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The longevity of the antimicrobial response in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) supplemented diet
Fish & Shellfish Immunology 44 (2015) 316e320
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
The longevity of the antimicrobial response in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) supplemented diet lez Vecino b, Elisa Casadei a, Steve Bird a, 1, Simon Wadsworth b, Jose L. Gonza Christopher J. Secombes a, * a b
Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK EWOS Innovation A.S., Dirdal, N-4335, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 December 2014 Received in revised form 24 February 2015 Accepted 26 February 2015 Available online 5 March 2015
This study builds upon previous work studying antimicrobial peptide (AMP) gene expression in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) enriched diet. The aims here were 1) to evaluate how long AMP expression is elevated in skin with continuous feeding of fish with the PG enriched diet for 21 or 28 days, and 2) to assess the impact of stopping PG feeding at day 14 when sampled at day 21 or 28. The rainbow trout were divided into 6 groups, with two fed a control commercial diet for the duration of the experiment and the other four given the same diet enriched with 10 mg PG/Kg for 14 days (PG 1e14) or continuously (PG continuous), the former reverting back to the commercial diet at day 14. No mortalities occurred during the study and there were no significant differences in growth among the fish in the different diet groups. The expression of six AMP genes was studied by real-time PCR in the skin, since these genes were shown to be induced in response to the PG enriched diets in a previous experiment. We show that continuous PG treatment for 21 or 28 days maintained high levels of AMP expression, although in general the levels decreased with time on the diets. Withdrawal of the PG diets at day 14 resulted in a fall in expression level especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated levels (x10) in comparison to fish given a control diet. These results confirm that orally administered PG clearly enhances the trout innate immune system and could be used as a means to boost fish defences. Future studies should be conducted to verify the impact on survival after pathogen challenge in trout fed PG enriched diets under these regimes. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial peptides Peptidoglycan Rainbow trout Aquaculture Innate immunity
1. Introduction In aquaculture, pathogen associated molecular patterns (PAMPs) have been routinely used as immunostimulants (IS) to boost the fish immune system and fight infections. They are easily administered, by supplementation of fish diets, thus avoiding any stress of the treatment [1e3]. Orally administered immunostimulants have been shown to enhance macrophage activation [4], lysozyme activity [5,6], phagocytosis [7,8] and cytokine expression [9,10], and can directly interact with the host mucosal and
* Corresponding author. University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, UK. Tel.: þ44 (0)1224 272872. E-mail address: [email protected] (C.J. Secombes). 1 Present address: Department of Biological Sciences, School of Science and Engineering, University of Waikato, Hamilton 3240, New Zealand. http://dx.doi.org/10.1016/j.fsi.2015.02.039 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
gut-associated lymphoid tissue (MALT and GALT) leading to an improvement in overall fish health. PAMPs that have been shown to have the greatest effects on fish natural defences include b-glucan, alginic acid and peptidoglycan (PG) [9e13]. PG is a major component of the cell walls of Gram-positive and Gram-negative bacteria and its use as an immunostimulant has been reported in many fish species. For example, enhanced resistance against Enterococcus seriolicida and Vibrio anguillarum has been described after administration of diets supplemented with PG to yellowtail kingfish and Japanese flounder respectively [8,14]. The specific PG receptors (PGRPs) have been characterized in fish [15,16], however the cascade of events that lead to an increase in macrophage activity, and enhanced complement and lysozyme production are not fully understood [17]. In fish, antimicrobial peptides (AMPs) are well known, with 3e4 major families present in most species. Whilst some fish possess
E. Casadei et al. / Fish & Shellfish Immunology 44 (2015) 316e320
piscidin genes [18], these are absent in salmonids which possess bdefensins (DB), cathelicidins (CATH) and liver expressed antimicrobial peptides (LEAP) [19e23]. The main characteristic of AMPs is their cationic charge that allows them to interact directly with the negatively charged microbial surface, causing disruption of the membrane integrity and pathogen lysis [24,25]. In addition, these peptides are rich in cysteine residues, which form intra- and extramolecular disulphide bridges, conferring more stability to these molecules and enhancing their microbicidal capacity [26,27]. AMPs have a broad range of activity against a number of pathogenic organisms and little or no toxicity toward host cells has been reported [28,29]. These features have led to their potential application as new antimicrobial agents to replace antibiotics where microbial resistance has developed [30]. Previously, we have demonstrated the efficacy of PG supplemented diets to up-regulate AMPs in rainbow trout mucosal tissues in a short-term (2 weeks) feeding trial [1]. In this trial four dietary inclusion levels were studied (5, 10, 50 and 100 mg PG/ Kg), and in general terms the effects on AMP expression were very similar. In the current work, we extend these studies using a single PG inclusion level (10 mg PG/Kg) and examine if the AMP response persists if feeding continues and what happens when the PG diet is withdrawn after two weeks of feeding. We have focused this work on the skin, which showed some of the largest changes in AMP expression relative to the other tissues studied (gut, gills, liver) and is therefore deemed a sensitive tissue to assess these effects. Six AMPs (omDB-1, omDB-3, omDB-4, omCATH-1, omCATH-2 and omLEAP-2a) were shown previously to be significantly up-regulated at day 14 during PG treatment. We now show that continuous PG treatment after 21 or 28 days maintains high levels of AMP expression, although the levels do fall with time. Withdrawal of the PG diet at day 14 results in a dramatic fall in expression level, especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated (x10) levels in comparison to fish given a control diet.
