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JOURNAL OF THE WORLD AQUACULTURE SOCIETY

Vol. 40, No. 5 October, 2009

Use of Modied 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

Abstract
Vaccination is an important disease management strategy used to maintain human and animal health worldwide. Vaccines developed for aquaculture have reduced antibiotic use in sh production. Original sh 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 modied live vaccines for use in sh. A modied live vaccine is a live pathogen that has been rendered non-pathogenic or avirulent by physical, chemical, or genetic engineering methods. The modied 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). Modied live vaccines are advantageous in that they can be easily delivered (i.e., by immersion to young sh) and stimulate both humoral and cellular immunity of long duration. Disadvantages include issues with modied live vaccine safety to the host and environment. A successful modied 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 rst known vaccine to protect humans against small pox was derived from a live virus used as a vaccine. Jenner (1961) demonstrated that vaccinia protected humans against the small pox virus by passage of the virus from horse, to cow udder, to humans. Tizard (1999) provides an excellent review of early events in vaccinology of humans and veterinary animals. Interestingly, many of the early vaccines used were modied live vaccines based on the success of the small pox vaccine. Intensication of sh reared in captivity for use as food and for remedial stocking programs has resulted in an increased presence of disease. Disease is manifested under these
1

Corresponding author.

intense conditions because of many factors, including inadequate maintenance of environmental conditions (i.e., poor water quality) and the ease of transmission of pathogens in sh reared at high stocking densities. Duff (1942) was the rst to report on a vaccine trial in cutthroat trout, Oncorhynchus clarki, that were fed a killed Aeromonas salmonicida vaccine. In aquaculture, most early vaccine work focused on use of killed vaccines. The rst commercially licensed vaccine for sh was a killed vaccine delivered by immersion against Yersinia ruckeri the causative agent of enteric redmouth disease (Plumb 1999). Following the success of this product, formalin-killed immersion vaccines for vibriosis of trout and salmon were developed. 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

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develop the early salmonid vaccines (Elvelyn 1997) to be delivered by immersion. Immersion vaccines against A. salmonicida were not effective in the eld and thus the rst injection 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 following injection (Bricknell et al. 1997). ODowd et al. (1999) used formalin-killed bacterins generated from A-layer positive or A-layer negative A. salmonicida grown under iron-restricted conditions to immunize Atlantic salmon. These preparations resulted in signicant antibody responses that were enhanced following booster immunization; however, the sh were not challenged. Bricknell et al. (1999) using a combination EPS and A-layer negative bacterin demonstrated antibodies and protection at 8 wk postinjection 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 (bacterial and viral) in oil-adjuvant delivered in one injection (Sommerset et al. 2005). These vaccines are successful and have reduced the use of environmentally unfriendly chemicals, especially 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 on the use of vaccines in the aquaculture industry in Chile indicated that both immersion and injection vaccination strategies are used (Bravo and Midtlyng 2007). Injection vaccination of sh that must be vaccinated at a young age (1012 d post-hatch) or small size (12 g smolt) is not practical. The exploration and use of attenuated or modied live bacterial vaccines in aquaculture was initiated in the 1990s (Norqvist et al. 1989; Vaughan et al. 1993; Thornton et al. 1994; Lawrence et al. 1997; Hernanz Moral et al. 1998; Marsden et al. 1998; Klesius and Shoemaker 1999;

Thune et al. 1999). Presently, three modied live vaccines are licensed for use in the USA. These include the vaccine against bacterial kidney disease (Renogen1 ), enteric septicemia of catsh disease (AQUAVAC-ESC ) and columnaris disease (AQUAVAC-COL ). This review focuses on attenuation strategies for the development of modied live vaccines, advantages and disadvantages of using modied live vaccines in aquaculture and presents an example of a successfully commercialized modied live vaccine currently used in warmwater aquaculture.

