魚 病 研 究 Fish Pathology,38(4),151-161,2003.12 2003 The Japanese Society of Fish Pathology

Virulence Properties of a Newly Identified Species

Aeromonas sp. T8 Group Isolated from

EUS-affected Fish

M.M. Rahman1,T. Somsiri2, K. Tajima1*and Y. Ezura1

1Laboratory of Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan

2Aquatic Animal Health Research Institute , Kasetsart University Campus, Jatujak, Bangkok 10900, Thailand

(Received May 30,2003)

ABSTRACT-The present study was conducted to determine the virulence and virulence proper

ties of 4 bacterial strains belonging to a newly identified species Aeromonas sp. T8 group, isolated from epizootic ulcerative syndrome (EUS)-affected fish of Southeast Asian countries. Virulence of

a representative strain T8 to fish was investigated through intramuscular injection to silver barb Barbodes gonionotus, striped snakehead Channa striatus and tilapia Oreochromis sp., where the

LD50 value was determined to be 1.5×105,3.2×105 and 2.9×105 CFU/fish, respectively.

However, all strains were found non-virulent for mice upon intraperitoneal or intravenous injections

at a concentration of 3×107 CFU/mouse. Live bacterial cells of these strains showed lipase ,

protease and hemolytic activities in agar plate assays. The hemolytic activity of the extracellular

products(ECPs)was found to be higher for fish blood cells than for mammalian blood cells. Heat

stability, EDTA and PMSF sensitivities of β-hemolytic activity of the ECPs differed from that of A.

hydrophila and A. caviae reference strains. ECP of only one strain(P2)showed cytotoxicity

against both fish(RTG-2, FHM, EPC, BF-2 and SNN-1)and human(HeLa)cell lines, while none of

the strains exhibited enterotoxicity to suckling mice. All of the strains also failed to amplify

aerolysin and cytolytic enterotoxin(AHCYTOEN)genes by PCR.

Key words:Aeromonas sp. T8, virulence factor, protease, hemolysin, cytotoxin, enterotoxin, EUS

Bacteria of the genus Aeromonas have been reported as pathogens of poikilothermic animals for more than one hundred years (Carnahan and Altwegg, 1996). Recently, several motile species are also increasingly recognized as primary, opportunistic disease agents in human (Janda, 1991; Janda and Abbott, 1996). Although several Aeromonas species have been found in association with healthy or diseased fish

(Sugita et al., 1994, 1995; Esteve, 1995; Iqbal et al., 1998) and human clinical samples (Huys et al., 1996; Valera and Esteve, 2002), the importance of recently identified species in fish and human pathology is mostly unknown. During the last decade, the taxonomy of the genus

Aeromonas has undergone major changes due to the application of extended DNA-DNA hybridization and

other molecular techniques (Kozinska et al., 2002). At

present, the genus Aeromonas comprises 16 wellrecognized genomic species (Valera and Esteve, 2002). Recently, a new species designated as Aeromonas sp. T8 has been reported (Iqbal, 1999). The first identified strain T8 (IAM 14920T, JCM 11177T) of this species possesses distinct phenotypic properties, which represents a cluster with Aeromonas caviae in the phylogenetic dendogram constructed on the basis of 16S rDNA sequences. However, it comprises discrete DNA-DNA homologies with all recognised Aeromonas genomic species (Iqbal, 1999). Recently, we identified three more strains of the new species on the basis of phenotypic properties, phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization studies

(Unpublished). All of these strains were isolated from epizootic ulcerative syndrome (EUS)-affected fish of Southeast Asian countries. Evaluation of the patho

genic importance of these strains for fish and mammals

*Corresponding author

E-mail:ktajima@fish.hokudai.ac.jp

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

is needed.

In the recent years, various putative virulence fac

tors have been ascribed to explain the process of patho

genicity of Aeromonas spp.(Krovacek et al.,1994).

Such factors include the production of extracellular pro

tease,α-andβ-hemolysins, cytotoxin, enterotoxin, leci

thinase etc. Crude extracellular products(ECPs)of

motile Aeromonas spp. have been suggested to play a

role in virulence process(Allan and Stevenson,1981;

Rodriguez et al.,1992).

The present study was conducted to determine

the pathogenesis of the strain (s) belonging to the

Aeromonas sp. T8 group to fish and mouse, characterize

in vitro enzymatic activities of bacterial cells and ECPs

as putative virulence factors, and screen for the pres

ence of aerolysin and cytolytic enterotoxin genes by

polymerase chain reaction as susceptive virulence mark

ers.

