AN ABSTRACT OF THE THESIS OF
Antonio Amandi
for the degree of
Fisheries and Wildlife
in
Master of Science
presented on
August 10, 1977
Title: EFFECTS OF NANOPHYETUSSALMINCOLA, THE SALMON
POISONING TREMATODE, ON GROWTH AND HEMATOLOGI-
CAL CHARACTERISTICS OF COHO SALMON AND STEELRedacted for privacy
Abstra
Growth and hematology of coho salmon (Oncorhynchus kisutch)
and steelhead trout (Salmo gairdneri) infected with different numbers
of the salmon poisoning trematode (Nanophyetus salmincola) and fed
five rations of Rubifex sp. worms were measured.
Growth rates were not affected by the numbers of trematodes
recovered from the infected fish. Minor effects-were found on
hematological characteristics of infected fish as measured by hernatocrits, hemoglobin concentration (Hb), red blood (RBC) and white blood
cell (WBC) numbers, thrombocyte-numbers, mean corpuscular voiume (MCV), mean corpuscular hemoglobin (MCH) and-mean corpuscu-
lar hemoglobinconcentration (MCHC). Hematology was affected by
handling. Coho salmon hematocrits, Hb, RBC numbers, MCV and
MCH- values increased while WBC and thrombocyte numbers
decreased. In steelhead trout RBC numbers, HB, MCH and MCHC
values increased while hematocrits, WBC and thrombocyte numbers
and MCV values decreased after handling.
Lighter infections were found in fish exposed daily to the
trematode compared with those exposed only once. This may be
related to a defense system in the fish.
e
Effects of Nanophyetus salmincola, the Salmon Poisoning
Trematode, on Growth ahd Hematological
Characteristics of Coho Salmon
and Steelhead Trout
by
Antonio Amandi
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed August 1977
Commencement June 1978
APPROVED:
Redacted for privacy
Professx of Microiology
in charge of major
Redacted for privacy
Chairman of Department of Fisheries and Wildlife
Redacted for privacy
Dean of Graduate School
Date thesis is presented
August 10, 1977
Typed by Mary Jo Stratton for
Antonio Amandi
ACKNOWLEDGEMENTS
I would like to thank Dr. Raymond E. MiUemarm for his help
before and during the research and for later reviewing the manuscript.
My special thanks to Dr. John L. Fryer for giving me the chance to
complete my program, which I was unable to finish under Dr.
Millemann, and for his great patience, assistance, and review of the
manuscript.
The Oregon Department of Fish and Wildlife supplied the fish
and gave me access to the worms used in the experiments. I am
indebted to Oregon Aquafoods Inc. for providing fish food.
I am grateful to Dr. Richard A. Tubb for his assistance.
My thanks go to the people of the fish pathology laboratorywho
readily gave me assistance and information.
Above all, I want to thank mywife Betty for her love and patience
and for always being there when I needed reassurance.
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
2
MATERIALS AND METHODS
Collectionand Maintenance of
Experimental Animals
Fish
Tubificid Worms
Snails
Experimental Procedure
Hematological Procedures
RESULTS
12
12
12
12
13
14
16
18
Parasite Recovery
Fish Growth
21
Hematology
Coho Salmon
Steelhead Trout
24
24
27
18
DISCUSSION
30
SUMMARY AND CONCLUSIONS
39
LITERATURE CITED
41
APPENDIX
46
LIST OF TABLES
Page
Table
1
Food rations, exposure levels and numbers of
Nanophyetus sairaincola recovered from -coho
salmon.
2
19
Food rations, exposurelevels and numbers of
Nano phyetus s almincola re cove red from steelhe ad
trout.
3
Food rations, numbers of parasites recovered,
weight and length changes and food consumed by
coho salmon infected with Nanophyetus salmincola.
4
6
22
Food rations, numbers of parasites recovered,
weight and length changes and food consumed by
steelhead trout infected with Nanophyetus
salmincola.
5
20
23
Food rations for andmean numberof parasites
re covered, hematocrits, hemoglobin concentrations (I-]Jb), numbers of red (RBC) and white blood
cells (WBC) and thrombocytes, mean corpuscular
volume (MCV), meancorpuscular hemoglobin
MCH) and mean corpuscular hemoglobin concentration (MCHC) from coho salmon infected
with Nanophyetus salmincola. -
25
Food rations for and mean numbers of parasites
recovered, hemato crit s, hemoglobin conce ntrations (Hb), numbers of red (RBC) and white
blood cells (WBC) and thrombocytes, mean corpuscularvolume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin
concentration MCHC) from steelhead trout
infected with Nanophyetus salmincola.
28
LIST OF FIGURES
Page
Figure
2
3
4
5
6
7
8
Meanweight of groups (10 fish per group) of
coho salmon on a 100% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
46
Meanweight of groups (10 fishper group) of
coho salmon on a 75% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
47
Mean weight of groups (10 fish per group) of
coho salmon on a 50% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
48
Mean weight of groups (10 fish per group) of
coho salmon on a 25% rationof Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
50
Mean weight of groups (10 fish per group) of
coho salmonon a 12. 5%rationof Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
50
Total length of groups (10 fish per group) of
coho salmon on a 100% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
52
Total lengthof groups (10 fish pergroup) of
coho salmon on a 75% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salrnincola cercariae.
52
Total length of groups (10 fish per group) of
coho salmonon a 50% ration of Tubifex sp.
worms and expOsed to different numbers of
Nanophyetus salmincola cercariae.
54
List of Figures (Continued)
Page
Figure
9
10
11
12
13
14
15
16
Total length of groups (10 fish per group) of
coho salmon on a 25% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
54
Total length of groups (10 fish per group) of
coho salmon on a 12.5% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
54
Mean weight of groups (10 fishper group) of
steelhead trout on a 100% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
56
Mean weight of groups (10 fish per group) of
steelhead trout on a 75% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
58
Mean weight of groups (10 fish per group) of
steelhead trout on a 50% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
59
Mean weight of groups (10 fish per group) of
steelhead trout on a 25% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
61
Meanweight of groups (10 fish per group) of
steelhead trout on a 12. 5% ration of Tabifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola ce rcariae.
61
Total length of groups (10 fish per group) of
steelhead trout on a 100% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
63
List of Figures (Continued)
Page
Figure
17
18
19
20
21
22
23
24
Total length of groups (10 fish per group) of
steelhead trout on a 75% ration of Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
63
Total length of groups (10 fish per group) of
steelhead trout on a 50% rationof Tubifex sp.
worms and exposed to different numbers of
Nanophyetus salmincola cercariae.
65
Total length of groups (10 fish per group) of
steelhead trout on a 25% ration of Tubifex. sp.
worms andexposed to different numbers of
Nanophyetus salmincola cercariae.
65
Total length of groups (10 fish per group) of
steelhead trout on a 12. 5% ration of Tubifex sp.
worms and exposed to different numbers of
Nano phyetu s s almincola ce rcariae.
65
Red blood cell numbers of coho salmon
infected with different numbers of Nanophyetus
salmincola metacercariae and fed different
rations of Tubifex sp. worms.
66
White blood cell numbers of coho salmon
infected with different numbers of Nanophy
salmincola metacercariae and fed different
rations of Tubifex sp. worms.
67
Thrombocyte numbers of coho salmon infected
with different numbers of Nano.phyetus saLrnincola
metacercariae and fed different rations of
Tubifex sp. worms.
68
Red blood cell numbers of steelhead trout
infected with different numbers of Nanophyetus
salmincola rnetacercariae and fed different rations
of Tubifex sp. worms.
69
List of Figures (Continued)
Page
Figure
25
26
White blood cell numbers of steelhead trout
infected with different numbers of Nano phyetus
salmincola metacercariae and fed different rations
of Tubifex sp. worms.
70
Thrombocyte numbers of steelhead trout infected
with different numbers of Nanophyetus salmincola
metacercariae and fed different rations of
Tubifex sp. worms.
71
EFFECTS OF NANOPHYETUS SAUSVfINCOLA, THE SALMON
POISONING TREMATODE, ON GROWTH AND
HEMATOPOIESIS OF COHO SALMON
AND STEELHEAD TROUT
INTRODUCTION
Parasites may kill fish or they may produce sublethal effects
such as interference with fish reproduction, food conversion, resistance to other parasitic and infectious agents, behavior and hematopoiesis.
Nano phyetu s s almincola, the "salmon poisoning" t re matode, can
cause the death of fingerling salmonid fish with heavy infections.
Also, they i-nay affect adversely the swimming ability of infected fish.
Large numbers of metacercaria.e encysted in the kidney of infected
fish conceivably could interfere with the renal and hematopoietic
functions of the organ. Such sublethal effects could weaken heavily
infected fish making them more susceptible to secondary infection
or predation.
