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Article

Assessment of Egg Yolk IgY Antibodies Against Live or Inactivated Aeromonas hydrophila for Polyvalent Passive Immunization in Goldfish (Carassius auratus)

by
Pan Cui
1,2,
Jing Chen
1,
Huihui Xiao
1,2,
Xixian Che
1,2,
Shujun Sun
1,2,
Zijian Ma
1,2,
Juan Lu
2,
Gaoxiao Xu
1,* and
Xiang Liu
1,*
1
Anhui Province Key Laboratory of Embryo Development and Reproductive Regulation, Fuyang Normal University, Fuyang 236041, China
2
Rural Revitalization Collaborative Technology Service Center of Anhui Province, Fuyang Normal University, Fuyang 236041, China
*
Authors to whom correspondence should be addressed.
Fishes 2025, 10(10), 491; https://doi.org/10.3390/fishes10100491
Submission received: 18 June 2025 / Revised: 19 September 2025 / Accepted: 30 September 2025 / Published: 1 October 2025
(This article belongs to the Section Welfare, Health and Disease)
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Versions Notes

Abstract

Egg yolk IgY antibody has significant application potential in aquaculture as a form of passive immunotherapy against various bacterial infections owing to its capacity for large-scale and cost-effective production. In this research, laying hens were immunized with live or inactivated Aeromonas hydrophila to produce IgY antibodies. Following this, experiments were carried out to assess the passive immune protection rates of the two types of IgY antibodies when used to immunize goldfish (Carassius auratus), which were then infected with A. hydrophila or Aeromonas veronii. ELISA experiments were conducted to demonstrate the interaction between the IgY antibodies and the bacteria. The kidneys of C. auratus were coated on a Luria–Bertani (LB) medium to evaluate bacterial content. The leukocyte phagocytosis was detected by a cell phagocytosis assay. The serum of C. auratus was used to assess the expression of antioxidant factors, and a qRT-PCR was conducted to evaluate the mRNA expression of inflammatory factors in visceral tissue. Furthermore, histopathology and immunofluorescence analysis were performed to evaluate the structural integrity, apoptosis, and DNA damage of visceral tissues. The results indicated that the live or inactivated A. hydrophila IgY antibodies exhibited passive immune protection rates against A. hydrophila and A. veronii and could recognize these two bacteria in vitro. Additionally, these two IgY improved the phagocytic ability of leukocytes, diminished renal bacterial concentration, and decreased the levels of antioxidant factors and mRNA expression of inflammatory factors. Meanwhile, the two IgY antibodies did not cause any pathology of the kidney, spleen, and intestine, and decreased the levels of DNA damage factor (γH2A.X) and cell apoptosis factor (p53) in renal tissue. Therefore, live and inactivated A. hydrophila IgY antibodies can resist bacterial infections, with live bacteria IgY providing greater protection than inactivated bacteria IgY. Further, A. hydrophila is an aquatic pathogen that causes minimal damage to laying hens, and the immunity of live A. hydrophila conforms to animal welfare. Altogether, live A. hydrophila IgY antibody can serve as a polyvalent passive immune vaccine candidate in aquaculture.
Keywords:
Aeromonas hydrophila; Aeromonas veronii; IgY antibody; multivalent vaccine; passive immunization; aquaculture
Key Contribution: This research proposed that egg yolk IgY antibody against live A. hydrophila taken from the eggs of immunized laying hens as a multivalent vaccine against bacterial infections in fish.

1. Introduction

With the increasing demand for high-quality protein, the consumption of aquatic products has risen rapidly [1]. However, the presence and spread of pathogenic bacteria pose a serious threat to the healthy development of the aquaculture industry. These pathogens can cause various diseases in farmed aquatic animals, leading to stunted growth, reduced reproductive capacity, and even mortality, resulting in significant economic losses for the global aquaculture industry annually [2]. The main pathogenic bacteria in goldfish (Carassius auratus) include Aeromonas hydrophila and A. veronii [3]. Aeromonas hydrophila and A. veronii belong to the family Aeromonadaceae and the genus Aeromonas, characterized as a Gram-negative bacterium that has a single polar flagellum and lacks spores or capsules. It is an opportunistic pathogen that causes the diseases of enteritis and septicemia in C. auratus [4].
The treatment of A. hydrophila or A. veronii infection primarily involves the use of antibiotics, among which sulfonamides (sulfadiazine and sulfamethoxydiazine), β-lactams (amoxicillin), and quinolones are the most mentioned and utilized [5]. However, the misuse of antibiotics causes leftover drug residues, environmental issues, and bacterial resistance [6]. Chinese herbal medicines, such as astragalus polysaccharides and saponins (from Astragalus), rhein (from Rhubarb), and andrographolide (from Andrographis), also have certain antibacterial and immune-enhancing functions, but their properties take effect more slowly, and they are challenging to apply in large-scale epidemics [7,8]. Additionally, certain probiotics, such as lactic acid bacteria, nitrifying bacteria, and photosynthetic bacteria, can have a preventive effect on A. hydrophila by secreting antibacterial substances and improving water quality [9,10]. However, the cost of high-quality beneficial bacterial preparation is relatively high, and these bacteria have poor environmental adaptability. Vaccines have attracted much attention due to their lack of residues and limited toxic side effects. Vaccine-based animal disease prevention and control constitute an essential path for green aquaculture [11]. Currently, the focus of vaccine development is on attenuated vaccines, inactivated vaccines, protein subunit vaccines, and mRNA vaccines [12]. Due to the adverse reactions and secondary infection risks of a few vaccines, as well as the high cost of vaccine development and application, most vaccines are in the laboratory research stage. Due to the wide variety of pathogens in aquaculture, multivalent vaccines capable of preventing multiple bacterial infections are considered ideal for the aquaculture industry and have garnered significant research attention [13,14].
Immunoglobulin Y (IgY) is an antibody obtained from the yolks of eggs from hens immunized with an immunogen, and it can be used for the prevention and treatment of diseases. IgY has significant application potential in the prevention and control of pathogens as a passive immunotherapy owing to its capacity for large-scale and cost-effective production, and no drug residue [15]. Additionally, IgY antibodies can improve the capacity of animals and humans to combat pathogenic infections and are expected to become one of the alternatives to antibiotics [16,17,18]. Studies have shown that IgY antibodies targeting PirA-like toxin not only neutralize the Vibrio parahaemolyticus strain but also alleviate acute hepatopancreatic necrosis disease in shrimp [19]. Moreover, IgY antibodies have also demonstrated potential in combating antibiotic-resistant pathogens. By preparing IgY antibodies against Vibrio parahaemolyticus and administering them orally or adding them to feed, researchers found that these antibodies could protect shrimp against V. parahaemolyticus infection and improve their survival rates [20]. Liang et al. [21] prepared an IgY antibody of Nervous Necrosis Virus (NNV) by immunization of laying hens and found that the IgY antibody reduced the mortality rate of mandarin fish (Siniperca chuatsi) induced by NNV. Further, an IgY antibody against the intracellular pathogen Piscirickettsia salmonis can enhance the resistance of fish to P. salmon infection [22]. Therefore, IgY antibody has potential application value in the prevention and control of fish pathogens. However, there is a lack of research reports on IgY against major aquatic pathogen infections (A. hydrophila and A. veronii).
In this study, live or inactivated A. hydrophila were used to immunize laying hens, enabling the large-scale and cost-effective preparation of IgY antibodies against live or inactivated A. hydrophila. The two IgY antibodies were used in the passive immunization of Carassius auratus, which were then infected with two major aquatic pathogens (A. hydrophila and A. veronii). The immunological functions of the two IgY antibodies were comprehensively compared using immune protection rates, bacterial counts in fish kidneys, antioxidant and inflammatory response, the histopathology of visceral organ tissues, and cell viability analysis (Supplementary Figure S1). This study provides technical support for the study of passive multivalent IgY antibody vaccines.

2. Materials and Methods

2.1. Strains and Animals

The strains Aeromonas hydrophila ATCC 7966, Aeromonas veronii ATCC 33809, and Staphylococcus aureus ATCC 15305 were stored in the Molecular Biology Laboratory of Fuyang Normal University, Fuyang, China.