Attenuation Strategies
Laboratory Passage Multiple methods have been used to attenuate pathogens for successful development of modied live vaccines in human and veterinary medicine. The methods used thus far on pathogens of sh will be highlighted. One of the earliest techniques was simple laboratory passage of organisms on media or in tissue culture that resulted in attenuation of the pathogenic microorganism. An early patent describes use of this technique with channel catsh, Ictalurus punctatus, virus (CCV) (Hartman and Noga 1980). They created an attenuated CCV by passage of the virus in tissue culture using a cell line derived from the walking catsh, Clarius batrachus. More recently, Daly et al. (2001) used laboratory attenuated Renibacterium salmoninarum strains to immunize Atlantic salmon. They isolated two strains that grew readily on common bacterial medium and 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. However, the isolate was not completely attenuated in yellowtail, Seriola quinqueradiata, the host animal.
1 Mention of trade names or commercial products in this publication is solely for the purpose of providing specic information and does not imply recommendation or endorsement by the United States Department of Agriculture.

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Environmental Bacterium A second technique involves passage of the virulent pathogen in an alternative host (i.e., one not susceptible to that pathogen) or the use of an environmental bacterium that mimics the pathogens antigenic structure. The most successful use of this strategy in aquaculture is use of Arthrobacter davidanieli delivered by injection to protect salmon from bacterial kidney disease caused by R. salmoninarum (Grifths et al. 1998; Salonius et al. 2005). The authors showed a close phylogenetic relationship between A. davidanieli and R. salmoninarum and the ability of antiserum to cross-react with surface carbohydrates (Grifths et al. 1998). More recently, Itano et al. (2006) attempted to use a similar strategy (i.e., phylogenetic relatedness and antigenic cross-reactivity) to identify vaccine candidates against N . seriolae. In this study, the authors used environmental Nocardia species and evaluated the ability of these isolates (N . soli and N . uminea) to induce protective immunity against N . seriolae in injected yellowtail. Unfortunately, the isolates provided minimal protection to challenge with virulent N . seriolae. Chemical or Physical Mutagenesis The third strategy involves chemical or physical mutagenesis that results in random mutation(s) in the pathogen, with the vaccine strain being selected because of the lack of virulence in 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 gramnegative bacteria by inducting changes in the lipopolysaccharide (LPS), an important virulence 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 in the example provided in this review. This strategy has been one of the most practiced to date in generation of successful veterinary vaccines

(Linde et al. 1990; Schurig et al. 1991; Klesius 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 catsh exposed to this mutant were shown to be protected following challenge with virulent wildtype parent isolate. A more recent attempt was use of transposon mutagenesis to generate an Opolysaccharide decient isolate of E. ictaluri to be used as a modied live vaccine (Lawrence et al. 2001; Lawrence and Banes 2005). The authors were successful in generation of Opolysaccharide decient isolate of E. ictaluri but failed to demonstrate protection in immersion immunized catsh (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 challenge. Leung et al. (1997) also generated miniTn5 (transposon mutants) induced growth and protease-decient A. hydrophila for use as modied live vaccines in blue gourami (Trichogaster trichopterus). The generated vaccine strains 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 phosphoglucomutase gene (Buchanan et al. 2005). The phosphoglucomutase enzyme is believed to be important for polysaccharide capsule formation in bacteria. Presence or absence of a capsule has been suggested to be important for virulence (Barnes et al. 2003; Buchanan et al. 2005). This isolate was attenuated in hybrid

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striped bass and shown to provide protection upon lethal challenge if the modied live S. iniae was delivered by intraperitoneal injection (Buchanan et al. 2005). One of the more common strategies is use of 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 the need 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 sh.

