International Journal of Aquaculture, 2013, Vol.3, No.17, 92
-
100
http://ija.sophiapublisher.com
98
The present study reveals the prevalence of motile
aeromonads in ornamental fishes and associated
carriage water. Most of the motile aeromonads were
capable of producing extracellular virulence factors
and had acquired multidrug resistance. Poor water
management practices could trigger infections caused
by these potential pathogens/opportunists which might
spell doom for this emerging industry in Kerala.
Prudent use of antibiotics and proper water
management practices may be promoted for
sustainable development of ornamental fish industry,
which can sustain the livelihoods of large number of
rural people, who are currently engaged in this
industry.
3
Materials and Methods
3.1
Collection of Samples
Live, healthy ornamental fish samples
(
Poecilia
sphenops
and
Poecilia reticulate)
and associated
carriage water samples were collected from three
different aquarium vendors in and around Cochin. The
fish samples were transported to the laboratory in
sterile polythene bags and water samples in sterile
bottles. The samples were then analyzed for
aeromonads within 4 hours of collection.
3.1.1
Isolation and identification of
Aeromonas
Different parts of the body (body surface, gill and
intestine) of fish samples were analyzed for motile
aeromonads. The body surface and the gill of the
fishes were repeatedly swabbed using sterile cotton
swabs. Using a pair of scissors, an incision was made
near the vent of the fish facilitating the swabbing of
intestine. The swabs were then transferred to alkaline
peptone water (APW pH 8.4) which was used as the
enrichment medium. After incubation at 37
℃
for 18
h, a loopful of the APW culture was streaked on
Starch
Ampicillin Agar (Ampicillin 10 mg/L) which
was used as the selective isolation medium and
incubated at 37
o
C for 18-24 h (Palumbo et al., 1985).
The water samples were serially diluted and bacterial
isolation was done by spread plate method using
Starch
Ampicillin Agar plates. The plates were
incubated at 37
℃
for 18-24 h and then flooded with
approximately 5 ml of Lugol’s iodine solution and
amylase positive yellow to honey colored colonies
were isolated. The isolated cultures were then purified
by repeated streaking on nutrient agar plates. Those
strains that were gram negative bacilli, motile, oxidase
and catalase positive, glucose fermenting, nitrate
reducing, urease negative and which do not grow in
6%
NaCl were further tested for arginine dihydrolase,
lysine decarboxylase, and ornithine decarboxylase
activity. Additional tests include acid production from
mannitol, sucrose, arabinose, Voges-Proskauer reaction,
hydrolysis of esculin and indole production. The
isolates were identified to the species level according
to Aerokey II (Carnahan et al., 1991).
3.2
Study of Extracellular Virulence Factors
3.2.1
Production of gelatinase
Pure cultures of the isolates were spot inoculated on
gelatin agar plates (2%w/v gelatin), and the plates
were incubated at 28
℃
for 24-48 hrs. Zone of
clearance around the colonies, after the plates were
flooded with saturated solution of ammonium sulphate
indicated that gelatin has been hydrolyzed.
3.2.2
Production of caseinase
The test organisms were heavily spot inoculated on
skim milk agar plates and the plates incubated at 28
℃
for 24 hrs -48 hrs. Caseinase production was detected
by the presence of clear zones around the test
colonies.
3.2.3
Production of lipase
Tributyrin or glyceryl tributyrate is commonly used
for studying lipolytic activities. The test organisms
were heavily spot inoculated on tributyrin agar plates
and the plates incubated at 28
℃
for 24 hrs -48 hrs. A
positive result was indicated by a zone of clearance
around the colonies of lipolytic organisms, where the
tributyrin has been hydrolyzed (Rhodes, 1959).
3.2.4
Production of DNase
A plate test for the demonstration of bacterial
decomposition of nucleic acid was performed. The test
organisms were heavily spot inoculated on DNA agar
plates and the plates incubated at 28
℃
for 24 hrs -48
hrs. After incubation the plates were flooded with 1M
HCl. The appearance of clear zone around the
colonies indicated that the bacteria has elaborated
DNase and hydrolyzed the DNA. The rest of the plate
with the intact DNA turned opaque white, on addition
of 1M HCl.
3.2.5
β– Haemolytic assay
Haemolytic activity was determined using blood agar