MODULE 10: PHYSICAL, ENVIRONMENTAL AND CHEMICAL METHODS OF DISEASE

PREVENTION AND CONTROL

Introduction

The environment determines the balance between the host and the disease agent.
Microorganisms are always present in the water and some of them cause disease only when the host had
been weakened through exposure to stressful environmental conditions. Thus, the fish culturist must be
able to maximize the environment and make it favorable to the cultured species.

This chapter deals with the general principles involved in the physical/environmental and chemical
aspects of disease prevention and control that are applicable in the hatchery and the grow-out stages of
shrimps and fishes. Specific chemical treatments against a particular disease are discussed in their
respective modules. The principles involved in biological assay tests are also discussed in this module.

Physical Methods

Physical methods of disease prevention are based on the physiological tolerance of disease agents to
adverse conditions such as increased or low temperature, absence of moisture, or presence of deleterious
irradiation (Kabata, 1985). Another principle is based on the removal of the pathogen source or putting
up of physical barriers to prevent contact between the disease agent and the host. The following are some
methods by which potential pathogens may be inactivated or removed in a culture facility:

o Inactivation of pathogens by means of exposure to ultraviolet radiation;

o Removal of pathogens by means of microfiltration;
o Elimination of chemical pollutants by means of carbon filtration, bio-filtration and water dilution;
o Exposure of tank or pond to heated water and sun drying can also eliminate some microbial
flora; and
o Infected fish must be removed quickly and destroyed.

Quarantine

Quarantine is defined as a period of isolation of newly transported fish and aquatic invertebrates
until the possibility of introducing any pathogens they may harbor can be eliminated (Arthur, 1987; Lio-
Po et al., 1982; Davy and Chouinard, 1982). Quarantine measures are very important in preventing the
spread of diseases of aquatic organisms from one farm to another and from one country to another.
Quarantine protocols involves the inspection or examination of animals for disease agents, certification,
and the issuance of a certificate stating that a particular lot of animals or a production facility has been
inspected and is free from infection by a particular pathogen or pathogens.

Quarantine and health certification form part of the control disease program addressed to prevent
the spread of disease by movement of fish, eggs, and in certain cases, movement of water between
different fish farms. These preventive measures must also be a first line of defense against possible
adverse effects resulting from the introduction or transfer of exotic fish and shellfish. Health certification
is a prerequisite for international movement of live aquatic animals and an important integral part of the
quarantine process. These are documents issued by a Competent Authority which is used for the purpose
of applying quarantine measures in transboundary trade of live aquatic animals and for minimizing the
risk of spread of infectious diseases. There are model health certificates provided in the Aquatic Animal
Health Code of the Office International des Epizooties or the OIE Aquatic Code (OIE, 2008).
 The following are guidelines on health certification requirements (OIE, 2008):

o When importing live fish of any susceptible species of a listed disease, or their eggs or gametes,
the importing country should require that the consignment be accompanied by an international
aquatic animal health certificate issued by the Competent Authority of the exporting country or a
certifying official approved by the exporting country.
o This certificate must certify on the basis of an official health surveillance scheme comprising
inspection and laboratory tests on susceptible species conducted according to the procedures
described in the Manual of Diagnostic Tests for Aquatic Animals or OIE Aquatic Manual (OIE,
2006), whether or not the place of production of the consignment is a country, zone or
aquaculture establishment officially declared free from one or more of the listed diseases.
o Health certification for freedom of one or more of the diseases may also be required for imports
of live or dead uneviscerated fish of a susceptible species or their eggs or gametes, by a country
with an official control policy for the disease(s) in question.
o Exporting countries should only export live aquatic animals or eggs or gametes destined for a
country or zone or aquaculture establishment officially declared free of one or more diseases
listed by the OIE when the exporting country or zone or aquaculture establishment of origin is
itself officially declared free of the same disease(s).

Arthur (1987) discussed properly designed quarantine programs at different levels – national and
international, in order to minimize the risks of pathogens/diseases associated with aquatic animal
movements. These are summarized below:

Quarantine at the Farm Level

Quarantine should be practiced to minimize risk of disease among local species. Fish imported
from abroad, or fish moved from one place to another within a 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. Fish markets can become centers for the dispersal
of pathogens. To avoid this danger, fish should be disinfected upon arrival at the market (Kabata, 1985).
The quarantine period for incoming stock must be observed for at least 2-3 weeks. Legislation of
quarantine requirements should be imposed on all imported and exported fish to minimize the spread of
disease, both within a country and outside. Quarantine ponds must be safely isolated and must be located
downstream from all other ponds on the farms to minimize the danger of pathogen penetration.

