8. c5. modified live vaccine

8/2/2019 8. C5. Modified Live Vaccine

1/13

J OU RN AL OF T HE Vol. 40, No. 5

W O RL D AQ U AC UL T UR E SO CI ETY October, 2009

Use of Modified Live Vaccines in Aquaculture

Craig A. Shoemaker1

and Phillip H. Klesius

Aquatic Animal Health Research Laboratory, United States Department of

Agriculture-Agricultural Research Service, 990 Wire Road, Auburn, Alabama 36832 USA

Joyce J. Evans

Aquatic Animal Health Research Laboratory, United States Department of

Agriculture-Agricultural Research Service, 118 B S. Lynchburg Street, Chestertown, Maryland

21620 USA

Covadonga R. Arias

Department of Fisheries and Allied Aquacultures, Auburn University, Alabama 36849 USA

AbstractVaccination is an important disease management strategy used to maintain human and animal

health worldwide. Vaccines developed for aquaculture have reduced antibiotic use in fish production.

Original fish vaccines were bacterins (formalin-killed bacteria) delivered through immersion or

injection that induced humoral (antibody) immunity. Next generation vaccines relied on multiple

killed antigens delivered with an adjuvant to enhance vaccine effectiveness. Work in the 1990s

showed the use of various strategies to develop modified live vaccines for use in fish. A modified live

vaccine is a live pathogen that has been rendered non-pathogenic or avirulent by physical, chemical,

or genetic engineering methods. The modified live vaccine typically retains its ability to infect the

host which allows for effective presentation of protective antigens to generate cellular immunity

(CD4 or CD8 T-cell responses). Modified live vaccines are advantageous in that they can be easily

delivered (i.e., by immersion to young fish) and stimulate both humoral and cellular immunity of long

duration. Disadvantages include issues with modified live vaccine safety to the host and environment.A successful modified live vaccine for use in warm water aquaculture is used to highlight the live

vaccine strategy.

Vaccination is an effective strategy used

worldwide for controlling infectious diseases

in all animal species. The first known vac-

cine to protect humans against small pox was

derived from a live virus used as a vaccine.

Jenner (1961) demonstrated that vaccinia pro-

tected humans against the small pox virus by

passage of the virus from horse, to cow udder,

to humans. Tizard (1999) provides an excel-

lent review of early events in vaccinology of

humans and veterinary animals. Interestingly,

many of the early vaccines used were modified

live vaccines based on the success of the small

pox vaccine.

Intensification of fish reared in captivity for

use as food and for remedial stocking pro-

grams has resulted in an increased presenceof disease. Disease is manifested under these

1 Corresponding author.

intense conditions because of many factors,

including inadequate maintenance of environ-

mental conditions (i.e., poor water quality) and

the ease of transmission of pathogens in fish

reared at high stocking densities. Duff (1942)

was the first to report on a vaccine trial incutthroat trout, Oncorhynchus clarki, that were

fed a killed Aeromonas salmonicida vaccine. In

aquaculture, most early vaccine work focused

on use of killed vaccines. The first commer-

cially licensed vaccine for fish was a killed vac-

cine delivered by immersion against Yersinia

ruckeri the causative agent of enteric redmouth

disease (Plumb 1999). Following the success

of this product, formalin-killed immersion vac-

cines for vibriosis of trout and salmon weredeveloped. The same principle for inactivation

(i.e., formalin treatment) of bacterial pathogens

of Atlantic salmon, Salmo salar, was used to

Copyright by the World Aquaculture Society 2009

573


2/13

574 SHOEMAKER ET AL.

develop the early salmonid vaccines (Elvelyn

1997) to be delivered by immersion. Immer-

sion vaccines against A. salmonicida were not

effective in the field and thus the first injec-

tion bacterins were developed. Bricknell et al.(1997) and ODowd et al. (1999) studied the

immune response to different A. salmonicida

killed vaccines in Atlantic salmon without the

use of adjuvants. Extracellular polysaccharide

(EPS) vaccines induced an antibody response

and were protective for about 2 mo follow-

ing injection (Bricknell et al. 1997). ODowd

et al. (1999) used formalin-killed bacterins gen-

erated from A-layer positive or A-layer nega-

tive A. salmonicida grown under iron-restrictedconditions to immunize Atlantic salmon. These

preparations resulted in significant antibody

responses that were enhanced following booster

immunization; however, the fish were not chal-

lenged. Bricknell et al. (1999) using a combina-

tion EPS and A-layer negative bacterin demon-

strated antibodies and protection at 8 wk post-

injection immunization (90% relative percent

survival [RPS]). This same vaccine formulation

yielded protection (60 RPS) for up to 9 mo.

Most vaccines presently used in the Atlantic

salmon industry rely on multiple antigens (bac-

terial and viral) in oil-adjuvant delivered in one

injection (Sommerset et al. 2005). These vac-

cines are successful and have reduced the use

of environmentally unfriendly chemicals, espe-

cially antibiotics, even as commercial salmon

production has increased (Markestad and Grave

1997). The higher-valued product has allowed

the use of the injection vaccination strategy

in the salmon industry. A recent review onthe use of vaccines in the aquaculture indus-

try in Chile indicated that both immersion

and injection vaccination strategies are used

(Bravo and Midtlyng 2007). Injection vacci-

nation of fish that must be vaccinated at a

young age (1012 d post-hatch) or small size

(12 g smolt) is not practical. The explo-

ration and use of attenuated or modified live

bacterial vaccines in aquaculture was initiated

in the 1990s (Norqvist et al. 1989; Vaughanet al. 1993; Thornton et al. 1994; Lawrence

et al. 1997; Hernanz Moral et al. 1998; Mars-

den et al. 1998; Klesius and Shoemaker 1999;

Thune et al. 1999). Presently, three modified

live vaccines are licensed for use in the USA.

These include the vaccine against bacterial kid-

ney disease (Renogen1), enteric septicemia of

catfish disease (AQUAVAC-ESC) and colum-naris disease (AQUAVAC-COL). This reviewfocuses on attenuation strategies for the devel-

opment of modified live vaccines, advantages

and disadvantages of using modified live vac-

cines in aquaculture and presents an example

of a successfully commercialized modified livevaccine currently used in warmwater aquacul-

ture.

