Characterization and production of IgY antibodies anti-Photobacterium damselae subsp. piscicida: Therapeutic and prophylactic use in Rachycentron canadum

Journal Pre-proof Characterization and production of IgY antibodies anti-Photobacterium damselae subsp. piscicida: Therapeutic and prophylactic use in Rachycentron canadum

Silas Fernandes Eto, Dayanne Carla Fernandes, Jefferson Yunis-Aguinaga, Gustavo da Silva Claudiano, Marina Tie Shimada, Rogério Salvador, Flávio Ruas de Moraes, Julieta Rodini Engracia de Moraes PII:

S0044-8486(19)30510-1

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734424

Article Number:

734424

Reference:

AQUA 734424

To appear in:

Aquaculture

Received Date:

28 February 2019

Accepted Date:

27 August 2019

Please cite this article as: Silas Fernandes Eto, Dayanne Carla Fernandes, Jefferson YunisAguinaga, Gustavo da Silva Claudiano, Marina Tie Shimada, Rogério Salvador, Flávio Ruas de Moraes, Julieta Rodini Engracia de Moraes, Characterization and production of IgY antibodies antiPhotobacterium damselae subsp. piscicida: Therapeutic and prophylactic use in Rachycentron canadum, Aquaculture (0), https://doi.org/10.1016/j.aquaculture.2019.734424

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Journal Pre-proof Characterization and production of IgY antibodies anti-Photobacterium damselae subsp. piscicida: therapeutic and prophylactic use in Rachycentron canadum Silas Fernandes Etoa; Dayanne Carla Fernandesa; Jefferson Yunis-Aguinagab; Gustavo da Silva Claudianoc; Marina Tie Shimadad; Rogério Salvadore; Flávio Ruas de Moraesa,b; Julieta Rodini Engracia de Moraesa,b* a

Department of Veterinarian Pathology, Faculty of Agrarian and Veterinarian Sciences, São Paulo

State University, Unesp, Brazil. E-mail: [email protected]; [email protected]; [email protected] b

Aquaculture Center of UNESP, Jaboticabal, São Paulo, Brazil. E-mail: [email protected];

[email protected] c

Institute of Biodiversity and Forests, Federal University of Western Pará, UFOPA, Pará, Brazil. E-

mail: [email protected] d

State University of Northern Paraná, Campus ‘Luiz Meneghel’, Bandeirantes, Paraná, Brazil. E-

mail: [email protected] *Corresponding author. Tel. +55 16 99756-8822. E-mail address: [email protected] (J. R. E. de Moraes).

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Journal Pre-proof Abstract Pseudotuberculosis is caused by Photobacterium damselae subsp. piscicida (Phdp), an important pathogen in marine fish culture. In this study, Phdp isolated from a Rachycentron canadum farm was used to produce anti-Photobacterium damselae IgY and its protective effect were evaluated in R. canadum challenged with the homologous strain. The IgY was obtained from egg yolks of White Leghorn hens using polyethylene glycol precipitation method. Anti-Phdp IgY showed strongly specific reaction against Phdp proteins. Finally, it was performed the antibody efficacy in vivo to determinate their prophylactic and therapeutic effects during sepsis in R. canadum. For this, fish received anti-Phdp IgY 48 hours and 15 days before the challenged with the homologous bacteria of Phdp. In these groups, it was observed less liver and kidney lesions comparing to control group, which was also correlated to the biochemical profile. In this study, it was also developed an immunohistochemistry test that presented highly specificity and affinity to Phdp that could be used as screening method for the diagnosis and analysis of pathogenesis of this disease. It was concluded that the IgY produced effectively inhibited the dissemination of this bacteria that could be helpful for the treatment and prophylaxis of this disease. Keywords: teleost; cobia fish; marine fish culture; polyclonal antibody; immunohistochemistry

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Journal Pre-proof 1. Introduction Marine fish bacteria are a serious issue in fish farm industry due to the high risk of disease transmission, fish mortality, and economic losses (Dunn et al.,1990; Shimada et al., 2014). Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of acute septicemia, pseudotuberculosis or pasteurellosis in marine fish (Tsai et al., 2015). Antibiotics are commonly used in the feed or added into the water to control this disease. However, the misuse of these substances results in drug residues in fillet, increase bacterial resistance (Syed et al., 2012), changes in the aquatic microbial populations and fish immune system (Zhang et al., 2010). Live attenuated vaccines and bacterins of membrane and/or cytoplasm proteins provide limited protection against this disease and have high production cost (Barnes et al., 2005, Gudding et al., 2014). Chicken egg yolk immunoglobulin (IgY) antibodies are environment friendly and, unlike antibiotics, elicits no undesirable side effects, disease resistance, or occurrence of toxic residues (Coleman et al., 1999). The production of IgY is relatively inexpensive, can be produced in large quantities and it is not necessary to sacrifice the hen to obtain it (Chacana et al., 2004). One egg produces on average 100 mg of IgY in the yolk, in 30 days it can reach more than two grams per hen (Carlander, 2002). IgY therapy is largely used in diseases of mammals as in the diarrhea of post-weaning pigs by E. coli, gastric ulcer by Helicobacter pylori, distemper in dogs, and against toxins of venoms animals (Carlander, 2002, Chacana et al., 2004). In fish, there are reports of the use of antiEdwarsiella sp IgY administered directly into the water that protected Japanese eel (Hatta et al., 1994) and anti-Yersinia ruckeri IgY administered in the feed that protected rainbow trout against the experimental infection of the homologous bacteria (Lee et al., 2002). It was also reported that an intra-coelomic administration of anti-Vibrio anguillarum IgY protected rainbow trout against the challenged of V. anguillarum with 83% of survival rate in the treated group compared to 18% in