2.2. Animals and Breeding

Laying hens (20-week-old) were obtained from Chongqing Tengxin Biochemistry Co., Ltd. (Xian, China). Carassius auratus (20 ± 0.5 g) were purchased from Fuyang Economic Fish Farming Co., Ltd. (Fuyang, China).
Laying hens were housed in a clean and ventilated environment with a daily photoperiod of 15 h light/9 h dark, a temperature of 20 °C, and a humidity of 60%. Each laying hen was kept in a cage measuring 80 cm × 60 cm × 70 cm (L × W × H) (Yujie Stainless Steel Cage Co., Ltd., Yangjiang, China). Chicken feed (23% protein, 5% fat, and 60% carbohydrate) was provided by Chongqing Tengxin Biotechnology Co., Ltd. (Chongqing, China) with feeding three times a day at intervals of 5 h. The drinking water for hens was ultrapure water. The henhouse was disinfected once a week by spraying with 2000 mg/L potassium peroxymonosulfate. The excreta of the laying hens were cleaned three times a day to ensure environmental hygiene.
Carassius auratus were immersed in a 0.5% potassium permanganate solution for 10 min to kill pathogens on the fish’s surface. Then, 15 fish from each group were placed into a fish tank measuring 70 cm × 60 cm × 50 cm (L × W × H) (Xiaomi Technology Co., Ltd., Beijing, China), and cultured using dechlorinated water. Fish food was provided by Beiyipin Pet Products Co., Ltd. (Zhongshan, China), and the fish were fed using a method of frequent small meals. A filter was used to purify the water, and an air pump was use to aerate the water. One quarter of the water in the fish tank was replaced with fresh water daily to control fish waste and metabolic products. Fish were kept in an environment with a daily light exposure time of 12 h, a temperature of 22 °C, and water of pH = 7.0.
All animal experiments were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” and approved by the Institutional Animal Care and Use Committee of Fuyang Normal University, China (No. 2024-03-008).

2.3. Preparation of IgY Antibodies

Aeromonas hydrophila was cultured overnight at 30 °C in Luria–Bertani (LB) medium, and collected using centrifugation at 9100× g for 2 min. Then, the bacteria were preserved in formalin solution and placed in a water bath with 80 °C for 90 min to produce inactivated bacteria. For sterilization verification, the sample of A. hydrophila was plated on Luria–Bertani (LB) medium and placed in a constant-temperature incubator overnight at 30 °C. There were no colonies on the LB medium, indicating successful inactivation of A. hydrophila. Furthermore, live A. hydrophila antigen was obtained directly from overnight cultured bacteria by centrifugation. Then, laying hens were immunized via breast muscle injection with 400 μL of live or inactivated A. hydrophila (2 × 107 CFU) or 400 μL normal saline to produce blank IgY as the nature control (NC) group. Bacterial immunization was conducted three times with a 14-day interval between each immunization, and eggs were preserved 10 days after the third immunization. The egg yolk was separated from the eggs and added to 20 mL of phosphate-buffered saline (PBS) solution while stirring. Further, the solution was added to powdered PEG6000 with a concentration of 3.5%. After centrifugation at 21,367× g for 20 min, the supernatant was added to 8.5% powdered PEG6000 while stirring. Then, 10 mL of PBS solution was used to dissolve the supernatant, and 12% powdered PEG6000 was added to the sample. After centrifugation, 2 mL of PBS solution was used to dissolve the supernatant, and the solution was transferred into a dialysis bag and placed in PBS solution for 36 h at 4 °C to obtain the IgY solution [23]. The concentration of IgY antibody was evaluated in accordance with the instructions provided in the bicinchoninic acid protein assay kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., Nanjing, China), and the purity of IgY was determined by SDS-PAGE gel electrophoresis [23].

2.4. Titer Analysis of IgY

The titer of live or inactivated A. hydrophila IgY was detected by an enzyme-linked immunosorbent assay (ELISA) as previously described by Liu et al. [23]. Briefly, A. hydrophila was cultured overnight at 30 °C in LB medium, and 200 μL bacterial solution was placed in an ELISA plate per well. After washing the plates three times with PBS solution, 5% bovine serum albumin solution was added to the 96 wells and incubated for 1 h at 37 °C in a constant-temperature incubator. Using PBS solution as the diluent, the gradient-diluted IgY antibody (1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400) was added to the wells and incubated at 37 °C in a constant-temperature incubator for 2 h, respectively. After washing three times with PBS, goat anti-IgY secondary antibody (1:1000) (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China) was added to the plate wells at 37 °C for 1 h. Then, chromogenic solution was put into the wells, and reaction termination solution was added to the wells to terminate the reaction. OD450 absorbance was read with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

2.5. Passive Immunization and Challenge Experiments with IgY Antibodies

To detect the infection dose of the bacteria, the lethal dose (LD50) value of A. hydrophila or A. veronii was determined in C. auratus using four groups with 10 fish per group for the two bacteria, respectively. A group of fish without infection was also maintained as a negative control. Briefly, each group was passively immunized with 20 μL (40 μg) of blank IgY antibody. Two hours after immunization with the IgY antibody, the concentrations of A. hydrophila (2 × 109, 4 × 109, 6 × 109, and 8 × 109 CFU) or A. veronii (5 × 108, 1 × 109, 1.5 × 109, and 2 × 109 CFU) were intraperitoneally administered to fish, and the mortality rate was observed for up to one week. The LD50 values of the bacteria were calculated for subsequent bacterial challenge tests of immune protection rate.
The C. auratus were divided into two groups: an A. hydrophila challenge group and an A. veronii challenge group. Each group was further divided into three subgroups, with 15 fish in each subgroup, namely, a nature control group (NC) (blank IgY group), a live bacteria IgY group, and an inactivated bacteria IgY group. The control group was intraperitoneally immunized with blank IgY antibody (immunization with normal saline for laying hens), while the experimental groups were intraperitoneally immunized with live bacteria IgY or inactivated bacteria IgY antibodies. Moreover, each group of fish was administered with 20 μL (40 μg) of IgY antibodies. Two hours after injection of IgY antibodies, the fish were infected with A. hydrophila (3.8 × 109 CFU) or A. veronii (1.5 × 109 CFU) according to the LD50 detection of the infecting dose. Then, the C. auratus were observed continuously for 14 days to evaluate their mortality. The protection rate (RPS) was obtained according to the following formula: RPS = (1 − [% mortality rate of IgY group/% mortality rate of NC group]) × 100% [23].

2.6. The Interaction Between IgY Antibodies and Bacteria as Well as Fish Serum and Bacteria

Aeromonas hydrophila or A. veronii were cultured in an LB medium at 30 °C. When the bacterial concentration (OD600) reached 1.0, the bacteria were collected by centrifugation and 200 μL of bacterial solution was transferred into an ELISA plate per well. Then, the bacterial solution was incubated overnight at 4 °C. After washing the plates three times with PBS solution, 5% bovine serum albumin solution was added to the 96 wells, and the plates were sealed at 37 °C for 1 h. Using PBS solution as the diluent, gradient-diluted IgY antibody (1: 200, 1: 400, 1: 800, 1: 1600, 1: 3200, and 1: 6400) or gradient-diluted fish serum (immunized with IgY and challenged to bacteria) (1: 200, 1: 400, 1: 800, 1: 1600, 1: 3200, and 1: 6400) were added to the wells at 37 °C for 2 h. After washing with PBS solution, the second antibody, at a 1: 1000 dilution, was added to the ELISA plate wells at 37 °C for 1 h. After chromogenic solution was put into the wells at 37 °C for 10 min, the reaction termination solution was added to the wells to terminate the reaction, after which we immediately read the results on a microplate reader at a wavelength of OD450 [24].

2.7. Kidney Bacterial Content

Two days after the passive immunization of C. auratus with live bacteria IgY and inactivated bacteria IgY, followed by a challenge with A. hydrophila and A. veronii, the kidney tissues of the C. auratus were collected after euthanasia with 200 mg/L ethyl 3-aminobenzoate methanesulfonate (MS–222) (Sangon Biotechnology Co., Ltd., Shanghai, China). Under sterile conditions, the kidney tissues were thoroughly ground to prepare homogenate using 400 μL of physiological saline solution. Then, 200 μL of the homogenate tissue solution was spread on LB solid medium and cultured in a constant-temperature incubator for 37 °C overnight. After photographing the colonies, bacterial counts were detected with Image J 1.47 software (National Institutes of Health, Bethesda, Maryland, USA) [23].

