The immune protection induced by a serine protease from the Trichinella spiralis adult against Trichinella spiralis infection in pigs

Abstract

Trichinellosis is a major foodborne parasitosis caused by Trichinella spiralis. In the present study, a serine protease gene from an adult T. spiralis (Ts-Adsp) cDNA library was cloned, expressed in Escherichia coli and purified by Ni-affinity chromatography. Previous studies of our laboratory have found that mice vaccinated with recombinant Ts-Adsp protein (rTs-Adsp) exhibited partial protection against T. spiralis infection. In this study, the protective effect of rTs-Adsp against T. spiralis infection in pigs was further explored. The cell-mediated and humoral immune responses induced by rTs-Adsp were measured, including the dynamic trends of specific antibody levels (IgG, IgG1, IgG2a and IgM), as well as the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) in the serum. Moreover, the changes in T lymphocytes, B lymphocytes, and neutrophils were measured to evaluate cellular immune responses in pigs vaccinated with rTs-Adsp. The results indicated that a Th1-Th2 mixed immune response with Th1 predominant was induced by rTs-Adsp after vaccination. Flow cytometric analysis showed that the proportions of CD4+ T cells, B cells, and neutrophils in the immunized groups were significantly increased. Furthermore, pigs vaccinated with rTs-Adsp exhibited a 50.9% reduction in the muscle larvae burden, compare with pigs from the PBS group five weeks after challenged. Our results suggested that rTs-Adsp elicited partial protection and it could be a potential target molecule for preventing and controlling Trichinella transmission from pigs to human.

Fig 1. Purification and identification of recombinant rTs-Adsp by SDS-PAGE and western blot.

(A) Purified rTs-Adsp was analyzed by SDS-PAGE. Lane M: protein molecular weight marker, Lane 1: purified rTs-Adsp by Ni-affinity chromatograph. (B) The antigenicity of rTs-Adsp was identified by western blot. Lane 1: rTs-Adsp incubated with serum from pig infected with T. spiralis at 60 dpi, Lane 2: rTs-Adsp incubated with serum from Trichinella–negative pig.

Fig 2. Immune responses from the vaccinated pigs.

(A) The levels of IgG against rTs-Adsp were measured by ELISA. (B) The levels of IgM in the sera of immunized pigs were measured by ELISA. (C) IgG subclass responses to the rTs-Adsp were measured by ELISA. (D) The IgG1 subclass responses against rTs-Adsp were evaluated at different time points. (E) The IgG2a subclass responses against rTs-Adsp were evaluated at different time points. The values shown for each group are the mean ± SD of the antibody levels. Significant differences were as follows: *p< 0.05; **p< 0.01; ***p< 0.001.

Fig 3. Cytokine production from sera were evaluated by ELISA.

The levels of (A) IFN-γ (B) IL-2 (C) IL-4 (D) IL-10 are presented as the mean ± SD (n = 6). Asterisks indicate that the production of cytokines from immunized group are significantly different from (*p<0.05, **p<0.01, ***p< 0.001) that of PBS control group.

Fig 4. Flow cytometry analysis of T lymphocytes in peripheral blood.

The gating strategy are shown in (A) and (B). The percentage change of CD4⁺ and CD8⁺ T cells in peripheral blood lymphocytes from (C) PBS-control group (D) IMS1313 adjuvant group (E) rTs-Adsp group. Statistical analysis of (F) the percentage of CD4⁺ T lymphocytes (G) the percentage of CD8⁺ T lymphocytes. The values are shown as mean ± SD. Significant differences are as follows: *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Flow cytometry analysis of B lymphocytes in peripheral blood.

The gating strategy are shown in (A) and (B). The populations change of B lymphocytes in peripheral blood from (C) PBS-control group (D) IMS1313 adjuvant group (E) rTs-Adsp group. Statistical analysis of (F) the percentage of CD3⁺ T lymphocytes (G) the percentage of CD21⁺ B lymphocytes. The values are shown as mean ± SD. Significant differences are as follows: *p<0.05, **p<0.01, ***p<0.001.

Fig 6. Flow cytometry analysis of neutrophils in peripheral blood.

The gating strategy are shown in (A) and (B). The percent change of neutrophils in peripheral blood from (C) PBS-control group (D) IMS1313 adjuvant group (E) rTs-Adsp group. Statistical analysis of (F) the percentage of neutrophils. The values are shown as mean ± SD. Significant differences are as follows: *p<0.05, **p<0.01, ***p<0.001.

Fig 7. Protective immunity of rTs-Adsp-vaccinated pigs after being challenged with 5000 Trichinella spiralis larvae.

The results are shown as the mean ± SD (n = 6). Asterisks indicate that muscle larvae burden of immunized group is significantly different from (*p<0.05) that of PBS control group.