Bacterial strains, including P. anguilliseptica MatS1, A. veronii ATCC 35622, and Staphylococcus aureus ATCC 6538, were preserved in the Microbiology Laboratory of Fuyang Normal University, Anhui Province, China. Twenty-week-old laying hens were purchased from Chongqing Tengxin Biotechnology Co., Ltd. (Chongqing, China). C. auratus (20 ± 1.0 g) individuals were purchased from Fuyang Economic Fish Farming Co., Ltd. (Fuyang, China). 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. 2024-04-006).

P. anguilliseptica was cultured in 600 mL of Luria–Bertani (LB) liquid medium at 30°C overnight. Bacterial cells were collected via centrifugation, and a portion of the cells was dissolved in a 1% formaldehyde physiological saline solution and inactivated in an 80°C water bath for 90 min to prepare inactivated bacteria. Then, the bacteria were spread on LB solid medium and cultured overnight at 30°C. No bacteria grew on the plates, which proved that they were inactivated. Both live or inactivated P. anguilliseptica [2 × 10⁷ colony-forming unit (CFU)] (200 μL each) and 200 μL of normal saline as a blank IgY antibody group (control) were intramuscularly injected into laying hens. Immunization was performed three times, with an interval of 14 days between each immunization. Eggs were collected 35 days after immunization and stored at 4°C. After 35 days, the eggs were taken, the yolks separated, and an equal volume of a phosphate-buffered saline (PBS) solution was added to the yolk solution. While stirring, 3.5% of powdered PEG6000 was added, mixed thoroughly, and centrifuged at 10,000 r/min for 20 min to collect the supernatant. Then, while stirring, 8.5% of powdered PEG6000 was continuously added to the supernatant, which was then centrifuged, and the precipitate was collected. The precipitate was dissolved in 10 mL of the PBS solution, and while stirring, 12% of powdered PEG6000 was added. After another centrifugation, the precipitate was dissolved in 2 mL of the PBS solution. The solution was dialyzed in PBS at 4°C for 36 h. The main component of the solution was IgY antibodies against live P. anguilliseptica (L-PA-IgY) or inactivated P. anguilliseptica (I-PA-IgY), and the two IgY antibodies were prepared (Xiao et al., 2025a). The purified production of IgY antibodies was detected using the method of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The concentration of IgY antibody was adjusted to 2 μg/μL using a bicinchoninic acid (BCA) protein analysis kit (Shanghai Beyotime Biotech Co., Ltd., Shanghai, China).

Enzyme-linked immunosorbent assay (ELISA) was used for IgY titer detection as described previously (Xiao et al., 2024). P. anguilliseptica was cultured in LB liquid medium at 30°C to an OD ₆₀₀ = 1.0. The bacteria were centrifuged and adjusted to OD ₆₀₀ = 1.0 with normal saline. The bacterial solution was then placed in a 96-well plate with 200 μL per well and coated overnight at 4°C. The wells were washed three times with the PBS solution. A total of 200 μL of 5% skimmed milk was added to the wells to seal at 37°C for 2 h. Then, L-PA-IgY, I-PA-IgY, and the IgY (without bacterial immunization) as a negative control (NE-C) were diluted in multiples with the PBS solution (1:200, 1:400, 1:800, 1:1,600, 1:3,200, and 1:6,400). Each dilution gradient was repeated six times. The wells were added with 200 μL of the IgY solution. After incubation at 37°C for 2 h, the wells were washed three times with the PBS solution. Furthermore, goat anti-IgY secondary antibody (1:500) (Sanying Biotechnology Co., Ltd., Wuhan, China) was added to the wells and incubated at 37°C for 1 h. After washing with PBS three times, the chromogenic solution was added to the wells and incubated at 37°C for 15 min. Absorbance at OD ₄₅₀ was immediately read using a microplate reader (Bio-Rad, Hercules, CA, USA).

