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Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachus—a carnivore model
Fish & Shellfish Immunology 16 (2004) 613e619 www.elsevier.com/locate/fsi
Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachusda carnivore model D.K. Nayak, A. Asha, K.M. Shankar), C.V. Mohan Department of Aquaculture, Fish Pathology and Biotechnology Laboratory, College of Fisheries, University of Agricultural Sciences, Mangalore 575 002, India Received 23 June 2003; accepted 29 September 2003
Abstract Biofilm of Aeromonas hydrophila was evaluated for oral vaccination of walking catfish (Clarias batrachus L.). Fish were fed with fish paste incorporating biofilm (BF) or free cells (FC) of A. hydrophila for 20 days and monitored for serum antibody production up to 60 days post-vaccination. Serum agglutinating antibody titre and relative percent survival (RPS) following challenge were found to be significantly higher in catfish fed with BF vaccine compared to that with FC. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Biofilm vaccine; Aeromonas hydrophila; Clarias batrachus
1. Introduction Oral vaccination is regarded beneficial in aquaculture as it is non-stressing and accessible to fish of any size, age and number [1]. The very first attempt of oral vaccination in trout against furunculosis was highly encouraging [2]. However, the later attempts were either unsuccessful [3,4] or produced variable results [5e8]. One of the suggested reasons for these poor and inconsistent responses to oral vaccination was the gastric destruction of orally introduced antigens before they could reach and be absorbed into the immune responsive areas of the hindgut [9]. Strategies developed for improvements of oral vaccines have attempted to avoid this gastric destruction [10e12] especially by the use of encapsulated antigen microspheres [13e15]. Our laboratory has developed a biofilm of Aeromonas hydrophila to facilitate improved antigen delivery in oral vaccination of fish. It induced significantly higher humoral and protective responses in Indian carps compared to free cell vaccine of the pathogen [16e18]. Studies on retention of biofilm (BF) and free cell (FC) vaccines in the gut lumen and their uptake and processing have been carried out by antigen localisation employing monoclonal antibody [19]. Biofilm vaccine was retained in larger quantities than
) Corresponding author. E-mail address: kalkulishankar@rediffmail.com (K.M. Shankar). 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2003.09.012
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that of free cell vaccine and for a longer duration in the tissues of foregut, hindgut, spleen and kidney after a single oral delivery. The evaluation of biofilm vaccine remains incomplete without supporting data from cultured carnivorous species, which possess a stomach with a quite different anatomy and digestive physiology. In general, reports of oral vaccination trials in carnivorous fishes are fewer. Culture of carnivorous fishes such as clarids, snakeheads and sea bass is becoming popular in India and elsewhere in Asia. The walking catfish, Clarias batrachus, represents a high priced food fish and a great deal of attention is now being paid for popularisation of its mass culture. By virtue of its preferential habitat in bottom zones of swampy waters, where the bacterial population may be 10e20 times higher than in the water column [20] it might be at a higher risk of getting infected when cultured at high stocking densities as well as in composite cultures. In this communication, we present successful oral immunisation of C. batrachus with a biofilm vaccine of A. hydrophila.
2. Materials and methods 2.1. Bacterial isolate Pure culture of a virulent A. hydrophila (AAH 2/96) isolated from ulcerated Clarias sp. was maintained in sterile Tryptic Soya Broth (TSB) (Hi Media, India) with 15% glycerol at 20 (C. For use, the culture was revived on Nutrient Agar (NA) (2.8% w/v, Hi Media) slants at room temperature and subsequently stored at 4 (C till used. 2.2. Preparation of biofilm and free cells of A. hydrophila Preparation of biofilm cells of A. hydrophila was according to Azad et al. [16]. In brief, A. hydrophila was promoted to grow as biofilm on chitin flakes provided as substrate (0.3% w/v) in TSB (0.225% w/v). Chitin with 4-day-old biofilm cells on it was harvested for use as biofilm vaccine. For free cells, 1-day-old culture grown in TSB (1.5% w/v) was centrifuged at 3000 g for 10 min, washed thrice with sterile phosphate buffered saline (PBS 0.01 M, pH 7.2) and then resuspended in PBS to the desired optical density (O.D.) at 540 nm. Biofilm and free cells were heat inactivated at 100 (C for 50 min and 90 (C for 10 min, respectively and incorporated into a minced fish paste, prepared from minced fish meat (70%) blended with wheat flour (30%) to constitute biofilm vaccine (BF) and free cell vaccine (FC). A control fish paste (C) was prepared without biofilm or free cells but with PBS (pH 7.2). All vaccine incorporated and control fish pastes were stored at 20 (C before use. 2.3. Fish stock maintenance Catfish, C. batrachus (25e35 cm, 140e260 g) were collected from the local fish market and acclimatised in fibreglass tanks (1!1!0:5 m) filled with stored ground water (26 (C) (30G2 cm deep). For the first 30 days of acclimatisation, water was replenished to the extent of 80e90% on each alternate day and reduced to 50% thereafter. Fish were fed with the minced fish paste at the rate of 3% body weight per day. Initially, as the fish did not accept feed readily, they were force fed employing a wide mouth 2 ml syringe for 30 d. Once acclimatised, the fish paste was offered as feed balls (8e10 mm dia) dispensed into the water. Feeding was done once a day between 16.30 and 17.30 h. Holding tanks and fish were surface disinfected with KMnO4(10 ppm) for 5 min once a week.
