Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis.

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis 1 s1, R. Sandoval1, J.P. Pontigo1, C. Alvarez C. Oliver1, K. Valenzuela1, H. Silva1, R.E. Haro1, M. Corte , 1,2 2,3,4 5 1,2 ~ o-Herrera n ~ ez J.E. Figueroa , R. Avendan , J.M. Troncoso and A.J. Ya

1 Instituto de Bioquımica y Microbiologıa, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile n, Chile 2 Interdisciplinary Center for Aquaculture Research (INCAR), Concepcio gicas, Facultad de Ciencias 3 Laboratorio de Patologıa de Organismos Acuaticos y Biotecnologıa Acuıcola, Departamento de Ciencias Biolo gicas, Universidad Andres Bello, Vin ~a del Mar, Chile Biolo n Marina Quintay (CIMARQ), Quintay, Chile 4 Centro de Investigacio 5 EWOS Innovation Chile, Calbuco, Chile

Keywords chicken egg yolk, growth inhibition, IgY, immunoglobulin, Piscirickettsia salmonis, piscirickettsiosis. Correspondence ~ez, Universidad Austral de Alejandro J. Y an Chile, Campus Isla Teja, Valdivia, Chile. E-mail: [email protected] 2015/0568: received 26 March 2015, revised 14 May 2015 and accepted 18 May 2015 doi:10.1111/jam.12857

Abstract Aims: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. Methods and Results: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDSPAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 lg ml1 in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. Conclusions: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. Significance and Impact of the Study: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.

Introduction Piscirickettsia salmonis, a facultative intracellular Gramnegative, is the etiologic agent of piscirickettsiosis, which causes significant mortality and economic losses in fish

farming in Chile. This pathogen was originally identified in southern Chile in 1989, isolated from farmed Coho salmon (Oncorhynchus kisutch) (Bravo and Campos 1989; Branson and Diaz-Mu~ noz 1991). After this first report, piscirickettsiosis was described in Norway (Olsen et al.

Journal of Applied Microbiology 119, 365--376 © 2015 The Society for Applied Microbiology

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1997), Ireland (Rodger and Drinan 1993), Scotland (Grant et al. 1996), eastern Canada (Cusack et al. 2002) and southern California (USA) (Arkush et al. 2005). Nowadays, vaccination and antibiotics are used in aquaculture to prevent and treat bacterial infections. Vaccination requires considerable management and is stressful for fish; current vaccine trials have used bacterins as antigens for vaccination against P. salmonis. However, these preparations do not provide the expected protection against piscirickettsiosis (Smith et al. 1997; Kuzyk et al. 2001). On the other hand, treatment with oxytetracycline, a bacteriostatic agent commonly used in the salmon industry, has not been effective in controlling piscirickettsiosis. Thus, the indiscriminate use of antibiotics has led to problems with drug residues in animal products and contributes to the development of drug ~ez et al. 2014). Therefore, resistance in the bacteria (Yan alternatives to antibiotics and vaccination are urgently needed. A wide range of products has been tested as potential alternatives to antibiotics. Among these, passive immunization with antibodies is highly attractive and effective as an alternative approach due to its high specificity. Recently, chicken egg yolk immunoglobulin (IgY) has attracted significant attention as a potential method to prevent and control diseases. The use of IgY on a large-scale offers several advantages; principally, IgY can be easily obtained noninvasively from the egg yolk, it is a polyclonal antibody that can recognize multiple epitopes and, the amount of antibodies produced by a single hen is similar to that of a large mammal such as a sheep or goats, whereas maintenance costs are much lower (Zhang 2003; Schade et al. 2005). The amount of the IgY varies between 100 and 250 mg per egg (Schade et al. 1996). Thus, a large amount of antibody (up to 40 g of total IgY per chicken per year) can be produced from just one hen, of which 1–10% is expected to be specific to the antigen of interest (Mine and KovacsNolan 2002). The efficacy of IgY has been demonstrated in several applications such as, treatment and prevention of human and veterinary diseases (Larsson and Carlander 2003). In aquatic species, (Lee et al. 2000) reported that intraperitoneal injection of IgY anti-Yersinia ruckeri was effective against an immersion challenge of rainbow trout (Oncorhynchus mykiss) with this pathogen. Similarly, IgY against Edwardsiella tarda was administered orally to passively immunize Japanese eels (Anguila japonica), demonstrating that IgY could be effective in controlling Edwardsiellosis (Mine and Kovacs-Nolan 2002). However, the production of IgY against P. salmonis and its antibacterial activity has not been reported so far. Therefore, the objective of this study was to produce and characterize IgY 366