C. auratus individuals were divided into two groups: challenge with P. anguilliseptica and A. veronii. Each group included a blank IgY group as a nature control (NC), an L-PA-IgY group, and an I-PA-IgY group, with 15 fish in each group. Each fish was intraperitoneally injected with 20 μL of IgY antibodies (40μg). According to the preliminary experiment, the median lethal dose (LD50) of P. anguilliseptica was determined to be 8 × 10⁸ CFU, and that of A. veronii was 1 × 10⁹ CFU. Two hours after IgY passive immunization, the fish were intraperitoneally challenged with P. anguilliseptica (8 × 10⁸ CFU) and A. veronii (1 × 10⁹ CFU). The mortality of the fish was observed for 14 consecutive days. The RPS was calculated using the following formula: RPS (%) = (1 − [mortality rate in the experimental group %/mortality rate in the control group %]) × 100 (Xiao et al., 2024).

P. anguilliseptica and A. veronii were cultured in 300 mL of LB liquid medium at 30°C to an OD ₆₀₀ = 1.0. The bacteria were centrifuged and adjusted to OD ₆₀₀ = 1.0 with normal saline. The bacterial solution was then placed in a 96-well plate, with 200 μL per well, and coated overnight at 4°C. The wells were washed three times with a tris-buffered saline with tween-20 (TBST) solution for 10 min each time. A total of 200 μL of 5% skimmed milk was added to the wells; then, the plate was sealed and incubated at 37°C for 2 h. Meanwhile, C. auratus was passively immunized with L-PA-IgY, I-PA-IgY, and blank IgY as a nature control (NC). Additionally, the fish without IgY immunization or bacterial infection were used as a negative control (NE-C). After 2 h of passive immunization with IgY, C. auratus was challenged with P. anguilliseptica or A. veronii. After 2 days, C. auratus serum was obtained from the tail vein. Then, gradient dilutions (1:200, 1:400, 1:800, 1:1,600, 1:3,200, and 1:6,400) of C. auratus serum were sequentially added to the wells and incubated at 37°C for 2h. Three wells were used per dilution, and the experiment was repeated three times. After washing, rat anti-fish IgM secondary antibody (1:400) (Sanying Biotechnology Co., Ltd., Wuhan, China) was added to the wells and incubated at 37°C for 1h. After washing with TBST, goat anti-rat tertiary antibody (1:2,500) (Sanying Biotechnology Co., Ltd., Wuhan, China) was added to the wells and incubated at 37°C for 1h. After washing with TBST three times, the chromogenic solution was added and incubated at 37°C for 15 min to terminate the reaction. Absorbance at OD ₄₅₀ was immediately read using a microplate reader (Bio-Rad, Hercules, CA, USA) (Xiao et al., 2024).

The kidney count was used to evaluate the effect of IgY antibody on bacterial clearance, as previously described (Xiao et al., 2025b). Briefly, C. auratus individuals were divided into two groups: challenge with P. anguilliseptica and A. veronii. Each group included a blank IgY group as a nature control (NC), an L-PA-IgY group, and an I-PA-IgY group, with 10 fish in each group. Additionally, to detect bacterial contamination, the fish without IgY immunization or bacterial challenge were taken as a negative control group (NE-C). Each fish was intraperitoneally injected with 20 μL of IgY antibodies (40μg). After 2 h, the fish were intraperitoneally challenged with P. anguilliseptica (8 × 10⁸ CFU) and A. veronii (1 × 10⁹ CFU). Six kidney tissues for each group were taken from C. auratus under anesthesia on the second day after being challenged with bacteria. For subsequent testing, 50 mg of kidney per fish was weighed. After thoroughly grinding, the kidney tissue was added to 200 μL of the physiological saline solution. Then, the kidney homogenates were diluted in serial 10-fold dilutions with the physiological saline solution, and 200 μL of diluent was spread onto selective culture media for cultivation overnight at 28°C. The selective culture medium that suppressed the growth of background flora for P. anguilliseptica was Pseudomonas CFC Selective Agar HB8689, and the selective culture medium for A. veronii was Aeromonas Medium Base (Ryan) Selective Agar HB7023 (Haibo Biotechnology Co., Ltd., Qingdao, China). The negative control group (NE-C) was used fully nutritious culture medium (LB) to detect the contamination status of bacteria in kidney tissues. Photographs were taken for a medium plate, and the bacterial colony count was determined. The dilution ratio of the kidney homogenates was reported as CFU values exclusively from plates with 30–300 countable colonies. Furthermore, five colonies were randomly selected from each selective culture medium for expansion culture, and 16S rDNA sequencing was performed by Sangon Biotech Co., Ltd. (Shanghai, China) to identify bacterial species to exclude the growth of background bacteria.

