Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2013, 3 (3):41-45
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ISSN : 2231 3168
CODEN (USA) : JMBRB4

41
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Presumptive and definitive identification of Pseudomonas from infected
Pangasius hypophthalmus in culture ponds of West Godavari and Krishna
districts of Andhra Pradesh

M. Phani Kumar and K. Sree Ramulu

Department of Zoology, Andhra University, Visakhapatnam, Andhra Pradesh, India
_____________________________________________________________________________________________

ABSTRACT

The paper deals with the presumptive identification of Pseudomonas and definitive identification of
Pseudomonas up to species level i.e. Pseudomonas fluorescens in various organs of Pangasius hypophthalmus
infected with red disease in culture ponds of West Godavari and Krishna districts of Andhra Pradesh.

Key words: Pangasius hypophthalmus, Red disease, Pseudomonas fluorescens
_____________________________________________________________________________________________

INTRODUCTION

Andhra Pradesh, fifth largest state in India, primarily an agrarian state is endowed with rich aquatic resources,
comprising Godavari, Krishna and Penna river systems, 114 major as well minor reservoirs and lakes, 45,528
perennial and seasonal tanks and well developed canals. This state called the Fish Bowl of India. The state ranks
first in the country in coastal aquaculture and second in fresh water fish production. The major part of fish
production of Andhra Pradesh is from West Godavari and Krishna districts. The culture of Pangasius
hypophthalmus can be considered as a unique aquatic farming system in many ways. Production is the fastest
growth recorded in any aquaculture sector, ever, based on a single species, superseding the production per unit for
any form of primary production [1]. It appears that the higher the increase of fish production, the higher the risk of
disease outbreak. The main reason is a high density of cultured fish in the intensive system. The waste from the
unconsumed feed, excretion and the poor pond management leads to fish diseases. Bacteria always present in the
water environment, multiply and invade to fish and spread the disease when reaching the suitable condition [2]. The
investigation on the appearance of bacteria that causes disease in the organs of the fish is important in terms of
providing the guidelines for prevention, control and treatment of disease problems.

P. fluorescens was originally described as the causative agent of Bacterial Hemorrhagic Septicemia disease of pond-
cultured fish [3]. It is considered as a primary pathogen of freshwater fish and opportunistic pathogen for different
fish species cultured in marine and brackish waters worldwide [4]. In Bangladesh, P. fluorescens has been isolated
from eye surface and mouth lesions of diseased Rajpunti (Barbodes gonionotus) along with some other fish
pathogenic bacteria [5]. Isolation and characterization of P. fluorescens from gills of silver carp
(Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idella), African magur (Clarias gariepinus) and Nile
tilapia (Oreochromis niloticus) has also been reported elsewhere [6].



M. Phani Kumar et al J. Microbiol. Biotech. Res., 2013, 3 (3):41-45
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MATERIALS AND METHODS

Pangasius fish samples were collected from culture ponds of Kaikaluru and Mudinepalli mandals of Krishna district
in Andhra Pradesh, India, after examining the gross clinical signs and external characteristics of fish. The collected
fish exhibited red ulcerations/lesions on the body followed by reddening at the tips of paired and unpaired fins. In
some of the samples there was loss of scales appearing reddish white patches with deep wound like appearance. The
vital organs like kidney and liver were observed for structural changes and later cut for further examination along
with body slime on Pseudomonas selective agar plates. Tests used for identification of Pseudomonas are
categorized as

Presumptive identification tests: the test includes Pseudomonas isolation media, Growth in Nutrient broth,
Motility test, Grams staining, Oxidase test, Catalase test.

Definitive identification tests: This involves the identification of Pseudomonas, up to Species level from
samples found positive using a set of biochemical tests.

Tests related to carbohydrate Metabolism: Sugar fermentation, Methyl red test, proskauer Voges test, TSI.

Tests related to Amino acid & protein metabolism: Indole test, Lysine, ornithine decarboxylase & arginine
dehydrolase test.