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Journal Pre-proof control group (Arasteh et al., 2004). Some studies have highlighted the efficiency of hen IgY as biomarkers for the diagnosis of infectious diseases (Chalghoumi et al., 2009; Fernandes et al., 2019) and neoplasms (Xiao et al., 2008). R. canadum (cobia) is an important fish in marine aquaculture due to presents fast growth rate, good market demand, feed conversion rate, and easy adaptation to captivity (Shimada et al. 2014). Thus, develop diagnostic biomarkers and studying the pathophysiology of Photobacterium damselae in R. canadum are important for a better understanding of this disease (Remuzgo-Martínez et al. 2014). The aim of this study was to characterize and produce a specific anti-Photobacterium damselae subsp. piscicida IgY and determinate their prophylactic and therapeutic effects against the homologous bacterium on experimental infection of Rachycentron canadum. It was also developed an immunohistochemistry assay for this experiment that could be used as screening method to detect this bacterium.

2. Material and Methods 2.1 Identification and isolation of bacteria Photobacterium damselae subsp. piscicida strain was isolated from an outbreak occurred in cage-reared cobias on the northern coast of Sao Paulo State, Brazil (23° 48' 54" S 45° 22' 14" O) (Shimada et al., 2014). Briefly, after euthanasia, fish were necropsied and tissues of brain, kidney and blood were aseptically sampled for bacteriological culture. Bacteria were characterized and identified biochemically in accordance of Popoff (1984) and with the API 20E kit (BioMerieux® SA, France), according to manufacturer recommendations. Bacterial mass derived from pure colonies culture was submitted to a DNA extraction process, according to the manufacturer suggested methodology (Genomic DNA purification kit - Wizard®). DNA concentration was 1780.9 ng-1 and absorbance ratio 260/280 and 260/230, varying between 2.07 and 2.10. After obtaining the DNA, ribosomal gene 16 S RNA was amplified according to

4

Journal Pre-proof Zappulli

et

al.

(2005).

Sequences

were

analyzed

by

the

BLAST

algorithm

(http://www.ncbi.nlm.nih.gov), which presented 99% similarity with Photobacterium damselae subsp. piscicida (Phdp).

2.2 Production of the anti-Phdp IgY 2.2.1 Purification of fractions of Phdp, protein determination and electrophoresis on polyacrylamide gel (SDS-PAGE) in the presence of SDS of bacterial protein fractions Phdp bacterium were seeded in 500 ml BHI (Disk, Detroit, USA) supplemented with 3% NaCl for five days at 29.0 °C. Soluble and insoluble fractions were purified (Chart, 1994). Protein content in the yolk, soluble and insoluble fractions were determined by the method of Hartree (1972) using BSA (bovine serum albumin) and fraction V as a standard. The proteins present in each fraction were separated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDSPAGE) (Laemmli, 1970).

2.2.2 Immunization, collection of IgY samples and quantification of anti-Phdp IgY White Leghorn hens (n=7 / 32 weeks old) accommodated in individual cages were immunized with the total fraction (cytoplasmic and membrane) of Phdp at concentration of 0.5 µg -1

diluted in 0.15 M PBS pH 7.4 and incomplete Freud's adjuvant (Sigma-Aldrich, USA) (1:1,

vol/vol). Additional booster doses were administered every 15 days for 90 days. Serum and eggs were collected 15 days after each challenged (Ethical Committee of Animal Welfare of State University of Sao Paulo, protocol nº 03183/14). A microplate-ELISA was established for anti-Phdp IgY. First, microplates were sensitized with 0.05% (vol/vol) poly-L-lysine/ 0.15 M PBS pH 7.2, incubated for 60 minutes at room temperature, and washed twice with PBS-0.005% Tween-20. Then, it was added 100 µL of the total fraction incubated at 4°C, for 18 h, fixed with 100 uL of 0.005% (vol/vol) glutaraldehyde/0.15 M

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Journal Pre-proof PBS pH 7.2, and incubated for 20 minutes at 22 °C. Then, each well was washed three times with PBS-Tween and added 250 µL of PBS containing 5% skim milk (Molico, Nestle, Brazil) and incubated for 1 hour at room temperature. In each well was adding 100 µL of serum (1: 1000 vol / vol) or yolk (1: 2000 vol / vol)/ PBS- skim milk 1%, incubated for one hour at room temperature, washed with PBS-0.05% Tween20, added 100 µl/well of peroxidase anti-IgY, and incubated for one hour at room temperature. After washing with PBS-0.05% Tween 20, it was added the peroxide substrate (ABST) followed by incubation at room temperature for 15 minutes. The reaction was stopped with 5% SDS solution and the absorbance was determined at 640 nm with an ELISA reader (Labsystems Multiskan, Helsinki, Finland). IgY antibodies were purified according to the method described by Pauly et al. (2011).

2.2.3 Determination of anti-Phdp IgY avidity, western blot assay and anti-Phdp IgY half-life in R. canadum It was used similar procedures as described above, except that, after incubation of the samples, each well was washed with PBS-0.05% Tween-20, added 100 µL of 2M MgCl2 and in the control group 100 µL of PBS-0.05% Tween-20. The electrophoresis was performed at 30 V for 18 h. The membrane was incubated in 5% skim milk-PBS at room temperature for one hour, washed 5 times with 0.1% PBS Tween-20, and incubated for one hour with a 1mg/ml(w/vol) IgY solution/ PBS-1% skim milk, washed 5 times with 0.1% PBS Tween-20. The peroxidase conjugated anti-IgY diluted in 1% skim milk PBS was added to the membrane and incubated for one hour at room temperature (Fernandes et al., 2019). Forty fish were used to analyze the half-life of the anti-Phdp IgY. It was used ELISA method as described above with modifications in the dilution and time of incubation of the primary antibody.