Attenuation StrategiesLaboratory Passage

Multiple methods have been used to atten-

uate pathogens for successful development of

modified live vaccines in human and veteri-

nary medicine. The methods used thus far onpathogens of fish will be highlighted. One

of the earliest techniques was simple labo-

ratory passage of organisms on media or in

tissue culture that resulted in attenuation of

the pathogenic microorganism. An early patentdescribes use of this technique with channelcatfish, Ictalurus punctatus, virus (CCV) (Hart-

man and Noga 1980). They created an atten-

uated CCV by passage of the virus in tissue

culture using a cell line derived from the walk-

ing catfish, Clarius batrachus. More recently,

Daly et al. (2001) used laboratory attenuated

Renibacterium salmoninarum strains to immu-

nize Atlantic salmon. They isolated two strains

that grew readily on common bacterial mediumand demonstrated protection following injection

vaccination. Itano et al. (2006) also suggest the

use of a low virulence Nocardia seriolae isolate

as a potential vaccine strain and demonstrated

protection following virulent challenge. How-

ever, the isolate was not completely attenuated

in yellowtail, Seriola quinqueradiata, the host

animal.

1

Mention of trade names or commercial products inthis publication is solely for the purpose of providing spe-cific information and does not imply recommendation orendorsement by the United States Department of Agricul-

ture.


3/13

MODIFIED LIVE VACCINES IN AQUACULTURE 575

Environmental Bacterium

A second technique involves passage of the

virulent pathogen in an alternative host (i.e.,

one not susceptible to that pathogen) or theuse of an environmental bacterium that mim-

ics the pathogens antigenic structure. The most

successful use of this strategy in aquacul-

ture is use of Arthrobacter davidanieli deliv-

ered by injection to protect salmon from bac-

terial kidney disease caused by R. salmoni-

narum (Griffiths et al. 1998; Salonius et al.

2005). The authors showed a close phylo-

genetic relationship between A. davidanieli

and R. salmoninarum and the ability of anti-serum to cross-react with surface carbohydrates

(Griffiths et al. 1998). More recently, Itano

et al. (2006) attempted to use a similar strat-

egy (i.e., phylogenetic relatedness and anti-

genic cross-reactivity) to identify vaccine can-

didates against N. seriolae. In this study, the

authors used environmental Nocardia species

and evaluated the ability of these isolates (N.

soli and N. fluminea) to induce protective

immunity againstN

. seriolae in injected yel-lowtail. Unfortunately, the isolates provided

minimal protection to challenge with virulent

N. seriolae.

Chemical or Physical Mutagenesis

The third strategy involves chemical or phys-

ical mutagenesis that results in random muta-

tion(s) in the pathogen, with the vaccine strain

being selected because of the lack of virulencein the host animal (Linde et al. 1990; Klesius

and Shoemaker 1999; Shoemaker et al. 2007).

The best example is the use of rifampicin, an

antibiotic that results in attenuation of gram-

negative bacteria by inducting changes in the

lipopolysaccharide (LPS), an important viru-

lence factor (Schurig et al. 1991; Klesius and

Shoemaker 1999; Arias et al. 2003; Zhang et al.

2006). Details on the changes to LPS induced

by rifampicin passage are discussed in detail inthe example provided in this review. This strat-

egy has been one of the most practiced to date

in generation of successful veterinary vaccines

(Linde et al. 1990; Schurig et al. 1991; Kle-

sius and Shoemaker 1999; Gantois et al. 2006;

Shoemaker et al. 2007).

Genetic Engineering

The fourth strategy is genetic engineering

typically by insertion, deletion, or disruption

of metabolic pathway(s) or virulence gene(s)

that result in pathogen attenuation. Cooper et al.

(1996) used a mini-transposon to disrupt the

production of chondroitin sulfatase (suspected

virulence factor) in E. ictaluri. Channel catfish

exposed to this mutant were shown to be pro-

tected following challenge with virulent wild-type parent isolate. A more recent attempt was

use of transposon mutagenesis to generate an O-

polysaccharide deficient isolate ofE. ictaluri to

be used as a modified live vaccine (Lawrence

et al. 2001; Lawrence and Banes 2005). The

authors were successful in generation of O-

polysaccharide deficient isolate of E. ictaluri

but failed to demonstrate protection in immer-

sion immunized catfish (Lawrence and Banes

2005).

Igarashi and Iida (2002), using a similar

technology, reported the development of an

attenuated E. tarda vaccine by creating an

E. tarda mutant (transposon mutagenesis) with

lower siderophore production that protected

tilapia, Oreochromis niloticus, upon lethal chal-

lenge. Leung et al. (1997) also generated mini-

Tn5 (transposon mutants) induced growth and

protease-deficient A. hydrophila for use as

modified live vaccines in blue gourami (Tri-

chogaster trichopterus). The generated vaccinestrains were not completely attenuated in blue

gourami using this strategy (Leung et al. 1997).

Random transposon (Tn917) mutagenesis

and subsequent screening in hybrid striped bass

(Morone chrysops X M. saxatilis) produced

a Streptococcus iniae with a disrupted phos-

phoglucomutase gene (Buchanan et al. 2005).

The phosphoglucomutase enzyme is believed

to be important for polysaccharide capsule for-

mation in bacteria. Presence or absence of acapsule has been suggested to be important for

virulence (Barnes et al. 2003; Buchanan et al.

2005). This isolate was attenuated in hybrid


4/13


striped bass and shown to provide protection

upon lethal challenge if the modified live S.

iniae was delivered by intraperitoneal injection

(Buchanan et al. 2005).

One of the more common strategies is useof auxotrophic mutants (Hoiseth and Stocker

1981; Smith et al. 1984; Vaughan et al. 1993;

Lawrence et al. 1997; Hernanz Moral et al.

1998; Thune et al. 1999; Temprano et al. 2005).

Typically, the aroA gene of the pathogen is

inactivated by insertion of a DNA cassette

containing an antibiotic resistance gene marker

for selection upon allelic exchange using a

suicide vector. Inactivation of this gene does not

allow survival within the host because of theneed for aromatic metabolites. However, high

doses of these mutants may induce mortality in

the host animal (Lawrence et al. 1997; Thune

et al. 1999). Although this strategy creates

attenuated vaccine isolates, often times, these

attenuated isolates persist for short duration

(2472 h) and thus fail to stimulate adequate

immunity in young fish.

Advantages and Disadvantages of the Useof Modified Live Vaccines

Safety

The major disadvantage of using modified

live vaccines is safety. Killed vaccines or vac-

cine products are generally considered safe

for use in aquatic animals because the dis-

ease agents are killed or inactivated (chemical

or heat treatment). The potential safety issues

with killed vaccines are inadequate killing of

the vaccine (i.e., delivery of viable pathogen).Another problem associated with killed vac-

cines is the adhesions seen in salmonids fol-

lowing injection of the vaccine antigens with

the oil-adjuvants. Occasionally, this results in

decreased growth in the vaccinated animal

and loss of product because of the adhesions

affecting fillet quality (Evensen et al. 2005).