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2.3. Real time PCR (qPCR) Expression of 6 selected AMP genes (omDB-1, omDB-3, omDB-4, omCATH-1, omCATH-2, omLEAP-2a), as well as the housekeeping gene elongation factor (EF) -1a, was assessed by real time PCR, using the primer pairs reported in our previous study [1]. An immolase DNA polymerase kit (Bioline) was used for qPCR, where the working solution was prepared using 7 ml of 10X ImmoBuffer (160 mM (NH4)2SO4, 670 mM TriseHCl pH 8.3, 0.1% Tween-20), 4.48 ml of 50 mM MgCl2, 0.56 ml 25 mM dNTPs, 140 ml SYBR Green dye, 300 ml of Immolase (1500 U) and 36.17 ml sterile water. Into each well was placed 14 ml immolase working solution, 2 ml of each forward and reverse primer (2 mM), 4 ml cDNA (10 ng) or 4 ml of standard (consisting of a pool of RNA from all samples collected that was reverse transcribed at the same time as the individual samples). PCR was performed as follows: 95 C for 11 min, then 40 cycles (25 cycles for the EF-1a) of 95 C for 30 s, 60e65 C for 30 s and 72 C for 30 s, with a final extension of 72 C for 5 min. The fluorescence of the plate was recorded after every cycle and the melting curve ranged from 65 C to 95 C. To be able to calculate the primer efficiency, the threshold and the melting curve, 3 dilutions of standards (in triplicate) were run for all genes within the same plates as the samples. The reaction was performed in a 96 well plate using the Engine Opticon™ system (MJ Research, Inc.). Data were analysed using the Pfaffl method [31] and fold changes were calculated using the control diet as a baseline. 2.4. Statistical analysis A t-test was performed to calculate the difference between control and treatment groups, and values greater or lower than þ2/-2 were considered statistically significant if p 0.05. Oneway analysis of variance (ANOVA) with a post-hoc Tukey's HSD test (if the ANOVA was significant), was performed using SPSS Statistics v.19 to compare rainbow trout AMP expression in the PG continuous and PG 1-14 groups within and between time points. 3. Results
2. Materials and methods 3.1. Growth 2.1. Fish rearing and experimental feeding trials Rainbow trout (Oncorhynchus mykiss) (~100g) were acquired from Almondbank fish farm (Perthshire, UK) and acclimatised for 15 days within the School of Biological Sciences aquarium at the University of Aberdeen before starting the feeding trial. The fish were kept at 14 C, with DO levels at 90% and a flow rate into the tanks of ~8 L/min. The fish were then fed with two different diets, a control commercial diet (EWOS) and the same diet enriched with peptidoglycan (PG) at 10 mg PG/Kg (EWOS). Initially two tanks were fed with the control diet and four with the 10 mg PG/Kg enriched diet. After 14 days two tanks from the immunostimulated groups (PG 1e14) were transferred to the control diet, while the fish in the other 2 tanks were kept on the PG diet (PG continuous) until sampling at day 21 or 28.
2.2. Sample collection Sampling was performed 21 or 28 days from the start of the experiment. Six trout from each group (control, PG 1e14 and PG continuous) were killed by a schedule 1 method and their weight and length recorded before collecting skin samples for subsequent RNA extraction and cDNA synthesis, performed as described previously [1].
In all experimental groups growth (Table 1) and survival were unaffected by the administration of the PG diets. The fish continued to grow throughout the experiment, from a starting mean weight of ~100 g at day 0 to ~170 g at day 28. 3.2. AMP expression In the skin after 21 days of feeding (Fig. 1) omDB-3 was the most significantly (p 0.01) up regulated AMP compared to the control diet group in fish given PG continuously (123-fold). It was also increased significantly in the PG 1e14 fed group (31-fold) albeit at a lower level when compared to the PG continuous group. The expression of omDB-4, omCATH-1, omCATH-2 and omLEAP-2a were also increased in the two groups but little or no change in omBD-1 was detected. Whilst in all cases the fold increase was higher in the PG continuous groups vs the PG 1-14 group (ie 40 fold vs 22 fold for omDB-4, 53 fold vs 24 fold for omCATH-1, 11 fold vs 3 fold for omCATH-2), it was only for omLEAP-2a that there was a significantly reduced level in the latter (ie 41 fold vs 6.5 fold). After 28 days of feeding (Fig. 2), there were more significant differences found between the PG continuous and PG 1e14 fish. Whilst the levels of AMP up-regulation had fallen in the PG continuous fish (significantly so in the case of omDB-3 and omDB4) compared to fish sampled at day 21, they were still significantly
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E. Casadei et al. / Fish & Shellfish Immunology 44 (2015) 316e320
Table 1 Average weight and length of rainbow trout sampled during the feeding trial. Results are means ± SD. N ¼ 6. Day 14
Control PG continuous PG 1e14
Day 21
Day 28
Weight (g)
Length (cm)
Weight (g)
Length (cm)
Weight (g)
Length (cm)
145 ± 36 152 ± 15 132 ± 26
20.3 ± 1.8 20.6 ± 1.1 20.5 ± 1.2
161 ± 30 156 ± 31 160 ± 33
20.7 ± 1.1 21.2 ± 1.1 22.1 ± 2.5
170 ± 25 165.7 ± 25 169.5 ± 27
22.7 ± 1.5 23.8 ± 2.1 22.4 ± 1.4
Fig. 1. Expression of six AMPs in rainbow trout skin after 21 days of feeding a control diet, a peptidoglycan (PG) enriched diet containing 10 mg PG/Kg (PG continuous) or the PG diet for the first 14 days and then the control diet for 7 days (PG 1e14). Data are presented as means ± SD and were analysed using the Pfaffl method (ie the fold changes were calculated using the control diet as a baseline). Asterisks indicate significant differences (* ¼ p 0.05, ** ¼ p 0.01) between control vs treated fish. Comparison between the PG continuous and PG 1e14 fish are shown above the bars when significant.
