427

WHIRLING DISEASE OF TROUTS
CAUSED BY Myxosoma cerebralis
IN THE UNITED STATES

;CT 1 0 1962
WOODS HOLE, pass. )

SPECIAL SCIENTIFIC REPORT-FISHERIES Na427

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now and in the future .

UNITED STATES DEPARTMENT OF THE INTERIOR, STEWART L . UDALL, SECRETARY
Fish and Wildlife Service, Clarence F. Pautzke, Commissioner
Bureau ofSport Fisheries and Wildlife, Daniel H . Janzen, Director

WHIRLING DISEASE OF TROUTS CAUSED BY MYXOSOMA

CEREBRALIS IN THE UNITED STATES
By
Glenn L. Hoffman, Clarence E. Dunbar, and Arthur Bradford

Special Scientific Report --Fisheries No. 427

Washington, D. C ,
June 1962

CONTENTS

Page

Introduction 1

Historical and Geographical 2

Occurrence in the United States 2

Symptoms and the course of the disease 3

Transmission and host specificity 5

Life cycle 5

Development stages in trout 8

Histopathology 10

Diagnosis 11

Control and treatment 12

Summary 13

Literature cited 13

WHIRLING DISEASE OF TROUTS CAUSED BY MYXOSOMA
CEREBRALIS IN THE UNITED STATES

By

Glenn L. Hoffman, Clarence E. Dunbai;-
and Arthur Bradford2^/

ABSTRACT

This disease has recently appeared in North America for the
first time. It appeared in a Pennsylvania trout hatchery and may have
spread from there to the Lamar National Fish Hatchery which is on
the same watershed, and from Lamar to the Kensington State Hatchery
in Connecticut in transferred fish. The parasite develops in the car-
tilage, primarily of the head of very small trout. Symptoms of black-
tail, whirling, spinal curvature and misshapen heads follow SKeletal
damage .

It was not possible to infect rainbow trout fry experimentally
with the spores although the incidence was very high in one of the
hatcheries . The development of the parasite was studied in infected
fish brought from the Benner Spring Hatchery to Leetown. In histo-
logical sections the parasite can be seen as a small multinucleate
trophosoite at 3 months after infection. The first spores can be seen
at 4 months and persist for at least 3 years. Spores can be found in
wet mounts prepared from head cartilage .

A program for control has been started, and the incidence
appears to be declining in the Benner Spring Hatchery. The spring
water reservoir was chlorinated, the ponds cleaned and potassium
cyanamide applied. Acetarsone (Stovarsol) was fed to one lot of
small trout at high concentrations with no noticed side effects, but
control of whirling disease was not determined.

This disease, apparently of central repeatedly to form a much larger organism

European origin, has more recently appeared which finally produces the spores. During the

in Russia, Italy, and the United States . Trout growth of the parasite much host cartilage is

usually become infected during the first few eroded and the skeleton weakened, resulting

weeks of feeding, mortalities ensue and most in the symptoms -- whirling, black tail, gaped

of the surviviors exhibit disease symptoms for jaws, misshapen heads and trunks. The whirl -

3 or more years. TTie spores gain entrance to ing is caused by damage to the cartilaginous

the fish, presumably through accidental ingestion, capsule of the organ of equilibrium; the black -

and the sporoplasm of the spore emerges and tail is caused by damage to the skeleton in the

migrates to the cartilage, mainly that of the region of the nerves which control the posterior

head. The very small sporoplasm, now called pigment cells,
a trophosoite, grows and its nuclei divide

- Eastern Fish Disease Laboratory, Leetown (P.O. Kearneys ville), West Virginia.

2/ Benner Spring Research Station, Pennsylvania Fish Commission, Bellefonte, Pennsylvania.

To the best of our knowledge no other
group of parasites has representatives which
develop in cartilage or bone of the host, but
among the Myxosporidia there are 6 other
species which have been so recorded. They are:
Myxobolus aegelefini from the head skeleton of
Gadus, Pleuronectes, Molva, Merluccius (marine)
(Kabata, 1957); Myxobolus dentium from the base
of the teeth of Esox masquinongy (Fantham et al.
1939); Henneguya brachyura from the fin ray of
Notropis (Ward, 1919); Henneguya sp. in the
cartilage of gill of Pomoxis (Davis, 1923); and
Myxosoma sp. in the head cartilage of Lepomis
macrochirus (Hoffman, 1961, unpub. research).
To date the pathogenicity of cartilage parasites
has been studied only in M. cerebralis infections.

During the past 3 years, we have studied
the symptoms, the cause, and have attempted to
infect fish experimentally to study methods of
control and treatment. Our results are here
incorporated in a review of the subject.

HISTORICAL AND GEOGRAPHICAL

The parasite and disease were first
described as Lentospora cerebralis by Hofer
(1903) in Germany. His associate, Marianne
Plehn (1904, 1924) described the disease, the
parasite, and the histopathology in greater
detail. SchSperclaus (1931, 1954) also described
the disease in detail and outlined a method of
control. Heuschman (1940, 1949), Tack (1951),
and Luling (1952) in Germany have also studied
whirling disease and its control. Vanco (1952)
records it from France, Kocylowski (1953) from
Poland, Scolari (1954) from Itally, Dyk (1954)
and Volf (1957) from Czechoslovakia, Uspenskaya
(1955, 1957) in trout and salmon from Russia,
SchSperclaus (1959, pers. comm.) from Denmark,
and Bogdanova (1960) in salmon from S.E. Russia.
Hoffman and Dunbar (1961) briefly reported on it
from the United States.