environment. Frey (2007) provides an excellent review of biological safety concepts of genetically modied live bacterial vaccines used in veterinary medicine. The basic principles apply to the use of modied live vaccines in aquaculture. In the USA, the United States Department of Agriculture (USDA)-Animal Plant Health Inspection Services (APHIS) Center for Veterinary Biologics provides the regulatory authority for licensing and registering veterinary biologics (www.aphis.usda.gov). Safety studies consist of experiments using 10 times the immunizing dose and direct sh-to-sh passage. Also, a risk analysis should consider the release of the vaccine into the environment. Presence of the 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 modied live vaccines is the ability to stimulate cell-mediated, humoral (antibody) and mucosal immunity (Clark and Cassidy-Hanley 2005). Modied live vaccines survive and replicate within the host, which results in a strong cellular immune response that confers protection of long duration. Induction of cellular immunity (CD4+ and CD8+ T-cell responses) is responsible for providing protection against intracellular infections (Seder and Hill 2000). Recent studies have demonstrated the relevant major histocompatibility complex (MHC) class I (Antao et al. 2001) and class II molecules (Goodwin et al. 2000) in sh. Presentation of antigen with the correct MHC allows for the response and recognition by the appropriate subpopulations of Tand B-cells. Seder and Hill (2000) suggest that modied live vaccines can induce Th1 and CD8 T-cell responses. Marsden et al. (1996) were the rst to demonstrate the ability of modied live aroA deletion mutant A. salmonicida vaccine to preferentially enhance T-cell over B-cell responses in modied live vaccinated rainbow trout, Oncorhynchus mykiss. Recent transcriptome analysis of gene expression in catsh liver tissue suggest that upon exposure to live bacteria, MHC class I genes along with other acute

Advantages and Disadvantages of the Use of Modied Live Vaccines


Safety The major disadvantage of using modied live vaccines is safety. Killed vaccines or vaccine products are generally considered safe for use in aquatic animals because the disease 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 vaccines is the adhesions seen in salmonids following 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 llet quality (Evensen et al. 2005). The concept of safety in the use of modied live vaccines is with respect to the inability of the vaccine strain to cause disease in the vaccinated animal. Furthermore, environmental issues become a concern because of the potential release of the vaccine strain into the

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phase response genes are upregulated suggesting 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. Subsequent stimulation of a subpopulation of T-cells may induce interferon gamma (Milev-Milovanovic et al. 2006) production that can mediate intracellular pathogen destruction (Seder and Hill 2000). Thus, assumptions can be made to the nature of the immune response following modied live vaccine administration in sh. Seder and Hill (2000) further point out that killed or subunit vaccines are not effective at stimulating cellular responses. Killed vaccines, or delivery of killed antigens, results in the induction of humoral (antibody mediated) immunity. Killed Edwardsiella ictaluri vaccine previously used in aquaculture was shown not to enter the sh (Nusbaum and Morrison 1996) and thus, failed to induce immunity. 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 inactivated vaccine failure and no protection in young sh 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 delivered by immersion, however, the best protective effect has been shown following injection (Norqvist et al. 1989). Duration of immunity induced by killed vaccines is often less than 4 mo and only effective on extracellular pathogens or pathogens producing toxins. Extending the length of immunity following administration of killed vaccines often relies on multiple immunizations and/or use of adjuvant(s). Immunity following exposure to live bacteria (cell mediated immunity) has been demonstrated for greater than 4 mo in single bath immunized catsh (Klesius and Shoemaker 1997). Vaccine Delivery Modied 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 sh in water (Norqvist et al. 1989; Klesius et al. 2004). Modied live vaccines developed for use in warmwater aquaculture are delivered by immersion exposure to the youngest life stage (i.e., prior to release into ponds). Modied live vaccines retain their ability to colonize and infect the host which allows for effective immunity to develop following immersion delivery. Modied 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 modied live vaccine strain was able to replicate in the host and thus increased the vaccine signal. Cost Vaccine cost is a major question for manufacture constraints and farmer acceptance. Modied live bacterial vaccines, for the most part, have a low-to-moderate cost (Seder and Hill 2000; Klesius et al. 2004). A single attenuated bacterium can be used to produce many liters of vaccine in commercial-scale fermentors. The major cost is in purity, safety, immunodose determination, potency (efcacy) testing, and packaging (e.g., lyophilization). For formalinkilled vaccines, the cost is similar and killed Y. ruckeri vaccines for trout are extensively used. However, the need to handle and inject individual sh in the Atlantic salmon industry adds a signicant 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 investment in isolation and characterization of the proper gene(s) to provide protective immunity in the vaccinated host. DNA vaccination technology has been successfully used in Atlantic salmon to protect against infectious hematopoietic necrosis virus (IHNV) (Simard et al. 2006) and rainbow trout to protect against viral hemorrhagic septicemia virus (VHSV) (Lorenzen et al. 1999). The acceptance of DNA vaccines