This studywas done to determine if N. salmincola could affect
the growthand hematological characteristics of coho salmon
(Qncorhynchus kisutçh) and stelhead trout (Salmo gairdneri),
2
LITERATURE REVIEW
Nanophyetus salmi,ncola, the ttSalmOfl poisoningt' trematode,
requires three hosts for completion of its life cycle. Oxytrema
silicula, a stream snail, the first intermediate host is parasitized by
the redial and cercarial stages of the trematode. Gebhardt et al.
(1966) showed that in addition to salmonid fish, which are the principal
intermediate hosts, several other species of fishes and the Pacific
giant salamander (]jcamptodonensatus) can also serve as second
intermediate hosts for the metacercarial stage of the trematode. The
definitive hosts are various fish-eating mammals and birds in which
the trematode reaches maturity and lays eggs (Millemann and Knapp
1970a). Bennington and Pratt (1960) described the life cycle of
N. salmincola, and Millemann and Knapp (1970a) reviewed the biology
of the parasite and the course of "salmon poisoning" disease.
The enzootic area of N. salmincolais determined by the geographic distributionof its snail hDst, which inhabits the Pacific
coastal region west of the Cascade Mountains extending from the
Olympic Peninsula in Washingtonto the Smith River in northern
California (Sirnms et al. 1931a).
Salmonid fish within the enzootic area are often heavily
infected. Simms et al. (193 la, b) recovered 14, 062 encysted meta.e from one coastal cutthroat trout (Salmo clarki clarki) 12 cm
3
long, and in another fish of the same species 36 cm long they counted
2, 676 cysts in 65 rng of kidney tissue. Donham et al. (1926) estimated that approximately one million metacercariae were contained in
1 lb (0. 45 kg) of muscle tissue from a spawned out salmon. Ward and
Mueller (1926) counted up to 1, 600 parasites in a brook trout
(a1velinusfontinalis) 45 mm long obtained from Willamette Trout
Hatchery on the Willamette River during a N. salmincola epizootic.
In a study of natural infections of coho salmon and steelhead
trout from three Oregon streams, Bender (unpublished data cited by
Millemarmand Knapp 1970a) found that coho salmon and steelhead
trout 50-60 mm long collected du.ringthe fall months had from 400 to
1, 565 and from 1, 735 to 2, 002 parasites, respectively. He also
determined that the daily infection level in coho salm9n under natural
conditions ranged from 6 to, 143 parasites.
Baldwin et al. (1967) determiied experimentallythe 24-hr
lethal exposure levels (24-hr LE50
the exposure level, in numbers
of cercariae, lethal to 50% of the exposed fish in 24 hr) and the 24-hr
lethal dosage levels (24-hr LD50
the infection level, innumbers of
rnetacercariae, that results in 50% mortality of the infected fish in
24 hr) for six species of salmonid fish. The 24-hr
LE50,5
for koka-
nee salmon (Q. nerka), Montanablack-spotted cutthroat trout
.
clarki lewisi), Atlantic salmon (. sal,ar), coho salmon, rainbow
trout
.
gairdneri), and coastal cutthroat trout were 94, 135, 157,
315, 440 and 700, and the 24-hr LD
50 s
were 58, 74, 110, 200, 295
and 430, respectively.
Farrel and Lloyd (1962) showed that rainbow trout could
accumulate large numbers of metacercariae if they were gradually
infected during a period of one month, but thisnumber killed fish
during a 12-hr exposure period. Millemannand Knapp (1970b) also
reported that fish tolerated daily exposure to small numbers of
cercariae and accumulated over a period of time numbers of meta-
cercariae that in a 24-hr period would be lethal. These findings may
explain the occurrence of heavy infections inwild fish as reported by
others.
Encysted rnetacercariae occur in almost every tissue of infected
fish. They were found in the kidneys, muscles, gills, hearts, livers,
eyes, gall bladders, intestines and tongues of yearling coho salmon
from a hatchery population by Wood and Yasutake (1956a). Ward and
Mueller (1926) also recovered parasites from the optic nerves and
brains of infected brook trout.
Bennington and Pratt (1960) found extensive lesions in the fins,
gills, tails, retinas and corneas of infected wild fish. They concluded
that these lesions were caused by the migration of cercariae during
penetration. Millemann and Knapp (1970a) repoited that salmonid fish
died 24 to 48 hr after peritoneal injection of homogenized cercariae.
The large hemorrhagic areas that developed were similar to those
5
produced by living cercariae. They postulated that proteolytic
enzymes or other substances released by cercariae to aid in their
migration could be toxic to fish and cause tissue damage.
Obstruction and mechanical injury to the ventricle of the heart,
retinas, kidney tubules, pancreatic tissue and gall bladder wall were
observed by Wood and Yasutake (1956a) in yearling coho salmon
infected with metacercariae of N. salmincola. They concluded that
practically every parasitized organ of the fish was weakened physiologically. If metacercariae injure the organs as suggested above,
then the increase in size of encysted metacercariae with increasing
age of the infection as demonstrated by Gebhardt et al. (1966) may
enhance this damage.
In laboratory experiments, Baldwin et al. (1966) found that
60-76% of the cercariae used in exposure tests penetrated and encysted
in salmonid fish. Thus, when a high number of infected snails is
present during periods of low water the pathogenic effects to young
fish may be increased (Bennington 1951).
Anadromous fish retain the parasite during their ocean residence. Farrell et al. (1964) studied coho salmon kept in salt water
ponds at Bowman's Bay Station near Anacortes, Washington for 33.5
months and found viable metacercarlae in the fish at the end of this
time. Millemann et al. (1964) found viable metacercariae in 24 of 43
adult coho salmon nd three of four adult chinook salmon
(Q.. tshawytscha) caught one to six miles (1.6 to 9.6 1cm) off the Oregon
coast near Newport. These fish had not been in fresh water during the
previous two to four years; therefore salinity has no effect on rnetacercarial viability as previOusly believed.
Butler and Millemann (1971) studied the swimming performance
of coho salmon and steelhead trout 57-60 mm in total length infected
with known numbers of parasites. The fish were exposed either once
for 1 hr to 1, 500 cercariae or daily for 1 hr to 100 cercariae for 15
days. Those fish exposed only once were tested either immediately
after exposure or after 6, 12, 24, 96 hr and 15 days, and those
exposed dailywere tested immediately after the last exposure. The
swimming ability of fish exposed once and tested 15 days later was
slightly affected, but a marked reduction in swimming abilitywas
observed for those fish exposed once and tested 0-96 hr later, and for
those exposed repeatedly and tested after their last exposure. The
percent reductions in swimming abilities for steelhead trout and coho
salmon were 10-85% and 4-95%, respectively.
Simms et al. (1931a) studied the effects of metacercariae on
naturally parasitized eastern brook trout with light infections. They
divided the fish into two groups and placed them in separate tanks.
One tank contained snails infected with N. salmincola. During the
experiment, they found no appreciable differences in size, growth
rate, general appearance or activity between the two groups, although
7
at the end of the experiment they found that the number of parasites
was greater in all fish reared in the tank with the snails.
Millemann and Knapp (1968) in a preliminary study determjned
the effect of the parasites on the growth of coho salmon that were fed
unrestricted amounts of tubificid worms. The fish were exposed daily
for 1 hr to 25, 75, or 225 cercariae for 24 days. They found that the
weights of the fish exposed to 0, 25, 75 and 225 parasites daily
increased by 140, 73, 57 and 48%, respectively.
Heniatological data as indicators of disease in fish have not been
widely used. The blood characteristics of several species of fish
infected with Aeromonas salmonicida, the causative agent of furunculo-
sis, have been studied by several investigators. Field et al (1944)
reported that hemoglobin concentration and white and red blood cell
numbers in infected carp (Cyprinus carpio) were slightly affected, but
a rapid fall in blood sugar and increases in total blood nonprotein
nitrogen occurred. The blood of brook trout with furunculosis had a
faster sedimentation rate compared with noninfected individuals
(Schumacher et al. 1956). A decrease in hematocrits, from 3550%
to 5-15%, and hemoglobin, from 9.5 gJiOo ml to 2.7 g/100 ml, was
found by Foda (1973) for Atlantic salmon infected with A. salmonicida.
The blood of infected fish also had a faster sedimentation rate than
that of normal fish. Shieh and MaClean (1976) found that total blood
protein and hemoglobin from brook trout with furunculosis decreased
while amino acids and glucose values increased.
Hematological effects of Vibrio anguillarum, the causative agent
of vibriosis, in fish have been reported. Yamashita (1967) found
decreased hematocrits, blood serum protein levels and blood specific
gravity values in rockfish (ebasticus marmoratus) that developed
ulcers during use in experiments on the influence of chlorinity on the
blood. He did not determine the etiology of the ulcers but Anderson
and Conroy (1970) stated that V. anguillarum was the cause. Anderson
and Conroy (1970) reported decreases in hematocrits, hemoglobin concentration and red blood cell counts in cod (Gadus morhua), lemon
sole (Pleuronectes microcephalus), plaice (Pleuronectes platessa)
and turbot (ombus inaximus) infected with V. apgu.illarum. Cardwell
and Smith (1971) found decreases in hernatocrit, hemoglobin concentra
tion, total cell count and total blood dissolved solIds in vibriosis
infected chinook salmon.
