studies on fungus infection in striped catfish (pangasianodon hypophthalmus) on grow out stage

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES STUDIES ON FUNGUS INFECTION IN STRIPED CATFISH Pangasianodon hypophthalmus ON GROW - OUT STAGE By NGO THI MONG TRINH A thesi

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

STUDIES ON FUNGUS INFECTION IN

STRIPED CATFISH (Pangasianodon hypophthalmus)

ON GROW - OUT STAGE

By

NGO THI MONG TRINH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of science in Aquaculture

Can Tho, January 2013

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

STUDIES ON INFECTION OF FUNGUS IN

STRIPED CATFISH (Pangasianodon hypophthalmus)

ON GROW - OUT STAGE

By

NGO THI MONG TRINH

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of science in Aquaculture

Supervisor

Dr PHAM MINH DUC

Can Tho, January 2013

ABSTRACT

This study was carried out the challenge test, the effects of salt and temperature on the

growth of fungus, histopathology of the fungus on striped catfish (Pangasianodon hypophthalmus) The fungus which was used for the experiment was the isolation in the

natural The total strains which isolating were 10 strains at An Giang province There were 5 strains of fungi that were used for the challenge test that has designed with two series The infection rate is 42.2% for 105 of concentration, and 45.6% for the 106 of concentration The re-isolation result showed that the fungi which were injected on fish were mainly occurred in swimming bladder 17.2%-25.9%, kidney 10%-22.5%, and seldom in gills 0%-6.7% of 105, and 23.3%-34.5% in swimming bladder, 13.3%-23.8%

in the kidney, and 0%-6.9% in gill of 106 for all strains The fungus can be grown differently when the temperature was different in the temperature test The result showed that the temperature was one of factor which infected to the growth of fungi colony The temperature which helped the fungi was good growth at 25oC-30oC, and the best growth was 28oC, and the fungi were lower growth at 20oC, and stopped growth at 35oC Beside that, the sodium chlorine was also infected to the growth of fungi The fungi were grown well on the GYA media with 0%-8% of sodium chlorine The histopathology was showed that the different between of healthy fish and fish which infected by fungi The healthy fish was not found the fungus on the sample of histopathology The mycelia were appeared on the fish which affected by fungal The result showed that was ensured the occurring of fungi on the striped catfish which isolated on the nature, and re-isolated on the challenge test

CONTENTS

Acknowledgements i

Abstract ii

Contents iii

List of tables vi

List of figures vii

Chapter I: Introduction 1

1.1 Introduction 1

1.2 Objective 2

1.3 Research contents 2

Chapter II: Literature review 3

2.1 Status of parasites disease 3

2.2 Disease caused by fungus 4

2.2.1 Fungus diseases on aquatic animal 4

2.2.2 Fungus diseases on fish 5

2.3 Methods using to study fungus 7

Chapter III: Materials and Methods 9

3.1 Place and time study 9

3.2 Materials 9

3.2.1 Fish samples 9

3.2.2 Equipments 9

3.2.3 Medium and chemicals 9

3.3 Methods 10

3.3.1 Observation on wet-mount 10

3.3.2 Making the wet-mount 10

3.3.3 Isolation and identification 10

3.3.3.1 Isolation method 10

3.3.3.2 Identification method 11

3.4 Examination the effect of temperature and sodium chlorine on fungus 11

3.4.1 The effect of sodium chlorine 11

3.4.2 The effect of temperature 11

3.5 The challenge test 11

3.5.1 Collecting zoospores method 11

3.5.2 The challenge experiment 12

3.6 Examine the histopathology 12

Chapter IV: Results and Discussions 13

4.1 Clinical sign 13

4.2 Isolation and Identification 14

4.2.1 Observation on the wet-mount 14

4.2.2 Isolation 14

4.2.3 Identification 14

4.3 The effect of sodium chlorine on growth of fungus 17

4.4 The effect of temperature on growth of fungus 18

4.5 The challenge test 19

4.5.1 The clinical sign 19

4.5.2 The challenge test result 19

4.6 Histopathology 22

Chapter V: Conclusions and Recommendations 24

References 25

Appendices 28

LIST OF FIGURES

Figure 4.1: The healthy fish and fungus disease fish in pond culture 13

Figure 4.2: Morphology characteristic of Fusarium sp 16

Figure 4.3: The characteristic of fish which using in the challenge test 21

Figure 4.4: The histopathology 23

CHAPTER I INTRODUCTION

1.1 Introduction

Striped catfish (Pangasianodon hypophthalmus) is an importance species which is

a high economy value, however, the farmers culture it with high density, and it is easy to make their fish get diseases There are many studies to search what kind of diseases which can infect to catfish General research is about pathology, the results that are catfish can be infected by bacterial, virus, parasites, or fungi Tu

