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. 2022 Jan 25;3:100053. doi: 10.1016/j.fsirep.2022.100053

Modulation of expression of proinflammatory genes and humoral immune response following immunization or infection with Aeromonas hydrophila in silver catfish (Rhamdia quelen)

Lucas de Figueiredo Soveral 1, Paola Aparecida de Almeida 1, Yasmin Kreutz 1, Vitoria Agnoletto Ribeiro 1, Rafael Frandoloso 1, Luiz Carlos Kreutz 1,⁎
PMCID: PMC9680053  PMID: 36419599

Highlights

  • •

    Vaccination induces expression of TNF-α and IL-1β in silver catfish.

  • •

    Immune-related gene expression profile in vaccinated and infected fish is similar.

  • •

    Vaccination increases innate immune parameters.

  • •

    Production of anti-A.hydrophila antibody correlates to protection.

Keywords: Silver catfish, Vaccination, Aeromonas, Cytokine expression, Antibody

Abstract

The early immune-related events arising from the interaction of antigen and innate immune cells are central to modulating the acquired immune response. Ideally, the immunizing antigen should elicit immunological changes similar to that observed after infection with the wild type pathogen. Here, we evaluated early changes on the expression of selected proinflammatory genes (TNF-α, IL-1β, IRAK4 and myeloperoxidase) and innate immune parameters (serum myeloperoxidase, lysozyme and complement hemolytic activity) in silver catfish vaccinated or infected with Aeromonas hydrophila, the etiological agent of hemorrhagic septicemia. The humoral immune response and resistance to challenge were also evaluated in vaccinated and placebo inoculated fish. We found that the expression of TNF-α and IL-1β genes was higher (p<0.05) in vaccinated or infected fish at 24 h post inoculation (p.i) compared to the control group but returned to basal levels at 72 h p.i. The expression of IRAK4 gene, however, was not altered by vaccination or infection. In addition, the natural hemolytic activity of complement was higher (p<0.05) at 24 h and 72 h p.i. in the vaccinated and infected groups; serum myeloperoxidase was higher (p<0.05) in these groups but only at 24 h p.i. and lysozyme activity was higher (p<0.05) only in the infected group at 72 h p.i. Furthermore, vaccination induced the production of IgM-like antibodies and protection to challenge with the A. hydrophila. Our results indicate that the vaccine formulation induces an immune response similar to that induced by the infecting pathogen and might be a valuable tool in the prophylaxis of hemorrhagic septicemia in silver catfish.

1. Introduction

Silver catfish (Rhamdia quelen) is an omnivorous fish species commonly found in rivers and lakes in Central and South America [1] and is suitable to farming alone or comingled with other fish species [2]. However, stressful situations caused by farming practices, high density rearing, sudden changes on water temperature, reduced water quality or water contamination with immune toxigenic agrichemical reduce the fish ability to cope with viral and bacterial infections. The Gram-negative bacterium Aeromonas hydrophila, for instance, is ubiquitous on aquatic environment [3] and might cause hemorrhagic septicemia in stressed silver catfish leading to high mortality rates mostly in fingerlings and juvenile fish [4].

In a previous work we found that the resistance of silver catfish to A. hydrophila challenge could be improved by feeding a β-glucan enriched diet [5] or by prior intraperitoneal inoculation of synthetic oligodeoxynucleotides (ODNs) containing cytidine-phosphate-guanosine (CpG) motifs [6]. However, when administered in the absence of an antigen, the immunomodulating effects of such molecules are restricted to innate immune cells and the anti-bacterial resistance provided is short lived [7], [8]; thus, the use of immunomodulating molecules might be recommended mostly prior to stressful events.

