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In ovo methods for utilizing the modified live Edwardsiella ictaluri vaccine against enteric septicemia in channel catfish
Aquaculture 203 Ž2002. 221–227 www.elsevier.comrlocateraqua-online
In ovo methods for utilizing the modified live Edwardsiella ictaluri vaccine against enteric septicemia in channel catfish Craig A. Shoemaker a,) , Phillip H. Klesius a , Joyce J. Evans b a
Aquatic Animal Health Research Laboratory, United States Department of Agriculture — Agricultural Research SerÕice, PO Box 0952, Auburn, AL 36830, USA b Aquatic Animal Health Research Laboratory, United States Department of Agriculture — Agricultural Research SerÕice, 151 Dixon Dr., Suite 4 Chestertown, MD 21620, USA Received 9 January 2001; received in revised form 26 March 2001; accepted 28 March 2001
Abstract Eyed channel catfish Ž Ictalurus punctatus . eggs were vaccinated by immersion exposure Ž10 min. with either the modified live Edwardsiella ictaluri isolate RE-33 grown in brain heart infusion broth Žtrial 1. or the lyophilized AQUAVAC-ESCe vaccine Žtrial 2.. AQUAVAC-ESCe is the modified live E. ictaluri isolate ŽRE-33. that was licensed, produced and marketed as a vaccine against enteric septicemia of catfish by Intervet. Trial 1 consisted of vaccinated ŽRE-33 E. ictaluri at 1 = 10 5 CFUrml. and non-vaccinated Žcontrols. treatments with the eyed eggs hatching into fry 4 days following treatment. No mortality was recorded in these fish for 33 days post vaccination. In trial 2, three treatments were used: single vaccinated, vaccinated and fry booster vaccinated 7 days following initial immunization; and non-vaccinated Žcontrols.. The vaccination in trial 2 was carried out according to the manufacturer’s recommendation for use on channel catfish fry Ži.e. AQUAVAC-ESCe label.. In trial 2, the eyed eggs hatched 24 h post vaccination and no control fish or single-vaccinated fish died following vaccination. Three fry died in the booster vaccinated treatment. No rifampicin-resistant E. ictaluri was isolated from these dead fish. Thirty-three Žtrial 1. and sixty days Žtrial 2. following vaccination, fish were challenged with E. ictaluri Žisolate AL-93-75. at 1 = 10 7 CFUrml. In trial 1, the relative percentage survival ŽRPS. of vaccinates was 87.9; however, data did not fit Amend’s criteria of 60% mortality in the non-vaccinated treatment and no significant difference was seen between mortality of the vaccinates or control treatments in trial 1 wDev. Biol. Stand. 49 Ž1981. 447x. In trial 2, mean percentage mortality in the non-vaccinated treatment Žcontrols. Ž64.2 " 5.8. was significantly higher Ž p s 0.003. than mean percentage mortality in the single vaccinated treatment and booster vaccinated treatment Ž25.8 " 5.1 and 46.7 " 0.8, respectively.. Relative percentage
)
Corresponding author. Tel.: q1-334-887-2983; fax: q1-334-887-2983. E-mail address: [email protected] ŽC.A. Shoemaker..
0044-8486r02r$ - see front matter. Published by Elsevier Science B.V. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 6 3 1 - 7
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survival was 59.7 in the single vaccinated treatment and 27.3 in the booster vaccinates. Safety and efficacy of the modified live E. ictaluri vaccine ŽAQUAVAC-ESCe. was demonstrated in eyed channel catfish eggs following single vaccination. Booster vaccination did not enhance vaccine efficacy; however, timing of the booster may have been too soon following initial vaccination of eyed eggs. Published by Elsevier Science B.V. Keywords: In ovo vaccination; Eyed eggs; Modified live vaccine; Edwardsiella ictaluri; AQUAVAC-ESCe; Channel catfish; Ictalurus punctatus
1. Introduction Edwardsiella ictaluri, the causative agent of enteric septicemia in catfish ŽESC., is responsible for US$60 million in annual losses to the cultured channel catfish industry in the US. Successful vaccination of juvenile channel catfish with the modified live E. ictaluri vaccine has been well established ŽKlesius and Shoemaker, 1999; Wise et al., 2000.. Shoemaker et al. Ž1999. demonstrated that vaccination of channel catfish, as young as 7–12 days post hatch, with the modified live E. ictaluri vaccine was possible with a relative percentage survival ŽRPS. rate greater than 50%. Ricks et al. Ž1999. reviewed the technology of in ovo vaccination of chicken eggs. In ovo vaccination with live viruses is a vaccination strategy that has been successfully used in the poultry industry ŽSharma and Burmester, 1983.. This vaccination strategy using live, attenuated viral vaccines has resulted in a protective immune response that is equal to or greater than immunity to viral pathogens in chickens vaccinated by killed products using conventional methods after hatch ŽSharma and Burmester, 1982; Johnston et al., 1997.. Recently, a US patent was awarded for in ovo vaccination of eyed fish eggs ŽKlesius et al., 2000.. The objective of this study was to determine if egg vaccination and fry booster vaccination of channel catfish with the modified live E. ictaluri vaccine ŽAQUAVAC-ESCe. was safe and efficacious. 