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Journal Pre-proof Samples were diluted at a concentration of 1:1 in a final volume of 200 µl and incubated for 1 h at room temperature and 18 h at 4 °C. Before and after the experimental infection, the concentration of anti-Phdp IgY was determined in serum. In the second case, it was detected Y immunoglobulins, free and opsonized to bacterial antigens in organs and tissues.

2.3 Evaluation of the protective efficacy of immunoglobulin Y (anti- Phdp IgY) in acute photobacteriosis in Rachycentron canadum 2.3.1 Fish and induction of infection Cobia fish, R. canadum (~380 g), were randomly allocated in four 10 000 L tanks (50 fish per tank) (dissolved oxygen = 5.2 ± 1.5 mg/L; temperature = 20.4 ± 5.2 ºC). All fish were maintained in tanks with aeration during the course of the experiment with 100% of the tank seawater refreshed daily and fed commercial marine fish food 2 times a day. This study was approved by the Ethical Committee of Animal Welfare of State University of Sao Paulo (protocol nº 03183/14). The groups were distributed randomly (n=10): Group 1 – fish not immunized and injected with PBS (C-); Group 2 – fish not immunized and inoculate with Phdp (C+); Group 3 – fish injected 15 days before challenged with one single dose of anti-Phdp IgY (IgY 15 days); Group 4 – fish injected 48 hours before challenged with one single dose of anti-Phdp IgY (IgY 48 h). The groups treated with IgY were injected via a coelomatic cavity with 200 µL of 14 mg/mL (w/vol)/ PBS (determined in a previous test, data not shown). DL50 of Phdp was previously determined according to Claudiano et al. (2019). Finally, fish were anesthetized (benzocaine solution 1: 20 000 in alcohol 98°, 0.1 g/mL) until the surgical plane (Ross and Ross 2008). Fish were distributed into two groups (completely randomized design), the first injected with 1.0 mL of PBS (C-) and the other inoculated with 1.8 x 106 CFU / mL (corresponding to LD50%) dissolved in the same vehicle and volume per animal (n = 50 tank).

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Journal Pre-proof The quantification and determination of activity of IgY in fish plasma were tested by ELISA and Western blot analysis as described above. Samples were taken at 6, 24 and 48 h post-infection (HPI) of groups by puncture of the caudal vessel (3.0 mL blood / sample) and was collects tissue samples for morphological analyzes. Another aliquot was centrifuged at 1 200 g, for 5 min at 4 ºC, to obtain the serum that was stored at - 80 ºC.

2.3.2 Hematology, peritoneal leukocytes, biochemical profile and leukocyte respiratory burst Blood samples were collected from the caudal vein into heparinized tubes (10%) and stained with May-Grünwal-Giemsa-Wright method to carry out white blood cells and thrombocytes counts (Tavares-Dias and Moraes, 2003). The RBC and blood measurement indices were performed according to Yunis-Aguinaga et al. (2016). Peritoneal exudate was collected using 3.0 mL of 10% EDTA / PBS at 4ºC into the coelomic cavity, and then collected again in microtubes for total and differential cells count according the methodology using by Castro et al. (2014). It the same blood samples it was measured creatine kinase activity (CK), aspartate transaminase (AST), alanine transaminase (ALT), glucose concentration, and iron concentration according to Brito et al. (2015). The leukocyte respiratory burst was performed by turbidometric assay using nitroblue tetrazolium according to Yunis-Aguinaga et al. (2015).

2.3.3 Bacterial dissemination, histopathology, and immunohistochemistry Bacterial spread (heart, liver, spleen, and kidney) was evaluated according to Galeotti et al. (2013). Histopahologic changes were evaluated in heart, liver, spleen and kidney. Immunohistochemistry was used to detect the microorganism in heart, liver, spleen, and kidney (1994). In the first test, it was used 1.0 mg of the IgY anti-Phdp previously produced at 4 °C, for 18 hours (1:1000 dilution in PBS) (primary antibody) and goat anti-chicken IgY- heavy chain Fc

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Journal Pre-proof (Bethyl Laboratories, Inc. USA) (1:1000 dilution in PBS) (secondary antibody). In the second test, it was used goat anti-Chicken IgY- heavy chain Fc (Bethyl Laboratories, Inc. USA) (1:1000 dilution in PBS) (primary antibody) and rabbit anti-goat IgG-heavy chain Fc (Bethyl Laboratories, Inc. USA) (1:1000 dilution in PBS) (secondary antibody) at room temperature for one hour. In both cases, after incubation, sections were washed in Tris buffer HCL (pH 7.4) for five minutes and then chromogen DAB (DAKO, CA, USA) was applied for 90 seconds. Finally, sections were rinsed, counterstained with Harris hematoxylin, and routinely dehydrated through a series of alcohol baths and coverslipped. The mounted slides were examined under light microscope (Olympus BX51) and were photographed using Olympus DP72 camera and Cell Sens software v. 1.5.

2.4 Statistical analysis The results were subjected to analysis of variance and comparison of means through Tukey test at a significance level of 5%. The normal distribution was verify with Kolmogorov-Smirnov and Shapiro-Wilk's W normality test (when necessary). R software was used as statistical program for the calculations.

