FINAL REPORT TRAINING COURSE ON TROPICAL FISH HEALTH

at the

FACULTY OF FISHERIES AND MARINE
SCIENCE, UNIVERSITY PERTANIAN
MALAYSIA

(UNDER NACA/FAO PROGRAMME)

4 July 1988 to 3 March 1989

BALBIR SINGH

CENTRAL INLAND CAPTURE FISHERIES RESEARCH INSTITUTE
(Indian Council of Agricultural Research)
BARRACKPORE-743101, WEST BENGAL, INDIA

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EXECUTIVE REPORT IN RESPECT OF Dr. BALBIR SINGH, SCIENTIST (SG) ON TROPICAL FISH HEALTH PROGRAMME AT MALAYSIA FROM 4 JULY to 3 MARCH 1989

Various protozoans diseases, helminth infestations and crustacean ectoparasites were dealt within sufficient depth in order to have a broad knowledge of parasitic disease affecting cultured fishes and shellfishes. The major bacterial diseases of tropical fishes and shellfishes i.e. haemorrhagic septicaemias, vibriosis, myxobacteriosis were studied in detail. Other bacterial diseases of less importance were also briefly reviewed.

Comprehensive studies of the fungal diseases of fish and shellfish, with emphasis on integumentary and systemic mycoses, including fish mycotoxicoses were done. The toxicoses of fishes were described with an emphasis on pathology and diagnosis. The course included an extensive study of the non-infectious diseases of fishes including nutritional diseases, malformations and deformations, toxic algal blooms, genetic diseases, malformations and deformations, toxic algal blooms, genetic disease, traumatic injuries, diseases caused by physical and chemical factors in the water, neoplasias and diseases of unknown aetiology, Diagnostic techniques and pathology applicable to invertebrate diseases, pharmacology, chemotherapy, anaesthesia, and the management aspects of disease prevention were studied in detail.

A review of current fish quarantine regulations and systems throughout the world was undertaken. The “ideal” quarantine system, diagnostic tests, recommended procedures and laboratory systems needed to impliment such a system were discussed.

The knowledge gained by the training will help in greater understanding of the relationship between the environment and ulcerative disease syndrome in fish and development of much needed facilities. Training in research methodology and techniques will enable me to conduct research into new methods and techniques of rapid diagnosis and improved methods in treatment and control of fish disease. The gained knowledge will also help in developing fish quarantine regulations and systems in India.

( Balbir Singh )

1. Introduction

One of the constraints identified in the rapidly expanding aquaculture industry of the Southeast Asian region is the lack of skilled resource personnel in fish health. The implimentation and management of fish quarantine and health extention services is dependant on the availability of specialists in fish health. The programme was offered as an attempt to meet present and future requirement for specialist fish health workers in government and private sectors. Throughout the programme emphasis was laid on the aetiology, epizootiology, pathogenesis, diagnosis, treatment and prevention of health problems affecting commonly cultured tropical food fishes.

The following aspects were studied during the course

2. Aquatic Biology

Reviewed the principles and concepts in ecology, water chemistry and their applied implication in aquaculture and fish disease management. Studied the ecosystem concept with special reference to energy flow, food chain/web, water quality criteria, air-water-soil chemistry, fertilizers, and accumulated waters in modified ecosystems and physical, chemical & biological phenomena. The general taxonomy and biology of cultured fish and shellfish of the region were discussed at length.

A detailed examination of the integument, skeletal, muscular, circulatory, respiratory, heamatopoietic, excretory, digestive, reproductive and central nervous systems and organs of special sense were studied. Basic histology and histology of the different organ systems of the fish, functions and adaptation of fish particular to the aquatic environment osmoregulation, respiration, circulation, excretion and endocrinology were also studied.

3. Aquaculture Science

The role of water and soil in aquaculture and its implication were focussed on aquaculture systems and practices in the Southeast Asian region in general and Malaysia in particular. Applied aspects of fish nutrition and genetics were also studied.

4. Biology of pathogens

Studied the procaryotic cell, its morphology, growth characteristics and requirements. Bacterial taxonomy and the classification of fish bacteria, microbiological methods and their application in the laboratory to identify bacteria isolated from disease fishes were also studied.

Various protozone diseases, helminth infestations and crustacean ectoparasites were dealt with in sufficient depth in order to have a brood knowledge of parasitic diseases affecting cultured fishes and shellfishes. The major bacterial diseases of tropical fishes and shellfishes, i.e. haemorrhagic septicaemias, vibriosis, myxobacteriosis and mycobacteriosis were studied in detail. Other bacterial diseases of less importance were also briefly reviewed.

Studies were also undertaken on principles of parasitology with emphasis on fishes, structure, mode of life and classification of viruses, tissue culture techniques and laboratory methods in fish virology.

5. Fish pathology

Comprehensive studies of the fungal diseases of fish and shellfish, with emphasis on integumentary and systemic mycoses, including fish mycotoxicoses were done. Toxicology and toxiconts important to aquatic organisms were also studied in detail. The toxicoses of fishes were described with an emphasis on pathology and diagnosis. The course included on extensive study of the non-infectious diseases of fishes including nutritional diseases, malformations and deformations, toxic algal blooms, genetic diseases, traumatic injuries, diseases caused by physical and chemical factors in the water, neoplasias and diseases of unknown aetiology. Diagnostic techniques and pathology applicable to invertebrate diseases, pharmacology, chemotherapy, anaesthesia, and the management aspects of disease prevention were also given due emphasis in the course.

6. Fish diseases

The importance, characteristics, cause, transmission control and therapy of some of the important tropical diseases were studied in detail in theory as well as in practical classes.

6.1 The importance of fish diseases

1. The infectious diseases are most important. These are caused by the:

   1. bacteria
   2. fungi
   3. protozoa

   4. viruses

2. Of these the non-treatable forms are of greatest concern - example:

   1. virus

   2. drug resistant bacteria

3. Economic loss due to disease

   1. not much detailed information available but it is large.
   2. may not be just death, but loss of weight resulting from parasitism.

   3. the whole future of fin mariculture is subject to control of vibriosis.

4. Why does disease occur

   1. it is a relationship between pathogen, host and the environment.
   2. This can be diagrammed as follows:
      
   3. A fish population (1) a pathogen (2) environment (3).
   4. Serious losses only occur when factors (1) and (2) are present in an environment (3) which favors the disease.
   5. When the pathogen and host are present (1–2) but the environment isn't favourable for the disease, no outbreak occurs.
   6. Also, when the environment is favourable for disease and the host is present (1–3), no outbreak occurs unless the pathogen is also present.
   7. With bacterial gill disease, the causative bacterium is almost always present.
   8. As long as the environment remains unsuitable for the pathogen, no disease occurs.
   9. With this disease, all that remains is for the environment to deteriorate and disease will occur.
   10. Under conditions of (1– 2 –3), the outbreak would be impossible to prevent without adequate drug or chemical therapy.
   11. What if one treats with drugs at this point.
   12. Temporarily eliminates factor (2), the bacterium.
   13. Bacterium is indigenous and treatment only provides short term solution.
   14. Without constant treatment with drugs, losses to disease will reoccur.

6.2. Microbial Etiology of Infectious Diseases

1. Koch's Postulates

   1. One must demonstrate a particular agent is the cause of a disease.
   2. This is done by Koch's Postulates.
      • The organism must be observed in association with the disease.
      • The microorganism must be isolated and grown in pure culture.
      • Disease must be reproduced in a susceptible animal.
      • Recovery of the organism.

2. What it takes for infection to occur.

   1. Entrance
   2. Establishment and multiplication
   3. Exit

   4. Establish new infection

3. How to best express disease

   1. A simple formula can be established for this

   2. This states

      Disease or health = Virulence of pathogen × no. present
      Resistance of host

7. BACTERIAL DISEASES OF FISH

7.1 Vibrio anguillarum and Vibrio ordalii

1. Introduction

   1. Vibriosis - the disease caused by V. anguillarum and V. ordalli primarily affect fish in salt water

   1. Control of this disease is necessary for the rearing of salmonids and other species in brackish or salt water
   2. Can result in 90% mortality if not controlled.

   3. Pen - rearing of salmon was not successful until vaccines against this disease were developed.

2. Because of its economic importance, V. anguillarum is probably one of the most extensively studied bacterial pathogens of fish.

   2. Characteristics of V. anguillarum

   Morphology	Short curved rods. Motile by single polar flagellum Endopores absent. Nonencapsulated.
   Staining	Gram negative
   Culture	Many standard media (TSA, BHI) colonies are cream-colored, circular and convex. Optimum growth 18–30°C. Generation time of 45 minutes.
   Biochemical	Facultative anaerobe Catalase positive Oxidase positive Acid, but no gas from certain sugars.
   G+C contact of DNA	43–45%
   Serology	Six serotypes are known, 3 are major pathogens of cultured fish.
   Pathogenicity	Highly virulent, can survive outside the host.
   Disease	Usually causes an acute Gram negative septicemia, can occur subacutely, rarely a chronic disease.
   Distribution	World wide in salt and brackish water can be found in some fresh water areas.
   Species infected	Probably most species of fin fish. Some crustaceans and shellfish.

   3. Transmission

   1. The disease agent is transmitted horizontally through the water in most instances.
   2. There is no evidence of vertical transmission.