The chronology of the reports indicates
that the disease originated in Central Eurpoe.
In support of this hypothesis is the fact that the
brown trout, a native of Europe, may become
infected but is resistant to the disease, whereas
the imported rainbow trout becomes seriously
diseased.

OCCURRENCE IN THE UNITED STATES

Whirling disease appeared in brook trout
at the Benner Spring Fish Research Station,
Bellefonte, Pennsylvania, in 1956; one pond of
fish was affected. European trout were circum-
stantially implicated in the appearance of whirl-
ing disease at Benner Spring; sample purchases
of frozen imported table fish may have been fed
accidentally, or their viscera discarded in
streams. The epizootic became severe in the
hatchery in 1957 and 1958 at which time a
tentative diagnosis was made by Dr. S. F .
Snieszko. This was verified histologically by
Dr . E . M . Wood, Fish Pathologist, Seattle,
Washington. In the spring of 1960 an attempt
was made to eradicate the parasite by using
calcium cyanamide (1/10 lb. per sq. ft.) in the
dirt ponds as recommended by Dr. Sch^perclaus
of Germany, and chlorine gas (max. 300 ppm) in
the spring water source and small reservoir.
A few trout were killed in the small reservoir,
but it was not certainly known if this was the
source of infection for the hatchery. There was
no recurrence of the disease in 1961 fingerlings
in the hatching building. We believe the disease
is under control and can be eliminated completely
in a year or so. Whirling disease did reappear
later in some of the dirt ponds, however. As
long as the spring water supply remains unin-
fested there probably will be no recurrence of
the disease in the hatching building. We hope
the spores presumably remaining in and around
the ponds will eventually be eliminated.

The National Fish Hatchery, Lamar, Pa.,
is on the same watershed as Benner Spring and
in 1960 Mr. Fred Howard discovered the disease
there. The disease was verified by the Eastern
Fish Disease Laboratory. There were only a
few fish affected, and we believe that only one
series of ponds was contaminated. The Lamar
Hatchery plans to treat the ponds, when drained,
with calcium cyanamide. All diseased fish were
incinerated.

In December 1961, Mr. Lyle Pettijohn
diagnosed Myxosoma cerebralis disease in
fingerling rainbow trout from the Kensington
State Fish Hatchery, Kensington, Connecticut.
The hatchery personnel had noticed symptoms.

apparently of M. cerebralis disease, about a
year earlier. The source of infection may have
been in one lot of trout transferred from Lamar
to Kensington in 1959.

To our knowledge whirling disease caused
by M. cerebralis has not been verified anywhere
else in North America. Dr. R. Bangham (pers.
comm.) recalls identifying M. cerebralis at a
northern Wisconsin hatchery about 1945 but no
specimens are available for our verification .
We have seen similar whirling at three other
hatcheries but could find no M_. cerebralis and
assume that some physiological disturbance or
other disease may also cause this symptom . No
other characteristic symptoms were present in
these fish.

If the spreading of this disease is not
halted shortly, it can be expected to show up in
trout and salmon hatcheries where infected fish
are transferred and particularly those which
have earthen ponds, trout in spring water reser-
voirs or stream water supply.

SYMPTOMS AND THE COURSE OF THE DISEASE

The symptoms are thoroughly discussed by
Plehn (1904), SchMperclaus (1954) and Uspenskaya
(1957) but will be reviewed here because of the
lack of a previous English discussion. The
symptoms we have seen are identical with those
reported in Europe.

Trout may beco me infected up to one year
of age (SchSperclaus, 1954) but usually become
infected during the first few weeks of life --the
earlier the infection, the more severe the
disease because of the greater amount of carti-
lage present in younger fish. As we have
pointed out, we were not able to prove that trout
become infected by ingesting the spores, but this
should be assumed until disproven. The disease
takes the following course:

Period of "incubation". After exposure to the
spores a lapse of 40-60 days ensues before the
symptoms of whirling disease are evident
(SchMperclaus, 1954). Our experimental fish
in which we were never able to demonstrate the
parasite exhibited symptoms in 12 to 16 days.
We mention it here because there may be other

conditions which simulate M. cerebralis whirl-
ing disease. We do not know for certain whether
the disease causes mortalities during the
"incubation" period. The parasites are so small
during this period that histological verification
is probably impossible or extremely difficult and
mortalities might be attributed to other factors.

Initial symptoms. The most obvious symptoms,
tail-chasing, whirling and black tail (fig. 1),
become evident at about 40 to 60 days and may
last about 1 year. The trophozoites have in-
vaded and eroded the cartilage of the developing
skeleton. Rather large "lesions" containing the
parasites and debris can be seen in histological
preparations. The cartilaginous capsule around
the auditory -equilibrium organ behind the eye is
usually invaded. Perhaps toxins released by the
parasite (Plehn, 1904) or simply weakening of
the capsule destroys the equilibrium of the fish
to such an extent that each time it is disturbed
or tries to feed it goes into a frantic tail-chasing
whirl. This tail-chasing type of whirling differs
from the horizontal spiraliqg of the fish along its
long axis which is characteristic of a virus
disease, infectious pancreatic necrosis (Snieszko
and Wolf, 1958) and possibly hexamitiasis
(octomitiasis) (Davis, 1953). Small fish, up to
3 months of age (about 2 inches long) may become
so exhausted that they fall to the bottom and re-
main on their sides until they regain their
strength. It is during this period that mortalities
are likely to occur. Other debilitating factors
such as other parasites, bacterial or viral
diseases or malnutrition will probably increase
mortalities. During the early part of this period
the whirling symptom is at its worst, but it tends
to subside gradually until it is only rarely seen
in fish one year post -infection.