3. Results 3.1 Production of anti-Phdp IgY It was verified significant increase in the production of specific anti-Phdp IgY (p<0.05) in the serum of the hens on the 15th day after the immunization. Titers remained constant for all sampling times (Figure 1A). The same occurred in the egg yolk (p<0.05) (Figure 1B). The potential of antibodies affinity in serum increased gradually until the 90th day after immunization (p<0.05) (Figure 1C).

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Journal Pre-proof Western blot specificity of anti-Phdp IgY showed the presence of eight proteins with molecular masses in the total fraction, seven in the cytoplasmic fraction, and four in the membrane fraction (Figure 2). Positive anti-Phdp IgY was observed on tissues, mainly in parenchymal organs opsonizing microorganisms associated with melanomacrophages in kidney (Figures 3A and 3B) and spleen (Figures 3B and 3C). It was found that anti-Phdp IgY 48 hours presented a significant increase (p <0.05) compared to the others groups in plasma (Figure 3E).

3.2 Efficacy of anti-Phdp IgY in Rachycentron canadum 3.2.1 Analysis of blood and plasma variables There were no differences (p>0.05) in red blood cell count between groups. The C+ group showed a significant increase (p<0.05) in the concentration of hemoglobin and in the mean corpuscular hemoglobin (MCH) (Table 1). It was observed in all threated groups enhances of the total leukocytes count compared to the negative control (C-). However, C+ group had a significant increase of these cells (p<0.05) 6 HPI compared to IgY (15 days) and IgY (48 hours) groups (Table 2). The number of granulocytes was higher in the group C+ compared to IgY (15 days) and IgY (48 hours) groups (p <0.05) after 6 HPI. However, after 24 HPI the number of granulocytes in the IgY (48 hours) group decreased significantly (p<0.05). Monocytes and lymphocytes of the C- group were lesser than the other groups in all sampling times (p<0.05) (Table 2). In peritoneal leukocytes, it was observed that C+ group fish presented hemorrhagic exudate in the coelomic cavity at six hours, which may have compromised the leukocytes count (Table 3). At 24 and 48 HPI, the number of leukocytes increased gradually in C+ compared to other groups. The IgY group (48 hours) showed lesser quantity of lymphocyte number at 6 and 24 HPI and thrombocytes at 48 HPI than other groups (p<0.05). The number of thrombocytes and macrophages increased at 24 HPI in the C+ group compared to IgY (15 days) and IgY (48 h) groups. 10

Journal Pre-proof The C+ Group showed an increase (p <0.05) in the enzymes CK, AST, and ALT in all sampling times in relation to IgY (15 days) and IgY (48 hours) groups (Table 4). Glucose levels were similar in all groups at 6 hours. However, at 24 and 48 hours all challenged groups presented lesser levels (Table 4). Respiratory activity of leukocytes increased in all challenged groups and both with the C+ group presented the highest levels in all sampling times (Figure 4).

3.2.2 Dissemination of P. damselae in tissues The C+ group presented extensive lesions in heart, kidney, liver, and spleen (Figure 5). The IgY (15 days) and IgY (48 hours) groups presented some lesion at 48 HPI. It was observed that the C + group presented progressively leukocyte infiltrate in the heart and at 48 HPI presented coagulative necrosis of myocardial fibers (Figure 5 A), with degenerative lesions and coagulative necrosis in the renal tubules (Figure 5 B). In the liver occurred vacuolization of hepatocytes with steatosis and spleen presented coagulative necrotic foci in the parenchyma of the organ mainly in the positive control group (Figure 5 C and D). The dissemination of the bacteria occurred in all organs and tissues studied. The C+ group presented the higher percentage of bacteria in all sampling times in all the organs studied. The kidneys presented the more percentage of bacteria in most sampling time (Figure 6). At 48 HPI, it was found that the group IgY (48 hours) presented no bacteria in liver and spleen (Figure 6). It was also observed that at 24 and 48 hours the IgY (15 days) group presented less quantity of bacteria compared to control group. However, presented more quantity of bacteria than the IgY (48 hours) group. Bacteria were successfully immnunostaining with the technique developed for this experiment, showing positive staining in bacterial cells, which were found in all challenged groups and in all the organs sampled. Bacteria were more abundant in the C+ group (Figure 7).

4. Discussion

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Journal Pre-proof Photobacterium damselae subsp. piscicida infection has produced high mortality in several species of saltwater fish in different countries (Zappulli et al., 2005) including Brazil (Shimada et al., 2014). Fish infected by Phdp can also be asymptomatic and becoming chronic with no host specificity (Magariños, 2003, Barnes et al., 2005). Prophylactic measures such as passive immunization with IgY is an effective strategy for protection against pathogens in fish (Arasteh et al., 2004, Wu et al., 2011, Jin et al., 2013, Li et al., 2014, Gan, 2015). Although some studies highlight the advantages of oral administration of specific IgY for immunity to fish against bacteria, some studies have demonstrated the protective effect of intraperitoneal injection (Jin et al., 2013, Li et al., 2014). In the present study, it was established a new prophylaxis and therapeutic strategies using specific anti-Phdp IgY and developed an immunochemistry assay to detect Phdp in Rachycentron canadum. The results showed that vaccinated hens produced specific IgY for several antigenic epitopes, present in soluble and insoluble protein fractions of the bacterial extract. The avidity of immunoglobulins for antigen increased progressively and reached the peak on the 90th day after the last challenged. Andrade et al. (2013) observed that the avidity of anti-Bothrops IgY and Crotalus venom IgY increasing until 367 days after the challenged. Although this experiment was more extensive than the present study, the progressive curve presented similar results. Anti-Phdp IgY was highly specific to the bacteria, being immunoreactive for protein bands of soluble and insoluble fraction in Western Blot assay. Molecular mass differences in the proteins found in this study can be explained due to the different cellular compartments where they were found. In addition, they may have structural differences in the molecular isoform (Esteve and Birbeck, 2004). Phdp as any other microorganism depends on nutrients to the expression of virulence factors. The iron (Fe) is one of the most important of these nutrients (Costa-Ramos et al., 2011). The main source of Fe in higher organisms is the hemoglobin present in red blood cells. In the current study, it was observed that fish inoculated with Phdp presented sepsis. This was evidenced by the clinical

