以口服疫苗取代注射疫苗，誘發石斑魚體液性免疫反應，不但能節省疫苗注射時所需的人力與時間，並能降低注射造成的緊迫，提升存活率。口服奈米疫苗能誘發哺乳類動物體液性免疫反應，本研究探討口服奈米疫苗是否亦能在魚類誘發體液性免疫反應。研究以微量中和試驗(microneutralization assay)檢測出石斑虹彩病毒不活化病毒疫苗(grouper iridovirus vaccine)，可產生具有抑制虹彩病毒及保護細胞不受病毒破壞能力的抗體(Anti-GIV)，再以此抗體選擇親合力較佳之兩段蛋白質片段當作抗原。根據親合力選擇二段20 a.a.長度之虹彩病毒外鞘蛋白片段並結合牛血清蛋白成為免疫原(immunogen)，並以聚氰基丙烯酸(poly- alkylcyanoacrylate)包覆成粒徑小於200奈米膠囊，另加枯草桿菌(Bacillus subtilis)為佐劑，灌食點帶石斑並檢測血液中之抗體力價。實驗結果顯示口服投餵虹彩病毒外鞘蛋白合成胜肽之奈米膠囊，能夠誘發點帶石斑體液性免疫反應，產生石斑虹彩病毒外鞘蛋白合成胜肽片段之抗體，抗體力價隨著投餵次數增加而增強。顯示這項技術有潛力成為製備養殖魚類口服病毒疫苗之基礎。

To replace invasive vaccination by oral vaccination can reduce labor and time cost and improve fish survival rate owing to reduced stress on fish. In mammals, it has been shown that oral nano-vaccine can induce humoral immune response. The objective of this study was to investigate whether oral nano-vaccine can similarly induce humoral immune response in grouper (Epinephelus coioides). The experiment protocol include: (1) development of grouper anti-GIV by GIV vaccine, this antibody was confirmed effectively against GIV by micro-nutralization assay (2) selection of possible peptide fragments from iridovirus capsid protein for oral vaccine manufacturing based on their affinity to anti-GIV (3) two fragments of synthetic peptide (20 a.a. length) conjugated to BSA carrier and encapsulated by PACA to capsule size less than 200 nm (4) nanocapsule were oral-administered to grouper with Bacillus subtilis as adjuvant (5) Anti-synthetic peptide titer were determined by dot blot assay. The results showed: oral-administered nanocapsule of synthetic peptide of grouper iridovirus capsid protein can effectively induce humoral immune response in groupers and the antibody titer increased over time after each additional booster. This provides a promising opportunity for future development of oral virus vaccine technology in aquaculture.

目錄
章次
壹、緒論+1
貳、材料方法+9
一、實驗材料+9
1. 實驗用魚+9
2. 實驗藥品+9
二、實驗方法+10
1. 陽性品質管制(Positive Quality Control, PQC)血漿樣本+10
2. 點漬法(Dot blot)+11
3. 石斑虹彩病毒外鞘蛋白合成胜肽製備+12
4. 奈米顆粒製備+12
5. 實驗設計+13
6. 分析與統計方法+14
參、結果+15
1. Anti-GIV以micro-nutralization檢測+15
2. 點漬法陽性品質管制血清樣本+15
3. 石斑虹彩病毒外鞘蛋白合成胜肽之製備及檢測+16
4. 口服合成石斑虹彩病毒外鞘蛋白胜肽奈米膠囊活體實驗+16
肆、討論+18
伍、參考文獻+24
表次
表一、以微量中和試驗(micro-neutralization assay)檢測Anti-GIV血漿樣本中抗體力價之變化表+39
圖次
圖一、Anti-GIV與石斑虹彩病毒外鞘蛋白合成胜肽(SP1、SP0)親合力之比較+40
圖二、Anti-GIV與石斑虹彩病毒外鞘蛋白合成胜肽(SP1、SP2、SP3、SP4)親合力之比較+41
圖三、陽性品質管制血漿樣本點漬圖+42
圖四、口服SP3-BSA奈米膠囊活體實驗血漿中抗SP3-BSA之點漬圖+43
圖五、口服SP4-BSA奈米膠囊活體實驗血漿中抗SP4-BSA之點漬圖+44
圖六、口服SP4-Acrylo奈米膠囊活體實驗血漿中抗SP4-BSA之點漬圖+45
圖七、投餵SP3-BSA、SP4-BSA、SP4-Acrylo奈米膠囊，對抗體力價影響之比較+46
圖八、SP4胜肽片段結合不同的載體蛋白，對抗體力價影響之比較+48
圖九、BSA結合不同序列片段(SP3、SP4)，對抗體力價影響之比較+49

林建邦，2013。口服蛋白質奈米膠囊誘發點帶石斑魚體液性免疫反應之效果。
國立中山大學海洋生物研究所碩士論文。
Alpar, H. O., W. N. Field, R. Hyde, and D. A. Lewis. 1989. The transport of microspheres from the gastro-intestinal tract to inflammatory air pouches in the rat. Journal of Pharmacy and Pharmacology 41: 194-196.
