Veterinary Parasitology 205 (2014) 28–37

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Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA) Galit Sharon a,1 , Pulak R. Nath b,1 , Noah Isakov b , Dina Zilberg a,∗ a b

The Jacob Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Midreshet Ben Gurion, Israel The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben Gurion University of the Negev, Beer Sheva, Israel

a r t i c l e

i n f o

Article history: Received 3 April 2014 Received in revised form 3 July 2014 Accepted 6 July 2014 Keywords: Guppy Tetrahymena ELISA Immunization Anal intubation

a b s t r a c t Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse antiguppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10 ␮L) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund’s adjuvant (15,000 Tetrahymena/fish in total a volume of 20 ␮L). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from pathogen challenge by immersion. The results suggest a direct correlation between the anti-Tetrahymena Ab response and fish resistance to i.p.-injected Tetrahymena, but not to infection by immersion preceded by cold stress. © 2014 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author. Tel.: +44 972 8 6596818; fax: +972 8 6596742. E-mail address: [email protected] (D. Zilberg). 1 These authors contributed equally to this work and share first authorship. http://dx.doi.org/10.1016/j.vetpar.2014.07.007 0304-4017/© 2014 Elsevier B.V. All rights reserved.

Guppies (Poecilia reticulata Peters) are the most popular fish among hobbyists due to their vibrant colors and the fact that they are easy to breed and maintain (Harpaz

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et al., 2005). Although they are successfully produced in intensive systems, this practice increases the occurrence of disease outbreaks. One of the most serious diseases affecting guppies is caused by Tetrahymena sp., a ciliated protozoan that is also known as the guppy killer parasite. There are many different species of Tetrahymena, with over 40 documented (Simon et al., 2008). They are facultative parasites that infect a large range of fish species, and they appear to be highly pathogenic, particularly in guppies (Imai et al., 2000; Ponpornpisit et al., 2000; Kim et al., 2002; Shenberg, 2003; Sharon et al., 2014a). In this study, we used a Tetrahymena strain reported by Pimenta Leibowitz and Zilberg (2009), which was identified as a new strain by Chantangsi et al. (2007), based on the sequence of its mitochondrial cytochrome c oxidase subunit 1 (cox 1), a gene proposed as a DNA barcode for the identification of animal species. Initial immunization against this strain of Tetrahymena was reported by Chettri et al. (2009), demonstrating protection from experimental infection following intraperitoneal (i.p.) immunization with adjuvant. The in vivo protection from infection correlated with in vitro immobilization of the parasite by body homogenates of immunized fish, suggesting that the immunization induced the production of immobilizing antibodies (Abs) (Chettri et al., 2009). Iglesias et al. (2003) reported that immunization against another invasive parasitic ciliate, Philasterides dicentrachi, causing systemic scuticociliatosis in turbot [Scophthalmus maximus (Linnaeus)], induces antibody (Ab) production which correlates with protection from infection. In that work, crude extract and ciliary Ag fractions of the parasite were used as primary Ags, and Ab response was evaluated by ELISA. Elevated OD readings of the tested antiserum in the enzyme-linked immunosorbent assay (ELISA) correlated with the antiserum’s ability to mediate parasite agglutination in vitro, demonstrating the effectiveness of the immunization and the expression of a surface-immobilizing Ag(s) on the parasite (Iglesias et al., 2003). In the present study, we used two different routes to immunize guppies against Tetrahymena: anal intubatation and i.p. injection, and developed an ELISA to compare the efficiency of the consequent Ab response. Anal intubation was selected since the hindgut segment of teleosts has been found to be the main site of Ag uptake (Georgopoulou and Vernier, 1986). Introduction of Ag at this site was aimed at obtaining a proof of concept for the feasibility of immunization via the gastrointestinal tract. IgM, the major Ab subclass in fish, is involved in the immune response against pathogens (Wilson and Warr, 1992; Pilström and Bengtén, 1996). However, IgM differs between fish species, and due to a lack of commercial guppy immunoglobulin (Ig) Abs, we prepared these Abs in mice to use them in a quantitative ELISA. The presence of IgM has previously been demonstrated in guppies by Lim et al. (2009), who also developed guinea pig anti-guppy Ig antiserum for an ELISA. In the present study, we used several different preparations of guppy Igs as immunogens, and following mouse immunization, compared the efficiency and specificity of the resulting antisera.