2.8. Leukocyte Phagocytosis Analysis

After passive immunization and bacterial challenge, blood from each C. auratus specimen’s caudal vessels was collected using anticoagulant (ethylenediaminetetraacetic acid, EDTA) centrifuge tubes under anesthesia with 40 mg/L MS–222. Staphylococcus aureus (1 × 106 CFU) was inactivated in advance with formalin solution and then mixed equally with fish blood (200 μL) in the centrifuge tubes. The mixture was incubated in a water bath at 25 °C for 60 min. Then, the mixture solution was placed in a glass slide to prepare blood smears. After methanol fixation, the smears were stained using a Giemsa staining kit (Sangon Biotech Co., Ltd., Shanghai, China) and observed under a microscope for counting. The phagocytic percentage (PP %) and phagocytic index (PI %) were calculated. The calculation methods employed were as follows: PP % is the count of cells involved in phagocytosis out of 100 phagocytic cells, and PI % is the count of bacteria within phagocytic cells/the count cells involved in phagocytosis × 100 [24].

2.9. Antioxidant Factor Analysis

Passive immunization with IgY antibodies was carried out, and serum was obtained from the caudal vessels of each C. auratus specimen after intraperitoneally challenging with bacteria under sedation [23]. The antioxidant indices were measured according to the instructions of the catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) kits (Sangon Biotechnology Co., Ltd., Shanghai, China).

2.10. Analysis of mRNA Expression of Inflammatory Factors

A real-time quantitative PCR method (qRT-PCR) was performed to detect the mRNA expression of inflammatory factors as described previously [24]. Briefly, on day 2, after passively intraperitoneally immunizing the C. auratus with IgY antibodies (40 μg) and challenging them with A. hydrophila (4 × 109 CFU) or A. veronii (1.5 × 109 CFU), their kidneys and spleens were harvested after euthanasia with 200 mg/L MS–222. The visceral tissues were thoroughly ground with liquid nitrogen, and RNA was extracted according to the instructions of the RNA extraction kits (TAKARA, Beijing, China). Further, the mRNA was reverse-transcribed to cDNA according to the method provided by the reverse transcription kit (Sangon Biotech Co., Ltd., Shanghai, China). A qRT-PCR was performed using a SYBR® kit (TAKARA, Beijing, China) with a qRT-PCR System instrument (ABI Applied Biosystems, Waltham, Massachusetts, USA) and synthesized primers of inflammatory factors (Supplementary Table S1). The inflammatory factors include interleukin-6 (IL-6), IL-8, IL-1β, and tumor necrosis factor-α (TNF-α). The ΔCt (cycle threshold change) was calculated with the Ct values of the genes, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. Then, the ΔΔCt was obtained by calculating the ΔCt values of the IgY group and NC group. Finally, mRNA expression was calculated with the 2−(ΔΔCt) formula [23].

2.11. Histopathological Analysis of Visceral Tissue Organs

After the fish specimens were intraperitoneally infected with bacteria, the kidneys, spleens, and intestines were collected after euthanasia with 200 mg/L MS–222 and immersed in Davidson’s fixative for 24 h, followed by transfer to a formalin solution for 24 h to fixate the tissues. Then, the tissue samples were dehydrated in a graded series of ethanol. Embedding was performed in paraffin at 65 °C, and sections with a 4 μm thickness were cut using a paraffin microtome. The sections were placed on glass slides and dried overnight at 37 °C in a constant-temperature incubator. Hematoxylin and eosin (H&E) were used to stain the sections. After being cleared with xylene, the sections were placed in neutral resin and observed with a histopathological microscope (Leica, Wetzlar, Germany) for photography. The histopathological parameters mainly include the morphology of glomeruli and renal tubules, splenic cells, and the intestinal mucosal lamina propria [24].

2.12. Immunofluorescence Analysis of C. auratus Kidney Tissue

The prepared kidney tissue sections were dewaxed in xylene and hydrated in ethanol with gradient reduction concentration. After antigen retrieval, the tissue periphery was circled with an immunohistochemical pen in the section and 5% bovine serum albumin solution was added in the circle, blocking the tissue at room temperature for 1.5 h. Rabbit monoclonal antibodies against p53 and γH2A.X (1: 200 dilution) (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China) were added to the tissue and incubated at 37 °C for 2 h. After a wash with PBST solution (phosphate-buffer saline with 0.05% Tween-20), a donkey anti-rabbit (1: 250 dilution) secondary antibody solution was added and incubated at 37 °C for 1 h. After a wash with PBST solution, the nuclei were stained with DAPI at room temperature for 10 min and subsequently mounted and imaged under a fluorescence microscope (Leica, Wetzlar, Germany) [23].

2.13. Statistical Analysis

All the experiments were repeated at least three times, and the research data were performed as mean ± SD. Significant differences between groups were evaluated with one-way analysis of variance and Tukey’s multiple comparison test by Statistical Package for the Social Sciences 19.0 (SPSS19.0) software. Statistical significance was considered when the value of p < 0.05 [23].

3. Results

3.1. The Passive Protection and Passive Cross-Protection Rates of IgY in C. auratus

A total of 2 mL of high-purity IgY antibodies (2 μg/μL) against live or inactivated A. hydrophila were obtained (Supplementary Figure S2), and a total of 4000 μg of IgY antibodies was obtained by the PEG6000 purification method. According to the ELISA method, it was found that the titer of live or inactivated A. hydrophila IgY reached 1: 1600 (Supplementary Figure S3).
To detect the differences in the protective rates of the live and inactivated bacterial IgY against aquaculture bacterial infections in the C. auratus specimens, the fish were passively immunized with IgY antibodies and then subjected to a challenge with bacteria. Subsequently, the fish displayed slow swimming, abdominal swelling, epidermal hemorrhage, and death, with the mortality rate stabilizing after 4 days (Figure 1). Additionally, the passive immunization protection rates of the live or inactivated bacterial IgY antibodies against A. hydrophila were 84.62% (p < 0.001) and 38.47% (p < 0.05), respectively, while the passive cross-protection rates against A. veronii were 58.34% (p < 0.05) and 50.00% (p < 0.05), respectively (Supplementary Table S2). It is evident that both live and inactivated bacteria IgY antibodies have immunoprotective effects, and the protective efficacy of live bacteria IgY antibody is superior to that of inactivated bacteria IgY antibody.

3.2. Bacterial Counts in Fish Kidneys

To quantify the bacterial load in fish, kidney samples were collected and streaked in LB medium following immunization with antibodies and challenge with bacteria. Compared to the control group, the bacterial counts in the two IgY antibody groups decreased (p < 0.05), with the live bacteria IgY group showing fewer bacteria than the inactivated bacteria IgY group (Supplementary Figure S4). The results indicate that both live and inactivated bacteria IgY antibodies can reduce the bacterial load in the kidneys, with live bacteria IgY being more effective at immunologically clearing bacteria from the kidneys.

3.3. Phagocytic Activity of C. auratus Blood

To assess the phagocytic activity of leukocyte, C. auratus were intraperitoneally immunized with IgY and infected with pathogenic bacteria, and C. auratus blood was collected for phagocytosis assays. The phagocytic index and phagocytic percentage of leukocytes increased in the fish (p < 0.05) after their immunization with IgY antibodies and challenge with A. hydrophila or A. veronii (Table 1). This indicated that live or inactivated IgY antibodies stimulated the phagocytic activity of leukocytes, with no significant difference observed between the two types of IgY antibodies.

3.4. Antioxidant Factors in C. auratus Serum

To assess the content of antioxidant factors in the serum of the C. auratus specimens, serum was collected for antioxidant factor detection after an immune challenge. Compared to the nature control group (blank IgY group), the expression levels of most antioxidant factors (SOD, CAT, and GSH-Px) in the immune IgY antibody groups were lower (p < 0.05), and the most members of the live bacterial IgY group was lower than that of the inactivated bacterial IgY group (Figure 2). These results indicate that both types of IgY antibodies exhibit antioxidant activity against bacterial infection, with the live bacteria IgY antibody showing slightly greater antioxidant effects than the inactivated bacteria IgY antibody.

3.5. Detection of Inflammatory Gene Expression in C. auratus

The C. auratus specimens were subjected to IgY passive immunization, followed by a challenge with A. hydrophila and A. veronii. The mRNA expression of inflammation factors (IL-6, IL-8, TNF-α, and IL-1β) in the kidney and spleen was detected. Compared with the NC group, the mRNA expression of IL-6, IL-8, TNF-α, and IL-1β in the kidney and spleen was decreased (p < 0.05) (Figure 3). The results showed that both live and inactivated bacterial IgY antibodies reduced the inflammatory response caused by A. hydrophila and A. veronii in fish, with no significant differences between these two IgY antibodies.