After passive immunization with IgY antibodies and challenge for 2 days, six blood samples were taken from C. auratus under anesthesia. S. aureus was transferred to 100 mL of LB liquid medium and incubated overnight at 37°C with shaking. The bacterial cells were then collected and dissolved in a 1% formaldehyde saline solution, followed by inactivation in a water bath at 80°C for 90 min. After the inactivation, the bacteria were adjusted to an OD ₆₀₀ = 0.6 with normal saline. A mixture of 200μL of anticoagulated blood and S. aureus suspension (1 × 10⁶ CFU) was prepared, incubated in a 25°C water bath for 60 min, and gently inverted every 10 min to ensure thorough mixing. Blood smears were then prepared. After methanol fixation, staining was performed using a rapid Giemsa staining kit (Sangon Biotech Co., Ltd., Shanghai, China), and observation and counting were conducted under a microscope to calculate the phagocytic percentage and phagocytic index. The calculation methods were as follows. Phagocytic percentage (PP): number of cells involved in phagocytosis among 100 phagocytic cells/100 × 100%. Phagocytic index (PI): number of bacteria within phagocytic cells/number of counted cells involved in phagocytosis × 100% (Xiao et al., 2024).

Antioxidant factors and reactive oxygen species (ROS) were assessed in C. auratus serum as described previously (Xiao et al., 2025b). Briefly, on the second day of passive immunization with IgY antibodies and bacterial challenge, six sera in each group were obtained from the tail vein of C. auratus under anesthesia. The group passively immunized with blank IgY was used as a nature control (NC), and the group without IgY or bacteria was used as a negative control (NE-C). The antioxidant factor levels of catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were assessed in accordance with the instructions provided in the detection kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China). Additionally, the ROS of C. auratus serum was detected according to the kit manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China).

Real-time fluorescence-based quantitative PCR (qRT-PCR) was used to evaluate the expression of inflammatory factor mRNA, as previously described (Xiao et al., 2024). Briefly, C. auratus individuals were passively immunized with IgY antibodies and challenged with P. anguilliseptica or A. veronii. The kidneys and spleens of six C. auratus individuals for each group were collected under anesthesia on the second day after challenge. The group passively immunized with blank IgY was used as a nature control (NC), and the group without IgY or bacteria was used as a negative control (NE-C). The tissues were thoroughly ground with liquid nitrogen, and RNA was extracted using an RNA extraction kit (Sangon Biotech Co., Ltd., Shanghai, China). The RNA was then converted to cDNA using a reverse transcription kit (Servicebio Biotechnology Co., Ltd., Wuhan, China). qRT-PCR was performed using the SYBR^® Green Premix kit (Servicebio Biotechnology Co., Ltd., Wuhan, China) and synthesized primers ( Supplementary Table S1 ). The ΔCt (cycle threshold change) was determined by comparing the Ct values of the factor genes with those of the reference gene (gapdh), and then the ΔΔCt was determined by comparing the ΔCt values of the experimental group with those of the control group. Subsequently, mRNA expression was analyzed using the 2^−(ΔΔCt) formula.

