Skip to main content
NCBI home page
Poultry Science logoLink to Poultry Science
. 2023 May 19;102(8):102780. doi: 10.1016/j.psj.2023.102780

Construction of recombinant SAG22 Bacillus subtilis and its effect on immune protection of coccidia

Chen Zifan *,1, Zheng Chaojun *,1, Peng Qiaoli *, Zhou Qingfeng †, Du Yunping †, Zhang Huihua *,2
PMCID: PMC10258495  PMID: 37276704

Abstract

Avian coccidiosis causes huge economic losses to the global poultry industry. Vaccine is an important means to prevent and control coccidiosis. In this study, Bacillus subtilis was selected as the expression host strain to express anti Eimeria tenella surface protein SAG22. The synthesized surface protein SAG22 gene fragment of E. tenella was ligated with Escherichia coli-bacillus shuttle vector GJ148 to construct the recombinant vector SAG22-GJ148. And then the recombinant Bacillus strain SAG22-DH61 was obtained by electrotransfer. The results of SDS-PAGE and Western Blot showed that the recombinant protein SAG22 was successfully expressed intracellularly. The immunoprotective effect of recombinant Bacillus strain SAG22-DH61 on broiler chickens was evaluated in 3 identically designed animal experiments. The birds were infected with E. tenella on d 14, 21, and 28, respectively. Each batch of experiments was divided into 4 groups: blank control group (NC), blank control group + infected E. tenella (CON), addition of recombinant SAG22-DH61 strain + infected with E. tenella (SAG22-DH61), addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella (GJ148-DH61). The animal experiments results showed that the average weight gain of the SAG22-DH61 group was higher than that of the infected control group, and the difference was significant in the d 14 and 28 attack tests (P < 0.05); the oocyst reduction rate of the SAG22-DH61 group was much higher than that of the GJ148-DH61 group (P < 0.05); the intestinal lesion count score of the SAG22-DH61 group was much lower than that of the GJ148-DH61 group (P < 0.05). In addition, the SAG22-DH61 group achieved highly effective coccidia resistance in the d 14 attack test and moderately effective coccidia resistance in both the d 21 and 28 attack tests. In summary, recombinant SAG22 B. subtilis has the potential to become one of the technological reserves in the prevention and control of coccidiosis in chickens in production.

Key words: E. tenella, SAG22 protein, immunological effect, Bacillus subtilis

INTRODUCTION

Chicken coccidiosis remains a common parasitic disease in the poultry industry, and its enormous economic damage to the poultry industry cannot be ignored, especially Eimeria tenella is the most pathogenic (Lopez-Osorio et al., 2020). Infection can damage the gastrointestinal tract of chickens, leading to malnutrition absorption, weight loss, and increased susceptibility to infection by other pathogens, and in severe cases, bleeding and death (Chapman, 2014; Song et al., 2017). Currently, the prevention and treatment of coccidiosis in chickens is focused on vaccines and chicken coccidiostats. Although coccidiostats are the most powerful means of treating chicken coccidiosis, the emergence of drug resistance problems has led to a significant reduction in the effectiveness of treatment (Clarke et al., 2014). The application of traditional vaccines such as live coccidial vectors to immunize poultry is indeed effective in preventing coccidiosis, but the risk of contamination of the environment or outbreaks of coccidiosis due to improper use exists at the same time (Chapman, 1998). Therefore, there is an urgent need for the development of new safe and effective anticoccidial vaccines for poultry farming. The major steps in the development of anticoccidial resistance are the exploration of vectors capable of expressing immunogenic Eimeria antigens and antigens with immunogenicity. Novel anticoccidial vaccines have been developed such as NAΔ3-1E protein delivered by Enterococcus faecalis (Chen et al., 2020); Etmic3 (eMama1) delivered by attenuated Salmonella typhimurium (Zhao et al., 2020a) and IMP1 protein delivered by Lactococcus lactis (Ma et al., 2021). The immune responses induced by the Eimeria antigens expressed by the above new expression systems and the immunoprotective effect against Eimeria infected are encouraging.

