ae y3 STON a
Ry ents

Sas
8
“9

~
3
Se
oe)

SPOROZOON PARASITES OF CERTAIN FISHES IN
THE VICINITY OF WOODS HOLE, MASSACHUSETTS
By C. W. Hahn

From BULLETIN OF THE BUREAU OF FISHERIES, Volume XXXIII, 1913

Pocumentie NG hk GlO ume ree me Ge a eh See GS 2 Sine Issued April 29, 1915

——_—_—_——————————O————————————————————————
WASHINGTON : : : : : : GOVERNMENT PRINTING OFFICE : : : : : : : : ¢ 1915

Menegraph

ADDITIONAL COPIES

OF THIS PUBLICATION MAY BE PROCURED FROM
THE SUPERINTENDENT OF DOCUMENTS

GOVERNMENT PRINTING OFFICE
WASHINGTON, D. C.
AT
10 CENTS PER COPY

Vv

“
OS 2.
oS

SPOROZOON PARASITES OF CERTAIN FISHES IN THE VICINITY
OF WOODS HOLE, MASSACHUSETTS

ad

By C. W. Hahn

IgI

CONTENTS:

*
Page.

Oecurrenceiof disease. 275... oie an cteereiaete cle ele cielo eerola ne eiiseGertcre mince eine ee et ebsites 193
Methods iofistid y-fi3 4. < 1 . 2 wieder. ALE Se. TES. See Se Se BES. Se ae eee 194
Eexperimertits to determine character of infections. <i .sae 1 ones cnele sicieisis co eniaia sistent 195
Pathological condition of the tissuesss.,. sac eoattaceeg. ocutectte tie eee eee TES eee cael ence 197
Bacteria associated with atrophied tisstiestcsn... case asic eee ae in Oil teis oe ere 199
Sporozoalassociated with} atrophied tisstles ina wtianae case otf lerctsteeetetePteie resales siele ie ow tkert ies eteietete ln 201

Stagesiof Myxobolus musculi...... sos Pa Ae Pe cre ceva Pes cara vo Seats avs Slottiaems aloe eros ereeineteless 202

Chloromyxum funduli..... EAA Hoe Cao cA, SCOR aor MOO OMe ASO OOS AS00000 6 205
Protozoa:related to those here:described IeseMe 2.x. 8 x ards pte oie 5s wia:s vitve isso evens = mints aa eleiareee rae 206
Prevalence.of myxospordian infection ta fskujss.s cleric acme icleeicts eicteele ie siciete eiaiore clei vere eee eee 208
General! comelusions 03725, shea stce oan Oe St eee PORTE oe ee See enact iets ea cvele CL MCT Ae Reece 209
Bib liogralp lays rf resect A ctevct Stoves ove 5 biased upe ceil ate tet earn Menai sic tanet a aes See oes tages occ A otal aot ieee eet ae 210
Explanation ofiplates so chins. si. ceips cic opiase oie eieerise enisttoie cieieteac treiereneichete leet aie etc roars 212

192

SPOROZOON PARASITES OF CERTAIN FISHES IN THE VICINITY
OF WOODS HOLE, MASSACHUSETTS.

a
By C. W. HAHN.
od

While studying the Sporozoa in different species of fish at Woods Hole, Mass., in
1909, the myxospore of one was observed in diseased killifish, Fundulus heteroclitus
and Fundulus majalis. Additional material was obtained and some special experiments
were carried out during the seasons of 1910, 1911, 1912, and 1913, the United States
Bureau of Fisheries providing the facilities for this and other similar studies at its
Woods Hole biological laboratory. @

OCCURRENCE OF DISEASE.

When a number of Fundulus of either of the common species (hetferoclitus or
majalis) are confined in aquaria for a few days during the warm season, one or more
thickened white or pink areas appear upon the integument of some of the fishes. The
scales of these patches are more or less loosened. They increase in size and number, and
the number of afflicted fishes also increases. The fins when involved become bloody and
the fin-rays are exposed. Elsewhere the integument disintegrates and the flesh is laid
bare. Considerable excavations into the body muscle are not uncommon. The largest
cavity of this kind observed was in the head region, measuring about ro to 12 mm. in
diameter and 2 to 3 mm. in depth. Such excavations expose large areas of the skull.
When other parts are attacked, loss of blood or penetration of vital parts causes death
before the lesion becomes conspicuous externally. The integument is thickened around
the sores where the scales are loose. Its color is pink or white. The scales fall out at
the edge of the sores. The caudal fin may be completely removed, also the flesh and
integument of the tail, thus exposing the vertebre, before the fish succumbs to the
disease. Fish frequently give evidence of weakness and depression even before the
flesh has been exposed. There is nothing peculiar about the locomotion except a dimin-
ished activity. In certain cases where there is conspicuous inflammation of the integ-
ument, especially under the head, the fish may be observed to dart downward, and,
with a slight rotation or twist of the body, to scrape the ventral or lateral portion of
the head upon the bottom of the aquarium. The fish slowly lose strength, the smaller
ones first, and the larger ones not until they are greatly mutilated. Apparently all
afflicted fish die unless special care is given to cleanliness, water, and food.

The proportion of fish that are diseased when caught has not been ascertained.
The ratio of those that develop integumentary sores in the first day or two to those
that are healthy depends to a great measure upon the injuries received in handling the

@ Valuable assistance from Dr. Edward Linton and Mr. Vinal E. Edwards is gratefully acknowledged.
193

194 BULLETIN OF THE BUREAU OF FISHERIES.

catch. Sometimes 50 per cent of the Fundulus that have been roughly handled, as
when stripped for eggs, will become diseased in 24 hours. Of these, half may be dead
within 12 hours. If a few crabs happen to be confined in the same aquarium with a
large number of Fundulus, they inflict injuries upon practically all the fish and all are
soon diseased. Uninjured Fundulus develop the disease infrequently. (See p. 196.)
Roughly speaking, 3 to 4 per cent of the Fundulus that are brought into the laboratory
at this season (July and August) and confined in small aquaria having but a liter or
two of water for each fish, will be found diseased within two days. Within another
day or two some of these fish die and a large number die in the course of a week. Dis-
eased Fundulus are therefore almost constantly available.

METHODS OF STUDY.

Both fresh and preserved tissues were examined microscopically, the method of
handling the tissues being as follows: The scales having been removed with forceps,
the edge of a slide is drawn over a diseased area with a little pressure, and the mucus
and cellular material thus obtained is spread evenly over the surface of another slide;
or, a portion of integument or muscle which has been removed with a scalpel is ground
between two thick slides by giving to the upper slide.a circular motion. It is necessary
to use considerable pressure, and at times cut tough fragments with the sharp edge of
the upper slide. Under these conditions, both slides may be preserved for observation
and still others made from the ground-up material. Some of the smear preparations
made in this manner were examined while fresh and others were fixed and stained.
Altogether about 85 fish were examined microscopically. Fresh smears which were
sometimes supplied with bile and serum were sealed with vaseline, and could then be
examined from time to time, during a period of 24 hours.

For sectioning, tissues were fixed in a saturated solution of corrosive sublimate in
35 per cent alcohol with 0.2 per cent acetic acid and 6 per cent formaldehyde; also in
the ether-formalin-alcohol mixture given below. Some of the smears were fixed in the
same sublimate mixture; others in a solution of corrosive sublimate in 2 parts abso-
lute alcohol and 1 of ether; still others in a mixture of absolute alcohol (60 per cent),
ether (35 per cent), and strong formaldehyde (5 per cent). The mercury preparations
are stained with a modification of Mayer's hematein. (See Hahn, Archiv ftir Protis-
tenkunde, bd. xvit, no. 3, p. 316, footnote.) Usually the alcohol and formalin prepa-
rations are stained in methylene blue or Giemsa’s stain. The methylene blue was
extracted in a saturate alcoholic (7o per cent) solution of both eosin and orange G.
The Giemsa was washed in water, allowed to dry, and decolorized in carbol-xylol,
without the use of alcohol. Some of the more recent preparations fixed by either of
the above fluids have been more successfully stained by first treating with hamatein
for several hours, then decolorizing in 70 per cent alcohol with 1 per cent HCl, returning
through the alcohols to water, and staining in methylene blue or toluidin blue. After
dehydration they were left in a contrast stain (eosin and orange G) for a few minutes
and rapidly run into 95 per cent alcohol, carbol-xylol, and two changes or xylol. Both
smear preparations and sections are mounted in Canada balsam without cover glasses.

Searching is most satisfactorily carried on with an ocular of 1 inch and an objective
of one-fifth inch focal distance. A one-twelfth inch oil immersion objective combined
with the same ocular for ordinary observation is supplemented, when occasion requires,
with a no. 2 compensating ocular. The one-fifth inch objective is not too high to be

SPOROZOON PARASITES OF FISHES. 195

used without a cover glass and reveals most of the details necessary to recognize the
presence of Protozoa or other unusual histological conditions. A mechanical stage is
indispensable.

Three organisms are involved in most of the Fundulus ulcers, rarely a fourth. A
thick, short bacillus is the most abundant. A long, slender bacillus is less common.
The Sporozoa are represented by a species of Myxobolus, and in one case a species of
Chloromyxum. From the evidence in the following account it will be learned that the
primary attack upon healthy tissues, in a certain proportion of the diseased fish, is prob-
ably made by the long bacillus. At least a few and probably many of the diseased fish
are primarily attacked by Myxosporidia. The short bacillus is more or less incapable
of rapid growth in living cells of any kind. While it is not within our province to make
an exhaustive study of the fungus diseases, it has been necessary to ascertain to what
extent they participate in bringing about these pathological conditions.