against P. salmonis and to evaluate the antibacterial activity of IgY in vitro as well as the availability in the serum of Atlantic salmon (Salmo salar) immunized orally. Materials and methods Bacterial isolates and growth conditions In this study, three Chilean P. salmonis isolates recovered in 2008 from diseased farmed rainbow trout (AUSTRAL005), Coho salmon (AUSTRAL-006) and Atlantic salmon (AUSTRAL-010) were used. The type strain ATCCâ VR1361TM (equivalent to LF-89T) was included for comparative purposes. The identity of each isolate was confirmed as P. salmonis by phenotyping procedures and PCR assays (Karatas et al. 2008). For all experiments, P. salmonis was routinely grown on Austral-TSHem agar plates for ~ez et al. 2013) or in AUSTRAL-SRS 10 days at 18°C (Yan ~ez et al. broth for 6 days at 18°C with agitation (Yan 2012). Preparation of Piscirickettsia salmonis antigens The isolate AUSTRAL-005 was chosen to prepare the antigens. Bacterial cells were harvested by centrifugation at 5000 g at 4°C for 10 min. Then, cells were washed with phosphate-buffered saline (PBS) followed by centrifugation at 5000 g at 4°C for 10 min and the resulting cell pellet was resuspended in 50 mmol l1 Tris-HCl pH 75 buffer containing 1% Terginolâ type NP-40, 05% sodium deoxycholate, 150 mmol l1 sodium chloride, 1% SDS, 01 mmol l1 phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Thermo Fisher Scientific Inc, Rockford, IL). This bacterial suspension was then subjected to mechanical disruption at 4°C for 20 min followed by incubation at 4°C for 30 min. Finally, after centrifugation at 12 000 g for 30 min at 4°C, the supernatant containing the whole protein extract, was collected, aliquoted and stored at 80°C until use. The immunization emulsion was prepared by mixing 500 ll (1 mg ml1) of P. salmonis AUSTRAL-005 antigens in PBS with 500 ll of Titermax Gold (Sigma, St. Louis, MO) as adjuvant. Immunization of hens Two 21-week-old laying White Leghorn hens, maintained in the Avian Pathology Laboratory facility at the Institute of Animal Pathology of Universidad Austral de Chile, were immunized by injecting the antigen solution (protein extract mixed with Titermax) into the pectoral muscle. The primary immunization was made by intramuscular injection of 200 mg of whole protein extract in


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400 ml of emulsion. Then, two booster inoculations were given after three and 7 weeks, respectively, by the same route and volume as the first. Eggs from immunized and nonimmunized hens were collected daily and stored at 4°C until antibody extraction. Isolation and purification of IgY Eggs from immunized and nonimmunized (control group) hens were collected and pooled in 1-week intervals after the first immunization. The crude antibody from yolk was extracted using the water-soluble fraction protocol described by Akita and Nakai (1992) with some modifications. Egg yolks were physically separated from the white, diluted at 1 : 9 ratios with distilled water at pH 53 and the mixture was incubated overnight at 4°C. The liquid phase containing the IgY was filtered through a 02 lm membrane filter. After centrifugation at 10 000 g for 30 min at 4°C, the water-soluble fraction was collected and further purified using a 33% (v/v) saturated ammonium sulphate (SAS) solution. The pellet was resuspended with 65% (v/v) SAS and centrifuged again. Finally, the pellet containing the IgY was resuspended in antibody buffer and dialysed against PBS (pH 74) at 4°C and thereafter stored at 20°C before use. Electrophoresis and Western-blot analysis To identify IgY, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli 1970) and Western-blot assays were performed as follows: preparations of IgY were diluted at 5, 10 and 20 lg ml1 of proteins in sample buffer (625 mmol l1 Tris-HCl, pH 68, 6 mol l1 urea, 2% SDS, 5% b-mercaptoethanol, 10% glycerol, 000125% bromophenol blue). The samples were separated by 12% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue R (Sigma) or transferred onto a polyvinylidene difluoride membrane. Western-blot membranes were blocked with blocking buffer (5% w/v instant nonfat milk powder, 01% Tween-20 in PBS buffer, pH 74) for 3 h. Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-chicken IgY antibody (1 : 5000) for 2 h. Finally, the membranes were developed using DAB/H2O2 system and digitally recorded using a gel documentation system (Ultralum, Cambridge, UK).


