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Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida
Fish & Shellfish Immunology 23 (2007) 747e759 www.elsevier.com/locate/fsi
Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida I.E. Mulder a,1, S. Wadsworth b, C.J. Secombes a,* a
Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen AB24 2TZ, Scotland, UK b Ewos Innovation, N-4335 Dirdal, Norway Received 30 November 2006; revised 12 February 2007; accepted 15 February 2007 Available online 24 February 2007
Abstract Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1b, IL-8, TNF-a and IFN-g was increased in the proximal intestine. TGF-b was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1b, IL-8, TNFa and TGF-b. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Rainbow trout; Intestine; Cytokines; Aeromonas salmonicida
1. Introduction The mucosa-associated lymphoid tissues in teleost fish (gut, skin and gills), with their layer of mucus and array of non-specific immune defences, provide an initial barrier to the entry of pathogens [1,2]. Mucus secreted by goblet cells reduces the ability of bacteria to adhere to underlying enterocytes [3]. The digestive function of the gut provides an extremely hostile environment to potential pathogens because of the low pH and the presence of digestive enzymes and bile [1]. The gut-associated lymphoid tissue (GALT) is present in teleost fish as individual cells and small aggregations in both the epithelium and lamina propria [4]. GALT in fish lacks Peyer’s patches and M cells [5], however, some evidence exists that the distal intestine contains functional analogues of mammalian M cells. These cells seem to be specialised in the uptake and processing of antigens [6]. Cells associated with the intestinal immune system of * Corresponding author. Tel.: þ44 1224 272870; fax: þ44 1224 272396. E-mail address: [email protected] (C.J. Secombes). 1 Present address: Gut Immunology Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK. 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.02.002
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fish are diverse and different from those recovered from the spleen and the head kidney [5,7], and include intraepithelial cells, macrophages and eosinophilic granular cells [1]. The largest numbers of lymphocytes and plasma cells are found dispersed in the connective tissue of the lamina propria and within the epithelial layer [5,8]. IgM and IgMproducing cells have been found in the mucosal epithelium, the submucosa and the lamina propria [9,10]. IgM is transported across the epithelial cells and released into the lumen [11]. Fish lack secretory antibody similar to mammalian IgA, which is resistant to proteolytic degradation in the gut [12], and serum IgM added to gut mucus is rapidly degraded [13]. Cytokines play an important role in the immune system by binding to specific receptors at the cell membrane, setting off a cascade that leads to induction, enhancement or inhibition of a number of cytokine-regulated genes in the nucleus. Many important cytokines have been identified in teleost fish in the last number of years, including interleukin-1b (IL-1b), an important pro-inflammatory cytokine [14], tumour necrosis factor-a (TNF-a) [15], interferon (IFN) [16], transforming growth factor-b (TGF-b) [17] and several chemokines [18]. Numerous other cytokines have recently been discovered in teleosts, including amongst others IL-2 [19], IL-6 [20], IL-10 [21], IL-12 [22], IL-15 [23] and IL-21 [19], as well as the first non-mammalian interleukin-11 (IL-11) gene from rainbow trout (Oncorhynchus mykiss) [24]. The non-motile, Gram-negative bacterium Aeromonas salmonicida, the causative agent of furunculosis, is commonly used as a model pathogen for studies of hostepathogen interactions in salmonids. A. salmonicida leads to whole body infection and acute mortality in susceptible salmonids. Clinical signs of relevance to this study include hemorrhagic and necrotic lesions in the gut [25] and destruction of the stratum compactum of the distal intestine [26,27]. Also, because the infected fish stop feeding, the lumen of the intestine may contain sloughed epithelial cells, mucus and blood. The outcome of infection locally may be mediated in large part by the cytokine response elicited. Little is known about the cytokine profile of cells in the fish gut, and in this study their ability to express a number of important cytokine genes is examined in control fish and those exposed to a virulent strain of A. salmonicida in the water. The key components of the classical cytokine cascade induced by Gram-negative bacterial infection, TNF-a, IL-1b and the chemokine IL-8, together with a major activator (IFN-g) and down-regulator (TGF-b) of cell-mediated immune responses were chosen for study. 2. Materials and methods 2.1. Animals Rainbow trout, weighing approximately 70 g, were purchased from Almondbank (Perthshire, UK) and stocked in two 350 L tanks, with each tank containing approximately 60 fish. The fish were allowed a two-week acclimatisation period before the start of the experiment. They were fed on a standard commercial feed (EWOS Ltd) throughout the experiment. 2.2. Bacteria The pathogenic A. salmonicida strain MT423 (Marine Lab, Aberdeen) was grown for 16 h in tryptic soy broth (TSB, Fluka Biochemika) at 22 C in a shaking incubator at 200 rpm. The concentration of the bacterial suspension was determined using a bacterial counting chamber. Fish were exposed to the bacteria by bath infection. The bacterial suspension was added to the water to give a final concentration of 1 105 bacteria/mL tank water. The fish were exposed to the bacteria for 24 h, during which time the tanks were isolated from the ingoing and outgoing water flow. After the incubation time, the water flow was turned on again. Wastewater was treated by ozonation to ensure no live bacteria were released. Fish were regularly checked throughout the experiment and dead fish were recorded. 2.3. Tissue sampling, total RNA isolation and RT-PCR analysis Six fish were killed for sampling at day 0, before bath challenge was commenced, and six further fish were killed for sampling at days 2, 4, 6 and 8, the last sample taken as mortalities were beginning to occur. The head kidney, the