EXPERIMENTS TO DETERMINE CHARACTER OF INFECTION.

The following experiments were carried out in order to gain some accurate infor-
mation as to the conditions whereby healthy fish are infected and the possibilities of
their recovery. At the time it was not possible to discriminate between fish that were
infected by a fungus and those that were infected by a sporozoén. It will be apparent
that the experiments are not vitally affected by the kind of parasite present.

Forty fish were divided equally and placed in two 5-gallon aquaria. These fish
had been seined in the usual manner and brought to the laboratory on board the steamer
Phalarope in large milk cans. The trip from the collecting grounds (Menemsha Bight)
usually requires about one and one-half hours. The cans accommodate from 200 to
300 fish each. A hose supplies them with fresh water. The 4o fish used in this case
were examined carefully and found to be free from all visible integumentary disturbance.

First stage-—Aquarium no. 1 was carefully cleaned and sterilized. Aquarium no. 2
had contained diseased fish, and 2 diseased fish were allowed to remain with the 20
fish used in the experiment. Contaminated fish from other sources were always kept
in this jar. Both groups were fed about every 48 hours. After a period of 11 days
none of the fish in the clean jar showed any signs of disease. From this fact we con-
cluded that they were free from the disease and suitable for experimentation of a dif-
ferent kind. After the same period (11 days) the contaminated jar had one fish with a
conspicuous sore. It died a day later.

Second stage.—On the eleventh day one of the fish in each of the two jars was
operated upon. A scale or two was removed and the integument pierced with a scalpel
just back of and dorsal to the opercle. More diseased fish were introduced into aquarium
no. 2. Five days later the fish in aquarium no. 1 which had been operated upon died.
The integument, at the point where the incision had been made, had developed a typical
sore. At this time the fish with the pierced integument in no. 2, being a large fish, had
not developed a sore of noticeable extent.

Third stage.—On the sixteenth day of the experiment, all fish having recovered in
both no. 1 and no. 2, scales were removed and the integument of all the fish was pierced
in the same manner as was done with the two above mentioned. ‘Two days later almost
all of those in jar no. 2 had developed marked diseased patches at the very spot where
the integument had been pierced. No noticeable change had taken place in the fish of

the clean jar. Four days later one fish in jar no. 1 died from the effects of the rapidly
64023°—15—2

196 BULLETIN OF THE BUREAU OF FISHERIES.

advancing disease. Subsequent examinations of the tissues showed that the probable
cause of this disease is a myxosporidian belonging to the genus Chloromyxum, being
unique in this respect. (See p. 205.) Four dead fish taken from jarno. 2 at this time
included two that had been introduced for the purpose of spreading the disease. After
seven days the fish in jar no. 1 were all recovering. The incised integument had closed
and appeared a little white. Of those in jar no. 2, two were dead, three were seriously
diseased and died within 24 hours, and the others had conspicuous sores. The remaining
14 fish from this time began to show signs of recovery, probably because they were not
subjected to contamination and they were fed more regularly. Twelve fish remained
in jar no. 1 and had completely recovered before the experiment was discontinued.

In the above experiment the treatment given to the two jars was as far as possible
the same. Some fish escaped from both jars by jumping out.

The first stage of this experiment, which corresponds with the first 11 days, was not
conclusive. One fish, having contracted a fatal disease from a contaminated environ-
ment, demonstrates the possibility that fish with apparently healthy integument may
acquire the ulcers. The second stage of the test, covering six days, was still less con-
clusive. But the third stage, covering seven days, showed beyond doubt that the infec-
tion enters a lesion of the integument, that contamination favors its entrance, that
some of these diseases may be contracted in tolerably pure water, and that lesions
which are not contaminated heal completely.

Another experiment of this character was then started, making use of some of all
the lots of fish that had been under observation. All were in good condition. Eight
fish of fair size were carefully removed from this stock and, by means of a small steril-
ized scalpel, an incision was made back of the head and a pocket then made under the
integument so as to disturb the tissues as little as possible. Into this pocket was inserted
a bit of the diseased flesh from sores of four fish taken from different aquaria. As a
control, eight more fish of the same size were similarly cut, but nothing was introduced
into the pockets. Of the contaminated fish, four died from the disease in two days,
the balance in four days. In this case the disease spread over the whole upper part of
the body and assumed the characteristic appearance usually encountered. Only one of
the controls died. The others healed and recovered completely. From time to time the
diseased fish which were introduced into the contaminated jar and those used for the
inoculation experiments were examined microscopically. All were infected with bacteria.

This last experiment, covering a period of four days, confirms the results of the
previous experiments as to the infectious nature of the disease as well as the inability
of the fish to throw off strong cultures of the causal agents. We also learn that when
the fish is well nourished and in a wholesome environment, it has considerable natural
immunity and recovers readily from the affliction.

In order to prevent the customary mortality from this-kind of affliction, care
should be taken not to injure the fish while collecting; no crabs or other carnivorous
enemies should be confined in the same tanks with the Fundulus, and after establishing
them in an aquarium without crowding, they should be fed on alternate days. The
aquarium should be kept free from dead and diseased fish. With proper circulation of
water, this treatment will no doubt reduce the mortality to a negligible quantity and
preserve the fish for several months.

SPOROZOON PARASITES OF FISHES. 197
PATHOLOGICAL CONDITION OF THE TISSUES.

Those typical sores in which Sporozoa can not be positively demonstrated, and of
which a part may be due to bacteria, present the following histological conditions. They
are probably primarily exogenous ulcers in which there is at times abundant granular
degeneration derived both from lymphocytes and hemocytes. Sometimes at the nidus
of the necrotic area there are small cysts or abcesses containing small lymphocytes.
Usually the vascular tissues abound and erythrocytes preponderate. There is a decided
tendency at times for the epidermis to form a cicatrix. Again it gives evidence of
sloughing off. But sofar as the muscle tissue is concerned universal necrosis is common.

The involved epidermis contains numerous nonstaining globules or masses of
variable size (fig. 36, pl. Xx1), as to the exact nature of which we are yet in doubt. They
are also to be found in the connective tissue of the dermis and in certain partly atrophied
muscle fibers when adjacent to degenerate tissue. They seem to be more numerous in
the epidermal cells wherein there are obvious signs of disintegration (pp. 198, 201, 203).
Inasmuch as there is a nonstaining zodgloea or secretion about some of the bacilli that are
commonly found in these parts, which frequently prevents them from staining (see p.
200), it is possible that these bodies are of the same nature and contain one or more of the
bacilli. No doubt many are fat globules, but some are certainly not. Some of these
bodies in sections of muscle containing myxoplasms possess a well-defined nucleus.
(Fig. 12, pl. xx.)

In smears of integument, it is occasionally possible to find fragments of considerable
size having the epidermal cells more or less filled with the short bacillus referred to above.
It is not difficult to prove, by the observation of fresh material or by comparison of tis-
sues of different stages of degeneration, that the short bacillus is seldom found in normal
living cells. It is therefore not probable that the primary attack upon the epidermis is
caused by this particular organism. The long slender bacillus is less commonly en-
countered in the dermis and epidermis. ‘There is but little evidence in support of the
view that it is the initial cause of epidermal decadence.

The muscle fibers beneath these infected areas present an interesting condition. To
the naked eye there seem to be numerous white threads running parallel with the muscle
cells. This is especially true of well-advanced ulcers. When seen under the microscope,
such flesh has but few normal fibers with fibrille and cross strie. Most of them have the
sarcolemma and interfibrillar connective tissue still sufficiently intact to retain the general
external structure of the separate fibers, but the myoplasm is in various stages of de-
generation. We conclude, therefore, that the parasite is intracellular and does not pass
readily from one fiber to another. The muscle fibers sometimes undergo degeneration
more or less uniformly throughout their length. In some cases it is more rapid in the
immediate vicinity of the parasites. This we know from sections where the fibrillae show
in places adjacent to degenerate myoplasm in which Sporozoa are numerous. One side
or the middle may be far more degenerate than the rest of the fiber. The parasites have
probably passed through these regions. The first indication of change is the loss of
fibrillation. It is rather difficult to find a parasitized fiber showing normal fibrillation
(fig. 13, pl. xx). The pale bands of muscle fibers next become granular (figs. 1 and 2,
pl. xx) and at length the sarcous elements break up into large pieces. Eventually there
is total granular atrophy of the fiber within the sarcolemma. In certain cases, usually

198 BULLETIN OF THE BUREAU OF FISHERIES.

when the atrophy is hyalin, there are considerable clefts in the sarcoplasm. (Fig. 4,
pl. xx). These spaces may come to be more or less closely packed with erythrocytes or
leucocytes, or both, so that when the cytoplasm of the blood cells has degenerated a
third and common condition is encountered. The nuclei in various stages of degenera-
tion become densely packed and enlarged. They assume amceboid shapes, large alveoli
appear in them, and eventually they fall a prey to the short bacillus (fig. 5 and 6, pl. xx)
elsewhere encountered.

