Download pathogenic bacteria isolated from tiger prawn

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International Journal of Research in Biological Sciences
Universal Research Publications. All rights reserved
ISSN 2249–9687
Original Article
PATHOGENIC BACTERIA ISOLATED FROM TIGER PRAWN PENAEUS
MONODON IN SHRIMP CULTURE PONDS AT EAST COAST OF
THANJAVUR DISTRICT TAMIL NADU, INDIA.
N.NARASIMHAN *1, T.RAVIMANICKAM *3, M.SUKUMARAN *1, R.RAVICHELVAN *2,
R.RAVICHANDRAN *2, D.MADHAVAN *2
*1 PG and Research Department of Zoology, Rajah Serfoji Government College, Thanjavur - 613 005,
Tamil Nadu, India.
*2 PG Department of Zoology, Govrnment Arts College, Ariyalur - 621 704, Tamil Nadu, India.
*3 PG Department of Bio –Technology and Gene Technology, JJ College,Pudukkottai ,Tamilnadu, India
Received 25 April 2013; accepted 20 May 2013
Abstract
Pathogenic bacterial flora was isolated from muscle tissues of black tiger shrimp, Penaeus monodon. The infected shrimp
samples were collected from Omsakthi Aquafarm at Sedhubhavasatram in Thanjavur District, Tamil Nadu, India, during
December 2012 to February 2013. Bacterial species were isolated by serial dilution technique method were plated in
nutrient agar medium (Jolt et al., 1994). Totally eleven different bacterial strains such as Aureobacterium faciens,
Aeromicrobium erythreum, Bacillus subtilis, Escherichia coli, Vibrio cholarae, Enterobacter aerogens, Micrococcus
Luteus, Pseudomonas putida, Pseudomonas aeruginosa and Enterococcus pseudo avium were isolated from muscle tissues
of P.monodon. The culture plates were incubated at 37°C for 24 to 48 hours. The different species of bacteria were
identified using the biochemical tests namely, Motility test, Indole test, Methyl red test, Voges - proskauer test, Citrate
utilization test, Catalase test, Oxidase test, Triple sugar Iron test, Urease test and Nitrate test. Broth cultures were observed
the colour formation, ring formation and gas production which indicated positive and no colour change, no ring formation
and no gas production which indicated nagative results.
© 2013 Universal Research Publications. All rights reserved
Key Words : Penaeus monodon, Biochemical tests, Pathogenic bacteria, Shrimp culture ponds.
INTRODUCTION
Bacterial infection is one of the major disease problems in
shell fish and fin fish aquaculture. Disease cause the
largest economic losses in aquaculture and bacterial
infections are second only to fungal diseases in economic
importance. Bacterial infections are generally restricted to
chronic, steady losses (Intesar, 2003). Vibrio cholerae is
one of the important etiological agents in mass mortalities
of Penaeus monodon rearing systems (Tanasomwang et al.,
1998.)
Gram positive bacteria is based on the general models of
infection and disease that are caused by the pathogenic
bacteria can be grouped into three categories, extra cellular
invasive pathogens produce toxin, but the disease that these
pathogen cause are primary due to their invasion and
growth within tissue, rather than production and
dissemination of a toxin. Food security is a complex issue,
where shell fish and fishery products are generally
98
regarded as high risk commodity is respect of pathogen
contents, natural toxins and other possible contaminants
and adulterants. Although there has been a significant
increase in the knowledge of shrimp disease (Nash, 1988 ;
Mayer, 1991 ; Maeda and Liao, 1992).
Gram negative bacteria are cocci or coccibacilli whose
associated disease usually involve the accumulation of
copious amounts to pus frequently affect the respiratory
tract.
Although studies have been performed on
microbiological disease in Indian prawns, there is a paucity
of detailed investigations on the effects of microbial
pathogens. The research in shrimp pathology has been with
reference to disease in shrimps collected from the shrimp
farms at the time of occurrence of disease. There have
been laboratory, based studies on shrimps (Brock and
Lightner, 1990). Shrimp affected by this bacterial disease
were than taken from the culture ponds and microbial
pathogens was controlled in the laboratory conditions.