   4. Control

   Prevention

   1. Some fish diseases can be controlled by avoidance of the pathogen.
   2. This is not a feasible alternative for control of vibriosis.
   3. Several vaccine preparations and methods for their administration have been effective in the control of vibriosis.
      1. Injection of killed cells.
      2. Injection of TCA extracts (0 antigens).
      3. Oral administration of lyophilized sonicates.
      4. Oral administration of lyophilized whole cells.
      5. Oral administration of wet pacted whole cells.
      6. Hyperosmotic infiltration of bacterin.
      7. Ommersion in bacterin containing solution.
      8. Spray or shower of bacterin containing solution.

   4. presently there are several commercially available bacterin preparations.

   5. Therapy

   1. Many bacterial pathogens are controlled by oral administration of antibiotics. It is possible to control vibriosis by feeding drugs.

      1. Oxyterracycline (terramycin, TM 50).
      2. Sulfonamide drugs may be effective.
      3. Drug resistance is possible and does occur. Is very evident in Japanese fish culture.

7.2. Aeromonas salmonicida

1. Introduction

1. The disease caused by Aeromonas salmonicida is known as furunculosis.

   1. This is a misnomer which has persisted even through the lessions are not true “furuncules”.

   2. The lesions involve no outstanding leukocytic infilteration.

2. Aeromonas salmonicida is probably the most commonly encountered, bacterial fish pathogen in the culture of salmonid species.

   1. In the past, nearly all hatcheries had some level of management.

   2. More recently, this problem has decreased because of better management.

3. Because of its importance in freshwater culture of salmonids, Aeromonas salmonicida and its disease furunculosis have been extensively studied.

2. Chracteristics of A. salmonicida

3. Transmission

1. The primary mode of transmission seems to be by horizontal methods.

   1. Can occur through contaminated water.

   2. Contact with diseased or carrier fish.

   3. Contaminated eggs.

   4. Contact with contaminated equipment or clothing.

   5. Surface of aquatic birds, not through faces because of the thermal sensitivity of A. salmonicida.

2. Vertical transmission may occur, but has not been shown definitively.

4. Control

1. Prevention

1. The most effective method of prevention is AVOIDANCE of the pathogen.

2. Because A. salmonicida is an obligate fish pathogen, one can avoid infection by obtaining fish eggs or live fish from sources certified free of furunculosis.

   1. Such sources have been established in USA and Canada.

   2. Also must have no carrier fish in water supply.

3. Another useful method is avoidance by disinfection of eyed eggs. This is most commonly done with iodophors.

4. pathogen free fish are not useful if water supply is contaminated with fish pathogens.

5. Vaccines have been tested for the control of A. salmonicida.

6. There have been efforts in selective breeding to produce furunculosis resistant strains of fish.

   1. These have been somewhat successful in producing more resistant animals.

   2. However, the fish lost desirable traits or became more susceptible to other pathogens (i.e. Renibacterium salmoninarum).

5. Therapy

1. Furunculosis was the first disease of fishes to be treated with modern drugs - sulfonimides and nitrofurans. Some of these were effective.

2. Oxytetracycline (terramycin, TM 50) is also effective.

3. It is a USDA requirement that treatment with drugs or chemicals be terminated 3 weeks prior to marketing or release of the fish.

   1. This allows for clearance of the drug.

   2. Clearance usually taken place very rapidly (within a few days levels are down).

4. Bacteria can become resistant to drugs.

   1. There are numerous reports of drug resistant A. salmonicida.

   2. Usually this resistance trait is carried on a plasmid of the bacterium and is called on “R factor”.

5. Many drugs have been used to control A. salmonicida. Only two are now FDA approved in the USA. These are terramycin and sulfamerzaine.

   1. A potentiated sulfonamide (R05–0037) is being tested and shows great promise. The criteria for FDA approved have been met.

6. It is well to remember that drugs are effective only in the treatment of outbreaks.

7. Recurrence of furunculosis are likely as long as A. salmonicida is present and environmental conditions are suitable for its growth.

7.3 PSEUDOMONAS

1. Introduction

   1. The etiology of Pseudomonas septicemia is not precisely defined. Generally caused by P. fluorescens, but other species are probably also involved.

2. Characteristics of Pseudomonas fluorescens

   Morphology	Motile rods Endospores absent May be encapsulated, not usually
   Staining	Gram negative
   Culture	Many standard media May produce fluorescent green pigment Grows well at temperatures from 18–37°C
   Biochemical	Strict aerobe Oxidative in O/C medium Oxidase positive
   G+C content of DNA	59–61%
   Serology	Not defined
   Pathogenicity	Opportunistic pathogen - disease is stressmediated
   Distribution	Worldwide in freshwater - have been some saltwater isolations.
   Species infected	Probably most species of freshwater fish

   
   

3. Control

   1. Because the disease is stress-mediated, the best method of control is to provide good environmental conditions.

   2. Drug therapy is similar to that for other Gram negative fish pathogens. Streptomycin has been reported to be superior in some cases.

   3. There has been no interest in producing bacterins against Pseudomonas for use in aquaculture.

7.4 Aeromonas hydrophila

1. Introduction

   1. There are numerous biotypes and serotypes of A. hydrophila which are some times referred to as Aeromonas hydrophila complex.

   2. This group of organisms causes disease which is called motile Aeromonas septicemia (MAS)

   3. Aeromonas hydrophila is primarily a pathogen of warm water species of fish. In all species it is usually considered an opportunistic pathogen.

2. Characteristics of Aeromonas hydrophila

   Morphology	Motile rods Endospores absent Nonencapsulated
   Staining	Gram negative
   Culture	Many standard media, Rimler-Shotts can be used Colonies are white circular and convex Usually is nonpigmented Grows well at 37°C
   Biochemical	Facultative anaerobe Oxidase positive Acid and gas from carbohydrates Fermentative in O/F medium
   G+C content: of DNA	57–63%
   Serology	Many serotypes which are not well defined
   Pathogenicity	Usually considered a opportunistic pathogen
   Distribution	Widely distributed in freshwater
   Species infected	Many species of freshwater fish are susceptible.

   
   

3. Transmission

   1. The disease agent is transmitted horizontally

      1. From intestinal tract and external lesions

      2. Through water

      3. External parasites (trematodes, copepods and leeches) may be vectors.

   2. Vertical transmission has not been demonstrated.

4. Control

   1. Prevention

   1. Avoidance of the pathogen is always the best method of control. Avoid the transfer of fish from areas where infected animals are found.

   2. A. hydrophila is an opportunistic pathogen and it has been stated that prevention of its disease is a matter of good house keeping.

   3. In European carp culture, prophylactic injections of Chloromphenicol are given to fish in the spring after overwintering.

   4. No effective vaccines are available.

5. Therapy

   1. Drugs

      1. Tarramycin is available for use in the USA.

      2. Chloramphenicol has been used in Europe.

      3. Furance has been used in Japan.

7.6 Bacterial gill disease

1. Introduction

1. Characterised by presence of large numbers of cytophagal bacteria on swollen gill lamellae.

2. Common disease- Causative agent unknown. Could be more than one bacterium.

3. Occasional disease of warmwater pondfish.

B. Etiology: Etiology not fully understood, induction of disease with bacterium isolated has never been achieved. Koch's postulates never completed.

1. Long filamentous, gram negative rods

2. Filamentous forms grow on cytophaga agar. Will also grow well in broth cultures.

3. Mechanisms of pathogenicity.

   1. Is generally accepted as cytophagal disease.

   2. Cytophagal bacteria (causing gill disease) not adequately described.

4. Serotypes

   1. Bacteria isolated from salmonids with BGD

      1. Differ serologically from those causing colummaris disease.

      2. Those causing low temperature disease.

   2. While a particular bacterial type may predominate in an outbreak of bacterial gill disease, serological studies have shown that different types may be involved

C. Epizootiology

1. Host: infects wids range of hosts - all cultured salmonids.

   1. Common disease among these fish.

   2. Disease occurs among warm water fish but may not be same bacterium.

2. Source and reservoir of infection BGD

   1. Not established.

   2. Likely fish with chronic infections.

   3. Mud and silt in water.

3. Mode of transmission: assumed to be transmitted in water.

4. Incubation period: about 3 to 7 days.

5. Period of communicability

   1. Unknown.

   2. Likely as long as infected fish in water supply.

6. Susceptibility and resistance

   1. Common and acute among hatchery-raised salmonids.

   2. Yearing and older fish less susceptible then fry.

   3. Most hatchery trout have a low antibody titar against bacteria isolated from gill disease.

7. Range

   1. Apparently worldwide in salmonids.

D. Pathology- (gross external)

1. Fish with BGD show

   1. Lack of appetite

   2. Loss of orientation with current

2. Gill lamellae show proliferation of epithelium, which in advanced cases results in clubbing and fusing of

   1. Lamellae

   2. Filaments

3. Examination of affected lamellae shows

   1. Long, thin bacteria

   2. Covering gill epithelium

4. Some bacteria attached by just one end.

5. Stained smeare show filamentous, Gram-negative bacteria about 0.5 um × 5–10 um.

6. Differentation

   1. Combination of large numbers of filamentous bacteria about-0.5 and gill epithelial proliferation differentiates BGD from

      1. Columnaris
      2. Nutritional gill disease
      3. Haemorrhagic (aneurysmal) gill disease
      4. Infestation of gills by higher parasites
      5. Fungal gill disease
      6. Irritation of gills by chemical agents.

