A Brief History Of The Leetown West Virginia Laboratory

[Graham L. Bullock]{.underline}

The Conservations Fund's Freshwater Institute, 1098 Turner Road,
Shepherdstown, West Virginia 25443

In 1931, a United States Fisheries Center was developed on the site of a
gristmill at Leetown, a small community in the eastern panhandle of West
Virginia. A stone building was constructed that housed fish culture
facilities, laboratories, office space, and a library. Circular pools,
raceways, and warm water ponds were constructed to rear cold and warm
water fishes. During these early days, Dr. H. S. Davis was the
laboratory pathologist and he researched bacterial and parasitic
diseases. Davis authored *Culture and Diseases of Game Fishes*, which
became the first book on fish diseases that was published in the United
States. In 1947, Dr. S. F. Snieszko became director of the laboratory
and his leadership catalyzed the development of a comprehensive program,
which brought the facility international recognition for its many
contributions to fish health research and instruction. It was Dr.
Snieszko's foresight that brought Dr. Ken Wolf and Dr. Glenn Hoffman to
Leetown. It was Dr. Wolf 's who established the first continuously
growing trout cell line (RTG-2 cells), isolated he first trout viral
pathogen (IPNv), and published a definitive textbook on fish viruses and
viral diseases. Together with Mrs. Maria Markiw, Dr. Wolf also completed
the highly elusive life cycle of the whirling disease parasite
*Myxobolus cerebralis*, a major parasite of salmonid fishes. Doctor
Glenn Hoffman also became renowned for his work in fish parasitology.
During his tenure at Leetown, Dr. Hoffman diagnosed the first outbreak
of salmonid whirling disease in the United States and demonstrated its
transmission. He would later publish an exhaustive compendium, which is
used as a standard reference on parasites of North American fishes.
Studies conducted within this facility were vitally important to the
registration of the three antibiotics that were federally licensed for
use on food fish in the United States. Throughout its history, the
laboratory's staff has capably blended applied and basic science to
resolve national issues associated with the identification, control,
diagnosis and prevention of bacterial, parasitic, and viral diseases.
Additionally, both short and long-term courses covering all aspects of
fish health and pathology were developed. The excellence of this program
is evident in more than 3,400 publications, to date.

**Pathologies In Demersal Fishes Of The Barents Sea And Adjacent
Wate**rs

[Tatiana A. Karaseva]{.underline} and Vladimir V. Donetskov

Polar Research Institute of Marine Fisheries and Oceanography (PINRO), 6
Knipovich Street, Murmansk, 183763, Russia

The Barents Sea and areas adjoining it to the north are zones of
international fisheries. Although the Barents Sea fisheries are well
researched, little information exists on the pathologies and diseases of
marine organisms from this area. To work out a system that evaluated the
health of commercial fish and the quality of their habitats, the Polar
Research Institute of Marine Fisheries and Oceanography began to
investigate the epizootic situation in the Northern seas and instituted
a Russian database on pathologies of marine demersal fishes. Samples
were performed on board research and fishing vessels. Techniques of
clinical analysis including pathological and anatomical obduction plus
histopathology were used. Some 89,516 were assayed that included plaice
(*Pleuronectes platessa*), long rough dab (*Hippoglossoides
platessoides*), Greenland halibut (*Reinchardtius hippoglossoides*),
haddock (*Melanogrammus aeglefinus*), cod (*Gadus morhua morhua*), and
wolffishes (*Anarchihas denticulatus*, *A. lupus*, A. *minor*). Ulcers,
necroses, tumors and skeletal deformations were found and different
forms of necrosis prevailed. Their proportion compared to other
pathologies increased from 4% in 1999 to 80% in 2002. Tumors in fish
occurred least often. Ppapillomas were most often evident in wolffishes
with a level of morbidity of 3-8%. About 1% of plaices had pathologies.
The next group consisted of cod and haddock, in which the prevalence of
pathologies was less than 1%. An inter-annual increase of quantity of
fish with pathologies from 0.5% in 1999 to 2.2% in 2002 was reported in
the Barents and eastern Norwegian Seas. The morphological diversity and
low occurrence of pathologies suggested that their appearance was caused
by various endogenic and exogenic factors.

**The Volga Delta And Northern Caspian Sea: Fishing, Fish Health And
Disease**

[Lubov V. Lartseva]{.underline} and Victoria V. Proskurina

FSUE Caspian Fisheries Research Institute, 1 Savushkina Street,

Astrakhan, 414056, Russia

The Volga-Caspian basin contains one of the most important fisheries in
Russia and yields 40.0% of the nation's freshwater (zander, common carp,
bream, vobla, catfish, pike, etc.) and 70.0% of its sturgeon fisheries.
There are also about 390,000 seals inhabiting the Caspian Sea. Russian
and stellate sturgeons are the principal catches in the Volga River, and
the stellate sturgeon is the predominant catch in the Ural River. Nine
fish hatcheries operate around the Caspian Sea basin in the Russian
Federation and their annual production averaged 55 million fry from 1997
through 2001. Hatchery production accounts for 98.0% of total beluga
stocks, 56.0% of Russian sturgeon stocks, and 36.6% of stellate
sturgeons. The most significant changes occurred in the second half of
the 20^th^ century when construction of hydropower stations caused sea
level fluctuations. Today, the sea level is 192 cm higher than it was in
1997, which creates favorable habitat conditions for hydrobiont diseases
caused by cestodes, nematodes and crustacea found in commercial catches
from the Volga delta. Because the prevalence of these diseases was not
high, records were not taken after 2000. Bacterial diseases were not
noted, but viral fibrosarcomas in zander as well as cutaneous and fin
papillomas in sheatfish were observed. The most important parasites
included: *Anisakis schupakovi, Eustrongylides excisus, Apophallus
muehlingi,* *Rossicotrema donicum, Paracoenogonimus ovatus,
Pseudamphistomum truncatum, Opisthorchis felineus, Corynosoma
strumosum*. Each of these with the exception of *C. strumosum* was
detected in muscles. Unfavorable winter conditions in 1999-2000
initiated a mixed infection with Canine distemper virus, salmonellosis
and pasterellosis that killed about 20,000 Caspian seals. Although
viruses were not detected in stellate sturgeon brood fish used in
aquaculture, bacteria were present in their gonads and they also had
infestations of C*occonema sulci, Polypodium hydriforme, Amphilina
foliacea,* and *Eustrongylides excisus* that were clinically
insignificant.

**Health Of Pacific Salmonids Held Under Natural And Artificial
Conditions Of Reproduction In Kamchatka**

[Irina V. Karmanova]{.underline}, Svetlana L. Rudakova., Elena A.
Ustimenko, Nataliya V. Sergeenko, Galina P. Liniova, Svetlana A.
Korneeva, Tatjana V. Gavriuseva

Kamchatka Pacific Research Institute of Fisheries and Oceanography,

18 Naberezhnaya Street, Petropavlovsk-Kamchatskiy, 683000, Russia

# Results of bacteriological, virological, parasitological, histological and histochemical investigations of Pacific salmon (chum, pink, sockeye, Chinook and Coho) were compared among fish held under natural and artificial conditions of reproduction. Initiated in 1998, these investigations are ongoing in an effort to monitor salmon health and apply preventive treatments for infectious diseases. A total number of 100 eggs and 11,635 fingerlings were investigated at hatcheries. Another 2,110 fingerlings and 1,600 adult salmon were investigated in natural waters. Carriers of Infectious Hematopoietic Necrosis virus (IHNv) were detected among wild and hatchery adult sockeye salmon at the southwest rivers of the Kamchatka peninsula in 2001-2002 and an IHNv epizootic occurred among sockeye salmon fingerlings at one hatchery in 2002. Viral Erythrocytic Necrosis and Erythrocytic Inclusion Body Syndrome were also found. Eleven species of bacteria were isolated from wild and hatchery fish. *Aeromonas salmonicida* subsp. *salmonicida* and non-motile *A. salmonicida* subsp. *masoucida* were the most problematic but nine other secondary pathogens were noted. Four species of protozoan parasites *Ichthyobodo necator, Chilodonella* *piscicola*, *Apiosoma conicum* and *Trichodina truttae* were found among hatchery fingerlings of Pacific salmon. Thirty-one species of parasites from 15 classes were detected among young feral salmon. Nine of these species persisted within the fish during their time at sea. Thirty-six species of parasites representing eight classes were detected among adult fish from natural reservoirs. The F~1~ generations that were produced by infected parents among nine of these species were also affected. 

#  **Diseases Of Whitefish (*Coregonidae*) In The Russian North-West And North Caucasus**

[Elena V. Kuznetsova]{.underline} and Vladimir N. Voronin

Laboratory of Fish Diseases, State Research Institute of Lake and River
Fisheries, Makarov Embankment, 26, St. Petersburg, 199053, Russia

The 1960\'s through the 1990's was a period of time in which whitefish
(*Stenodus leucichthys leucichthys* Gueldenstaedt) were widely cultured
within Russia. Infections produced by numerous diplostomatids, protozoa,
cestodes and crustacea were often described in these fishes, but our
data showed that there was actually a low prevalence of these parasites
in coregonid fishes from the Russian North-West and North Caucasus.
Today, diplostomosis is not considered to be a serious disease among
whitefish in these latter regions. Although the prevalence of infection
ranges between 1.3 to 89.2 %, parasite abundance is rather low and
generally only 1 -- 12 specimens may be found on an infected fish. The
cause of the reduction in the severity of these parasitic infestations
was a break in the parasite's development that led to reduced contact
between cultured whitefish and other hosts, such as birds or mollusks.
Toxicoses, alimentary disorders, and infectious diseases are currently
the most problematic issues among whitefish. Round black tumor-like
lesions have been found on the skin and in kidney of cultured juvenile
whitefish at a fish farm located in North Caucasus. Light microscopy
revealed that these growths were surrounded by a rim of hyperplastic
epidermis that contained numerous branched, septate fungal hyphae and
various host cells including erythrocytes, macrophages, and fibroblasts.
The fungus was preliminarily identified as *Exophiala* sp.
(*Hyphomycetes*) and this was the first record of the genus *Exophiala*
that was reported as a pathogen in whitefish.

**Aquaculture And Fish Diseases In Russia And Former Soviet Union:**

**Recent History And Current** **Situation**

# Igor S. Shchelkunov

All-Russia Research Institute of Freshwater Fisheries,

Rybnoe, Dmitrov Region, Moscow Province, 141821 Russia

The last decade of the 20^th^ century was a critical time for
aquaculture in what formerly was the USSR. All aspects of the industry
(fish production, disease control and applied science) were affected and
output dropped from 418,000 tons in 1990 to 108,000 tons in 1997. The
decrease in Russia went from 260,000 tons in 1990 to 70,000 tons in
2000. Today, Russian aquaculture is poorly diversified when compared to
that of the former Soviet Union. Although signs of revived activity are
evidenced by the culture of sturgeons, salmonids and shellfish, cyprinid
culture comprises about 84% of the total aquaculture production in
Russia. Fisheries in Russia are oriented toward wild catches and there
are no laws or national legislation that regulate the industry. Although
many aquaculture enterprises have become joint stock companies, most are
in urgent need of investment. The state veterinary service controls fish
diseases in Russia and there is some specific veterinary legislation for
certain fish disease issues. Unfortunately, few veterinary laboratories
in the country can perform disease diagnostics and such tests are mostly
done in research laboratories or universities. Even fewer of these
facilities perform diagnostics certified to international standards.
Although veterinary certificates normally accompany fish or egg
movements, these are rarely based on laboratory data. Furthermore,
federal programs for fish disease monitoring, reporting, risk analysis
and contingency planning do not exist in Russia. Only six of ten
research laboratories that dealt with infectious fish diseases in the
former Soviet Union still exist today; - five of these facilities are in
Russia. Twenty-one viral and virus-like agents or conditions and seven
principal bacterial agents are present in wild and cultured fish. The
most economically important viral diseases are SVC, VHS, IHN, The most
serious bacterial diseases include vibriosis, furunculosis,
streptococcosis, motile aeromonad septicemia, bacterial hemorrhagic
septicemia and *Columnaris* disease. Contraction of aquaculture due to
the national economy improved the epizootic situation for many
infectious diseases, but it worsened some parasitic and bacterial
epizootics caused by ubiquitous organisms (e.g.-aeromonads,
pseudomonads, and flavobacteria). Current trends emphasize development
and use of probiotics and vaccines, bur more efforts are needed to
develop molecular diagnostic techniques.

**Atlantic Salmon Restoration In New England: Significance Of Certain
Diseases**

# Rocco C. Cipriano

U.S. Geological Survey, National Fish Health Research Laboratory, 11700
Leetown Road, Kearneysville, West Virginia 25430

Atlantic salmon (Salmo salar) once thrived in the northeastern part of
the United States and ranged from Maine to the Housatonic River in
Connecticut. Despite the large populations that once thrived in the
Connecticut, Merrimack and Penobscot Rivers, Atlantic salmon essentially
disappeared from New England by the mid 1880's. This decline was
attributed to the construction of dams, pollution and overfishing; -
problems associated with unfettered industrialization of the northeast.
In the latter half of the 20^th^ century, however, efforts began in
earnest to restore the salmon based on legal justification provided by
the U.S. Anadromous Fish Conservation Act of 1965. The act provided
stability for a long-range restoration program involving the cooperation
of numerous federal agencies, state department of fish and wildlife
agencies, industry, and conservation groups. The current restoration
program utilizes river-specific brood stock to maximize genetic
diversity within the Connecticut, Merrimack, Penobscot and several other
smaller rivers on the downeast coast of Maine. Following two to three
years at sea off the coast of Greenland, mature Atlantic salmon return
(around May) to spawn in their natal rivers. These fish are captured at
dams or weirs, transported to holding facilities, and maintained as
non-feeding captive brood until they become gravid (September/November).
Thus, holding and production facilities are the cornerstones of
restoration. Gametes, obtained from captive brood, are used to produce
quality fry, parr and smolts that are stocked to enhance restoration.
These fish spend their first two years of life in their natal rivers
before they migrate to the ocean. Due to the unique genetic composition
and depleted numbers of Atlantic salmon along the downeast coast of
Maine, federal authorities have listed those stocks as an endangered
species. In support of this listing several disease issues were cited
that could deleteriously impact survival. Predominant among these
concerns are: significant mortalities associated with Furunculosis,
caused by *Aeromonas salmonicida*; vertical transmission of Bacterial
Coldwater Disease, caused by *Flavobacterium psychrophilum*; the
potential introduction of Infectious Salmon Anemia virus within
restoration fishes; and the deleterious effects of Swimbladder Sarcoma
Virus. Each of these issues will be discussed within this presentation.