The conditions thus presented are such as to suggest an amoeboid parasite which
has demolished a muscle fiber and simultaneously broken up into innumerable bacillus-
shaped spores by schizogony. (See fig. 10, pl. xx.) The connective tissue nuclei of the
flesh and integument and the nuclei of the gill epithelium give rise to the same degen-
eration phenomena. Such nuclei may be about equally hypertrophied and massed in
such a manner as to completely disguise their true nature. Both muscle and vascular
nuclei may occur in abnormal numbers under the sarcolemma of fibers which are in
almost any state of atrophy but without clefts. (Fig. 5, pl. xx.)

In both fresh and stained muscle the evolution of a curious artifact was observed.
It appears as a dense hyalin body in the sarcolymph, between fibrille. (Fig. 3, pl. xx.)
Assuming an ameceboid form it resembles a rapidly growing organism. (Fig. 2 and 7,
pl. xx.) But the regular distribution (fig. 2 and 3) and numerous variations toward a
crystalline rosette structure are conclusive evidence of their lifeless nature.

Whatever the active cause of the degeneration of muscle fiber, be it bacteria or
Protozoa, the atrophy advances far into one or more muscle fibers without causing any
damage to the adjacent fibers. In cross sections of such tissues there may be a small
group of normal fibers cut in section amongst numerous others that are wholly degenerate.
Capillaries, arteries, veins, and sheets of connective tissue, entirely normal in appear-
ance, may also penetrate these necrotic masses. This is no doubt due to the restraining
influence of the sarcolemma upon either the parasite or toxin. As we have already
noted, the sarcolemma retains its normal relations in completely atrophied fibers.

Restricting our statements to tissues known to be infected by Sporozoa, there are
but two kinds where their action has been observed, namely, muscle, and the connective
tissue of the gill. The pathological condition of the muscle tissue, in such cases, is
not distinguishable, as far as we know, from that which results from the action of
bacteria; but if the pathological changes are to be considered as characteristic of a
parasite when it is known to be the cause of the atrophy, a careful study of those cases
where bacteria are a negligible factor is important. The myxospores, which are the
most easily recognized stages of the Myxosporidia, are common only in smear prepara-
tions and only those which include more or less diseased muscle fibers. These same
smear preparations also contain cells identical in appearance to myxoplasms, pansporo-
blasts, and sporoblasts, which happen to be the only representatives of the Sporozoa that
we have encountered in sectioned material, thus suggesting their myxosporidian character.

Several fragments of tissue, the integument of which was slightly diseased, were
sectioned. They give no evidence of myxospores, but the muscle fibers present prac-
tically the same degenerative changes to be seen elsewhere. The dermis contains
numerous minute unstained lens-shaped structures similar to those described on
page 197. These extend into the ends of the adjacent muscle fibers, becoming less
numerous in the deeper parts. Such fibers show obvious signs of atrophy. Elsewhere

SPOROZOON PARASITES OF FISHES. 199

there are numerous deep fibers containing many large cells, which vary in size and have
conspicuous nuclei. (Fig. 18, pl. xx1.) These are confined by the sarcolemma to a
very few fibers and extend for a long distance through them. A small cavity only is
excavated about each cell. They are usually isolated, though two or more may occupy
the same cavity. The sarcoplasm in such cases is much atrophied, being uniformly
granular or homogeneous. A sharp line of demarcation exists between the infected
and uninfected parts of the muscle fiber, the former being degenerate and the latter
striated and normal. Situated amongst the fibers containing the Protozoa are others
lacking them but atrophied in a typical manner, the sarcoplasm being broken into
irregular fragments. There are several other foreign and unnatural structures in the
sections just referred to, about which the details are given on page 203. Muscle fibers
packed with blood tissues and degenerate nuclei have not been found in any of the
sectioned tissues which contain unmistakable cases of Myxosporidia; but no special
significance has been attributed to this fact.

Smears of gill filaments stained with Giemsa stain present the following conditions:
Both normal and degenerate tissues are encountered. In some places the cartilage
supporting structures have been attacked and are partly disintegrated. The general
external form of the supporting tissue, including the surrounding connective tissue and
epithelium, are, as a rule, partly maintained; but elsewhere the degeneration is com-
plete. Epithelium and connective tissue cells disappear completely, leaving the elastic
fibers and blood elements. Here, as elsewhere, the nuclei of the latter are most persistent,
especially those of the erythrocytes. A large portion of the expressed fluids is composed
of an acidophile substance containing odd-shaped portions of the fused nuclei. The
spaces between the chromatin threads of the latter having become much dilated, fuse
and form large masses of network. These are mechanically separated on crushing the
tissue. Such masses of nucleic acid or degenerate chromatin have unbroken connections
with the normal blood in the arteries or veins of the less disturbed tissue. Where the
blood emerges from partly degenerated blood vessels, they are filled with atrophied
erythrocyte nuclei. It seems probable that very large masses of homogeneous eosinophil
material, which are constantly associated with the degenerate gill tissue, are derived
from hemoglobin, lecithin, etc., of the stroma.

Myxospores abound in these degenerate gill tissues, especially in the purulent
residues of degeneration where nothing else remains recognizable. They also occur
deep in the connective tissue near the cartilage and amongst the capillaries. The
spores, developing spores, sporoblasts, and pansporoblasts, in all stages, are clearly
defined, apparently unaffected by the conditions where tissue cells have become wholly
atrophied. This fact, together with the great abundance of myxospores and developing
myxospores, both occurring in considerable clusters, prove beyond question that the
primary cause of necrosis in this case is the Myxobolus. No bacteria or other possible
agents have been encountered.

BACTERIA ASSOCIATED WITH ATROPHIED TISSUES.

The small bacillus above referred to (p. 195) varies greatly in size. The smallest
(fig. 8, pl. xx) measure less than 0.74 in thickness and 1.54 in length. The large ones
(fig. 9, pl. xx) average 1.5 in thickness and 74 in length. The former are homogeneous
when stained. The latter frequently appear to have very conspicuous granules just

200 BULLETIN OF THE BUREAU OF FISHERIES.

inside the cell wall. These are probably artifacts. The older bacilli (fig. 9, pl. xx) taper
at one end. They were at first taken for protozoan spores. These bacilli occur by
thousands in and near degenerate epidermis and muscle tissue. It is not unusual to find
them grouped in the form of the cell which they have completely destroyed. They are
then of nearly uniform size (fig. 10, pl. xx); but between individuals of separate groups,
there is often a great difference in size. They stain, as a rule, with methylene blue,
gentian violet, toluidin blue, and Giemsa stain. Inside the host cells (fig. 6, pl. xx) and
when first set free from them they stain, if at all, with great difficulty. This may no
doubt be due to a zoégleeic condition. In smears, the stretching of this secretion causes
the bacilli to be drawn into long parallel rows. The secretion then resembles elastic
connective tissue fibers and the bacteria replace the connective tissue nuclei. At times
the zodgloea is not noticeable. (Fig. 8 and ro.)

To what extent toxins emanating from the short bacillus are the cause of the death
and disintegration of the host tissues we can judge from the following facts: As already
stated, this bacillus is not to be found throughout large areas of atrophied muscle and
integument. If the toxin emanating by diffusion from a localized organism brought
about the decadence of a tissue, one would expect the evidences of such decadence
to indicate a uniform advance of said toxin in the same direction through a given tissue;
but, as we have seen, the atrophy of muscle fibers is limited to a certain few in a large
number of normal cells, or there may even be a few normal fibers extending through and
far into a necrotic region. The same relations prevail more or less in the epidermis.
If the short bacillus is to be regarded as a saprophyte, then some more virulent primary
organism must be present. In the diseased gills the abundance of M. musculi and the
extent of injury in its immediate presence point to the sporozo6n as the primary agent.
There are a few places in the gill tissue where the short bacillus is abundant, but, as
would be expected of a saprophyte, in very degenerate tissue only. Such seems to be
its relation to all the tissues.

There are also tissues in which nothing but the long bacillus can be recognized as
the agent of primary degeneration. While never abundant, it may be observed more
frequently than the short bacillus in fresh smears of infected tissue. After about 24 hours
the latter appear in clusters in the muscle fibers occupying excavations of regular ovoid
contour. The long type occurs less frequently in tissues that are completely atrophied
than in those which just begin to show signs of decadence. Fresh muscle, in the latter
condition, may have the long bacilli more or less abundantly distributed under the
sarcolemma, but never in compact groups, a condition which is characteristic of the
short form. In sections, the long type has been encountered, one or two at a time,
in muscle fibers at or near the region of advancing degeneration, and occupying irregular
transverse clefts in the scarcoplasm (similar to those in fig. 4, pl. xx). But these cavities
seem to be much too large to be considered the excavations of so few of these minute
organisms. Their toxins may precede them and the transverse cleavage of the muscle
fiber may be due to subsequent mechanical forces. On the other hand, the bacillus is
quite as likely to creep into the crevices in the sarcoplasm as are the blood tissues
(p. 198). Its presence is therefore not necessarily evidence that it is the cause of the
crevices. In one stained smear, some of the muscle fibers of which are completely
hypertrophied, the long bacillus is very abundant, especially in the connective tissue.
There is no evidence of the admixture of fluid from purulent tissue such as is frequently

SPOROZOON PARASITES OF FISHES. 201

common when the short bacillus occurs abundantly; nor are there any of the short
bacilli. The normal striated fibers possess few if any of the germs and they seem to be
numerous in proportion as the sarcoplasm is degenerate. These are not the conditions
we would expect of a virulent parasite unless its primary attack is through the agency
of a toxin. There is a second factor to be considered, however, inasmuch as numerous
myxoplasms and autospores of M. musculi occur in some of the less decomposed por-
tions of the same tissue. With the evidence at hand bearing upon the virulence of the
two bacilli, the most natural conclusion is that the short bacillus is a saprophyte, that
the long bacillus is either a facultative parasite upon the post tissues, which has been
reduced in vigor by the Sporozoa already established therein, or perhaps a true parasite,
in which case there are frequent double infections, the long bacillus and Myxosporidia
together preparing the way for the saprophytic short bacillus.