Quarantine at the National Level

Quarantine is viewed as one of the component of a national aquatic animal health strategy. The
international spread of serious pathogens of aquatic animals has been a concern of Southeast Asian
countries for many years (Davy and Chouinard, 1982; Arthur and Shariff, 1991). In Southeast Asia, the
components of such a national program have been defined through a regional FAO/NACA TCP project and
one of the major inputs was the “Asia Regional Technical Guidelines on Health Management for the
Responsible Movement of Live Aquatic Animals and the Beijing Consensus and Implementation
Strategy” (FAO/NACA, 2000). These guidelines have been officially adopted as a policy document by the
Association of South East Asian Nations (ASEAN) and these act as a platform for greater implementation
of aquatic animal health management measures within the region. The components of a National Strategy
for Aquatic Animal Health are:

o National pathogen list

o Disease diagnosis
o Health certification and quarantine measures
 o Disease zoning
o Disease surveillance and reporting
o Contingency planning
o Import risk analysis
o National strategies and policy framework
o National and regional capacity building

Quarantine at the International level

The International Council for the Exploration of the Seas (ICES) has recommended policy measures
dealing with the introduction of aquatic species and guidelines for implementation, including methods to
minimize the possibility of disease transfers. The recommendations are embodied in the Revised Code of
Practice to Reduce the Risks of Adverse Effects Arising from the Introduction and Transfers of Marine
Species (Sinderman and Lightner, 1988). The ICES Code of Practice includes the following:

o A recommended procedure for all species prior to reaching a decision regarding new
introductions;
o Recommended action if the decision is taken to proceed with the introduction;
o A suggestion that regulatory agencies use the strongest possible measures to prevent
unauthorized introductions;
o A recommended procedure for introduced or transferred species which are part of current
commercial practice; and
o A note recognizing that countries will have different attitudes toward the selection of the place of
inspection and control of the consignment.

With the emerging impact of infectious diseases on live aquatic animals many countries are in the
process of undertaking risk analyses to prevent the entry and spread of pathogens. Import risk analysis
(IRA) is the process by which importing authorities determine whether live aquatic animal imports or their
products pose a threat to the aquatic resources of their country. This is usually undertaken by the
Competent Authority for the importing country, but risk analyses apply equally to the individual who wants
to import live aquatic animals onto the farm or site (OIE, 2008). The four components of an import risk
analysis are:

o Hazard identification: This identifies the pathogens of concern in the context of the commodity to
be imported, and the possible countries of origin of the commodity.
o Risk assessment: This must be able to accommodate the variety of animal commodities, the
multiple hazards that may be identified with an importation and the specificity of each disease,
detection and surveillance systems, exposure scenarios and types and amounts of data and
information.
o Risk management: This is the process of deciding upon and implementing measures to achieve
the Member’s appropriate level of protection, while at the same time ensuring that negative
effects on trade are minimized.
o Risk communication: This is the process by which information and opinions regarding hazards
and risks are gathered from potentially affected and interested parties during a risk analysis, and
by which the results of the risk assessment and proposed risk management measures are
communicated to the decision makers and interested parties in the importing and exporting
countries.

Some examples of risk management measures for importations of living aquatic animal (Arthur
et al., 2004) are to:
 o source from stocks of known disease status, including the use of specific pathogen free (SPF)
stocks;
o import eggs only;
o require quarantine and inspection in the country of origin;
o require quarantine and testing within the receiving country;
o use International Council for the Exploration of the Sea (ICES) protocols;
o require the use of specific diagnostic tests and standards; and
o require pre-shipment and/or post-shipment treatments.

Fish Health Classification Scheme

Sanitary classification of farms can be instituted such that exchanges of fish occur only among
farms of similar fish health status. This sanitary classification of fish farms can be used as basis for
issuance of permits for fish import, export, exchange or restocking. The following is the fish health
classification scheme (Ghittino and de Kinkelin, 1975; OIE, 2008).

Fish free of specific pathogenic organisms (SPF): This category refer to fish free of all species-
specific pathogens. The water supply in such a facility must be completely sterile. At present, exchange
of fish is possible only between establishments classified as specific pathogen-free. Fish free of coded
pathogenic organisms (CPF): This category include fish free of all diseases appearing in a list drawn up
by an international agreement. Such a list has yet to be made for Southeast Asian countries. The
water supply should have to be pretreated. CPF farms can receive SPF or CPF fish but cannot dispatch
fish to SPF farms.

Fish free of specified diseases (SDF): Fish are reared in water supplies in which pathogens could
exist, multiply or be disseminated by wild fish. Disease could occur but is readily controlled by therapy. A
certification of freedom from certain diseases can be issued but guarantees only for the disease(s) listed
in the document. A farm classified thus can receive fish from SPF or SDF farms as well as enterprises of
similar sanitary level or classification.

Uncontrolled fish consist of those not checked for the presence of disease or pathogens. Fish
exchange is possible only with farms of similar category but can receive fish from the three foregoing
ones. Specific pathogen resistant (SPR) describes a genetic trait of a stock that confers some resistance
against one specific pathogen. An SPR stock usually results from a specific breeding program designed to
increase resistance to a particular virus.

Environmental Methods

Monitoring of the environment is extremely important for success in fish culture. The primary
objective of environmental methods is to protect the host by intercepting the pathogen or cutting its
pathway to the host. The sections that follow will attempt to describe some of the ways this is carried out.