International Journal of Research in Biological Sciences 2013; 3(2): 98-101
Vibrio sp., Bacillus sp., Pseudomonas sp., E.coli., and
Enterobacter sp., were the common bacterial pathogens
(Moriarty, 1998).
The bacteria causing the most serious diseases of the post
larval and adult stages of P.Monodon of the genus Vibrio,
Bacillus, Pseudomonas and Aeromonas (Lightner and
Redman, 1998). This paper reports on pathogenic bacteria
associated with mortalities of P.monodon in rearing ponds,
with a view to provide potential approaches for improving
the quality assurance and create awareness among the
consumers.
MATERIALS AND METHODS
Study area: Infected shrimp Penaeus monodon were
collected from Omsakthi Aquafarm at Sedhubhavasatram,
Thanjavur district, Tamil Nadu, India.
Study period: Samples were collected from December
2012 to February 2013. To avoid further contamination,
during transportation from the source to laboratory,
Samples were carried by special sterile bags packed in
insulated box with ice to maintain the temperature around 5
to 6 C.
Isolation of shrimp bacteria:
The muscle samples of affected Penaeus monodon were
collected and homogenized were used as a sample for
bacterial isolation (Aneja, 2001). The homogenated each
tissue sample was serially diluted from 10-1 from 10-9 from
the diluted sample taken 0.1 ml of sample from 10-5, 10-6
and 10-7
and spread over on nutrient agar medium
separately, one plate maintain as a control without sample.
The plates were incubated at 37 C for 24 to 48 hours
observed the bacterial colonies.
Identification of shrimp bacteria
The isolated bacterial species were identified by the
following
the
morphological
and
biochemical
characteristics of the individual colony was recorded. The
individual colony was transferred to nutrient agar. The
isolates were subjected to following different biochemical
test for example Gram staining, Motility test, Indole test,
Methyl red test, Voges Proskauer test, catalase test, Nitrate
test and Carbohydrate fermentation test as described by Jolt
et al., (1994).
RESULTS AND DISCUSSION
The different species of pathogenic bacteria were identified
using the physical and chemical tests. They are presented in
Table -1.
Physical Analysis:
Motility test: Hanging drop method was carried out for
motility test. The bacterial culture plate BCP2 and BCP8
resulted were showed non motile and other cultures were
motile.
Chemical Analysis
Gram staining: All the bacterial cells have been stained by
counter stain safranin and appeared red in colour. The
culture BCP1, BCP2, BCP3 and BCP7 were resulted in
gram positive and other cultures were negative. Among the
species five bacterial were rod shaped (BCP3, BCP4,
BCP5, BCP8 and BCP9) two bacterial species (BCP1 and
BCP2) were rod or cocci shaped and four bacterial were
cocci shaped (BCP6, BCP7, BCP10 and BCP11).
99
Indole test: Ring formation was observed only BCP2,
BCP4, BCP6, BCP8, BCP 10 and BCP11 and other culture
were indicate no ring formation. Broth cultures showed no
ring formation so it indicates the negative result.
Methyl red test. Addition of methyl red indicated to the
MR broth cultures of such as BCP2, BCP4 and BCP7
showed the presence of pink colour, which indicated
positive results and there was no colour change and other
broth culture were showed negative results.
Voges Proskauer test: Addition of methyl red indicated to
the VP broth cultures of such BCP2, BCP3, BCP6 and
BCP8 showed the presence of pink red colour, which
indicated positive results and there was no colour change
in other culture strains which showed negative results.
Citrate Utilization test: The broth cultures such as BCP4
showed negative results and other culture showed green to
blue colour, which indicated positive results.
Urease test: The both cultures BCP1, BCP2, BCP6,
BCP10 and BCP11 showed yellow to pink colour, which
indicated positive results and other cultures were showed
no colour changed, which indicated negative results.
Catalase test: The appearance of gas bubbles was observed
in all the broth cultures, which indicated positive results.
The negative results were not showed.
Oxidase test: The broth cultures such as BCP4, BCP6 and
BCP9 were showed negative results and other cultures were
showed positive results.