2. Materials and methods 2.1. Experimental animals and Õaccination In trial 1 Ž1999., two USDA-103 channel catfish egg masses Ž500 g wet weight. at the eyed egg stage were obtained from the Catfish Genetics Research Laboratory in Stoneville, MS. Eggs were held in baskets in 57-l aquariums supplied with flow-through water at 0.5 lrmin and aeration via airblower and airstones. Daily water temperature was 25 " 18C and dissolved oxygen ranged from 6 to 8 mgrl. A 12:12 h lightrdark schedule was maintained. Upon hatching, fry were fed a commercial ration. A sample of 50 eggs from each egg mass was homogenized and cultured for the presence of E. ictaluri ŽKlesius, 1992.. No E. ictaluri was isolated. One egg mass was vaccinated with E. ictaluri RE-33 vaccine ŽKlesius and Shoemaker, 1999. at a concentration of 1 = 10 5 CFU RE-33rml for 10 min at a density of about 50 g of eggsrl water. The vaccine was prepared according to Klesius and Shoemaker Ž1999.. Briefly, one vial of E. ictaluri RE-33 passaged two times from the master seed was used to inoculate brain heart infusion broth. The RE-33 broth culture Ž250 ml. was incubated in a water bath at 258C
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with shaking at 50 rpm for 24 h prior to vaccination. The second egg mass Žcontrol. was held in water only at the same density for the 10-min exposure. Vaccinated and control eggs hatched 4 days after treatment. In trial 2 Ž2000., 600–900 eyed USDA-103 channel catfish eggs were obtained from the same source as above and held under similar conditions. Prior to experimentation, a sample of 50 eggs was homogenized and cultured for E. ictaluri ŽKlesius, 1992.. No E. ictaluri was isolated. Eyed channel catfish eggs were divided into 200–300 eggs per treatment. The treatments consisted of vaccinated, vaccinated and booster vaccinated as fry at 7 days following initial immunization and non-vaccinated Žcontrols.. The vaccine was made by adding one lyophilized vial of AQUAVAC-ESCe as per vaccine label to 4 l of water ŽCFUsrml is proprietary information of Intervet.. Briefly, eyed eggs were added to 1 l of water containing AQUAVAC-ESCe vaccine and allowed to remain in vaccine for the 10-min immersion exposure. Non-vaccinated controls were placed in 1 l of water without the vaccine for 10 min. Eyed eggs Žvaccinated and control. hatched 24 h following treatment. The booster immunization was given 7 days following initial vaccination. The booster vaccine was prepared the same as described above. Following preparation, the fish were caught and placed into 1 l of vaccine for a 10-min exposure. 2.2. Experimental challenge In trial 1, a random sample of 90 fish was selected from each treatment Žvaccinated and non-vaccinated. and divided into three replicate 57-l aquariums of 30 fish each. In trial 2, a random sample of 120 fish was selected from each treatment Žsingle vaccinated, vaccinated plus booster at 7 days and non-vaccinated. and divided into three replicate 57-l aquariums of 40 fish each. The groups of fish in trials 1 and 2 were challenged by immersion exposure for 30 min in water containing E. ictaluri ŽAL-93-75. at a concentration of 1 = 10 7 CFUsrml ŽKlesius and Shoemaker, 1997.. Challenge occurred 33 and 60 days post vaccination for trials 1 and 2, respectively. Mortalities were monitored twice daily for 14 days after challenge ŽKlesius and Shoemaker, 1999.. Dead or moribund fish exhibited signs of enteric septicemia, as described by Plumb Ž1999., which included hemorrhage on the operculum, at the base of fins and abdomen, as well as abdominal distension. Necropsies were performed and anterior kidney tissue from dead fish was cultured to confirm death due to E. ictaluri ŽKlesius, 1992.. 2.3. Statistical analysis Data were analyzed by one-way analysis of variance using Duncan’s multiple range test ŽSAS Institute, 1997. to determine differences in mean percentage mortality. Significant differences were determined at p - 0.05. Relative percentage survival was calculated according to Amend Ž1981.. 3. Results 3.1. Safety Fish in trials 1 and 2 were observed daily for mortality or signs of ESC following vaccination. In trial 1, no fry died for 33 days following treatment in either the
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Table 1 Trial 1, efficacy of E. ictaluri RE-33 isolate delivered to eyed channel catfish eggs Fish were challenged with E. ictaluri ŽAL-93-75. 33 days post immunization. Treatment
Replicate Žnumber dead ) r number total.