In contrast, long-term specific immunity to pathogens can be induced by injectable antigens. In aquaculture, several infectious diseases have been controlled by the systematic use of vaccination [9], [10], [11]. Different types of vaccines have been evaluated in several fish species but vaccines formulated with inactivated, adjuvanted-pathogens are still considered safer and most widely used [12], [13]. Intraperitoneal injection of inactivated vaccines induces a robust antibody response that is correlated to protection. In addition, by using adjuvants that differently modulate the maturation of Th cells, these vaccines might induce both humoral and cellular-mediated immunity [14], [15], [16]. The humoral immune response of silver catfish to inoculated bacteria [17] or purified antigens mixed with different types of adjuvant, including β-glucan, Montanide, Freund's complete and incomplete adjuvant, ODNs CpGs [18], and inactivated Parapoxvirus ovis [39] has also been investigated; antigen-specific antibodies peak from 3 to 4 weeks post vaccination. However, there is no data regarding the early events following vaccination and protection to challenge with A. hydrophila.

The initial interaction of antigen with innate immune cells is a key event that orchestrates the development of acquired immunity [12], [19], [20]; and, in this sense, the early immune-related changes induced by a vaccine antigen should ideally mimic that one induced by the pathogen. With this in mind, here we aimed to evaluate early selected immune-related events in silver catfish after vaccination and/or infection with A. hydrophila and to evaluate antibody production and resistance to challenge in vaccinated fish. We found that the immune-related events induced by the vaccine or by infection are similar and that vaccination induces a strong humoral response and protects against challenging.

2. Material and methods

2.1. Fish

Juveniles mixed sex silver catfish (30±10 g) were equally divided into indoor tanks with a natural source of running water (23/25 °C) and acclimatized for at least 10 days before the experiment. Water parameters of dissolved oxygen (5.5–7.0 mg/L), pH (7.1 ± 0.3), hardness and alkalinity (45 ± 5 mg CaCO3/L) and total ammonia (< 0.7 mg/L) were within accepted range for the species. Fish were feed with commercial pelleted food (crude protein 30%; fiber 3%; fat 8%; and ash 14%; Supra, Brazil). All inoculation and sampling procedures were carried out with anesthetized fish (Eugenol, 50 mg/L−1) and the study was approved by the Institutional Ethical and Animal Welfare Committee (protocol number 017/2017).

2.2. Preparations of bacterial inoculum and vaccine

A pathogenic strain of A. hydrophila [6] was inoculated in Luria BertaniTM (LB) media and incubated with shaking (200 rpm/37 °C) until reaching optical density of 0.5 at 600 nm. For the vaccine, the bacteria was formalin-inactivated (18 h at 37 °C with agitation at 200 rpm), collected by centrifugation (600xg for 20 minutes) and washed 3 times with sterile PBS (137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 1.8 mM KH2PO4; pH 7.2). An aliquot was labelled with propidium iodide (Sigma-Aldrich) and then counted using Flow Cytometry (FACSVerse Cytometer–Becton Dickinson, USA). Vaccine doses were prepared to contain 5 × 108 bacteria in 100 µL of sterile PBS containing 20% of montanide Gel PR1TM (Seppic, USA). For the infection, the bacteria were cultivated and washed as described above and the number and percentage of viable bacteria was determined by flow cytometry. The infecting inoculum was prepared to contain 2.5 × 108 bacteria/ 100 µL of sterile PBS [21].

2.3. Vaccination, infection and sampling procedures

To evaluate innate immune parameters fish were allocated into three 500L-tanks and the vaccine, infecting inoculum and PBS inoculations were made intraperitoneally in anesthetized fish. Each tank represented a different groups and different treatments: a) the control group consisted of 20 fish inoculated with sterile PBS pH 7.2; b) the vaccinated group had 20 fish inoculated with inactivated bacteria (5 × 108 bacteria/ fish + 20% Montanide Gel PR1™); c) the infected group had 20 fish inoculated with 2.5 × 108 viable bacteria/fish. Blood samples and cranial kidney (n=6 per time point/group) were collected from anesthetized fish 24 hours before vaccination or infection to obtain the basal values for the immunological parameters, and then at 24 h and 72 h post inoculations. For sampling, fish were anesthetized and blood samples were collected from the caudal vein and allowed to clot on ice to obtain the serum used to measure the myeloperoxidase, lysozyme and complement hemolytic activity. After that, the fish were then euthanized and the cranial kidney were collected under aseptic conditions and kept in RNA later solution™ (Sigma, USA) at -80 °C for RNA extraction procedures.