Nitrate test: After incubation few drops of a
Naphthylamine and Sulphanilic acid were added. The red
colour formation was indicated as positive results. The
broth cultures were showed the red colour in BCP2, BCP3,
BCP8 and BCP9. The absence of red colour were showed
BCP1, BCP4 and BCP7 indicates the negative results and
the red colour was reductase in BCP5, BCP6, BCP10 and
BCP11.
Carbohydrate fermentation test: All the broth cultures
were showed positive results. This indicated the gas
production.
Identification of bacteria:
The isolated different bacterial strains were identified based
on the culture morphology and biochemical characteristics.
The table results were compared with Bergey’s manual of
systemic bacteriology.
Based on the results were
confirmed as Aureobacterium faciens, Aeromicrobium
erythreum, Bacillus subtilis, Escherichia coli, Vibrio
cholarae, Enterobacter aerogens, Pseudomonas putida,
Pseudomonas
aeruginosa,
Brucella
canis
and
Enterococcus pseudo avium.
In the present study bacterial species were isolated from
infected shrimp muscle tissues. Totally eleven bacterial
species were isolated by diluting plating technique. The
isolated all the bacterial species was harmful pathogen.
Bacterial infections are caused penaeid species and the
overviews of diseases in cultured prawn were studied
(Bright et al., 1988). Epizoobiology and pathogenicity of
bacterial infections caused by many problems in cultured
giant tiger prawn Penaeus monodon (Chen et al., 1992).
Vibrio species are considered as part of the normal flora of
seawater and can invade marine animals (Olsen et al.,
1995). In general, the results of our study indicate that
International Journal of Research in Biological Sciences 2013; 3(2): 98-101
VP
TST
G
F
Catalase
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
-
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
cocci
+
-
+
+
+
+
+
-
+
+
+
+
+
cocci
-
+
-
+
-
+
+
+
+
+
+
-
-
-
Rod
+
-
+
+
-
+
+
+
+
+
+
+
9
-
+
Rod
-
-
-
+
-
+
+
-
+
+
+
10
-
+
cocci
+
-
-
+
+
+
+
+
+
+
+
+
11
-
+
cocci
+
-
-
+
+
+
+
+
+
+
+
+
Shape
2
+
-
3
4
5
+
-
+
+
+
6
-
+
7
+
8
=
=
=
=
Voges Proskauer Test
Trible Sugar Iron Test
Glucose Test
Fructose Test
bacterial load of shrimp pond water and sediment increases
with increasing organic load, which is in agreement with
previous reports by (Horsley, 1997; Dalmin et al., 2002).
In the context of aquaculture, the muscle microbiota is
constantly influenced by the aquatic microflora. Thus, a
modified definition of probiotics for aquaculture has been
proposed (Verschure et at, 2006). Experimental infections
performed by Lavilla - pitogo et al., (1990) also showed
high pathogenicity of the isolated strain.
Usually in prawn culture water or even in entrophicated
coastal water bacterial numbers are less than 10 6 cells /ml
because protozoa feed on bacterial cells which results in
maintaining a certain level of bacterial populations in water
(Maeda and Nogami, 1989).
Based on the present study, it is concluded that the
pathogenic bacteria are perhaps that most important
pathogens in shrimp culture ponds causing servere
mortalities and financial losses. There is also a need to
develop the shrimp culture practice and control the
pathogenic microbes is very essential.
ACKNOWLEDGEMENT
The authors will express their gratitude for the staff of
Gowri Biotech Research Laboratory for their help to this
work.
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100
+
-
Carbohydrate
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G
F
S
=
=
Nitrate
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Oxidase
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+
TSI
+
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Urease
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1
Rod/cocc
i
Rod/cocc
i
Rod
Rod
Rod
Citrate
VP
-
Indole
-
Motility
-
Gram
staining
Methyl red
Bacterial
Culture
Plate (BCP)
Table.1. Bio-Chemical Characterization of bacterial isolates from infected shrimp Penaeus monodon.
Identification of
Bacteria.
Aureobacterium
faciens
Aeromicrobium
erythreum
Bacillus subtilis
Escherichia coli
Vibrio cholerae
Enterobacter
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Micrococcus
luteus
Pseudomonas
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Enterococcus
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Source of support: Nil; Conflict of interest: None declared
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International Journal of Research in Biological Sciences 2013; 3(2): 98-101