Vaccinated
1 2 3 1 2 3
Non-vaccinated Žcontrols.
5r30 0r30 0r30 24r30 6r30 11r30
Mean percentage mortality Ž"S.E.M.. ) )
Relative percentage survival ) ) )
5.5 Ž"5.5. a
87.9
45.5 Ž"17.9. a
–
)
Dead fish were culture-positive for E. ictaluri. Standard error of the means is presented in parentheses. Means were not significantly different. ))) Relative percentage survival was calculated according to the method of Amend Ž1981.. However, data do not fit Amend’s criteria as only 45.5% mortality was observed in the non-vaccinated treatment Žcontrols.. ))
vaccinated or non-vaccinated eyed eggs. In trial 2, no fry died for 60 days following treatment in the single vaccinated or non-vaccinated treatment. Three fry of the vaccinated and booster treatment died, one at day 7 and two at day 11, following the booster vaccination Žmonitored for 53 days following booster immunization.. Rifampicin-resistant E. ictaluri Ži.e. the vaccine strain. was not isolated from these dead fish. Gram-negative cytochrome oxidase-positive bacteria were recovered. No abnormal behavior or signs of ESC were observed in trial 1 or 2 prior to challenge.
Table 2 Trial 2, efficacy of AQUAVAC-ESCe in channel catfish Ž Ictalurus punctatus . following immunization of eyed eggs Fish were challenged with E. ictaluri ŽAL-93-75. 60 days post vaccination. Treatment
Replicate Žnumber dead ) r number total.
Mean percentage mortality Ž"S.E.M.. ) )
Relative percentage survival ) ) )
Single vaccinated
1 2 3 1 2 3 1 2 3
25.8 Ž"5.1. a
59.7
46.7 Ž"0.8. b
27.3
64.2 Ž"5.8. c
–
Vaccinated and booster vaccinated at 7 days Non-vaccinated Žcontrols.
)
7r40 14r40 10r40 19r40 19r40 18r40 30r40 25r40 22r40
Dead fish were culture-positive for E. ictaluri. Standard error of the means is presented in parentheses. Means with different letters are significantly different Ž ps 0.003.. ))) Relative percentage survival was calculated according to the method of Amend Ž1981.. ))
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3.2. Efficacy Efficacy of the E. ictaluri RE-33 vaccine strain was demonstrated in trial 1 following a single immersion exposure as demonstrated by an RPS of 87.9% ŽTable 1.. However, the data did not fit Amend’s criteria of at least 60% mortality ŽAmend, 1981. in the control treatment. Efficacy of AQUAVAC-ESCe vaccination Žtrial 2. of eyed channel catfish eggs is shown in Table 2. Mortality in the non-vaccinated controls was 64.2 " 5.8 and was significantly higher Ž p s 0.003. than mortality in either of the vaccinated treatments. Mortality in the single vaccinated treatment was 25.8 " 5.1, whereas mortality in the booster vaccinated treatment was 46.7 " 0.8. Relative percentage survival was 59.7 in the single vaccinated treatment and 27.3 in the booster vaccinated treatment.
4. Discussion The results demonstrate that in ovo vaccination of eyed channel catfish eggs is possible with the modified live E. ictaluri vaccine ŽAQUAVAC-ESCe.. Previously, it was suggested that channel catfish did not obtain immunocompetence Žantibody-mediated. until about 4 weeks post hatch ŽPetrie-Hanson and Ainsworth, 1999.. Passive transfer of anti-E. ictaluri channel catfish immunoglobulin failed to protect channel catfish from enteric septicemia following challenge ŽKlesius and Sealey, 1995.. Immunity to enteric septicemia of catfish has been shown to be cell-mediated in nature ŽAntonio and Hedrick, 1994; Klesius and Shoemaker, 1997. and relies on the macrophages’ ability to kill E. ictaluri for acquired immunity ŽShoemaker and Klesius, 1997; Shoemaker et al., 1997.. The results of this study suggest that cellular immunity developed in eyed channel catfish eggs upon exposure to the modified live E. ictaluri vaccine. Presumably, immunity, rather than immunological tolerance, develops due to the presence of macrophages or antigen presenting cells ŽRidge et al., 1996.. Aguirre et al. Ž1997. demonstrated acquired resistance to Cryptococcus neoformans in adult mice following vaccination when immunologically immature. This suggests that even in young fish without mature immune systems, as in other animals, the immune system will respond protectively against pathogens ŽRidge et al., 1996.. Mast and Goddeeris Ž1999. suggested two possibilities for the success of in ovo vaccination in poultry: Ž1. live viral vaccine strains survive or persist that results in stimulation of the immune system at a time when immune system is responsive andror Ž2. the live vaccine causes expression of antigens with the correct MHC on host cells and thus results in the activation of immunocompetent cells. Awad et al. Ž1989. were also successful in the vaccination of 3–4-day-old eggs of channel catfish with the soluble channel catfish virus envelope. Protection was demonstrated 8 weeks following vaccination against channel catfish virus ŽCCV. disease following challenge with CCV in their study. Our results suggest that a booster vaccination did not enhance vaccine performance as compared to the single immersion exposure of the eyed eggs. However, Awad et al. Ž1989. found enhanced protection against CCV disease when a booster vaccination was given 14 days following initial
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immunization of eggs Ž31% vs. 82% survival.. We booster vaccinated the fish in our trial 7 days following initial vaccination with the modified live E. ictaluri vaccine. Booster vaccination too soon following initial immunization may have resulted in the low vaccine efficacy as seen in our study. Alternatively, booster immunization 7 days following initial immunization may have caused tolerance due to the presence of excess antigen in relation to antigen presenting cells as suggested by Ridge et al. Ž1996. in newborn mice. Successful in ovo vaccination of channel catfish with the modified live E. ictaluri vaccine will be another tool for use in the health management plans of catfish producers.