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2. Materials and methods 2.1. Fish Three-month-old guppies were obtained from commercial ornamental fish farms in the Arava valley, Israel. Fish were pre-examined to ensure absence of Tetrahymena sp. before initiating the study. Naïve fish were kept in 100L tanks supplied with biological filters and aeration, and fed daily with commercial guppy food (Ocean Nutrition, Newark, CA) at about 2% of their body weight until the end of the experiments. Water quality was monitored weekly; ammonia and nitrite levels were measured by Visocolor® kits (Macherey-Nagel, Düren, Germany) and maintained at 80% saturation. All procedures were conducted on anesthetized fish (250 ␮L clove oil/L of water) for >3 min). Experimental protocols were carried out in compliance with the principles of biomedical research involving animals, set up by the Ben Gurion University Committee for the Ethical Care and Use of Animals, authorization number: IL-51-8-2008. 2.2. Isolation and maintenance of Tetrahymena The Tetrahymena sp. used in the experiments was originally diagnosed in guppies imported from Singapore at the quarantine stage. Comparative DNA barcode analysis of the cox 1 gene carried out by Chantangsi et al. (2007), indicated that the isolated Tetrahymena is a new species. Tetrahymena was maintained in vivo and in vitro as described by Pimenta Leibowitz and Zilberg (2009). Briefly, infected fish were maintained in 100-L containers, and naïve fish were added regularly to replace mortalities. For in vitro culture, Tetrahymena sp. isolated from infected fish organs (skin, gills or tail) was placed in RM9 medium [containing 5 g protease peptone, 5 g tryptone, 2 g glucose, 0.1 g liver extract and 0.2 g dipotassium hydrogen phosphate in 1 L of double-distilled water (DDW); ATCC, 1999]. Subculturing was conducted once a week and a passage through fish was performed once every 8–10 in vitro passages, by i.p. injection of ca. 900 Tetrahymena in 20 ␮L RM9 medium per fish, to maintain the parasite’s pathogenicity, followed by reisolation and culture. 2.3. Demonstrating Igs in guppy serum and body homogenate 2.3.1. Blood withdrawal and serum preparation Blood was withdrawn from large (1.5–2.0 g) female guppies, immunized (70 fish) or not (70 fish) with glutathione S-transferase (GST; GE Healthcare Bio-Sciences, Uppsala, Sweden), a well-established antigen, which is known to induce a high Ab response in fish (Oshima et al., 1996). For immunization, female guppies were injected i.p. with 350 ␮g GST in PBS (pH 7.2, 0.05 M) emulsified in complete Freund’s adjuvant (CFA; Sigma, St. Louis, MO), at a ratio of 1:2 (final volume of 20 ␮L/fish). After 3 weeks, fish were boosted with GST emulsified in incomplete Freund’s adjuvant (IFA; Sigma) and blood was withdrawn 4 weeks

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after the booster. Blood was withdrawn from the caudal vein of anesthetized fish using a 1-mL heparinized syringe with a 30G needle. About 10 ␮L blood was collected per fish, and all blood samples within each of the two groups were pooled. After centrifugation at 9000 × g for 10 min at 4 ◦ C (Eppendorf A6 centrifuge, 5417R, Hamburg, Germany), sera were collected and stored at −80 ◦ C. For complement inactivation, sera were incubated at 56 ◦ C for 30 min. 2.3.2. Preparation of guppy body homogenates Fish body homogenates were prepared as described by Chettri et al. (2009) with slight modifications. Fish were euthanized (250 ␮L clove oil/L of water for >3 min) and dissected to remove the gut, gallbladder and tail fin. Individual fish were then placed in a flat-bottom tube containing 2 mL DDW and homogenized on ice, using a commercial homogenizer (Ystral GmbH Maschinenbau, Ballrechten-Dottingen, Germany). Homogenates were centrifuged at 4000 × g for 10 min at 4 ◦ C (Eppendorf A6 centrifuge, 5417R) and supernatants were transferred to 2-mL tubes and centrifuged at 13,000 × g for 15 min at 4 ◦ C. The process was repeated 2–3 times, until the supernatant samples were clear. Samples were lyophilized for 12–18 h (VirTis, Gardiner, NY), and resuspended in 200 ␮L DDW. Protein concentration was measured in each sample by Bradford assay (Bradford, 1976) and adjusted to a final concentration of 14 ␮g/mL, unless otherwise specified. Body homogenates were diluted in PBS (pH 7.2, 0.05 M), for subsequent use in gel electrophoresis, or in Tris-buffered saline (TBS; pH 7.6) for subsequent ELISA. 2.3.3. Gel electrophoresis of guppy sera and body homogenates Guppy sera and body homogenates were fractionated by Polyacrylamide gel electrophoresis (PAGE) under reducing conditions on a 10% gel. In parallel, guppy sera or antisera (25 ␮L/group) or body homogenates (1.2 mg/25 ␮L) were incubated with 10 ␮L protein A/G covalently immobilized on sepharose beads (Santa Cruz Biotechnology, Inc., Dallas, TX). After 3 h of incubation at room temperature (RT), the protein A/G beads were washed three times in PBS, centrifuged at 14,000 g (MIKRO 22 R, Tuttlingen, Germany) for 1 min at RT and the supernatant was discarded. For gel electrophoresis, samples were diluted 4:1 in 5× sample buffer (10% SDS, 50% glycerol, 300 mM Tris pH 6.8, 10% ␤-mercaptoethanol and 0.005% bromophenol blue), boiled for 5 min, and loaded on the gel. The gel was electrophoresed at 150 V and 400 mA for 60 min (PowerPacTM 300, Bio-Rad Laboratories, Inc., Hercules, CA), followed by staining with Coomassie brilliant blue (Roth, Karlsruhe, Germany). Protein bands were quantified by comparison to a standard reference (Precision Plus Protein Dual Color Standards, Bio-Rad), revealing that 1 ␮L guppy serum contained ∼10 ␮g of IgH. 2.4. Preparation of mouse anti-guppy Igs 2.4.1. Mouse immunization with guppy Igs Mice were immunized with three different preparations of guppy Ig: (i) gel preparation–whole guppy serum (∼150 ␮L containing ∼1.5 mg of IgH) was electrophoresed,