Amanna, I. J., N. E. Carlson, and M. K. Slifka. 2007. Duration of humoral immunity to common viral and vaccine antigens. New England Journal of Medicine 357: 1903-1915.
Askonas, B. A., and J. Humphrey. 1958. Formation of specific antibodies and y-globulin in vitro. A study of the synthetic ability of various tissues from rabbits immunized by different methods. Biochemical Journal 68: 252–261.
Askonas, B. A., and R. White. 1956. Sites of antibody production in the guinea-pig. The relation between in vitro synthesis of anti-ovalbumin and γ-globulin and distribution of antibody-containing plasma cells. British Journal of Experimental Pathology 37: 61-74.
Barnes, A. G., V. Cerovic, P. S. Hobson, and L. S. Klavinskis. 2007. Bacillus subtilis spores: a novel microparticle adjuvant which can instruct a balanced Th1 and Th2 immune response to specific antigen. European Journal of Immunology 37: 1538-1547.

Brandt, E. R., K.S. Sriprakash, R. I. Hobb, W. A. Hayman, W. Zeng, M. R. Batzloff, D. C. Jackson, and M. F. Good.. 2000. New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population. Nature Medicine 6: 455-459.
Briand, J.P., S. Muller, S. Plaué, and M. H. V. van-Regenmorte. 1988. Synthetic polypeptides as antigens. Laboratory Techniques in Biochemistry and Molecular Biology 19: 1-227
Cáceres, V. M., and R. W. Sutter. 2001. Sabin monovalent oral polio vaccines: review of past experiences and their potential use after polio eradication. Clinical Infectious Diseases 33: 531-541.
Carcaboso, A. M., R. M. Hernández, M. Igartua, A. R. Gascón, J. E. Rosas, M. E. Patarroyo, and J. L. Pedraz. 2003. Immune response after oral administration of the encapsulated malaria synthetic peptide SPf66. International Journal of Pharmaceutics 260: 273-282.
Carter, J. M. 1996. Conjugation of peptides to carrier proteins via glutaraldehyde. The Protein Protocols Handbook. Springer 1996: 679-687
Cheng, C. A., J. A. John, M. S. Wu, C. Y. Lee, C. H. Lin, C. H. Lin, and C. Y. Chang. 2006. Characterization of serum immunoglobulin M of grouper and cDNA cloning of its heavy chain. Veterinary Immunology and Immunopathology 109: 255-265.
Clem, L. W. 1971. Phylogeny of immunoglobulin structure and function IV. immunoglobulins of the giant grpuper (Epinephelus itaira). The Journal of Biological Chemistry 246: 9-15.
Couvreur, P., B. Kante, M. Roland, P. Guiot, P. Bauduin, and P. Speiser. 1979. Polycyanoacrylate nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties. Journal of Pharmacy and Pharmacology 31: 331-332.
