Evaluation of humoral immune status in porcine epidemic diarrhea virus (PEDV) infected sows under field conditions

Abstract

Porcine epidemic diarrhea virus (PEDV) is an economically devastating enteric disease in the swine industry. The virus infects pigs of all ages, but it cause severe clinical disease in neonatal suckling pigs with up to 100% mortality. Currently, available vaccines are not completely effective and feedback methods utilizing PEDV infected material has variable success in preventing reinfection. Comprehensive information on the levels and duration of effector/memory IgA and IgG antibody secreting B cell response in the intestines and lymphoid organs of PEDV-infected sows, and their association with specific antibody levels in clinical samples such as plasma, oral fluid, and feces is important. Therefore, our goal in this study was to quantify PEDV specific IgA and IgG B cell responses in sows at approximately 1 and 6 months post-infection in commercial swine herds, including parity one and higher sows. Our data indicated that evaluation of both PEDV specific IgA and IgG antibody levels in the plasma and oral fluid (but not feces) samples is beneficial in disease diagnosis. PEDV specific B cell response in the intestine and spleen of infected sows decline by 6 months, and this associates with specific antibody levels in the plasma and oral fluid samples; but the virus neutralization titers in plasma remains high beyond 6 months post-infection. In conclusion, in sows infected with PEDV the presence of effector/memory B cell response and strong virus neutralization titers in plasma up to 6 months post-infection, suggests their potential to protect sows from reinfection and provide maternal immunity to neonates, but challenge studies are required to confirm such responses.

Figure 1

Quantification of PEDV-specific IgA and IgG antibody titers in clinical samples of PEDV infected sows. Samples were collected from the uninfected and post PEDV-infected sows as indicated in the Table 1. PEDV specific IgA (A–E) and IgG (F–J) antibody titers were quantified by ELISA, with plates coated either using PEDV whole virus-derived antigen (A–C and F–H), S protein (D, I) or M protein (E, J); in plasma (A, D–F, I, J), oral fluid (B, D, E, G, I, J), and fecal (C–E and H–J) samples. Each bar represents mean OD₄₅₀ value ± SEM from six sows. Lowercase alphabet indicates statistically significant difference between uninfected and PEDV-infected corresponding parity sow groups, and asterisk indicates statistical difference between the indicated PEDV-infected sow groups (“a” or *P < 0.05, “b” or **P < 0.01 and “c” or ***P < 0.001).

Figure 2

Representative pictures showing virus neutralizing antibody response in PEDV infected sows plasma samples. A Fifty TCID₅₀ of virus was used in the NA assay which showed appreciable quantity of infected cells by immunofluorescence (IFA) assay at 24 h post-infection. B Each of representatives experimental plasma sample of PEDV uninfected and infected sows were twofold serially diluted from 1:8 to 1:1024 and mixed with 50 TCID₅₀ of PEDV; and Vero cells treated with that mixture were subjected to IFA and examined under 200×magnification. VN titer was expressed as the reciprocal of the highest dilution ratio of test samples that caused greater than 90% reduction in virus induced fluorescence foci units compared to that of virus control.

Figure 3

Virus neutralizing antibody titers in PEDV infected sows. (A) Plasma, (B) Oral fluid and (C) fecal samples were analyzed for the VN titers against PEDV by immunofluorescence assay. The VN titer was determined as the reciprocal dilution of the test sample that induced greater than 90% inhibition of viral infection. Each bar represents mean VN titers ± SEM from six sows. Lower case alphabet indicates a statistically significant difference (“a” P < 0.05 and “b” P < 0.01) between mock-uninfected and PEDV-infected corresponding primiparous and multiparous sow groups.

Figure 4

Flow cytometry analyses to determine the frequency of IgA ⁺ and IgG ⁺ B cells in mucosal tissues of PEDV infected sows. Mononuclear cells were isolated from ileum (A, B, G, H), mesenteric lymph nodes (C, D, I, J), and spleen (E, F, K, L), and stimulated ex vivo with PEDV whole virus-derived antigen (B, D, F, H, J, L) or medium control (A, C, E, G, I, K) for 6 days. The frequency of B cells positive for IgA (A–F) and IgG (G–L) antibodies were determined by flow cytometry. Each bar represents the mean of percent of lymphocytes positive for IgA or IgG positive B cells ± SEM from six sows. Lowercase alphabet indicates statistically significant difference between uninfected and PEDV-infected corresponding parity sow groups, and asterisk indicates statistical difference between the indicated PEDV-infected sow groups (“a” or *P < 0.05, “b” or **P < 0.01 and “c” or ***P < 0.001).

Figure 5

Analysis of PEDV specific IgA and IgG antibodies secreted by stimulated MNCs of ileum, mesenteric lymph nodes, and spleen. Culture supernatants were harvested on day six from MNCs stimulated ex vivo with PEDV whole virus-derived antigen (B–D, F–H) or control unstimulated (A, E). PEDV specific IgA (A–D) and IgG (E–H) levels were determined by ELISA, with plates coated either with PEDV whole virus-derived antigen (A, B and E, F), S protein (C, G) or M protein (D, H). Each bar represents mean OD₄₅₀ value ± SEM from six sows. Lowercase alphabet indicates statistically significant difference between uninfected and PEDV-infected corresponding parity sow groups, and asterisk indicates statistical difference between the indicated PEDV-infected sow groups (“a” or *P < 0.05, “b” or **P < 0.01 and “c” or ***P < 0.001).

Figure 6

ELISPOT analyses to quantify PEDV specific IgA and IgG antibody secreting cell population in ileum, mesenteric lymph nodes, and spleen of PEDV infected sows. MNCs isolated from ileum (A, B, G, H), MLN (C, D, I, J), and spleen (E, F, K, L) were stimulated ex vivo with PEDV whole virus-derived antigen (B, D, F, H, J, L) or medium control (A, C, E, G, I, K) for 6 days. PEDV specific IgA (A–F) and IgG (G–L) antibody secreting cells (ASCs) were detected by ELISPOT. Each bar represents mean number of PEDV-specific ASCs per 5 × 10⁵ MNCs from six sows. Lowercase alphabet indicates statistically significant difference between uninfected and PEDV-infected corresponding parity sow groups, and asterisk indicates statistical difference between the indicated PEDV-infected sow groups (“a” or *P < 0.05, “b” or **P < 0.01 and “c” or ***P < 0.001).