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Journal Pre-proof alterations, laboratory findings and dissemination in the tissues, including hemolysis mainly at early stages of infection and lesser Fe concentration in C+ group at 48 HPI comparing to other groups. Hsu et al. (2014) described a 43 kDa exotoxin (phospholipase A2 – PLA2), isolated and purified from extracellular products of Phdp with hemolytic action. In addition, it was described other exotoxins with cytolytic effects and molecular weight of 48.3, 79, 132, and 175 kDa in extracellular products of Phdp by Bakopoulos et al. (2004). The IgY produced in this study was specific for the cytoplasmic proteins described by these authors likely due to blocking hemolysis by binding to PLA2. This explain the hemorrhagic exudate in the coelomatic cavity, reduction of serum iron and hemolysis in the experimental infection with this bacterium in the positive control group and absence in the treated groups. The activation of receptors (FcR) and complement receptor 3 (CR3) induces the production of ROS and phagocytosis. However, polysaccharide capsule (PC) blocks the action of ROS, increasing the production of these molecules without elimination of the pathogen. By contrast, in fish pretreated with IgY, IgY Fc did not activate FcR and CR3 of phagocytic cells reducing the production of ROS. The receptor that recognizes IgY and its fractions had not been elucidated in this study. Opsonization of the pathogen neutralized the protective effect of PC, increasing effective phagocytosis, reducing the production of ROS by phagocytes. Proposed mechanism of the effects of LPS opsonization, the anti-Phdp IgY may inhibited the activation of Toll like receptor-4 (TRL-4) and CD14. In this study, the specificity of IgY for lipopolysaccharide (LPS) and other proteins of Phdp in membranes was observed in the protein insoluble fraction (30, 42, and 53 kDa), similar to that observed by Magariños et al. (2003). The clinical effects of the therapy and prophylaxis with anti-Phdp IgY in Rachycentron canadum were similar to those described in mammals (Zhen et al., 2011). The opsonization of the proteins of bacterial membrane facilitated phagocytosis and the elimination of the pathogen, which could reduce the acute infection and the migration of leucocytes to the coelomic cavity.

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Journal Pre-proof The burst activity was enhanced in pre-immunized groups. However, it was observed a reduction, likely due to bacterial opsonization neutralized the effect of LPS reducing the bioenergetic cost of phagocytes (Brieger, 2012). In this study, it was no observed proteolysis of anti-Phdp IgY in Rachycentron canadum serum. This can be explained due to the differences of C3b molecule or Fc receptor (FcR) of fish related to the phylogenetic distance between species (Urbaczek et al., 1990). Animals pretreated with IgY presented less ROS production likely due to the neutralization of the bacteria and by not activation of FcR and complement receptor 3 (CR3) of fish phagocytes. Opsonization improved the elimination of pathogens and reduced the bioenergetic cost of phagocytes avoiding the deleterious effects of an excessive production of ROS (Nascimento et al., 2007). The challenged using live Phdp in Rachycentron canadum also produced structural lesions in fish organs. Necrosis was observed in all sampling times and groups. However it was observed that when IgY was present, both free or opsonizing bacteria were in less quantity and organs presented only minor lesions. The reduction of AST, ALT and CK enzymes in the threated groups supported the findings in the histopathology. The treatment reduced the damage causing by the bacteria due to probably the block of bacteria spread to kidneys, liver, and spleen as observed in the threated groups. IgY protection was also observed in the immunohistochemistry assay. The bacteria were successfully opsonized with anti-Phdp IgY in the spleen and kidney blocking its growth. It is possible that the IgY mechanism of action was due to a multifactorial action of polyclonal antibodies recognizing and blocking the virulence factors of Phdp. The best response of the IgY (48 hours) group suggests that this response was correlated with the antibodies half-life. These results support the hypothesis that IgY improves the response against bacteria and can be used in prophylaxis. Arasteh et al. (2004) observed the prophylactic effect of anti-Vibrio anguillarum IgY in Oncorhynchus mykiss 14 days before experimental infection. They found similar results to those found in this study including reduced mortality rate in the immunized group compared to non-immunized (70%), showing the protection conferred by the IgY.

