Species Diversity, Existence of Virulence Gene Profile and in-vivo Pathogenicity Study of Aeromonas spp. Isolated from Diseased Catla catla

Chethurajupalli Lavanya1*, Tambireddy Neeraja1, Thatavarthi Venkata Ramana2, Arumugam Balasubramanian3 and Ogirala Sudhakar2

1Department of Aquatic Animal Health Management, College of Fishery Science, Andhra Pradesh Fisheries University, Muthukur, Andhra Pradesh, India

2Andhra Pradesh Fisheries University, Vijayawada, Andhra Pradesh, India

3Department of Fisheries Resource Management, College of Fishery Science, Andhra Pradesh Fisheries University, Muthukur, Andhra Pradesh, India

ABSTRACT

This study aimed to isolate Aeromonas species from hemorrhagic septicemia diseased Catla catla freshwater fish in Andhra Pradesh, India, in order to learn more about them, locate its virulence genes, and evaluate their pathogenicity. A total of nine Aeromonas spp., were isolated and identified by API 20E kit. Out of nine Aeromonas spp., A. veronii bv veronii (28%) was most prevalent species than other Aeromonas spp. The isolated species were tested for frequency of six virulence genes viz., aerolysin, enterotoxin, elastase, hemolysin, lipase and serine protease. We found heterogeneous distribution pattern of virulence genes among the Aeromonas spp. with dominance of elastase (87%). Furthermore, catla were challenged with nine Aeromonas spp. with various virulence gene profile by intraperitoneal injection at 108cfu/ml and studied for 96 h to determine co-relation between the number of virulence genes and their pathogenicity. In-vivo pathogenicity test revealed that the mortality did not depend on number of genes rather it depended on the type of combination of aerolysin, elastage and protease genes. The study concluded that isolated Aeromonas spp. harbored various virulence genes indicating their pathogenicity for fishes.

Article Information

Received 25 May 2024

Revised 05 July 2024

Accepted 16 July 2024

Available online 5 December 2024

(early access)

Published 13 December 2025

Authors’ Contribution

CL conducted the study and wrote the manuscript. TN planned the work, analysed the data and supervised the project. TVR, AB and OS edited the manuscript.

Key words

Aeromonas species, Biochemical characterization, Virulence genes, In-vivo, Pathogenicity, Catla catla

DOI: https://dx.doi.org/10.17582/journal.pjz/20240525064332

* Corresponding author: [email protected]

0030-9923/2026/0001-0071 $ 9.00/0

Copyright 2026 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The Gram-negative, facultative anaerobic bacteria known as aeromonads are seriously risky. They may be found in many natural aquatic habitats (Igbinosa et al., 2012). Fresh water fishes especially carps are the important food sources and study models all around the world (Sanyal et al., 2018). Most popular amongst carps is Catla catla which is a native fish of regions of riverine systems (Shahzad et al., 2014). The genus Aeromonas has experienced a number of revisions to its classification, adding complexity to an already convoluted subject. A. hydrophila, A. caviae, A. salmonicida, and A. sobria are the four Aeromonas spp. reported in 1980s (Hu et al., 2012). Then, recently, the number of Aeromonas spp. has increased to 41 (Fernandez-Bravo and Figueras, 2020). These species are responsible for the diseases furunculosis, hemorrhagic septicemia, skin ulcers, fin/tail rot, dropsy, and skin ulcer-causing hemorrhagic septicemia (Reith et al., 2008). In humans, some Aeromonas species, including A. caviae, A. hydrophila, A. veronii, A. trota, A. schubertii, and A. jandaei, cause gastroenteritis (Sinha et al., 2004), septicemia, peritonitis, meningitis and eye infections (Kelly et al., 1993).