Tests for both carbohydrate & protein metabolism: citrate Utilization
Other tests Salt tolerance test, Nitrate reduction test

RESULTS

Tab: 1 Presumptive Identification of Pseudomonas in infected Pangasius hypophthalmus

Culture
plate
GROWTH ON
Pseudomonas MEDIUM
(colony characteristics)
BROTH
CHARACTERS
MOTILITY STAINING OXIDISE CATALASE RESULT
T SF
1
round yellowish entire
pinpoited colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
2
round yellowish undulate
pinpointed colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
3
irregular, yellowiish slimy
nature big colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
4
round yellowish entire
pinpoited colonies
+ ve + ve + ve - ve + ve - ve
Pseudomonas
present
5
round yellowish
pinpointed colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
6
round yellowish umbonate
pinpointed colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
7
white round irregular big
colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
8
yellowish small slimy
nature colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
9
white round yellowish
undulated colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
10
irregular, yellowiish slimy
nature big colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present
11
round yellowish entire
pinpoited colonies
+ ve + ve + ve - ve + ve - ve
Pseudomonas
present
12
white mucoid round
irregular big colonies
+ ve + ve + ve - ve + ve + ve
Pseudomonas
present


M. Phani Kumar et al J. Microbiol. Biotech. Res., 2013, 3 (3):41-45
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Table-2 DEFINITIVE IDENTIFICATION OF PSEUDOMONAS

SL.
NO
DECARBOXYLASE TSI SF ST
MOF I MR VP CIT SPECIES
ARG LYS ORNI ACID BULT ALK SALT H2S G S M 0% 3% 8% 11%
1 `+' ve `+' ve `+' ve `-' ve `+' ve `-' ve A+G+ A+G+ A+G+ `+' ve `+' ve `-' ve `-' ve O `-' ve `-' ve `-' ve `+' ve P.flourescens
2 `+' ve `+' ve `+' ve `+' ve `+' ve `-' ve A+G- A+G- A+G- `+' ve `+' ve `-' ve `-' ve O `-' ve `-' ve `-' ve `+' ve P.flourescens
3 `+' ve `+' ve `+' ve `+' ve `+' ve `-' ve A+G- A+G- A+G- `+' ve `+' ve `+' ve `-' ve O `-' ve `-' ve `-' ve `+' ve P.flourescens
4 `+' ve `+' ve `-' ve `+' ve `+' ve `+' ve A+G- A+G- A+G- `+' ve `+' ve `+' ve `-' ve O `-' ve `+' ve `+' ve `+' ve P.flourescens
5 `+' ve `+' ve `-' ve `+' ve `+' ve `+' ve A+G- A+G- A+G- `+' ve `+' ve `+' ve `-' ve O `+' ve `+' ve `+' ve `+' ve P.flourescens
6 `+' ve `+' ve `-' ve `+' ve `+' ve `+' ve A+G- A+G- A+G- `+' ve `+' ve `+' ve `-' ve O `-' ve `+' ve `+' ve `-' ve P.flourescens
SF: Sugar fermentation, G: Glucose, S: Sucrose, L: Lactose, M: Maltose, A
+
: acid production, G
+
: gas production, MR: Methyl red test, VP: Proskauer Voges test, TSI: Triple sugar iron I:
Indole test, LYS and ORNI: Lysine, Ornithine decarboxylase, ARG: arginine dehydrolase test. C: citrate Utilization, MOF: Marine oxidation fermentation (I: inert) i.e. Neither Oxidation nor
Reduction, ST: Salt tolerance test, NIT: Nitrate reduction test.
M. Phani Kumar et al J. Microbiol. Biotech. Res., 2013, 3 (3):41-45
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Through presumptive identification, Pseudomonas is identified and through definitive identification, Pseudomonas
Species Pseudomonas fluorescens is identified.

