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This article has been accepted for publication and undergone full peer review but has not been DR. CARLO LAZADO (Orcid ID : 0000-0002-2823-2669)
Article type : Original Article
Full-length article
Pathogenic characteristics of Aeromonas veronii isolated from the liver of a diseased guppy (Poecilia reticulata)
Carlo C. Lazadoa,b and Dina Zilberga*
a The French Associates Institute for Agriculture and Biotechnology of Drylands, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, Israel
b Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, Ås, Norway
Running title: Aeromonas veronii in guppy
*Corresponding author:
D. Zilberg
Email: [email protected] Tel: +972-8-6596818 Fax: +972-8-6596742
Significance and impact of the study
Aeromonad infections continue to affect the fish farming industry. Several new species of Aeromonads in freshwater ornamental fish have been identified in the last years. In this study, we have characterized an Aeromonas veronii isolate from a diseased guppy. The series of experiments identified the intrinsic and extrinsic factors contributing to the pathogenic characteristics of the isolate. It has been shown to be pathogenic to both guppy and zebrafish.
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The results offer foundational knowledge in the development of preventive and therapeutic measures to combat this pathogen in the ornamental fish industry.
Abstract
Despite the significant development on their diagnoses and control, aeromonad infection is still a problem in aquaculture. The present study described the key bacteriological and pathogenic features of a presumptive Aeromonas sp. isolated from the liver of a diseased guppy (Poecilia reticulata). Molecular identification revealed that the isolate was an
Aeromonas veronii (A. veronii PR). It was able to grow in a wide range of temperatures and salt concentrations, and was capable of auto-aggregation and biofilm formation, with
temperature as an influencing factor. Some of the extracellular enzymes that may be involved in its virulence include caseinase, gelatinase and lipase. The infection rate was relatively progressive, and fish with prior infection showed marginal resistance to secondary infection.
Handling stress differentially influenced the infection kinetics at the early stages; however, the final mortality rates did not significantly differ between groups. A comparative infection trial revealed that zebrafish (Danio rerio) were more susceptible to A. veronii PR than guppy.
The presented intrinsic and extrinsic factors influencing the pathogenicity of A. veronii PR lay the foundation for future research to better understand this pathogen in freshwater ornamental fish aquaculture.
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outbreaks. At present, there are at least 25 species in the genus Aeromonas that have been described (Janda and Abbott 2010), and those that infect fish are highly diverse (Dong et al.
2017). The prominence of these Gram-negative rods in aquaculture can be attributed to their widespread distribution in the aquatic environment (Cipriano et al. 1984). Motile aeromonad infection in freshwater aquaculture is often described with Aeromonas hydrophila as the etiologic agent; hence, knowledge on the pathogenicity and virulence of this species has been considerably well documented compared with other pathogens from the group (Austin and Austin 2012). In the last years, several other Aeromonas species have been described to cause significant threats to aquaculture, including A. sobria, A. caviae, A. jandaei, A. dhakensis and A. veronii (Cipriano et al. 1984; Carriero et al. 2016; Dong et al. 2017). Amongst these pathogenic Aeromonas, the motile, mesophilic Aeromonad, A. veronii, seems to exhibit the broadest host range in virulence (Rahman et al. 2002; Janda and Abbott 2010; Song Y. et al.
2017). Species ranging from invertebrates to aquatic vertebrates to mammals, including humans, have been shown to be susceptible to this pathogen (Lu et al. 2016; Havixbeck et al.
2017). Nonetheless, the factors contributing to the pathogenicity of A. veronii are not well explored.
Aeromonas sp. was isolated from the liver of a diseased guppy (Poecilia reticulata) from a commercial ornamental fish farm that reported on-going chronic mortality. The present paper identified and characterized the isolate to provide valuable knowledge for ornamental aquaculturists in developing strategies to control this pathogen. The isolate was identified as Aeromonas veronii. It was proven pathogenic to guppy and zebrafish (Danio rerio). Intrinsic and extrinsic factors contributing to the pathogenicity of the isolate were characterized and are described in this paper.
