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Research Article
 

Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia



M.M. Rahman, T. Somsiri , K. Tajima and Y. Ezura
 
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ABSTRACT

Distribution pattern of a collection of 106 Aeromonas strains isolated from both healthy and epizootic ulcerative syndrome (EUS)-affected fish, septicaemic disease affected frog and turtle in Bangladesh, Japan, Malaysia, Philippines and Thailand was investigated. The study was conducted through the physio-biochemical characterization of the strains and subsequent confirmation by analysis of the 16S rDNA sequences of some randomly chosen representative strains from all identified phenotype. Special emphasis was given to confirm a group of strains, which belonged to a newly identified species Aeromonas sp. T8 by DNA-DNA hybridization method. The newly identified species Aeromonas sp. T8 group was particularly found in different species of EUS-affected fish in Philippines and Thailand. A. hydrophila subsp. hydrophila and A. hydrophila subsp. ranae was recovered from EUS of fish and septicaemic diseases of frog and turtle. A. hydrophila subsp. hydrophila was distributed in Bangladesh and Thailand while A. hydrophila subsp. ranae was found only in Thailand. A. veronii biotype sobria and A. veronii biotype veronii was found to be dispersed mostly in EUS-affected fish in different countries. A. jandaei was obtained from EUS-positive fish in Bangladesh and Malaysia but A. media from healthy fish in Bangladesh.

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M.M. Rahman, T. Somsiri , K. Tajima and Y. Ezura , 2004. Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia. Pakistan Journal of Biological Sciences, 7: 258-268.

DOI: 10.3923/pjbs.2004.258.268

URL: https://scialert.net/abstract/?doi=pjbs.2004.258.268

INTRODUCTION

Bacteria of the genus Aeromonas are wide spread in fresh, brackish, estuarine and marine water[1]. They are frequently isolated from both healthy and diseased fish as well as from other aquatic animals. They are also considered to be one of the most important bacteria among the etiological agents of fish diseases[2]. Particularly, motile aeromonads are associated with tail and fin rot, hemorrhagic septicaemia and epizootic ulcerative syndrome (EUS) in a variety of freshwater and marine fish of the world[3,4]. Moreover, they are repeatedly reported to cause diseases in amphibians and reptiles[5,6]. The outbreaks of motile Aeromonas associated diseases can reach epidemic proportions among the aquatic animals, leading to massive mortality rates[7]. Therefore, knowledge on the distribution of Aeromonas species in diseased fish and other aquatic animals is necessary for establishing the epidemiological pattern involved in the diseases of aquatic animals.

The taxonomy of the genus Aeromonas is changing continuously due to identification of newly described species. At present, the genus Aeromonas comprises 14 well-recognized genospecies[8]. Recently, Iqbal[9] identified an atypical motile Aeromonas strain (T8) isolated from EUS-affected fish of Thailand. It possessed distinct phenotypic characteristics and represented a cluster with Aeromonas caviae in the phylogenetic tree constructed on the basis of 16S rDNA sequences. However, it exhibited discrete DNA-DNA hybridization homologies with all recognized Aeromonas genospecies[9]. Thus, it was considered as a new species and designated as Aeromonas sp. T8. Isolation, identification and distribution of the bacteria in the Southeast Asian region is essentially needed.

The objective of this study was to determine the distribution pattern of a collection of 106 aeromonad strains, isolated from both healthy and EUS-affected fish, septicaemic disease affected frog and turtle in Southeast Asian countries by means of physio-biochemical and molecular identification methods.

MATERIALS AND METHODS

Bacterial strains: A total of 106 Aeromonas strains collected from fish, frog and turtle of Bangladesh, Japan, Malaysia, Philippines and Thailand were investigated in this study (Table 1). Most of them were collected from EUS-positive fish and only five (B33, J1, J2, J3 and J4) from healthy fish specimen. In addition, several strains were collected from frog (Rana tigrina and Rana rugulosa) and turtle (Chrysemys scripta and Trionyx sinensis) suffering from septicaemic disease.

