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Journal of Environmental Biology �July, 2007�
Effect of Pseudomonas aeruginosa on the giant freshwater prawn, Macrobrachium
rosenbergii - histopathological and electron microscopic study
K. Ramalingam*1 and S. Ramarani2
1PG (Aquaculture) and Research Department of Advanced Zoology and Biotechnology,
Government Arts College, Nandanam, Chennai-600 035, India2Chellamal College for Women, Guindy, Chennai-600 035, India
(Received: August 04, 2005 ; Revised received: February 10, 2006 ; Accepted: March 29, 2006)
Abstract: The giant freshwater prawn Macrobrachium rosenbergii was injected with an inoculum containing LD50 96 hr dose of 107 Pseudomonas
aeruginosa (MTCC 1688) to determine the histopathological effects in vivo. The comparison of tissues of both the control and the bacterial endotoxin
treated prawns after 96 hr revealed significant degenerative changes in treated prawns. Both light microscopic and electron microscopic observations
revealed the infiltration of the tissues of Pseudomonas sp in the muscular and hepatopancreatic tissues of prawn. The muscular tissue changes in the
myofibrillar arrangement with blockage at the gap junctions and necrotic lesions were observed. The hepatopancreatic cells were vacuolated with
hypertrophied nucleus. Atrophy of hepatopancreatic tubules was conspicuous. The pathogenicity of Pseudomonas aeruginosa is attributed to its infiltration
and multiplication inside the tissues and the consequent release of extra-cellular enzymes for its metabolism. The degeneration of host tissues is also
attributed to the latter.
Key words: Macrobrachium rosenbergii, Pseudomonas aeruginosa, Extracellular proteases, EndotoxinPDF file of full length paper is available with author
Introduction
In shrimp culture, the disease outbreaks in growout ponds
have been a perennial threat to their growth and productivity. Such
diseases also bring heavy economic loss for the culturists and
investors due to mass mortality. Both bacterial and viral diseases
have been established in prawn/shrimp culture (Sugita et al., 1987;
Anderson, 1989; Mukherjee and Chandra, 1991 and Ra’-anan et
al., 1991; Ramalingam and Ramarani, 2006). The literature has
revealed that both pathogenic and non pathogenic bacterial
population exist in natural freshwater and marine water sources.
The complex nature of these disease outbreaks in crustaceans
have necessitated the need for an understanding of the causative
pathogens and their metabolism and the progress of their infection
in vivo in order to apply control measures and managerial strategies.
Several investigations have been carried out to understand
the different aspects of commercially important prawns in India.
However, the cases documented at present are not comprehensive,
and considerable research efforts are required to survey the diseases
that occur in growout ponds and to understand the host-pathogen
relationship, pathogenesis, disease diagnosis, biochemical profile of
the host and on specialized aspects like virology, bacteriology,
mycology and immunology.
The gross and histopathological findings, both at macro
(gross) and micro level, are of diagnostic value in the identification
of the nature of infection. Such identification/prognostic analysis of
symptoms in shrimp culture farms would go a long way in minimizing
the risks involved or in eliminating the pathogenic population from
water.
Towards this end, a time bound analysis of tissues of prawns,
after the inoculation of the pathogen would be of value in assessing
the pathological effects over a specific period. Since mortalities have
been reported to begin towards the end of the grow out period
when the shrimps approach the market size, the diagnosis of
pathogenic infection is crucial and prudent at this stage. In the present
study, the giant freshwater prawn inoculated with an LD50 96 hr
dose of Pseudomonas aeruginosa MTCC 1688 was subjected to
histopathological analysis. The period of observation was after 96
hours.
