Major Diseases Encountered in Rainbow Trout Reared in Recirculating Systems
Alicia C. Noble
The Conservation Fund's Freshwater Institute
Shepherdstown, West Virginia
Abstract
Recirculation systems create unique environments for fish culture. Opportunistic microorganisms
may cause disease due to favorable conditions occurring in recirculation systems. The biofilters used
may become reservoirs of infectious microorganisms. Stressful conditions such as suspended solids
in culture tanks and high rearing densities may contribute to disease outbreaks. Additionally, noninfectious problems such as high nitrite, elevated carbon dioxide or high ozone levels may cause
mortalities or produce stress. The major infectious diseases which have been encountered in trout
reared in recirculation systems include: bacterial gill disease, furunculosis, amoebic infestation, Colegs
sp. infestation, and the parasites Gyrodactylus, Chilodonella, Triichnoodaiina, Ichthyopthirius, and
Ichtyobodo. Because eradication is particularly difficult in a water reuse culture system, to prevent
introduction of infectious diseases and to reduce disease occurrence, it is important to check the
fingerlings before stocking and to maintain optimum environmental conditions in the system. Disease
problems are usually controllable if action is taken at the onset to stop spread of infection and steps
taken to correct the stress that may have triggered the outbreak. Treatments in recirculation systems
may affect biofilter function.
Introduction
This overview discusses personal experiences at The Freshwater Institute and at other facilities where
trout are reared in recirculation systems. There are a limited number of recirculation culture systems
where trout are reared, so experience and information about disease problems characteristic of these
conditions are not widespread. Information that has been published is also reported.
Recirculation systems are becoming more common in fish culture. These systems create highly
artificial environments with unique characteristics which can cause fish health problems. Contrasts
in both system management and treatment process design may produce different fish health problems,
or problems which may occur in one system, will be absent or a minor problem in another.
Additionally, treatment of recirculation system water with chemicals poses the problem of possible
damage to the nitrifiers in the biofilter, while treatment by-passing the biofilter may allow the disease
organisms to remain in the biofilter and reinfect fish. To prevent the introduction of infectious
diseases into a recirculation system the best recommendation is to hatch eggs at the facility, or buy
fingerlings from a certified disease-free source and quarantine fingerlings before introduction into the
system and rearing them in a pathogen-protected environment. Because each recirculation facility
is different, it will be necessary to design a fish health protocol that is specific for that facility.
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Diseases encountered in rainbow trout reared in recirculation systems have been: infectious diseases
and parasites, and non-infectious problems.
Infectious Diseases and Parasites
The infectious diseases and parasites which have caused mortalities in trout in recirculation systems
include: bacterial gill disease, furunculosis, amoebic gill infestation, Colegs sp. infestation, and
parasite infections with Gyrodactylus, Chilodonella, Trichodina, Ichthyophthirius, and Ichtyobodo.
Bacterial diseases
Bacterial gill disease. Bacterial gill disease (BGD) is common among cultured salmonids and
produces high mortality if not treated early in an epizootic. The causative bacterium is
Flavobacterium branchiophilum (Von Graevenitz, 1990) and the disease has been experimentally
transmitted with this bacterium (Ferguson et al., 1991). Although stressors are not required for
disease outbreaks, they probably contribute to the number and severity of BGD outbreaks. At the
Freshwater Institute (FI) BGD has been a recurring problem. Each time new fingerlings were added
to the system they experienced BGD within 6-8 days. The fingerlings were treated and mortality
subsided until new fingerlings were again added. Although newly added trout showed some
hyperplasia before being stocked they had no detectable F. branchiophilum. It was postulated that
the continuous presence of suspended solids from the biofilter, feces, and uneaten food may have
contributed to the outbreaks. The design of the cross-flow culture tanks may have been at least partly
responsible for the regular outbreaks (Bullock et al. 1994). It is interesting to note that only trout
newly added to the reuse system became infected. Trout already residing in the system and which
had previously experienced BGD, did not experience subsequent additional outbreaks. This suggests
that there was some form of acquired immunity in trout which had recovered from BGD and suggests
that the recovered trout may have acted as carriers transmitting the bacterium to the newly stocked
trout.
Other recirculation systems have had outbreaks of BGD but only on a sporadic basis (Hinshaw, North
Carolina State University, personal communication). The circular tanks used (rather than cross-flow
tanks) reduce the suspended solids in the tanks and the use of batch culture may have prevented
carry- over of gill disease in these systems.
Furunculosis. Furunculosis is a bacterial infection of salmonids and other fishes caused by
Aeromonas salmonicida. An outbreak of furunculosis at the Freshwater Institute in March 1994 was
stress-related. The ozone in the system had been abruptly turned off and resulting nitrite levels were
high (0.46 mg/L) for several days prior to mortalities. A few fish started dying each day with clinical
symptoms of furunculosis including furuncles and swollen kidneys. Aeromonas salmonicida was
isolated from the kidneys. Two five day RometTM treatments at 50 mg/Kg trout/day controlled
mortalities (Warren 1991). Subsequent samplings from the continuously operated system have
demonstrated the presence of the bacteria in the mucus but not in the kidneys of asymptomatic fish
in the system. The bacterium was not isolated from the biofilter or tank water after the outbreak
ended (Cipriano et al. 1996).
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Aeromonas salmonicida has been isolated from the kidneys of sick fish which are seen occasionally
in the system to the present day. Because of the continuous production method utilized, bacteria have
become established in carrier fish.
The fingerlings are quarantined upon arrival at the Freshwater Institute and checked for parasites and
for bacteria by kidney sample cultures. Bacteria were never isolated from fingerlings in quarantine
during five years of sampling. It appears that carrier fish were not detected, allowing the organism
to enter the system with the fish. No clinical disease was observed until the trout were severely
stressed. Observations of other investigators have indicated that normal bacteriological examination
of kidney samples is often inadequate to ensure reliable detection of A. salmonicida among
asymptomatic carrier fish (Cipriano et al. 1996). This situation highlights the importance of
prevention and the need to improve detection of asymptomatic carriers.
Parasites
Several parasites caused problems in trout cultured in recirculation systems at the FT and other
facilities. It has been generally assumed that the parasites entered the system with infected fish.
Control sometimes created a problem and disinfection of the system was necessary.
Gyrodactylus appears to be one of the most common parasite infestations in rainbow trout
in recirculation systems causing mortalities if heavy infestation occurs (Noble, FT unpublished data;
Hinshaw, North Carolina State University, and Bowser, Cornell University, personal communication).
Gyrodactylus occurs in skin and fins multiplying when water quality conditions are less than optimal.
The problem is easily controlled with a formalin treatment at 167 mg/L for 1 h (Warren, 1991).
Most of the time at the FI the parasite was detected during quarantine and treated before trout were
moved to the recirculation system.
Chilodonella is a small, oval, colorless ciliated protozoan that infects the skin, fins and gills
of salmonids and warm water species (Warren 1991). A mixed infection of Chilodonella with
Ichthyopthirius occurred in one system (Hinshaw, North Carolina State University, personal
communication).
Trichodina is a ciliated protozoan parasite that infests the gills, skin and fins of trout.
Trichodina have been seen at FI in conjunction with Gyrodactylus or alone. Treatment with formalin
at 167 mg/L for 1 h has been successful in controlling the parasite. Infestations appeared to be
triggered by water quality deterioration.
Ichthyopthirius multifilis (Ich) is a large ciliated protozoan parasite of the skin, fins, and gills
of fish. Repeated treatments with formalin at 167 mg/L are usually necessary to control the infection
(Warren, 1991). Ichthyopthirius has been a problem at some recirculating facilities (Hinshaw, North
Carolina State University, Bowser, Cornell University, personal communication). Several treatments
with formalin at 167 mg/L for 1 h did not control the infection (Hinshaw, North Carolina State
University, personal communication). Subsequently, a treatment regimen was established where the
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system was treated continuously at a rate of 25-30 mg/L formalin for 20-25 days. This continuous
treatment controlled Ich, and was economical in a small system. In a large system, Ich was controlled
with a continuous treatment with copper sulfate at a rate of 1% of water alkalinity for 30-35 days.
The fish continued to be fed during treatment and no detrimental effect on biofilter function was
detected. This treatment is not recommended if alkalinity levels are below 50 mg/L. In one facility,
several days of chlorine disinfection of the system were necessary to prevent reinfection of newly
introduced trout with Ich (Hinshaw, North Carolina State University, personal communication).
Ichthyobodo is a small protozoan which infests the skin and gills of trout and other fishes
(Warren 1991). This parasite has been reported from one facility (Hinshaw, North Carolina State
University, personal communication) where control with formalin or salt did not seem to work even
though the whole system including the biofilter was treated. An alternate treatment with copper
sulfate metered to the tanks for 30 days controlled the parasite. The level of free copper ion in the
water had to be monitored to avoid toxic levels. The safe level of copper sulfate will depend on the
total alkalinity of the water, so the dosage has to be calculated for each facility where it is to be used.
Opportunistic microorganisms
Mortalities caused by microorganisms that normally are not pathogenic have been reported in
recirculation systems. Conditions develop in the recirculation system environment that seem to favor
the reproduction of these microorganisms and they can subsequently infect fish. Two such
infestations have been described in the literature, both caused by protozoans (Bowser 1994; Bullock
et al. 1994).
Coleps spp. a freshwater ciliated protozoan not normally considered pathogenic, was
associated with mortalities of 5-10 rainbow trout/d maintained in a densely stocked water
recirculation system in central New York State (Wooster and Bowser 1994). The protozoan was
found in wet mounts of skin scrapings. Histologically, severe multifocal interlarnellar tissue
proliferation was observed on the gills and multifocal areas in the skin had eroded or missing
epidermis (Wooster and Bowser 1994).
Wooster and Bowser (1994) reported that microscopic examination of water samples showed
a dense bloom of the ciliated protozoan Coleps sp. with a density estimated to be 7000 organisms per
ml. When the infestation appeared nitrites were high in the system and salt was routinely added to
maintain a chloride ion concentration of 190 mg/L. Treatment with an additional 0. 15 % sodium
chloride did not reduce the Coleps sp. counts in the water after 48 hours. The total mortality reached
35%. Potassium permanganate at 25 mg/L destroyed Coleps sp. in laboratory bioassays, but
permanganate could not be used due to toxicity to the biofilter. Mortalities may have been due to
excessive skin irritation caused by overwhelming numbers of protozoans. Conditions within the
recirculation system probably provided an ideal environment for Coleps to multiply and thus become
a problem (Wooster and Bowser 1994).
Amoebic gill infestation. An amoebic gill infestation followed bacterial gill disease in five
consecutive lots of fish stocked in the recirculation system at the Freshwater Institute (Bullock et al.
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1994). The unidentified amoeba appeared on the gills after several treatments with Chloramine-T.
The amoeba appears to be an opportunistic microorganism causing a secondary infestation (Noble
et al., submitted 1995). The amoeba caused chronic mortalities that lasted up to two months if left
untreated. Formalin treatment at 167 mg/L for 1 h, every other day, three times, controlled
mortalities due to the amoeba. Histologically, the amoeba was found in large numbers associated
with severe hyperplasia of the gills and was 10 gm in diameter (Bullock et al. 1994). The mortalities
may have been caused by asphyxiation due to large numbers of amoebae on the gill surfaces (Noble
et al., submitted 1995). The amoeba was probably introduced with the fish as it was observed in one
lot during the quarantine period. Because consecutive lots were kept in the same tank, the amoebae
may have become established in the population and also in the biofilter. The environmental conditions
within the system probably promoted the establishment and reproduction of the amoeba in numbers
that caused problems.