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2.4. Oral immunisation of fish After 30 days of acclimatisation, fish were fed once a day with fish paste incorporating the BF and FC vaccine at 4:29!1010 and 6:79!1010 bacterial cells (g fish)1d1, respectively for a period of 20 d. More than 150 fish were assigned to each treatment group. 2.5. Estimation of agglutinating antibody titre After completion of the 20 day vaccination, serum samples were collected from the 0 day postvaccination (dpv) i.e. 20th day of vaccination, onwards up to 60 dpv at 10 day intervals. Fish were anaesthetised and bled from their caudal veins with non-heparinised disposable syringes. Blood was allowed to clot at room temperature for 5 min, followed by 1 h at 4 (C. The clot free serum was separated following centrifugation at 3000 g for 10 min and stored at 20 (C. Antibody agglutination titre against the homologous isolate of A. hydrophila was determined for each fish by a modified agglutination assay [21]. Briefly, 90 ml of immune/control serum was serially diluted with 50 ml PBS pH 7.2 (dilution factor 1.55) and then 50 ml of heat-inactivated free cell suspension of A. hydrophila (OD 0.7 at 540 nm, approx. 109cfu/ml) was added. Plates were incubated at room temperature overnight prior to microscopic examination (!40) for agglutination. Antibody titre was expressed as the reciprocal of the highest dilution of serum that was positive for agglutination. Comparison of agglutinating antibody titre was done through a 3-way ANOVA followed by Duncan’s multiple range test. A value of P ! 0:05 was considered to be significant. 2.6. Estimation of protection Orally immunised catfish from BF, FC and control groups were divided into three challenge groups in separate tanks and injected intramuscularly (0.5 ml) with 24 h old live free cells of A. hydrophila (107cfu g1fish) on the 60th day post vaccination (dpv) to assess the overall protection. Appearance of gross external lesions and mortality pattern was monitored for 7 days. From the freshly dead and moribund specimens, whole blood and kidney inoculants were aseptically plated on nutrient agar and Aeromonas selective medium (Hi Media) to confirm specific cause of death and morbidity. Relative percent survival (RPS) was calculated according to Amend [22].
3. Results and discussion In commercial aquaculture, oral vaccination is the most preferred means to immunise fish of all size and age groups with least stress. However, poor responses to oral vaccines [3e8], possibly due to destruction of antigenic epitopes [9], pose major hindrance for developing an effective oral vaccine. Oral vaccination of Indian major carps (catla, rohu) and common carp with biofilm of A. hydrophila was highly successful in eliciting antibody titre and protection [16e18]. Carps are usually planktophagous and lack a distinct stomach. Therefore, the current study was conducted in order to evaluate the performance of the biofilm vaccine in a carnivorous fish model, C. batrachus. Dietary habits of the catfish have been described as predator but not piscivore [23], scavenger [24], omnivore [25], insectivore [26], larvivore [27], with great affinity for a carnivorous diet [28]. More or less it shows preference for food of animal origin and possesses a well-demarcated stomach unlike carp. Kinetics of the catfish serum antibody response to BF and FC oral vaccines is presented in Fig. 1. The duration of oral vaccination was 20 days and the antibody response was monitored from 0 dpv, i.e. the 20th day since the beginning of daily immunisation. The biofilm-vaccinated fish had significantly higher
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Fig. 1. Agglutinating antibody response in catfish, control (C) and biofilm (BF) and free cell (FC) vaccine. Titre values are the highest serum dilutions (mean with SE, n ¼ 15) causing agglutination. 0 day post vaccination corresponds to the 20th day of vaccination.