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Journal Pre-proof Several studies showed the efficacy of IgY treatment in fish disease and the advantages over antibiotics and anti-inflammatory drugs (Arasteh et al., 2004, Wu et al., 2011, Jin et al., 2013, Li et al., 2014, Gan, 2015). Although these drugs present important mechanism to block bacterial damage as bacteriostatic and bactericidal action, and inhibiting the cyclooxygenase enzyme activity, they have no pharmacological effect on the LPS molecules released by bacterial lysis during infection. In contrast, anti-LPS IgY had effect on the bacteria and their by-products, modulating inflammatory process (Zhen et al., 2008) as observed in the current study. Thus, it was concluded that the use of anti-Phdp IgY was able to control the dissemination of Phdp in Rachycentron canadum, decreased the effects of exacerbated inflammatory response and induced an antigen-specific antibody response helping to protect against this disease. However, field studies are still needed to analyze the effectiveness of fish farmers. The immunohistochemistry assay development for this experiment presented highly specificity and affinity for this bacterium and it would be used as screening method to detect this bacterium in early stages analysis of pathogenesis of the pseudotuberculosis in Rachycentron canadum. 5. Acknowledgments This work was supported by the Coordination for the Improvement of Higher Education Personnel “Edital CAPES-Ciências do Mar” N0 09/2009-Proc. 300613/2009-1 and Fapesp-Proc. 2012/10090-4. In Memoriam: A Great Friend and Mentor, Professor Flávio Ruas de Moraes.

6. References Adams, A., Marin-Mateo, M., 1994. Immunohistochemical detection of fish pathogens. In: Stolen JS, Fletcher TC, Rowley AF, Anderson DP, Kaattari SL, Zelikoff JT, Smith SA, editors. Techniques in Fish Immunology, New Jersey: SOS publications, p. 133-144.

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Journal Pre-proof Andrade, F. G., Eto, S. F., Navarro, A. C., Marioto, D. T., Vieira, N. J., Cheirubim, A. P., Ramos, S. P., Venâncio, E. J., 2013. The production and characterization of anti-bothropic and anti-crotalic IgY antibodies

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Journal Pre-proof Chacana, P. A., Terzolo, H. R., Gutiérrez, C. E., Schade, R., 2004. Tecnología IgY o aplicaciones de los anticuerpos de yema de huevo de gallina. Rev. Bras. Med. Vet. 85, 179-189. Chart, H. 1994. Bacterial fractionation and membrane protein characterization. Pages 1-10 In: Chart, H., editor. Methods in Practical Laboratory Bacteriology, Boca Raton: CRC Press (p 1-10). Chalghoumi, R., Beckers, Y., Portetelle, D., Théwis, A., 2009. Hen egg yolk antibodies (IgY), production and use for passive immunization against bacterial enteric infections in chicken. Biotechnol. Agron. Soc. Environ. 13, 295–308. Claudiano, S.C., Yunis-Aguinaga, J., Marinho-Neto, F.A., Miranda, R.L., Martins, I.M., Otani, F.S., Mundim, A.V., Marzocchi-Machado, C.M., Moraes, J.R.E., Moraes, F.R. 2019. Hematological and immune changes in Piaractus mesopotamicus in the sepsis induced by Aeromonas hydrophila. Fish Shellfish Immunol. 88, 259-265. https://doi.org/10.1016/j.fsi.2019.01.044 Coleman, M. 1999. Using egg antibodies to treat diseases. In: Sim JS, Nakai S, Guenter W, editors. Egg Nutrition and Biotechnology, Wallingford: CABI Publishing CAB International (p. 351– 370). Costa-Ramos, C., Vale, A. D., Ludovico, P., Santos, N. M., Silva, M. T. 2011. The bacterial exotoxin AIP56 induces fish macrophage and neutrophil apoptosis using mechanisms of the extrinsic and intrinsic pathways. Fish Shellfish Immunol. 30, 173-181. doi: 10.1016/j.fsi.2010.10.007. Dunn, E. J., Polk, A., Scarrett, D. J., Olivier, G., Lall, S., Goosen, M. F. A., 1990. Vaccines in aquaculture: the search for an efficient delivery system. Aquacult. Eng. 9, 23–32. https://doi.org/10.1016/0144-8609(90)90009-O Esteve, C., Birbeck, T. H., 2004. Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metalloprotease (Elastase). J. Appl. Microbiol. 96, 994-1001. doi: 10.1111/j.1365-2672.2004.02227.x Fernandes, D. C., Eto, S. F., Funnicelli, M. I. G., Fernandes, C. C., Charlie-Silva, I., Belo, M. A. A., Pizauro J. M. 2019. Immunoglobulin Y in the diagnosis of Aeromonas hydrophila infection in Nile

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Figure 1: Production of specific IgY antibodies in serum (A) and egg yolk (B) of hens immunized with cytoplasmic proteins and membrane of Phdp. Potential of antibodies affinity in serum (C).

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Figure 2. Western blot specificity of anti-Phdp IgY. (A) SDS-PAGE gel (SDS 9%) protein fraction samples. (P= Bio-Rad Precision Plus protein standard; 1= Total fraction, cytoplasm and membrane; 2= Cytoplasmic fraction; 3= Membrane fraction. (B) Western blot, nitrocellulose membrane (0.45 μm), SDS-PAGE protein bands were transferred and incubated with purified anti-Phdp IgY, 90 days after the fourth immunization at the diluted 1 mg / mL concentration in 1: 500 ratio.

Figure 3. Immunostaining of opsonized anti-Phdp IgY in Phdp bacterial cells in the spleen (A-B) and caudal kidney (C-D). Anti-Phdp IgY concentration in Rachycentron canadum serum prior to immunization, 15 days and 48 hours after immunization (E). Vertical columns represent the means of each group in different evaluation times. Vertical bars represent the standard error of the mean. (*) Statistical difference (p <0.05) 5% level by Tukey test (n=10).

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Figure 4 - Leukocyte respiratory activity of blood phagocytes of Rachycentron canadum (n=10) injected with saline (control -), inoculated with Phdp without antibody therapy (control +), inoculated 15 days prior to challenge with specific anti-Phdp IgY (IgY 15 days), and inoculated 48 hours before bacterial challenge with specific anti-Phdp IgY (IgY 48 hours).