Proper Site Selection and Farm Design

Proper site selection and farm design are very important in providing the fish or shrimp with
appropriate good water quality management. The following are some considerations involved in proper
site selection and farm design (Herman, 1970; Kabata, 1985; Lio-Po et al., 1989):

 The farm should have access to good water supply free from any type of pollution (Fig. 1).
 An independent water supply and drainage canals should be provided to each individual part of an
aquaculture grow-out facility (Fig. 2) to ensure that water emerging from one pond compartment
does not enter the other ponds or compartments.
 The farm should be kept free of wild fish and other potential carriers of infectious agents such as
invertebrates, pests, and predators.
 The farm should be accessible by road to avoid excessively long transport time of the larvae or fish.
 Pond development, wherever possible, should be adjacent to mangrove areas for protection from
erosion, and to provide a natural filter for farm effluents (Primavera 2000). Mangrove to pond ratio
of 7.82:2.7 ha for intensive shrimp culture ponds is recommended to efficiently treat the effluent.
 Well-designed farms as well as trained personnel are important requirements in ensuring that fish
health management practices can be incorporated in the routine operations of an aquaculture system.

Figure 1. An ideal (a) and poor (b) aquaculture site: (a) A potential aquaculture area that is likely to have
water of good quality. The site has lush vegetation and distant from industrial and domestic
pollution; (b) Note the presence of houses and factories suggesting domestic and industrial
pollution (Lio-Po et al. 1989).

Good Water Quality

Good water quality is crucial in the hatchery or pond. It can spell the difference between success
and failure of the aquaculture enterprise. The lower the water quality, the lower the fish/shrimp biomass
it can support; the higher the water quality, the higher the production potential will be. Poor water quality
can lead to poor growth, disease, bacterial, viral, fungal and parasitic outbreaks. Good water quality
appears to be the best defense against all diseases.

The following are some procedures used to obtain good water quality (Kabata, 1985; Lio-Po et
al., 1989; Post, 1983):

 Aside from being pathogen-free, the water must meet the specific quality requirements of the cultured
species. Table 1 below gives optimum level of water parameters for the successful grow-out culture
of P. monodon.
 Rearing water quality parameters such as salinity, pH, dissolved oxygen, ammonia, temperature, etc.
must be monitored regularly.
 When used properly, ultraviolet radiation and filtration systems can eliminate potential pathogens.
 Sand filters or filter bags will remove most of the debris. The water may be filtered with fine net (Fig.
3), cloth or cartridge filters, which come in various mesh sizes from 1-5 μm before delivery to tanks
and before stocking the fish. Filters should be cleaned regularly.
 Biological filtration is also an essential requirement of any re-circulating fish holding facility. It breaks
down toxins excreted by fish. Biofilters are designed to remove these compounds from water by the
use of nitrifying bacteria, where ammonia and nitrite are broken down by the bacteria.
 Rearing water should be aerated (Fig. 4) and changed regularly.
 Bottom sediments should be siphoned off regularly to remove feces, organic debris and unused feed
(Fig. 5).
 Paddle wheels and other aerators should be installed in a settling reservoir to reduce the organic and
particulate load before the water is directed into ponds.
 Provision of central drain system made of perforated pipes and placed horizontally at the center of
the pond connected to a pipe leading to the outlet to remove wastes.
 The pond bottom must be cleaned any time throughout the entire culture period.
 Use a screen of small mesh size to cover the drain for the first 50 days of culture and remove it to
allow for easy removal of water when the fish/shrimps are larger than the diameter of the pipe.

Table 1. Water quality suitable for the grow-out culture of black tiger shrimp, Penaeus
monodon (Chanratchakool et al., 1995).
Figure 2. Layout of a 10-ha semi- intensive shrimp pond
system (Lio-Po et al. 1989): A main supply canal
serves as the main conduit for water from the
main source. The water is then allowed in the main
gate, which further enters through the
secondary gate. This will impound the water in the
supply canal from which the pond compartments
obtain their water supply by opening individual
gates. The bold arrow represents the direction of
the water in the canal. Broken lines represent the
trench canal, which provides for easy drainage
during water changes. Effluents from the
individual compartments are collected in the drain
canal. Finally, all the effluents are released to the
main drain canal. The drain canal is lower in
elevation than the main supply canal for easy
draining.
 Figure 3. Filtration of rearing water using a fine
net filter bag,
simultaneously delivering it to the
rearing tank (Lio-Po et al., 1989).

Sanitary Practices

Sanitation is an important phase of any animal husbandry program to prevent the development
of pathogens. Cleanliness improves the general standard of health in aquaculture facilities. It also prevents
or retards the development of disease agents.

The following are the common sanitary practices in fish culture (Kabata, 1985; Lio-Po et al., 1989):

 Tanks and ponds should be drained of water between culture periods (Fig. 6). Tanks need to be
exposed to the sun for them to dry completely, while the pond soil also needs to be dried completely.
 Filters should be backwashed or cleaned regularly (Fig. 7).
 Day-to-day hygienic measures should include:
o siphoning out of organic materials that accumulate in the tank bottom (Fig. 5);
o immediate removal of any sick or dead fish; and
o careful control of aquatic vegetation in ponds.
 Gear such as scoop nets, buckets and pails for exclusive use in individual facilities or sections should
have proper labels and markings.
 Only non-toxic plastic pipes, (such as polyvinyl chloride or PVC), pails, and other equipment parts
should be used.
 Workers should disinfect their hands with soap and water before preparing and administering feed,
and before performing other jobs.
Figure 4. Rearing water especially in hatcheries are provided with adequate aeration
Figure 5. Bottom sediments that accumulate in the tank
bottom are siphoned out at regular intervals
Figure 6a and b. Draining, cleaning and drying of
aquaculture facility: a) After the tank is drained of its water, the
sides are cleaned with foam or brush using a detergent solution
to remove algal growth and bacterial flocs. Rinsing with
clean water follows (Lio-Po et al., 1989); b) After complete
draining, the pond soil is exposed to sunlight for several days or
weeks to dry completely
Figure 7. Sand filter system showing operational inlet flow (a) and reverse flow or backwashing (b)
(Lio-Po et al., 1989)