In this study, the Aeromonas sp. strain T8 (IAM 14920, JCM 11177) and 14 reference strains representing different Aeromonas DNA hybridization groups[10] were also included. The reference strains were as follows: A. hydrophila ATCC7966T, A. bestiarum CDC5933-76, A. salmonicida subsp. salmonicida ATCC14174,A. salmonicida subsp. masoucida ATCC27013T, A. caviae ATCC15468T, A. media JCM2385T, A. eucrenophila NCMB74T, A. sobria JCM2139T, A. veronii biotype sobria ATCC9071T, A. jandaei JCM8316T, A. veronii biotype veronii JCM7375T, DNA hybridization group 11 CDC1306-83, A. schubertii JCM7373T and A. torta JCM8315T.

Culture condition: All bacterial strains were periodically cultured in nutrient agar (NA; 1% Poly peptone, 0.5% Beef extract, 0.12% Sodium chloride and 1.5% Agar powder; pH 7.2). The stock cultures were maintained at –80°C in nutrient broth medium supplemented with 10% glycerol (V/V).


Table 1:
Origin of Aeromonas spp. strains used in this study
Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia
*: Isolated from healthy fish
-: Unknown

Phenotypic characterization: Each of the Aeromonas strains was tested for 44 physio-biochemical properties. Among these urea hydrolysis, nitrate reduction, gas production from glucose, utilization of acetate, malonate, Christensen’s citrate, Jordan’s tartrate and phenylalanine, decarboxylations of lysine and ornithine and arginine dihydrolase tests were conducted according to the “Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria”[11]. Gelatin hydrolysis, esculin hydrolysis, methyl red (MR), Voges-Proskauer (VP) tests were performed according to the “Cowan and Steel’s Manual for the Identification of Medical Bacteria”[12]. Lipase (tributirin) activity was tested following the method described in the “Bacterial Culture Media”[13]. Acid production from various carbohydrates was examined as described by Iqbal[9]. All of the tests were performed at 25°C and the results were observed after 24 h unless otherwise indicated. Strains that differed by only 1 to 5 characteristics from the reference strains was placed into the same species according to the criteria described by Iqbal[9].

Sequence of 16S rDNA: All strains that possessed similar or very close phenotypic properties with the Aeromonas sp. strain T8 (IAM 14920; JCM 11177) and 1-2 randomly chosen bacterial strains representing each phenotye were further investigated for the 16S rDNA sequence similarity and subsequent phylogenetic analysis. Bacterial cells were grown in nutrient broth and genomic DNA was extracted by using Wizard Genomic DNA Purification Kit (Promega, USA) following the manufacturer’s instructions.

PCR for amplification of the targeted gene was performed with the universal primer set 24 F (forward primer) and 1540 R (reverse primer). Each PCR mixture contained 6 μl of 25 mM MgCl2, 10 μl of 10xPCR buffer, 2.0 μl of each 10 mM deoxyribonucleotide tri-phosphate, 5.0 μl of 20 μM solution of each primers, 100-200 ng of DNA template, 0.5 μl of Taq DNA polymerase (Promega) and sterile double-distilled water in a total volume of 100 μl. The PCR amplification was performed with a Gene Amp 9700 PCR system (PE Applied Bio systems). The thermal profile for PCR amplification was followed as described by Iqbal[9]. The PCR amplicons were visualized on 1.5% agarose gel stained with ethidium bromide solution. The amplified PCR products were purified by using Wizard PCR Purification System (Promega, USA) following the manufacturer’s instructions. The purified PCR products were then subjected to cycle sequencing. The primer set used for cycle sequence reaction was the same as mentioned by Iqbal[9]. The sequence reaction mixture contained 2 μl of Big Dye V 3.0 matrix standard (Applied Biosystems), 3 μl of 5 x sequencing buffer, 1 μl of primer (1.6 pmole), 100-200 ng of purified PCR product and sterile double-distilled water in a total volume of 20 μl. The cycle sequence was performed with a Gene Amp 9700 PCR system (PE Applied Biosystems) following the program described by Iqbal[9]. The extended products were precipitated and vacuum dried. The dried pellets were resuspended in 20 μl of template suppression reagent (Applied Biosystems), heated at 95°C for 2 min and immediately placed on ice. The solution was transferred in capillary tube and sequencing was done with an automated capillary type sequencer (Applied Biosystems).