Cui, M., Q. Zhang, Z. Yao, Z. Zhang, H. Zhang, and Y. Wang. 2010. Immunoglobulin M gene expression analysis of orange-spotted grouper, Epinephelus coioides, following heat shock and Vibrio alginolyticus challenge. Fish and Shellfish Immunology 29: 1060-1065.
Damgé, C., C. Michel, M. Aprahamian, and P. Couvreur. 1988. New approach for oral administration of insulin with polyalkylcyanoacrylate nanocapsules as drug carrier. Diabetes 37: 246-251.
Damge, C., C. Michel, M. Aprahamian, P. Couvreur, and J. Devissaguet. 1990. Nanocapsules as carriers for oral peptide delivery. Journal of Controlled Release 13: 233-239.
Damge, C., H. Vranckx, P. Balschmidt, and P. Couvreur. 1997. Poly (alkyl cyanoacrylate) nanospheres for oral administration of insulin. Journal of Pharmaceutical Sciences 86: 1403-1409.
DeLisi, C., and J. A. Berzofsky. 1985. T-cell antigenic sites tend to be amphipathic structures. Proceedings of the National Academy of Sciences 82: 7048-7052.
Des R. A., V. Fievez, M. Garinot, Y. J. Schneider, and V. Préat. 2006. Nanoparticles as potential oral delivery systems of proteins and vaccines: A mechanistic approach. Journal of Controlled Release 116: 1-27.
Dhar, A. K., and F. C. T. Allnutt. 2011. Challenges and opportunities in developing oral vaccines against viral diseases of fish. Journal of Marine Science: Research and Development 1: 1-6.
DiMarchi, R., G. Brooke, C. Gale, V. Cracknell, T. Doel, and N. Mowat. 1986. Protection of cattle against foot-and-mouth disease by a synthetic peptide. Science 232: 639-641.
Driks, A. 1999. Bacillus subtilis spore coat. Microbiology and Molecular Biology Reviews 63: 1-20.
Elliott, W. L., C. J. Stille, L. J. Thomas, and R. E. Humphreys. 1987. An hypothesis on the binding of an amphipathic, alpha helical sequence in Ii to the desetope of class II antigens. The Journal of Immunology 138: 2949-2952.
Emini, E. A., B. A. Jameson, and E. Wimmer. 1985. Antigenic structure of poliovirus. Immunochemistry of Viruses. Elsevier. Amsterdam: 281-294.
Emini, E. A., B. A. Jameson, and E. Wimmer. 1982. Priming for and induction of anti-poliovirus neutralizing antibodies by synthetic peptides. Nature 304: 699-703.
Emini, E. A., B. A. Jameson, and E. Wimmer. 1984. Identification of a new neutralization antigenic site on poliovirus coat protein VP2. Journal of Virology 52: 719-721.
Fehling, H., W. Swat, C Laplace, R. Kühn, K. Rajewsky, U. Müller, and H. von Boehmer. 1994. MHC class I expression in mice lacking the proteasome subunit LMP-7. Science 265: 1234-1237.
Feng, L.N., D. Q. Lu, J. X. Bei, J. L. Chen, Y. Liu, Y. Zhang, X. C. Liu, Z. N. Meng, L. Wang, and H. R. Lin.. 2009. Molecular cloning and functional analysis of polymeric immunoglobulin receptor gene in orange-spotted grouper (Epinephelus coioides). Comparative Biochemistry and Physiology, Part B 154: 282-289.
Florence, A. T., A. M. Hillery, N. Hussain, and P. U. Jani. 1995. Nanoparticles as carriers for oral peptide absorption: studies on particle uptake and fate. Journal of Controlled Release 36: 39-46.
Foo, D. G. W., S. Alonso, M. C. Phoon, N. P. Ramachandran, V. T. Chow, and C. L. Poh. 2007. Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides. Virus Research 125: 61-68.
Frangione-Beebe, M., B. Albrecht, N. Dakappagari, R. T. Rose, C. L. Brooks, S. P. Schwendeman, M. D. Lairmore, and P. T. Kaumaya. 2000. Enhanced immunogenicity of a conformational epitope of human T-lymphotropic virus type 1 using a novel chimeric peptide. Vaccine 19: 1068-1081.