Materials and Methods

Bacterial strains

Four bacterial strains of Aeromonas sp. T8 group

collected from Thailand(T4, T8 and T27)and the Philip

pines(P2), and some reference strains were used

(Table 1). Strains of Aeromonas sp. T8 group were iso

lated from EUS-affected fish and previously identified

through their phenotypic characteristics, phylogenetic

analysis based on 16S rDNA sequences and DNA-DNA

homology studies(Unpublished). In the present study,

reference strains Aeromonas hydrophila ATCC 7966T, A.

caviae ATCC 15468T and A. veroniibiotype sobria ATCC

9071T were included either to confirm the results or to

compare the putative virulence properties with those of

the experimental strains. All bacterial strains were rou

tinely sub-cultured on nutrient agar(NA;pH 7.2)at

25℃. The composition of NA was 1.0% polypepton

(Nihon Seiyaku, Japan),0.5% beef extract(Wako,

Japan),0.12% NaCl and 1.5% agar(Wako,Japan)dis

solved in distilled water. Stock cultures were preserved

in nutrient broth supplemented with 10% glycerin at

-80℃.

V

irulence for fish The virulence assay was conducted with a repre

sentative strain(T8)to fingerlings(average weight 10g)

of three tropical fish species, silver barb Barbodes

gonionotus, striped snakehead Channa striatus and tila

pia Oreochromis sp. The bacterial strain was cultured on

tryptic soy agar(Difco, USA)and then suspended in

sterile physiological saline(0.85% NaCl). Desired dilu

tions were adjusted by the ten-fold dilution method.

The fish were injected intramuscularly just below the

anterior part of the dorsal fin with 100μL of serially

diluted concentrations of bacterial suspensions. Each

suspension was injected into 10 fish for each challenge

group, and the fish was kept separately in 10 L aquaria

at 28℃ for 4 days. No feed was given to fish during the

experiment. The clinical symptoms and mortality of arti

ficially infected fish were recorded, and the median lethal

dose(LD50)was calculated by the method of Reed and

Muench(1938).

Virulence for mouse

Virulence of all of the strains belonging to the

Aeromonas sp. T8 group was assayed in albino mice

(10-12 wk old)by intraperitoneal and intravenous injec

tions following the methods of Janda et al.(1985)and

Mateos et al.(1993). Bacterial strains were grown in

nutrient broth at 37℃ for 18 h with shaking(160

rpm). Cells were harvested by centrifugation at

4,000×g and 4℃, and suspended in sterile saline

(0.85% NaCl). The cell suspensions were adjusted to

approximately 3×108 CFU/mL and confirmed by dupli-

Table 1. Bacterial strains used in this study

Virulence properties of Aeromonas sp. T8 group153

cate plate counts. For each assay, 5 albino mice of

both sexes (average weight 30 g) were injected with 100

,ƒÊL of the bacterial cell suspension and observed for 7

days. A. hydrophila ATCC 7966T was used as a posi

tive control and sterile saline was injected into control

mice. Mice died within the experimental period were

immediately autopsied, and portions of the kidney, liver

and spleen were aseptically collected for isolation of

bacteria on NA. All surviving mice were sacrificed after

the experimental period, internal organs were visually

examined, and attempts were made to re-isolate the

bacteria from kidney, liver, spleen and cardiac

blood. Bacteria grown on the plates were tested by

physiological and biochemical characterization.

Enzymatic and hemolytic activities of live bacterial cells

Exoenzyme activities of bacterial cells were evalu

ated by spot inoculations onto NA plates supplemented

with various proteins, lipid and phospholipid as

substrates. Albumin (from bovine serum), casein, gela

tin and hemoglobin were obtained from Wako, Japan;

elastin (from bovine neck ligament) from Nakarai, Japan;

Tween-20 from Bio-Rad, UK. and egg-yolk coagulates

from Difco, USA. The composition of the supplements

for protease assay was the same as described by Shotts

et al. (1985). To determine lipase and phospholipase

activity, 1% Tween-20 and 5% egg-yolk coagulates were

supplemented into NA, respectively. All assay plates

were incubated at 25•Ž for 7 days. After incubation,

the colony size and diameter of hydrolytic halo around

the inocula were measured, and the ratios were calcu

lated as mentioned by Shotts et al. (1985).

Hemolytic activities of live bacterial cells were deter

mined against erythrocytes of both poikilothermic (com

mon carp and rainbow trout) and homoeothermic (horse,

human, rabbit and sheep) sources by the plate assay

method as described by Esteve et al. (1995). Five per

cent washed blood cells were added into NA. Spot

inoculations of the bacteria were done. Plates contain

ing fish or mammalian blood cells were incubated at

25•Ž or 37•Ž for 48 h, respectively. After incubation,

clear zones surrounding the inocula were considered a

positive result and categorized as ƒ¿ or ƒÀ as mentioned

by Janda (1991). The zone ratios were calculated as

mentioned by Esteve et al. (1995).