References Aguirre, K.M., Garvy, B.A., Johnson, L.L., 1997. Acquired resistance to Cryptococcus neoformans in adult mice vaccinated as newborns. Infect. Immun. 65, 1688–1694. 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. Anim. Health 6, 44–52. Awad, M.A., Nusbaum, K.E., Brady, Y.J., 1989. Preliminary studies of a newly developed subunit vaccine for channel catfish virus disease. J. Aquat. Anim. Health 1, 233–237. Johnston, P.A., Liu, H., O’Connel, T., Phelps, R., Bland, M., Tyzkowski, J., Kemper, A., Harding, T., Avakian, A., Haddad, E., Whitfill, C., Gildersleeve, R., Ricks, C.A., 1997. Application of in ovo technology. Poult. Sci. 76, 165–178. Klesius, P.H., 1992. Carrier state of channel catfish infected with Edwardsiella ictaluri. J. Aquat. Anim. Health 4, 227–230. Klesius, P.H., Sealey, W.M., 1995. Characteristics of serum antibody in enteric septicemia of catfish. J. Aquat. Anim. Health 7, 205–210. Klesius, P.H., Shoemaker, C.A., 1997. Heterologous isolates challenge of channel catfish, Ictalurus punctatus, immune to Edwardsiella ictaluri. Aquaculture 157, 147–155. Klesius, P.H., Shoemaker, C.A., 1999. Development and use of modified live Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In: Schultz, R. ŽEd.., Advances in Veterinary Medicine, vol. 41. Academic Press, San Diego, CA, USA, pp. 523–537. Klesius, P.H., Shoemaker, C.A., Evans, J.J., 2000. In ovo methods for utilizing live Edwardsiella ictaluri against enteric septicemia in channel catfish. US Patent Number 6,153,202. Mast, J., Goddeeris, B.M., 1999. Development of immunocompetence of broiler chickens. Vet. Immunol. Immunopathol. 70, 245–256. Petrie-Hanson, L., Ainsworth, A.J., 1999. Humoral immune response of channel catfish Ž Ictalurus punctatus . fry and fingerlings exposed to Edwardsiella ictaluri. Fish Shellfish Immunol. 9, 579–589. Plumb, J.A., 1999. Health Maintenance and Microbial Diseases of Cultured Fishes. Iowa State Univ. Press, Ames, IA, USA. Ricks, C.A., Avakian, A., Bryan, T., Gildersleeve, R., Haddad, E., Ilich, R., King, S., Murray, L., Phelps, P., Poston, R., Whitfill, C., Williams, C., 1999. In ovo vaccination technology. In: Schultz, R. ŽEd.., Advances in Veterinary Medicine, vol. 41. Academic Press, San Diego, CA, USA, pp. 495–522. Ridge, J.P., Fuchs, E.J., Matzinger, P., 1996. Neonatal tolerance revisited: turning on newborn T cells with dendritic cells. Science 271, 1723–1726. SAS Institute, 1997. SAS User’s Guide, version 6.12. SAS Institute, Cary, NC. Sharma, J.M., Burmester, B., 1982. Resistance to Marek’s disease at hatching in chickens vaccinated as embryos with the turkey herpesvirus. Avian Dis. 26, 134–149. Sharma, J.M., Burmester, B.R., 1983. Embryo vaccination against Marek’s disease with serotypes 1, 2 and 3 vaccines administered singly or in combination. Avian Dis. 27, 453–463. Shoemaker, C.A., Klesius, P.H., 1997. Protective immunity against enteric septicemia in channel catfish,
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Ictalurus punctatus ŽRafinesque., following controlled exposure to Edwardsiella ictaluri. J. Fish Dis. 20, 101–108. Shoemaker, C.A., Klesius, P.H., Plumb, J.A., 1997. Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicemia of catfish. Vet. Immunol. Immunopathol. 58, 181–190. Shoemaker, C.A., Klesius, P.H., Bricker, J.M., 1999. Efficacy of a modified live Edwardsiella ictaluri vaccine in channel catfish as young as seven days post hatch. Aquaculture 176, 189–193. Wise, D.J., Klesius, P.H., Shoemaker, C.A., Wolters, W.R., 2000. Vaccination of mixed and full-sib families of channel catfish Ictalurus punctatus against enteric septicemia of catfish with a live attenuated Edwardsiella ictaluri isolate ŽRE-33.. J. World Aquacult. Soc. 31 Ž2., 206–212.