increased compared to the control fish (except omDB-1). However, up-regulation in the PG 1e14 fish was now only seen for omDB-3 (12 fold) and omDB-4 (10 fold), which were approximately 50% of the level at day 21, and no up-regulation was seen for the other genes (omCATH-1, omCATH-2, omLEAP-2a), giving significantly lower expression levels compared to the PG continuous fish. 4. Discussion This study investigated the effect of administering a PG supplemented diet to rainbow trout for 3e4 weeks on the expression of selected antimicrobial peptide (AMP) genes at an important mucosal site, the skin, that acts as a first line of defence against
microbial infection [32,33]. The study attempted to address the following two questions: 1) does elevated AMP expression continue with continuous feeding of the PG containing diet, and 2) does elevated AMP expression continue if the PG diet is stopped, and if so for how long. Few studies have evaluated the expression of immune genes immediately following cessation of feeding a PG enriched diet and none in the case of AMP gene expression. In a previous short-term experiment (2 weeks), the efficacy of 5, 10, 50 and 100 mg PG/Kg diets as enhancers of AMP expression in rainbow trout was shown (Casadei et al., 2013) and the importance of AMP expression at mucosal sites as potential molecular markers to test novel immunostimulants validated. In this study, we selected the 10 mg/kg PG diet for a further feeding trial, in which fish were given
Fig. 2. Expression of six AMPs in rainbow trout skin after 28 days of feeding a control diet, a peptidoglycan (PG) enriched diet containing 10 mg PG/Kg (PG continuous) or the PG diet for the first 14 days and then the control diet for 14 days (PG 1e14). Data are presented as means ± SD and were analysed using the Pfaffl method (ie the fold changes were calculated using the control diet as a baseline). Asterisks indicate significant differences (* ¼ p 0.05, ** ¼ p 0.01) between control vs treated fish. Comparison between the PG continuous and PG 1e14 fish are shown above the bars when significant.
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either a control diet continuously for up to 21 or 28 days, the PG containing diet continuously (PG continuous) for up to 21 or 28 days or the PG containing diet for the first 14 days and then transferred back to the control diet (PG 1e14) for up to 21 or 28 days. AMP expression was determined at day 21 and day 28, with the focus on six genes shown in our previous trial to be upregulated in skin by the PG diet [1]. However, one of these genes (omDB-1) was not found to be modulated in this study. The PG supplementation did not affect the growth of the fish, and no significant difference in weight and length were found between the three groups, suggesting the PG in the diet does not impact on palatability or other physiological pathways, such as protein synthesis. The addition of other immunostimulants, such as beta glucan and alginic acid, to diets is known to be well tolerated by fish, as described previously [3,34]. The PG diet did have a clear impact on AMP expression, with the induction of AMPs generally higher in the PG continuous group compared to the PG 1-14 group. This effect was most marked at day 28, where several AMP genes were no longer elevated (omCATH-1, omCATH2, omLEAP-2a) in fish taken off the PG containing diet two weeks earlier. However, interestingly the two defensin genes that responded to the PG diet in this study (omDB-3 and omDB-4) were still elevated significantly at day 28 in fish that had been taken off the PG treatment. Indeed, their expression level was no different to that in fish given PG continuously, since in the latter group omDB expression had fallen by day 28 despite the continuous administration. This shows that the longer-term administration of PG containing diets had a benefit, assuming AMP expression impacts on disease resistance (see below), in terms of maintaining some enhanced AMP expression although that this was not equal for all AMP genes studied. However, compared to the day 14 results seen in our previous study [1], where omDB-4, omCATH-1 and omCATH-2 were highly up-regulated (several hundred fold) it appears that such high levels are quite transitory and are not maintained. The present study also shows that in some cases AMP expression can remain elevated for at least two weeks after cessation of feeding the PG diet, as seen with omDB-3 and omDB-4. Why variable expression patterns of induction [1] and decline are seen is interesting to consider. This may reflect differences in the cells that synthesise and secrete the different AMPs, or differences in cell activation at different times perhaps mediated by other immune factors. Whatever the explanation it seems that in trout defensins are induced early by PG containing diets, and stay elevated longer than other AMPs when the diets are removed. The secretion of AMPs at mucosal sites such as the skin is considered key to provide a protective immune shield against microbial infection [33]. AMPs commonly increase in expression in the skin following infection [35,36], and in addition to their ability to directly kill microbes they can have an immunomodulatory role [37], eliciting cytokine release to enable a more effective response. Individual fish AMPs have also been shown to provide disease