. hydrophila isolates from aquaculture may show the following resistance patterns:

| Antimicrobial Class | Representative Agent | Resistance Prevalence | | :-, | :-, | :-, | | Tetracyclines | Oxytetracycline | 30-60% | | Sulfonamides | Trimethoprim-sulfamethoxazole | 20-50% | | Fluoroquinolones | Enrofloxacin | 10-40% | | Phenicols | Florfenicol | 5-20% | | Beta-lactams | Amoxicillin | 60-80% (intrinsic) | | Aminoglycosides | Gentamicin | 15-35% |

Data adapted from multiple surveillance studies [30, 31, 32].

Treatment Options and Therapeutic Protocols

Treatment strategies must be tailored to the severity of the outbreak, the fish species, water temperature, and regulatory constraints.

Antimicrobial Therapy

  • Medicated Feed: This is the preferred route for treating large populations. Oxytetracycline is administered at 50-75 mg per kg of fish body weight per day for 10 days. Florfenicol is dosed at 10 mg per kg per day for 10 days [33].
  • Bath Treatments: For external infections or when fish are not feeding, bath treatments with potassium permanganate (2-4 mg/L for 1 hour) or formalin (25-50 mg/L for 1 hour) can reduce bacterial load on skin and gills. These treatments are non-specific and may cause environmental toxicity [34].
  • Injectable Antibiotics: For high-value individual fish (e.g., broodstock), injectable enrofloxacin (5-10 mg/kg) or gentamicin (2-4 mg/kg) can be administered intramuscularly or intraperitoneally.

Supportive Care

  • Water Quality Management: Immediate improvement of water quality through increased aeration, partial water changes, and reduction of organic load is critical.
  • Reduction of Stocking Density: Decreasing fish density reduces stress and limits horizontal transmission.
  • Nutritional Support: Supplementation of feed with vitamin C (500-1000 mg/kg feed) and vitamin E (200-400 mg/kg feed) enhances immune function [35].

Vaccination

Vaccination is a preventive strategy that reduces the reliance on antimicrobials. Both inactivated (killed) whole-cell vaccines and live attenuated vaccines have been developed.

  • Inactivated Vaccines: Formalin-killed bacterins are administered via injection (intraperitoneal) or immersion. They induce a humoral immune response and provide moderate protection against homologous serotypes [36].
  • Live Attenuated Vaccines: Strains with targeted deletions in virulence genes (e.g., aroA, aerA) have shown promise in experimental trials, eliciting both humoral and cell-mediated immunity [37].
  • Subunit and DNA Vaccines: Recombinant outer membrane proteins (e.g., OmpA, OmpW) and DNA vaccines encoding aerolysin are under investigation [38].

Vaccination is most effective when combined with good management practices. For a broader discussion of vaccine development in aquaculture, see Aeromonas hydrophila in Aquaculture: Pathogenesis, Antimicrobial Resistance, and Vaccine Development.

Biosecurity Measures in Aquaculture

Prevention of A. hydrophila outbreaks relies on robust biosecurity protocols that minimize pathogen introduction and transmission.

Facility Design and Management

  • Quarantine: New fish stocks should be quarantined in separate systems for a minimum of 2 to 4 weeks. During quarantine, fish should be observed for clinical signs and screened for bacterial pathogens.
  • Disinfection: Equipment (nets, buckets, tanks) should be disinfected between uses. Effective disinfectants include chlorine compounds (200 mg/L free chlorine for 30 minutes), iodophors, and quaternary ammonium compounds.
  • Water Source Management: Use of UV sterilizers or ozone treatment on incoming water can inactivate A. hydrophila. Recirculating aquaculture systems (RAS) should have effective biofiltration and solids removal.
  • Separation of Age Classes: Different age groups should be reared in separate units to prevent pathogen transmission from older, potentially carrier fish to naive juveniles.

Health Monitoring and Surveillance

  • Regular Clinical Inspections: Daily observation for signs of lethargy, anorexia, or external lesions.
  • Sentinel Fish: Placement of naive sentinel fish in the system can provide early warning of pathogen presence.
  • Microbiological Surveillance: Periodic culture of water and fish samples (e.g., gill and skin swabs) can detect subclinical infections.

