臺灣博碩士論文加值系統

莫三比克吳郭魚是一種廣鹽性硬骨魚，可直接從淡水轉移至千分之二十五海水中而存活。對吳郭魚的研究，大部分集中在其滲透壓調節機制，而對於轉移到高滲透壓後，吳郭魚免疫反應的研究相當少。當吳郭魚面對緊迫或是更嚴重的緊迫時，其免疫能力的改變可能會導致魚群的生病或死亡。硬骨魚類的專一性免疫系統發展的並不完全，因此對於抵抗外來病原菌先天免疫系統包括體液性（humoral）和細胞性（cellular parameter）免疫就顯得格外重要。
在in vivo實驗中，吳郭魚直接由淡水轉移至千分之二十五海水後，測定其溶菌酶酵素活性（lysozyme activity）、補體活性（alternative complement pathway activity, ACP activity）、細胞吞噬作用（phagocytosis）和呼吸爆炸活性（respiratory burst activity）等先天免疫指標，並利用蛋白質體分析尋找在血清中含有哪些免疫相關因子，以探討吳郭魚在滲透壓緊迫後，免疫指標及免疫相關因子的改變及其調節。吳郭魚血清及頭腎（head kidney）溶菌酶活性，在海水轉移1小時後明顯上升，然而補體活性卻在轉移8小時後才明顯上升。頭腎及脾臟呼吸爆炸活性，在海水轉移8小時後也有顯著的差異。而血清蛋白質體的分析顯示complement component C3, Mg2+-dependent neutral sphingomyelinase和caspase 3在轉移海水後，蛋白質量皆被提昇，這些分子會調節細胞吞噬作用和呼吸爆炸活性。故推測溶菌酶先活化補體系統，而補體和其他免疫相關因子共同調節呼吸爆炸活性，造成呼吸爆炸活性在轉移後8小時有顯著的上升。在in vitro實驗中，將淡水吳郭魚頭腎及脾臟免疫細胞分離，並在300 和500 mOsm medium 滲透壓下培養4，8和24小時後，測定其細胞吞噬作用和呼吸爆炸活性。在500 mOsm medium高滲透壓培養下，其細胞吞噬能力和呼吸爆炸的活性皆比300 mOsm medium等滲透壓活性高。在500 mOsm medium高滲透壓培養4小時，細胞吞噬能力上升，但在in vivo實驗只有慢慢上升的趨勢，顯示高滲透壓直接影響免疫細胞增強其吞噬能力。將轉移海水後4個不同時間點，吳郭魚血清蛋白質體分析顯示，有17個蛋白質量提昇，9個蛋白質量下降。而這些受高滲透壓緊迫下而提昇蛋白點包含semaphorin, C3, Mg2+-dependent neutral sphingomyelinase and caspase 3等免疫相關因子。綜合上述結果顯示，吳郭魚受急遽高滲透壓緊迫後，將增加其先天免疫能力，並透過一些受滲透壓調控的免疫相關因子，更增強先
免疫系統。

Tilapia (Oreochromis mossambicus) are one of euryhaline teleosts which can be directly transferred from isosmolality (FW) to hyperosmolality (SW). Most studies in tilapia are focused on the mechanism of osmoregulation; however the understanding of innate immune response in fish facing hyperosmolality is rare. Once fish facing stress, the immunity of fish may be mediated to prevent the bacteria or microbial invasion. In teleost, specific immune system does not well develop so that innate immune system such as humoral and cellular responses might play an important role for protecting from foreign molecules infection.
In present study, in vivo and in vitro assays were used to investigate the effects of innate immunity and whether more factors or molecules are regulated after tilapia faced osmotic stress by proteomic analysis combined with ESI-Q-TOF MS/MS. In vivo, the lysozyme activity of serum and HK were increased at 1 h and 24 h after SW transfer, and decreased at 8 h. Surprisingly, the alternative complement pathway (ACP) activity in serum increased at 8 h after SW transfer. The phagocytic capacity of immune cells was increased at 4 h in HK and at 1 h in spleen. The respiratory burst activity of immune cells from HK and spleen was increased at 8 h after SW transfer. These results reveal that the innate immunity of tilapia facing hyperosmotic condition is increased and the lysozyme activity of serum activates the complement system. Complement component C3, Mg2+-dependent neutral sphingomyelinase and Caspase 3 of serum in tilapia after SW transfer were up-regulated followed proteomic analysis, suggesting that these proteins might mediate the phagocytosis and the respiratory burst activity after SW transfer. In vitro, the cellular parameters (phagocytic ability and respiratory burst activity) of HK and spleen at 500 mOsm osmolality medium cultured for 4, 8, or 24 h were higher than that those of 300 mOsm/kg osmolality medium in each corresponding group. It reveals that the immune cells of spleen and HK in hyperosmolality medium express the osmoregulatory factors not only mediate the osmolality but also the innate immunity of phagocytosis and respiratory burst. The phagocytic ability in immune cells from HK and spleen was rapidly increased at 500 mOsm/kg osmolality medium cultured for 4 h; however, the phagocytic ability was gradually increased in HK and spleen of tilapia after SW transfer. The result suggests that the direct effects of hyperosmolality can immediately enhance the phagocytic ability of immune cells. In proteomic analysis of serum, 17 proteins were up-regulatedand 9 proteins were down-regulated at four tested times compared to FW. These up-regulated proteins in serum after SW transfer contain semaphorin, C3, Mg2+-dependent neutral sphingomyelinase and caspase 3 Combinations of in vivo, in vitro and proteomic analysis, these results suggest that the innate immunity is enhanced via the immunoregulatory factors of serum after tilapia transfer to hyperosmotic condition

