Abstract
To evaluate the effect of thermal and microbial stress on the immune response of common carp (Cyprinus carpio L.), relative mRNA expression level of pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β] and other genes related to immune or stress response [inducible nitric oxide synthase (iNOS), heat shock protein 70 (Hsp70), superoxide dismutase one (SOD1), and glucocorticoid receptor (GR)] was measured by quantitative PCR (qPCR). In addition, total protein and total immunoglobulin level in blood plasma of experimental common carp was also assayed. All the above parameters were estimated 24 h post-challenge with Gram-negative bacterium, Aeromonas hydrophila. Common carp (54.89 ± 6.90 g) were initially exposed to 20 °C (control group) and 30 °C (thermal stress group) water temperature for 30 days, followed by experimental challenge with 2.29 × 108 colony forming unit/mL (CFU/mL; LD50 dose) of A. hydrophila. Exposure of fish to thermal stress and subsequently challenge with A. hydrophila significantly (P < 0.05) increases the IL-1β mRNA expression in head kidney and spleen of common carp by ~ 39.94 and ~ 4.11-fold, respectively. However, TNF-α mRNA expression in spleen decreased ~ 5.63-fold in control fish challenged with A. hydrophila. Thermal stress and challenge with bacterium suppresses the iNOS and GR mRNA expression in spleen of common carp. Moreover, significant (P < 0.05) increase in total protein content of blood plasma (~ 43 mg/g) was evident in fish exposed to thermal stress and challenged with A. hydrophila. In conclusion, our study highlights the importance of elevated temperature stress and microbial infection in differential regulation of expression of several immunogenes in common carp.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ackerman PA, Iwama GK (2001) Physiological and cellular stress responses of juvenile rainbow trout to vibriosis. J Aquat Anim Health 13(2):173–180. https://doi.org/10.1577/1548-8667(2001)013<0173:PACSRO>2.0.CO;2
Benli ACK, Yildiz HY (2004) Blood parameters in Nile tilapia (Oreochromis niloticus L.) spontaneously infected with Edwardsiella tarda. Aquac Res 35(14):1388–1390. https://doi.org/10.1111/j.1365-2109.2004.01158.x
Biswas G, Korenaga H, Takayama H, Kono T, Shimokawa H, Sakai M (2012) Cytokine responses in the common carp, Cyprinus carpio L. treated with baker’s yeast extract. Aquaculture 356:169–175
Biswas G, Korenaga H, Nagamine R, Takayama H, Kawahara S, Takeda S, Kikuchi Y, Dashnyam B, Kono T, Sakai M (2013) Cytokine responses in the Japanese pufferfish (Takifugu rubripes) head kidney cells induced with heat-killed probiotics isolated from the Mongolian dairy products. Fish Shellfish Immunol 34(5):1170–1177. https://doi.org/10.1016/j.fsi.2013.01.024
Bly JE, Clem W (1992) Temperature and teleost immune functions. Fish Shellfish Immunol 2(3):159–171. https://doi.org/10.1016/S1050-4648(05)80056-7
Campos-Perez JJ, Ward M, Grabowski PS, Ellis AE, Secombes CJ (2000) The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the Gram-positive pathogen Renibacterium salmoninarum. Immunology 99(1):153–161. https://doi.org/10.1046/j.1365-2567.2000.00914.x
Chen DC, Pan JQ, Du B, Sun DX (2005) Induction of heat shock response in vivo inhibits NF-kappa B activity and protects murine liver from endotoxemia-induced injury. J Clin Immunol 25(5):452–461. https://doi.org/10.1007/s10875-005-5636-3