The concept of safety in the use of modified

live vaccines is with respect to the inability

of the vaccine strain to cause disease in thevaccinated animal. Furthermore, environmen-

tal issues become a concern because of the

potential release of the vaccine strain into the

environment. Frey (2007) provides an excellent

review of biological safety concepts of genet-

ically modified live bacterial vaccines used in

veterinary medicine. The basic principles apply

to the use of modified live vaccines in aquacul-ture. In the USA, the United States Department

of Agriculture (USDA)-Animal Plant Health

Inspection Services (APHIS) Center for Veteri-

nary Biologics provides the regulatory authority

for licensing and registering veterinary biolog-

ics (www.aphis.usda.gov). Safety studies con-

sist of experiments using 10 times the immuniz-

ing dose and direct fish-to-fish passage. Also,

a risk analysis should consider the release of

the vaccine into the environment. Presence ofthe pathogen in the natural environment and

the ability of the pathogen to infect people are

among the important considerations.

Protective Immunity

A major advantage of modified live vac-

cines is the ability to stimulate cell-mediated,

humoral (antibody) and mucosal immunity

(Clark and Cassidy-Hanley 2005). Modified

live vaccines survive and replicate within the

host, which results in a strong cellular immune

response that confers protection of long dura-

tion. Induction of cellular immunity (CD4+ and

CD8+ T-cell responses) is responsible for pro-

viding protection against intracellular infections

(Seder and Hill 2000). Recent studies have

demonstrated the relevant major histocompat-

ibility complex (MHC) class I (Antao et al.

2001) and class II molecules (Goodwin et al.

2000) in fish. Presentation of antigen with thecorrect MHC allows for the response and recog-

nition by the appropriate subpopulations of T-

and B-cells. Seder and Hill (2000) suggest that

modified live vaccines can induce Th1 and CD8

T-cell responses. Marsden et al. (1996) were

the first to demonstrate the ability of modified

live aroA deletion mutant A. salmonicida vac-

cine to preferentially enhance T-cell over B-cell

responses in modified live vaccinated rainbow

trout, Oncorhynchus mykiss. Recent transcrip-tome analysis of gene expression in catfish liver

tissue suggest that upon exposure to live bacte-

ria, MHC class I genes along with other acute


5/13


phase response genes are upregulated suggest-

ing active antigen processing and presentation

(Peatman et al. 2008). Induction of MHC class I

genes suggests a CD8 T-cell response following

infection with intracellular bacteria. Subsequentstimulation of a subpopulation of T-cells may

induce interferon gamma (Milev-Milovanovic

et al. 2006) production that can mediate intra-

cellular pathogen destruction (Seder and Hill

2000). Thus, assumptions can be made to the

nature of the immune response following mod-

ified live vaccine administration in fish.

Seder and Hill (2000) further point out that

killed or subunit vaccines are not effective

at stimulating cellular responses. Killed vac-cines, or delivery of killed antigens, results

in the induction of humoral (antibody medi-

ated) immunity. Killed Edwardsiella ictaluri

vaccine previously used in aquaculture was

shown not to enter the fish (Nusbaum and Mor-

rison 1996) and thus, failed to induce immu-

nity. In some cases, the killing process (e.g.,

formalin treatment) has been demonstrated to

alter important surface antigens (Bader et al.

1997). These two factors can lead to inacti-vated vaccine failure and no protection in young

fish against intracellular pathogens (Thune et al.

1997). Most killed vaccines are delivered by

injection in the presence of adjuvant to be

effective. Early Vibrio bacterins were deliv-

ered by immersion, however, the best protec-

tive effect has been shown following injec-

tion (Norqvist et al. 1989). Duration of immu-

nity induced by killed vaccines is often less

than 4 mo and only effective on extracellu-

lar pathogens or pathogens producing toxins.

Extending the length of immunity following

administration of killed vaccines often relies

on multiple immunizations and/or use of adju-

vant(s). Immunity following exposure to live

bacteria (cell mediated immunity) has been

demonstrated for greater than 4 mo in sin-

gle bath immunized catfish (Klesius and Shoe-

maker 1997).

Vaccine Delivery

Modified live vaccines have the advantage

of being easier to deliver to the animals in that

vaccination can occur through the oral route in

feed or water (Frey 2007) or through immersion

of fish in water (Norqvist et al. 1989; Klesius

et al. 2004). Modified live vaccines developed

for use in warmwater aquaculture are deliv-ered by immersion exposure to the youngest life

stage (i.e., prior to release into ponds). Modified

live vaccines retain their ability to colonize and

infect the host which allows for effective immu-

nity to develop following immersion delivery.

Modified live Vibrio anguillarum vaccines were

shown to induce high degrees of protection with

a small dose of vaccine following immersion

immunization (Norqvist et al. 1989). Norqvist

et al. (1989) suggest the modified live vaccinestrain was able to replicate in the host and thus

increased the vaccine signal.

Cost

Vaccine cost is a major question for manufac-

ture constraints and farmer acceptance. Modi-

fied live bacterial vaccines, for the most part,

have a low-to-moderate cost (Seder and Hill

2000; Klesius et al. 2004). A single attenuatedbacterium can be used to produce many liters

of vaccine in commercial-scale fermentors. The

major cost is in purity, safety, immunodose

determination, potency (efficacy) testing, and

packaging (e.g., lyophilization). For formalin-

killed vaccines, the cost is similar and killed

Y. ruckeri vaccines for trout are extensively

used. However, the need to handle and inject

individual fish in the Atlantic salmon industry

adds a significant labor cost. Newer strategies,such as recombinant DNA technologies and

DNA vaccination techniques, are potentially

more expensive because of the need to purify

the recombinant antigens and the initial invest-

ment in isolation and characterization of the

proper gene(s) to provide protective immunity

in the vaccinated host. DNA vaccination tech-

nology has been successfully used in Atlantic

salmon to protect against infectious hematopoi-

etic necrosis virus (IHNV) (Simard et al. 2006)and rainbow trout to protect against viral hem-

orrhagic septicemia virus (VHSV) (Lorenzen

et al. 1999). The acceptance of DNA vaccines


6/13


in some countries (i.e., food safety and geneti-

cally modified organism issues) may be a limit-

ing factor in the commercial realization of these

products (Babiuk et al. 2000).

Example of a Successful Modified Live

Vaccine in Warm Water Aquaculture

Background

Enteric septicemia of channel catfish (ESC)

is caused by E. ictaluri, a gram-negative bac-

terium that was initially described in 1976

(Hawke et al. 1981). Today, losses to ESC cost

the US catfish industry $4060 million, annu-ally. Development of formalin-killed E. ictaluri

bacterins was attempted, but they did not pro-

vide protection against ESC (Shoemaker and

Klesius 1997; Thune et al. 1997). Failure of

the killed products was because of stimula-

tion of only an antibody response (Klesius and

Sealey 1995; Klesius and Shoemaker 1997) and

the fact that killed bacteria were not entering

the fish following immersion exposure (Nus-

baum and Morrison 1996). Methods to controlESC relied on feeding antibiotic medicated diet;

however, this practice is ineffective because

disease reduces diet intake and antibiotic resis-

tance has been observed in the bacterium (Walt-

man and Shotts 1986).