In ovo methods for utilizing the modified live Edwardsiella ictaluri vaccine against enteric septicemia in channel catfish Craig A. Shoemaker a,) , Phillip H. Klesius a , Joyce J. Evans b a
Aquatic Animal Health Research Laboratory, United States Department of Agriculture — Agricultural Research SerÕice, PO Box 0952, Auburn, AL 36830, USA b Aquatic Animal Health Research Laboratory, United States Department of Agriculture — Agricultural Research SerÕice, 151 Dixon Dr., Suite 4 Chestertown, MD 21620, USA Received 9 January 2001; received in revised form 26 March 2001; accepted 28 March 2001
Abstract Eyed channel catfish Ž Ictalurus punctatus . eggs were vaccinated by immersion exposure Ž10 min. with either the modified live Edwardsiella ictaluri isolate RE-33 grown in brain heart infusion broth Žtrial 1. or the lyophilized AQUAVAC-ESCe vaccine Žtrial 2.. AQUAVAC-ESCe is the modified live E. ictaluri isolate ŽRE-33. that was licensed, produced and marketed as a vaccine against enteric septicemia of catfish by Intervet. Trial 1 consisted of vaccinated ŽRE-33 E. ictaluri at 1 = 10 5 CFUrml. and non-vaccinated Žcontrols. treatments with the eyed eggs hatching into fry 4 days following treatment. No mortality was recorded in these fish for 33 days post vaccination. In trial 2, three treatments were used: single vaccinated, vaccinated and fry booster vaccinated 7 days following initial immunization; and non-vaccinated Žcontrols.. The vaccination in trial 2 was carried out according to the manufacturer’s recommendation for use on channel catfish fry Ži.e. AQUAVAC-ESCe label.. In trial 2, the eyed eggs hatched 24 h post vaccination and no control fish or single-vaccinated fish died following vaccination. Three fry died in the booster vaccinated treatment. No rifampicin-resistant E. ictaluri was isolated from these dead fish. Thirty-three Žtrial 1. and sixty days Žtrial 2. following vaccination, fish were challenged with E. ictaluri Žisolate AL-93-75. at 1 = 10 7 CFUrml. In trial 1, the relative percentage survival ŽRPS. of vaccinates was 87.9; however, data did not fit Amend’s criteria of 60% mortality in the non-vaccinated treatment and no significant difference was seen between mortality of the vaccinates or control treatments in trial 1 wDev. Biol. Stand. 49 Ž1981. 447x. In trial 2, mean percentage mortality in the non-vaccinated treatment Žcontrols. Ž64.2 " 5.8. was significantly higher Ž p s 0.003. than mean percentage mortality in the single vaccinated treatment and booster vaccinated treatment Ž25.8 " 5.1 and 46.7 " 0.8, respectively.. Relative percentage
)
Corresponding author. Tel.: q1-334-887-2983; fax: q1-334-887-2983. E-mail address: [email protected] ŽC.A. Shoemaker..