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proximal intestine and the distal intestine were removed, rinsed in cold phosphate buffered saline, pH 7.2 (PBS, Gibco) and stored in 1 mL Trizol (Gibco) at 80 C. Tissue samples were used for RT-PCR to ascertain the gene expression levels of b-actin, IL-1b, IFN-g, TNF-a, IL-8 and TGF-b. The samples were homogenised with a sonicator prior to extraction, and RNA was extracted from the homogenised tissues using Trizol (Invitrogen). Reverse transcription to cDNA was carried out. Five microgram RNA in 11 mL DEPC-water was incubated with 1 mL oligo (dT) 12e18 (500 mg/mL, Invitrogen) for 10 min at 70 C and chilled on ice for at least 3 min. One microlitre Moloney Murine Leukaemia Virus (MMLV) reverse transcriptase (Bioline), 4 mL 5 first strand buffer (Bioline), 1.25 mL 10 mM dNTPs (Bioline) and 1.75 mL water were added, and the reaction was incubated at 42 C for 90 min followed by 5 min at 95 C. The resultant cDNA was dissolved in water and stored at 20 C. PCR was carried out using different primer sets and conditions (Table 1). cDNA (1 mL) was added to an RNAse free tube, followed by 24 mL of a mastermix containing 2.5 mL 10 NH4 Buffer (Bioline), 1 mL 2.5 mM dNTPs (Bioline), 0.75 mL 50 mM MgCl2 solution (Bioline), 17.625 mL PCR H2O (Bioline), 1 mL forward primer and 1 mL reverse primer (Sigma, Table 1) and 0.125 mL 5 U/mL Taq DNA Polymerase (Bioline). The solution was mixed and briefly centrifuged and then subjected to a gene-specific heat-protocol (Table 1) in a Techne thermal cycler. A b-actin PCR was initially performed, at 26 cycles, and the amount of template added adjusted to give approximately equal PCR products between samples. During this procedure the reproducibility of the products obtained from individual fish was confirmed. The same amount of cDNA was then used for all immune gene PCRs as a way of normalising the data in order to give a more quantitative result. Optimal cycling number of the other genes was determined to Table 1 Oligonucleotide primers used, and PCR heat-protocol of each gene investigated Gene
Primer sequence
Temp. ( C)
Time
Cycles
b-Actin
F 50 -ATCGTGGGGCGCCCCAGGCACC-30 R 50 -CTCCTTAATGTCACGCACGATTT-30
94 94 56 72 72
2 min 30 s 30 s 30 s 10 min
1 26 26 26 1
IL-1B
F 50 -ACAGACATGGATTTTGAGTCA-30 R 50 -CTCATACTGTGATGTACTGCTGA-30
94 94 57 72 72
2 min 30 s 30 s 10 s 10 min
1 35 35 35 1
TNF-aa
F 50 -CAAGAGTTTGAACCTCATTCAG-30 R 50 -TGGCAACGATGCAGGACGGAA-30
94 94 62 72 72
2 min 25 s 25 s 25 s 10 min
1 37 37 37 1
IL-8
F 50 -TCCAGACAAATCTCCTGACCG-30 R 50 -GGATGTCAGCCAGCCTTGTC-30
94 94 56 72 72
2 min 30 s 30 s 30 s 10 min
1 32 32 32 1
TGF-b
F 50 -GAAGAAACGACAAACCACTAC-30 R 50 -GACATGTGCAGTAATTCTAGC-30
94 94 50 72 72
2 min 30 s 30 s 30 s 10 min
1 35 35 35 1
IFN-g
F 50 -GTGAGCAGAGGGTGTTGATG-30 R 30 -GATGGTAATGAACTCGGACAG-30
94 94 59 72 72
2 min 30 s 30 s 10 s 10 min
1 35 35 35 1
a
These primers amplified the rainbow trout TNF-a2 gene, as described in Ref. [19].
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generate a product that was visible but not saturated, and found to be between 32 and 37 cycles, depending on the specific gene. All PCR products were visualized on a 2% agarose gel containing 100 ng/mL ethidium bromide. The products were run on the gel for 1 h at 100 V, using a 100 bp DNA ladder (Bioline) as a size marker. Each gel was then examined using a UVP gel imaging system. 2.4. Data analysis UVP Gel-works ID advanced software was used to calculate ratios of cytokine gene expression relative to b-actin expression using the pixel density for each product. This enabled the evaluation of differential expression of the selected genes between different sample groups. The data were plotted as box plots with median values. Statistical analysis of gene expression results was performed using the Kruskal-Wallis test (P < 0.05) followed by the ManneWhitney test (P < 0.05) if significance was found.
3. Results 3.1. IL-1b gene expression Low level, constitutive expression of IL-1b was observed in the head kidney, distal intestine and proximal intestine of uninfected fish at day 0 (Fig. 1). Bath challenge with A. salmonicida caused a small, but significant decrease in IL1b mRNA levels at days 2, 4 and 8 in the head kidney (P < 0.01). At day 6, IL-1b expression was high in three out of six fish, but due to the large variation within the group, this was not significantly different from expression levels in uninfected fish. No significant differential expression was observed in the distal intestine. However, a large increase in IL-1b expression was observed at day 6 in the proximal intestine in three out of six fish (P < 0.01). Expression was still high in two out of six fish at day 8 post-infection, but this was not significantly different from uninfected fish. 3.2. IL-8 gene expression Constitutive IL-8 gene expression was observed in uninfected fish, especially in the head kidney (Fig. 2). Expression in this tissue during infection was decreased at days 2 and 4 (P < 0.01). After this time, IL-8 expression returned to levels similar to those found in non-infected fish. A significant trend in IL-8 expression was also found in the distal intestine (P < 0.05), but no significant differences in IL-8 levels compared to uninfected fish were found. Strong IL-8 induction was observed in the proximal intestine (P < 0.05) during A. salmonicida infection, with expression increased up to 11-fold compared to uninfected tissue. 3.3. TNF-a gene expression The proximal intestine of uninfected fish showed quite strong TNF-a gene expression, compared to the head kidney and the distal intestine (Fig. 3). Infection with A. salmonicida by bath challenge had most effect on TNF-a mRNA production in the proximal intestine. However, a statistically significant decrease of expression was seen in the head kidney (P < 0.01) at days 4, 6 and 8. TNF-a was significantly decreased at days 4 and 6 post-infection in the proximal intestine, but significantly increased at day 8 (P < 0.0001), with an approximately 5-fold increase in mRNA levels compared to uninfected fish. 3.4. TGF-b gene expression TGF-b was strongly expressed in all three tissues of uninfected fish, especially in the head kidney (Fig. 4). Infection with A. salmonicida by bath challenge caused a significant decrease in TGF-b expression in the head kidney (P < 0.0001) and the distal intestine (P < 0.01) at all timepoints.