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Fig 5 – Histopathology findings of R. canadum (n=10) inoculated with Phdp without antibody therapy (control +). (A) Cardiac tissue, dotted arrow shows dissociation of muscle fibers due to edema and necrosis, and inflammatory infiltrate in the myocardial interstitial space (arrow). (B) Kidney tissue, arrows show tubular necrosis and degeneration. (C) Liver tissue, asterisk shows steatosis areas associated with necrosis. (D) Spleen tissue, black arrow shows inflammatory infiltrate and dotted arrow shows necrosis and loss of parenchyma (asterisk). Bar=10 µm.

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Figura 6. Percentage (n = 10) of bacterial dissemination in Rachycentron canadum organs. Control (+), anti-Phdp IgY (IgY 15) and (IgY 48). K – kidney; S – spleen; L – liver. HPI – Hours post-infection

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Fig 7. Positive immunostaining Phdp bacterial cells in R. canadum (n=10), 48 hours after experimental infection. (A) heart, (B) kidney, (C) liver, and (D) spleen. Bar = 10 µm.

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Highlights



IgY anti-Photobacterium damselae subsp. piscicida as prophylactic and therapeutic



IgY effectively inhibited the dissemination of anti-Photobacterium damselae subsp. piscicida in Rachycentron canadum



Developed an immunohistochemistry assay with highly specificity and affinity for the Photobacterium damselae subsp. piscicida

Table 1. Hematology. Hct: hematocrit; Hb: hemoglobin; MCHC: mean corpuscular hemoglobin concentration; MCH: Mean corpuscular hemoglobin, and MCV Mean corpuscular volume. Means ± SD (n=10). Variables

Hct (%)

MCV (fl)

MCH (pg)

MCHC (g/dl)

Erythrocytes (million/mm³)

Treatments

Time 6 hours

24 hours

48 hours

Control (-)

29.7

±

1.6

A

32.0

±

1.2

A

33.0

±

1.6

A

Control (+)

30.0

±

2.0

A

30.0

±

1.4

A

31.2

±

1.9

A

IgY (15 days)

31.8

±

1.1

A

27.0

±

1.5

A

31.4

±

1.8

A

IgY (48 hours)

31.8

±

3.4

A

24.8

±

2.0

A

27.2

±

1.9

A

Control (-)

2.1

±

0.1

A

2.2

±

0.2

A

2.3

±

0.2

A

Control (+)

1.7

±

0.1

A

2.3

±

0.1

A

3.5

±

0.1

A

IgY (15 days)

2.3

±

0.1

A

1.8

±

0.1

A

3.1

±

0.4

A

IgY (48 hours)

2.8

±

0.3

A

1.7

±

0.1

A

3.9

±

0.4

A

Control (-)

5.7

±

0.4

A

5.9

±

0.1

A

6.7

±

0.8

B

Control (+)

4.7

±

0.1

A

7.1

±

0.1

A

12.9

±

0.8

A

IgY (15 days)

7.1

±

0.8

A

5.0

±

0.1

A

10.6

±

0.9

B

IgY (48 hours)

7.0

±

0.6

A

5.5

±

0.1

A

7.5

±

1.0

B

Control (-)

28.2

±

2.6

A

28.6

±

0.1

A

28.7

±

2.4

A

Control (+)

27.6

±

1.0

A

30.3

±

0.1

A

36.9

±

2.6

A

IgY (15 days)

30.4

±

4.0

A

27.4

±

0.1

A

32.8

±

1.5

A

IgY (48 hours)

32

±

3.0

A

29.7

±

0.1

A

26.5

±

1

A

Control (-)

148

±

16

A

153

±

0.1

A

143

±

13

A

Control (+)

174

±

6.4

A

128

±

0.1

A

88

±

3.9

B

IgY (15 days)

137

±

7.8

A

143

±

0.1

A

99

±

8.7

B

IgY (48 hours)

136

±

4.1

A

101

±

0.1

A

125

±

1.6

A

Hemoglobin (g/dl)

Control (-)

8.3

±

0.6

A

9.1

±

0.1

A

9

±

0.4

B

Control (+)

8.2

±

0.4

A

9.0

±

0.1

A

11

±

0.4

A

IgY (15 days)

9.6

±

0.9

A

8.0

±

0.1

A

10

±

0.4

B

IgY (48 hours)

9.8

±

0.7

A

7.5

±

0.1

A

9

±

0.5

B

Table 2. Total and differential leukocyte count in blood. Means ± SD (n=10). Time Variables (cells/mm³)

Total leukocytes

Absolute lymphocyte

Absolute granulocyte

Treatment

6 hours

24 hours

48 hours

Control (-)

8.267 ±

1.038

C

9.354 ±

420

B

8.144 ±

627

C

Control (+)

18.492 ±

1.016

A

14.092 ±

805

A

9.983 ±

654

B

IgY (15 days)

16.583 ±

1.075

A

11.292 ±

523

A

9.800 ±

768

B

IgY (48 hours)

14.433 ±

577

B

12.883 ±

993

A

12.550 ±

494

A

Control (-)

7.604 ±

292

B

7.918 ±

305

B

7.080 ±

225

B

Control (+)

15.423 ±

617

A

13.924 ±

676

A

15.574 ±

168

A

IgY (15 days)

13.568 ±

771

A

12.607 ±

328

A

12.175 ±

793

A

IgY (48 hours)

12.203 ±

521

A

10.776 ±

628

A

10.628 ±

166

A

89

B

565 ±

168

C

674 ±

356

B

Control (-)