Hunn (1964) reported a 44% reduction in hematocrit and a drop of
52% in the total plasma protein of brook trout with bacterial kidney
disease. He attributed the loss of plasma protein to three factors:
loss via external open lesions, from reduced liver function and via the
urine due to kidney damage. Electrophoresis of the plasma protein
showed that the albumin fraction was the most drastically affected.
Kusuda (1975) observed that the cultured yellowtails (iola
guingueradiata and S. purpurascens) infected with Nocardia kampachi
had lower hematocrits, hemoglobin concentrations and red blood cell
numbers compared with healthy individuals. Young (1949) found that
hematocrits of the opaleye (Qirella nigricans) were lowered when the
fish became infected with tail rot.
Hematological changes in fish infected with viruses have been
reported. Watson et a1 (1956) observed an increase in hematocrits
at the onset of disease (agent not specified, but it was most likely
infectious hematopoietic necrosis virus or IHN) in sockeye salmon
0. nerka); then a three-fold decrease on the eighth day postinfection.
Basophilia and prolonged clotting time also were attributed to the
infection. Wood and Yasutake (1956b) observed an increase in the
number of immature erythrocytes in 90% of sbckeye salmon infected
with fl-IN. Decreased red blood cell and leukocyte numbers, hemo-
globin concentration and hematocrits were seen by Amend and Smith
(1975) in rainbow trout infected with IHN virus. Mean corpuscular
volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration values remained normal in diseased fish.
Arnlacher (1961, cited by Reichenbach-Klinke 1966) found
de creases in red blood cell numbers from l 3 million to 300, 000/mm3
and hemoglobin concentration from 80 to 10% in rainbow trout infected
with viral hemorrhagic septicemia. The numbers of red blood cells
10
and hemoglobin concentration were lowered in diseased carp with
dropsy (viral etiology at least partly responsible for the disease)
(Aml.acher and MtIler 1961, cited by Reichenbach-Klinke 1966).
Mulcahy (1967, 1969, 1975) showed that blood protein concentra-
tion decreased in Atlantic salmon with ulcerative dermal necrosis
(TJDN).
Electrophoretic patterns of diseased fish differed from those
of healthy ones. Mulcahy (1971) also found similar decreases in blood
protein content and changed electrophoretic patterns in brown trout
(. trutta) with UDN. Wild northern pike (sox lucius) with spontaneous lymphomas (presumed viral etiology) had lowered red blood
cell numbers, hematocrits, hemoglobin concentrations and serum
total protein concentrations (Mulcahy 1975).
Reichenbach-Klinke (1966) found in wild coregonids (Gogonus
wartmanni) that with anincrease in numbers of the tapeworm Proteocephalus percae there was a decrease in red blood cell numbers and
hemoglobin concentration while the number of leukocytes increased
in some fish. He also found a correlation between increased numbers
of tapeworm cysts of Triaenophorus crassus in the liver of the arctic
char (. alpinus) and decreased red blood cell numbers in fish
simultaneously carrying heavy infe ctions of the trematode Disco cotyle
sagitta.
Evans (1974) experimentally infected cutthroat trout with the
blood trematode Sanguinicola kiamathensis and found a decrease in
11
hemoglobin concentration from 10.4 g/100 ml to 3.8 g/100 ml and
lowered hematocrits (from 40 to 16%) in the fish. Smitherman (1964)
reported decreased hematocrits in bluegills (Lepomis macrochirus)
infe cted with the t re matode Po sthodi plo stomum minimum.
RiedmUller (1966) observed that blood proteins of carp can
change in quantity and composition when fish become diseased. She
found in serum from diseased fish that the albumin fraction was
reduced or absent, the total protein was decreased and in some cases
gamma globulin was increased.
12
MATERIALS AND METHODS
Collection and Maintenance of Experimental Animals
Fish
Steelhead trout eggs and coho salmon fingerlings were obtained
from the Alsea River Trout Hatchery and the Fall Creek Hatchery,
respectively, of the Oregon Department of Fish and Wildlife (ODFW).
The trout' eggs were hatched in Heath incubators and the salmon fry,
which were 40 mm long, were maintained in 190-liter fiberglass tanks
with flowing dechlorinated water at 8-12 C. Because infected snails
are present in the creek supplying water to the hatchery, 100 coho
salmon fry were examined and found free of natural infections.
Tubificid Worms
Tubifex sp. were collected from earthen-bottom raceways of the
Roaring River Trout Hatchery (ODFW) and from the river below the
hatchery. The worms were kept in the laboratory in a trough 1.22 m
long by 0.61 m wide by 0. 15 m deep provided with chilled (8-12 C)
flowing, dechlorinated water and river mud and organic debris that
served as a substrate.
To separate the worms from the mud and debris, handfuls of
the material were placed in a 38 cm longby 19 cm wide by 2 cm deep
13
rack with a fiberglass screen bottom of 6 meshes/cm and thoroughly
rinsed by running a stream of water over the mixture. This elimi-
nated the fine silt leaving the worms and debris on the screen. Rocks,
leaves and large pieces were then removed. The rack was placed on
top of a 19-liter styrofoam cooler chest supplied with flowing, cooled,
dechlorinated water and under a 100 watt light bulb. The water level
was maintained 9 cm below the bottom of the rack to keep fine debris
from entering the water in the chest due to worm movements.
To avoid the heat or light, the worms pass through the screen
and fall into the water in small masses. They were left in the chest
for at least 24 hr before being fed to fish to allow for defecation and
weight changes due to osmotic effects.
Snails
Snails were collected from Marys River, Big Elk Creek,
Dixon Creek and Oak Creek in western Oregon. To determine if the
snails were infected, they were placed in water in individual con-
tainers at room temperature; the water was examined for
salmin-
cola cercariae 3-4 hr later. Tjninfected snails and those with mixed
infections were discarded.
The infected snails were kept in troughs 1.22 mlong by 0.61 m
wide by 0. 15 m deep, which were supplied with running, dechlorinated
water at 8-12 C. The low water temperature inhibits cercarial
release. Clam shells in the troughs served as a calcium source. The
snails were fed lettuce once each week.
perimental Procedure
Before beginning each experiment, 11 fish were put into each of
25 19-liter styrofoam containers provided with flowing, aerated water
at 17 C. They were acclimated to these test conditions for 14 days.
The coho salmon were 65-68 mm in total length and weighed 2.2-3 6
g; the steelhead trout were 80-8 5 mm in length and weighed 4. 1-7. 1 g.
During the acclimation period the fish were fed worms adlibitum. On
the fifteenth day, one fish was removed from each container and used
for determination of preinfection hematological levels. At this time
weights and lengths of each of the remaining fish were recorded.
Five groups, each with 50 fish, were used. Fish in one group
were exposed once to 1, 500 parasites and those in three of the remain-
ing groups to 225, 75 or 25 parasites daily for 20 or 24 days. The
fifth group served as uninfected controls. Each group was divided
into five equal subgroups. Each subgroup was put on one of the following five feeding rations: 100, 75, 50, 25 or 12.5%. The daily worm
consumption of fish was determined in preliminary experiments, and
the 100% ration exceeded this amount. The remaining rations were
calculated daily using the amount of food eaten on the preceding day by
uninfected fish on the 100% ration as the basis for the calculations. If
15
fish died during the experiment, then the food ration was adjusted
accordingly for that group, except for fish on 100% ration, so that the
amount of food eaten dailyby each fish remained proportionatelythe
same. Before worms were weighed they were dried with a blotter to
remove any excess water. They were then dispersed byhand in the
fish tanks. After 24 hr, the uneaten worms were removed, dried with
a blotter and weighed.
When cercariae were needed to infect fish, 60 infected snails
were placed in beakers of water under a lamp for 30-60 mm. The
released cercariae were pooled and counted under a microscope. The
same snails were used every fourth day.
Fish were exposed for 1 hr to cercariae in 1,000 ml beakers
containing 150 ml of aerated water. On each day, the first fish were
exposed approximately 3 hr after feeding. These fish were exposed
last on the following day. By such adjustment of the times of exposure, effects on digestion and assimilation of food by fish due to
handling were assumed to be approximately the same for all fish
during the experiment. At the time of exposure, individual weights
and lengths were recorded using the apparatus described by Baldwin
(1967). The coho salmon were infected for 24 days, but steelhead
trout for only 20 days because not enough parasites were available for
longer infections.
i6
Fish were kept for 10 days after their last exposure because this
is the minimum time required for complete encystment of metacer-
cariae. During this time, the fish were fed and their weights and
lengths recorded as described previously. At the end of the 10 days,
the walls of the metacercariae are sufficiently developed to resist the
effects of homogenization of host tissue, which is required for their
recovery for counting. The homogenization- sedimentation procedure
described by Gebhardt et al. (1966) was used for recovery and enu-
meration of metacercariae. When fish were killed, blood samples
were taken from the caudal peduncle as suggested by Hesser (1960).