16S rDNA sequence similarity: The 16S rDNA sequences of the representative strains were compared with the sequences of other experimental strains as well as with the sequences of reference strains found from the gene bank sequences of NCBI. The 16S rDNA sequence homology was determined following the method described by Iqbal[9].

Phylogenetic analysis: The 16S rDNA sequence data were aligned and phylogenetic analyses were performed by using neighbour-joining[14] method of CLUSTAL X 1.8 software program. The sequences determined in this study and the sequences obtained from NCBI database (Table 2) were used for the analysis. The phylogenetic tree was constructed by using the tree-view program for the windows.

DNA-DNA hybridization: Aeromonad strains that formed a cluster with Aeromonas sp. strain T8 were further investigated for DNA-DNA hybridization homology. Bacterial cultures were grown to mid log-phase at 28°C in 500 ml nutrient broth in a shaking incubator. DNAs were extracted according to the procedure of Altwegg et al.[15]. Purified DNA from Aeromonas sp. strain T8 was labelled with photobiotin (Vector Laboratories, USA) and DNA-DNA microplate hybridization was done at stringent condition following the method described by Iqbal[9].

RESULTS

Phenotypic identification: All bacterial strains were Gram-negative, rod shaped motile with polar flagella, positive for catalase, cytochrome oxidase, D-glucose fermentation and were resistant against vibriostatic agent 0/129 (Table 3). All of the strains were able to grow without the presence of NaCl and showed positive reaction for gelatin hydrolysis and nitrate reduction tests.


Table 2:
Gene Bank accession number of the 16S rDNA sequences of Aeromonas species used for the sequence similarity and phylogenetic analysis
Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia
ATCC: American Type Culture Collection, Rockville, MD, USA
CDC: Centers for Disease Control, Atlanta, GA, USA
CETC: Coleccion Espanola de Cultivos Tipo, Valencia, Spain
CIP: Collection bacterienne de l’Institut Pasteur, Paris France
DSMZ: Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH, Germany
LMG: Culture Collection of the Laboratorium voor Microbiologie, Ghent, Belgium
NCIMB: National Collection of Indusrtial and Marine Bacteria, Scotland
T: Type strain

Table 3: Common phenotypic properties of Aeromonas strains
Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia

On the other hand, all strains were negative for urea hydrolysis and malonate utilization tests. All strains produced acid from glucose, D-galactose, glycerol, maltose and threhalose but did not produce acid from adonitol, D-arbitol, dulcitol, D-sorbitol and D-xylose.

Differentiable phenotypic characteristics of the strains are shown in Table 4. These characteristics were compared with the characteristics of Aeromonas sp. strain T8 as well as with other reference strains.

Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia
Fig. 1:
16S rDNA sequence-based phylogenetic relationship of Aeromonas strains. The unrooted phylogenetic tree was drawn using the neihbor-joining method in Clustal X. Scale bar 0.01 accumulated changes per uncleotide

Among the aeromonads, four strains possessed very close phenotypic characteristics with the Aeromonas sp. strain T8. The strains T4 and T27 revealed complete similarity with the phenotypic properties of strain T8. Whereas, strain P2 and B30 exhibited variable results with the strain T8 for 2 and 3 phenotypic characteristics, respectively. The rest of the aeromonad strains were identified as A. hydrophila subsp. hydrophila (37 strains), A. hydrophila subsp. ranae (22 strains), A. media (1 strain), A. veronii biotype sobria (24 strains), A. veronii biotype veronii (10 strains) and A. jandaei (8 strains).