French, V. I., J. Stark, and R. White. 1970. The influence of adjuvants on the immunological response of the chicken: II. Effects of Freund's complete adjuvant on later antibody production after a single injection of immunogen. Immunology 18: 645.
Freund, J., J. Casals, and E. P. Hosmer. 1937. Sensitization and antibody formation after injection of Tubercle bacilli and paraffin oil. Experimental Biology and Medicine 37: 509-513.

Freund, J., E. M. Schryver, M. B. McGuiness, and M. B. Geitner. 1952. Diphtheria antitoxin formation in the horse at site of injection of toxoid and adjuvants. Experimental Biology and Medicine 81: 657-658.
Ge, W., Y. Li, Z. S. Li, S. H. Zhang, Y. J. Sun, P. Z. Hu, X. M. Wang, Y. Huang, S. Y. Si, X. M. Zhang, and Y. F. Sui. 2009. The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following peroral administration route. Cancer Immunology, Immunotherapy 58: 201-208.
Hart, S., A. Wrathmell, and J. Harris. 1986. Gut associated lymphoid tissue in the dogfish Scyliorhinus canicula: a light microscopic study. Journal of the Marine Biological Association of the United Kingdom 66: 721-729.
Henriques, A. O., and C. P. Moran Jr. 2000. Structure and assembly of the bacterial endospore coat. Methods 20: 95-110.
Hillyer, J. F., and R. M. Albrecht. 2001. Gastrointestinal persorption and tissue distribution of differently sized colloidal gold nanoparticles. Journal of Pharmaceutical Sciences 90: 1927-1936.
Hoa, N. T., L. Baccigalupi, A. Huxham, A. Smertenko, P. H. Van, S. Ammendola, E. Ricca, and A.S. Cutting. 2000. Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinl disorders. Applied and Environmental Microbiology 66: 5241-5247.


Hodges, G. M., E. A. Carr, R. A. Hazzard, C. O'reilly, and K. E. Carr. 1995. A commentary on morphological and quantitative aspects of microparticle translocation across the gastrointestinal mucosa. Journal of Drug Targeting 3: 57-60.
Hong, H. A., L. H. Duc, and S. M. Cutting. 2005. The use of bacterial spore formers as probiotics. Federation of European Microbiological Societies. Microbiology Reviews 29: 813-835.
Hossain, M., J. Y. Song, S. I. Kitamura, S. J. Jung, and M. J. Oh. 2008. Phylogenetic analysis of lymphocystis disease virus from tropical ornamental fish species based on a major capsid protein gene. Journal of Fish Diseases 31: 473-479.
Huang, S. M., C. Tu, C. H. Tseng, C. C. Huang, C. C. Chou, H.C. Kuo, and S. K. Chang. 2011. Genetic analysis of fish iridoviruses isolated in Taiwan during 2001-2009. Archives of Virology 156: 1505-1515.
Huguette, P. A., A. Malam, J. Danielle, C. Patrick, and V. Christine. 2003. Visualization of insulin-loaded nanocapsules: in vitro and in vivo studies after oral administration to rats. Pharmaceutical Research 20: 1071-1084.
Hunter, R. L., M. R. Olsen, and B. Bennett. 1995. Copolymer adjuvants and Titermax. John Wiley & Sons 1995: 51-94
Husby, S., J. Mestecky, Z. Moldoveanu, S. Holland, and C. O. Elson. 1994. Oral tolerance in humans. T cell but not B cell tolerance after antigen feeding. The Journal of Immunology 152: 4663-4670.

Jameson, B. A., and H. Wolf. 1988. The antigenic index: a novel algorithm for predicting antigenic determinants. Computer Applications in the Biosciences 4: 181-186.
Janet, M., Thornton, and B. L. Sibanda. 1983. Amino and carboxy-terminal regions in globular proteins. Journal of Molecular Biology 167: 443-460.