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Results and Discussion
Bacteriological characteristics of A. veronii PR
On the tryptic soy agay (TSA) plate following a 24-h incubation, the colony of the bacterial isolate was round (diameter of 0.9–1.1 mm) and smooth with a slight cross-sectional elevation. The colonies exhibited tan to buff coloration. The partial 16S rRNA sequencing revealed that the isolate was an Aeromonas veronii. From here onwards, the isolate will be referred to as Aeromonas veronii PR. A sequence analysis showed that A. veronii PR shared 99% identity with 100% coverage with the A. veronii strain CiAVO2 (KF530822) and the A.
veronii strain WAB1882 (AM184224). The partial sequence was deposited in GenBank under accession no. MF276645. The biogram of antibiotic susceptibility showed that A. veronii PR was highly sensitive to trimethoprim/sulphamethoxazole and florfenicol but resistant to oxytetracycline, neomycin and norfloxacin. Its resistance to oxytetracycline was in agreement with previous studies on the negligible effect of this antibiotic on this pathogen (Yu et al.
2010; Jagoda et al. 2014). The presence of multiple genes coding for tet resistance in A.
veronii may be ascribed to the observed resistance to oxytetracycline (Skwor et al. 2014;
Chung et al. 2017).
There were no significant differences in the growth of the bacteria when incubated at temperatures between 21–27°C (Fig. 1a). Temperatures ≥ 29°C and ≤ 19°C significantly affected the growth of A. veronii GP, especially when compared with its growth at 25°C. The growth rates of A. veronii PR in salt concentrations ranging from 0–20 ppt did not
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Biofilm formation is an emblematic example of the adaptive trait of microorganisms for niche construction (Odling-Smee et al. 2003). Biofilm formation in Aeromonads enables them to persist in and resist stressful conditions including salinity, antimicrobial substances, and oxidative stress, compared to the planktonic lifestyle (Van Acker et al. 2014; Talagrand- Reboul et al. 2017). Aeromonas veronii PR was shown to form biofilm in both static and mobile conditions, with temperature affecting the process contrastively (Fig. 1c). The lowest biofilm formation in all set-ups was observed at 30°C and under mobile conditions. At 20°C, biofilm formation was quite stable regardless of the dynamics of the culture system and this may be potentially influenced by the strong auto-aggregation (AG) potential that was similarly observed at this temperature (Fig. 1c), since these cell-to-cell properties are
interrelated (Lazado et al. 2018). The A-layer of Aeromonas is a key feature that plays a part in bacterial auto-aggregation (Johnson et al. 1985), and its structural function has been shown to be negatively impacted by higher temperatures (Ishiguro et al. 1981).
A. veronii PR could hydrolyse casein and was capable of producing gelatinase and lipase. These factors could contribute to the virulence repertoire of A. veronii PR, since earlier studies pointed out the
importance of these extracellular factors in the virulence of Aeromonas, including A. veronii (Pemberton et al. 1997; Sun et al. 2016). No acidic extracellular factors have been detected.
B.
Pathogenicity of A. veronii PR in guppy
The pathogenicity of A. veronii PR in guppy was successfully demonstrated by experimental infection at varying dose levels, with at least 50% mortality documented at injection concentrations of 107 CFU ml-1 and higher (Fig. 2a). Fourteen days post-injection (dpi), cumulative mortality was recorded at 33% for 105 CFU ml-1, 38% for 106 CFU ml-1,
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54% for 107 CFU ml-1, and 71% for 108 CFU ml-1. In infection doses of 107 and 108 CFU ml-1, mortality was progressive until 11 dpi and then remained relatively stable until termination.