326 ±

Control (+)

1.592 ±

563

A

1.965 ±

147

A

1.052 ±

535

A

IgY (15 days)

858 ±

364

AB

1.944 ±

606

A

1.417 ±

779

A

IgY (48 hours)

717 ±

260

AB

969 ±

182

B

1.424 ±

876

A

Absolute thrombocytes

Absolute monocytes

Control (-)

295 ±

171

B

346 ±

25

A

432 ±

200

B

Control (+)

640 ±

220

A

463 ±

73

A

693 ±

720

A

IgY (15 days)

1.008 ±

107

A

317 ±

35

A

739 ±

267

A

IgY (48 hours)

217 ±

67

B

357 ±

59

A

790 ±

430

A

Control (-)

329 ±

107

B

306 ±

77

B

384 ±

85

B

Control (+)

531 ±

230

A

1.877 ±

459

A

1.222 ±

198

A

IgY (15 days)

613 ±

331

A

1.044 ±

490

A

1.250 ±

563

A

IgY (48 hours)

680 ±

143

A

1.654 ±

410

A

1.173 ±

383

A

Table 3. Total and differential leukocyte count in the coelomic cavity (n=10). Time (hours) Variable (cells/mm³)

Treatment

Absolute lymphocyte

24 hours

48 hours

0.00 ± 0.00

∆

3.308 ± 699

A

3.063 ±

333

A

IgY (15 days)

1.652 ± 176

A

1.477 ± 271

C

1.919 ±

101

A

IgY (48 horas)

2.276 ± 706

A

2.386 ± 395

B

2.411 ±

184

A

Control (+)

0.00 ± 0.00

∆

460 ± 211

C

591 ±

105

B

IgY (15 days)

994 ± 940

A

1081 ± 146

B

1186 ±

212

A

IgY (48 hours)

737 ± 655

B

1594 ± 273

A

1461 ±

240

A

Control (+) Total leukocytes

6 hours

Absolute granulocyte

Absolute thrombocytes

Absolute monocytes

Control (+)

0.00 ± 0.00

∆

452 ± 211

C

1140 ±

333

A

IgY (15 days)

657 ± 164

A

812 ± 286

B

441 ±

691

C

IgY (48 hours)

366 ± 835

B

1479 ± 587

A

602 ±

197

B

Control (+)

0.00 ± 0.00

∆

1275 ± 556

A

1237 ±

501

A

IgY (15 days)

681 ± 161

A

403 ± 794

B

1328 ±

402

A

IgY (48 hours)

474 ± 159

A

579 ± 137

B

761 ±

158

B

Control (+)

0.00 ± 0.00

∆

935 ± 430

B

1199 ±

212

A

IgY (15 days)

533 ± 191

A

1769 ± 338

A

1459 ±

141

A

IgY (48 hours)

561 ± 101

A

1743 ± 337

A

1763 ±

367

A

Values (means ± SD) with different capital letters compare treatments (6, 24, and 48 h) (P < 0.05).

Table 4. Means and standard deviation (SD) of serum enzyme and metabolites levels (n=10). Variable

CK (U/L)

AST (U/L)

Treatment

Time (hours) 6 hours

24 hours

48 hours

Control (-)

132 ± 49

C

128 ± 49

C

130 ± 49

C

Control (+)

454 ± 15

A

865 ± 45

A

1.402 ± 92

A

IgY (15 days)

241 ± 63

B

347 ± 24

B

476 ± 71

B

IgY (48 hours)

124 ± 74

C

198 ± 35

C

210 ± 20

C

Control (-)

7.8 ± 0.4

B

8.5 ± 0.4

C

8 ± 0.4

Control (+)

15 ± 0.6

A

A

15 ± 1.3

19.8 ± 1

BC A

ALT (U/L)

Glucose (g/dL)

Fe (µg/dL)

IgY (15 days)

9.5 ± 0.6

B

13.5 ± 0.8

B

10 ± 1.1

B

IgY (48 hours)

10.5 ± 0.6

B

12.3 ± 1.2

B

6.7 ± 1

C

Control (-)

4.2 ± 1

A

5.2 ± 0.8

A

4.9 ± 0.8

BC

Control (+)

3.9 ± 0.4

A

4.6 ± 0.9

A

12 ± 1.2

A

IgY (15 days)

6.5 ± 0.6

A

5.6 ± 1.3

A

6 ± 0.9

B

IgY (48 hours)

5.5 ± 0.6

A

4.7 ± 1.2

A

3.8 ± 0.4

C

Control (-)

104 ± 1.87

A

101 ± 1.05

A

96 ± 2.03

A

Control (+)

100 ± 1.35

A

34.8 ± 3.35

B

25 ± 2.70

B

IgY (15 days)

99 ± 1.14

A

33.0 ± 4.06

B

26 ± 2.93

B

IgY (48 hours)

106 ± 2.44

A

34.8 ± 4.54

B

24 ± 2.69

B

Control (-)

32 ± 1.7

B

28 ± 1.6

B

22 ± 1.6

A

Control (+)

59 ± 3.9

A

90.0 ± 4.8

A

13 ± 1.8

C

IgY (15 days)

33 ± 2.3

B

42.0 ± 3.2

B

16 ± 4.0

B

IgY (48 hours)

28 ± 2.7

B

36.0 ± 3.8

B

27 ± 2.0

A

Values (means ± SD) with different capital letters compare treatments (6, 24, and 48 h) (P < 0.05). Creatinine kinase (CK), glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT).