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Fish & Shellfish Immunology xxx (2015) 1e8

Contents lists available at ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Full length article

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Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes

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M. Makesh a, b, *, Ponnerassery S. Sudheesh a, Kenneth D. Cain a a b

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Department of Fish and Wildlife Sciences, College of Natural Resources, University of Idaho, Moscow, ID 83844-1136, USA Aquatic Environment and Health Management Division, Central Institute of Fisheries Education, Versova, Mumbai 400061, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 September 2014 Received in revised form 2 February 2015 Accepted 3 February 2015 Available online xxx

Teleosts possess three immunoglobulin (Ig) heavy chain isotypes viz., IgM, IgT and IgD and all three isotypes are reported in rainbow trout. The expression of these Ig isotypes in response to different immunization routes was investigated and results provide a better understanding of the role these Igs in different tissues. Rainbow trout (Oncorhynchus mykiss) were immunized with an attenuated Flavobacterium psychrophilum strain, 259-93-B.17 grown under iron limiting conditions, by intraperitoneal, anal intubation and immersion routes. Serum, gill mucus, skin mucus and intestinal mucus samples were collected at 0, 3, 7, 14, 28, 42 and 56 days post immunization by sacrificing four fish from each treatment group and the unimmunized control group, and the IgM levels were estimated by an enzyme linked immunosorbent assay (ELISA). In addition, blood, gill, skin and intestinal tissue samples were collected for Ig gene expression studies. The secretory IgM, IgD and IgT gene expression levels in these tissues were estimated by reverse transcription quantitative real time PCR (RT-qPCR). Levels of IgM in serum, gill and skin mucus increased significantly by 28 days after immunization in the intraperitoneally immunized group, while no significant increase in IgM level was observed in fish groups immunized by other routes. Secretory IgD and IgT expression levels were significantly upregulated in gills of fish immunized by the immersion route. Similarly, secretory IgT and IgD were upregulated in intestines of fish immunized by anal intubation route. The results confirm mucosal association of IgT and suggest that IgD may also be specialized in mucosal immunity and contribute to immediate protection to the fish at mucosal surfaces. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Rainbow trout Oncorhynchus mykiss Systemic and mucosal immune response IgM IgT IgD

1. Introduction Aquaculture of salmonid species is a high value fish production activity in Europe, North America, Chile, Japan, Australia and other parts of the world. Bacterial cold water disease (CWD) and rainbow trout fry syndrome (RTFS) caused by Flavobacterium psychrophilum are major diseases very often resulting in severe production and economic losses in salmonid aquaculture facilities, especially those producing rainbow trout Oncorhynchus mykiss (Walbaum), coho salmon Oncorhynchus kisutch (Walbaum) and steelhead [1,2]. Trout farming often suffers substantial economic loss due to problems

* Corresponding author. Aquatic Environment and Health Management Division, Central Institute of Fisheries Education, Versova, Mumbai 400061, India. Tel.: þ91 22 2636 1446; fax: þ91 22 2636 1573. E-mail address: [email protected] (M. Makesh).

associated with CWD epizootics including high mortality, increased susceptibility to other diseases, use of chemotherapeutants, high costs of treatment and skeletal deformities resulting in quality reduction in recovering fish [1]. Despite tremendous efforts, a vaccine to control CWD is yet to be approved and available. A major constraint to the development of an efficacious CWD vaccine is our inadequate understanding of the innate and adaptive immune responses of the host fish to the pathogen, especially at the mucosal surfaces. Immunoglobulins (Igs) comprised of heavy and light chain molecules, are the mediators of adaptive immunity in fish and other vertebrates. Mammals have five different heavy chain isotypes of Ig namely IgM, IgG, IgA, IgD and IgE each with distinct functions while teleosts were long considered to have only two Ig isotypes, IgM and IgD. Teleosts were thought to lack specialized mucosal antibodies equivalent to IgA of mammals. However, recent

http://dx.doi.org/10.1016/j.fsi.2015.02.003 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Makesh M, et al., Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/ j.fsi.2015.02.003