**Mycobacteriosis And Chesapeake Bay Striped Bass: An Integrated
Cooperative Research Program**

[Christopher A. Ottinger]{.underline}

U.S. Geological Survey, Leetown Science Center, National Fish Health
Research Laboratory, 1700 Leetown Road, Kearneysville, WV, 25430

Mycobacteriosis is a bacterial disease of striped bass (*Morone
saxatilis*), which can be disfigured by skin ulcers and internal
lesions. Bass may also be emaciated by the chronic nature of this
wasting disease. Recent evidence of polymicrobial infections in some
striped bass suggested that the observed condition of infected fish
might be caused by somewhat complex disease progression. While the
extent of mortality associated with mycobacteriosis in wild striped bass
is unknown, laboratory studies indicated that the bacteria are lethal
when injected in biologically relevant doses. In the Chesapeake Bay and
other regions of the United States, striped bass are a prized species
for recreational anglers and commercial fishermen. The economic impact
of mycobacterial infection could be significant. Data collected from
1998 to 2002 revealed that 70% of the bass taken from some locations
within the Chesapeake Bay were infected and that the disease had
persisted in these populations for at least five years. A cooperative
research program to address research issues surrounding this
mycobacteriosis epizootic began in 1999. This program, involving both
state and federal entities, addressed issues associated with
pathogenesis and immunity. Understanding the epidemiology of this
disease is important to resource managers who must responsibly manage
the striped bass and other fisheries in the Chesapeake Bay. It may also
be important to those managers responsible for the overall health of the
bay and interagency efforts to address concerns for environmental
quality and the safety of bay resources for recreation.

**Diagnosis And Management Of Bacterial Diseases In Warmwater
Aquaculture**

# John P. Hawke

Department of Pathobiological Sciences, Louisiana State University,
School of Veterinary Medicine, Baton Rouge, Louisiana, USA 70803

Warmwater aquaculture, particularly within the catfish industry,
experienced tremendous growth in the last 25 years. Mariculture (marine
aquaculture) is being examined as a means to compensate for over-fished
natural stocks of marine fishes. Extensive culture practices, that were
common before 1980, have yielded to more intensive pond culture and
closed recirculating system culture. This increase in technology and
production capability has also caused an increased incidence of disease
in warmwater aquaculture. In this presentation, I shall describe methods
to diagnose and manage important bacterial, viral, and parasitic agents
that affect channel catfish *Ictalurus punctatus*, hybrid striped bass
*Morone saxatilis*, and tilapia *Oreochromis niloticus*. Methods of
diagnosis and control will be discussed based on procedures practiced at
the Louisiana Aquatic Diagnostic Laboratory.

**Health And Disease Issues Associated With Fish Populations In The
Great Lakes Basin**

[Mohamed Faisal]{.underline}

Colleges of Veterinary Medicine and Agriculture and Natural Resources,
Michigan State University, East Lansing, Michigan 48824

In the Great Lakes basin, conservation and restoration efforts of
fisheries depends primarily upon increasing the existing fish through
stocking of hatchery-propagated fish. This practice requires collecting
gametes from feral fish species and raising the offspring to a stage,
which if released in the Great Lakes' environment, would yield a high
survival rate. Maintaining the health of these fish is instrumental to
the success of these efforts. This study describes a number of emerging
fish diseases that threaten the success of the Great Lakes fisheries
restoration efforts such as infection with the Largemouth Bass Virus
(Ranavirus, Iridoviridae). This viral infection is spreading rapidly
among the inland lakes of Michigan's lower peninsula. Infection with a
*Piscirickettsia* sp has caused epizootic mortalities in the muskellunge
population of Lake St. Claire. Visceral mycosis, primarily caused by a
unique strain of *Phoma herbarum*, causes heavy losses in
hatchery-reared Chinook salmon. Efforts to control the vertical
transmission of bacterial kidney disease and coldwater disease have been
successful.

**Disease Transmission From Cultured Salmonids To Wild Fish Stocks:
Perspectives On The Alaskan Hatchery Program**

# Theodore R. Meyers^1^

^1^Alaska Department of Fish and Game, Commercial Fisheries Division,
Juneau Fish Pathology Laboratory, P.O. Box 25526, Juneau, AK 99802-5526

Geographic isolation, coldwater temperatures and protective fish health
legislation have probably accounted for fewer indigenous salmonid
pathogens in Alaska and have prevented the introduction of exotic
disease agents through prohibition of fish imports. Nonetheless,
prevalences of indicator pathogens such as infectious hematopoietic
necrosis virus (IHNv) and *Renibacterium salmoninarum* (Rs) have
approached 100% in wild fish stocks that have not had any exposure to
hatchery fish or hatchery practices. In Alaska, all hatchery broodstocks
have originated from wild salmonids. Disease interactions between
hatchery and wild fish are minimized by stringent fish disease and
genetics policies, hatchery and release site selection, and other
management criteria. Examination has shown that there were no
significant differences in IHNv or Rs prevalences between hatchery and
wild stocks of salmonids. Furthermore, isolates of IHNv from hatchery
and wild fish appear evolutionarily constrained with a low genetic
diversity and restriction to sockeye salmon as a host species. Hence,
these diseases in Alaskan salmonids have been relatively static with
cyclical peaks and declines. Thus, there were no significant increases
in pathogen levels and prevalences that could be attributed to hatchery
practices and/or interactions with hatchery fish stocks.

United States Department of Agriculture Services Related To Infectious
Salmon Anemia And Spring Viremia Of Carp Aquatic Animal Health Programs

[Otis Miller, Jr]{.underline}.

# United States Department of Agriculture, Animal and Plant Health Inspection Service, 4700 River Road, Unit 46, Riverdale, Maryland 20737

The Office of the Secretary of Agriculture announced that an emergency
threatens the livestock industry of this country, and United States
Department of Agriculture (USDA) funds were used to establish Infectious
Salmon Anemia (ISA) and Spring Viremia of Carp (SVC) programs to address
the threat to the U.S. salmonid and ornamental fish industries.
Approximately \$8.3 million and 11.7 million was authorized for the
Animal Plant Health Inspection Service (APHIS) Veterinary Services (VS)
to implement an ISA and SVC control and indemnity program for
farm-raised salmon and ornamental Koi carp in the United States. In
addition to the payment of indemnity, these funds are to be used to
assist the states of Maine, North Carolina and Virginia with program
activities such as: depopulation and disposal, clean-up and
disinfection, establishment of surveillance programs, epidemiology and
diagnostic support, and training for producers and veterinarians.
Federal assistance is deemed necessary to effectively control this
disease, which poses a threat to animal health and the U.S. economy. Our
goal is to control and contain ISA and SVC viruses through rapid
detection and depopulation of salmon and koi that have been infected
with or exposed to ISA or SVC. It is believed that these viruses can be
controlled within high-risk zones through surveillance, and best
management practices. This paper will outline those steps required for
ISA and SVC indemnity, control, depopulation, cleaning and disinfection,
surveillance and restocking of pens or ponds of commercial production
operations.

**Theory And Practice Of Establishing Long-Term Cell Lines**

From Fish And Shellfish

[Igor S. Shchelkunov]{.underline} and Tatiana I. Shchelkunova

All-Russian Research Institute of Freshwater Fisheries, Rybnoe,

Dmitrov Region, Moscow Province, 141821 Russia

Potentially immortal continuous cell lines are of both scientific and
pragmatic interest in relation to lower vertebrates and invertebrates.
Since the 1960's, cell lines have been established from 300 finfish, 500
terrestrial invertebrates (insects) and soft-bodied or calcareous
corals. However, the development of only one cell line from an aquatic
invertebrate, (the freshwater mollusk, *Biomphalaria glabrata*), clearly
indicates that success is still based largely on empirical findings
rather than theoretical background. Experiences suggest that best
results are achieved when cells are gently excised from donor organisms
followed by placement in a comfortable environment that favors *in*
*vitro* growth. There are at least five "bottlenecks" in which the most
vulnerable of the explanted cells are lost. Such barriers include the
donor animal itself, method of donor tissue decontamination, tissue
dissociation techniques, composition of growth media, and quality of
cell culture glass and plastic ware. In this context, the significance
of the donor's immuno-physiological state, tissue selection, type of
sample (individual or pooled), dissociation techniques, contributions of
synthetic media (and sera), and glassware processing are reviewed.
Additionally, this presentation assesses the importance of cell seeding
densities, allogenic serum and conditioned cell culture media, role of
"minicultures" and "chess-board" cell seeding, seeding of mixed cell or
separated cell suspensions, and procedures for subculture. The work
predominantly concentrates on spontaneously established cell lines, but
new approaches for directed immortalization of primarily cultured cells
will also be discussed.

**Retroviral Pathogens And Tumors In Coolwater Fish - Walleye Dermal
Sarcoma**

[Paul R. Bowser^1^]{.underline}, Sandra L. Quackenbush^2^, Rodman G.
Getchell^1^, Gregory A. Wooster^1^, Rufina N.Casey^1^, and James W.
Casey^1^

Aquatic Animal Health Program^1^, Department of Microbiology and
Immunology, College of Veterinary Medicine, Cornell University, Ithaca,
New York; ^2^Department of Molecular Biosciences, The University of
Kansas, Lawrence, Kansas

Walleye dermal sarcoma is a dermal lesion found on walleyes
*Stizostedion* *vitreum* during the cooler months of the year. The
lesion develops in the fall, is carried by the fish through the winter
and typically regresses in the early summer. Our laboratories have been
investigating this tumor over the past 18 years in an effort to
understand the mechanism(s) by which this retroviral tumor develops and
regresses on a seasonal basis. The tumor can be transmitted in the
laboratory by intramuscular injection, feeding or topical application of
cell-free tumor filtrates from tumors collected in the spring. Attempts
to transmit the tumor using filtrates from fall-collected tumors have
met with little success. Tumors considered to be malignant have only
been found on rare occasions. When laboratory challenges were performed
with very young walleye (\< 8 weeks post-hatch), invasive tumors were
observed. During our studies at the New York State Department of
Environmental Conservation Oneida Fish Hatchery, Oneida Lake, New York,
typically 30-40,000 adult walleyes are brought to the facility. During
that time, we have only observed three adult walleyes with tumors that
were invasive. In the overwhelming majority of the cases, tumors will
regress on fish. Field and laboratory studies suggest that, in most
cases, a walleye that is tumor-positive in a given year will be
refractory to development of a tumor in a subsequent year. The walleye
dermal sarcoma virus is the first retrovirus of fish to have its
complete sequence determined.

**Immune Sensor Based On Surface Plasmon Resonance For Express Control
Of Fish Retroviral Infections**

[L.P. Buchatsky^1^]{.underline}, N.F. Starodub^2^

^1^Taras Shevchenko National University, 64 Vladimirska Str., 00017,
Kyiv, Ukraine; ^2^A.V. Palladin Institute of Biochemistry of National
Academy of Sciences, 9 Leontovicha Street, 01030 Kyiv, Ukraine

Solving the problem of early diagnostics for retroviral infection is
very important today because diseases induced by this infection are
widespread among fishes, animals and humans. Bovine leucosis, AIDS, and
some fish tumors are induced by retroviruses, which are identical in
some conservative regions. We had earlier developed immune biosensors
based on the surface plasmon resonance for express diagnostics of bovine
leucosis. Some proteins of retrovirus were used as sensitive elements in
this biosensor. In this report we intended to present the experimental
results about our approaches for the development of the immune biosensor
based on the above-mentioned transducer that detects retroviral
infection among fishes in some reservoirs. In the Ukraine we have found
tumors in pike \[2\], bream, walleye and mullet. At first, we
investigated the possibility of obtaining retroviral antigens from some
tumors of fish. In the course of these investigations, it was observed
that retroviral antigens of pike were a complexed with antigen-specific
antibodies. Second, it was revealed that these antigens cross-reacted
with antibodies, which were induced by retroviruses that caused bovine
leucosis. In all cases bovine leucosis virus antigen or purified fish
retroviral antigen were spontaneously adsorbed on bare thin gold surface
or preliminary lectin-treated one. After the selective binding of
specific antibodies with the immobilised antigens, the specific signal
of surface plasmon resonance was observed. At last, it was discovered
that preliminary treatment of transducer surface by lectin allowed
reception of a more stable and distinguished signal of the immune
sensor. These investigations showed that the immune sensor could be used
to diagnose retroviral infections among fishes. Moreover, such
diagnostics may be done in field conditions as well as in real time. Our
next experiments will investigate effective algorithm of fish retroviral
antigen purification to study level of cross reactivity of fish and
bovine retroviral antigens and to analyse the level of specific
antibodies in the blood of fish and different stages of disease
progression.

**Isolation Of Infectious Pancreatic Necrosis Virus From Young Atlantic
Salmon (*Salmo salar* L.) In Russia**

Tatjana D. Pitchugina, [Maria N. Borissova]{.underline}, Lev P.
Dyakonov, Konstantin P. Yurov, Igor Y. Tkachev, Grigory A. Nadtochey
^1^, and Sergey S. Yakovlev ^2^

^1^The Kovalenko All Russian Institute of Experimental Veterinary
Medicine (VIEV); ^2^Department of Veterinary of Russian Federation

Viral diseases cause significant economic losses and serious problems
for Atlantic salmon (*Salmo salar L.*) raised in aquaculture. Infectious
Pancreatic Necrosis virus (IPNv) is one of our more significant
concerns. In this study, the viral cytopathogenic agent was isolated
from young salmon at a fish farm of the Murmansk region in Russia. Chum
salmon heart (CHH-1) and Chinook salmon embryo (CHSE-214) cell cultures
were used to isolate the virus. Cytopathic alterations typically
associated with IPNv were observed with the third passage after cell
culture viral transformation. Distinctive inclusions in cell cytoplasm
and larger pink inclusions typical of the RNA viruses were detected. The
cells widened, displayed symplast formations, and had increased nuclear
and cytoplasmic densities by the second day and cell monolayers were
completely degraded by day three. The titer of virus in CHH-1 cell
culture was up to10^3^ TCD~50/~mL and 10^6.5^TCD~50/m~L in CHSE-214
cells. The titer was as high as 10^8^ TCD~50/~mL by the eleventh passage
in CHH-1 cells. The optimal temperature for viral propagation was 15^o^C
and the virus could be stored frozen at either --70^o^C or --20^o^C. Two
types of viral particles were detected using electron microscopy; one
resembled the family Birnaviridae. Serological identification of the
virus was accomplished with an IPNv antigen-based enzyme linked
immunosorbent assay that was conducted at the National Veterinary and
Food Research Institute, Helsinki. Their data confirmed the presence of
IPNv in the samples that were tested.

