causal pathogen in grouper

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INVESTIGATION OF CAUSAL

PATHOGEN IN GROUPER, Epinephelus

spp. BY SCANNING ELECTRON

MICROSCOPE (SEM) ANDHISTOLOGY

MOHD FAZRUL HAFIZUL BIN MAHAMAD NON

UK23504B.Sc of AGROTECHNOLOGY (AQUACULTURE)

SUPERVISOR: DR SANDRA C. ZAINATHAN


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Background study

Groupers are widespread in warm and temperatewaters of all the seas and oceans of the planet. They areof considerable economic value, especially in the coastalfisheries in subtropical and tropical areas (Pierre et al.,2008)

The expanding trade and demand in live groupers ofvarious ages and stages, whether for aquaculture orseafood restaurants, has increased since 2006 (FAO,2012)

In Malaysia, the production of grouper includes wild-

capture and aquaculture industry. The main countries that involve in grouper culture

include Pulau Pinang, Perak, Johor, and Selangor.

Generally, cultured fish is more exposed to the infectionof virus, bacteria and parasites.


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Production of grouper in Malaysia. (DOF, 2012)


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Problem Statement

The cultured grouper in Besut, Terengganu havebeen affected by an unknown pathogen.

The infection of the unknown pathogen is causing

mortalities in the grouper culture in Besut.

High mortality rate can cause significant losses to

the production of grouper.


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Significant of study

The causal pathogen in grouper can be detectedand the prevention or treatment can be applied

in the future.

This study will provide awareness aboutdiseases in grouper.

By using scanning electron microscope, the

causal pathogen can be identified quickly.


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Objective of the study

To detect the causal pathogen in grouper usingScanning Electron Microscope (SEM) and to

observe histological changes by histology.


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Literature Review

Taxonomy of grouper, Epinephelus fuscoguttatus

Kingdom : Animalia

Phylum : Chordata

Class : Actinopterygii

Order : Serranidae

Family : Serranidae

Genus : Epinephelus

Species :Epinephelus fuscoguttatus


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Scanning electron microscope

Electron microscopy (EM) has long been used in thediscovery and description of pathogen.

Scanning Electron Microscopy (SEM) are extremely

useful tools for the ultrastructural examination of

prokaryotic cells as well as for the study of theinteraction between pathogens and host cells (Mendez,

2007).

The instrument combines the advantages of viewing

larger areas of the specimen with a magnification range(x50 to x50, 000) and resolving power (200 A).


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Scanning electron microscope


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Species Pathogen

References

Cromileptes altiveli,

Epinephelus bontoides, E. coioides, E. malabaricus,

E. tauvina, E. bleekeri, E. malabaricus, E. suillus, E.

tauvina.

Protozoan Nagasawa et al.,

2004

Epinephelus bleekeri, E. bontoides, E. coioides, E.

malabaricus, E. tauvina, E. fuscoguttatus, E.

lanceolatus and E. tauvina, Cromileptes altivelis,.

Monogeneans Nagasawa et al.,

2004

Epinephelus coioides, E. malabaricus,

E. tauvina.

Digeneans Nagasawa et al.,

2004

Epinephelus coioides, E. malabaricus, Cromileptes

altivelis and

Plectropomus leopardus

Nematodes Nagasawa et al.,

2004

Epinephelus coioides, E. fuscoguttatus, E.

malabaricus, Cromileptes altivelisand Plectropomus leopardus

Copepod Nagasawa et al.,

2004

Epinephelus coioides and E. malabaricus Isopods Nagasawa et al.,

2004

Epinephelus bleekeri,

E. coioides, E. fuscoguttatus, E. lanceolatus, E.malabaricus and Cromileptes altivelis

Leeches Nagasawa et al.,

2004


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Histology Method

Sample fixationin Davidsons

fixative.Dehydration

Sampleembedding

Sample slicingStaining

Mounting Observation


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SEM MethodSample Preparation

Fixation

Rinse

Postfixation

Rinse

Dehydration

CPD

Mounting

Sputter Coater

Scanning

Samples scanning

Sputter coater


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Results

20 samples were used in this study.

Most of the samples showed the clinical

sign such as darkened body color,

excessive mucous secretion, skin ulcersand pale gills.


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Sample Clinical sign

Darkened body color

Excessive mucous secretion


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Sample Clinical sign

Skin ulcers

Pale gills


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Histology

Ten samples were processed.

No pathological changes were found in

the liver and heart samples.