This dose response was relatively comparable to the previous studies on the experimental infection of A. veronii in tilapia and the loach (Zhu et al. 2016; Dong et al. 2017). It was apparent in all the infection trials that IP injection of A. veronii PR (107 CFU ml-1) resulted in a gradual mortality pattern. This finding was quite a striking contrast to earlier reports
showing severely acute mortality following A. veronii infection, with at least 50% mortality at 3 dpi (Dong et al. 2015; Zhu et al. 2016; Dong et al. 2017). The identified LD50 for infection, i.e., 107 CFU ml-1 in 5-µl delivery volume by IP injection, was found to be a reproducible route and infection dose, as cumulative mortality rates were reasonably similar between the trials.
Survivors of the primary infection demonstrated a small reduction in mortality (15%) compared with the control (i.e., no prior infection) upon re-infection, with no significant differences between naïve and re-infected fish (Fig. 2b). The previous report in tilapia (Dong et al. 2017) demonstrated a marked increase in resistance to A. veronii following re-infection.
Since the bacterial isolate originated from a commercial farm, as did the experimental fish, it is possible that the fish used in the trial were previously exposed to this bacterial pathogen;
thus, some immunological memory might have also been present in the control fish. Still, in the present study, prior infection history appeared to delay the mortality associated with A.
veronii PR infection by at least three days (Fig 2b).
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Handling stress and the susceptibility of guppy to A. veronii PR
Handling stress in itself was not very detrimental to guppy (Fig. 3). The fish with stress history showed a relatively lower mortality than the fish that had not been subjected to handling stress, though the difference at the termination of the experiment was not statistically significant. The two groups infected with the pathogen displayed the typical progression of A.
veronii PR infection in guppy (as in Figs 2,4); however, it appeared that the mortality rate in the first 10 days progressed relatively slower in the group with stress history than in the group without prior stress incidents. This finding reveals a slightly positive effect of the stressful episode that increased the resistance of guppy to the pathogen. Earlier report on the stimulatory effect of an acute and short-term stressor on the fish immune response lend support to this suggestion (Caipang et al. 2014).
Comparative susceptibility of guppy and zebrafish to A. veronii PR
There was a clearly disparate pattern in the two fish species’ susceptibility to A.
veronii PR, and the survival analysis showed significant differences between the groups (Fig.
4). Aeromonas veronii PR was highly pathogenic towards zebrafish as evidenced by the 100%
mortality at end of the observation period. This was two-fold higher than the mortality
observed in guppy at the same time point. Nearly 50% of zebrafish were dead at 2 dpi, and the rate of mortality sharply increased until mortality reached 100% at 6 dpi. The contrasting findings in the mortality rate suggest that although A. veronii is pathogenic in both fish species, zebrafish are likely a more susceptible host. We believe that this is the first report to demonstrate the pathogenicity of A. veronii in zebrafish. This observation supports the previous evidence indicating that A. veronii infectivity amongst fish hosts varied significantly (Dong et al. 2017).
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In conclusion, this study presents the pathogenic characteristics of Aeromonas veronii isolated from fish. The challenge trials revealed the susceptibility of the fish host to A.
veronii PR and the kinetics of infection. The intrinsic and extrinsic properties underscoring the pathogenicity of the isolate lay the foundation for future works aiming at further exploration of the infection mechanisms associated with this pathogen in freshwater ornamental fish farming. Likewise, the findings provide valuable knowledge in developing prophylactic and therapeutic measures to circumscribe and mitigate potential outbreaks.
Materials and Methods
Bacterial origin and culture conditions
The bacterium characterized in the present study was isolated from the liver of a diseased guppy during routine bacteriological work conducted by the Fish Health Laboratory of the Jacob Blaustein Institutes for Desert Research at Ben-Gurion University of the Negev for a commercial ornamental fish farm in southern Israel. The farm had suffered ongoing chronic mortality. Diseased fish appeared lethargic with no other evident clinical signs.
Preliminary morphological and biochemical analyses of the isolate revealed that it was a presumptive Aeromonas sp. (Internal case number: DL-04-14, isolate a).