System Response to Chemical Treatment
Chemical treatment of the water in a recirculation system presents special considerations, the main
one is whether the biofilter will be treated and how the chemical could affect its function. If not
treated, the biofilter could become a reservoir for the disease organism. The biofilter at the FI
tolerated successive treatments with formalin without disruption of nitrification, due in part to excess
capacity of the biofilter (Heinen et al. 1995). However, a final indefinite treatment at 70 mg/L was
followed by high nitrite levels. These repeated treatments were far in excess of usual therapeutic use.
Routine treatments for disease control at the FI were bath treatments for 1 hour by-passing the
blofilter. The tanks were flushed with spring water after treatment ended. Other chemotherapeutants
may have a much larger effect on biofilter performance. Bullock (FI, personal communication) found
that 100 mg/L hydrogen peroxide completely stopped biofilter performance.
The key to treating a recirculation system for parasites may be a constant treatment at a lower
concentration of chemical for a longer period of time, as in this manner the chemical will eventually
reach all parts of the system where parasites may be present (Hinshaw, North Carolina State
University, personal communication).
Control Through Biosecurity
The best method to prevent infectious disease outbreaks is to avoid the introduction of infected fish
into a recirculation system. Several measures may be taken to achieve this goal. The ideal situation
is to bring only eggs into the facility, and disinfecting and hatching them on site. If this is not
possible, buy fingerlings from a certified disease-free source, quarantine fingerlings and check for
parasites and bacteria before introduction into the recirculation system. The fish should then be
reared in a pathogen protected environment.
Non-infectious Problems
The non-infectious problems encountered were caused by adverse conditions in the fish culture water.
These were: high nitrite levels, high carbon dioxide levels and ozone toxicity. Water quality needs
5
to be monitored closely in a recirculation system so that problems can be detected early and
corrected.
High nitrites
The most common non-infectious problem may be high nitrite levels caused by biofilter malfunction
(Durant, FI and Hinshaw, North Carolina State University, personal communication). Nitrite toxicity
is pH-dependent and dependent, in varying degrees, on the presence of chloride, sulfate, phosphate,
and nitrate ions in the water (Russo et al. 1981). Wedemeyer and Yasutake (1978) reported a
reduction in nitrite toxicity as pH increased. Russo and Thurston (1977) reported that the presence
of chloride ion suppressed nitrite toxicity in rainbow trout and Bowser et al. (1989) found chloride
to have a protective effect against nitrite toxicity in Atlantic salmon (Salmo salar). Sodium chloride
is used to control nitrite toxicity in fish culture systems as the chloride ion is taken up by the gills
preferentially to nitrite ion (Perrone and Meade 1977).
The 96-h LC50 value of nitrite to rainbow trout is 0.20 to 0.40 mg/L as N02-N (Russo and Thurston,
1977). However, the toxicity depends upon the concentration of calcium or chloride ions, which can
increase the tolerance of rainbow trout to nitrite by a factor of 20 to 30 times (Colt and Armstrong,
1981). The desirable level of nitrite for trout culture in a reuse system is <0.1 mg/L (Hankins, Fl,
personal communication).
At the FI, the biofilter was broken-in naturally by stocking the tanks at low density and allowing the
ammonia produced by the fish to increase the nitrifying bacteria population. Nitrobacter multiply
more slowly at the lower water temperatures (Kaiser and Wheaton 1983) found in the FI system (e.g.,
15EC), so there was a lag period when nitrites accumulated. Once the biofilter reached a steady state
the problem disappeared. At the FI palliative measures were taken when nitrite reached a level of 0.3
mg/L. Salt was added directly to the culture tanks at 10 times the nitrite concentration to reduce
toxicity. Abrupt increases in salinity may impair nitrification and biofilter recovery.
Elevated nitrite levels also occurred on several occasions that may be attributed to biofilter
defluidization, exposure of the biofilter to chemotherapeutants, or large and rapid increases in
fish/feed loading on the biofilter. In one particular example, during a biofilm stripper study, too much
biofilm was sheared from the biofilter. Both ammonia and nitrite levels increased and the ensuing
situation was similar to breaking-in a new biofilter with a lag in the Nitrobacter population. In this
case, the levels of nitrite reached 0.8 mg/L and clinical signs of methemoglobinemia were present in
the fish. The fish were lethargic and had flared gills that were brownish in color. Some fish with
clinical signs of furunculosis also were detected. Mortalities due to nitrites have been low at FI, but
it appears that high nitrite acts as a stressor that has precipitated outbreaks of infectious diseases
(Noble, FI, unpublished data).
Another example of high nitrite levels occurred at FI during system ozonation. Ozone
stoichiometrically oxidizes nitrite to nitrate (Bablon et al. 1991); therefore, addition of ozone to
recirculating systems is beneficial because it reduces nitrite levels. The fact that ozone decreases the
levels of nitrite is a substantial benefit on those occasions when nitratification (bacterial conversion
6
of nitrite to nitrate) in the biofilter is lost. However, because ozone actually reduces the nitrite
concentration going to the biofilter, it also reduces the quantity of nitratifying bacteria in the biofilter
and, thus, reduces the total nitrite removal capacity of the biofilter (Summerfelt et al. 1996). The
drawback to ozonation occurs when addition of ozone is interrupted. Because ozone is responsible
for removing a fairly large fraction of the total nitrite produced in the recirculating system, when
ozone addition is interrupted, nitrite rapidly accumulates within these systems (Summerfelt et al.
1996).
Carbon dioxide
Carbon dioxide is toxic to fish because it significantly reduces the capacity of their blood to transport
oxygen at levels of 20-40 mg/L (Basu, 1959). Carbon dioxide can accumulate to toxic levels in a
recirculation system with pure oxygen addition, due to low water gas exchange and high fish loading,
as reviewed by Summerfelt (1993). The level of free carbon dioxide is dependent on water pH,
alkalinity, and temperature (APHA et al. 1989). Fish eliminate carbon dioxide produced by
metabolism through their gills. Carbon dioxide unloading is a function of both ventilation rate and
concentration gradient (Walsh and Henry 1991). If levels of carbon dioxide in the water are elevated,
less carbon dioxide can be transferred from the gills into the water. Trout control the rate water is
pumped through their gills based upon the blood's oxygen content (Randall and Daxboeck 1984).
High carbon dioxide levels in the water result in a reduction of blood oxygen in the fish (Basu 1959).
Additionally, estimates of upper safe levels for chronic exposure to carbon dioxide in rainbow trout
range from <9 mg/L - 30 mg/L (Billard 1990; Piper et al 1982; Klontz 1993; Bromage et al. 1988;
Colt and Orwicz 1991; Colt 1994; and Piedrahita and Grace 1991). Levels of 55 mg/L reduce
growth in rainbow trout (Smart et al. 1979). The 20 mg/L recommended safe level may be
conservative, especially if oxygen in the water is high (Colt and Orwicz 1991). Due to buffering and
acid-base chemistry, water with high alkalinity or pH, or both, may enable fish to tolerate higher
concentrations of free carbon dioxide (Schaperclaus 1991). This may account for the varying levels
reported as safe in the literature (Heinen et al. 1996).
At the FI, pure oxygen was added to the water and fish loadings were high (2.96 Kg/L/min). When
carbon dioxide was first identified as a fish health problem at FI (Hankins, FI, personal
communication), there was insufficient gas exchange to strip off the carbon dioxide from the incoming
water (24-34 mg/L) and that produced by fish metabolism. This caused an accumulation of carbon
dioxide in the water to levels of 32 mg/L. In high alkalinity water toxic levels of carbon dioxide can
occur at higher pH levels than in low alkalinity water. Small-scale tests at FI indicated that rainbow
trout mortality could occur in the FI high alkalinity water at pH levels less than about 6.8 (Heinen et
al. 1996). A carbon dioxide stripper was installed at the FI in 1992 to correct this problem.
Ozone toxicity
Roselund (1974) reported gill epithelial damage and death to rainbow trout exposed to an estimated
0.010-0.060 mgo3/L. Wedemeyer et al. (1979) reported a lethal threshold level of about 8 mg/L with
death apparently resulting from massive destruction of the gill lamellar epithelium and a severe
hydromineral imbalance. The maximum safe level for chronic exposure for salmonids was
7
provisionally set at 2 mg/L. Sparrow (1973) stated that rainbow trout fingerlings exposed to water
containing 0.020 to 0.4mg O3/L all died within 195 minutes and 32% within 195 minutes when
exposed to approximately 0.010 mg O3/L. Oakes et al. (1979), in a pilot study at Dworshak
National Fish Hatchery found that most of the steelhead trout (Salmo izairdneri) exposed to
concentrations of 0.20 mg O3/L or greater died within four hours while those fish exposed to 0.008
mg/L did not die during 50 hours of exposure.
Ozone was added to the water at the Fl facility just before it entered the culture tanks at a rate of
0.025-0.039 kg ozone per kilogram of feed fed (Bullock et al. 1996; Summerfelt et al. 1996).
Mortality events caused by ozone residual in the tanks at the Fl occurred on five occasions causing
a total of 3.9% and 5.0% mortalities in two lots of trout. ORP probes and controllers were used to
limit ozone toxicity. However, tank locations away from the probes did accumulate toxic
concentrations. The first signs of exposure to toxic concentrations of dissolved ozone were
noticeable changes in fish behavior. Fish stopped feeding and congregated near the surface of the
water and sometimes "gasped" for air. Eventually, erratic swimming behavior occurred and became
progressively worse. Attempts to jump out of the tank increased, and some fish showed darting
behavior followed by listless swimming. Fish then lost equilibrium and became pale, with vertical
patches of dark pigment on the sides of the body. Fish which reach this latter condition rarely
survived. Gills of fish exposed to high levels of ozone showed excess mucus, hyperplasia and
aneurysms (Bullock et al. 1996).
Summary
Once a disease enters a recirculation system it could spread rapidly throughout the population due
to the high loading density. If continuous production is used, a disease may establish itself in the
population and infect newly introduced fish. In a sequencing batch production system, disinfection
may be possible after an infected lot has been harvested. Disease problems can generally be
controlled with prompt diagnosis and treatment. Steps should be taken to reduce or eliminate the
stress which may have triggered the outbreak.
Each facility should design their own protocol for prevention and control of fish diseases based on
the generally accepted principles of fish health management.
Acknowledgments
I thank Dr. Paul Bowser and Dr. Michael Timmons at Cornell University and Dr. Jeffrey Hinshaw
at the Department of Zoology, North Carolina State University for sharing information on disease
problems they have encountered in trout reared in recirculation systems. I also thank Dr. Pete
Bullock, Dr. Steven Summerfelt, and Joseph Hankins at the Freshwater Institute for reviewing the
manuscript and giving suggestions. Parts of this material were based upon research conducted at The
Freshwater Institute using support provided by the United States Department of Agriculture,
Agricultural Research Service, under agreement number 591931-3-012.
8
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von Graevenitz, A. "Revised Nomenclature of Campylobacter laridis, Enterobacter intermedium, and
Flavobacterium branchluhila." International Journal of Systematic Bacteriology, 40 (1989):
211.