resistance, whether in transgenic fish overexpressing AMPs in muscle [38] or after peptide administration [39]. Thus, their upregulation by PG containing diets has the potential to provide enhanced resistance for several weeks, with levels remaining 10e20 fold higher than in control fish. Importantly, even after cessation of feeding the PG diet enhanced AMP expression remains for 1e2 weeks in the case of some genes. However, the need for simultaneous secretion of different AMPs to achieve disease resistance is completely unknown, and so it can only be speculated as to the degree of protection that might be seen if, for example, b-defensins remain elevated (as seen 2 weeks after termination of the PG treatment in this study). Indeed, it is possible that total AMP production is a more important measure to establish, but is
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dependent upon the identification of the complete AMP repertoire in an individual species. In conclusion, PG containing diets elevate AMP expression at important mucosal sites such as the skin. Elevated AMP expression is seen during 4 weeks of continuous feeding of the diet but does decrease with time. AMP expression also remains elevated to some degree when feeding of the PG diets stops, but in some cases (omCATH-1, omCATH-2, omLEAP-2a) expression rapidly returns to control levels. Acknowledgements The research was supported financially by an industrial studentship between the University of Aberdeen, Ewos Innovation and MSD Animal Health. References lez Vecino, S. Wadsworth, C.J. Secombes, The ef[1] E. Casadei, S. Bird, J.L. Gonza fect of peptidoglycan enriched diets on antimicrobial peptide gene expression in rainbow trout (Oncorhynchus mykiss), Fish Shellfish Immunol 34 (2013) 529e537. [2] A. Falco, P. Frost, J. Miest, N. Pionnier, I. Irnazarow, D. Hoole, Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with b-glucan supplements, Fish Shellfish Immunol 32 (2012) 1051e1057. [3] M. Bagni, N. Romano, M.G. Finoia, L. Abelli, G. Scapigliati, P.G. Tiscar, et al., Short- and long-term effects of a dietary yeast b-glucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax), Fish Shellfish Immunol 18 (2005) 311e325. [4] T. Kadowaki, Y. Yasui, O. Nishimiya, Y. Takahashi, C. Kohchi, G.-I. Soma, et al., Orally administered LPS enhances head kidney macrophage activation with down-regulation of IL-6 in common carp (Cyprinus carpio), Fish Shellfish Immunol 34 (2013) 1569e1575. [5] J. Xueqin, P.W. Kania, K. Buchmann, Comparative effects of four feeds types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum), J Fish Dis 35 (2012) 127e135. [6] J.H. Lauridsen, K. Buchmann, Effects of short- and long-term glucan feeding of rainbow trout (Salmonidae) on the susceptibility to Ichthyophthirius multifiliis infections, Acta Ichthyologia Piscatoria 40 (2010) 61e66. [7] A.-C. Cheng, Y.-Y. Chen, J.-C. Chen, Dietary administration of sodium alginate and k-carrageenan enhances the innate immune response of brown-marbled grouper Epinephelus fuscoguttatus and its resistance against Vibrio alginolyticus, Vet Immunol Immunopathol 121 (2008) 206e215. [8] J. Zhou, X.-L. Song, J. Huang, X.-H. Wang, Effects of dietary supplementation of A3a-peptidoglycan on innate immune responses and defense activity of Japanese flounder (Paralichthys olivaceus), Aquaculture 251 (2006) 172e181. [9] G. Gioacchini, P. Smith, O. Carnevali, Effects of Ergosan on the expression of cytokine genes in the liver of juvenile rainbow trout (Oncorhynchus mykiss) exposed to enteric red mouth vaccine, Vet Immunol Immunopathol 123 (2008) 215e222. [10] S. Peddie, J. Zou, C.J. Secombes, Immunostimulation in the rainbow trout (Oncorhynchus mykiss) following intraperitoneal administration of Ergosan, Vet Immunol Immunopathol 86 (2002) 101e113. [11] W.M. Sealey, F.T. Barrows, A. Hang, K.A. Johansen, K. Overturf, S.E. LaPatra, et al., Evaluation of the ability of barley genotypes containing different amounts of b-glucan to alter growth and disease resistance of rainbow trout Oncorhynchus mykiss, Animal Feed Sci Technol 141 (2008) 115e128. [12] R. Russo, R.P.E. Yanong, H. Mitchell, Dietary beta-glucans and nucleotides enhance resistance of red-tail black shark (Epalzeorhynchos bicolor, fam. Cyprinidae) to Streptococcus iniae infection, J World Aquac Soc 37 (2006) 298e306. [13] N. Sheikhzadeh, A. Karimi Pashaki, K. Nofouzi, M. Heidarieh, H. Tayefi-Nasrabadi, Effects of dietary ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss), Fish Shellfish Immunol 32 (2012) 407e410. [14] T. Itami, M. Kondo, M. Uozu, A. Suganuma, T. Abe, A. Nakagawa, et al., Enhancement of resistance against Enterococcus seriolicida infection in yellowtail, Seriola quinqueradiata (Temminck & Schlegel), by oral administration of peptidoglycan derived from Bifidobacterium thermophilum, J Fish Dis 19 (1996) 185e187. [15] X. Li, S. Wang, J. Qi, S.F. Echtenkamp, R. Chatterjee, M. Wang, et al., Zebrafish peptidoglycan recognition proteins are bactericidal amidases essential for defense against bacterial infections, Immunity 27 (2007) 518e529. [16] C.M.S. Ribeiro, T. Hermsen, A.J. Taverne-Thiele, H.F.J. Savelkoul, G.F. Wiegertjes, Evolution of recognition of ligands from Gram-positive bacteria: similarities and differences in the TLR2-mediated response between mammalian vertebrates and teleost fish, J Immunol 184 (2010) 2355e2368.