(P ! 0:05) antibody titre than those vaccinated with free cells on all days post-vaccination (dpv). The difference between BF and FC is quite distinct even at 0 dpv. Overall, orally vaccinated catfish produced higher antibody titres compared to unvaccinated controls. Similar trends of higher systemic agglutinating antibody response to biofilm oral vaccine have been previously reported in agastric carpsdcatla, rohu and common carp [16,17]. Biofilm glycocalyx, chemically a polymer of neutral hexoses [29] encapsulates and possibly protects cells/antigens in the stomach/foregut [17] and thus provides a large pool of antigens to lymphoid organs compared to FC. This property of biofilm vaccine is reported to facilitate longer retention of antigens in the gut and lymphoid tissue [19] and hence, might have resulted in the early and heightened primary antibody response. The magnitude of humoral response in fish appears to be dependent on the nature, dose and presentation route of antigen. High dose priming is normally believed to elicit higher humoral response and formation of memory in carp [30] and the extent of immune memory response in fish depends on the route and dose of primary immunisation and persistence of antigens [31]. Availability of antigens in large quantities with BF vaccine due to the protective nature of the glycocalyx, might have facilitated its protracted presence in the lymphoid organs. As fish do not possess physiologically distinct memory cells, rather it appears to rely upon the increasing size of the pools of antigen sensitive cells [32], vaccination with biofilm may induce long term memory response helping the fish to fight the disease for a longer duration. Lower antibody response to FC vaccine might be due to the destructive processes in the catfish gut with acid secretion and activity of acidic proteolytic enzymes. The unvaccinated control groups showed some titre values even though significantly lower. This might be the background level reaction corresponding to the occurrence of natural agglutinin in fish serum [33,34]. There was an elevated level of protection in biofilm vaccinated catfish to the homologous injection challenge (Table 1). A. hydrophila was reisolated from all the moribund and dead fish. Protection was higher with BF than with FC. The unvaccinated control group registered 24e35% survival. Biofilm vaccinated (BF) fish were fully protected with 93e100% survival while those vaccinated with free cell (FC) had only 46e56% survival. The protective immunity calculated as relative percent survival (RPS) was higher for BF than FC. Higher and complete protection in BF vaccinated catfish compared to that with FC vaccine observed here, is consistent with the findings of Azad et al. [18] in omnivore common carp. In their experimental conditions, common carp fed with biofilm vaccine (1010 bacterial cells/fish/day) for a period of 15 days recorded RPS of 100% to both immersion and injection challenge, while the values for free cell vaccine were 76.46 and 80.86% to both these challenge routes, respectively. The percentage of survival assessed to homologous injection challenge was also higher in biofilm-vaccinated catla, rohu and common
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D.K. Nayak et al. / Fish & Shellfish Immunology 16 (2004) 613e619 Table 1 Protection level in vaccinated catfish challenged with A. hydrophila at 107CFU (g fish)1 BF vaccine
FC vaccine
Control
Challenge-1 No. of fish challenged Specific mortality Survival (%) RPS
25 0 100 100
25 11 56 42.1
25 19 24 e
Challenge-2 No. of fish challenged Specific mortality Survival (%) RPS
29 2 93.1 90.8
30 16 46.6 28.8
28 21 25 e
Challenge-3 No. of fish challenged Specific mortality Survival (%) RPS
20 0 100 100
20 9 55 30.7
20 13 35 e
carp than with the free cell vaccine [16]. The LD50values were also enhanced in biofilm vaccinated common carp measuring 107.67 compared to 106.3 and 106.0 for FC and control respectively. The higher protection here is in contrast to poor or variable protection with oral vaccines obtained in carnivorous fish such as salmonids [2,3], channel catfish [4], plaice [35] and ayu [36]. Improved protection and antibody response in the catfish further support the concept of biofilm vaccine as an effective oral delivery method. There exists a difference in protective immunity with free cell vaccine as studied in carp [16,18] and the present study. Protection with FC is lower in catfish than in carp. Therefore, BF vaccine has more relevance for oral vaccination of carnivorous fish. It appears that differences exists in degradation, uptake and processing of antigen in carp and catfish as indirectly indicated by the antibody response and protection level. Degradation of antigen in the gut is likely to be more in carnivorous catfish. Furthermore, the extent of proteolysis in a carnivorous gut is many times higher than that in a herbivore and is influenced by the secretory rates of HCl and pepsin, food retention times, stomach distensibility, food bulk, specific activity of pepsin and the temperature [37]. Thus the orally introduced antigens are more vulnerable to alteration in a carnivore gut than that in a herbivore. Assessment of immune status after oral vaccination may not be complete by restricting measurement to serum parameters. Immunity assessment needs to be carried out at the integumental/entero-mucosal surfaces considered as the primary sites of pathogen attack and entry. Moreover, the interaction of fish gut associated lymphoid tissue (GALT) and oral vaccines needs to be evaluated in terms of eliciting secretory as well as systemic responses to an enteric pathogen. The concept of biofilm oral vaccination, besides having application in aquaculture, can have potential for oral prophylaxis for enteric diseases in veterinary and human medicine.