Table 4: Differentiable phenotypic characteristics of Aeromonas strain
Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia
Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia
1: Esculin hydrolysis, 2: Acetate utilization, 3: Gas from glucose, 4: Hydrogen sulphide, 5: VP, 6: MR, 7: Phenyl alanine, 8: Christensen’s citrate, 9: Jordan’s tartrate, 10: Lipase (tributyrin, 11: Arginine dihydrolase, 12: Lysine decarboxylase, 13: Ornithine decarboxylase, 14: Acid from: a Cellobiose, b: Myo-inositol, c: Lactose, d: D-Mannose, e: Raffinose, f: L-Rhamnose, g: Sucrose, h: Salicin, i: Arabinose
* Results from Hyus et al.[38], Nt: Not tested

DNA sequence similarity: The 16S rDNA sequence similarity of the selected strains with other experimental and reference strains have been shown in Table 5. In the present study, the strains phenotypically belonged to any species showed highest sequence similarity with their respective type or reference strains. Among them, strains T4, T27 and P2 exhibited 100% sequence similarity while strain B30 exhibited 99.8% similarity with Aeromonas sp. strain T8 (Table 5).

Phylogenetic analysis: The aeromonad strains that were phenotypically identified as Aeromonas sp. T8 group, A. hydrophila subsp. hydrophila, A. hydrophila subsp. ranae, A. media, A. veronii biotype sobria, A. veronii biotype veronii and A. jandaei formed clusters with their respective type or reference strains in the phylogenetic tree (Fig. 1).

DNA-DNA hybridization: In the DNA-DNA hybridization experiment, the strains T4, T27 and P2 revealed 98, 111 and 70% DNA-DNA hybridization homology, respectively, whereas the strain B30 exhibited 36% hybridization homology with the Aeromonas sp. strain T8.

Table 5:
Level of homology of the 16S rDNA sequences of representative strains with other Aeromonas species
Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia
Homology of one representative strain (except strain B30) from each species is presented in the Table. Homology of the representative strains belonged to a species was completely similar to each other

Table 6:
Distribution pattern of Aeromonas species in Southeast Asian countries
Image for - Distribution of Aeromonas spp. Emphasizing on a Newly Identified Species Aeromonas sp. T8 Isolated from Fish and Aquatic Animals in Southeast Asia
HG1A: A. hydrophila subsp. hydrophila, HG1B: A. hydrophila subsp. ranae, HG5: A. media, HG8: A. veronii biotype sobria, HG9: A. jandaei, HG10: A. veronii biotype veronii, Aero. sp. T8: Aeromonas sp. T8 group, * : Aeromonas sp. (close to Aeromonas sp. T8 group), n: Number of strains

Distribution of Aeromonas spp.: A distribution pattern of the Aeromonas species identified in the present study has been summarized in Table 6. The newly identified species Aeromonas sp. T8 group was found to be distributed in Philippines and Thailand in EUS-affected fish. Among the other species, A. veronii biotype sobria and A. veronii biotype veronii were determined to be extensively distributed particularly in EUS-positive fish in Southeast Asia.

DISCUSSION

EUS is an extremely damaging fish disorder that has swept through the South and Southeast Asian region with varying intensity since 1980[16]. The aetiology of EUS is very complex and for a long time, the specific aetiological agent of the disease remained unclear. Recently, a fungus Aphanomyces invadans is widely considered as the primary causative agent of the disease[17]. However, the fungus alone cannot initiate the disease because it is unable to break the skin barrier of fish[18]. In addition, Lilley and Roberts[19] argued that the fungus could not be considered as the primary cause of EUS unless the infective zoospore stage can be shown to breach the fish’s skin unaided. Thus, a conclusive primary factor responsible for the outbreak of EUS is still unclear. On the other hand, Aeromonas spp. is frequently isolated from EUS-affected fish[20-23]. Some reports suggested that Aeromonas spp. might contribute to the pathogenesis of the disease[24]. Moreover, Aeromonas strains isolated from EUS-affected fish are often found virulent for fish upon artificial challenge experiments[9,25]. Although, the role of Aeromonas spp. in EUS-affected fish is still unclear, it’s pathological importance in EUS should keep in concern.