Very often the cartilage of the vertebral
column, posterior to the 26th vertebra, is
simultaneously affected (Plehn, 1904; Schaperclaus,
1954). The sympathetic nerves which control
the caudal pigment cells have their origin at about
the 26th vertebra. Apparently the weakened
skeleton at this point causes pressure on the
caudal nerves and pigment cell control is lost,
hence the black tail. The black tail tends to
dissappear earlier than the whirling symptoms.
One lot of about 4, 000 rainbows at Kensington
became infected when brought to the hatchery at

Figure 1: — Photograph of black -tail
rainbow trout caused by t^^yxosoma
cerebralis - about 3 months post-
infection. Photograph by S. F.
Snieszko.

Figure 2: — Photograph of eastern brook
trout with spinal curvature and
black tail due to >^osoTna cerebralis
infection. Photograph by S. F.
Snieszko.

6 months of age; an estimated 100 percent of
these developed whirling symptoms, but no
black tail.

Survivors' symptoms (figs. 2 and 3). Those
infected fish which were not killedby the parasite
during the early stages of the disease tend to
recover althoug^i they may be misshapen, part-
icularly in the head. The black tail and whirling
usually disappear but tlie spinal curvature and
misshapen head may reflect permanent damage.
The two most common and obvious head symptoms
are the sunken areas behind the eyes and the
permanently open or twisted lower jaw. All of
these symptoms are caused by the loss of carti-
lage during bone formation when the skeleton is
weakened and support is lost. EXjring the latter
part of the first year much of the damaged area
is filled in with an epitheloid granuloma type of
tissue which tends to proliferate in many instances
and cause secondary damage. Plehn (1904) p. 163
has an excellent illustration of such a tissue pro-
liferating from a vertebra and causing pressure
on a sympathetic nerve. Presumably these
"recovered" fish can live for a long period of
time so long as they are not crippled too badly

Figure 3' — Photograph of yearling rainbow
trout with spinal curvatvire due to
J^^QSoma cerebral is infection.

to feed. In spite of apparent recovery, however,

the spores remain in the tissues of the fish for
at least 3 years.

TRANSMISSION AND HOST SPECIFICITY

It has long been assumed that, if ingested,
the spores taken directly from infected trout
will infect other trout (Plehn, 1904; SchMperclaus,
1931, 1954). As previously discussed, we were
not able to do this experimentally at Leetown,
and to our knowledge no one else has . Therefore,
all knowledge concerning transmission and life
cycle has come from studies on material collect-
ed during epizootics.

Trout fry can be infected by exposing them
to water containing the silt, and presumably the
spores, of ponds from a hatchery epizootic
(Schaperclaus, 1931). One assumption is that
the spores are ingested accidentally, but the
possibility of an invertebrate transport host has
not been ruled out. Another assumption is that
spores are freed from infected fish when they
decompose or are crushed. Uspenskaya (1957)
has found isolated spores in various organs of
the fish and suggests that they may be carried
away from the site of infection by blood or lymph
and be deposited in other organs . If this in-
cludes the intestine, they could be shed while the
fish is still alive. However, it has not been
determined whether fish can be infected by this
method. Apparently the freed spores accumulate
in the ponds (particularly earthen ponds) and the
severity of the epizootic depends on the number
so accumulated. Small trout up to four months
are most severely affected. The disease can be
controlled therefore, but perhaps not eliminated,
by keeping the trout in spore -free water until
they are four months or more of age. Older fish
may become infected, but are usually not serious-
ly affected because ossification of the skeleton
prevents massive infection. Such fish, however,
may act as "carriers".

On the basis of epidemilogical evidence
the spores are apparently very resistant to dry-
ing, freezing, and survive a long period of time
(Plehn. 1904, 1924; Schaperclaus, 1931, 1954).
We have kept 2 vials of spores for 3 years, one
at room temperature, the other at about 6° C.
At the end of 22 months all spores appeared
normal, but at 3 years the sporoplasm has com-
pletely disappeared from 85 percent and did not
appear normal in the remaining 15 percent. We
believe they were 100 percent non-viable.

It is likely that the spores can be carried
from pond to pond or hatchery to hatchery on
boots and other equipment. Schaperclaus (1931)
found myxosporidean spores in the feces of
kingfishers at an affected hatchery and believes
that the disease can be spread in this manner.

Most myxosporidean species show varying
degrees of specificity for certain species or
closely related species of fish . They are also
usually specific for a certain organ or tissue.
Myxosoma cerebraUs is no exception- -it has
been found in rainbow trout (Salmo gairdneri);
eastern brook trout (Salvelinus fontinalis); brown
trout (Salmo trutta) and recently in salmon
(Salmo salar) (Uspenskaya, 1957), grayling
(Volf, 1957), and in Salvelinus leucomaenis,
S. malma, Oncorhynchus keta and O. masu
(Bogdanova, 1960). Rainbow trout are most
seriously affected by the disease, brook trout
somewhat less severely, and brown trout show
no symptoms at all but may act as "carriers".
Likewise, any symptom -free but infected rainbow
or brook trout may be serious "carriers". The
initial infection in a fish is always in cartilage,
but loss of cartilage and proliferation of tissue
may leave the spores outside of the skeleton in
little cyst-like structures.