E. tenella contains secretory organelles that secrete secretory proteins that mediate parasite invasion and survival (Liu et al., 2018), such as surface antigen (SAG), and SAG proteins play an important role in adhesion and invasion in host–parasite interactions (Lopez-Osorio et al., 2020), inducing immune responses such as Th1 and Th2 in chickens against Eimeria increasing body weight, and reducing oocyst numbers (Chow et al., 2011; Liu et al., 2018) and is highly immunogenic, making it a good target for host innate and acquired immune responses (McDonald et al., 1988; Constantinoiu et al., 2008) and therefore recognized as a potential vaccine antigen (Palmieri et al., 2017; Zhao et al., 2020b). In addition, E. tenella is immunologically vulnerable and energetic at the cleistogamous stage, which is important for the life cycle of E. tenella (Rafiqi et al., 2018). Therefore, screening for antigens with high expression in the lytic phase is a reasonable way to develop an anticoccidial vaccine (Clark et al., 2016). SAG22 is one of SAG B gene with peak expression in both second-generation lytics, and it has been shown that SAG22 triggers moderate immune protection (Zhao et al., 2021), so SAG22 could be an effective candidate vaccine antigen against Eimeria crassa.

Gram-positive Bacillus subtilis is usually considered as a probiotic with a high resistance to environmental stress, stable, safe and can promote the growth of beneficial bacteria in the animal intestine, reduce pathogenic bacteria colonization, and maintain intestinal health (Jeong and Kim, 2014; Park and Kim, 2014). In addition, studies have shown that the bacteriophages formed under nutrient deficiency or other stress conditions are highly resistant and immunological (Henriques and Moran, 2007). These advantages may suggest that B. subtilis could be used in the development of live vector vaccines.

Therefore, in this study, B. subtilis with good probiotic performance was selected as the expression host to construct a recombinant strain of B. subtilis that could efficiently express the E. tenella-specific antigenic protein SAG22, and the immunogenicity of the recombinant anticoccidial protein and its protective effect on broiler chickens infected with E. tenella at 3 different ages were initially investigated, providing a technical reserve for the prevention and control of chicken coccidiosis in production. The results showed that the recombinant anticoccidial protein was effective in protecting E. tenella-infected broiler chickens at different ages.

MATERIALS AND TEST METHODS

Primer Design for SAG22 Gene

The SAG22 gene was optimized according to the codon preference of the host bacterium B. subtilis based on the gene sequence of the E. tenella-specific protein SAG22 on NCBI (NW_013543465.1), and a 6 × His tag was added at the end. Shanghai Biotechnology Co., Ltd. synthesized the gene and cloned it into the pUC57 vector, thereby constructing a recombinant plasmid pUC57-SAG22. Snapgene software was applied to design primer pairs SAG22-F and SAG22-R based on the optimized SAG22 gene fragment. The amplified fragment length was estimated to be 707 bp. The primer sequences were as follows: SAG22-F: 5′-CCGgaattcGCACTTTCACTTAGAT-3′; SAG22-R: 5′-CGggatccATGATGATGATGATGAT-3′.

Amplification of SAG22 Gene

The SAG22 fragment with the corresponding enzymatic sites (EcoRI, BamHI) was amplified according to the above primer sequence using pUC57-SAG22 as the template, and the reaction conditions were: predenaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 10 s, 34 cycles of amplification; extension at 72°C for 5 min. After the PCR reaction, all products were electrophoresed on 1% agarose gel (A9539, Sigma, CHN, Darmstadt, Germany) and the amplification results were detected under the gel imaging system (ChemiDocXRS+, BIO-RAD, CA), and if the band size was correct, the products were purified and recovered using the gel recovery kit (DP214, Tiangen Biochemical Technology (Beijing) Co., Beijing, China)