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in some countries (i.e., food safety and genetically modied organism issues) may be a limiting factor in the commercial realization of these products (Babiuk et al. 2000).

Example of a Successful Modied Live Vaccine in Warm Water Aquaculture


Background Enteric septicemia of channel catsh (ESC) is caused by E. ictaluri, a gram-negative bacterium that was initially described in 1976 (Hawke et al. 1981). Today, losses to ESC cost the US catsh industry $4060 million, annually. Development of formalin-killed E. ictaluri bacterins was attempted, but they did not provide protection against ESC (Shoemaker and Klesius 1997; Thune et al. 1997). Failure of the killed products was because of stimulation of only an antibody response (Klesius and Sealey 1995; Klesius and Shoemaker 1997) and the fact that killed bacteria were not entering the sh following immersion exposure (Nusbaum and Morrison 1996). Methods to control ESC relied on feeding antibiotic medicated diet; however, this practice is ineffective because disease reduces diet intake and antibiotic resistance has been observed in the bacterium (Waltman and Shotts 1986). Protective Immunity Early work on E. ictaluri suggested that antibody was important for the protective immune response (Vinitnantharat and Plumb 1993). Klesius and Sealey (1995) demonstrated through passive transfer that specic antibody was not protective. Antonio and Hedrick (1994) were the rst to suggest cell-mediated immunity was needed for protection; however, the method they used was indirect by administering an anti-inammatory drug (Kenalog) and showing that this increased susceptibility of catsh to a second exposure to E. ictaluri. Shoemaker and Klesius (1997) were the rst to demonstrate cellular immunity was responsible for protection. Fish used in their studies

were immunized by exposing them to low numbers of virulent E. ictaluri. Upon immunization, macrophages were harvested from surviving sh and assessed for the ability to kill E. ictaluri in vitro. Macrophages from sh vaccinated with live E. ictaluri were able to kill E. ictaluri (85.9%); whereas, macrophages from non-vaccinated sh or sh immunized with a killed bacterin were signicantly less effective at killing (68.1 and 71.4%, respectively) (Shoemaker and Klesius 1997). Furthermore, sh that survived a low dose exposure to E. ictaluri were resistant upon subsequent challenge with homologous and sometimes heterologous isolates (Klesius and Shoemaker 1997). Taken together, the above research demonstrated the need for a vaccine that would result in a cellmediated immune response in young catsh. Modied Live E. ictaluri Development Klesius and Shoemaker (1999) developed a modied live E. ictaluri vaccine by passage of a virulent isolate on media (brain heart infusion agar) supplemented with rifampicin. This technique was used with other gram-negative bacteria 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 vaccine. 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 LPS when compared to the parent isolate (EILO) (Fig. 1) by immunoblot (Klesius and Shoemaker 1999; Arias et al. 2003). This change resulted in an attenuated E. ictaluri that was capable of entering and persisting in catsh to allow for the proper immune response but without causing disease (Klesius and Shoemaker 1999). Additional tests were conducted to determine whether the vaccine isolate (RE-33) differed in biochemical characters or fatty acid proles. Initial information suggested the parent isolate (EILO) and vaccine mutant did not differ in biochemical parameters except that the