LIFE CYCLE

The complete life cycle of M3rxosoma
cerebralis (Lentospora c .) has never been
determined experimentally (fig. 4). We know
of no one who has demonstrated the experimental
life cycle of any Myxosoma or related Myxobolus
or Henneguya species. These are all histozoic
parasites. Plehn (1904, 1924), SchSperclaus
(1954), and Uspenkaya (1957) assume that the
spores are ingested, the sporoplasm leaves the
spore, penetrates the intestinal mucosa and
migrates to the cartilage. However, this has
never been verified experimentally. Kudo (1930),
p. 313, reviews experimental infections and cites
two workers who successfully infected fish with
three coelozoic m yxospori deans- -Myxidium,
Chloromyxum, and Leptotheca; but none of the
histozoic Myxosoma, Myxobolus, or Henneguya
are mentioned.

The various tissue stages from hatchery
epizootics have been described by Plehn (1904)

INFECTED SALMONID CONTAINS SPORES
i^. MO TO AT LEAST 3 YRS AFTER
BEING INFECTED

SIDE

FRONT

^

SPORE RELEASED UPON DEATH
OF FISH

SPORES MAY BE FOUND IN LESIONS
IN BONE AND GRANULOMAS FROM ABOUT
k MO AFTER INFECTION TILL AT LEAST
3 YRS

SMALL FISH PROBABLY BECOMES
INFECTED BY INGESTING SPORES
— NOT PROVEN EXPERIMENTALLY

MULTINUCLEATE SPOROPLASM, NOW
CALLED TROPHOZOITE, MAY BE FOUND
IN CARTILAGE FROM J+O DAYS TO 3 MO
AFTER INFECTION

POLAR FILAMENTS EXTRUDE IN INTESTINE.
SPOROPLASM EMERGES, PROBABLY INVADES
INTESTI1S,NAL WALL AND MIGRATES TO HEAD
CARTILAGE

Figure h' — Life cycle of Myxosoma cerebralis . (Note that
experimental infection of fish has never been achieved).

and SchSperclaus (1931). In neither instance,
however, was the material from experimental
infections, i.e., the fish were not exposed to
infection under controlled conditions. SchHper-
claus (1931) held newly feeding fry in infested
ponds for 11 days and then observed them in
aquaria supplied with uninfested water. Symp-
toms of black-tail disease (tail -chasing whirl
and black tail) developed at 35-46 days. The
fish recovered in about 2 months but the infec-
tions were not verified histologically (SchSper-
claus, 1961, pers. comm.). It is not known
for certain exactly how they acquired the para-
sites .

The sporoplasm becomes the multinucleate
amoeboid trophozoite that can be seen in the his-
tological sections of cartilage from about 40 days
post infection (SchSperclaus, 1954) to 3-4 months.
The trophozoite grows and the nuclei divide and
differentiate to produce units known as pansporo-
blasts, containing 12 nuclei each which event-
ually produces 2 spores at about 4-6 months .
Spores are probably released after the death
and disintegration of the fish . However,
Uspenskaya (1957) has found spores in various
organs other than skeleton and believes that
some make their way to the outside through the
intestine during the 4-9 month phase of the
disease. Spores have been found in fish up to
3 years of age (Uspenskaya, 1957). It is assumed
that newly feeding trout fry become infected by
ingesting spores which have been released from
the skeleton and associated lesions of older fish
that have died or been crushed. However, no
one has reported on establishing the disease
experimentally by feeding fresh spores to fry
or by holding the fry in water to which fresh
spores have been added.

In an attempt to reproduce the life cycle
and to test disinfectants, we set up 47 experi-
ments in 1959. Spores were fed to 47 lots of
6-24 rainbow and brook trout each. The fish
ranged from 2 weeks to 4 months of age. In-
dividual lots of the spores were treated with
one of the following: sodium hypochlorite,
sodium hydroxide, formalin, phenol, calcium
oxide, zephiran chloride (Roccal*^), calcium
cyanamide, drying, heating, freezing, and others
(94 fish) served as controls. The aquarium
water was not changed until absolutely necessary

in order to retain the spores. Compressed air
was supplied and the aquaria cooled by immer-
sion in running 54° F . spring water . Of the
entire group of fish, 17, including two controls
in uncontaminated, but otherwise similar aquaria,,
developed whirling symptoms at 12 to 19 days
post exposure. Only one developed black tail.
Whirling (tail chasing type) alone cannot be used
as proof of JM. cerebralis infection because we
have seen whirling at two hatcheries where no
spores could be found. Presumably other con-
ditions, perhaps certain types of malnutrition,
can cause whirling. Therefore, we attempted
to verify our studies by identifying the develop-
ing stages of the parasite in histological section^
but we were not able to demonstrate any para-
sites.