The long and short bacilli are easily distinguishable by their size, shape, and habits.
The long bacillus is 0.74 in diameter and usually at least 2.54 long, but it may be 22p
long, without any noticeable increase in diameter. (Fig. 11, pl. xx.) They have tapered
ends, especially those which have but recently divided. Sometimes the long type
divides, forming short rods, but they are then in chains. They never occur in clusters
asin figure 10, platexx. Theshort typeis never coiled, never so long, and always thicker
than the long bacillus. They are both encountered in smears which include the fluids
of completely broken-down tissues, but the short form is always abundant in such
fluids, while the former is rare. One is frequently clustered and in regular pockets, the
other isolated or scattered and, if in cavities at all, they are irregular crevices.

SPOROZOA ASSOCIATED WITH ATROPHIED TISSUES.

From the evidence in the foregoing pages and borne out by that which is to follow,
it is certain that a sporozoén causes the primary degeneration of muscle, gill, and pos-
sibly integumentary tissues, resulting in pathological conditions which are quite as
characteristic as when the bacillus is the primary parasite. In one tissue which was
sectioned (fig. 18, pl. xx1) the degeneration of the muscle fibers is identical to that where
bacteria alone have been observed (p. 200). The atrophied fibers, which contain numerous
scattered Sporozoa (p. 198, 203), occur in groups of two or three here and there through-
out the fragment of flesh. Frequently, in both sections and smears, degenerate muscle
fibers occur in which there are cells similar to the above but with neither nucleus nor
cytoplasm stained; also large amceboid masses of granular cytoplasm without any visible
nucleus (fig. 13, pl. xx). Usually such foreign cells occur in tissues when either myxo-
spores or multiplicative stages are more or less abundant.

In one or the other of the above stages the sporozo6n has been positively identified
with the disease in 18 of the 85 fish which have been examined. On the other hand,
many degenerating fibers have been encountered both in smears and sections in which
neither Protozoa nor bacteria could be found. In such cases there is about equal
lack of evidence that either of the above are the causal agents of such disintegration.
While it is probable that the majority of the sores are caused by the inoculation of a
wound by a germ, there is less evidence of a primary attack upon the tissues by the
bacteria, except through a widespread toxin, than by Sporozoa. In this connection there
is probably a significant difference in the external appearance of diseased tissues which
are primarily due to the sporozoén attack and those which are caused by bacteria.

202 BULLETIN OF THE BUREAU OF FISHERIES.

Certain fish in which the diseased parts were conspicuously congested (ventral part of
the head, around the anus, and about the eyes) were almost invariably found to con-
tain a large number of myxospores. When we consider the unknown stages of the
Sporozoa which, according to the cyclic habit of these organisms, advance from stage
to stage in a given culture at nearly the same rate, there is reason to attribute to them
more destruction than our observations warrant. Our present lack of knowledge is no
doubt due in part to the inadequate stains that have been employed and in part to the
confusion of tissue cells with certain stages of the myxosporidian cycle. (See also p. 205.)

STAGES OF MYXOBOLUS MUSCULI.

Mention is made in the literature of but one other case of mxyosporidian disease
of the integument and flesh which is closely allied to that of the Fundulus, namely M.
lintoni of Cyprinodon variegatus (Linton, 1889-1891). With this one exception, similar
diseases in other American and European salt-water minnows, as far as we can learn,
have not been described. The M. lintoni of the Cyprinodon was at first supposed to be
identical to the M. musculi of Fundulus. But very recently a tumor of the variegated
minnow was encountered. (See p. 206.) Both the spore and the tumor are markedly
different from the common condition of Fundulus.

The myxoplasm of M. musculi produces a great many pansporoblasts, each with a
single spore. There is a large vacuole in most of the spores which is the characteristic
iodinophilous vacuole of the genus Myxobolus, to which the parasite undoubtedly belongs.

Of the life history we have the spore, pansporoblast, possibly the myxoplasm,
schizont, and multiplicative or autospore. In but 3 of the 18 fish which harbor Sporozoa
have we stages (figs. 20, 21, 26, 27, pl. Xx1) that can be unmistakably connected with the
spore. By association in the same tissue or by the appearance and staining reaction
we have probably identified the myxoplasms and autospores.

According to Auerbach’s (1910) description of M. bergense, the spore terminates
the life cycle in a given host and starts a new cycle in a new host. We can but assume
that the trophoplasm of M. musculi likewise arises in some way from a primary myxo-
spore. The trophoplasm (fig. 12, pl. xx) is difficult to stain, and therefore its sporozo6n
properties are not always certain. (See also Chloromyxum properties, p. 205.) Spherical
or oval spaces in the diseased myoplasm and in the epidermal cells (possibly identical,
fig. 36, pl. xx1,and p. 197) are very abundant. These are probably multiplicative tropho-
plasms, unless we have confused them with fat or other nonstaining substances. Some-
times these bodies have nuclei (fig. 12) which, though usually faint, may stain deeply.
It is not impossible that some of these small trophoplasms may be those of the Chloro-
myxum. When large, the trophoplasms have a granular structure (fig. 13, pl. xx) and are
doubtless preparing to undergo schizogony. We have encountered but five or six
such schizonts. In one series of sections they are associated in diseased muscle fibers
with cysts containing many spores. (Fig. 14, pl. xx.) The amceboid form of the mature
schizont is characteristic and distinguishes it from the smaller forms. The schizont
in figure 13 is 33 wide by 74 in length. Some of the cysts are about this size, but
figure 14, which is 19 wide and 24» long, is a section through the small end of a cyst
of only moderate size. The cysts are found both within and between the muscle fibers.
They contain several hundred spores, the nucleus of which, like that of the trophoplasm,
has at times little affinity for the stains we have employed. The spores sometimes

SPOROZOON PARASITES OF FISHES. 203

appear to be spherical in form (fig. 14, pl. xx) and vary somewhat in size. They have
a small faintly-staining nucleus and hyalin nonstaining cytoplasm. Isolated spores
and masses of spores recently discharged from the cysts also occur in the smear prepa-
rations associated with the intrafibrillar masses of material that appear to be equivalent
to schizonts. These spores also occur in small numbers in the diseased gill where
myxospores and sporoblasts are to be found in very great numbers.

The occurrence of a multiplicative process of reproduction amongst the Myxo-
sporidia in the manner here described is not uncommon. We have authentic cases
in gall parasites of the flounder, and they have been described in M. pjetffert (Keys-
selitz, 1908) and in Henneguya gigantea (Nemeczek, 1911). While there is no question
but that there are multiplicative spores, our evidence that the spore here described
is such is, as with the trophoplasm, far from conclusive. Judging from the meager
evidence at our disposal, there is about equal reason for considering it a young sporo-
blast or a young trophoplast. It is more harmonious to regard these amceboid spores
as the progenitors of both multiplicative and propagative trophoplasts and the oval
spores, which are described below (p. 204) as sporoblasts, more especially since they
apparently arise by free cell formation and in smaller numbers.

The propagative and sporoblast stages have been encountered frequently in both
sections and smear preparations. One series of sections of diseased integument and
muscle (referred to on p. 198), which were cut approximately at right angles to the body
surface, contains numerous large cells (fig. 18, pl. xxr) with small well-stained (with
hematein) nuclei. Some of the muscle fibers are cut obliquely. They lie in the midst of
healthy tissue and under integument which is apparently healthy. These myxoplasms
are of oblong or spherical form with more or less even surface. The cytoplasm is
tolerably homogeneous and does not retain the stains. The karyoplasm is also unstained,
but the chromatin is somewhat conspicuous. These cells occur abundantly throughout
60 or more sections, in 6 to 8 adjacent fibers, also in other distant fibers. Upwards
of a hundred perfectly normal fibers around them have not a single foreign cell. Such
cells are always intracellular. The sarcoplasm is considerably modified. The fibrillar
structure is lost and the appearance is almost homogeneous.

A cell similar in every respect to the myxoplasm just referred to, occurs abundantly
in smears of muscle tissues. It stains less readily than do leucocytes and has smaller
nuclei with less conspicuous chromatin. Myxospores and pansporoblasts have been
found in their midst, in fact are to be found on slides where this type of cell occurs and
not elsewhere. In this connection it is interesting, and perhaps additional evidence of
relationship, that the same sore from which the sections containing these myxoplasms
(fig. 18, pl. xx) were made also supplied a smear preparation containing numerous myxo-
spores and pansporoblasts represented in figure 26, plate xx1. The sporoblast resembles
the myxoplasm of smear preparations in shape, clear, nonstaining cytoplasm, size, and
feebly staining (with methylene blue) nucleus. It is for the above reasons that this
type is assumed to belong in the propagative cycle.