Materials and Methods
Specimens of Macrobrachium rosenbergii were collected
from the commercial shrimp farm at Kanathur, along the coastal area
of Chennai. After being brought to the laboratory, the giant prawns
were acclimated to the laboratory conditions in the stocking glass
tanks (salinity 2 ppt, temperature 28 + 2oC). The prawns were kept
for a minimum of 15 days prior to the experimentation as suggested
by Drach (1939) for aquatic crustaceans. Pseudomonas aeruginosa
is ubiquitous in the environment and is found in the water, soil and
on plants (Sabath, 1980). Pseudomonas aeruginosa was selected
as the biotoxin for the study, in view of its pathogenicity on
Macrobrachium rosenbergii. The bacterial strain was brought from
the Institute of Microbial Type Culture Collection and Gene Bank,
Chandigarh, India. Culture was done as presecribed by the above
Institute. The bacterial inoculum was prepared by the procedure
adopted by Lightner and Lewis (1975). The live bacteria were
harvested from 24 hr culture using sterile bacterial loop and mixed
with double distilled water. This was then diluted to two-fold serial
Journal of Environmental Biology July 2007, 28(3) 627-635 (2007)
©Triveni Enterprises, Lucknow (India) For personal use only
Free paper downloaded from: www. jeb.co.in Commercial distribution of this copy is illegal
Journal of Environmental Biology �July, 2007�
628 K. Ramalingam and S. Ramarani
dilutions of the bacterial suspension, which was made into different
dilutions viz., 108, 107, 106, and 105. About 0.05 ml of the inoculum
of the different dilutions was taken in 1 ml tuberculin syringe and
injected in between 5th and 6th abdominal segment of Macrobrachium
rosenbergii. The LD50 concentration of inoculum was 107. The
bacterial count for LD50 value of inoculum was calculated as 2.41 x
107 CFU/0.05 ml. The above LD50 dose of 107 was taken for the
study and injected into the prawns. The muscular, hepatopancreatic
tissues were taken for both light microscopic and electron microscopic
observations.
The muscular and hepatopancreatic tissues of control and
test groups were selected for histological studies. Tissues were
separated and immediately fixed in aqueous Bouin’s fluid (saturated
solution of picric acid -75 ml, Formaldehyde -20 ml and Acetic acid
5 ml) for a period of 24 hr. After fixing, they were washed overnight
in running water to remove the fixative. To remove the water content
present in the tissues, dehydration was carried out by transferring
them to a series of gradually increasing percentages of alcohol in
water. The materials were then cleared in xylol and embedded in
wax (melting point 52oC). Sections were cut at 7µ thickness and
stained with haematoxylin and eosin (Culling, 1957; Humason,
1972).
For electron microscopic study, the cuticle, muscular and
hepatopancreatic tissues of both control and test groups of
Macrobrachium rosenbergii were fixed for 24 hr at 4oC in
glutaraldehyde buffered to pH 7.2 with 0.1 M sodium cacodylate-
HCl containing 3% sucrose and 0.55 sodium chloride (Ramasamy,
1995). They were washed and stored at 4oC in 4% sodium
cacodylate buffer until required further. The tissues were post fixed
for one hour with 15% aqueous osmium tetraoxide, dehydrated
through ethanol to propylene oxide and embedded in Epoxy resin.
The section of 60-90 nm were cut using a Richter E Ultramicrotome
with diamond/glass knives. Sections were collected on copper grids
stained with uranyl acetate and lead citrate and examined using a
Philips Transmission Electron Microscopy operating at 100 KV.
Results and Discussion
In the uninfected control (0 hr) forms of shrimps, the muscular
tissue showed normal longitudinal arrangement of myofibrillar
filaments without any pathogenic infection. After 96 hr, muscle tissues
revealed the presence of melanized haemocytic granulomas in the
connective tissue around the haemal sinuses together with haemocytic
aggregation in necrotic musculature and broken myofibrillar
arrangement (Fig. 1).
The hepatopancreas revealed the presence of normal
hepatopancreatic tubules with F and B cells and normal brush
border in the control group of Macrobrachium rosenbergii. After 96
hr, multiple haemocytic nodules in the hepatocytes and increased
number of melanised haemocytes markedly affecting the tissue was
noticed (Fig. 2).
In the electron microscopic sections, the body muscle of
Macrobrachium rosenbergii showed the regular arrangement of
myofibrillar filament of the muscle tissue with the presence of
sarcoplasm and interstitial matrix in between the muscle bundles in
the control group (Fig. 3).
After 96 hr of inoculation with Pseudomonas aeruginosa,
MTCC 1688, the significant changes observed were, i) localization
of bacteria invading the muscle; ii) longitudinal shape of the muscle
being constricted, iii) loosened myofibrils with gap regions, iv)
disappearance of nuclear materials, and v) large necrotic lesions
with bacterial colonies incorporated in them (Fig. 5).
The hepatopancreas of uninfected shrimps showed the
presence of hepatopancreatic cells with intact nuclear membrane,
less number of nuclear pores and vacuoles, eccentric positions of
nucleolus and more number of microvilli (Fig. 3, 4).
After 96 hr of inoculation with Pseudomonas aeruginosa,
MTCC 1688, the changes observed were more number of vacuoles
and nuclear pores; hypertrophied nucleus; disappearance of
nuclear membrane and loss of microvilli at the apical surface of the
tubules (Fig. 6).