Walsh, P. J., and R. P. Henry. "Carbon Dioxide and Ammonia Metabolism and Exchange." Pages
181-207 In P. W. Hochachka and T. P. Mommsen, Editors. Biochemistry and Molecular
Biology of Fishes, Phylogenetic and Biochemical Perspectives. Elsevier, New York. 1991.
Warren, J. W. Diseases of Hatchery Fish. Sixth Edition. U.S. Fish and Wildlife Service Pacific
Region. 1991.
Wooster, G. A., and P. R. Bowser. "Unusual Infection of Rainbow Trout Oncholynchus mykiss by
the Protozoan Coleps sp. (Ciliophora: Prostomatea) in a Closed Recirculation System."
Journal of the World Aquaculture Society, 25 (1994): 566-570.
Wedemeyer, G., N. C. Nelson, and W. T. Yasutake. "Physiological and Biochemical Aspects of
Ozone Toxicity to Rainbow Trout (Salmo gairdneri)." Journal of the Fisheries Research
11
Board of Canada, 36 (1979): 605-614.
Wedemeyer, G. and W. T. Yasutake. "Prevention and Treatment of Nitrite Toxicity in Juvenile
Steelhead Trout (Salmo gairdneri)." Journal of the Fisheries Research Board of Canada, 35
(1978): 822-827.
12
Mycobacteriosis in Intensively Cultured Foodfish
Stephen A. Smith
Assistant Professor of Aquatic Animal Medicine/Fish Health
Department of Biomedical Sciences and Pathobiology
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute and State University
Introduction
Mycobacteriosis, commonly known as "piscine tuberculosis" or "granuloma disease", is an important
bacterial infection of fish. Bacterial species of the genus Mycobacterium are Gram-positive, nonmotile, pleomorphic bacilli with strong acid-fast staining properties. Infections have been reported
worldwide from over 150 different species of marine, brackish and freshwater fishes representing at
least 40 piscine families (Nigrelli and Vogel, 1963). The most frequently isolated species of
Mycobacterium from fishes are M. marinum, M. fortuitum and M. piscium (Ashbumer 1977; Bragg
et al. 1990; Hedrick et al. 1987; Humphery et al. 1987; Lansdell et al. 1993; Shamsudin et al. 1990
and Snieszko 1978), and most of these can commonly be isolated from the aquatic environment,
especially from marine and estuarine waters.
The prevalence of infections may reach 15% in some wild fish populations (Parisot and Wood 1970),
and as high as 100% in fish populations under intensive culture practices. Infection with any one of
these species of bacteria can cause a chronic, progressive localized or systemic disease in fish.
Clinical manifestations of a mycobacterial infection in fish include ulcerated skin lesions to tumor-like
nodules of the body and/or internal organs. The spleen, kidney and liver are generally the visceral
organs most commonly affected, but the intestinal tract, gills, muscle and gonads may also be
infected. Other clinical signs may include loss of scales, depigmentation or hyperpigmentation,
exophthalmia, fin and tail rot, abnormal swimming behavior, lethargy, stunted growth and "wasting"
syndrome.
Transmission of the disease between fish is probably by ingestion of contaminated food, fish tissues
or debris in the water. The infectious agent could also potentially invade directly through damaged
skin or gill tissue. The water may become contaminated by the rupture of skin or gill lesions, the
decay of infected fish and through the gonadal fluids released by infected spawning fish.
In addition to often causing high mortality in cultured species of finfish, mycobacteriosis renders the
carcass of the fish unsightly and unwholesome for human consumption, resulting in an unmarketable
product. Further, these particular bacterial organisms also have a documented zoonotic potential for
humans, especially in immunosuppressed individuals (Nigrelli and Vogel 1963). Infections of humans
with these aquatic species of Mycobacterium generally produce localized, cutaneous granulomas
commonly termed "fish handlers disease" or "swimming pool granulomas". Though the majority of
human infections involve the skin, mycobacterial organisms have sometimes been isolated from the
1
lungs, lymph nodes and other internal organs of humans (Inglis et al. 1993). Thus, an outbreak of
mycobacteriosis in an aquaculture system is a serious zoonotic problem for both the personnel
handling and processing the infected fish.
Commercial production of finfish for food in the United States generally consists of raising fingerlings
to marketable size in raceways, ponds, cages or closed recirculation systems. Fish in any of these
culture situations can potentially be exposed to these infectious organisms. In particular, fish
maintained in the marine or estaurine environment during any portion of their life cycle are often more
frequently exposed to the mycobacterial agents. In addition, fish maintained in intensive aquaculture
systems, where the stress of current culture practices frequently predispose the fish to infectious
diseases, often develop an overwhelming clinical infection of mycobacteriosis. Infections of
Mycobacterium have already been documented in salmonids (Ashbumer 1977), yellow perch (Daoust
et al, 1989) and both feral (Sakanari et al. 1983) and cultured striped bass (Hedrick et al. 1987). In
addition, at least two commercial firms raising hybrid striped bass have condemned several million
dollars worth of fish over the past several years due to mycobacterial infections (R. Thune, per
comm).
Current diagnostic assays for fish mycobacteriosis involve either the culture of the bacterial agent on
a specific culture medium or the histological identification of the agent with special staining
techniques (Kent and Kubica 1985). Culture of the organism on the recommended culture media,
such as Lowenstein-Jensen or Middlebrook 7H1O media, can be extremely difficult and time
consuming. These organisms are extremely slow growing and may take as long as 28-45 days to
grow (Austin and Austin, 1987). Similarly, histological evaluation for a mycobacterial infection with
special stains such as a Ziehl-Neelsen stain is also time consuming and often non-productive. Stains
for acid-fast mycobacterial organisms are usually not included in the standard histological protocol
for most diagnostic evaluations of fish. Therefore, obvious pathology with mycobacterial-like lesions
is required before special staining techniques are generally considered. In addition, the use of these
special histological stains identify all acid-fast bacterial and are not specific for identifying the genus
Mycobacterium and sometimes mycobacterial lesions do not contain the acid-fast bacteria. Recently,
interest in molecular diagnostic techniques has led to the development of a polymerase chain reaction
(PCR) specific for the detection of mycobacteriosis in fish (Knibb et al. 1993).
Once fish in an aquaculture facility become infected with mycobacteriosis, it is an extremely difficult
problem to manage. There are no FDA-approved chemotherapeutics available for the treatment of
mycobacteriosis in foodfish, thus the standard recommendation is to depopulate and sterilize the
facility (Plumb 1994). Therefore, testing and monitoring fish on a regular basis for the presence of
a mycobacterial infection is of both a practical and economical benefit to the aquaculturists in
avoiding the potential loss of investment, time and effort with an infected group of fish, as well as,
reducing the potential health risk of a zoonotic infection in fish culturists and fish processors.
2
References
Ashbumer, L. D. "Mycobacteriosis in hatchery-confined chinook salmon (Oncorhynchus tshawytscha
Walbaum)in Australia." Journal of Fish Biology 10(1977):523-528.
Austin, B. and Austin, D.A. Bacterial Fish Pathogens: Disease in Farmed and Wild Fish. John
Wiley and Sons. 1987.
Bragg, R.R., Huchzermeyer, H. and Hanisch, M.A. "Mycobacterium fortuitum isolated from three
species of fish in South Africa." Onderstepoort Journal of Veterinary Research
57(1990):101-102.
Daoust, P.Y., Larson, B.E. and Johnson, G.R. "Mycobacteriosis in yellow perch (Perca flavescens)
from two lakes in Alberta." Journal of Wildlife Diseases 25(1989):31-37.
Hedrick, R.P., McDowell, T. and Groff, J. "Mycobacteriosis in cultured striped bass from California."
Journal of Wildlife Diseases 23 (1987):391-395.
Humphrey, J.D., Lancaster, C.E., Gudkovs, N. and Copland, J. W. "The disease status of Australian
salmonids: bacteria and bacterial diseases." Journal of Fish Diseases 10(1987):403-410.
Inglis, V., Roberts, R. J. and Bromage, N. R. Bacterial Diseases of Fish. John Wiley and Sons.
1993.
Kent, T.P. and Kubica, P. G. Public health microbiology, a guide for the level III laboratory. U.S.
Department of Health and Human Service, Centers for Disease Control, Atlanta, Georgia.
1985.
Knibb, W., Colomi, A.,. Ankaoua, M., Lindell, D., Diamant, A. and Gordin, H. "Detection and
identification of a pathogenic marine mycobacterium from the European seabass
Dicentrarchus labrax using polymerase chain reaction and direct sequencing of 16S rDNA
sequences." Molecular Marine Biology and Biotechnology 2(1993):225-232.
Lansdell, W., Dixon, B., Smith, N. and Benjamin, L. "Isolation of several Mycobacterium species
from fish." Journal of Aquatic Animal Health 5(1993):73-76.
Nigrelli, R.F. and Vogel, H.. "Spontaneous tuberculosis in fishes and in other coldblooded vertebrates
with special reference to Mycobacterium fortuitum Cruz from fish and human lesions."
Zoologica (New York) 48(1963):131-144.
Parisot, T.J. and Wood, J.N. Fish mycobacteriosis (tuberculosis). Fish Disease Leaflet Number 7,
U.S. Fish and Wildlife Service. 1970.
3
Plumb, J.A. Health Maintenance of Cultured Fishes: Principal Microbial Diseases. CRC Press,
Inc. 1994.
Sakanari, J.A., Reilly, C.A. and Moser, M. "Tubercular lesions in Pacific coast populations of striped
bass." Transactions of the American Fisheries Society 112(1983):565-566.
Shamsudin, M., Tajima, K., Kimura, T., Shariff, M. and Anderson, I.G. "Characterization of the
causative organism of ornamental fish mycobacteriosis in Malaysia." Fish Pathology
25(1990):1-6.
Snieszko, S.F. Mycobacteriosis (tuberculosis) of fishes. Fish Disease Leaflet Number
55. U.S. Fish and Wildlife Service. 1978.
4
Streptococcal Infections of Hybrid Striped Bass and Tilapia
Ana M. Baya
Fish Health Scientist
Animal Health Laboratory
Maryland Department of Agriculture
University of Maryland, College Park
Evan Kotopoulis
Laboratory Scientist
Animal Health Laboratory
Maryland Department of Agriculture
Rosangela B. Navarro
Laboratory Scientist
Animal Health Laboratory
Maryland Department of Agriculture
Introduction
Aquaculture is an emerging industry in Maryland. To date, the main emphasis has been on the
culture of striped bass (Morone saxatilis) and striped bass-white bass (M. saxatilis x
M.chrysops) hybrids, however during the last few years, tilapia (Oreochromis niloticus x 0.
aureus) hybrids have become an increasingly popular aquaculture species. Both fish have a high
market value.
Outbreaks of streptococcosis have been commonly reported in a wide variety of fish species
around the world (2). This disease is a particular problem in Japan where it causes serious
economic damage especially among cultured yellow tail (Seriola quinqueradiata) (9, 11), eels
(Anguffla japonica) (10), rainbow trout (Oncorhynchus mykiss) and Nile tilapia (Oreochromis
nfloticus) (7, 13).
This study describes the isolation, biochemical characteristics, and antimicrobial sensitivity
patterns of Streptococcus species isolated from striped bass and tilapia and compares these new
isolates to those described in the literature.