Abstract...........................................I
中文摘要.................................................III
目次.................................................V
表目錄.................................................VII
圖目錄.................................................VIII
1. Introduction.......................................1
1.1. Tilapia (Oreochromis mossambicus).............1
1.2. Stress........................................1
1.3. Teleost immunity..............................2
1.4. Lysozyme......................................3
1.5. Phagocytosis..................................4
1.6. Respiratory burst.............................4
1.7. The complement System.........................5
2. Specific aims......................................7
3. Materials and Methods..............................9
3.1. Experimental designs..........................9
3.2. Experimental fish and osmotic stress..........10
3.3. Serum collection..............................11
3.4. Spleen and head kidney total protein
extraction....................................11
3.5. Lysozyme activity.............................12
3.6. Preparation of the immune cells from fish
spleen and HK.................................12
3.7. Phagocytosis..................................12
3.8. Respiratory burst assay.......................13
3.9. ACP activity..................................13
3.10. Hyperosmotic and Isomotic treatment..........14
3.11. Immune cells from spleen or HK phagocytosis
and burst activity in vitro..................14
3.12. 2-D electrophoresis..........................14
3.13. In-gel digestion.............................15
3.14. ESI-Q-TOF MS/MS..............................15
3.15. Statistics analysis..........................16
4. Results............................................17
4.1. In vivo.......................................17
4.2. In vitro......................................17
4.3. Serum 2D gel analysis.........................19
4.4. Immune cells of HK and spleen 2D gel
analysis......................................20
5. Discussion.........................................23
5.1. In vivo.......................................23
5.2. In vitro......................................24
5.3. In vivo and in vitro compared.................25
5.4. Proteomics....................................26
6. Conclusions........................................29
7. Future work........................................31
8. References.........................................33

表目錄
Table 1. Total protein spots is analyzed by
ImageMaster 2D elite software......43
Table 2. The changes (Ratio to FW control)
of individual protein spot at various
times after SW transfer. Each spot is
identified by ESI-Q-TOF MS/MS......44
Table 3. The protein identification and protein
name corresponds to Fig. 17........45
Table 4. Total protein spots after cultured
immune cells in HK and spleen
analyzed by ImageMaster 2D elite
software...........................47
Table 5. The numbers of protein spot are
regulated (up and down) from immune
cells of HK or spleen culture in 300
and 500 mOsm/kg media analyzed by
ImageMaster 2D elite software......48

