Aquacutture

Aquaculture 157 (1997) 147-155

ELSEVIER

Heterologous isolates challenge of channel catfish,

Ictalurus punctatus, immune to
Edwardsiella ictaluri
P.H. Klesius *, C.A. Shoemaker
Fish Diseases and Parasites Research Laboratory, Agricultural Research Service, United States Department
ofAgriculture, P.O. Box 952, Auburn, AL 36830.0952, USA

Received 8 December 1996; revised 3 March 1997; accepted 24 March 1997

Abstract
It is generally believed that channel catfish, Zctulurus punctutus, immunized with one isolate of
ictaluri would be protected against different isolates of E. ictaluri. In this study six
different isolates of E. ictuluri cultured from channel catfish having enteric septicemia of catfish
(ESC) and one isolate from the walking catfish, Clurius batruchus,
were evaluated for cross
protection. Channel catfish immunized by live exposure with isolate EILO were significantly
protected against ESC following challenge with E. ictuluri isolates EILO, ATCC-33202, S-941017, S-94-629 and AL-93-75, but not against isolate AL-93-58. Immunization with isolates
AL-93-75, EILO, AL-93-58, S-94-1017 and S-94-629 induced protection against AL-93-75.
Immunization
with isolates ATCC-33202 and S-94-1051 did not induce protection against
AL-93-75. Channel catfish immunized with two isolates EILO and AL-93-58 (together) were
protected upon challenge with the isolates AL-93-75, EILO, AL-93-58 and S-94-1017. Challenge
of EILO and AL-93-58 immunized fish with ATCC-33202 resulted in partial protection. These
results show that differences exist between E. ictuluri isolates in their ability to induce protective
immunity against ESC. Catfish immunized at 26C and held at 18C remain protected against
heterologous challenge for four months. These findings are important to the development of
efficacious ESC vaccines. 0 1997 Elsevier Science B.V.
Edwardsiella

Keywords:

Live vaccination; Edwardsiella

ictaluri; Acquired immunity

* Corresponding author. Tel.: (334) 887-3741; Fax: (334) 887-2983; E-mail: Klesiph@vetmed.aubum.edu.
0044~8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved.
PII SOO44-8486(97)00076-S

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P.H. Klesius, C.A. Shoemker/Aquaculture

1.57 (1997) 147-155

1. Introduction
Infection of channel catfish, Ictalurus punctatus, with the bacterium Edwardsiella
ictaluri causes enteric septicemia of catfish (ESC), which seriously affects intense
farming of catfish in the southeastern United States (Hawke, 1979; Hawke et al., 1981;
Klesius, 1992; Plumb and Vinitnantharat,
1993). The use of vaccination to control E.
ictuluri infection is desirable. Development of effective vaccines against ESC must rely
on serological relatedness among isolates of E. ictuluri. Researchers have determined
there is little or no antigenic variability among isolates and that most isolates of E.
ictuluri represent a single antigenic serotype (Rogers, 1981; Plumb and Vinitnantharat,
1989; Bertolini et al., 1990). Plumb and Vinitnantharat
(1993) suggested that this
antigenic homogeneity
makes E. ictuluri an excellent candidate for ESC vaccine
development.
Vaccination
with killed E. ictaluri vaccines have not resulted in strong acquired
immunity that protects channel catfish against E. ictuluri infection. Researchers working
with other facultative intracellular pathogens and host systems have found similar results
(Eisenstein et al., 1984; Montaraz and Winter, 1986; Schurig et al., 1991). A formalinkilled ESC bacterin based on a single isolate was developed and tested (Thune et al.,
1994). Field trial results indicated that relative percent survival (RPS) was 35.1% for the
bacterin vaccinates naturally exposed to E. ictuluri. Plumb et al. (1994) found no
significant effect of oral vaccination on survival of catfish stocked at different densities
in ponds. One reason for the low efficacy may have been due to the failure of a single
isolate bacterin to stimulate immunity against all isolates of E. ictuluri present in
production ponds. Newman (1993) reviewed vaccination
of fish and suggested that
bacterins used for immunization
against ESC were ineffective. Recently, Nusbaum and
Morrison (1996) demonstrated that killed 35S-labelled E. ictuluri does not enter the
fish. All immunizations
to date have been with killed preparations of E. ictuluri.
In recent studies, we documented that vaccination
by a live E. ictaluri vaccine
conferred strong acquired protection against homologous
challenge with the same
isolate. Vaccination with a low concentration of live E. ictaluri was found to produce
better protection against homologous E. ictuluri infection than did immunization
with
killed vaccines (unpublished observation). Our results also showed that strong acquired
protection against E. ictuluri infection was dependent on the development of a cellular
immune response, specifically the activation of macrophages (Shoemaker et al., in
press). Other researchers, Antonio and Hedrick (1994) and Klesius and Sealey (1995)
demonstrated that antibody alone was not protective against E. ictuluri infection and
suggested a role for cell-mediated
immunity in protection. The protective immune
response to heterologous challenge with E. ictaluri isolates has not been performed.
The first objective of this study was to determine if immersion vaccination with a low
dose of a single isolate of E. ictuluri provided acquired protection against homologous
virulent E. ictaluri infection. A second objective was to determine if acquired protection
against heterologous isolates of E. ictaluri was provided by vaccination with a live
isolate of E. ictuluri. The third objective was to examine acquired protection against
virulent E. ictuluri isolates produced by immunization
with a combination of two live

