In ovo vaccination using Eimeria profilin and Clostridium perfringens NetB proteins in Montanide IMS adjuvant increases protective immunity against experimentally-induced necrotic enteritis

Abstract

Objective: The effects of vaccinating 18-day-old chicken embryos with the combination of recombinant Eimeria profilin plus Clostridium perfringens (C. perfringens) NetB proteins mixed in the Montanide IMS adjuvant on the chicken immune response to necrotic enteritis (NE) were investigated using an Eimeria maxima (E. maxima)/C. perfringens co-infection NE disease model that we previously developed.

Methods: Eighteen-day-old broiler embryos were injected with 100 μL of phosphate-buffered saline, profilin, profilin plus necrotic enteritis B-like (NetB), profilin plus NetB/Montanide adjuvant (IMS 106), and profilin plus Net-B/Montanide adjuvant (IMS 101). After post-hatch birds were challenged with our NE experimental disease model, body weights, intestinal lesions, serum antibody levels to NetB, and proinflammatory cytokine and chemokine mRNA levels in intestinal intraepithelial lymphocytes were measured.

Results: Chickens in ovo vaccinated with recombinant profilin plus NetB proteins/IMS106 and recombinant profilin plus NetB proteins/IMS101 showed significantly increased body weight gains and reduced gut damages compared with the profilin-only group, respectively. Greater antibody response to NetB toxin were observed in the profilin plus NetB/IMS 106, and profilin plus NetB/IMS 101 groups compared with the other three vaccine/adjuvant groups. Finally, diminished levels of transcripts encoding for proinflammatory cytokines such as lipopolysaccharide-induced tumor necrosis factor-α factor, tumor necrosis factor superfamily 15, and interleukin-8 were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with IMS 106, and profilin plus NetB toxin in combination with IMS 101 compared with profilin protein alone bird.

Conclusion: These results suggest that the Montanide IMS adjuvants potentiate host immunity to experimentally-induced avian NE when administered in ovo in conjunction with the profilin and NetB proteins, and may reduce disease pathology by attenuating the expression of proinflammatory cytokines and chemokines implicated in disease pathogenesis.

Keywords: Clostridium perfringens; Eimeria; Immunity; In ovo Vaccination; Necrotic Enteritis; Profilin.

Figure 1

Effects of in ovo immunization with recombinant profilin plus necrotic enteritis B-like proteins plus Montanide IMS adjuvants on body weight gains. Chicken were immunized and were uninfected or co-infected with Eimeria maxima and Clostridium perfringens as described in Table 1. Body weight gains were measured between days 14 and 20. Each bar represents the mean±standard deviation value (n = 20). Bars not sharing the indicated letters are significantly different according to the Duncan’s multiple range test (p<0.05).

Figure 2

Effects of in ovo immunization with recombinant profilin plus necrotic enteritis B-like (NetB) proteins plus Montanide IMS adjuvants on gut lesions. Chicken were in ovo injected with PBS (NE control) or profilin plus NetB plus IMS 106 or IMS 101 with the indicated protein/adjuvant combinations. The birds were co-infected with Eimeria maxima (EM) on day 14 and Clostridium perfringens (CP) on day 18, and lesion scores were assessed on day 20 using a scale from 0 (none) to 4 (high). Each bar represents the mean±standard deviation. value of the combined observations on 5 chicken by three independent observers (n = 15). Bars not sharing the indicated letters are significantly different according to the Duncan’s multiple range test (p<0.05).

Figure 3

Effects of in ovo immunization with recombinant profilin plus NetB proteins in combination with Montanide adjuvants on necrotic enteritis B-like (NetB) antibody levels after necrotic enteritis (NE) infection. Chicken were in ovo immunized and were uninfected or co-infected with Eimeria maxima (EM) and Clostridium perfringens (CP) as described in Table 1. The concentration of serum antibodies against NetB toxin were measured by enzyme-linked immunosorbent assay at the indicated days post-hatch. Each bar represents the average antibody concentration in ng/mL (n = 3). The letters (a, b) indicate significantly increased NetB antibody concentrations comparing the profilin+NetB/IMS 106 and profilin+NetB/IMS 101 groups with profilin alone or NE groups (p<0.05). Numerical values shown below the figure are average serum antibody concentrations.

Figure 4

Effects of in ovo vaccination with profilin plus necrotic enteritis B-like (NetB) proteins plus IMS 101 or IMS 106 on IL-8, and TNFSF15 transcript levels in intestinal IELs. Chicken were in ovo immunized and co-infected with Eimeria maxima and Clostridium perfringens as described in Table 1. Intestinal IELs were isolated at day 20, and the levels of (A) IL-8 and (B) TNFSF15 mRNAs were measured by quantitative RT-PCR at day 20 and normalized to GAPDH transcript levels. Each bar represents the mean±SD. value (n = 4). Within each graph, bars not sharing the indicated letters are significantly different according to the Duncan’s multiple range test (p<0.05). Numerical values below the figures show the averages normalized RT-PCR measurements. IL-8, interleukin-8; TNFSF15, tumor necrosis factor superfamily 15; IELs, intraepithelial lymphocytes; qRT-PCR, quantitative real time polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.