The structural morphology of cuticle in both uninfected
(control) and infected shrimps at 96 hr revealed marked difference
with reference to the intactness of the layer and also the permeability
as envisaged by the translucent appearance in the infected cuticle
(Fig. 4, 6).
The results of the histopathological study in the
Pseudomonas aeruginosa inoculated prawns revealed the
characteristic degenerative changes in both body muscle and
hepatopancreas. The abnormalities were greatly discernible
after 96 hr of inoculation. In the muscle the changes included, i)
muscular lesions containing bacteria, ii) changes in the
myofibrillar arrangement from longitudinal disposition to diagonal
direction, iii) necrotic changes, iv) melanised haemocytic
granulomas around the haemal sinuses, v) haemocytic
aggregation in the necrotic musculature and vi) broken
myofibrillar arrangements, etc. Likewise in the hepatopancreas,
the changes included i) melanised hepatopancreas tubules, ii)
haemocytic nodules and iii) irregular luminal cavity of the tissues.
Earlier workers have reported similar destructive changes in
the different tissues of shrimps due to both bacterial and viral
infections. Lightner (1984) reported the presence of a large
number of lesions in the tissues of hepatopancreas, antennal
gland and mandibular organs in the penaeid prawns infected
with bacteria. Leone et al. (1994) have reported haemocytes
infiltration alongside with nodule and granuloma formation in the
epidermis and connective tissues of Penaeus vannamei infected
with vibriosis. Jiravanichpasial et al. (1994) reported the
occurrence of bacteria phagocytosing haemocytes in the tissues
of prawns infected with Vibrio harveyi. Cai and Cai (1996)
Journal of Environmental Biology �July, 2007�
629Histopathology and EM studies on giant freshwater prawn
reported multi-organ necrosis affecting hepatopancreas, gills,
heart and gonads in their histopathological findings in
Macrobrachium rosenbergii infected with Torulosis. Esteve and
Herrera (1997), also revealed the tissues destruction and
accumulation of bacteria in the hepatopancreatic tubular lumen
of Penaeus vannamei infected with Vibrio anguillarum. Cheng
et al. (1998), reported the presence of melanized haemocytic
granulomas in the connective tissue around haemal sinuses
together with haemocytic aggregation in necrotic musculature in
Macrobrachium rosenbergii infected with Enteroccocus
bacterium. Similar to bacterial infection, Arcier et al. (1999)
reported various histopathological changes due to viral infection
in the post larvae of the giant freshwater prawn Macrobrachium
rosenbergii which were similar to bacterial infection.
Electron microscopic study in control and inoculum injected
groups of prawns revealed that in control group muscles, regular
arrangement of myofibrillar filaments of the muscle tissue was clear.
By contrast the muscles of prawns inoculated with Pseudomonas
aeruginosa, showed the following changes; viz., i) localization of
bacteria invading the muscle; ii) longitudinal shape of the muscle
being constricted, iii) loosened myofibrils, with gap regions; iv)
disappearance of nuclear materials; v) large necrotic lesions with
bacterial colonies infiltrated in them. Similarly in control group, the
hepatopancreas of Macrobrachium rosenbergii showed a normal
arrangement with intact nuclear membrane, less number of nuclear
pores and vacuoles, eccentric position of nucleolus and more
number of microvilli etc. By contrast, the hepatopancreas of prawns
inoculated with Pseudomonas aeruginosa showed the following
Fig. 1: Photomicrographs of body muscle of Macrobrachium rosenbergii
(MF = Myofibrillar filament, S = Sarcoplasm, G = Granuloma, B = Broken filament, Ne = Necrosis)
B
Control
D
Inoculated
A
400 x
C
400 x
200 x 100 x
Journal of Environmental Biology �July, 2007�
630
Fig. 2: Photomicrographs of hepatopancreas of Macrobrachium rosenbergii
(Lum = Lumen, Brb = Brush border, HPC = Hepatopancreatic cell, V = Vacuole, WL = Widened lumen,
M = Melanized hemocytes, Ht = Hepatopancreatic tubule, S = Sinus)
significant changes: i) more number of vacuoles and nuclear pores,
ii) hypertrophied nucleus, iii) disappearance of nuclear membrane,
iv) loss of microvilli at the apical surface of the tubules, v) atrophied
hepatopancreatic tubules and vi) more number of free ribosomes in
the cytoplasm. The ultrastructural changes revealing the invasion
of the bacterium Pseudomonas aeruginosa and the infiltration and
multiplication into colonies, which are discernible as occluded
particles seem to suggest that the complete immune mechanism of
host failed to resist their multiplication in vivo. Moreover, this bacterial
form Pseudomonas aeruginosa endowed with their own intrinsic
enzyme systems would have facilitated the degeneration of the
tissues and further extractions of the nutrient elements of the host
tissues for their own growth and multiplication. In this context, the
disassemblage of myofibrils in the Pseudomonas aeruginosa infected
muscle may be attributed to the extra cellular proteases characteristic
of the chitinoclastic bacteria and the consequent protein disintegration.