Protective Immunity

Early work on E. ictaluri suggested that anti-

body was important for the protective immuneresponse (Vinitnantharat and Plumb 1993). Kle-

sius and Sealey (1995) demonstrated through

passive transfer that specific antibody was not

protective. Antonio and Hedrick (1994) were

the first to suggest cell-mediated immunity was

needed for protection; however, the method

they used was indirect by administering an

anti-inflammatory drug (Kenalog) and show-

ing that this increased susceptibility of catfish

to a second exposure to E. ictaluri. Shoe-maker and Klesius (1997) were the first to

demonstrate cellular immunity was responsi-

ble for protection. Fish used in their studies

were immunized by exposing them to low num-

bers of virulent E. ictaluri. Upon immuniza-

tion, macrophages were harvested from surviv-

ing fish and assessed for the ability to kill E.

ictaluri in vitro. Macrophages from fish vacci-nated with live E. ictaluri were able to kill E.

ictaluri (85.9%); whereas, macrophages from

non-vaccinated fish or fish immunized with a

killed bacterin were significantly less effective

at killing (68.1 and 71.4%, respectively) (Shoe-

maker and Klesius 1997). Furthermore, fish that

survived a low dose exposure to E. ictaluri

were resistant upon subsequent challenge with

homologous and sometimes heterologous iso-

lates (Klesius and Shoemaker 1997). Takentogether, the above research demonstrated the

need for a vaccine that would result in a cell-

mediated immune response in young catfish.

Modified Live E. ictaluri Development

Klesius and Shoemaker (1999) developed a

modified live E. ictaluri vaccine by passage of

a virulent isolate on media (brain heart infusion

agar) supplemented with rifampicin. This tech-

nique was used with other gram-negative bac-

teria to produce rough mutants (Schurig et al.

1991) that were used as vaccines. The rough

phenotype is characterized by a change in the

LPS of the parent isolate. This change was

thought to be the lack of an O-side chain of

LPS as seen in the RB-51 Brucella abortus vac-

cine. Recent work to characterize the mutant

E. ictaluri RE-33 demonstrated the change

was in the LPS. The mutant was shown to

lack high molecular weight bands of the LPSwhen compared to the parent isolate (EILO)

(Fig. 1) by immunoblot (Klesius and Shoe-

maker 1999; Arias et al. 2003). This change

resulted in an attenuated E. ictaluri that was

capable of entering and persisting in catfish to

allow for the proper immune response but with-

out causing disease (Klesius and Shoemaker

1999). Additional tests were conducted to deter-

mine whether the vaccine isolate (RE-33) dif-

fered in biochemical characters or fatty acidprofiles. Initial information suggested the parent

isolate (EILO) and vaccine mutant did not dif-

fer in biochemical parameters except that the


7/13


210 KD

110 KD

56 KD

37 KD

21 KD

A B

M 1 2 3 4 1 2 3 4

Figure 1. Immunoblot using anti-EILO (A) and anti-RE-33 (B) sera with lipopolysaccharide prepared from both the

parent isolate (EILO) and vaccine isolate (RE-33). Lane 1, EILO aqueous phase; lane 2, RE-33 aqueous phase; lane 3,

EILO phenol phase; lane 4, RE-33 phenol phase. M= molecular standard.

vaccine isolate grew on media supplemented

with rifampicin (Klesius and Shoemaker 1999).However, using the microbial identification sys-

tem (Microbial ID, Inc., Newark, DE, USA)

differences in fatty acid profiles were deter-

mined. Overall, quantitative differences were

seen in fatty acid profiles between the iso-

lates and some fatty acids were present only

in the chromatograph from the parent iso-

late EILO (3-hydroxy hexadecanoid acid) or

mutant isolate RE-33 (hepatdecanoic acid, 3-

hydroxy-heptadecanoic acid, and 10-methyl-

octodecanoid acid), allowing for accurate iden-

tification (Arias et al. 2003). Biochemical dif-

ferences were detected with Biologs (Biolog,

Hayward, CA, USA) carbon utilization sys-

tem for bacterial identification. Biologs system

demonstrated a percentage similarity of 73%

(Pearson product moment correlation) and thus

allowed the differentiation of the vaccine and

parent isolates (Arias et al. 2003).

Safety

The vaccine isolate (RE-33) was demon-

strated to be safe following direct fish-to-fish

passage (i.e., back passage) indicating an inabil-

ity to revert to the virulent form. Ten timessafety tests were also performed with no

adverse reactions following vaccination (Kle-

sius and Shoemaker 1999). Field trials were

conducted in 2.2 million 10- to 30-d-old chan-

nel catfish following state veterinarian and

USDA-APHIS approved protocols in Missis-

sippi and Alabama in 1997 with no adverse

effects of vaccination reported (Klesius and

Shoemaker 1999). Including field safety tri-

als conducted by Intervet, Inc. (1998 and

1999) more than 57 million channel catfish fry

and/or fingerlings were vaccinated to satisfy the

requirements for a safe product. No problems

were reported in fish following immersion vac-

cination from locations where the trials were

conducted.

Efficacy

Efficacy has been demonstrated in 3- to

9-mo-old channel catfish against E. ictaluri fol-lowing vaccination with the modified live vac-

cine (Klesius and Shoemaker 1999; Lawrence

and Banes 2005). Vaccine dose was variable


8/13


with a range of 1 105 to 1 107 colony-

forming units (CFUs) RE-33/mL being admin-

istered to fish by immersion exposure for

230 min. Wise et al. (2000) demonstrated the

vaccine to be effective in fish immunized at

1 107 CFU/mL in both laboratory and field

challenges (i.e., fish in cages) with E. ictaluri.

Wise et al. (2000) also demonstrated that vac-

cine efficacy was related to genetic differences

of families of channel catfish. Six full sibling

families of channel catfish were tested and RPS

was found to range from 67.1 to 100% with a

mean across all families of about 80%. These

results suggest catfish lines may be developed

that can respond better to vaccination.Another source of variability in efficacy stud-

ies is the isolate of E. ictaluri used to chal-

lenge immunized catfish (Klesius and Shoe-

maker 1997). Antigenic variation exists in E.

ictaluri with respect to acquired immunity.