P.H. Klesius, C.A. Shoemaker/Aquaculture

157 (1997) 147-155

149

isolates. Finally, we examined the influence of duration and temperature on acquired

immunity to E. ictuluri using a live isolate of E. ictuluri to vaccinate fish.

2. Material and methods

2.1. Channel catfish
Marion strain of channel catfish raised from eggs and free of E. ictaluri were used at
about 10 to 12 months of age, 25 to 30 g and 12.5 to 14.0 cm total length. The fish were
stocked into triplicate 55-l aquaria at a density of 30 fish each. The aquaria were
supplied with 26C flow-through water at a rate of 0.5 l/h. The fish were fed at 5%
body weight daily with trout chow. A light and dark schedule 12: 12 h was maintained.
2.2. E. ictaluri isolates used as vaccines and for infection challenge
Six of the seven isolates of E. ictuluri (Table 1) were obtained from channel catfish
suffering from ESC. The sixth isolate, EILO, was originally cultured from the walking
catfish, Clarius batruchus (Kasomchandra
et al., 1987) and then re-isolated from an
infected channel catfish suffering from ESC in our laboratory.
2.3. Liue vaccination
Fish were immersed for 2 min in water containing
1 ml bacterial culture/l
(approximately 6 X lo5 CFU of live E. ictuluri). The E. ictuluri isolates were cultured in
brain-heart
infusion broth (BHI) for 24 h before vaccination.
Non-vaccinates
were
immersed in 1 ml uninoculated
BHI/l water for 2 min. The water temperature was
26C. The vaccinates and non-vaccinates
were returned to aquaria and infected with E.
ictaluri 14 days post-vaccination
(described below). We also conducted an experiment
in a similar fashion using Escherichia coli J5 to vaccinate channel catfish. The E. coli
vaccinates were infected with E. ictaluri 14 days post-vaccination
to determine possible
non-specific effects of vaccination.

Table 1
E. ictaluri isolates used in experiments
Isolate

Source

AL-93-15
AL-93-58
ATCC-33202
s-94-1017
s-94-1051
S-94-629
EILO

channel catfish
channel catfish
American Type
channel catfish
channel catfish
channel catfish
walking catfish

Original

Location
with ESC
with ESC
Culture Collection
with ESC
with ESC
with ESC

isolate was from a diseased catfish in Georgia.

Alabama
Alabama
Georgia
Mississippi
Mississippi
Mississippi
Thailand

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P.H. Klesius, C.A. Shoemaker/Aquaculture