Extraction of GJ148 Plasmid

Take the seed solution of Escherichia coli carrying the empty vector of GJ148 (Microecology Laboratory of Winslow Research Institute, Guangdong, China), inoculate it with LB medium (Sigma, L3522-250G) at 1% inoculum, incubate it overnight in a shaker at 37°C, take 5 mL of the bacterial solution and centrifuge it at 12,000 rpm/min for 1 min, discard the supernatant, and extract the plasmid from the bacterial precipitate according to the instructions of OMEGA Small Extraction Kit (D101-01, Zhongmei Taihe Biotechnology Co., Ltd., Beijing, China)

Enzyme Digestion of Recovered Product and Vector GJ148

The above recovered SAG22 gene amplification product and the extracted plasmid GJ148 were double digested with QuickCutTM EcoRI and QuickCutTM BamHI (Takara Bio, Dalian, China) for 15 min at 37°C. After the digestion reaction, all the products were taken and electrophoresed on 1% agarose gel and placed under gel imaging system to detect the digestion. If the band size was correct, the product was purified and recovered using the gel recovery kit. The recovered products were stored at −20°C.

Ligation and Identification of SAG22 Gene and Cloning Vector GJ148

The above purified and recovered SAG22 gene was ligated overnight at 16°C with the linearized vector GJ148 to construct the recombinant plasmid SAG22-GJ148, and then transformed into DH5α receptor cells (Vidi), and single colonies were picked and cultured the next day for PCR identification, double digestion identification, and gene-sequencing verification.

Electrotransformation of Recombinant Plasmid SAG22-GJ148 Into DH61 Receptor Cells

Prepared B. subtilis receptor cells DH61 (Animal Nutrition Laboratory of Foshan University) were gently blown and mixed with the correctly sequenced positive clone plasmid, added into 0.22 mm electrotransformation cup and left on ice for 30 min, electrotransformed according to the electrotransformation conditions (2 kv/mm, 25 F, 200Ω), 1 mL of resuscitation medium was added immediately after the electrotransformation was completed for recovery; the culture was incubated at 37°C, 225 rpm/min for 3 h. The tube was removed and centrifuged at 5,000 rpm for 2 min; the supernatant was discarded, and the remaining 100 μL of supernatant was mixed with the bacterium by pipetting, spread onto LC agar plates, and incubated overnight at 37°C upside down; single colonies were picked and cultured for subsequent PCR identification, double digestion identification, and gene-sequencing verification.

Induction of SAG22 Protein Expression

The above positive clone was inoculated into LC solid medium and incubated overnight at 37°C in inverted position; the monoclonal strain was inoculated into LC liquid medium and incubated overnight at 37°C at 200 rpm/min; it was transferred to LC liquid medium at 2% and incubated at 37°C at 200 rpm/min for 4 h; the final concentration of 2% maltose was added to induce the culture for about 16 h.

After the above induction, remove the medium, add 3 mL of PBS to resuspend the bacteria, and use a cell ultrasonic crusher (JY92-ⅡDN, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) to ultrasonically crush the bacteria. The crushed supernatant and the crushed precipitate were stored at −80°C.

SDS-PAGE Analysis

Recombinant strains of the above in 40 μL were mixed with 5×Protein Loading Buffer (Amersco) and the samples were in a boiling water bath for 10 min. After the samples had cooled, 25 μL of each was sampled. The electrophoresis program is: 80 v, 20 min; 120 v, 60 min. After the electrophoresis, the gels were stained in Kemas Brilliant Blue staining solution (6104-58, Biofroxx, DE, North Rhine-Westphalia, Germany) for 1 h. The staining solution was discarded, and the gels were completely submerged by adding decolorizing solution and decolorized in a shaking table at room temperature. After decolorization, the gel was placed on a gel developer (Bio-RadXRS+) for observation and record keeping.