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
The longevity of the antimicrobial response in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) supplemented diet lez Vecino b, Elisa Casadei a, Steve Bird a, 1, Simon Wadsworth b, Jose L. Gonza Christopher J. Secombes a, * a b
Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK EWOS Innovation A.S., Dirdal, N-4335, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 December 2014 Received in revised form 24 February 2015 Accepted 26 February 2015 Available online 5 March 2015
This study builds upon previous work studying antimicrobial peptide (AMP) gene expression in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) enriched diet. The aims here were 1) to evaluate how long AMP expression is elevated in skin with continuous feeding of fish with the PG enriched diet for 21 or 28 days, and 2) to assess the impact of stopping PG feeding at day 14 when sampled at day 21 or 28. The rainbow trout were divided into 6 groups, with two fed a control commercial diet for the duration of the experiment and the other four given the same diet enriched with 10 mg PG/Kg for 14 days (PG 1e14) or continuously (PG continuous), the former reverting back to the commercial diet at day 14. No mortalities occurred during the study and there were no significant differences in growth among the fish in the different diet groups. The expression of six AMP genes was studied by real-time PCR in the skin, since these genes were shown to be induced in response to the PG enriched diets in a previous experiment. We show that continuous PG treatment for 21 or 28 days maintained high levels of AMP expression, although in general the levels decreased with time on the diets. Withdrawal of the PG diets at day 14 resulted in a fall in expression level especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated levels (x10) in comparison to fish given a control diet. These results confirm that orally administered PG clearly enhances the trout innate immune system and could be used as a means to boost fish defences. Future studies should be conducted to verify the impact on survival after pathogen challenge in trout fed PG enriched diets under these regimes. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Antimicrobial peptides Peptidoglycan Rainbow trout Aquaculture Innate immunity
1. Introduction In aquaculture, pathogen associated molecular patterns (PAMPs) have been routinely used as immunostimulants (IS) to boost the fish immune system and fight infections. They are easily administered, by supplementation of fish diets, thus avoiding any stress of the treatment [1e3]. Orally administered immunostimulants have been shown to enhance macrophage activation [4], lysozyme activity [5,6], phagocytosis [7,8] and cytokine expression [9,10], and can directly interact with the host mucosal and
* Corresponding author. University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, UK. Tel.: þ44 (0)1224 272872. E-mail address: [email protected] (C.J. Secombes). 1 Present address: Department of Biological Sciences, School of Science and Engineering, University of Waikato, Hamilton 3240, New Zealand. http://dx.doi.org/10.1016/j.fsi.2015.02.039 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
gut-associated lymphoid tissue (MALT and GALT) leading to an improvement in overall fish health. PAMPs that have been shown to have the greatest effects on fish natural defences include b-glucan, alginic acid and peptidoglycan (PG) [9e13]. PG is a major component of the cell walls of Gram-positive and Gram-negative bacteria and its use as an immunostimulant has been reported in many fish species. For example, enhanced resistance against Enterococcus seriolicida and Vibrio anguillarum has been described after administration of diets supplemented with PG to yellowtail kingfish and Japanese flounder respectively [8,14]. The specific PG receptors (PGRPs) have been characterized in fish [15,16], however the cascade of events that lead to an increase in macrophage activity, and enhanced complement and lysozyme production are not fully understood [17]. In fish, antimicrobial peptides (AMPs) are well known, with 3e4 major families present in most species. Whilst some fish possess
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piscidin genes [18], these are absent in salmonids which possess bdefensins (DB), cathelicidins (CATH) and liver expressed antimicrobial peptides (LEAP) [19e23]. The main characteristic of AMPs is their cationic charge that allows them to interact directly with the negatively charged microbial surface, causing disruption of the membrane integrity and pathogen lysis [24,25]. In addition, these peptides are rich in cysteine residues, which form intra- and extramolecular disulphide bridges, conferring more stability to these molecules and enhancing their microbicidal capacity [26,27]. AMPs have a broad range of activity against a number of pathogenic organisms and little or no toxicity toward host cells has been reported [28,29]. These features have led to their potential application as new antimicrobial agents to replace antibiotics where microbial resistance has developed [30]. Previously, we have demonstrated the efficacy of PG supplemented diets to up-regulate AMPs in rainbow trout mucosal tissues in a short-term (2 weeks) feeding trial [1]. In this trial four dietary inclusion levels were studied (5, 10, 50 and 100 mg PG/ Kg), and in general terms the effects on AMP expression were very similar. In the current work, we extend these studies using a single PG inclusion level (10 mg PG/Kg) and examine if the AMP response persists if feeding continues and what happens when the PG diet is withdrawn after two weeks of feeding. We have focused this work on the skin, which showed some of the largest changes in AMP expression relative to the other tissues studied (gut, gills, liver) and is therefore deemed a sensitive tissue to assess these effects. Six AMPs (omDB-1, omDB-3, omDB-4, omCATH-1, omCATH-2 and omLEAP-2a) were shown previously to be significantly up-regulated at day 14 during PG treatment. We now show that continuous PG treatment after 21 or 28 days maintains high levels of AMP expression, although the levels do fall with time. Withdrawal of the PG diet at day 14 results in a dramatic fall in expression level, especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated (x10) levels in comparison to fish given a control diet.