We found that protection was not conferred

against all isolates tested. Protection (RPS >

50%) was seen against 813 isolates following

vaccination with the RE-33 E. ictaluri (Kle-

sius and Shoemaker 1999). Efficacy was alsodetermined using other rifampicin generated E.

ictaluri mutants (B-21909; B-21910; B-21911;

Agricultural Research Service Culture Collec-

tion, National Center for Agricultural Utiliza-

tion Research, Peoria, IL, USA). Table 1 shows

that the other vaccine isolates were able to

provide protection against homologous and het-

erologous challenge (Klesius and Shoemaker

2000). A polyvalent vaccine trial using com-

binations of rifampicin-attenuated mutantE

.ictaluri s (ATCC 202058 = RE-33; B-21909;

B-21910; B-21911) at various ratios to yield

a total vaccine dose of 1 107 CFU/mL of

immersion water (Table 2) (Klesius and Shoe-

maker 2000) was also conducted. Results of

the polyvalent trials demonstrated that a vac-

cine based on multiple rifampicin-attenuated

mutants ofE. ictaluri is possible and effective.

This is important if the present single isolate

vaccine becomes less effective. The formula-

tion may be changed or modified by addition

of one or more of the additional vaccine iso-

lates to provide solid protection if another anti-

genic type becomes predominant in the catfish

industry.

Vaccine effectiveness in young catfish (712

d post-hatch) and eyed eggs has been demon-

strated (Shoemaker et al. 1999; Wise and Ter-

hune 2001; Klesius et al. 2002; Shoemaker

et al. 2002; Shoemaker et al. 2007). Petrie-

Hanson and Ainsworth (1999) suggested that

catfish do not become immunocompetent (anti-

body mediated) until 4 wk post-hatch. Petrie-

Hanson and Ainsworth (2001) demonstrated

the presence of immune cells in the renal

hematopoietic tissue at hatch and in the spleen

by Day 3 post-hatch. Warr (1997) detected

functional lymphocytes in channel catfish lym-

phoid organs a few days after hatching. Most

work suggests cell-mediated immunity is

responsible for protection against E. ictaluri

(Shoemaker and Klesius 1997; Shoemaker et al.

1997). Development of immunity to the mod-

ified live vaccine is probably because of the

presence of macrophages or antigen presentingcells (in the young fry) or to the persistence of

Table 1. Efficacy of rifampicin-resistant Edwardsiella ictaluri mutants B-21909, B-21910, and B-21911 in channel

catfish challenged with E. ictaluri.

Treatmenta Challenge isolate (type) Percent mortality Relative percent survivalb

Non-vaccinated AL-93-75 46.7

B-21909c AL-93-75 (Homologous) 2.7 94.3

B-21910 AL-93-75 (Heterologous) 9.3 80.0

B-21911 AL-93-75 (Heterologous) 22.7 51.4

CFU = colony-forming unit; ESC = enteric septicemia of catfish.aAll fish were alive and free of signs of ESC for 21 d after vaccination.bRelative percent survival (RPS) was determined as by Amend (1981).cFish were vaccinated with 1 107 CFU of each vaccine isolate/mL of immersion water.


9/13


Table 2. Polyvalent vaccine trial using multiple rifampicin-attenuated strains of Edwardsiella ictaluri in various

combinations and ratios.

Ratioa Vaccine strains or controls Percent mortalityb Relative percent survivalc

Control (not vaccinated) 84.0 None ATCC 202058 24.0 71.4

None B-21909 20.0 76.2

1:1 ATCC 202058:B-21909 20.0 76.2

1:2 ATCC 202058:B-21909 56.0 33.3

2:1 ATCC 202058:B-21909 8.0 90.5

1:2 ATCC 202058:B-21911 16.0 80.9

1:1 ATCC 202058:B-21911 24.0 71.4

1:1 B-21910:B-21909 8.0 90.5

1:1 B-21910:B-21911 24.0 71.4

2:1 B-21911:ATCC 202058 20.0 76.2

1:1:1:1 ATCC 202058:B-21909: B-21910:B-21911 12.0 85.7

CFU = colony-forming unit.aTwenty-four hour cultures of the attenuated vaccine strains were mixed at the following ratios. The vaccine dose used

was equivalent to 1 107CFU of a single mutant or of the mixed attenuated mutants/mL of immersion water for 2 min.

Fish were returned to respective tanks and held for 16 d with no adverse effect of vaccination being seen.bMortality was determined after challenges with E. ictaluri isolate AL-93-58 (1 107 CFU/mL) for 30 min immersion

exposure.cRelative percent survival (RPS) was determined as by Amend (1981).

the live vaccine strain until the immune sys-

tem is responsive. This has been demonstrated

following in ovo administration of live viral

vaccines in poultry (Mast and Goddeeris 1999).

Knowledge that fish can be successfully vac-

cinated at 710 d post-hatch or as eyed fish

eggs (vaccinated 2472 h) prior to hatch is

important if other modified live vaccines (virus

or bacteria) become available for use in aqua-

culture. Utilizing this strategy will allow for

the earliest possible vaccination of fish (i.e., in

the hatchery) prior to release into a production

environment.

The modified live E. ictaluri RE-33 waspatented (US Patent no. 6,019,981) and licensed

to Intervet, Inc. by the USDA-Agricultural

Research Service. Intervet, Inc. (Millsboro, DE,

USA), marketed the modified live ESC vaccine

(2001 to present) under the label AQUAVAC-

ESC as an USDA-APHIS-CVB licensed vac-

cine for immersion vaccination of 710 d post-

hatch channel catfish. In 2006, the product was

changed from a freeze-dried to a frozen formu-

lation. Efficacy was re-evaluated and demon-strated with the frozen product for at least

65 d post-vaccination (Wise 2006). Field tri-

als suggested use of the vaccine results in

larger fingerlings, improved feed conversion,

and an improved return of $3900$4900 per

ha for vaccinated fish versus non-vaccinated

fish (Intervet Inc. 2003; Wise 2006). Carrias

et al. (2008) recently reported on the use of

the modified live vaccine in addition to an

extended hatchery/nursery phase. Results sug-

gest an improved return (money and survival)

for vaccinated fish held in a nursery setting

prior to release into fingerling ponds (Carrias

et al. 2008). Reports from the USA catfish

industry indicate greater than 25% of the fry

and/or fingerlings produced each year are vac-

cinated with AQUAVAC-ESC. Channel catfish

producers can and are using this vaccine in

health management plans to effectively manage

ESC.

Conclusions

Modified live vaccines are successfully used

in human, veterinary, and aquatic animal

medicine to prevent disease. Use of modifiedlive vaccines in aquaculture is an appropri-

ate strategy if potential risks to the animal,

environment and people are low or negligible.


10/13


Regulatory authorities need to seriously con-

sider the potential release of these vaccines

into the environment without strong scientific

data documenting safety and reversion to viru-

lence potential. Vaccines are tools to be used inassociation with sound health management and

biosecurity plans to result in the greatest benefit

to aquaculture producers.