0044-8486r02r$ - see front matter. Published by Elsevier Science B.V. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 6 3 1 - 7
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survival was 59.7 in the single vaccinated treatment and 27.3 in the booster vaccinates. Safety and efficacy of the modified live E. ictaluri vaccine ŽAQUAVAC-ESCe. was demonstrated in eyed channel catfish eggs following single vaccination. Booster vaccination did not enhance vaccine efficacy; however, timing of the booster may have been too soon following initial vaccination of eyed eggs. Published by Elsevier Science B.V. Keywords: In ovo vaccination; Eyed eggs; Modified live vaccine; Edwardsiella ictaluri; AQUAVAC-ESCe; Channel catfish; Ictalurus punctatus
1. Introduction Edwardsiella ictaluri, the causative agent of enteric septicemia in catfish ŽESC., is responsible for US$60 million in annual losses to the cultured channel catfish industry in the US. Successful vaccination of juvenile channel catfish with the modified live E. ictaluri vaccine has been well established ŽKlesius and Shoemaker, 1999; Wise et al., 2000.. Shoemaker et al. Ž1999. demonstrated that vaccination of channel catfish, as young as 7–12 days post hatch, with the modified live E. ictaluri vaccine was possible with a relative percentage survival ŽRPS. rate greater than 50%. Ricks et al. Ž1999. reviewed the technology of in ovo vaccination of chicken eggs. In ovo vaccination with live viruses is a vaccination strategy that has been successfully used in the poultry industry ŽSharma and Burmester, 1983.. This vaccination strategy using live, attenuated viral vaccines has resulted in a protective immune response that is equal to or greater than immunity to viral pathogens in chickens vaccinated by killed products using conventional methods after hatch ŽSharma and Burmester, 1982; Johnston et al., 1997.. Recently, a US patent was awarded for in ovo vaccination of eyed fish eggs ŽKlesius et al., 2000.. The objective of this study was to determine if egg vaccination and fry booster vaccination of channel catfish with the modified live E. ictaluri vaccine ŽAQUAVAC-ESCe. was safe and efficacious. 2. Materials and methods 2.1. Experimental animals and Õaccination In trial 1 Ž1999., two USDA-103 channel catfish egg masses Ž500 g wet weight. at the eyed egg stage were obtained from the Catfish Genetics Research Laboratory in Stoneville, MS. Eggs were held in baskets in 57-l aquariums supplied with flow-through water at 0.5 lrmin and aeration via airblower and airstones. Daily water temperature was 25 " 18C and dissolved oxygen ranged from 6 to 8 mgrl. A 12:12 h lightrdark schedule was maintained. Upon hatching, fry were fed a commercial ration. A sample of 50 eggs from each egg mass was homogenized and cultured for the presence of E. ictaluri ŽKlesius, 1992.. No E. ictaluri was isolated. One egg mass was vaccinated with E. ictaluri RE-33 vaccine ŽKlesius and Shoemaker, 1999. at a concentration of 1 = 10 5 CFU RE-33rml for 10 min at a density of about 50 g of eggsrl water. The vaccine was prepared according to Klesius and Shoemaker Ž1999.. Briefly, one vial of E. ictaluri RE-33 passaged two times from the master seed was used to inoculate brain heart infusion broth. The RE-33 broth culture Ž250 ml. was incubated in a water bath at 258C
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with shaking at 50 rpm for 24 h prior to vaccination. The second egg mass Žcontrol. was held in water only at the same density for the 10-min exposure. Vaccinated and control eggs hatched 4 days after treatment. In trial 2 Ž2000., 600–900 eyed USDA-103 channel catfish eggs were obtained from the same source as above and held under similar conditions. Prior to experimentation, a sample of 50 eggs was homogenized and cultured for E. ictaluri ŽKlesius, 1992.. No E. ictaluri was isolated. Eyed channel catfish eggs were divided into 200–300 eggs per treatment. The treatments consisted of vaccinated, vaccinated and booster vaccinated as fry at 7 days following initial immunization and non-vaccinated Žcontrols.. The vaccine was made by adding one lyophilized vial of AQUAVAC-ESCe as per vaccine label to 4 l of water ŽCFUsrml is proprietary information of Intervet.. Briefly, eyed eggs were added to 1 l of water containing AQUAVAC-ESCe vaccine and allowed to remain in vaccine for the 10-min immersion exposure. Non-vaccinated controls were placed in 1 l of water without the vaccine for 10 min. Eyed eggs Žvaccinated and control. hatched 24 h following treatment. The booster immunization was given 7 days following initial vaccination. The booster vaccine was prepared the same as described above. Following preparation, the fish were caught and placed into 1 l of vaccine for a 10-min exposure. 2.2. Experimental challenge In trial 1, a random sample of 90 fish was selected from each treatment Žvaccinated and non-vaccinated. and divided into three replicate 57-l aquariums of 30 fish each. In trial 2, a random sample of 120 fish was selected from each treatment Žsingle vaccinated, vaccinated plus booster at 7 days and non-vaccinated. and divided into three replicate 57-l aquariums of 40 fish each. The groups of fish in trials 1 and 2 were challenged by immersion exposure for 30 min in water containing E. ictaluri ŽAL-93-75. at a concentration of 1 = 10 7 CFUsrml ŽKlesius and Shoemaker, 1997.. Challenge occurred 33 and 60 days post vaccination for trials 1 and 2, respectively. Mortalities were monitored twice daily for 14 days after challenge ŽKlesius and Shoemaker, 1999.. Dead or moribund fish exhibited signs of enteric septicemia, as described by Plumb Ž1999., which included hemorrhage on the operculum, at the base of fins and abdomen, as well as abdominal distension. Necropsies were performed and anterior kidney tissue from dead fish was cultured to confirm death due to E. ictaluri ŽKlesius, 1992.. 2.3. Statistical analysis Data were analyzed by one-way analysis of variance using Duncan’s multiple range test ŽSAS Institute, 1997. to determine differences in mean percentage mortality. Significant differences were determined at p - 0.05. Relative percentage survival was calculated according to Amend Ž1981.. 3. Results 3.1. Safety Fish in trials 1 and 2 were observed daily for mortality or signs of ESC following vaccination. In trial 1, no fry died for 33 days following treatment in either the
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Table 1 Trial 1, efficacy of E. ictaluri RE-33 isolate delivered to eyed channel catfish eggs Fish were challenged with E. ictaluri ŽAL-93-75. 33 days post immunization. Treatment
Replicate Žnumber dead ) r number total.