E. Detection and laboratory diagnosis

1. Large numbers of long filamentous bacteria.

2. Swollen gills (clubbing and fusion of gills).

3. Growth on cytophaga agar.

F. Method of control

1. Water supply free from adult fish, silt, and mud.

   1. Crowding, resulting in accumulation of fish metabolic products.

      1. Most important factor contributing to disease.

      2. Avoid crowding at all costs.

   2. If water reused should be

      1. Filtered

      2. Amonia oxidized by nitrifying bacteria

      3. Keep ponds clean (remove unused food and feces).

5. Chemoprophylaxis with quaternary ammonium and organic mercurial disinfectants were used.

3. 3. Treatment: Oxytetracycline.

7.7 Fin rot

1. Introduction

1. Fin rot occurs among population of fishes often where intensive culture is practiced.

2. Bacteria are found in the fin lesion only.

B. Etiological agent

1. Causative agent unknown but often gliding bacteria are observed.

2. May be cytophagal, myxobacteria or flavobacteria.

3. In addition, Aeromonas salmonicida, A. hydrophila and V. anguillarum have been known to cause fin rot condition.

2. Morphology

1. May observe gliding bacteria or more well-known fish pathogens such as A. salmonicida

3. Cultivation

1. Gliding bacteria grown on cytophaga medium.

4. Mechanisms of Pathogenicity

1. Not known but associated with ponds which have not been kept clean and in which fish are crowded.

5. Serotypes- unknown.

C. Epizootiology

1. Attacks a variety of hosts, particularly salmonids; geographic range worldwide.

2. Reservoirs of infection.

   1. Perhaps fish

   2. Often the organism isolated from the fin rot lesion is the predominant organism in the incoming water supply.

   3. Not known if it is a primary or secondary involvement.

3. Transmission

   1. Carrier fish

   2. Waterborne infection

4. Environmental factors.

   1. Poor sanitary conditions associated with occurrence of disease.

   2. Also crowding and nipping.

   3. Nutrition may play a role in predisposing fish to disease.

D. Pathology

1. Bacteria found in fin lesions only.

2. Advanced cases of fin rot of the caudal fin resemble peduncle disease.

3. Signs of fin rot may occur during outbreaks of other diseases- Example- furunculosis.

4. Fins become opaque at the margine.

5. Opacity usually progresses towards the base of the fin.

6. Fins become thickned (proliferation of epithelium).

7. In advanced cases- are frayed with rays protruding.

8. In advanced cases of the tail fin, the whole caudal fin may be lost.

9. This may be followed by erosion of peduncle.

E. 1.   Prevention - Control

1. Clean ponds

2. Avoid crowding

2. Therapy

   1. Treat with oxytetracycline added to water 10 to 30 parts per million.

8. Viral diseases of fish

8.1 Tissue culture

Introduction

In the 1050s a major advance in virology was made with the development of methods for establishing and maintaining cells in vitro. The ability to culture cells provided a system for the isolation and propagation of many viral agents. In the late 1950s these techniques found application in fish virology. To date, over 60 cell lines of fish have been reported and many more have been established. The use of these cell lines has led to the isolation of over 20 viral agents from fish. Many fish viruses are important disease agents and the use of cell cultures has allowed the completion of Rivers' postulates in establishing the viral etiology of these diseases.

Cell Culture

1. Growth media

   1. Formulations of various complexity depending on cells.

   2. Defined medium.

      1. Vitamins and confactors.
      2. Nucleic acid precursors.
      3. Essential amino acids.
      4. Energy source-glucose.
      5. Salts.

      6. Buffer.

   3. Undefined medium

      1. Serum 5–20%

      2. Exact components not known.

   4. Sterility is essential - antibiotics may be employed.

2. Establishing cell cultures

   1. Tissue of choice.

      1. Embryos.

      2. Rapidly proliferating tissues.

         1. Gonads, etc.

         2. Neoplastic tissue.

   2. Enzymatic treatment.

      1. Trypsinization of tissues.

      2. Plating of single cells in growth medium.

   3. Explant technique

      1. 1 mm minced tissue.
      2. Plating of explants in growth medium.

      3. Growth of cells from explants to form monolayers.

   4. Mophology and cultured cells.

      1. Fibroblastic.

      2. Epithelial.

3. Handling cultured cells

   1. Sterility.
   2. Trypsinization of monolayers.
   3. Splitting cells.

   4. Culture vessels- flasks, microplates, etc.

Virus Isolation and Propagation

1. Viruses as intracellular parasites.

   1. Cell line specificity.

   2. pH and temperature for replication.

2. Choice of material for isolation.

   1. Whole fry.
   2. Visceral organs.

   3. Sexual fluids.

3. Preparation of samples.

   1. Homogenization.
   2. Antibiotics or filtration.

   3. Toxicity.

4. Inoculation of cells.

   1. Cytopathic effects (CPE) in 1–7 days in susceptible cells.

   2. Depend on type of virus- somewhat characteristic.

Virus Identification

1. Serum neutralization.

   1. Block infectivity of virus.

   2. Prevent CPE.

2. Fluorescent antibody.

   1. Antibody binds to infected cells.

   2. Fluorescence in UV microscope.

8.2 Rhabdovirus carpio

1. Introduction

The most serious cause of loss among cultured cyprinid fishes in Europe is attributed to a disease complex variously known as infectious dropsy, hoemorrhagic septicemia, rubells and infectious ascites. The etiology of this complex has been controversial. A rhabdovirus (R. carpio) has been isolated and shown to reproduce symptoms of the acute stage of the disease now termed spring viremia of carp. A bacterial etiology (Aeromonas sp.) is generally accepted for the chronic or subacute form of the complex with the proposed name carp erythrodermatitis.

Additionally, a contageous and lethal disease of carp termed swim bladder inflammation has been described. The pathology of this condition is quite different and the disease has an acute and a chronic form. A virus has been isolated from fish with the acute form and shown to reproduce the disease. This virus is morphologically and serologically identical to R. carpio.

2. Characteristics of Rhabdovirus carpio.

3. Transmission

1. Natural route not determined. Waterborne infection has been experimentally demonstrated and is the most likely source.

2. Experimental transmission by the carp louse has been reported.

3. Vertical transmission in or on eggs is unlikely.

4. Experimentally, the virus has been transferred by injection and bath but not feeding.

4. Control

1. Prevention.

   1. Avoidance by use of virus-free broodstock and water source.

   2. Destruction of infected stocks and hatchery disinfection.

2. Carp surviving infection are resistant to R. carpio.

   1. Attenuated vaccines have been developed which show promise.

   2. IP inoculation of killed virus also stimulated immunity.

   3. Some carp strains are more resistant to challenge and have been selected for culture.

3. Chloramphenicol has been reported to be bebeficial in treating SBI. This has confused the etiology and suggests secondary bacterial infection may be important.

9. Protozoan disease of fish

CILIOPHORANS

9.1 Ichthyophthirius multifiliis

1. Introduction

   1. This ciliate causes ichthyophthiriasis (“ich”) or white spot disease in freshwater fishes throughout the world with the possible exception of Antarctica. Can parasitize the gills, eyes, skin and fins.

2. Characteristics of Ichthyophthirius multifiliis, the causative agent of ichthiophthiriasis or white spot disease.

   Morphology	Uniformly ciliated Temites oval 30–45 um Trophozoites oval to round 50 μm -1mm
   Culture	Can be maintained alive for a period of time but cannot complete life cycle in fish mucus or pulverized skin and gills.
   Pathogenicity	Obligate pathogen occurring in the epidermis.
   Disease	Highly temperature dependent and characterized by white spots caused by hyperplasia of cells around parasite.
   Distribution	Worldwide in freshwaters.
   Species infected	Most freshwater fishes.

   
   

3. Transmission

   1. Fish to fish. Trophozoites drop off fish, encapsulate and divide. Release tomites which penetrate and infest fish.

4. Control

   1. Prevention

   1. Avoidance

      1. Change to ground water where surface supply contaminated.
      2. Quarantine new fish.

      3. Disinfect equipment.

   2. Alterations of environment

      1. Decrease crowding
      2. Clean ponds

      3. Increase water temperature if fish species allows.

   3. Vaccines

      1. Vaccine using Tetrahymena pyriformis cilia has given promising results. Natural immunity is short lived unless fish in constant challenge and community probably due to antibody being secreted in the fish's mucus thus stopping the infective stage before penetration.

   4. Prophylactic agents

      1. Malachite green at 1 ppm/1 hr or 0.15 ppm indefinite.
      2. Formalin at 200 ppm/1 hr or 25 ppm indefinite.
      3. Salt at 0.2% indefinite.
      4. Temperature 27°C or greater if tolerated by fish species.
      5. U.V. irradiation of incoming water.

      6. Chlorination-dechlorination of water supply.

5. Therapy

   1. Drugs

      1. Chloramine T 5 ppm day 1 and 3 ppm daily thereafter.

      2. Quinine sulfate 1 ppm daily

   2. Chemical agents

      1. Malachite green, formalin and salt.
      2. Combination of malachite green at 0.1 ppm and formalin at 25 pp.

9.2 Other ciliophorans

Ciliophorans bear cilia during some stage of their development and all possess two types of nuclei, a macronucleus and a micronucleus. Reproduction is both a sexual and sexual which consists of conjugation with micronucleus exchange. Ciliates bear cilia throughout their life while sucturians possess them only during early developmental stages. Most ciliophorans are freeliving but several symbiotic forms, including many parasites, exist. In fishes the majority of the parasitic ciliophorans are ectocommensal, but a few endocommensal species have been described.

The majority of these parasites do not feed on fish tissues but merely use the fishes' surface as enchors or feeding grounds. The primary food sources for these organisms are bacteria and other protozoans. Problems arise when large numbers attach to the respiratory or skin surfaces of fish or grase over these surfaces creating respiratory blockage and irritation.

