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. 2021 Jun 4:8:684818.
doi: 10.3389/fvets.2021.684818. eCollection 2021.

Oral Delivery of Bacillus subtilis Expressing Chicken NK-2 Peptide Protects Against Eimeria acervulina Infection in Broiler Chickens

Samiru S Wickramasuriya  1 , Inkyung Park  1 , Youngsub Lee  1 , Woo H Kim  1   2 , Chris Przybyszewski  3 , Cyril G Gay  4 , Jolieke G van Oosterwijk  3 , Hyun S Lillehoj  1
Affiliations

Oral Delivery of Bacillus subtilis Expressing Chicken NK-2 Peptide Protects Against Eimeria acervulina Infection in Broiler Chickens

Samiru S Wickramasuriya et al. Front Vet Sci. .

Abstract

Chicken NK-lysin peptide 2 (cNK-2) is a natural lytic peptide with direct cytotoxicity against many apicomplexan parasites including Eimeria. Developing an effective oral delivery strategy to express cNK-2 in the intestine, where Eimeria parasites interact with the host's gut epithelial cells, may effectively reduce the fecundity of parasites and minimize intestinal damage. Furthermore, cNK-2 modulates gut immune responses to decrease local inflammation elicited by Eimeria infection in the intestine. Therefore, we developed a stable strain of Bacillus subtilis (B. subtilis) that carries cNK-2 to the gut to determine its effectiveness in ameliorating the negative impacts of coccidiosis and to replace the use of antibiotics in controlling coccidiosis in commercial broiler chicken production. Chickens were randomly allocated into eight treatment groups: two control groups (NC: E. acervulina infected non-B. subtilis control; CON: non-infected control); three B. subtilis-empty vector (EV) groups (EV6: 106 cfu/day/bird; EV8: 108 cfu/day/bird; EV10: 1010 cfu/day/bird), and three B. subtilis-cNK-2 groups (NK6: 106 cfu/day/bird; NK8: 108 cfu/day/bird; NK10: 1010 cfu/day/bird). All chickens, except those in the CON group, were challenged with 5,000 freshly sporulated E. acervulina oocysts through oral gavage on day 15. Chickens were given an oral dose of B. subtilis on days 14, 15, and 16. Body weight, weight gains, and fecal oocyst shedding were measured. To investigate the efficacy of oral B. subtilis-cNK-2 against coccidiosis, gene expression of gut health-related biomarkers was measured using RT-PCR. Markers included SOD1, CAT, and HMOX1 for oxidative stress in the spleen and intestinal mucosa, OCLN, ZO-1, and JAM2 for tight junction proteins, and MUC2 for mucin gene expression in the gut. The results showed that oral treatment of young chickens with B. subtilis-cNK-2 improved growth performance, enhanced gut integrity, and reduced fecal oocyst shedding. Altogether, these results confirm B. subtilis-cNK-2 treatment as a promising and effective alternative strategy to replace antibiotics against coccidiosis based on its ability to reduce parasite survival, to reduce coccidiosis-induced body weight loss, and to decrease gut damage based on the enhanced expression of proteins associated with gut integrity and intestinal health.

Keywords: Bacillus subtilis; NK-lysin; antimicrobial peptide; chicken; coccidiosis; growth performance; gut health; oxidative stress.

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Conflict of interest statement