In 1960 seven lots of 10-75 (total 290)
newly feeding rainbow fry were placed in
aquaria at 54° F. Suspensions containing many
spores were prepared by homogenizing infected
yearling trout heads with the Waring blendor or
macerating with mortar and pestle. This mat-
erial was added to the aquaria just after the fish
had begun to feed and the water was not changed
in order to retain the spores. Another lot was
also fed such a suspension for 6 days. All fish
were kept in the "contaminated" water for 5 to
14 days and then transferred to running spring
water. Whirling was seen in a very few at about
2 weeKs but no infections have been verified and
no symptoms persisted. From these experiments
we assume that a transport host, pond envoron-
ment, or different water condition is necessary
for transmission of the disease. Another possi-
bility for our negative results is that we may
have rendered the spores non-viable during
handling. We were not able, however, to as-
certain any damaging factors and the spores
appeared normal microscopically.

We have likewise been unable to infect
very young bluegills with the spores of a dif-
ferent Myxosoma sp. that occurs in bluegill
cartilage .

In an attempt to determine whether fry
can become infected prior to feeding, 4 lbs. of
rainbow sac -fry were kept at the affected Benner
Spring Hatchery for 10 days and then brought to
Leetown, before feeding, for rearing in uncon-

taminated water. They were observed for 5
months; except for one which developed whirl-
ing symptoms, growth and behavior were normal.
The affected fish was sectioned at 3 months of
age and the developing stages of M. cerebralis
were readily recognized in the cartilage.

DEVELOPMENT STAGES IN TROUT

Prior to 3 months (fig. 5)

Presumably the sporoplasm has made its
way through the intestinal wall and migrated via
blood or lymph channels to the cartilage, mainly
of the head. This seldom affects fish over 12
months of age (Plehn, 1904, 1924; Schaperclaus,
1931, 1954). Our attempts to infect fish experi-
mentally failed, so we could not demonstrate this
stage. Schaperclaus (1954) p. 379 (our fig. 5)
has an excellent photomicrograph of the develop-
ing trophozoite which has "eroded" a cavity for
itself in the cartilage. As near as we can deter-
mine this is from a 40-day-old fry, so the para-
site must be 40 days or less of age.

Figure ^'. — Trophozoite of ?tp:osonia
cerebralis in cartilage at about
UO days post-infection (drawn from
photo of Schaperclaus, 19$h) >

Three months (figs. 6, 7, and 8)

Our only specimen of known age is this
one. It was from one of many sac -fry brought
from Benner Spring, Pa., during the epizootic,
to Leetown for rearing in water free of M.
cerebralis. Since it must have become infected

at Benner Spring, we know the approximate age
of the parasite. At this stage the parasites are
multinucleate ameboid trophozoites and in cross
sections range from 5 x 5 to 30 x 8u in diameter;
the smaller ones are possibly cross sections of
elongate trophozoites. There are at least 18
nuclei about 1-1/2 - 2uin diameter in each tro-
phozoite. These exist in lesions measuring
about 300 X 100 u in the cartilage. Also in the
lesions are freed cartilage cells and remnants
of disintegrating cells and cartilage matrix.
Apparently in normal osteogenesis in the trout
studied, the cartilage of the skeleton in certain
places is eroded from within by vascular action
at the same time that bone is being laid down on
the outside of the skeletal structures. Blood
vessels penetrate the cartilage, and pockets of
it are eroded, presumably by enzymes released
from the capillaries. These pockets of carti-
lage erosion (fig. 9) resemble M_. cerebralis
lesions, but contain blood vessels, disintegra-
ting cartilage cells and cartilage matrix and
sometimes multinucleate host cells but no
parasites. The multinucleate cells resemble
phagocytic giant cells, but Ruth-i suggested
that they might be tissue cells that have failed
to divide.

Four months (figs. 10, 11, and 12)

The multinucleate trophozoite has grown
considerably but is still in the cartilage. Some
of the nuclei have divided rqjeatedly to form
groups of nuclei (or cells?) which are now known
as pansporoblasts. These distinct units produce
the spores, usually two each. Kudo (1960) be-
lieves that myxosporideans are distinctly multi-
cellular at this stage and, therefore, much dif-
ferent from other protozoa. Kudo (1930) and
Noble (1944) have reviewed pansporoblast form-
ation in other Myxosporidia . At this age some
of the pansporoblasts have already produced
spores. The trophosoite (entire parasite) has
probably reached its maximum size at this stage;
those measured were up to 1 mm in greatest di-
ameter although we do not Know for certain
whether two or more trophozoites may be in such
close association that their boundaries are not
discernible.

3/ Ruth, Delbert. Anatomy Department, John
Hopkins Medical School, Baltimore, Maryland,
pers. comm., 1961.

» -J.*', * •..,•»

Figure 6: — Photomicrograph of >yxosoma
cerebralis in cartilage at 3 months
post-infection (x 100). See fig. 7
for labelling.

TROPHOZOITE, '^

\». •■

r>

INTACT C^PTIL/lGE

Figure 8: — Photomicrograph of %xosoma
cerebralis in cartilage at 3 months
post-infection (x U30).

^ikbHVt"

|.— BONE

CART I LAGE
OEBR I S

TROPHOZOITES

INTACT
CARTILAGE

CARTILAGE
CELL

^ <^ (5^ C5)

Figure 1: — Drawing of I^osoma cerebralis
in cartilage at 3 months post-infection.
(Made from same slide as fig. 6) Drawn
with aid of microprojection.