Aeromonad’s pathogenesis is very complex because of its wide range of virulence factors that interact with one another in a multifactorial manner. Bacterial pathogenesis has been linked to a variety of proteases, including aerolysin, hemolysin, enetrotoxin, temperature-sensitive protease and serine protease (Albert et al., 2000; Nawaz et al., 2010; Hu et al., 2012; Li et al., 2020). Aeromonad isolates pathogenicity varies widely in terms of the amount, and presence of virulence genes within and between species. Perhaps there is a geographical explanation for the disparities that exist (Ghenghesh et al., 2014). Therefore, the continuous monitor about these species in fish culture is necessary to improved disease management and preventative approaches, as it understanding that Aeromonas are highly virulent in nature. Therefore, the present study was carried out on Aeromonas species identification and characterization of virulence genes with main focus on their pathogenicity.

MATERIALS AND METHODS

Fish sampling

Thirty-two infected Catla catla fish were collected from farms in the West Godavari and SPSR Nellore districts of Andhra Pradesh, India. All of the collected samples were brought to the lab at the College of Fishery Science in Muthukur, Nellore district.

Isolation and identification of Aeromonas spp.

Infected catla fish showed symptoms of bacterial hemorrhagic septicemia such as hemorrhages on body surface and internal organs, dropsy, pale gills, fin and tail rot, and discoloration of internal organs. Based on severity of infections, inoculums were collected from kidney, liver and gill tissues and were streaked on a rimler shotts (RS) medium. After 18-24 h of streaking on RS plates, the most prominent colonies were sub cultured on trypticase soy agar (TSA). Initially, the standard technique of identifying bacterial isolates relied on biochemical assays through API 20E test kit (Hi-Media, India). Taxonomic keys proposed by Abbott et al. (2003), Martinez-Murcia et al. (2008) and Beaz-Hidalgo et al. (2010) were followed for identification of Aeromonas spp.

Virulence genes detection in Aeromonas spp.

The DNAs of all the isolated strains were extracted by a DNA extraction kit (Bangalore Genei, Bangalore) according to the company’s guidelines with minor adjustments. Briefly, each strain mixed in 110 µl of extraction solution, incubated for 20 min at 35oC then centrifuged for 10 min at 10,000 rpm. 100 µl supernatant collected into another tube having 100 µl 100% ethanol, then centrifuged for 5 min at 10,000 rpm. The DNA pellet was washed for two times at 5000 rpm for 5 min with 100 µl of 95% ethanol. Next allow to dry DNA pellet at room temperature and dissolved in 50 µl of nucleus free water. Aerolysin, cytotonic enterotoxin, elastase, lipase, hemolysin and serine protease virulence genes were amplified by PCR. The virulence genes primers and their thermal cycling conditions are given in Table I. PCR amplification of virulence genes was performed in a reaction volume of 25 µl using a thermal cycler (BioRad, T100, Germany). The reaction mixture consists: 1 µl of template DNA, 2 µl of F and R primer, 10 µl of master mix (Taq DNA polymerase, 2.0 x master mix red, MgCl2 2.0 mM, Thermo Scientific) and 12 µl of molecular grade water. The amplified product was checked on a 1.2% agarose gel.

In-vivo pathogenicity study of Aeromonas spp.

To determine the correlation between the number of virulence genes and the pathogenicity mortality rates, In-vivo test was conducted. A total of 330 catla (average weight 25 ± 2 g) were used for this study. Before acclimatization, fishes were treated with 2 ppm potassium permanganate for 10 min (Barkoh et al., 2010) and maintained for 15 days. During that period fishes were fed with 35% protein commercial pellet (CP, Chennai) @ 3% of body weight.

 

Table I. Primers used in PCR for virulence genes expression of Aeromonas.