2.4. Temperature

and duration

157 (1997) 147-155

One thousand channel catfish (approximately

8 g each) were vaccinated with E.
ictaluri isolate EILO at 26C following the procedure described above to examine the
influence of temperature on and duration of protection following live vaccination. The
water temperature of 500 live vaccinated fish and 500 controls was lowered to 18C
(over a 24 h period after vaccination)
and these fish were held at 18C. Water
temperature was lowered to determine if acquired immunity developed and remained in
fish held at lower temperature. If acquired immunity remained, it would suggest channel
catfish may be protected over a wider range of temperature. Fish (approximately
30)
were placed into 55-l aquaria (in triplicate) and infected as described below, monthly for
4 months. The fish at 18C were placed into aquaria and the water temperature raised
(over a 24-h period) to 26C. Fish were then allowed to acclimate for 5 d before
infection. The E. ictaluri isolate used for infection was AL-93-75.
2.5. Infection
The vaccinates and non-vaccinates
were infected by immersion in separate tanks
using the same inoculum of bacteria, following the procedure of Klesius and Sealey
(1995). Briefly, fish were infected by immersion in 1 X IO7 CFU/ml
of E. ictaluri at
26C for 1 h. The cumulative mortality due to ESC was determined 10 d post-infection.
RPS values were calculated according to Amend (1981).
2.6. Statistical

analysis

Data were analyzed by one-way analysis of variance using Duncans multiple-range

test (SAS Institute Inc., 1985). Significant differences were determined at P < 0.05.

3. Results
Vaccination
of channel catfish with six out of seven live isolates resulted in
significant protection against a virulent isolate of E. ictaluri AL-93-75 (Table 2). The
RPS of these vaccinates ranged from 40.8 to 90.6%. Amend (1981) indicated a positive
effect of a vaccine was suggested by RPS values greater than 50.0%. Vaccination with
the reference isolate ATCC-33202 failed to produce protection against AL-93-75. The
RPS was only 15.7%, with the exception of this isolate, vaccination with live bacteria
conferred excellent protection. Vaccination of channel catfish with E. coli J5 resulted in
an RPS of 2.6%, against E. ictaluri AL-93-75.
Vaccination
of channel catfish with the live EILO isolate resulted in significant
protection against AL-93-75, ATCC-33202, S-94-1017, S-94-629. Table 3 shows RPS
of 84.2 to 98.4%. Live vaccination with EILO produced RPS of 92.6% against infection
with EILO. Live vaccination with EILO failed to protect against AL-93-58 (RPS of

P.H. Klesius, CA. Shoemaker/Aquaculture

Table 2
Survival of heterologous
isolate of E. ictaluri

isolate immunized

Immunogen

Challenge

isolate

isolate

and non-immunized
Cumulative

channel

catfish to challenge

survival (fish challenged)b

AL-93-75
AL-93-75
AL-93-75
AL-93-75
AL-93-75
AL-93-75
AL-93-75
AL-93-75

with AL-93-75

Relative percent
survival (RPS)

Tanks

AL-93-75
AL-93-58
ATCC-33202
s-94-1017
s-94-105 1
S-94-629
EILO
Non-immunized

151

157 (19971 147-155

30(301
25(28)

23(24)
23(28)
18(30)
19(21)
lOi21)
27(27)
27(29)
1 l(30)

1 l(16)
26(30)

6(29)
21(26)
15(27)
26(28)
18(22)
9(30)

lo(30)
17(20)
18(28)
17(19)
20(25)
4(30)

88.3
80.9
15.7
79.7c
40.8
90.6
80.2
_

dTen days post-challenge

with 1 X 10 CFU/ml
E. ictduri AL-93-75 and 14 days post-immunization.
bNumber of fish surviving immunization at 14 days post-immunization.
RPS values were significantly different (P < 0.05) from the non-immunized catfish.

15.6%). Vaccination with S-94-629 provided protection against itself (S-94-629) with an
RPS of 93.0% (data not shown). Again, excellent vaccinate survival was obtained with
the live vaccination practice.

Table 3
Survival of EILO isolate
isolates of E. ictaluri
Immunogen

isolate

immunized

Challenge

and non-immunized

isolate

Cumulative

channel

catfish

to challenge

survivala (fish challenged)b

AL-93-75
AL-93-75
AL-93-58
AL-93-58
ATCC-33202
ATCC-33202
s-94-1017
s-94-1017
S-94-629
S-94-629
EILO
EILO

Relative percent
survival (RPS)

Tanks

EILO
Non-immunized
EILO
Non-immunized
EILO
Non-immunized
EILO
Non-immunized
EILO
Non-immunized
EILO
Non-immunized

with heterologous

28(30)

28(30)