Jung, T., W. Kamm, A. Breitenbach, E. Kaiserling, J. X. Xiao, and T. Kissel. 2000. Biodegradable nanoparticles for oral delivery of peptides: is there a role for polymers to affect mucosal uptake? European Journal of Pharmaceutics and Biopharmaceutics 50: 147-160.
King, A. M., M. J. Adams, E. B. Carstens, and E. J. Lefkowitz. 2012. Virus taxonomy: classification and nomenclature of viruses : ninth report of the international committee on taxonomy of viruses. Elsevier 9: 1-1327.
Kai, Y., H. Su, K. Tai, and S. Chi. 2010. Vaccination of grouper broodfish (Epinephelus tukula) reduces the risk of vertical transmission by nervous necrosis virus. Vaccine 28: 996-1001.
Karplus, P. A., and G. E. Schulz. 1985. Prediction of chain flexibility in proteins. Naturwissenschaften 72: 212-213.
Klaus, G. G. B., and A. M. Cross. 1974. The influence of epitope density on the immunological properties of hapten-protein conjugates: I. Characteristics of the immune response to hapten-coupled albumen with varying epitope density. Cellular Immunology 14: 226-241.
Kreuter, J. 1983. Evaluation of nanoparticles as drug-delivery systems. III: materials, stability, toxicity, possibilities of targeting, and use. Pharmaceutica Acta Helvetiae 58: 242-240.
Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157: 105-132.
Leonard, F. 1966. Medical applications of plastics. Journal of Dental Research 45: 1657-1659.
Li, F. L., Z. X. Li, Y. X. Zhai, and M. M. Guo. 2012. Adaptive immune system in fish. Fisheries Science 31: 240-244.
Liang, M., N. M. Davies, and I. Toth. 2008. Increasing entrapment of peptides within poly (alkyl cyanoacrylate) nanoparticles prepared from water-in-oil microemulsions by copolymerization. International Journal of Pharmaceutics 362: 141-146.
Liu, C. H., C. H. Chiu, S. W. Wang, and W. Cheng. 2012. Dietary administration of the probiotic, Bacillus subtilis E20, enhances the growth, innate immune responses, and disease resistance of the grouper, Epinephelus coioides. Fish and Shellfish Immunology 33: 699-706.
Liu, W., C. H. Hsu, C. Y. Chang, H. H. Chen, and C. S. Lin. 2006. Immune response against grouper nervous necrosis virus by vaccination of virus-like particles. Vaccine 24: 6282-6287.
Lowe, P. J., and C. S. Temple. 1994. Calcitonin and insulin in isobutylcyanoacrylate nanocapsules: protection against proteases and effect on intestinal absorption in rats. Journal of Pharmacy and Pharmacology 46: 547-552.
Mauriello, E. M., H. Duc-le, R. Isticato, G. Cangiano, H. A. Hong, M. De-Felice, E. Ricca, and S.M. Cutting. 2004. Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine 22: 1177-1187.
Meloen, R. H. 1995. Design of synthetic peptides for oral vaccination. Advanced Drug Delivery Reviews 18: 91-100.
Miller, M. D., S. Gould-Fogerite, L. Shen, R. M. Woods, S. Koenig, R. J. Mannino, and N. L. Letvin. 1992. Vaccination of rhesus monkeys with synthetic peptide in a fusogenic proteoliposome elicits simian immunodeficiency virus-specific CD8+ cytotoxic T lymphocytes. The Journal of Experimental Medicine 176: 1739-1744.
Moudallal, Z. A., J. Briand, and M. Van Regenmortel. 1985. A major part of the polypeptide chain of tobacco mosaic virus protein is antigenic. The EMBO Journal 4: 1231.
Müller, R. H., C. Lherm, J. Herbert, and P. Couvreur. 1990. In vitro model for the degradation of alkylcyanoacrylate nanoparticles. Biomaterials 11: 590-595.