Virulence gene	Primer sequence (5’-3’)	Product size (bp)	Denaturation	Annealing	Extension	Reference
Aerolysin	CCTATGGCCTG-AGCGAGAAG CCAGTTCCAGT-CCCACCACT	431	94oC, 30 s	55.5oC, 30 s	72oC, 30s	Mansour et al. (2019)
Enterotoxin	TGACCCAGTCC-TGGCACGGC GGTGATCGAT-CACCACCAGC	442	94oC, 30 s	63oC, 50 s	72oC, 30s	Nawaz et al. (2010)
Haemolysin	GGCCGGTGGC-CCGAAGATACGGG GGCGGCGCCG-GACGAGACGGGG	597	94oC, 30 s	62oC, 30 s	72oC, 2 min	Sreedharan et al. (2012)
Lipase	GACTCCCTCA-AGGACAGCAG AGAGGCTTTCA-GGGCATTG	594	94oC, 45 s	58oC, 30 s	72oC, 30s	U-taynapun et al. (2020)
Elastase	ACACGGTCAA-GGAGATCAAC CGCTGGTGTTG-GCCAGCAGG	540	94oC, 30 s	60.6oC, 30s	72oC, 30s	Mansour et al. (2019)
Serine protease	ATTGGATCCCT-GCCTATCGCTTCAGTTCA GCTAAGCTTGC-ATCCGTGCCGTATTCC	911	94oC, 30 s	55oC, 30s	72oC, 30s	Zheng et al. (2012)

 

For bacterial cell suspension preparation, we used the protocol described by Sasmal et al. (2014). Initially, a pure culture of Aeromonas spp. was obtained from tyrpticase soya agar (TSA) slants, then streaked onto the TSA plate. After 24 h of culture at 32 ± 1°C the young colonies were transferred into 10 ml of trypticase soya broth (TSB). The bacterial cell suspensions were then inoculated into a 250 ml conical flask containing 90 ml of TSB for mass culture and incubated on a shaker at 32°±1°C for 24 h. The cultures were centrifuged at 7500 rpm for 20 min. After three washes with saline, the resulting cell pellet was resuspended in 10 ml of the solution. We used the spread plate method (Collins et al., 1989) to determine the number of bacterial cells per ml of neat suspension on TSA after 24 h of incubation at 32 oC.

For challenge test, healthy fish were divided into three categories viz., first category for Aeromonas spp. injection, second category for sterile normal saline injection as sham control and third category as control with no injection and maintained as triplicates. Prior to injection, fish were anaesthetized by tricaine methanesulfonate (MS222, Sigma, 150 mg/L) (Das et al., 2019). Each catla fish in the first group was injected I/P with 0.2 ml of a bacterial cell solution of Aeromonas spp. containing 108 cfu/ml. A 0.2 ml I/P injection of sterile normal saline was administered to the second group of fish. All of the fishes in the experimental category were monitored for a full 96 h after the challenge to analyze any changes in behavior, any clinical symptoms, and any deaths that happened. The mortality rate (total number of dead fishes in a time period/ total number of fishes stocked x 100) was calculated based on Sung et al. (2000). To confirm Koch’s postulates, the moribund/freshly dead fishes with obvious clinical indications were randomly chosen for re-isolation of challenged Aeromonas spp.

Statistical analysis

R studio software (3.6.1 version) was used for analysing pathogenicity studies difference by multivariant ANOVA at 5% level of significance.

RESULTS and DISCUSSION

The motile Aeromonads are the opportunistic Gram negative bacteria responsible to cause various diseases in fish like septicaemia, fin rot, haemorrhages, and dropsy, commonly called motile aeromonas septicaemia (Lewbart, 2001) and in humans, they cause gastrointestinal diseases, peritonitis, meningitis, and eye infections (Reith et al., 2008). The infected samples of the present study also recorded various motile aeromonas septicaemia disease symptoms such as pinpoint haemorrhages on the kidney, on body and fin bases, fluid discharge from a reddened vent, excess fluid in the gut and visceral cavity, pale gills, fin and tail rot, and discoloration of internal organs like the kidney, liver, and spleen.

 

Table II. Prevalence and distribution of Aeromonas spp. between two sampling locations.

S. No	Aeromonas species	Prevalence (N=56)	Location wise prevalence
			W. Godavari (N=37)	SPSR Nellore (N=19)
1	A. veroniibv.veronii	28% (n=16)	n = 9	n =7
2	A. veroniibvsobria	17% (n=10)	n = 6	n = 4
3	A. trota	10% (n=6)	n = 3	n = 3
4	A. tecta	10% (n=6)	n = 4	n = 2
5	A. schubertii	7% (n=4)	n = 2	n = 2
6	A. popoffii	7% (n=4)	n = 3	n = 1
7	A. media	7% (n=4)	n = 4	0
8	A. aquariorum	7% (n=4)	n = 4	0
9	A. allosacharophila	3% (n=2)	n = 2	0

 

Characterization of Aeromonas spp.