Specific activity of IgY against Piscirickettsia salmonis Determination of specific activity of IgY against AUSTRAL-005 was tested using an indirect ELISA. Ninetysix-well microtitre plates (Nunc Immunoplate MaxiSorp, Nunc Co., Roskilde, Denmark) were coated with 100 ll per well of 1 lg ml1 P. salmonis AUSTRAL-005 whole protein extract solution and the plates were incubated overnight at 4°C. After incubation, the plates were washed three times with 003% Tween-20, pH 7 (PBS-T) and 200 ll of 05% bovine serum albumin in PBS (blocking solution) were added to each well and incubated for 2 h at room temperature. After one wash, wells were loaded with 200 ll of 6-fold serially diluted IgY against P. salmonis extracts or IgY from nonimmunized hens and incubated at 37°C for 2 h. Previously, both antibody extracts were properly diluted to give the same starting protein concentration (45 mg ml1). Plates were then washed and incubated with HRP-conjugated goat antichicken IgY (1 : 5000 in blocking solution) for 1 h at 37°C. The plates were then washed with PBS-T and 50 ll of 3,30 ,550 -tetramethylbenzidine (TMB) liquid substrate (Sigma) was added to each well. After 30 min, the reaction was stopped by addition of 50 ll of 01% sulphuric acid solution and optical density (OD) was read at 450 nm using a Bio Tek Microplate reader. Three different negative controls were used in each plate: (i) wells loaded with IgY anti-FBPase instead of IgY against P. salmonis, (ii) wells without P. salmonis antigens and, (iii) wells without IgY. All samples and controls were tested in triplicate. The same method described above was used to evaluate the reactivity of IgY against other three P. salmonis. Several protein concentrations from 0001 to 1 lg ml1 of whole protein extracts from the P. salmonis AUSTRAL006, AUSTRAL-010 and LF-89T, were loaded into the wells. The plates were incubated overnight at 4°C and then, washed, blocked and incubated with IgY anti-P. salmonis AUSTRAL-005 (1 : 100) for 2 h at 37°C. After three washes with PBS-T, the plates were incubated with HRP-conjugated goat anti-chicken IgY (1 : 5000) for 1 h at 37°C and developed as described above. The background was defined by the wells without IgY and wells loaded with IgY from nonimmunized hens were used as negative controls. All samples were tested in triplicate. Growth inhibition assay

Protein quantification Protein concentration in the whole protein extract of P. salmonis and filtered IgY extract was determined using Pierce BCA Protein assay kit (Thermo Scientific) according to the manufacturer’s recommendation.

IgY against P. salmonis was added to AUSTRAL-SRS broth at final concentrations of 64, 128 and 256 lg ml1. IgY from nonimmunized hens was also added to additional bacterial cultures a final concentration of 256 lg ml1 as a negative control. All IgY preparations


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were sterilized with a 02 lm filter. Oxytetracycline at final concentration of 1 lg ml1 was used as control of ~ez et al. 2014) and media bacterial growth inhibition (Yan without any inhibitor was the positive control of bacterial growth. To prepare the inoculum, P. salmonis AUSTRAL005 was grown in broth for 6 days and then diluted in the same medium to final OD at 600 nm between 008 and 01. Then, 20 ll of this bacterial suspension was used to inoculate a 96-well plate loaded with 180 ll of the media solutions described above. Plates were incubated at 18°C with shaking for 10 days. Growth was measured every 2 days using a Bio Tek Microplate reader (OD at 600 nm). To confirm purity, at the end of the incubation period, samples from the bacterial cultures were Gram stained and observed under the microscope. Inhibitory activity of IgY in SHK-1 cells infected with Piscirickettsia salmonis Atlantic salmon head kidney (SHK)-1 cell line with twenty passages was used in this study. SHK-1 cells were cultured at 20°C in 75 cm2 flask, in Leibovitz’s L-15 medium supplemented with 10% FBS (Gibco BRL), 6 mmol l1 L-glutamine (Hyclone Laboratories Inc., UT) and 40 lmol l1 2-mercaptoethanol (Gibco, Invitrogen Laboratories, Grand Island, NY). Confluent flasks were subcultured every 15 days by dividing cells between two flasks and adding an equal volume of new media to each flask. The SHK-1 cells (1 9 105 cells per well) were seeded onto poly-L-lysine-coated coverslips in 24- well culture plates with L-15 medium supplemented with 2% FBS and then cultivated for 3–4 days to 70–80% confluence. For infection experiments, monolayers containing adherent cells were pre-incubated for 1 h with IgY against P. salmonis AUSTRAL-005 (1 lg ll1) and then, infected with P. salmonis AUSTRAL-005 for a period of 3 h, time in which the pathogen should be within the host cell (Smith et al. 2010). Bacteria in exponential growth phase were used at multiplicity of infection 20 bacteria per cell. After this infection period, the cells were washed twice with PBS and culture medium was replaced with L-15 with 2% heat-inactivated foetal bovine serum. SHK-1 cells treated with IgY pre-immune or IgY against fructose-16bisphosphatase 1 (FBPase) as well as nontreated cells with IgY and noninfected cells were used as controls. The cytopathic effect (CPE) was evaluated in SHK-1 cell up to 10 days postinfection (dpi) by inverted microscopy. In addition, possible differences in the permeability of the membrane in SHK-1 cells exposed to P. salmonis were determined by evaluating the liberation level of cytosolic enzyme lactate dehydrogenase (LDH) in the supernatant using a commercial LDH Cytotoxicity Detection kit (Takara Bio Inc., Japan) according to the manu368