**Infectious Hematopoietic Necrosis Virus: Repetitive Isolation From
Adult Sockeye Salmon (*Oncorhynchus nerka)* From River Bolshaya-Bistraya
(Kamchatka)**

# Svetlana L. Rudakova

Kamchatka Pacific Research Institute of Fisheries and Oceanography, 18
Naberezhnaya Street, Petropavlovsk-Kamchatski, 683000, Russia

This study was conducted to detect the prevalence of Infectious
Hematopoietic Necrosis virus (IHNv) among prespawning sockeye salmon
*(Oncorhynchus nerka)* from the river Bolshaja-Bistraya. In 2002, 217
adult feral salmon were examined that were either caught on their
natural spawning grounds or held as captive brood stock at a fishpond
prespawning holding pool at the Malkinskiy Salmon Hatchery. Even though
the IHNv carrier prevalence was 50% among the sockeye salmon from this
population, pathological signs were not evident by visual examination.
Samples of kidneys, spleens, brains and ovarian fluids were collected
for virological examination. Samples were inoculated on CHSE-214 and EPC
cell lines and cytopathic effects generally became apparent six days
after inoculation. The virus was confirmed by neutralization assays
using rabbit antiserum to IHNv obtained from the United States. The
first sampling of the adult sockeye salmon revealed that 10% of the fish
were infected, but the number of carriers increased to 75% before
spawning ended. The titers of infectious virus in organs and ovarian
fluid of the examined fishes was always at an epizootically significant
level (0.4х10^5^ to 0.4х10^8.6^ TCD~50~/mL). Bullet-shaped viral
particles, which measured approximately 150-220 x 60-80 nm were observed
by electron microscopy. Morphological attributes indicated that the
virus belonged to the family *Rhabdoviridae*.

Genetic Typing of Infectious Hematopoietic Necrosis Virus

[Gael Kurath]{.underline}, Kyle A. Garver, Evi J. Emmenegger, and Ryan
M.Troyer

United States Geological Survey, Biological Resources Division, Western
Fisheries Research Center, 6505 NE 65th St., Seattle, Washington, 98115

Infectious hematopoietic necrosis virus (IHNv) is a rhabdovirus that
causes severe disease and is prevalent in salmon and trout species in
the northwestern region of North America. Since the 1950\'s IHNv has
been known to cause epidemics in hatchery and farmed salmonids, and
outbreaks in wild salmonids have also been reported. Genetic typing
methods have been developed to characterize IHNv isolates on the basis
of partial gene nucleotide sequences within the glycoprotein (G) and
nucleocapsid (N) genes. Phylogenetic analyses of sequences from over 400
IHNv isolates from throughout the North American range of the virus have
shown that there are three major genogroups of IHNv. These genogroups
correlate strongly with geography, are they are therefore designated U,
M, and L, to indicate that they are found in the upper, middle, and
lower portions of the virus range. The IHNv genogroups vary in genetic
diversity and evolutionary rate, suggesting varied evolutionary
histories. Genetic typing also provides epidemiological insights,
revealing complex virus traffic patterns and identifying potential
sources of disease outbreaks. A subset of this data has been selected to
generate phylogenetic trees of partial G (303 nucleotides) and partial N
(412 nucleotides) sequences from 29 IHNv isolates. These trees were
designed to include representatives of all IHNv genogroups and
sub-groups that have been identified, so they can be used by other
researchers to determine how new IHNv isolates fit into this family of
IHNv.

**Diagnosis and Specific Prevention of Spring Viremia of Carp**

[Svetlana F. Oreshkova^1^,]{.underline} Igor S. Shchelkunov^2^, Olga S.
Voronova^1^, Anastasiya G. Popova^1^, Galina N. Nikolenko^1^, Tatiana I.
Shchelkunova^2^, and Alexander A. Ilyichev^1^

^1^Research Institute of Bioengeneering of State Research Center of
Virology and Biotechnology \"Vector\", Koltsovo, Novosibirsk region,
630559, Russia

^2^All-Russian Research Institute of Freshwater Fisheries, Rybnoe,
Moscow Province, 141821, Russia

Spring Viremia of Carp virus (SVCv) is a rhabdovirus that causes severe
disease, but remains largely untreatable. This research aimed to develop
methods to detect and identify different SVCv isolates and evaluate
recombinant vaccines that would prevent the disease. For diagnosis, an
RT-PCR was developed that used primers to several SVCv genes. Most
isolates of the virus were detected with three pairs of primers to the N
gene. For detection of virus in clinical samples (tissues from fish,
infected ZL4 strain SVCv), a semi-nested PCR was developed and the
sensitivity of viral detection was 10^2^TCID~50~/g. The N1-N2 fragments
were amplified, cloned, and sequenced. The aliments of N1-N2 fragments
of the SVCv N gene and intergenic regions from a reference isolate
(courtesy of Nikolai Fijan) and isolates from Czechia, Hungary and
countries of Western Europe were very homologous. However, there were
many differences in the N gene and intergenic region of Russian
isolates. Three nucleotide substitutions lead to the amino acid
placements that allowed differentiation between Russian and European
isolates by PCR. Based on the sequence comparisons, restriction fragment
length polymorphism (RFLP) analysis was used to type subgroups. A
recombinant vaccine against SVCv, based on the viral glycoprotein gene,
was evaluated. In initial experiments, a DNA vaccine (plasmid pcDNA3-G,
constructed by means of insertion of the full-length viral G protein
from an American isolate) was supplied by Joanne Leong. The vaccine was
very protective and produced a relative percent survival of 49% among
fish challenged at eight weeks post-vaccination and 80% among fish
challenged after 10 weeks. Another DNA-vaccine based on G-gene of the
Russian ZL4 strain under control of CMV or carp-beta-actin promoters
(courtesy of N. Lorenzen) was constructed but there have been problems
with efficacy due to impurities in the plasmids. Vaccine-candidates
purified by different methods were prepared and their efficacy is under
further study.

**First Report Of Spring Viremia Of Carp Virus At A Fish Farm In Moscow
Province, Russia**

[Tatiana D. Pichugina]{.underline}^1^, Maria N. Borisova^1^, Tatiana I.
Shchelkunova^2^,

Igor S. Shchelkunov ^2^, and Elena A. Zavyalova^1^

^1^All-Russia Research Institute of Experimental Veterinary Medicine
(VIEV), Kuzminki, Moscow, 109472; ^2^ All-Russia Research Institute of
Freshwater Fisheries, Rybnoe, Dmitrov Region, Moscow Province, 141821
Russia

Although a cytopathogenic agent was first isolated from carp with
infectious dropsy syndrome in the USSR during the early 1960's, viral
etiology was confirmed eight years later and after the disease was first
described in Yugoslavia. Since then, dozens of Spring Viremia of Carp
virus (SVCv) isolations have been made in the Russian Federation,
Ukraine, Belarus, Moldova, Georgia and Lithuania. These isolations
geographically overlap natural areas where carp, *Cyprinus carpio*, are
found in eastern Europe. Most of the Russian Federation isolates came
south European Russia and other areas were considered to be free of
SVCv. In May 2003, a heavy epizootic occurred among broodstock, 1+ and
2+ carp at the Lotoshinsky fish farm located in the northwestern part of
Moscow Province. Mass mortality began soon after transfer of fish from
wintering to summer brood and grow out ponds. Disease was triggered by
water temperature fluctuations: from low to 20^o^C and back to 15^o^C in
few days. Affected fish displayed pale gills, scale protrusion,
exophthalmia, inflamed and protruded anus and skin ulcers. At necropsy,
the liver and kidney were pale and swollen and ascitic fluid was
present. Poloed liver, spleen and kidney samples were assayed from 11
diseased fish for virological examination and inoculated onto EPC cells.
The isolated cytopathogenic agent was identified as SVCv using
neutralization tests with rabbit antiserum (neutralization index
10^4.25^ at serum dilution 1:40). The disease was reproduced in yearling
carp by intraperitoneal injection of the virus after its seventh *in
vitro* passage. At water temperatures of 14.5--15.5^o^C, eight of nine
infected fish had clinical signs of SVC and six of these fish died. More
than an 80% mortality was observed on the farm at that same time in
8-day-old fry. Unfortunately, the diseased farm fish were not sampled.
This was the first case of SVC detected in the Russian Federation after
almost a decade-long collapse of national aquaculture, but carp culture
is now an active industry in Moscow Province. The central position of
the Province, the revival of aquaculture, weakened veterinary controls
on fish movements, and peculiarities of the local watershed can
contribute to the spread of SVCv into other regions.

Intracytoplasmic Inclusions In Erythrocytes Of Pacific Salmon

Nina A. Golovina and [Pavel P. Golovin]{.underline}

All -- Russian Research Institute of Freshwater Fisheries (VNIIPRKh),
Rybnoe, Dmitrov Region, Moscow Province, 141821 Russia

Investigations have been conducted for two years that revealed two types
of intra-erythrocytic cytoplasmic inclusions from northern Okhotsk sea
populations of chum, pink, and Coho salmon. These conditions were
similar to those observed with Viral Erythrocytic Necrosis (VEN) and
Erythrocytic Inclusion Body Syndrome (EIBS), which have been described
in detail as among salmon abroad. Our investigations in northern
Okhotsk-sea populations suggested that VEN was present in the chum, pink
and Coho salmon, but EIBS was restricted to chum salmon fingerlings and
brood fish. The number of affected breeders (of chum and coho salmon)
increased rapidly during their maintenance within fish plants during the
fall and just before spawning. To that extent, a seasonal character of
infection in chum salmon was revealed. In the summer, the inclusions
EIBS inclusions were predominant within fingerlings and breeders of chum
salmon, but VEN inclusions became more prevalent in the fall. Such
infections did not produce acute mortality, but compromised the
immuno-physiological condition of chum fingerlings. This dehabilitation
could potentially affect the survival of young fish after they are
released into the river. Such infections should be considered to be
epidemiologically dangerous to salmon aquaculture in the Far Eastern
region of Russia.

The United States Fish And Wildlife Service's National Wild Fish Health
Survey: A New Tool For Fisheries Management

# Thomas A. Bell

U.S. Fish and Wildlife Service; Division of the National Fish Hatchery
System - Fish Culture Operations Branch, 4401 North Fairfax Drive,
Arlington, Virginia 22203

The United States Fish and Wildlife Service\'s (USFWS) National Wild
Fish Health Survey (NWFHS) has existed since 1996 and became available
to the public in September 2001. The NWFHS comprises field collection,
laboratory analysis and web-accessible database components, and is
primarily a result of Federal, State and Tribal cooperative efforts. All
field collections are conducted in accordance with published NWFHS
standardized procedures. All laboratory procedures are, likewise, in
accordance with the same published procedures, but are only conducted by
USFWS fish health biologists stationed at our nine Fish Health Centers.
The database can be queried relative to national fish pathogen
distributions in free-ranging fish, the results of which can be
displayed graphically and/or in tabular form, and overlaid on United
States Geological Survey-based watershed maps. As of August 2003, the
database contains entries from greater than 62,000 individual fish
samples collected from more than 1,700 distinct geographic locations
(each with an exact latitude and longitude based on global positioning
satellite, or GPS, information) located in 44 States. The fish sampled
are from 183 different species. Samples are routinely assayed for as
many as 28 fish pathogens. The database has already been used by the
USFWS and other aquatic resource managers to formulate, and implement,
well-informed and more scientifically-based fishery management plans. As
each year\'s samples are collected, analyzed and entered into the
database, the NWFHS progresses closer to achieving its full potential.
In addition to a review of the NWFHS, several examples of how the NWFHS
has been successfully used by aquatic resource managers will be
presented.

***Aphanomyces invadans (= A. piscicida)*: A Widespread Problem In Wild
And Cultured Fishes**

[Vicki S. Blazer]{.underline}^1^, Melba B. Bondad-Reantaso^2^, Richard
B. Callinan^3^, Kishio Hatai^4^, and C. V. Mohan^5^

^1^U.S. Geological Survey, National Fish Health Research Laboratory,
11700 Leetown Road, Kearneysville, West Virginia 25430; ^2^Cooperative
Oxford Laboratory, Maryland Department of Natural Resources, 904 S.
Morris Street, Oxford, Maryland 21654; ^3^New South Wales Fisheries,
Regional Veterinary Laboratory, Wollongbar, New South Wales 2477
Australia; ^4^Division of Fish Diseases, Nippon Veterinary and Animal
Science University, 1-7-1 Kyonan-cho, Musashino, Tokyo 180-8602 Japan;
^5^Network of Aquaculture Centres in Asia Pacific, Department of
Fisheries Compound, Kasetsart University Campus, Ladyao, Jatujak,
Bangkok, Thailand

The disease now known by most investigators as epizootic ulcerative
syndrome (EUS) has caused major losses in many countries for over three
decades. It was first described in Japan as mycotic granulomatosis, a
year later in Australia as red spot disease, throughout Southeast and
South Asia as epizootic ulcerative syndrome and in the United States as
ulcerative mycosis. These syndromes include the presence of ulcerative,
dermal lesions in which invasive fungal (oomycete) hyphae have elicited
a characteristic granulomatous response. In many cases, lesions extend
deep into the musculature. Many species of freshwater and estuarine
fishes are susceptible. Much controversy has occurred over the "cause"
of these lesions. The role of other fungi or oomycetes, bacteria,
viruses and toxic dinoflagellates in ulcer formation has been disputed.
The presence of many opportunistic organisms in the open ulcers of
affected fish, complicates isolation of a primary infectious agent; the
lack, until recently, of molecular diagnostic techniques and
reproducible models for experimental induction; and the lack of
consistent methods for confirmatory diagnosis of EUS have contributed to
this controversy. However, most investigators now agree that the cause
is *Aphanomyces invadans (=A. piscicida)*, an aquatic oomycete. The
state of our knowledge on the phylogenetic characterization of the
organism, histopathological presentation of the disease, models for
experimental induction of disease, environmental influences on the
growth and sporulation of the organism, epidemiology and advances in
molecular diagnostic techniques will be discussed. In addition, the
outcomes of an expert consultation on EUS held during the Fifth
Symposium on Diseases in Asian Aquaculture, Brisbane, Australia,
November 2002, will be reviewed.