Two days after passive immunization with IgY and bacterial challenge, C. auratus kidney, spleen, and intestinal tissues were obtained and immersed in Davidson’s fixative for 18 h and then transferred to a 10% formaldehyde solution for fixation for more than 24 h. The tissues were then dehydrated in a gradually increasing concentration gradient of ethanol (80%, 90%, 95%, and 100%), followed by clearing in xylene. Afterward, the samples were embedded in paraffin at 65°C to form paraffin blocks. The blocks were then sectioned into 4-μm-thick slices using a microtome, and the slices were placed on adhesive slides and dried overnight at 37°C. The slides were sequentially immersed in xylene and a gradient of decreasing ethanol concentrations (100%, 95%, 80%, and 70%) to dewax and rehydrate them. After rinsing with tap water, the sections were stained with hematoxylin and eosin, dehydrated with ethanol, and cleared with xylene. Finally, the slides were mounted with a neutral resin. The structural integrity of the glomeruli and renal tubules in the kidney tissue, along with the cellular compactness of the spleen tissue and the structural changes in the mucosal layer cells of the intestine of the six C. auratus individuals for each group, was observed under a microscope (Leica, Wetzlar, Germany) (Xiao et al., 2025a).

The six prepared renal tissue sections for each group were dewaxed in xylene, hydrated in a decreasing ethanol gradient (100%, 95%, 80%, and 50%), and washed twice with phosphate buffered saline with tween-20 (PBST). After permeabilizing the cells with 1% Triton X-100, the slides were placed in a carbonate buffer solution (CBS) solution and boiled for 2 min for antigen retrieval. After natural cooling, the slides were immersed in 3% H₂O₂, washed twice with PBST, and circled with an immunohistochemical pen around the tissue. Then, 10 μL of a blocking solution (5% bovine serum albumin) was added within the circle and incubated at room temperature for 1.5 h, followed by three washes with PBST. p53 or γH2A.X antibodies (1:200) were added to the tissue section and incubated at 37°C for 2 h. After PBST washing, a donkey anti-rabbit (1:250) secondary antibody solution was added to the tissue section, and the section was incubated at 37°C for 1 h. After PBST washing, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min and washed three times with PBST; the slides were mounted, and the expression of p53 and γH2A.X was observed under a fluorescence microscope (Leica, Wetzlar, Germany) (Xiao et al., 2024).

All the experimental data were expressed as mean ± SD, and all experiments were repeated at least three times. The significant difference from the respective control in all experiments was assessed by one-way analysis of variance (ANOVA) using the software of Statistical Package for the Social Sciences 19.0 (SPSS 19.0). Values of p < 0.05 were considered statistically significant (Dudley et al., 2004).

According to PEG6000 antibody purification technology, high-purity L-PA-IgY and I-PA-IgY were obtained ( Supplementary Figure S1 ). The titer of L-PA-IgY and I-PA-IgY was 1:1,600 via the ELISA method ( Supplementary Figure S2 ).

C. auratus individuals were passively immunized with L-PA-IgY or I-PA-IgY and then challenged with P. anguilliseptica and A. veronii. The results showed that the mortality of the fish stabilized after 4 days ( Figure 2 ). The RPS values of live or inactivated bacterial IgY antibodies against P. anguilliseptica were 66.67% (p < 0.05) and 73.33% (p < 0.05), respectively, and their RPS values against A. veronii were 75.00% (p < 0.05) and 58.34% (p < 0.05), respectively. Using SPSS 19.0 for the chi-square test, it was found that there was no significant difference in RPS values (p > 0.05) between L-PA-IgY and I-PA-IgY. It is evident that both L-PA-IgY and I-PA-IgY possess passive immunoprotective effects.

To determine the bacterial count within C. auratus, kidney tissues from the fish were coated on LB medium on day 2 after passive immunization with IgY and bacterial challenge. Compared to those in the control group, the bacterial counts in both L-PA-IgY and I-PA-IgY groups were reduced (p < 0.05) ( Figure 3 ). This indicates that L-PA-IgY and I-PA-IgY can reduce the number of bacteria in the kidneys, and the difference in effectiveness between the two IgY antibodies is not significant.