4(30)
17(30)
14(30)
28(30)
lO(30)
28(30)
22(30)
19(20)

30(30)
6t30)
28(30)
26(30)
30(30)
22(30)
29(30)
25(30)
24(24)

5(29)
30(30)
19(30)

o(301
3000)
23(30)

9(30)
28(30jd
21(30)

4(30)
18(301d
18(30)
30(30)d
12(30)
30(30Y
24(30)
29(29)

94.7c
15.6
95.7
84.2
98.4
92.6

Ten days post-challenge

with 1 X 10 CFU/ml
E. ictaluri isolates and 14 days post-immunization.
bNumber of fish surviving immunization at 14 days post-immunization.
RPS values were significantly different (P < 0.05) from the non-immunized catfish.
dData do not fit Amends criteria (less than 60% mortality among controls) for calculating RPS values,

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P.H. Klesius, CA. Shoemaker/Aquaculture

Table 4
Survival of EILO and AL-93-58 combined isolates
challenge with heterologous isolates of E. ictaluri
Immunogen

isolate

Challenge

isolate

157 (1997) 147-155

immunized

Cumulative

and non-immunized

survivala (fish challengedlb

Tanks

EILO/AL-93-58
Non-immunized
EILO/AL-93-58
Non-immunized
EILO/AL-93-58
Non-immunized
EILO/AL-93-58
Non-immunized
EILO/AL-93-58
Non-immunized

AL-93-75
AL-93-75
AL-93-58
AL-93-58
ATCC-33202
ATCC-33202
s-94-1017
s-94-1017
EILO
EILO

channel

catfish

to

Relative percent
survival (RPS)

30(30)
20(30)
24(29)

29(30)
24(30)
28(29)

21(27)
20(30)*
25(30)

l(301
14(191
27(30)
29(30)

3(30)
18(23)*
30(30)
29(30)

3(301
26(30)

2(301
25(29)
O(30)
30(301
25(30)
30(30)

l(30)
24(30)

o(301

O(30)

o(301

73.0
86.6c
46.2
96.7
88.9

Ten days post-challenge

with 1 X 10 CPU/ml
E. ictaluri and 14 days post-immunization
93-58.
bNumber of fish surviving immunization at 14 days post- immunization.
RPS values were significantly different (P < 0.05) from the non-immunized catfish.
*Data do not lit Amends criteria (less than 60% mortality among controls) for calculating

with EILO/AL-

RPS values.

Channel catfish vaccinated with a combination of EILO and AL-93-58 isolates (Table
4) produced RPS of 46.2 to 88.9% against infection with isolates AL-93-75, ATCC33202, S-94-1017, EILO and AL-93-58.
Considerable
variation was seen in the data generated from the temperature and
duration study (Table 5). Nevertheless, only some times and treatments showed this
variation. RPS at all times ranged from 50.6% (lowest) to 100% (highest). The RPS of

Table 5
Relative percent survival of channel catfish following
for 4 months
Month

Immunized cumulative
(fish challengedlb

survival

Tanks

live E. ictaluri immunization

Non-Immunized cumulative
(fish challengedlb

at 26C and held at 18C

survivala

Tanks

Relative
percent
survival
(RPS)

15(32)

2
3
4

30(321
34(34)
3Ot301

32(32)
29(32)
27(35)
30(30)

32(321
32(32)
31(351
30(301

12(32)
12(32)
29(33)
12(30)

3(321
o(32)
27(35)
23(30)

32(321d
l(32)
23(35)*
2Oi30)*

65.3
94.0c
50.6
100.0

Ten days post-challenge with 1 X 10 CPU/ml

E. ictaluri.
bNumber of fish stocked per tank following immunization and holding at 18C.
RPS values represent significant differences (P < 0.05) between immunized and non-immunized
that month.
dData do not fit Amends criteria (less than 60% mortality among controls) for calculating RPS

groups for

values.