Vaccinated
1 2 3 1 2 3
Non-vaccinated Žcontrols.
5r30 0r30 0r30 24r30 6r30 11r30
Mean percentage mortality Ž"S.E.M.. ) )
Relative percentage survival ) ) )
5.5 Ž"5.5. a
87.9
45.5 Ž"17.9. a
–
)
Dead fish were culture-positive for E. ictaluri. Standard error of the means is presented in parentheses. Means were not significantly different. ))) Relative percentage survival was calculated according to the method of Amend Ž1981.. However, data do not fit Amend’s criteria as only 45.5% mortality was observed in the non-vaccinated treatment Žcontrols.. ))
vaccinated or non-vaccinated eyed eggs. In trial 2, no fry died for 60 days following treatment in the single vaccinated or non-vaccinated treatment. Three fry of the vaccinated and booster treatment died, one at day 7 and two at day 11, following the booster vaccination Žmonitored for 53 days following booster immunization.. Rifampicin-resistant E. ictaluri Ži.e. the vaccine strain. was not isolated from these dead fish. Gram-negative cytochrome oxidase-positive bacteria were recovered. No abnormal behavior or signs of ESC were observed in trial 1 or 2 prior to challenge.
Table 2 Trial 2, efficacy of AQUAVAC-ESCe in channel catfish Ž Ictalurus punctatus . following immunization of eyed eggs Fish were challenged with E. ictaluri ŽAL-93-75. 60 days post vaccination. Treatment
Replicate Žnumber dead ) r number total.
Mean percentage mortality Ž"S.E.M.. ) )
Relative percentage survival ) ) )
Single vaccinated
1 2 3 1 2 3 1 2 3
25.8 Ž"5.1. a
59.7
46.7 Ž"0.8. b
27.3
64.2 Ž"5.8. c
–
Vaccinated and booster vaccinated at 7 days Non-vaccinated Žcontrols.
)
7r40 14r40 10r40 19r40 19r40 18r40 30r40 25r40 22r40
Dead fish were culture-positive for E. ictaluri. Standard error of the means is presented in parentheses. Means with different letters are significantly different Ž ps 0.003.. ))) Relative percentage survival was calculated according to the method of Amend Ž1981.. ))
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3.2. Efficacy Efficacy of the E. ictaluri RE-33 vaccine strain was demonstrated in trial 1 following a single immersion exposure as demonstrated by an RPS of 87.9% ŽTable 1.. However, the data did not fit Amend’s criteria of at least 60% mortality ŽAmend, 1981. in the control treatment. Efficacy of AQUAVAC-ESCe vaccination Žtrial 2. of eyed channel catfish eggs is shown in Table 2. Mortality in the non-vaccinated controls was 64.2 " 5.8 and was significantly higher Ž p s 0.003. than mortality in either of the vaccinated treatments. Mortality in the single vaccinated treatment was 25.8 " 5.1, whereas mortality in the booster vaccinated treatment was 46.7 " 0.8. Relative percentage survival was 59.7 in the single vaccinated treatment and 27.3 in the booster vaccinated treatment.
4. Discussion The results demonstrate that in ovo vaccination of eyed channel catfish eggs is possible with the modified live E. ictaluri vaccine ŽAQUAVAC-ESCe.. Previously, it was suggested that channel catfish did not obtain immunocompetence Žantibody-mediated. until about 4 weeks post hatch ŽPetrie-Hanson and Ainsworth, 1999.. Passive transfer of anti-E. ictaluri channel catfish immunoglobulin failed to protect channel catfish from enteric septicemia following challenge ŽKlesius and Sealey, 1995.. Immunity to enteric septicemia of catfish has been shown to be cell-mediated in nature ŽAntonio and Hedrick, 1994; Klesius and Shoemaker, 1997. and relies on the macrophages’ ability to kill E. ictaluri for acquired immunity ŽShoemaker and Klesius, 1997; Shoemaker et al., 1997.. The results of this study suggest that cellular immunity developed in eyed channel catfish eggs upon exposure to the modified live E. ictaluri vaccine. Presumably, immunity, rather than immunological tolerance, develops due to the presence of macrophages or antigen presenting cells ŽRidge et al., 1996.. Aguirre et al. Ž1997. demonstrated acquired resistance to Cryptococcus neoformans in adult mice following vaccination when immunologically immature. This suggests that even in young fish without mature immune systems, as in other animals, the immune system will respond protectively against pathogens ŽRidge et al., 1996.. Mast and Goddeeris Ž1999. suggested two possibilities for the success of in ovo vaccination in poultry: Ž1. live viral vaccine strains survive or persist that results in stimulation of the immune system at a time when immune system is responsive andror Ž2. the live vaccine causes expression of antigens with the correct MHC on host cells and thus results in the activation of immunocompetent cells. Awad et al. Ž1989. were also successful in the vaccination of 3–4-day-old eggs of channel catfish with the soluble channel catfish virus envelope. Protection was demonstrated 8 weeks following vaccination against channel catfish virus ŽCCV. disease following challenge with CCV in their study. Our results suggest that a booster vaccination did not enhance vaccine performance as compared to the single immersion exposure of the eyed eggs. However, Awad et al. Ž1989. found enhanced protection against CCV disease when a booster vaccination was given 14 days following initial
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immunization of eggs Ž31% vs. 82% survival.. We booster vaccinated the fish in our trial 7 days following initial vaccination with the modified live E. ictaluri vaccine. Booster vaccination too soon following initial immunization may have resulted in the low vaccine efficacy as seen in our study. Alternatively, booster immunization 7 days following initial immunization may have caused tolerance due to the presence of excess antigen in relation to antigen presenting cells as suggested by Ridge et al. Ž1996. in newborn mice. Successful in ovo vaccination of channel catfish with the modified live E. ictaluri vaccine will be another tool for use in the health management plans of catfish producers.