Choi JH, Roche H, Caquet T (2000) Effects of physical (hypoxia, hyperoxia) and chemical (potassium dichromate, fenitrothion) stress on antioxidant enzyme activities in Chironomus riparius Mg. (Diptera, Chiromonidae) larvae: potential biomarkers. Environ Toxicol Chem 19:495–500
Dash P, Patel S, Dixit A, Garg LC, Sahoo PK (2015) Four pro-inflammatory cytokines of rohu (Labeo rohita) during early developmental stages, their tissue distribution and expression by leucocytes upon in-vitro stimulation. Fish Shellfish Immunol 47(2):913–922. https://doi.org/10.1016/j.fsi.2015.10.034
Deane EE, Li J, Woo NYS (2004) Modulated heat shock protein expression during pathogenic Vibrio alginolyticus stress of sea bream. Dis Aquat Org 62(3):205–215. https://doi.org/10.3354/dao062205
Eddy FB (2005) Role of nitric oxide in larval and juvenile fish. Comp Biochem Phys A 142:221–230
Engelsma MY, Stet RJ, Saeij JP, Verburg-van Kemenade BM (2003) Differential expression and haplotypic variation of two interleukin-1beta genes in the common carp (Cyprinus carpio L.) Cytokine 22(1-2):21–32. https://doi.org/10.1016/S1043-4666(03)00102-9
FAO Yearbook (2012) Fisheries and Aquaculture Statistics, Rome, FAO, Rome, Italy, 76 pp
Forsyth RB, Candido EPM, Babich SL, Iwama GK (1997) Stress protein expression in coho salmon with bacterial kidney disease. J Aquat Anim Health 9(1):18–25. https://doi.org/10.1577/1548-8667(1997)009<0018:SPEICS>2.3.CO;2
Froese R, Pauly D (2011) FishBase. World Wide Web electronic publication, version (02/2011) (available at: www.fishbase.org/summary/speciessummary.php, 2011 (accessed 18.03.16)
Gonzalez SF, Buchmann K, Nielsen ME (2007) Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses caused by the ectoparasite Ichthyophthirius multifiliis. Fish Shellfish Immunol 22(6):641–650. https://doi.org/10.1016/j.fsi.2006.08.011
Grayson TH, Cooper LF, Wrathmell AB, Roper J, Evenden AJ, Gilpin ML (2002) Host responses to Renibacterium salmoninarum and specific components of the pathogen reveal the mechanisms of immune suppression and activation. Immunology 106(2):273–283. https://doi.org/10.1046/j.1365-2567.2002.01420.x
Hong S, Zou J, Crampe M, Peddie S, Scapagliati G, Bols N, Cunningham C, Secombes CJ (2001) The production and bioactivity of rainbow trout (Oncorhynchus mykiss) recombinant IL-1β. Vet Immunol Immunopathol 81(1-2):1–14. https://doi.org/10.1016/S0165-2427(01)00328-2
Jackson SE (2013) Hsp90: structure and function. Molecular Chaperones 328:155–240
Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG (2010) Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8(6):e1000412. https://doi.org/10.1371/journal.pbio.1000412
Kim KY, Lee SY, Cho YS, Bang IC, Kim KH, Kim DS, Nam YK (2007) Molecular characterization and mRNA expression during metal exposure and thermal stress of copper/zinc- and manganese-superoxide dismutases in disk abalone, Haliotis discus discus. Fish Shellfish Immunol 23(5):1043–1059. https://doi.org/10.1016/j.fsi.2007.04.010
Lindenstrom T, Secombes CJ, Buchmann K (2004) Expression of immune response genes in rainbow trout skin induced by Gyrodactylus derjavini infections. Vet Immunol Immunopathol 97(3-4):137–148. https://doi.org/10.1016/j.vetimm.2003.08.016
Low C, Wadsworth S, Burrell C, Secombes CJ (2003) Expression of immune genes in turbot (Scophthalmus maximus) fed a nucleotide-supplemented diet. Aquaculture 221(1-4):23–40. https://doi.org/10.1016/S0044-8486(03)00022-X