Iqbal[9] identified an atypical Aeromonas strain, considered to be a new species Aeromonas sp. T8, which was isolated from a EUS-affected giant gourami (Osphronemus goramy) of Thailand. In a recent investigation, the strain was proved to be virulent for several tropical fish species; reproduced hemorrhagic and necrotic lesions and mortality in fish upon intramuscular challenge, but it was non-virulent for mammal (mouse) upon artificial challenge. Additionally, it exhibited higher hemolytic activity against fish blood cells compared to mammalian blood cells. Thus, whether the new species possess any significance in EUS and/or other Aeromonas-associated disease in aquatic animals is a new research concern. In the present study, the distribution pattern of a total of 106 Aeromonas spp. isolated mostly from different disease affected aquatic animals in Southeast Asian countries were determined emphasizing on the distribution of a new species Aeromonas sp. T8.

The experimental strains examined in this study, obviously fulfilled the basic morphological and biochemical properties of the genus Aeromonas as mentioned by Popoff[26]. Aeromonad strains can be classified up to species level by biochemical characteristics like esculin hydrolysis, gas production from glucose, lysine and ornithin decarboxylation, fermentation of arabinose, salicine and sucrose etc.[7,27,28]. In this study, the aeromonad strains isolated from healthy and EUS-affected fish, septicaemic disease affected frog and turtle were phenotypically identified as Aeromonas sp. T8 group, A. hydrophila subsp. hydrophila, A. hydrophila subsp. ranae, A. media, A. veronii biotype sobria, A. veronii biotype veronii and A. jandaei.

Identification of bacterial species in most ichthyo-pathological laboratories is still depended on the phenotypic identification method. This method is quite successful, but several researchers stressed on the necessity of using molecular methods, in addition to biochemical markers for more accurate identification[9,29,30]. Presently, a direct comparison of ribosomal RNA gene sequence is considered as a powerful tool for identification of many bacterial groups[31]. It is also a standard method for the investigation of phylogenetic relationship of bacteria[32]. Even though some closely related species might have only a few differences in their 16S rDNA sequences, a phylogenetic tree can be established to give them an exact taxonomic position[33]. Thus, we determined the 16S rDNA sequence similarity and subsequent phylogenetic analysis of all phenotypically identified Aeromonas sp. T8 like strains and representative strains from each phenotypes to confirm the accuracy of the phenotypic identification. During the investigation, it has been noticed that the strains phenotypically identified as Aeromonas sp. T8 formed a cluster with the Aeromonas sp. strain T8 in the phylogenetic tree constructed on the basis of 16S rDNA sequences. Among them, three strains (T4, T27 and P2) possessed 100% sequence similarity while, the strain B30 exhibited 99.8% similarity with the strain T8. The representative strains phenotypically belonged to A. hydrophila subsp. hydrophila, A. hydrophila subsp. ranae, A. veronii and A. jandaei revealed highest sequence similarity with only one nucleotide difference with their corresponding type or reference strains and also formed clusters with the respective type or reference strains in the phylogenetic tree. No nucleotide difference was observed among the strains phenotypically belonged to A. veronii biotype sobria and A. veronii biotype veronii and these strains took place in the cluster with the type strain of A. veronii. Martinez-Murcia et al.[34] also reported similar findings.

However, the strain B33, which phenotypically resembled to A. media, exhibited closest similarity with the sequence of type strain of A. media with 6 nucleotide differences. It formed a cluster with A. media but in a separate line in the phylogenetic tree. Most recently, Yanez et al.[35] reported a similar type of finding. Therefore, determination of the accurate taxonomic position of the mentioned strain will be worthwhile.

Although, 16S rDNA sequence analysis is an important tool for bacterial identification, DNA-DNA hybridization study is considered to be the best-suited method for identification of closely related species or strains of bacteria within a single species. Since, Aeromonas sp. T8 is a new species, special emphasis was given to confirm the Aeromonas sp. T8 related strains. On the basis of DNA-DNA hybridization study, strains T4, T27 and P2 were confirmed to be the member of the new species Aeromonas sp. T8. Whereas, the strain B30, which was phylogenetically near to Aeromonas sp. strain T8, did not show significant DNA-DNA hybridization homology with Aeromonas sp. strain T8. As a result, the strain was assumed to be a different species that might be close to Aeromonas sp. T8. Thus, confirmation of the taxonomic status of this strain will be sensible.