There is a wide range of conditions to be seen in the nuclei of these myxoplasms,
as well as some variations in size. Some densely-staining, cigar-shaped bodies (fig. 17,
pl. xx1) almost devoid of protoplasm are embedded in the sarcoplasma, and others are
closely applied to the myxoplasms (fig. 18, pl. xx1, near right-hand upper corner). The

204 BULLETIN OF THE BUREAU OF FISHERIES.

conditions suggest conjugation, but the stages are too few to indicate a succession of
events. One myxoplasm contains two oblong spores. Elsewhere, replacing a degen-
erated muscle fiber, are numerous small cysts (12 in diameter) with eccentric nuclei,
which contain from four to ten or a dozen clearly defined oblong spores (fig. 16, pl. xx).
These spores are found abundantly in other fish. (Fig. 19, pl. xx1.) They appear to
arise by free cell formation. They are characterized by a transparency and a failure to
stain that recall both the trophic stages and the sporoblasts. The nucleus, however,
does stain faintly. It is quite large when the spores are set free. The latter measure
4p by 2.54 and sometimes assume a spherical or amoeboid form. Between this condi-
tion and the mature sporoblast we lack recognizable connecting stages. They are
not far removed, however, from the latter, which are spherical cells with very large
nuclei. (Fig. 35, pl.xxr.) These occur in the gill above mentioned and have there been
definitely connected with the myxospore. The pansporoblast has been encountered,
along with spores and sporoblasts, in fresh smears of muscle. These are apparently
identically homologous to those described for M. pjfeifferi in the gills of the barbel
(Keysselitz, 1908). If so, the sporogenesis there related would appropriately apply to
M. musculi. Many stages in the genesis of the spore are represented in one of our
smear preparations. These have propagative stages (Keysselitz, 1908) as follows:
First the sporoblast with large nucleus (fig. 35, pl. xx1) and two-parted pansporoblast
(sporocyst) (fig. 22, 23, pl. xx1), which, according to Keysselitz, arises after a process of
autogamous conjugation. The sporocyst apparently sets free the sporocytes -before
sporogenesis has proceeded far (fig. 21, pl. xx1). Giemsa stain does not reveal all the
nuclei concerned in sporogenesis. Valve cells are formed (fig. 24, pl. xx1) before the
polar capsules appear as large spherical bodies (fig. 25, pl. xxz). Later the myxospore
becomes elongated and tapered (fig. 20, 26, pl. xxt). Two preparations have multitudes
of immature spores. They are all free from the sporocyst protoplasm and have thick
valves. It is therefore rather perplexing to explain figures 20 and 26. Perhaps the
spore is about to be discharged in figure 20. Considerable variation in this respect
occurs amongst some of the gall Myxosporidia.

There are myxospores in 12 of the 85 fish examined. In but 3 of these do they
occur in great numbers. With two exceptions (in diseased gills), the myxospores are
not assembled in a manner that would suggest their origin from cysts or masses of
pansporoblasts, as is common in other species of Myxosporidia. The two cases referred
to may not be interpreted as evidence of this condition, but rather that the pansporo-
blasts, where very numerous, have been packed close together. There are at least a
thousand well-stained spores in the preserved tissues. Not one occurs in the ro tissues
of which sections have been made. But those same tissues which contain spores have
supplied all the propagative myxoplasms.

The myxospores are very small (fig. 28, 29, pl. xxi). They average 14.3y in length
and 6.74 in width. In one fixed individual the plane at right angles to that passing
through the polar capsules is presented. It measures 6.74 in thickness, from which
we conclude that they are approximately circular in section. But another fresh spore
was flattened in a plane perpendicular to that of the polar capsules and sutures to about
two-thirds its width (fig. 30, pl. xxr). The polar capsules of myxospores average 6.5 in
length and 2 in thickness. When extruded the filament is three to four times the length
of the spore (fig. 29, pl. xx1). Coiled within the polar capsule, the filament makes from

SPOROZOON PARASITES OF FISHES. 205

10 to 14 turns (fig. 26, 28, pl. xxr). In young spores the valves are quite thick and may
be seen at the edges as a pale border to the spore, but in mature spores they are thin
and almost invisible. Young spores are shorter (12) and wider (7.5) than the
mature spores and the polar capsules are not so long (6”). They lengthen out as they
approach maturity. When young, the nuclei stain with great difficulty, if at all. The
sporoplasm occupies all the space at the large end of the spore. A large vacuole is nearly
always visible in the sporoplasm. There are also dense areas and from 1 to 1o nuclei.
(Fig. 20, 28, pl. xx1.) The nuclei are unstained in figure 29, plate xx1. There are some-
times seven greenish-blue nuclei (fig. 28, pl. xxr) and three rather irregular dark-blue
bodies between the polar capsules. It is not possible to be sure that this (1o) is the
maximum number as some of these are ill defined. A number of spores have their
nuclei attached near the large end of each polar capsule, thus identifying them as the
“polar capsule” nuclei. Probably the remaining four belong to the sporoplasm. It is
not possible to recognize the “‘wall nuclei’’ at this stage, and the “ resting nuclei” of
the pansporoblast are doubtless lost.

CHLOROMYXUM FUNDULI.

The Chloromyxa which have been observed in the muscle of other fish (p. 208) are not
identical to that found in Fundulus. C. quadratum, which resembles the latter, has
myxospores measuring 6 in diameter by 5 along the polar axis, while the Chloro-
myxum of the Fundulus measures 7.54 in diameter and 6 along the polar axis and
differs in shape. The spore of C. quadratum, when seen in line with the polar axis, has
the sides deeply concave, and in the other plane it is more pointed. The polar capsules
are also much shorter. They also differ in the relation of the spore to the pansporoblast
and in the pathological effects. (For description of spore of C. funduli see p. 208.) No
reference to a myxospore of this character has been found by the writer. The name
C. junduli has therefore been applied to this species.

The myxospores of C. junduli have been encounteredin but one fish. If the myxo-
plasm occurs in other preparations, it has not been possible to identify it, although many
suspected myxoplasms exist. It is not very probable that they are at all uncommon.
They do not take up a particle of such stains as we have employed. The single slide
containing this species is a smear preparation made from the diseased flesh of a fish
which died in jar no. 1 of the experiment reported on page 196. It is stained with
Giemsa stain.

The muscle of this fish is in an advanced stage of decomposition. When the fresh
slides were examined no myxospores were noticed, being difficult to see without a stain,
but the sporoblasts were observed without recognizing their importance.

Bacteria are present on the slide but lacking in the muscle fibers. The decadence
of the muscle must in this case be ascribed to the Chloromyxum, which is abundant in
the hypertrophied muscle. The muscle is full of cavities containing unstained myxo-
plasms and sporoblasts which are identical in appearance to those of many other prepara-
tions of diseased Fundulus. While this case introduces the possibility that many of the
Fundulus cancers may be caused by Chloromyxum fundult, it gives very substantial sup-
port to the agency of Sporozoa as the cause of these diseases. Since Chloromyxum and
Myxobolus are not uncommon in muscle tissue, double infections are to be expected.
But having failed to encounter myxospores of the Chloromyxum in over 100 stained

206 BULLETIN OF THE BUREAU OF FISHERIES.

preparations that have been examined, we are inclined to consider the Myxobolus more
abundant and therefore the more common causal agent.

The myxospore of C. funduli is about 7.54 in diameter, with a polar axis somewhat
shorter (64). At right angles to the polar axis, it is circular. There are four polar
capsules, which taper to the apex of the spore, curving so as to conform to the constric-
tion of the spore, which provides it with a blunt pointed apex (fig. 31, 34, pl. xxr). There
are four conspicuous nuclei (black), one near the base of each polar capsule. The sporo-
plasm is stained a pale blue by the Giemsa. The polar capsules do not stain (fig. 31, 34,
pl.xx1). There are occasional myxospores of considerable size to be seen inside the sporo-
blasts when the latter do not take up a particle of stain. Such clear hyalin amceboid
pansporoblasts are numerous throughout the sarcoplasm. They vary in size from a
diameter of about 2 to four or five times the diameter of the spore.

PROTOZOA RELATED TO THOSE HERE DESCRIBED.

Numerous Myxosporidia parasitic upon either integument, gill epithelium, con-
nective tissue, or muscle of fish have been described by other authors. About most of
them we have very meager information. M. lintom (Linton, 1889; Gurley, 1893) of
Cyprinodon variegatus (short minnow), as already stated (p. 202), more closely resembles
the parasites of Fundulus than any other species of which we know. The difference
at first seemed to be slight and to be easily accounted for by a difference in the age
of the spores. But when a case of the Cyprinodon tumor was finally obtained and
examined, the indentity of the parasites in the two hosts, as well as the nature of
the lesions, was found to be different. The “irregular fungoid elevations,’’ described
and figured by Linton and observed again by the writer, are of the nature of cysts con-
taining spores, located in the integument, whereas the elevated scales in Fundulus are
due to an infection of the epidermis by bacilli and a subdermal atrophy of the muscle.
No tumor or spore-filled cyst has ever been encountered. The Cyprinodon tumor which
we examined developed in a comparatively short time, probably less than a week,
though the period can not be accurately stated. It caused the death of the fish the day
following that on which it was first noticed. After the death of the host, the tumor was
8 mm. wide by 10 mm. long, and caused a conspicuous elevation from the back of the
fish anterior to the dorsal fin, about 2 to2!4 mm. thick. It wasof a yellowish-pink color
when seen through the slightly pigmented integument. The scales were practically
undisturbed and the integument was completely intact, in this respect differing remark-
ably from the Fundulus sores. Beneath the tumor, the flesh contained intrafibrillar
myxoplasms and sporoblasts with occasional spores, while the tumor itself was almost
wholly a mass of myxospores, the latter numbering millions. We have already described
(p. 197) a totally different condition in the Fundulus, resulting from the M. musculi.