Hematological Procedures
Blood was collected in heparinized microcapillary tubes. Tubes
for hematocrit determination were centrifuged for 3 mm and read on a
Spiracrit microhematocrit computer. Blood cell counts were determined with a hemocytorneter with dilutions and staining carried out in
Unopette vials as described by Meyers (1975). Hemoglobin concentra-
tions were determined using Unopette vials
To 4.98 ml (1:250 dilu-
tion of Uno-heme solution) 0.02 ml of blood were added. The mixture
was allowed to stand for 30-60 mm to allow for breakage of red blood
cells. Hemoglobin concentrations were determined colorimetri cally.
17
The following values were calculated (Miale 1967):
Hematocrit x 10
Meancorpuscularvolume (MCV)
RC count (millions/mm3)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin
concentration (MCHC)
Hb (g%) x 10
RBC count
(millions 1mm3)
Hb (g%) x 100
Hematocrit
RESULTS
As noted previously N. salmincola was not found in 100 coho
salmon sampled from Fall Creek Salmon Hatchery stock. The number
of fish examined provides a 95% confidence of detecting parasites
with an assumed incidence of parasitism at or greater than 5% (Amer.
Fish. Soc. Fish Health Sec. 1975). The fish were obtained in mid-
May and cercarial release prior to this time does not occur because
of low water temperatures; therefore, it is unlikely that the test fish
had natural infections.
Parasite Recovery
As expected, there was generally an increase in the numbers of
parasites recovered from fish with increases in exposure levels
(Tables 1 and 2). The percentages of metacercariae recovered from
coho salmon ranged from 2-27% and for steelhead trout from 12-34%.
and corresponding parasite numbers ranged from 14-1, 017 and from
15-1, 478. Experimental errors in counting the parasites, in exposing
fish to the parasites and in recovery of parasites undoubtedly explain
thevariations among comparable exposure groups. My recovery rate
was much lower than the 61-76% reported by Baldwin et al. (1967) for
coho salmon obtained from the same hatchery and infected in a simi
lar manner.
The percent of encysting parasites was higher for fish
Table 1. Food rations, exposure levels and numbers ofaNanophyetus salmincola recovered from coho salmon,
Rationb
100
Exposure levelC
75
225
25
75
225
12.5
(
(
94-528)
40 23-64)
217 (108-526)
648 (507-935)
339
(
(
25
75
225
12
8
14
23
7
12
12
0
346 (249-634)
36-133)
85
(
63-325)
645 (456-806)
158
(
0
219 (134-348)
48 20-102)
231 (153-346)
499 (216-946)
(
23
14
9
12
-
15
8
13
9
0
0
1500
26
0
0
1500
25
(%)
0
0
1500
25
75
225
d
395 (154-545)
73 45-90)
151 70-260)
745 (607-1017)
0
1500
25
50
(No.)
0
1500
25
75
225
75
Metacercariae recovered
407 (276-569)
47 14-72)
27
50-182)
41-142)
6
2
(
114
83
(
(
8
aTh fish were held at 17 C.
b
Tubifex sp.
cFSh were exposed once to 1500 parasites on day 1 of the experiment
or daily to 25, 75 or 225 parasites for the first 24 days of the
34-day test period.
dMean numbers; range in parentheses.
20
Table 2. Food rations, exposure levels and numbers of Nanophye-
tus salmincola recovered from steelhead trout, a
Rationb
Exposure leveiC
Metacercariae recovered
(No.)d
(%)
100
75
225
75
75
225
12
17
(
75
225
303 (202-449)
82 38-118)
268 (201-306)
916 (4a8-1478)
(
20
16
18
20
0
461 (325-565)
60-105)
80
217 (130-324)
689 (458-962)
(
31
16
14
15
0
382 (226-572)
76 48-114)
215 98-401)
794 (645-1020)
25
(
15
(
14
18
0
0
75
462
89
266
225
918
1500
25
24
0
0
1500
25
12.5
34
15-99)
258 (186-402)
1092 (896-1268)
60
0
1500
25
25
517 (418-667)
0
1500
25
75
225
50
0
0
1500
25
(%)
(322-679)
23-143)
(183-346)
(672-1130)
(
31
18
18
20
aThe fish were held at 17 C.
b
Tubifex sp.
CFish were exposed once to 1500 parasites on day 1 of the experiment
ordailyto 25, 75 or 225 parasites for the first 20 days of the
30-day test period.
dMean numbers- range in parentheses.
21
exposed only once whencompared with those exposed daily (Tables
1 and 2).
Fish Growth
As expected, the weights and lengths of fish increased with
increased rations (Appendix).
Apparently, the parasites had little
effect on growth of coho salmon (Table 3, & Appendix). Two possible
exceptions were fish on a 75% ration with an average of 217 and
parasites (Table 3). They had weight gains of
increases of 17 and
16%
62
648
and 59% and length
compared with 75 and 20%, respectively, for
the controls, but the food consumption of all these fish was the same.
The loss of weight of fish on a 12.5% ration with an average of 83
parasites cannot be attributed to the infection because fish in the same
ration group with higher parasite numbers lost less weight.
As with coho salmon, there was apparently little effect of the
parasites on growth of steelhead trout except possiblywith fish on the
100% ration (Table 4, & Appendix).
In this group, fish with a mean
of 258 and 1,092 parasites consumed more food, 1.52 and 1.50
g/day/fish, respectively, than the controls did (1.37 g/day/fish), but
they gained slightly less weight than the controls (Table 4). This
stimulation of appetite with lower food conversion of stressed fish on
unrestricted rations has also been observed by Leduc
(1966)
for the
22
Table 3. Food rations, numbers of parasites recovered, weight and
length changes and food consumed by coho salmon infected
with Nanophyetus salmi,ncola. a
Mean weight Mean length
b
changed
changed
Food consumed
No. of
Ration
parasitesc
(g/day/fish)
(mm)
(g)
(%)
(%)
(%)
0.61
22
16.2
3.4
83
100
0
0.68
23
16.9
395
95
3.8
0.61
22
16.0
82
73
3.4
21
0.59
15.3
151
3.4
79
21
0.57
15.0
3.3
83
745
75
3.0
3.0
3.0
2.6
75
77
70
62
648
2.5
0
0
339
40
217
50
346
85
158
645
25
0
219
48
231
499
12.5
0
407
47
114
83
0.49
0.48
0.49
0.48
0.50
20
59
14.8
13.6
13.9
12.4
11.8
2.0
1.9
2.0
2.0
2.0
53
11.5
16
46
7.4
12.6
10.5
10
18
15
9.9
14
0.5
0.6
0.5
0.7
0.9
11
14
12
4.8
0.17
0.17
0.17
19
22
6.0
4.1
7
7
7
8
6
-0.04
-0.1
-0.2
-0.2
-0.4
-1
-2
-5
-5
-8
1.6
2.8
2.9
2.7
2.0
2
0.09
0.09
0.09
0.09
0.09
52
51
50
4.9
5.4
19
19
17
16
4
4
4
3
0.34
0.34
0.33
0.35
0.35
0. 18
0.17
aThe fish were held at 17 C.
bTubifex sp.
CParasites recovered from fish exposed to 0, 1500, 25, 75 and 225
in descending order. Fish were exposed once to 1500 parasites on
day 1 of the experiment or daily to 25, 75 or 225 parasites for
24 days.
dDiffererlce between weights and lengths of fish on day 1 and day 34 of
the experiment. Each group at each ration level was composed of
10 fish.
23
Table 4. Food rations, numbers of parasites recovered, weight and
length changes and food consumed bysteelhead trout infected
with Nanophyetus salmincola.a
Ration
(%)
100
b
No. of
parasitesC
0
517
60
258
1092
75
0
303
82
268
916
50
0
461
80
217
689
25
0
382
76
215
794
12.5
0
462
89
266
918
Mean weiht
Mean length
change°
(g)
(%)
(mm)
(%)
(g/day/fish)
66
6o
18
40
38
35
48
32
17.6
16.2
17.3
18.2
16.9
17.6
16.2
17.3
18.2
16.9
25
11.5
23
21
11.4
14
10.0
12
12
10
11
9
1.37
1.38
1.32
1.52
1.50
1.04
1.05
1.01
1.01
1.04
0.68
changea
6.2
5.2
5.4
5.9
5.2
3.8
3.8
3.3
4.5
2.9
2.4
2.1
2.0
1.3
2.2
0.6
0.3
0.5
0.6
0.7
-0.4
-0.2
-0.3
-0.1
-0.7
61
63
53
22
7
4
5
9.4
8.5
5.5
3.4
4.2
17
18
19
18
i6
15
14
17
12
6
4
4
7
8
4.2
5.6
4
-5
1.5
-2
-3
-1
-8
2.7
2.9
3.8
2
3
3
1.3
7
4
1
Food consumed
0.69
0.68
0.68
0.68
0.34
0.34
0.34
0.34
0.35
0.18
0.17
0.17
0.17
0.17
aTh fish were held at 17 C.
b
Tubifex sp.