Trichodina spp. are ectoparasite and endoparasitic in many fish species. These ciliates move about freely over the surface of the skin, gills or within the digestive, urinary, or urogenital tracts.

Epistylis spp. are bell-shaped, stalked, sessile, facultative ectoparasites of fish usually occurring in colonial arrangements. This ciliate has been implicated as a factor in red sore disease of warm water fishes.

Chilodonella (Chilodon) spp. are ectoparasitic on the skin of several fishes, including salmonids. These ciliates are ovoid and move about freely over the surface of the fish.

Tricophrya spp. are attached suctorians which possess cilia only during their swarmer stage. As the parasite matures the cilia are lost and tentacles develop which are used for obtaining food. Primarily attached to gills of fish.

Treatment for ciliophorans consists of one of the following:

1. Malachite green (0.1 ppm)

2. Formalin (167–250 ppm for one hour)

3. Copper sulfate (concentration variable depending on water hardness

4. Malachite green-formalin (0.1 ppm-25 ppm).

9.3 ADDITIONAL PARASITIC DISEASES OF FISHES

The following parasites may be very destructive to fish. Infected fish should not be transferred to fisheries facilities where such parasites do not exist. Highlights of host range, diagnosis, and other pertinent facts for these diseases are given below.

1. Monogenea (gill and body flukes)

   1. Gyrodactylus

      Many species, usually species-specific for fish host. Possesses two large posterior anchors, no eye spots, and an embryo with anchors and hooks in utero.

   2. Dactylogyrus (gill flukes)

      Many species, usually on cyprinids. Goldfish Dactylogyrus is probably the most important. Possesses two large posterior hooks, four anterior eye spots, and egg(s) in utero.

   3. Cleidodiscus (gill flukes)

      Many species, on many fishes. Catfish Cleidodiscus can be dangerous to fry. Possesses four large posterior anchors, four anterior eye spots, and egg(s) in utero.

2. Digenetic trematodes

   Adults or metacercariae in fish.

   1. Diplostomulum spathaceum (eye fluke)

      Found in many fishes. The metacercaria is not encysted, possesses forebody and hindbody, oral sucker, ventral sucker, ventral holdfast organ, and two anterolateral pseudosuckers. May cause blindness.

   2. Ornithodiplostomum ptychocheilus (brain fluke of fathead minnow)

      Found in the viscera of many cyprinids, but in the cranial cavity and brain of Pimephales promelas, Notropis heterolepis, and Notropis cornutus. Can be serious in cultured fatheads. Is encysted, has forebody and hindbody, anterior oral sucker, ventral sucker, and ventral holdfast.

   3. Clinostomum complanatum (Clinostomum marginatum, yellow grub)

      Unsightly, usually yellowish grub. Body with large ventral sucker and anterior “shoulders”. Encysted; large, 1–2 mm long.

   4. Sanguinicola sp.

      1. Adult Worms

         Sanguinicola davisi, fully developed individuals are flattened and spindleshaped flukes, having a length of 8.5 mm and a width of 0.21 mm. There is no oral sucker, acetabulum or pharynx. The mouth is subterminal and leads into a long slender esophagus that ends in an X-shaped cecum with four short, rounded lobes. The single testis is large, irregular in shaped and approximately equoatorial in position, the sperm duct leads to a crook like thick-walled cirrus sac. The bilobed ovary located at the posterior margin of the testis opens by means of a short oviduct into a gourd-shaped ootype. Genital pores open separately near the posterior end of the body with the female pore being anterior to the male pore. Yolk glands fill the entire body. Eggs are oval, nonoperculate, and measure 63 by 35 μm.

         Major gill arteries, dorsal aorta, and heart chambers from at least 60 fish should be examined for blood flukes fitting the above description. Squeeze contents from blood vessels, splitting large ones if necessary.

3. Cestodes (tapeworms)

   Adults in intestine, plerocercoids in viscera and musculature.

   1. Proteocephalus ambloplltis (Bass tapeworm)

      Adult in intestine of Micropterus spp., plerocercolds in viscera of bass and small fish. Globular scolex has four suckers and an anterior vestigial apical sucker. The eggs are dumbell-shaped.

4. Nematodes (roundworms)

   Goezia, a spined nematode, produces modules in the intestine of Micropterus salmoides in the Southeastern United States. Eustrongylides sp. cause swollen abdomen in tropical fish and small balt fish in extreme South-western and Southeatern United States.

5. Acanthocephala (thorny headed worms)

   1. Acanthocephalus jacksoni

      Acanthocephalus jacksoni of trout in Northeastern United States may cause concern among fishermen finding them in lower intestine of fish.

6. Copepods

   1. Lernaea elegans (L. cyprinacea, anchor parasite) No host specificity. Adult female with large Y-shaped dorsal arms.

   2. Ergasilus spp. (hay fork claspers)

      On gills of many fish. Ergasilus labracis of striped bass (Morone saxatilis) can be lethal.

7. Branchiura

   Argulus (fish louse). Occasionally causes problems. Try to avoid Asian Argulus japonicus and European Argulus follaceus and Argulus glordanii.

10. MYCOTIC DISEASES OF FISH

10.1 OOMYCETE FUNGI

Saprolegina and Achlya

1. Introduction

   1. Oomycetes are the most common and widely distributed fungi infecting fishes.

   2. The ability to produce motile spores separates the Oomycetes from the other fungi.

   3. Produce a cottony mycelium on the surface of the affected animal.

2. Characteristics of Oomycete fungi producing infections in fish.

   Morphology	Mycelia composed of hyphae with few septae. Zoosporangia which produce biflagellated zoospores and gemmae. Antheridial cells and oogonia which produce oospores upon fertilization.
   Staining	Polyvinyl Lactophenol blue to stain hyphae and reproductive stages.
   Culture	Sabouraud dextrose agar, nutrient agar, hemp seeds. Development of white mycelial mats.
   Cell wall	Some cellulose present.
   Pathogenicity	Some species pathogenic, others saprotrophs.
   Diesease	Epidermal and dermal tissue destruction with expanding edges from site of infection. Destruction of gill epidermal layer.
   Distribution	Worldwide.
   Species Infected	Many species of fish.

   
   

3. Transmission

   1. Fish or substratum to fish via zoospores, gemmules, or oospores.

4. Control

   1. Prevention

   1. Avoidance

      1. Reduce handlings.
      2. Maintain good rations.

      3. Remove dead fish and debris from ponds, remove dead eggs.

   2. Alterations of environment

      1. Lower water temperature if possible.

      2. Use ground water.

   3. Vaccines, mechanism of immunity

      1. None available.

      2. Little or no inflammatory response against fungi has been observed.

   4. Prophylactic agents

      1. Malachite green.
      2. Formalin.
      3. Salt.

      4. Ultraviolet irradiation of water supply.

5. Therapy

   1. Drugs

      1. None

   2. Physical, chemical and biological agents

      1. Malachite green
      2. Formalin.
      3. Salt.
      4. Removal of dead fish, eggs and organic debris.
      5. Ultraviolet irradiation of water supply.

      6. Biological control by crustaceans.

10.2 BRANCHIOMYCES SPP.

1. Introduction

   This fungus is the causative agent of gill rot in fish. Two species described B. sanguinis and B. demigrams.

2. Characteristics of Branchiomyces sp., the causative agent of branchiomycosis disease of fish

   Morphology	Hyphae 8–30 μm in diameter with spores ranging from 5–17 μm in diameter.
   Staining	Polyvinyl lactophenol blue
   Culture	Sabouraud maltose agar, other mycological media. Brownish pellicle-like colonies with no aerial or submerged hyphae.
   Pathogenicity	Appears to be an obligate pathogen, can occur intravascular or on the surface of cell lamellae.
   Disease	Acute or chronic, characterized by obstruction of blood circulation and necrosis of gill tissue.
   Distribution	Eastern and Western Europe, Mediterranean countries, Japan and United state.
   Species infected	Several species of fish, mostly cyprinids.

   
   

3. Transmission

   1. Fish to fish via spores or gemmae.

4. Control

   1. Prevention

   1. Avoidance

      1. Do not introduce infected fish.

   2. Alterations of environment.

      1. Cooler temperatures.

      2. Reduce organic load in the water.

   3. Vaccines, mechanism of immunity

      1. None available.
      2. Inflammatory response to the fungus is available

      3. Some species appear to be immune in certain situations.

   4. Prophylactic agents.

      1. Salt baths, 3–5%, for one hour.
      2. Reduce organic load.
      3. Cool water down.
      4. Use of copper sulfate.

      5. Quicklime.

5. Therapy.

   1. Drugs.

      1. None

   2. Physical and chemical agents.

      1. 0.3 mg/l malachite green for 24 hours.
      2. 1–4 ppm benzalkonium chloride for one hour.
      3. 100 mg/l copper sulfate for 10–30 minutes.
      4. 1.5 mg/l formalin.
      5. Feeding methylene blue to fish.
      6. Stop feeding fish when they become infected and remove dead fish.

11. VITAMIN DEFICIENCY SYNDROMES

12. PRAWN DISEASES

1. Viral disease

1. Monodon Baculovirus (MBV)

2. Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV)

3. Hepatopancreatic parvo-like Virus (HPV)

4. Reo-like Virus.

1. Monodon Baculovirus (MBV)

   • Found in PL5 to adult.

   • Bright green spherical bodies in hyperthrophied nuclei.