圖目錄
Fig. 1. In vivo humoral parameter of lysozyme
activity that tilapia were exposed to
25 ppt SW for 1, 4, 8 or 24 h on (A)
serum and (B) HK
(pool sample n=3)...................49
Fig. 2. In vivo humoral parameter of serum ACP
activity that tilapia were exposed to
25 ppt SW for 1, 4, 8 or 24 h.......50
Fig. 3. In vivo cellular parameters of (A) HK
phagocytosis ability, (B) phagocytosis
capacity, (C) the percentage of
respiratory burst and (D) respiratory
burst capacity after tilapia were
exposed to 25 ppt SW for 1, 4, 8
or 24 h.............................51
Fig. 4. In vivo cellular parameter of (A)
spleen phagocytosis ability, (B)
phagocytosis capacity, (C) the
percentage of respiratory burst and
(D) respiratory burst capacity after
tilapia were exposure to 25 ppt SW
for 1, 4, 8 or 24 h.................52
Fig. 5. In vitro HK phagocytic ability in (A)
300 mOsm/kg, and (B) 500 mOsm/kg
osmolality media after cultured for
4, 8 or 24 h........................53
Fig. 6. In vitro HK phagocytosis ability
compared between 300 mOsm/kg and
500 mOsm/kg osmolality media after
cultured for 4, 8, 24 h.............54
Fig. 7. In vitro HK respiratory burst in
(A) 300 mOsm/kg, and (B) 500 mOsm/kg
osmolality media after cultured for
4, 8 or 24 h........................55
Fig. 8. In vitro HK respiratory burst compared
between 300 mOsm/kg and 500 mOsm/kg
osmolality media after cultured for
4, 8, 24 h..........................56
Fig. 9. In vitro spleen phagocytic ability in
(A) 300 mOsm/kg and (B) 500 mOsm/kg
osmolality media after cultured for
4, 8 or 24 h........................57
Fig. 10. In vitro spleen phagocytosis ability
compared between 300 mOsm/kg and
500 mOsm/kg osmolality media after
cultured for 4, 8, 24 h............58
Fig. 11. In vitro spleen respiratory burst
in (A) 300 mOsm/kg and in (B)
500 mOsm/kg osmolality media after
cultured for 4, 8 or 24 h..........59
Fig. 12. In vitro spleen respiratory burst
compared between 300 mOsm/kg and
500 mOsm/kg osmolality media after
cultured for 4, 8, 24 h............60
Fig. 13. Two-dimensional gel analysis of
tilapia serum after 25 ppt SW
transfers for 1 and 8 h
(300 μg total protein).............61

Fig. 14. Two-dimensional gel analysis of
tilapia serum in FW control and
after 25 ppt SW transfers for 1 h
(300 μg total protein).............62
Fig. 15. Two-dimensional gel analysis of
tilapia serum after 25 ppt SW
transfers for 4 and 8 h
(300 μg total protein).............63
Fig. 16. Two-dimensional gel analysis of
tilapia serum after 25 ppt SW
transfers for 24 h
(300 μg total protein).............64
Fig. 17. Numbers represent the proteins that
were identified ESI-QUAD-TOF MS/MS
ion search and peptide mass
fingerprinting.....................65
Fig. 18. Two-dimensional gel analysis of
tilapia HK immune cells without
culture (A) and after 300 (B) or
500 (C) mOsm/kg osmolality cultured
for 24 h (300 μg total protein)....66
Fig. 19. Two-dimensional gel analysis of
tilapia spleen immune cells without
culture (A) and after 300 (B) or 500
(C) mOsm/kg osmolality cultured for
24 h (300 μg total protein)........67

Al Amoudi M, El-Sayed A, Ghobashy A. Effects of thermal and thermo-haline shocks on survival and osmotic concentration of the tilapias Oreochromis mossambicus and Oreochromis aureus X Oreochromis niloticus hybrids. J World Aquacult Soc. 1996. 27:456-61.

Alcorn SW, Murra AL, Pascho RJ. Effects of rearing temperature on immune functions in sockeye salmon (Oncorhynchus nerka). Fish Shellfish Immunol. 2002. 12:303-34.

Alishahi M and Buchmann K. Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts. Dis Aquat Organ. 2006. 72:269-73.

Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol. 1993. 54:1-78.

Amar EC, Kiron V, Satoh S, Watanabe T. Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products. Fish Shellfish Immunol. 2004. 16:527-37.

Amaro MJ, Bartolome J, Pardo M, Cotonat T, Lopez-Farre A, Carreno V. Decreased nitric oxide production in chronic viral hepatitis B and C. J MedVirol. 1997. 51:326-31.

Bagni M, Archetti L, Amadori M, Marino G. Effect of long-term oral administration of an immunostimulant diet on innate immunity in sea bass (Dicentrarchus labrax). J. Vet. Med. B. Infect. Dis. Vet Public Health. 2000. 47:745-51.

Barabash RD, Ermakova TA, Bondarenko VS, Bondarenko EI, Vakulenko LB. Cooperation of antigens, immunoglobulins, complement and antimicrobial enzymes in the regulation of blood granulocyte locomotion. Biull Eksp Biol Med. 1980. 89:40-2.

Bjornson AB. Role of complement in host resistance against members of the Bacteroidaceae. Rev Infect Dis. 1984. 1:34-9.

Boshra H, Li J, Sunyer JO. Recent advances on the complement system of teleost fish. Fish Shellfish Immunol. 2006. 20:239-62.

Brekke OL, Christiansen D, Fure H, Fung M, Mollnes TE. The role of complement C3 opsonization, C5a receptor, and CD14 in E. coli-induced up-regulation of granulocyte and monocyte CD11b/CD18 (CR3), phagocytosis, and oxidative burst in human whole blood. J Leukoc Biol. 2007. 81:1404-13.

Briggs RT, Drath DB, Karnnovsky ML, Karnnovsky MJ. Localization of NADH oxidase on the surface of numan polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol. 1975. 67:566-86.

