Progress Report Series No. 95
Agricultural Experiment Station
AUBURN UNIVERSITY
Channel Catfish Virus Research
at Auburn University
JOHN 
A. PLUMB
2
C HANNEL CATFISH VIRUS DISEASE (CCVD) was first
diagnosed in 1968 by Fijan et al. (2) and has now
been diagnosed in 11 epizootics from six South-
ern States. It may cause up to 90 per cent mortality
among infected, susceptible, fingerling channel cat-
fish, thus is a serious disease in some catfish opera-
tions. The degree of mortality depends upon the
condition of the fish, water temperature and prob-
ably other environmental factors.
Primary interest at Auburn University is in learn-
ing more about the virus disease and its association
with the host. The areas of research are: (1) age and
size of fish affected, (2) other affected species of cat-
fish, (3) what the reservoir is and how the disease is
passed from one individual to another, (4) methods of
detecting carrier fish, (5) determination of primary
target tissues and, (6) determination of the immune
response stimulated by the virus. Although some
data have been accumulated in all these areas, these
comments will be restricted to those of greatest im-
portance to the catfish producer.
METHODS OF IDENTIFYING CARRIER
BROODSTOCK
Before arriving at any meaningful conclusion about
the extent of the distribution of CCVD, methods of
detecting carrier or reservoir fish must be developed.
This is dependent ipon the theory that the disease is
carried by the parent fish and can be passed from the
adult to the offspring via the reproductive products.
Evidence for such a method of vertical transmission
is only circumstantial. However, it is known that the
disease can be transmitted horizontally from infected
fish to uninfected fish via the water or b contact
Fijan et al. (2).
Research supported by the Southeastern Cooperative Fish Dis-
ease Unit.
2 Research Associate, Department 
of Fisheries and Allied Aqua-
cultures.
The geographical range of CCVD has been de-
termined by isolating the virus from infected fish in
active epizootics. It appears that only a small per-
centage of the outbreaks is recognized as many are
not revealed because of the reluctance on the grow-
ers' part to report them. Such attitudes further com-
plicate any hopeful measures of reducing the effects
of the disease. This in itself has probably led to its
increased distribution because there is circumstantial
evidence to indicate that survivors of epizootics may
be carriers of the virus.
Methods used in identifying carrier fish with in-
fectious pancreatic necrosis (IPN) virus (Wolf and
Quimby (5)), and viral hemmorrhagic septicemia
(VHS) (Hoffman et al. (3)) of trout have been tried
experimentally with CCV, however, none of these
methods have afforded satisfactory results. Internal
organs, peritoneal wash or fecal samples from fish
artificially infected with virus and sampled several
weeks after injection did not yield virus. IPN, or
VHS of trout may be detected in carrier fish by at
least one of these methods. However, the only
method of detecting carriers that holds any promise
for CCV at present is immunological, that is to dem-
onstrate a previous exposure to the disease. Two-year
old channel catfish that were injected with CCV
demonstrated an immune response to the virus sev-
eral weeks after injection. Also virus neutralizing an-
tibodies have been found in channel catfish brood-
stock that produced CCV diseased offspring 2 out of
3 previous years.
The attempts to isolate 
CCV from the artificially
infected fish was done in the laboratory by injecting
2-year-old channel catfish (200 g. to 400 g.) with virus
and holding them in tanks with water temperature
at 750 F to 80' F. These fish were checked periodi-
cally by taking fecal and peritoneal wash samples
and gill swabs, then assaying these samples for virus
in brown bullhead (BB) cell cultures. The only sam-
1. V. Smith, Director
Auburn, Alabama
April 1971
ple that was positive for CCV was the peritoneal
wash, 12 days post injection, when 3 of 7 
sampled
fish were positive for virus. No other subsequent
samples were positive, therefore, it is thought 
that
the recovered virus was actually residual particles
from the inoculations.
In July, 1970, a federal fish hatchery decided to
liquidate their channel catfish broodstock because
these fish had produced CCV diseased offspring.