Stress Avoidance

Stress is defined as physical or chemical factors that cause body reactions that may contribute to
disease and death. It is among the major predisposing factors for disease epidemics in aquaculture
(Walters and Plumb, 1980). Kabata (1985) reported that stress plays a major role in the susceptibility of
fish to disease. Most diseases are stress-related. The following are stress inducing factors: poor water
quality; inadequate food; overstocking; handling; grading; and transfer and transport of animals. Regular
monitoring of the health status of the stock can detect early signs or onset of diseases before they become
uncontrollable.

Termination procedures

Termination is the most drastic measure against the spread of disease agents but may also be
used to control fish diseases (Kabata, 1985; Lio-Po et al., 1989). This includes destruction of infected
individuals, by burning, cooking or burying in limed pits. Related measures are:

 Disposal of infected individuals should be in areas that will not affect the culture system. Dead fish
are to be burnt or buried into deep pits (approximately 2 m depth) with a distance of at least 20 m
from the pond bank. The bottom of this pit and dead fish must be covered by burnt or chlorinated
lime. A layer of at least 60-80 cm of soil must cover the contents of the pit.
 Contact between diseased and normal individuals must be avoided at all times.
 The water supply system that may have carried the pathogen must be disinfected and drained of
water, and dried. Contaminated facilities may be disinfected with chlorine or lime.
 Alternative management techniques, such as using the distribution canal as such and not as a reservoir
and requiring that it be empty for significant periods during grow-out cycle is effective in controlling
viral transmission from surroundings to the ponds. Some farmers switch to: (a) crop rotation using
other commodities such as milkfish and tilapia in order to improve pond production or to reduce
disease occurrences; (b) long dry-out period where a reduced number of growing cycles for each
pond for a given year is apparently compensated by a reduced risk of production failure and increased
yield and some growers extend drying period of ponds for 2-3 months to benefit good yield.
 Paraphernalia used to handle infected individuals must be disinfected.
 The pond bottom must be drained and dried at the end of the production cycle. The pond is drained
and left to dry in the sun for a period of 10-30 days. Then the waste is removed, either manually or
 mechanically, and transported to the waste dumping area. Once the pond is cleaned, it is then filled
with water and left overnight before flushing out to remove debris and elevate the pH. This process
should be repeated until the pH of the water remains above 7, and only then the lime is applied.

The amount of lime to be used should be carefully calculated to avoid inducing an excessively
high water pH which may increase ammonia toxicity and result in the mortality of fish/shrimps. During the
application, lime should be spread throughout the pond bottom and up to the top of the dike. It should
be applied within 3 or 4 days after the ponds are drained and before the bottom soil becomes
extremely dry. After liming, the pond should be filled to the maximum depth through screen with
fine mesh (24 per square inch) to prevent the predators and competitors from entering the pond. The pH
of the water in treated pond must be checked before stocking fish.

Biosecurity Measures

Biosecurity is defined as the practice that prevents the spread of pathogens from infected animals
to susceptible animals or that prevents the introduction of infected animals into a region or country in
which the infection has not yet been identified (Lee and Bullis, 2003; Lightner, 2002). The primary goals
of a biosecurity program in aquaculture are to maintain healthy animals and to prevent the introduction
of any infectious organisms into an aquaculture facility.

Horowitz and Horowitz (2003) described physical, chemical and biological precautionary measures
to be taken as well as a second line of defense against potential disease outbreaks. Physical measures are
those that aim at preventing the intrusion of disease-carrying vectors to the farm site. The following are
some of the more commonly implemented biosecurity measures among fish/shrimp growers (Cruz-
Lacierda et al., 2008; Horowitz and Horowitz, 2003):