3.6. In Vitro Interactions of IgY Antibodies or Fish Serum with Bacteria

To investigate the interaction between IgY antibodies and aquaculture bacteria in vitro, ELISA experiments were conducted using live or inactivated bacteria IgY antibodies. The results showed that the binding capacity of the two IgY to A. hydrophila or A. veronii decreased as the dilution of IgY antibodies increased, and recognition was assessed at a dilution of 1: 6400 for A. hydrophila or A. veronii (Figure 4A,C).
Additionally, ELISA experiments were performed using C. auratus serum (immunized with IgY and infected with bacteria), and the results indicated that the fish serum could combine with A. hydrophila or A. veronii, with the absorbance decreasing as the dilution of C. auratus serum increased; recognition was displayed at a dilution of 1: 6400 for A. hydrophila or A. veronii (Figure 4B,D).
Overall, the two IgY antibodies could interact with A. hydrophila or A. veronii in vitro, and the binding interaction of live bacteria IgY antibody was greater than that of inactivated bacteria IgY antibody.

3.7. Histopathological Observation of C. auratus

To assess the protective effect of live or inactivated A. hydrophila IgY on the visceral structures, C. auratus were intraperitoneally immunized with live or inactivated IgY or blank IgY (control) and infected with the pathogenic bacteria. In addition, fish that had not undergone any experimental treatment were used as the baseline for the experiment and served as the negative control. The kidneys, spleens, and intestines of the fish were used for histopathological examination. The results indicated that, in the NC group (blank IgY), the structure of kidney tissues was loose and necrotic, with degeneration and atrophy of renal tubules and glomeruli; the spleen tissue was necrotic, with reduced cell density and cell apoptosis; in addition, the lamina propria of the intestinal mucosa had atrophied and exhibited a necrotic structure and apoptosis. In the live or inactivated bacteria IgY immunization groups and negative control groups, the kidney, spleen, and intestine exhibited intact tissue architecture with no abnormal cellular morphology observed (Figure 5).

3.8. Immunofluorescence Analysis of Kidneys

To assess apoptosis in the kidney cells of the C. auratus specimens, immunofluorescence analysis was employed on the kidney tissues. Red fluorescence represents the expression of p53 and γH2A.X proteins, and blue fluorescence represents DAPI-stained nuclei. Compared to the NC group (blank IgY), the expression of p53 and γH2A.X was diminished in the live and inactivated bacteria IgY groups (p < 0.05) (Figure 6). The results showed that immunization with live or inactivated bacteria IgY could reduce apoptosis and DNA damage in the kidney cells, with no significant differences between these two IgY antibodies.

4. Discussion

The survival rate obtained from animal challenge tests is the most direct indicator of the protective effect of vaccines in animals [25]. Zhang et al. [26] found that adding a certain quantity of antimicrobial peptides to the feed of red claw crayfish enhanced their resistance to A. hydrophila and increased their survival rates. Paola et al. [27] confirmed that Hericium erinaceus extract improved the survival and embryonic development rates of zebrafish embryos exposed to aflatoxin B1. In this study, it was found that both live or inactivated A. hydrophila IgY provided high immune protection efficiency against different bacterial species. Interestingly, we found that live and inactivated A. hydrophila IgY antibody had passive cross-protection abilities against A. veronii infection in fish. Nevertheless, the underlying mechanisms remain underexplored. Liu et al. [24] found that the IgY of outer membrane proteins (PF1380 and ExbB) of P. fluorescens hold a passive cross-protection activity against A. hydrophila in fish and indicated that PF1380 and ExbB may possess homologous antigens or epitopes corresponding to the proteins of A. hydrophila. In addition, researchers indicated that live and inactivated Vibrio fluvialis hold cross-protection activity against A. hydrophila in goldfish, and 18 outer membrane proteins (OMPs) of V. fluvialis emerged as antibodies in the two IgY antibodies [23]. It may be that the 18 OMPs had conserved epitopes between V. fluvialis and A. hydrophila to perform cross-protection activity against A. hydrophila in goldfish [28]. Thus, there is a possible existence of cross-antigens or conserved epitopes between A. hydrophila and A. veronii.
Non-specific immune factors exhibit the body’s antioxidant status. The evaluation metrics in this regard include the phagocytic activity of the leucocytes, analysis of immune-related factors, bacterial count of visceral tissues, and the immune recognition between antibody and pathogen in vitro [29]. Mohammadi et al. [30] investigated the synergistic effects of pistachio shell polysaccharides and Lactobacillus plantarum on the immune activity of Nile tilapia. The results indicated that the combined supplementation enhanced complement alternative pathway hemolytic activity, lysozyme (LYZ), and alkaline phosphatase (AKP) activity in tilapia. Liu et al. [31] obtained specific IgY of outer membrane proteins of A. hydrophila. Through passive immunization of C. auratus and bacterial challenge experiments, it was found that the kidney bacterial count in the IgY immunization group was lower than that of the control group, and the phagocytosis of white blood cells was enhanced. Thus, the IgY immunization may activate phagocyte-mediated pathways in fish. In this research, after intraperitoneal immunization of live and inactivated A. hydrophila IgY antibodies and bacterial challenge in C. auratus, the phagocytic activity of leukocytes increased, and C. auratus serum could interact with the bacteria in vitro. Additionally, the bacterial count in their kidneys decreased. These results suggest that live or inactivated A. hydrophila IgY antibodies enhance the non-specific immune activity of fish and improve defensive ability to resist exogenous pathogen invasion in fish.
Antioxidant factors and inflammatory factors can serve as important biomarkers for assessing the extent of body damage [23]. Among them, the detection indicators for oxidative factor levels mainly include SOD, CAT, GSH-Px, and MDA, while the detection indicators for inflammatory response levels encompass IL-6, IL-8, IL-10, IL-1β, and TNF-a. Li et al. [32] investigated the relieving effects of sodium butyrate (SB) on the inflammatory response and oxidative stress induced by glycine in carp, finding that supplementation with a certain amount of SB reduced the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum. Simultaneously, it downregulated the mRNA expression of IL-1β, TNF-α, and nuclear transcription factor-κB (NF-κB) in the hepatopancreas. Furthermore, Zhou et al. prepared a high-purity Fab fragment of IgY to assess its anti-inflammatory activity in the inflammatory macrophage system. The Fab fragment reduced the translocation of NF-κB and the phosphorylation of mitogen-activated protein kinase proteins and inhibited the mRNA expression levels of toll-like receptor-4 and integrin αvβ3. Thus, the Fab fragment of IgY shows anti-inflammatory activity by blocking the NF-κB and MAPKs pathways in macrophages [33]. In this research, after immunization of C. auratus with live or inactivated A. hydrophila IgY antibodies and infection by bacteria, the expression levels of antioxidant and inflammatory factors decreased. These results indicate that these two IgY antibodies have antioxidant and anti-inflammatory effects.
Histological observation can reveal pathological changes in tissues and cells, and the examination of histological sections is the most intuitive method for observing the damage pathogens inflict on an organism [34]. Adewoyin et al. [35] injected outer-membrane vesicles (OMVs) from Porphyromonas gingivalis into the abdominal cavity of adult zebrafish and observed lesions in the brain tissue of the zebrafish, such as coagulation, edema, and spongy hyperplasia, between 6 and 24 h later. Maceda-Veiga et al. [36] utilized the histopathological method to investigate the metal content and visceral histopathology of wild native fish exposed to sewage in a Mediterranean river. Dong et al. [37] injected turtles with varying concentrations of cadmium and found through histopathological examination that the renal tissues of the cadmium-treated turtles exhibited varying degrees of lesions depending on the dose concentration. Destaw et al. [38] evaluated the prevalence of Ligula intestinalis in Lake Tana, Ethiopia through histopathological observations of fish gonads, livers, and spleens. In this study, after immunization of C. auratus with live or inactivated A. hydrophila IgY antibodies and challenge with pathogenic bacteria, internal organ structure in fish was intact and undamaged. The results indicate that the two IgY antibodies have a protective effect in regard to maintaining the integrity of internal organ morphology.
The extent of DNA damage and apoptosis is related to the intensity of pathogen invasion in the host. Immunofluorescence staining allows for the direct observation of DNA damage and apoptosis [39]. Xiao et al. [40] immunized crucian carp with the VF14355 protein vaccine of Vibrio fluvialis and challenged them with the pathogen. Using immunofluorescence analysis, they found that the contents of p53 and γH2A.X in the kidneys of fish were reduced (p < 0.05), indicating that the VF14355 vaccine can reduce apoptosis and DNA damage of fish visceral tissue cells induced by bacterial infection. Additionally, many studies have analyzed changes in γH2A.X expression in zebrafish embryos induced by PM2.5 by immunofluorescence analysis, investigating the impact of PM2.5 on DNA damage in fish [41,42]. In this study, after immunization of C. auratus with live or inactivated A. hydrophila IgY antibodies and challenge with pathogenic bacteria, the immunofluorescence intensities of p53 and γH2A.X were reduced, which indicated that the expression of p53 and γH2A.X decreased. The results indicate that the two IgY antibodies can reduce apoptosis and DNA damage induced by aquaculture bacteria in fish.
Overall, the immunoprotective activity of live A. hydrophila IgY antibody is higher than that of inactivated bacteria IgY antibody. Liu et al. [43] found that live Vibrio parahemolyticus immunization could generate more antibodies of outer membrane proteins of V. parahemolyticus compared to immunization by inactivated bacteria, and the immunoprotective ability of live V. parahemolyticus immunization is higher than that of inactivated bacteria in mice. It may be that live bacteria have a wider antigenic spectrum and a spatial conformation closer to natural proteins compared to inactivated bacteria. Additionally, A. hydrophila is a pathogenic bacterium in aquaculture and exhibits weak virulence in laying hens [44]. By controlling the immunization dosage of live bacteria in laying hens, the harm to them can be minimized. Certainly, IgY antibodies obtained from live bacterial immunization, along with the potential presence of pathogenic bacteria, may pose a latent infection risk to fish. Further, from the perspective of the efficiency of immune protection, that of the live A. hydrophila IgY antibody is higher than that of inactivated bacteria IgY antibody.
IgY antibody is a type of passive immunization vaccine. Passive immunization vaccines provide rapid but relatively short-lived immune protection by directly introducing antibodies (such as immunoglobulins, antitoxins, etc.) and are suitable for emergency infection prevention, especially for outbreak pathogens in aquaculture. Most of the immune recognition between antibody and bacteria is considered to be generated shortly after animals are immunized with antibodies. Additionally, passive immunization antibodies in animals are metabolized and cleared by the body, typically providing protection for only 2 weeks to several months. Therefore, it is crucial to understand how long antibody molecules remain in the body after administration and how long their effects persist. In previous experiments, it was found that the optimal time for bacteria to infect fish was 2 or 2.5 h after IgY antibody was administrated to fish [23,24]. In this research, it was found that 2 h was the optimal time in a preliminary experiment after the IgY antibody was intraperitoneally administered to fish, as fish may need some time to adapt to the antibody for the antibody to exert immune abilities. Moreover, in this research, the assessment of the potential immunologic activity of the IgY passive immunization was conducted by measuring mortality of fish in the experiment over 14 days. Although the fish survival rate test experiment required observation for 14 days according to internationally recognized testing standards [31,40], passive immunization vaccines result in rapid but relatively short-lived immune protection. Additionally, the mechanism of passive immunization vaccines differs from that of active immunization; it does not activate the body’s own immune system but instead neutralizes pathogens through exogenous antibodies. They do not form immune memory cells and cannot offer long-term protection. However, it is necessary to conduct evaluation of long-term (over 14 days) immunoprotective ability in future work for the development of vaccines.
In aquaculture, the vaccine can be encapsulated in chitosan or sodium alginate to prepare encapsulation particles or be incorporated into feed to achieve oral immunization in fish, which is more feasible and cost-effective, making it a preferred method for large-scale aquaculture. Further, it does not require professional equipment or advanced technical personnel [40,45]. Recent work highlights the interplay between mucosal and systemic immunity in teleost fish, which is relevant for future oral IgY delivery [46]. As highlighted by Rathor and Swain, ongoing advances in fish vaccination emphasize innovative delivery methods and adjuvants that complement passive immunization approaches like IgY [47]. However, in this study, the use of intraperitoneal injection for IgY delivery, although effective in controlled laboratory settings, is impractical and costly in large-scale aquaculture. Therefore, it will be necessary to conduct subsequent research on oral immunization approaches for IgY.
In summary, this study evaluated the polyvalent passive immune activity of IgY antibodies of live or inactivated A. hydrophila in goldfish, laying the foundation for research on IgY passive immunization vaccines in aquaculture. However, the application of IgY in aquaculture still requires further study. Proteases in the fish digestive tract can degrade IgY antibody vaccines, necessitating the development of oral immunization vaccines using microencapsulation protection technology [48,49]. Additionally, the separation and purification process of IgY from egg yolk is complex, with a recovery rate of approximately 65%, requiring optimization of IgY purification techniques to reduce costs [50]. Therefore, broader aquaculture application of IgY needs to focus on the stability of IgY antibodies, achieving low-cost mass production and long-term efficacy assessments, as well as safety.