There is such a difference in the appearance (Linton, 1889, fig. 3) of the spores that
they are readily distinguished. One can not be certain, however, that such differences
are not due to the comparison of different stages in the development of spores of the same
species. We have shown that the spores of M. musculi grow longer as they mature and
the spore wall becomes thinner (p. 205). This fact would explain in part the discrepancy
in the dimensions of the spores from the Fundulus and Cyprinodon. But, since the
spore of M. lintoni measures 13.9 in length, 11 in width, and 8y in thickness (at right

SPOROZOON PARASITES OF FISHES. 207

angles to the planes of the two polar capsules), and the mature spore of M. musculi
measures 14.34 in length by 6.7y thick, and from 4 to 6.74 in width (see p. 204), in indi-
viduals of apparently the same stage of development, it still seems that a sufficient
discrepancy in size exists to supplement the marked differences in the pathological con-
ditions. It may yet prove that the latter are due to the influence of different hosts, inas-
much as we have one case of a Fundulus with a typical M. musculz lesion, but having
spores indistinguishable from those of M. /intoni in either size or appearance.

M. lintom presents another contrast to the conditions in Fundulus. In the former,
calcareous bodies were observed amongst the spores by Linton (1889) and the writer,
whereas nothing of the kind has ever been encountered in the hundreds of Fundulus
tissues which we have examined.

Although the name M. dintoni was for a time retained for the Fundulus parasite, the
present state of our knowledge will not permit of thisassumption. The species “musculi”’
has been adopted because of the interesting and characteristic attack which the tropho-
plasm makes upon muscle fibers.

The spore of Myxobolus ovijormis (Thelohan) resembles M. musculi very much in
appearance, but is less tapered and shorter (Thelohan, 1894).

The following, for one reason or another, are also of interest in their bearing upon
M. muscult. A “Myxosporidian”’ of unknown genus and species was found by Linton
(1899) in the connective tissue of the entire body of Notropis megalops Rafinesque
(albeolus Jordan), the shiner. The epidermis is marked by dark purplish blotches. The
scales are absent in most cases. A “Myxosporidian” of unknown genus and species was
observed by Lieberktihn (1854) in the connective tissue of Gastereosteus aculeatus (stickle-
back). The skin is said to have contained cysts. The conditions seem to be unlike those
in Fundulus. Cyprinus leuciscus (Miller, 1841) has been observed with tumors in the
integument caused by a species of Myxobolus. M. oblongatus Gurley produces cysts
under the scaleless skin of the head region in Catostomus tuberculatus Le Sueur (Gurley,
1891, 1893, p. 234). M. transovalis Gurley (1893) of Phoxinus (Clinostomus) funduloides
Girard, occurs under the scales and external to the epidermis. “It forms a thin dis-
coidal mass situated in the center of the concave undersurface of the scale.” That it
is not identical with /. musculi is certain from the dimensions of the myxospore (length
6y, breadth 8), the diameter of which, at right angles to the polar axis, is greater than
through the polar axis. We have very scanty information concerning the M. strongylurus
(Gurley, 1893, p. 247), which is found encysted in the skin of the head of Synodontis
schal; of M. momurus (Gurley, 1891, p. 416), known from cysts in the subcutaneous inter-
muscular tissue of A phredoderus sayanus Gilliams; of Henneguya niisslini Schuberg und
Schréder (Leger, 1906), which is found in the connective tissue of the dorsal fin of the
trout; of M. gigas Auerbach (1907), which thickens the integument at the ventral angle
of the gill in Abramis brama Linnzus (bream); and of a Myxobolus of unknown species
described by Borne in 1886 (Gurley, 1893, p. 244), which causes great tumors over the
surface of Leuciscus rutilis.

In Coregonus jera there occurs a common disease of the integument caused by a
species (M. zschokket, Gurley, 1893; Zschokkei, 1884), the myxoplasm of which is not
known. The cysts lie in the subcutaneous connective tissue and between the muscles.
It causes irregular thick patches on the skin, from which the scales drop.

208 BULLETIN OF THE BUREAU OF FISHERIES.
PREVALENCE OF MYXOSPORIDIAN INFECTION IN FISH.

The infection of muscle tissue by Myxosporidia is quite common in fish. A parasite
belonging to the genus Chloromyxum occurs in the flesh of the young herring and young
alewife (Linton, 1891). Both the pansporoblast and spores of a Chloromyxum have been
found abundantly by the writer inside the fibers, and the spores also assembled else-
where in large cysts. A fuller account of this species will be published later. The
muscle cells of Callionymus lyra are also subject to an intracellular parasite (Glugea
destruens Thelohan, 1891; Henneguy et Thelohan, 1892; Gurley, 1893), the myxoplasm
of which has not been observed. It causes the muscle fibers to undergo degeneration.
Chloromyxum quadratum (Thelohan, 1894) also occurs in the muscles of this fish. It is
also reported in the flesh of Corts julis, Syngnathus acus, Trachurus trachurus (Minchin,
1903) and Nerophis equoris. In Cottus scorpio the muscle tissue is attacked by Plezsto-
phora typicalis (Thelohan, 1890, and 1891; Gurley, 1893). Both pansporoblast and spores
have been found, but they are intercellular in position. The muscle fibers are displaced
but do not degenerate. Leptotheca perlata (Gurley) occurs in the muscles of Acerina
cernua Linneus. Of these species there are none that closely resemble M. muscult.
Numerous cases of Myxoboli are known to inhabit gill tissues. Auerbach (rgrr) lists
22 species of Myxobolus which have been described in the gills of fish. But we have
encountered nothing that might be considered identical to M. muscult.

The disease of Fundulus is remarkably like that which has so frequently caused
epidemics amongst the barbel (Barbus barbus Linnzus) of European rivers. The latter
is caused by M. pfeiffert Thelohan (Raillet, 1890; Ludwig, 1888; Thelohan, 1894). It
produces both tumors and ulcers and occurs encysted and free in muscle, liver, kidney,
spleen, and connective tissue. The tumor when formed does not at all times break
through, either into the body cavity or to the outside. It is not an integumentary
parasite at the beginning as those of Fundulus seem to be. The tumor commonly occurs
amongst the connective tissue and the muscles of the body wall. The parasite may be
encysted in a thin restraining membrane produced by the host. Numerous individuals
of about the same age tend to gather in groups and become isolated in tube-like cysts.
The muscle fiber is invaded and undergoes a ‘‘vitreous alteration’’ (Thelohan, 1893)
leaving ‘‘ yellow granulations as degeneration products” (Keysselitz, 1908). Thelohan’s
figure 5, plate vi (Thelohan, 1894), representing a muscle fiber containing myxoplasms
in transverse crevices recalls, very vividly the appearances we have encountered in the
degenerate muscle of Fundulus (fig. 4, pl. xx). The tumors may soften and become a
“stinking abscess containing spores” (Ludwig). M. pjeiffert passes through distinct
cycles of development which is no doubt the case in M. musculi. In April it is in a vege-
tative stage in which the multiplicative reproduction prevails; later propagative repro-
duction is encountered and myxospores are developed. The rate of advance of the dis-
ease depends upon the temperature (Keysselitz).

Both Keysselitz and Thelohan describe bacteria in tissues of diseased barbel.
Keysselitz says bacteria contribute liberally to the formation of the tumors. These
bacilli are found only in the tissues infected by Myxosporidia. They prevent the
growth of connective tissue and bring about degeneration (gangrene) of the tissue.
These bacilli are ‘‘as long as the spore” (Pfeiffer, 1890) (6, Thelohan) and stain easily
with methylene blue and gentian violet. (This is also true of the bacilli of Fundulus

SPOROZOON PARASITES OF FISHES. 209

diseases.) Pfeiffer mentions threads attached to these bacilli. A coccus is also occa-
sionally found. The presence of bacteria is therefore not necessarily an indication that
they are primary as causal agents of disease since IM. pfetfferi is known to be the cause
of the barbel disease.

GENERAL CONCLUSIONS.

I. The sores of Fundulus are usually caused primarily by lesions. These may
occasionally be due to parasites such as leeches, distomes, and copepods, but usually
to rough handling and carnivorous enemies.

II. At least four kinds of germs invade these lesions and bring about hypertrophy
of the tissue elements and decomposition, namely, two species of bacteria and two species
of Myxosporidia.

III. There is doubt as to the virulence of the bacteria. One species at least is
saprophytic. There is no doubt as to the virulence of the Myxosporidia when present.

IV. Cleanliness, careful feeding, and aeration bring about recovery in practically
all injured fish. It can not be claimed that fish which are known to have Myxosporidia
are curable.

V. The trophoplasm of both species of Myxosporidia attacks the muscle fibers,
that of the M. musculi also attacks the gill connective tissue.