**Marine Mammal Stranding Response: Conservation Through Cooperation**

Kathleen M. Touhey, Kristen Patchett, and [Donald A. Abt]{.underline}

Cape Cod Stranding Network, Inc., Post Office Box 287, Buzzards Bay,

Massachusetts 02532

The southeastern region of Massachusetts, with more than 700 miles of
coastline including Cape Cod, is a major area for the stranding of
marine mammals. The Cape Cod Stranding Network, Inc. (CCSN) responds to
strandings of marine mammals in this region. Our mission is to provide
rapid response, assessment and humane care to stranded marine mammals;
to effect appropriate release; and to conduct research and education in
order to reduce suffering and prevent avoidable mortality. The CCSN is a
non-profit organization formed by a group of like-minded animal welfare
and scientific organizations functioning in a cooperative manner to
provide effective responses to marine mammal stranding events with the
added benefits of enhancing education about and conservation of marine
mammals through innovative methodologies. As the holder of a Letter of
Authorization from the National Marine Fisheries Service for the Cape
Cod region, the CCSN is responsible for all strandings of marine mammals
common to the many different habitats found along our shores. In 2002,
the CCSN responded to approximately 300 stranded pinnipeds and cetaceans
representing 16 species. In carrying out our mission, we work closely
with federal, state, and local officials as well as other stranding
networks in the Northeast Region. While our staff usually deals with
single strandings, mass strandings require extensive cooperation between
the CCSN, governmental agencies, and other networks in the region. The
small permanent staff of the CCSN is enormously augmented with the
contributions of more than 400 trained volunteers who assist at
strandings of all magnitudes. Without the faithful cooperation of the
staff of the various networks, the trained volunteers, and the several
governmental agencies with jurisdiction over marine mammals in our
waters, our mission could not be fulfilled.

**A Brief Review Of Some Important And Emerging Bacterial Diseases Of
Pinnipeds And Cetaceans**

# J. Lawrence Dunn

Department of Research and Veterinary Services, Mystic Aquarium, 55
Coogan Boulevard, Mystic, Connecticut 06355

Bacterial diseases continue to play an important role in mortalities of
both captive and wild marine mammals. Various authors have attributed up
to 30% of wild marine mammal deaths and up to 50% of captive held marine
mammal deaths to bacterial etiologies. A number of bacterial pathogens
involved in mortalities in early zoological collections of pinnipeds and
cetaceans continue to be recognized as the cause of death in
contemporary captive collections. This presentation reviews the major
bacterial diseases most frequently encountered in captive and stranded
marine mammals. The clinical signs and courses of these diseases will be
reviewed as well as the associated clinical pathologic changes and
common treatment and prophylactic regimens. In addition, emphasis will
be placed on two zoonotic bacterial diseases, either newly recognized,
or emerging, in marine mammal populations (Brucellosis and
Mycobacteriosis).

#  

# **Emerging Challenges In Public Aquaria**

[Brent R. Whitaker, M.S., D.V.M.]{.underline}

National Aquarium in Baltimore, 501 E. Pratt St., Baltimore, Maryland
21202

In the past 25 years, public aquaria have gained popularity worldwide.
Many major cities now host aquariums that educate and entertain
visitors. Modern facilities house diverse aquatic and terrestrial
collections requiring advanced life support systems, specific
nutritional requirements, development of customized quarantine and
husbandry programs, and the ability to recognize, diagnose and treat
disease. Excellent water quality is required for fish health and clarity
is also needed for public viewing. Providing water changes to large
closed-water systems is expensive and, in some cases, not permitted due
to elevated levels of phosphate, nitrates, or other pollutants.
Alternate methods to manage potential toxins are in development.
Additionally, fish utilize trace and macro elements from the water such
as iodine, which is required to prevent goiter. The use of ozone, a
common oxidant that disinfects and improves water clarity, alters the
form of iodine that potentially decreases its absorption. Maintaining
healthy elasmobranchs in captivity presents numerous challenges. There
is a lack of standardized analytical methods for hematology and
leukocytes morphologically differ among species. Because cell function
is not understood, its relevance in health assessment is under
investigation. Large sharks at some facilities have developed spinal
deformities and there is a collaborative effort among clinicians and
pathologists to determine its cause. Suitable anesthetic and surgical
techniques are continually being developed for elasmobranchs. Oxygen
carrying replacement fluids such as Oxyglobin^^, a polyionic colloidal
fluid made with ultra pure bovine hemoglobin, have been used in teleosts
and elasmobranchs and show promise in emergency treatments. Seahorses
and sea dragons (sygnathids) have become popular exhibits, but many of
their diseases are not understood or described. For example,
subcutaneous gas bubbles occur in many species, which often respond to
treatment with antibiotics and a carbonic anhydrase inhibitor, but the
cause is unclear. Mycobacteriosis is also a problem among captive
sygnathids. Head and lateral line disease is commonly seen in captive
teleosts. Although the cause is unknown, a new treatment offers hope for
valuable specimens and may help determine its etiology. Everyday brings
challenges to aquarium veterinary and husbandry staff, which present
opportunities for collaborative research.

DDT In Bottom Sediments And In The Liver Of Barfin Plaice *Pleuronectes
pinnifasciatus*: A Factor Influencing Health Of Fishes From Amursky Bay
(Peter The Great Bay, Sea Of Japan)

#### [Marina A. Vaschenko]{.underline}^1^, Iraida G. Syasina^1^, and Petr M. Zhadan^2^

^1^Institute of Marine Biology, Far East Branch, Russian Academy of
Sciences,

ul. Pal'chevskogo 17, Vladivostok 690041, Russia; ^2^Pacific
Oceanological Institute, Far East Branch, Russian Academy of Sciences,
ul. Baltiiskaya 43, Vladivostok 690041, Russia

In August andSeptember 2001, 15 samples of bottom sediments were
collected in inner, middle and open areas of Amursky Bay near
Vladivostok, Russia. Barfin plaice *Pleuronectes pinnifasciatus* were
also sampled from the inner and middle parts of the bay. In the
sediments and in the fish liver, the contents of chlorinated
hydrocarbons, *p,p′*- DDT and its metabolites (*p,p′*- DDD and *p,p′-*
DDE) as well as α-, β- and γ-isomers of hexachlorocyclohexane (HCH) were
determined using gas-liquid chromatography. The sediments contained
negligible amounts of HCHs (0.2--0.8 ng/g dry weight), while DDT
concentrations were quite high (1.7--16.3 ng/g dry weight). There were
no differences in levels of pesticide contamination between the
locations and our results resembled those reported for the Amursky Bay
in the 1990s. Surprisingly, "fresh" DDT comprised 70--85% of total DDT
content in the sediments from all locations studied in 2001. The DDT/DDE
ratios were high (4.3--11.5). In fish livers, total DDT concentrations
were 212.8 and 122.5 ng/g wet weight for the inner and the middle
locations, respectively, and "fresh" DDT comprised 35 and 64% of DDTs,
respectively. These results provide evidence for a recent input of DDT
from an unknown source into Amursky Bay. Fish livers were examined for
histopathological changes, which are biomarkers of toxic contaminants.
Vacuolization of hepatocytes, coagulative necrosis of hepatocytes,
necrosis of epithelial cells of bile ducts, and inflammatory reactions
were evident. Diffuse vacuolization of hepatocytes was the most common
type of pathology that was observed. On the whole, liver morphology as
well as the occurrence of histopathological alterations were similar in
the plaices from both locations. The only difference was that 23% of the
plaices from the middle site had pathological changes of the bile ducts
that were not detected in the fish from the inner site. This pathology
is probably connected with an intensive metabolism of DDT by the fish.

Number range CHAPTER**Global Climate And Large-Scale Environmental
Influences On Aquatic Animal Health**

[William S. Fisher]{.underline}^1^

United States Environmental Protection Agency, Office of Research and
Development, National Health and Environmental Effects Research
Laboratory, Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze,
Florida 32561

In the last 3 decades, numerous large-scale mortality events have
occurred that affected a broad diversity of aquatic organisms in North
America. Short-term forensic investigations of these events have
sometimes characterized a causative agent or condition, but they have
rarely provided insight sufficient to predict and manage animal health.
Although traditional tools (microbiology, parasitology and pathology)
are essential, technological advances in remote sensing, satellite
imagery, and information management are required to understand these
local biological phenomena in context to environmental conditions that
change across broad regional and global scales. These additional tools
will be invaluable for exploring the influences of climate, weather
(e.g., precipitation), ultraviolet light exposure, Aeolian dust
(transporting microorganisms and nutrients), and regional land use
patterns on disease and host susceptibility. Ultimately, regional
observing systems could forecast health risks to aquatic resources. One
such system, under development in the Gulf of Mexico, will forecast red
tide events that have extended along the Gulf coastal states of the
United States and Mexico. But to further this approach, a meaningful
understanding of large-scale environmental effects on disease is needed.
Data collected from diseases of fish, corals, sea urchins, marine
mammals, oysters, lobsters, and abalone can be examined in an integrated
manner within broad temporal and spatial contexts for possible links
with regional environmental factors. A pilot for this approach has been
introduced but has not been utilized. Success will also require a
comprehensive monitoring and data collection network, rather than the
case-by-case, species-by-species reporting that exists. Disease and
mortality data for multiple species must be gathered into an inclusive
and comprehensive format. Again, a pilot for such a database exists, but
has not been widely implemented. Together, application of these tools
will allow development of epizootiological models to aid in
understanding large-scale environmental influences on aquatic disease
and will ultimately lead to management alternatives.

**Tumors Of Coregonid Fishes In The River Pechora**

[Anatoly A. Lukin]{.underline}

Northern Water Problem Institute, Karelian Science Center, Russian
Academy of Science, 50 A. Nevsky Street, Petrozavodsk,

Karelia, 185000, Russia

The Pechora River is one of the largest watersheds in Northwest Russia,
which is anthropogenically affected by oil pollution, heavy metal
pollution, nuclear explosions, intensive navigation, and uncontrollable
poaching. This investigation was based on fieldwork performed on the
Pechora River and its tributary, the Usa River, which began in 1994 and
continues to this day. Coregonid fish (*Coregonus lavaretus L.*, *C
albula L*., *C. peled Gmel.*, *C. autumnalis Pall*., and *C. nasus
Pall*.) were used as a test subjects and pathologies were hitologically
observed in functionally important organs. Fatty degeneration and
vacuolar degeneration of the nuclei of hepatic cells were found in the
livers of fish. Hemorrhage and necrosis were diagnosed in the kidneys.
Neoplasias (hyperplasia and tumors) were the most dangerous form of
pathology that was observed. Hyperplasia was found in epithelial cells
of the gills. Skin, which had bactericidal properties, did not change.
Tumors developed mainly in the kidneys. Pathological changes in blood
vessels and circulation of the blood were found in all of the organs and
tissues that were investigated. Infiltration properties and rapid growth
testified to the malignant nature of tumors, which resulted in dystrophy
and necrosis of the tissues of fish. All tumors were in the early stage
of oncogenesis and their etiology was unknown. The pathological changes
described were only diagnosed among Coregonid fish that were examined
between 1994 and 1997. It was hypothesized that these pathologies were
appearently caused by oil pollution.

**Disturbance Of Reproduction In Bottom Invertebrates And Fish
Inhabiting Polluted Coastal Waters Near Vladivostok (Amursky Bay, Sea Of
Japan)**

#### [Marina A. Vaschenko]{.underline}^1^, Valentina B. Durkina^1^, and Petr M. Zhadan^2^

^1^Institute of Marine Biology, Far East Branch, Russian Academy of
Sciences,

ul. Pal'chevskogo 17, Vladivostok 690041, Russia; ^2^Pacific
Oceanological Institute, Far East Branch, Russian Academy of Sciences,
ul. Baltiiskaya 43, Vladivostok 690041, Russia

The coastal zone of Amursky Bay adjacent to Vladivostok city is one of
the most polluted areas of Peter the Great Bay (Sea of Japan).
Contaminants (heavy metals, oil hydrocarbons, pesticides etc.) have
accumulated in surface bottom sediments. Bottom invertebrates and fish
inhabiting bottom layers of the water column come into direct contact
with these contaminated sediments and have been negatively impacted by
such pollution. We studied the reproductive function in two common
species of bottom invertebrates (the sea urchin *Strongylocentrotus
intermedius* and the scallop *Mizuhopecten yessoensis*) and the barfin
plaice *Pleuronectes pinnifasciatus* inhabiting Amursky Bay. In *S.
intermedius*, reproductive function was examined using gonad index,
gonad maturity index, frequency of pathological changes and heavy metal
contents in the gonads, and quality of offspring (number of normal
embryos and larvae and growth of larvae). In the sea urchins in the
"near city" zone, severe anomalies were observed in the gonadal and
offspring development. There was decreased gonadal maturity, a high
frequency of histopathological changes in the gonads, increased
concentrations of heavy metals, abnormal development, low growth rate
and death of offspring at the early stages development. In the scallop
*M. yessoensis* from the "near city" zone, similar disturbances were
observed. In the ovaries of barfin plaice from the inner part of Amursky
Bay, a mass destruction of the large vitellogenous oocytes was observed
during the spawning period. This process was accompanied by degeneration
of follicular epithelium; thus, follicular epithelial cells did not
participate in oocyte resorption. This phenomenon is evidence of the
loss of an important link in the process 'oogenesis -- oocyte
maturation'. We concluded that ecological conditions in the "near city"
zone of Amursky Bay were greatly unfavorable for the reproduction of
bottom invertebrates and plaices. Increased concentrations of heavy
metals and DDT in bottom sediments and in the gonads apparently are the
factors that negatively impacted animal reproduction.