Acknowledgments

We thank Lisa Biggar for editing and format-

ting this article. We also gratefully acknowl-

edge John Grizzle (Department of Fisheries

and Allied Aquacultures, Auburn University),Richard Shelby and Benjamin LaFrentz

(Aquatic Animal Health Research Laboratory,

USDA-ARS, Auburn, Alabama) for critical

review of this article.

Literature Cited

Amend, D. F. 1981. Potency testing of fish vaccines.

Pages 447 454 in D. P. Anderson and W. Hennessen,

editors. Fish biologics: serodiagnostics and vaccines

Developments in biological standardization, volume

49. Karger, Basel, Switzerland.

Antonio, D. B. and R. P. Hedrick. 1994. Effects of the

corticosteroid Kenalog on carrier state of juvenile

channel catfish exposed to Edwardsiella ictaluri . Jour-

nal of Aquatic Animal Health 6:4452.

Antao, A. B., M. Wilson, J. Wang, E. Bengten, N. W.

Miller, L. W. Clem, and V. G. Chinchar. 2001.

Genomic organization and differential expression of

channel catfish MHC class I genes. Developmental and

Comparative Immunology 25:579595.

Arias, C. R., C. A. Shoemaker, J. J. Evans, and

P. H. Klesius. 2003. A comparative study of

Edwardsiella ictaluri parent (EILO) and E. ictaluririfampicin-mutant (RE-33) isolates using lipopolysac-

charides, outer membrane protiens, fatty acids,

Biolog, API 20E and genomic analyses. Journal of

Fish Diseases 26:415421.

Babiuk, L. A., S. L. Babiuk, B. I. Loehr, and S. van

Drunnen Little-van den Hurk. 2000. Nucleic acid

vaccines: research tool or commercial reality. Veteri-

nary Immunology and Immunopathology 76:123.

Bader, J. A., S. Vinitnantharat, and P. H. Klesius. 1997.

Comparison of whole-cell antigens of pressure- and

formalin-killed Flexibacter columnaris from channel

catfish (Ictalurus punctatus ). American Journal of

Veterinary Research 58:985 988.

Barnes, A. C., F. M. Young, M. T. Horne, and

A. E. Ellis. 2003. Streptococcus iniae: serologi-

cal differences, presence of capsule and resistance to

immune serum killing. Diseases of Aquatic Organisms

53:241247.

Bravo, S. and P. J. Midtlyng. 2007. The use of fish

vaccines in the Chilean salmon industry 19992003.

Aquaculture 270:3642.

Bricknell, I. R., T. J. Bowden, J. Lomax, and

A. E. Ellis. 1997. Antibody response and pro-

tection of Atlantic salmon (Salmo salar) immunised

with an extracellular polysaccharide of Aeromonas

salmonicida . Fish and Shellfish Immunology 7:116.

Bricknell, I. R., J. A. King, T. J. Bowden, and

A. E. Ellis. 1999. Duration of protective anti-

bodies, and the correlation with protection in Atlantic

salmon (Salmo salar L.), following vaccination

with Aeromonas salmonicida vaccine containing

iron-regulated outer membrane proteins and secretory

polysaccharide. Fish and Shellfish Immunology

9:139151.

Buchanan, J. T., J. A. Stannard, X. Lauth, V. E.

Ostland, H. C. Powell, M. E. Westerman, and

V. Nizet. 2005. Streptococcus iniae phosphogluco-

mutase is a virulence factor and target for vaccine

development. Infection and Immunity 73:69356944.

Carrias, A. A., J. S. Terhune, C. A. Sayles and

J. A. Chappel. 2008. Effects of an extended

hatchery phase and vaccination against enteric sep-

ticemia of catfish on the production of channel catfish,

Ictalurus punctatus, fingerlings. Journal of the World

Aquacutlure Society 39:259266.

Clark, T. G. and D. Cassidy-Hanley. 2005. Recombi-

nant subunit vaccines: potentials and constraints. Pages

153163 in P. J. Midtlyng, editor, Progress in fish

vaccinology. Developments in biological standardiza-

tion, volume 121, Karger, Basel, Switzerland.

Cooper II, R. K., E. B. Shotts, Jr., and L. K. Nolan.

1996. Use of a mini-transposon to study chondroitinase

activity associated with Edwardsiella ictaluri . Journal

of Aquatic Animal Health 8:319324.

Daly, J. G., S. G. Griffiths, A. K. Kew, A. R. Moore,

and G. Olivier. 2001. Characterization of attenu-

ated Renibacterium salmoninarum strains and their

use as live vaccines. Diseases of Aquatic Organisms

44:121126.

Duff, D. B. B. 1942. The oral immunization of trout

against Bacterium salmonicida. Journal of Immunol-

ogy 44:8794.

Elvelyn, T. P. T. 1997. A histortical review of fish vacci-

nology. Pages 312 in R. Gudding, A. Lillehaug, P. J.

Midtlyng, and F. Brown, editors. Fish vaccinology.

Developments in biological standardization, volume

90, Karger, Basel, Switzerland.

Evensen, O., B. Brudeseth, and S. Mutoloki. 2005. The

vaccine formulation and its role in inflammatory

processes in fish effects and adverse effects. Pages

117125 in P. J. Midtlyng, editor, Progress in fish

vaccinology. Developments in biological standardiza-

tion, volume 121, Karger, Basel, Switzerland.


11/13


Frey, J. 2007. Biological safety concepts of genet-

ically modified live bacterial vaccines. Vaccine

25:55985605.

Gantois, I., R. Ducatelle, L. Timbermount, F. Boyen, L.

Bohez, F. Haesebrouck, F. Pasmans, and F. van

Immerseel. 2006. Oral immunization of laying hens

with live vaccine strains of TAD Salmonella vac

E and TAD Salmonella vac T reduces internal egg

contamination with Salmonella Enteritidis. Vaccine

24:62506255.

Goodwin, U. B., M. Flores, S. Quiniou, M. R. Wil-

son, N. W. Miller, L. W. Clem, and T. J.

McConnel. 2000. MHC class II A genes in the

channel catfish (Ictalurus punctatus ). Developmental

and Comparative Immunology 24:609622.

Griffiths, S. G., K. J. Melville, and K. Salonis. 1998.

Reduction of Renibacterium salmoninarum culture

activity in Atlantic salmon following vaccination

with avirulent strains. Fish and Shellfish Immunology

8:607619.

Hartman, J. X. and E. J. Noga. 1980. Channel catfish

virus disease vaccine and method of preparation

thereof and method of immunization therewith. US

Patent No. 4,219,543.

Hawke, J. P., A. C. McWhorter, A. G. Steigerwalt, and

D. J. Brenner. 1981. Edwardsiella ictaluri sp. nov.

the causative agent of enteric septicemia of cat-

fish. International Journal of Systematic Bacteriology

31:396400.

Hernanz Moral, C., Flano, E., Lopez Fierro, P.,

Villena, A., Anguita, J., Cascon, A., Sanchez

Salazar, M., Razquin Peralta, B., and Naharro, G.