During the investigation, the aeromonad strains isolated from EUS-positive fish were identified as Aeromonas sp. T8 group, A. hydrophila subsp. hydrophila, A. hydrophila subsp. ranae, A. veronii biotype sobria, A. veronii biotype veronii and A. jandaei. Iqbal[9] identified similar type of Aeromonas species, except A. hydrophila subsp. ranae from the EUS-affected fish. Several articles reported on frequent isolation of A. hydrophila and A. sobria from EUS-affected fish since the epizootic incidence of the disease[21,22,36]. At present, A. sobria phenospecies contains A. veronii biotype sobria, A. veronii biotype veronii, A. jandaei and four other genospecies. However, Iqbal[9] first reported on identification of the Aeromonas sp. strain T8 isolated from a EUS-affected fish of Thailand. The most important finding of this study is identification of more strains belonging to the new species from EUS-positive fish of Philippines and Thailand.

The septicaemic disease in frog, also occasionally known as ‘red leg’ disease, referring to hemorrhages in the leg muscles is reported to cause by motile Aeromonas species[5]. Recently, a new phenon A. hydrophila subsp. ranae has been reported as the causative agent of a septicaemic disease in frog of Thailand[37,38]. In the present investigation, the strains isolated from frog were identified as A. hydrophila subsp. hydrophila and A. hydrophila subsp. ranae. Until the present study, A. hydrophila subsp. ranae was reported to be isolated only from frogs of Thailand[37]. In this study, we also identified the bacteria from EUS-affected siamese tiger fish (Datnioides microlepis) and striped catfish (Pangasius sutchi) and from soft shell turtle (Trionyx sinensis). Since, A. hydrophila subsp. ranae is distributed in a wide range of hosts, determination of the pathological importance of the bacteria to fish and other animals will be of grate interest.

Motile Aeromonas spp. is also known to cause disease in turtle[5,6]. During this study, 2 aeromonad strains collected from red-eared turtle (Chrysemys scripta) and soft shell turtle (Trionyx sinensis) were identified as A. hydrophila subsp. hydrophila and A. hydrophila subsp. ranae, respectively.

Through the investigation, A. hydrophila subsp. hydrophila was detected as the dominant group followed by A. hydrophila subsp. ranae and A. veronii biotype sobria. However, A. hydrophila subsp. hydrophila and A. hydrophila subsp. ranae were found to be associated with different types of diseases like EUS in fish and septicaemic disease in frog and turtle. A. hydrophila subsp. hydrophila was distributed in Bangladesh and Thailand while A. hydrophila subsp. ranae was found only in Thailand. On the other hand, A. veronii biotype sobria and A. veronii biotype veronii were determined to be extensively distributed particularly in EUS-positive fish in Southeast Asia. A. jandaei was obtained from EUS-positive fish in Bangladesh and Malaysia but A. media from healthy fish in Bangladesh.

The newly identified species Aeromonas sp. T8 group was detected in EUS-affected fish collected from Philippines and Thailand. The strains of Aeromonas sp. T8 group were isolated from giant gourami (Osphronemus goramy), hybrid catfish (♂ Clarias macrocephalus x ♀ Clarias gariepinus) and walking catfish (Clarias batrachus). Giant gourami and walking catfish are commonly found in natural freshwater bodies in the tropical region especially in the Southeast Asian countries. All of these fish are also cultivated as popular commercial freshwater fish species in this area. Thus, we assume that the newly identified species Aeromonas sp. T8 might be widely distributed in Southeast Asia. To the best of our knowledge, this is the first report about the distribution of Aeromonas hydrophila subsp. ranae found in different diseased aquatic animals and also the distribution of a new species Aeromonas sp. T8 in EUS-affected fish in Southeast Asia.

ACKNOWLEDGEMENTS

The authors would like to express their sincere thanks to Dr. Md. Bazlur Rashid Chowdhury (Professor, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh) and Dr. A.N. Hasna Banu (Senior Scientific Officer, Bangladesh Fisheries Research Institute, Mymensingh, Bangladesh) for providing bacterial strains for the study. The first author also acknowledges the Japanese Ministry of Science, Education, Culture and Sports for his scholarship support.

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