**Tumors In Fish From Peter The Great Bay, Sea Of Japan**

[^1^Iraida G. Syasina]{.underline} and ^2^I. -S. Park

^1^Institute of Marine Biology, Far East Branch, Russian Academy of
Sciences, Vladivostok 690041, Russia; ^2^Division of Ocean Science,
Korea Maritime University, Bussan 606791, Korea

A total of 2,000 fish belonging to 20 species commonly found in Peter
the Great Bay (Sea of Japan) were examined for neoplastic lesions
between 1995 and 2001. Tumors were detected in 60 individuals
representing six species: black plaice *Pleuronectes obscurus*, striped
flounder *P. pinnifasciatus*, longsnout flounder *P. punctatissimus*,
Pacific needlefish *Strongylura anastomella*, sculpin *Myoxocephalus
yaok*, and gizzard shad *Konosirus punctatus*. Only in three species,
*P. obscurus, P. pinnifasciatus,* and *S. anastomella*, did morbidity by
neoplasm develop a permanent character; tumor occurrence in the
remaining species was sporadic. Tumors of skin, liver, and gills were
detected. Among these neoplasia, eight different tumors were classified
histologically: epidermal papillomas, rhabdomyosarcomas, hepatocellular
adenomas, scirrhous carcinomas, chondromas, chondrosarcomas,
mucoepidermoid carcinomas, and guanophoromas. The most frequently found
tumors in fish from Peter the Great Bay were from the skin (4%) and
gills (8%) of flatfish from Amursky Bay. Skin tumors were a complex of
two heterogeneous tumors (epidermal papilloma and rhabdomyosarcoma) and
Х-cells. Despite more than 30 years of studing Х-cells and Х-cellular
lesions in fishes, the etiology of such pathology remains unknown. Gill
tumors in flatfish resulted from the growth and neoplastic
transformation of cartilage, which sometimes exhibited malignancies in
adjoining tissues and metastases in other organs. Microscopic parasites
of unknown etiology were found in gills that had neoplastic alterations.
The parasites were localized in connective tissue or cartilage of
filaments. A dense connective tissue capsule formed around the parasites
and became large enough to deform gill filaments and lamellae. Cartilage
grew around the encapsulated parasites and in the injured areas, which
gave rise to tumors that were classified as chondromas and
chondrosarcomas. When only one parasite was present in the gills, growth
of cartilage occurred in several places and not just around the
parasite, itself. In *P. punctatissimus*, which displayed such
pathology, a tumor metastasis was detected as a nodule in the kidney
that consisted of mature chondrocytes.

Endocrine Disruption in Aquatic Environments: Effects on Fish
Populations

[Vicki S. Blazer]{.underline}

U.S. Geological Survey, National Fish Health Research Laboratory, 11700
Leetown Road Kearneysville, WV 25430 USA

It has been recognized that the endocrine system of animals is affected
by many environmental contaminants, often at very low concentrations.
Wildlife, as well as human, health effects have been demonstrated as a
result of exposure to these contaminants. The endocrine system is a
multi-organ, multifaceted regulatory system that influences
reproduction, embryonic development, growth, behavior and survival.
Hormones are produced in an endocrine organ and move from their site of
production into the bloodstream, usually bound to plasma proteins. There
is a complex system of receptor-mediated feedback mechanisms that
control release of these hormones. Once in the bloodstream the
bioavailability of hormones is controlled by several factors, including
plasma or tissue concentrations, sequestration by binding proteins,
clearance and hepatic metabolism. Once hormones arrive at the target
cells, they cross the cell membrane and elicit actions by binding to
specific protein receptors. The receptor-hormone complex then binds to
chromatin in the cell nucleus, stimulating the synthesis of specific
RNAs and proteins. Endocrine-disrupting effects can occur along many
sites of this complex, regulatory system. The rate of steroid excretion
and biotransformation is also important to the regulation of hormone
levels. A wide range of substances, both natural and synthetic, has been
reported to be potential endocrine disruptors. These substances either
interfere with the production, release, action or excretion of hormones
or act as hormone mimics. Much of the research in this area has focused
on the reproductive system. Biomarkers for reproductive effects include
alterations in secondary sex characteristics (morphological changes),
alterations in gonadal development and intersex (cellular changes),
alterations in circulating sex steroid levels and induction of
vitellogenin production by males (biochemical changes). It is believed
these changes can and do translate to impaired reproductive capabilities
and hence, population effects. Two other hormone systems that have begun
to receive attention are the thyroid (thyroxine T~4~ and
triiodothyronine T~3~) and the adrenal (glucocorticoids or stress
hormones). These systems are involved in intermediary metabolism and
hence growth, metamorphosis, osmoregulation and immunity. The
differences between endocrine systems of mammals and fish, difficulties
in assessing endocrine disruption and examples of studies in fish will
be presented.

# **Myxosporidia And Microsporidia From Salmon On Kamchatka**

### Irina V. Karmanova

Kamchatka Pacific Research Institute of Fisheries and Oceanography,

18 Naberezhnaya Street, Petropavlovsk-Kamchatskiy, 683000, Russia

Parasitological investigations were conducted within the five reservoir
watersheds on the Kamchatka peninsula and in the Karaginsky Gulf of the
Bering Sea. Five species of salmon belonging to the genus *Oncorhynchus*
(Walbaum) were examined. These included chum salmon (*O. keta*), pink
salmon (*O. gorbuscha*), sockeye salmon (*O. nerka*), Chinook salmon
(*O.* *tschawytscha*) and Coho salmon (*O. kisutch*). Altogether 1,110
fry and 1,628 adult salmon were examined parasitologically from 1990
through 2001. Six species of myxo- and microsporideans were observed:
*Henneguya zschokkei, Myxosoma dermatobia, Myxobolus arcticus,
Chloromyxum wardi, Zschokkella orientalis, Leptotheca krogiusi*
(*Myxosporea*) and *Pleistophora* spp. (*Microsporea*). *Henneguya
zschokkei* formed ugly cysts in somatic muscle that spoiled the economic
value of affected fish. It was found predominantly in Coho salmon, which
had prevalences of infection from 1.7 to 20% in different years.
*Myxosoma dermatobia* infected scale pockets of coho and chum salmon
with prevalences from 6.7 to 13.3%. This parasite caused multiple, small
dermal lesions in the skin that affected market values. *Myxobolus
arcticus* was a nonpathogenic parasite of the central nervous system
that ranged in prevalence from 33.3 to 80% in sockeye, 13.3 to 20% in
Chinook and 13.3 to 40% in Coho salmon. It was also found in freshwater
smolts at a prevalence of 33.3-36.7%, 6.7-10% and 6.7% in the same three
species, respectively. *Chloromyxum* *wardi, Z. orientalis* and *L.
krogiusi* were observed in chum and Coho salmon. The first two species
were found in the gall, and the last species was observed in the urinary
bladder at prevalences of 6.7 to 20%. They were not considered to be
pathogenic. The microsporidians *Pleistophora* spp. Were found in chum
and pink salmon in spawning reservoirs with prevalences ranging from
24.6 to 43.7%. It was found in pink salmoin in the Karaginsky Gulf of
the Bering Sea. Individual meronts, sporonts, sporoblasts and spores of
*Pleistophora* sp*.* were found in the kidneys of some fishes without
visible pathology, and also in fish with superficial ulcers. A lot of
plazmodias at different stages of development and mature spores were
found in fishes with tumors or among those affected with Vibriosis.

**Whirling Disease in the United States: A Shifting Perspective in the
21^st^ Century**

# Christine L. Densmore

United States Geological Survey, Leetown Science Center, National Fish
Health Research Laboratory, 11700 Leetown Road, Kearneysville, WV 25430

Whirling disease, caused by the myxosporean *Myxobolus cerebralis*,
affects a number of salmonid fishes worldwide. In the United States, *M.
cerebralis* has been recognized as a salmonid pathogen since the 1950's.
As whirling disease spread within the United States during the latter
20^th^ century, much research focused upon the means of dissemination,
fundamental host-parasite interactions, potential methods of control and
treatment, and elucidation of the parasite's life history. Because
whirling disease was perceived to be problematic predominantly, if not
exclusively, among populations of cultured salmonids, management
techniques designed to reduce *M. cerebralis* transmission were
developed and utilized among hatcheries. With the advent of these
largely effective control measures, the associated level of scientific
interest in whirling disease diminished. In the mid-1990's, however,
population declines among wild rainbow trout in regions of the
intermountain West. were associated with the occurrence of whirling
disease. This discovery brought about a resurgence of scientific
interest and correspondingly widened the scope of research efforts. Over
the past decade, relatively novel avenues of whirling disease research
include ecological and epidemiological approaches such as those that
incorporate risk assessment and dynamic modeling tools to aid in our
understanding of the occurrence of whirling disease in the natural
environment. Molecular, genetic, and immunological techniques are also
being increasingly incorporated to advance evaluations of disease
pathogenesis and host immune responses as well as to recognize taxonomic
differences in salmonid and oligochaete host susceptibility. At the
National Fish Health Research Laboratory, a team of investigators has
participated in whirling disease research for approximately six years.
Specific investigations have examined species and strain-based
differences in salmonid host susceptibility, physiological and
immunological responses of rainbow trout to infection with *M.
cerebralis*, environmental factors associated with infection of the
intermediate host, and development of molecular techniques for
evaluating infection status and host susceptibility.

**Myxosporian-Induced Alterations In Kidneys Of Siberian Roach (*Rutilus
rutilus lacustris)* From Lake Baikal**

[Marina D.-D. Badmaeva ^2^]{.underline}, Pronin N.M.^2^, Pronina S.V.^1^

Buryat state university^1^, The institute of general and experimental
biology SB RAS^2^

This study reports the occurrence of five myxosporean species from the
kidneys of Siberian roach (*Rutilus rutilus lacustris)* in Lake Baikal
(Chiviyrkuy Bay). *Myxidium rhodei* was localized in renal corpuscles,
but rarely in the interstitium. In single infections, the parasite
occurred in proximal segments of nephrons. The prevalence of infection
was 71.4-100%; intensity was 3-15,070; and abundance was 40-2,209.
Infection was characterized by hypertrophy, associated with atrophy of
surrounding renal parenchyma. The glomeruli were gradually compressed
and the parietal layer of Bowman's capsules was modified into a thin
membrane. When very abundant (\>3,000 plasmodia), the parasite was
atypically present in the interstitium and a granulomatous inflammation
was provoked in that tissue. The parasite destroyed renal corpuscles and
reduced the volume of renal tubules. *Myxobolus ellipsoides* was located
in the interstitium, but seldom under the parietal layer of Bowman's
capsule. Prevalence of infection was 9-86,6%; intensity was 1-400; and
abundance was 2--80. Plasmodia in the interstitium were surrounded with
extra thin fibrillar capsules. Co-infection with *M. rhodei* under the
parietal layer of Bowman's capsule provoked some degeneration of M*.
ellipsoides* plasmodia*. Myxobolus muelleri* was found in surface
connective tissue membranes of the kidney. Prevalence of infection was
6-87%; intensity was 1- 52; and abundance was 1--12. The ectoplasm of
plasmodia was thin and difficult to observe and endoplasmic vacuoles
contained considerable amounts of melanin. Capsules around parasites
were absent. The parasites damaged the surface connective tissue
membrane and other structures of the kidney. *Henneguya cutanea* was
located in the interstitium of the kidney. Prevalence of infection was
7-10 % and intensity consisted of single plasmodia. The plasmodia were
large (300µm) and not surrounded by a layer of connective tissue.
Pathomorphological alterations were similar to M*. muelleri.
Spirosuturia* sp. was present in renal tubules; prevalence of infection
was 6-13% and intensity was that of single specimens. Early vegetative
stages were situated intracellularly in the epithelium of renal tubules.
Late stages were found in the lumina of tubules and caused dystrophy and
destruction of epithelial cells.

# **Parasites Of Fish In The Volga-Caspian Region: Organisms Pathogeneic For Humans And Animals**

[Lubov V. Lartseva]{.underline}, Victoria V. Proskurina

FSUE Caspian Fisheries Research Institute, 1 Savushkina Street,

Astrakhan, 414056, Russia,

Results of research conducted in 1996-2002 showed that eight
epidemiologically important helminthes are parasitic on commercial fish
in the Volga-Caspian region: *Anisakis schupakovi, Eustrongylides
excisus, Apophallus muehlingi,* *Rossicotrema donicum, Paracoenogonimus
ovatus, Pseudamphistomum truncatum, Opisthorchis felineus, and
Corynosoma strumosum*. The acanthella *C. strumosum* were noted only in
the body cavity of zander, catfish, asp, black-backed shad, big-eye
shad, Caspian shad, and sturgeon. The most widespread parasites were *A.
schupakovi* and *E. excisus* that invaded gonads and body cavities.
However, *A. schupakovi* larvae also parasitized muscles of 3.3% of the
asp, 1.6% of the bream, and 14.3% of the big-eye shad that were
examined. *Eustrongylides excisus* was found in 2.0% of zander, 11.5% of
pike, 78.6% of perch, 3.3% of asp, 5.0% of catfish and 2.1% of the
sturgeon that were examined. The trematode metacercaria of *A.
muehlingi* invaded the muscle tissue of rudd (11.5%), vobla (1.0%),
bream (4.0%); *R. donicum* (28.0%) and zander (8.3%), but more often
they parasitized cutaneous coverings and fins of those fish.
Metacercaria of *P. ovatus* were found in muscles of tench (100%), rudd
(92.2%), bream (55.2%), vobla (77.0%), asp (73.9%), common carp (54.2%),
crucian carp (26.5%), zander (94.3%), pike (86.1%) and black-backed shad
(0.3%). Metacercaria *O. felineus* (*Opisthorchidae* family) were
revealed in muscles of tench (4.6%), bream (1.6%) and vobla (5.8%) while
*P. truncatum* (*Opisthorchidae* family) was found in rudd (21.0%),
bream (11.2%), tench (34.0%), and vobla (31.1%). Most of the
aforementioned parasites, except for the trematodes of the family
*Opisthorchidae*, rarely occur in humans. Diseases initiated by these
parasites are often difficult to determine and diagnosed inaccurately.
This necessitates a requirement for strict processing controls and
decontamination of row fish and fish products.