To evaluate the phagocytosis of leukocytes, blood from C. auratus individuals passively immunized with two types of IgY antibodies and then challenged with bacteria was subjected to a leukocyte phagocytosis assay. The blood smears are shown in Supplementary Figure S3 . The results showed that the phagocytic percentage (PP%) and phagocytic index (PI%) of leukocytes in the IgY groups increased (p < 0.05) ( Table 1 ). This indicates that both L-PA-IgY and I-PA-IgY can activate leukocyte phagocytosis, with no significant difference between the two types of antibodies.

After passive immunization with two IgY antibodies and pathogen challenge, C. auratus serum was collected for antioxidant factor detection. In the negative control group (NE-C), antioxidant-related factors (SOD, CAT, and GSH-Px) were the lowest compared to those in other groups. Compared with those in the control group (NC), the levels of most antioxidant-related factors (SOD, CAT, and GSH-Px) were decreased (p < 0.05) in the L-PA-IgY and I-PA-IgY groups ( Figure 4 ). Additionally, after passive immunization with the two IgY antibodies and challenge with P. anguilliseptica, the levels of ROS in C. auratus serum were also decreased (p < 0.05) in the L-PA-IgY and I-PA-IgY groups ( Supplementary Table S2 ). These results indicate that L-PA-IgY or I-PA-IgY immunization reduced oxidative pressure due to pathogen suppression in fish, the reduced enzyme activity likely results from lower ROS generation, and there is no difference between the two IgY antibodies.

After passive immunization of C. auratus individuals with L-PA-IgY or I-PA-IgY and challenge with P. anguilliseptica and A. veronii, their kidneys and spleens were collected to evaluate the mRNA expression of inflammation-related factors (IL-6, IL-8, TNF-α, and IL-1β). In the negative control group (NE-C), the mRNA expression of inflammatory factors (IL-6, IL-8, TNF-α, and IL-1β) was the lowest compared to that in other groups. Compared with that in the control (NC) group, the mRNA expression of IL-6, IL-8, TNF-α, and IL-1β in the kidneys and spleens was reduced (p < 0.05) in the L-PA-IgY and I-PA-IgY groups ( Figure 5 ). The results indicate that L-PA-IgY and I-PA-IgY can reduce the inflammatory response induced by P. anguilliseptica and A. veronii in C. auratus, and the effects of the two IgY antibodies are not different.

To detect the in vitro interaction between C. auratus serum and bacteria, the serum of C. auratus passively immunized with IgY and post-challenge bacteria underwent an ELISA. The results showed that the serum could bind to P. anguilliseptica and A. veronii in the L-PA-IgY or I-PA-IgY group. Additionally, the absorbance decreased with the increase in dilution of C. auratus serum, and the recognition was assessed at a dilution of 1:6,400 ( Figure 6 ).

An organ pathological section experiment was conducted to evaluate the visceral damage in C. auratus after passive immunization with IgY antibodies and challenge with pathogenic bacteria. The results showed that the renal tissue structure in the control group (NC) was incomplete, with a higher degree of atrophy and degeneration in the glomeruli and renal tubules; the cellular density of the splenic tissue was reduced, and the tissue structure was incomplete; the intestinal tissue structure was loose, with atrophy and apoptosis observed in some areas of the mucosal layer. The renal, splenic, and intestinal structures in the L-PA-IgY, I-PA-IgY, and NE-C groups were more intact compared to those in the NC group, with no significant differences between the two types of IgY antibodies ( Figure 7 ).

Immunofluorescence analysis was performed to evaluate the apoptosis and DNA damage in C. auratus kidney cells. Red represents p53 and γH2A.X, and blue represents DAPI-stained nuclei. In the negative control group (NE-C), the expression of p53 and γH2A.X was the lowest compared to that in other groups. Compared to that in the control group (NC), the expression of p53 and γH2A.X was reduced in the L-PA-IgY or I-PA-IgY group (p < 0.05) ( Figure 8 ). The results indicate that passive immunization with L-PA-IgY or I-PA-IgY can reduce apoptosis and DNA damage in the kidney cells of C. auratus.