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2.3. Real time PCR (qPCR) Expression of 6 selected AMP genes (omDB-1, omDB-3, omDB-4, omCATH-1, omCATH-2, omLEAP-2a), as well as the housekeeping gene elongation factor (EF) -1a, was assessed by real time PCR, using the primer pairs reported in our previous study [1]. An immolase DNA polymerase kit (Bioline) was used for qPCR, where the working solution was prepared using 7 ml of 10X ImmoBuffer (160 mM (NH4)2SO4, 670 mM TriseHCl pH 8.3, 0.1% Tween-20), 4.48 ml of 50 mM MgCl2, 0.56 ml 25 mM dNTPs, 140 ml SYBR Green dye, 300 ml of Immolase (1500 U) and 36.17 ml sterile water. Into each well was placed 14 ml immolase working solution, 2 ml of each forward and reverse primer (2 mM), 4 ml cDNA (10 ng) or 4 ml of standard (consisting of a pool of RNA from all samples collected that was reverse transcribed at the same time as the individual samples). PCR was performed as follows: 95 C for 11 min, then 40 cycles (25 cycles for the EF-1a) of 95 C for 30 s, 60e65 C for 30 s and 72 C for 30 s, with a final extension of 72 C for 5 min. The fluorescence of the plate was recorded after every cycle and the melting curve ranged from 65 C to 95 C. To be able to calculate the primer efficiency, the threshold and the melting curve, 3 dilutions of standards (in triplicate) were run for all genes within the same plates as the samples. The reaction was performed in a 96 well plate using the Engine Opticon™ system (MJ Research, Inc.). Data were analysed using the Pfaffl method [31] and fold changes were calculated using the control diet as a baseline. 2.4. Statistical analysis A t-test was performed to calculate the difference between control and treatment groups, and values greater or lower than þ2/-2 were considered statistically significant if p 0.05. Oneway analysis of variance (ANOVA) with a post-hoc Tukey's HSD test (if the ANOVA was significant), was performed using SPSS Statistics v.19 to compare rainbow trout AMP expression in the PG continuous and PG 1-14 groups within and between time points. 3. Results
2. Materials and methods 3.1. Growth 2.1. Fish rearing and experimental feeding trials Rainbow trout (Oncorhynchus mykiss) (~100g) were acquired from Almondbank fish farm (Perthshire, UK) and acclimatised for 15 days within the School of Biological Sciences aquarium at the University of Aberdeen before starting the feeding trial. The fish were kept at 14 C, with DO levels at 90% and a flow rate into the tanks of ~8 L/min. The fish were then fed with two different diets, a control commercial diet (EWOS) and the same diet enriched with peptidoglycan (PG) at 10 mg PG/Kg (EWOS). Initially two tanks were fed with the control diet and four with the 10 mg PG/Kg enriched diet. After 14 days two tanks from the immunostimulated groups (PG 1e14) were transferred to the control diet, while the fish in the other 2 tanks were kept on the PG diet (PG continuous) until sampling at day 21 or 28.
2.2. Sample collection Sampling was performed 21 or 28 days from the start of the experiment. Six trout from each group (control, PG 1e14 and PG continuous) were killed by a schedule 1 method and their weight and length recorded before collecting skin samples for subsequent RNA extraction and cDNA synthesis, performed as described previously [1].
In all experimental groups growth (Table 1) and survival were unaffected by the administration of the PG diets. The fish continued to grow throughout the experiment, from a starting mean weight of ~100 g at day 0 to ~170 g at day 28. 3.2. AMP expression In the skin after 21 days of feeding (Fig. 1) omDB-3 was the most significantly (p 0.01) up regulated AMP compared to the control diet group in fish given PG continuously (123-fold). It was also increased significantly in the PG 1e14 fed group (31-fold) albeit at a lower level when compared to the PG continuous group. The expression of omDB-4, omCATH-1, omCATH-2 and omLEAP-2a were also increased in the two groups but little or no change in omBD-1 was detected. Whilst in all cases the fold increase was higher in the PG continuous groups vs the PG 1-14 group (ie 40 fold vs 22 fold for omDB-4, 53 fold vs 24 fold for omCATH-1, 11 fold vs 3 fold for omCATH-2), it was only for omLEAP-2a that there was a significantly reduced level in the latter (ie 41 fold vs 6.5 fold). After 28 days of feeding (Fig. 2), there were more significant differences found between the PG continuous and PG 1e14 fish. Whilst the levels of AMP up-regulation had fallen in the PG continuous fish (significantly so in the case of omDB-3 and omDB4) compared to fish sampled at day 21, they were still significantly
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Table 1 Average weight and length of rainbow trout sampled during the feeding trial. Results are means ± SD. N ¼ 6. Day 14
Control PG continuous PG 1e14
Day 21
Day 28
Weight (g)
Length (cm)
Weight (g)
Length (cm)
Weight (g)
Length (cm)
145 ± 36 152 ± 15 132 ± 26
20.3 ± 1.8 20.6 ± 1.1 20.5 ± 1.2
161 ± 30 156 ± 31 160 ± 33
20.7 ± 1.1 21.2 ± 1.1 22.1 ± 2.5
170 ± 25 165.7 ± 25 169.5 ± 27
22.7 ± 1.5 23.8 ± 2.1 22.4 ± 1.4
Fig. 1. Expression of six AMPs in rainbow trout skin after 21 days of feeding a control diet, a peptidoglycan (PG) enriched diet containing 10 mg PG/Kg (PG continuous) or the PG diet for the first 14 days and then the control diet for 7 days (PG 1e14). Data are presented as means ± SD and were analysed using the Pfaffl method (ie the fold changes were calculated using the control diet as a baseline). Asterisks indicate significant differences (* ¼ p 0.05, ** ¼ p 0.01) between control vs treated fish. Comparison between the PG continuous and PG 1e14 fish are shown above the bars when significant.