Cparasites recovered from exposure levels of 0, 1500, 25, 75 and 225
in descending order. Fish were exposed once to 1500 parasites on
day 1 of the experiment or daily to 25, 75 or 225 parasites for
20 days.
dDifference betweenweights and lengths of fish on day 1 and day 30 of
the experiment. Each group at each ration level was composed of
10 fish.
cichlid fish Cichiasoma bimaculatum exposed to different concentrations of cyanide.
Fish on the 75% ration with a mean of 916 parasites gained less
weight than the controls, 32 and 40% respectively, but their daily food
consumption was the same as the controls (Table 4). Also, their
length did not increase as much as the controls.
Fish on the 50% ration with a mean of 217 parasites gained less
weight than the controls, 14 and 25% respectively, but they consumed
the same amount of food as the controls (Table 4).
Initially, all steelhead trout lost weight (Figs. 11-15). This
species is more ttnervoustt than coho salmon in captivity so an initial
weight loss due to handling is not unexpected.
lIe matology
Coho Salmon
The hematocrits and hemoglobin (Hb) concentrations were higher
in postexposure controls compared with values obtained from pre-
exposure control fish (Table 5). When the same comparison is made,
red blood cell (RBC) numbers were higher in postexposure control
fish on 100, 75 and 50% rations, but white blood cell (WBC) numbers
were lower in all postexposure controls and thrombocyte numbers
were lower in four of the five postexpo sure controls (Table 5). Mean
Table 5. Food rations for and mean number of parasites recovered, hematocrits, hemoglobin concentrations (Hb), numbers of
red (RBC) and white blood cells (WEC) and thrombocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) from coho salmon infected with Nanophyetus
salmincola. a, b
Ration'
(%)
No. of
parasitesd
Hematocrit
Hb
(%)
(g%)
RBC
(x
106/mm)
WBC
(x l04/mm)
Thrombocytes
(x 104/mm)
MCV
(Cm3)
MCH
(pg)
MCHC
209
41.9
45.8
57.6
49.8
52.2
51.7
55.2
55.0
48.7
47.7
55.9
43.7
61.2
60.6
55.1
20.0
18.7
(%)
Preexposure
controlse
100
75
50
28
5.6
1.34
0
38
395
73
151
745
44
36
1.55
1.34
1.43
1.44
1.41
0
339
40
217
648
42
36
43
40
38
0
41
346
39
39
7.1
7.7
7.1
7.5
7.3
8.0
7.7
6.7
7.4
8.1
7.1
7.5
8.1
7.1
8.0
6.3
7.2
7.0
7.1
7.2
6.4
6.3
6.7
7.0
6.1
85
158
645
25
0
219
48
231
499
12.5
0
407
47
114
83
aThe fish were held at 17 C.
41
40
38
39
32
36
33
35
38
34
34
33
33
33
1.45
1.40
1.38
1.55
1.45
1.63
1.23
1.34
1.29
1.30
1.34
1.30
1.39
1.43
1.38
1.28
1.24
1.40
1.23
1.88
9.75
7.19
6.31
7.94
7.56
5.13
4.38
5.69
7.50
6.13
5.50
7.13
4.00
4.38
2.88
2.94
2.88
3.44
2.63
2.88
2.81
4.75
3.13
3.81
3.94
6.63
4.06
10.75
6.75
7.25
4.81
7.13
4.44
5.19
9.25
4.88
4.19
4.13
7.75
6.88
7.38
5.63
4.75
6.88
3.50
6.63
5.06
6.38
5.25
4.44
3.94
4.00
245
329
253
285
283
290
257
313
258
262
252
318
292
295
300
239
277
238
246
276
267
275
236
269
278
61.5
47.1
55.4
50.4
49.8
52.4
50.2
50.9
47.9
57.1
51.4
17.5
19.7
18.3
18.3
19.0
21.4
15.6
18.5
21.3
17.3
18.3
20.8
18.7
20.5
19.7
20.0
21.2
20.3
18.9
18.8
18.5
20.3
21.2
18.5
bMean hematological values were determined on day 34 of the test period.
CTubifex sp.
dparasites recovered from exposure levels of 0, 1500, 25, 75 and 225 in descending order. Fish were exposed once to 1500
parasites on day 1 of the experiment or daily to 25, 75 or 225 parasites for the first 24 days of the 34-day test period. Each
e
group at each ration level was composed of 10 fish.
Mean values determined one day prior to the start of the experiment from 20 fish acclimated to experimental conditions
for 14 days.
jl
z6
corpuscular volumes (MCV) and mean corpuscular hemoglobins (MCH)
were higher in all postexposure controls, but the mean corpuscular
hemoglobin concentrations (MCHC) were lower. These changes are
probably due to stress on the fish from handling.
When infected fish are compared with control fish, there is little
change in hematocrits, Hb concentration and MCHC (Table 5). All
fish exposed once to 1, 500 parasites and all infected fish on the 100
and 50% rations had lower RBC numbers than their respective controls
(Table 5, Fig. 21). There was no apparent effect of the parasites on
REC numbers in other infected fish when they are compared with their
controls.
As with RBC numbers, there was variation in WBC numbers
among groups (Table 5, Fig. 22). All infected fish on the 75% ration
and fish in three of the four infection groups on the 25% ration had
higher numbers than their respective controls, but infected fish on the
12.5% ration had lower numbers. Fish in two of the infection groups
in 50 and 100% rations had higher numbers while the remaining infected
fish had lower numbers than their controls.
Generally, thrombocyte numbers were higher in infected fish
thanin controls (Table 5, Fig. 23). The greatest increases occurred
ininfected fish on 12.5, 50 and 25% rations. Most of the infected fish
on all except the 75% ration had higher MCV and MCIri values than their
ive controls (Table 5).
27
Steelhead Trout
As was true with coho salmon, comparison of blood cell values
of pre- and postexposure controls shows the effects of handling on the
fish (Table 6). Most of the postexposure controls had lower hematocrits, WBC and thrombocyte numbers and MCV values, but higher
RBC numbers and MCH and MCHC values than the preexposure controls.
There was little difference in hernatocrit values between control
and infected fish except for the fish with the heaviest infections and on
the 12.5% ration (Table 6). Their mean value was 29% compared with
34% for the controls.
Infected fish on the 100% ration had higher Fib concentrations
than the controls but those on the 12.5% ration had lower values (Table
6).
There were little differences between the other infected fish and
their controls. Infected fish on the 100 and 75% rations had higher
RBC numbers than their respective controls but those on the 25 and
12.S% rations had lower numbers (Table 6, Fig. 24).
Most of the infected fish had higher WBC numbers than their
respective controls (Table 6, Fig. 25). The greatest difference
occurred between the most heavily infected fish on the 100 and 75%
rations and their controls. The respective numbers for the infected
fish were 6.63 x
5x
1O4
and 4.44 x lO. Controls had 4.38 x
WBC/rnm3, respectively (Table 6).
1O4
and
Table 6. Food rations for and mean numbers of parasites recovered, hematocrits, hemoglobin concentrations (Nb), numbers of
red (RBC) and white blood ceLLs (WBC) and thrombocytes, mean corpuscular volume (MCV), mean corpuscuLar hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) from steelhead trout infected with Nanophyetus
salmincola. a, b
Rationc
MCHC
Thrombocytes MCV
MCH
No. of
Hematocrit
Hb
RBC
WBC
parasitesd
Preexposure
controlse
100
40
-
0
517
60
258
1092
75
0
303
82
268
916
50
a
42
39
45
42
40
80
217
689
38
37
0
794
33
34
36
36
32
382
76
215
12.5
37
41
43
40
43
36
36
39
0
461
25
(%)
0
34
462
89
266
33
33
918
29
The fish were held at 17 C.