1998. Molecular characterization of the Aeromonas

hydrophila aroA gene and potential use of an aux-

otrophic aroA mutant as a live attenuated vaccine.

Infection and Immunity 66:18131821.

Hoiseth, S. K. and B. A. Stocker. 1981. Aromatic-

dependent Salmonella typhimurium are non-virlulent

and effective as live vaccines. Nature 291:238239.

Igarashi, A. and T. Iida. 2002. A vaccination trial using

live cells ofEdwardsiella tarda in tilapia. Fish Pathol-

ogy 37:145148.

Intervet Inc. 2003. AQUAVAC Technical Bulletin-

Control of Enteric Septicemia of Catfish, Millsboro,

Delaware, USA.

Itano, T., H. Kawakami, T. Kono, and M. Sakai. 2006.

Live vaccine trials against nocardiosis in yellowtail

Seriola quinqueradiata . Aquaculture 261:11751180.

Jenner, E. 1961. An enquiry into the causes and effects

of the variolae vaccinae, a disease discovered in

some of the western counties of England, particularly

Gloucestershire, and known by the name of The Cow

Pox. Pages 121125 inT. D. Brock, editor. Milestones

in microbiology, Prentice-Hall, London.

Klesius, P. H. and W. M. Sealey. 1995. Characteristics of

serum antibody in enteric septicemia of catfish. Journal

of Aquatic Animal Health 7:205210.

Klesius, P. H. and C. A. Shoemaker. 1997. Heterologous

isolates challenge of channel catfish, Ictalurus punc-

tatus, immune to Edwardsiella ictaluri. Aquaculture

157:147155.

Klesius, P. H. and C. A. Shoemaker. 1999. Development

and use of modified live Edwardsiella ictaluri vaccine

against enteric septicemia of catfish. Pages 523537

in R. Schultz, editor. Advances in veterinary medicine,

volume 41. Academic Press, San Diego, California,

USA.

Klesius, P. H. and C. A. Shoemaker. 2000. Modified live

Edwardsiella ictaluri against enteric septicemia in

channel catfish. U.S. Patent No. 6,019,981.

Klesius, P. H., C. A. Shoemaker, and J. J. Evans. 2002.

In ovo methods for utilizing live Edwardsiella ictaluri

against enteric septicemia in channel catfish. U.S.

Patent No. 6,153,202.

Klesius, P. H., J. J. Evans, and C. A. Shoemaker. 2004.

Warmwater fish vaccinology in catfish production.

Animal Health Research Reviews 5:305311.

Lawrence, M. L., R. K. Cooper, and R. L. Thune. 1997.

Attenuation, persistence, and vaccine potential of

an Edwardsiella ictaluri purA mutant. Infection and

Immunity 65:46424651.

Lawrence, M. L., M. M. Banes, and M. L. Williams.

2001. Phenotype and virulence of a transposon-

derived lipopolysaccharide O side-chain mutant strain

of Edwardsiella ictaluri. Journal of Aquatic Animal

Health 13:291299.

Lawrence, M. L. and M. M. Banes. 2005. Tissue per-

sistence and vaccine efficacy of an O polysaccha-

ride mutant strain of Edwardsiella ictaluri . Journal of

Aquatic Animal Health 17:228232.

Leung, K. Y., L. S. Wong, K. W. Low, and Y. M. Sin.

1997. Mini-Tn5 induced growth- and protease-

deficient mutants of Aeromonas hydrophila as live

vaccines for blue gourami, Trichogaster trichopterus

(Pallas). Aquaculture 158:1122.

Linde, K., J. Beer, and V. Bondarenko. 1990. Stable

Salmonella live vaccine strains with two or more atten-

uating mutations and any desired level of attenuation.

Vaccine 8:278 282.

Lorenzen, N., E. Lorenzen, K. Einer-Jensen, J. Hep-

pell, and H. L. Davis. 1999. Genetic vaccination of

rainbow trout against viral haemorrhagic septicaemia

virus: small amounts of plasmid DNA protect against

a heterologous serotype. Virus Research 63:1925.

Markestad, A. and K. Grave. 1997. Reduction of antibac-

terial drug use in Norwegian fish farming due to

vaccination. Pages 365369 in R. Gudding, A. Lille-

haug, P. J. Midtlyng, and F. Brown, editors. Fish vac-

cinology. Developments in biological standardization,

volume 90, Karger, Basel, Switzerland.

Marsden, M. J., L. M. Vaughan, R. M. Fitz-

patrick, T. J. Foster and C. J. Secombes. 1998.

Potency testing of a live genetically modified vaccine

for salmonids. Vaccine 16:10871094.


12/13


Marsden, M. J., L. M. Vaughan, T. J. Foster and

C. J. Secombes. 1996. A live (aroA) for furuncu-

losis preferentially stimulates T-cell responses relative

to B-cell responses in rainbow trout (Oncorhynchus

mykiss ). Infection and Immunity 64:38633869.

Mast, J. and B. M. Goddeeris. 1999. Development of

immunocompetence of broiler chickens. Veterinary

Immunology and Immunopathology 70:245256.

Milev-Milovanovic, I., S. Long, M. Wilson, E.

Bengten, N. W. Miller and V. G. Chinchar. 2006.

Identifiction and expression analysis of interferon

gamma genes in channel catfish. Immunogenetics

58:7080.

Norqvist, A., A. Hagstrom and H. Wolf-Watz. 1989. Pro-

tection of rainbow trout against vbriosis and furun-

culosis by the use of attenuated strains of Vibrio

anguillarum . Applied and Environmental Microbiol-

ogy 55:14001405.Nusbaum, K. E. and E. E. Morrison. 1996. Entry of

35 S-labeled Edwardsiella ictaluri into channel cat-

fish (Ictalutus punctatus ). Journal of Aquatic Animal

Health 8:146149.

ODowd, A. M., I. R. Bricknell, C. J. Secombes, and

A. E. Ellis. 1999. The primary and secondary anti-

body responses to IROMP antigens in Atlantic

salmon (Salmo salar L) immunised with A+ and A-

Aeromonas salmonicida bacterins. Fish and Shellfish

Immunology 9:125138.

Peatman, E., J. Terhune, P. Baopraserkul, P. Xu, S.

Nandi, S. Wang, B. Somridhivej, H. Kucuktas, P.

Li, R. Dunham, and Z. Liu. 2008. Microarray anal-

ysis of gene expression in the blue catfish liver

reveals early activation of the MHC class I pathway

after infection with Edwardsiella ictaluri . Molecular

Immunology 45:553566.

Petrie-Hanson, L. and A. J. Ainsworth. 1999. Humoral

immune response of channel catfish (Ictalurus punc-

tatus ) fry and fingerlings exposed to Edwardsiella

ictaluri . Fish and Shellfish Immunology 9:579589.

