[en] Monitoring the immune response in fish over the progression of a disease is traditionally carried out by experimental infection whereby animals are killed at regular intervals and samples taken. We describe here a novel approach to infectiology for salmonid fish where blood samples are collected repeatedly in a small group of PIT-tagged animals. This approach contributes to the reduction of animals used in research and to improved data quality. Two groups of 12 PIT-tagged Atlantic salmon (Salmo salar) were i.p infected with Infectious Salmon Anaemia Virus (ISAV) or culture medium and placed in 1 m3 tanks. Blood samples were collected at 0, 4, 8, 12, 16, 21 and 25 days post infection. The viral load, immune and stress response were determined in individual fish by real-time quantitative PCR (QPCR) on the blood cells, as well as the haematocrit used as an indicator of haemolysis, a clinical consequence of ISAV infection. "In-tank" anaesthesia was used in order to reduce the stress related to chase and netting prior to sampling. The data were analysed using a statistical approach which is novel with respect to its use in fish immunology. The repeated blood collection procedure did not induce stress response as measured by HSP70 and HSP90 gene expression in the un-infected animals. A strong increase in viraemia as well as a significant induction of Mx and gammaIP gene expression were observed in the infected group. Interleukin 10 was found induced at the later stage of the infection whereas no induction of CD8 or gamma IFN could be detected. These results and the advantages of this approach are discussed.
Turner PV, Brabb T, Pekow C, Vasbinder MA. Administration of Substances to Laboratory Animals: Routes of Administration and Factors to Consider. J Am Assoc Lab Anim Sci. 2011; 50: 600-613. PMID: 22330705
Diehl K-H, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, et al. A Good Practice Guide to the Administration of Substances and Removal of Blood, Including Routes and Volumes. J Appl Toxicol. 2001; 21: 15-23. PMID: 11180276
Wiles S, Hanage WP, Frankel G, Robertson B. Opinion: Modelling infectious disease-time to think outside the box? Nat Rev Microbiol. 2006; 4: 307-312. PMID: 16518420
Peters LL, Robledo RF, Bult CJ, Churchill GA, Paigen BJ, Svenson KL. The mouse as a model for human biology: a resource guide for complex trait analysis. Nat Rev Gen. 2007; 8: 58-69.
Baker RF, Blanchfield PJ, Paterson MJ, Flett RJ, Wesson L. Evaluation of Nonlethal Methods for the Analysis of Mercury in Fish Tissue. Trans Am Fish Soc. 2004; 133: 568-576.
Kelly MH, Hagar WG, Jardine TD, Cunjak RA. Non-lethal Sampling of Sunfish and Slimy Sculpin for Stable Isotope Analysis: How Scale and Fin Tissue Compare with Muscle Tissue. North American J Fish Management 2006; 26: 921-925.
Bruno D, Collet B, Turnbull A, Kilburn R, Walker A, Gubbins M, et al. Examination of diagnostic methods for Renibacterium salmoninarum causing bacterial kidney disease (BKD) in the UK. Aquaculture 2007; 269: 114-122.
Drennan JD, Lapatra SE, Samson CA, Ireland S, Eversman KF, Cain KD. Evaluation of lethal and nonlethal sampling methods for the detection of white sturgeon iridovirus infection in white sturgeon, Acipenser transmontanus (Richardson). J Fish Dis. 2007; 30: 367-379. PMID: 17498180
Ellis AE, Cavaco A, Petrie A, Lockhart K, Snow M, Collet B. Histology, immunocytochemistry and qRTPCR analysis of Atlantic salmon, Salmo salar L., post-smolts following infection with infectious pancreatic necrosis virus (IPNV). J Fish Dis. 2010; 33: 803-818. doi: 10.1111/j.1365-2761.2010.01174.x PMID: 20561142
Raida MK, Holten-Andersen L, Buchmann K. Association between Yersinia ruckeri infection, cytokine expression and survival in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2011; 30: 1257-1264. doi: 10.1016/j.fsi.2011.03.022 PMID: 21501689
Fluttert M, Dalm S, Oitzl MS. A refined method for sequential blood sampling by tail incision in rats. Lab Anim. 2000; 34: 372-378. PMID: 11072857
Murray AG, Smith RJ, Stagg RM. Shipping and the Spread of Infectious Salmon Anaemia in Scottish Aquaculture. Emerg Infect Dis. 2002; 8: 1-5. PMID: 11749740
Conte FP, Wagner HH, Harris TO. Measurement of blood volume in the fish (Salmo gairdneri gairdneri). Am J Physiol. 1963; 205: 533-540. PMID: 14065906
Billett HH. Hemoglobin and Hematocrit. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. Boston: Butterworths; 1990. pp 718-719.