P.H. Klesius, C.A. Shoemker/Aquaculture

157 (19971 147-155

all times and treatments were above 50% which is considered

vaccination (Amend, 1981).

a favorable

153

effect of

4. Discussion
Vaccination with a low dose of live E. ictaluri resulted in strong acquired immunity
against infection with most E. ictaluri isolates. However, differences exist between E.
ictuluri isolates in their ability to induce protection against infection with all isolates of
E. ictaluri. The basis for this difference is unknown, but differences in antigens between
isolates are suggested. However, Bertolini et al. (1990) indicated isolates of E. ictuluri
were antigenically
homologous
when examined using formalin and heat killed E.
ictaluri and that the isolates composed a single antigenic serotype based on antibodies
generated in rabbits. Rogers (19811, Waltman et al. (1986) and Plumb and Vinitnantharat (1989) indicated that E. ictuluri is a serologically homogenous bacterium. Plumb
and Klesius (1988) found no major antigen differences between isolates using a panel of
monoclonal antibodies. The antigenic homogeneity as determined in the previous studies
(i.e., using mouse or rabbit serum) may or may not translate into antigenic homogeneity
as seen by fish. Mutheria et al. (1993) and Hastings and Ellis (1988) demonstated that
differences exist in the way fish and mammals distinguish antigens. Thus, the variability
seen in the protective ability of the isolates indicates antigenic heterogeneity which may
be important to differential recognition of the isolates by channel catfish.
Killed vaccines against heterologous and homologous infections have proved ineffective. Live vaccination practices that provide greater protection probably resulting from
induction of cell mediated responses were more vigorous and/or were directed against
protective antigens restricted to live bacteria. Work on Brucella abortus, a facultative
intracellular pathogen of mice and cattle suggests that non-living vaccines (killed B.
abortus or antigen preparations, i.e., peptidoglycan
antigen-PG 3 1, lipopolysaccharideLPS, or outer membrane proteins-OMPs) produce protection of short duration (Montaraz
and Winter, 1986). Protection of long duration (> 1 week) was not demonstrated. In
contrast, Montaraz and Winter (1986), Schurig et al. (199 1) and Winter et al. (1996)
demonstrated mice vaccinated with live Brucellu (attenuated rough strains) were protected from l-4 weeks post-infection. In the latter study (Winter et al., 1996) mice were
protected against homologous and heterologous species of Brucella. Dickerson et al.
(1993) demonstrated immunity to Zchthyopthirius multi$liis infection of channel catfish
which survived infection with I. multijiliis. They demonstrated survivors were immune
to homologous and heterologous isolates of I. multifiliis.
Fish immunized by controlled exposure to live E. ictaluri and challenged up to 4
months after initial exposure were protected against a heterologous E. ictuluri isolate.
Plumb et al. (1986) suggested that catfish immunized at 25C (with formalin-killed
E.
ictduri) and water temperature lowered to 12C were partially protected upon challenge
with the homologous bacteria. Bly and Clem (1991) demonstrated in vitro immunosuppresion in channel catfish held at low water temperatures.
However, they did not
challenge the fish to look at the protective immune response to E. ictuluri at low
temperature. The results show that acquired immunity is long lasting at 18C.

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P.H. Klesius, C.A. Shoemaker/Aquaculture

157 (1997)