31
(g%)
7.2
7.8
8.0
9.5
8.5
9.0
9.4
8.2
9.9
8.9
8.3
9.2
8.4
8.7
9.3
8.9
7.8
8.0
8.4
7.3
8.1
8.4
7.9
8.1
7.0
6.1
(x l06/rnm)
(x l0/mrn3)
1.28
6.63
4.38
4.19
4.25
4.56
6.63
3.75
2.81
3.75
3.69
4.44
3.50
3.13
4.06
3.81
3.88
2.44
2.63
2.94
2.88
2.81
2.25
3.44
2.69
2.81
2.25
1.31
1.46
1.51
1.63
1.41
1.48
1.58
1.59
1.51
1.53
1.44
1.39
1.41
1.46
1.40
1.48
1.38
1.35
1.43
1.36
1.43
1.20
1.39
1.28
1.38
(x 10/mm3)
4.69
2.50
2.88
1.81
3.25
3.75
(urn3)
314
282
280
284
246
304
3.13
3.31
1.38
3.44
1.38
285
1.75
1.31
1.38
250
259
276
260
264
1.63
1.63
2.25
2.69
3.69
224
247
267
253
235
1.25
1.13
239
275
238
243
211
1.69
2.81
1.44
1.13
2.94
248
283
278
262
(pg)
(%)
56.4
59.4
54.7
62.8
52.3
63.7
63.7
52.1
62.4
58.8
54.4
64.0
60.5
61.6
63.6
63.6
52.9
58.2
62.2
51.2
59.4
58.9
65.8
58.4
54.9
44.4
18.0
21.1
19.5
22.1
21.3
20.9
22.4
21.0
22.0
21.2
20.8
25.6
23.3
22.3
24.5
24.1
23.6
23.5
23.3
20.3
25.3
24.7
23.9
24.5
22.6
21.0
bMean hematological values were determined on day 30 of the test period.
CTubifex sp.
dparasites recovered from exposure levels of 0, 1500, 75, and 225 in descending order. Fish were exposed once to 1500
parasites on day 1 of the experiment or daily to 25, 75 or 225 paraiites for the first 20 days of the 30-day test period.
Each group at each ration level was composed of 10 fish.
CMean values determined one day prior to the start of the experiment from 20 fish acclimated to experimental conditions for
14 days.
29
The majority of the infected fish on the 100 and 12. 5% rations
had higher numbers of thrornbocytes than their controls (Table 6,
Fig. 26). Differences between infected fish and their controls in the
remaining ration groups were inconsistent.
Therewas no apparent trend among the differences inMCV,
MCH and MCHC values between infected fish and their respective
controls (Table 6).
30
DISCUSSION
As expected, the numbers of metacercariae recovered from
fish increased as cercarial exposure numbers increased. The reason
for the low mean number of metacercariae recovered from coho
salmon exposed to 225 cercariae daily and fed a 12. 5% ration is not
known. Only 83 metacerca.riae or 2% of the number of cercariae used
for exposure were recovered from this group, which is 4. 5 to 7 times
less than the percentage recovery for the other groups exposed to 225
cercariae daily. Fish variation probably does not account entirely for
these differences because the ranges of recovered parasites were also
low. Similar differences were found between coho salmon on a 25%
ration and exposed to 1, 500 cercariae on day L and those fish exposed
to the same number of parasites but on different rations. Fish varia-
tionin susceptibility to N. salmincola also is apparent from the ranges
of metacercariae recovered from fish exposed to the same numbers of
cercariae and on the same food rations.
Fewer parasites were recovered from fish exposed daily cornpared with those exposed only once. These differences in numbers of
recovered parasites could be due to an enhanced humorai, cellular or
skin mucus response by fish to the cercariae or the proteolytic
enzymes that they are presumed to have and use for penetration and
migration. Also, one exposure of fish to large numbers of cercariae
31
could augment penetration because of cercarial attraction to substances
released by earlier penetrating cercariae or use by later parasites of
penetration channels created by earlier parasites.
Kennedy and Walker (1969) reported an immune response by dace
Leuciscus leuciscus to the cestode Caryophyllaeuslaticeps. Rejection of established parasites and decreased numbers after reinfection
were found. Although the authors were unable to demonstrate circulating antibody against the cestode, they found that the time required for
rejection decreased with an increase in temperature which is consistent with an antibody mechanism for rejection.
Occurrence of natural antibodies in fish against helminths has
been reported. Harvey and Meade (1969) found cercaricidal activity
in the serum of four species of cntrarchids against the digenetic
trematode P. minimum. Cercariae exposed to the serum of either
infected or uninfected fish showed an initial hyperactive period and all
were dead within a few hours. The serum had the same effect on
excysted but not on encysted metacercariae.
Harris (1972) found antibodies in both the serum and the intestinal mucus of the chub Leuciscus cephalus infected with the acantho-
cephalanPomphorhynchus laevis. He postulated that some damage to
the intestinal epithelium may be necessary for antigen access to the
host's defense system.
32
Di Conza and Halliday (1971) found imm.unoglobins, which
appeared to be produced locally, against injected bovine albumin and
Salmonella enteritidis in skin mucus secretions of the catfish
Tashysurus australis. Bradshaw et al. (1971) found normal 1gM antibody against sheep erythrocytes in the mucus of the gar Lepisosteus
platyrhynchus. Antibody production increased with immunization.
These investigators also detected antibody activity against erythrocytes in the skin mucus secretions of unimmunized snapper Lutjaneus
griseus and the bowfin Amia calva. Fletcher and Grant (1969) found
similar antibodies in the skin mucus of the plaice. Hildeman (1962)
reported secretions of antibodies in skin mucus of discus fish
Symphysodon discus. Their fry feed on this mucus and seem to acquire
some immunity as they cannot be raised without their parents unless
their water s.ipply contains high antibiotic concentrations. Hines and
Spira (1973, cited by Corbel 1975) reported that mirror carp exposed
to sublethal infections with Ichthyophthirius multifilis developed
immobilizing antibodies in the skin mucus to the protozoan.
O'Roarke (1961) found species specific antigens in the skin
mucus of several fish species and he postulated that these antigens
could be partly responsible for host detection by the parasites.
Chemotactic response of cercariae to such antigens could be
strengthened after host injury, which could cause an increase in antigen release. However, this rnecha.nism does not accoiint completely
33
for the higher infection rates in fish exposed once compared with
those exposed daily because the long exposure time, small water
volume and water movement from aeration alone probably provided
maximal conditions for cercarial detection and penetration of fish
exposed once. Therefore, the more likely mechanism is one based on
an enhanced response in resistance by fish to penetrating parasites.
The parasites did not affect growth of most of the fish. The
infection levels may have been too low to produce a measurable effect.
Baldwin et al. (1967) reported LE50 and LD50 values of j. salrnincola of 315 and 200 for coho salmon 37 mm fork length, and for rain-
bow trout 33 mm in fork length the respective values were 440 and
295.
The coho salmon and steelhead trout I used were 65-68 and
80-85 mm in total length, respectively and they had mean parasite
numbers of 219-407 and 303-517 for those exposed only once. Thus,
these larger fish probably were able to tolerate the relatively low
infection levels without production of discernible effects.
Mi.11emann and Knapp (1968) reported a decreased growth rate
of coho salmon, which weighed 1.5 g (lengths not given), exposed to
25, 75 and 225 N. salmincola cercariae daily for 24 days. My coho
salmon weighed from 2.2 to 3. 6 g and thus, as noted above, the
infection levels may have been too low to produce effects in fish this
size.
34
This relationship between fish size and Infection levels may also
explain the results-with fish exposed once to 1, 500 cercariae. Again,
there was no effect of parasites on fish growth.
In-an experiment similar to mine, Smitherman (1964) reported
decreased growth rates of bluegills infected with the trematode
minimum. Throughout a 32-week test period he found growth rates
increased at a low-er rate for-fish infected with an average of 353
parasites, than for control fish. He-also observed that fish with
lower numbers of parasites (103) showed no discernible change in
growth rate compared with the controls. Perhaps if I have exposed
my fish for a longer time to higher numbersof parasites, their growth
might also have been affected adversely.
Handling coho salmon-affected hematological characteristics.
Hematocrits, HB concentrations, RBC numbers, MCV and MCI-I
values increased 'but WBC and thrombocyte numbers decreased after
handling. Thus, in postexposure fish more mature RBC1s were present with larger-volumes and they contained more Hb than those from
preexposure controls. In all postexposure control fish MCI-IC values
-decreased; therefore, the amount of Hb-was higher in these cells than
in those from preexposure fish but the RBC volumes were larger and
thus the Hb concentration per RBC w-as reduced.
A decrease in food rations for-the controls-was associated with
decreased hematocrits, Hb concentrations and RBC and WBC numbers.
L
35
These reductions were probably due to lower food consumption by the
fish and to handling stress.
The parasites did not affect heniatocrits, Hb concentrationand
MCHC values of coho salmon. Only fish exposed to 1,500 cercariae
on day 1 had lower RB C nmbe r s than their re s pe ctive cont ro is.
Because parasites were in the fish longer and were larger perhaps
they had a greater effect on the hematopoietic system than those in
more recently exposed fish.
There were slight increases in WBCnumbers of infected fish on
100, 75, 50 and 25% rations. Arrilacher (1961, cited by Reichenbach-
Klinke 1966) reported an increase in the ratio of leucocytes to erythrocytes from 1% on normal fish to 3- 18% in rainbow trout infected with
viral hemorrhagic septicemia.
The increase in thrombocyte numbers in parasitized fish independent of their infection levels may have been a compensation for
thrombocyte losses incurred during clot formation of hemorrhages
caused by penetrating and migrating parasites.