Unless otherwise specified, the isolate was cultured in tryptic soy agar/broth (TSA/TSB, pH 7.5 ± 0.2; Oxoid, Hampshire, UK) and incubated at 25°C for 24 h. Bacterial liquid cultures were subjected to orbital agitation (100 rpm) during incubation. Stock cultures
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Molecular identification
A 24-h culture on TSA was sent to Hy Laboratories Ltd. (Rehovot, Israel) for 16S rRNA sequencing. The resulting sequence was subjected to comparative sequence analysis in NCBI (National Center for Biotechnology Information) by employing nucleotide BLAST (Basic Local Alignment Search Tool).
Susceptibility to antibiotics
The susceptibility of the isolate to several antibiotics was determined by the disc diffusion method on Mueller-Hinton (MH) agar. Commercially available antibiotic discs, including trimethoprim/sulphamethoxazole, oxytetracycline and florfenicol, which are permitted for use in the food fish industry; as well as neomycin, and norfloxacin (BBL™
Sensi-Disc™, BD, NJ) were used to evaluate antibiotic susceptibility. The response to
antibiotics was scored as either susceptible or resistant based on the diameter of the inhibition halo using the Interpretive Chart of BBL™ as a reference. The test was performed on two separate plates on two independent occasions.
Effects of temperature and salt on bacterial growth
The growth of the isolate was evaluated under varying incubation temperatures and salt (i.e., sodium chloride, NaCl) concentrations in the culture media. Freshly inoculated bacterial culture in TSB was incubated at temperatures ranging from 17 to 33°C for 24 h with constant orbital rotation. For evaluation of growth in response to salt concentration, TSB was augmented with NaCl at varying levels (i.e., 25, 10, 15, 20, 30, 40 and 50 ppt). Thereafter, the inoculated media were incubated at 25°C for 24 h with constant orbital rotation. In both culture manipulations, bacterial growth was determined by measuring the optical density at
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620 nm in a spectrophotometer. All tests were performed in three independent trials with three replicates each.
Detection of extracellular virulence factors
Casein hydrolysis and the activities of gelatinase and lipase were tested on nutrient agar containing 10% skimmed milk, 8% gelatin and 1% Tween 20, respectively. The presence of a transparent zone surrounding the colonies after 24 h at 25°C indicated the presence of casein hydrolysing enzymes. All tests were performed in three independent trials with three plates each time. The ability to produce acid was determined by the change in pH of the culture media following incubation in TSB at 25°C for 24 h.
Biofilm formation and auto-aggregation
The ability to form biofilms was determined by a modified crystal violet assay (Lazado et al. 2010). The plates were incubated at three different temperatures (i.e., 20, 25 and 30°C), under either static or mobile conditions. The mobile condition was achieved by incubating the seeded plate on top of an orbital shaker (100 rpm). The formation of biofilm was quantified by the absorbance at 570 nm.
The assay protocol for auto-aggregation was adopted from a previously modified method (Lazado et al. 2011). Auto-aggregation of the bacterial isolate was tested under different incubation temperatures (i.e., 20, 25, 30°C). The percentage of auto-aggregation was
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Fish
Guppies (average weight 8.03±0.33 g) were obtained from a commercial ornamental fish farm in the Negev Desert, Israel. The fish were kept in 100-L tanks (80-100 fish per tank), supplied with biological filters and aeration, fed (Ocean Nutrition, Essen, Belgium) at about 2% of their body weight (BW) per day and maintained at 26°C until used for
experimentation. Water exchange (10% of the total volume) was carried out every 2–3 days.
Oxygen level was kept at > 80% saturation. A representative number of fish was subjected to routine bacteriological and parasitological examinations (i.e., swabs from internal organs for bacterial isolation and direct microscopic analysis of wet mounts from skin, gills and gut) to ensure that only apparently healthy fish were used in the experiment.
All fish handling procedures involved in the study complied with the principles for biomedical research involving animals. The experimental protocol was approved by the Ben- Gurion University Committee for the Ethical Care and Use of Animals in Experiments, authorization no. IL-51-8-2008.