The biochemical characterization of isolated Aeromonas spp. are given in Supplementary Table I. Based on the severity of disease condition, inoculums were taken from kidney, liver and gills. A total of 56 of nine Aeromonas species were identified from of 32 diseased catla. Table II shows the prevalence and distribution of Aeromonas spp. among the two districts. Many researchers isolated Aeromonas species from fishes, including A. hydrophila, A. veronii, A. sobria, A. caviae, A. salmonicida, A. allosaccharophila, A. media and A. jandaei (Nawaz et al., 2010; Hu et al., 2012; Sanayal et al., 2018). Among them, A. veronii is a significant cause of infections in fish (Silver et al., 2011; Nawaz et al., 2010; Li et al., 2020). Our study reports also suggested that A. veronii bv. veronii and A. veronii bv. sobria are the major pathogenic bacteria causing disease in freshwater fishes. In the present study, we found that A. veronii bv. veronii (28%, 16/56) was more prevalent spp. when compared to others species. This is completely in accordance with several studies (Hu et al., 2012; Li et al., 2020; U-taynapun et al., 2020; Sayuti et al., 2021). Moreover, we have seen variation in species distribution among the two sampling locations. From W. Godavari district, 37 isolates of nine Aeromonas spp. recovered, while 19 isolates of six Aeromonas spp. found in SPSR Nellore district. Which might be influenced by type of water intake, because in W. Godavari district

 

Table III. Mortality rate (Mean±SD) of catla fish challenged with Aeromonas species.

Aeromonas species	No. of virulence genes	Virulence gene profile	Bacterial injection (cfu/ml) (10-1 dilution)	Mortality (mean number of fish died)					Mortality (%)
				0-12 h	12- 24 h	24 - 48 h	48 - 72 h	72 - 96 h
A. schubertii	3	Aer, Ela, AhyB	2.0 x108	-	2.00a±0.57	3.00a±0.57	2.33 b±0.57	2.67b± 1.00	100.0a± 0.00
A. tecta	4	Aer, Ela, AhyB, Lip	2.1 x108	-	2.33a±0.57	2.67a±0.57	2.33b±0.57	2.67b ±0.57	100.0a± 0.00
A. aquariorum	4	Aer, Ela, AhyB, Lip	2.3 x108	-	3.00a±0.57	3.33b±0.57	2.67b±0.57	1.00a ±1.15	100.0a± 0.00
A. allosaccharophila	5	Aer, Ela, AhyB, Lip, Alt	1.4 x108	-	2.67a±0.57	3.67b±0.57	2.67b±0.57	1.00a ±0.00	100.0a± 0.00
A. media	5	Aer, Ela, AhyB, Lip, Hly	1.2 x108	-	2.33a±0.57	3.33b±0.57	2.67b±0.00	1.67a ±0.57	100.0a± 0.00
A. trota	6	Aer, Ela, AhyB, Lip, Alt, Hly	1.4 x108	-	5.00b±0.57	2.67a±0.57	1.33a±0.57	1.00a ±1.00	100.0a± 0.00
A. popoffii	6	Aer, Ela, AhyB, Lip, Alt, Hly	2.1 x108	-	5.33b±0.57	3.67b±0.57	1.00a±1.00	-	100.0a± 0.00
A. veronii bv. Sobria	6	Aer, Ela, AhyB, Lip, Alt, Hly	1.2 x108	-	5.33b±0.57	3.33b±0.57	1.33a±0.57	-	100.0a± 0.00
A. veronii bv. veronii	6	Aer, Ela, AhyB, Lip, Alt, Hly	2.4 x108	-	5.67b±1.00	3.33b±0.57	1.00a±1.00	-	100.0a± 0.00
Sham control group	-	-	-	-	-	-	-	-	00.00
Control group	-	-	-	-	-	-	-	-	00.00

 

Aer, Aerolysin; Ela, Elastase; AhyB, Serine protease; Lip, Lipase; Alt, Enterotoxin; Hly, Hemolysin.