   • Necrosis and cytolysis of hepatopancreas and gut epithelium.

   • PL small, dark, weak, lose appetite reduced preening and mortality.

   Avoidance

   Proper sanitation, proper nutrition, eradication.

   Treatment

   No treatment but to eradicate.

2. Infectious Hypodermal and Haematopoietic Necrosis Virus (IHHNV)

   • Found in PL and juvenile-possible latent infection in larvae.

   • Transferred from P. stylirostis.

   • Cowdry type interamuscular inclusion bodies.

   • Necrosis of hypodermis, hemocytes, haematopoietic organs, connecting tissues.

   • 80–90% commulative mortality.

   Avoidance

   Absolute quarantine and eradication.

3. Hepatopancreatic Parvo-like Virus (HPV)

   • Found in juveniles.

   • Necrosis/atrophy of hepatopancreas

   • Poor growth rate, anorexia.

   • Reduced preening and mortalities.

4. Reo-like Virus

   Sign of disease in P. japonicus

   • Poor growth

   • Anorexia, reduced preening.

   • Gill/surface fouling.

   • Opacity of muscles.

   Effect on host

   • Necrosis and atrophy of hepatopancreas.

   Prevention

   • Avoidance by quarantine.

   • Destruction of contaminated stocks, depopulation and disinfection of contaminated facilities.

   Treatment

   Not known

   Rickettsial infection

   Cause - Rickettsia or Rickettsia like organism.

   Stages affected - Juvenile

   Sign of disease (P. merguiensis)

   • Heavily infected shrimp lethargic, off feed with atrophy, pale colouration of hepatopancreas.

   Sign of disease (P. monodon)

   • Heavy infected shrimp lethargic, off feed, swim along side of pond, brown gills, deffused opacity of abdominal muscle and mushy texture of hepatopancreas.

   Effect on host

   • Impaired digestion and absorption of nutrients.

   • Reduced growth rate and high mortality.

   Prevention

   • Avoidance by quarantine.

   • Destruction of contaminated stocks and disinfection of contaminated facilities.

   Treatment

   None responded.

2. Bacterial diseases

1. Necrosis in appendages (in PL & larvae).
2. Luninous bacterial disease.
3. Filamentous bacterial disease.

4. Shell disease.

1. Necrosis of appendages

   • Found in larvae/PL

   • Necrosis/erosion of appendages

   Chemoprophylaxis/Chemotheraphy

   Erythromycin phosphate
   Streptomycin - Bipencillin
   Tetracyclin - chlorohydrate
   Sulphamethazone, Furanace and chloromphenicol.

2. Shell disease

   Cause : Chitinolytic (Chitin degrading) species of Vibrio and Aeromonas.

   Stages affected - Post larvae to adult

   Sign of disease - Brownish/blackish area on shell

   Effect on host - Erosion of shell, underlying tissues may be affected.

   Prevention

   Avoid over crowding and minimise handling to prevent injuries to prawn.

3. Luminous bacterial infection

   Cause - Vibrio harveyi and V. splendidus

   Stages affected - Larvae and PL.

   Sign of disease - Prawn weak & luminace in dark

   Effect on host - 100% mortality.

   Prevention

   • Disinfection of rearing water, rigid water management and sanitation.

   Treatment

   Furazolidine at 10 ppm for 12 hrs. for 4–5 consecutive days.

4. Filamentous bacteria

   Cause - Leucothrix mucor and other filamentous spp.

   Stages affected - PL to adult

   Sign of disease - Fine filamentous growth on gills, setae, shell and appendages.

   Effect on host - Hypoxia, impaired moulting and mortalities.

   Treatment

   - Cutrine plus at 0.1 mg cu/l for 24 h. or 0.25–0.5 mg Cu/l 4–8 h.

3. Fungal disease

1. Larval mycosis

   Cause - Lagenidium callinectes, Lagenidium sp. Haliphthors phillipinsis and Sirolpidium sp.

   Stages affected - Eggs, larvae and PL

   Sign of disease - Prawn whitish and weak, mortalities.

   Effect on host - Fungal mycelia replace internal tissues mortality 100% in 2 days.

   Prevention

   • Disinfection of rearing facilities with Tide detergent at 100 ppm for 24 h.

   • Chemical prophylaxis of spawners with Treflin at 5 ppm for 1 h. or eggs with tide detergent at 20 ppm for 2 h.

   Treatment

   • Treflin or trifluratin at 0.2 ppm for 24 h.

2. Fussarium infection (Black gill disease)

   Cause - Fussarium solani

   Stages affected - Adult

   Sign of disease - Scanty white cottony growth on prawn, black gills, reduced feeding and preening.

   Effect on host - Destruction of gill tissue and mortality.

   Prevention

   Disinfection by Chlorination

   Treatment

   Harvest prawn if size is adequate

4. Protozoan disease

1. Ciliate infestation.
2. Gregarine disease.

3. Microsporidiosis.

1. Ciliate infestation

   Cause - Zoothamnium, Epistylis, Vorticella Acineta, Ephelota.

   Stages affected - PL to adult

   Sign of disease - Fuzzymat on shell, dark brown gills.

   Effect on host - Respiratory/locomotory difficulties, loss of appetite, mortality

   Prevention

   • Avoid build up of organic matter, silt and sediment.

   • Maintain optimum dissolved oxygen.

   Treatment

   Chloroquin diaphosphate at 1.1 ppm for 2 days.

   For Zoothamnium - 50–100 ppm. formalin for 30 min.

   For Epistylis - 30 ppm formalin for juveniles only.

2. Gregarine disease

   Cause - Gregarine.

   Stages affected - Larvae and adult.

   Sign of disease - Under microscope gliding through the digestive tract or attached to the intestinal wall.

   Effect on host - Large number of protozoans may interfere with particle filteration through digestive tract.

   Prevention

   Eliminate molluscans which act as intermediate host.

   Treatment

   None

3. Microsporidiosis

   Cause - Microsporidia

   Stage affected - Adult

   Sign of disease - Whitening of affected areas, e.g. ovaries & abdominal muscles.

   Effect on host - Microsporidian spores and other stages of parasite replace affected tissue, sterility in female spawners with white ovaries.

   Prevention

   Disinfect with chloride or iodine containing compounds.

4. Dinoflagellate infestation

   Cause - Peridinium sp.

   Stage affected - PL

   Sign of disease - Shrimp moribund and depigmented microorganism fill ventral sinus and hemolymph spaces.

   Effect on host - Mortality

5.    Ectozoic algal bloom

Cause - Schizothrix calcicola

Stages infected - Mysis, PL, juvenile, adult.

Sign of disease - Sluggishness, inactivity, surface fouling.

Effect on host - Locomotory and respiratory difficulties, gill disease, mortality.

6.    Nematodes infestation

Cause - unidentified nematodes.

Stages affected - Mysis, PL, adult.

Sign of disease - Presence of nematodes.

Effect on host - mortalities.

Prevention

• Maintain good water quality and reduce organic load.

7.    Copepod infestation

Cause - Caligus epidemicus

Stages affected - Adult

Sign of disease - Presence of ectoparasite, on pleopods and tail.

Effect on host - Morphological deformatics.

WATER QUALITY DISEASES

1. Temperature

This is probably the single most important factor in both the farming situation and in fancy fish keeping. Fish are ectotherms (poikilotherms) and with few exceptions (muscles of fast swimming species) mirror the temperature of the surrounding water. Nevertheless, each species has its own preferred range. Sustained periods at the extremes of this range, or probably more important, rapid changes (even very small ones sometimes) within this range, represent stressful conditions. While metabolic rates alter with varying water temperature (roughly doubling for each 10°C) this does not happen immediately. Advantage is taken of this enhanced metabolic rate in aquaculture where waste heat is available from eg. power stations. Thus the fish will grow faster in warmer water.

In general terms, fish will tolerate a temperature drop better than a rise. Some species are more susceptible to temperature stress than others and this is thought to be due to a poorer ability to osmoregulate at the new temperature. Higher temperatures cause an increased metabolic rate and hence an increased O₂ demand necessitating increased “irrigation” rates. The increased water flow past the gills causes an increased water influx and thus possible compromise of the osmoregulatory mechanisms.

Fish also appear much more susceptible to bacterial diseases in conditions of rising water temperatures. Even a small change from as low as 5°C can be sufficient to promote overt furunculosis in Atlantic salmon fry. The reason for this susceptibility is unknown in precise terms but it is thought to be the result of an enhanced rate of replication or enzymic production by the pathogen which is not matched by that of the fish's immune mechanisms.

Increasing water temperature in general reduces the survival time of fish in pollutants. With heavy metals, their toxicity is enhanced, but the fish also has an increased respiratory rate and therefore increased exposure - a synergistic effect.

2. Oxygen

The efficiency of extraction by the gills is high, and it has to be when one considers that at 15°C one litre of freshwater contains 7 cc O₂ and seawater even less as compared to almost 21 volumes per cent in air. In warm or polluted waters, where O₂ levels are low, the cost of extraction may be too high i.e. where the energy required for ventilation exceeds that released by the O₂ so obtained. Under these circumstances, the fish enter Respiratory Distress Syndrome. Different species of course have different requirements for O₂: salmonids for example have a high requirement, catfish a lower one. Some species have an accessory breathing apparatus, such as a modified swimbladder, and in some cases rely heavily upon them although they are able to survive in even totally anoxic conditions eg. snakeheads. Small fish have higher requirements than older larger ones: this translates in an aquaculture situation into placing small fish in the best quality water available.