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CARTILAGE

Figure 9= — Photomicrograph of normal
cartilage resorption in trout about
3 months of age. These "lesions"
grossly resemble Myxosoma cerebralis
infections .

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Figure 10: — Photomicrograph of l"^os oina
cerebralis in cartilage at U months
post-infection. Note that two spores
develop in each pansporoblast. Stained
with Giemsa's to show spores (x U30).

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Figure 11: — Photomicrograph of Myxosoma
cerebralis in cartilage at U months
post-infection. Stained with Giemsa's
to show spores (x 970).

Figure 12: — Free-hand drawing of t^osoma
cerebralis pansporoblasts containing
two spores each. Four months post-
infection.

Eight months and older (figs. 13 and 14)

At this time all of the pansporoblasts have
produced their spores, and these can be seen in
the "lesion". Apparently the rest of the para-
site disintegrates. The spores appear to re-
main in the lesion site permanently- -Uspenskaya
(1957) found them in 3-year-old fish. We have
found many spores in 2 -year -old fish and pre-
sume that they may remain much longer. It has
been assumed that the spores remain in the site
of the lesion, but she cites evidence that some of
them are transported, presumably by blood, to
other organs including the liver and lumen of the
intestine. We believe that this is not the usual
means of spore dissemination.

HISTOPATHOLOGY

There is no evidence of histopathology
during the incubation period of 40 to 60 days .
However, after this, as the trophozoite grows,
cartilage is eroded, skeletal support is weaKen-
ed, and simultaneously there is a proliferation
of epithelioid-type granuloma about the parasite
and its spores.

10

1. Forty days. Slight erosion of carti-
lage. SchSperclaus (1954) p. 379 (our fig. 5).

2. Sixty -five days. Still trophozoite
only; no tissue proliferation. Schaperclaus
(1931) p. 542, 554.

3. Three months (figs. 6, 7, and 8) .
Cartilage still being eroded by trophozoites in
a lesion -like cavity in cartilage; cellular and
cartilage debris present in lesion, bone being
formed at periphery of skeleton. No tissue
proliferation or inflammation.

4. Four months (figs. 10, 11, and 12).
Some spores now present, but still no prolifer-
ation.

5. Eight - 12 months (fig. 13). Spores
present. Trophozoites no longer present. Epi-
thelioid proliferation of fish tissue now surrounds
the mass of spores. This cyst-like structure is
sometimes referred to as a granuloma which may
cause pressure on vital organs (Plehn, 1904).

DIAGNOSIS

Before development of the spores (about
four months) the disease can be tentatively
diagnosed on the basis of the symptoms- -tail -
chasing, whirling, and black tail. Verification
can be made only by finding the ameboid stages
in the cartilage of the head in histological sec-
tion. Very early stages are more difficult to
find but at three months they are often easily
found and consist of trophozoites 5 to 30|ain
size with many nuclei about 1-1/2 to 2|Jin
diameter.

From 4 months post infection to at least
2 years, the spores can be found easily in wet
mounts or histological section. Wet material
may be prepared by dissecting out the auditory
capsule and crushing it (Plehn, 1904) or split-
ting the head lengthwise and scraping the poste-
rior part of the cranium with a scalpel to free
the spores. A more reliable method for fish,
up to at least 2 years of age, is to cut up the
head with scissors and macerate in a Waring
blendor in about 50 ml of water- -the spores can
usually be found in a random drop.

SPORE

*■!?•* # •

t%

EPITHELIOID
HOST TISSUE

c,^'

Figure 13: — Photomicrograph of Myxosoma
cerebralis spores in an epithelioid
granuloma at 12 months post-infection.
Stained with Giemsa's to show spores
(x U30).

Figure lU: — Photomicrograph of spores of
f^osoma cerebralis from rainbow trout
at 12-15 months post-infection. Stained
with Giemsa's (x 970). Photo by Dr.
E. M. Wood.

II

In histological section the early stages
(1-1/2 to 3 month post infection) can be recog-
nized by their many small nuclei in the lesions
in the head cartilage (figs. 5, 7, and 8). Le-
sion-like structures in normal bone formation
(fig. 9) resemble whirling disease lesions.
Sometimes these contain large multinucleate
host cells (giant cells) which may be confused
with parasites.

In histological section of the head the
spores stain blue with Giemsa's (figs. 10, 11,
13, and 14), blue with carbol toluidin blue
(SchSperclaus, 1931) and red with aniline water-
saffranin (Plehn, 1904) .

CONTROL AND TREATMENT

A modified version of SchSperclaus'
(1954) and Tack's (1951) recommendations for
control follows:

1. Destroy all fish from ponds contain-
ing fish known to be infected. Incineration or
deep burial is recommended.

4. Disinfection of concrete ponds . Drain
ponds and immediately apply calcium cyanamide
at about 0.04 lb. /sq. ft. (1780 lb. /acre). Allow
to stand 3-4 weeks, clean thoroughly, wet down
and repeat treatment. Some of the chemicals
listed in 3 above would probably be satisfactory
for small ponds . It would take about 10 times
as much chemical if used in water-filled ponds.
Quicklime and hydroxides are strongly caustic
chemical agents and personnel using them
should wear protective clothing and use goggles
and respirators.