In a second experiment, 80 silver catfish (40 fish/tank) were used to evaluate resistance to challenge with A. hydrophila. One group (n = 40 fish) was vaccinated (5 × 108 inactivated bacteria + 20% Montanide Gel PR1™/fish) and the other (n = 40) inoculated with sterile PBS. Blood samples were collected prior to inoculation and then again 21 days later immediately prior to challenge with A. hydrophila (2.5 × 108 viable bacteria/fish). The number of dead fish was registered daily up to 7 days post infection.

2.4. RNA extraction and analysis of cytokine genes expression

Total RNA was extracted from cranial kidney tissue fragments (20 mg) using RNeasy® Mini Kit (Qiagen) followed by on column treatment with DNAse I. The RNA quality and concentration were evaluated by spectrophotometry (Nano-photometer Pearl®, IMPLEN, Germany), and stored at -80 °C. The synthesis of cDNA was carried out using 1 µg of total RNA as template, SuperScript® III Reverse Transcriptase (Invitrogen, EUA) and random primers. cDNA samples were then diluted in nuclease-free water (1:100) prior to mRNA quantitation by real time quantitative polymerase chain reaction (qPCR) analysis. The expression of selective cytokine genes was carried out as reported previously [22] . In brief, the qPCR mix contained 400 nM of each primer pair, 7,5 µL of PCR Evagreen Master mix (Jena bioscience, Germany) and 5 µL of cDNA samples, in a final reaction volume of 14 µL. The cycling steps were carried out in a Rotor Gene Q® (Qiagen, EUA) equipment with the following conditions: initial denaturing (10 min/95 °C) followed by 40 cycles 30 s at 95 °C for denaturing, 30 s at [55 °C (β-actin); 60 °C (IL-1β, TNF-α, MPO and IRAK4)] and 30 s at 72 °C for primer extension. Fluorescence levels at the end of cycles were used to estimate threshold cycle values (Ct) of each sample. Fold changes in transcript levels were determined using the mathematical model of relative expression ratio [23] of each gene versus control in comparison with β-actin levels as reference gene.

2.5. Myeloperoxidase, lysozyme, complement hemolytic activity and detection of anti-A. hydrophila antibodies

Serum myeloperoxidase activity assay was carried out as described previously [24] with minor modification. 10 µL of fish serum was diluted with 90 ul of Ca++, Mg++, and phenol red free Hank's solution in flat bottomed 96 well plates. Then, 35 µL of OPD (o-phenylenediamine dihidrochloride), in citrate (0, 2 M) and phosphate buffer (0, 1 M, pH 5, 3) were added. The peroxidase reaction was stopped after 5 min by adding 35 µL of hydrochloric acid (HCl, 3 M). Plates were read with a spectrophotometer at 492 nm (Synergy H1®, Biotek, EUA). Samples without serum were added as negative control.

Serological lysozyme activity was evaluated as described previously [24] with some modifications. Briefly, 20 µL of fish serum diluted 1:20 in PBS was mixed with 180 µL of Micrococcus lysodeikticus (O.D 0.5 at 450 nm) in flat bottom plates. The samples were incubated at 23 °C and then the optical density was measured at 450 nm (Synergy H1® (Biotek, EUA) after 1 and 5 minutes. Standard curve of lysozyme white chicken egg (Sigma, USA) was used as positive control. Then, O.D values of samples in 1 minute was compared with O.D values in 5 minutes of incubation time using the follow formula: Enzyme Units/mL = (ΔO.D450/5 – ΔO.D450/1).(Dilution factor) / (0.001).(0.1). The enzyme activity was expressed in units/ml−1, so that one unit of enzyme corresponds to 0.001 reductions in O.D. The natural hemolytic activity of serum complement was accessed as described in a previous work [25]

The production of anti-A.hydrophila antibodies was evaluated in serum samples collected from all fish prior to inoculation procedures and again at the day of challenge (21 days p.i). Antibodies were detected by indirect ELISA carried out as reported in a previous work [26] except that here we used sonicated bacteria (500 ng/well) to coat the plates.