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discovery of other isotypes viz. IgT/IgZ [3,4] and IgM-IgZ chimera [5], the functions of which are not fully characterized, has thrown open the research to elucidate the role of these newly discovered Ig isotypes. IgM is the most ancient and the only isotype functionally conserved in all jawed vertebrates (reviewed by Flajnik [6]). Serum IgM, a pentamer in mammals, birds and cartilaginous fish, is secreted as a tetramer in teleosts [7] in response to infections. IgM is considered to protect the fish against pathogens both systemically and at mucosal surfaces [8]. Recent studies provide evidence that IgD is also a primordial antibody isotype present in all jawed vertebrates including elasmobranchs [9], acipenseriformes [10], and teleosts except birds [11] and some mammalian species. Among teleosts, IgD is reported in almost all species examined including channel catfish (Ictalurus punctatus) [12], Atlantic salmon (Salmo salar) [13], Atlantic cod (Gadus morhua) [14], Atlantic halibut (Hippoglossus hippoglossus) [15], Japanese flounder (Paralichthys olivaceus) [16], fugu (Takifugu rubripes) [17], grass carp (Ctenopharyngodon idella) [18], threespined stickleback (Gasterosteus aculeatus) [19,20], and rainbow trout (O. mykiss) [21]. IgD exists as multiple structural variants and splice forms exist in different vertebrates. Teleost IgD is characterized by long chimeric molecules consisting of repeated domains compared to shorter hinge containing molecules in mammals [22]. It is monomeric [21] and is found as membrane bound on B cells as well as a secreted form [23]. In humans, IgD secreted by IgDþ cells in the upper respiratory mucosa mediate mucosal immunity by binding to respiratory pathogens. In addition, IgD activated basophils trigger innate antimicrobial response. Binding of IgD to channel catfish granulocytes has been reported [24], and this function is believed to be evolutionarily conserved. However, the exact function of IgD in teleost is not clearly understood. Another antibody isotype, IgT is the latest antibody class discovered in vertebrate species [25]. The IgT or its equivalent (IgZ) is reported in many teleosts including Zebrafish (Danio rerio) [4], common carp (Cyprinus carpio) [5], fugu (T. rubripes) [26], rainbow trout (O. mykiss) [3], grass carp (C. idella) [18], three-spined stickleback (G. aculeatus) [19], and Atlantic salmon (S. salar) [27]. Most of these species possess more than one subclass of IgT [28]. Evidences show that IgT is involved in gut [29] and gill [30] mucosal immunity. The finding has challenged the paradigm that the specialization of immunoglobulin isotypes into mucosal and systemic arose during tetrapod evolution [28]. IgT is expressed as a monomer in serum and as a tetramer in gut mucus [29]. All the three Ig isotypes (IgM, IgD and IgT) are expressed both as membrane bound and secretory form [21,23,28]. Measurement of IgM, IgD and IgT expression in teleosts will improve our understanding of the role of these immunoglobulins in combating invading pathogens not only through the systemic circulation, but at specific mucosal surfaces as well. A live attenuated strain of F. psychrophilum (259-93-B.17) has been developed at the Department of Fish and Wildlife Sciences at the University of Idaho [31]. The efficacy of this vaccine has been improved by growing the bacteria under iron limiting conditions and has recently been shown to provide significant protection against CWD in coho salmon [32]. A deeper understanding of the immune response of fish to F. psychrophilum especially at the mucosal surfaces, which is the natural route of infection is important for further fine tuning the efficacy of the CWD vaccine and for better health management of farmed salmonids. The expression of different immunoglobulin isotypes of fish in response to infection and vaccination is not fully understood. In this study the expression of all three isotypes of Ig was characterized in blood and different mucosal organs following immunization with F. psychrophilum by different routes.