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A M 210 KD 110 KD 1 2 3 4

B 1 2 3 4

56 KD

37 KD

21 KD

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 identication system (Microbial ID, Inc., Newark, DE, USA) differences in fatty acid proles were determined. Overall, quantitative differences were seen in fatty acid proles between the isolates and some fatty acids were present only in the chromatograph from the parent isolate EILO (3-hydroxy hexadecanoid acid) or mutant isolate RE-33 (hepatdecanoic acid, 3hydroxy-heptadecanoic acid, and 10-methyloctodecanoid acid), allowing for accurate identication (Arias et al. 2003). Biochemical differences were detected with Biologs (Biolog, Hayward, CA, USA) carbon utilization system for bacterial identication. 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 demonstrated to be safe following direct sh-to-sh

passage (i.e., back passage) indicating an inability to revert to the virulent form. Ten times safety tests were also performed with no adverse reactions following vaccination (Klesius and Shoemaker 1999). Field trials were conducted in 2.2 million 10- to 30-d-old channel catsh following state veterinarian and USDA-APHIS approved protocols in Mississippi and Alabama in 1997 with no adverse effects of vaccination reported (Klesius and Shoemaker 1999). Including eld safety trials conducted by Intervet, Inc. (1998 and 1999) more than 57 million channel catsh fry and/or ngerlings were vaccinated to satisfy the requirements for a safe product. No problems were reported in sh following immersion vaccination from locations where the trials were conducted. Efcacy Efcacy has been demonstrated in 3- to 9-mo-old channel catsh against E. ictaluri following vaccination with the modied live vaccine (Klesius and Shoemaker 1999; Lawrence and Banes 2005). Vaccine dose was variable

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with a range of 1 105 to 1 107 colonyforming units (CFUs) RE-33/mL being administered to sh by immersion exposure for 230 min. Wise et al. (2000) demonstrated the vaccine to be effective in sh immunized at 1 107 CFU/mL in both laboratory and eld challenges (i.e., sh in cages) with E. ictaluri. Wise et al. (2000) also demonstrated that vaccine efcacy was related to genetic differences of families of channel catsh. Six full sibling families of channel catsh 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 catsh lines may be developed that can respond better to vaccination. Another source of variability in efcacy studies is the isolate of E. ictaluri used to challenge immunized catsh (Klesius and Shoemaker 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 (Klesius and Shoemaker 1999). Efcacy was also determined using other rifampicin generated E. ictaluri mutants (B-21909; B-21910; B-21911; Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA). Table 1 shows that the other vaccine isolates were able to provide protection against homologous and heterologous challenge (Klesius and Shoemaker 2000). A polyvalent vaccine trial using combinations of rifampicin-attenuated mutant E. 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 Shoemaker 2000) was also conducted. Results of the polyvalent trials demonstrated that a vaccine based on multiple rifampicin-attenuated mutants of E. ictaluri is possible and effective. This is important if the present single isolate vaccine becomes less effective. The formulation may be changed or modied by addition of one or more of the additional vaccine isolates to provide solid protection if another antigenic type becomes predominant in the catsh industry. Vaccine effectiveness in young catsh (712 d post-hatch) and eyed eggs has been demonstrated (Shoemaker et al. 1999; Wise and Terhune 2001; Klesius et al. 2002; Shoemaker et al. 2002; Shoemaker et al. 2007). PetrieHanson and Ainsworth (1999) suggested that catsh do not become immunocompetent (antibody mediated) until 4 wk post-hatch. PetrieHanson 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 catsh lymphoid 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 modied live vaccine is probably because of the presence of macrophages or antigen presenting cells (in the young fry) or to the persistence of

Table 1. Efcacy of rifampicin-resistant Edwardsiella ictaluri mutants B-21909, B-21910, and B-21911 in channel catsh challenged with E. ictaluri.

Treatmenta
Non-vaccinated B-21909c B-21910 B-21911

Challenge isolate (type)


AL-93-75 AL-93-75 (Homologous) AL-93-75 (Heterologous) AL-93-75 (Heterologous)

Percent mortality
46.7 2.7 9.3 22.7

Relative percent survivalb


94.3 80.0 51.4

CFU = colony-forming unit; ESC = enteric septicemia of catsh. a All sh were alive and free of signs of ESC for 21 d after vaccination. b Relative percent survival (RPS) was determined as by Amend (1981). c Fish were vaccinated with 1 107 CFU of each vaccine isolate/mL of immersion water.

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

Ratioa
None None 1:1 1:2 2:1 1:2 1:1 1:1 1:1 2:1 1:1:1:1

Vaccine strains or controls


Control (not vaccinated) ATCC 202058 B-21909 ATCC 202058:B-21909 ATCC 202058:B-21909 ATCC 202058:B-21909 ATCC 202058:B-21911 ATCC 202058:B-21911 B-21910:B-21909 B-21910:B-21911 B-21911:ATCC 202058 ATCC 202058:B-21909: B-21910:B-21911

Percent mortalityb
84.0 24.0 20.0 20.0 56.0 8.0 16.0 24.0 8.0 24.0 20.0 12.0

Relative percent survivalc


71.4 76.2 76.2 33.3 90.5 80.9 71.4 90.5 71.4 76.2 85.7

CFU = colony-forming unit. a Twenty-four hour cultures of the attenuated vaccine strains were mixed at the following ratios. The vaccine dose used was equivalent to 1 107 CFU 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. b Mortality was determined after challenges with E. ictaluri isolate AL-93-58 (1 107 CFU/mL) for 30 min immersion exposure. c Relative percent survival (RPS) was determined as by Amend (1981).

the live vaccine strain until the immune system is responsive. This has been demonstrated following in ovo administration of live viral vaccines in poultry (Mast and Goddeeris 1999). Knowledge that sh can be successfully vaccinated at 710 d post-hatch or as eyed sh eggs (vaccinated 2472 h) prior to hatch is important if other modied live vaccines (virus or bacteria) become available for use in aquaculture. Utilizing this strategy will allow for the earliest possible vaccination of sh (i.e., in the hatchery) prior to release into a production environment. The modied live E. ictaluri RE-33 was patented (US Patent no. 6,019,981) and licensed to Intervet, Inc. by the USDA-Agricultural Research Service. Intervet, Inc. (Millsboro, DE, USA), marketed the modied live ESC vaccine (2001 to present) under the label AQUAVACESC as an USDA-APHIS-CVB licensed vaccine for immersion vaccination of 710 d posthatch channel catsh. In 2006, the product was changed from a freeze-dried to a frozen formulation. Efcacy was re-evaluated and demonstrated with the frozen product for at least 65 d post-vaccination (Wise 2006). Field trials suggested use of the vaccine results in

larger ngerlings, improved feed conversion, and an improved return of $3900$4900 per ha for vaccinated sh versus non-vaccinated sh (Intervet Inc. 2003; Wise 2006). Carrias et al. (2008) recently reported on the use of the modied live vaccine in addition to an extended hatchery/nursery phase. Results suggest an improved return (money and survival) for vaccinated sh held in a nursery setting prior to release into ngerling ponds (Carrias et al. 2008). Reports from the USA catsh industry indicate greater than 25% of the fry and/or ngerlings produced each year are vaccinated with AQUAVAC-ESC. Channel catsh producers can and are using this vaccine in health management plans to effectively manage ESC.

Conclusions
Modied live vaccines are successfully used in human, veterinary, and aquatic animal medicine to prevent disease. Use of modied live vaccines in aquaculture is an appropriate strategy if potential risks to the animal, environment and people are low or negligible.

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Regulatory authorities need to seriously consider the potential release of these vaccines into the environment without strong scientic data documenting safety and reversion to virulence potential. Vaccines are tools to be used in association with sound health management and biosecurity plans to result in the greatest benet to aquaculture producers.

Acknowledgments
We thank Lisa Biggar for editing and formatting this article. We also gratefully acknowledge 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.

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