2.6. Statistical analysis

The relative expression ratio of each gene was first transformed to its natural logarithm (In) prior to statistical analysis. The data normality distribution was validated by the Brown-Forsythe and Bartlett's test (gene expression data) and by the Shapiro-Wilk test (serum immune parameters). Then, the difference amongst groups was analyzed by one-way Anova followed by Dunnett's (gene expression data) or Tukey's post hoc test (GraphPad Prisma 7.0 USA). Differences were considered significant when p < 0.05.

3. Results

Vaccination or infection of silver catfish with A. hydrophila similarly modulated the expression of immune-related genes and the production of serum molecules with innate immune functions. Here, the intraperitoneal inoculation of a formalin-killed, bacterin-adjuvanted vaccine, or viable bacteria, induced a significant (p < 0.05) upregulation on the expression of TNF-α, IL1-β and Myeloperoxidase genes at 24 h p.i. but not at 72 h p.i. (Fig. 1A, B and C); in contrast, the expression of IRAK4 gene was not altered by inoculating the vaccine or by the infecting bacteria (Fig. 1D).

Fig. 1.

Fig. 1

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Comparative expression of selected immune-related genes on head kidney leukocytes obtained from fish inoculated with PBS, vaccinated or infected with A. hydrophila. The expression levels are expressed as fold changes compared to the expression of β-actin gene. Basal expression refers to the expression of each gene on fish samples collected 24 h prior to the inoculation. The mRNA level of the control group was used as reference value at each time point. Each data is represented by the mean ± SEM of 6 fish. Significant differences (p < 0.05) within groups at the same time point are represented by small letters and the capital letters indicate differences observed on different days.

The production of molecules central do innate immune defense against bacteria were also altered in vaccinated or infected fish. The natural hemolytic activity of the complement system was higher (p < 0.05) at 24 h and 72 h post vaccination or infection (Fig. 2A). The activity of total blood myeloperoxidase was higher (p < 0.05) but only at 24 h post vaccination or infection (Fig. 2B), and serum lysozyme activity was higher (p < 0.05) at 72 h but only in fish infected with A. hydrophila (Fig. 2C).

Fig. 2.

Fig. 2

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Analysis of innate immune parameters on silver catfish serum samples collected from fish inoculated with PBS, vaccinated of infected with A. hydrophila. Each data is represented by the mean ± SEM of serum samples collected from 6 fish. Significant differences (p < 0.05) within groups at the same time point are represented by small letters and the capital letters indicate differences observed on different days.

In the second experiment, the effectiveness of the vaccine was assessed by measuring the production of antibodies and the resistance of fish to intraperitoneal challenge with the bacteria. Serum samples collected prior to vaccination had no antibodies to A. hydrophila and the inoculation of the bacterin induced anti-Aeromonid antibodies that were detected by ELISA at 21 days post vaccination (Fig. 3A). In addition, the rate of survival after intraperitoneal challenge with A. hydrophila (Fig. 3B) was significantly higher (p < 0.05) in vaccinated fish (78.0%) compared to non-vaccinated (13.8%), indicating a central role of antibodies in protecting against infection.

Fig. 3.

Fig. 3

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Effectiveness of vaccine on antibody production and protection against challenge with A. hydrophila. A) production of IgM-like antibodies against A.hydrophila was analyzed by indirect ELISA in samples collected form all fish prior to and 21 days after vaccination or inoculation with PBS. B) Survival rate of silver catfish challenged with A. hydrophila (2.5 × 108 bacteria/fish). Fish were monitored twice a day up to the seventh day when the experiment was finished. Both groups consisted of 40 fish and the data is represented as daily survival rate ± SEM. Differences amongst groups are represented by different small letters (p <0.05).