Acknowledgements Authors acknowledge the funding support from International Foundation for Science, Sweden. DKN thanks Indian Council of Agricultural Research, New Delhi for Junior Research Fellowship and AA thanks Council of Scientific and Industrial Research, New Delhi for a Research Associateship.
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References [1] Quentel C, Vegneulle M. Antigen uptake and immune responses after oral vaccination. In: Gudding R, Lillehaug A, Midtlying P, Brown F, editors. Fish vaccinology. Developments in Biological Standards 1997;90:69e78. Basel: Karger. [2] Duff DCB. The oral immunisation of trout against Bacterium salmonicida. J Immunol 1942;44:87e94. [3] Post G. Response of rainbow trout (Salmo gairdneri) to antigens of Aeromonas hydrophila. J Fish Res Board Can 1966;23:1487e94. [4] Schachte JH. Immunisation of channel catfish, Ictalurus punctatus against two bacterial diseases. Mar Fish Rev 1978;40:18e24. [5] Spence KD, Fryer JL, Pilcher KS. Active and passive immunization of certain salmonid fishes against Aeromonas salmonicida. Can J Microbiol 1965;11:397e405. [6] Fryer JL, Rohovec JS, Tebbit GL, Michael JS, Pilcher KS. Vaccination for control of infectious diseases in Pacific salmon. Fish Pathol 1976;10:155e64. [7] Fryer JL, Rohovec JS, Garrison RL. Immunization of salmonids for control of vibriosis. Mar Fish Rev 1978;40:20e3. [8] Amend DF, Johnson KA. Current status and future needs of Vibrio anguillarum bacterin. Dev Biol Stand 1981;49:403e17. [9] Rombout JHWM, Lamers CHJ, Helfrich MH, Dekker A, Taverne-Thiele JJ. Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications. Cell Tissue Res 1985;239:519e30. [10] Davina JHM, Parmentier HK, Timmermans LPM. Effect of oral administration of Vibrio-bacterin on the intestine of cyprinid fish. Dev Comp Immunol 1982;2:157e66. [11] Johnson KA, Amend DF. Efficacy of Vibrio anguillarum and Yersinia ruckeri bacterins applied by oral and anal intubation of salmonids. J Fish Dis 1983;6:473e6. [12] Rombout JHWM, Blok LJ, Lamers CHJ, Egberts E. Immunization of carp with Vibrio anguillarum bacterin: Indications for a common mucosal immune system. Dev Comp Immunol 1986;10:341e51. [13] Piganelli JD, Zhang JA, Christensen JM, Kaattari SL. Enteric coated microspheres as an oral method for antigen delivery to salmonids. Fish Shellfish Immunol 1994;4:179e88. [14] Polk AE, Amsden B, Scarrat DJ, Gonzal A, Okahmafe AO, Goosen MFA. Oral delivery in aquaculture: Controlled release of protein from chitosan-alginate microcapsules. Aquacult Eng 1994;13:311e23. [15] Dalmo RA, Leifson RM, Bogwald J. Microspheres as antigen carriers: Studies on intestinal absorption and tissue localisation of polystyrene microspheres in Atlantic salmon, Salmo salar L. J Fish Dis 1995;18:87e91. [16] Azad IS, Shankar KM, Mohan CV. Evaluation of an Aeromonas hydrophila biofilm for oral vaccination of carp. In: Flegel TW, Macrae IH, editors. Diseases in Asian aquaculture III. Manila: Fish Health Section, Asian Fisheries Society; 1997. p. 181e6. [17] Azad IS, Shankar KM, Mohan CV, Kalita B. Biofilm vaccine of Aeromonas hydrophiladstandardisation of dose and duration for oral vaccination of carps. Fish Shellfish Immunol 