These fish were made available to Auburn University
for study and with the cooperation of The Division
of Fish Hatcheries, U.S. Bureau of Sport Fisheries
and Wildlife some interesting information is emerg-
ing from the study. It 'must be added, however, that
so far much of the information is negative. Initially
30 fish were studied in July and an additional 22 in
September, 1970. The sampling techniques were di-
vided into two catagories: (1) sampling techniques
without sacrificing the fish, and (2) sampling tech-
niques by sacrificing the fish, Table 1.
TABLE 1. TISSUES, ORGANS, AND PRODUCTS ASSAYED FOR
CHANNEL CATFISH VIRUS FROM ADULT CHANNEL CATFISH
Techniques without
sacrificing the fish
Blood
Kidney-blood'
Gill-swab
Urine
Feces
Not checked for virus in September.
Techniques by
sacrificing the fish
Liver
0
Intestive
s
Kidney
Ovaries
Sera were also collected from the blood 
of each fish
to determine if CCV specific neutralizing 
antibodies
were present.
RESULTS
Virus was not isolated from any of the samples
from the fish in July or September. There was poor
survival of virus in the July samples where CC V was
added with the exception of one urine sample where
the survival was good, Table 2.
A positive CCV neutralization index 
(Casals (1))
was found in the serum of each of the 30 
fish ex-
amined in July, however, the antibody 
level for fe-
males differed from that of the males, Table 3. The
average neutralization index for 21 females was 20,-
559 (range '56 to 177,800), whereas the average
for 9 males was 55,503 (range 103 to -177,800).
TABLE 3. CCV NEUTRALIZATION INDEX OF SERUM FROM
CHANNEL CATFISH BROODSTOCK SUSPECTED OF
CARRYING CCV
Sex Number 
Neutralization index
Max Min Mean
Female 21 -177,800 -56 :20,859
Male 9 >177,800 103 -55,503
The data collected thus far are primarily negative,
although they do reveal some interesting facts. It is
assumed that the CCV infected channel catfish fin-
gerlings in question contacted the disease from the
TABLE 2. SURVIVAL OF CCV IN TISSUE SAMPLES AND EXCRETORY PRODUCTS FROM ADULT CHANNEL CATFISH
Fish Days post
number inoculation Feces
Virus survivaP (expressed as TCIDs
0
per ml.)
Urine Kidney- G ill swab
blood
Kidney Liver Spleen 
Intestine 
index (serumtrizaton
6 11 Neg. Neg. Neg. Neg. 1,778 Neg. 
660
12 17 Neg. 177 Neg. Neg. Neg. 354 
316
18 24 Neg. Neg. Neg. Neg. Neg. Neg. Neg. --31,620
24 36 Neg. Neg. 18 Neg. Neg. Neg. 
112 :n177,800
30 38 Neg. -1,000 Neg. 56 Neg. Neg. Neg. Neg. 17,780
1 17,380 TCID virus were added per ml. of sample.
Blood was taken with a 10 ml. syringe from the
dorsal aorta in the caudal peduncle; kidney-blood
was secured by injecting physiological saline into the
kidney and withdrawing a sample; gill-swabs were
taken by swabbing the gill with a Q-tip soaked in
saline; urine was aspirated from the urinary bladder
with a Pasteur pipette; and feces were taken by
squeezing intestinal content into a test tube. Internal
organs were obtained by sacrificing the fish and re-
moving the organs. These samples were homogen-
ized, diluted, filtered (0.45 u), and assayed in BB cell
cultures. These samples were not assayed for 48
hours after being taken, therefore, a known amount
of CCV was added to the samples from 1 out of 6
fish to determine the survival of virus in the samples.
broodstock, however, no viable virus was isolated in
surveys of the adult fish during July and September,
1970. Several factors may contribute to the lack of
recovered virus: (1) the broodstock may have lost
their infection, during the year, (2) the infection is so
slight that the level of viable virus is below our de-
tectioR methods, (3) the virus released from infected
cells may be neutralized by the high level of circulat-
ing antibody in the blood, or (4) viable virus may
only be released during certain metabolic cycles such
as during spawning activity, or (5) there may be some
disease reservoir other than channel catfish. Any of
these factors will present problems in virus detection
and continued work during the next spawning season
may answer some of these questions.