2. Materials and methods 2.1. Experimental animals Rainbow trout (O. mykiss) (mean weight 35 g) were procured from the University of Idaho's Aquaculture Research Institute (ARI) and were maintained in 500 L tanks with continuous aeration in a flow through water system supplied with dechlorinated municipal water maintained at 15 ± 1  C. The fish were fed a commercial pellet feed (Rangen EXTR 450 1/16) at 1% of body weight divided into two equal doses. All experimental procedures with live fish were carried out with prior approval from the Institutional Animal Care and Use Committee, University of Idaho (IACUC # 2012-30). 2.2. Bacteria A live attenuated vaccine strain of F. psychrophilum, 259-93-B.17 [31] maintained at Department of Fish and Wildlife Sciences, College of Natural Resources, University of Idaho, Moscow, USA was used for immunizing the fish. 2.3. Culture of F. psychrophilum Glycerol stocks of F. psychrophilum strain 259-93-B.17 were revived by inoculating in to tryptone yeast extract salts broth (TYES; 0.4% tryptone, 0.04% yeast extract, 0.05% MgSO4, 0.05% CaCl2, pH 7.2) and incubated at 15  C shaking at 80 rpm. Once visible turbidity was observed in the broth, the culture was streaked on TYES agar (1.5% agar in TYES broth) and incubated at 15  C. The vaccine strain F. psychrophilum 259-93-B.17 was grown under iron limited condition (B.17-ILM) in TYES broth in the presence of an iron chelator, 20 ,20 Bipyridine (50 mM) under shaking conditions at 15  C for 4 days. Bulk culture was produced by inoculating a single colony of F. psychrophilum in TYES broth containing 20 ,20 Bipyridine in 10 mL and scaled up to produce the desired volume of culture. The bacterial cells were pelleted by centrifuging at 5000  g for 20 min at 4  C. The cells were washed twice in phosphate buffered saline (PBS), finally resuspended in desired volume of PBS and stored at 4  C. The colony forming units (CFU) in the culture was estimated by standard plate count method. The plate count of the vaccine strain, F. psychrophilum B.17-ILM obtained was 3  108 CFU mL1. 2.4. Immunization Fishes were divided into four groups and were immunized with F. psychrophilum B.17-ILM as shown in Table 1. Fishes were anaesthetized with tricaine methanesulfonate (100 mg L1) before immunization. Intraperitoneal (IP) injection was administered using tuberculin syringe and anal intubation (AI) was done using a micropipette attached with a 200 mL microtip and bacteria were administered into the hind gut. A 100 mL dose of the stock culture containing 3  107 CFU was used to immunize each fish by IP and AI Table 1 Table showing different treatment groups, route and dose of immunization of rainbow trout. Group

Number of fish immunized

Route of immunization

Dose

A

55

Nil

B C D

40 40 40

Unimmunized control Intraperitoneal Anal intubation Bath

3.0  107 CFU fish1 in 100 mL 3.0  107 CFU fish1 in 100 mL 3.0  106 CFU mL1 water for 1 h

Please cite this article in press as: Makesh M, et al., Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/ j.fsi.2015.02.003

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route. For bath immunization the stock culture was diluted 1:100 in the rearing water and the fish were held for 1 h with aeration. The control group was maintained without immunization. 2.5. Sampling Fish were sampled prior to immunization (0 day) and then divided into four groups and immunized. Subsequently, fish were sampled at 3, 7, 14, 28, 42 and 56 days post-immunization (DPI). The following samples were collected from four fish from each group at each time point. For all sampling, fish were anesthetized using tricaine methanesulfonate. 2.5.1. Whole blood Whole blood was collected from the caudal vein using a 5 mL syringe and needle. Part of the blood was immediately mixed with equal volume of Alsever's solution (0.1 M Dextrose, 70 mM NaCl, 30 mM Sodium citrate; pH 7.2) in a 15 mL centrifuge tube for separation of lymphocytes and the rest of the blood was allowed to clot at room temperature and stored at 4  C for serum separation. For lymphocyte separation, the RBCs were lysed following the protocol of Crippen et al. [33] with modifications. Nine millilitres of sterile distilled water was added to one millilitre of blood and mixed gently for 20 s. One millilitre of 10X PBS was added to the tube and mixed immediately. The tube was allowed to stand on ice for 10 min. The cell debris and nuclear material precipitated and the clear supernatant containing the leukocytes, which is rich in lymphocytes, were transferred to another tube using a serological pipet and centrifuged at 750  g for 10 min at 4  C. The pelleted cells were washed twice in PBS by centrifuging at 750  g for 10 min at 4  C and stored in RNAlater® (Life Technologies, USA). Serum was separated from clotted blood by centrifuging at 4000  g for 5 min at 4  C. The separated serum was stored at 20  C. 2.5.2. Gill mucus Gill mucus was collected using a sterile cotton swab by rubbing gently over the gills on both the sides of the fish and vortexed thoroughly in a microcentrifuge tube containing 0.2 mL of PBS and protease inhibitor cocktail (Sigma, USA). The cotton swab was squeezed with a pair of forceps to collect the adsorbed liquid. The mucus samples were centrifuged at 10,000  g for 5 min at 4  C and the supernatant was stored at 20  C. 2.5.3. Skin mucus The skin mucus was collected following the method of Valdenegro-Vega et al. [34] with minor modifications. The fish was placed inside a polythene bag containing 0.5 mL of PBS and protease inhibitor cocktail and rubbed gently. The mucus samples were collected in a microcentrifuge tube and were centrifuged at 10,000  g for 5 min at 4  C. The supernatant was stored at 20  C. 2.5.4. Intestinal mucus For collecting the intestinal mucus, approximately 2 cm length of hind gut was excised and the gut contents were removed by striping with a pair of forceps. The mucus was collected in a microcentrifuge tube containing 0.2 mL of PBS with protease inhibitor cocktail by stripping with a pair of forceps. The samples were vortexed briefly and centrifuged at 10,000  g for 10 min at 4  C. The supernatants were separated and stored at 20  C. 2.5.5. Tissues Gill, skin (about 2 cm2 on the lateral side) and intestine (about 2 cm of hind gut) samples were collected in RNAlater® and stored at 20  C until processed.