DISCUSSION

Red disease often occurs during the change from the dry to rainy season and during the flood season in MRD [7].in
the present findings more number of infections was recorded in rainy seasons. This is similar with the findings of
[7].In the present study, it was observed that the signs of Red disease associated with often fraying and reddening of
fins, accompanied by irregular, variably sized areas of de-pigmentation as well as reddish pigmentation that can
develop anywhere on the body surface. The skin overlying these sites is eventually lost, exposing the muscle below.
These open sores or ulcers may remain superficial or they can be extensive and invade deeply into muscle, revealing
underlying bone in some cases. These ulcers often have ragged white margins bordered by a narrow zone of
hemorrhage. Infections can occur in any age fish, but losses are usually most severe in fry and small fingerlings.
Similar conditions of Red Disease were also reported in the rural carp culture by several authors [8]

Samples found positive for Pseudomonas were selected for species identification and accordingly bio-chemical tests
were conducted as per Bergey's Manual of Determinative Bacteriology. The putative isolates so selected were short
rods, gm negative and were lactose fermenators. It is further noted that all isolates were identified as P.florescens
standing in agreement with the work carried by Graham L. Bullock. The study indicates that motile aeromonad
septicemias are generally mediated by stress. Elevated water temperature [9], a decrease in dissolved oxygen
concentration, or increases in ammonia and carbon dioxide concentrations have been shown to promote stress in fish
and trigger motile aeromonad and Pseudomonas infections coinciding the work of [10].

P. fluorescens was originally described as the causative agent of Bacterial Hemorrhagic Septicemia disease of pond-
cultured fish [3]. It is considered as a primary pathogen of freshwater fish and opportunistic pathogen for different
fish species cultured in marine and brackishwaters worldwide [4]. In Bangladesh, P. fluorescens has been isolated
from eye surface and mouth lesions of diseased Rajpunti (Barbodes gonionotus) along with some other fish
pathogenic bacteria [5]. Isolation and characterization of P. fluorescens from gills of silver carp
(Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idella), African magur (Clarias gariepinus) and Nile
tilapia (Oreochromis niloticus) has also been reported elsewhere [6]. Concerning the clinical sings and postmortem
examinations of naturally bacterial affected fish, present study revealed that, the clinical signs were darkening of the
skin, and the most common finding is hemorrhage in skin, fins. Superficial ulceration of the epidermis in the oral
cavity and muscles, exophthalmia and as cites are commonly observed. Chronic motile aeromonad infections
manifest themselves primarily as ulcerous forms of disease, in which dermal lesions with focal hemorrhage and
inflammation are apparent. Both the dermis and epidermis are eroded and the underlying musculature becomes
severely necrotic [11].The liver may become pale or have a greenish coloration while the kidney may become
swollen and friable. These organs are apparently attacked by bacterial toxins and lose their structural integrity [11].
The liver of infected fish showed the same symptoms as discussed by [11]. Internally enlarged gall bladder, enlarged
kidney and congested, enlarged spleen, Haemorrhage on the skin of catfish and enteritis. The results nearly agree
with that obtained by [12-14].

With intensive fish farming systems, whether these systems are outdoor ponds or indoor aquaria and tanks,
predisposing factors are primarily responsible for the precipitation of this disease. Stress is the single most important
predisposing factor associated with this disease. Stress is due to poor management and/or poor water quality.
Management factors include: Nutrition, handling, transportation and over-crowding of fish. Water quality must be
excellent to prevent this disease. This means that the dissolved oxygen (DO), pH, temperature and alkalinity of the
water must be satisfactory, and that the ammonia, nitrite and CO
2
levels must be kept to a minimum.

REFERENCES

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Aquaculture. Springer, NACA and IDRC, Dordrecht, Bangkok and Ottawa, 2009, 133-149.
[2] Valerie, I., Roberts, R.J and N.R. Bromage,. Bacterial disease of fish, 1
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e d i t i o n . Institute of Aquaculture, Oxford,
Blackwell Scientific Publications, U.K. 1993.
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