1999;9:519e28. [18] Azad IS, Shankar KM, Mohan CV, Kalita B. Protective response of common carp orally vaccinated with biofilm and free cells of Aeromonas hydrophila challenged by injection and immersion routes. J Aquacult Tropics 1999;15:65e70. [19] Azad IS, Shankar KM, Mohan CV, Kalita B. Uptake and processing of biofilm and free cell vaccines of Aeromonas hydrophila in Indian major carps and common carp following oral vaccination-antigen localization by a monoclonal antibody. Dis Aquat Organ 2000;43:103e8. [20] Lewis WM, Bender M. Free-living ability of a warm-water fish pathogen of the genus Aeromonas and factors contributing to its infection of the golden shiner. Prog Fish Culturists 1961;23:124e6. [21] Roberson BS. Bacterial agglutination. In: Stolen JS, Fletcher GC, Anderson DP, Roberson BS, van Muiswinkel WB, editors. Techniques in fish immunology. Fair Haven (NJ): SOS Publications; 1990. p. 137e54. [22] Amend DF. Potency testing of fish vaccines. Dev Biol Stand 1981;49:447e54. [23] Alikunhi KH. Fish culture in India. Farming Bulletin No. 20. New-Delhi: ICAR, 1957, p. 144. [24] Sidthimunka A, Sanglert J, Pawapootanon O. The culture of catfish (Clarias spp.) in Thailand. FAO Fish Rep 1968;44:196e204. [25] Hora SL, Pillay TVR. Hand book on fish culture in the Indo-Pacific region. FAO Fish Biol Tech Pap 1962;14:203. [26] Chaudhuri H. Culture of air-breathing fishes. First workshop report on the All India Coordinated Research Project on culture of air-breathing fishes: Cuttack; 1971. 6 pp. [27] Varghese TJ, Devraj KV, Rao GPS. Food of juvenile of the catfish, Clarias batrachus (Lin.). J Inland Fish Soc India 1973;5:78e81. [28] Lehri GK. The walking catfish. Sci Rep 1971;8:586e7. [29] Costerton JW, Irvin RT, Cheng KJ. The bacterial glycocalyx in nature and disease. Annu Rev Microbiol 1981;35:299e324. [30] Lamers CHJ, de Haas MJM, van Muiswinkel WB. Humoral response and memory formation in carp after injection of Aeromonas hydrophila bacterin. Dev Comp Immunol 1985;9:65e75. [31] Press CM, Evensen O, Reitan LJ, Landsverk T. Retention of furunculosis vaccine components in Atlantic salmon, Salmo salar L. following different routes of vaccine administration. J Fish Dis 1996;19:215e24. [32] Kaattari SL. Development of a piscine paradigm of immunological memory. Fish Shellfish Immunol 1994;4:447e57.
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[33] Yin Z, Lam TJ, Sin YM. The role of specific antiserum of catfish, Clarias gariepinus, as a defence against Aeromonas hydrophila. Fish Shellfish Immunol 1996;6:57e69. [34] Sinyakov MS, Dror M, Zhevelev HM, Margel S, Avtalion RR. Natural antibodies and their significance in active immunization and protection against a defined pathogen in fish. Vaccine 2002;20:3668e74. [35] Fletcher TC, White A. Antibody production in the plaice (Pleuronectes platessa L.) after oral and parenteral immunisation with Vibrio anguillarum antigens. Aquaculture 1973;1:417e28. [36] Kawai K, Kusuda R, Itami T. Mechanism of protection in ayu orally vaccinated for vibriosis. Fish Pathol 1981;15:257e62. [37] Ash R. Protein digestion and absorption. In: Cowey CB, Mackie AM, Bell GR, editors. Nutrition and feeding in fish. Florida: Academic Press; 1985. p. 69e93.
Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachusda carnivore model D.K. Nayak, A. Asha, K.M. Shankar), C.V. Mohan Department of Aquaculture, Fish Pathology and Biotechnology Laboratory, College of Fisheries, University of Agricultural Sciences, Mangalore 575 002, India Received 23 June 2003; accepted 29 September 2003
Abstract Biofilm of Aeromonas hydrophila was evaluated for oral vaccination of walking catfish (Clarias batrachus L.). Fish were fed with fish paste incorporating biofilm (BF) or free cells (FC) of A. hydrophila for 20 days and monitored for serum antibody production up to 60 days post-vaccination. Serum agglutinating antibody titre and relative percent survival (RPS) following challenge were found to be significantly higher in catfish fed with BF vaccine compared to that with FC. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Biofilm vaccine; Aeromonas hydrophila; Clarias batrachus
1. Introduction Oral vaccination is regarded beneficial in aquaculture as it is non-stressing and accessible to fish of any size, age and number [1]. The very first attempt of oral vaccination in trout against furunculosis was highly encouraging [2]. However, the later attempts were either unsuccessful [3,4] or produced variable results [5e8]. One of the suggested reasons for these poor and inconsistent responses to oral vaccination was the gastric destruction of orally introduced antigens before they could reach and be absorbed into the immune responsive areas of the hindgut [9]. Strategies developed for improvements of oral vaccines have attempted to avoid this gastric destruction [10e12] especially by the use of encapsulated antigen microspheres [13e15]. Our laboratory has developed a biofilm of Aeromonas hydrophila to facilitate improved antigen delivery in oral vaccination of fish. It induced significantly higher humoral and protective responses in Indian carps compared to free cell vaccine of the pathogen [16e18]. Studies on retention of biofilm (BF) and free cell (FC) vaccines in the gut lumen and their uptake and processing have been carried out by antigen localisation employing monoclonal antibody [19]. Biofilm vaccine was retained in larger quantities than
) Corresponding author. E-mail address: kalkulishankar@rediffmail.com (K.M. Shankar). 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2003.09.012
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that of free cell vaccine and for a longer duration in the tissues of foregut, hindgut, spleen and kidney after a single oral delivery. The evaluation of biofilm vaccine remains incomplete without supporting data from cultured carnivorous species, which possess a stomach with a quite different anatomy and digestive physiology. In general, reports of oral vaccination trials in carnivorous fishes are fewer. Culture of carnivorous fishes such as clarids, snakeheads and sea bass is becoming popular in India and elsewhere in Asia. The walking catfish, Clarias batrachus, represents a high priced food fish and a great deal of attention is now being paid for popularisation of its mass culture. By virtue of its preferential habitat in bottom zones of swampy waters, where the bacterial population may be 10e20 times higher than in the water column [20] it might be at a higher risk of getting infected when cultured at high stocking densities as well as in composite cultures. In this communication, we present successful oral immunisation of C. batrachus with a biofilm vaccine of A. hydrophila.
2. Materials and methods 2.1. Bacterial isolate Pure culture of a virulent A. hydrophila (AAH 2/96) isolated from ulcerated Clarias sp. was maintained in sterile Tryptic Soya Broth (TSB) (Hi Media, India) with 15% glycerol at 20 (C. For use, the culture was revived on Nutrient Agar (NA) (2.8% w/v, Hi Media) slants at room temperature and subsequently stored at 4 (C till used. 2.2. Preparation of biofilm and free cells of A. hydrophila Preparation of biofilm cells of A. hydrophila was according to Azad et al. [16]. In brief, A. hydrophila was promoted to grow as biofilm on chitin flakes provided as substrate (0.3% w/v) in TSB (0.225% w/v). Chitin with 4-day-old biofilm cells on it was harvested for use as biofilm vaccine. For free cells, 1-day-old culture grown in TSB (1.5% w/v) was centrifuged at 3000 g for 10 min, washed thrice with sterile phosphate buffered saline (PBS 0.01 M, pH 7.2) and then resuspended in PBS to the desired optical density (O.D.) at 540 nm. Biofilm and free cells were heat inactivated at 100 (C for 50 min and 90 (C for 10 min, respectively and incorporated into a minced fish paste, prepared from minced fish meat (70%) blended with wheat flour (30%) to constitute biofilm vaccine (BF) and free cell vaccine (FC). A control fish paste (C) was prepared without biofilm or free cells but with PBS (pH 7.2). All vaccine incorporated and control fish pastes were stored at 20 (C before use. 2.3. Fish stock maintenance Catfish, C. batrachus (25e35 cm, 140e260 g) were collected from the local fish market and acclimatised in fibreglass tanks (1!1!0:5 m) filled with stored ground water (26 (C) (30G2 cm deep). For the first 30 days of acclimatisation, water was replenished to the extent of 80e90% on each alternate day and reduced to 50% thereafter. Fish were fed with the minced fish paste at the rate of 3% body weight per day. Initially, as the fish did not accept feed readily, they were force fed employing a wide mouth 2 ml syringe for 30 d. Once acclimatised, the fish paste was offered as feed balls (8e10 mm dia) dispensed into the water. Feeding was done once a day between 16.30 and 17.30 h. Holding tanks and fish were surface disinfected with KMnO4(10 ppm) for 5 min once a week.