and a thin slice of gel containing the ∼75-kDa protein band corresponding to guppy IgH (Wilson and Warr, 1992; Lim et al., 2009) was excised, chopped into small pieces, mixed with PBS, and emulsified in CFA or IFA (1:1, v/v ratio) for primary and secondary immunization of the mice, respectively; (ii) protein A/G preparation – guppy serum (100 ␮L) was incubated with protein A/G beads (40 ␮L) for 2 h at RT. The beads where washed three times in PBS, resuspended in PBS at a final volume of 200 ␮L and emulsified 1:1 in CFA or IFA, as in preparation (i); (iii) protein A/G beads + gel preparation – guppy serum was incubated with protein A/G beads, as in preparation (ii). After washing in PBS, the proteins adsorbed on the beads were fractionated by gel electrophoresis; a gel slice containing the guppy IgH was excised, chopped, and emulsified in CFA or IFA, as in preparation (i). Three ∼8-week-old BALB/c mice per group were immunized s.c. with 200 ␮L guppy Ig preparation. A total of three immunizations were applied at 3-week intervals. All mice were initially immunized with guppy Igs from the gel preparation in CFA (emulsified 1:1). Mice were boosted with (i) guppy Igs from the gel preparation, (ii) guppy Igs from the protein A/G preparation, or (iii) guppy Igs from the protein A/G preparation + gel preparation. All booster immunizations were in IFA. Ten days after the third immunization, mice were anesthetized using a mixture of 2% xylazine (100 mg/kg; Proxylaz® ) and ketamine HCl (10 mg/kg; USP, Fort Dodge® , Iowa), and blood was withdrawn from the facial vein. 2.4.2. Western blot analysis Samples of guppy serum or body homogenates were fractionated by gel electrophoresis as described in Section 2.3.3 and gel proteins were transblotted onto a nitrocellulose membrane by electrophoresis (1 h at 100 V, 250 mA; PowerPacTM 300, Bio-Rad). The membrane was stained with Ponceau S (Sigma), scanned and recorded, washed in PBS-T (PBS with 0.1% Tween-20), and blocked in 3% Bovine serum albumin (BSA)/PBS-T + 0.01% sodium azide (Western blot blocking solution) for 1 h at RT. The membrane was incubated on a shaker and washed three times in PBS-T for 5 min each time, followed by incubation with serum from a mouse immunized with guppy Ig from a protein A/G preparation (primary Ab) or non-immunized mouse as a control, diluted in blocking solution, for 1 h at RT. Unbound Abs were removed and the membrane was washed as before and incubated with peroxidaseconjugated goat anti-mouse IgG (Sigma) diluted 1:5000 in PBS-T for 1 h at RT. Following the washes, the membrane was incubated with ECL (enhanced chemiluminescence; Thermo Scientific, Rockford, IL) for 1 min, exposed to X-ray film in the dark, and developed by autoradiography. 2.4.3. ELISA Flat-bottom microtiter plate wells (96-well, NuncMaxisorp, Roskilde, Denmark) were coated with 50 ␮L of guppy serum (10-fold dilutions, in the range of 10−4 –10−8 ) or guppy body homogenate (twofold dilutions, in the range of 1/2–1/32) diluted in TBS by overnight incubation at 4 ◦ C. Wells were washed three times with TBS-T (TBS + 0.05% Tween-20) and once with TBS, blocked with 200 ␮L TBS + 5% BSA (ELISA blocking solution) for 2 h