Thanh Dung, et al (2002) was found the bacteria disease on catfish that is very

dangerous disease because it make more risk for farmers That bacteria is

Edwarsiella ictaluri, it infect on liver, kidney of catfish and make many death fish

Beside that, they also do research to find the way to treat this disease There are many studies to search about this kind of bacteria, and show way how to know

how many Edwarsiella ictaluri are infected on catfish (Nguyen Ngoc Dung and

Dang Thi Hoang Oanh, 2010) Beside that, Nguyen Thi Thu Hang and Dang Thi

Hoanh Oanh (2011) also found that catfish can be infected by Microsporidia; it is

a group of parasite

On the other hand, catfish can get disease from fungus, but there is no more research to study about that There is some research to find-out how fungi infect

on aquatic animals In 2004, Kanit Chukanhom and Kishio Haitai were isolated

freshwater fungi from eggs of common carp (Cyprinus carpio) in Thailand They were isolated some kind of fungus: Saprolegnia diclina, and Allomyces arbuscula Fungi can be caused disease for many species of fish such as: Snakehead (Channa striata), Climbing perch (Anabas testudineus) On study of Nguyen Thi Thuy Hang (2011), finding about the fungi which cause disease on Channa triata in

fingerling stage with purposing is identify and put the forward to solve problem In

2010, Tran Ngoc Tuan and Pham Minh Duc have the study on fungus which infected on climbing perch That study focus on biology characteristic of fungal

Study on fungus in Vietnam is not more, that is the reason why the research

“Studies on fungus infection on striped Catfish (Pangasianodon hypophthalmus)

in grow – out stage” was done

1.2 Objective of study

The aim is to describe isolation and identification of causative agents of fungal

infection found in the striped catfish (Pangasianodon hypophthalmus) in grow-out

stage Beside that, the research also determines the probability to cause disease of fungus which is the basic to prevent the fungus disease on striped catfish

1.3 Research contents of study

o To isolate and identify fungal infection in striped catfish disease

o To examine the effects temperature and sodium chlorine on the growth of fungus strains

o To observe histological characteristics of striped catfish with fungus infection

CHAPTER II LITERATURE REVIEW

2.1 The status of parasites diseases

According to Bui Quang Te (2011); Tran Thi Minh Tam et al (2003); Tran Anh

Dung (2005), they had the similar result that parasites disease can occur every time, but the pathological can breakout when the season change about the

temperature suddenly; and the main agents are Myxobolus, Trichodina, Ichthyophthyrius multifiliis, etc In study of Nguyen Thi Thu Hang and Dang Thi Hoang Oanh (2011), they found that striped catfish (Pangasianodon hypophthalmus) can be infected by one species of parasites with some white spots

in the body, especially focusing on belly area or lateral line when having more opalescent or milky oval shaped cysts attacked On first stage of this disease, it is difficult to identify because of no symptoms This disease is caused by

Microsporidia Besides that, there are many agents of parasites can cause

disease on aquatic animals Some species of Trichodina were infected on tilapia in pond culture of Mekong Delta When Trichodina spp infected on fish, there are

some symptom to recognize, such as: the mucus has opalescent fluid, the body change to grey color, fish swimming is lazily, stay on surface The disease can occur and develop after the temperature is decline, especially at rainy season

Trichodina spp is less caused disease on adult stage, but usually infected on

juvenile stage of fish (Vemedim Company) According to Bui Quang Te (2006),

Ichthyophthyrius multifiliis is one of dangerous agent to cause disease of fish The

clinical sign of this disease that is gill has more parasites attacked with small opalescent spot which can call “white spot” Skin and gill has more mucus with

pale color, fish come to lethargic Ichthyophthyrius mutifiliis which attacked on

gill can be break-out the filament and fish cannot get oxygen; fish can be sinking

on the bottom

2.2 Diseases caused by fungi

2.2.1 Fungi diseases on aquatic animals

Fungus is one of importance agent which is infection on aquatic animal Following

the study of Pham Minh Duc et al (2010) is about systematic fungus disease on aquatic animals This study focus on some genus of fungi: Saprolegnia, Achlya, Leptolegnia, and Aphanomyces (Lower fungus); and study on the group of Fusarium, Acremonium, Plectosporium, Ochroconis, Phoma, and Exophiala

(Higher fungus) That are some genus fungi which can infect on aquatic animals (Ishikawa, 1968; Egusa and Ueda, 1972; Lightner and Fontaine, 1975; Hatai and