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Fig. 1. Gene expression of IL-1b as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 2. Gene expression of IL-8 as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 3. Gene expression of TNF-a as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 4. Gene expression of TGF-b as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 5. Gene expression of IFN-g as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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3.5. IFN-g gene expression Low level, constitutive IFN-g gene expression was observed in all three tissues of uninfected fish at day 0 (Fig. 5). Variation in tissue expression was observed in the head kidney and distal intestine during infection, but this was not significant at any timepoint. Differential expression of IFN-g was observed in the proximal intestine. One fish showed very strong IFN-g expression at day 2, but the other fish in this group did not show any differential expression. A significant decrease at days 4 and 6 post-infection was followed by a 3-fold increase at day 8 post-infection (P < 0.01). 4. Discussion It has been proposed that the gastrointestinal tract is the principal infection route of A. salmonicida in various salmonid species, such as Arctic charr (Salvelinus alpinus) [28], due to the fact that A. salmonicida can be detected in the submucosa of the midgut and diverse dietary treatments cause different mortality figures. Other studies further illustrate the role of the intestine as an important route of infection with A. salmonicida. Atlantic salmon (Salmo salar L.) and rainbow trout fed a diet containing the probiotic bacterium Carnobacterium sp. showed reduced mortality after challenge with A. salmonicida, possibly due to the ability of the probiotic to prevent potential pathogens from colonising the gut [29]. The gut has also been shown to respond to A. salmonicida, particularly in studies using killed bacteria within vaccines [30] or administered extracellular products [31]. Such responses may be crucial to the outcome of the infection, and in part will be regulated by the local cytokine response elicited within the gastrointestinal tract. In the current study, rainbow trout were infected with A. salmonicida by bath challenge and the cytokine expression profile in the intestine post-challenge established. Differential expression mostly occurred in the proximal intestine. In this region, gene upregulation of the pro-inflammatory cytokines IL-1b, IL-8, TNF-a and IFN-g was observed. The finding that IL-1b was increased during A. salmonicida infection is not surprising, as this cytokine plays an important role in the regulation of immune and inflammatory processes. IL-1b induces the growth and proliferation of T and B lymphocytes, macrophages, vascular endothelial cells and tissue cells [32]. It has also been shown in rainbow trout that challenge with an attenuated strain of A. salmonicida induces IL-1b transcription in a range of tissues, including blood, gills, kidney, liver and spleen [33]. Expression of IL-1b can also be induced in head kidney leukocytes and macrophages by stimulation with lipopolysaccharide (LPS) [14,34]. In vitro stimulation of cells with recombinant IL-1b, as well as intraperitoneal injection with recombinant IL-1b, enhances phagocytosis and increases expression of IL-1b, COX-2, lysozyme and the MHC class II b chain [35,36]. TNF-a was also significantly increased in the proximal intestine during bath challenge. TNF-a2 specifically was monitored in this study, which is the predominant TNF-a isoform expressed in trout upon stimulation [37]. TNF-a, like IL-1b, has a variety of functions in the regulation of inflammation and cellular immune responses including the stimulation of respiratory burst activity and phagocytosis [38]. It is produced by a diverse range of cells, but mainly by macrophages/monocytes and T lymphocytes [15,38]. TNF-a is constitutively expressed in the gill and kidney of rainbow trout but not in the blood, brain and liver. Co-expression of IL-1b and TNF-a is not unexpected, since these cytokines have similar functions in the initiation of immune responses. Head kidney leukocytes and RTS-11 cells stimulated with recombinant IL-1b produce TNF-a [15], while addition of recombinant TNF-a to the same cells leads to expression of IL-1b, together with TNF-a1, TNF-a2, IL-8 and COX-2 [38]. Concomitant expression of IL-8 was observed in the proximal intestine during infection. IL-8 has chemotactic ability, and attracts neutrophils but not monocytes and macrophages [18]. Thus, the expression of IL-8 in the proximal intestine at days 2, 6 and 8 indicates a mechanism to attract and maintain neutrophil infiltration at the site of infection. IL-8 production is stimulated by the expression of IL-1b and TNF-a, so it is not surprising to see the simultaneous expression of these three cytokines. TGF-b showed no differential expression in the proximal intestine of fish infected by A. salmonicida; however, IFN-g was increased at this site post-challenge. IFN-g is a type II IFN and is produced by activated T lymphocytes and NK cells [16]. IFN-g is a strong activator of macrophages and the key cytokine of type 1 T helper (Th1) cell immune responses during infections with intracellular pathogens, autoimmune diseases and anti-tumour defence [39]. IFN-g is not expressed constitutively in vitro in head kidney cells from rainbow trout, but is inducible by addition of PHA or poly (I:C). In vivo expression of IFN-g is observed in the head kidney and spleen after i.p. injection of poly I:C [16]. It is perhaps surprising that these increases in cytokine expression are observed mostly at days 6 and
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8, as it is presumed that the gut surface is exposed to the bacteria in the first few days of the challenge. However, largescale infection does not occur until days 6 and 7, when the first mortalities take place in this challenge model. Little differential cytokine expression was observed in the distal part of the intestine during infection, apart from decreased expression of TGF-b. This lack of differential expression could be due to the absence of pathogens in the distal part of the intestine. A. salmonicida ingested by the fish travels through the stomach to reach the intestine and is then probably lysed by digestive enzymes and immune reactions. Therefore, few bacteria may be able to infect the distal intestine. Whilst intuitively the stomach is a place for this to occur, in fact Bøgwald et al. [30] found that the stomach has relatively low degradative powers on the bacterium and that A. salmonicida orally intubated into the stomach of Atlantic salmon are not lysed here but travel into the intestine. However, the capacity of the pylorus, midgut and hindgut to lyse A. salmonicida is much greater. Nevertheless, Aeromonas spp. can survive in the piscine gut, as evidenced by the isolation of the bacteria from the healthy gut of rainbow trout [40] and catfish [41]. Another possible reason for the lack of differential cytokine expression in the distal intestine could be due to the fact that the sampling timepoints were too late to detect an acute response that could have occurred in