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at RT and washed again. Fifty ␮L of antiserum from a mouse immunized with protein A/G-adsorbed guppy Ig (protein A/G preparation) was then added as the first Ab (diluted 1:10−5 ) in TBS-T + 3% BSA, and naïve mouse serum was used as a negative control. The wells were washed again and 50 ␮L of peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) diluted 1:3000 in TBS-T + 3% BSA was added and incubated for an additional 2 h at RT. After the last wash, 100 ␮L TMB peroxidase substrate (3,3 ,5,5 tetramethylbenzidine solution plus hydroxide; Bio-Rad) was added to each well for an additional 30 min of incubation at RT in the dark with gentle shaking. The reaction was terminated by the addition of 50 ␮L 1 N H2 SO4 , and plates were read at 450 nm using a microplate absorbance reader for 96-well plates (Tecan SunriseTM , Salzburg, Austria). Samples were tested in tetraplicates and OD values of the negative control (TBS only) were subtracted from all wells. 2.5. Tetrahymena Ag preparation Preparation of Tetrahymena Ag followed the protocol described by Iglesias et al. (2003) for P. dicentrachi crude extract preparation, with some modifications. Cultured Tetrahymena were collected 4 days postinoculation in fresh culture medium, centrifuged (328 × g for 6 min at 10 ◦ C), resuspended in fresh RM9 medium, and adjusted to a concentration of 750,000 Tetrahymena/mL. Tetrahymena were centrifuged again and resuspended in the same volume of PBS (0.015 M phosphate buffer, 0.15 M NaCl, pH 7.2) containing protease inhibitors [0.1 mM pepstatin A, 0.02 mM N-(trans-epoxysuccinyl)-l-leucine 4 guanidinobutylamide (E-64), 1 mM phenylmethanesulfonyl fluoride (PMSF) and 2 mM EDTA; all from Sigma]. The parasites were lysed ultrasonically (Branson Digital Sonifier, Wilmington, NC) on ice, and the lysate was centrifuged at 15,000 × g for 20 min at 4 ◦ C (Eppendorf A6 centrifuge, 5417R). Supernatants containing the Ag were collected and stored at −80 ◦ C. Protein concentration was measured using the Bradford assay (Bradford, 1976), and adjusted to 450 ␮g/mL for use as a primary Ag in ELISA. 2.6. Fish immunization against Tetrahymena and analysis of Ab production 2.6.1. Fish immunization, sample collection and challenge with Tetrahymena Two trials were conducted to test different routes of immunization. In trial 1, guppies were immunized by i.p. injection (20 ␮L) of sonicated Tetrahymena (750,000 Tetrahymena/mL) emulsified in CFA. Fish were boosted after 4 weeks using a similar Tetrahymena preparation emulsified in IFA (Chettri et al., 2009). Two groups of control fish were injected in parallel with PBS emulsified in adjuvant or PBS only. Details of the experiment are given in Chettri et al. (2009), in which the vaccinated fish were protected from i.p. challenge with live Tetrahymena and there was no protection from infection in any of the control groups. Fish samples were frozen (after removal of gut and gallbladder) and kept at −80 ◦ C until further analysis for anti-Tetrahymena Ab titer. Body homogenates were prepared as described in Section 2.3.2. A total of eight

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Table 1 Sample preparation for anal intubation.a Reagent

Control group (␮L)

Experimental group (␮L)

PBS DOMb DOCc FAAd Sonicated Tetrahymena Total volume (␮L)

925 25 30 20 0 1000

0 25 30 20 925 1000

Each fish was intubated with 10 ␮L of the preparation. 13.5 mg domperidone dissolved in 1 mL 1 M citric acid. 9.8 mg deoxycholic acid dissolved in 50 mL absolute EtOH. d Free amino acids: 5 mg valine, 5.7 mg leucine, 5.7 mg isoleucine, 10.7 mg phenylalanine, 4.7 mg tryptophan in 15 mL DDW. a

b

c

Tetrahymena-immunized fish were analyzed in two pools of four fish each, and control groups included pools of three fish per group. Samples were analyzed using the ELISA described in Section 2.6.2. In trial 2, guppies were stocked at 30–35 fish per 10-L aquarium, in two to four replicates. Fish were immunized against Tetrahymena by anal intubation, using a solution containing 2,000,000 sonicated Tetrahymena/mL. The solution was mixed with a carrier composed of domperidon (DOM), deoxycholic acid (DOC) and free amino acids (FAA) (based on Tandler et al., 2007; Table 1). Control fish were intubated with PBS plus carrier. For intubation, fish were anesthetized, turned on their backs and a 24G venflon (without the needle) connected to a 1-mL syringe was inserted through the anal orifice to the far end of the colon. Each fish was intubated with a volume of 10 ␮L containing 40,000 Tetrahymena. Positive control fish were immunized by i.p. injection of 20 ␮L sonicated Tetrahymena (750,000 parasite/mL) emulsified in CFA (at a ratio of 2:1; after Chettri et al., 2009). A negative control consisting of untreated fish was also included. Three weeks after the initial immunization, anally intubated fish were boosted using the same preparation as in the initial immunization, and i.p.-injected fish were boosted with the same Ag, emulsified in IFA instead of CFA. After 3 additional weeks, fish were challenged with live Tetrahymena, either by i.p. injection or by immersion, as previously described (Sharon et al., 2014b). Briefly, for i.p. injection, a dose of 900 Tetrahymena in 20 ␮L RM9 was used per fish. For immersion, preapplied cold stress was carried out by gradually decreasing the water temperature to 17 ◦ C over 48 h from the original 27 ◦ C. Fish were kept at 17 ◦ C for 48 h and then the temperature was raised back to 21 ◦ C over 3 h. Exposure to Tetrahymena by immersion (10,000 Tetrahymena/mL, in plastic containers filled with 600 mL water, for 6 h with aeration) was carried out at 21 ◦ C, after which the temperature was elevated to 27 ◦ C over 48 h and maintained at 27 ◦ C throughout the experiment. Mortality was monitored daily and dead fish were examined postmortem for the presence of Tetrahymena in the skin, gills and internal organs. Treatment types and number of replicates are presented in Table 2. Two days before challenge (before the temperature decrease), three fish from each replicate aquarium were collected, and their gut and gallbladder were removed (dissected on ice) and stored at −80 ◦ C until analysis for