facturer’s recommendation. To calculate the percentage of cytotoxicity, the control SHK-1 cell were run simultaneously with the addition of Triton X-100. Oral delivery of IgY antibodies in Atlantic salmon blood Chitosan-alginate microcapsules were evaluated as a method of delivery of IgY antibodies using the coating procedure described by Li et al. (2007). IgY was added to a mix (1 : 1) of 1% (w/v) chitosan and 15% (w/v) sodium alginate (Sigma) solution at a loading rate of 25% (w/w) and dissolved with 100 mmol l1 CaCl2 (w/v, final concentration) by stirring (500 g) for 30 min at 4°C. Finally, microcapsules containing IgY against P. salmonis AUSTRAL-005 were stored in PBS at 4°C before their use in fish. To determine the incorporation of the IgY antibody in fish serum, oral delivery trials on Atlantic salmon were conducted. Healthy P. salmonis-free Atlantic salmon (weighing 2830 g) obtained from a fish farm with no history of diseases were used. Fish were randomly allocated in groups of 15 per 50 l tank and acclimatized for 10 h prior to bacterial challenge and fed with 2 mm pellets at 12% of their body weight per day. All fish were maintained in tanks at 15 1°C with aeration during the course of the experiments (2 days), with 50% of the tank seawater refreshed daily. Then, the fish were kept in starvation condition for 4 days, and a group of 15 fish were orally intubated with 3 ml (8 mg ml1) of PBS containing chitosan-alginate coated IgY. Nonintubated fish were used as negative controls and were processed in the same manner described above. Blood samples were taken 2, 4, 8, 12 and 24 h postoral intubation (poi) by caudal vein puncture with a heparinized syringe, centrifuged at 5000 g for 5 min and stored as pools of blood at –20°C until use. To obtain the blood, all fish were anaesthetized by immersion in 30 lg ml1 BZ-20 (Veterquımica) according to the Ethics Committee for Animal Experiments of Universidad Austral de Chile. The determination of IgY activity in the fish pooled plasma was tested by Western-blot analysis as described above, except that the membranes were incubated in a 1 : 40 dilution of the pooled plasma in the blocking solution. In addition, absorbed antisera against the whole protein extract of P. salmonis were employed as control. Determination of IgY concentration in fish serum In order to determine the amount of IgY that reaches the fish blood stream, an ELISA test was performed. All steps of the reaction were carried out in a 96-well microtitre plate, which was coated overnight with a rabbit antibody against whole protein extract of P. salmonis diluted


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1 : 5000. Following a washing step, the plate was blocked for 2 h with 05% skimmed milk in PBS and then 5 lg per well of total protein extract of P. salmonis were added to each well and incubated overnight. After washing three times, 100 ll of each fish serum was added to the plate and incubated for 2 h. In addition, a calibration curve was assessed by adding 50–1000 ng of IgY per well in triplicate. Following washing, each plate was incubated with a 1 : 5000 dilution HRP-conjugated goat anti-chicken IgY for 2 h and finally developed by adding 50 ll of 3,30 ,550 TMB Liquid Substrate (Sigma) per well. After 30 min, the reaction was stopped by addition of 50 ll of 01% sulphuric acid solution and OD was measured at 490 nm.


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Production and purification of IgY against Piscirickettsia salmonis Figure 1a shows the protein profile of P. salmonis AUSTRAL-005 extracts used for hen immunization, while Fig. 1b shows the protein concentration of the IgY extracts obtained from hens in the immunization period. The protein concentration was relatively constant between the second to the ninth week after the first immunization and the highest concentration of 1754 lg ll1 was found 3 weeks after the second immunization. Protein concentration diminished 3 weeks after the third booster (1371 lg ll1). The control nonimmunized hens (IgY-Pi) showed lower protein concentrations ranging from 432 to 476 lg ll1, indicating that the protein levels founded in the immunized hens could be due to increased production of IgY. In fact, analysis of the IgY extracts by SDS-PAGE and Western-blot showed three predominant proteins in the IgY extract (Fig. 2), two of them represent the heavy chain (67 kDa) and light chain (27 kDa) of IgY respectively. Specific activity of IgY against Piscirickettsia salmonis The antibody titre in the IgY extracts was evaluated by an indirect ELISA (Fig. 3a). The IgY extract showed a strong

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Statistical analysis The results were subjected to an analysis of variance (Sokal and Rohlf 1980). Student’s t-test was used to test the significance of differences between the experimental and control groups. Data are presented as means standard deviation. A probability level of P < 0001 was considered statistically significant. ELISA assay validation was performed for some parameters, such as comparison of the samples with the negative and positive controls (Petrie and Watson 1999).