4. Discussion

Here we found that early changes on selected innate immune parameters in silver catfish after vaccination with an A. hydrophila-adjuvanted vaccine or infected with the wild pathogen are similar. Vaccination is a powerful tool to protect fish against pathogens but the early immune events after vaccination with bacterial antigens or bacterial infection in fish have been poorly explored. Vaccine or pathogen-derived antigen are firstly recognized by innate immune cells which are central in orchestrating both innate and acquired immunity. Upon bacterial infection, a suit of immune-related genes and molecules are upregulated and, ideally, from an immunological perspective, the same profile of cells, cytokines and molecules should be activated after vaccination with bacterial antigens. Here, the expression of the classical proinflammatory cytokines TNF-α and IL1-β genes was upregulated by the vaccine or infecting bacteria. The way these cytokines contribute to fish resistance to bacterial infection is yet unclear, but their expression during infection highlight their central role in driving a protective immune response. A higher expression of IL-1β was reported in the head kidney, spleen and intestine of Asian Seabass (Lates calcarifer) vaccinated with outer membrane protein K of Vibrio harveyi [27]. In rainbow trout (Oncorhynchus mykiss), a higher expression of IL-1β was found in the spleen and gills after immunization with a formalin-killed Yersinia ruckeri vaccine [28] and also in head kidney and peritoneal cells after vaccination with a formalin-killed bacterin of Aeromonas salmonicida [16]. IL-1β and TNF-α are cytokines produced by several leukocytes in response to pathogen-associated molecular patterns (PAMPs) [29]. The expression of TNF-α triggers the expression of IL-1β and vice-versa and their pleiotropic, mostly overlapping effects on immune cells are central to triggering a proinflammatory immune response suited to face bacterial infection [29]. Early during infection, TNF-α drives phagocytes migration to the site of antigen injection and enhances their phagocytic and killing activity which is accompanied by increased production of reactive oxygen species [30]. In turn, IL-1β is a strong leukocyte chemoattractant and improves the expression of IL-17 family members, known mostly for their anti-bacterial effects [31]; in addition, IL-1β might improve antibody production when injected along with antigen [32]; therefore, its expression early during antigen processing should contribute to humoral response.

In addition, we found a higher expression of the myeloperoxidase (MPO) gene in both vaccinated and infected groups; the higher expression of this gene indicates that neutrophils and monocytes are actively processing engulfed microorganism and producing myeloperoxidase, which acts upon hydrogen peroxidase resulting in the formation of hypochlorous acid, a strong oxidant involved in killing invading pathogens [33]. In the other hand, myeloperoxidase produced early during infection might also modulate the inflammatory response. For instance, in mice MPO-deficient and intranasally inoculated with zymosan, a higher neutrophilic inflammatory reaction was observed on lungs suggesting that MPO is also involved in modulating the progression of tissue inflammation. In contrast, the expression of IRAK4 mRNA was not affected by vaccination or bacterial infection. In mammals, IRAK4 is a central kinase on Toll-like receptor (TLR) signaling pathway and as such is involved in the regulation of innate and adaptive immune response [34]. In silver catfish we already demonstrated that the expression of IRAK4 mRNA is reduced in monocytes exposed in vitro to atrazine [22], an immune suppressing agrichemical. Because of its role on TLR signaling pathway in mammals, we were expecting an upregulation of IRAK4; however, the role of IRAK4 in the innate immune response in fish is yet unclear and its role on bacterial infection deserves further investigation.

The complement system has a major role on innate resistance to several pathogens. Here we found that the natural hemolytic activity of complement system was significantly higher (p<0.05) in vaccinated and infected fish. The complement system is one of the most important innate immune defense mechanisms and in the absence of pathogen-specific antibodies, it might be activated by the natural deposition of C3b on pathogen surface [35]. During complement activation several anaphylatoxin are produced (e.g. C3a and C5a) and contribute to inflammation and antibody production. C5a, for instance, is a potent chemoattractant and drives leukocytes to the site of infection, improves respiratory burst and the production of IL-1β [35], and might act as adjuvant to antibody production [36]. Furthermore, in mammals, C5a induces the expression of TNF-α and IL-1β and, altogether with TNF-α, induces maturation of monocytes [37]. Thus, early during exposure to antigen, the complement glycoproteins and their cleavage byproduct are important to control progression to infection and act as modulators of innate and acquired immune response.