Preparation of ECPs

NA plates overlaid with sterile cellophane sheets

were used to collect the concentrated preparations of

ECPs as described by Esteve et al. (1995). Plates

were inoculated with 100 ƒÊL of overnight culture suspen

sion and incubated at 25•Ž for 48 h. After incubation,

ECPs on the cellophane sheet were washed off with 3

mL of phosphate buffered saline (PBS, pH 7.2) and

centrifuged. The supernatant fluids were filter-sterilized

through 0.22 ƒÊm pore-sized filters•@ (Millex-GV, Millipore,

Japan), and the aliquots were either directly used or pre

served at 4•Ž for no more than 2 days. The protein

concentration of ECPs was determined by the method of

Lowry et al. (1951), using bovine serum albumin (Wako,

Japan) as a standard.

Protease activity of ECPs

Protease activity of ECPs was determined as

described by Gudmundsdottir (1996) using azocasein

(Sigma, USA) as a substrate.

The protease activity was characterized on the

basis of the inhibition effect of different treatments on

ECPs. For this purpose, portions of ECPs were heated

at 60 and/or 80•Ž for 15 min. Other portions were

mixed with equal volume of 0.020 mol/L ethylenedi

amine-N,N,N,N-tetraacetic acid (EDTA; Dojindo, Japan)

and/or 0.050 mol/L phenyl methyl sulphonyl fluoride

(PMSF; wako, Japan) for 15 min. Then protease activ

ity was assayed as mentioned above, and the percent

age of inhibition was determined as described by

Gudmundsdottir (1996). ECPs of A. hydrophila ATCC

7966T and A. caviae ATCC 15468T were also incorpo

rated in the experiment.

Hemolytic activity of ECPs

Hemolytic activity of the ECPs (prepared as men

tioned above) against fish and mammalian erythrocytes

was assayed after Gunnlaugsdottir and Gudmundsdottir

(1997) with a little modification. Briefly, 100 ƒÊL of two

fold serial dilutions of ECPs in PBS were added to an

equal volume of 1% (v/v) blood cell suspension in micro

centrifuge tubes and incubated at 25•Ž. Fish blood

cells were incubated for 12 h, whereas mammalian

blood cells were incubated for 24 h. PBS was used

instead of ECPs as a control. The hemolytic activity

was estimated by eye observation as well as by micro

scopic observation of the titration mixture. Hemolytic

titre was expressed as the reciprocal of the highest dilu

tion that gave 50% hemolysis. Hemolytic activity of the

ECPs of A. hydrophila ATCC 7966T was also assayed at

the same time.

In order to characterize the hemolytic activity, differ

ent portions of ECPs were treated (heating at 60 and

80•Ž, and addition of EDTA and PMSF) as mentioned in

protease characterization. After the treatments,

hemolytic activity was estimated against rabbit blood

cells as mentioned above. ECPs of A. hydrophila

ATCC 7966T and A. caviae ATCC 15468T were also

included in the characterization experiment.

Cytotoxicity of ECPs

Both poikilothermic and homoeothermic cell lines

were used to evaluate the cytotoxic effect of the ECPs of

the Aeromonas sp. T8 group. The fish cell lines

included RTG-2, EPC, BF-2, FHM and SNN-1. HeLa

was used as mammalian cell line. The cytotoxicity

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

assay was carried out with a slight modification of the

method described by Gunnlaugsdottir and Gudmundsdottir

(1997). Briefly, confluent monolayers of the cells were

grown in flat-bottomed 96-well tissue culture plates in

minimal essential medium(MEM)supplemented with

10% fetal bovine serum and 1% penicillin-streptomycin

except for the SNN-1 cell line. SNN-1 cell line was

grown in L-15 medium. The reaction system was pre

pared by removing 100 mL of medium from each well

and adding serial two fold dilutions of 100μL of ECPs

(ECP content in the first dilution was adjusted to 450μg

protein)in PBS (pH 7.2). PBS was used as a

control. The fish cells were incubated at 20℃ for 2 d,

and the mammalian cells were incubated at 37℃ in a

5%CO2 atmosphere for 1 d. The cytotoxic activity was

examined under an inverted microscope. Toxicity titres

were expressed as the reciprocal of the highest dilution

of ECPs that caused complete or partial destruction of

cell monolayers. Heat stability of the cytotoxicity was

investigated by heating the ECPs at 80℃ for 15

min. ECPs collected from A. hydrophila ATCC 7966T

was used as a positive control.