Isolation
The streptococcal disease in both striped bass and tilapia is a subacute to chronic infection. Affected
individuals lose appetite gradually, show erratic swimming, and exhibit exophthalmia, corneal opacity,
darkening of the skin, hemorrhages around the jaws, eyes, opercula, bases of fins and tails and on the
body surface. Abdominal swelling is present in some fish. Moribund animals lie on their side and
finally die. The disease was chronic in nature with greater losses occurring after stress related
situations such as handling, partial harvesting etc. A large number of protozoan and trematodal
parasites in the gills and skin of the fish and additional stressors like high density (crowding) and poor
water quality probably increased the risk of infection and disease in these fish.
1
Isolates were obtained from the head kidney, spleen, liver, brain and eyes on Trypficase Soy Agar
plates (TSA) and in Trypficase Soy broth (TSB) incubated at 25E C for 48 to 72h. Pure cultures
were recovered from all organs examined.
Identification of the organism
On agar plates the colonies were small (0.5mm diameter), smooth, white and translucent. API Rapid
Strep (bio Merieux Vitek, Inc. Hazelwood, MO) and other biochemical characterizations permitted
the identification of the isolates as members of the genus Streptococcus. Results of serotyping with
a commercial Streptoccocal grouping kit (Wellcome Diagnosfic, Dartford, England) indicated that
our isolates did not belong to Lancefield serotypes A to G.
The Gram positive non-motile cocci were catalase and oxidase negative and either b-hemolytic or ahemolytic . Other biochemical characteristics are shown in Table 2.
Representative strains that were isolated from tilapia and striped bass in Maryland (Table 1) from
different farms over a three year period were chosen for a study to determine the relationship of our
isolates with other streptococci described in the literature.
Table 1. Sources of the isolates from Striped bass and Tilapia
Strain designation
Fish Species
Year of Isolation
FL 3-8
Tilapia
1993
FL 3-35
Tilapia
1993
FL 5-43
Striped bass
1995
FL 5-12
Tilapia
1995
FL 5-25
Striped bass
and Tilapia
1995
FL 5-42
Striped bass
1995
Maryland Isolate #1
Maryland Isolate #2
2
Table 2. Comparison of phenotypic characteristics of Streptococcus species isolated Tilapia
and Striped bass with those reported in the literature
Characteristic
Maryland
Isolate
#1
Maryland
Isolate #2
Ceschia
et al
(5)a
Kitao
et al
(7)a
Al Harbi
(1)a
Ming-Chen
et al
(12)a
Perera et
al (15)a
Austin
et al (3)a
Gram stain
+
+
+
+
+
+
+
+
Motility
-
-
-
-
-
-
-
-
Oxidase
-
-
-
-
-
-
-
-
Catalase
-
-
-
-
-
-
-
-
Haemolytic type
b
a
ND
b
a
b
b
b
0% NaCl
+
+
ND
+
ND
ND
ND
+
3% NaCl
-
-
ND
+
ND
ND
ND
ND
6.5% NaCl
-
-
ND
-
ND
-
-
ND
4EC
-
-
ND
ND
ND
ND
ND
ND
10EC
+
+
ND
+
ND
-
+
ND
25EC
+
+
+
+
ND
+
-
ND
37EC
+
+
+
+
ND
+
+
ND
45EC
-
-
ND
-
ND
-
+
ND
-
-
ND
ND
-
-
ND
+
-
-
ND
-
-
-
ND
-
H2S Production
-
-
ND
-
-
ND
ND
ND
Methyl Red
-
-
ND
+
ND
-
ND
+
Voges-Proskauer
-
-
+
-
-
-
-
+
Arginine
dihydrolase
-
+
-
+
+
-
+
+
Growth in
Growth at
Growth on
MacConkey
Agar
Indole
3
Characteristic
Maryland
Isolate
#1
Maryland
Isolate #2
Ceschia
et al
(5)a
Kitao
et al
(7)a
Al Harbi
(1)a
Ming-Chen
et al
(12)a
Perera et
al (15)a
Austin
et al (3)a
Lysine
-
-
ND
-
-
ND
ND
ND
Ornithine
-
-
ND
-
-
ND
ND
ND
Pyrrolidonyl
arylamidase
-
+
+
ND
+
ND
+
+
"-Galastosidase
-
-
-
ND
-
ND
ND
-
$-Galactosidase
-
-
-
ND
-
ND
ND
-
$-Glucuronidase
+
+
-
ND
+
ND
ND
-
Alkaline
phosphtase
+
+
-
ND
+
ND
ND
-
+
+
+
ND
+
ND
ND
+
-
-
F
ND
-
F
ND
F
Hippurate
-
-
ND
-
-
-
-
-
Esculin
+
+
ND
+
+
-
ND
-
Robose
+
+
+
ND
-
ND
ND
-
Arabinose
-
-
-
-
-
-
-
-
Mannitol
+
+
+
+
+
-
+
-
Sorbitol
-
-
-
-
-
-
-
-
Lactose
-
-
-
-
-
-
-
-
Decarboxylation
of
(PYR)
(PAL)
Leucine
arylamidase
(LAP)
O/F (Leifson)
Hydrolysis of
Acid from
4
Characteristic
Maryland
Isolate
#1
Maryland
Isolate #2
Ceschia
et al
(5)a
Kitao
et al
(7)a
Al Harbi
(1)a
Ming-Chen
et al
(12)a
Perera et
al (15)a
Austin
et al (3)a
Trehalose
+
+
+
+
+
+
+
+
Inulin
-
-
-
-
-
-
-
-
Raffinose
-
-
-
-
-
-
-
-
Starch
+
+
+
+
ND
ND
ND
+
Glycogen
-
+
-
-
+
ND
ND
-
a
Publication describing the isolate (see reference)
+: Positive reaction, -: Negative reaction or no growth, ND: No data provided, F: Fermentative
Selected characteristics were chosen for comparison (Table 2). The two Maryland isolates appear
to be different biotypes with the major differentiating characteristics being that isolate #1 is negative
for arginine dihydrolase, arylamidase, and acid production from glycogen and isolate #2 is positive
in these tests. Maryland isolate #2 is very similar to the one described by Al-Harbi (1). However the
Maryland isolate #1 is more difficult to relate to any of them even though it has some characteristics
similar to the ones described in Table 2. Although many characteristics are shared by the majority of
the strains tested, there is also some variance in the overall descriptions reported by the different
groups of workers (2, Table 2). Such differences may reflect either the lack of standarization in the
testing procedures or the heterogeneity in the group.
Antimicrobial susceptibility pattern
Antimicrobial susceptibility patterns were determined by the disk diffusion method using MullerHinton agar (Oxoid). There were no differences in the antibiotic sensitivity patterns of the two
isolates (Table 3); both tested sensitive to Ampicillin, Bacitracin, Chloramphenical, Erythromycin,
Nitrofurazone, Novobiocin, Oxytetracycline, Penicillin, Romet, Tetracycline, Trimethoprim and
Vanomycin but were resistant to Nalidixic acid, Oxolinic acid, and Streptomycin. Some strains were
sensitive to Furazolidone, Gentamycin and Kanamycin and some were not.
5
Table 3. Comparison of antibiotic sensitivities of Streptococcus species isolated from Tilapia
and Striped bass with those in the literature
Resistance/
Sensitive to
Maryland
Isolate #1
Maryland
Isolate #2
Ceschia et
al
(5)a
Kitao
et al
(7)a
Al-Harbi
(1)a
MingChen et
al
(12)a
Perera
et al
(15)a
Austin
et al
(3)a
Penicillin (10)
S
S
ND
ND
S
R
S
ND
Ampicillin (10)
S
S
ND
ND
S
R
R
S
Erythromycin
(15)
S
S
ND
S
S
S
S
ND
Chloramphenic
ol (30)
S
S
S
S
S
S
S
ND
Tetracycline
(30)
S
S
S
S
ND
ND
S
S
Oxytetracycline
(30)
S
S
S
ND
S
ND
S
ND
Streptomycin
(10)
R
R
ND
S
ND
ND
ND
R
Bacitracin (10)
S
S
ND
ND
ND
ND
S
ND
Gentamycin
(10)
R/S
R/S
ND
ND
S
S
S
R
Kanamycin (30)
R/S
R/S
ND
S
ND
R
ND
ND
Nalidixic acid
(5)
R
R
ND
R
ND
S
ND
R
Novobiocin
(30)
S
S
ND
ND
ND
R
ND
ND
Sulfadimethoxi
ne/Ormetoprim
(25)
S
S
ND
S
ND
S
S
ND
Trimethoprim
(5)
S
S
ND
S
S
ND
ND
ND
S: Sensitive
R: Resistant
ND: No data provided
a
Publication describing the isolate (see reference)
6
Fatty acid analyses
Fatty acid methyl esters (FAME) from bacterial cells were prepared following the method of Moss
et al.(14). Briefly, saponification with 15% NAOH in 50/o methanol in boiling water for 30 min was
followed by methylation with 25% HCl in methanol for 10 min at 85E C. The methyl esters were then
extracted with methyl-tert-butyl ether and hexane(l: 1), followed by washing in phosphate buffer.
One milliliter of each sample containing the extracted FAME was analyzed by gas chromatography
(GLC) with a Shimadzu 14A chromatograph equipped with a fused silica capillary column (30 m by
0.25 mm internal diameter with a 0.25 uM film thickness), consisting of a 5% phenyl-methylsilicone(DB-5) stationary phase(J & W Scientific), and fitted with an AOC-9 automatic sampler
connected to a CR6A data processor (Shimadzu). Helium was employed as the carrier gas at a speed
of 37 cm/s at 150E C. The split ratio was approximately 100: 1.
The analysis utilized a temperature program of 150E C for the initial 4 min, followed by a
temperature increase of 4E C/min to a maximum temperature of 240E C. The injector was maintained
at 250E C and the detector was operated at 300E C. Peak identifications were made by calibration
of the data processor with the retention times of a standard FAME mix (Matreya Inc., Pleasant Gap,
PA).
The fatty acid profiles of the streptococcal isolates are shown in Fig 1 and Table 4. Analysis of the
strains by GLC revealed that the predominant fatty acids for Streptococcus species were the
saturated and unsaturated fatty acids of 16 carbon atoms (hexadecanoate (C16:0)and hexadecenoate
(C16:1)); the dodecanoate (C12:0); the tetradecanoate (C14:0); the unsaturated cis-11 octadecenoate
(C18:IC) the octadecanodeinoate(C18:2) and the octadecanoate (C18:0).
Table 4. Composition of cellular fatty acid methyl esters (FAME) of the Streptococcus
included in this study.
Bacterial
species
Isolate 1
Percentage of total fatty acid composition
10:0a
12:0
14:0
w7c
16:1
16:0
18:2
w9c
18:1
w7c
18:1
1.0b
8.4
8.3
19.8
40.8
2.9
5.9
10.0
Isolate 2 Tc
4.7
11.8
17.4
46.5
T
2.9
7.1
a
Numbers indicate the number of carbon atoms, w7c, and w9c indicates double bond position from
hydrocarbon end of carbon chain. c is cis isomer.
b
Number is the arithmetic mean of the relative percentage of the fatty acid.
c
Trace amounts (less than 1.0%)
7
8
Discussion
A number of freshwater and saltwater fish have been reported to be infected by a Streptococcus
species. This is the first report documenting the occurrence of Streptococcosis in fresh water fish in
Maryland; however Streptococcus species were previously isolated from moribund or freshly dead
bluefish (Pomatomus saltatrix), striped bass, and sea trout (Cynoscion regalis) in the Chesapeake
Bay and some of its tributaries during the unusually hot and dry summer of 1988 (4).