VI. Blood elements, especially nuclei, give rise to abundant artifacts which are
closely associated with the parasite involved.

VII. Sporogenesis of the Myxobolus occur infrequently in the muscle and gill tissues.

VIII. Multiplicative spores are probably formed in M. musculi in addition to pri-
mary sporocytes. :

IX. The myxoplasm of both C. funduli and M. musculi are stained with difficulty
and are therefore not easily found.

BIBLIOGRAPHY.
AUERBACH, MAX.

1907. Weitere Mitteilungen iiber Myxobolus eglefini Auerbach. Zodlogischer Anzeiger, bd.
XXXI, p. 386-391. Leipsig.
1910. Biologische und morphologische Bemerkungen iiber Myxosporidien. Zodlogischer Anzeiger,
bd. xxxv, p. 57-64. Leipsig.
rgt1. Unsere heutigen Kenntnisse iiber die geographische Verbreitung der Myxosporidien.
Zoologischer Jahrbucher, Abtheilung fiir Systematik, bd. 30, p. 471-494.
BORNE, MAX VON DEM.
1886. Handbuch der Fischzucht und Fischerei, p. 21t, fig. 215.
Gur.ey, R. R.
1891. On the classification of the Myxosporidia, a group of protozoan parasites infesting fishes.
Bulletin United States Fish Commission, vol. x1, 1891, p. 407-420. Review in Central-
blatt fiir Bakteriologie und Parasitenkunde, bd. 15, p. 86-88.
1893. The Myxosporidia or psorosperms of fishes, and the epidemics produced by them. Report
U. S. Commission of Fish and Fisheries, 1892, p. 65-304, pl. 1-47.
Hennecuy, F., Et THELOHAN, P.
1892. Sur un Sporozoaire parasite des muscles de l’ecrevisse. Comptes rendus hebdomadaire
Société de Bioiogie Paris, t. 4, p. 748-749.
KeysseE.iiz, G.
1908. Die Entwicklung von Myxobolus pfeifferi Thelohan, Theil 1 und 2, Archiv fiir Protisten-
kunde, bd. 11, p. 252, 276-308. Jena.
LEGER, L.
1906. Sur une nouvelle myxosporidian de la tauche commune et de la truite indigene. Comptes
rendus de 1’Academie des Sciences, Paris, t. 142, p. 655.
LiEBERKUHN, N.
1854. Uber die Psorospermien. Miiller’s Archiv, p. 9-10, 22, 24, 354, taf. 2, fig. 28; taf. 14, fig. 9-12.
Linton, Epwin.
1889-1891. On certain wart-like excrescences occurring on the short minnow, Cyprinodon variegatus,
due to psorosperms. Bulletin United States Fish Commission, vol. rx, 1889, p. 99-102,
pl. xxxv; ibid. p. 359-361, pl. cxx, fig. 1-3. Review in Centralblatt fiir Bakteriologie
und Parasitenkunde, 1892, bd. 11, p. 475.
Lupwic, H.
1888. Uber die Myxosporidien krankheit der Barben in der Mosel. Jahrbuch des rheinischen
Fischerei Vereins, Bonn, p. 27-36.
Mincuin, E. A.
1903. A Treatise on Zodlogy. Edited by E. Ray Lankester, pt. 1, fase. 2, p. 150-361.
MULLER, J.
1841. Ueber Psorospermien, Miiller’s Archiv fiir Anatomie und Physiologie, p. 477-496, pl. 16.
Abstract in Microscopical Journal, London, 1841-2, p. 123-124.
PFEIFFER, L.
1890. Die Protozoen als Krankheitserreger, Jena, 1 ed.
PLEHN, M.
1905. Uber die Drehkrankheit der Salmoniden (Lentospora cerebralis) Archiv fiir Protistenkunde,
bd. 5, p. 145-166, taf. 1, fig. 7. Jena.
Raruet, M. A.
1890. La maladie des Barbeaux de la Marne. Bulletin Société Centrale d’Aquiculture, Paris,

t. 2, p. 117-120.
210

SPOROZOON PARASITES OF FISHES. 211

THELOHAN, P.

1890. Contribution a 1’étude des Myxosporidies. Annales de Micrographie specialement consacrees
a la bacteriologie, aux protophytes et aux protozoaires. P.1, t. 2, p. 193-213. Paris.

1890. Recherches sur le developpement des spores chez les Myxosporidies. Comptes rendus heb-
domadaire de la Société de Biologie. Paris, t. 2, p. 602-604. Abstract in Journal Royal
Microscopical Society, 1890, pt. 2, p. 194-195.

1891. Sur deux sporozoaires nouveaux parasites des muscles des poissons. Ibid., t. 62, p. 168-172.

1893. Alterations du tissu musclaire dues a la présence de Myxosporidies et de microbes chez le
barbeau. Ibid., t. 5, p. 267-270. Abstract in Centralblatt fiir Bakteriologie und Para-
sitenkunde, bd. 14, p. 532.

1894. Recherches sur les Myxosporidies. Bulletin Scientifique de la France et de la Belgique,
t. 26, p. 100-394, pl. 7-9. Paris.

WIERZEJSEI, A.

1898. Uber Myxosporidien des Karpfens. Anzieger der Akademie der Wissenchaft in Krakau,
Marz. Résumé in Bulletin International de 1’Academie des Sciences de Cracovie, Comptes
rendus des seances, 1898, p. 129-145. Cracovie.

ZSCHOKKE, F.
1884. Psorosperms de Coregonus fera, Archives de Biologie, t. 5, p. 234-235, pl. 10.

EXPLANATION OF PLATES.

With the exception of figures 20, 27, 30, and 35, the drawings were made with the
aid of acamera lucida. For figures 3, 5 to 11, 15 to 17, 19, 21 to 26, 28, 29, 31 to 34, and
36 a no. 12 Bausch & Lomb compensating ocular and one-twelfth inch oil immersion
objective were used. For figures 1, 2, 12, 14, and 18 a Bausch & Lomb 1-inch occular
was employed with the same objective. The 1-inch occular and a one-fifth inch objec-
tive were used in figures 4 and 13. All figures have been reduced to two-thirds the size
of the camera images. The tube length was 160 mm. and the camera arm 90 mm.

The figures are numbered approximately in the order of development. Figures 2,
3, 5, 6, 7, 8, 9, and 10 are made from the same slide, and figures 13, 14, 16, 17, 18, 26,

and 28 are from the same fish.
PLATE XX.

Fic. 1. A bit of infected muscle from a smear of a sore on the side of a small Fundulus heterclitus in
the first stage of disintegration. Fixed in corrosive sublimate and acetic acid and stained with Mayer’s
hematein. The pale bands of the fiber are beginning to become granular at one end. Fibrin threads
have been spread over it in making the smear preparation. (>86o0.)

Fic. 2. A bit of degenerating muscle fiber. Numerous artifacts and a degenerate erythrocyte
nucleus occur in the sarcoplasm. The granular striz are degenerated sarcolymph. Note the sarco-
plasm is also becoming granular. ( 860.)

Fic. 3. From a smear of a bit of degenerating muscle in a sore on the side of Fundulus majalis.
The integument more or less disintegrated, scales entirely absent. Fixed in absolute alcohol, ether,
and formaldehyde. Stained in methylene blue, orange G, and eosin. Sarcous elements have lost
their sharp rectangular form and are becoming granular. A characteristic muscle artifact is distributed
between the sarcostyles and some are just beginning to become ameeboid in form. ( 2000.)

Fic. 4. A characteristic appearance of a degenerating muscle fiber which may or may not be a later
stage than those represented in figures 2 and 3. Neither bacteria nor Myxosporidia are necessarily
present in these spaces. Both have been encountered there. ( 400.)

Fic. 5. A fragment of degenerating muscle upon and into which erthrocytes and leucocytes have
entered. The cytoplasm of the latter is disintegrated and the nuclei are in an advanced stage of
degeneration. (X2000.)

Fic. 6. Atypical mass of degenerate nuclei containing unstained bodies which are probably zoéglcea
containing the short bacillus. There are cords of this material in which the bacilli are faintly visible.
Such white areas are not merely transparent spaces but thick masses with stainable protoplasm above
or below. ( 2000.)

Fic. 7. Artifacts from decomposing muscle fibers. In fresh muscle these are common after 10 to
12 hours, appearing first between the sarcostyles. Older stages assume a more compact form. (See
figures 3 and 2.) The stain is a homogeneous pale blue. Maximum length 8.9u. (X2000.)

Fic. 8. The short bacillus. An isolated group near which are located cells containing white oval-
shaped bodies like those in figure 6. Note the variation in size and shape. That one near the “ X”’
sign measures 1.5 by 7.4; that near the ‘‘+’’ sign measures 1.84 by 1.1. (2000.) (See also fig. 10.)

Fic. 9. Short bacillus older than figure 8. Nearly the maximum size. Note the taper toward
one end and the stainable granules. The latter are probably artifacts. Left-hand upper one measures
5-24 by 1.4". (X2000.) |

Fic. to. A cluster of long bacilli which have caused the complete breakdown of a tissue cell and
rest in situ. (X2000.)

Fic. rr. Several of the long type of bacilli which are located just under the sarcolemma of a muscle
fiber that shows the first signs of degeneration. The small individual in the middle below has dimen-
sions as follows: Length, 4.84; thickness, 0.7. (> 2000.)

