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INVESTIGATION OF CAUSAL

PATHOGEN IN GROUPER, Epinephelus


spp. BY SCANNING ELECTRON
MICROSCOPE (SEM) AND
HISTOLOGY
MOHD FAZRUL HAFIZUL BIN MAHAMAD NON
UK23504
B.Sc of AGROTECHNOLOGY (AQUACULTURE)
SUPERVISOR: DR SANDRA C. ZAINATHAN

Background study

Groupers are widespread in warm and temperate


waters of all the seas and oceans of the planet. They are
of considerable economic value, especially in the coastal
fisheries in subtropical and tropical areas (Pierre et al.,
2008)
The expanding trade and demand in live groupers of
various ages and stages, whether for aquaculture or
seafood restaurants, has increased since 2006 (FAO,
2012)
In Malaysia, the production of grouper includes wildcapture 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 infection
of virus, bacteria and parasites.

Production of grouper in Malaysia. (DOF, 2012)

Problem Statement

The cultured grouper in Besut, Terengganu have


been 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.

Significant of study
The causal pathogen in grouper can be detected
and the prevention or treatment can be applied
in the future.
This study will provide awareness about
diseases in grouper.
By using scanning electron microscope, the
causal pathogen can be identified quickly.

Objective of the study

To detect the causal pathogen in grouper using


Scanning Electron Microscope (SEM) and to
observe histological changes by histology.

Literature Review

Taxonomy of grouper, Epinephelus fuscoguttatus


Kingdom

: Animalia

Phylum

: Chordata

Class

: Actinopterygii

Order

: Serranidae

Family

: Serranidae

Genus

: Epinephelus

Species

:Epinephelus fuscoguttatus

Scanning electron microscope


Electron microscopy (EM) has long been used in the
discovery 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 the
interaction 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).

Scanning electron microscope

Species
Pathogen
Cromileptes altiveli,
Protozoan
Epinephelus bontoides, E. coioides, E. malabaricus,
E. tauvina, E. bleekeri, E. malabaricus, E. suillus, E.
tauvina.

References
Nagasawa et al.,
2004

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


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

Nagasawa et al.,
2004

Epinephelus coioides, E. malabaricus,


Digeneans
E. tauvina.
Epinephelus coioides, E. malabaricus, Cromileptes Nematodes
altivelis and
Plectropomus leopardus

Nagasawa et al.,
2004
Nagasawa et al.,
2004

Epinephelus coioides, E. fuscoguttatus, E.


malabaricus, Cromileptes altivelis
and Plectropomus leopardus

Copepod

Nagasawa et al.,
2004

Epinephelus coioides and E. malabaricus

Isopods

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

Leeches

Nagasawa et al.,
2004
Nagasawa et al.,
2004

Materials & Methods

The experimental animals for this study is the grouper,


Epinephelus spp. The samples were obtained from Besut,
Terengganu.
Two methods that were used in this study which are: Histology
Scanning electron microscope

The organs that were used in this study are gills, liver
and heart.

Histology Method
Sample fixation
in Davidsons
fixative.

Staining

Mounting

Dehydration

Sample
embedding

Sample slicing

Observation

Sample Preparation

SEM Method

Fixation

Rinse
Postfixation
Rinse

Sputter coater
Dehydration

CPD

Mounting

Samples scanning
Sputter Coater
Scanning

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 ulcers
and pale gills.

Sample

Clinical sign

Darkened body color

Excessive mucous secretion

Sample

Clinical sign

Skin ulcers

Pale gills

Histology
Ten samples were processed.
No pathological changes were found in
the liver and heart samples.
10 out of 20 gills showed pathological
changes such as hyperplasia, gills diffusion
and gill necrosis.

Specimen

Description

Organ: Gills

Observation: No pathological
changes
Total magnification: 100X

Specimen

Description

Organ: Gills

Observation: Gill filament


diffusion.
Total magnification: 400X

Specimen

Description

Organ: Gills

Observation: Gill hyperplasia


Total magnification: 400X

Specimen

Descrition

Organ: Gills
Suspected organism:
Diplectanum sp.
Total magnification: 100X

Specimen

Description

Organ: Gills

Suspected organism:
Pseudorhabdosynochus spp
Total magnification: 100X

Specimen

Description

Organ: Heart
Observation: No pathological
changes.
Total magnification: 4x

Specimen

Description

Organ: Liver
Observation: No pathological
changes.

Total magnification: 100X

Scanning Electron Microscope


For scanning electron microscope, five samples were
processed.
Liver and heart: no causal pathogen was detected in
liver and heart.
For gills, one species of parasite which is Trichodina sp.
were found on the samples.
Trichodina sp. was found attached on the gill arch and
gill filament.

Specimen

Description

Organ: Gill
Observation: Trichodina sp.
attached to gills filament.
The Trichodina sp. size is about
30 m.

Specimen

Description

Organ: Gill
Observation: Trichodina sp.
attached to gill arch.

Discussion
Trichodina sp.

Trichodina sp. 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 the
suction 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.

Diplectanum sp.

Other pathogen that found on the gills is parasite


which is Diplectanum sp.
Diplectanum sp. were observed to penetrate in the
basal membrane of primary lamella where they
induced a hyperplastic response (Purivirojkul,
2012).
The infected fish will have: Difficulty in breathing.
Fish swimming in jerky motions.
Gills hyperplasia and increased mucus.

Pseudorhabdosynochus spp.

The third suspected parasite that found on the fish


gills is Pseudorhabdosynochus spp.
The signs of the Pseudorhabdosynochus spp. infection
is same with the Diplectanum sp. infection.
The infection of the parasites being considered as
the source of stress to cultured fishes and the
major contributing factor to disease outbreaks
(Leong, 1997).

Conclusion
The causal pathogen that cause mortality in
grouper were found.
Prevention and treatment can be made to
reduce the mortality rate in the farm.

Further Research

In the future, large scale of samples can be


used to confirm the parasites infection in
the farm that cause mortality.

References
Abdel-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 n-shes 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-recentadvances/
Pierre, S., Gaillard, S., Prvot-D'Alvise, N., Aubert, J., Leung-Tack, D., Grillasca, J-P. and RostaingCapaillon, 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

Thank You