Mean corpuscular volume values increased in most infected fish.
Thus, they had more mature erythrocytes than the controls or at least
the ratios of mature to immature RBC's were higher. The MCH values
also increased in infected fish. This result was expected because the
parasites had no effect on the Hb concentration and therefore, RBCs
with larger volumes would contain more Hb.
Handling of steelhead trout was associated with decreased hema-
tocrits in four of the five postexpo sure control groups compared with
the preexposure fish, although RBC numbers had increased in all
controls. The increases in RBC numbers were small and the
decreased hematocrits could have been due to large decreases in WBC
and thrombocyte numbers of postexposure controls. Mean RBC
volumes were smaller in controls. This and the slight increase in
RBC numbers suggests that a large number of immature RBC's must
have been present inthese fish. Hemoglobin concentration and MCH
values were higher in most postexposure ccntrols, possibly due to
handling, thanin the preexposure fish. Thus, the fishwouldbe able
to transport more o:xygen during the time theywere being handled.
Higher MCHC values were found in all controls, which resulted in
increases in the amount of Hb per RBC. These observations again
suggest that a large number of smaller immature erythrocytes was
present in the blood of postexposure controls.
Decreased food consumption in steelhead trout was associated
with decreases in hematocrits, Hb concentrations, RBC, WBC and
thrombocyte numbers and MCV values in controls. Thus, hemato-
logical characteristics in fish on the low food rations was affected by
the handling and by food availability.
Most of the effects that could be attributed to parasitism
appeared to be associated with food rations. Large decreases in Nb
37
concentration were onlyfound in fishon 25 and 12.5% rations.
Numbers of RBC's in infected fish showed a close relationship with
food consumption. Infected fish on high rations had more RBC's than
their controls and the reverse relationship was found for fish on low
rations. Thus, a synergistic action from the effects of the parasites,
low food rations and handlingwas apparent.
Most infected fish had higher WBC numbers than their respec-
tive controls, which is a common finding in parasitic infections. The
parasites had no apparent effect on thrombocyte numbers and MCV,
MCH and MCHC values.
Any short term effects on hematological characteristics of fish
exposed once to 1, 500 parasites could not be determined. It is
possible that the parasites could have damaged the hetnatopoietic
system, but such damage could have been repaired between the time
of infection and the time of hematological. determinations so only
slight changes, if any, would be detected at the end of the experiments.
Also, because of the experimental design, hematological measurements could not be made until 10 days after the last exposure. Again,
repair of damaged tissue could have taken place during this time.
Not all of the N. salmincola metacercariae occur in the kidney.
Therefore, as in the growth experiments, perhaps insufficient parasites were present in the kidney to cause measurable damage to the
hematopoietic system. Reichenbach- KJ.inke (1966) reported that lower
than lethal numbers of the tapeworm P. per cae and ergasilid copepods
on coregonids and of the tapeworm cysts of Triaenophorus crassus in
the liver of the arctic char resulted in small hematological changes
and that hematopoietic repair masked large changes. When large
alterations in the blood components of infected fish have been reported
by other investigators, extensive hemorrhages were usually one of the
causes (Hunn 1964; Cardwell and Smith 1971; Foda 1973; Evans 1974;
Amend and Smith 1975). I observed only small hemorrhages in the
caudal peduncle of the exposed fish and they usually disappeared
between exposures.
39
SUMMARY AND CONCLUSIONS
1.
A study was done to determine if the trematode parasite
Nanophyetus salmincola affected the growth and hematopoiesis of
coho salmon and steelhead trout.
2.
Fish were fed five different food rations and exposed to different
parasite numbers.
3.
Different infection levels were achieved by either exposing the
fish once or daily to known numbers of parasites
during
the
experiments.
4.
Weights of parasitized and control fish were recorded daily and
total lengths were recorded every fourth day during the experiment S.
5.
Ten days after the last exposure, hematocrits, hemoglobin
concentrations, red and white blood cell and thrombocyte num-
bers were recorded. Also, mean corpuscular volume, mean
corpuscular hemoglobin and mean corpuscular hemoglobin concent ration values we re calculated.
6.
Fewer parasites were found in fish exposed daily compared with
those exposed only once. The lighter infections may be related
to some type of defense system in the fish.
7.
The parasites did not affect the weights or lengths of fish
exposed once to 1, 500 parasites and they had only a slight effect
on growth of some of the other fish.
8.
Handling and differences in food availability were associated
with some hematological changes.
9.
Parasites appeared to cause some hematological changes, but
generally exposure levels were too low to cause detectable
damage.
41
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46
8.0-
0
7.5-
....
I?,
P
g,o'/
//
if
I.
I
(D
I
5.5
......./
...,
0/f
/ /
' /
/) 7"
Controls
0
......
4.5
.-'
1500 Cercariae on Day I
--25 Cercariae Doily
(days 1-24)
00075
Cercariae Daily
(days 1-24)
/
e0
0
t/J/
4.0-
oI/
°"I
0
50-
£°"/
225 Cercariae Daily
I
(days 1-24)
'
I
5
I
I
I
9
13
17
21
25
29
34
TIME (days)
Figure
1.
Mean weight of groups (10 fish per group) of coho salmon
on a 100% ration of Tubifex sp. worms and exposed to
diffe rent numbers of Nanophyetus salmincola ce rcariae.
1%
'
/
7.0-
_,,,.
//
,-.
6.5-
'I
/
/
I
/
6.0-
0
/
I.
=
CD
Lu
...0
/
0.
1
/
/
55...
,
/
(0
/
.0
,"
-
.0
0.0
/
5.0/
...*.*..
°.1
f4
i
'
45_i
Contro's
" 1500 Cercariae on Day I
--25 Cercariae Daily
(days 1-24)
.....
00075
0
100
-
.......
4.0-
Cercariae Daily
(days (-24)
225 Cercariae Daily
(days 1-24)
3.5-
I
I
5
9
13
17
21
25
29
34
TIME (days)
Figure 2.
Mean weight of groups (10 fish per group) of coho salmon
on a 75% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus salniincola cercariae.
Controls
1500 Cercarioe on Day I
6.5
--25 Cercariae Daily
000
6.0
(days 1-24)
75 Cercariae Daily
(days 1-24)
- 225 Cercariae Daily
(days 1-24)
/
'
-- 0..o
A
0.
.Q.
/
S.-
5,0
0
w.... ..
4.5
......o
/
i0
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0
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,/ \,'
4.0
3.5j
I
I
5
9
13
I?
21
25
I
29
34
TIME (days)
Figure 3.
Mean weight of groups (10 fish per group) of coho salmon
on a 50% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus saLrnincola cercariae.
49
Figure 4.
Mean weight of groups (10 fish per group) of coho salmon
on a 25% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus salrnincola cercariae.
Figure 5.
Mean weight of groups (10 fish per group) of coho salmon
on a 12. 5% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus salmincola cercariae.
50
5.0
a
a.
II
0
-
4.5
4.0
3.5
9
13
25
21
17
29
34
TIME (days)
Figure 4
Controls
1500 Cercariae on Day I
-- 25 Cercariae Daily
(days 1-24)
000 75
Cercarioe Daily
(days 1-24)
- 225 Cercariae Daily
4.5
a
(days 1-24)
....
-
I
3.0
13
17
21
TIME (days)
Figure 5
25
29
34
Figure 6. Total length of groups (10 fish per
group) of coho salmon on a 100% ration
of Tubifex sp. worms and exposed to
different numbers of Nanophyetus
salmincola ce r cariae.
Figure 7. Total length of groups (10 fish per
group) of coho salmon on a 75%
ration of Tubifex sp. worms and
exposed to different numbers of
Nanophyetus salmincola ce rcariae.
Controls
92
92
90
E
E
I
000
=
I
85
-
1500 Cercariae on Day I
25 Cercariae Daily
(days 1-24)
75 Cercariae Daily
(days 1-24)
225 Cercariae Daily
(days 1-24
85
z.---CD
CD
z
LU
LU
/
-J
-J
80
.j
-
80
,,
0
/
I
/
/
75
70
0
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I-..
I-
00
,
4
4
I0
.0
0
o°/
70
I
5
9
(3
(7
21
TIME (days)
Figure 6
25 29
34
I
5
9
(3
17 21
25 29 34
TIME (days)
Figure 7
Ui
53
Figure 8.
Total length of groups (10 fish per group) of coho salmon
on a 50% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus salniincola cercariae.
Figure 9.
Total length of groups (10 fish per group) of coho salmon
on a 25% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus salmincola cercariae.
Figure 10. Total length of groups (10 fish per group) of coho salmon
on a 12. 5% ration of Tubifex sp. worms and exposed to
diffe rent numbers of Nano phyetus salmincola cer cariae.