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Time (week) Figure 1 Chicken egg yolk antibody (IgY) against Piscirickettsia salmonis. (a) SDS-PAGE of whole protein extract of P. salmonis AUSTRAL-005 used to immunize hens against P. salmonis. Lane 1, molecular weight marker; 2, 5 lg ml1; 3, 10 lg ml1 and 4, 20 lg ml1. (b) Total protein concentration of the IgY extracts from immunized hens (IgY-PT) and nonimmunized hens (IgY-Pi) was measured and expressed as mg ml1. The arrowheads indicate the immunization week. (b) ( ) IgY-PT and ( ) IgY-Pi.

binding activity against P. salmonis AUSTRAL-005. The ELISA titres of the IgY from immunized hens (IgY-PT) were significantly higher than IgY extracts from the nonimmunized hens (IgY-Pi), regardless of the dilutions of antibody assessed (P < 0001). These results indicate that whole protein extract of P. salmonis can induce the generation of high levels of IgY in hens.


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Subsequently, we evaluated the heterologous sensitivity of the anti-P. salmonis IgY antibody against the AUSTRAL-006 and AUSTRAL-010 as well as the type strain LF89T by indirect ELISA (Fig. 3b). The IgY extract was diluted at 1 : 1000 and then tested against increasing concentrations of whole protein extracts from the different isolates. The antibody was able to detect P. salmonis antigens as low as 01 lg ml1 of total proteins of the three isolates analysed, indicating that anti-P. salmonis IgY is able to recognize low antigen concentrations from different isolates of this fish pathogen. As expected, the reactivity increases at higher antigen concentrations. Growth inhibition assay The growth inhibition assay, performed to evaluate IgY activity against P. salmonis under in vitro conditions, showed that anti-P. salmonis IgY was able to inhibit bacterial growth of the isolate AUSTRAL-005 in AUSTRALSRS broth (Fig. 4a). The growth inhibition rate varied according to the protein concentration of IgY extract added to the medium. Interestingly, bacterial growth was completely inhibited by the addition of 256 lg ml1 of IgY anti-P. salmonis similar to wells containing oxytetracycline (1 lg ml1). Growth inhibition of P. salmonis was statistically significant, when 128 and 256 lg ml1 of IgY anti-P. salmonis (IgY-PT) were added to the medium, compared with medium containing IgY extract from nonimmunized hens (IgY-Pi) (Fig. 4b). Antibody concentration of 64 lg ml1 showed no inhibitory effect on P. salmonis growth in liquid medium. In addition, as expected, the growth of S. aureus and E. coli was not inhibited when the highest concentration of IgY-P. salmonis (256 lg ml1) was used (data not shown). 370

Figure 2 Analysis of purified IgY against Piscirickettsia salmonis. (a) SDS-PAGE of purified IgY against total protein of P. salmonis. Lane 1, molecular weight marker; 2, 20 lg ml1; 3, 10 lg ml1 and 4, 5 lg ml1. (b) Western-blot of purified IgY against total protein of P. salmonis. H and L indicate high and light chains respectively. Lane 1, 20 lg ml1; 2, 10 lg ml1 and 3, 5 lg ml1.

Inhibitory activity of IgY in SHK-1 cells infected with Piscirickettsia salmonis To evaluate whether IgY against P. salmonis would be able to prevent the CPE in vitro, we carried out an inhibitory assay in which SHK-1 cells, pre-treated or not with IgY against P. salmonis after 3 h of infection. As expected, in SHK-1 cells not exposed to IgY and infected with P. salmonis, the CPE was evident from the 4th day post infection (Fig. 5a). Moreover, no protective effects were detected for the SHK-1 cells with P. salmonis and treated with IgY pre-immune or IgY against FBPase (data not shown). It is important to note that pretreated SHK-1 cells with IgY against P. salmonis, no CPE was observed during the 10 days of the experiment. Thereby, there was no increase in the number and size of cytoplasmic vacuoles. Therefore, SHK-1 cells remained similar to uninfected control cells. Interestingly, it was possible observing large accumulations of bacteria bound to the antibody in the culture medium during the period of infection (Fig. 5b), which disappeared when the medium was removed. LDH values from infected cells without pretreatment with IgY was 15% at 4 dpi, after which LDH values began to increase, indicating disruption of cell membranes of most cells at 10 dpi, reaching a cytotoxicity of 70% (Fig. 5c). Similar LDH values from infected cells treated with IgY preimmune were obtained, reaching 55% at 8 dpi (data not shown). It is important to denote that the cytotoxicity of treated cells with IgY against P. salmonis and infected with the pathogen remained low (between 17 and 25%), very similar to observed in uninfected control cells at 10 dpi.