Calibration of infection dose
Prior to the challenge test, the bacterial isolate (from storage in -80°C) was streaked directly onto TSA plates and incubated at 25°C for at least 20–48 h. A pure single colony that typified an Aeromonas sp. was isolated and cultured in TSB at 25°C for 24 h, with constant agitation. A bacterial pellet was collected by centrifugation at 3000 x g for 10 min. The supernatant was discarded, and the bacterial pellet was resuspended in sterile phosphate- buffered saline (PBS; pH 7.4). Four concentrations of the bacterial isolate were prepared: 108, 107, 106 and 105 CFU ml-1. There were two replicate tanks per concentration. The actual bacterial count was retroactively verified by the spread plate method. For each concentration, 12 anesthetized fish (clove oil, 250 μL L−1) were intraperitoneally (IP) injected, in a delivery
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volume of 5 µl using a 30-G needle fitted to a 100-μL syringe attached to an automated injection chamber. A group injected with sterile PBS served as the control. The fish were then transferred to 10-L glass aquaria, each supplied with a submerged biological filter and
aeration. Mortality was recorded over a 14-day period. Representatives of freshly dead or moribund fish were subjected to bacterial isolation and identification. The isolated colonies were sent to Hy Laboratories (Israel) for 16S rRNA sequencing. The controlled infection was repeated in two independent trials.
Re-challenge experiment
The survivors from the calibration dose trial (i.e., 105, 106 and 107 CFU ml-1) were combined and transferred to a 60-L aquarium with biofilters and aeration. Feeding and water quality standards were maintained as detailed above. The fish were allowed to recover for 25 days. Twenty (20) of the previously infected guppies were injected intraperitoneally with the freshly prepared bacterial inoculum (107 CFU ml-1), in a delivery volume of 5 µl and
thereafter divided into two tanks (i.e., 10 fish per tank). A separate group of naïve guppies was also injected with the same concentration of the bacterial isolate. Mortality was recorded for 14 days.
Influence of handling stress
Fish from the holding container were transferred to 10-L aquaria at a density of 10
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resting interval for each air exposure. One hour after the stress challenge, Groups A and B were IP injected with the bacterial isolate (i.e., 107 CFU ml-1). Groups D and E were injected with sterile PBS. The trial was repeated twice, with two aquaria dedicated for each treatment in every trial.
Comparative infection experiment
The susceptibility of guppy and zebrafish to the bacterial isolate was compared.
Wild-type zebrafish from the AB line was gifted to the Fish Health Laboratory by Dr.
Birnbaum (BGU, Israel). Fish were distributed into 10-L aquaria at a density of 10 fish per aquarium. Each fish species was represented with three replicate tanks. They were allowed to acclimatize for 10 days to the same experimental conditions as in the guppy experiment.
Thereafter, the fish were challenged with the bacterial isolate (107 CFU ml-1) following the protocol employed in the guppy trials. Mortality was recorded for 14 days.
Statistics
The responses to different culture conditions were subjected to a one-way ANOVA followed by Tukey’s multiple comparison test to identify significant differences. The level of significance was set at P < 0.05. The difference in the biofilm-forming activity between the static and mobile conditions at a particular incubation temperature was determined by the Student’s t-test for independent samples. The Kaplan-Meier survival analysis was performed to compare differences in mortality rates between groups in various infection trials. The level of significance was set at P < 0.05. All statistical tests were performed in the SigmaStat statistical package (Systat Software, London, UK).
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Acknowledgments
The study was supported by research grants from the Ramat Negev Research and
Development Center, Israel, and the Central and Northern Arava Research and Development Center, Israel. C. Lazado would like to thank the Jacob Blaustein Center for Scientific Cooperation for his postdoctoral fellowship. We acknowledge the assistance of Tamar Sinai, Isabel Portugal, Sagar Nayak and Attila György Hadnagy during the infection trials and samplings. We thank Dr. Christian R. Karlsen for his help in the survival analysis.
Conflict of Interest
There is no conflict of interest to declare.