Enterotoxicity of ECPs

Enterotoxic activity of bacterial culture supernatant

was evaluated by the suckling mouse test using three

types of culture media to screen whether the growth

medium had any effect on the assay. These media

included double-strength nutrient broth, double-strength

brain heart infusion broth(Difco, USA)and double

-strength glucose-free tryptic soy broth(Difco, USA). A

5 mL portion of each growth medium in an Erlenmeyer

flask was inoculated with bacteria and incubated at 37

℃ with shaking(175 rpm)for 24 h. Cell-free supernatant

was prepared by centrifugation at 10,000×g for 15 min at

4℃ and subsequent filtration through a 0.22μm pore

sized filter(Millex-GV, Millipore, Japan). Culture super

natant solution(100μL)containing sterile 0.02%(w/v)

trypan blue was administered orally into the stomachs of

2-4 day-old infant mice with a fine silicon tube con

nected with a pipette tip attached to a micropipette.

Three mice were used per challenge group. The infant

mice were then individually placed on a layer of tissue

paper in a petridish and left at 30℃ for 3-4 h. The ani

mals were then sacrificed, and entire intestinal tracts

were removed and weighed. The remaining body

weight was also measured. The ratio of intestinal

weight to remaining body weight was calculated, and

enterotoxicity was considered positive if the ratio

exceeded 0.08. Supernatant from A. hydrophila ATCC

7966T and sterile growth media were used as positive

and negative controls. Supernatant from A. caviae

ATCC 15468T was also included.

PCR for detection of virulence genes

PCR was conducted in order to detect aerolysin and

cytolytic enterotoxin(AHCYTOEN)genes in the strains

of Aeromonas sp. T8 group. Bacterial cells were grown

in nutrient broth, and genomic DNA was extracted

by using the Wizard Genomic DNA Purification Kit

(Promega, USA)following the manufacturer's instruc

tions. The aerolysin gene was amplified using forward

primer 5'-GC(A/T)GA(A/G)CCC(A/G)TCTATCC(A/T)G-

3'and reverse primer 5'-TTTCTCCGGTAAGACCATTG-

3'(Santos et al.,1999). The primer combination

AHCF1(5'-GAGAAGGTGACCACCAAGAACA-3')and

AHCR1(5'-AACTGACATCGGCCTTGAACTC-3')were

used to amplify the AHCYTOEN gene as described by

Kingombe et al.(1999).

A final volume of 50 mL PCR mixture contained 1.25

μL of each 0.2 mM deoxyribonucleotide tri-phosphate,5

μL of 25 mM MgCl2,100-300 ng genomic DNA,5μL of

10×PCR buffer,2.5μL of a 20μM solution of each prim

ers,0.3μL of Taq DNA polymerase(Promega SUSA)at

5U/μL and double-distilled sterile water. The PCR am

plification was performed with a Gene Amp 9700 PCR

system(PE Applied Bio Systems). The aerolysin gene

was amplified following the protocol described by

Gonzalez-Serrano et al.(2002), and the AHCYTOEN

gene was amplified following the temperature profile de

scribed by Rahman et al.(2002). The PCR amplicons

were separated by electrophoresis in a 1.5% agarose

gel and visualised after ethidium bromidestaining. DNA

templates from A. hydrophila ATCC 7966T and A. veron

ii biotype sobria ATCC 9071T were used as positive con

trols, whereas the template from A. caviae ATCC 15468T

was used as a negative control.

Results

Virulence for fish

Strain T8 showed a high degree of virulence for a

ll fish species. The LD50 of the strain for silver barb,

striped snakehead and tilapia was 1.5×105,3.2×105

and 2.9×105 CFU/fish, respectively(Table 2). In all of

the challenged fish, swelling and inflammation of the tis

sue surrounding the injection site were observed within

12 h after injection. Hemorrhagic and necrotic lesions

developed in the muscle adjacent to the injection site

within 24 h. Lesions became more pronounced at the

2nd and 3rd day after inoculation. The internal clinical

symptoms that occurred after the injection are

summarised in Table 2. Bacteria were re-isolated from

the moribund or dead fish and confirmed by the slide

agglutination test against anti-T8 serum.