K. Ramalingam and S. Ramarani
B
Control
D
Inoculated
Az
400 x
C
400 x
200 x100 x
zzzzzzzzzzzzzzzz
Journal of Environmental Biology �July, 2007�
631
Fig. 3: Transmission electron micrographs of body muscle and hepatopancreas
(INM = Interstitial matrix, MF = Myofibrilar filament, S = Sarcoplasm, Hc = Heterochromatin,
NM = Nuclear membrane, M = Mitochondria, ER = Endoplasmic reticulum, V = Vacuole)
Histopathology and EM studies on giant freshwater prawn
Control - Body muscle
3000 x
15000 x 10000 x
Control - Hepatopancreas
3000 x
Journal of Environmental Biology �July, 2007�
632
Fig. 4: Transmission electron micrographs of cuticle and hepatopancreas
(MV = Microvilli, Control - Cuticle - Intactness of the layers)
Similar views were extended by Liu et al. (1997), Singh et al.
(1998), Herrera et al. (1999) and Gooday et al. (1999). Singh et
al. (1998), revealed that the growth and multiplication of vibrio
colonies in the muscles of Penaeus indicus occurs due to action of
extracellular enzymes and degradation of the metabolic nutrients in
the muscle tissue and the consequent uptake by the bacteria. Gooday
et al. (1999) have reported the destruction in the shape of the
longitudinal myofibrillar filament and the granulation of the myofibrils
inoculated with vibrios leading to muscle degradation. Liu et al.
(1997) have reported that the inoculated vibrios producing extra
cellular enzymes are the causative factor of cell lysis and their
components. Lamas et al. (1994), have reported that the host
K. Ramalingam and S. Ramarani
Control - Cuticle
1500 x
7000 x
Control - Hepatopancreas
7000 x
Journal of Environmental Biology �July, 2007�
633
Fig. 5: Transmission electron micrographs of body muscle
(DMF = Denatured myofibrillar filament, HC = Heterochromatin, Bc = Bacterial colony, NP = Nuclear pore,
V = Vacuole, Ne = Necrosis, L = Lesion, D = Diagonal)
Histopathology and EM studies on giant freshwater prawn
Body muscle - 96 hr
7000 x
Body muscle - 96 hr
4500 x
1000 x 7000 x
immune response was not able to resist the bacterial multiplication,
which results in the proliferation of bacterial colonies in the host
tissues. In this context it is of interest to note that in shrimps
infected with pathogens experimentally, phenoloxidase activity
and the total haemocyte count were drastically reduced.
Increased number of vacuoles noticed in the present study may
represent the degeneration of cells due to lack of lipid reserves,
which could have been used for bacterial metabolism. Similar
reports of tissue and cellular damage with reference to giant
freshwater prawn species are found mostly meager except a
few (Owens et al., 1992). The tissue degenerative changes in
both muscle and hepatopancreas as elucidated by both light
microscopic and TEM study in Macrobrachium rosenbergii
inoculated with Pseudomonas aeruginosa reveal the
pathogenicity of the bacterial population in vivo after 96 hr. The
significant changes as noticed in the permeability of the cuticle
by the translucent appearance in the inoculated prawns also
suggest that the exoskeletal barrier layer of the cuticle is being
Journal of Environmental Biology �July, 2007�
634
Fig. 6: Transmission electron micrographs of hepatopancreas and cuticle
(NPI = Nucleoplasm, DNM = Disappeared nuclear membrane, NP = Nuclear pore
V = Vesicle, M = Mitochondria, Cuticle – translucent appearance)
K. Ramalingam and S. Ramarani
Hepatopancreas 96 hr
3000 x
Cuticle - 96 hr
3000 x
4500 x7000 x
Journal of Environmental Biology �July, 2007�
635Histopathology and EM studies on giant freshwater prawn
disrupted by the extracellular proteases of the bacteria
thus enabling their in vivo infiltration.
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