Cecchini S, Terova G, Caricato G, Saroglia M. Lysosome activity in embryos and larvae of sea bass (Dicentrarchus labrax L.), spawned by broodstocks fed with vitamin C enriched diets. Bull Eur Assoc Fish Pathol. 2000. 20:120-4.

Chan LW. The functionalgenomics and proteomic analysis of splenic genes from tilapia Oreochomis mossambicus, facing osmotic stress. Master thesis. Department of Life Science and Institute of Biotechnology, National Dong Hwa Uuniversity, Hualien, Taiwan. 2006.

Chomarat P, Banchereau J, Davoust J, Palucka AK. IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol. 2000. 1:510-4.

Clark EC, Nath KA, Hostetter TH, Hostetter MK. Hyperosmolality impairs ammonia-mediated inflammation: implications for the renal medulla. Am J Physiol. 1992. 263:R148-55.

Craig S, Lopez A, Hoskin D, Markham F. Meconium inhibits phagocytosis and stimulates respiratory burst in alveolar macrophages. Pediatr Res. 2005. 57:813-8.

Crawshaw LI. Responses to rapid temperature change in vertebrate ectotherms. Am Zool. 1979. 19:225-37.

Cruchaud A. Specific and non-specific defense mechanisms against infection. Schweiz Med Wochenschr. 1977. 107:1721-8.

Cuesta A, Laiz-Carrión R, Del Río MP, Meseguer J, Mancera JM, Esteban MA. Salinity influences the humoral immune parameters of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol. 2005. 18:255-61.

Espelid S, Løkken GB, Steiro K., Bøgwald J. Effects of cortisol and stress on the immune system in Atlantic salmon Salmo salar L. Fish Shellfish Immunol. 1996. 6:95-110.

Esteban MA, Mulero V, Munoz J. Meseguer J. Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue Res. 1998. 293:133-41.

Evans DL, Taylor SL, Leary JH III, Bishop GR, Jaso-Friedmann L. In vivo activation of nonspecific cytotoxic cells by Streptococcus iniae and amplification with apoptosis regulatory factors. Fish Shellfish Immunol. 1999. 11:231-40.

Evans JJ, Shoemaker CA, Klesius PH. Experimental Streptococcus iniae infection of hybrid striped bass (Morone chrysops X Morone saxatilis) and tilapia (Oreochromis niloticus) by nares inoculation. Aquaculture. 2000. 189:197-210.

Gasque P. Complement: a unique innate immune sensor for danger signals. Mol Immunol. 2004. 41:1089-98.

Ghosh D, Bhattacharya S, Mazumder S. Perturbations in the catfish immune responses by arsenic: organ and cell specific effects. Comp Biochem Physiol C Toxicol Pharmacol. 2006. 143:455-63.

Graves SS, Evans DL, Dawe DL. Antiprotozoan activity of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus). J Immunol. 1985. 134:78-85.

Grinde B. Lysozyme from rainbow trout, Salmo gairdneri Richardson, as an antibacterial agent against fish pathogens. J Fish Dis. 1989. 12:95-104.

Grinde B, Lie O, Poppe T, Salte R. Species and individual variation in lysozyme activity in fish of interest in aquaculture. Aquaculture. 1988. 68:299-304.

Handeland SO, Berge A, Bjornson BT, Lie O, Stefansson SO. Seawater adaptation by out-of-season Atlantic salmon (Salmo salar L.) smolts at different temperatures. Aquaculture. 2000. 181:377-96.

Hansen NE. Lysozyme in haematology. Pathophysiology and clinical use. Scand J Haematol. 1975. 14:160-5.

Hayman JR, Bly JE, Levine RP, Lobb CJ. Complement deficiencies in channel catfish (Ictalurus punctatus) associated with temperature and seasonal mortality. Fish Shellfish Immunol. 1992. 2:183-92.

Hinkovska-Galcheva V, Kjeldsen L, Mansfield PJ, Boxer LA, Shayman JA, Suchard SJ. Activation of a plasma membrane-associated neutral sphingomyelinase and concomitant ceramide accumulation during IgG-dependent phagocytosis in human polymorphonuclear leukocytes. Blood. 1998. 91:4761-9.

Holladay SD, Smith SA, El-Habback H, Caceci T. The influence of chlorpyrifos, anorganophosphate insecticide, on the immune system of tilapia (Oreochromis niloticus). J Aquatic Anim Health. 1996. 8:104-10.

Holland MC, Lambris JD. The complement system in teleosts. Fish shellfish Immunol. 2002. 12:399-420.