References
Austin, B. and Austin, D.A. (2012) Characteristics of the pathogens: Gram-negative bacteria.
In Bacterial Fish Pathogens. Diseases of Farmed and Wild Fish ed. Austin, B. and Austin, D.A. pp. 81-150. Netherlands: Springer.
Caipang, C.M.A., Fatira, E., Lazado, C.C. and Pavlidis M. (2014) Short-term handling stress affects the humoral immune responses of juvenile Atlantic cod, Gadus morhua. Aquac Int 22, 1283-1293.
Carriero, M.M., Mendes Maia, A.A., Moro Sousa, R.L. and Henrique-Silva, F. (2016) Characterization of a new strain of Aeromonas dhakensis isolated from diseased pacu fish (Piaractus mesopotamicus) in Brazil. J Fish Dis 39, 1285-1295.
Chung, T.H., Yi, S.W. and Shin, G.W. (2017) Antibiotic resistance and repetitive-element PCR fingerprinting in Aeromonas veronii isolates. J Fish Dis 40, 821-829.
Cipriano, R.C., Bullock, G.L. and Pyle, S.W. (1984) Aeromonas hydrophila and motile
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jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia, Oreochromis niloticus (L.). J Fish Dis DOI: 10.1111/jfd.12617.
Havixbeck J.J., Rieger A.M., Churchill L.J. & Barreda D.R. (2017) Neutrophils exert protection in early Aeromonas veronii infections through the clearance of both bacteria and dying macrophages. Fish Shellfish Immunol 63, 18-30.
Ishiguro, E.E., Kay, W.W., Ainsworth, T., Chamberlain, J.B., Austen, R.A., Buckley, J.T. and Trust, T.J. (1981) Loss of virulence during culture of Aeromonas salmonicida at high temperature. J Bacteriol 148, 333-340.
Jagoda, S., Wijewardana, T.G., Arulkanthan, A., Igarashi, Y., Tan, E., Kinoshita, S., Watabe, S. and Asakawa, S. (2014) Characterization and antimicrobial susceptibility of motile aeromonads isolated from freshwater ornamental fish showing signs of septicaemia. Dis Aquat Organ 109, 127-137.
Janda, J.M. and Abbott, S.L. (2010) The Genus Aeromonas: Taxonomy, pathogenicity, and infection. Clin Microbiol Rev 23, 35-73.
Johnson, C.M., Tatner, M.F. and Horne, M.T. (1985) Autoaggregation and extracellular A- layer protein in Aeromonas salmonicida. Aquaculture 46, 163-166.
Lazado, C.C., Caipang, C.M.A., Brinchmann, M.F. and Kiron V. (2011) In vitro adherence of two candidate probiotics from Atlantic cod and their interference with the adhesion of two pathogenic bacteria. Vet Microbiol 148, 252-259.
Lazado,, C.C., Caipang, C.M.A., Rajan, B., Brinchmann, M.F. and Kiron V. (2010) Characterization of GP21 and GP12: Two potential probiotic bacteria isolated from the gastrointestinal tract of Atlantic cod. Probiotics Antimicrob Proteins 2, 126-134.
Lazado, C.C., Fridman, S., Sinai, T. and Zilberg, D. (2018) First report of Streptococcus parauberis in a cultured freshwater ornamental fish, the ram cichlid Mikrogeophagus ramirezi (Myers & Harry, 1948). J Fish Dis 41, 161-164.
Lu, A., Yajiao， S., Xiucai， H., Jingfeng， S., Li，L., Chao，P., Chao，Z. and Guoxing, N. (2016) Aeromonas veronii, associated with skin ulcerative syndrome, isolated from the goldfish (Carassius auratus) in China. Isr J Aquac 68, 1-10.
Odling-Smee, F.J., Laland, K.N. and Feldman, M.W. (2003) Niche Construction: The Neglected Process in Evolution (MPB-37). Princeton University Press.