*Figures having different super scripts are significantly different

 

culture is mainly using creek water as intake where as ground water is a major intake source for Nellore district. Our findings are supported by Altwegg et al. (1989) who suggested that the prevalence of Aeromonas is likely to vary with geographical locations.

Determination of virulence genes in Aeromonas spp.

There is a heterogeneous distribution of virulence genes pattern was observed among the nine Aeromonas species. It believed that virulence factors contributing to the severity of many diseases through the release of a wide range of toxins Sen and Rodgers (2004). However, all the Aeromonas species cannot produce all the toxins (Chopra and Houston, 1999). The Aeromonas pathogenesis process was rather complicated, since no one suspected virulence-associated factor could be definitively linked to any given set of symptoms or disease (Albert et al., 2000). Major virulence factors in Aeromonas were aerolysin, cytotonic enterotoxin and serine protease (Chopra et al., 1996; Sha et al., 2002; Nawaz et al., 2010; Tomas, 2012). The present study findings showed that, 87% of the isolates possessed at three or more virulence gene. Of the six virulence genes, elastase gene was found to be dominant with prevalence of 87% (49/56) followed by serine protease 73% (41/56), aerolysin 62% (35/56), cytotonic enterotoxin 48% (27/56), lipase 46% (26/56) and haemolysin 39% (22/56).

Similar to our findings, Shuang et al. (2020) also found elastase in 100% of Aeromonas. Elastase is a zink metalloprotease enzyme that involved in pathogenesis (Tomas, 2012). Further, Serine proteases participate in β-hemolysin precursor activation, which includes the stimulation of aerolysin and other cellular enzymes, which may have an impact on the total virulence of Aeromonas (Nawaz et al., 2010). In the present study, we found serine protease in 73% of the isolates and aerolysin in 62% of the isolates. The aerolysin gene participates in the secretion of adhesins, hemagglutinins, and several hydrolytic enzymes, all of which are important in pathogenesis (Sreedharan et al., 2012). More or less similar frequencies of virulence genes are found by (El-Gohary et al., 2020; Li et al., 2020; Nawaz et al., 2010). The present study isolates had enterotoxin in 48%, which is very similar to (Gashgari and Selim, 2015) who fond 42% in A. veronii isolated from sea bream. These enterotoxins are implicated in tissue destruction and the release of fluid in infected fish intestines (Sha et al., 2002). Further, lipase gene recorded in 46% of present study isolates, similarly (U-taynapun et al., 2020) found 46% of lipase in Aeromonas spp. Lipase enzymes involving in the modification of the animal cell membrane, increasing the seriousness of the disease (Tomas, 2012). In our isolates haemolysin gene was found in 39% of the strains, which are involving in lysis of the erythrocytes (Wang et al., 2008).

In-vivo pathogenicity of Aeromonas spp. to Catla catla

The mortality pattern of catla challenged with nine Aeromonas spp. is shown in Table III. In-vivo pathogenicity studies showed that the bacteria were able to induce severe infections in catla fishes. We found 100% mortality in challenged fishes with Aeromonas spp. possessed three (elastase, serine protease and aerolysin) to six virulence genes (elastage, serine protease, aerolysin, enterotoxin, lipase and haemolysin). Indicating that, even presence of only three virulence genes such as elastase, serine protease and aerolysin could cause 100% mortality. The In-vivo pathogenicity results were supported by the prevalence of virulence genes in isolated Aeromonas spp. of the present study. The results completely agreed with those of Nawaz et al. (2010); Hu et al. (2012) and Li et al. (2011, 2020), who suggested that aerolysin, enterotoxin elastase, and protease in Aeromonas pose a high threat to the animals. Moreover, we have observed prominent pathological signs after 24 h of post injection, such as hemorrhages on various body parts, tail rot and internal fluid accumulation, which might be due to presence of aerolysin, enetrotoxins and proteases. In addition, mortality was not found in sham control and control groups. Our findings suggested that a bacterial strain’s pathogenicity was actually associated with the type of virulence genes possessed rather than its number.