5. Conclusions

This research prepared IgY of live and inactivated A. hydrophila. These two IgY exhibited good protective rates in resisting A. hydrophila and A. veronii challenges in C. auratus, and there was mutual immune recognition between the two IgY antibodies and these bacteria. Additionally, the phagocytic activity of C. auratus leukocytes was improved. Furthermore, kidney bacterial content was reduced; levels of inflammatory or antioxidant factors diminished; the morphology of the kidney, spleen, and intestine remained intact; and apoptosis and DNA damage in kidney cells were diminished. Overall, the immunoprotective activity of live A. hydrophila IgY antibody is higher than that of inactivated bacteria IgY antibody. Furthermore, live bacteria IgY antibody can be used as a multivalent passive vaccine against various bacterial infections in aquaculture.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fishes10100491/s1, Figure S1: Experimental design; Figure S2: The purity of IgY antibodies was detected by SDS-PAGE gel electrophoresis. 1 and 2 represent live bacteria IgY antibodies. 3 and 4 represent inactivated bacteria IgY antibodies. HC and LC represent the heavy and light chain of IgY antibody, respectively; Figure S3: The titer of IgY by ELISA; Figure S4: Bacterial counts in C. auratus kidneys on LB medium. (A) Challenged with A. hydrophila, (B) challenged with A. veronii. (d) Control (blank IgY), (e) Live bacteria IgY, (f) Inactivated bacteria IgY and (g) negative control (the fish without infection by bacteria). Labels a-c indicate statistically different groups (p < 0.05); Table S1: Information on primers used for qRT-PCR; Table S2: Passive protection and passive cross-protection rates of IgY antibodies in C. auratus.

Author Contributions

Methodology, P.C., J.C., and H.X.; investigation, P.C., J.C., and H.X.; validation, P.C. and J.C.; software, X.C., S.S., and Z.M.; formal analysis, X.C., S.S., and Z.M.; data curation, J.L.; conceptualization, G.X. and X.L.; funding acquisition, G.X. and X.L.; investigation, G.X. and X.L.; writing—original draft preparation, X.L.; writing—review and editing, X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the University Collaborative Innovation Project of Anhui Province (GXXT-2023-077), the Key Projects of Scientific Research Plan of Colleges and Universities of Anhui Province (2022AH051330), the Biological and Medical Sciences of Applied Summit Nurturing Disciplines in Anhui Province (Anhui Education Secretary Department [2023]13), the Outstanding Innovative Research Team for Molecular Enzymology and Detection in Anhui Provincial Universities (2022AH010012), and the University Synergy Innovation Program of Anhui Province (GXXT-2022-067).