the first two days after bacterial challenge. Following intraperitoneal injection of A. salmonicida, expression of IL-1b, TNF-a and IL-8 occurs quickly (in the first 24 h) in the liver and kidney of pink salmon (Oncorhynchus gorbuscha) and chum salmon (Oncorhynchus keta) [42]. Differential expression of IL-1b, IL-8, TNF-a and TGF-b is also observed within 12 h of exposure of isolated head kidney cells to probiotic bacteria [43]. However, in the present study a bath challenge was used and could be expected to result in significantly different kinetics of exposure. Indeed, peaks in gene expression took place mostly at days 6 and 8 post-infection and were therefore not obviously decreasing from earlier sampling points. A. salmonicida has effects on the epithelial layer of the distal and proximal intestinal regions. In the distal intestine, A. salmonicida causes little detachment of enterocytes but cellular damage is observed involving the microvilli, desmosomes and tight junctions. Substantial detachment of enterocytes is observed in the proximal intestine, and all the enterocytes can be seen detached from the epithelium in certain regions and the basement membrane is exposed [44]. This suggests that the proximal intestine is a more likely infection route for A. salmonicida. However, since loosening of cell junctions is observed in the distal intestine, any bacteria that are able to survive gut lysis and reach the distal intestine will be able to take advantage of this fact. The head kidney showed a very different cytokine profile compared to the intestine. There was a general trend toward a decrease in expression (IL-1b, IL-8, TNF-a and TGF-b), with minimum levels reached at day 4 post-infection. However, at the later stages expression of TNF-a and IL-8 were increased again. This could be due to depletion of immune cells from the head kidney in the earlier stages of the experiment, to sites of infection and contact, such as the mucosal surfaces of the gills, skin and gut. The immune response of fish against A. salmonicida depends on the co-operation of both humoral and cellular immune responses. The outcome of the current study suggests the presence of a cellular response to A. salmonicida in the intestine of rainbow trout due to the differential expression of numerous cytokines. Further work is needed to determine the function of this pro-inflammatory cytokine expression during A. salmonicida infection, in terms of the effects on eventual survival and recovery from infection. Acknowledgments This study was supported financially by EWOS Ltd. References [1] Dalmo RA, Ingebrigtsen K, Bøgwald J. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). J Fish Dis 1997;20:241e73. [2] Press CM, Eversen O. The morphology of the immune system in teleost fishes. Fish Shellfish Immunol 1999;9:309e18. [3] Ellis AE. Innate host defence mechanisms of fish against viruses and bacteria. Dev Comp Immunol 2001;25:827e39. [4] Doggett A, Harris JE. The ontogeny of the gut-associated lymphoid tissue in Oreochromis mossambicus. J Fish Biol 1987;31:23e7. ˚ , Bakke-McKellep AM. The intestines of carnivorous fish: structure and functions and the relations with diet. [5] Buddington RK, Krogdahl A Acta Physiol Scand 1997;161:67e80. [6] Rombout JHWM, Bot HE, Taverne-Thiele JJ. Immunologic importance of the second gut segment of carp. II. Characterization of mucosal leucocytes. J Fish Biol 1989;35:167e78.
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Fish Disease Leaflet 2001;66:1e32. [26] Scott M. The pathogenicity of Aeromonas salmonicida in sea and brackish waters. J Gen Microbiol 1968;50:321e7. [27] Drinan EM, Donlon J, Molloy S, Smith PR. Observations on the role of the caseinase enzyme in the pathogenesis of furunculosis. J Fish Dis 1989;12:45e50. [28] Lødemel JB, Mayhew TM, Myklebust R, Olsen RE, Espelid S, Ringø E. Effect of three dietary oils on disease susceptibility in Arctic charr (Salvelinus alpinus L.) during cohabitant challenge with Aeromonas salmonicida ssp. salmonicida. Aquac Res 2001;32:935e45. [29] Robertson PAW, O’Dowd C, Burrells C, Williams P, Austin B. Use of Carnobacterium sp. as a probiotic for Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss, Walbaum). Aquaculture 2000;185:235e43. [30] Bøgwald J, Stensva˚g K, Stuge TB, Jørgensen TØ. Tissue localisation and immune responses in Atlantic Salmon, Salmo salar L., after oral administration of Aeromonas salmonicida, Vibrio anguillarum and Vibrio salmonicidia antigens. Fish Shellfish Immunol 1994;4:353e68. [31] Powell MD, Briand HA, Wright GM, Burka JF. Rainbow trout (Oncorhynchus mykiss Walbaum) intestinal eosinophilic granule cell (EGC) response to Aeromonas salmonicida and Vibrio anguillarum extracellular products. Fish Shellfish Immunol 1993;3:279e89. [32] Bird S, Zou J, Wang T, Munday B, Cunningham C, Secombes CJ. Evolution of interleukin-1b. Cytokine Growth Factor Rev 2002;13: 483e502. [33] Zou J, Cunningham C, Secombes CJ. The rainbow trout Oncorhynchus mykiss interleukin-1b gene has a different organization to mammals and undergoes incomplete splicing. Eur J Biochem 1999;259:901e8. [34] Zou J, Holland J, Pleguezuelos O, Cunningham C, Secombes CJ. Factors influencing the expression of interleukin-1b in cultured rainbow trout (Oncorhynchus mykiss) leucocytes. Dev Comp Immunol 2000;24:575e82. [35] Peddie S, Zou J, Cunningham C, Secombes CJ. Rainbow trout (Oncorhynchus mykiss) recombinant IL-1b and derived peptides induce migration of head-kidney leucocytes in vitro. Fish Shellfish Immunol 2001;11:697e709. [36] Hong S, Peddie S, Campos-Perez JJ, Zou J, Secombes CJ. The effect of intraperitoneally administered recombinant IL-1b on immune parameters and resistance to Aeromonas salmonicida in the rainbow trout (Oncorhynchus mykiss). Dev Comp Immunol 2003;27:801e12. [37] Zou J, Wang T, Hirono I, Aoki T, Inagawa H, Honda T, et al. Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss. Dev Comp Immunol 2002;26:161e72. [38] Zou J, Peddie S, Scapigliati G, Zhang Y, Bols NC, Ellis AE, et al. Functional characterisation of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss. Dev Comp Immunol 2003;27:813e22. [39] Bogdan C, Mattler J, Schleicher U. 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[40] Spanggaard B, Huber I, Nielsen J, Nielsen T, Appel KF, Gram L. The microflora of rainbow trout intestine: a comparison of traditional and molecular identification. Aquaculture 2000;182:1e15. [41] DePaola A, Peeler JT, Rodrick GE. Effect of oxytetracycline-medicated feed on antibiotic resistance of Gram-negative bacteria in catfish ponds. Appl Environ Microbiol 1995;61:2335e40. [42] Fast MD, Johnson SC, Jones SR. Differential expression of the pro-inflammatory cytokines IL-1beta-1, TNFalpha-1 and IL-8 in vaccinated pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon juveniles. Fish Shellfish Immunol 2006;22:403e7. [43] Kim DH, Austin B. Cytokine expression in leucocytes and gut cells of rainbow trout, Oncorhynchus mykiss Walbaum, induced by probiotics. Vet Immunol Immunopathol 2006;114:297e304. [44] Ringø E, Jutfelt F, Kanapathippillai P, Bakken Y, Sundell K, Glette J, et al. Damaging effects of the fish pathogen Aeromonas salmonicida ssp. salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar K.). Cell Tissue Res 2004;318:305e11.
Cytokine expression in the intestine of rainbow trout (Oncorhynchus mykiss) during infection with Aeromonas salmonicida I.E. Mulder a,1, S. Wadsworth b, C.J. Secombes a,* a
Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen AB24 2TZ, Scotland, UK b Ewos Innovation, N-4335 Dirdal, Norway Received 30 November 2006; revised 12 February 2007; accepted 15 February 2007 Available online 24 February 2007
Abstract Gene expression of a number of cytokines in the intestine of rainbow trout (Oncorhynchus mykiss) was investigated after challenge with a pathogenic strain of Aeromonas salmonicida. Fish were exposed to A. salmonicida by immersion in a bacterial suspension (bath challenge) and tissue samples of the distal and proximal intestine were collected at days 0, 2, 4, 6 and 8 post-exposure. Head kidney tissue was also collected to assess the effect in a systemic immune tissue. A classic profile of pro-inflammatory cytokine upregulation was observed in the proximal intestine of fish infected by bath challenge, as determined by semi-quantitative RT-PCR. Expression of IL-1b, IL-8, TNF-a and IFN-g was increased in the proximal intestine. TGF-b was significantly decreased in the distal intestine. In the head kidney, infection with A. salmonicida by bath challenge caused decreased expression levels of IL-1b, IL-8, TNFa and TGF-b. The results are discussed in the context of potential immune mechanisms in the gut to prevent infection. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Rainbow trout; Intestine; Cytokines; Aeromonas salmonicida
1. Introduction The mucosa-associated lymphoid tissues in teleost fish (gut, skin and gills), with their layer of mucus and array of non-specific immune defences, provide an initial barrier to the entry of pathogens [1,2]. Mucus secreted by goblet cells reduces the ability of bacteria to adhere to underlying enterocytes [3]. The digestive function of the gut provides an extremely hostile environment to potential pathogens because of the low pH and the presence of digestive enzymes and bile [1]. The gut-associated lymphoid tissue (GALT) is present in teleost fish as individual cells and small aggregations in both the epithelium and lamina propria [4]. GALT in fish lacks Peyer’s patches and M cells [5], however, some evidence exists that the distal intestine contains functional analogues of mammalian M cells. These cells seem to be specialised in the uptake and processing of antigens [6]. Cells associated with the intestinal immune system of * Corresponding author. Tel.: þ44 1224 272870; fax: þ44 1224 272396. E-mail address: [email protected] (C.J. Secombes). 1 Present address: Gut Immunology Group, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, UK. 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.02.002
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fish are diverse and different from those recovered from the spleen and the head kidney [5,7], and include intraepithelial cells, macrophages and eosinophilic granular cells [1]. The largest numbers of lymphocytes and plasma cells are found dispersed in the connective tissue of the lamina propria and within the epithelial layer [5,8]. IgM and IgMproducing cells have been found in the mucosal epithelium, the submucosa and the lamina propria [9,10]. IgM is transported across the epithelial cells and released into the lumen [11]. Fish lack secretory antibody similar to mammalian IgA, which is resistant to proteolytic degradation in the gut [12], and serum IgM added to gut mucus is rapidly degraded [13]. Cytokines play an important role in the immune system by binding to specific receptors at the cell membrane, setting off a cascade that leads to induction, enhancement or inhibition of a number of cytokine-regulated genes in the nucleus. Many important cytokines have been identified in teleost fish in the last number of years, including interleukin-1b (IL-1b), an important pro-inflammatory cytokine [14], tumour necrosis factor-a (TNF-a) [15], interferon (IFN) [16], transforming growth factor-b (TGF-b) [17] and several chemokines [18]. Numerous other cytokines have recently been discovered in teleosts, including amongst others IL-2 [19], IL-6 [20], IL-10 [21], IL-12 [22], IL-15 [23] and IL-21 [19], as well as the first non-mammalian interleukin-11 (IL-11) gene from rainbow trout (Oncorhynchus mykiss) [24]. The non-motile, Gram-negative bacterium Aeromonas salmonicida, the causative agent of furunculosis, is commonly used as a model pathogen for studies of hostepathogen interactions in salmonids. A. salmonicida leads to whole body infection and acute mortality in susceptible salmonids. Clinical signs of relevance to this study include hemorrhagic and necrotic lesions in the gut [25] and destruction of the stratum compactum of the distal intestine [26,27]. Also, because the infected fish stop feeding, the lumen of the intestine may contain sloughed epithelial cells, mucus and blood. The outcome of infection locally may be mediated in large part by the cytokine response elicited. Little is known about the cytokine profile of cells in the fish gut, and in this study their ability to express a number of important cytokine genes is examined in control fish and those exposed to a virulent strain of A. salmonicida in the water. The key components of the classical cytokine cascade induced by Gram-negative bacterial infection, TNF-a, IL-1b and the chemokine IL-8, together with a major activator (IFN-g) and down-regulator (TGF-b) of cell-mediated immune responses were chosen for study. 