**Similarities And Differences In Fauna Of Parasites Of Chars In
Palaearctic (Eurasia) And Newarctic (North America)**

## 

## Tamara E. Boutorina

## The Far Eastern State Technical Fishery University, 690950, Lugovaya str. 

## 52 b, Vladivostok, Russia

Chars of the genus *Salvelinus* are widely distributed through Eurasia
and North America. In the Palaearctic there are *S. malma, S. albus, S.
taranetzi, S. leucomaenis, S. levanidovi*, *S. alpinus, S. czerskii, S.
boganidae*, *S. elgyticus, S. kronocius, S. schmidti, S. krogiusae, S.
neiva, S. andriashevi, and Salvethymus svetovidovi*. Dolly Varden,
arctic char, *S.fontinalis* and *S. namaycush* are commonly found in
North America. The similarity index of Jaccard equaled 30% for
comparisons of the parasitic composition of chars between Eurasia and
North America. Fifty-two species of parasites were identified, which
were also common to other fish that are closely related to chars. Data
suggested that parasites have been actively exchanged between chars of
Eurasia and North America and within each continent. Within the genus,
*Salvelinus*, Dolly Varden and white spotted char (an anadromous Pacific
species) are the most similar, but have diverse parasitofaunas. The next
cluster included the lake endemic chars along the Asian coast, the
Krogius char and the neiva (of Arctic origin). A third cluster was
comprised of Arctic char and lake trout, mainly from lakes. The endemic
brook trout and Schmidt char from Lake Kronotskoe in Kamchatka were more
different from all of the other species. Dolly Varden had 132 species of
parasites and its fauna was most diverse in Kamchatka, Chukotsky
Peninsula and Primorye. The poorest parasitofauna was found in *S.
malma* from Kuril Island (11 species), Alaska (8 species) and Oregon (3
species). Most parasites of Dolly Varden in Canada were found on both
sides of the Pacific. An Asian origin of *S. malma* was postulated and
most likely in Northern Asia on Kamchatka (probably in Chukotka). We
could also distinguish between northern and southern subspecies of Dolly
Varden. *Salvelinus leucomaenis* had 98 species of parasites throughout
the area. The parasitofauna of white spotted char was most diverse in
Kamchatka and Primorye. In Japan it had 33 species of parasites, which
was twice as much as reported on Dolly Varden therein. The origin of
*S.* leucomaenis was postulated to be somewhere within the Sea of Japan
basin. Arctic char had 59 species of parasites and its parasitofauna was
most diverse in Canada and the Kola Peninsula (Eastern Murman).

Health Protection Of Fish In The European North Of Russia

## Tatiana A. Karaseva

Polar Research Institute of Marine Fisheries and Oceanography (PINRO), 6
Knipovich Street, Murmansk , 183763, Russia

The Polar Research Institute of Marine Fisheries and Oceanography
(PINRO) is the principal scientific institution of the fishing industry
in the European North of Russia that studies diseases of marine and
cultured fishes. When investigations are conducted microbiologic,
histopathologic, hematological and physiologic techniques are used. In
1992-1999, mycosis of Atlantic-Scandian herring *Clupea* *harengus* L.
in the Norwegian and Barents Sea caused by *Ichthyophonus hoferi* was
investigated. The etiology, epizootilogy, mechanism and diagnosis of the
Ichthyophoniasis problem were examined. This provided information on the
impact of mycosis within the Atlantic-Scandian herring population. In
2001 -- 2003, experience gained from studying Ichthyophoniasis was
applied to diagnose disease in Atlantic salmon *Salmo* *salar* returning
to spawn in rivers of the Kola Peninsula. In 1999, PINRO also monitored
epizootics among populations of commercial ground fish in the Barents,
Norwegian and Greenland Seas. Such investigations developed biological
monitoring programs that evaluated fish health and habitat quality.
Thus, the pathological status of eight species of marine fish has been
achieved. Traditionally, research on diseases of cultured salmonids
remains important but, until now, quarantine measures have not been
enacted within the Northern region. Streptococcal and bacterial
infections caused by relatively pathogenic microorganisms, some mycotic
phenomena, alimentary diseases, and papillomatosis are commonly found in
this area. Because of the severe climate in the Northern Region,
epizootics mainly occur as chronic infections that persist for six
months. The epidemiological sources of infectious agents in this area
appear to come from natural reservoirs of pathogens in the water,
itself.

**Disease On Coral Reefs -- What Do We Know?**

[Virginia H. Garrison^1^]{.underline}, Laurie L. Richardson^2^ and
Garriet W. Smith^3^

^1^U.S. Geological Survey, Center for Coastal and Watershed Studies, 600
Fourth Street South, St. Petersburg, Florida, 33701; ^2^ Department of
Biology, Florida International University, Miami, Florida, 33199; ^3^
Biology Department, University of South Carolina -- Aiken, 471
University Parkway, Aiken, South Carolina, 29801

Diseases on coral reefs were first reported in the early 1970's.
Incidence of disease in coral reef organisms has greatly increased in
the past 30 years, as has disease-related mortality, particularly in
reef-building corals. Surveys have confirmed these observations
throughout the world's oceans. Disease is now considered an important
factor in the ecology of coral reefs. Of the coral diseases currently
being monitored worldwide, most are in the Caribbean region, but
increasing reports from the Pacific have shown this has become a global
problem. Pathogens have been identified for some diseases: aspergillosis
of gorgonians (*Aspergillus sydowii*); white plague of stony corals
(*Aurantimonas coralicida*); white pox of acroporid corals (*Serratia
marcescens*), all in the Caribbean; bacterial bleaching of *Oculina
patagonica* (*Vibrio shiloi*) in the Mediterranean and *Pocillopora
damicornis* (*Vibrio coralliilyticus*) from the Red Sea; black band of
gorgonians and stony corals in the Pacific and Atlantic (cyanobacterial
mat); and ciliate diseases of hard corals from the Great Barrier Reef.
Fulfilling Koch's Postulates has successfully confirmed the etiology of
a number of these conditions, but others remain elusive. Fungi and
bacteria have been implicated in the majority of Caribbean diseases of
coral reef organisms. What basic processes are driving the increases in
incidence and number of diseases on coral reefs? Are biotic/abiotic
interactions involved and if so, how? More questions remain than have
been answered, even for those diseases with identified pathogens.

# **Recent Observations On Aspergillosis Of Gorgonians**

### 

### Garriet W. Smith^1^ and Kiho Kim^2^

^1^ Biology and Geology Department., University of South Carolina Aiken,
471 University Parkway, Aiken SC 29801; ^2^ Biology Department.,
American University, 4400 Massachusetts Ave. NW, Washington DC,
20016-8066

## 

## 

Aspergillosis of Caribbean sea fans (*Gorgonia ventalina* and *G.
flabellum*) has been known for almost a decade and the pathogenic agent
has been identified as *Aspergillus sydowii*. Aspergillosis in the genus
*Gorgonia* has now been reported from all areas of the Caribbean and is
commonly considered in coral disease surveys. The impact of this disease
has been significant in a number of locations, resulting in population
shifts and overgrowth of competing organisms. *A. sydowii* that are
gorgonian pathogens produce sydowinol and other secondary metabolites
not produced by nonpathogenic strains. Sporogenesis does not occur in
seawater, but does at the sea surface-to-air interface. Recently, a
number of other gorgonians have been identified with aspergillosis. This
has been confirmed in species of the genus *Pseudoterogorgia* and
morphological evidence that the disease is much more widespread among
gorgonians than previously thought. Gorgonian responses to infection by
*A. sydowii* include nonspecific galling and the production of
antifungal compounds. The relative severity of infection among various
species and clones of gorgonians may be due to differences in their
ability to react to infection.

**Integrated Research Into Species Of The *Ctenophora* Family: *Beroe
Ovata* And *Mnemiopsis Leidyi***

[Lubov V. Lartseva^1^]{.underline}, Irina A. Lisitskaya^1^, Igor S.
Shchelkunov^2^,

and Svetlana Yu. Kasaeva^1^

^1^FSUE Caspian Fisheries Research Institute 1 Savushkina Street,

Astrakhan, 414056 Russia; ^2^All-Russian Research Institute of
Freshwater Fisheries Russia, Moscow Oblast, Dmitrovskiy District,
Rybnoye

Introduction of live organisms in new geographic areas can damage the
indigenous fauna. An example an incidental introduction was the invasion
and expansion of the comb-jelly *Mnemiopsis leidyi* within the Caspian
Sea. In order to curtail the expansion of *M. leidyi,* the state
introduced *Beroe ovata* from the Black Sea to the Caspian Sea.
Virological, parasitological and microbiological investigations were
conducted on members of the *Ctenophora* family *(specifically, Beroe
ovata and* *Mnemiopsis leidyi)* to guard against any detrimental
effects. Viral cytopathogenic agents and parasites were not observed in
specimens of *Beroe* that were examined. Results of microbiological
investigations showed that total bacterial counts of the Black Sea
comb-jellies and their habitats was about 10^4^ colony forming units
(cfu)/g or mL. Microbial colonization of *Mnemiopsis* in the Caspian Sea
was 1-2 orders lower than those in the Black Sea, which was attributed
to differences in salinity and chemical composition. During the
adaptation period (7 days) in mixed water, indices of quantity of
mesophilic aerobes and facultative-anaerobes of *Beroe* decreased and
averaged 3.8x10^3^ cfu/g. Decrease in microbe contamination of *B.
ovata* was related to the displacement of certain bacterial species by
others as a result of changes in salinity (from 18.0 to 12.0 ^0^/~00~)
and temperature (from 24.0 to 20.5^o^С). Qualitative composition of the
*Ctenophora* microflora and water consisted mainly of vibrios and
aeromonads in the Black Sea and of pseudomonads and acinetobacteria in
the Caspian. Indigenous species of the Caspian microflora prevailed in
microbiocenosis of *Beroe* after 7 and 12 days of acclimatization under
laboratory conditions. Caspian ichthyofauna (*Acipenseridae, Gobiiedae,
Mugilidae, Rutilus ritilus*) that were challenged with virulent and
non-virulent strains of *Aeromonas hydrophila, Vibrio alginolyticus,
Pseudomonas putida* and *Acinetobacter calcoaceticus* showed that
microorganisms isolated from *Beroe* after 7 days of adaptation were not
dangerous.

**Introduced Invasive Species: Shrimp, Shrimp Diseases, And Their
Impacts On Native And Cultured Shrimp**

[Frederick G. Kern]{.underline}

National Oceanic and Atmospheric Administration, National Ocean Service,
Center for Coastal Environmental Health and Biomolecular Research,
Cooperative Oxford Laboratory, Oxford, Maryland 21654

During the early 1980's, production of shrimp from aquaculture
facilities increased worldwide. At the same time, reports of mortalities
in shrimp aquaculture facilities were increasingly associated with newly
described viral diseases. The developing multibillion-dollar shrimp
aquaculture industry began to search for bigger and better brood stocks
to produce a more palatable and larger product. This encouraged the vast
international network of shrimp growers to exchange stocks during a
variety of life cycle stages. Such unregulated exchanges brought about a
concomitant exchange of viral agents that resulted in significant
mortalities within the shrimp aquaculture industry. The resulting
impacts of contagion within aquaculture facilities also raised concerns
about the potential impact of these newly emerging diseases on native
wild populations. The United States Joint Subcommittee on Aquaculture,
therefore, convened a work group in 1996 charged with the responsibility
to evaluate potential shrimp virus impacts on cultured and wild shrimp.
This report will review the four viral species that were targeted in
that study. These include Taura Syndrome Virus (TSV), Infectious
Hypodermal and Hematopoietic Necrosis Virus (IHHNV), White Spot Syndrome
Virus (WSSV), and Yellow Head Virus (YHV). The concerns identified by
the Shrimp Virus Work Group, as well as the current status of natural
shrimp populations and aquaculture production in the United States will
be discussed.

**Impacts Of Hypoxia On Resistance Of Penaeid Shrimp To Bacterial
Pathogens**

[Karen G. Burnett]{.underline} and Louis E. Burnett

Grice Marine Laboratory, College of Charleston, 205 Fort Johnson,
Charleston, South Carolina 29412

Levels of dissolved oxygen in aquatic production systems and natural
habitats can vary on a daily, seasonal and annual basis. Hypoxia (low
dissolved O~2~) is often accompanied by an increase in dissolved carbon
dioxide, called hypercapnia. The reaction of CO~2~ with water produces
carbonic acid that dissociates to bicarbonate ion and H^+^. As a
consequence, hypercapnia usually leads to a decrease in water pH.
Dissolved O~2~ levels are routinely monitored in well-managed
aquaculture ponds, because hypoxia can be lethal. The effects of
sub-lethal or moderate hypoxia (20--50% air saturation), although
immediately less apparent, can have dramatic consequences on animal
health. For example, moderate hypercapnic hypoxia or HH (low O~2~, high
CO~2~, low pH) significantly increased pathogenicity of *Vibrio* sp.
90-69B3 administered intramuscularly into the penaeid shrimp
*Litopenaeus vannamei*. Evidence suggests that the effects of HH on
disease resistance may be mediated by suppression of cellular and
humoral mechanisms of immune defense. In addition, the effects of HH on
disease resistance may be aggravated by physiological responses that
maintain blood gas and acid-base homeostasis in HH. We have shown that
HH reduces the rate at which live 90-69B3 are cleared from the hemolymph
of the blue crab, *Callinectes sapidus*. Preliminary data suggests that
there is a similar effect of HH in *L. vannamei*. Hypercaapnic hypoxia
decreases respiratory burst and bactericidal activity of fish
macrophages as well as oyster and shrimp hemocytes. Crustaceans, unlike
fish and mollusks, also rely on the prophenoloxidase (PPO) cascade as an
immediate humoral defense against pathogens. On exposure to bacteria or
fungi, the PPO cascade is rapidly activated to produce melanin. Melanin
and other products of the PPO cascade help sequester and eliminate
pathogens. We have now shown that levels of O~2~ and pH that occur in
the hemolymph of crustaceans held in moderate to severe HH reduce the
activity of phenoloxidase, the terminal enzyme of the PPO cascade. These
results suggest that HH may target both humoral and cellular components
of the immune system in crustaceans, crippling both immediate and long
term mechanisms of resistance to disease. (NSF-REU DBI-0244007,
NSF-IBN-0212921, USDA CSREES 99-35204-8555).

