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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Specimen Description
Organ: Gills
Observation: Gill filament
diffusion.
Total magnification: 400X
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Specimen Description
Organ: Gills
Observation: Gill hyperplasia
Total magnification: 400X
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Specimen Descrition
Organ: Gills
Suspected organism:
Diplectanum sp.
Total magnification: 100X
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Specimen Description
Organ: Gills
Suspected organism:
Pseudorhabdosynochus spp
Total magnification: 100X
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Specimen Description
Organ: Heart
Observation: No pathological
changes.
Total magnification: 4x
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Specimen Description
Organ: Liver
Observation: No pathological
changes.
Total magnification: 100X
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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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Specimen Description
Organ: Gill
Observation: Trichodinasp.
attached to gills filament.
The Trichodina sp.size is about
30 m.
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Specimen Description
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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Thank You
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