Virulence for mouse

Neither mortality nor clinical disease symptom was observed in any mice challenged with strains T4, T8,

T27 and P2. The internal organs of the mice, sacrificed

after the experimental period, seemed apparently nor

mal, and no expected bacteria were re-isolated from any

Virulence properties of Aeromonas sp. T8 group 155

Table 2. Virulence and clinical symptoms expressed by the fish challenged with Aeromonas sp. strain T8

of the mice. When mice were challenged with A.

hydrophila ATCC 7966T, 20% mortality was observed.

The kidney, liver, spleen and other internal organs of the

infected mice became pale, and slight proliferation of

bacteria was found in the organs of infected

mouse. The internal organs of the surviving mice

seemed normal, and no bacterium was re-isolated from

the internal organs.

Enzymatic and hemolytic activities of bacterial cells

Exoenzymatic activities of bacterial cells based on

the ratio of the hydrolytic halo zone diameter to colony

diameter are illustrated in Table 3. All strains of the

Aeromonas sp. T8 group showed moderate to strong

hydrolysis activity against casein, gelatin, hemoglobin

and lipid. These strains showed weak hydrolytic activ

ity against albumin, and no activity against elastin and

phospholipid. Exoenzyme activities exhibited by A.

hydrophila ATCC 7966T and A. caviae ATCC 15468T

were very similar to those of the strains of Aeromonas

sp. T8 group except for hydrolysis against albumin,

which was negative in both strains.

All strains of the Aeromonas sp. T8 group exhibited

hemolytic activity against all of the erythrocytes except

sheep erythrocytes, which was susceptible only to strain

P2 (Fig. 1). Among the strains, Thailand strains (T4, T8

and T27) showed a clear hydrolytic zone in blood agar

plates, hence categorized as ƒÀ-hemolytic. On the other

hand, the Philippines strain (P2) showed an opaque,

incomplete type of hemolysis in blood agar plates, and

therefore was considered as ƒ¿-hemolytic strain. A.

hydrophila ATCC 7966T showed ƒÀ-hemolytic activity for

all of the erythrocytes tested.

Protease activity of ECPs The protease activities of ECPs of the Aeromonas

sp. T8 group and the reference strains are summarized in Table 4. Among the experimental strains, strain P2 had the highest activity (2865 units/mg protein) and strain T4 had the lowest activity (282 units/mg

protein). Strains T8 and T27 produced 461 and 447 protease units/mg protein, respectively. The protease activities of A. hydrophila ATCC 7966T and A. caviae ATCC 15468T were 325 and 171 units/mg, respectively.

The protease activity of all experimental and reference strains was almost completely inhibited by heating

Table 3. Protease, lipase and phospholipase activities of bacterial cells at 25℃

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

at 80•Ž for 15 min (Table 4). When ECPs were heated

at 60•Ž for 15 min, protease activities of A. hydrophila

ATCC 7966T and A. caviae ATCC 15468T were inhibited

92 and 80%, respectively, but the activities of the

Aeromonas sp. T8 group remained stable. Protease

activities of the strains T4, T8, and T27 were inhibited

38, 43 and 44%, respectively, by EDTA treatment and

87, 88 and 88%, respectively, by PMSF treatment.

Both EDTA and PMSF inhibited the activity of strain P2

at 19 and 24%, respectively. PMSF inhibited 91% of

the protease activity of A. hydrophila ATCC 7966T but

EDTA had little effect (9%). A. caviae ATCC 15468T

protease possessed similar characters exhibited by theThailand strains (T4, T8 and T27).

Hemolytic activity of ECPsThe hemolytic activities of bacterial ECPs are sum-

marized in Fig. 2. Although hemolytic activities of ECPsvaried among the strains of the Aeromonas sp. T8 groupagainst different blood cells, generally higher activity wasobserved against fish blood cells compared to mamma-lian blood cells. On the other hand, hemolytic activity of

Fig. 1. Hemolytic activity of bacterial cells on blood agar plates, prepared by 5% blood cells from different fish and mammaliansources. Hemolytic activity was expressed as zone ratio (ratio of hemolytic zone diameter to colony diameter) . Strains T4,T8, T27 and P2 belong to the Aeromonas sp. T8 group and ATCC 7966T is the type strain of A. hydrophila .

Table 4. Protease activity of ECPs

*

% inhibition: (difference between the protease activity of untreated ECPs and treated

ECPs •€ activity of untreated ECPs)•~ 100

Abbreviations: EDTA: Ethylenediamine-N,N,N,N-tetraacetic acid

PMSF: Phenyl methyl sulphonyl fluoride

Virulence properties of Aeromonas sp. T8 group 157

the A. hydrophila ATCC 7966T was found to be higher

against mammalian blood cells than against fish blood

cells. Between the two kinds of fish blood cells, carp

blood cells were more susceptible than rainbow trout

blood cells for the strains of Aeromonas sp. T8 group.