Western Blot Identification

After the recombinant strain was induced and expressed, the sample was purified by Ni-NTA, and the purified supernatant and precipitate were mixed with 5×Protein Loading Buffer for 10 min in a boiling water bath. The membrane was incubated with mouse anti-His (1:5,000) as primary antibody and goat anti-mouse IgG (1:6,000) as secondary antibody for 1 h. The membrane was then washed 5 to 6 times with 1× TBST (T1081, Solarbio, Beijing, China) repeatedly for 5 min each time; color developing solution (34580, Thermo, CHN, Waltham, Massachusetts, MA) was added to cover the whole membrane and placed on a gel visualizer for observation and record keeping.

Immunoprotective Test Design for E. tenella Infection

The immune protection of recombinant Bacillus strain SAG22-DH61 on broilers was evaluated through 3 animal experiments. The birds were infected with E. tenella on d 14, 21, and 28, respectively. The dose of challenge was 5.5 × 104, 6.0 × 104, and 8.3 × 104 coccidia oocysts per chicken, respectively. The E. tenella coccidia were provided by (Foshan Zhengdian Biotechnology Co., Foshan, China). The design of the 3 experiments was the same, that is, a total of 96 healthy dwarf yellow broilers (Wenshi Food Group Co., Ltd., Guangdong, China) with similar weights were randomly divided into 4 groups, namely blank control group (NC), blank control group + infected E. tenella (CON), addition of recombinant SAG22-DH61 strain + infected with E. tenella (SAG22-DH61), addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella (GJ148-DH61), with 24 chickens in each group, as shown in Table 1.

Table 1.

Grouping of immune protection against coccidia.

Batch Group Immunization dose Immunization age(d) Immunization pathway Age of infection(d) Infection dose
A NC1 — — — — —
CON2 — — — Day 14 5.5 × 104 oocyst of E.tenella
SAG22-DH613 2 × 108 CFU/bird Daily Drinking water
GJ148-DH614
B NC — — — — —
CON — — — Day 21 6.0 × 104 oocyst of E.tenella
SAG22-DH61 2 × 108 CFU/bird Daily Drinking water
GJ148-DH61
C NC — — — — —
CON — — — Day 28 8.3 × 104 oocyst of E.tenella
SAG22-DH61 2 × 108 CFU/bird Daily Drinking water
GJ148-DH61
Open in a new tab
1

Blank control group.

2

Blank control group + infected E. tenella.

3

Addition of recombinant SAG22-DH61 strain + infected with E. tenella.

4

Addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella.

Evaluation of Test Immune Effect

  • 1)

    Scoring of cecum lesions

The cecum was dissected between days 5 and 7 after infection and the lesions were scored by 4 independent observers in a blinded manner on a scale of 0 (absent) to 4 (high) with reference to the method of Johnson and Reid (1970). In case of inconsistent lesions on both sides of the cecum, the severe side was used (Johnson and Reid, 1970).

  • 2)

    RLS (Rate of reduction in cecum lesion score)

RLS = (mean lesion score in the infected-not-vaccinated group − mean lesion score in the infected-not-vaccinated group) / mean lesion score in the infected-not-vaccinated group × 100%.

  • 3)

    OPG (number of oocysts per gram of feces)

The OPG values of the feces were counted by wheat counting method after taking the chicken cecum feces mixed in equal proportions on the fifth, sixth, and seventh day after the infected.

  • 4)

    ROP (relative oocyst production)

ROP = total number of oviposited eggs in the infected with vaccine group / total number of oviposited eggs in the infected without vaccine group × 100%.

  • 5)

    ACI (anticoccidial index)

ACI = (survival rate + relative weight gain rate) - (lesion value + oocyst value), where ACI below 120 is ineffective anticoccidial; between 120 and 160 is ineffective anticoccidial; between 160 and 180 is moderately effective anticoccidial; above 180 is highly effective anticoccidial (McManus et al., 1968).