 All incoming fish/shrimp should be obtained from reputable suppliers and accompanied by a health
certificate verifying their disease-free status.
 Installation of physical barriers to prevent entry of disease-carrying animals, thus preventing disease
transmission from the outside. These include fences against the migration of crabs into the ponds
(Fig. 8) and bird scaring device to minimize birds from dropping their fecal matter into the ponds (Fig.
9).
 Filtration of incoming rearing water using membrane ultrafiltration filters, cartridges and carbon filters
(Fig. 3), as discussed above.
 Physical disinfection of water using ultraviolet (UV) light can be used as effective sterilizer for small
volume facilities.
 All farm personnel and visitors should follow biosecurity measures when entering a biosecured facility
such as washing and disinfection of hands (Fig. 10) with soap, water and alcohol. Footwear are to be
disinfected by placing 200 ppm chlorine or 3% Lysol solution in disinfecting rugs/trays at the entrance
of aquaculture facility (Fig. 11a and b). Rugs should be washed and changed regularly. Disinfectants
should also be changed regularly.
 Access to the facility is limited to a minimum and/or only to farm personnel.
 Fence the facility and use locked gates.
 Use tire baths containing a strong solution of chlorine or formalin or chemical sprays to disinfect tires
of incoming and outgoing vehicles and other mobile equipment that enters the facility (Fig. 11-12a
and b).
 The use of “green water” culture techniques appears to reduce pathogen-related problems. Green
water technique is an innovative technique in which the shrimp stocks are cultured in water where
microalgae such as Chlorella sp. grow abundantly (Tendencia and de la Peña, 2003). In this method,
the reservoir is stocked with fish, with Tilapia hornorum as the most frequently used species that
produce “green water” and subsequently pumped to shrimp grow-out ponds. In some cases, the fish
are cultured in cages inside shrimp ponds. Lio-Po et al. (2005) showed that the effectiveness of the
“green water” in preventing outbreaks of luminous vibriosis in shrimp grow-out ponds can be
 attributed to the presence of anti-luminous Vibrio factors in the bacteria, fungi, phytoplankton
microbiota, and the skin mucus of tilapia.

Figure 8. Crab fence: polyethylene sheets and nylon screen nets are lined along sides of pond dikes as
fence to prevent entry of disease-carrying organisms such as crabs and other crustaceans
Figure 9. Bird scaring device: polyethylene ropes 2-3 m above the water surface (arrow), with 30 cm
distance in between ropes are installed to prevent birds from dropping their fecal matter to the
ponds
Figure 10. Washing with soap and water and disinfection of hands with alcohol are
required for all farm personnel and visitors at the entrance of a
biosecured facility
Figure 11a and b. Disinfection rugs/trays
containing 200 ppm chlorine or 3%
Lysol solution are placed at the entrance of
a biosecured facility for all incoming
farm personnel and visitors
 Figure 12a and b. Tire bath containing a strong solution of chlorine (a) or
formalin to disinfect tires of incoming and outgoing vehicles (b) are provided
at the entrance/exit gate of a biosecured shrimp brackishwater grow-out
farm

Chemical Methods

The intensification of aquaculture resulted to frequent occurrence of several infectious diseases,

and consequently the use of chemicals has become inevitable. Among the many uses of chemicals in
aquaculture the most notable are to: enhance natural aquatic productivity; maintain optimum physico-
chemical parameters required for growth of the cultured animal; and prophylaxis and treatment of disease
problems (Cruz-Lacierda et al., 2000, 2008; Schnick, 1991; Schnick et al., 1997; Subasinghe et al., 2000).

Chemicals are routinely used as prophylactic or disinfectant to prevent disease, or as fish

therapeutics to treat and control disease outbreaks (Arthur et al., 2000).

Prophylactic Methods

Prophylactic treatment methods are protective or defensive measures designed to prevent a

disease from occurring. These are used to combat external parasites and stress-mediated bacterial
diseases. There is a common saying that an ounce of prevention is worth a pound of cure. Preventive
measures have always big advantage over curative practices.

Disinfection of Culture Facilities

Tanks: Rearing tanks should be disinfected in between rearing periods. Tanks bottom and
sidewalls are drained and scrubbed using powdered detergent and plastic brush to remove attached
debris, and rinsed thoroughly to remove soap suds and loosened contaminants. Disinfection follows with
200 ppm chlorine for 1 h or with 100 ppm chlorine for several hours (Lio-Po et al., 1989). Tank bottoms
and sidewalls are scrubbed again and rinsed several times with clean freshwater and allowed to dry for
several days, under the sun if possible.

Earthen ponds: The pond is drained and then dried. Lime at 0.5-1 ton/ha CaCO3 or agricultural
lime is applied with 20 ppm tea seed cake, or any of the following: 600-ppm Roccal (benzalkonium
chloride), Hyamine 1622 and Hyamine 3500 (quaternary compounds) (Herwig, 1972; Kabata, 1985; Post,
1983; Roberts, 1978).

Disinfection of Rearing Water

Chlorination method: Chlorine is the cheapest disinfectant. One of the best and commonly used
is calcium hypochlorite (powder form) or ordinary household bleach (Purex, Chlorox). The water must be
filtered first. It is also reduced by organic matter such as mud, slime and plant matter, and must be
covered as chlorine loses its strength when exposed to air. Disinfect sand-filtered water with 5-20 ppm
chlorine for 12-24 h, then neutralize with sodium thiosulfate until residual chlorine becomes zero (Lio-Po
et al., 1989) using portable kits available in the market. Chlorinated, neutralized water must be used within
6 h as bacterial load increases after 12h (Baticados and Pitogo, 1990).

Ozonation method: Ozone (O3-triatomic oxygen) is a more powerful oxidizing agent than
hypochlorite. It can deactivate or destroy viruses and bacteria that might be transmitted through the water
supply system (Munro and Fijian, 1980; Post, 1983). At 90 mg/L concentration and exposure for 20 min,
ozone can control bacterial and viral fish pathogens in water supplies, although this level may not eliminate
100% of pathogens (Yoshimizu et al., 1995). Like chlorine, ozone is toxic to aquatic organisms. Oxygen
(O2) is a breakdown product of ozone, and oxidizing action may result in oxygen super-saturation or gas
bubble disease. The concentration of 0.005 ppm ozone is the upper limit for continuous exposure.
Ozonated water must be re-aerated before it is used.