5. Disinfection of earthen ponds. This
is the most difficult aspect because organic
matter usually interferes with disinfectants.
Drain the ponds and immediately apply calcium
cyanamide as in 4 above. Allow to stand a
month or more and clean out silt as thorou^ly
as possible. Haul out to a farm field for plow-
ing under or bury deeply. Fill pond with water,
drain and immediately apply calcium cyanamide
as before. Flush out and refill ponds 2 weeks
or more later and stock with fish; Tack (1951)
allowed 6 weeks before adding fingerlings.

2. Water supply. That which supplies
the hatching house fry and early fingerlings (up
to 8 months at least) should be spore -free spring
or well water. There should be no fish in the
water system before it reaches the hatching
house. Stream water may be used only if the
stream contains no fish. No satisfactory fil-
tering device for whirling disease has been
described for hatcheries in the United States,
but we have heard that sand-charcoal filters
have been used successfully in France.

3. Disinfection of hatching house. Clean
thoroughly (this is more important than chemical
disinfection). We have found that overnight soak-
ing with the following will dissolve the spores or
cause them to extrude their polar filaments and
probably render them uninfective: sodium hypo-
chlorite (commercial or home laundry bleaches)
as per instructions on container; zephiran chlor-
ide (RoccalR) 800 ppm; sodium hydroxide 1/2
percent, calcium oxide (quicklime) 1/2 percent.
It is imperative to have spore-free facilities for
fry because the younger the fish the more seri-
ously they are affected by whirling disease.

Schaperclaus (1954) recommends quick-
lime as the chemical of second choice to be
used at the same rate as calcium cyanamide.
Snow and Jones (1959) have used quicklime
successfully in treating bluegill ponds for fish
diseases. We believe that the hot reaction of
quicklime which produces a high transient pH
is effective in killing spores- -a pond so treated
is safe to use 10 days after treatment with no
flushing necessary. Sodium hydroxide is prob-
ably more effective than Lime but may be more
expensive.

6. Restocking hatchery. Eggs or fry
should be obtained from a known uncontam-
inated source. Fry should be kept in hatching
house as long as possible (3-8 months) because
it is usually easier to control the disease here.
It is advisable to maintain 2 series of ponds
until it is certain that the disease is eradicated.

The first series of ponds should be con-
crete raceways supplied with spore-free water.
Use great care not to contaminate with spores
or infected fish. Keep fish here only 3 months.

12

It takes 4 months for the spores to develop,
therefore, these ponds could not be contamin-
ated by fish as long as the hatchery house, ponds,
and water supply are spore-free. Pick off mor-
talities twice daily; incinerate or bury deeply.

The second series of ponds may be con-
crete or earthen . For best results the fish
should be 8-12 months or more of age when
stocked here. The older the fish when infected,
the less serious the disease and it is doubtful
that 13-month -old fish can be infected- -certain-
ly not heavily .

Following this routine it is expected that
some disease will show up in the last series of
ponds. If whirlers and mortalities are inciner-
ated, however, one can expect the disease to
disappear in 2-3 years. It may be necessary to
treat earthen ponds annually for 2-3 years.

7. No fish from an affected hatchery
should be transferred to an unaffected hatchery.
However, the disease probably cannot be trans-
mitted by eggs (Schiiperclaus, 1931). It must
be kept in mind that infected brown trout and
lightly infected rainbow and brook trout may
serve as carriers although they show no symp-
toms.

8 . Fish from an affected hatchery should
not be stocked in fishing waters unless there is
no other hatchery on that watershed and no
natural trout reproduction or stocked fingerlings.
It is better to destroy the infected fish than to
take a chance on spreading the disease which has
apparently spread from Central Europe to Russia
Italy, and now North America in the last decade.
Disposal or transport by fishermen of spore -
bearing fish is an ever-present threat to spread
of the disease.

Suppression of the disease, but not elimin-
ation, with Acetarsone (Stovarsol) at 10 mg per
kilogram of fish daily on 3 consecutive days with
weekly intervals between each course was re-
ported by Scolari (1954). He recommends that
it be used for 6 months for (partial) prophylaxi s.
Apparently the drug has no serious side effects
on the fish . We fed it to several thousand
rainbow trout for eleven months at Benner Spring
at concentrations as high as 100 times that rec-

ommended by Scolari (1954) . Althou^ these
Acetarsone experiments have not been completed
as of this writing, the preliminary results are
not promising. We thus believe it should be
tested further before recommending it for pro-
phylaxis of whirling disease.

SUMMARY

Whirling disease (black -tail), caused by
the myxosporidean, Myxosoma cerebralis, is
reported from the United States. It has made
its appearence at 2 trout hatcheries located on
the same watershed in Pensylvania and at an-
other one in Connecticut. Young rainbow and
Eastern brook trout were severly affected.
European brown trout were not severely dis-
eased, but probably served as "carriers".

Many attempts to transmit the disease
in the laboratory failed.

The developmental stages of the parasite,
histopathology, diagnosis, and control are dis-
cussed. The severity of the disease in the
hatcheries has been reduced, but not eliminated,
by removing fish from the water source and
treating the ponds with calcium cyanamide.

Preliminary experimaits with Acetarsone
(Stovarsol), although inconclusive, indicate that
the drug is not vary toxic to trout .

LITERATURE CITED

Bogdanova, E . A .

1960. Natural habitat of the myxospor-
idean (Myxosoma cerebralis -whirl-
ing disease) at Sakhalin (S. E. Russia).
Rep. USSR Academy of Science, Vol.
134, no. 6, pp. 1501-1503.