Among the mammalian blood cells, rabbit blood cells

were the most sensitive for all of these strains followed

by human and horse blood cells. No hemolytic activity

was observed for sheep blood cells for any tested

strains. Among the 4 strains of the Aeromonas sp. T8

group, P2 had much higher hemolytic activity than the

other strains.

Like protease activity, hemolytic activities of all of

the strains were also completely inhibited by heating the

ECPs at 80•Ž for 15 min. On the other hand, heating at

60•Ž for 15 min did not inhibit the activity for the strains

of the Aeromonas sp. T8 group, though it partially inhib-

ited the activity of the reference strains (Table 5). The

hemolytic activity of A. hydrophila ATCC 7966T and A.

caviae ATCC 15468T was inhibited at 50% by PMSF

treatment, but EDTA inhibited 33% activity for A. caviae

and showed no inhibition for A. hydrophila. On the other

hand, the activity of ƒÀ-hemolytic strains (T4, T8 and T27)

were inhibited at 25% by EDTA, but not inhibited by

Fig. 2. Hemolytic activity of ECPs against blood cells from different sources. Strains T4, T8, T27 and P2 belong to the Aeromonas

sp. T8 group and ATCC 7966T is the type strain of A. hydrophila.

Table 5. Hemolytic activity of ECPs against rabbit blood cells

* %

inhibition: (difference between the hemolytic titre of untreated ECPs and treated

ECPs •€ hemolytic titre of untreated ECPs) •~ 100

Abbreviations: EDTA: Ethylenediamine-N,N,N,N-tetraacetic acid

PMSF: Phenyl methyl sulphonyl fluoride

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

PMSF treatment.

Cytotoxi city of ECPs

Among the strains of the Aeromonas sp. T8 group,

only P2 showed evidence of cytotoxicity for all of the

tested cell lines. The cytotoxicity titres of the ECPs of

strain P2 for HeLa, BF-2, SNN-1, EPC, FHM, and RTG-

2 cell lines were 4, 8, 8, 16, 64 and 64, respectively.In

general, the degenerative changes manifested by the

ECPs were vacuolization, shrinking, dendritic elongation

of the cells, cell detachment and final monolayer

destruction. The cytotoxicity of the ECPs heated at

80℃ was completely inhibited for all tested cell

lines. A. hydrophila ATCC 7966T, which was used as a

positive control for the assay, showed cytotoxic effect against all the tested cell lines. The degenerative

changes were almost similar to that observed for the strain P2 against all tested cell lines.

Enterotoxicity of ECPs

No enterotoxic activity was observed in any of the samples from the strains belonging to the Aeromonas

sp. T8 group. No fluid accumulated in the intestinal tract, and diarrhoea was not observed. The culture supernatant of A. hydrophila ATCC 7966T cultured in any

of the media, gave positive response for enterotoxic assay: fluid accumulated in the intestinal tract, and diar

rhoea occurred in the infant mice. The culture supernatant of A. caviae ATCC 15468T exhibited enterotoxicity

(both fluid accumulation and diarrhoea) only when double strength glucose-free TSB was used as a growth medium.

Detection of virulence genes by PCR

No strains belonging to the Aeromonas sp. T8 group amplified the targeted aerolysin or AHCYTOEN genes by PCR amplification. Amplified PCR products were observed in A. hydrophila ATCC 7966T for both genes.

A. veronii biotype sobria ATCC 9071T was positive for AHCYTOEN but negative for aerolysin gene, whereas A. caviae ATCC 15468T was negative for both genes.

Discussion

EUS is a severe disease of freshwater and some brackishwater fish that has extended rapidly in South and Southeast Asia over the past 25 years (Supranee

and Roberts, 1999). Although the aetiology of EUS is considered to be complex, recently, a fungus

Aphanomyces invadans has been reported as a primary causative agent and bacteria including Aeromonas spp.

are considered to be a secondary invader of the disease

(Supranee and Roberts, 1999). However, Aeromonas spp. is frequently isolated from EUS-affected fish

(Subasinghe et al., 1990; Roberts et al., 1990; lqbal et al, 1998). Some reports suggested that Aeromonas

spp. might contribute to the pathogenesis of the disease

(Costa and Wejeyaratne, 1989). Since several species

of Aeromonas are virulent for fish, and possess severa

l virulent properties, the role of Aeromonas spp. when

exist in EUS-affected fish is still unclear. The

Aeromonas sp. T8 is a newly identified species and all

strains of the group were isolated from EUS-affected

fish. Thus, whether the Aeromonas sp. T8 group has

any significance in EUS is a new research interest.