**Possible Roles Of The Enzymes Produced By The Intestinal Obligate
Anaerobic Microflora Isolated From Shrimp *Litopenaeus vannamei***

[Beverly Dixon]{.underline}^1^, Leslie Reiman^1^, Tamas Torok^2^, and
Shaun Moss^3^

^1^California State University, Hayward, California 94542; ^2^Lawrence
Berkeley National Laboratory, Berkeley, California 94704; ^3^The Oceanic
Institute, 41-202 Kalanianaole, Waimanalo, Hawaii 96795

The intestinal obligate anaerobic bacterial flora of shrimp remains
relatively unknown. Our studies have isolated several obligate anaerobic
genera from shrimp, which produce enzymes that possibly aid digestion.
Previous studies in our lab have shown that diet and environmental
factors influence the composition of the anaerobic intestinal flora of
shrimp. The purpose of this on-going research is to continue to
characterize this group of bacteria in order to determine their role in
the digestive process of shrimp. Intestinal samples from shrimp were
obtained from facilities in Hawaii. Samples were acquired from shrimp
that were maintained on different feeding regimes and cultured in
salinities ranging from 2-31 ppt. Shrimp were harvested at weekly
intervals; mid- and hindguts were excised for recovery of anaerobic
bacteria. Samples were grown in an anaerobe chamber supplied with a
mixture of 5% H~2~, 5% CO~2~ and 90% N~2~, plated on pre-reduced
anaerobically sterilized media, and incubated at 28^o^C. Cellular and
colonial morphology, Gram stain, aerotolerance testing, and biochemical
reactions were performed to identify isolates. Anaerobic bacterial
isolates also were identified by fatty acid methyl ester (FAME). The
majority of the isolates thus identified belonged to two species of the
genus *Clostridium;* *C. bifermentans* GC subgroup A and *C. butyricum*
GC Subgroup A. Enzyme analyses on the isolates were performed using the
API ZYM panel. Anaerobic isolates produced *α*-galactosidase, *α*-
glucosidase, *β*-glucuronidase, *α*-fucosidase, trypsin,
aminopeptidases, phosphohydrolase, esterases, and phosphatases. Several
enzymes were similar to those produced by shrimp for the digestion and
re-absorption of the exoskeleton. The possible roles of bacterial
enzymes in the digestive process of shrimp will be discussed.

**Microbial Diseases And Population Status Of The American Lobster
(*Homarus americanus*)**

[Richard A. Robohm]{.underline}

National Oceanic and Atmospheric Administration - Fisheries, Northeast
Fisheries Science Center, 212 Rogers Avenue, Milford, Connecticut 06460

The American lobster (*Homarus americanus)*, a crustacean of high
commercial value, occupies coastal and marine areas of the northwest
Atlantic along the northeastern coast of North America from southern
Labrador (Canada) to Cape Hatteras (North Carolina). Canadian commercial
landings reached an all-time high of 44 million kg in 1991 and declined
to 36.7 million kg by the year 2000. United States landings peaked at
39.7 million kg in 1999 and declined to 33.4 million kg by 2001. Prior
to 1973, the only recorded diseases in the American lobster were shell
disease, that was expressed as an erosion of the exoskeleton putatively
caused by several bacteria (first reported in 1937), and gaffkemia, a
bacterial infection caused by *Aerococcus viridans*, which competes for
the lobster's energy reserves and results in death (first reported in
1946). Shell disease affects the marketability of lobsters and since
1998 has become a serious problem in eastern Long Island Sound --as
prevalent as 70% in April of 2001. *Aerococcus viridans* is always
present in 5-7% of feral lobsters and sometimes causes deaths in up to
58% of pound-held lobsters. "Bumper-car disease", caused by the ciliated
protozoan, *Anophryoides haemophila,* was recognized in 1973. It results
in tissue destruction and death in up to 25% of lobsters during winter
impoundment and is found in 0.4 to 20% of feral lobsters. Three fungal
infections and one bacterial infection caused deaths in cultured larvae
and post-larval juveniles (*Halipthoros milfordensis* and *Fusarium* sp.
in 1975, *Lagenidium* sp. in 1976, and *Vibrio sp.* in *1979*). They
were not proven to be serious infections in feral lobsters. Recently two
additional diseases of adult American lobsters have emerged. From 1997
through 1999, lobsters valued at \$2.5 million died from a bacterial
disease in Maine coastal waters. It is unclear whether mortalities
resulted from two different bacteria or if there was a conflict in
classification; both *Vibrio fluvialis* and *Hyphomicrobium indicum*
were implicated. In 1998 and 1999,1.0 to 1.4 million kg of lobsters died
in western Long Island Sound from an amoeba infection multiple organs,
but especially in nervous tissues. Confirmation of pathogenicity for
this agent is complicated because it cannot be cultured; however,
several studies are in progress.

**Pathogen And Disease Issues Associated With Imperiled Populations of
Freshwater Mussels**

> [Clifford E. Starliper]{.underline}.
>
> United States Geological Survey, National Fish Health Research
> Laboratory, 11700 Leetown Road, Kearneysville, West Virginia, 25430
>
> Freshwater mussels are important contributors to the health of aquatic
> ecosystems and are excellent indicators of system health. Because they
> are filter feeders, mussels are integral components of nutrient
> cycling and certain species can filter as much as 1.25 gallons of
> water/hour per individual. Their combined numbers can have a
> substantial impact on a given body of water, for example, filtering
> nearly 100% of the total volume daily. The life cycle of freshwater
> mussels involves a vertebrate host, i.e. fish, for transformation of
> glochidial larvae into juveniles; therefore, adverse impacts on
> mussels or their hosts affect mussel survival. Unfortunately, such
> impacts have made mussels one of the most imperiled fauna in the
> aquatic environment. There are nearly 1,000 species of freshwater
> mussels in the family Unionidae. Although the greatest diversity is in
> North America (300 species), more than 70% of the mussels from this
> region are categorized as endangered, threatened or of special
> concern. Federal and state agencies have expanded mussel conservation
> efforts to include the captive propagation of imperiled populations
> within disease-free refugia. Natural mussel mortalities in rivers have
> been reported with increased frequency and it is imperative to
> implement strategies that minimize the risk of pathogen introduction
> and transmission within such refugia. This research was initiated to
> prevent such disease progression. Methods were developed to isolate
> bacteria from mussels and it was found that their bacterial flora was
> quite stable in terms of total numbers, but species composition varied
> in response to environmental changes. The ability of mussels to harbor
> fish pathogens was also demonstrated by the isolation of
> *Flavobacterium columnare* (cause of Columnaris disease in fish)
> within two species of mussels that were obtained from the Ohio and
> Holston Rivers. Laboratory studies further indicated that the mussels
> *Amblema plicata* and *Fusconaia ebena* readily served as vectors for
> the fish pathogen *Aeromonas salmonicida*. If these mussels were
> depurated in specific pathogen-free water for less than 15 d, they did
> not act as vectors. Current studies are in progress to determine the
> etiology of natural mussel mortalities and develop non-lethal
> monitoring techniques.

**Monitoring *Flexibacter-Flavaobacterium* Infection In Sturgeon**

[Natalia V. Guseva]{.underline} ^1^, Pavel P. Golovin ^2^, Nina A.
Golovina ^2^

^1^Department of Microbiology, J.N. Quillen College of Medicine, East
Tennessee State University, Johnson City, Tennessee, 37614;
^2^Laboratory of Ichthyopathology, All-Russian Research Institute of
Freshwater Fisheries, Rybnoe, Dmitrov Region, Moscow province, 141 821
RUSSIA

During the last decade, sturgeons became major objects of interest in
Russian fisheries. Restoration, food marketing, and sport-fishing
industries induced growth of many hatcheries increase and increased
total production in different regions of Russia. High-density
industrial-type facilities that often use power plant cooling waters
were predominantly used for such culture. This study was conducted at
one of the largest fish hatcheries in Russia, the Konakovo Sturgeon
Center, which concentrated on freshwater sturgeon species such as
Siberian sturgeon (*Acipenser baeri*), starlet (*A. ruthenus*) and their
hybrids. Analysis of fish production over the decade showed the high
level of mortality (up to 70 % loss of fry) in young sturgeons during
the spring and summer months. We performed microbiological monitoring
that showed the presence of bacteria in the water, mucus, gills, and
kidney samples of affected fish. The number and prevalence of
*Flexibacter-Flavobacterium* bacteria in the samples were correlated
with water temperatures. In the absence of treatment, infections became
septic. *Flavobacter johnsonae* was the predominant microorganism and it
was isolated from 80 % of the kidney simples as a pure cultures.
Virulence of the isolates was confirmed using challenge experiments on
bacteria-free fish of the same species. About 50-60 % of the fish became
infected via immersion challenges. In all cases symptoms of disease were
similar to those that were observed among clinically ill fish at the
hatchery and *F. johnsonae* was re-isolated as a "pure" culture.
Oxytetracycline and Chloramine T treatments were recommended and such
treatments effectively decreased (up to 100 times) the number of
bacteria present in the water and on the gills of sturgeon. In addition,
*Flexibacter-Flavobacterium* bacteria were totally eliminated from
internal organs of the affected fish.

Bacterial Diseases Of Herbivorous Fishes In Belarus

Evelina K. Skourat, Valentina A. Sivolotskaya, [Raisa L.
Asadchaya]{.underline}

Fish Industry Institute, National Academy of Sciences, 22, Stebeneva
Street., Minsk, 220024, Republic of Belarus

Enhancement of aquaculture production in Belarus under the current
economic situation is possible only by increasing the share of
herbivorous fish culture. Investigations conducted in fish farms showed
that during bacterial epizootics in carp, *Cyprinus carpio* L., other
herbivorous fishes grown in polyculture with carp were also infected.
When pseudomonad infections occurred under wintering ponds conditions in
February and March, clinical disease symptoms in grass carp
*Ctenopharyngodon idella* were identical to those in carp. These were
somewhat different in bighead carp *Aristichthys nobilis*, and
manifested itself as whitish cover on gills and petechial hemorrhages in
fins. Pathogenic *Pseudomonas fluorescens* was isolated from all of the
diseased fish (carp, grass carp and bighead carp). To treat bacterial
infections during the winter period, we modified a method that directed
the formation of bacterial plankton in ponds. A biological preparation
containing a live bacterial culture of *Azotobacteraceae* was used as a
bacterial fertilizer before fish were released into the ponds. After
introduction in ponds, these bacteria grow well due to their high
ecological plasticity and resistance to changes in environmental
conditions. Introduced azotobacteria occupy a dominant position in pond
bacterial plankton (about 30%) and suppress the growth of opportunistic
bacteria like pseudomonads and aeromonads. The treatment reduces
contagion, thereby decreasing the severity of bacterial disease.

**Characterization Of *Aeromonas Salmonicida* subsp*. salmonicida*
Isolated From Salmon Spawned In Hatcheries On Kamchatka**

[Nataliya V. Sergeenko]{.underline} and Elena A. Ustimenko

Kamchatka Pacific Research Institute of Fisheries and Oceanography, 18
Naberezhnaya Street, Petropavlovsk-Kamchatskiy, 683000, Russia

The bacterial pathogen of Pacific salmon *A. salmonicida* subsp.
*salmonicida* was originally isolated on Kamchatka in 1989, from chum
salmon (*Oncorhynchus keta*) of the Bering Sea. In 1990, the bacterium
was also isolated from Coho salmon (*Oncorhynchus kisutch*) in the river
Bistraya. Clinical signs of furunculosis were evident in both cases.
Since 2000, bacterial control was developed for captive salmon spawners
at hatcheries, because the pathogen can be transmitted via surface
contamination of fertilized eggs. Five incidents of carriers of *A.
salmonicida* subsp. *salmonicida* among spawning salmon populations were
detected at two hatcheries in 2001-2002. One strain was isolated from
chum salmon from river Paratunka and four strains were obtained from
chum salmon and sockeye salmon (*Oncorhynchus nerka*) from the river
Bistraya. Strains of *A. salmonicida* subsp. *salmonicida* were
identified using the АРI 20Е identification system. All of the strains
were identical and produced acid from sucrose. This latter, somewhat
"atypical" characteristic, was similar to reports of atypical strains
isolated in Japan. Our investigation collaborates the assumption of T.
Wiklund, who proposed a common origin for Kamchatka and Japanese
isolates. In order to test the pathogenicity of the strain, an isolate
from a chum salmon obtained in 2002 was inoculated into chum salmon
fingerlings by two routes; oral and perbranchial deliveries. The
pathogen was re-isolated from all dead fish and the highest mortality
was observed among fingerlings that were infected through the gills.

**Molecular Mechanisms Of Host-Pathogen Interactions Between *Aeromonas
salmonicida* And Atlantic Salmon, *Salmo salar***

[Laura L. Brown]{.underline}, E. Altman, J. Boyd, A. Dacanay, S.
Douglas, R. Ebanks, K. V. Ewart, Y. Han, S. C. Johnson, N. Mattatall, S.
Nikumb, D. Pinto, M. Reith, N. Ross, R. Singh, K. Soanes, and S. Tsoi

National Research Council Canada, Institute for Marine Biosciences,
Halifax, Canada, 1411 Oxford Street, Halifax, NS, B3H 3Z1, Canada.

We are studying the molecular nature of host-pathogen interactions
between Atlantic salmon (*Salmo salar*) and *Aeromonas salmonicida*. We
are sequencing the entire genome of the bacterium, the analysis and
assembly being carried out through the Canadian Bioinformatics Resource.
Information gained from this large sequencing project will enable us to
target potential virulence factors and other genes that may have
application as vaccines or immunomodulatory candidates. Knockout mutants
of *A. salmonicida* will help identify key virulence factors. We are
also examining the functional genomics and proteomics of the *A.
salmonicida* - *S. salar* relationship by examining expressed genes and
their protein products of both the pathogen and the host. Salmon are
challenged with *A. salmonicida* and samples of the bacterium and host
are taken at selected times during the infection. From these samples,
mRNA and proteins are isolated to determine which genes and proteins are
differentially expressed and produced during the infection process. At
the same time, immunological responses are measured. By linking genome
sequencing, functional genomics, proteomics, carbohydrate analysis, and
the immunological assays we are taking an integrated and innovative
approach to pathogenesis research.

**Immune Parameters of Common Species of Aquatic Animals**

[Irina A Kondratieva^1^]{.underline}, Аlexandra А. Kitashova^2^, Andrei
V. Kitashov ^2^

^1^ International Biotechnology Center, ^2^ Faculty of Biology;
Lomonosov Moscow State University, Vorobjevy Gory, Moscow 119992,
Russia.