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Dilutions of antigen (Log µg ml–1) Figure 3 Analysis of activity of anti-Piscirickettsia salmonis IgY extracts by ELISA. (a) The titre of IgY in the egg yolk of immunized hens (IgY-PT) was significantly higher than in those without antigen treatment (IgY-Pi). The asterisks (***) indicate a significant difference (P < 0001). (b) The cross-reactivity of IgY against different P. salmonis isolates was tested by ELISA. The IgY extracts were able to react against three other P. salmonis isolates. Results are the mean standard deviation of three independent experiments. (a) ( ) IgY-PT; ( ) IgY-Pi and (b) ( ) LF89; ( ) AUSTRAL-010; ( ) AUSTRAL006.

Oral delivery of IgY antibodies in Atlantic salmon blood The Western-blot analysis revealed that active anti-P. salmonis IgY reaches the blood stream of fish and it was able to recognize large immunoreactive proteins from whole protein extracts of P. salmonis (Fig. 6). In fact, the pooled plasma strongly recognized a large number of proteins with molecular masses between 10 and 100 kDa, particularly at 2, 4 and 8 h poi. In the case of the pool of plasma obtained from nonintubated fish, it was only able to recognize slightly bands of approx. 72 and approx.

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Figure 4 Evaluation of growth inhibitory properties of IgY against Piscirickettsia salmonis. (a) The growth inhibitory activity of IgY to P. salmonis was tested using different concentrations of IgY against P. salmonis extracts. Bacterial growth without IgY (w/o IgY) was used as control. Vertical bars indicate the standard deviation. (b) Growth inhibitory effect of the three concentrations of IgY (IgY-PT) used was compared with the effect of IgY from nonimmunized hens (IgY-Pi) at the end of the logarithmic phase of growth (day 8th). Growth inhibition is represented as percentage SD. The asterisks (***) indicate a significant difference (P < 0001). (a) ( ) w/o IgY; ( ) Igy-Pi; ( ) Oxytetracycline; ( ) IgY 64 lg ml1; ( ) IgY 128 lg ml1 and ( ) IgY 256 lg ml1.

14 kDa. Similar results were found when the absorbed serum was employed (data not shown). The ELISA revealed IgY titres between 200 and 250 ng ml1 for IgY after 4 h post inoculation with 20 and 200 mg of IgY against P. salmonis (Fig. 7). As expected, serum IgY levels decreased over time in both groups of fish, obtaining 100–150 ng ml1, respectively, 24 h after inoculation of the beads.


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Figure 5 Evaluation of protective effect of IgY against Piscirickettsia salmonis. (a) Salmon head kidney (SHK)-1 cells infected with P. salmonis (SHK-1 + Ps) for 0, 4 and 8 days, noninfected cells (SHK-1), and pretreated with IgY against P. salmonis (IgY-Ps). (b) Accumulations of bacteria bound to IgY against P. salmonis in the culture medium 2 h post infection. (c) Cytotoxicity of SHK-1 cells was measured by LDH release. The data are the mean SD from three independent experiments. (c) ( ) SHK-1 + Ps; ( ) IgY-Ps and ( ) SHK-1.

Discussion The use of chicken egg yolk antibodies has attracted researchers’ interest because of the absence of considerable side effects. An alternative method to the antibiotic 372

use is the passive immunization with IgY. In fish farming, several studies indicated that this therapy has been successfully used to treat a variety of bacterial infections, including Y. ruckeri (Lee et al. 2000), Edw. tarda (Mine and Kovacs-Nolan 2002), Vibrio anguil-


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~15 ~10 Figure 6 Analysis of IgY activity obtained from Atlantic salmon plasma against whole protein extract of Piscirickettsia salmonis using Western-blot assays. The pooled plasma corresponds to 2, 4, 8, 12 and 24 h after inoculation of IgY-PT. Lane C: total protein incubated with serum from fish noninoculated with IgY; N: total protein incubated with secondary antibody (without serum); P: fish serum absorbed with 40 lg of whole protein from P. salmonis. Std: molecular masses.

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0·6 0·4 0·2 0·0 4 24 Hours post treatment Figure 7 Quantification of IgY concentration obtained from Atlantic salmon plasma against whole protein extract of Piscirickettsia salmonis using ELISA test. The titre of IgY in the fish inoculated with 200 lg kg1 was slightly higher than in those inoculated with 20 lg kg1. Vertical bars indicate the standard deviation. ( ) Control; ( ) 200 lg kg1 and ( ) 20 lg kg1.