The Maryland isolates from cultured striped bass and tilapia may be classified as Streptococcus
species based on their morphological and biochemical characteristics. All the strains were from
different fish cases and were made on different dates. Our isolates were sensitive to most of the
antibiotics tested including the few approved in the USA for use in food fish.
The taxonomy of this heterogenous group is confusing, and there is some variance in the reported
descriptions by different researchers (2, 8, Table 2). Many shared characteristics are evident in the
majority of piscine streptococci, but in most published reports the bacteria were identified as
Streptococcus species because they were similar to an isolate described in some earlier cited
reference. Further work is needed to determine the precise taxonomic position of all isolates.
The results of the fatty acid analysis are in qualitative agreement with other descriptions of FAME
in Streptococcus species (6). We are not able to separate the two groups by FAME analysis. Minor
differences between the groups occurred in the relative areas 14:0 and 16:0. The Streptococcus
group #2 contained higher levels of these two fatty acids than the cultures of Streptococcus group
#1; however these differences are probably not significant enough to provide reliable a differentiation
scheme. Growth conditions, esterification, extraction procedures and several other factors could
account for differences between the reported fatty acid patterns of the streptococci.
Ideally, any new fish being added to an aquaculture system should be inspected and have a health
certificate. Also, it would be prudent to inquire if any bacterium, virus or parasite has been isolated
recently from the fish or the facility, even if the fish appear clinically healthy. We believe that some
fish may be carriers of streptococci, and could introduce it into recirculating systems. Isolations of
Streptococcus species from striped bass cultured in freshwater were made only when striped bass
and tilapia were present together in a recirculating system facility. This suggests that the tilapia may
have been the source of the infection. In the absence of some stressor(s) being introduced into the
aquaculture system, the organism may remain undetected. Since intensive culturing systems are prone
to episodes of stress, preventing the introduction of a potential pathogen is the most effective way
of avoiding the problem.
9
References
(1)
Al-Harbi, A. "First isolation of Streptococcus sp. from hybrid tilapia (Oreochromis x O.
aureus) in Saudi Arabia." Aquaculture 128 (1994): 195-201.
(2)
Austin, B. and Austin, D. Fish Pathogens. Disease in farmed and wild fish. Chichester:
Ellis Horwood. 1993.
(3)
Ausfin, B. and Robertson, P. "Recovery of Streptococcus milleri from ulcerated koi carp
(Cypinus carpio L.) in the U.K." Bulletin of the European Association of Fish Pathologists 13
(1993): 207-208.
(4)
Baya, A., Lupiani, B., Hetrick, F., Roberson, B., Lukacovic, R., May, E. and Pouldsh, C.
"Associafion of Streptococcus sp. with fish mortalities in the Chesapeake bay and its tributaries."
Journal of Fish Diseases 13 (1990): 251-253.
(5)
Ceschia, G., Giorgetfi, R., Giavenni, R. and Sarti, M. "A new problem for Italian trout
farms: Streptococcosis in Rainbow trout (Oncorhynchus mykiss). " Bulletin of the European
Association of Fish Pathologist 12 (1992): 71-72.
(6)
Drucker, D. "Chemotaxomonic fatty-acid fingerprints of some streptococci with
subsequent statistical analysis." Canadian Journal of microbiology 20 (1974): 1723 -1728.
(7)
Kitao, T., Aoki T., and Sakoh, R. "Epizootic caused by b-haemolytic Streptococcus
species in cultured freshwater fish. Fish Pathology 15 (1981): 301-307.
(8)
Kusuda, R. and Komatsu, I. "A comparative study of fish-pathogenic Streptococcus
isolated from saltwater and freshwater fishes." Bulletin of the Japanese Society of Scientific
Fisheries 44 (1978): 1073-1078.
(9)
Kusuda, R-, Kawai, K., Toyoshima, T. and Komatsu, I. "A new pathogenic bacterium
belonging to the genus Streptococcus, isolated from an epizootic of cultured yellowtail." Bulletin
of the Japanese Society of Scientific Fisheries 42 (1976): 1345-1352.
(10) Kusuda, R., Komatsu, I. and Kawai K. "Streptococcus sp. isolated from an epizootic of
cultured eels. Bulletin of the Japanese Society of Scientific Fisheries 44 (1978): 295.
(11) Minami, T. "Streptococcus sp. pathogenic to cultured yellowtail, isolated from fishes for
diets. " Fish Pathology 14 (1979): 15-19.
(12) Ming-Chen, T., Chen, S. and Tsai S. "General Septicemia of Streptococcal infections in
cage-cultured Tilapia, Tilapia mossambica, in Southern Taiwan." COA Fisheries series No. 4,
Fish Disease Research (VII) 12 (1985): 95-105.
10
(13) Myazaid, T., Kubota, S., Kaige, N. and Myashita, T. "A histopathological study of
streptococcal disease in tilapia. " Fish Pathology 19 (1984); 167-172.
(14) Moss, C., Wallace, P., Hoffis, D., Weaver, R. "Cultural and chemical characterization of
CDC groups groups EO-2, M-5 and M-6, in MoraxeRa species, Oligefla urethralis,
Acinetobacter species, and Psychrobacter immobilis. " Journal of Clinical Microbiology 26
(1988): 484-492.
(15) Perera, R. and Johnson, S. "Streptococcus iniae associated with mortality of Tilapia
nilotica x T.aurea Hybrids. "Journal of Aquatic Animal Health 6 (1994): 335-340.
11
Nitrate Toxicity: A Potential Problem of Recirculating Systems
Terry C. Hrubec
Research Scientist
Aquatic Medicine Laboratory
Department of Biomedical Science and Pathobiology
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute and State University
Stephen A. Smith
Assistant Professor of
Aquatic Animal Medicine
Department of Biomedical
Science and Pathobiology
Virginia-Maryland Regional College of
Veterinary Medicine
Virginia Polytechnic Institute and
State University
John L. Robertson
Associate Professor of Pathology
Department of Biomedical
Science and Pathobiology
Virginia-Maryland Regional College of
Veterinary Medicine
Virginia Polytechnic Institute and
State University
Introduction
Rearing fish successfully depends on a number of factors, including the maintenance of good water
quality. Adverse water quality can decrease production, reduce growth, and decrease a fish's
resistance to disease (Nicholson et al., 1990). Ammonia is the major nitrogenous excretory product
of fishes and is also formed from the breakdown of uneaten feed. The conversion of ammonia by
bacterial oxidation to nitrate (NO3-) is termed nitrification. Bacteria of the Nitrosomonas species
convert NH3 to nitrite (NO2-) which is acted upon Nitrobacter species to form N03-. Nitrates are
taken up by plants, converted anaerobically to nitrogen gas, or removed by regular water changes.
Ammonia and nitrite are both toxic to fishes. Ammonia toxicity is thought to occur from
osmoregulatory imbalance causing renal failure and gill epithelial damage resulting in suffocation,
decreased excretion of endogenous ammonia, and general neurological and cytological failure
(Meade, 1985). Elevated nitrite levels cause methemoglobinemia (brown blood disease). Nitrate is
generally considered nontoxic to fishes (Bromage et al. 1988). In most aquaculture systems, nitrate
levels are below 50 mg/L, but in intensive culture systems, nitrate levels often exceed 100 mg/L.
Nitrate levels, in recirculating systems that have limited fresh water input, can be 200 mg/L or greater.
The fact that fish are often raised for extended periods of time in water with elevated nitrate levels
prompted research into the effects of prolonged nitrate exposure.
Results
Presented here are summaries of studies conducted to investigate the effects of prolonged exposure
1
to elevated nitrate concentrations. The first study examined the effect of elevated nitrate on the
humoral immune response (antibody production) of hybrid striped bass to a formalin killed
Aeromonas salmonicida bacterin (Hrubec et al. 1996a). Hybrid striped bass were acclimated to the
experimental conditions over a two week period by slow addition of sodium nitrate to the tank until
levels reached 200 mg/L N03-N. An identical tank with untreated water was used as a control. Fish
were maintained at the elevated nitrate level for 4 weeks prior to immunization. Nitrate treated and
control fish were immunized and the antibody response monitored for three months. Antibody
response was determined with an ELISA and expressed as a percent of the positive ELISA control.
The maximum antibody response in control and nitrate treated fish was 72% and 48% of the positive
control respectively. Randomized control block analysis of variance (ANOVA) showed significant
(p < 0.0001) differences in the antibody response between the nitrate exposed and control groups.
Hematological and serum biochemical parameters were also monitored in nitrate treated fish. Hybrid
striped bass were acclimated to elevated nitrate levels as described above. After five weeks at
elevated nitrate levels fish were bled for hematologic and biochemical determinations following
procedure determined previously (Hrubec et al. 1996b). There were notable changes in the blood of
nitrate exposed fish as compared to control fish. Numbers of reticulocytes (immature red blood cells)
were increased in the nitrate exposed fish. In control fish, there were one to two reticulocytes per
microscope field under oil immersion, while in the elevated nitrate treated fish there were five to six
reticulocytes per field. Nitrate exposed fish had higher monocyte and neutrophil counts and lower
thrombocyte-like cell (TLC) counts. The TLC is an unidentified cell type that superficially resembles
a thrombocyte, and is probably an immature form of leukocyte. Serum biochemical changes included
higher creatinine, and calcium values, and lower chloride concentration than controls. All of the
changes seen in the blood parameters were statistically significant.
In addition to the changes in humoral immune function and blood parameters, the nitrate treated fish
appeared to become blind three weeks into the experiment, one week after the nitrate concentration
reached 200 mg/L. The fish would often swim into objects and the walls of the tank. The skin color
became darker, but there were no obvious gross lesions, or changes to the eyes. After seven weeks
at the elevated nitrate level, the fish began to die. The fish were found moribund, swimming weekly
upside down or lying on the bottom. About 50% of the moribund fish were icteric (jaundiced) around
the operculum, gills, ventral abdomen, and in the viscera and fat. The other moribund fish showed
no clinical signs. The icteric fish were anemic with a packed cell volume (PCV) of 20 - 25%, while
the other moribund fish had a PCV of 30 - 45%. The serum biochemical profile of icteric fish
demonstrated elevated creatinine (1.8 mg/dL), total bilirubin (2.5 mg/dL), and AST (585 mU/mL),
and decreased sodium (114 mEq/L) concentrations. This hematological profile is consistent with an
intravascular lysis of red cells. Histopathological changes included inflammation, hyperplasia and
fusion of the gills. The spleen was hypocellular with erythrocytic and lymphoid depletion, and
multifocal necrosis. The liver was vacuolated with necrotic foci and the head kidney was depleted
of lymphoid tissue. The posterior kidney was edematous with dilated tubules; and there was
mineralization and necrosis of the tubules.