increased compared to the control fish (except omDB-1). However, up-regulation in the PG 1e14 fish was now only seen for omDB-3 (12 fold) and omDB-4 (10 fold), which were approximately 50% of the level at day 21, and no up-regulation was seen for the other genes (omCATH-1, omCATH-2, omLEAP-2a), giving significantly lower expression levels compared to the PG continuous fish. 4. Discussion This study investigated the effect of administering a PG supplemented diet to rainbow trout for 3e4 weeks on the expression of selected antimicrobial peptide (AMP) genes at an important mucosal site, the skin, that acts as a first line of defence against
microbial infection [32,33]. The study attempted to address the following two questions: 1) does elevated AMP expression continue with continuous feeding of the PG containing diet, and 2) does elevated AMP expression continue if the PG diet is stopped, and if so for how long. Few studies have evaluated the expression of immune genes immediately following cessation of feeding a PG enriched diet and none in the case of AMP gene expression. In a previous short-term experiment (2 weeks), the efficacy of 5, 10, 50 and 100 mg PG/Kg diets as enhancers of AMP expression in rainbow trout was shown (Casadei et al., 2013) and the importance of AMP expression at mucosal sites as potential molecular markers to test novel immunostimulants validated. In this study, we selected the 10 mg/kg PG diet for a further feeding trial, in which fish were given
Fig. 2. Expression of six AMPs in rainbow trout skin after 28 days of feeding a control diet, a peptidoglycan (PG) enriched diet containing 10 mg PG/Kg (PG continuous) or the PG diet for the first 14 days and then the control diet for 14 days (PG 1e14). Data are presented as means ± SD and were analysed using the Pfaffl method (ie the fold changes were calculated using the control diet as a baseline). Asterisks indicate significant differences (* ¼ p 0.05, ** ¼ p 0.01) between control vs treated fish. Comparison between the PG continuous and PG 1e14 fish are shown above the bars when significant.
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either a control diet continuously for up to 21 or 28 days, the PG containing diet continuously (PG continuous) for up to 21 or 28 days or the PG containing diet for the first 14 days and then transferred back to the control diet (PG 1e14) for up to 21 or 28 days. AMP expression was determined at day 21 and day 28, with the focus on six genes shown in our previous trial to be upregulated in skin by the PG diet [1]. However, one of these genes (omDB-1) was not found to be modulated in this study. The PG supplementation did not affect the growth of the fish, and no significant difference in weight and length were found between the three groups, suggesting the PG in the diet does not impact on palatability or other physiological pathways, such as protein synthesis. The addition of other immunostimulants, such as beta glucan and alginic acid, to diets is known to be well tolerated by fish, as described previously [3,34]. The PG diet did have a clear impact on AMP expression, with the induction of AMPs generally higher in the PG continuous group compared to the PG 1-14 group. This effect was most marked at day 28, where several AMP genes were no longer elevated (omCATH-1, omCATH2, omLEAP-2a) in fish taken off the PG containing diet two weeks earlier. However, interestingly the two defensin genes that responded to the PG diet in this study (omDB-3 and omDB-4) were still elevated significantly at day 28 in fish that had been taken off the PG treatment. Indeed, their expression level was no different to that in fish given PG continuously, since in the latter group omDB expression had fallen by day 28 despite the continuous administration. This shows that the longer-term administration of PG containing diets had a benefit, assuming AMP expression impacts on disease resistance (see below), in terms of maintaining some enhanced AMP expression although that this was not equal for all AMP genes studied. However, compared to the day 14 results seen in our previous study [1], where omDB-4, omCATH-1 and omCATH-2 were highly up-regulated (several hundred fold) it appears that such high levels are quite transitory and are not maintained. The present study also shows that in some cases AMP expression can remain elevated for at least two weeks after cessation of feeding the PG diet, as seen with omDB-3 and omDB-4. Why variable expression patterns of induction [1] and decline are seen is interesting to consider. This may reflect differences in the cells that synthesise and secrete the different AMPs, or differences in cell activation at different times perhaps mediated by other immune factors. Whatever the explanation it seems that in trout defensins are induced early by PG containing diets, and stay elevated longer than other AMPs when the diets are removed. The secretion of AMPs at mucosal sites such as the skin is considered key to provide a protective immune shield against microbial infection [33]. AMPs commonly increase in expression in the skin following infection [35,36], and in addition to their ability to directly kill microbes they can have an immunomodulatory role [37], eliciting cytokine release to enable a more effective response. Individual fish AMPs have also been shown to provide disease resistance, whether in transgenic fish overexpressing AMPs in muscle [38] or after peptide administration [39]. Thus, their upregulation by PG containing diets has the potential to provide enhanced resistance for several weeks, with levels remaining 10e20 fold higher than in control fish. Importantly, even after cessation of feeding the PG diet enhanced AMP expression remains for 1e2 weeks in the case of some genes. However, the need for simultaneous secretion of different AMPs to achieve disease resistance is completely unknown, and so it can only be speculated as to the degree of protection that might be seen if, for example, b-defensins remain elevated (as seen 2 weeks after termination of the PG treatment in this study). Indeed, it is possible that total AMP production is a more important measure to establish, but is
