Feeding with 4,4'-diaponeurosporene-producing Bacillus subtilis enhances the lactogenic immunity of sow

Abstract

Specific antibodies produced sow by oral porcine epidemic diarrhea virus (PEDV) vaccines would transfer to newborn piglets via colostrum, and it is an effective strategy to prevent porcine epidemic diarrhea (PED). However, there is a lag in the development of corresponding vaccines due to the rapid mutation of PEDV, which could increase the difficulty of PED prevention and control in pig farms. Hence, congenital lactogenic immunity was assessed by feeding 4,4'-diaponeurosporene-producing Bacillus subtilis (B.S-Dia) to sow on the 80th day of gestation in order to protect newborn piglets from PEDV infection. Firstly, we found that the quantities of T lymphocytes and monocytes in the blood and colostrum after oral administration of B.S-Dia were significantly increased as observed by flow cytometry, whereas the proliferative activity of T lymphocytes in colostrum was also markedly increased. Furthermore, enzyme-linked immunosorbent assay (ELISA) results revealed that levels of TGF (Transforming growth factor) -β, Interleukin (IL) -6, lysozyme and lactoferrin were significantly increased. Finally, it was found in the piglets' challenge protection test that offspring pigs of the sows feeding B.S-Dia during pregnancy did not develop diarrhea symptoms and intestinal pathological changes at 48 h after infection with PEDV, and PEDV load in the jejunum and ileum was significantly reduced, but offspring pigs of the sows taking orally PBS during pregnancy developed pronounced diarrhea symptoms and extensive PEDV colonization was noted both in the jejunum and ileum. In summary, sow by oral administration of B.S-Dia substantially increased congenital lactogenic immunity, thereby preventing newborn piglets from being infected with PEDV.

Keywords: 4,4′-diaponeurosporene-producing Bacillus subtilis (B.S-Dia); Congenital lactogenic immunity; Cytokines; Lysozyme and lactoferrin; PEDV.

Fig. 1

(A) Schematic diagram showing oral administration of B.S-Dia, B.S, and PBS to sows; (B) Blood was collected from the auricular vein and analyzed by an automated cell analyzer, n = 5; (C-D) CD14⁺ monocytes and CD3⁺T lymphocytes in blood were detected by flow cytometry, n = 5; (E) The number of positive cells in blood was counted. Differences between the different treatment groups (B, E) were analyzed by one-way ANOVA, n = 5, *P < 0.05

Fig. 2

(A-B) The number of CD14⁺ monocytes and CD3⁺T lymphocytes in colostrum were detected and counted, and significant differences between the different treatment groups were analyzed by one-way analysis of variance, n = 5, *P < 0.05, **P < 0.01; (C-E) Detection of lymphocyte proliferation. Significant differences between different treatment groups (B, E) were analyzed by one-way ANOVA, n = 5, *P < 0.05, **P < 0.01

Fig. 3

(A) Contents of milk fat, lactose, and milk protein in colostrum were analyzed using a milk composition analyzer; (B-D) IgA, IgG, cytokines, and CCL2 levels in colostrum were measured by ELISA; (E) Antiviral proteins in colostrum were evaluated by ELISA. Significant differences between different treatment groups were analyzed by one-way ANOVA, n = 5, *P < 0.05, **P < 0.01

Fig. 4

(A) Anti-PEDV properties of whey after oral administration of B.S-Dia, B.S, and PBS. PBS, whey from sow colustrum in the PBS group was used to treat cells; B.S, cells pretreated with colostral whey in the B. subtilis group; B.S-Dia, the colostral whey from sows in B.S-Dia group was used to treat cells. RT-qPCR (B) and plaque tests (C and D) were used to assess antiviral effects. Differences between different treatment groups were assessed by one-way ANOVA, **P < 0.01. (E) Anti-TGEV effects of whey in the B.S-Dia, B.S., and PBS groups. (F) Plaque tests were used to assess the antiviral effects

Fig. 5

(A) Schematic diagram showing oral infection of piglets with PEDV; (B) Piglet feces were scored by consistency, n = 5; (C) Viral loads in feces were measured by RT-qPCR, n = 5; (D) Clinical symotoms in piglets. Statistical analysis was performed using the two-way ANOVA with repeated measures and Bonferroni’s correction for multiple comparisons, n = 5, *P < 0.05, **P < 0.01

Fig. 6

(A-C) Viral loads in the duodenum, jejunum, and ileum of piglets were measured by RT-qPCR, and differences between the different treatment groups were analyzed by one-way ANOVA, n = 5; (D-E) Viral loads in the jejunum and ileum of piglets were verified by immunofluorescence; Original magnification×100, ×400. scale bars = 50, 20 μm, *P < 0.05, **P < 0.01

Fig. 7

(A) Immunohistochemical assessment of lymphocyte (CD3) numbers in the intestinal tract of piglets, magnification×100, ×400, scale bar = 50 μm; (B) The numbers of CD3⁺T cells were counted in random fields (×400) and differences were analyzed by one-way ANOVA, **P < 0.01. (C) The detection of cytokines and antiviral proteins in the blood of piglets, and differences between the different treatment groups were determined by one-way ANOVA, *P < 0.05, **P < 0.01