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Table 2 Treatments applied in the guppy immunization studies. Primary immunization and boostera

Challengeb

Number of replicate aquaria

Intubation, Tetrahymena Intubation, Tetrahymena Intubation, Control Intubation, Control i.p., Tetrahymena i.p., Tetrahymena i.p., Control i.p., Control

Immersion i.p. injection Immersion i.p. injection Immersion i.p. injection Immersion i.p. injection

4 4 4 4 4 2 2 2

a b

Booster was applied 3 weeks after the primary immunization. Challenge took place 3 weeks after the booster.

anti-Tetrahymena Ab titer by ELISA. Body homogenates were prepared from individual fish of each treatment group, as described in Section 2.3.2. Eight fish were analyzed per group, except for the Tetrahymena-intubated fish, which consisted of 14 individuals.

IgG (50 ␮L of a 1:3000 dilution in TBS-T + 3% BSA) for 2 h at RT, washed again, and supplemented with TMB peroxidase substrate (100 ␮L) for 30 min at RT in the dark with gentle shaking. Reactions were stopped by the addition of 1 N H2 SO4 (50 ␮L), and plates were read at 450 nm. Samples were tested in tetraplicates and OD values of the negative control (TBS only) were subtracted from all wells. 2.7. Statistics Statistical analyses were conducted using Sigma Stat 3.1 (Systat Software Inc. 2004). Infection rates were compared by one-way analysis of variance. Differences that were found to be significant were reanalyzed by Tukey’s post hoc test or Dunn’s method for multiple-comparison procedure. Mortality rates were compared using Kaplan–Meier survival analysis and Log Rank test. Differences were considered significant at P < 0.05, unless otherwise specified. 3. Results

2.6.2. Analysis of guppy anti-Tetrahymena Ab titer by ELISA ELISA was carried out based on Sitjà-Bobadilla et al. (2004), with a few modifications. Flat-bottom microtiter plate wells (96-well Nunc-Maxisorp) were coated with 50 ␮L of Tetrahymena Ag at 4 ◦ C overnight. After three washes with TBS-T and one wash with TBS, a blocking solution was added for 2 h of incubation at RT. The wells were washed again and body lysate samples (50 ␮L) were added to the wells at dilutions ranging from 1:1 to 1:16 in TBS and incubated for 3 h at RT. After additional washing, mouse anti-guppy Ig (50 ␮L), obtained by mouse immunization with guppy Ig adsorbed to protein A/G beads, diluted 1:100,000 in TBS-T + 3% BSA, was added to the wells and incubated for 2 h at RT. The wells were washed again, supplemented with peroxidase-conjugated goat anti-mouse

3.1. Presence of guppy Ig in the serum and body homogenates To determine the presence of Igs in guppy serum and body homogenates, samples were subjected to PAGE and the gels were stained with Coomassie blue. The results (Fig. 1) showed that all serum samples preadsorbed onto protein A/G beads included one major protein band of ∼75 kDa, which is likely to correspond to the guppy IgM H chain, and a second, less intense protein band of ∼24 kDa, which is likely to represent the guppy IgM L chain (Wilson and Warr, 1992; Lim et al., 2009). The same protein bands were also observed in whole-serum samples, which included many other protein bands as well. The amount of IgH and IgL observed in the serum samples of naïve and

Fig. 1. Gel electrophoresis of guppy sera and body homogenates. Individual lanes were loaded with the following samples: untreated (2) or complementinactivated (3) normal guppy sera; untreated (4) or complement-inactivated (5) antisera from GST-immunized guppies, and guppy body homogenate (6). Lanes 8–12: the same samples as in lanes 2–6 after adsorption to protein A/G beads. Lanes 1 and 7: MW markers. Molecular mass of standard proteins are indicated on the left (in kDa). Position of the protein bands representing the IgH and L chain (∼75 and ∼24 kDa, respectively) on the Coomassie blue stained gel are indicated by arrows.