The expression of myeloperoxidase gene on immune cell coincided with a significantly higher (p<0.05) serum myeloperoxidase activity in vaccinated and infected fish, suggesting higher phagocytosis activity of neutrophils and monocytes that migrated to the site of antigen inoculation or infection. Unfortunately, we could not evaluate the expression of the lysozyme gene; however, the activity of serum lysozyme was significantly higher (p < 0.05) but only in infected fish at 72 hours p.i.. The effect of lysozyme is directed mainly towards Gram positive bacteria and here we were not expecting to detect higher levels of lysozyme in serum. However, bacteria replication, but not inactivated bacterial antigen, might have stimulated the production of lysozyme which was coincident with a reduction in the expression of TNF-α and IL-1β. In mouse peritoneal macrophages activated with lipopolysaccharides (LPS), the addition of lysozyme suppressed the expression of TNF-α [38] but not macrophage phagocytosis, indicating its key role in regulating inflammatory response and likely contributing to lessen tissue damage. Thus, complement derived proteins, myeloperoxidase and lysozyme might contribute to control the initial replication of bacteria and also to modulating the cytokine-induced inflammatory reaction.

The production of TNF-α and IL-1β in the initial phase of immune response along with complement C5a are essential to generate a robust innate and adaptative immunity. The central role of antibody in protecting fish against bacterial pathogens is well documented. Here, the production of anti-A. hydrophila IgM-like antibodies was evaluated by an indirect ELISA in samples collected prior to or 21 days after vaccination, or placebo (PBS) inoculation. The mean optical density (OD492nm) value obtained by testing all pre-vaccination serum samples was similar to that obtained in fish inoculated with PBS . And, as expected, the mean OD492nm obtained with serum samples from vaccinated fish was significantly (p < 0.05) higher indicating the presence of IgM-like antibodies specific to A. hydrophila. Antibodies contribute to protection by activating the complement system via classical pathway, opsonization and agglutination of bacteria and are correlated with resistance against the infecting bacteria. Indeed, here we also demonstrated that the survival rate (78.0%) of vaccinated fish was higher compared to PBS-inoculated control fish (13.8%) and the kinetics of mortality observed in the control group was similar to that observed in our previous challenging studies [24]. However, the rate of vaccine-induced protection was lower than expected; we hypothesize that the lack of full protection amongst the vaccinated group might be explained by the challenging dose. In our previous study we found that the LD50 of A. hydrophila inoculated intraperitonially to silver catfish was 1.63 × 108 bacteria/fish [24]. Here, vaccinated and placebo inoculated silver catfish were challenged intraperitoneally with 2.5 × 108 bacteria/fish (equivalent to 1.5 times de LD50) and this might also explain the lower survival rate (13.8%) amongst non-vaccinated fish.

The mechanisms underlying the success of bacterial immunogens in preventing bacterial disease is yet unknown for most vaccines. Here, the inoculation of vaccine induced the expression of immune-related genes and the production of soluble factors similar to those found in infected fish. In addition, vaccinated fish produced a robust humoral immune response to the vaccine antigen. Our data indicate that the vaccine formulae triggers the same profile of immune-related genes and the production of similar molecules which induce a protective immune response to the invading pathogen.

5. Conclusion

Altogether, our results demonstrate that A. hydrophila is pathogenic to silver catfish but fish mortality might be prevented by intraperitoneal inoculation of a formalin-killed bacterin-adjuvanted vaccine. Although limited, our analysis of the expression of immune-related genes and anti-bacterial soluble factors indicate that the resulting immune response to the vaccine antigen is similar to that one derived from infection and highlight the role of vaccine in preventing outbreaks of A. hydrophila..

Declaration of Competing Interest

None to declare

Acknowledgments

Author contribution

Conceptualization, LFS and LCK; Methodology, LFS, PAO, YK and VAR; Formal Analysis, LFS and LCK; Investigation, LFS, LCK; Resources, LCK;

Writing –Original Draft Preparation, LFS and LCK; Writing – Review & Editing, LFS, RF and LCK; Visualization, LFS; Supervision, LCK; Project Administration, LCK.

Acknowledgements

Lucas de Figueiredo Soveral was a Master Students supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) – Finance Code 001. Yasmin Kreutz and Vitoria Agnoletto Ribeiro are undergraduate student supported by the Fundação Universidade de Passo Fundo (Y.K.) and FAPERGS (V.A.R). Luiz Carlos Kreutz holds a fellowship from CNPq (PQ 307900/2016–9).

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