Stock Management

  • Optimal Stocking Densities: Adherence to species-specific stocking density guidelines reduces stress.
  • Nutrition: Provision of a balanced diet with appropriate levels of immunostimulants (e.g., beta-glucans, mannan-oligosaccharides) can enhance disease resistance [39].
  • Stress Reduction: Minimizing handling, transport, and temperature fluctuations is essential.

Decision Tree for Outbreak Management

The following Mermaid diagram outlines a clinical decision algorithm for managing a suspected A. hydrophila outbreak in a freshwater aquaculture facility.

flowchart TD
    A[Observe clinical signs: hemorrhages, ulcers, lethargy], > B{Collect moribund fish}
    B, > C[Necropsy and sample collection: kidney, spleen, liver]
    C, > D[Microbiological culture on TSA or selective media]
    D, > E{Isolate Gram-negative, oxidase-positive rods?}
    E, No, > F[Consider other pathogens: Flavobacterium, Pseudomonas]
    E, Yes, > G[Biochemical or molecular identification: A. hydrophila]
    G, > H[Perform antimicrobial susceptibility testing (AST)]
    H, > I{Identify effective antimicrobials}
    I, > J[Administer medicated feed or bath treatment]
    J, > K[Improve water quality and reduce stocking density]
    K, > L{Clinical improvement within 48-72 hours?}
    L, Yes, > M[Continue treatment course; monitor for relapse]
    L, No, > N[Re-culture and re-test AST; consider mixed infection]
    N, > O[Adjust antimicrobial therapy based on new AST]
    O, > P[Implement enhanced biosecurity measures]
    P, > Q[Document outbreak and review protocols]

Conclusion

Aeromonas hydrophila remains a formidable pathogen in freshwater aquaculture, capable of causing devastating outbreaks of hemorrhagic septicemia. The pathogenesis of the disease is driven by a sophisticated arsenal of virulence factors, including pore-forming toxins, proteases, and biofilm formation capabilities. Effective management requires a multipronged approach that integrates accurate diagnosis, targeted antimicrobial therapy guided by susceptibility testing, and rigorous biosecurity protocols. The increasing prevalence of multidrug resistant strains underscores the urgent need for alternative control strategies, including improved vaccines, immunostimulants, and probiotic interventions. A holistic health management approach, emphasizing environmental optimization and stress reduction, is essential for sustainable control of A. hydrophila infections in freshwater fish populations.

References

[1] Austin B, Austin DA. Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. 6th ed. Springer; 2016.

[2] Janda JM, Abbott SL. The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev. 2010;23(1):35-73.

[3] Martin-Carnahan A, Joseph SW. Aeromonadaceae. In: Brenner DJ, Krieg NR, Staley JT, editors. Bergey's Manual of Systematic Bacteriology. 2nd ed. Springer; 2005.

[4] Beaz-Hidalgo R, Figueras MJ. Aeromonas spp. whole genomes and virulence factors implicated in fish disease. J Fish Dis. 2013;36(4):371-388.

[5] Kirov SM. The public health significance of Aeromonas spp. in foods. Int J Food Microbiol. 1993;20(4):179-198.

[6] Howard SP, Buckley JT. Activation of the hole-forming toxin aerolysin by extracellular processing. J Bacteriol. 1985;163(1):336-340.

[7] Wong CY, Heuzenroeder MW, Flower RL. Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model. Microbiology. 1998;144(Pt 2):291-298.

[8] Leung KY, Stevenson RM. Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish. Infect Immun. 1988;56(10):2639-2644.

[9] Merino S, Aguilar A, Nogueras MM, et al. Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. Infect Immun. 1999;67(8):4008-4017.

[10] Byers BR, Massad G, Barghouthi S, Arceneaux JE. Iron acquisition and virulence in the motile aeromonads: siderophore-dependent and -independent systems. Experientia. 1991;47(5):416-418.

[11] Lynch MJ, Swift S, Kirke DF, et al. The regulation of biofilm development by quorum sensing in Aeromonas hydrophila. Environ Microbiol. 2002;4(1):18-28.

[12] Talagrand-Reboul E, Jumas-Bilak E, Lamy B. The social life of Aeromonas through biofilm and quorum sensing systems. Front Microbiol. 2017;8:37.