These results demonstrate hematological and immunological changes in fish exposed to nitrate, but
do not preclude that the changes could be due to other factors. A possible explanation for the
2
responses seen in the nitrate exposed fish was contamination of the sodium nitrate with some other
toxicant. However, analytical examination of the sodium nitrate used in the experiment, revealed no
evidence of contamination. Another possible explanation was that the changes observed were due
to increased sodium and not nitrate since the addition of sodium nitrate increases sodium levels as
well as nitrate. To determine whether the changes in blood parameters were produced by nitrate we
examined the hematology and blood chemistry of hybrid striped bass in recirculating systems with
elevated nitrate levels.
Hybrid striped bass, raised in recirculating systems with nitrate concentrations above 200 mg/L, were
bled. Changes in blood parameters from these fish were compared to hybrid striped bass raised in
low density tanks with a nitrate level less than 10 mg/L (Hrubec et al. 1996c, 1996d). Fish from the
recirculating system had higher plasma protein values higher leukocyte, lymphocyte, neutrophil,
monocyte and reticulocyte counts, and lower TLC counts than fish in tanks. Serum chemistry
changes included higher concentrations of total protein, albumin, globulin, creatinine, phosphorus and
calcium, and lower chloride values. Many of these changes were the same as seen in the sodium
nitrate treated fish. Although these data suggested that the blood changes were caused by elevated
nitrate levels, they were not conclusive. Many high density recirculating systems have pH fluctuations
that are controlled by addition of sodium bicarbonate to increase alkalinity. This was the case with
the systems used in our study. The amount of sodium, added as sodium bicarbonate to the
recirculating system, was approximately equivalent to the amount added as sodium nitrate in the
original study. Although this study showed similar changes in sodium nitrate treated fish and fish
exposed to nitrate produced by nitrification in the tank, the question of whether these changes were
due to nitrate or sodium was still not resolved.
To address the nitrate versus sodium issue, a study was conducted where hybrid striped bass were
exposed to 200 mg/L nitrate produced by three different salts of nitrate and to elevated bicarbonate
levels. Nitrate levels were increased in three separate tanks over a two week period by addition of
sodium, calcium and potassium salts of nitrate into the respective tanks. Sodium bicarbonate was
added to a fourth tank at the same rate of sodium addition as in the sodium nitrate tank. Fish in a
tank with untreated water were used as controls. Differences between the water treatments for each
blood value were determined by ANOVA. When a significant difference was detected (P < 0.05),
the means were compared with a Tukey's means comparison test. The results are shown in Table 1.
In most instances, there was little difference between the control and sodium bicarbonate treated fish.
Consistent differences were seen in all nitrate fish for many of the blood parameters. All nitrate
treated fish had significantly higher plasma protein, monocyte and reticulocyte counts, and lower TLC
counts than control fish. Additionally, total protein, albumin, globulin, and calcium levels were
elevated and chloride values depressed when compared to control or bicarbonate treated fish. Fish
exposed to the sodium nitrate and potassium nitrate began to die after six weeks at the elevated
nitrate levels.
3
Table 1. Hematologic and serum biochemistry values for fish maintained at different salts of
nitrate (200 mg/L N-Nitrate) elevated sodium bicarbonate (NaHCO,) and control water.
Analyte
Control
NaHCO3
NaNO3
CaNO3
KNO3
Plasma Protein
(g/dL)
4.9
a1
4.5
b
5.4
c
5.4
c
5.6
c
Leukocytes
(103/mL)
52.73
a
54.40
a
48.63
a
50.41
a
51.56
a
Lymphocytes
(103/mL)
40.96
a
40.60
a
26.55
a
40.18
a
40.44
a
Neutrophils
(103/mL)
1.32
a
1.37
a
2.16
a
2.35
a
1.79
a
Monocytes
(103/mL)
2.55
a
2.66
a
4.84
a,b
3.79
a,b
5.87
b
TLC2 (103/mL)
2.74
a
2.22
a
0.95
b
0.97
b
1.11
b
Thrombocytes
(103/mL)
54.77
a
47.60
a
48.32
a
44.33
a
48.06
a
Total Protein
(g/dL)
2.7
a
2.6
a
3.73
b
3.2
c
3.3
c
Albumin (g/dL)
1.0
a
1.1
a
1.6
b
1.3
c
1.3
c
Globulin (g/dL)
1.6
a
1.5
a
2.0
b
1.9
b
2.0
b
Creatinine
(mg/dL)
0.2
a
0.2
a
1.0
b
0.3
a
0.3
a
T. Bilirubin3
(mg/dL)
0.0
a
0.0
a
0.4
b
0.0
a
0.0
a
Sodium (mEq/L)
152
a,b
148
c
147
c
149
a,c
154
b
Potassium
(mEq/L)
3.5
a
3.4
a
3.5
a
3.5
a
6.0
b
Chloride (mEq/L)
150
a
143
b
52
c
103
d
113
e
Calcium (mg/dL)
9.71
a
9.41
a
11.49
b,c
10.44
b
12.12
c
Phosphorus
(mg/dL)
8.1
a,b
8.1
a,b
7.7
a
8.7
b
9.1
c
1) values with different letters are statistically different; 2) Thrombocyte-Like cell; 3) Total Bilirubin
4
Discussion
Fishes are exposed to environmental nitrate from nitrification of excretory waste, runoff from
agricultural lands, and waste water effluent from sewage treatment plants. Nitrate concentrations in
aquaculture are not usually elevated due to high water flow through a system or removal of nitrate
by plants. In recirculating systems, however, fresh water exchange is often limited. Bonn (1976)
reported that adult striped bass tolerated nitrate levels up to 800 mg/L, while fry showed signs of
stress at 200 mg/L, however, both adult and fry fed and grew better at levels below 38 mg/L.
Unfortunately, the original study was not referenced; and the duration of exposure to the nitrate and
clinical signs are not known. There is no information available on the nitrate tolerance of hybrid
striped bass. The fish in our study were maintained at elevated levels for extended time periods,
which may have enhanced any pathophysiology associated with nitrates. Water quality conditions
for rearing striped and hybrid bass have been proposed (Warren et al., 1990). With the exception of
nitrate concentrations purposefully outside these ranges, our water quality was within the proposed
ranges for all water quality parameters except for nitrite. When nitrate levels were elevated due to
addition of a nitrate salt or from nitrification, increased nitrite levels were observed. This increase
in nitrite was most likely due to enhanced reduction of nitrate to nitrite caused by the elevated nitrate
concentrations.
The effects of ammonia and nitrate on serum enzymes have been investigated using domestic waste
water which has high concentrations of these two compounds (Weiser and Hinterleitner, 1980,
Bucher and Hofer 1990). Domestic waste water did not increase serum enzymes, but did cause
histopathological changes characterized by heavy hyaline droplet degeneration of the kidney tubular
cells and progressing to necrosis of the kidney tubules and hepatocytes (Bucher and Hofer 1990).
These histopathologic lesions are similar to our observations of nitrate treated fish. The elevated
creatinine levels seen in nitrate exposed fish may be an indication of compromised renal function as
is observed in mammals. In fishes, creatinine is excreted by the kidneys, but it is not known if blood
levels become elevated with impaired renal function (McDonald and Milligan 1992). The results of
this study indicate that creatinine may reflect renal function.
The cause of the severe hypochloremia observed in all nitrate treated fish is unknown. Severe
hypochloremia has been demonstrated in response to handling stress, (Tomasso et al. 1980) and a
mild hypochloremia occurs with gill injury (Byme et al. 1989) The hypochloremia is due to increased
loss of chloride across the gills. Whether the hypochloremia demonstrated in the nitrate exposed fish
is in response to stress, gill damage or some other mechanism is unclear. A possible explanation is
that chloride moved passively out of the fish to balance the sodium added to the water as sodium
nitrate. However the hypochloreiffla was also observed in calcium nitrate and potassium nitrate
treated fish and no hypochloremia was noted in the sodium bicarbonate treated fish.
Electrolyte concentrations are indicative of a fish's ability to osmoregulate. This ability is often
compromised with stress, disease, or gill lesions that increase gill permeability to ions (McDonald and
Milligan 1992). Blood electrolytes appear to be influenced to a greater extent than other biochemical
parameters in the nitrate exposed fish. This may be because nitrate damages the gills and kidneys
affecting osmoregulatory ability. Alternatively, the pathology is caused by an electrolyte imbalance
5
in the water (from the added salts of nitrate) affecting the physiology of the fish. The fact that similar
biochemical changes were produced in fish from all three salts of nitrate but not elevated sodium
bicarbonate provides evidence that the biochemical changes seen are due to elevated nitrate and not
the added cation. At the same time, however, the added cation does affect electrolyte values as the
potassium concentration is elevated in fish from the potassium nitrate tank.
The changes observed in the nitrate treated fish most likely represent a pathological response as
apposed to a generalized stress response. In mammals the stress response is well characterized.
There are three stages; an initial alarm phase, a stage of resistance and a final stage of exhaustion.
Exhaustion is reached if the stress is sufficiently prolonged or severe, and is characterized by
decreased levels of cortisol, depletion of liver glycogen, immunosuppression and other changes
reducing the survivorship of the organism. This progression has been demonstrated in fishes with
long term exposure to environmental pollutants (Hontela et al. 1992). Fish from polluted sites had
decreased cortisol and atrophied pituitaries, indicating an exhaustion of the cortisol producing system.
Hematologic changes associated with stress include a leukopenia characterized by a lymphopenia,
and a neutrophilia (Ellsaesser and Clem 1986, 1987). Serum biochemical changes with stress include
a hyperproteinemia, hypercortisolemia, hyperglycemia, hyponatremia, hypochloremia, and a
hypocalcemia from increased permeability of the gills (McDonald and Milligan 1992). The
hematologic and biochemical profiles of the nitrate treated fish were not indicative of a stress
response as no lymphopenia, hyperglycemia, hypercortisolemia, hyponatremia nor hypocalcemia was
noted (Hrubec et al. 1996b, 1986c, 1986d). It is possible that the nitrate treated fish had reached a
state of exhaustion and were unable to elevate glucose and cortisol levels, however, both values were
well within the reference interval (Hrubec et al. 1986a, 1996c). Thus it is unlikely that the changes
seen in the nitrate treated fish were solely stress induced.
The cause of the clinical signs and mortality in the nitrate treated fish is unclear. Some fish suffered
from a hemolytic crisis, becoming anemic and icteric prior to death, but whether this was a direct
result of exposure to nitrate is not known. There is a report of jaundice in cultured eels associated
with a hemolytic crisis, although, excessive destruction of erythrocytes was not the primary cause of
jaundice as the bilirubin was mainly conjugated (Endo et al. 1992). The cause of the cholestasis in
eels was not determined, but based on histologic changes, the authors felt there was an intrahepatic
disorder. The water quality in this report was not mentioned, so it is not known if the nitrate
concentrations were elevated.
Conclusion
The data presented here support the theory that prolonged exposure to elevated levels of nitrate may
decrease the immune response, induce hematological and biochemical changes indicative of a
pathologic response, and may increase mortality. If elevated nitrate levels are responsible for the
pathologic changes seen in these fish, then management of recirculating systems must change to lower
nitrate levels. The pathologic changes are sufficient to affect the normal physiology of the fish and
will probably result in decreased growth and increased susceptibility to disease. These results
however do not conclusively show that elevated nitrate levels are responsible for the pathology seen.
Further studies demonstrating a dose response to nitrate levels should be conducted prior to making
6
major management changes in a recirculating system.