CONCLUSION

Catla fish farms often have problems with Aeromonas spp., especially by A. veronii bv veronii and A. veronii bv sobria. In addition, the present study isolates found with elastase, enterotoxin, protease and aerolysin genes, indicating the pathogenic potential of our isolates. The in-vivo pathogenicity test also confirmed the virulence of Aeromonas spp. to catla. It was suggesting that pathogenicity was related to the type of virulence genes possessed by Aeromonas spp. but not on number of genes. Hence, our findings suggesting that, Aeromonas spp. pose more serious threat to the freshwater fishes.

Declarations

Funding

The study was funded by College of Fishery Science, Muthukur.

Ethical approval

The catla fishes were used in this study handled with very care as per Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) and study was approved by Sri Venkateswara Veterinary University committee, 2019.

Statement of conflict of interest

The authors have declared no conflict of interest.

References

Abbott, S.L., Cheung, W.K. and Janda, J.M., 2003. The genus Aeromonas: Biochemical characteristics, atypical reactions, and phenotypic identification schemes. J. clin. Microbiol., 41: 2348–2357. https://doi.org/10.1128/JCM.41.6.2348-2357.2003

Albert, M.J., Ansaruzzaman, M., Talukder, K.A., Chopra, A.K., Kuhn, I., Rahman, M., Faruque, A.S., Islam, M.S., Sack, R.B. and Mollby, R., 2000. Prevalence of enteroxin genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, and the environment. J. clin. Microbiol., 38: 3785-3790. https://doi.org/10.1128/JCM.38.10.3785-3790.2000

Altwegg, M., Geiss, H.K. and Freij, B.J., 1989. Aeromonas as a human pathogen. CRC Crit. Rev. Microbiol., 16: 253-286. https://doi.org/10.3109/10408418909105478

Barkoh, A., Smith, G.D. and Southard, M., 2010. Prymnesium parvum control treatments for fish hatcheries. J. Am. Water Resour. Assoc., 46: 161-169. https://doi.org/10.1111/j.1752-1688.2009.00400.x

Beaz-Hidalgo, R., Alperi, A., Bujan, N., Romalde, J.L. and Figueras, M.J., 2010. Comparison of phenotypical and genetic identification of Aeromonas strains isolated from diseased fish. Syst. appl. Microbiol., 33: 149-153. https://doi.org/10.1016/j.syapm.2010.02.002

Chopra, A.K. and Houston, W.C., 1999. Enterotoxins in Aeromonas associated gastroenteritis. Microb. Infect., 1: 1129–1137. https://doi.org/10.1016/S1286-4579(99)00202-6

Chopra, A.K., Peterson, J.W., Xu, X.J., Coppenhaver, D.H. and Houston, C.W., 1996. Molecular and biochemical characterization of heat-stable cytotonic enterotoxin from Aeromonas hydrophila. Microb. Pathogen., 21: 357-377. https://doi.org/10.1006/mpat.1996.0068

Collins, C.H., Lyne, P.M. and Grange, G.M., 1989. Collins and lyne microbiological methods, 6th Ed. Butterworth, London.

Das, S., Aswani, R., Jasim, B., Sebastian, K.S., Radhakrishnan, E.K. and Mathew, J., 2019. Distribution of multi-virulence factors among Aeromonas spp. isolated from diseased Xiphophorus hellerii. Aquacult. Int., 28: 235–248. https://doi.org/10.1007/s10499-019-00456-5

El-Gohary, F.A., Zahran, E., El-Gawad, A., Eman, A. and El-Gohary, A.H., 2020. Investigation of the prevalence, virulence genes, and antibiogram of motile aeromonads isolated from Nile Tilapia fish farms in Egypt and assessment of their water quality. Animals, 1432: 10. https://doi.org/10.3390/ani10081432

Fernandez-Bravo and Figueras, 2020. An update on the genus Aeromonas: Taxonomy, epidemiology, and pathogenicity. Microbiology, 8: 1-39. https://doi.org/10.3390/microorganisms8010129