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2.4. Oral immunisation of fish After 30 days of acclimatisation, fish were fed once a day with fish paste incorporating the BF and FC vaccine at 4:29!1010 and 6:79!1010 bacterial cells (g fish)1d1, respectively for a period of 20 d. More than 150 fish were assigned to each treatment group. 2.5. Estimation of agglutinating antibody titre After completion of the 20 day vaccination, serum samples were collected from the 0 day postvaccination (dpv) i.e. 20th day of vaccination, onwards up to 60 dpv at 10 day intervals. Fish were anaesthetised and bled from their caudal veins with non-heparinised disposable syringes. Blood was allowed to clot at room temperature for 5 min, followed by 1 h at 4 (C. The clot free serum was separated following centrifugation at 3000 g for 10 min and stored at 20 (C. Antibody agglutination titre against the homologous isolate of A. hydrophila was determined for each fish by a modified agglutination assay [21]. Briefly, 90 ml of immune/control serum was serially diluted with 50 ml PBS pH 7.2 (dilution factor 1.55) and then 50 ml of heat-inactivated free cell suspension of A. hydrophila (OD 0.7 at 540 nm, approx. 109cfu/ml) was added. Plates were incubated at room temperature overnight prior to microscopic examination (!40) for agglutination. Antibody titre was expressed as the reciprocal of the highest dilution of serum that was positive for agglutination. Comparison of agglutinating antibody titre was done through a 3-way ANOVA followed by Duncan’s multiple range test. A value of P ! 0:05 was considered to be significant. 2.6. Estimation of protection Orally immunised catfish from BF, FC and control groups were divided into three challenge groups in separate tanks and injected intramuscularly (0.5 ml) with 24 h old live free cells of A. hydrophila (107cfu g1fish) on the 60th day post vaccination (dpv) to assess the overall protection. Appearance of gross external lesions and mortality pattern was monitored for 7 days. From the freshly dead and moribund specimens, whole blood and kidney inoculants were aseptically plated on nutrient agar and Aeromonas selective medium (Hi Media) to confirm specific cause of death and morbidity. Relative percent survival (RPS) was calculated according to Amend [22].
3. Results and discussion In commercial aquaculture, oral vaccination is the most preferred means to immunise fish of all size and age groups with least stress. However, poor responses to oral vaccines [3e8], possibly due to destruction of antigenic epitopes [9], pose major hindrance for developing an effective oral vaccine. Oral vaccination of Indian major carps (catla, rohu) and common carp with biofilm of A. hydrophila was highly successful in eliciting antibody titre and protection [16e18]. Carps are usually planktophagous and lack a distinct stomach. Therefore, the current study was conducted in order to evaluate the performance of the biofilm vaccine in a carnivorous fish model, C. batrachus. Dietary habits of the catfish have been described as predator but not piscivore [23], scavenger [24], omnivore [25], insectivore [26], larvivore [27], with great affinity for a carnivorous diet [28]. More or less it shows preference for food of animal origin and possesses a well-demarcated stomach unlike carp. Kinetics of the catfish serum antibody response to BF and FC oral vaccines is presented in Fig. 1. The duration of oral vaccination was 20 days and the antibody response was monitored from 0 dpv, i.e. the 20th day since the beginning of daily immunisation. The biofilm-vaccinated fish had significantly higher
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Fig. 1. Agglutinating antibody response in catfish, control (C) and biofilm (BF) and free cell (FC) vaccine. Titre values are the highest serum dilutions (mean with SE, n ¼ 15) causing agglutination. 0 day post vaccination corresponds to the 20th day of vaccination.