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Fig. 2. Western blot analysis of mouse anti-guppy Igs. Lanes: (1) molecular weight standard, (2) guppy serum preadsorbed onto protein A/G beads, (3) guppy whole serum, (4) guppy body homogenate, (5) guppy body homogenate preadsorbed onto protein A/G beads. Samples were fractionated by SDSPAGE and proteins were transblotted onto nitrocellulose membranes, which were blotted with anti-guppy Ig obtained from mice immunized with: (A) gel preparation of guppy Ig, (B) gel preparation of guppy Ig + guppy Ig adsorbed to protein A/G beads, and (C) guppy Ig adsorbed to protein A/G beads. Molecular mass of standard proteins are indicated on the left (in kDa), and IgH and IgL are indicated by arrows. (D) ELISA of mouse anti-guppy Ig (corresponding to the antiserum tested in panel C) tested in microtiter wells coated with guppy serum or guppy body homogenate. Serum of naïve, nonimmunized mice served as a negative control.

immune guppies was almost identical. The position of IgH and IgL in the sample of guppy body homogenate was not clear (Fig. 1, lane 6), but IgH was the major protein band observed in the homogenate following adsorption onto protein A/G beads (Fig. 1, lane 12).

3.2. Preparation of anti-guppy Igs in mice BALB/c mice were immunized s.c. with three different preparations of guppy Ig: (i) a minced gel slice containing guppy IgH, excised from whole-guppy serum electrophoresis, emulsified in CFA, (ii) protein A/G beadadsorbed guppy Ig (from guppy serum) emulsified in CFA, and (iii) a mixture of preparations (i) and (ii). All mice were boosted twice, after 3 and 6 weeks, with the same Ag preparation used initially, except that it was emulsified in IFA. To evaluate the specificity of these antisera, we used them to develop Western blots containing guppy serum or body homogenates, and the same types of samples after their adsorption to protein A/G beads. All three types of antisera contained relatively high anti-guppy IgH Ab titers,

which immunoreacted in the Western blot with the guppy IgH derived from all four samples tested (Fig. 2A–C). The antiserum obtained from group (iii) was the most specific, and when interacted with protein A/G bead-adsorbed guppy serum on the gel, it immunoreacted predominantly with the ∼75-kDa and ∼24-kDa protein bands, likely representing guppy IgH and IgL, respectively (Fig. 2C). Use of this mouse antiserum in an ELISA demonstrated a specific response against guppy whole serum, and body homogenate, which was significantly higher than that of the normal mouse serum (Fig. 2D).

3.3. Evaluation of the guppy anti-Tetrahymena Ab response in immunized fish In trial 1, body homogenate from fish that were protected from Tetrahymena following immunization by i.p. injection (Chettri et al., 2009) was analyzed by ELISA for the presence of anti-Tetrahymena Abs. In this assay, an antiserum from a mouse immunized with protein A/Gadsorbed guppy Ig was used as a source of secondary Abs.

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Fig. 3. Evaluation of the guppy anti-Tetrahymena Ab response in immunized fish. (A) Guppies were immunized with Tetrahymena in adjuvant (Tet + adjuvant, two similar groups, each representing a pool of antisera from four fish), PBS in adjuvant (Adjuvant; a pool of antisera from three fish), and untreated control (Control; a pool of sera from three fish). (B) Guppy fish were intubated with (Intubation + Tet, n = 14), or without (Intubation – Tet, n = 8) Tetrahymena, or injected i.p. with Tetrahymena in adjuvant (i.p. Tet + adjuvant, n = 8), or untreated (Control, n = 8). Body homogenates of individual fish were tested at the indicated dilutions. *P < 0.05, statistical difference between treatment groups within individual dilutions.

Maximal OD readings obtained in the ELISA were markedly higher in body homogenates of immunized fish (OD of 1.23 at a 1:1 dilution) compared to control, non-immunized fish (OD of 0.79 and 0.6; Fig. 3A). The difference between the immunized and control fish remained higher at all tested dilutions, up to 1/16 (Fig. 3A). In trial 2, guppies immunized against Tetrahymena by intubation and i.p. injection were evaluated for antiTetrahymena Ab production by ELISA. Body homogenates from fish immunized by i.p. injection presented significantly higher OD readings, suggesting higher Ab titer than in all other treatment groups (Fig. 3B). OD levels of fish that were immunized by intubation (with and without Tetrahymena) and nonimmunized control groups did not differ significantly. A reduction in OD readings in all treatment groups was obtained at higher dilutions of body homogenate. Although not statistically significant, at a dilution of 1:2, the group intubated with Tetrahymena had a slightly higher OD reading (average of 0.15) than the intubated and non-treated controls (OD of 0.11 and 0.09, respectively; Fig. 3B). 3.4. Challenge with Tetrahymena Survival rates of fish immunized against Tetrahymena by different routes and subsequently challenged with the parasite were monitored. Cumulative mortality following challenge with Tetrahymena by i.p. injection is presented in Fig. 4A. In both anally intubated groups (with and without Tetrahymena) and in the untreated control group, mortality reached about 50%. Although not statistically significant, the i.p.-vaccinated group had a lower mortality, of approximately 31%. Mortality rates in the groups challenged by immersion exposure preceded by cold stress are presented in Fig. 4B. Mortality in the control groups (anally intubated and non-treated controls) was significantly lower (8% and 2%, respectively) than that in the treatment groups that were immunized by anal intubation or i.p. injection with Tetrahymena (17% and 20%, respectively). Tolerance induction by