Fish will normally demonstrate an O₂ lack by gathering at inlets or by gasping at the surface and by decreased activity. Although O₂ levels may be higher in surface waters, the gills collapse on contact with the air, so reducing surface area.

Increasing water temperature cause an increased demand for O₂ for the increased metabolic rate so produced, but a decrease in the holding capacity of water for O₂. Appetite also increases, but this extra intake of food may place a burden on the ability of the fish to extract enough O₂ from the reduced levels available to metabolize the food properly. At 28°C (a high temperature, but is encountered in some trout farms in Europe for short periods of time) O₂ consumption just about matches that which is obtainable and there is thus no O₂ left over to properly metabolize food. In general terms therefore, with high water temperatures, a reduced food intake is advisable, and a complete cessation of feeding is indicated if furunculosis or other bacterial diseases appear. In some situations it may be necessary to match feeding frequencies with O₂ levels: these are normally highest at mid-day due to photosynthesis, but surprising variations do occur - best therefore to directly measure.

Fish can acclimate to lower O₂ levels to a certain extent by increased hematocrit. Diel variations, however, depress appetite and growth.

The LC50 of some toxicants is lower at high O₂ levels, largely due to the reduced irrigation rate over the gills. Similarly, uptake of many toxicants correlates positively with O₂ uptake: thus methyl mercury or endrin uptake increase with increased swimming speed, due to the increased oxygen uptake. The rate of uptake depends on the physiochemical properties of the toxicant: Lipid solubility, small molecular size and un-ionized molecules favour uptake.

High fecal levels or other organic detritus utilize available O₂.

A minimum of 5 mg/L is considered a satisfactory level for most stages under most conditions, although higher is certainly preferable for salmonids.

3. Supersaturation

Rapid increases in water temperature or reduced pressure may lead to a situation of supersaturation. Both of these may be seen when water from a tap is used to fill a domestic aquarium. Supersaturation may also be seen in the wild associated with very rapid photosynthesis by plants and algae, or more commonly in an intensive culture operation, associated with leaky valves or pumps, in those farms where pumping is a feature (air is forced under pressure into the water supply - so called Venturi principle). Water which is supersaturated with gas, either O₂ or N₂, may cause the condition gas-bubble disease. This is the fish equivalent of divers “bends”. Fish normally equilibrate quickly with supersaturated water and it should therefore cause little problem. The reason for the gas in the blood coming out of solution is not therefore completely understood but is thought to be associated with the great drop in pressure experienced by the blood when crossing the gills. Whatever the reason, bubbles of gas cause emboli in the vessels of the gills pseudobranch, choroid gland and elsewhere. These are often difficult to see, and a “candling” procedure may be found advantageous in diagnosing the condition. An apparent contraindication, vigorous aeration or agitation of the incoming water, or replacement of the leaky valves, are considerations in curing the condition.

4. Suspended solids

• there are various types; organic such as feed fines, fecal waste, pulp mill effluent and inorganic such as clays, which may be inert (kaolin) or non-inert (bentonite). Non-inert inorganics may absorb substances such as heavy metals, pesticides or ammonia, and present these to the gill surface.
• in general, there appears to be evidence suggesting that organic particles are more of a problem than inorganics, but whether this is a direct effect, or an indirect one is less clear.
• Our work with young rainbow showed that fish kept for 3 months at roughly 6,000 ppm kaolin (almost the consistency of whey!) had no significant lesions on the gills. They were smaller than controls because they ate less - they couldn't see the food! They may also have been stressed more because they did succumb to a protozoan infection (Ichthyobodo) but they recovered despite continued exposure. In the same set of experiments, 10–12,000 ppm kaolin did kill fish, by clogging the gills.
• some species appear to “tolerate” or prefer muddy bottoms than others, and the type of bottom may dictate species found.
• definite effects of suspended solids include (1) settling on eggs and young larvae and possibly suffocating them, (2) reducing light penetration and therefore abundance of food, (3) modifying behaviour patterns and natural movements.
• evidence for a direct damaging effect on fish is less convincing.

5. Ammonia

• fish excrete this via the gills, and where there is plenty of water to remove it there is no problem. Toxicity arises however, in situations of overcrowding, or where for example, chicken slurry is added to the water. Young fish are relatively quite susceptible.

  
• free ammonia or NH₃ is highly toxic, whereas bound ammonia NH₄ + is much less so. Free ammonia combines with water as shown in the above equation and dissociates or recombines as shown. In acid water, most ammonia is in the bound or non-toxic form whereas in alkaline water, free ammonia may be more of a problem. Similarly, as water temperature increases, the amount of free NH₃ also increases, whereas elevated calcium levels (seawater, hard water areas) increase the tolerance of fish to ammonia, possibly by decreasing the permeability of the gills to the toxin. The gills also give off CO₂ and due to its combination with water to form carbonic acid, in the local environment of the gills, the lower pH so produced may afford a degree of protection.
• high ammonia levels cause epithelial hyperplasia of the gills, thus effectively increasing the diffusion distance to O₂. At the same time, it is thought that NH₃ affects the ability of haemoglobin to bind O₂, but the precise toxic action of NH₃ is unknown. Chronic levels have been shown to cause meningeal proliferation in young minnows.
• this then is the “party line” on ammonia toxicity.
• we conducted experiments exposing rainbow trout try to unionized (free) ammonia of 0.4 ppm (20 times established “maximum”) continuously for 3 months. Levels in the fish were high enough to produce coma, probably by interfering with neurotransmitters in the brain. After 3 weeks, the fish started acclimating, and feeding. No gill lesions were seen at any time up to 3 months. The only abnormality detected histologically was increased protein droplets in urinary tubular epithelium (ammonia is known to increase glomerular filtration rate, and this may help explain the observation). Thus we question the significance of ammonia per se. There is little doubt however, that its measurement in water does provide a good indicator for an overall increase in metabolic waste products and these do correlate with branchial hyperplasia: included however, are other products such as urea, trimethylamine and fecal proteases. In young heavily-fed rainbow trout, fecal protease levels are roughly 10 times those seen in a dog. Thus if looking for agents which might induce branchial proliferation in a crowded pond, other products seem more likely candidates than ammonia.

6. Nitrite

• this is an intermediate in the oxidation of ammonium to nitrate, a process which is carried out naturally and by the bacteria in biological filters. Nitrosomonas spp. convert ammonium to nitrite. Nitrobacter spp. convert nitrite to nitrate.
• as we have already seen, alkaline conditions lead to a higher amount of un-ionized ammonia, and this in turn has a greater inhibiting action on Nitrobacter than on Nitrosomonas spp. Thus alkaline water (seawater) see an increase in nitrite levels. Other parameters which affect the bacteria differently also have an impact.
• nitrite levels may be high under some types of aquacultural operations, and in some effluent discharges, notably sewage, metals, dyes and celluloid manufacturing.
• nitrite is actively taken up by the gills (chloride cells probably) and blood levels may be 10 times those of the surrounding water
• nitrite oxidizes the iron in hemoglobin → methemoglobin and this pigment Lacks the ability to bind reversibly to oxygen. Grossly, high levels of methemoglobin result in a brown colour to the blood. So-called “chocolate blood disease” of channel catfish farms.
• by contrast with mammals where methemoglobin levels should not exceed 1%, fish may normally have levels of 10%. Levels of 50% or more are considered too high.
• erythrocytes of fish have a reductase which reconverts methemoglobin to hemoglobin but this takes 24–48 hours if fish moved to normal water.
• nitrite also causes a leukopenia, and although in the short term this is probably inconsequential, in the long term with chronic low level exposure, it may increase susceptibility to disease.
• under most situations, nitrite levels in freshwater should not exceed 100 ppb in soft water, 200 ppb in hard (EPA).
• it is crucial to realize that chloride competes with nitrite for transport across the gills. Thus in saltwater, nitrite levels 50 to 100 times greater than in freshwater can be tolerated.
• similarly bromide and to a lesser extent bicarbonate have a similar protective action.
• oxygen levels affect nitrite toxicity because of the reduced carrying capacity of the blood. Similarly, temperature has an affect because of the increased demand for oxygen with a decreasing availability.

7. Nitrate

• as we have already seen, nitrate is formed by the complete oxidation of ammonia.
• it is naturally present in sometimes high concentrations in surface waters and in fish farms.
• they are considered essentially non-toxic although they do enhance the net productivity of aquatic systems, and under some situations, may promote massive algal blooms.

8. Carbon dioxide

• CO₂ is of course excreted by the gills, and with water, it forms carbonic acid, which reduces the pH in the microenvironment of the gills.
• increasing levels of CO₂ in the blood (or a decreasing pH) decreases the affinity of hemoglobin for O₂ (Bohr effect). Fish hemoglobin is very sensitive to CO₂ (large Bohr effect) by comparison with mammals, which are relatively tolerant of CO₂.
• in high enough levels, CO₂ will → anesthesia (> 100 ppm)
• high environmental levels correlate with nephrocalcinosis in intensively cultured salmonids. This is a condition which is common when liquid O₂ is used to boost holding capacities of water. The lesions comprise mineral within renal interstitium and tubules, often leading to severe granulomatous inflammation and cystic dilation of tubules. Muscles dorsal to the kidney are also involved in severe cases. Lamina propria of stomach is usually involved, often before renal lesions. The condition reduces feed conversion efficiency, but causes apparently little else. The major concern is with the esthetics.
  In Canada, we have encountered situations on salmonid farms where nephrocalcinosis is present, and in the acute stages also a suppurative pyelonephritis. This correlates with large numbers of Gramnegative bacteria seen only on tissue impression smears. The significance of this observation is unknown at present.
• CO₂ is utilized by plants during photosynthesis in daylight and in some ponds and surface water systems, this may cause a rise in pH. Similarly, during respiration at night, CO₂ is produced, causing a drop in pH. This diurnal pH change may be as high as 1 pH unit, especially in soft waters.