Collet B, Ganne G, Bird S, Collins C. Isolation and expression profile of a gene encoding for the Signal Transducer and Activator of Transcription STAT-2 in Atlantic salmon (Salmo salar). Dev Comp Immunol. 2009; 33: 821-829. doi: 10.1016/j.dci.2009.01.007 PMID: 19428483
Collet B, Bain N, Prevost S, Besinque G, McBeath A, Snow M, et al. Atlantic salmon Salmo salar Signal Transducer and Activator of Transcription STAT-1: a contrasting kinetics of expression upon ISAV or IPNV infection. Fish Shellfish Immunol. 2008; 25: 861-867. doi: 10.1016/j.fsi.2008.01.011 PMID: 18996723
Collet B, Collins C. Comparative gene expression profile in two Atlantic salmon cell lines TO and SHK-1. Vet Immunol Immunopathol. 2009; 130: 92-95. doi: 10.1016/j.vetimm.2008.12.022 PMID: 19162334
McBeath AJA, Snow M, Secombes CJ, Ellis AE, Collet B. Expression kinetics of interferon and interferon-induced genes in Atlantic salmon following infection with IPNV and ISAV. Fish Shellfish Immunol. 2007; 22: 230-241. PMID: 16806972
Fisher RA. Statistical methods for research workers. Edinburgh: Oliver & Boyd; 1934.
R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2014.
Yee TW. The VGAM package for categorical data analysis. Journal of Statistical Software. 2010; 32 (10): 1-34.
Searle SR. Linear Models. New York: John Wiley and Sons; 1971.
Steiner E, Balmelli C, Gerber H, Summerfield A, McCullough K. Cellular adaptive immune response against porcine circovirus type 2 in subclinically infected pigs. BMC Vet Res. 2009; 5: 45. doi: 10.1186/1746-6148-5-45 PMID: 20028550
Grant CFJ, Lefevre EA, Carr BV, Prentice H, Gubbins S, Pollard AJ, et al. Assessment of T-dependent and T-independent immune responses in cattle using a B cell ELISPOT assay. Vet Res. 2012; 43: 68. doi: 10.1186/1297-9716-43-68 PMID: 23050495
Cornwell E, Bellmund C, Groocock G, Wong P, Hambury K, Getchell R, et al. Fin and gill biopsies are effective non-lethal samples for detection of Viral haemorrhagic septicaemia virus genotype IVb. J Vet Diagn Invest. 2013; 25: 203-209. doi: 10.1177/1040638713476865 PMID: 23404480
McCormick SD. Method for nonlethal Gill Biopsy and measurement of Na+, K+-ATPase activity. Can J Fish Aquat Sci. 1993; 50: 656-658.
Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell. 1994; 76: 241-251. PMID: 7904900
Collet B. Innate immune responses to viral infections of salmonid fish. Dev Comp Immunol. 2014; 43: 160-173. doi: 10.1016/j.dci.2013.08.017 PMID: 23981327
Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Moser K, Ortmann WA, et al. Expression levels for many genes in human peripheral blood cells are highly sensitive to ex vivo incubation. Genes Immun. 2004; 5: 347-353. PMID: 15175644
Gingerich WH, Pityer RA, Rach JJ. Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri). Comp Biochem Physiol. 1987; 87: 251-256.
Carragher JF, Sumpter JP. Corticosteroid Physiology in Fish. In: Epple A, Scanes CG, Stetson MH, editors. Progress in Comparative Endocrinology. New-York: Wiley-Liss; 1990. pp 487-92.
