Академический Документы
Профессиональный Документы
Культура Документы
ELSEVIER
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:
* 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
148
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
149
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
Alabama
Alabama
Georgia
Mississippi
Mississippi
Mississippi
Thailand
150
2.4. Temperature
and duration
analysis
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
isolate immunized
Immunogen
Challenge
isolate
isolate
and non-immunized
Cumulative
channel
catfish to challenge
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
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
_
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
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
152
Table 4
Survival of EILO and AL-93-58 combined isolates
challenge with heterologous isolates of E. ictaluri
Immunogen
isolate
Challenge
isolate
immunized
Cumulative
and non-immunized
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
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
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
groups for
values.
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.
154
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.
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.