212

SPOROZOON PARASITES OF FISHES. 213

Fic. 12. A section cut diagonally through a muscle fiber. This fiber is adjacent to the dermis.
On the inner side the sarcoplasm is hypertrophied, on the outer side it retains the fibrillation. The
oval bodies are interpreted as trophoplasms of the M. musculi. The large one has several spherical
bodies which take a deep hematein stain, presumably nuclei. (X8o0.)

Fic. 13. A muscle fiber in which there are the first evidences of disintegration. It contains two
or more large trophoplasts or schizonts. The appearance of the cytoplasm is like that of other
stages, pale and unstained, there being no sign of the nucleus. There is evidence of a complex system
of pseudopodial extensions of the cytoplasm which is characteristic of the Myxosporidia. Large indi-
vidual 84.74 by 192.54. (X400.)

Fic. 14. Multiplicative spores of M. musculi, presumably derived from a large trophoplasm such
as figure 13. There isno cyst wall. In adjacent sections are fragments of the schizont nuclei mingled
with the spores. The spores stain feebly with eosin and orange G. The nuclei are not stained deeply.
Ig.3: in diameter. (> 860.)

Fic. t5. A myxoplasm of M. musculi in muscle from a smear preparation fixed with absolute alcohol
and ether and stained with methylene blue. One side overlies a nucleus of the muscle fiber. The
pale bands of the muscle fiber may be seen. The muscle stained deeply and the parasite pale. The
protoplasm is finely granular and there is only a suggestion of a cytoplasmic network. The nucleus is
vaguely stained. 13.4 by 18.6. ( 2000.)

Fic. 16. Formation of sporoblasts of M. musculi. This cyst is one of a mass numbering several
hundred which occupy a position where a muscle fiber has been completely destroyed. The to spores
stain very feebly. They lie in slight cavities of the protoplasm. Diameter of cyst 12”; length of

spore 4. (2000.)
PLATE XXI.

Fic. 17. A possible microgamete of M. musculi from amongst the numerous myxoplasms of muscle
fibers adjacent to that shown in figure 18. The motile shape of several such structures, the small
amount of cytoplasm, and close approximation to some of the large myxoplasms are noteworthy. (See
right-hand upper region of fig. 18.) 6.54 by 2.24. (X2000.)

Fic. 18. A section of a muscle fiber of Fundulus heteroclitus cut crosswise at a slight angle. The
scales in the region of this infection had dropped off, and the area was almost white, being slightly
discolored by blood. The tissue was fixed in corrosive sublimate and acetic acid and stained first in
Mayer’s hematein, then in methylene blue, later in eosin and orange G. One of the structures in the
sarcoplasm, that to the left in the middle, is the nucleus of a muscle fiber. The others are stages in
the propagative cycle of M. musculi, primary and secondary sporoblasts. The large one in the middle,
at the top, is 12.64 in length and 5.9” in width. (X860.)

Fic. 19. Three young sporoblasts of M. musculi from the smaller type of cysts represented in
figure 16, plate xx. Note the increase in the size of the nuclei. They are typically free from cyto-
plasmic stain. (See C. funduli, fig. 31.) Lower individual 4y by 2.54. (X2000.)

Fic. 20. A fresh sporoblast of M. musculi containing a spore which is almost mature. From a deep
cavity in the flesh back of the head. Interesting in connection with figure 26. (Free-hand drawing,
not to scale.)

Fic. 21. Sporocyte of M. musculi expelled from pansporoblast. It forms the first stage in the
series represented by figures 23, 24, and 25. The nucleus is small and faintly stained, as is the rest of
the cytoplasm. It has no external envelope. Diameter 11.94. (2000.)

Fic. 22. A pansporoblast of M. musculi (sporocyst) with two daughter cells, the nuclei of which
are undergoing autogamous conjugation. ( 2000.)

Fic. 23. A pansporoblast of M. musculi after the autogamous conjugation and subsequent division
of the nuclei.

Fic. 24. A sporocyst of M. musculi which has been set free from the pansporoblast. Apparently
the sporoplasm remains attached to one myxospore (fig. 20), and the other is almost devoid of external
protoplasm. The two wall cells are clearly visible, but without nuclei. The capsule nuclei are prob-
ably formed but donot stain. Oneof the 12 nuclei happens to be in a suitable condition to take the stain.
Il.ou by 13.4u. (X2000.)

Fic. 25. A myxospore of M. musculi with a remnant of protoplasm. Two polar capsules are
beginning to form. (>2000.)

214 BULLETIN OF THE BUREAU OF FISHERIES.

Fic. 26. A sporocyst of M. musculi from a smear of diseased integument of the mouth and head
in front of the eyes. Elsewhere the sporocysts have less cytoplasm. It is the only one encountered
in this condition. The failure of the nuclei to take the stain is characteristic. The myxospore is
immature, being less slender than older myxospores. The details of the polar capsules are very trans-
parent and stain dark blue, while the spore wall is a very pale blue. ‘The vacuole and sporoplasm are
prominent, but the nuclei of the spore can not be clearly discerned. Sporocyst, 17.8 by 23.84; spore,
14.8 by 7.4; polar capsule, 7.4 by 2.24. There are 13-14 spirals in the filament. Fixation: Absolute
alcohol, ether, corrosive sublimate, acetic acid. Stain: Mayer’s hematein, methylene blue, orange G.,
eosin. (> 2000.)

Fic. 27. Asporoblast of M. musculi from a fresh smear of degenerated muscle taken from a deep
cavity (the same as fig. 20). Easily distinguished from tissue cells by the three nuclei. Protoplasm
contains much coarsely granular matter. (Drawn free-hand, not to scale.)

Fic. 28. Myxospore from the same slide as figure 26. The mature spore, when compared with that
in the pansporoblast, is longer and more pointed at the polar end. The vacuole is probably an iodi-
nophilous structure. The coiled filaments make 11 to 12 turns. The polar capsule wall is visible, but
the spore wall can not be clearly seen. The valves and suturesare also indistinguishable. While there
are as many as 12 blue and green bodies present, one can not be sure that allof them arenuclei. Seven
or eight bodies are moderately conspicuous. ‘Two lie in the wall of the polar capsules and are doubtless
the capsule nuclei. 14.84 by 6.2/1. (X2000.)

Fic. 29. A myxospore of M. musculi from large sores on each side of the tail of a Fundulus heteroclitus,
caudal fin entirely gone. Fixed in absolute alcohol and ether, stained with methylene blue. Six
unstained nuclei in the sporoplasm and one large vacuole. Filament discharged. Spore, 7.4 by 16.4m.
Polar capsule, 2.211 by 7.44. (X2000.) 3

Fic. 30. Diagram of the cross section of a fresh myxospore of M. musculi as if seen from the end.
The specimen was lying so as to present the edge of the valves to view. It is obviously flattened. The
polar capsules also appeared to be, but one can not be certain about this. The sutures are straight and
symmetrical. Fixation: Alcohol, ether, formalin; Giemsa stain. (This drawing not made to scale.)

[Figures 31 to 34 are all from the same smear preparation of diseased muscle from a dead fish, being
one of those taken from jar no. 1 (see pp. 195, 196).]

Fic. 31. Pansporoblast of Chloromyxum funduli embedded in a degenerated muscle fiber. The
contained myxospore has taken up the stain, but the protoplasm of the pansporoblast is absolutely
devoid of visible structure. Note the even contour of the characteristic lobose pseudopodia. 15.24 by
1241. (X2000.)

Fic. 32. One of a group consisting of free young myxospores of C. funduli. Like the mature
myxospores, they stain readily, but their nuclei are not differentiated. They are, as a rule, not quite
so irregular, but the pseudopodia are always small and angular. Note the contrast between these and
the pansporoblasts. 3.74 by 4.5. (X2000.)

Fic. 33. Myxospore of C. funduli. The outline is approximately circular. The sporoplasm is
homogeneous but dense around the four polar capsules, doubtless because of the greater thickness at
this point. The four nuclei are always associated with the polar capsules, hence are doubtless capsule
nuclei. Diameter, 8.94. (X2000.)

Fic. 34. Myxospore of C. funduli seen from the side. Note the sporoplasm is not much denser
about the polar capsules. The sporoplasm tapers to a blunt apex. In many it is more pointed. The
polar capsules have long, curved, tapering necks with the large ends far apart. The capsule nuclei
alone stain. 8.24 by 6.74. (X2000.)

Fic. 35. A fresh sporoblast of M. musculi from the same slide as figures 20 and 27. The cyto-
plasm is rich in granules. The nucleus is very large and has a conspicuous karyosome. (Not drawn
to scale.)

Fic. 36. An isolated epidermal cell derived from a mass near the margin of an advanced ulcer,
most of which have numerous unstained bodies like those in the muscle fibers (fig. 12, pl. xx). From
sections. The epidermal cell is not typical in appearance, but the unstained bodies are, and are iden-
tical to those in the adjacent slightly atrophied epidermis. (X2000.)

*

Burr Wes. B:
LL Se Bees, Lon. PLATE XX.

awe

ee ee
o> ow

obi 3 .
Weeeoet in

r

IPE NGNS; NOMI

Bieri, Wi, So Wy 1s, Mewas

Gaylord Bros.
Makers
Syracuse, N. Y.
PAT, JAN, 21, 1908

WW
Hin}

Ni

=< =
asapa ee ee ee

SS

i.