Gashgari, R.M. and Selim, S.A., 2015. Detection and characterization of antimicrobial resistance and putative virulence genes in Aeromonas veronii Biovar Sobria isolated from Gilthead sea bream (Sparus aurata L.). Fd. Borne Pathog. Dis., 12: 620, 806-811. https://doi.org/10.1089/fpd.2015.1976

Ghenghesh, K., Ahmed, S.F., Cappuccinelli, P. and Klena, D.J., 2014. Genospecies and virulence factors of Aeromonas species in different sources in a North African country. Libyan J. Med., 9: 25497. https://doi.org/10.3402/ljm.v9.25497

Hu, M., Wang, N., Pan, Z.H., Lu, C.P. and Liu, Y.J., 2012. Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China. Lett. appl. Microbiol., 55: 224-233. https://doi.org/10.1111/j.1472-765X.2012.03281.x

Igbinosa, I.H., Igumbor, E.U., Aghdasi, F., Tom, M. and Okoh, A.I., 2012. Emerging Aeromonas species infections and their significance in public health. Sci. World J., pp. 1-13. https://doi.org/10.1100/2012/625023

Kelly, K.A., Koehler, J.M. and Ashdown, L.R., 1993. Spectrum of extraintestinal disease due to Aeromonas species in tropical Queensland, Australia. Clin. Infect. Dis., 16: 574–579. https://doi.org/10.1093/clind/16.4.574

Lewbart, G.A., 2001. Bacteria and ornamental fish. Semin. Avian Exotic Pet Med., 10: 48-56. https://doi.org/10.1053/saep.2001.19543

Li, J., Ni, X.D., Liu Y.J. and Lu, C.P., 2011. Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophilaand their relationship with actual virulence to zebraish. J. appl. Microbiol., 110: 823-830. https://doi.org/10.1111/j.1365-2672.2011.04944.x

Li, T., Raza, S.Z., Bintong, Y., Yufeng, S., Wang, J., Sun, W., Qian, A., Wang, C., Yuanhuan, K. and Shan, K., 2020. Aeromonas veronii infection in commercial freshwater fish: A potential threat to public health. Animals, 10: 608. https://doi.org/10.3390/ani10040608

Mansour, A., Mahfouz, N.B., Husien, M. and El-Magd, M.A., 2019. Molecular identification of Aeromonas hydrophila strains recovered from Kafrel Sheikh fish farms. Slov. Vet. Res., 56: 201-208. https://doi.org/10.26873/SVR-758-2019

Martinez-Murcia, A.J., Saavedra, M.J., Mota, V.R., Maier, T., Stackebrandt, E. and Cousin, S., 2008. Aeromonas aquariorum sp. nov., isolated from aquaria of ornamental fish. Int. J. Syst. Evol. Microbiol., 58: 1169–1175. https://doi.org/10.1099/ijs.0.65352-0

Nawaz, M., Khan, S.A., Khan, A.A., Sung, K., Tran, Q., Kerdahi, K. and Steele, R., 2010. Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Fd. Microbiol., 27: 327-331. https://doi.org/10.1016/j.fm.2009.11.007

Reith, M.E., Singh, R.K., Curtis, B., Boyd, J.M., Bouevitch, A., Kimball, J., Munholl, J., Murphy C., Sarty, D., Williams, J., Nash, J.H., Johnson, S.C. and Brown L.L., 2008. The genome of Aeromonas salmonicida subsp. salmonicida, insights into the evolution of a fish pathogen. BMC Genom., 9: 427. https://doi.org/10.1186/1471-2164-9-427

Sanyal, K.B., Mukherjee, D., Guchhait, A. and Dash, G., 2018. Phenotypic and molecular identification of bacterial species in Indian major carps and exotic carps from south 24 Parganas, West Bengal, India. Int. J. Curr. Microbiol. appl. Sci., 7: 534-547.

Sasmal, D., Surendhra, B.C. and Abaham, T.J., 2014. Effect of garlic (Alliumsativum) extract on the growth and disease resistance of Carassius auratus (Linnaeus, 1758). Indian J. Fish, 52: 207-214.