9. Chlorine

• commonly added to domestic water supplies to destroy pathogens and improve taste and “slugs” of the chemical frequently found in systems after plumbing disturbances. Also discharged from industrial processes, especially textile and paper industries and sewage treatment. Used in experimental conditions to disinfect effluents discharged to surface water. Also used in cooling towers and swimming pools to control bacterial numbers and algal “slime”.
• 
  At pH 6, 96% of dissolved chlorine is present as the acid HOCl (hypochlorous acid). At pH 9, 97% of this acid is dissociated. HOCl is more toxic than the hypochlorite ion OCl^- but this can dissociate to O and Cl^-, the atomic oxygen so produced being a strong oxidizing agent which rapidly produces gill necrosis.
• species such as salmonids are much more sensitive to chlorine than coarse fish.
• unlike some other poisons, recovery does not occur, possibly due to necrosis.
• in the presence of nitrogen compounds such as ammonia, hypochlorous acid forms chloramines which are highly toxic, probably mainly due to the formation of methemoglobin.
• 0.003 ppm is regarded (EPA, 1973) as the upper limit for continuous exposure.
• removal of chlorine and chloramines is accomplished by (1) activated carbon although in the early stages, with fresh charcoal, some ammonia will be produced, (2) sodium thiosulphite and hydrogen sulphide will also remove chlorine although once again, ammonia is produced: this is often removed by the addition of natural clays (zeolites), (3) merely aerating the water vigorously will often remove most chlorine and a large quantity of chloramines. These methods (1) - (3) are most commonly employed in recirculating systems, or in those experimental systems where domestic water is used.

10. Alkalinity, hardness, salinity and pH

• alkalinity, or the buffering ability of water, is due to carbonates, bicarbonates and hydroxides and freshwater from limestone areas typically is well buffered, as in seawater. Waters with a good buffering capacity generally have a stable pH, and are able to resist agricultural or domestic pollution.
• hardness is a measure of the quantities mainly of calcium and magnesium ions, and is expressed as the equivalent of calcium carbonate. Different countries and areas within countries have different ideas as to what constitutes “soft” water, but in general:
    0–50 ppm - soft
    50–150 ppm - medium hard
    150–300 ppm - hard
  Soft water is usually acidic, and hard alkaline. Some species such as the S. American freshwater tropicals seem to prefer soft water whereas salmonids generally do better in harder water (remember the coupled pump mechanisms at the gill surface). Another benefit of hard water is that heavy metals tend to precipitate out of solution. Some diseases seem to occur more frequently in soft water eg. BKD.
• salinity measures the total salts dissolved in water and is expressed as parts per thousand (0/00). Full-strength seawater is approximately 30–40 0/00. Some species naturally can adapt from fresh to seawater eg. salmon. Other species such as rainbow trout can also be made to adapt, but in some situations, such fish may struggle to maintain their osmotic balance. An example of this is seen at low water temperatures in full-strength seawater - rainbow trout become dehydrated so that their muscle is dry, but their stomach full of seawater. Towing the cages to an estuarine area, or running freshwater onto the surface of the water is the only remedy, (increasing the freshwater content of the pellets helps a bit).
• a pH range of 5–9 is generally considered the “safe” range for fish, although maximum productivity is seen from 6.5–8.5. Fish populations are found naturally however from 4–10.
  The addition of acid waste to hard water results in the liberation of CO₂ from bicarbonate, and this may be sufficient to be acutely toxic. Heavy rain may flush out peat bogs (humic acids) or stripmining areas leading to such an acid flush.
  “Acid-rain” is of course a major problem in various parts of the world and is the result of sulphuric and nitric acid aerosols produced by fossil fuel combustion, metal smelting and other industrial processes. In areas where the buffering capacity of water is low, the pH of lakes and streams has decreased and all levels of aquatic life affected, decomposers, primary producers, primary and secondary consumers. Some sensitive species have disappeared altogether. The effects have been acute mortality, reduced growth, skeletal deformities but especially reproductive failure. Spring run-off from melted snow may cause a flush of acid and metals eg. in S. Ontario from 5.5 to 4.5 and aluminum levels from roughly 0.2 mg/L to 1 mg/L. Aluminum toxicity (in the hydroxyl from) is highest at pH 5.0 and causes increased branchial mucus production and hyperplasia. The first run-off may contain most of the acid or metal, thought to be due to a process of ion separation or freeze concentration.
  Acute toxicity of acid results from gill damage, with lamellar epithelial edema and necrosis, plus possibly plasma electrolyte imbalance due to a failure of ionoregulation.

11. Light intensity

• very important but poorly understood
• eggs and fry usually kept shaded, leads to increased hatchability
• shaded fish seen to have lower incidence of disease, both bacterial and protozoal
• deep sea species displayed in high intensity aquaria may lead to retinal degeneration
• sunburn lesions seen in several species

12. Heavy Metals

• can occur in high concentrations in natural waters near deposits, but usually present only in trace amounts. The salts are soluble but precipitate out in hard water and in addition may be absorbed to some of the non-inert clay particles.
• cadmium and copper are extremely toxic to fish, especially in soft water of low oxygen content. Damage to the gills is prominent with lamellar fusion, and edema as early as 24 hours after exposure. Zinc has a similar action but in addition, scoliosis is seen, possibly due to a myelopathy, which causes darkening as well.
• mercury causes branchial changes but also renal tubular changes due to increased permeability of cell membrane and impaired mitochondrial ATP production → cell death. Cadmium has a similar action.

13. Pesticides and Herbicides

• any discussion of these is considered outside the scope of these notes. It should be appreciated however that many herbicides eg. 2,4-D often find their way into waters containing fish, and should always be borne in mind in cases of large fish kills.
• water chemistry should always be borne in mind whenever assessing significance of analytical results, as factors such as hardness, temperature and pH have a profound affect on toxicity of chemicals.
• age and size of fish also have a marked influence as increased surface area/body size increases uptake, as does increase respiration (smaller fish have a higher respiratory rate). Similarly very young fish may have poorly developed organ/enzyme systems to handle toxicant presumably this could be beneficial if a degradation product is the specific toxicant.

• species of organism is also important. In general, fish are more resistant than invertebrates, although there are exceptions (the herbicide trifluralin).

  Comparison of 96 hr LC50's to OP's given in ug/L
  Pesticide	Inverts	Fish
  Malathion	49	162
  Ethyl parathion	24	1391
  Methyl parathion	11	5411
  Diazinon	7	640
  Chlorpyrifos	4	81

14. Algal blooms (include dinoflagellates, phytoflagellates and blue-green algae)

• these may proliferate in large numbers in warm weather so that shading occurs and there is an increase in photosynthesis in deeper water and hence lower O₂ levels. Anaerobic conditions may eventually develop especially at night and fish die, algae die, and various anaerobic products accumulate such as H₂S or methane which are themselves toxic to fish.
• certain algal species however, also produce toxins which are lethal to fish and other vertebrates. Implicated species include Anabaena, Microcystis (a blue-green alga), Gymnodinium and Prymnesium. Many deaths of fish under field conditions occur during sunlight (as opposed to darkness for O₂ utilization) suggesting the toxin production accompanies photosynthesis. Gymnodinium brevis is one of the “red-tide” organisms. Prymnesium parvum (a phytoflagellate) has been responsible for pond mortalities in brackish waters. The toxin (prymnesin) is thought to alter gill membrane permeability and hence cause loss of ionoregulation.
• Ciguatera toxin accumulates in high trophic species such as groupers and barracuda probably due to their eating coral species which graze on coral algae. While not toxic to fish, this toxin can kill man although vomiting, cramps and peripheral tingling are common symptoms (atropine helps control symptoms).
• paralytic shellfish poisoning of man (and other animals) is also a dinoflagellate-produced neurotoxin which causes muscular paralysis and respiratory failure.

15. Electrocution

Occasionally encountered with badly insulated pumps, or aerators, or due to improper use of electro-shocking equipment.

Lightning may cause massive fish kills: the amount of current needed to kill fish depends on several factors, but water hardness is one of the most important. Soft waters are poorer conductors than hard, and thus the fish's body with its higher conductance will be a selective path. Thus, lethal voltages need to be higher in hard than soft waters.

Outer ponds can be affected, but indoor facilities are not immune, as lightning can enter via pipes or drains.

Fish so affected may be killed outright; but survivors may show hyperesthesia or spinal damage due to overcontraction of muscles. A common location is the fulcrum just beneath the dorsal fin in salmonids.

FISH HEATLH AND NECROPSY EXAMINATION

Examination Outline

1. HISTORY

2. SPECIMEN EXAMINATION

   1. External Examination:

      1. growths, deformities, abnormalities

      2. skin - coloration, mucus production, scale examination, scraping and wet mount, vital staining, parasites

      3. fins - color, condition, wet mount preparation

      4. gills - color, condition, mucus production, foreign bodies or parasites, wet mount preparation

      5. eye, opacities, exophthalmia.