Oggioni, M. R., A. Ciabattini, A. M. Cuppone, and G. Pozzi. 2003. Bacillus spores for vaccine delivery. Vaccine 21: 96-101.
Ozin, A. J., C. S. Samford, A. O. Henriques, and C. P. Moran. 2001. SpoVID guides SafA to the spore coat in Bacillus subtilis. Journal of Bacteriology 183: 3041-3049.
Pakingking, J. R., N. B. Bautista, E. G. D. Jesus-Ayson, and O. Reyes. 2011. Protective immunity against viral nervous necrosis (VNN) in brown-marbled grouper (Epinephelus fuscogutattus) following vaccination with inactivated betanodavirus. Fish and Shellfish Immunology 28: 525-533.
Pani, K., G. Gladieux, G. Brandes, R. Kulkarni, and F. Leonard. 1968. The degradation of n-butyl alpha-cyanoacrylate tissue adhesive. II. Surgery 63: 481-489.
Patarroyo, M. E., P. Romero, M. L. Torres, P. Clavijo, A. Moreno, A. Martínez, R. Rodríguez, F. Guzman, and E. Cabezas. 1987. Induction of protective immunity against experimental infection with malaria using synthetic peptides. Nature 328: 629-632.
Paterson, W., and J. Fryer. 1974. Immune response of juvenile coho salmon (Oncorhynchus kisutch) to Aeromonas salmonicida cells administered intraperitoneally in Freund's complete adjuvant. Journal of the Fisheries Research Board of Canada 31: 1751-1755.
Pfaff, E., M. Mussgay, H. Böhm, G. Schulz, and H. Schaller. 1982. Antibodies against a preselected peptide recognize and neutralize foot and mouth disease virus. The EMBO Journal 1: 869.
Pinchuck, P., and P. H. Maurer. 1965. Antigenicity of polypeptides (poly alpha amino acids) XV. studies on the immunogenicity of synthetic polypeptides in mice. The Journal of Experimental Medicine 122: 665-671.
Rammensee, H. G. 1995. Chemistry of peptides associated with MHC class I and class II molecules. Current Opinion in Immunology 7: 85-96.
Robert-Neurath, A., B. Seto, and N. Strick. 1989. Antibodies to synthetic peptides from the preS1 region of the hepatitis B virus (HBV) envelope (env) protein are virus-neutralizing and protective. Vaccine 7: 234-236.
Rock, K. L., C. Gramm, L. Rothstein, K. Clark, R. Stein, L. Dick, D. Hwang, and A.L. Goldberg. 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78: 761-771.
Sato, A., and N. Okamoto. 2010. Induction of virus-specific cell-mediated cytotoxic responses of isogeneic ginbuna crucian carp, after oral immunization with inactivated virus. Fish and Shellfish Immunology 29: 414-421.
Scherer, D., J. Robinson, and J. Kreuter. 1994. Influence of enzymes on the stability of polybutylcyanoacrylate nanoparticles. International Journal of Pharmaceutics 101: 165-168.
Schwarz, G., and G. Beschiaschvili. 1989. Thermodynamic and kinetic studies on the association of melittin with a phospholipid bilayer. Biochimica et Biophysica Acta Biomembranes 979: 82-90.

Semete, B., L. I.. J. Booysen, L. Kalombo, J. D. Venter, L. Katata, B. Ramalapa, J. A. Verschoor, and H. Swai. 2010. In vivo uptake and acute immune response to orally administered chitosan and PEG coated PLGA nanoparticles. Toxicology and Applied Pharmacology 249: 158-165.
Shukla, A., B. Singh, and O. P. Katare. 2011. Significant systemic and mucosal immune response induced on oral delivery of diphtheria toxoid using nano-bilosomes. British Journal of Pharmacology 164: 820-827.
Slifka, M. K., and R. Ahmed. 1996. Long-term humoral immunity against viruses: revisiting the issue of plasma cell longevity. Trends in Microbiology 4: 394-400.