larum (Arasteh et al. 2004) and Aeromonas hydrophila (Li et al. 2006). Large amounts of anti-P. salmonis egg yolk antibodies were produced by immunization of hens with P. salmonis AUSTRAL-005 whole cell extracts that showed a similar protein profile to those reported by Barnes et al. (1998) ~ez et al. (2012). Methods for large-scale producand Yan tion of antibody from egg yolk are well established and we used the water dilution method (Hatta et al. 1990; Akita and Nakai 1992). The analysis of the IgY

anti-P. salmonis extracts by SDS-PAGE resulted into two main bands identified as heavy and light chains of IgY, along with other proteins contained in the fraction. As reported in the literature, the water-soluble fraction of egg yolk is composed of 86% of low-density lipo-protein (LDL) and 14% of livetins (McCully et al. 1962). Livetins are divided into three classes: a-, b- and c-livetin (IgY). The molecular weights of a-, b- livetin are 70 and 45 kDa respectively (Martin et al. 1957; Williams 1962). Our SDS-PAGE analyses showed two bands of 70 and 45 kDa that could correspond to these proteins as well as a major band of 35 kDa that could correspond to vitellogenin, which is precursor of vitellin, lipovitellin and phosvitine into the egg yolk. In addition, the molecular weight of the unknown anti-adhesive factor(s) previously reported in the water-soluble egg yolk fraction should be higher than 100 kDa (Klimentzou et al. 2006). However, the second step in the isolation of the IgY from the water-soluble fraction was by ultrafiltration using a membrane with a cut off of approximately 100 kDa. Thereby, these minor proteins could be omitted by other IgY purification protocols, such as polyethylene glycol precipitation (Goldring and Coetzer 2003) or ion-exchange chromatography (Ko and Ahn 2007; Wang et al. 2012). Nevertheless, for this work, the crude protein extract was sufficient for the subsequent experiments on the reactivity against P. salmonis and antibacterial effect of IgY. In other related studies, two bands of heavy and light chains of both chicken and quail IgYs at the same molecular weights on SDS-PAGE analysis under reducing conditions were obtained (Bae et al. 2009). In our study, the activity of IgY anti-P. salmonis was evaluated by ELISA using total protein extracts as the coating antigen. The produced IgY anti-P. salmonis showed high avidity to P. salmonis AUSTRAL-005 antigens, demonstrated by detectable signal using a dilution of 1 : 3200 in the ELISA test. Moreover, the antibody was effective in recognize antigens from other 3 P. salmonis isolates (AUSTRAL-006, AUSTRAL-010 and LF-89T). This cross-reactivity might be attributed to similarities between the epitopes of the P. salmonis species. We also demonstrate that IgY anti-P. salmonis inhibits the replication in vitro of this pathogen. The highest concentration (256 lg ml1) of IgY anti-P. salmonis used in the growth inhibition assay was able to completely inhibit bacterial growth in liquid medium during the course of the experiment, and lower concentrations (128 lg ml1) showed partial inhibition compared with the IgY from nonimmunized hens. The dose-dependent pattern of the inhibition effect observed in this study is supported by similar findings on other pathogenic bacteria also inhibited by IgY, such as Escherichia coli (Sunwoo et al. 2002), Streptococcus aureus (Guimar~aes et al. 2009), Vibrio


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vulnificus (Kassim et al. 2012) and Aer. hydrophila (Li et al. 2006). However, the IgY concentration required to inhibit P. salmonis growth in liquid medium was higher than those reported to inhibit these pathogens, which may be due to the IgY purification method used in this study, which does not allow us to obtain pure IgY extracts. Thus, an undetermined amount of contaminating proteins masked the real IgY concentration. The mechanism of growth inhibition by IgY antibodies is not entirely understood yet. However, bacterial agglutination was found when the cultures were observed under the microscope; this phenomenon seems to be due to the interaction between the IgY and multiple epitopes of the bacterial membrane, resulting in the formation of visible clumps (data not shown). Sadziene et al. (1993) reported that agglutination between antibodies and bacterial cells might lead to a decrease in the acquisition of key nutrients such as iron/siderophores (Pintor et al. 1996) by several intracellular bacteria. Likewise, Sim et al. (2000) found that the specific binding between antibodies and bacterial surface components exposed on the Gram-negative bacteria surface, such as, outer membrane proteins, lipopolysaccharide, fimbriae (or pili) and flagella, potentially leads to a reduction in biological functions of the pathogen. Thus, we hypothesize that anti-P. salmonis IgY may block the functions of growth-related bacterial components by agglutination of bacterial cells leading to growth inhibition. The bactericidal and bacteriostatic effects of specific IgY has also been associated with a mechanism of bacterial opsonization which enhances phagocytosis or activation of complement system as well as the ability of the antibody to inhibit growth (Chalghoumi et al. 2009). However, further work must be performed to elucidate the mechanism by which IgY inhibits P. salmonis growth. Interestingly, in an inhibition assay, IgY demonstrated to be highly effective in the protection against the pathogen after 3 h of infection. Similarly, pretreated cells remained similar to control uninfected cells even 10 days after the infection, although the mechanism of this effect is still poorly understood. These results suggest that bacteria that entered during the period of infection produce the CPE and IgY antibodies against P. salmonis appear to prevent the entry of the pathogen to the host cell by agglutination. Thereby, these findings substantiate the potential use of IgY in food applications, owing to its ability to inhibit or neutralize pathogenic activities associated with infectious diseases. Therefore, applicability of chitosan-alginate microcapsules for oral delivery of IgY was examined. The results showed that the IgY against P. salmonis encapsulated with chitosan-alginate could maintain the IgY activity up to 12 h in serum of Atlantic salmon orally inoculated with the immunoglobulins. 374