Hong S, Zou J, Crampe M, Peddie S, Scapigliati G, Bols N, Cunningham C, Secombes CJ. The production and bioactivity of rainbow trout (Oncorhynchus mykiss) recombinant IL-1 β. Vet Immunol Immunopathol. 2001. 81:1-14.

Hoogerwerf M, Weening RS, Hack CE, Roos D. Complement fragments C3b and iC3b coupled to latex induce a respiratory burst in human neutrophils. Mol. Immunol. 1990. 27:159-67.

Huang YL. Stress-activated protein kinase pathways of tilapia astrocytes respond to osmotic shock. Master thesis. Department of Life Science and Institute of Biotechnology, National Dong Hwa Uuniversity, Hualien, Taiwan. 2006.


Huising MO, Guichelaar T, Hoek C, Verburg-van Kemenade BM, Flik G, Savelkoul HF, Rombout JH. Increased efficacy of immersion vaccination in fish with hyperosmotic pretreatment. Vaccine. 2003. 21:4178-93.

Hwang P, Sun CM, Wu SM. Changes of plasma osmolarity, chloride concentration and gill Na+–K+-ATPase activity in tilapia (Oreochromis mossambicus) during seawater acclimation. Mar Biol. 1989. 100:295-99.

Imsland AK, Gunnarsson S, Foss A, Stefansson SO. Gill Na+, K+-ATPase activity, plasma chloride and osmolality in juvenile turbot (Scophthalmus maximus) reared at different temperatures and salinities. Aquaculture. 2003. 218:671-83.

Iyer GYN, Islam MF, Quastel JH. Biochemical aspects of phagocytosis. Nature. 1961. 192:535-41.

Jang SI, Hardie LJ, Secombes CJ. Elevation of rainbow trout Oncorhynchus mykiss macrophage respiratory burst activity with macrophage-derived supernatants. J Leukoc Biol. 1995. 57:943-7.

Jaso-Friedmann L, Evans DL. Mechanisms of cellular cytotoxic innate resistance in tilapia (Oreochromis nilotica). Dev Comp Immunol. 1999. 23:27-35.

Jaso-Friedmann L, Ruiz J, Bishop GR, Evans DL. Regulation of innate immunity in tilapia: activation of nonspecific cytotoxic cells by cytokine-like factors. Dev Comp Immunol. 2000. 24:25-36.

John MB, Chandran MR, Aruna BV, Anbarasu K. Production of superoxide anion by head-kidney leucocytes of Indian major carps immunised with bacterins of Aeromonas hydrophila. Fish Shellfish Immunol. 2002. 12:201-7.

Kakuta, I. Effect of sewage on blood parameters and the resistance against bacterial infection of goldfish, Carassius auratus. Environ Toxicol Water Qual. 1997. 12:43-51.

Kang HR, Lee CG, Homer RJ, Elias JA. Semaphorin 7A plays a critical role in TGF-β1-induced pulmonary fibrosis. J Exp Med. 2007. 204:1083-93.

Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: a regulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends Immunol. 2003. 24:25-9.


Kishimoto T. Interleukin-6. In: Thomson AN, Lotze MT, editors. The cytokine handbook. 4th ed. San Diego, CA: Academic Press. 2003. 281-304.

Kishore U, Reid KB. C1q: structure, function, and receptors. Immunopharmacology. 2000. 49:159-70.



Laing KJ, Wnag T, Zou J, Holland J, Hong S, Bols N, Hirono I, Aoki T, Secombes CJ. Cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) tumour necrosis factor-alpha. Eur J Biochem. 2001. 268:1315-22.

Lappin D, Hamilton AD, Morrison L, Aref M, Whaley K. Synthesis of complement components (C3, C2, B and C1-inhibitor) and lysozyme by human monocytes and macrophages. J. Clin. Lab. Immunol. 1986. 20:101-5.

Lauber K, Bohn E, Kröber SM, Xiao YJ, Blumenthal SG, Lindemann RK, Marini P, Wiedig C, Zobywalski A, Baksh S, Xu Y, Autenrieth IB, Schulze-Osthoff K, Belka C, Stuhler G, Wesselborg S. Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell. 2003. 113:717-30.

Lee JS, Nauseef WM, Moeenrezakhanlou A, Sly LM, Noubir S, Leidal KG, Schlomann JM, Krystal G, Reiner NE. Monocyte p110 (alpha) phosphatidylinositol 3-kinase regulates phagocytosis, the phagocyte oxidase, and cytokine production. J Leukoc Biol. 2007. 81:1548-61.

Lee TH, Tsai JC, Fang MJ, Yu MJ, Hwang PP. Isoform expression of Na+–K+-ATPase alpha - subunit in gills of the teleost Oreochromis mossambicus. Am J Physiol. 1998. 275:R926-932.