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2.6. Enzyme linked immunosorbent assay (ELISA) The serum, gill mucus, skin mucus, and intestinal mucus IgM level were assessed by ELISA following the protocol described by LaFrentz et al. [35] with modifications. The 96 well ELISA plates were coated with 100 mL of inactivated F. psychrophilum B.17-ILM (containing 1  108 CFU mL1 before inactivation) in bicarbonate buffer (pH 9.6) and incubated overnight at 4  C. On the following day the plates were washed three times with PBS containing 0.05% Tween 20 (PBST) and the samples were added to the first well in the column and serially diluted two fold in PBST containing 0.02% sodium azide. The initial dilution of the samples were 1:200 for serum, 1:8 for gill mucus, undiluted for skin mucus and intestinal mucus samples. Positive and negative rainbow trout serum controls were also included in each plate. The plates were incubated at 15  C overnight. The plates were washed three times in PBST and mouse monoclonal antibody (MAb 1.14) [36] against trout IgM was added at a dilution of 1:400 containing 0.1% non-fat dry milk at 100 mL well1. The plates were incubated for 1 h at room temperature (RT) followed by three washings with PBST. The plates were incubated with 100 mL well1 of goat anti-mouse Ig HRP conjugate (Bio-rad, USA) diluted 1:4000 in PBS containing 0.1% non-fat dry milk for 1 h at RT. The plates were washed 3 times with PBS and 50 mL ABTS peroxidase substrate (KPL Inc., USA) was added. The plates were incubated at RT for 15 min in the dark and the reaction was stopped by adding 50 mL of 1% SDS solution and the plates were read at 405 nm in an ELISA plate reader (Biotek, USA). The antibody titre in serum was determined as the reciprocal of the highest dilution of the serum having twice the OD of the negative controls. For gill, intestinal and skin mucus samples the OD values of the initial dilution were used as such for analysis as reference negative and positive controls were not available. The serum IgD level was assessed by ELISA using the same protocol described above using an initial dilution of 1:50. The secondary antibody, the mouse monoclonal anti-trout IgD antibody was kindly provided by Dr. John Hansen, WFRC-USGS Biological Resources Division through the U. S. Veterinary Immunological Reagent Network (http://www.umass. edu/vetimm/trout/index.html). The plates were read at 405 nm in an ELISA plate reader and the OD values of the initial dilutions were used for the analysis. All the data was further subjected to one way analysis of variance using SPSS 16.0 software. The ELISA titres were considered statistically significant if p values were

Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes.

Teleosts possess three immunoglobulin (Ig) heavy chain isotypes viz., IgM, IgT and IgD and all three isotypes are reported in rainbow trout. The expre...
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