54
E
E
I
I-
z
ILl
-J
-J7
I-
0
I.-
7C
Figure 8
TIME (days)
80
I
I-
w
-J
-J
I.-
0
Figure 9
1
5
9
13
17
21 25 29 3
TIME (days)
Controls
1500 Cercariae on Day
80
.
0
75
---25 Cercariae Daily
(days 1-24)
000
75 Cercorioe Daily
(days 1-24)
225 Cercariae Doily
(days 1-24)
7o
I
5
9
17 21 25 29 34
TIME (days)
13
Figure 10
I
55
Figure 11. Mean weight of groups (10 fish per group) of steelhead
trout on a 100% ration of Tubifex sp. worms and exposed
to different numbers of Nanophyetus sairnincola cercariae.
56
6.0
15.5
15.0
14.5
14.0
I 3.5
13.0
12.5
I
C.')
12.0
11.5
Lii
11.0
rol S
10.5
Cercariae on Day I
Cercariae Daily
(days 1-20)
Cercariae Daily
(days 1-20)
Cercariae Daily
(days 1-20)
10.0
9.5
9.0
8.5
8.0
I
5
9
TIME
13
17
21
(days)
Figure 11
25
30
57
Figure 12. Mean weight of groups (10 fish per group) of steelhead
trout on a 75% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus salmincola cercariae.
58
14.0
0
0
0.0
0
0
'3.5
'I
I .--'
13.0
,
r ,'
12.5
I
/
I
,
12.0
''.5
11.0
I
(D
10.5
IJ
-
9.5
:
000
Controls
1500 Cercariae on Day I
9.0
25
Cercariae
Daily
(days 1-20)
Cercariae
75
-
(days 1-20)
225 Cercarioe Daily
(days 1-20)
8.0I
5
9
17
TIME
(days)
Figure 12
Daily
000
21
25
30
59
Controls
12.0-
1500 Cercariae on Day I
-- 25 Ce.rcariae Daily
(days 1-20)
000 75
Cercariae Daily
II .5
(days 1-20)
225 Cercariae Daily
(
:.
,.
ays 1-
HO
/I
00
0
k
/
0
0
f
10.5
1
I
../..
(9
/
0
0
00
0
0
0
0
.,/
/
0
0
I:I..
0
0
I/fl/if,...
/
.,
10.0-
0
0
0
o0o
0
Li
.,
L000
.to
0
lJ0
9.5-
0
00
9.0-
0V0 0 0
8.51
I
5
9
13
TIME
I?
21
25
30
(days)
Figure 13. Mean weight of groups (10 fish per group) of steelhead
trout on a 50% ration of Tubifex sp. worms and exposed
to different levels of Nanophyetus salmincola cercariae.
Figure 14. Mean weight of groups (10 fish per group) of steelhead
trout on a 25% ration of Tubifex sp. worms and exposed to
different numbers of Nanophyetus salmincola cercariae.
Figure 15. Mean weight of groups (10 fish per group) of steelhead
trout on a 12.5% ration of Tubifex sp. worms and
exposed to different numbers of Nanophyetus salmincola
ce rcariae.
61:
I
I'
9.5
j
8.0-
00
000000000
5
9
13
17
21
25
30
TIME (days)
Figure 14
Controls
1500 Cercariae on Day I
9
25 Cercariae Daily (days 1-20)
0000 75
Cercariae Daily (days 1-20)
225 Cercarioe Doll (do s 1-20)
8.5-
00000
° I
I
I
5
9
I
13
TIME
1
I
17
21
(days)
Figure 15
0 0 0
25
30
Figure 16. Total length of groups (10 fish per
group) of steelhead trout on a 100%
ration of Tubifex sp. worms and
exposed to different numbers of
Nanophyetus salmi.ncola cercariae.
Figure 17. Total length of groups (10 fish per
group) of steelhead trout on a 75%
ration of Tubifex sp. worms and
exposed to different numbers of
Nanophyetus salmincola cercariae.
114
114
110
E
I
ICD
z
105
Ui
-J
- Controls
-J
4 100
1500 Cercariae on Day I
I-
0
25
I-
0000
95
100
I-
Cercariae Daily
(days 1-20)
75 Cercariae Daily
(days 1-20)
95
225 Cercariae Daily
(days 1-20)
92
I
5
9
13
17
21
TIME (days)
Figure i6
25
30
92
I
5
9
13
17
21
TIME (days)
Figure 17
25 30
64
Figure 18. Total length of groups (10 fish per group) of steelhead
trout on a 50% ration of Tubifex sp. worms and exposed
to different numbers of Nanophyetus salmincola cercariae.
Figure 19. Total length of groups (10 fish per group) of steelhead
trout on a 25% ration of Tubifex sp. worms and exposed
to different numbers of Nanophyetus salmincola cercariae.
Figure 20. Total length of groups (10 fish per group) of steelhead
trout on a 12. 5% ration of Tubifex sp. worms and exposed
to different numbers of Nanophyetus salmincola cercariae.
65
1o1
I05
I
I-
Controls
1500 Cercariae on Day
---25 Cercariae Daily (days 1-20)
000 75 Cercariae Daily (days 1-20)
- 225 Cercariae Daily (days 1-20)
100
I
J
4
095
I.-
92-J-I
i
5
91317212530
TIME
(days)
Figure 12
I
'::i:::::;:::
;
92-f---Figure 19
I
i
i
9
5
'
13
I
17 21
TIME (days)
I
25 30
1000000
oo0.000000.
:::11:t,
13 I? 21 25 30
I
Figure 20
5
9
TIME (days)
o
*
18-
°°°o I00%RATION
75% RATION
50%RATION
t
E
I
25%RATION
i
I 2.5%RATION
I
5
0
0
100
200
300
400
500
600
700
PARASITE NUMBERS
Figure 21. Red blood cell numbers of coho salmon infected with different numbers of
Nanophyetus salmincola metacercariae and fed different rations of Tubifex sp.
worms. Mean numbers obtained from five fish.
800
8.0.
0
00
00
0
0
o
0
00
0
7 0
0
0
0
00
0
0
0
-J
-J
6.0-
I\
/
I.
I.
W
()
a
0
\
I'
/
I'\
'\
'
ILl
\
00
..//
--
V
I.
..-\
/
000.
/..
\
/
/
/
/
/
0000 100% RATION
.
4.0-
/
%/
/
\
..
I.
,
00
\
/..
/:
*d..#
I
\
I.
p \
-J
0 0
0 0
75%
50%
25%
.
RATION
RATION
RATION
12.5% RATION
3.1
0
100
200
300
400
500
PARASITE NUMBERS
600
700
Figure 22. White blood cell numbers of coho salmon infected with different numbers of
Nanophyetus salmincola metacercariae and fed different rations of Tubifex sp.
worms. Mean numbers obtained from five fish.
800
ii:
0
9
E
E
8
S...
(1)
I
Ui
>-
6
C)
0
5
0
I
4
a:
0
100
200
300
400
PARASITE
500
NUMBERS
600
700
Figure 23. Thrombocyte numbers of coho salmon infected with different numbers of
Nanophyetus salmincola metacercariae and fed different rations of Tubifex sp.
worms. Mean numbers obtained from five fish.
800
'7
(.0
0
0
0
0
00
0
0
0
/o00
K)
E
E
00000000.00
I .5
0000
C/)
0
:1I:.:°:
-J
-j
IiJ
C-)
0
0
0
-j
1.3
0000 100 % RATION
.........
RATION
75
50% RATION
1.2
25 % RATION
Lii
12.5% RATION
0
tOO
200
300
400
500
600
700
800
900
1000
1100
PARASITE NUMBERS
Figure 24. Red blood cell numbers of steelhead trout infected with different numbers of
Nanophyetus salmincola metacercariae and fed different rations of Tubifex sp.
worms. Mean numbers obtained from five fish.
6.8
0000 100 % RATION
o
0
75% RATION
0
0
0
- 50% RATION
6.0-
0
0
25% RATION
0
0
0
12.5% RATION
0
0
0
r)
E
E
.
0
0
0
o0
5.0
0
0
0
Cl,
0
0
_.I
ILl
o
0
J
00400
0000
000
000
0
0
0
0000
00
S
4.O
/
3.0 -
........
2.0 0
100
200
300
400
500
600
700
800
900
1000
1100
PARASITE NUMBERS
Figure 25. White blood cell numbers of steelhead trout infected with different numbers of
Nanophyetus salmincola metacercariae and fed different rations of Tubifex sp.
worms. Mean numbers obtained from five fish.
.
4.0
If)
E
E
3.0
AT ION
C')
w
AT ION
I.
>-
AT ION
0
AT ION
2.0
ATION
0
a:
I
I
La,]
0
tOO
200
300
400
500
PARASITE
600
700
NUMBERS
800
900
1000
1100
Figure 26. Thrombocyte numbers of steelhead trout infected with different numbers of
Nanophyetus salmincola metacarcariae and fed different rations of Tubifex sp.
worms. Mean numbers obtained from five fish.