Likewise, numerous reports indicate a rapid decrease in IgY activity within of the fish stomach. For example, Lee et al. (2000) demonstrated that anti-Y. ruckeri IgY activity was completely loss after 5 h in the stomach, while Hatta et al. (1993) also observed a 63% or total loss of anti-rotavirus IgY activities when exposed to pepsin at pH 4 or 2 respectively. Interestingly, IgY titres were between 200–250 and 100–150 ng ml1 after 4 and 24 h of inoculation respectively. On the basis of the facts presented, our findings suggest that encapsulated IgY is able to resist digestive tract degradation, being absorbed across the intestine epithelium into the fish bloodstream. Nevertheless, further work must be performed to estimate the efficacy of this IgY in vivo condition. In conclusion, our study demonstrates that the method used to immunize hens with P. salmonis and then purify IgY from egg yolk is effective, yielding a good titre of antibodies with high reactivity to their corresponding antigens. Moreover, IgY against P. salmonis showed a significant inhibitory effect on the multiplication of this bacterium in liquid medium and a strong protective effect on the infection of P. salmonis in SHK-1 cells. Having observed this strong antibacterial effect of IgY against P. salmonis in vitro, and the capacity to be transported by fish intestine and maintain the immunoglobulin activity we can propose that anti-P. salmonis IgY have a strong biological potential. Acknowledgements This work was supported by grants FONDAP INCAR No 15110027 and DID-UACh 1330-32-79 from Universidad Austral de Chile. CO acknowledges the receipt of CONICYT PhD scholarship No 21090889 and 24121415. Conflict of Interest No conflict of interest exists. References Akita, E.M. and Nakai, S. (1992) Immunoglobulins from egg yolk: isolation and purification. J Food Sci 57, 629–634. Arasteh, N., Aminirissehei, A.H., Yousif, A.N., Albright, L.J. and Durance, T.D. (2004) Passive immunization of rainbow trout (Oncorhynchus mykiss) with chicken egg yolk immunoglobulins (IgY). Aquaculture 231, 23–36. Arkush, K.D., McBride, A.M., Mendonca, H.L., Okihiro, M.S., Andree, K.B., Marshall, S., Henriquez, V. and Hedrick, R.P. (2005) Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis. Dis Aquat Organ 63, 139–149.


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Piscirickettsia salmonis gen. nov., sp. nov., the causative agent of an epizootic disease in salmonid fishes.

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model.

Identification of the clpB and bipA genes and an evaluation of their expression as related to intracellular survival for the bacterial pathogen Piscirickettsia salmonis.

Disease resistance in Atlantic salmon (Salmo salar): coinfection of the intracellular bacterial pathogen Piscirickettsia salmonis and the sea louse Caligus rogercresseyi.

The Genome Sequence of an Oxytetracycline-Resistant Isolate of the Fish Pathogen Piscirickettsia salmonis Harbors a Multidrug Resistance Plasmid.

Protective effect of an egg yolk-derived immunoglobulin (IgY) against Prevotella intermedia-mediated gingivitis.

Protection Efficacy of Multivalent Egg Yolk Immunoglobulin against Eimeria tenella Infection in Chickens.

Piscirickettsiosis and Piscirickettsia salmonis in fish: a review.

Electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element.

Effect of Piscirickettsia salmonis inoculation on the ASK continuous cell line.

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

Anti-obesity activity of hen egg anti-lipase immunoglobulin yolk, a novel pancreatic lipase inhibitor.

Infectivity of Piscirickettsia salmonis in immersion-bath exposed rainbow trout Oncorhynchus mykiss (Walbaum) fry.

Comparative Analysis of Membrane Vesicles from Three Piscirickettsia salmonis Isolates Reveals Differences in Vesicle Characteristics.

Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP.

Egg yolk IgY against RHDV capsid protein VP60 promotes rabbit defense against RHDV infection.

Patterns of Piscirickettsia salmonis load in susceptible and resistant families of Salmo salar.

Selective extraction of phospholipids from egg yolk.

Polymorphism in gyrA is associated to quinolones resistance in Chilean Piscirickettsia salmonis field isolates.

Transcriptional response of Atlantic salmon families to Piscirickettsia salmonis infection highlights the relevance of the iron-deprivation defence system.

Occurrence of glycosphingolipids in chicken egg yolk.

Chicken egg yolk immunoglobulin (IgY) developed against fusion protein LTB-STa-STb neutralizes the toxicity of Escherichia coli heat-stable enterotoxins.

B and their passive protection in mouse models.

Effects of oral moisturising gel containing egg yolk antibodies against Candida albicans in older people.