Disinfection of Materials

Materials like pails, brushes, scoop nets, secchi disk, glasswares, hose, etc. may be disinfected in
between use in different culture facilities. The materials should be dipped in 400 ppm chlorine solution for
a few seconds, and rinsed thoroughly with clean water (Lio-Po et al., 1989).
Disinfection of Feeds

Artemia cysts: Artemia cysts may be decapsulated in 30 ppm chlorine solution or 10 ppm formalin
1 h before hatching (Lio-Po et al., 1989).

Disinfection of Penaeus Monodon

Spawners: Penaeus monodon spawners may be disinfected with 5 ppm Treflan for 1 h or 50-100
ppm formalin for 30-60 min (Lio-Po et al., 1989). After disinfection, the spawners must be rinsed
thoroughly in clean water.

Eggs: Penaeus monodon eggs may be disinfected with 20 ppm detergent for 2-4 h (Lio-Po et al.,
1982, 1985; Lio-Po and Sanvictores, 1986). Disinfection should be done at least 6 h before hatching. After
disinfection, the eggs must be rinsed thoroughly. The water in the hatching tank must be changed
completely.

Larvae: Penaeus monodon larvae may be disinfected with 0.1 ppm Treflan (trifluralin) once every
other day (Lio-Po et al., 1982, 1985; Lio-Po and Sanvictores, 1986).

Chemotherapy

Chemotherapy involves the use of drugs or chemicals for treating infectious diseases. It is
considered as the method of “last resort” in any disease control program. There are four key factors that
are of utmost importance in chemotherapy. These are the water, the fish, the causative agent and the
chemical (Austin, 1985; Herman 1972; Horner, 1983; Munro, 1980; Plumb, 1995; Sindermann and
Lightner, 1988). Complete information about each of these factors must be known before any therapy is
planned. Knowledge about the fish includes the species affected, the number and size of the fish.
Information about the causative agent of the disease is based upon correct diagnosis and its life cycle.
Data on the physicochemical characteristics of the culture water are also required for the selection of the
most suitable chemical. Selection of the therapeutic compound must be based on the following
considerations:

 Tolerance of the fish to the chemical: Tolerance of fish to a chemical varies with age, size, species,
and health condition. Younger or smaller fish are more sensitive than bigger or older ones. Some
species are better able to tolerate chemicals than others are. Fish weakened by disease become less
tolerant to stress and environmental fluctuations.
 Efficiency of the chemical: The choice of what chemical to use is based on differential toxicity, that is,
the chemical must be lethal to the target microorganism but harmless to the host. It is essential to
know the properties of the chemical such as the active ingredient, solubility and application method.
Also, the chemical must not bring harm to the environment.
 Restrictions on the use of chemicals to treat food fish: Only chemicals that break down rapidly and
are eliminated quickly from all fish tissues to avoid tissue residue problems should be used. Strict
implementation of withdrawal period before the cultured stock is harvested. In the tropics, this usually
takes 2-3 weeks.
 Consequences of drug resistance: The indiscriminate use of antibiotics may lead to the development
of drug-resistant strains.
 Economics: Chemicals are expensive, and so one should know the value of the stock and the cost of
treatment to determine the benefits that may be derived from their use.
Methods of Disease Treatment

There are several methods of disease treatment (Herman, 1972; Horner, 1983; Kabata, 1985;
Munro, 1980; Plumb, 1995; Sindermann and Lightner, 1988). Selection of the best treatment method
depends upon the specific disease situation and the chemical used in treatment. Generally, the treatment
methods described below require either the fish to be removed from the culture area and then returned
after treatment or instead of treating the culture water to remove a pathogen, the fish themselves are
treated with a chemical. This is usually done through incorporation of the chemical into the fish diet or is
injected directly onto the diseased fish. The following treatment methods can be used:

External Methods

External methods of treatment are used to control ectoparasites and other microorganisms outside
the fish. They are employed to reduce or eliminate potential pathogens from tanks, ponds, and from other
materials. The external method may either be topical or immersion type.

Topical: Topical method is the most direct and simplest method for treating individual fish with
localized external infection. The fish is anaesthetized before being taken out of the water for treatment,
after which the chemical in high concentration is applied directly on the infected area. This method is
labor-intensive and should be used only for high-value fish.

Immersion: Immersion method is the most common method of administering therapeutic agents
to fish. Immersion in water soluble compounds is primarily used for surface infections involving the skin
or gills of fish. Depending on the available facilities, severity and nature of the disease and local conditions,
there are several types of immersion treatments:

 Dip. The fish are collected with a scoop net and immersed in a high concentration of chemical solution
for a specified time, usually from a few seconds to a few minutes. They are rinsed immediately in
clean water after treatment and returned to the holding facility (Fig. 13).

 Short bath. The chemical solution is added to the holding facility where the fish are to be treated,
allowing the fish to remain in the chemical and water mixture for a designated time, usually a few
hours or less. After treatment, the water is immediately removed and replaced with clean water (Fig.
14).

