Multivalent Immune-Protective Effects of Egg Yolk Immunoglobulin Y (IgY) Derived from Live or Inactivated Shewanella xiamenensis Against Major Aquaculture Pathogens

Abstract

Egg yolk immunoglobulin Y (IgY) possesses advantages such as low cost, easy availability, simple preparation, high antigen specificity, absence of drug residues, and compliance with animal welfare standards, making it an environmentally friendly and safe alternative to antibiotics. This research utilizes IgY antibody technology to develop a multivalent passive immune vaccine for major pathogenic bacteria in aquaculture. In this study, IgY antibodies against live Shewanella xiamenensis (LSX-IgY) and inactivated S. xiamenensis (ISX-IgY) were prepared by immunizing laying hens, and passive immunization protection experiments were conducted in Carassius auratus infected with S. xiamenensis and Aeromonas hydrophila. The passive immunization protection rates of LSX-IgY and ISX-IgY against S. xiamenensis were 63.64% and 72.73%, respectively, and the passive cross-protection rates against A. hydrophila were 50% and 71.43%, respectively. Further, C. auratus sera could specifically bind to S. xiamenensis or A. hydrophila in vitro, and the phagocytic activity of leukocytes was increased. LSX-IgY and ISX-IgY could reduce the bacterial load in the C. auratus kidneys. Meanwhile, they could significantly reduce the levels of antioxidant factors in serum and inhibit the mRNA expression of inflammation-related factors in the kidneys and spleens. Additionally, histopathology and immunofluorescence analysis showed that both IgY preparations preserved tissue integrity and reduced the expression of apoptosis factor (p53) and DNA damage factor (γH2A.X) of visceral organs, respectively. In summary, LSX-IgY and ISX-IgY can combat various bacterial infections, with no significant difference between the two. Additionally, inactivated bacterial immunization is more aligned with animal welfare standards for laying hens. Therefore, ISX-IgY is expected to serve as a multivalent vaccine against major aquaculture pathogens.

Keywords: Aeromonas hydrophila; IgY antibody; Shewanella xiamenensis; multivalent vaccine; passive immunity.

Figure 1

Survival rate of C. auratus infected with pathogenic bacteria. (A,B) represent infection with S. xiamenensis and A. hydrophila, respectively. NC, the nature control (blank IgY antibody).

Figure 2

Kidney bacterial count statistics of C. auratus. (A) represents kidney bacterial colonies of C. auratus challenged with S. xiamenensis; (B) represents the bacterial colony count of C. auratus kidney challenged with S. xiamenensis; (C) represents kidney bacterial colonies of C. auratus challenged with A. hydrophila; (D) represents the bacterial colony count of C. auratus kidney challenged with A. hydrophila. a, b and c represent kidney bacterial colonies of C. auratus immunized with blank IgY antibodies as the nature control (NC), LSX-IgY, ISX-IgY, respectively. d represents an uninfected kidney (negative control). **** p < 0.0001.

Figure 2

Kidney bacterial count statistics of C. auratus. (A) represents kidney bacterial colonies of C. auratus challenged with S. xiamenensis; (B) represents the bacterial colony count of C. auratus kidney challenged with S. xiamenensis; (C) represents kidney bacterial colonies of C. auratus challenged with A. hydrophila; (D) represents the bacterial colony count of C. auratus kidney challenged with A. hydrophila. a, b and c represent kidney bacterial colonies of C. auratus immunized with blank IgY antibodies as the nature control (NC), LSX-IgY, ISX-IgY, respectively. d represents an uninfected kidney (negative control). **** p < 0.0001.

Figure 3

The expression levels of antioxidant-related factors. (A,B) represent S. xiamenensis and A. hydrophila challenge, respectively. * p < 0.05, ** p < 0.01. ns indicates no significant difference (p > 0.05). Compared to the NC group, the expressions of CAT, SOD, and GSH-Px decreased (p < 0.05) in LSX-IgY or ISX-IgY groups, with no significant differences between the two IgY.

Figure 4

mRNA expression levels of inflammatory factors (IL-6, IL-8, TNF-α, and IL-1β) in internal organs. (A,B) represent challenges with S. xiamenensis and A. hydrophila, respectively. * p < 0.05, ** p < 0.01. ns indicates no significant difference (p > 0.05). Compared to the NC group, the mRNA expressions of IL-6, IL-8, TNF-α, and IL-1β decreased (p < 0.05) in LSX-IgY or ISX-IgY groups. The indicators of the LSX-IgY group were lower than that of the ISX-IgY group.

Figure 5

Immune recognition between the IgY or C. auratus serum and pathogens. (A) represents the recognition between IgY and S. xiamenensis. (B) represents the mutual recognition between the serum of C. auratus (immunized with IgY and challenged with S. xiamenensis) and S. xiamenensis. (C) represents the recognition between IgY and A. hydrophila; (D) represents the mutual recognition between the serum of C. auratus (immunized with IgY and challenged with A. hydrophila) and A. hydrophila.

Figure 5

Immune recognition between the IgY or C. auratus serum and pathogens. (A) represents the recognition between IgY and S. xiamenensis. (B) represents the mutual recognition between the serum of C. auratus (immunized with IgY and challenged with S. xiamenensis) and S. xiamenensis. (C) represents the recognition between IgY and A. hydrophila; (D) represents the mutual recognition between the serum of C. auratus (immunized with IgY and challenged with A. hydrophila) and A. hydrophila.

Figure 6

Histopathological sections of the kidney, spleen, and intestinal tissues of C. auratus. (A) represents infection with S. xiamenensis; (B) represents infection with A. hydrophila. (I) demonstrates loose renal tubular structure; (II) demonstrates glomerular atrophy; (III) demonstrates renal cell apoptosis; (IV) demonstrates low density of splenic cells; (V) demonstrates splenic cell apoptosis; (VI) demonstrates incomplete structure of splenic tissue; (VII) demonstrates intestinal villus necrosis; (VIII) demonstrate intestinal mucosal necrosis; (IX) demonstrates intestinal cell apoptosis.

Figure 6

Histopathological sections of the kidney, spleen, and intestinal tissues of C. auratus. (A) represents infection with S. xiamenensis; (B) represents infection with A. hydrophila. (I) demonstrates loose renal tubular structure; (II) demonstrates glomerular atrophy; (III) demonstrates renal cell apoptosis; (IV) demonstrates low density of splenic cells; (V) demonstrates splenic cell apoptosis; (VI) demonstrates incomplete structure of splenic tissue; (VII) demonstrates intestinal villus necrosis; (VIII) demonstrate intestinal mucosal necrosis; (IX) demonstrates intestinal cell apoptosis.

Figure 7

Immunofluorescence detection of p53 and γH2A.X proteins in the kidney of C. auratus. (A,B) represent the immunofluorescence of C. auratus kidneys infected with S. xiamenensis. (C,D) represent the immunofluorescence of C. auratus kidneys infected with A. hydrophila; (A,C) show the expression of p53. (B,D) show the expression of γH2A.X. ns indicates no significant difference. * p < 0.05, ** p < 0.01, **** p < 0.0001.