Institutional Review Board Statement

All animal experiments were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” and approved by the Institutional Animal Care and Use Committee of Fuyang Normal University, China (No. 2024-03-008, date: 4 February 2024).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Garlock, T.M.; Asche, F.; Anderson, J.L.; Eggert, H.; Anderson, T.M.; Che, B.; Chávez, C.A.; Chu, J.; Chukwuone, N.; Dey, M.M.; et al. Environmental, economic, and social sustainability in aquaculture: The aquaculture performance indicators. Nat. Commun. 2024, 15, 5274. [Google Scholar] [CrossRef]
  2. Sasikumar, R.; Saranya, S.; Lourdu Lincy, L.; Thamanna, L.; Chellapandi, P. Genomic insights into fish pathogenic bacteria: A systems biology perspective for sustainable aquaculture. Fish Shellfish Immunol. 2024, 154, 109978. [Google Scholar] [CrossRef]
  3. Zhang, Z.; Li, J.; Wang, G.; Ling, F. The oral protective efficacy of magnolol against Aeromonas hydrophila and A. veronii infection via enhancing anti-inflammatory ability in goldfish (Carassius auratus). J. Fish Dis. 2023, 46, 1413–1423. [Google Scholar] [CrossRef] [PubMed]
  4. Liang, R.; Cui, Z.; Abbas Raza, S.H.; Li, T.; Zhang, Z.; Huang, Q.; Bai, H.; Cheng, Y.; Du, B.; Li, J.; et al. Protective immune-response of Aeromonas hydrophila phage lysate in crucian carp against direct virulent challenge with A. hydrophila-TPS. Fish Shellfish Immunol. 2024, 155, 110011. [Google Scholar] [CrossRef] [PubMed]
  5. Thaotumpitak, V.; Sripradite, J.; Atwill, E.R.; Jeamsripong, S. Emergence of colistin resistance and characterization of antimicrobial resistance and virulence factors of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from hybrid red tilapia cage culture. Peer J. 2023, 11, 14896. [Google Scholar] [CrossRef] [PubMed]
  6. Liang, Y.; Zhao, H.; Li, Y.; Gao, F.; Qiu, J.; Liu, Z.; Li, Q. Joint effects about antibiotics combined using with antibiotics or phytochemicals on Aeromonas hydrophila. Mar. Environ. Res. 2024, 199, 106594. [Google Scholar] [CrossRef]
  7. Wang, T.; Tian, X.L.; Xu, X.B.; Li, H.; Tian, Y.; Ma, Y.H.; Li, X.F.; Li, N.; Zhang, T.T.; Sheng, Y.D.; et al. Dietary supplementation of probiotics fermented Chinese herbal medicine Sanguisorba officinalis cultures enhanced immune response and disease resistance of crucian carp (Carassius auratus) against Aeromonas hydrophila. Fish Shellfish Immunol. 2022, 131, 682–696. [Google Scholar] [CrossRef]
  8. Hou, T.; Liu, H.; Li, C. Traditional Chinese herb formulas in diet enhance the non-specific immune responses of yellow catfish (Pelteobagrus fulvidraco) and resistance against Aeromonas hydrophila. Fish Shellfish Immunol. 2022, 131, 631–636. [Google Scholar] [CrossRef]
  9. Torres-Maravilla, E.; Parra, M.; Maisey, K.; Vargas, R.A.; Cabezas-Cruz, A.; Gonzalez, A.; Tello, M.; Bermúdez-Humarán, L.G. Importance of probiotics in fish aquaculture: Towards the identification and design of novel probiotics. Microorganisms 2024, 12, 626. [Google Scholar] [CrossRef]
  10. Wang, Y.; Ren, Z.; Fu, L.; Su, X. Two highly adhesive lactic acid bacteria strains are protective in zebrafish infected with Aeromonas hydrophila by evocation of gut mucosal immunity. J. Appl. Microbiol. 2016, 120, 441–451. [Google Scholar] [CrossRef]
  11. Du, Y.; Hu, X.; Miao, L.; Chen, J. Current status and development prospects of aquatic vaccines. Front. Immunol. 2022, 13, 1040336. [Google Scholar] [CrossRef]
  12. Irshath, A.A.; Rajan, A.P.; Vimal, S.; Prabhakaran, V.S.; Ganesan, R. Bacterial pathogenesis in various fish diseases: Recent advances and specific challenges in vaccine development. Vaccines 2023, 11, 470. [Google Scholar] [CrossRef]
  13. Uchuwittayakul, A.; Thompson, K.D.; Thangsunan, P.; Phaksopa, J.; Buncharoen, W.; Saenphet, K.; Kumwan, B.; Meachasompop, P.; Saenphet, S.; Wiratama, N.; et al. Evaluation of a hydrogel platform for encapsulated multivalent Vibrio antigen delivery to enhance immune responses and disease protection against vibriosis in Asian seabass (Lates calcarifer). Fish Shellfish Immunol. 2025, 160, 110230. [Google Scholar] [CrossRef] [PubMed]
  14. Ahangarzadeh, M.; Houshmand, H.; Torfi Mozanzadeh, M.; Kakoolaki, S.; Nazemroaya, S.; Sepahdari, A.; Peyghan, R.; Ajdari, A.; Sadr, A.S. Effect of killed autogenous polyvalent vaccines against Vibrio harveyi, V. alginolyticus and Streptococcus iniae on survival and immunogenicity of Asian seabass (Latescal carifer). Fish Shellfish Immunol. 2023, 143, 109226. [Google Scholar] [CrossRef] [PubMed]
  15. Winkelbach, A.; Günzel, D.; Schulz, C.; Wuertz, S. Differences in IgY gut absorption in gastric rainbow trout (Oncorhynchus mykiss) and agastric common carp (Cyprinus carpio) assessed in vivo and in vitro. Comp. Biochem. Physiol. Part C Toxicol. Pharmacol. 2015, 167, 58–64. [Google Scholar] [CrossRef] [PubMed]
  16. El-Kafrawy, S.A.; Abbas, A.T.; Oelkrug, C.; Tahoon, M.; Ezzat, S.; Zumla, A.; Azhar, E.I. IgY antibodies: The promising potential to overcome antibiotic resistance. Front. Immunol. 2023, 14, 1065353. [Google Scholar] [CrossRef]
  17. Chen, W.; Xiao, H.; Lin, M.; Zhou, J.; Xuan, Q.; Cui, X.; Zhao, S. Preparation and evaluation of IgY against human papillomavirus. J. Virol. Methods 2025, 334, 115115. [Google Scholar] [CrossRef]
  18. Zhang, L.; Bai, Y.; Tao, J.; Yang, S.; Tu, C.; Liu, L.; Huang, X.; Li, L.; Qin, Z. Effects of feeding chicken egg yolk antibodies on intestinal cell apoptosis, oxidative stress and microbial flora of tilapia (Oreochromis niloticus) infected with Streptococcus agalactiae. Fish Shellfish Immunol. 2024, 150, 109596. [Google Scholar] [CrossRef]
  19. Nakamura, R.; Pedrosa-Gerasmio, I.R.; Alenton, R.R.R.; Nozaki, R.; Kondo, H.; Hirono, I. Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease. J. Fish Dis. 2019, 42, 1125–1132. [Google Scholar] [CrossRef]
  20. Kumaran, T.; Beulashiny, D.; Sujithra, S.; Uma, G.; Michael Babu, M.; Kesavan, K.; Citarasu, T. The production and influence of anti-Vibrio parahaemolytics IgY against experimental infection of V. parahaemolyticus in cultured Fenneropenaeus indicus. Fish Shellfish Immunol. 2022, 128, 246–259. [Google Scholar] [CrossRef]
  21. Liang, Z.; Ning, Y.; Cao, J.; Liu, S.; Liang, X.; Peng, X.; Huang, Y.; Wei, J.; Xiao, S.; Qin, Q.; et al. The protective effect of specific yolk antibody against nervous necrosis virus infection in Mandarin fish (Siniperca chuatsi). Fish Shellfish Immunol. 2024, 155, 109996. [Google Scholar] [CrossRef]
  22. Oliver, C.; Valenzuela, K.; Silva, H.; Haro, R.E.; Cortés, M.; Sandoval, R.; Pontigo, J.P.; Álvarez, C.; Figueroa, J.E.; Avendaño-Herrera, R.; et al. Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis. J. Appl. Microbiol. 2015, 119, 365–376. [Google Scholar] [CrossRef] [PubMed]
  23. Liu, X.; Xiao, H.H.; Cui, P.; Chen, J.; Chao, J.; Wu, X.Q.; Lu, J.; Zhang, X.; Xu, G.X.; Liu, Y. Differential polyvalent passive immune protection of egg yolk antibodies (IgY) against live and inactivated Vibrio fluvialis in fish. Fish Shellfish Immunol. 2024, 151, 109751. [Google Scholar] [CrossRef] [PubMed]
  24. Liu, X.; Xiao, H.H.; Chao, J.; Jian, S.J.; Wu, X.Q.; Lu, J.; Wang, J.; Chen, C.L.; Liu, Y. Polyvalent passive vaccine candidates from egg yolk antibodies (IgY) of important outer membrane proteins (PF1380 and ExbB) of Pseudomonas fluorescens in fish. Fish Shellfish Immunol. 2023, 143, 109211. [Google Scholar] [CrossRef] [PubMed]
  25. Mansour, S.; Bakry, K.A.; Alwaleed, E.A.; Ahmed, H.; Al-Amgad, Z.; Mohammed, H.H.; Emeish, W.F.A. Dietary nanocurcumin impacts blood biochemical parameters and works synergistically with florfenicol in African catfish challenged with Aeromonas veronii. Fishes 2023, 8, 298. [Google Scholar] [CrossRef]
  26. Zhang, Z.L.; Meng, Y.Q.; Li, J.J.; Zhang, X.X.; Li, J.T.; Xu, J.R.; Zheng, P.H.; Xian, J.A.; Lu, Y.P. Effects of antimicrobial peptides from dietary Hermetia illucens larvae on the growth, immunity, gene expression, intestinal microbiota and resistance to Aeromonas hydrophila of juvenile red claw crayfish (Cherax quadricarinatus). Fish Shellfish Immunol. 2024, 147, 109437. [Google Scholar] [CrossRef]
  27. Paola, D.D.; Iaria, C.; Capparucci, F.; Cordaro, M.; Crupi, R.; Siracusa, R.; D’Amico, R.; Fusco, R.; Impellizzeri, D.; Cuzzocrea, S.; et al. Aflatoxin B1 toxicity in zebrafish larva (Danio rerio): Protective role of Hericium erinaceus. Toxins 2021, 13, 710. [Google Scholar] [CrossRef]
  28. Peng, B.; Lin, X.P.; Wang, S.N.; Yang, M.J.; Peng, X.X.; Li, H. Polyvalent protective immunogens identified from outer membrane proteins of Vibrio parahaemolyticus and their induced innate immune response. Fish Shellfish Immunol. 2018, 72, 104–110. [Google Scholar] [CrossRef]
  29. Zhang, Z.; Li, X.; Huang, M.; Huang, Y.; Tan, X.; Dong, Y.; Huang, Y.; Jian, J. Siglec7 functions as an inhibitory receptor of non-specific cytotoxic cells and can regulate the innate immune responses in a primitive vertebrate (Oreochromis niloticus). Int. J. Biol. Macromol. 2024, 278, 134851. [Google Scholar] [CrossRef]
  30. Mohammadi, G.; Hafezieh, M.; Karimi, A.A.; Azra, M.N.; Van Doan, H.; Tapingkae, W.; Abdelrahman, H.A.; Dawood, M.A.O. The synergistic effects of plant polysaccharide and Pediococcus acidilactici as a synbiotic additive on growth, antioxidant status, immune response, and resistance of Nile tilapia (Oreochromis niloticus) against Aeromonas hydrophila. Fish Shellfish Immunol. 2022, 120, 304–313. [Google Scholar] [CrossRef]
  31. Liu, X.; Chao, J.; Xiao, H.H.; Chen, J.; Cui, P.; Wu, X.Q.; Lu, J.; Wang, J.; Chen, C.L.; Zhang, X.Y.; et al. Identification of polyvalent passive vaccine candidates from egg yolk antibodies (IgY) of important outer membrane proteins of Aeromonas hydrophila in fish. Aquacult. Rep. 2024, 35, 102002. [Google Scholar] [CrossRef]
  32. Li, D.L.; Liu, S.Y.; Zhu, R.; Meng, S.T.; Wang, Y.T.; Yang, Z.Y.; Li, L.; Wei, X.F.; Shang, G.J.; Wang, H.T.; et al. Potential protective effects of sodium butyrate on glycinin-induced oxidative stress, inflammatory response, and growth inhibition in Cyprinus carpio. Fish Physiol. Biochem. 2024, 50, 273–293. [Google Scholar] [CrossRef]