Lepelletier Y, Smaniotto S, Hadj-Slimane R, Villa-Verde DM, Nogueira AC, Dardenne M, Hermine O, Savino W. Control of human thymocyte migration by Neuropilin-1/ Semaphorin-3A mediated interactions. Proc Natl Acad Sci USA. 2007. 104:5545-50.

Lepelletier Y, Moura IC, Hadj-Slimane R, Renand A, Fiorentino S, Baude C, Shirvan A, Barzilai A, Hermine O. Immunosuppressive role of semaphorin-3A on T cell proliferation is mediated by inhibition of actin cytoskeleton reorganization. Eur J Immunol. 2006. 36:1782-93.
Levy JR, Stevens W. Plasma hyperosmolality stimulates leptin secretion acutely by a vasopressin-adrenal mechanism. Am J Physiol Endocrinol Metab. 2004. 287:E263-8.

Lewis CE, McCarthy SP, Lorenzen J, McGee JO. Differential effects of LPS, IFN-γ and TNF-α on the secretion pf lysozyme by individual human mononuclear phagocytes: relationship to cell maturity. Immunol. 1990. 69:402-8.

Liberek T, Topley N, Jörres A, Coles GA, Gahl GM, Williams JD. Peritoneal dialysis fluid inhibition of phagocyte function: effects of osmolality and glucose concentration. J Am Soc Nephrol. 1993. 3:1508-15.

Lie O, Evensen O, Sorensen A, Froysadal E. Study on lysozyme activity in some fish species. Dis Aquat Org. 1989. 6:1-5.

Long S, Wilson M, Bengten E, Bryan L, Clem LW, Miller NW, Chinchar VG. Identification of a cDNA encoding channel catfish interferon. Dev Comp Immunol. 2004. 28:97-111.
Mackenzie S, Planas JV, Goetz FW. LPS-stimulated expression of a tumor necrosis factor-alpha mRNA in primary trout monocytes and in vitro differentiated macrophages. Dev Comp Immunol. 2003. 27:393-400.

Madsen LM, Inada M, Weiss J. Determinants of activation by complement of group II phospholipase A2 acting against Escherichia coli. Infect Immun. 1996. 64:2425-30.

Magnadottir B. Innate immunity of fish (overview). Fish Shellfish Immunol. 2006. 20:137-51.

Marc AM, Quentel C, Severe A, Le Bail PY, Boeuf G. Changes in some endocrinological and non-specific immunological parameters during seawater exposure in the brown trout. J. Fish. Biol. 1995. 46:1065-81.

Matsumoto T, Kumazawa J, van der Auwera P. Suppression of leukocyte function and intracellular content of ATP in hyperosmotic condition comparable to the renal medulla. J Urol. 1989. 142:399-402.

Matsumoto T, Takahashi K, Mizunoe Y, Ogata N, Tanaka M, Kumazawa J, Nozaki M, van der Auwera P. Inhibition of chemiluminescent response of human polymorphonuclear leukocytes by hyperosmolality comparable to renal medulla and preservation by ofloxacin. Eur Urol. 1991. 20:232-7.

Metz JR, van den Burg EH, Wendelaar-Bonga SE, Flik G. Regulation of branchial Na+-K+-ATPase in common carp (Cyprinus carpio) acclimated to different temperatures. J Exp Biol. 2003. 206:2273-80.

Moody CE, Serrenze DV, Reno PW. Non-specific cytotoxic activity of teleost leucocytes. Dev Comp Immunol. 1985. 9:51-64.

Morgan JD, Sakamoto T, Grau EG, Iwama GK. Physiological and respiratory responses of the Mozambique tilapia (Oreochromis mossambicus) to salinity acclimation. Comp Biochem Physiol A. 1997. 117:391-8.

Murray CK, Fletcher TC. The immunohistochemieal localization of lysozyme in plaice (Pleuronectes. platessa L.) tissues. J Fish Biol. 1976 9:329-34.

Narnaware YK, Backer BI. Evidence that cortisol may protect against the immediate effects of stress on circulating leukocytes in the trout. Gen Comp Endocrinol. 1996. 103:359-66.

Neumann NF, Stafford JL, Barreda D, Ainsworth AJ, Belosevic M. Antimicrobial mechanisms of fish phagocytes and their role in host defense. Dev Comp Immunol. 2001. 25:807-25.

Ndong D, Chen YY, Lin YH, Vaseeharan B, Chen JC. The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures. Fish Shellfish Immunol. 2007. 22:686-94.
Ortuno J, Cuesta A, Esteban MA, Meseguer J. Effect of oral administration of high vitamin C and E dosages on the gilthead seabream (Sparus aurata L.) innate immune system. Vet Immunol Immunopathol. 2001. 79:167-80.