 Flush. A highly concentrated chemical solution is added at the water inlet, and allowed to be mixed
thoroughly and to pass through the water flow system and out of the effluent pipe (Fig. 15).

 Long bath. The fish is treated for a longer time, usually 12 h or more, in a chemical solution of low
concentration.

 Flow-through. The chemical is added at a fixed rate with a constant flow device to give a consistent
low concentration for the desired treatment time. The treated water moves through and out of the
container, and is eventually replaced by new clean water (Fig. 16).

Systemic Treatment

This method is applied for treatment of systemic infections by incorporation of the chemical into
the feed. The advantages of this method are that lesser chemicals are needed, environmental pollution is
lessened and labor input is low. The disadvantages are the non-feeding behavior of sick fish and that,
since some drugs are not stable in moist diets, this would require introduction of more palatable
components. Leeching of drug is another problem. If some of the water soluble drugs are properly mixed
with vegetable oil prior to its final mixing with the feed, such losses may be minimized.
Parenteral Treatment

This is a direct and the most effective route of drug administration. Its advantages are that
accurate dosage can be administered and pollution of the environment is avoided. The disadvantages are
that it is labor-intensive and contributes to handling stress. Injection is usually done only for big and very
valuable stocks, such as broodstock. The methods are:

Intramuscular injection: The needle is inserted posterior to the dorsal fin above the midline of the
body (Fig. 17a and b). Absorption is slow (not very effective) or, sometimes, does not take place at all.

Intraperitoneal. This is the most common method of injection. The needle is inserted into the visceral
cavity or belly of the fish (Fig. 18). The drug must be highly absorbable and should be able to pass through
either the intestinal wall or some other membrane to be absorbed into the fish system.

Intravenous. The needle is inserted by direct cardiac puncture, or through the caudal artery (Fig. 19).
This results in rapid dispersal and is the most effective route for administering antibiotics. The only
drawback is that this can be used only on large fish. This method is not commonly done.

As mentioned earlier, chemotherapy is considered as the last resort in any disease control program
because of the following disadvantages (Aoki, 1992; Austin and Austin, 1987; Baticados et al., 1990;
Herman, 1970, 1972; Horner, 1983; Plumb, 1995):

 If used in closed recirculating systems, chemicals may cause adverse effects or destroy the nitrification
processes in biofilters.
 Chemical may have adverse effect on natural food. They may induce oxygen depletion during their
degradation, or may destroy algal blooms whose decay then depletes the dissolved oxygen in the
water. They may also inhibit photosynthetic production of oxygen.
 Chemicals may leave harmful residues in the host tissues.
 Diseased animals are usually off-feed. Thus the drugs incorporated in the diet (medicated feeds) are
not taken at all.
 Immersion methods usually do not result in therapeutic tissue level and may be ineffective against
systemic infection.
 Prolonged or widespread use of chemicals may lead to development of antibiotic-resistant bacterial
strains.
 Some drugs have immunosuppresive effects.

The development of drug-resistant bacterial strains may be prevented by the following approaches:

 Correct diagnosis of the disease case;

 Use of the prescribed dosage for a given period;
 Restricted use of drugs;
 Simultaneous administration of two drugs that will not result in cross-resistance; and
 Strict observance and implementation of the clearance or withdrawal period before the fish/shrimp
stock is harvested or consumed.
Figure 13. Dip method: The fish are scooped out and suspended in the chemical
solution for a few seconds to 3 min, rinsed well with clean water and
returned to the holding facility

Figure 14. Short bath method: The chemical solution is added to the holding tank
with fish, allowing the chemical/water mixture to remain with the fish
for several minutes to 1 h. The solution is removed right after
treatment and replaced with new, clean water
Figure 15. Flush method: The chemical solution is added in concentrated form at the
inlet and allowed to disperse through the water flow system and out the
effluent pipe

Figure 16. Flow through method: The chemical is added at a constant rate by a constant
flow device. The chemical moves through and out of the container to be
eventually replaced by new, clean water
 Figure 17a and b. Intramuscular injection: The needle is
inserted into the space posterior to the
dorsal fin above the midline of
the body

Figure 18. Intraperitoneal injection: The needle is inserted into the visceral cavity or belly of the
fish
 Figure 19. Intravenous injection: The needle is inserted by (a) direct cardiac (h) puncture or (b)
through the caudal artery

Principles of Bioassay

A biological assay or bioassay is a procedure that involves the use of the responses of aquatic
organisms to detect or measure the presence or effect of one or more substances, wastes, or
environmental factors, alone or in combination (APHA, 1985; Hubert, 1980). In bioassay, test animals are
subjected to a series of concentrations of a known or suspended toxicant under controlled
conditions. Bioassays are conducted to: determine the tolerance of test animals to a particular chemical;
evaluate the toxicity of a chemical to a test animal; and determines the suitability of environmental
conditions for aquatic animals.

Types of Bioassay

Bioassays are characterized based on the duration of exposure to the chemical or toxicant and
the method of adding the chemical or toxicant (APHA, 1985; Hubert, 1980).