Survivalrate=(numberofsurvivingbirdsattheendofthetest/numberofbirdsinthetestgroup)×100%.
Relativeweightgainrate=weightgaininthetestorinfectedgroup/weightgaininthecontrolgroup×100%.
Lesionvalue=cecumlesionscore×10.

RESULTS

Amplification of SAG22 Gene of Coccidioides spp

A PCR amplification product fragment of approximately 707 bp in length was amplified by primer pair SAG22-F/R using pUC57-SAG22 as the template. The results of electrophoresis by 1.0% agar agglutination gel showed that the amplified fragment matched the expected size (Figure 1).

Figure 1.

Figure 1

Open in a new tab

The PCR amplification of gene SAG22. M:DL2000 marker;1: gene SAG22.

Recombinant Plasmid SAG22-GJ148 PCR Identification

Several monoclonal strains grown on resistant plates were selected and cultured overnight, and PCR amplified by the bacterial solution. Strains 1, 2, 3, 5, 6, 9, 10, 11, and 12 amplified fragments of approximately 707 bp in length, which were consistent with the expected size and were initially identified as positive strains. The results are shown in Figure 2.

Figure 2.

Figure 2

Open in a new tab

The PCR amplification of GJ148-SAG22.M;DL2000 marker;1-12: Recombinant plasmid GJ148-SAG22 Identification product.

Identification of Recombinant Plasmid SAG22-GJ148 by Enzyme Digestion

The above suspected positive strain was amplified and the extracted recombinant plasmid SAG22-GJ148 was incompletely digested by EcoR I and BamH I, and 3 bands of different sizes, one of about 707 bp (SAG22), one of about 3497 bp (GJ148), and one of about 4184 bp (SAG22-GJ148), were visible, which were consistent with the expected results (Figure 3).

Figure 3.

Figure 3

Open in a new tab

Enzyme digestion of GJ148-SAG22.M;DL5000 DNA marker;1: EcoRI/BamHI double digestion products;2: Negative control.

Sequencing and Identification of Recombinant SAG22-DH61 Bacillus subtilis

The above plasmids with the expected results were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the sequencing results were exactly as recombinant SAG22-DH61 Bacillus, indicating that the recombinant positive strain was successfully constructed. The positive strain with correct sequencing was named as recombinant SAG22-DH61 B. subtilis strain, hereinafter referred to as recombinant strain.

SDS-PAGE Analysis of Recombinant Protein SAG22

The lysate supernatant of recombinant strain, lysate precipitate of recombinant strain, lysate supernatant of vacuolated bacteria, and lysate precipitate of vacuolated bacteria were subjected to SDS-PAGE electrophoresis, and the results showed that the target bands appeared around 26 kD after Komas Brilliant Blue staining, as shown in Figure 4.

Figure 4.

Figure 4

Open in a new tab

Identification of recombinant SAG22 protein by SDS-PAGE.M.26616 marker;1:Recombinant strain lysis supernatant;2:Recombinant strain lysis bacteriophage precipitation;3:Vaccum lysis supernatant;4:Lysis of bacteriophage precipitated by unloaded bacteria.

Western Blot Analysis of Recombinant Protein SAG22

The recombinant strain and the null strain were crushed after the induction of expression, and the crushed supernatant and the crushed precipitate samples were purified using Ni-NTA and then subjected to Western Blot. In contrast, there was no corresponding band in the sample of the null strain. The results are shown in Figure 5.

Figure 5.

Figure 5

Open in a new tab

Identification of recombinant SAG22 protein by Western. 1:Maker;2:Recombinant strain lysis supernatant;3:Recombinant strain lysis bacteriophage precipitation;4:Vaccum lysis supernatant;5:Lysis of bacteriophage precipitated by unloaded bacteria.