Makrinos DL, Bowden TJ (2016) Natural environmental impacts on teleost immune function. Fish Shellfish Immunol 53:50–57. https://doi.org/10.1016/j.fsi.2016.03.008
Mohanty BR, Sahoo PK (2010) Immune responses and expression profiles of some immune-related genes in Indian major carp, Labeo rohita to Edwardsiella tarda infection. Fish Shellfish Immunol 28(4):613–621. https://doi.org/10.1016/j.fsi.2009.12.025
Monari M, Matozzo V, Foschi J, Cattani O, Serrazanetti GP, MG M (2007) Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina. Fish Shellfish Immunol 22(1-2):98–114. https://doi.org/10.1016/j.fsi.2006.03.016
Mulder IE, Wadsworth S, Secombes CJ (2007) Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida. Fish Shellfish Immunol 23(4):747–750. https://doi.org/10.1016/j.fsi.2007.02.002
Nielsen ME, Hoi L, Schmidt AS, Qian D, Shimada T, Shen JY, Larsen JL (2001) Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China? Dis Aquat Org 46(1):23–29. https://doi.org/10.3354/dao046023
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST(C)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36. https://doi.org/10.1093/nar/30.9.e36
Pleić IL, Secombes CJ, Bird S, Mladineo I (2014) Characterization of three pro-inflammatory cytokines, TNFα1, TNFα2 and IL-1β, in cage-reared Atlantic Bluefin tuna Thunnus thynnus. Fish Shellfish Immunol 36(1):98–112. https://doi.org/10.1016/j.fsi.2013.10.011
Pratt WB, Toft DO (2003) Regulation of signalling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Experimental Biology and Medicine (Maywood) 228(2):111–133. https://doi.org/10.1177/153537020322800201
Pridgeon JW, Klesius PH, Song L, Zhang D, Kojima K, Mobley JA (2013) Identification, virulence and mass spectrometry of toxic ECP fractions of West Alabama isolates of Aeromonas hydrophila obtained from a 2010 disease outbreak. Vet Microbiol 164(3-4):336–343. https://doi.org/10.1016/j.vetmic.2013.02.020
Ren P, Xu L, Yang Y, He S, Liu W, Ringø E, Zhou Z (2013) Lactobacillus planarum subsp. plantarum JCM 1149 vs. Aeromonas hydrophila NJ-1 in the anterior intestine and posterior intestine of hybrid tilapia Oreochromis niloticus ♀ × Oreochromis aureus ♂: an ex vivo study. Fish Shellfish Immunol 35(1):146–153. https://doi.org/10.1016/j.fsi.2013.04.023
Roberts RJ, Agius C, Saliba C, Bossier P, Sung YY (2010) Heat shock proteins (chaperones) in fishes and shellfishes and their potential role in health and welfare: a review. J Fish Dis 33(10):789–801. https://doi.org/10.1111/j.1365-2761.2010.01183.x
Rodríguez-Ramos T, Carpio Y, Bolívar J, Espinosa G, Hernández-López J, Gollas-Galván T, Ramos L, Pendón C, Estrada MP (2010) An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis. Fish Shellfish Immunol 29(3):469–479. https://doi.org/10.1016/j.fsi.2010.05.013
Saeij JPJ, Stet RJM, de Vries BJ, van Muiswinkel WB, Wiegertjes GF (2003) Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance? Dev Comp Immunol 27(1):29–41. https://doi.org/10.1016/S0145-305X(02)00064-2
Saeij JPJ, Stet RJM, Groeneveld A, Verburg-van Kemenade LBM, van Muiswinkel WB, Wiegertjes GF (2000) Molecular and functional characterization of a fish inducible-type nitric oxide synthase. Immunogenetics 51(4-5):339–346. https://doi.org/10.1007/s002510050628
Secombes CJ (2016) What’s new in fish cytokine research? Fish Shellfish Immunol 53:1–3. https://doi.org/10.1016/j.fsi.2016.03.035
Shahi N, Mallik SK, Sahoo M, Das P (2013) Characteristics and pathogenicity of a virulent Aeromonas hydrophila associated with ulcerative syndrome in farmed rainbow trout, Oncorhynchus mykiss (Walbaum). Isr J Aquacult Bamidgeh 65:926–936