References
Bonn, E.W., Bailey, W.M., Bayless, J.D., Erickson, K.E., and Stevens, R.E., 1976. Guidelines for
striped bass culture. Striped Bass Committee, Southern Division, American Fisheries Society,
Bethesda, Maryland, p. 69.
Bromage NR, Shepherd CJ, Roberts J. 1988. Farming systems and husbandry practice. In: Shepherd
CJ Bromage NR, eds. Intensive Fish Farming. Oxford: BSP Professional, pp. 94-95.
Byrne P, Speare D, and Ferguson HW. 1989. Effects of a cationic detergent on the gills and blood
chemistry of rainbow trout Salmo gairdneri. Dis Aquat Org 6:185-196.
Bucher F, and Hofer R. 1990. Effects of domestic waste water on the serum enzyme activities of
brown trout (Salmo truua). Comp Biochem Physiol 97C:381-385.
Ellsaesser CF, and Clem LW. 1986. Hematological and immunological changes in channel catfish
stressed by handling and transport. J Fish Biol 28:511-521.
Ellsaesser CF, and Clem LW. 1987. Cortisol-induced hematologic and immunologic changes in
channel catfish (Ictalurus punctatus). Comp Biochem Physiol 87A:405-408.
Endo M, Sakai T, Yamaguchi T, et al. 1992. Pathology of jaundice in the cultured eel Anguilla
japonica. Aquacult 103:1-7.
Hontela A, Rasmussen JB, Audet C, et al. 1992. Impaired cortisol stress response in fish from
environments polluted by PAHs, PCBs, and mercury. Arch Environ Contam Toxicol 22:278-283.
Hrubec, TC, Robertson JL, Smith SA, and Tinker MK. 1996a. The effect of temperature and water
quality on antibody response to Aeromonas salmonicida in sunshine bass (Morone chrysops X
Morone saxatilis). Veterinary Immunology and Immunopathology, in press.
Hrubec TH, Smith SA, Robertson JL, et al. 1996b. Comparisons of hematological reference intervals
between culture system and type of hybrid striped bass. American Journal of Veterinary Research
57:618-623.
Hrubec TH, Smith SA, Robertson JL et al. 1996c. Blood biochemical reference intervals for sunshine
bass (Morone chasgps x M saxatilis) in three culture systems. American Journal of Veterinary
Research 57:624-627.
Hrubec TH, Smith SA, and Robertson JL 1996d. The effects of water quality on the hematologic
and serum biochemical profiles of hybrid striped bass (Morone chrysops X Morone saxatilis).
Submitted to American Journal of Veterinary Research.
7
Meade, J.W., 1985. Allowable ammonia for fish culture. Prog. Fish. Cult. 47:135-145.
McDonald DG, and Milligan CL. 1992. Chemical Properties of Blood. In: Hoar WS, Randall DJ,
Farrell AP, eds. Fish Physiology Vol. 12 Part B, The Cardiovascular System. New York: Academic
Press, 56-135.
Nicholson, L.C., Woods, L.C., and Woiwode, J.G., 1990. Intensive culture techniques for the striped
bass and its hybrids. In: R.M. Harrell, J.H. Kerby, and R.V. Minton (Editors), Culture and
Propagation of Striped Bass and its Hybrids, American Fisheries Society, Bethesda, MD, pp. 141158.
Tomasso JR, Davis KB, and Parker NC. 1980. Plasma corticosteroid and electrolyte dynamics of
hybrid striped bass (white bass X striped bass) during netting and hauling. Proc World Maricult Soc
11:303-310.
Warren, H.J., Harrell, R.M., Geiger, J.G., and Rees, R.A., 1990. Design of rearing facilities for
striped bass and hybrid striped bass. In: R.M. Harrell, J.H. Kerby, and R.V. Minton (Editors),
Culture and Propagation of Striped Bass and its Hybrids, American Fisheries Society, Bethesda, MD,
pp. 17-28.
Weiser W, Hinterleitner S. 1980. Serum enzymes in rainbow trout as tools in the diagnosis of water
quality. Bull Environ Contam Toxicol 25:188-193.
8
Nutrition of Intensively Raised Foodfish
Brian C. Small
Graduate Research Assistant
Department of Animal Sciences
University of Maryland
Joseph H. Soares
Professor
Department of Animal Sciences
University of Maryland
INTRODUCTION
Growth of the aquaculture industry is currently rapid and extensive. Successful cultivation of
new species are announced regularly. How much this industry grows depends on the successful
development of the environmental and health technologies associated with growing and
reproducing fish. While there is considerable room for additional development of pond and net
cage technologies, there are desirable limits to these methods. Major concerns are the
containment of effluents that might lead to pollution of natural water resources, disease
prevention and sufficient control of the environment to allow maximal growth and reproduction.
This presentation will focus on the nutrition of several aquaculturally important foodfish i.e.
striped bass, tilapia and rainbow trout for comparative purposes. The former two species are
considered carnivorous and the latter species is an omnivore. Effective raising of these fish in
intensive culture systems requires several important considerations. One of the most important
considerations is matching the type of system to be employed with the species of fish to be
raised. If trout are to be fed, then cold water is needed and this is typically supplied via a flow
through system that allows continual supply of cold, highly oxygenated water with low organic
residue. Improved water chilling capacity may overcome this limitation. Tilapia, however,
because of their hardiness and warm water tolerance, are better suited for recirculating systems.
Formulation and manufacture of diets for intensively reared fish represents the major cost area in
aquaculture. Diet formulation requires more than satisfying nutrient requirements, it also means
paying close attention to palatability, rate of growth, bioavailability of nutrients in a feedstuff,
costs, whether there are regulatory requirements that must be met, etc. We shall attempt to
discuss some of the more costly nutrients needed in efficient diets for fish.
PROTEIN (AMINO ACIDS) and ENERGY.
When formulating diets for fish, it is important to recognize that certain nutrients are more costly
to add to diets while others may be added in excess and because of regulatory considerations,
may require expensive removal processes. It is generally recognized that protein, more correctly
amino acids, represent the most expensive part of the diet. Feed costs represent between 50 and
60 percent of the total cost of aquacultural fish production. Fifty percent or more of the cost of
feed is due to the protein component of a diet. All fish studied to date require 10 indispensable
amino acids i.e. arginine, histidine, isoleucine, leucine, lysine, methionine (up to one-third can be
1
supplied as cystine), phenylalanine (up to 20% can be supplied as tyrosine), threonine,
tryptophan and valine (NRC 1993). Unfortunately, the quantitative aspects of amino acid
requirements have not been determined for all the important foodfish. Dietary amino acid
requirements for trout and tilapia are published and are shown in Table 1. Also included in Table
1 are the preliminary estimates of the amino acid requirements for the striped bass, M. saxatilis
(Small and Soares 1996 unpublished).
Table 1. Amino Acid Requirements of Striped Bass, Rainbow Trout and Nile Tilapia.
Amino acid
Striped bass
Rainbow trout
Nile tilapia
% of Diet
Arginine
1.6
1.4
1.2
Histidine
0.8
0.6
0.5
Isoleucine
1.2
1.0
0.9
Leucine
2.2
1.8
1.0
Lysine
2.5
2.1
1.4
Methionine
1.1
0.7
0.8
Phenylalanine
2.0
1.2
1.1
Threonine
1.2
1.4
1.1
Tryptophan
0.3
0.3
0.2
Valine
1.4
1.2
1) Small and Soares (1996) - 37% protein diet.
2) Ogino (1980)- 40% protein diet.
3) Santiago and Lovell (1988) - 28% protein diet.
0.8
An important factor necessary for determining concentrations of amino acids needed in a diet is
energy level. This is because fish readily oxidize excess amino acids to energy. Since fish
excrete unused nitrogen as ammonia, there is very little energy cost to the animal when using
protein for energy. Like most other animals, fish eat to satisfy their energy needs. Consequently
a diet formulated to contain excess energy in relation to protein, will result in the fish under
consuming and not eating enough protein to satisfy their optimal amino acid needs. The result is
poor growth. If the protein-energy ratio is too high such that excess protein is fed, then the fish
will metabolize amino acids for energy. This situation could result in excessively high feed costs.
Fortunately there have been some studies in a number of species (NRC 1993) on protein calorie
ratios that give guidance as to what is necessary for efficient growth. Table 2 lists these ratios
for striped bass, trout and tilapia. It is noteworthy that the ratios are quite similar for all three
species ranging from 103 to 112 mg protein/kcal digestible energy (DE).
2
It is generally considered that protein, fat and carbohydrate result in the formation of 5.74, 9.32
and 4.06 kcal DE/g respectively, when completely oxidized by fish (Jobling 1995). Since fat
contains 60 % more energy than protein, it is usually the most cost efficient form of energy to be
added to the diet.
Table 2. Protein level and Protein-calorie Ratios Recommended for Striped Bass, Tilapia
and Rainbow Trout.
Species
% protein
Protein/Calorie
Striped bass
47
112
Nile tilapia
30
103
Rainbow trout
40
105
1) Millikin (1983); 2) Wang et al., (1985); 3) Satia (1974); 4) Nematipour et al., (1992); 5) ElSayed (1987) and 6) Cho and Woodward (1989).
In omnivorous fish such as tilapia energy can effectively be supplied as carbohydrate. At this
time, we know that carnivorous species can also digest and absorb carbohydrates but often they
appear not to be able to efficiently oxidize these substances to energy. Consequently it is
recommended that carnivore diets such as those fed to trout should be formulated to contain less
than 30% soluble carbohydrates. Cooking of carbohydrates enhances their utilization by most
fish.
Estimates of amino acid availability are difficult to determine because of the problems associated
with obtaining accurate samples of feces as well as the lack of sufficient reference feeds to use
for comparative quantification. Several studies conducted with salmon and catfish give us an
idea of the relative net absorption or digestion of some feedstuffs (Table 3). Work by Cho et al.
(1982) has indicated that the method of sampling feces needed to determine digestibility requires
direct removal from the fish to avoid erroneous estimations of digestibility. For example,
siphoning of feces from the tank results in severe leaching of feces and leads to over-estimation
of digestibility while underestimation can result by expressing feces too vigorously from the
rectum. Use of a low rate of suction by application of tubing to the anus is suggested as the
method of choice. A word of caution should be made about the liberal use of digestibility
coefficients as estimates of bioavailability of amino acids. Availability indicates that "utilization"
is occurring. While it is likely that most of the digested amino acids are being utilized, it is
possible that significant amounts are not able to be used for protein synthesis and may be energy
sources or excreted.
PHOSPHORUS. Phosphorus is usually the most expensive mineral that must be added to the
diet of fish. In diets where fishmeal is a major component, high levels of dietary phosphorus
exist. A 50% protein diet containing Menhaden fishmeal as the primary protein source can easily
contain 1.75-1.90% phosphorus. Therefore it is possible to be feeding 4-5 times as much of this
element as needed for optimal growth by most food fishes. Along with nitrogen, phosphorus has
3
become a nutrient that accumulates in our natural waters to the point of being a pollutant (Ketola
1985, Weismann etal 1988; Kendra 1991).
Table 3. True Amino Acid Availability or Digestibility of Selected Feedstuffs for Atlantic
Salmon, Channel Catfish and Striped Bass.