Weight Gain Results in Broilers Infected With E. tenella at Different Ages

In Figure 6, the infected test on d 14 showed the average end weight of the SAG22-DH61 cohort was significantly higher than that of the CON group (P < 0. 05), but not significantly different from the rest of the groups (P > 0.05); the average end weight of the GJ148-DH61 group was significantly higher than that of the NC group (P < 0.05) and significantly higher than that of the CON group (P < 0.01). The relative weight gain rates of the SAG22-DH61 group and GJ148-DH61 groups were 110.81% and 112.81%, respectively, compared with the NC group. The infected test on d 21 showed the weight gain of all groups in the test was lower than that of the NC group, but the difference between the groups was not significant (P > 0.05), with the highest weight gain rate of 94.12% in the SAG22-DH61 group and the lowest in the CON group, which was only 90.66%. The test on d 28 infected with E. tenella showed that there was no significant difference (P > 0.05) in average end weight between the NC group and the SAG22-DH61 group, but both were higher than the CON group and the GJ148-DH61 group, and the difference was significant (P < 0.05), with the relative weight gain rate of the SAG22-DH61 group reaching 97.90%.

Figure 6.

Figure 6

Open in a new tab

Weight gain results in broilers infected with E. tenella at different ages. All data were analyzed with ANOVA: **P < 0.01, and *P < 0.05. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.

Oocyst Reduction Rate

The infected test on d 14 showed (Figure 7A) that the oocyst volume in the GJ148-DH61 group was significantly lower than that of the CON group (P < 0.05); the oocyst volume in the SAG22-DH61 group was not significantly different from that of the NC group (P > 0.05) and was significantly lower than that of the CON group (P < 0.01). This indicated that SAG22-DH61 immunized chickens could better control the amount of fecal oocysts excreted from the cecum. The infected test on d 21 showed (Figure 7B) that the oocyst volume in the SAG22-DH61 group was significantly lower than that in the CON group (P < 0.05). However, the difference between the GJ148-DH61 group and CON group was not significant (P > 0.05). The infected test on d 28 showed (Figure 7C) that the oocyst volume in the SAG22-DH61 group was significantly lower than that in the CON group (P < 0.05), whereas there was no significant difference between the GJ148-DH61 group and the CON group (P > 0.05).

Figure 7.

Figure 7

Open in a new tab

Oocyst output results in broilers infected with E. tenella at different ages. Among them, d 14 broiler infect test is A, d 21 broiler infect test is B, d 28 broiler infect test is C. All data were analyzed with ANOVA: **P < 0.01, and *P < 0.05. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.

Cecum Lesion Scores

In Figure 8, the infected test on d 14 showed that the cecum lesion scores of all test groups were lower than those of the CON group, with the SAG22-DH61 group being the lowest, with a highly significant difference compared to CON group (P < 0.01); the GJ148-DH61 group had lower cecum lesion scores than the CON group, but there was no significant difference (P > 0.05), and the GJ148-DH61 group had lower cecum lesion scores than the CON group (P > 0.05). The infected test on d 21 showed that the cecum lesion scores of all groups were lower than those of the CON group, among which the cecum lesion scores of the SAG22-DH61 group were the lowest, with a highly significant difference compared with the CON group (P < 0.01) and no significant difference compared with the NC group (P > 0.05); the cecum lesion score of SAG22-DH61 group was significantly lower than GJ148-DH61 group (P < 0.05), whereas that of GJ148-DH61 group was lower than that of the CON group, but the difference was not significant (P > 0.05). The infected test on d 28 showed that the cecum lesion scores of all groups were lower and not significantly different (P > 0.05).

Figure 8.

Figure 8

Open in a new tab

Mean lesion scores results in broilers infected with E. tenella at different ages. All data were analyzed with ANOVA: **P < 0.01, and *P < 0.05. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.