anal intubation may explain the increased sensitivity of the fish to a challenge by live parasites, but further studies are required in order to verify this hypothesis. A microscopic examination of skin, gills and internal organs of 27 freshly dead fish from the i.p.-immunized group revealed restriction of Tetrahymena to the skin. On the other hand, examination of 10–16 freshly dead fish from each of the other treatment groups revealed the presence of Tetrahymena both on the skin and in the internal organs. 4. Discussion ELISA is the most common method for evaluating Ab responses. Due to heterogeneity in Ig structure among species, the secondary Ab required to develop an ELISA must immunoreact with the primary Abs of the species to be analyzed. To analyze the efficiency of immunizing guppies against the pathogen Tetrahymena, and to develop a suitable and efficient ELISA, we first had to prepare anti-guppy Ig Abs, as these are not commercially available. Protein A/G-coated beads were found to be a suitable method for isolating Ig from the serum and body homogenate of guppies, as clear bands at the expected MW for IgH and IgL, ca. 75 kDa and 24 kDa, respectively (Wilson and Warr, 1992; Lim et al., 2009), were demonstrated upon PAGE of anti-guppy Ig. Purification of fish Ig using protein A has been demonstrated to be effective in a variety of fish species, i.e. southern bluefin tuna (Thunnus maccoyii Castelnau), black rockfish (Sebastes schlegeli Higendorf) and turbot [S. maximus (Linnaeus)] (Estévez et al., 1993; Watts et al., 2001; Kang et al., 2006), despite the reported varying efficacy of protein A binding to Ig amongst different fish genera (Estévez et al., 1993). Purification of fish Igs using the protein A/G combination has never been reported. Protein G has two Fc binding domains, and its binding efficacy to Ig has been suggested to be higher than that of protein A, which has four Fc binding domains. Guppy IgH and IgL have been previously demonstrated by Lim et al. (2009) with reported MW of 74 kDa and 23 kDa, respectively. Our findings demonstrated similar sizes of IgH

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Fig. 4. Cumulative mortality of immunized and non-immunized fish following a challenge with Tetrahymena by i.p. injection or immersion. Immunization groups included: anal intubation with Tetrahymena (Intubation + Tet), anal intubation without Tetrahymena (Intubation – Tet), i.p. injection of Tetrahymena with adjuvant (i.p. Tet + adjuvant), and untreated control group (Control). (A) Mortality following challenge with Tetrahymena by i.p. injection (900 Tetrahymena/fish). (B) Mortality following challenge with Tetrahymena by immersion (10,000 Tetrahymana pare mL) preceded by cold stress (17 ◦ C for 48 h). Different lowercase letters indicate statistically significant differences at P < 0.05.

and IgL (75 and 24 kDa, respectively). The small difference might arise from interpretation based on the MW standards used in the gel electrophoresis. The standards used in the present study included 75 and 24 kDa, which enabled a more accurate interpretation. Different approaches for mouse immunization were tested, and following primary immunization, which was similar among the different mice (using guppy IgH eluted from gel electrophoresis), booster immunization with guppy Ig pre-adsorbed to protein A/G beads was the most specific, producing minimal background as compared to the other immunized mice. It is important to note that, based on these results, injecting mice with protein A/G beads preadsorbed with guppy Ig does not interfere with antiserum production.