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dependent upon the identification of the complete AMP repertoire in an individual species. In conclusion, PG containing diets elevate AMP expression at important mucosal sites such as the skin. Elevated AMP expression is seen during 4 weeks of continuous feeding of the diet but does decrease with time. AMP expression also remains elevated to some degree when feeding of the PG diets stops, but in some cases (omCATH-1, omCATH-2, omLEAP-2a) expression rapidly returns to control levels. Acknowledgements The research was supported financially by an industrial studentship between the University of Aberdeen, Ewos Innovation and MSD Animal Health. References lez Vecino, S. Wadsworth, C.J. Secombes, The ef[1] E. Casadei, S. Bird, J.L. Gonza fect of peptidoglycan enriched diets on antimicrobial peptide gene expression in rainbow trout (Oncorhynchus mykiss), Fish Shellfish Immunol 34 (2013) 529e537. [2] A. Falco, P. Frost, J. Miest, N. Pionnier, I. Irnazarow, D. Hoole, Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with b-glucan supplements, Fish Shellfish Immunol 32 (2012) 1051e1057. [3] M. Bagni, N. Romano, M.G. Finoia, L. Abelli, G. Scapigliati, P.G. Tiscar, et al., Short- and long-term effects of a dietary yeast b-glucan (Macrogard) and alginic acid (Ergosan) preparation on immune response in sea bass (Dicentrarchus labrax), Fish Shellfish Immunol 18 (2005) 311e325. [4] T. Kadowaki, Y. Yasui, O. Nishimiya, Y. Takahashi, C. Kohchi, G.-I. Soma, et al., Orally administered LPS enhances head kidney macrophage activation with down-regulation of IL-6 in common carp (Cyprinus carpio), Fish Shellfish Immunol 34 (2013) 1569e1575. [5] J. Xueqin, P.W. Kania, K. Buchmann, Comparative effects of four feeds types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum), J Fish Dis 35 (2012) 127e135. [6] J.H. Lauridsen, K. Buchmann, Effects of short- and long-term glucan feeding of rainbow trout (Salmonidae) on the susceptibility to Ichthyophthirius multifiliis infections, Acta Ichthyologia Piscatoria 40 (2010) 61e66. [7] A.-C. Cheng, Y.-Y. Chen, J.-C. Chen, Dietary administration of sodium alginate and k-carrageenan enhances the innate immune response of brown-marbled grouper Epinephelus fuscoguttatus and its resistance against Vibrio alginolyticus, Vet Immunol Immunopathol 121 (2008) 206e215. [8] J. Zhou, X.-L. Song, J. Huang, X.-H. Wang, Effects of dietary supplementation of A3a-peptidoglycan on innate immune responses and defense activity of Japanese flounder (Paralichthys olivaceus), Aquaculture 251 (2006) 172e181. [9] G. Gioacchini, P. Smith, O. Carnevali, Effects of Ergosan on the expression of cytokine genes in the liver of juvenile rainbow trout (Oncorhynchus mykiss) exposed to enteric red mouth vaccine, Vet Immunol Immunopathol 123 (2008) 215e222. [10] S. Peddie, J. Zou, C.J. Secombes, Immunostimulation in the rainbow trout (Oncorhynchus mykiss) following intraperitoneal administration of Ergosan, Vet Immunol Immunopathol 86 (2002) 101e113. [11] W.M. Sealey, F.T. Barrows, A. Hang, K.A. Johansen, K. Overturf, S.E. LaPatra, et al., Evaluation of the ability of barley genotypes containing different amounts of b-glucan to alter growth and disease resistance of rainbow trout Oncorhynchus mykiss, Animal Feed Sci Technol 141 (2008) 115e128. [12] R. Russo, R.P.E. Yanong, H. Mitchell, Dietary beta-glucans and nucleotides enhance resistance of red-tail black shark (Epalzeorhynchos bicolor, fam. Cyprinidae) to Streptococcus iniae infection, J World Aquac Soc 37 (2006) 298e306. [13] N. Sheikhzadeh, A. Karimi Pashaki, K. Nofouzi, M. Heidarieh, H. Tayefi-Nasrabadi, Effects of dietary ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss), Fish Shellfish Immunol 32 (2012) 407e410. [14] T. Itami, M. Kondo, M. Uozu, A. Suganuma, T. Abe, A. Nakagawa, et al., Enhancement of resistance against Enterococcus seriolicida infection in yellowtail, Seriola quinqueradiata (Temminck & Schlegel), by oral administration of peptidoglycan derived from Bifidobacterium thermophilum, J Fish Dis 19 (1996) 185e187. [15] X. Li, S. Wang, J. Qi, S.F. Echtenkamp, R. Chatterjee, M. Wang, et al., Zebrafish peptidoglycan recognition proteins are bactericidal amidases essential for defense against bacterial infections, Immunity 27 (2007) 518e529. [16] C.M.S. Ribeiro, T. Hermsen, A.J. Taverne-Thiele, H.F.J. Savelkoul, G.F. Wiegertjes, Evolution of recognition of ligands from Gram-positive bacteria: similarities and differences in the TLR2-mediated response between mammalian vertebrates and teleost fish, J Immunol 184 (2010) 2355e2368.
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