References Aguirre, K.M., Garvy, B.A., Johnson, L.L., 1997. Acquired resistance to Cryptococcus neoformans in adult mice vaccinated as newborns. Infect. Immun. 65, 1688–1694. 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. Anim. Health 6, 44–52. Awad, M.A., Nusbaum, K.E., Brady, Y.J., 1989. Preliminary studies of a newly developed subunit vaccine for channel catfish virus disease. J. Aquat. Anim. Health 1, 233–237. Johnston, P.A., Liu, H., O’Connel, T., Phelps, R., Bland, M., Tyzkowski, J., Kemper, A., Harding, T., Avakian, A., Haddad, E., Whitfill, C., Gildersleeve, R., Ricks, C.A., 1997. Application of in ovo technology. Poult. Sci. 76, 165–178. Klesius, P.H., 1992. Carrier state of channel catfish infected with Edwardsiella ictaluri. J. Aquat. Anim. Health 4, 227–230. Klesius, P.H., Sealey, W.M., 1995. Characteristics of serum antibody in enteric septicemia of catfish. J. Aquat. Anim. Health 7, 205–210. Klesius, P.H., Shoemaker, C.A., 1997. Heterologous isolates challenge of channel catfish, Ictalurus punctatus, immune to Edwardsiella ictaluri. Aquaculture 157, 147–155. Klesius, P.H., Shoemaker, C.A., 1999. Development and use of modified live Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In: Schultz, R. ŽEd.., Advances in Veterinary Medicine, vol. 41. Academic Press, San Diego, CA, USA, pp. 523–537. Klesius, P.H., Shoemaker, C.A., Evans, J.J., 2000. In ovo methods for utilizing live Edwardsiella ictaluri against enteric septicemia in channel catfish. US Patent Number 6,153,202. Mast, J., Goddeeris, B.M., 1999. Development of immunocompetence of broiler chickens. Vet. Immunol. Immunopathol. 70, 245–256. Petrie-Hanson, L., Ainsworth, A.J., 1999. Humoral immune response of channel catfish Ž Ictalurus punctatus . fry and fingerlings exposed to Edwardsiella ictaluri. Fish Shellfish Immunol. 9, 579–589. Plumb, J.A., 1999. Health Maintenance and Microbial Diseases of Cultured Fishes. Iowa State Univ. Press, Ames, IA, USA. Ricks, C.A., Avakian, A., Bryan, T., Gildersleeve, R., Haddad, E., Ilich, R., King, S., Murray, L., Phelps, P., Poston, R., Whitfill, C., Williams, C., 1999. In ovo vaccination technology. In: Schultz, R. ŽEd.., Advances in Veterinary Medicine, vol. 41. Academic Press, San Diego, CA, USA, pp. 495–522. Ridge, J.P., Fuchs, E.J., Matzinger, P., 1996. Neonatal tolerance revisited: turning on newborn T cells with dendritic cells. Science 271, 1723–1726. SAS Institute, 1997. SAS User’s Guide, version 6.12. SAS Institute, Cary, NC. Sharma, J.M., Burmester, B., 1982. Resistance to Marek’s disease at hatching in chickens vaccinated as embryos with the turkey herpesvirus. Avian Dis. 26, 134–149. Sharma, J.M., Burmester, B.R., 1983. Embryo vaccination against Marek’s disease with serotypes 1, 2 and 3 vaccines administered singly or in combination. Avian Dis. 27, 453–463. Shoemaker, C.A., Klesius, P.H., 1997. Protective immunity against enteric septicemia in channel catfish,
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