Moreover, as a newly identified microorganism, determi

nation of the pathological importance of the Aeromonas

sp. T8 group for fish and other animal, and screening of

the putative virulence properties are also essential.

This article describes the pathological importance of the

newly identified Aeromonas sp. T8 group and its putative

virulence properties.

The representative strain T8 showed a high degree

of virulence for silver barb, striped snakehead and tilapia

with median lethal doses of 1.5×105, 3.2×105 and 2.9×

105 CFU/fish, respectively. Hemorrhagic and necrotic lesions in the external tissue were observed in the artifi

cially infected fish. The lesions seemed apparently similar to the lesions usually observed in EUS-affected

fish. On the other hand, all strains of the Aeromonas

sp. T8 group failed to bring about any disease signs in the challenged mice, which suggests that these strains

are non-pathogenic for mammals. Mateos et al (1993) found a group of A. hydrophila isolates collected from fish farm water that were virulent for rainbow trout Oncorhynchus mykiss but did not cause any disease

signs in mice injected with the isolates. They assumed that the prolonged presence of these strains in aquatic environments might lead to loss or decrease in the viru

lence of those strains for homoeothermic animals. However, it is important to note that motile aeromonads

have long been considered as opportunistic pathogens. They have an affinity to cause disease in immune-com-

promised hosts. The present study is the first attempt on pathogenesis of the newly identified species the

Aeromonas sp. T8 group. More studies are needed to determine the pathological potency of the species for homoeothermic animals.

The exoenzyme activities of the live bacterial cells evaluated by the plate assay method showed that the strains of the Aeromonas sp. T8 group and reference

strains were positive for lipase and protease activity. For protease activity, all strains showed hydrolysis of

casein, gelatin and hemoglobin, but none of them hydrolyzed elastin. The extracellular proteases are considered to play an important rule in the pathogenesis of

Aeromonas spp. (Thune et al., 1982b; Tajima et al., 1983; Kanai and Wakabayashi, 1984). Although the effect of individual protease to virulence is unknown,

caseinase and elastase activities are frequently reported to co-relate with the virulence of Aeromonas spp. (Thune et al., 1982a; Esteve et al., 1995; Cascon et al.,

Virulence properties of Aeromonas sp. T8 group 159

2000). We assume that caseinase of the experimenta

l strains play an important role in the virulence of

fish. Other proteases like gelatinase and albuminase

probably provide nutrients for the growth of bacteria in

the host body by degrading host tissue protein. When

the representative strain T8 was injected to tropical fish,

necrotic lesion was observed in the muscles adjacent to

the injection site. We guess that extracellular protease

was responsible for tissue necrosis, Thune et al.

(1982a) reported that concentrated culture filtrate of A.

hydrophila that possessed proteolytic activity was lethal

and produced dermonecrotic symptom to channel catfish

lctalurus punctatus. This report supports our suspicion

. In this study, all strains of the Aeromonas sp. T8

group exhibited hemolytic activity against a wide range

of erythrocytes. The hemolytic titers determined using

different fish and mammalian blood cells were found to

vary among the strains. However, the ECPs of all

strains of the Aeromonas sp. T8 group showed higher

hemolytic activity to fish blood cells compared to mam

malian blood cells. Since the strain T8 was virulent for

different fish but all strains were non-virulent for a mam

mal, it can be presumed that there is a positive correla

tion between the virulence for fish and hemolytic activity

against fish erythrocytes. Esteve et al. (1995) also

found that fish virulent strains of the species A.

hydrophila and A. jandaei secreted hemolysins against

fish erythrocytes and concluded that hemolysins against

fish erythrocytes might play an important role in the

spread of the disease in eels. Moreover, we assume

that hemolysin was also responsible for hemorrhages

and necrosis observed in the fish artificially infected by

Aeromonas sp. strain T8. Allan and Stevenson (1981)

reported that ECP of protease-deficient but hemolysin

positive strain of A. hydrophila produced hemorrhages in

internal organs, excess fluid in the body cavity and lesion

in external muscles upon injection challenge to speckled

trout Salvelinus fontinalis. This report indicates the role

of hemolysin in the infection of fish and also justifies our

assumption.

The protease and hemolytic activities of experimen

tal and reference strains were completely inactivated by

heating the ECPs at 80℃ for 15 min, indicating that pro

tease and hemolysin of the strains are thermo

labile. Protease activity of the strains T4, T8, and T27

was highly inhibited by PMSF treatment and moderately

inhibited by EDTA treatment, indicating that these strains

produce serine protease as the major protease compo

nent and also produce some metalloprotease.