Stolte EH, Nabuurs SB, Bury NR, Sturm A, Flik G, Savelkoul HFJ, Verburg-van Kemenade BML (2008) Stress and innate immunity in carp: corticosteroid receptors and pro-inflammatory cytokines. Mol Immunol 46(1):70–79. https://doi.org/10.1016/j.molimm.2008.07.022
Stolte EH, Chadzinska M, Przybylska D, Flik G, Savelkoul HFJ, Verburg-van Kemenade BM (2009) The immune response differentially regulates Hsp70 and glucocorticoid receptor expression in vitro and in vivo in common carp (Cyprinus carpio L.) Fish Shellfish Immunol 27(1):9–16. https://doi.org/10.1016/j.fsi.2008.11.003
Uribe C, Folch H, Enriquez R, Moran G (2011) Innate and adaptive immunity in teleost fish: a review. Veterinarni Medicina 56:486–503
Vega VL, Rodriguez-Silva M, Frey T, Gehrmann M, Diaz JC, Steinem C, Multhoff G, Arispe N, de Maio A (2008) Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. J Immunol 180(6):4299–4307. https://doi.org/10.4049/jimmunol.180.6.4299
Yao J, Li C, Zhang J, Liu S, Feng J, Wang R, Li Y, Jiang C, Song L, Chen A, Liu Z (2014) Expression of nitric oxide synthase (NOS) genes in channel catfish is highly regulated and time dependent after bacterial challenges. Dev Comp Immunol 45(1):74–86. https://doi.org/10.1016/j.dci.2014.02.005
Zelck UE, Janje BO, Schneider O (2005) Superoxide dismutase expression and H2O2 production by hemocytes of the trematodes intermediate host Lymnaea stagnalis (Gastropoda). Dev Comp Immunol 29(4):305–314. https://doi.org/10.1016/j.dci.2004.09.002
Zelko IN, Marian TJ, Fola RJ (2002) Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structure, evolution, and expression. Free Radic Biol Med 33(3):337–349. https://doi.org/10.1016/S0891-5849(02)00905-X
Zou J, Cunningham C, Secombes CJ (1999) The rainbow trout Oncorhynchus mykiss interleukin-1b gene has a different organization to mammals and undergoes incomplete splicing. Eur J Biochem 259(3):901–908
Zhou Z, He S, Liu Y, Cao Y, Meng K, Yao B, Ringø E, Yoon I (2011) Gut microbial status induced by antibiotic growth promoter alters the prebiotic effects of dietary DVAQUA® on Aeromonas hydrophila-infected tilapia: production, intestinal bacterial community and non-specific immunity. Vet Microbiol 149(3-4):399–405. https://doi.org/10.1016/j.vetmic.2010.11.022
Acknowledgements
We gratefully acknowledge the help of Benkõ Lászlóné, Holp Józsefné, and Bogár Katalin of HAKI for their technical assistance and fish husbandry during our work.
Funding
During this work at the National Agricultural Research and Innovation Centre, Research Institute for Fisheries and Aquaculture (NARIC, HAKI), Szarvas, Hungary, the first author (Neetu Shahi) was supported by research stay grant from Tempus Public Foundation (TPF), Hungary. This research did not receive and specific grant from funding agencies in the public, commercial, or not for profit sectors.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Supplementary Table 1
(DOCX 15 kb)
Rights and permissions
About this article
Cite this article
Shahi, N., Ardó, L., Fazekas, G. et al. Immunogene expression in head kidney and spleen of common carp (Cyprinus carpio L.) following thermal stress and challenge with Gram-negative bacterium, Aeromonas hydrophila. Aquacult Int 26, 727–741 (2018). https://doi.org/10.1007/s10499-018-0250-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10499-018-0250-6