Effect of Pseudomonas aeruginosa rosenbergii ...Journal of Environmental Biology July, 2007 Effect...

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)

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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)


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


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


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


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


Control - Cuticle

1500 x

7000 x

Control - Hepatopancreas

7000 x


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


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)


Hepatopancreas 96 hr

3000 x

Cuticle - 96 hr

3000 x

4500 x7000 x


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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