Protease activity of strain P2 was inhibited by both

PMSF and EDTA, indicating the presence of both serine

and metalloprotease, though the inhibition rate was

lower than the three Thailand strains. PMSF highly

inhibited the protease activity of A. hydrophila ATCC

7966T, but EDTA had little effect, suggesting that serine

protease is the major protease of the strain, which also

produces a small amount of metalloprotease. Protease of A. caviae ATCC 15468T possessed similar characters

exhibited by the Thailand strains (T4, T8 and T27). The

inhibition patterns of the hemolytic activities of A

. hydrophila ATCC 7966T and A. caviae ATCC 15468T were found very similar to the protease activity of the

strains. However, the activity of β-hemolytic strains

(T4, T8 and T27) was partially (25%) inhibited by

EDTA, but no inhibition was detected by the PMSF

treatment. Thus, we assume, these strains probably

possess a novel hemolysin enzyme, which is different

from the hemolysins of A. hydrophila and A. caviae.

Although extracellular products of aeromonads are

often found to possess cytotoxicity against different cell

lines, there is probably little or no correlation with

virulence. Santos et al. (1988) reported that ECPs

obtained from both pathogenic and nonpathogenic A

. hydrophila strains displayed cytoxicity to different ce

ll lines, whereas some virulent A. sobria and A. caviae

strains did not produce any cytotoxic effect. Paniagua et al

. (1990) also reported the lack of correlation

between cytotoxic activity and virulence of Aeromonas

spp. In the present study, only strain P2 showed cyto

toxicity against all cell lines tested. Strain P2 also

exhibited high protease activity compared to other

strains of the Aeromonas sp. T8 group, which probably

enabled the strain to possess cytotoxic activity.

However, involvement of the protease in cytotoxicity is

yet to be known.

Clinical and environmental strains of motile

Aeromonas spp. are also known to produce extracellular

enterotoxin, an important virulence factor suspected to

be related with gastrointestinal diseases of humans. In

the present study, we used three types of growth media

for preparation of culture supernatant to examine

whether growth medium influences the enterotoxic

activity, because Namdari and Bottene (1990) reported

that A. caviae produced cytotoxin and enterotoxin only in

selected media such as glucose-free tryptic soy broth.

However, the present strains grown in any media did not

show enterotoxicity, although A. hydrophila ATCC 7966T

produced enterotoxin irrespective of the growth media

and A. caviae ATCC 15468T only in a selected medium

. Aerolysin is the well-characterized ECP responsible

for β-hemolysis exhibited by Aeromonas spp. It is con

sidered an important marker for virulence of motile

Aeromonas spp. Chakraborty et al. (1987) presented

genetic evidence that the structural gene aerA (pro

aerolysin) is found in all members of this genus. Using

a primer set, Santos et al. (1999), Gonzalez-Serrano et al

. (2002) and Gonzalez-Rodriguez et al. (2002)

detected the gene in several Aeromonas spp. strains

recovered from freshwater fish, cold-smoked fish and

diarrhoea of human. The cytolytic enterotoxin gene

(AHCYTOEN) in A hydrophila has been reported as a

multivirulence gene involved in lethality (in mice),

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

hemolysis, cytotoxicity and enterotoxicity, which are the

established virulence properties of Aeromonas spp.

(Chopra et al., 1993). Various strains of aeromonads from several geno-species viz., A. hydrophila, A.

bestiarum, A. salmonicida,A. caviae, A. eucrenophila,A.

sobria, A. veronii biotype sobria, A. veronii biotype

veronii, A. encheleia and A. torta were found to hold the

AHCYTOEN gene by investigation with the primer com

bination strategy of Kingombe et al. (1999). Rahman et

al. (2002) also detected the gene in several strains of A.

veronii biotype sobria isolated mostly from EUS-affected

fish and also from environmental and human clinical

samples. In the present study, we did not detect either

gene in the strains of the Aeromonas sp. T8 group. We suspect that the strains of the Aeromonas sp. T8 group

do not possess either gene.

In summary, the newly identified Aeromonas sp. T8

group was detected as a potential pathogen for tropical fish. Extracellular proteases, especially caseinase and

hemolysins are suspected to play a role in the virulence

of the species to fish. However, the role of specific

enzyme (s) is further object to be studied. Moreover,

the role of the Aeromonas sp. T8 group, when exists in

EUS-affected fish is also further object to be studied.

Acknowledgements

The authors would like to express their sincere thanks to Prof. M. Yoshimizu, Hokkaido University,

Japan for providing cell lines and valuable suggestions

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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