JO and CP are employed by US Biologic, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic outline of the experimental design.
Figure 2
Figure 2
Effect of B. subtilis expressing empty vector (B. subtilis-EV) or chicken NK2 (B. subtilis-cNK-2) in vitro. Eimeria acervulina sporozoites (1.0 × 107/mL) were incubated with culture supernatant from B. subtilis-EV or B. subtilis-cNK-2 for 3 h at 41°C. Chicken NK-lysin (cNK-2) was used as control at a concentration of 100 μg/mL. Sporozoites were stained with a fluorescence viability dye and viable sporozoites were counted microscopically. a, bBars with no common letter differ significantly (p < 0.05).
Figure 3
Figure 3
Fecal oocyst counts of Eimeria acervulina-infected chickens fed daily oral treatment with Bacillus subtilis-cNK-2. All chickens except CON were infected by oral gavage at day 15 with 5,000 oocysts/chicken of E. acervulina. B. subtilis were administrated by oral gavage at days 14–16. EV, B. subtilis-EV; NK, B. subtilis-cNK-2; NC, E. acervulina infected non-B. subtilis control; EV6, B. subtilis (empty vector) at 106 cfu/day; EV8, B. subtilis-EV at 108 cfu/day; EV10, B. subtilis-EV at 106 cfu/day; NK6, B. subtilis-cNK-2 at 106 cfu/day; NK8, B. subtilis-cNK-2 at 108 cfu/day; NK10, B. subtilis-cNK-2 at 1010 cfu/day. a−cBars with no common letter differ significantly (p < 0.05). Each bar represents the mean ± SEM (n = 8). Fecal samples were collected from 6 to 9 dpi to calculate the oocyst shedding.
Figure 4
Figure 4
Tight junction gene expression in duodenal mucosa of Eimeria acervulina-infected broiler chickens fed orally Bacillus subtilis expressing cNK-2 (13 dpi). All chickens except CON were infected by oral gavage at day 15 with 5,000 oocysts/chicken of E. acervulina. B. subtilis was administrated by oral gavage at days 14–16. EV, B. subtilis (empty vector), NK, B. subtilis-cNK-2; NC, E. acervulina infected non-B. subtilis control; EV6, B. subtilis-EV at 106 cfu/day; EV8, B. subtilis-EV at 108 cfu/day; EV10, B. subtilis-EV at 1010 cfu/day; NK6, B. subtilis-cNK-2 at 106 cfu/day; NK8, B. subtilis-cNK-2 at 108 cfu/day; NK10, B. subtilis-cNK-2 at 1010cfu/day. Transcript levels of (A) occludin (OCLN), (B) zonula occludens-1 (ZO1), (C) junctional adhesion molecule 2 (JAM2), and (D) Mucin-2 (MUC-2) in duodenal mucosa were measured by quantitative RT-PCR and genes expression were analyzed using the 2−ΔΔCt method. a−dBars with no common letter differ significantly (p < 0.05). Each bar represents the mean ± SEM (n = 5).
Figure 5
Figure 5
Anti-oxidant gene expression in duodenal mucosa of Eimeria acervulina-infected broiler chickens fed orally Bacillus subtilis expressing cNK-2 (13 dpi). All chickens except CON were infected by oral gavage at day 15 with 5,000 oocysts/chicken of E. acervulina. B. subtilis were administrated by oral gavage at days 14–16. EV, B. subtilis-EV; NK, B. subtilis-cNK-2; NC, E. acervulina infected non-B. subtilis control; EV6, B. subtilis-EV at 106 cfu/day; EV8, B. subtilis-EV at 108 cfu/day; EV10, B. subtilis-EV at 1010 cfu/day; NK6, B. subtilis-cNK-2 at 106 cfu/day; NK8, B. subtilis-cNK-2 at 108 cfu/day; NK10, B. subtilis-cNK-2 at 1010 cfu/day. Transcript levels of (A) superoxide dismutase 1 (SOD1), (B) catalase (CAT), (C) heme oxygenase (HMOX1) in duodenal mucosa were measured by quantitative RT-PCR and gene expression were analyzed using the 2−ΔΔCt method. a−cBars with no common letter differ significantly (p < 0.05). Each bar represents the mean ± SEM (n = 5).
Figure 6
Figure 6
Anti-oxidant gene expression in spleen of Eimeria acervulina-infected broiler chickens fed orally B. subtilis expressing cNK-2 (13 dpi). All chickens except CON were infected by oral gavage at day 15 with 5,000 oocysts/chicken of E. acervulina. B. subtilis were administrated by oral gavage at days 14–16. EV,vB. subtilis-EV; NK, B. subtilis-cNK-2; NC, E. acervulina infected non-B. subtilis control; EV6, B. subtilis-EV at 106 cfu/day; EV8, B. subtilis-EV at 108 cfu/day; EV10, B. subtilis-EV at 1010 cfu/day; NK6, B. subtilis-cNK-2 at 106 cfu/day; NK8, B. subtilis-cNK-2 at 108 cfu/day; NK10, B. subtilis-cNK-2 at 1010 cfu/day. Transcript levels of (A) superoxide dismutase 1 (SOD1), (B) catalase (CAT), (C) heme oxygenase (HMOX1) in the spleen were measured by quantitative RT-PCR and genes expression were analyzed using the 2−ΔΔCt method. a−dBars with no common letter differ significantly (p < 0.05). Each bar represents the mean ± SEM (n = 5).
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