2. Materials and methods 2.1. Animals Rainbow trout, weighing approximately 70 g, were purchased from Almondbank (Perthshire, UK) and stocked in two 350 L tanks, with each tank containing approximately 60 fish. The fish were allowed a two-week acclimatisation period before the start of the experiment. They were fed on a standard commercial feed (EWOS Ltd) throughout the experiment. 2.2. Bacteria The pathogenic A. salmonicida strain MT423 (Marine Lab, Aberdeen) was grown for 16 h in tryptic soy broth (TSB, Fluka Biochemika) at 22 C in a shaking incubator at 200 rpm. The concentration of the bacterial suspension was determined using a bacterial counting chamber. Fish were exposed to the bacteria by bath infection. The bacterial suspension was added to the water to give a final concentration of 1 105 bacteria/mL tank water. The fish were exposed to the bacteria for 24 h, during which time the tanks were isolated from the ingoing and outgoing water flow. After the incubation time, the water flow was turned on again. Wastewater was treated by ozonation to ensure no live bacteria were released. Fish were regularly checked throughout the experiment and dead fish were recorded. 2.3. Tissue sampling, total RNA isolation and RT-PCR analysis Six fish were killed for sampling at day 0, before bath challenge was commenced, and six further fish were killed for sampling at days 2, 4, 6 and 8, the last sample taken as mortalities were beginning to occur. The head kidney, the
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proximal intestine and the distal intestine were removed, rinsed in cold phosphate buffered saline, pH 7.2 (PBS, Gibco) and stored in 1 mL Trizol (Gibco) at 80 C. Tissue samples were used for RT-PCR to ascertain the gene expression levels of b-actin, IL-1b, IFN-g, TNF-a, IL-8 and TGF-b. The samples were homogenised with a sonicator prior to extraction, and RNA was extracted from the homogenised tissues using Trizol (Invitrogen). Reverse transcription to cDNA was carried out. Five microgram RNA in 11 mL DEPC-water was incubated with 1 mL oligo (dT) 12e18 (500 mg/mL, Invitrogen) for 10 min at 70 C and chilled on ice for at least 3 min. One microlitre Moloney Murine Leukaemia Virus (MMLV) reverse transcriptase (Bioline), 4 mL 5 first strand buffer (Bioline), 1.25 mL 10 mM dNTPs (Bioline) and 1.75 mL water were added, and the reaction was incubated at 42 C for 90 min followed by 5 min at 95 C. The resultant cDNA was dissolved in water and stored at 20 C. PCR was carried out using different primer sets and conditions (Table 1). cDNA (1 mL) was added to an RNAse free tube, followed by 24 mL of a mastermix containing 2.5 mL 10 NH4 Buffer (Bioline), 1 mL 2.5 mM dNTPs (Bioline), 0.75 mL 50 mM MgCl2 solution (Bioline), 17.625 mL PCR H2O (Bioline), 1 mL forward primer and 1 mL reverse primer (Sigma, Table 1) and 0.125 mL 5 U/mL Taq DNA Polymerase (Bioline). The solution was mixed and briefly centrifuged and then subjected to a gene-specific heat-protocol (Table 1) in a Techne thermal cycler. A b-actin PCR was initially performed, at 26 cycles, and the amount of template added adjusted to give approximately equal PCR products between samples. During this procedure the reproducibility of the products obtained from individual fish was confirmed. The same amount of cDNA was then used for all immune gene PCRs as a way of normalising the data in order to give a more quantitative result. Optimal cycling number of the other genes was determined to Table 1 Oligonucleotide primers used, and PCR heat-protocol of each gene investigated Gene
Primer sequence
Temp. ( C)
Time
Cycles
b-Actin
F 50 -ATCGTGGGGCGCCCCAGGCACC-30 R 50 -CTCCTTAATGTCACGCACGATTT-30
94 94 56 72 72
2 min 30 s 30 s 30 s 10 min
1 26 26 26 1
IL-1B
F 50 -ACAGACATGGATTTTGAGTCA-30 R 50 -CTCATACTGTGATGTACTGCTGA-30
94 94 57 72 72
2 min 30 s 30 s 10 s 10 min
1 35 35 35 1
TNF-aa
F 50 -CAAGAGTTTGAACCTCATTCAG-30 R 50 -TGGCAACGATGCAGGACGGAA-30
94 94 62 72 72
2 min 25 s 25 s 25 s 10 min
1 37 37 37 1
IL-8
F 50 -TCCAGACAAATCTCCTGACCG-30 R 50 -GGATGTCAGCCAGCCTTGTC-30
94 94 56 72 72
2 min 30 s 30 s 30 s 10 min
1 32 32 32 1
TGF-b
F 50 -GAAGAAACGACAAACCACTAC-30 R 50 -GACATGTGCAGTAATTCTAGC-30
94 94 50 72 72
2 min 30 s 30 s 30 s 10 min
1 35 35 35 1
IFN-g
F 50 -GTGAGCAGAGGGTGTTGATG-30 R 30 -GATGGTAATGAACTCGGACAG-30
94 94 59 72 72
2 min 30 s 30 s 10 s 10 min
1 35 35 35 1
a
These primers amplified the rainbow trout TNF-a2 gene, as described in Ref. [19].
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generate a product that was visible but not saturated, and found to be between 32 and 37 cycles, depending on the specific gene. All PCR products were visualized on a 2% agarose gel containing 100 ng/mL ethidium bromide. The products were run on the gel for 1 h at 100 V, using a 100 bp DNA ladder (Bioline) as a size marker. Each gel was then examined using a UVP gel imaging system. 2.4. Data analysis UVP Gel-works ID advanced software was used to calculate ratios of cytokine gene expression relative to b-actin expression using the pixel density for each product. This enabled the evaluation of differential expression of the selected genes between different sample groups. The data were plotted as box plots with median values. Statistical analysis of gene expression results was performed using the Kruskal-Wallis test (P < 0.05) followed by the ManneWhitney test (P < 0.05) if significance was found.