  33. Zhou, X.; Ahn, D.U.; Xia, M.Q.; Zeng, Q.; Li, X.M.; Cai, Z.X. Fab fragment of immunoglobulin Y modulates NF-kB and MAPK signaling through TLR4 and aVβ3 integrin and inhibits the inflammatory effect on R264.7 macrophages. J. Agric. Food Chem. 2021, 69, 8747–8757. [Google Scholar] [CrossRef] [PubMed]
  34. Rodrigues, M.d.S.; Toledo, V.P.B.; Nóbrega, R.H. Effects of maternal stress on the development of the somatotropic axis during the larval and tuvenile stages in Zebrafish (Danio rerio). Fishes 2025, 10, 37. [Google Scholar] [CrossRef]
  35. Adewoyin, M.; Hamarsha, A.; Akinsola, R.; Teoh, S.L.; Azmai, M.N.A.; Abu Bakar, N.; Nasruddin, N.S. Intraperitoneal injection of the Porphyromonas gingivalis outer membrane vesicle (OMV) stimulated expressions of neuroinflammatory markers and histopathological changes in the brains of adult zebrafish. Int. J. Mol. Sci. 2024, 25, 11025. [Google Scholar] [CrossRef] [PubMed]
  36. Maceda-Veiga, A.; Monroy, M.; Navarro, E.; Viscor, G.; de Sostoa, A. Metal concentrations and pathological responses of wild native fish exposed to sewage discharge in a Mediterranean river. Sci. Total Environ. 2013, 449, 9–19. [Google Scholar] [CrossRef] [PubMed]
  37. Dong, A.; Ma, Y.; Wang, X.; Jing, X.; He, H.; Zhang, T.; Dong, H.; Liu, W.; Fan, K.; Huo, J. Effect of cadmium on histopathological injuries and ultra-structural changes of kidney of the turtle Mauremys reevesii. Environ. Sci. Pollut. Res. Int. 2024, 31, 39774–39781. [Google Scholar] [CrossRef]
  38. Destaw, T.; Yessuf, M.; Mazengia, H.; Getnet, M.A.; Berihun, A.M. Ligula intestinalis in Labeobarbus fish species at Lake Tana, Ethiopia: Prevalence, histopathological investigation, and associated risk factors. BMC Vet. Res. 2025, 21, 392. [Google Scholar] [CrossRef]
  39. Ye, D.; Liu, T.; Li, Y.; Wang, Y.; Hu, W.; Zhu, Z.; Sun, Y. Identification of fish spermatogenic cells through high-throughput immunofluorescence against testis with an antibody set. Front. Endocrinol. 2023, 14, 1044318. [Google Scholar] [CrossRef]
  40. Xiao, H.; Chen, J.; Cui, P.; Che, X.; Wu, X.; Lu, J.; Zhu, G.; Liu, Y.; Liu, X. Evaluation of the multivalent immunoprotective effects of protein, DNA, and IgY vaccines against Vibrio fluvialis outer membrane protein VF14355 in Carassius auratus. Int. J. Mol. Sci. 2025, 26, 3379. [Google Scholar] [CrossRef]
  41. Huang, Y.; Tao, Y.; Cai, C.; Chen, J.; Ji, C.; Aniagu, S.; Jiang, Y.; Chen, T. Using immunofluorescence to detect PM2.5-induced DNA damage in zebrafish embryo hearts. J. Vis. Exp. 2021, 168, e62021. [Google Scholar]
  42. Chen, T.; Jin, H.; Wang, H.; Yao, Y.; Aniagu, S.; Tong, J.; Jiang, Y. Aryl hydrocarbon receptor mediates the cardiac developmental toxicity of EOM from PM2.5 in P19 embryonic carcinoma cells. Chemosphere 2019, 216, 372–378. [Google Scholar] [CrossRef]
  43. Liu, X.; Yang, M.J.; Wang, S.N.; Xu, D.; Li, H.; Peng, X.X. Differential antibody responses to outer membrane proteins contribute to differential immune protections between live and inactivated Vibrio parahemolyticus. J. Proteome Res. 2018, 17, 2987–2994. [Google Scholar] [CrossRef]
  44. Reyes-Becerril, M.; Maldonado-García, M.; López, M.G.; Silva-Jara, J.M.; Angulo, C. Microencapsulates of Moringa oleifera seed extracts enhance immunological parameters of Almaco jack (Seriola rivoliana) upon Aeromonas hydrophila challenge. Fish Shellfish Immunol. 2025, 161, 110239. [Google Scholar] [CrossRef] [PubMed]
  45. Jalali, S.A.H.; Sepehre Nia, S.; Beyraghdar Kashkooli, O.; Soleimanin-Zad, S. Oral immunization with attenuated Salmonella typhimurium as a carrier of DNA vaccine against infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2025, 158, 110127. [Google Scholar] [CrossRef] [PubMed]
  46. Miryala, K.R.; Swain, B. Advances and challenges in Aeromonas hydrophila vaccine development: Immunological insights and future perspectives. Vaccines 2025, 13, 202. [Google Scholar] [CrossRef] [PubMed]
  47. Rathor, G.S.; Swain, B. Advancements in fish vaccination: Current innovations and future horizons in aquaculture health management. Appl. Sci. 2024, 14, 5672. [Google Scholar] [CrossRef]
  48. Ruenkoed, S.; Pholoeng, A.; Nontasan, S.; Panprommin, D.; Mongkolwit, K.; Wangkahart, E. Assessing the impact of acidifiers on growth performance, innate immune capacity, response to ammonia nitrogen stress, digestive enzyme activity, intestinal histology, and gene expression of Nile tilapia (Oreochromis niloticus). Fish Shellfish Immunol. 2025, 162, 110315. [Google Scholar] [CrossRef]
  49. Yasmin, R.; Saharia, P.K.; Hussain, I.A.; Talukdar, A.; Baishya, S.; Bhagawati, K.; Sarma, D.K.; Patowary, A.N.; Kalita, K.; Das, U.K.; et al. Probiotic strain Lactobacillus plantarum GCLP4 derived from the gut of Ctenopharyngodon idella modulates growth, digestive enzymes and immune-biochemical parameters of Labeo rohita fingerlings. J. Anim. Physiol. Anim. Nutr. 2025, 109, 268–279. [Google Scholar] [CrossRef]
  50. Patel, D.; K, G.B.; Nayaka, S.; Gacem, A.; Kumar, P.; Sharma, A.; Yadav, K.K.; Mansour, L.; Kalasariya, H.S. Comprehensive analysis of the major IgY antibody extraction strategies from chicken egg yolk. Vet. Immunol. Immunopathol. 2025, 283, 110928. [Google Scholar] [CrossRef]
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Figure 1. The protection rates for the fish infected with pathogenic bacteria. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. (ns) represents no significant difference (p > 0.05). NC represents the blank IgY group. Live bacteria represent the live A. hydrophila IgY group. Inactivated bacteria represent the inactivated A. hydrophila IgY group.
Figure 1. The protection rates for the fish infected with pathogenic bacteria. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. (ns) represents no significant difference (p > 0.05). NC represents the blank IgY group. Live bacteria represent the live A. hydrophila IgY group. Inactivated bacteria represent the inactivated A. hydrophila IgY group.
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Figure 2. The expression levels of antioxidation factors in fish serum. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. Labels a–c display statistically different groups (p < 0.05). NC represents the blank IgY group. Live bacteria represents the live A. hydrophila IgY group. Inactivated bacteria represents the inactivated A. hydrophila IgY group.
Figure 2. The expression levels of antioxidation factors in fish serum. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. Labels a–c display statistically different groups (p < 0.05). NC represents the blank IgY group. Live bacteria represents the live A. hydrophila IgY group. Inactivated bacteria represents the inactivated A. hydrophila IgY group.
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Figure 3. The mRNA expression levels of inflammation factors in tissues. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. Labels a–c indicate statistically different groups (p < 0.05).
Figure 3. The mRNA expression levels of inflammation factors in tissues. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. Labels a–c indicate statistically different groups (p < 0.05).
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Figure 4. The recognition of IgY or fish serum with aquaculture bacteria. (A) The interaction between IgY and A. hydrophila, (B) the interaction between fish serum (immunization with IgY and infection by A. hydrophila) and A. hydrophila, (C) the interaction between IgY and A. veronii, (D) the interaction between fish serum (immunization with IgY and infection by A. veronii) and A. veronii.
Figure 4. The recognition of IgY or fish serum with aquaculture bacteria. (A) The interaction between IgY and A. hydrophila, (B) the interaction between fish serum (immunization with IgY and infection by A. hydrophila) and A. hydrophila, (C) the interaction between IgY and A. veronii, (D) the interaction between fish serum (immunization with IgY and infection by A. veronii) and A. veronii.
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Figure 5. Sections potentially with pathology of kidney, spleen, and intestinal tissues of fish. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. (a) Live bacteria IgY, (b) inactivated bacteria IgY, (c) control (blank IgY), (d) negative control (untreated fish used as the baseline for the experiment).
Figure 5. Sections potentially with pathology of kidney, spleen, and intestinal tissues of fish. (A) Challenge with A. hydrophila, (B) challenge with A. veronii. (a) Live bacteria IgY, (b) inactivated bacteria IgY, (c) control (blank IgY), (d) negative control (untreated fish used as the baseline for the experiment).
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Figure 6. Immunofluorescence analysis of the expression of p53 and γH2A.X proteins in kidney. (A,B) represent fish infected with A. hydrophila. (C,D) represent fish infected with A. veronii. (A,C) represent the immunofluorescence of p53 protein. (B,D) represent the immunofluorescence of γH2A.X protein. (a) Live bacteria IgY. (b) Inactivated bacteria IgY. (c) Control (blank IgY). * p < 0.05, ** p < 0.01. ns denotes no significant difference.
Figure 6. Immunofluorescence analysis of the expression of p53 and γH2A.X proteins in kidney. (A,B) represent fish infected with A. hydrophila. (C,D) represent fish infected with A. veronii. (A,C) represent the immunofluorescence of p53 protein. (B,D) represent the immunofluorescence of γH2A.X protein. (a) Live bacteria IgY. (b) Inactivated bacteria IgY. (c) Control (blank IgY). * p < 0.05, ** p < 0.01. ns denotes no significant difference.
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Table 1. The phagocytosis experiment conducted on bacteria in C. auratus.
Table 1. The phagocytosis experiment conducted on bacteria in C. auratus.
BacteriaIgY GroupPhagocytic Percentage (PP %)Phagocytic Index (PI %)
A. hydrophilaControl50.82 ± 9.64 b115.84 ± 4.64 b
Live bacteria77.50 ± 3.54 a156.04 ± 8.53 a
Inactivated bacteria74.00 ± 5.66 a145.01 ± 3.03 a
A. veroniiControl29 ± 2.00 b100.53 ± 8.68 b
Live bacteria50.33 ± 2.08 a155.00 ± 45.16 a
Inactivated bacteria45.00 ± 2.65 a144.56 ± 10.12 a
Labels a,b represent statistically different groups (p < 0.05).
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MDPI and ACS Style