Pemberton, J.M., Kidd, S.P. and Schmidt R. (1997) Secreted enzymes of Aeromonas. FEMS Microbiol Lett 152, 1-10.
Rael, R.M. and Frankenberger, W.T. (1996) Influence of pH, salinity, and selenium on the growth of Aeromonas veronii in evaporation agricultural drainage water. Water Res 30, 422- 430.
Rahman, M., Colque-Navarro, P., Kühn, I., Huys, G., Swings, J. and Möllby R. (2002) Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh. Appl Environ Microbiol 68, 650-
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Skwor, T., Shinko, J., Augustyniak, A., Gee, C. and Andraso, G. (2014) Aeromonas hydrophila and Aeromonas veronii predominate among potentially pathogenic ciprofloxacin- and tetracycline-resistant Aeromonas isolates from Lake Erie. Appl Environ Microbiol 80, 841-848.
Song, Y., Hu, X., Lü, A., Sun, J., Yiksung, Y., Pei, C., Zhang, C. and Li, L. (2017) Isolation and characterization of Aeromonas veronii from ornamental fish species in China. Isr J Aquac DOI: IJA_69.2017.1446.
Sun, J., Zhang, X., Gao, X., Jiang, Q., Wen, Y. and Lin, L. (2016) Characterization of virulence properties of Aeromonas veronii isolated from diseased gibel carp (Carassius gibelio). Int J Mol Sci 17, 496.
Talagrand-Reboul, E., Jumas-Bilak, E. and Lamy, B. (2017) The social life of Aeromonas through biofilm and quorum sensing systems. Front Microbiol 8, 37.
Van Acker, H., Van Dijck, P. and Coenye, T. (2014) Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms. Trends Microbiol 22, 326-333.
Yu, J.-H., Han, J.-J., Kim, H.-J., Kang, S.-G. and Park, S.-W. (2010) First report of Aeromonas veronii infection in farmed Israeli carp Cyprinus carpio in Korea. J Fish Pathol 23, 165-176.
Zhu, M., Wang, X.R., Li, J., Li, G.Y., Liu, Z.P. and Mo, Z.L. (2016) Identification and virulence properties of Aeromonas veronii bv. sobria isolates causing an ulcerative syndrome of loach Misgurnus anguillicaudatus. J Fish Dis 39, 777-781.
Figure captions
Figure 1. Growth, biofilm formation and auto-aggregation of A. veronii PR. Bacterial growth under different (a) incubation temperatures and (b) NaCl concentrations in the culture media. Different letters indicate significant differences. (c) Biofilm formation (bar; ■ mobile,
static) and auto-aggregation (line) under varying incubation temperatures. Different letters indicate statistically significant differences between incubation temperatures under mobile conditions; different numbers demonstrate significant differences under static conditions. An asterisk (*) represents a significant difference between mobile and static conditions at a particular temperature. The values presented show the mean + SEM from two independent experiments with three replicate set-ups on each occasion.
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day period post-challenge. n = 20. Significant differences are indicated by different letters (P
< 0.5).
Figure 3. Effects of handling stress on the susceptibility of guppies to A. veronii PR. Fish were subjected to a 10-min non-continuous handling stress prior to IP injection of A. veronii PR. Cumulative mortality was recorded over a 14-day period post-challenge. Different letters indicate significant differences (P < 0.5). Values presented are the mean of two independent experiments. In both trials, each treatment was represented with 20 fish equally distributed into two tanks. (■ A. veronii, □ A. veronii + stress, ◊ stress, ○ PBS + stress, ●PBS control) Figure 4. Comparative A. veronii PR infection trial between guppy and zebrafish. Both fish species were infected with A. veronii PR (i.e., 107 CFU ml-1), and cumulative mortality was recorded thereafter over a 14-day period. Different letters indicate significant differences (P < 0.5). Each treatment was allotted three replicate tanks, with a stocking density of 10 fish per tank. (■ zebrafish + A. veronii, ● guppy + A. veronii, □ zebrafish + PBS, ○ guppy + PBS)