Furthermore, in the infected test, the initial body weight of broilers was nonsignificantly different in all groups, whether 14, 21, or 28 days old, and there were no sick or dead chickens, with 0% morbidity and mortality.

Anticoccidial Index

In Figure 9, the infected test on d 14 showed that the anticoccidial indices of the SAG22-DH61 and GJ148-DH61 groups were 189.41 and 167.01, respectively, of which the SAG22-DH61 group reached the high efficiency anticoccidial level and the GJ148-DH61 group the medium efficiency anticoccidial level. The infected test on d 21 showed that the anticoccidial indexes of the SAG22-DH61 and GJ148-DH61 groups were 169.21 and 128.00, respectively, with the SAG22-DH61 group achieving a moderate anticoccidial level and the GJ148-DH61 group a low anticoccidial level. The infected test on d 28 showed that the anticoccidial indices of the SAG22-DH61 and GJ148-DH61 groups were 175.40 and 148.10, respectively, with the SAG22-DH61 group achieving a moderate anticoccidial level and the GJ148-DH61 group a low anticoccidial level.

Figure 9.

Figure 9

Open in a new tab

Anticoccidial index results in broilers infected with E. tenella at different ages. Abbreviations: CON, blank control group + infected E. tenella; GJ148-DH61, addition of recombinant empty vector GJ148-DH61 strain + infected with E. tenella; NC, blank control group; SAG22-DH61, addition of recombinant SAG22-DH61 strain + infected with E. tenella.

DISCUSSION

E. tenella is a specialized intracellular pathogen with high prevalence and disease potential, mainly due to the rapid growth of second-generation lytic colonies within the epithelial cells of the crypt of the cecum and their deep penetration into the lamina propria, as well as the rupture of these lytic infected cells to release second-generation lytic colonies (Fernando et al., 1983), which in turn triggers the exponential growth of coccidia and severely damages the intestine. As can be seen from the above, the invasion of E. tenella SAG is mainly manifested in the Merozoite stage (Kundu et al., 2017). Also cleavers contain proteins capable of inducing an immune response in the host and are highly immunogenic, thus cleavers antigens have potential as vaccine candidates for E. tenella (Song et al., 2021). The SAG protein belongs to a group of E. tenella surface antigens and consists of an N-terminal signal peptide and a C-terminal hydrophobic glycosylphosphatidylinositol (GPI) anchoring protein (Ajioka et al., 1998). All subfamilies encode GPI-anchored signal peptides and additive sites. Depending on the cysteine site, the homologous surface protein genes can be classified as multigene family A, multigene family B, and surface antigen type C, which was discovered in a later study (Reid et al., 2014). Among them, SAG 1-12 genes belong to multigene family A and SAG 13-23 genes belong to multigene family B. The expression of SAG is differentially regulated between oocyst/zygotes and second-generation cleavage stages, with only one being specifically expressed in the zygotes, a few in both stages, and most in the second generation cleavage (Tabares et al., 2004), with the SAG B gene being specifically expressed in the second-generation cleavage peak expression in the second generation of cleistogamy. In recent years, many researchers have investigated the immunogenicity of SAG proteins, such as SAG1 (Vo et al., 2021), SAG2, SAG4 (Zhao et al., 2020b), SAG6, SAG15 (Geng et al., 2022), SAG16, SAG22, SAG23 (Song et al., 2021; Zhao et al., 2021), among which SAG1, SAG4, SAG15, and SAG22 have candidate antigenic genes to be anticoccidial vaccines. In general, protective antigens will prefer proteins expressed in the intracellular phase or in the metabolically active phase of the worm, whereas SAG22 belongs to the SAG B gene, which is capable of efficient expression in the second generation of cleavers. In a study (Zhao et al., 2021), a truncated fragment encoding the surface antigen SAG22 of E. tenella was cloned into the pET-28 and expressed in the E. coli expression system, the recombinant protein SAG22 has immune protective effect on broilers. However, immune protection was only studied on d 14 chicks, and the immune protection was weak. Therefore, this study is the first to explore the use of B. subtilis, which is highly resistant, maintains intestinal health and immunological properties, as the expression host to construct a recombinant strain of B. subtilis that can efficiently express the specific antigenic protein SAG22 of Eimeria cepacia.