147-155

B. abortus and E. ictaluri are similar in that killed vaccines against these pathogens
(Montaraz and Winter, 1986; Thune et al., 1994) do not induce protection of long
duration. Work to date suggests cell-mediated immunity is important in development of
protective immunity to intracellular pathogens (North, 1974; North, 1975; Eisenstein et
al., 1984; Montaraz and Winter, 1986; Antonio and Hedrick, 1994; Shoemaker et al., in
press). It has also been demonstrated
that antibodies act as important opsonins for
Brucella (Araya and Winter, 1990) and E. ictaluri (Ainsworth and Dexaing, 1990;
Shoemaker et al., in press). Immunization
with live E. ictaluri results in production of
antibody to this pathogen (Klesius and Sealey, 1995). The development
of both
cell-mediated and humoral immunity which results from exposure to live bacteria results
in better protective immunity than would be present if only a single branch of the
immune system was stimulated (Araya et al., 1989; Jimenez deBaugues et al., 1994;
Marsden et al., 1996). This research suggests that protective immunity may be dependent on the antigenic composition of the E. ictaluri used to prepare a modified live
vaccine against ESC.
Acknowledgements
We wish to thank Wendy M. Sealey for technical laboratory assistance in performing
some challenges. Thanks to Dr. Stephen Kattarri (Virginia Institute of Marine Science,
Gloucester Point, VA, USA) and Dr. Harry W. Dickerson (Department of Medical
Microbiology,
University of Georgia, Athens, GA, USA) for critical reading of the
manuscript.
The authors also thank Dr. David Wise (Mississippi State Agricultural
Experiment Station, Stoneville, MS, USA) for providing some bacterial cultures used in
the experiments.
References
Ainsworth, A.J., Dexaing, C., 1990. Differences in the phagocytosis
of four bacteria by channel catfish
neutrophils. Dev. Comp. Immunol. 14, 201-209.
Amend, D., 1981. Potency testing of fish vaccines, Dev. Biol. Stand. 49, 447-454.
Antonio, D.B., Hedrick, R.P., 1994. Effects of the corticosteroid Kenalog on carrier state of juvenile channel
catfish exposed to Edwardsiella ictaluri. J. Aquat. Animal Health 6, 44-52.
Araya, L.N., Winter, A.J., 1990. Comparative protection of mice against virulent and attenuated strains of
Brucella ahortus by passive transfer of immune T-cells or serum. Infect. Immun. 58, 254-256.
Araya, L.N., Elzer, P.H., Rowe, GE., Enright, F.M., Winter, A.J., 1989. Temporal development of protective
cell-mediated and humoral immunity in BALB/c mice infected with Brucella abortus. J. Immunol. 143,
3330-3337.
Bertolini, J.M., Cipriano, R.C., Pyle, S.W., McLaughlin, J.J.A., 1990. Serological investigation of the fish
pathogen Edwardsiella ictaluri, cause of enteric septicemia of catfish. J. Wildl. Dis. 26, 246-252.
Bly, J.E., Clem, L.W., 1991. Temperature-mediated
processes in teleost immunity: In vitro immunosuppression induced by in vivo low temperature in channel catfish. Vet. Immunol. Immunopathol.
28, 365-377.
Dickerson, H.W., Clark, T.G., Leff, A.A., 1993. Serotypic variation among isolates of Ichthyopthirius
multifiliis based on immobilization.
J. Euk. Microbial. 40, 816-820.
Eisenstein, T.K., Killar, L.M., Sultzer, B.M., 1984. Immunity to infection with Salmonella yphimurium:
mouse-strain differences in vaccine-and serum-mediated protection. J. Infect. Dis. 150, 425-435.
Hastings, T.S., Ellis, A.E., 1988. The humoral immune response of rainbow trout, Salmo gairdneri Richardson, and rabbits to Aeromonas salmonicida extracellular products. J. Fish Dis. 11, 147-160.