Cui, P.; Chen, J.; Xiao, H.; Che, X.; Sun, S.; Ma, Z.; Lu, J.; Xu, G.; Liu, X. Assessment of Egg Yolk IgY Antibodies Against Live or Inactivated Aeromonas hydrophila for Polyvalent Passive Immunization in Goldfish (Carassius auratus). Fishes 2025, 10, 491. https://doi.org/10.3390/fishes10100491

AMA Style

Cui P, Chen J, Xiao H, Che X, Sun S, Ma Z, Lu J, Xu G, Liu X. Assessment of Egg Yolk IgY Antibodies Against Live or Inactivated Aeromonas hydrophila for Polyvalent Passive Immunization in Goldfish (Carassius auratus). Fishes. 2025; 10(10):491. https://doi.org/10.3390/fishes10100491

Chicago/Turabian Style

Cui, Pan, Jing Chen, Huihui Xiao, Xixian Che, Shujun Sun, Zijian Ma, Juan Lu, Gaoxiao Xu, and Xiang Liu. 2025. "Assessment of Egg Yolk IgY Antibodies Against Live or Inactivated Aeromonas hydrophila for Polyvalent Passive Immunization in Goldfish (Carassius auratus)" Fishes 10, no. 10: 491. https://doi.org/10.3390/fishes10100491

APA Style

Cui, P., Chen, J., Xiao, H., Che, X., Sun, S., Ma, Z., Lu, J., Xu, G., & Liu, X. (2025). Assessment of Egg Yolk IgY Antibodies Against Live or Inactivated Aeromonas hydrophila for Polyvalent Passive Immunization in Goldfish (Carassius auratus). Fishes, 10(10), 491. https://doi.org/10.3390/fishes10100491

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