[13] Merino S, Rubires X, Knochel S, Tomas JM. Emerging pathogens: Aeromonas spp. Int J Food Microbiol. 1995;28(2):157-168.

[14] Galindo CL, Gutierrez C, Chopra AK. Potential involvement of galectin-3 and SNAP23 in Aeromonas hydrophila-induced apoptosis of murine macrophages. Microb Pathog. 2006;40(2):76-85.

[15] Roberts RJ. Fish Pathology. 4th ed. Wiley-Blackwell; 2012.

[16] Ferguson HW. Systemic Pathology of Fish. 2nd ed. Scotian Press; 2006.

[17] Wedemeyer GA. Physiology of Fish in Intensive Culture Systems. Chapman & Hall; 1996.

[18] Blazer VS. Nutrition and disease resistance in fish. Annu Rev Fish Dis. 1992;2:309-323.

[19] Shotts EB, Rimler R. Medium for the isolation of Aeromonas hydrophila. Appl Microbiol. 1973;26(4):550-553.

[20] Abbott SL, Cheung WK, Janda JM. The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. J Clin Microbiol. 2003;41(6):2348-2357.

[21] Martinez-Murcia AJ, Benlloch S, Collins MD. Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations. Int J Syst Bacteriol. 1992;42(3):412-421.

[22] Wang G, Clark CG, Liu C, et al. Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR. J Clin Microbiol. 2003;41(3):1048-1054.

[23] Nawaz M, Khan SA, Khan AA, et al. Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food Microbiol. 2010;27(3):327-331.

[24] Hossain S, Wickramasinghe S, Dahanayake PS, Heo GJ. Comparative genomic analysis of Aeromonas hydrophila from diseased fish reveals novel virulence and antimicrobial resistance genes. Microb Pathog. 2020;149:104527.

[25] Jacobs L, Chenia HY. Characterization of integrons and tetracycline resistance determinants in Aeromonas spp. isolated from South African aquaculture systems. Int J Food Microbiol. 2007;114(3):295-306.

[26] Hernould M, Gagne S, Fournier D, et al. Role of the AheABC efflux pump in Aeromonas hydrophila intrinsic multidrug resistance. Antimicrob Agents Chemother. 2008;52(5):1559-1563.

[27] Goni-Urriza M, Arpin C, Capdepuy M, et al. Type II topoisomerase quinolone resistance-determining regions of Aeromonas caviae, A. hydrophila, and A. sobria complexes and mutations associated with quinolone resistance. Antimicrob Agents Chemother. 2002;46(2):350-359.

[28] Schmidt AS, Bruun MS, Dalsgaard I, Larsen JL. Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl Environ Microbiol. 2001;67(12):5675-5682.

[29] Clinical and Laboratory Standards Institute (CLSI). Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated from Aquatic Animals. CLSI guideline VET03/VET04. 2nd ed. 2020.

[30] Akinbowale OL, Peng H, Barton MD. Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia. J Appl Microbiol. 2006;100(5):1103-1113.

[31] Saavedra MJ, Guedes-Novais S, Alves A, et al. Resistance to beta-lactam antibiotics in Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss). Int Microbiol. 2004;7(3):207-211.

[32] Vivekanandhan G, Savithamani K, Hatha AA, Lakshmanaperumalsamy P. Antibiotic resistance of Aeromonas hydrophila isolated from marketed fish and prawn of South India. Int J Food Microbiol. 2002;76(1-2):165-168.

[33] Yanong RPE. Use of antibiotics in ornamental fish aquaculture. EDIS, University of Florida IFAS Extension. 2003.

[34] Noga EJ. Fish Disease: Diagnosis and Treatment. 2nd ed. Wiley-Blackwell; 2010.

[35] Wahli T, Verlhac V, Gabaudan J, et al. Influence of dietary vitamin C on the immune response of rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 1995;5(1):31-44.

[36] Karunasagar I, Ali A, Otta SK, Karunasagar I. Immunization with bacterial antigens: infections with motile aeromonads. Dev Biol Stand. 1997;90:135-141.

[37] Vivas J, Riano J, Carracedo B, et al. The auxotrophic aroA mutant of Aeromonas hydrophila as a live attenuated vaccine