   2. Internal Examination

      1. body cavities - color, condition, fluid accumulation (wet mount of body fluid)

      2. visceral organs - size, shape, color, location consistency, cut surface, impression smears, wet mounts

3. LABORATORY EXAMINATION

   1. Bacteria:

      1. tissue or organ submitted

      2. examination requested

      3. gram stain

   2. Blood

      1. hematology

      2. plasma chemistry

      3. parasite examination

   3. Virus

      1. tissue or organ submitted, method of preservation (do not fix)

   4. Parasita - tissue, organ, or preparation submitted, method of preservation gross description of organism, behavior of organism, location

   5. Histology - tissues submitted, method of fixation, principal findings, incidental findings

4. DIAGNOSIS

   1. Tentative Diagnosis

      Differential Diagnosis:

      1. principal findings

      2. supplemental findings

      3. proposed pathogenesis

      4. relationship of history, clinical findings, necropsy and laboratory results

5. RECOMMENDATIONS

   1. Therapy - type and amount of treatment and dosage schedule

   2. Prevention & Control - recommendations for shipping quarantine water quality control, disinfecting husbandry, record keeping

FISH EXAMINATION - HISTORY

Environmental Conditions:

PREPARATION OF A SKIN SCRAPING

(Figure 1)

1. Using a clean scalpel, scrape the skin from head to tall to prevent dislodging many scales. In this way, mucus mixed with epidermis cells and any skin parasites is obtained. The best skin smears are obtained from the lateral sides of the body, the caudal fin and the base of the fins.

2. The material collected is then smeared on a microscope slide and covered with a coverslip. The smear should not be too thick or identification of parasites may be difficult

3. Introduce freshwater (freshwater fish) or 1 to 2% saline (salt water fish) under the coverslip with a Pasteur pipette until the area beneath the coverslip is completely saturated. A well made preparation will be free of air bubbles, and will have no excess fluid at the edges of the coverslip. Methylene blue for contrast or a vital stain may be substituted for water or saline.

4. Examine under a light microscope. A duplicate smear may be airdried, fixed in methyl alcohol (3–5 minutes) for preparation of a permanent mount and stained with Wrights stain or Wright's Giemsa.

Figure 1

Preparation of a skin scraping

PREPARATION OF A GILL SMEAR

1. Remove part of a gill arch with scissors and holding the tissue in forceps, smear the lamellae on a microscope slide. The material which adheres to the slide may contain mucus, blood cells, epithelial cells and any bacteria present on the gills.

2. Allow the smear to air dry and pass the slide through an open flame 2–3 times to fix the smear on the slide.

3. Stain using the Gram stain method and examine under a compound microscope. Gill smears should be made when ever the gills are examined.

PREPARATION OF A GILL MOUNT

(Figure 2)

1. Remove a gill arch from the fish using forceps and scissors. Avoid damaging the filaments to be examined as much as possible.

2. Using a scalpel cut the cartilage away from the filaments.

3. Place a section of the gill on a microscope slide and coverslip it.

4. Introduce freshwater (freshwater fishes) or 1 to 2% saline (salt water fishes) under the coverslip until the area beneath the coverslip is saturated. If more contrast is desired, Wrights stain or a vital stain can be used in place of the saline or freshwater.

5. Examine under a light microscope for parasites, swollen gill lamellae or white spots on the filaments.

Figure 2.

Preparation of a gill wet mount

PREPARATION OF A TISSUE IMPRINT AND TISSUE SMEAR

(Figure 3)

1. Tissue Impriat:

   Cut a small block (1 cm₃) of the tissue and press it to a microscope slide Leaving an imprint. Repeat 2–3 times with each piece of tissue. Allow the imprint to air dry and then fix it to the slide by passing it through a flame 2–3 times. Dip the slide into 70% ethyl alcohol for about 5 minutes and when dry, stain with Wright's or Geimsa stain.

2. Tissue Smear:

   Scrape the tissue with a clean scalpel blade as shown in Figure 5.2. Smear the material on to a microscope slide and allow to air dry. Do not make the smear too thick or it will be difficult to see anything. When the smear is dry, pass it through a flame 2–3 times to adhere it to the slide and fix in 70% ethyl alcohol for 5 minutes. Stain with Wright's or Gram's stain when the smear is dry.

Figure 3.

Preparation of a tissue imprint (1) and a tissue smear (2)

PREPARATION OF A TISSUE SQUASH

(Figure 4)

1. Cut a small section of tissue and place on a microscope slide. Any cysts or abnormalities in the tissue should be examined.

2. Cover the tissue with a coverslip and press down gently allowing the tissue to spread out on the slide. If the tissue is hard or will not spread fairly easily, tease it apart carefully using two mounted needles.

3. Using a pasteur pipette, introduce saline (or fresh water) under the coverslip until the area under the coverslip is saturated. Examine under a dissecting or compound microscope. Bacterial cysts, sporozoa, fungal cysts and encapsulated larvae of various worms may be encountered.

Figure 4. Preparation of a tissue squash

Preparation of Tissue for Histopathology

Specimen Collection

Fixation is the process of killing and hardening tissue. Tissues from poikilotherms tend to decompose rapidly after death, and therefore should be placed in fixative immediately upon removal from the carcass. If fixative is not at hand then try to keep the tissue iced or refrigerated. Do not freeze, as this will destroy most of the cellular detail.

Small fish, up to 5 or 10 cm in length, may be fixed whole. Slit open the muscle along the ventral surface and remove the covering muscle on one side of the abdomen in the larger fish, to allow fixative to reach internal organs quickly. Keep fish flat - do not bend or twist while fixing.

If fish is larger than 10 cm, or in the case of large lesions, tissue sections should be cut thick enough so that the fixing fluid can penetrate within a reasonably short time. Tissues should be no more than 0.5 cm in thickness, and should be immersed in at least 20 times their volume in fixative. Smaller ratios may be required under field conditions, with some sacrifice in quality of the final product. If the fish is considered to be diseased, fix a portion of all tissues. Include some adjacent normal tissue with the lesion when fixing.

If possible photograph the condition and/or the lesion before processing the tissue.

The accompanying figure (page 61) lists the tissues which should be submitted routinely for histopathologic examination.

1. fin (s)

2. skin with underlying muscle

3. kidney (includes head kidney and posterior kidney)

4. spleen

5. intestine

6. pyloric caeca (includes pancreas)

7. stomach

8. liver

9. heart - (atrium and ventricle)

10. eye

11. gill

* Always submit one or two gill arches

  Submit brain when feasible.

Include with the specimen your name and address, your specimen identification number and species of fish. Supply any pertinent information that is available, eg.:

1. location, date, and method of catch

2. gross description of the lesion-where found etc.

3. size (length/weight), sex, and age of fish

4. evidence of abnormal behaviour

5. whether wild or cultivated fish

6. the number of fish with similar lesions

NOTE:

Bacteriological or virological identification cannot be undertaken on formalin-fixed tissue. However, it is possible to observe cellular changes which indicate infection by microorganisms.

FORMAL IN PRESERVATION OF TISSUES FOR HISTOPATHOLOGY

13. Quarantine

Quarantine, a period of isolation of newly transplanted stocks until the possibility of the introduction any pathogens they may carry can be eliminated, is the most potent weapon at the disposal of fish health authorities, as well as of every fish producer. Fish imported from abroad or moved from one locality to another within a single country should be placed in quarantine on arrival and should remain there until all danger has passed. The quarantine period should exceed the length of the longest: latent period of the pathogens. Russian authors recommend a period of one year's quarantine for all imported fish. However, under the higher temperatures occurring under tropical conditions this period can be much shorter.

Quarantine ponds must be safely isolated and must be located downstream from all other ponds on the farm to minimise the danger of penetration of pathogens into them.

Fish markets can become centres for the disposal of pathogens. To avoid this danger, fish should be disinfected upon arrival at the market. Disinfection should aim at destruction of the most dangerous ectoparasites (e.g. Lernaea, Gyrodactylus, etc.) and, so as not to impede normal market activities, should be organized to take as little time as possible. Short duration baths should be experimentally developed and bath techniques should be taught to market personnel. Water temperature/ bath duration charts should be developed and made available to market managers.

14. Regular prophylactic survey

Periodic checks on the status of health in fish production facilities can detect dangerous conditions before they become too great to be handled without incurring serious economic losses. Ideally, their frequency will be determined by the manpower and resources available.

Utilization of knowledge in the country

The knowledge gained by the training will help in greater understanding of the relationship between the environment and ulcerative disease syndrome in fish and development of much needed facilities. Training in research methodology and techniques will enable me to conduct research into new methods and techniques of rapid diagnosis and improved methods in treatment and control of fish disease.

February 27th to March 3rd 1989 (Thailand)

ACKNOWLEDGEMENT

I express my deep sense of gratitude to Dr. A.G. Jhingran, Director, Central Inland Capture Fisheries Research Institute, Barrackpore for deputing me to undergo such a valuable training. I acknowledge the financial aid rendered to me by FAO/NACA. I thank ICAR for permitting me to participate in the training programme. I am grateful to Dr. Mohamed Shariff, Coordinator, FPSS, University Pertanian, Malaysia for providing excellent facilities during the training period. Finally I remember with gratitude the whole hearted help extended by Dr. Chen Foo Yan, Coordinator, NACA and Dr. J. Richard Arthur, Programme Officer (Fisheries) IDRC.

ANNEXURE 1

Teaching Schedule for M.S. Tropical Fish Health Program 1988/89

ANNEXURE - II

(To be filled in by the Scientist deputed abroad to attend Training course/Seminar/Symposium/Conference)

( Balbir Singh )