Egusa, 1978; Alderman and Polglase, 1985; Kitancharoen et al., 1986; Hatai et al., 1986; Momoyama, 1987; Hatai and Kubota, 1989; Kitancharoen et al., 1995;

Kitancharoen et al., 1997; Hussein and Hatai, 1999; Diler and Bolat, 2001;

Chukanhom and Hatai, 2004; Khoa et al., 2005; Khoa and Hatai, 2005; Munchan

et al., 2009; Duc et al., 2009; Duc and Hatai, 2009; Duc, 2009) The main characteristic of lower fungus is that the hyphae are no having septate (de Hoog et al., 2000) Saprolegnia, Achlya, Aphanomyces are main agents to cause disease,

the hyphae of that fungi are thick and stick together; and can be reproduced with two ways: asexual and sexual (Neish and Hughes, 1980; Yanong, 2003) Higher fungi which can be identified by the characteristic are had the septum on hyphae

(de Hoog et al., 2000)

In the world, there are many studies on fungi that can cause diseases on aquatic animals, especially, the fungus isolated on crustaceans and mollusks Kitancharoen

et al (1994) isolated and identified the fungus Atikinsiella awabi which infecting

on abalone (Haliotis sieboldii), shrimp (Pandalus Hypsinotus) in Japan which is very sensitive with Atikinsiella awabi The other species of that was also caused disease on shell of abalone (Haliotis sieboldii), that species was Atikinsiella dubia which isolated by Nakumura and Hatai (1995); and Atikinsiella panulirata

infected on crayfish (Panulirus japonicus) identified by Kitancharoen and Hatai

(1995)

Freshwater prawn (Macrobrachium rosenbergii) also got fungus disease, causing

by Fusarium sp.; Black tiger shrimp (Penaeus monodon) and crayfish (Homarus americanus) were attacked on gill to cause black gill disease by Fusarium incarnatum (Burns et al., 1979; Lightner and Fontain, 1975; Khoa et al., 2004) There are some species of Fusarium genus which were agents to cause black gill

on kuruma prawn (Penaeus japonicus), that were Fusarium solani, Fusarium mooniliforme, and Fusarium oxyporum (Egusa and Ueda, 1972; Bian and Egusa, 1981; Khoa and Hatai, 2005) Fusarium solina also infected on turtle (Carreta carrete) (Hose et al., 1984; Cabanes et al., 1997) One agent which causing the black gill disease on Astacus leptodactylus; and causing brown disease on Oratosquilla oratoria, that is Acremonium spp.; in the laboratory condition, that fungi were infected on Penaeus japonicus (Diler and Bolat, 2001; Duc et al.,

on egg and larvae stage of swimming crab (Callinectes sapidus) and other species

of swimming crab (Portunus pelagicus); and caused the disease on larvae stage of mud crab (Scylla rerata), shrimp (Pandalus hysinotus) (Nakamura et al., 1994; Roza and Hatai, 1999; Nakamura and Hatai, 1995; Hatai et al., 2000)

2.2.2 Fungi diseases on fish

In 2010, Tuan study on fungus in climbing perch (Anabas testudincus); and the result showed that there were three genus which isolated Fusarium, Acremonium,

and Geotrichum In this case, Geotrichum has highest occurring frequency (60%), 30% is occurring frequency of Fusarium, and less occurring frequency was Acremonium (10%) The symptom of this disease that was more mucus on the fish body, and scale was lumpy The result of Saylor et al (2010) found that Aphanomyces invadans was the agent to cause Ulcerative on snakehead (Channa striata) Epizootic Ulcerative Syndrome (EUS) was the dangerous disease; it can

be transmissions very rapidly and make more risk for farmers in the world That disease caused by more agents: bacteria, parasites, and some lower fungi group

such as: Aphanomyces spp., Saprolegnia spp., and Achlya sp However, the main agent that causes ulcerative was Aphanomyces invadans If having suitability condition, Aphanomyces invadans will attack to the tissues on fish body, and

releasing the enzyme to breakup the protein to make the ulcerative on body of fish