REPLICATION OF CCV IN INTERNAL ORGANS
The purpose of this study was to determine from
which organs of infected channel catfish could one
most likely isolate CCV. Eight-month-old channel
catfish weighing 5 g. each were injected intraperi-
toneally with CCV and held in aquaria at 80'F. At
24-hour intervals for a period of 5 days, two samples
of fish (each sample consisting of 2 fish) were re-
moved from the aquaria for virus assay. The kidney,
liver, intestine, brain, and a sample of skeletal mus-
cle were removed from the fish in each sample and
the individual organs combined. These samples were
then homogenized and serially diluted 10 fold to 10
-2
from which cell free filtrates were prepared (0.45 u.)
The filtrates were then further serially diluted 10 fold
to 10
-5. 
Triplicate tubes of channel catfish gonad
(CCG) cells were inoculated with 0.1 ml. each of the
dilutions. The CCG cells were incubated at 25' C
and after 7 days were scored for cytopathic effect
(CPE) and the 50 per cent end point (TCIDo) de-
termined by the Reed-Muench -method (Reed and
Muench (4)).
CCV was isolated from all organs and tissues as-
sayed but at different levels and different frequen-
cies, Table 4. In live fish the kidney was the initial
organ demonstrating virus replication where virus
was recovered 24 hours after inoculation. Initial
CCV was isolated from the other organs as follows:
intestine-48 hours; liver-72 hours; brain and mus-
cle-96 hours.
TABLE 4. REPLICATION OF CHANNEL CATFISH VmIRus IN ORGANS
AND TISSUES OF LIVING CHANNEL CATFISH FINGERLINGS
INJECTED WITH CCV
Tissue culture infectious doseso
Hours 
(TCID,0) per ml. 
of tissue
i njetion Sample Kidney Intestine Liver 
Brain Muscle
24 1 -175 -----
2
48 1 562
2 5,620 1,000
72 1 31,620 562 17,400
-2 10,000 3,162 174
96 1 17,400 1,000 57,540
2 100,000 31,620 3,981 3,162 316
120 1 468 316,000 3,162 -10,000 316
2 3,162 5,623 -100,000 -10,000 58
1 Two fish in each sample.
It can be concluded from this study that the kid-
ney is the initial organ affected by virus and there-
fore is prime tissue for study, however, the intestine
and liver are only slightly less desirable.
Investigations into the effects of CCV and its im-
pact on the host have many unanswered questions.
It is hoped that continued research at Auburn Uni-
versity and other institutions will provide solutions
to some of these problems.
SUMMARY
Channel catfish virus (CCV) research at Auburn
University Agricultural Experiment Station primarily
involves development of methods for detecting the
disease in carrier populations. Data collected to date
indicate that methods used in detection of infectious
pancreatic necrosis (IPN) virus and viral hemorrhagic
septicemia (VHS) of trout are not applicable to CCV.
High CCV neutralization indices were found in 52
channel catfish broodstock suspected of being virus
carriers but no viable virus was isolated. The prin-
cipal organs involved in fingerling channel catfish
infected with CCV are kidney, liver and intestine
although the muscle and brain yielded detectable
virus.
LITERATURE CITED
(1) CASALS, J. 1967. Immunological Techniques for Animal
Viruses. In K. Maramorsch and H. Koprowski (Ed).
Methods in Virology, Academic Press, New York pp. 113-
198.
(2) FIJAN, N. N., T. L. WELLBORN, AND J. P. NAFTEL. 1970.
An Acute Viral Disease of Channel Catfish. Bur. of
Sport Fish. and Wildl. Tech. Pap. No. 43. 11 pp.
(3) HOFFMAN, C. L., S. F. SNIESZKO, AND K. WOLF. 1968.
Approved Procedures for Determining Absence of Viral
Hemorrhagic Septicemia, and Whirling Disease in Cer-
tain Fish and Fish Products. Bur. of Sport Fish. and
Wildl. FDL-9. 7 pp.
(4) REED, L. J. AND H. MUENCH. 1938. A Simple Method
of Estimating Fifty Per Cent Endpoints. Am. J. Hyg.
27:493-497.
(5) WOLF, K. AND M. C. QUIMBY. 1967. Infectious Pan-
creatic Necrosis (IPN): its Diagnosis, Identification, De-
tection and Control. Riv. Ital. Pisicolt. Ittiopatol. 2:76-80.