Sayuti, M.A., Salwany, M.D., Saad, M., Yusof, Annas, S., Yunus, N., Liles, M., Monir, S., Zaidi, Z. and Amal, Z., 2021. The prevalence, putative virulence genes and antibiotic resistance profiles of Aeromonas spp. isolated from cultured freshwater fishes in peninsular Malaysia. Aquaculture, 540: 736719-736728. https://doi.org/10.1016/j.aquaculture.2021.736719

Sen, K. and Rodgers, M., 2004. Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: A PCR identification. J. appl. Microbiol., 97: 1077-1086. https://doi.org/10.1111/j.1365-2672.2004.02398.x

Sha, J., Kozlova, E.V. and Chopra, A.K., 2002. Role of various enterotoxins in Aeromonas hydrophila-induced gastroenteritis: Generation of enterotoxin gene-deicient mutants and evaluation of theirenterotoxic activity. Infect. Immununol., 60: 1924-1935. https://doi.org/10.1128/IAI.70.4.1924-1935.2002

Shahzad, A., Iahtasham, K., Qurban, A., Safwan, U., Zahid, M. and Shamim, A., 2014. Isolation and characterization of Aeromonas sobria in catla catla affected with hemorrhagic septicemia. Bull. Eur. Assoc. Fish Pathol., 34: 35-44.

Shuang, Y., Yong, Lu, W., Chen, C., Jing, Y., Min, Y., Ning, N., Dong, D., Rong, L., Gang, C. and Juan, L., 2020. Genetic diversity, antimicrobial resistance, and virulence genes of Aeromonas isolates from clinical patients. Tap Water Syst. Fd. Biomed. Environ. Sci., 33: 385-395.

Silver, A.C., Williams, D., Faucher, J., Horneman, A.J. and Gogarten, J.P., 2011. Complex evolutionary history of the Aeromonas veronii group revealed by host interaction and DNA sequence data. PLoS One, 6: 16751.

Sinha, S., Shimada, T., Ramamurthy, T., Bhattacharya, S.K., Yamasaki, S., Takeda, Y. and Nair, G.B., 2004. Prevalence, serotype distribution, antibiotic susceptibility and genetic profiles of mesophilic Aeromonas species isolated from hospitalized diarrhoeal cases in Kolkata, India. J. med. Microbiol., 53: 527-534. https://doi.org/10.1099/jmm.0.05269-0

Sreedharan, K.R., Philip, S. and Singh, I.S.B., 2012. virulence potential and antibiotic susceptibility pattern of motile aeromonads associated with freshwater ornamental fish culture systems: A possible threat to public health. Braz. J. Microbiol., 43: 754-765. https://doi.org/10.1590/S1517-83822012000200040

Sung, H.H., Hwang, S. and Tasi, F., 2000. Responses of giant freshwater prawn (Macrobrachium rosenbergii) to challenge by two strains of Aeromonas spp. J. Invert. Pathol., 76: 278–284. https://doi.org/10.1006/jipa.2000.4981

Tomas, J.M., 2012. The main Aeromonas pathogenic factors. Int. Sch. Res. Netw. Microbiol., pp. 1-22. https://doi.org/10.5402/2012/256261

U-taynapun, K., Nganwisuthiphan, T. and Chirapongsatonkul, N., 2020. Species diversity and existence of virulence genes in clinical Aeromonas spp. causing motile Aeromonas septicemia (MAS) isolated from cultured Nile tilapia (Oreochromis niloticus). Int. J. Agric. Technol., 16: 749-760.

Wang, C.L., Cao, X., Liu, X.D., Guo, L.P., Hu, T., Ni, X.D., Liu, Y.J. and Lu, C.P., 2008. Detection of biochemical characters and extracellular proteases in the different isolates of Aeromonas hydrophila. Anim. Husband. Vet. Med., 40: 16–19.

Zheng, W., Cao, H. and Yang, X., 2012. Grass carp (Ctenopharyngodonidellus) infected with multiple strains of Aeromonas hydrophila. Afr. J. Microbiol. Res., 6: 4512-4520. https://doi.org/10.5897/AJMR11.1405