(Hoa et al., 2004) In 1984, Hatai isolated this fungus on fish which having ulcerative on body Saylor et al (2010) also found that Aphanomyces invadans was fungi which cause disease on snakehead (Channa triata) in United States Channa triata was very sensitive with Aphanomyces invadans When isolated the fungus on snakehead (Channa triata), Lilley et al (1997) also saw that fungi cause EUS disease Hoa et al (2004) found that some symptoms to recognize EUS

disease: less or no feeding, less active, when swimming the injury of fish stay in water surface The skin of fish change to dark color, the injury was grey and

widely to make large ulcerative, missing scales, and haemorrhage (Saylor et al.,

2010) When fish got seriously disease, the ulcerative was very large which can see the bone and the internal organs However, internal organs do not have any

disease symptoms, when surgery of fish (Hoa et al., 2004) EUS disease can be transmitting by water quality, fish transmission Actually, Aphanomyces invadans was only infected on fish when they have the primary disease which cause some necrosis on fish body because Aphanomyces invadans was secondary

agent to cause disease Therefore, protecting fish in good condition plays the

importance role when culturing fish In 2007, Panchai et al isolated the Achlya

bisexualis which infected on tilapia in Thailand Achlya bisexualis was white

colonies, it has two parts: one part was attached on body of fish; the other part was freed on environmental It can grow GY liquid medium, the result showed that the hyphae were touch, no septate; the suitability temperature was about 25-30oC, and cannot grow at 40oC, salinity was 15ppt, and grown at 20-40ppt There were some

studies in Singapore, Sinmuk et al (1996) was showed that Aphanomyces sp infected on Pelodiscus sinensis; and Hanjavanit et al (1997) also isolated Aphanomyces sp on ornamental fish

There were many species of fungi can cause diseases on egg and larvae stage of aquatic animals Following the result of the survey, there are four genuses of fungi

that can infect on snakehead (Channa triata): Acremonium, Geotrichum, Aphnomyces, and Fusarium They also known that the species was Aphanomyces frisgidofilus which made the egg of aquatic animal could not hatch In the other study, Czeczuga et al (1996) believed that salmon fish in egg stage were very sensitive with more genus of fungi Leptolegnia, Saprolegnia, and Achlya In

Thailand, the egg stage of tilapia also got diseases by some common species of

fungi that are Saprolegnia diclina, Achlya klebsiana, and Allomyces arbuscula

40oC and pH is 6-8 that are good condition for Allomyces arbuscula growth

2.3 Method using to study fungi

Fungus which was one of agent to cause disease on aquatic animals has more studies in the world The fungus diseases can study base on symptoms, morphology, and biochemical characteristic of isolated fungal Isolation, identification, and culturing on agar medium are the common using method (Hatai

and Egusa (1979); Neish and Hughes (1980); Khoa et al., 2004; Duc et al., 2009)

There are some medium usually use such as: Cherry-decoction agar, Cornmeal agar, Oatmeal agar, Potato-dextrose agar, Glucose-glutamate, Gys-tellurite, or

YpSs-tellurite (Gams et al., 1980) Beside that, GYA, and PGYSA media which

using with fungus caused disease on freshwater and brackish water (Hatai and

Egusa, 1979); Khoa et al., 2004; Duc et al., 2009) Some antibiotic which were

streptomycin and ampiciline were usually used to add in medium for isolating the

fungi (500µg/ml) (Khoa et al., 2004; Duc et al., 2009) The way to make pure

culture was that cutting the small part of agar which contents fungus and move to

other Petri dish, replacing on 2-3times (Gams et al., 1980; Ho and Ko, 1997)

The tradition method for studying the fungus was using popularly, nowadays, the biological molecular which development rapidly was also using to check the occurring of fungus on the sampling (Atkins and Clark, 2004) This method was popular for diagnostic on aquatic animals; some technical was used: PCR, restriction enzyme digestion, probe hybridization, and microarray (Altinok and Kurt, 2003; Atkins and Clark, 2004)

Histopathology is applying to evaluate the occurring of fungus in sampling There are some stain methods such as: Hematoxyline and Eosin (H&E), Grocott’s stain, PAS stain Grocott’s stain is used methenamine-silver to find fungus which their hyphae can take brown and black color Periodic – Schiff acid is only used for PAS stain to identify the fungus that hyphae can take pink and red color (Johnson

et al., 2004; Hanjavanit et al., 2008; Duc and Hatai, 2009)

CHAPTER III MATERIALS AND METHODS

3.1 Place and study time

The study was carried out in An Giang province from June to December in 2012 The experiments were done at the Department of Aquatic Biology and Pathology

of Can Tho University

3.2 Materials

3.2.1 Fish samples

The fish sample will be collected in one month with 2weeks/time The fish which has some characteristics: necrosis, spots, or injury on the body was collected The healthy fish was also collected because of making the comparative There were 65 fish which collected, in side; there were 55 diseases fish, and 10 healthy fish