Klontz GW. Care of Fish in Biological Research. J Animal Sci. 1995; 73: 3485-3492.
Wedemeyer G. Some Physiological Consequences of Handling Stress in the Juvenile Coho Salmon and Steelhead Trout. J Fisheries Res Board Canada. 1972; 29: 1780-1783.
Snow M, Raynard R, Bruno DW, van Nieuwstadt AP, Olesen NJ, Løvold T, et al. Investigation into the susceptibility of saithe Pollachius virens to infectious salmon anaemia virus (ISAV) and their potential role as a vector for viral transmission. Dis Aquat Org. 2002; 50: 13-18. PMID: 12152900
Collet B, Urquhart K, Noguera P, Larsen K, Lester K, Smail D, et al. A method to measure an indicator of viraemia in Atlantic salmon using a reporter cell line. J Virol Meth. 2013; 191: 113-117.
Aamelfot M, Weli SC, Dale OB, Koppang EO, Falk K. Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor. J Anat. 2013; 222: 547-557. doi: 10.1111/joa.12033 PMID: 23439106
Aamelfot M, Dale OB, Weli SC, Koppang EO, Falk K. Expression of the infectious salmon anaemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus. J Virol. 2012; 86: 10571-10578. doi: 10.1128/JVI.00047-12 PMID: 22811536
Workenhe ST, Wadowska DW, Wright GM, Kibenge MJ, Kibenge FS. Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon. Virol J. 2007; 4: 13. PMID: 17254352
Weli SC, Aamelfot M, Dale OB, Koppang EO, Falk K. Infectious salmon anaemia virus infection of Atlantic salmon gill epithelial cells. Virol J. 2013; 10: 5. doi: 10.1186/1743-422X-10-5 PMID: 23282149
Moneke E, Ikede BO, Kibenge FS. Viraemia during infectious salmon anaemia virus infection of Atlantic salmon is associated with replicating virus in leucocytes. Dis Aquat Org. 2005; 5: 153-157.
Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, et al. The Nature of the Principal Type 1 Interferon-Producing Cells in Human Blood. Science. 1999; 284: 1835-1837. PMID: 10364556
Sathiyaa R, Campbell T, Vijayan MM. Cortisol modulates HSP90 mRNA expression in primary cultures of trout hepatocytes. Comp Biochem Physiol. 2001; 129: 679-685.
Naito T, Momose F, Kawaguchi A, Nagata K. Involvement of Hsp90 in assembly and nuclear import of influenza virus RNA polymerase subunits. J Virol. 2007; 81: 1339-1349. PMID: 17121807
Chase G, Deng T, Fodor E, Leung BW, Mayer D, Schwemmle M, et al. Hsp90 inhibitors reduce influenza virus replication in cell culture. Virology. 2008; 377: 431-439. doi: 10.1016/j.virol.2008.04.040 PMID: 18570972
Jørgensen SM, Afanasyev S, Krasnov A. Gene expression analyses in Atlantic salmon challenged with infectious salmon anaemia virus reveal differences between individuals with early, intermediate and late mortality. BMC Genomics. 2008; 9: 179. doi: 10.1186/1471-2164-9-179 PMID: 18423000
Ejrnaes M, Filippi CM, Martinic MM, Ling EM, Togher LM, Crotty S, et al. Resolution of a chronic viral infection after interleukin-10 receptor blockade. J Exp Med. 2006; 203: 2461-2472. PMID: 17030951
Tsai T-T, Chuang Y-J, Lin Y-S, Wan S-W, Chen C-L, Lin C-F. An emerging role for the anti-inflammatory cytokine interleukin-10 in dengue virus infection. J Biomed Sci. 2013; 20:40. doi: 10.1186/1423-0127-20-40 PMID: 23800014
Ouyang P, Rakus K, van Beurden SJ, Westphal AH, Davison AJ, Gatherer D, et al. IL-10 encoded by viruses: a remarkable example of independent acquisition of a cellular gene by viruses and its subsequent evolution in the viral genome. J. Gen. Virol. 2014; 95: 245-262. doi: 10.1099/vir.0.058966-0 PMID: 24225498