Feedstuff
Species
Mean AA availability, %
Corn Gluten meal
Salmon
Catfish
Striped bass
93.1
91.4
Peanut meal
Salmon
Catfish
Striped bass
93.3
84.2
Fishmeal, Herring
Salmon
Catfish
Striped bass
92.2
90.8
Fishmeal, Menhaden
Salmon
Catfish
Striped bass
88.6
87.2
-
Soybean meal
Salmon
Catfish
Striped bass
1) Atlantic salmon - Anderson et al., (1992)
2) Channel catfish - Wilson et al., (1981)
3) Striped bass - Small and Soares (1995) unpublished
74.3
77.9
94.3
Currently many researchers are exploring the possibility of using alternative protein sources that
are not as high in phosphorus. Table 4 shows data recently obtained from studies of the
phosphorus requirement of striped bass at the University of Maryland. One of the most
difficult problems with determining a phosphorus requirement is the determination of how
much of the dietary phosphorus is actually available to the fish. In this case we have used the
average apparent absorption of the dietary phosphorus sources to abtain the available
phosphorus estimate of 0.4%. The phosphorus requirement for calcification is always higher
than that for optimal growth and is used as the dietary requirement. The latter is true because a
fish will use nutrients for growth before storage. A summary of the dietary phosphorus
requirements of some other species is shown in Table 5.
4
Table 4. Estimations of total dietary phosphorus requirements of striped bass, M.
saxatilis.
Variable
% total phosphorus
Noncalcification responses
Weight gain
.31
Feed efficiency
.28
Serum phosphorus
.29
Mean
.29
Calcificatinon responses
Scale ash
.64
Scale phosphorus
.65
Vertebral ash
.54
Vertebral calcium
.55
Vertebral phosphorus
Mean = 0.58% total P or estimated
From: Dougall et al., 1996.
.59
0.40% available P.
Estimates of phosphorus bioavailability are often made by determining apparent or true
absorption in fish. Ogino et al. observed that apparent P absorption for casein, fishmeal,
monobasic calcium phosphate and monobasic potassium phosphate were 90, 74, 94 and 98 %
respectively for rainbow trout. Ketola and Harland (1993) showed that defluorinated phosphate
was about 86% as effective as calcium phosphate in promoting growth and development in trout.
In our own studies with striped bass, potassium monophosphate, calcium phosphate and
defuorinated phosphate when fed as sole sources of phosphorus had absorption coefficients of
95, 92 and 65 % respectively.
5
Table 5. Dietary Phosphorus Requirements of Striped bass, Tilapia and Rainbow Trout.
Species
% dietary available phosphorus
Tilapia, O. aureus
.50
Rainbow trout
.43
Hybrid bass, M. chrysops x M. saxatilis
.40
Striped bass, M. saxatilis
.40
1) Robinson etal (I 987); 2) Ogino etal (I 979); 3) Brown et al. (I 993); 4) Dougall etal (1996).
Current studies in progress in our laboratory and in others are determining the amount of plant
phytate phosphorus that can be fed to fish effectively. Since fish do not have phytase in their
digestive tract, this enzyme is added to the diet after pelleting the diet. The potential for phytase use
is to make more plant protein feedstuffs available thereby allowing the formulation of fish diets that
are lower in phosphates while still suppling adequate available phosphorus and amino acids. It is
hoped that this potential will open the way to make less expensive diets for intensive fish culture a
reality in the near future.
REFERENCES
Anderson, J. S., J. P. Lall, D.M. Anderson and J. Chandrasoma. Apparent and true availability of
amino acids from feed ingredients for Atlantic salmon (salmo salar) Aquaculture 108:111-124. 1992.
Brown, M.L., F. Jaramillo, Jr. and D.M. Gatlin, III. Dietary phosphorus requirement of juvenile
sunshine bass, Morone chrysops x M. saxatilis. Aquaculture 113:355-363. 1993.
Cho, C. Y., and B. Woodward. Studies on the protein-to-energy ratio in diets for rainbow trout
(Salmo gairdneri) Pg 37-40 in Proceedings of the Eleventh Symposium on Energy Metabolism,
Lunteren 1988. European Assoc. for Animal Production 43. Wageningen, Netherlands: Pudoc
Wageningen. 1989.
Cho, C.Y., S. J. Slinger and H. S. Bayley. Bioenergetics of salmonoid fishes: Energy intake,
expenditure and productivity. Comp. Biochem. Physiol 73B: 25-41. 1982.
Dougall, D. S., L. Curry Woods III, L. W. Douglass and J. H. Soares Jr. Dietary phosphorus
requirement of juvenile striped bass Morone saxatilis. J World Aquaculture Soc. 27:(I)82-91. 1996.
El-Sayed, A. M. Protein and energy requirements of Tilapia zilli. Ph.D. dissertation. Michigan State
University. East Lansing, Mich. 1987.
Jobling, M. In: Fish Bioenergetics. Chapman and Hall. London. 1995.
6
Kendra, W. Quality of salmonid hatchery effluents during a summer low-flow season. Trans. Amer.
Fish. Soc. 120:43-51. 1991.
Ketola, H. G. Mineral nutrition: effects of phosphorus in trout and salmon feeds on water pollution.
Pgs 465-473 in C. B. Cowey, A. M. Mackie and J. G. Bell, editors Nutrition and feeding in fish.
Academic Press, London, England. 1985.
Ketola, H. G. and B. F. Harland. Influence of phosphorus in rainbow trout diets on phosphorus
discharges in effluent water. Trans. Amer. Fish. Soc. 122:1120-1126. 1993.
Millikin, M. R. Interactive effects of dietary protein and lipid on growth and protein utilization of
age-0 striped bass. Trans. Amer. Fish. Soc. 112:185-193. 1983.
Nematipour, G. R., M. L. Brown and D. M. Gatlin III. Effects of dietary energy: protein ratio on
growth characteristics and body composition of hybrid striped bass, Morone chrysops x M saxatilis.
Aquaculture. 107:359. 1992.
National Research Council. Nutrient Requirements of Fish. Washington D.C. National Academy
Press. 1993.
Ogino, C., L. Takeuchi, H. Takeda and T. Watanabe. Availability of dietary phosphorus in carp and
rainbow trout. Bull. Jpn. Soc. Sci. Fish. 45:1527-1532. 1979.
Ogino, C. Requirements of carp and rainbow trout for essential amino acids. Bull. Jpn. Soc. Sci.
Fish. 46:171-175. 1980.
Robinson, E. H., D. LaBomasascus, R. B. Brown and T. L. Linton. Dietary calcium and phosphorus
requirements of Oreochromis aureus reared in calcium-free water. Aquaculture. 64:267-276.
1987.
Santiago, C. B. and R. T. Lovell. Amino acid requirements for growth of Nile tilapia. J Nuir.
118:1540-1546. 1988.
Satia, B. P. 1974. Quantitative protein requirements of rainbow trout. Prog. Fish Cult. 36:8085.
Small, B. C. and J. H. Soares. Unpublished. Dept. Animal Sciences, University of Maryland,
College Park, MD. 1996.
Wang, K. W., T. Takuechi and T. Watanabe. Effect dietary protein levels on growth of tilapia
nilotica. Bull. Jpn. Soc. Sci. Fish. 51:133-140. 1985.
Watanabe, T., A. Murakami, L. Takeuchi, T. Nose and C. Ogino. Requirement of chum salmon held
in freshwater for dietary phosphorus. Bull. Jpn. Soc. Sci. Fish. 46:361-367. 1980.
7
Weismann, D., H. Scheid and E. Pfeffer. Water pollution with phosphorus of dietary origin by
intensively fed rainbow trout (Salmo gairdneri Rich). Aquaculture 69:263-270. 1988.
Wilson, R. P., E. H. Robinson and W. E. Poe. Apparent and true availability of amino acids from
common feed ingredients for channel catfish. J Nutr. 111: 923 -929. 1981.
8
Preventive Medicine for Intensively Cultured Foodfish in Recirculation Systems
Stephen A. Smith
Assistant Professor of Aquatic Animal Medicine/Fish Health
Department of Biomedical Sciences and Pathobiology
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute and State University
Introduction
Both time and monetary losses from disease and disease control efforts are highly significant in
the aquaculture industry. In a recent survey, Virginia aquaculture producers estimated finfish
losses of over 16% due to disease conditions (VDACS, 1994), with bacterial and parasitic
diseases probably causing more problems in the cultured fish than any other infectious
organisms. Thus, a primary goal of any aquaculture production facility should be the avoidance
and prevention of potential diseases rather than the control and treatment of actual disease
occurrences. The concept of preventive medicine or health maintenance in an aquaculture
facility encompasses the entire production management program for the fish including the
establishment and maintenance of optimal water quality and environmental conditions,
nutrition, genetics, and health (Plumb 1994).
Prevention
The prevention of disease in an aquaculture facility should start prior to delivery of fish to the
facility. Obtaining eggs or fish from a specific disease-free certified facility is desirable but not
always practical, since this is presently only applicable to salmonids and not to warmwater
species of fishes. A record of the health history and previous chemotherapeutic treatments
should be obtained from the seller by the purchaser. In addition, a sample of the lot of fish to be
obtained should be sent to a qualified diagnostic laboratory to evaluate the present health status
of the fish. When possible, this should include a complete evaluation of the bacterial, parasitic
and viral diseases of the fish. Unfortunately, many subclinical diseases are often undetectable
by current diagnostic methods.
Quarantine
It is extremely important that infectious agents are not introduced into an aquaculture facility
with new incoming stock. Thus, the incorporation of a standard quarantine program for all new
stock should be a part of the standard operating procedure for all aquaculture facilities. Fish
should be maintain in a facility completely isolated from the main aquaculture facility so that
diseases cannot become established in production tanks or transferred to other fish in the
facility. Though there are no set rules for how long fish need to be quarantined, many fish
health professionals suggest 30-45 days minimum. During this time the fish may be evaluated
for specific bacterial and viral diseases, and examined for external parasites. Since prophylactic
treatment of foodfish with most FDA approved chemotherapeutic agents is currently not legal, it
1
is extremely important that fish be examined during this isolation period. Depending on the
results of the diagnostic evaluations, the fish can then be treated appropriately for a specific
disease with a FDA approved chemotherapeutic.
Monitoring
Fish should be monitored for disease and health status at regular intervals throughout the entire
production cycle.
Monthly diagnostic evaluations, along with standard weights and
measurements, would provide the producer with up to date information on the health of the
stock in the aquaculture facilitate. This should include, like the initial diagnostic evaluation, an
evaluation of specific bacterial, parasitic and viral diseases of the fish. This should allow the
detection of subclinical disease in a population of fish prior to the development of overt clinical
disease. Thus, treatment with FDA approved chemotherapeutics are often more effective in
terminating a disease outbreak before substantial mortality occurs.
Obviously, accurate record keeping and attention to detail are essential to successfully
maintaining the health of fish in a production facility. This includes complete records of water
quality and other environmental parameters, diagnostic evaluations, and the health and
treatment history of the individual groups of fish in the facility. In addition, the nutritional
status and genetics of the acquired stock play an important role in maintaining fish health.
References
Myer, F.P., Warren, J.W. and Carey, T.G. A Guide to Integrated Fish Health Management in
the Great Lakes Basin. Great Lakes Fishery Commission. 1983.
Plumb, J.A. Health Maintenance of Cultured Fishes: Principal Microbial Diseases. CRC
Press. 1994.
VDACS. Virginia Aquaculture Survey Report, 1993. Virginia Department of Agriculture and
Consumer Services. 1994.
2