(P ! 0:05) antibody titre than those vaccinated with free cells on all days post-vaccination (dpv). The difference between BF and FC is quite distinct even at 0 dpv. Overall, orally vaccinated catfish produced higher antibody titres compared to unvaccinated controls. Similar trends of higher systemic agglutinating antibody response to biofilm oral vaccine have been previously reported in agastric carpsdcatla, rohu and common carp [16,17]. Biofilm glycocalyx, chemically a polymer of neutral hexoses [29] encapsulates and possibly protects cells/antigens in the stomach/foregut [17] and thus provides a large pool of antigens to lymphoid organs compared to FC. This property of biofilm vaccine is reported to facilitate longer retention of antigens in the gut and lymphoid tissue [19] and hence, might have resulted in the early and heightened primary antibody response. The magnitude of humoral response in fish appears to be dependent on the nature, dose and presentation route of antigen. High dose priming is normally believed to elicit higher humoral response and formation of memory in carp [30] and the extent of immune memory response in fish depends on the route and dose of primary immunisation and persistence of antigens [31]. Availability of antigens in large quantities with BF vaccine due to the protective nature of the glycocalyx, might have facilitated its protracted presence in the lymphoid organs. As fish do not possess physiologically distinct memory cells, rather it appears to rely upon the increasing size of the pools of antigen sensitive cells [32], vaccination with biofilm may induce long term memory response helping the fish to fight the disease for a longer duration. Lower antibody response to FC vaccine might be due to the destructive processes in the catfish gut with acid secretion and activity of acidic proteolytic enzymes. The unvaccinated control groups showed some titre values even though significantly lower. This might be the background level reaction corresponding to the occurrence of natural agglutinin in fish serum [33,34]. There was an elevated level of protection in biofilm vaccinated catfish to the homologous injection challenge (Table 1). A. hydrophila was reisolated from all the moribund and dead fish. Protection was higher with BF than with FC. The unvaccinated control group registered 24e35% survival. Biofilm vaccinated (BF) fish were fully protected with 93e100% survival while those vaccinated with free cell (FC) had only 46e56% survival. The protective immunity calculated as relative percent survival (RPS) was higher for BF than FC. Higher and complete protection in BF vaccinated catfish compared to that with FC vaccine observed here, is consistent with the findings of Azad et al. [18] in omnivore common carp. In their experimental conditions, common carp fed with biofilm vaccine (1010 bacterial cells/fish/day) for a period of 15 days recorded RPS of 100% to both immersion and injection challenge, while the values for free cell vaccine were 76.46 and 80.86% to both these challenge routes, respectively. The percentage of survival assessed to homologous injection challenge was also higher in biofilm-vaccinated catla, rohu and common
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D.K. Nayak et al. / Fish & Shellfish Immunology 16 (2004) 613e619 Table 1 Protection level in vaccinated catfish challenged with A. hydrophila at 107CFU (g fish)1 BF vaccine
FC vaccine
Control
Challenge-1 No. of fish challenged Specific mortality Survival (%) RPS
25 0 100 100
25 11 56 42.1
25 19 24 e
Challenge-2 No. of fish challenged Specific mortality Survival (%) RPS
29 2 93.1 90.8
30 16 46.6 28.8
28 21 25 e
Challenge-3 No. of fish challenged Specific mortality Survival (%) RPS
20 0 100 100
20 9 55 30.7
20 13 35 e
carp than with the free cell vaccine [16]. The LD50values were also enhanced in biofilm vaccinated common carp measuring 107.67 compared to 106.3 and 106.0 for FC and control respectively. The higher protection here is in contrast to poor or variable protection with oral vaccines obtained in carnivorous fish such as salmonids [2,3], channel catfish [4], plaice [35] and ayu [36]. Improved protection and antibody response in the catfish further support the concept of biofilm vaccine as an effective oral delivery method. There exists a difference in protective immunity with free cell vaccine as studied in carp [16,18] and the present study. Protection with FC is lower in catfish than in carp. Therefore, BF vaccine has more relevance for oral vaccination of carnivorous fish. It appears that differences exists in degradation, uptake and processing of antigen in carp and catfish as indirectly indicated by the antibody response and protection level. Degradation of antigen in the gut is likely to be more in carnivorous catfish. Furthermore, the extent of proteolysis in a carnivorous gut is many times higher than that in a herbivore and is influenced by the secretory rates of HCl and pepsin, food retention times, stomach distensibility, food bulk, specific activity of pepsin and the temperature [37]. Thus the orally introduced antigens are more vulnerable to alteration in a carnivore gut than that in a herbivore. Assessment of immune status after oral vaccination may not be complete by restricting measurement to serum parameters. Immunity assessment needs to be carried out at the integumental/entero-mucosal surfaces considered as the primary sites of pathogen attack and entry. Moreover, the interaction of fish gut associated lymphoid tissue (GALT) and oral vaccines needs to be evaluated in terms of eliciting secretory as well as systemic responses to an enteric pathogen. The concept of biofilm oral vaccination, besides having application in aquaculture, can have potential for oral prophylaxis for enteric diseases in veterinary and human medicine.
Acknowledgements Authors acknowledge the funding support from International Foundation for Science, Sweden. DKN thanks Indian Council of Agricultural Research, New Delhi for Junior Research Fellowship and AA thanks Council of Scientific and Industrial Research, New Delhi for a Research Associateship.
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