3. Results 3.1. IL-1b gene expression Low level, constitutive expression of IL-1b was observed in the head kidney, distal intestine and proximal intestine of uninfected fish at day 0 (Fig. 1). Bath challenge with A. salmonicida caused a small, but significant decrease in IL1b mRNA levels at days 2, 4 and 8 in the head kidney (P < 0.01). At day 6, IL-1b expression was high in three out of six fish, but due to the large variation within the group, this was not significantly different from expression levels in uninfected fish. No significant differential expression was observed in the distal intestine. However, a large increase in IL-1b expression was observed at day 6 in the proximal intestine in three out of six fish (P < 0.01). Expression was still high in two out of six fish at day 8 post-infection, but this was not significantly different from uninfected fish. 3.2. IL-8 gene expression Constitutive IL-8 gene expression was observed in uninfected fish, especially in the head kidney (Fig. 2). Expression in this tissue during infection was decreased at days 2 and 4 (P < 0.01). After this time, IL-8 expression returned to levels similar to those found in non-infected fish. A significant trend in IL-8 expression was also found in the distal intestine (P < 0.05), but no significant differences in IL-8 levels compared to uninfected fish were found. Strong IL-8 induction was observed in the proximal intestine (P < 0.05) during A. salmonicida infection, with expression increased up to 11-fold compared to uninfected tissue. 3.3. TNF-a gene expression The proximal intestine of uninfected fish showed quite strong TNF-a gene expression, compared to the head kidney and the distal intestine (Fig. 3). Infection with A. salmonicida by bath challenge had most effect on TNF-a mRNA production in the proximal intestine. However, a statistically significant decrease of expression was seen in the head kidney (P < 0.01) at days 4, 6 and 8. TNF-a was significantly decreased at days 4 and 6 post-infection in the proximal intestine, but significantly increased at day 8 (P < 0.0001), with an approximately 5-fold increase in mRNA levels compared to uninfected fish. 3.4. TGF-b gene expression TGF-b was strongly expressed in all three tissues of uninfected fish, especially in the head kidney (Fig. 4). Infection with A. salmonicida by bath challenge caused a significant decrease in TGF-b expression in the head kidney (P < 0.0001) and the distal intestine (P < 0.01) at all timepoints.
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Fig. 1. Gene expression of IL-1b as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 2. Gene expression of IL-8 as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 3. Gene expression of TNF-a as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 4. Gene expression of TGF-b as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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Fig. 5. Gene expression of IFN-g as a ratio relative to b-actin gene expression in the head kidney (A), distal intestine (B) and proximal intestine (C) of fish infected with A. salmonicida by bath challenge. The confidence interval box shows the 95% confidence interval for the median, with individual fish (N ¼ 6) depicted as black circles. Letters a, b and c denote statistical analysis using the ManneWhitney test (columns that have no letters in common are statistically different (P < 0.05)). Note the different scales on the y-axes.
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3.5. IFN-g gene expression Low level, constitutive IFN-g gene expression was observed in all three tissues of uninfected fish at day 0 (Fig. 5). Variation in tissue expression was observed in the head kidney and distal intestine during infection, but this was not significant at any timepoint. Differential expression of IFN-g was observed in the proximal intestine. One fish showed very strong IFN-g expression at day 2, but the other fish in this group did not show any differential expression. A significant decrease at days 4 and 6 post-infection was followed by a 3-fold increase at day 8 post-infection (P < 0.01). 4. Discussion It has been proposed that the gastrointestinal tract is the principal infection route of A. salmonicida in various salmonid species, such as Arctic charr (Salvelinus alpinus) [28], due to the fact that A. salmonicida can be detected in the submucosa of the midgut and diverse dietary treatments cause different mortality figures. Other studies further illustrate the role of the intestine as an important route of infection with A. salmonicida. Atlantic salmon (Salmo salar L.) and rainbow trout fed a diet containing the probiotic bacterium Carnobacterium sp. showed reduced mortality after challenge with A. salmonicida, possibly due to the ability of the probiotic to prevent potential pathogens from colonising the gut [29]. The gut has also been shown to respond to A. salmonicida, particularly in studies using killed bacteria within vaccines [30] or administered extracellular products [31]. Such responses may be crucial to the outcome of the infection, and in part will be regulated by the local cytokine response elicited within the gastrointestinal tract. In the current study, rainbow trout were infected with A. salmonicida by bath challenge and the cytokine expression profile in the intestine post-challenge established. Differential expression mostly occurred in the proximal intestine. In this region, gene upregulation of the pro-inflammatory cytokines IL-1b, IL-8, TNF-a and IFN-g was observed. The finding that IL-1b was increased during A. salmonicida infection is not surprising, as this cytokine plays an important role in the regulation of immune and inflammatory processes. IL-1b induces the growth and proliferation of T and B lymphocytes, macrophages, vascular endothelial cells and tissue cells [32]. It has also been shown in rainbow trout that challenge with an attenuated strain of A. salmonicida induces IL-1b transcription in a range of tissues, including blood, gills, kidney, liver and spleen [33]. Expression of IL-1b can also be induced in head kidney leukocytes and macrophages by stimulation with lipopolysaccharide (LPS) [14,34]. In vitro stimulation of cells with recombinant IL-1b, as well as intraperitoneal injection with recombinant IL-1b, enhances phagocytosis and increases expression of IL-1b, COX-2, lysozyme and the MHC class II b chain [35,36]. TNF-a was also significantly increased in the proximal intestine during bath challenge. TNF-a2 specifically was monitored in this study, which is the predominant TNF-a isoform expressed in trout upon stimulation [37]. TNF-a, like IL-1b, has a variety of functions in the regulation of inflammation and cellular immune responses including the stimulation of respiratory burst activity and phagocytosis [38]. It is produced by a diverse range