Short-term. This type of bioassay reveals in relatively less time (usually 8 days or less) the relative
toxicity of different toxicants to a selected test organism. It shows the relative sensitivity of various
organisms to different conditions or variables like temperature and pH. It also determines the median
lethal concentration (LC50), or the effective concentration values.

Intermediate. This bioassay is used when LC50 determination requires additional time (usually 8-90
days) for studies on the life stages of organisms with long life cycles, and to indicate toxicant
concentrations for life cycle tests.

Long-term. This bioassay procedure is almost always a flow-through test. It determines the maximum
allowable toxicant concentration, or safe concentration, for indication of water quality standards;

Static. The test animals remain in the same test concentration for the duration of the test.

Renewal. This is a static test where the test animals are transferred to a fresh test solution of the same
composition at periodic intervals, usually every 24 h.
Re-circulation. This static test involves the circulation of test solution through test chambers. The test
solution may be treated by aeration, filtration, sterilization or other means to maintain water quality.

Flow-through. Measured quantities of dilution water and the stock toxicant solution are mixed and
delivered periodically to the test containers to provide continuous flow-through of the test toxicant.

Bioassay Test Procedures

The procedures in conducting a bioassay test is outlined below based on APHA (1975) and Hubert
(1980). Some publications that dealt on experimental exposures of fish species to chemicals or toxicants
are also noted (Cruz, 1981; Cruz et al., 1988; Cruz-Lacierda, 1992, 1993; Cruz and Pitogo, 1989; Cruz and
Tamse, 1989; Pakingking et al., 2002; Pascual et al., 1994). Experimental fish. In conducting bioassay
experiments, a number of criteria are used in selecting and in preparing the test animals. These are:

 sensitivity to the materials under consideration;

 availability and abundance;
 recreational, economic and ecological importance;
 adaptability to laboratory conditions;
 suitability for bioassay tests;
 originating from a single common source;
 uniformity in size and of the same stage of maturity;
 healthy and free from disease and parasites;
 no previous exposure to heavy metals, pesticides, and other substances;
 acclimation to laboratory conditions for at least 10 days; and
 during acclimation, mortality should be less than 10% of the total population.

Experimental water. The water should not be polluted or contaminated with wastes from any
source. Experimental design. There should be a minimum of 5 test concentrations and control in at least
duplicate containers.

Test concentrations. Liquid waste concentrations are expressed in percentage on a volume to volume
basis. The concentrations of non-aqueous wastes and of individual chemicals are expressed as percentage
composition by weight. The resulting LC value is based on concentration of total material or active
ingredient.

Test containers. Glass aquaria should be clean and of uniform size and shape. They should measure 15-
30 cm deep and should be arranged at random in the testing area. If replicates are used, the series of
test containers should be randomized separately.

Number of test animals or biomass. Some of the requirements are: (1) There should be at least 10
animals per concentration; (2) Less than 10 animals may be used for the range finding test; (3) There
should be a maximum of 1 g fish/liter of water; (4) The animal are distributed at random by adding one
at a time to each container. Preparing test solutions. Test solutions must be freshly prepared. These should
not be unnecessarily exposed to air and light.

Feeding. The fish should not be fed for at least 2 days before the test. The fish should not be fed also
during the entire experimental period for short-term tests (96 h or less).

Biological data and observations. Mortality should be observed and recorded at periodic intervals.
The usual indicator for death is the absence of movement (no gill movement in fish) and absence of
reaction to gentle prodding. Dead organisms must be removed as soon as observed. Responses such as
erratic swimming, loss of reflex, discoloration, changes in behavior, excessive mucus production,
hyperventilation, opaque eyes, curved spine, and hemorrhaging should be recorded. Physical and
chemical water quality. Water quality parameters such as temperature, salinity, hardness, alkalinity, pH,
dissolved oxygen, ammonia and nitrite must be measured and recorded at the beginning of the test and
daily thereafter.

Calculation, analysis, and reporting of results. The recommended measures or indices of relative
toxicity are 48 and 96 h LC50. The 95% confidence limits for LC50 and EC50 values are always computed.

The most widely used methods for calculating an LC50 and confidence limit are probit, logit,
moving average and Lithcfield-Wilcoxon (1949). Other methods are straight line interpolation and the
Reed-Muench method (1938). The LC50 are reported together with the specified exposure time and the
confidence limits.

Descriptions of test organisms (species, source, size, weight), procedures for acclimation to test
conditions, and observations on behavior during the test are noted. Likewise, the source of experimental
water and its characteristics, source and properties of tested material, and concentrations of the test
solutions should also be described. The experimental temperature, test method, test conditions (type of
container with volume and depth of solution, number of organisms), and the criterion of response are also
indicated.

Summary

Disease prevention is a primary and cost-effective method in fish health management. It is more
effective and economical than attempting to stop a disease that has already set in. Preventive measures
have always big advantage over curative practices. Moreover, the drug may not provide remedies under
all circumstances. Also, the drug may not help the host survive the infection until the environment is
improved. Ideally, fish culturists should strive to decrease the stress-causing factors and eliminate and
prevent the entry of pathogenic organisms by strictly adhering to the fish health monitoring
programme. The recommendations given above will greatly reduce the possibility of disease outbreaks.

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