Ortuno J, Esteban MA, Meseguer J. Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L.) innate immune response. Fish Shellfish Immunol. 2001. 11:187-97.

Ortuno J, Esteban MA, Meseguer J. Effects of phenoxyethanol on the innate immune system of gilthead seabream (Sparus aurata L.) exposed to crowding stress. Vet Immunol Immunopathol. 2002. 89:29-36.

Panarelli M, Holloway CD, Mulatero P, Fraser R. Inhibition of lysozyme synthesis by dexamethasone in human mononuclear leukocytes: an index of glucocorticoid sensitivity. J. Clin Endocrinol Metab. 1994. 78:872-7.

Panigrahi A, Kiron V, Kobayashi T, Puangkaew J, Satoh S, Sugita H. Immune responses in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Vet Immunol Immunopathol. 2004. 102:379-88.

Parham P. The immune system. Garland publishing. 2000. 183-4.

Parry RM, Chandan RC, Shahani KM. A rapid and sensitive assay of muramidase. Proc Soc Exp Biol Med. 1965. 119:383-6.

Paul B, Sbarra AJ. The role of the phagocyte in host-parasite interactions.13. The direct quantitative estimation of H2O2 in phagocytizing cells. Biochim Biophys Acta. 1968 156:168-78.

Peddie S, Zou J, Cunningham C, Secombes CJ. Rainbow trout (Oncorhynchus mykiss) recombinant IL-1β and derived peptides induce migration of head-kidney leucocytes in vitro. Fish Shellfish Immunol. 2001. 11:697-709.

Pedrera IM, Collazos ME, Ortega E, Barriga C. In vitro study of the phagocytic processes in splenic granulocytes of the tench (Tinca tinca, L.). Dev Comp Immunol. 1992. 16:431-9.

Peveri P, Walz A, Dewald B, Baggiolini M. A novel neutrophil-activating factor produced by human mononuclear phagocytes. J Exp Med. 1988. 167:1547-59.


Pestka S, Krause CD, Walter MR. Interferons, interferon-like cytokines, and their receptors. Immunol Rev. 2004. 202:8-32.

Pickering AD. Cortisol-induced lymphocytopenia in brown trout, Salmo trutta L. Gen Comp Endocrinol. 1984. 53:252-9.



Plouffe DA, Hanington PC, Walsh JG, Wilson EC, Belosevic M. Comparison of select innate immune mechanisms of fish and mammals. Xenotransplantation. 2005. 12:266-77.

Plumb JH. Health Maintenance and Principal Microbial Diseases of Cultured Fish. Iowa State University Press, Ames. 1999. 328.

Prabakaran M, Binuramesh C, Steinhagen D, Dinakaran Michael R. Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent. Ecotoxicol Environ Saf. 2007. Epub ahead of print.

Price MA, Jurd RD, Mason CF. A field investigation into the effect of sewage effluent and general water quality on selected immunological indicators in carp (Cyprinus carpio L.). Fish Shellfish Immunol. 1997. 7:193-207.

Rasmussen F, Georgsen J, Antonsen S, Grunnet N. Phagocytic properties of granulocytes after intravenous injection of ioxaglate or iohexol. Acta Radiol. 1992. 33:271-4.

Robertson B, Bergan V, Rokenes T, Larsen R, Albuquerque A. Atlantic salmon interferon genes: cloning, sequence analysis, expression, and biological activity. J Interf Cytok Res. 2003. 10:601-12.

Saeij JPJ, Stet RJM, De Vries BJ, Van Muiswinkel WB, Wiegertjes GF. Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance? Dev Comp Immunol. 2003. 27:29-41.

Sakai M, Kobayashi M, Kawauchi H. In vitro activation of fish phagocytic cells by GH, prolactin and somatolactin. J Endocrinol. 1996b. 151:113-8.

Schorlemmer HU, Hofstaetter T, Seiler FR. Phagocytosis of immune complexes by human neutrophils and monocytes: relative importance of Fc and C3b receptors. Behring Inst Mitt. 1984. 76:88-97.

Schröder JM, Mrowietz U, Morita E, Christophers E. Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity. J Immunol. 1987. 139:3474-83.

Schröder K, Hertzog PJ, Ravasi T, Hume DA. Interferon gamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004. 75:163-89.

Seale AP, Riley LG, Leedom TA, Kajimura S, Dores RM, Hirano, T, Grau EG. Effects of environmental osmolality on the release of prolactin, growth hormone and ACTH from the tilapia pituitary. Gen. Comp. Endocrinol. 2002. 128:91-101.

Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson DP, Robertson BS, van Muiswinkel WB, editors. Techniques in fish immunology, vol. 1. Fair Haven, NJ: SOS Publications. 1990. 137-54.
Secombes CJ, Fletcher TC. The role of phagocytes in the protective mechanisms of fish. Ann Rev Fish Dis. 1992. 2:53-71.

Serada K, Moritomo T, Teshirogi K, Itou T, Shibashi T, Inoue Y, Nakanishi T. Comparison of respiratory burst activity of inflammatory neutrophils in ayu (Plecoglossus altivelis) and carp (Cyprinus carpio). Fish Shellfish Immunol. 2005. 19:363-73.

Shoemaker CA, Klesius PH, Plumb JA. Killing of Edwardsiella ictaluri by macrophages from channel catfish immune and susceptible to enteric septicemia of catfish. Vet Immunol Immunopathol. 1997. 58:181-90.

Shugar D. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim Biophys Acta. 1952. 8:302-9.

Slater CH, Schreck CB. Testosterone alters the immune response of chinook salmon, Oncorhynchus tshawytscha. Gen Comp Endocrinol. 1993. 89:291-8

Staurnes M, Sigholt T, Asgard T, Baeverfjord G. Effects of a temperature shift on seawater challenge test performance in Atlantic salmon (Salmo salar) smolt. Aquaculture. 2001. 201:153-9.

Sunyer JO, Lambris JD. Complement. In: Encyclopedia of life sciences. New York: Macmillan References Limited. 1999.

Takahashi Y, Itami T, Konegawa K. Enzymatic properties of partially purified lysozyme from the skin mucus of carp. Bull Jap Soc Scient Fish. 1986. 52:1209-14.

Takemura A, Takano K. Lysozyme in the ovary of tilapia (Oreochromics mossambicus): its purification and some biological properties. Fish Phys Biochem. 1995. 14:415-21.

Taylor SL, Jaso-Friedmann L, Allison AB, Eldar A, Evans DL. Streptococcus iniae inhibition of apoptosis of nonspecific cytotoxic cells: a mechanism of activation of innate immunity in teleosts. Dis Aquat Organ. 2001. 46:15-21.

Thelen M, Peveri P, Kernen P, von Tscharner V, Walz A, Baggiolini M. Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist. FASEB J. 1988. 2:2702-6.

Thompson KD, Tatner MF, Henderson RJ. Effects of dietary (n-3) and (n-6) polyunsaturated fatty acid ratio on the immune response of Atlantic salmon, Salmo salar L. Aqua Nutr. 1996. 2:21-31.

Uchida K, Kaneko T, Miyazaki H, Hasegawa S, Hirano T. Excellent salinity tolerance of Mozambique tilapia (Oreochromis mossambicus): elevated chloride cell activity in the branchial epithelia of the fish adapted to concentrated seawater. Zool Sci. 2000. 17:149-160.

Van der Heijden AJH, Verbost P, Eygensteyn J, Li J, Bonga S, Flik G. Mitochondria-rich cells in gills of tilapia (Oreochromis mossambicus) adapted to fresh water or sea water: quantification by confocal laser scanning microscopy. J Exp Biol. 1997. 200:55-64.

Weng CF, Chia-Chang C, Gong HY, Chen MHC, Lin, CJF, Huang, WT, Cheng CY, Hwang PP, Wu JL. Acute changes in gill Na+-K+ ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus). Physiol Biochem Zool. 2002. 75:29-37.

Yada T,Misumi I, Muto K, Azuma T, Schreck CB. Effects of prolactin and growth hormone on proliferation and survival of cultured trout leukocytes. Gen Comp Endocrinol. 2004. 136:298-306.

Yamamori T, Inanami O, Nagahata H, Cui Y, Kuwabara M. Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes. FEBS Lett. 2000. 467:253-8.

Yang MW. The alternations of gap junction-connexin 35 and connexin 43 in tilapia (Oreochromis mossambicus) brain responding to a seawater transition. Master thesis. Department of Life Science and Institute of Biotechnology, National Dong Hwa Uuniversity, Hualien, Taiwan. 2004.

Yousif AN, Albright LJ, Evelyn TPT. Occurrence of lysozyme in the eggs of coho salmon Oncorhynchus kisutch. Dis aquat Org. 1991. 10:45-9.

Zou J, Peddie S, Scapigliati G, Zhang Y, Bols NC, Ellis AE, Secombes CJ. Functional characterization of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss. Dev Comp Immunol. 2003. 271:813-22.

Zou J, Yoshiura Y, Dijkstra JM, Sakai M, Ototake M, Secombes C. Identification of an interferon gamma homologue in Fugu, Takifugu rubripes. Fish Shellfish Immunol. 2004. 17:403-9.