10 out of 20 gills showed pathologicalchanges such as hyperplasia, gills diffusion

and gill necrosis.


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Specimen Description

Organ: Gills

Observation: No pathological

changes

Total magnification: 100X


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Organ: Gills

Observation: Gill filament

diffusion.



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Organ: Gills

Observation: Gill hyperplasia



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Specimen Descrition

Organ: Gills

Suspected organism:

Diplectanum sp.



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Organ: Gills

Suspected organism:

Pseudorhabdosynochus spp



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Organ: Heart


changes.

Total magnification: 4x


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Organ: Liver


changes.



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Scanning Electron Microscope

For scanning electron microscope, five samples wereprocessed.

Liver and heart: no causal pathogen was detected in

liver and heart.

For gills, one species of parasite which is Trichodinasp.

were found on the samples.

Trichodina sp. was found attached on the gill arch and

gill filament.


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Organ: Gill

Observation: Trichodinasp.

attached to gills filament.

The Trichodina sp.size is about

30 m.


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Organ: Gill

Observation: Trichodinasp.

attached to gill arch.


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Discussion

Trichodina sp. Trichodinasp. is one of Trichodinids species that found in

cultured marine and freshwater fish (Abdel-Baki, 2011).

Trichodina sp. inhabit the surface of fish, adhere through thesuction on the epithelium may cause damage such as

dermatitis and hyperplasia. Fish with Trichodina sp.infection will have fin erosion or

ulcerations and respiratory and osmoregulatory difficulty(Smith, 2009).

Respiratory function can be impaired in gills infection.

The signs of infected fish is:- High respiration rate

Excess mucous on gills, skin and fins.

Flashing.


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Diplectanumsp.

Other pathogen that found on the gills is parasitewhich is Diplectanumsp.

Diplectanum sp. were observed to penetrate in thebasal membrane of primary lamella where theyinduced a hyperplastic response (Purivirojkul,

2012). The infected fish will have:-

Difficulty in breathing.

Fish swimming in jerky motions.

Gills hyperplasia and increased mucus.


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Pseudorhabdosynochusspp.

The third suspected parasite that found on the fishgills is Pseudorhabdosynochus spp.

The signs of the Pseudorhabdosynochus spp. infection

is same with the Diplectanumsp. infection.

The infection of the parasites being considered asthe source of stress to cultured fishes and the

major contributing factor to disease outbreaks

(Leong, 1997).


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Conclusion

The causal pathogen that cause mortality ingrouper were found.

Prevention and treatment can be made to

reduce the mortality rate in the farm.


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Further Research

In the future, large scale of samples can beused to confirm the parasites infection in

the farm that cause mortality.


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ReferencesAbdel-Baki, A.S., Sakran, T., Fayed, H. and Zayed, E. (2011). Trichidona fahaka in

Tertradon fahaka from Nile River, Egypt. Scientific Research and Essays

6:153-1587

FAO, 2012

Leong, T.S. (1997) Control of parasites in cultured marine fin-fishes in Southeast Asia

an overview. International Journal forParasitology, 27, 1,1771,184

Lio-Po, G., and de la Pena, L. (2004).Diseases of cultured groupers. Retrieved

http://rfdp.seafdec.org.ph/publication/manual/grouper/

Mendez-Vilaz, A., and Diaz, J. (2007). Modern Research and Educational Topics in

Microscopy. Formatex. p122-131

Nagasawa, K. and E. R. Cruz-Lacierda (eds.) (2004). Diseases of cultured groupers.

Southeast Asian Fisheries Development Center, Aquaculture Department, Iloilo,

Philippines. 81 p.

Purivirojkul, W. (2012). Histological Change of Aquatic by Parasitic Infection. Retrieved

from http://www.intechopen.com/books/histopathology-reviews-and-recent-

advances/

Pierre, S., Gaillard, S., Prvot-D'Alvise, N., Aubert, J., Leung-Tack, D., Grillasca, J-P. and Rostaing-

Capaillon, O. (2008) Grouper aquculture: Asian Success and Mediterranean Trials.

Aquatic conservation;18: 297-308

Smith, S. and Schwarz, M. S. (2009). Dealing with Trichidina sp. and Trichodina-like

species. Virginia Cooperative Extension. Publication 600-305

Vale, F. F., and Correia, A. C. (2010). Applications of electron microscopy to virus

detection and identification. Microscopy: Science, technology,applications and educations


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causal pathogen in grouper

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