Davis, H. S.

1923. Studies on the sporulation and de-
velopment of the cysts in a new
species of Myxosporidia, Lentospora
ovalis . Journal of Morphology, Vol.
37, no. 3, pp. 425-542.

1953. Culture and diseases of game
fishes . University of California
Press, 332 pp.

13

Dyk, V.

1954. Nemoci y.
in our fish) .

nasich ryb. (Diseases
Praha, Cezechoslovakia.

Fanthan, H. B., A. Porterjand L. R. Richardson

1939. Some Myxosporidia found in certain
freshwater fishes in Quebec Province,
Canada. Parasitology, Vol. 31, no.

1, pp. 1-77.

Heuschmann, O.

1940. Ober die Ausbreitung der Dreh-
krankheitung in Suddentschland.
Allgem. Fischereizeitung Vol. 65,
no. 3, pp. 17-19.

1949. Die Drehkrankheiten der Salmon-
iden. Allgem. Fischereizeitung
Vol. 74, no. 12, pp. 216-217.

Hofer, B.

1903. liber die Drehkrankheit der
Regenbogenforelle . Allgem .
Fischereizeitung Vol . 28, pp. 7-8.

Hoffman, G. L., andC. E. Dunbar,

1961. Studies on Myxosoma cerebralis
(Hofer) Plehn (Protozoa: Myxospor-
idia) the cause of whirling disease
of trout. Journal of Parasitology,
Vol. 47, no. 4, Sec. 2, 29 (abstract).

Kabata, Z.

1957. Note on a new host of Myxobolus
aeglefini . Journal of Parasitology,
Vol. 47, no. land 2, pp. 165-168.

Kocylowski, B.

1953. Choroby ryb i ich knalkanie.
Gespodarka rybna 5, No. 1, pp.
24-26.

Kudo, R . R .

1930. Myxosporidia. (In Problems and
methods of research in protozoology.
Ed. Hegner, R. W. and V. M.
Andrews. McMillan Co., New York,
pp. 303-324).

1960. Symposium on taxonomy in proto-
zoology. Talk given at annual meet-
ing of American Society of Proto-

zoologists at Stillwater, Oklahoma,
September, 1960.

Luling, K. H.

1952. Die Erreger der Knotchen-Baulen
und Drehkrankheit unserer Fische.
Ostereichs Fischerei, Vol. 5, no.
8, pp. 171-173.

Noble, E. R.

1944. Life cycles in the Myxosporidia.
Quarterly Review Biology, Vol. 19,
no. 3, pp. 213-235.

Plehn, M.

1904. Uber die Drehkrankheit der
Salmoniden. [Lentospora cere-
bralis (Hofer) PlehnTJ Archiv fur
Protistenkunde . Bd., Vol. 5, pp.
145-166.

1924. Praktikum der Fischkrankheiten.
Schweizerbart'sche, Stuttgart.
479 pp .

Schaperclaus, W.

1931. XXI. Die Drehkrankheit in der
Forellenzucht und ihre BekSmpfung.
Zeitschrift fur Fischerei. Bd. Vol.
29, pp. 521-567.

1954. Fischkrankheiten. Akademie-
Verlag, Berlin. 708 pp.

Scolari, C.

1954. Sull'impiego dello Stovarsolo
nella profilassi del "capostorno" O
"lentosporiasi" delle trote d'alleva-
mento. CUn. Veterin., Vol. 77,
no. 2, pp. 50-53.

Snow, J. R.,andR. O. Jones.

1959. Some effects of lime application
to warm -water hatchery ponds.
Given at 13th Annual Meeting S.E.
Association of Game and Fish
Commissioners, Duplicated, 15 pp.

Tack, E.

1951 . Bekampfung der Drehkrankheit
mit Kalkstickstoff. Fischwirt. Nr.
5, pp. 123-130.

14

Uspenskaya, A . V .

1955. Biology, distribution and eco-
nomic importance of Myxosoma
cerebralis, the causative agent of
the ovist disease of trout. Lec-
tures of the Academy of Science,
USSR. Vol. 105, no. 5, pp. 1132-
1135.

1957. The ecology and spreading of the
pathogen of Myxosoma cerebralis
(Hofer, 1903; Plehn, 1905) of trout
in the fish ponds of the Soviet Union.
(In Parasites and Diseases of Fish,
ed. byG. K. Petrusheveski. Vol.
42, Bulletin, Institute of Fresh-
water Fisheries. English Trans-
lation--Office of Technical Services,
U.S. Department of Commerce,
Washington, pp, 47-55).

Snleszko, S. F. and K. Wolf.

1958. Infectious pancreatic necrosis of
salmonid fishes . U.S. Fish and
Wildlife Service, Fishery Leaflet
453.
Vanco, R.

1952. Contribution a I'etude de la path-
ologic des alevins de truite. Paris,
92 pp.

Volf, F.

1957. The first cases of the whirling
disease among our salmonid fish in
our hatcheries. Sbornik Ceskosloveske
Akad. Zemedelskych ved. Zivocisna
Vyroba. Rocnik 2(XXX), pp. 425-428.

Ward, H.

1919. Notes on North American Myxo-
sporidia. Journal of Parasitology,
Vol. 6, no. 2, pp. 49-64.

15

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