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An ELISA was developed for the detection of antiTetrahymena Abs in guppies. Lim et al. (2009) described an ELISA to monitor Ab production in guppies in response to immunization with Pseudomonas fluorescens, using guinea pig antiserum against guppy Ig. Anti-Tetrahymena Ab was found in the fish serum and body homogenate using the developed ELISA, but the titer at which Abs could be detected in this study (1:8) was low compared to the titer of 1:2560 reported by Lim et al. (2009). Tetrahymena is known to evade the immune system, causing little or no inflammatory response in infected guppies (Pimenta Leibowitz and Zilberg, 2009; Sharon et al., 2014a). The titers obtained in the ELISA analysis were similar to the Tetrahymenaimmobilizing titers of 1:8 reported by Chettri et al. (2009). Ab production was examined following exposure to the Ag by anal intubation. To enhance uptake of Tetrahymena Ag from the hindgut, a carrier, based on Tandler et al. (2007) was used. The carrier contained DOM and DOC, which are known digestion and absorption enhancers, respectively. Sukumasavin et al. (1992) reported induced spawning in Thai carp (Puntius gonionotus Bleeker) by oral administration of salmon gonadotropin-releasing hormone (sGnRH) together with DOM, as compared to sGnRH alone, suggesting better uptake of the sGnRH hormone when DOM is used. Oral administration of human growth hormone with DOC to common carp (Cyprinus carpio Linnaeus) resulted in a 1000-fold increase in absorption as compared to oral intubation of the hormone without DOC (Hertz et al., 1991). Results of the present study suggest a possible low level of Ab production against Tetrahymena in the anally intubated fish (OD 0.15 at 1:2 dilution of body homogenate), although levels were not statistically different from those of the control groups (OD of 0.09 and 0.11) or the i.p.injected groups (OD of 0.29). In the latter experiment, Ab level was lower than that in fish samples from Chettri et al. (2009). A comparison of mortality levels in the i.p.immunized fish between the current study and Chettri et al. (2009) revealed a substantially higher mortality rate in the present study, 31% and 10%, respectively. It is possible that immunization was less effective in the present study, resulting in a lower Ab titer, and therefore reduced protection from infection. The success of oral immunization relies on the delivery of intact Ag in sufficient quantities to the hindgut and its uptake from that site. The transport of macromolecules, such as human IgG, from the hindgut to the blood was described by Georgopoulou and Vernier (1986). The hindgut is the main site for Ag uptake, containing Agtransporting epithelium, Ag-processing macrophages, and B and T lymphocytes (Georgopoulou and Vernier, 1986; Vervarcke et al., 2005). In this study, Ag was delivered directly to the hindgut, the site of absorption, thereby bypassing gastric degradation. Johnson and Amend (1983) showed that sockeye salmon Oncorhynchus nerka (Walbaum) and rainbow trout Salmo gairdneri (Richardson) that were immunized by anal intubation against Vibrio anguillarum and Yersinia ruckeri were better protected from infection with these pathogens by waterborne and bath challenges, as compared to oral- and immersionvaccinated fish. Vervarcke et al. (2005) reported that immunization of the African catfish Clarias gariepinus

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(Burchell) with V. anguillarum O2 Ag by i.p. injection resulted in higher serum Ab levels than anal intubation with the same Ag. Ab levels in oral- and immersionimmunized fish were even lower (Vervarcke et al., 2005). In the present study, immunization by anal intubation did not lead to protection from infection by either i.p. or immersion challenges. Unexpectedly, immunization by i.p. injection and anal intubation led to reduced protection from challenge by immersion. The calibrated challenge protocol included cold stress prior to the parasitic exposure, as without such a stress, infection occurs at very low levels (Sharon et al., 2014b). Low water temperature is known to suppress immune function in most fish (Fry, 1967; Morvan et al., 1998; Tanck et al., 2000) and cold water temperature was specifically shown to suppress primary Ab production in fish (Morvan et al., 1998). Moreover, the effect of stress on immune suppression is well documented. In humans, there is evidence of a relation between stress and reduced secondary Ab response. The potential importance of stress-related cortisol on Ab production in response to vaccination has been reviewed (Shan Wang et al., 2013; Cohen et al., 2001). The second pathway that can be considered is cold-induced oxidative stress in fish. Oxidative stress is known to adversely affect different factors of the immune response, including Ab production, following immunization (Kang-Le et al., 2014; Cannizzo et al., 2011; Ibarz et al., 2010). This could explain the absence of protection in i.p.-vaccinated fish, but not the increased mortality in immunized groups compared to controls. There seems to be no indication in the literature of a similar outcome, where immunization resulted in elevated mortality following challenge infection. It would be interesting to examine this further by exposing vaccinated fish to cold stress and challenging them by i.p. injection, to examine whether the observed phenomenon is related to the route of infection, i.e. by immersionvia the mucosal barrier, or is it a response to the cold stress, which would also be evident following i.p. infection. Interestingly, while examining freshly dead fish from the i.p. immunized group under a light microscope for the presence of Tetrahymena, parasites were found on the skin and gills but not in the internal organs, as opposed to high levels of internal Tetrahymena in all other treatment groups. It is possible that the presence of systemic Abs protected fish from internal infection, regardless of the cold stress. Infection of the skin and gills may have been the cause of mortality in the i.p.-immunized group, in contrast to both systemic and external infection in the other treatment groups. The present study suggests a correlation between Ab development, as determined by ELISA, and protection from systemic infection with Tetrahymena. Induction of protection from external challenge (via the skin and gills) has yet to be established and analyzed. Immunization by i.p. injection seems to be effective, but further research is needed to achieve administration of Ag via the gastrointestinal tract. References ATCC, 1999. ATCC Product Information Sheet. Collection of Protists 30327. Manassas, VA, available at: www.atcc.org

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