Sommerset, I., B. Krossøy, E. Biering, and P. Frost. 2005. Vaccines for fish in aquaculture. Expert Review of Vaccines 4: 89-101
Song, M., H. A. Hong, J. M. Huang, C. Colenutt, D. D. Khang, T. V. Nguyen, S. M. Park, B. S. Shim, H. H. Song, I. S. Cheon, J. E. Jang, J. A. Choi, Y. K. Choi, K. Stadler, and S. M. Cutting. 2012. Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine. Vaccine 30: 3266-3277.
Soppimath, K. S., T. M. Aminabhavi, A. R. Kulkarni, and W. E. Rudzinski. 2001. Biodegradable polymeric nanoparticles as drug delivery devices. Journal of Controlled Release 70: 1-20.
Spinosa, M. R., T. Braccini, E. Ricca, M. De-Felice, L. Morelli, G. Pozzi, and M. R. Oggioni. 2000a. On the fate of ingested Bacillus spores. Research in Microbiology 151: 361-368.
Spinosa, M. R., F. Wallet, R. J. Courcol, and M. R. Oggioni. 2000b. The trouble in tracing opportunistic pathogens: cholangitis due to Bacillus in a French hospital caused by a strain related to an Italian probiotic? Microbial Ecology in Health and Disease 12: 99-101.
Steward, M. W., C. M. Stanley, R. Dimarchi, G. Mulcahy, and T. R. Doel. 1991. High-affinity antibody induced by immunization with a synthetic peptide is associated with protection of cattle against foot-and-mouth disease. Immunology 72: 99-103.
Stoloff, G. A., W. Caparros, and Wilson. 2007. Synthetic multi‐epitope peptides identified in silico induce protective immunity against multiple influenza serotypes. European Journal of Immunology 37: 2441-2449.
Sun, Y. Z., H. L. Yang, R. L. Ma, and W. Y. Lin. 2010. Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides. Fish and Shellfish Immunology 29: 803-809.
Tanaka, S., K. Mori, M. Arimoto, T. Lwamoto, and T. Nakai. 2001. Protective immunity of seven-band grouper, Epinephelus septemfasciatus, against experimental viral nervous necrosis. Journal of Fish Diseases 24: 15-22.
Thuy, N. T. T., D. H. Nguyen, and H. I. Wergeland. 2013. Specific humoral immune response and protection against Vibrio parahaemolyticus in orange-spotted grouper Epinephelus coioides. International Journal of Aquatic Science 4: 24-35.
Tobar, J. A., S. Jerez, M. Caruffo, C. Bravo, F. Contreras, S. A. Bucarey, and M. Harel. 2011. Oral vaccination of Atlantic salmon (Salmo salar) against salmonid rickettsial septicaemia. Vaccine 29: 2336-2340.
Vauthier, C., C. Dubernet, E. Fattal, H. Pinto-Alphandary, and P. Couvreur. 2003. Poly (alkylcyanoacrylates) as biodegradable materials for biomedical applications. Advanced Drug Delivery Reviews 55: 519-548.

Wang, C. Y., T. Y. Chang, A. M. Walfield, J. Ye, M. Shen, S. P. Chen, M. C. Li, Y. L. Lin, M. H. Jong, P. C. Yang, N. Chyr, E. Kramer, and F. Brown. 2002. Effective synthetic peptide vaccine for foot-and-mouth disease in swine. Vaccine 20: 2603-2610.
Watnasirichaikul, S., N. M. Davies, T. Rades, and I. G. Tucker. 2000. Preparation of biodegradable insulin nanocapsules from biocompatible microemulsions. Pharmaceutical Research 17: 684-689.
Watnasirichaikul, S., T. Rades, I. G. Tucker, and N. M. Davies. 2002. In‐vitro release and oral bioactivity of insulin in diabetic rats using nanocapsules dispersed in biocompatible microemulsion. Journal of Pharmacy and Pharmacology 54: 473-480.
Weiner, H. L., A. P. Da-Cunha, F. Quintana, and H. Wu. 2011. Oral tolerance. Immunological Reviews 241: 241-259.