of cells, but mainly by macrophages/monocytes and T lymphocytes [15,38]. TNF-a is constitutively expressed in the gill and kidney of rainbow trout but not in the blood, brain and liver. Co-expression of IL-1b and TNF-a is not unexpected, since these cytokines have similar functions in the initiation of immune responses. Head kidney leukocytes and RTS-11 cells stimulated with recombinant IL-1b produce TNF-a [15], while addition of recombinant TNF-a to the same cells leads to expression of IL-1b, together with TNF-a1, TNF-a2, IL-8 and COX-2 [38]. Concomitant expression of IL-8 was observed in the proximal intestine during infection. IL-8 has chemotactic ability, and attracts neutrophils but not monocytes and macrophages [18]. Thus, the expression of IL-8 in the proximal intestine at days 2, 6 and 8 indicates a mechanism to attract and maintain neutrophil infiltration at the site of infection. IL-8 production is stimulated by the expression of IL-1b and TNF-a, so it is not surprising to see the simultaneous expression of these three cytokines. TGF-b showed no differential expression in the proximal intestine of fish infected by A. salmonicida; however, IFN-g was increased at this site post-challenge. IFN-g is a type II IFN and is produced by activated T lymphocytes and NK cells [16]. IFN-g is a strong activator of macrophages and the key cytokine of type 1 T helper (Th1) cell immune responses during infections with intracellular pathogens, autoimmune diseases and anti-tumour defence [39]. IFN-g is not expressed constitutively in vitro in head kidney cells from rainbow trout, but is inducible by addition of PHA or poly (I:C). In vivo expression of IFN-g is observed in the head kidney and spleen after i.p. injection of poly I:C [16]. It is perhaps surprising that these increases in cytokine expression are observed mostly at days 6 and
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8, as it is presumed that the gut surface is exposed to the bacteria in the first few days of the challenge. However, largescale infection does not occur until days 6 and 7, when the first mortalities take place in this challenge model. Little differential cytokine expression was observed in the distal part of the intestine during infection, apart from decreased expression of TGF-b. This lack of differential expression could be due to the absence of pathogens in the distal part of the intestine. A. salmonicida ingested by the fish travels through the stomach to reach the intestine and is then probably lysed by digestive enzymes and immune reactions. Therefore, few bacteria may be able to infect the distal intestine. Whilst intuitively the stomach is a place for this to occur, in fact Bøgwald et al. [30] found that the stomach has relatively low degradative powers on the bacterium and that A. salmonicida orally intubated into the stomach of Atlantic salmon are not lysed here but travel into the intestine. However, the capacity of the pylorus, midgut and hindgut to lyse A. salmonicida is much greater. Nevertheless, Aeromonas spp. can survive in the piscine gut, as evidenced by the isolation of the bacteria from the healthy gut of rainbow trout [40] and catfish [41]. Another possible reason for the lack of differential cytokine expression in the distal intestine could be due to the fact that the sampling timepoints were too late to detect an acute response that could have occurred in the first two days after bacterial challenge. Following intraperitoneal injection of A. salmonicida, expression of IL-1b, TNF-a and IL-8 occurs quickly (in the first 24 h) in the liver and kidney of pink salmon (Oncorhynchus gorbuscha) and chum salmon (Oncorhynchus keta) [42]. Differential expression of IL-1b, IL-8, TNF-a and TGF-b is also observed within 12 h of exposure of isolated head kidney cells to probiotic bacteria [43]. However, in the present study a bath challenge was used and could be expected to result in significantly different kinetics of exposure. Indeed, peaks in gene expression took place mostly at days 6 and 8 post-infection and were therefore not obviously decreasing from earlier sampling points. A. salmonicida has effects on the epithelial layer of the distal and proximal intestinal regions. In the distal intestine, A. salmonicida causes little detachment of enterocytes but cellular damage is observed involving the microvilli, desmosomes and tight junctions. Substantial detachment of enterocytes is observed in the proximal intestine, and all the enterocytes can be seen detached from the epithelium in certain regions and the basement membrane is exposed [44]. This suggests that the proximal intestine is a more likely infection route for A. salmonicida. However, since loosening of cell junctions is observed in the distal intestine, any bacteria that are able to survive gut lysis and reach the distal intestine will be able to take advantage of this fact. The head kidney showed a very different cytokine profile compared to the intestine. There was a general trend toward a decrease in expression (IL-1b, IL-8, TNF-a and TGF-b), with minimum levels reached at day 4 post-infection. However, at the later stages expression of TNF-a and IL-8 were increased again. This could be due to depletion of immune cells from the head kidney in the earlier stages of the experiment, to sites of infection and contact, such as the mucosal surfaces of the gills, skin and gut. The immune response of fish against A. salmonicida depends on the co-operation of both humoral and cellular immune responses. The outcome of the current study suggests the presence of a cellular response to A. salmonicida in the intestine of rainbow trout due to the differential expression of numerous cytokines. Further work is needed to determine the function of this pro-inflammatory cytokine expression during A. salmonicida infection, in terms of the effects on eventual survival and recovery from infection. Acknowledgments This study was supported financially by EWOS Ltd. References [1] Dalmo RA, Ingebrigtsen K, Bøgwald J. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). J Fish Dis 1997;20:241e73. [2] Press CM, Eversen O. The morphology of the immune system in teleost fishes. Fish Shellfish Immunol 1999;9:309e18. [3] Ellis AE. Innate host defence mechanisms of fish against viruses and bacteria. Dev Comp Immunol 2001;25:827e39. [4] Doggett A, Harris JE. The ontogeny of the gut-associated lymphoid tissue in Oreochromis mossambicus. J Fish Biol 1987;31:23e7. ˚ , Bakke-McKellep AM. The intestines of carnivorous fish: structure and functions and the relations with diet. [5] Buddington RK, Krogdahl A Acta Physiol Scand 1997;161:67e80. [6] Rombout JHWM, Bot HE, Taverne-Thiele JJ. Immunologic importance of the second gut segment of carp. II. Characterization of mucosal leucocytes. J Fish Biol 1989;35:167e78.
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