Petrie-Hanson, L. and A. J. Ainsworth. 2001. Ontogeny

of channel catfish lymphoid organs. Veterinary

Immunology and Immunopathology 81:113127.

Plumb, J. A. 1999. Health maintenance and principalmicrobial diseases of cultured fishes. Iowa State Uni-

versity Press, 328 p.

Salonius, K., C. Siderakis, A. M. MacKinnon, and

S. G. Griffiths. 2005. Use ofArthrobacter davidanieli

as a live vaccine against Renibacterium salmoni-

narum and Piscirickettsia salmonis in salmonids.

Pages 189197 in P. J. Midtlyng, editor. Progress in

fish vaccinology. Developments in biological standard-

ization, volume 121, Karger, Basel, Switzerland.

Schurig, G. G., R. M. Roop II, T. Bagchi, S. M. Boyle,

D. Buhrman, and N. Sriranganathan. 1991. Biolog-

ical properties of RB51; a stable rough strain of Bru-

cella abortus . Veterinary Microbiology 28:171188.

Seder, R. A. and A. V. S. Hill. 2000. Vaccines against

intracellular infections requiring cellular immunity.

Nature 406:793797.

Shoemaker, C. A. and P. H. Klesius. 1997. Protective

immunity against enteric septicemia in channel catfish,

Ictalurus punctatus (Rafinesque), following controlled

exposure to Edwardsiella ictaluri. Journal of Fish

Diseases 20:361368.

Shoemaker, C. A., P. H. Klesius and J. A. Plumb. 1997.

Killing ofEdwardsiella ictaluri by macrophages from

channel catfish immune and susceptible to enteric

septicemia of catfish. Veterinary Immunology and

Immmunopathology 58:181 190.

Shoemaker, C. A., P. H. Klesius, and J. M. Bricker.

1999. Efficacy of a modified live Edwardsiella ictaluri

vaccine in channel catfish as young as seven days post

hatch. Aquaculture 176:189193.

Shoemaker, C. A., P. H. Klesius, and J. J. Evans. 2002.

In ovo method for utilizing the modified live Edward-

siella ictaluri vaccine against enteric septicemia in

channel catfish. Aquaculture 203:221227.Shoemaker, C. A., P. H. Klesius, and J. J. Evans. 2007.

Immunization of eyed channel catfish, Ictalurus

punctatus, eggs with monovalent Flavobacterium

columnare vaccine and bivalent F. columnare and

Edwardsiella ictaluri vaccine. Vaccine 25:1126 1131.

Simard, N. C., C. Lyngoy, V. Funk, G. Traxler, S. LaP-

atra, and K. Salonius. 2006. Research to market:

meeting the safety and efficacy requirements for a

DNA vaccine used in Atlantic salmon. 4th Interna-

tional Veterinary Vaccines and Diagnostics Confer-

ence, Oslo, Norway. Abstract p. 46.

Smith, B. P., M. Reina-Guerra, B. A. Stocker, S. K.

Hoiseth, and E. Johnson. 1984. Aromatic-dependent

Salmonella dublin as a parenteral modified live vac-

cine for calves. American Journal of Veterinary

Research 45:22312235.

Sommerset, I., B. Krossoy, E. Biering, and P. Frost.

2005. Vaccines for fish in aquaculture. Expert Review

Vaccines 4:89101.

Temprano, A., J. Riano, J. Yugueros, P. Gonza-

lez, L. deCastro, A. Villena, J. M. Luengo, and

G. Naharro. 2005. Potential use of a Yersinia ruckeri

O1 auxotrophic aroA mutant as a live attenuated

vaccine. Journal of Fish Diseases 28:419427.

Thornton, J. C., R. A. Garduno, and W. W. Kay. 1994.The development of live vaccines for furunculosis

lacking the A-layer and O-antigen of Aeromonas

salmonicida . Journal of Fish Disease 17:194204.

Thune, R. L., L. A. Collins, and M. P. Pena. 1997. A

comparison of immersion, immersion/oral combina-

tion and injection methods for the vaccination of chan-

nel catfish Ictalurus punctatus against Edwardsiella

ictaluri . Journal of the World Aquaculture Society

28:193201.

Thune, R. L., D. H. Fernandez, and J. R. Battista. 1999.

An aroA mutant of Edwardsiella ictaluri is safe and

efficacious as a live, attenuated vaccine. Journal of

Aquatic Animal Health 11:358372.

Tizard, I. 1999. Grease, anthraxgate, and kennel cough: a

revisionist history of early veterinary vaccines. Pages

724 in R. Schultz, editor. Advances in veterinary


13/13


medicine, volume 41, Academic Press, San Diego,

California, USA.

Vaughan, L. M., P. R. Smith, and T. J. Foster. 1993.

An aromatic-dependent mutant of the fish pathogen

Aeromonas salmonicida is attenuated in fish and is

effective as a live vaccine against the salmonid disease

furunculosis. Infection and Immunity 61:21722181.

Vinitnantharat, S. and J. A. Plumb. 1993. Protection

of channel catfish Ictalurus punctatus following nat-

ural exposure to Edwardsiella ictaluri and effects of

feeding antigen on antibody titer. Diseases of Aquatic

Organisms 15:3134.

Waltman, W. D. and E. B. Shotts. 1986. Antimicro-

bial susceptibility of Edwardsiella ictaluri . Journal of

Wildlife Diseases 22:173177.

Warr, G. 1997. The adaptive immune system of fish.

Pages 1522 in R. Gudding, A. Lillehaug, P. J.

Midtyling and F. Brown, editors. Fish vaccinology,developments in biological standardization, volume

90, Karger, Basel, Switzerland.

Wise, D. J., P. H. Klesius, C. A. Shoemaker, and

W. R. Wolters. 2000. Vaccination of mixed and

full-sib families of channel catfish Ictalurus punc-

tatus against enteric septicemia of catfish with a

live attenuated Edwardsiella ictaluri isolate (RE-

33). Journal of the World Aquaculture Society

31:206212.

Wise, D. J. and J. Terhune. 2001. The relationship

between vaccine dose and efficacy in channel cat-

fish Ictalurus punctatus vaccinated as fry with a

live attenuated strain of Edwardsiella ictaluri (RE-

33). Journal of the World Aquaculture Society

32(19):177183.

Wise, D. J. 2006. ESC vaccination shown to improve

production efficiencies. The Catfish Journal 67.

Zhang, Y., C. R. Arias, C. A. Shoemaker and P. H.

Klesius. 2006. Comparison of lipopolysaccharide and

protein profiles between Flavobacterium columnarestrains from different genomovars. Journal of Fish

Diseases 29:657663.

8. c5. modified live vaccine

Documents