Comparative investigations were made of cellular and humoral immunity in
aquatic animals from pollution-free regions of Rugozyorskaya inlet,
Kandalakshskii Bay, White Sea within navaga *Eleginus navaga* Pallas,
cod *Gadus morhua maris-albi* Derjugin, common mussel *Mytilus edulis*
L., and starfish *Asterias rubens* L. Fish were assessed for 1)
morpho-physiological parameters, 2) intensity and extensiveness of the
acanthocephalan *Echinorhynchus gadi* Müller invasion, 3) bacterial
symbionts on gills, 4) blood corpuscles concentration, erythrocyte
sedimentation rate, hemoglobin concentration, blood colored index, serum
total protein concentration and serum lysozyme concentration. Mussels
were assayed for 1) morpho-physiological parameters, 2) bacterial
symbionts, 3) hemocyte concentration in hemolymph and liver, 4) total
protein concentration and lysozyme concentration. Starfishes were
evaluated for 1) morpho-physiological parameters, 2) amoebocyte
concentration in perivysceral fluid, 3) total protein concentration and
lysozyme concentration in perivysceral fluid. The dependence of
hematological, biochemical and immunological parameters on the intensity
of acanthocephalan invasion was demonstrated. In infected fishes, the
concentration of blood corpuscles was reduced two-fold. Blood hemoglobin
concentration and blood colored index did not change. The erythrocyte
sedimentation rate decreased to 2 mm/h in the most infected fishes. New
protein fractions were found in electrophoregrams of serum from infected
fishes, including a low-molecular weight fraction (approx. 14.5 kDa).
Total serum protein concentration increased by 50% in navagas and by
almost 100% in cod; serum lysozyme concentration increased 5-fold in
navagas and 2-fold in cods. Phagocytic activity of hemocytes in mussel
hemolymph and liver began to increase with 30 minutes of stimulation by
the addition of Indian ink into the medium. Hemocytes in mussel
hemolymph increased 1.5--3-fold after injection of BCA into mussels and
3-fold after the injection of HRBC. In liver, hemocyte concentration
increased 3-fold after the injection of HRBC. Injection of various
antigens into starfishes under normal conditions increased lysozyme
concentration, whereas concentration of amoebocytes and total proteins
did not change. Lower aeration reduced amoebocyte, total protein, and
lysozyme concentrations in experimental and control groups.

**Advances in Fish Immunology: Identification and Characterization of
Natural Killer-Like Cells in Channel Catfish (*Ictalurus punctatus*)**

Jason Evenhuis, Linling Shen, Eva Bengtén, Melanie Wilson, V. Gregory
Chinchar, L. Williiam Clem, and [Norman W. Miller]{.underline}

Department of Microbiology, University of Mississippi Medical Center,
2500 North State Street, Jackson, Mississippi

Cytotoxic effector cells have been cloned from alloantigen-stimulated
peripheral blood leukocytes of naïve channel catfish. These granular
cells not only kill the stimulating allogeneic cells, but also unrelated
allogeneic targets through a perforin/granzyme-mediated apoptosis
pathway. They do not express T cell receptors α,β,γ or immunoglobulin μ
transcripts, and are also negative for markers that define neutrophils,
monocytes/macrophages, and nonspecific cytotoxic cells (NCC). For these
reasons these clonal cytotoxic cells are operationally defined as
NK-like cells. Further characterization of these NK-like cells revealed
that some possess a putative Fcμ receptor, which can participate in
antibody dependent cell-mediated cytotoxicity. These NK-like cells
differentially express novel immune-type receptors (NITR), which have
either one or two extracellular immunoglobulin-like domains. Some of the
NITR have an immunotyrosine inhibitory motif (ITIM) in their cytoplasmic
tail, whereas others contain a charged residue in their transmembrane
domain, which suggests they may associate with an immunotyrosine
activation motif (ITAM) containing accessory molecule. It is notable
that each of the catfish NK-like cell lines expresses an ITAM-containing
FcεR γ chain homolog, which could potentially be used by activating
NITRs and/or the putative FcμR. The availability of these catfish
NK-like cells lines will facilitate future functional studies in ways
not currently possible with any other fish species.

# **Vaccine Design And The Analysis Of Long-Lived Plasma Cell Production And Regulation**

[Stephen L. Kaattari]{.underline}, Erin S. Bromage, and Ilsa M. Kaattari

Department of Environmental and Aquatic Animal Health, School of Marine
Science, Virginia Institute of Marine Science, College of William and
Mary, Gloucester Point, Virginia 23062

The importance of long-lived plasma cells (LLPC) in the generation
long-term immunity and memory in mammals has received considerable
attention. Recent studies suggested that long-term immunity to
infectious agents, such as viruses, may primarily be due to the
expression of antibody from this unique class of plasma cells that
reside within the bone marrow. These cells stand in distinct contrast to
those found within the periphery, whose antibody response appears to be
primarily generated by short-lived plasmablasts. Thus, the origin of
these LLPC, their regulation within the bone marrow, and the possible
qualitative differences in their antibody product may be of critical
importance for effective prophylactic immunity (in both primary and
memory responses). Of particular importance to fish vaccinologists is
recent evidence that these same cell types and functions exist in
rainbow trout (and likely in other teleosts as well). Our work has
demonstrated that greater than 90% of the Ig-secreting cells in the
periphery (blood and spleen) are plasmablasts, while the anterior kidney
harbors a population of non-replicating LLPC. These LLPC persistently
secrete specific antibody for greater than six months (and likely
longer) whereas antibody secretion ceases in the periphery within two
months after immunization. The implications of such B cell dichotomy in
fish are profound for vaccine design. Given that long-term prophylaxis
to certain pathogens may ultimately depend on the maintenance of a
subset of B cells (LLPC) that strictly reside within the anterior
kidney, the role that the supportive matrix may play within this tissue
is likely critical to the continued antibody production by LLPC.
Further, the anterior kidney is a complex organ that not only is
responsible for hematopoiesis and lymphopoiesis, but it also possesses
essential endocrine function. This dual role of endocrine function and
as a supportive matrix for LLPC may determine the capacity of this organ
to mount an effective response during vaccination.

# **Stress In Fishes: Diagnosis And Corrective Treatment with "Piscin"**

#  

Nina E. Lebedeva, Nina A. Golovina, and [Pavel P. Golovin]{.underline}

All -- Russian Research Institute of Freshwater Fisheries (VNIIPRKh),

Rybnoe, Dmitrov Region, Moscow Province, 141821 Russia

Stress is especially important in intensive aquaculture in which fish
are subject to numerous changes in physical, biological and
environmental parameters. The goal of this study was to develop methods
that diagnose and correct the negative influences of stress in different
taxonomic groups of fishes, including *Cyprinus carpio*,
*Hypophthalmichthys molitrix,* *Oncorhynchus mykiss,* hybrid Mozambique
tilapia and Nile tilapia *(Oreochromis mossambicus x O. niloticus),*
*Pterophillum scalare* and *Acipenser baeri*. The stress conditions that
were studied included handling, hypoxia, and pH alterations. To reduce
the negative influence of stress, a patented preparation made according
to the author's technology was applied. This preparation \"Piscin\" is a
water-soluble substance extracted from the skin of carp. Piscin was used
in ultra low concentrations (10^--15^) by means of its dilution in the
water with experimental fish. Normalization of changes in physical and
biochemical parameters caused by stressful conditions was monitored in
each of the aforementioned fishes treated with \"Piscin.\" The
development of ecological methods for prophylactic treatment of stress
presents new perspectives for future investigations in aquaculture.

**Aspects Of Application Of Vitamin-Coenzyme Fungal Complex At Early
Stages Of Fish Ontogenesis**

[Svetlana M. Suprun]{.underline}, Yulia M. Parkhomenko, Georgiy
Donchenko, Iryna V. Kuzmenko

A. V. Palladin Institute of Biochemistry of NAS of Ukraine, Leontovicha,
9, Kiev 01601, Ukraine

A vitamin-coenzyme complex was developed based on selected strains of
mytosporic fungi protein-vitamin producers. The complex contains
vitamins B~1~, B~3~, B~5~, E, ubiquinone Q~10~, essential amino acids,
and microelements, which was tested to see it use could increase the
survivability of fish at early stages of ontogenesis. The complex was
applied immediately after egg fertilization and was administered in
combination with standard methods to increase egg viability using
therapeutics of dye origin. The survivability of hatched larvae
increased and fungal contamination was almost non-existent. Percent
hatch was about 100%. The benefits of the complex were also studied in
one-year carp, in which body mass of treated fish increased by 17.3%.
Immunologically, the humoral titers of natural antibodies in serum of
treated fish decreased from 4.4 to 2.9, as did the concentration of
lysocine in serum. Immunological and parasitological studies showed that
application of fungal complex did not have any negative effects on the
fish. A simultaneous decrease of complement level suggested that the
complex intensified elements of the non-specific immune system.

**Metabolic Processes In The Livers Of Carp Cultured In Warm Water**

# Irina Yu. Chernysh, Tatyana Churilova, Anna Fomenko, Yulia M. Parkhomenko, and Georgiy V. Donchenko

A.V. Palladin Institute of Biochemistry of the National Academy of
Sciences of the Ukraine, 9 Leontovicha Street, Kiev 01601, Ukraine

The effects of increased water temperature on the contents of basic
vitamins and coenzymes and on the status of metabolic processes in carp
are discussed. Experiments were conducted on two-year-old carp, which
were divided into two groups. One group was kept in ponds at 37°C and
the other group was held in similar ponds under natural conditions where
the water temperature was 25°C. Growth inhibition, skeletal deformation,
and sporadic decrease of immunity were noted in the first group of fish.
Basic vitamin and coenzyme contents and activities of key enzymes on the
metabolism in the liver of fish of both groups were studied to analyse
causes of observed phenomena. We did not find any considerable changes
in the contents of vitamins B~1~, B~2~, PP and their coenzymes.
Simultaneous decrease of choline and reduced glutathione content was
observed in the liver of fish kept at 37°C. Explicit changes in the
activity of certain enzymes were revealed. Considerable increases were
noted in the activity of pyruvatdehydrogenase and acetyl-CoA-carboxilase
complexes, whereas decreases in the activities of oxidative enzymes of
electron transport chain, particularly of α-ketoglutaratdehydrogenase,
were observed. An increase of general lipid content free fatty acids was
noted in the livers of carp kept at 37°C. These changes provide
unambiguous evidence for an intensification of lipogenesis and for the
decrease in velocity of oxidative processes in carp that were maintained
at higher temperatures.

**Influence Of Fodder Additions That Lower Lipogenesis On The
Development And survival Of Fish Maintained In Warm Water**

# 

# Yulia M. Parkhomenko, Irina Yu. Chernysh, and Georgiy V. Donchenko

A.V. Palladin Institute of Biochemistry of the National Academy of
Sciences of the Ukraine, Leontovicha 9, Kiev 01601, Ukraine

Comparative studies of metabolic processes in tissues of carp maintained
under artificial and natural conditions show that the intensity of
oxidizing processes is reduced and lipogenesis is activated by warm
water cultivation. Changes in metabolism cause diminished growth,
reduced immunity, and accumulation of fat in tissues. In this study,
certain biologically active compounds were added to the diet and
attempted to correct the aforementioned metabolic. Based on experimental
data, a biologically active fodder additive was developed that contained
a vitamin complex and other biologically active compounds (aminoacid,
microelements) that promote activation of vitamin exchange and
synergistically act upon cellular metabolism. This fodder premix (and
also some of its modified variants) had been tested, particularly within
in Uzbekistan, on various age groups of fishes under differing climatic
regions. Research has confirmed that it was efficient to use the premix
to enhance the development of fish held under various conditions. When
this premix was tested on carp that were developing under artificial
conditions, the lipid content in tissues of all age groups decreased up
to 40 % and processes of protein synthesis were activated. Periodic
introduction of the premix in forage initiated when fish are young can
significantly prolong the active period of their development. Thus the
fish\'s survival and the efficiency of aquaculture were increased
significantly. Administration of the premix enhanced the quality of
inexpensive forages and, under certain conditions; it can replace
standard vitamin premixes.

**Number range CHAPTERThiamine Deficiency Complex In Fish And Aquatic
Animals**

[Dale C. Honeyfield]{.underline}^1^, John D. Fitzsimons^2^, Don
E.Tillitt^3^, Jim L. Zajicek^3^, Scott B. Brown^4^, Perran Ross^5^,
Timothy S. Gross^5,6^, Maria S. Sepúlveda^5,6^

^1^Northern Appalachian Research Laboratory, USGS, Wellsboro, PA, 16901;

^2^Department of Fisheries & Oceans, Burlington, ON, Canada Number range
CHAPTERL7R 4A6; ^3^Columbia Environmental Research Center, USGS,
Columbia, MO Number range CHAPTER65201;

^4^Environment Canada, Burlington, ON, Canada. Number range CHAPTERL7R
4A6; ^5^University of Florida, Gainesville, FL, 32610; ^6^Florida
Integrated Science Centers, USGS, Gainesville, FL 32653

Thiamine deficiency in fish and aquatic animals has been identified in
several geographical locations. Early life stage mortality in salmonids
in the Laurentian Great Lakes and New York Finger Lakes is such a
significant problem. The mortality of swim-up fry dying from early
mortality syndrome (EMS), a non-infectious disease, ranges from 0 to
100% depending upon the lake, the salmonid species, stock within a
species, and year. The syndrome is characterized by lack of vitamin B1
(thiamine). Adult salmonids feeding on prey species (e.g. -- alewife and
rainbow smelt) containing thiaminase produce eggs low in thiamine and
fry die between hatch and swim-up. Thiamine treatment of eggs or fry is
effective, but it is only practical in species that migrate to weirs
where eggs are collected and reared in the hatchery. Since lake trout
eggs are not collected, thiamine treatment is not an option.
Characteristic thiamine deficiency brain lesions have been observed in
lake trout that survived overt mortality and in Atlantic salmon from the
Baltic Sea, where the malady is called M74. Recent data suggested that
thiamine deficiency affects neural function (visual acuity). Both
predator avoidance and prey capture were impaired in lake trout fry with
low thiamine. Data also showed that adult Coho salmon, steelhead trout,
and lake trout are dying as a result of thiamine deficiency, which has a
negative impact on sustaining adequate numbers of spawning animals.
Thiaminase in alewives consumed by salmonids is highly variable and the
cause of that variability is under investigation. Naturally occurring
thiamine deficiency appears to occur in Florida alligators foraging on
gizzard shad. The data suggest that thiamine deficiency in wild aquatic
top predators has a significant impact on sustainable reproduction.