3.2.2 Equipments

There are many types of equipment which used in this study such as: Petri dish, test tube, surgical, alcohol lamp, micropipette, trays, cork borer No.2, fridge, microscope, and incubator These equipments which using to isolate the fungus on fish, culture the fungus to get pure culture, observe the characteristics of fungus, and keep the fungus in the long time

3.2.3 Medium and chemicals

The medium that used in this study was GYA media with the ingredients: Glucose (1%), Yeast extract (0.25%), and Agar (1.5%) That medium used to isolate the fungus on fish, and culture the fungus

This study was also used some the chemicals which was saline solution using to treat the specimen before isolation; cotton blue using to stain the sample to find out the mycelium on the fish or observing the characteristic of mycelium Some

Following the method of Gams et al., (1980); Hatai et al., (2000), using the naked

eyes to observe the samples They have ulcerative, or some white bunch on fish body; we will start to observe with wet-mount on microscope

3.3.2 Making the wet-mount

To take a part of sampling; drop one drop of saline solution; Cover the sample with cover-slip; Observing on microscope in 20X or 40X to find the occurring of mycelia or spore, and determination that the mycelia have or do not have septum

(Gams et al., 1980; Hatai et al., 2000)

3.3.3 Isolation and identification

3.3.3.1 Isolation method

Following Hatai et al., (2000), fresh specimen is observed on microscope If the

hyphae are occurred, isolation will start Washing that sampling 3 times with saline solution (0.85% NaCl), after that, inoculating into the GYA medium which added streptomycin (500µg/L) to inhibit other transmission; starting to incubate at

28oC, in 4 days in the incubator

After 4 days, the fungus grew on GYA medium, in this time, culturing the fungus which was 2-3 times to get pure culture Cutting a small part of colony with 3-5mm diameter of agar, bring it to the new GYA medium to take pure culture

(Hatai et al., 2000)

3.3.3.2 Identification method

The fungus culture was on the lame which followed to the Riddells’s that was

done in the sterilization condition (Gams et al 1980) The details showed on the

3.4.2 The effect of temperature

The temperature is a factor to affect about the growth of fungus This study was done the experiment to test the growth ability of fungus at differ temperature (Khoa and Hatai, 2003)

The pure culture of higher fungi were cultured on GYA medium at 280C in 5 days Next, cutting a small part of agar which content the fungal to the other GYA medium and incubating in five differ temperature: 20, 25, 28, 30, and 35oC This experiment is replaced 3 times Following and taking note about the diameter of colony in 5 days

3.5 The challenge test

3.5.1 Collecting zoospores method

The method to collect zoospores for challenge test was accorded to Pham Minh Duc, Shinpei Wada, Osamu Kutara, and Kishio Hatai; 2010 The details were showed on the appendix 3

The way to determine the number of zoospore (Duc, 2009)

M= B x 104

Where:

M is density of zoospore (spore/ml)

B is total spore in 25 plots

3.5.2 Challenge experiment

The challenge test is a way to test the transmission of disease The experiment was done with two treatments, two replications and one control The test took care in 4 weeks

The fish which was choice for challenge test must be good healthy with the body weight is about 25g and length is about 16-18cm Experiment was designed with

10 fish in one tank (60 litters/tank) with two replications The fish was cultured in the tank (0.5 m3) 15 days, and check the parasite and bacteria before was done in the experiment The fish was injected with 0.2ml of conidia per one fish in the treatment tank, and 0.2ml per one fish of sterile saline solution (0.85% NaCl) in the control tank

3.6 Examine histopathology

Fish samples were collected and fixed in neutral formalin solution 10%, expediting processing, casting blocks, cutting samples and staining samples follow H&E The details were showed on appendix 13, 14, and 15 There were 20 samples which used in this experiment; in case, 15 samples that infected by fungus and 5 samples did not cause disease by fungus

3.7 Data analysis

Excel was used to analyze the data in this study

CHAPTER IV RESULTS AND DISCUSSIONS

4.1 Clinical sign

Striped catfish (Pangasianodon hypophthalmus) was infected by fungus which

was not having clearly clinical sign, it was difficult to recognize This disease was occurring in the pond which cultured fish on 3-5 months, fish was no feeding, sluggish, and swimming on the surface When the fish was surgical, the main clinical sign was the swimming bladder that was swollen with white fluid and bubble (Fig 4.1)

Figure 4.1: The healthy fish and fungus disease fish in pond culture (A): The fish which non-fungus infection; (B): The fluid and bubble in the swimming bladder of stripped catfish; (C): The fungus colony in the GYA medium; (D): The macroconidia of fungus with cotton blue staining (arrows showing)