P.H. Klesius, C.A. Shoemaker/Aquaculture

157 (1997) 147-155

155

Hawke, J.P., 1979. A bacterium associated with disease of pond cultured channel catfish, Ictalurus punctatus.
J. Fish. Res. Board Can. 36, 1508-1512.
Hawke, J.P., McWhorter, A.C., Steigerwalt, A.G., Brenner, D.J., 1981. Edwardsielia ictaluri sp. nav., the
causative agent of enteric septicemia of catfish. hit. J. Syst. Bacterial. 31, 396-400.
Jimenez deBaugues, M.P., Elzer, P.H., Blasco, J.M., Marin, C.M., Gamazo, C., Winter, A.J., 1994. Protective
immunity to Brucella ovis in BALB/c
mice following recovery from primary infection or immunization
with subcellular vaccines. Infect. Immun. 62, 632-638.
Kasornchandra,
J., Rogers, W.A., Plumb, J.A., 1987. Edwardsiella ictuluri from walking catfish Clarias
hatrachus L., in Thailand. J. Fish Dis. IO, 137-138.
Klesius, P., 1992. Immune system of channel catfish: an overture on immunity to Edwardsiellu ictaluri. Annu.
Rev. Fish Dis. 2, 235-338.
Klesius, P.H., Sealey, W.M., 1995. Characteristics of serum antibody in enteric septicemia of cattish. J. Aquat.
Animal Health 7, 205-210.
Marsden, M.J., Vaughan, L.M., Foster, T.J., Secombes, C.J., 1996. A live (Delta-aroA) Aeromonas salmonicida vaccine for furunculosis preferentially
stimulates T-cell responses relative to B-cell responses in
rainbow trout (Oncorhynchus mykiss). Infect. Immun. 64, 3863-3869.
Montaraz, J.A., Winter, A.J., 1986. Comparison of living and nonliving vaccines for Brucella abortus in
BALB/c mice. Infect. Immun. 53, 245-25 1.
Mutheria, L.W., Raymond, T.B., Dekievit, T.R., Stevenson, R.M.W., 1993. Antibody specificities of polyclonal rabbit and rainbow trout antisera against Vibrio ordalii and serotype 0:2 strains of Vihrio
anquillarum. Can. J. Microbial. 39, 492-499.
Newman, S.G., 1993. Bacteria1 vaccines for fish. Annu. Rev. Fish Dis. 3, 145-185.
North, R.J., 1974. Cell-mediated
immunity and the response to infection. In: McCluskey, R.T.. Cohen, S.
(Eds.), Mechanisms of Cell-mediated Immunity. Wiley, New York, pp. 1X5-200.
North, R.J., 1975. Nature of memory in T-cell-mediated
antibacterial immunity: anamnestic production of
mediator T cells. Infect. Immun. 12, 754-760.
Nusbaum, K.E., Morrison, E.E., 1996. Entry of 5S-labe11ed Edwardsiella ictaluri into channel catfish
(Ictalurus punctatus). J. Aquat. Animal Health 8, 146- 149.
Plumb, J.A., Klesius, P., 1988. An assessment of the antigenic homogeneity of Edwardsiella ictaluri using
monoclonal antibody. I. Fish Dis. Il. 499-510.
Plumb. J.A., Vinitnantharat,
S., 1989. Biochemical, biophysical and serological homogeneity of Edwardsiella
ictuluri. J. Aquat. Animal Health 1, 51-56.
Plumb, J.A., Vinitnantharat,
S., 1993. Vaccination of channel catfish, Ictalurus punctatus, (Rafinesque), by
immersion and oral booster against Edwardsiella ictuluri. J. Fish Dis. 16, 65-71.
Plumb, J.A., Vinitnantharat,
S., Abe, V., Phelps, R.P., 1994. Density-dependent
effect on oral vaccination of
channel catfish against Edwardsiella ictaluri. Aquaculture 122, 91-96.
Plumb, J.A., Wise, M.L., Rogers, W.A., 1986. Modulary effect of temperature on antibody response and
specific resistance to challenge of channel catfish. Ictalurus punctatus, immunized against Edwardsiella
ictaluri. Vet. Immunol. Immunopath. 12, 297-304.
Rogers, W.A., 1981. Serological detection of two species of Edwardsiella infecting channel catfish. Dev. Biol.
Stand. 49, 169-172.
SAS Institute Inc.. 1985. SAS users guide: statistics, version 5 edn. SAS Institute, Cat-y, North Carolina.
Schurig, G.C., Roop II. R.M., Bagchi, T., Boyle, S., Buhrman, D., Sriranganathan,
N., 1991. Biological
properties of RB5 1; a stable rough strain of Brucella abortus. Vet. Microbial. 28, 171- 188.
Shoemaker, C.A., Klesius, P.H., Plumb, J.A., in press. Killing of Edwardsiella ictaluri by macrophages from
channel catfish immune and susceptible to enteric septicemia of catfish. Vet. Immunol. Immunopath.
Thune, R.L., Hawke, J.P., Johnson, M.C., 1994. Studies on vaccination
of channel catfish, lctalurus
punctofus, against Edwarsiella ictaluri. J. Appl. Aquaculture 3, 11-23.
Waltman, W.D., Shotts, E.B., Hsu, T.C., 1986. Biochemical characteristics
of Edwardsiella ictaluri. Appl.
Environ. Microbial. 51, 101-104.
Winter, A.J., Schurig, G.G., Boyle, S.M., Sriranganathan,
N., Bevins, J.S., Enright, F.M., Elzer, P.H., Kopec,
J.D., 1996. Protection of BALB/c
mice against homologous and heterologous
species of Brucella by
rough strain vaccines derived from Brucella melitensis and Brucella suis biovar 4. Am. J. Vet. Res. 57,
677-683.