Although at present, relatively few studies have been reported on the use of Bacillus as a carrier for coccidial antigen delivery. Unlike other traditional expression systems, B. subtilis, as a probiotic, is not toxic in itself and does not have the risk of inducing disease or returning to virulence; it does not require complicated processes and expensive costs for endotoxin removal or protein purification; and it also has lower requirements for culture media and shorter fermentation cycles, etc. In addition, Bacillus, as a microecological agent with immune adjuvant effect, has certain antimicrobial activity and growth-promoting effect, which can enhance the resistance to coccidial infection by competitively rejecting pathogenic bacteria, stimulating host mucin secretion (Yang et al., 2008), and activating immune response to resist coccidial invasion and prevent secondary infection of coccidiosis. Therefore, if B. subtilis can be used to construct new systems that can express anticoccidial proteins and achieve the dual advantages of delivering antigenic proteins and probiotics, a more efficient technological pathway for the control of coccidiosis may be found.

In general, coccidia tend to infect chickens from d 15 to 50, but few studies have been conducted to investigate the differences in infection of chickens at different ages. From the available literature, when researchers conducted protection tests against infection, the time of infection was generally chosen to be at d 14 (or 15) (Wickramasuriya et al., 2021), 21 (or 22) (Yang, 2015), or 28 (Liao, 2008; Tang, 2018). Wang (2021) investigated the protective effect of oral immunization with recombinant Lactobacillus lactis P32-PgsA-EtCab-NC8 on Hyland brown male chicks in chickens under coccidia-infected conditions at d 15. The results showed that the recombinant strain alleviated the problem of stunted growth caused by coccidial infection and reduced cecum lesions with an ACI value of 170.79, reaching a moderate level of anticoccidial resistance. Wickramasuriya et al. (2021) investigated the protective effect of immunization of chickens with different doses of B. subtilis expressing NK-2 peptide in broilers under coccidial infection conditions at d 15. It was shown that the immunization test group could increase the average daily weight gain and reduce the number of fecal oocysts to some extent. Yang (2015) investigated the protective effect of immunization with different species of probiotics on chicks under conditions of transoral infection with coccidial oocysts at d 21. The results showed a moderate level of anticoccidial resistance with some improvement in weight gain, lesion count score, and oocyst elimination. Tang (2018) investigated the protective effect of chickens immunized with different doses (50 μg, 100 μg) of the prokaryotic expressed protein EtSAG22 under the condition of oral administration of Eimeria coccidia oocysts at 5 × 104 at 28 d of age. The study showed that the relative weight gain rate could reach 94.51%, 87.72%; oocyst reduction rate was 21.09%, 46.26%; cecum lesion count was 2.41, 2.22; ACI value was 145.51, 152.52, and all the above indexes were significantly better than the infected non-immunized group. Therefore, in this research, based on the existing studies, the recombinant B. subtilis SAG22-DH61 was constructed to immunize chickens orally, and the E. tenella infection test was conducted at d 14, 21, and 28, respectively, and the indexes of weight gain, intestinal lesion count score, oocyst reduction rate, and anticoccidial index were counted to observe the immune protection of the recombinant strain. The effect of the recombinant strain was observed.

The invasion of foreign pathogens has a certain impact on the growth and development of poultry. Studies have shown that coccidial oocysts can cause damage to the intestinal tract of broilers, affecting the digestion and absorption of nutrients in chickens, or in the case of coccidial infection, making the body's distribution of nutrients from growth promotion to immune enhancement, thus stunting growth and development (Dalloul et al., 2005; Rochell et al., 2016). B. subtilis, one of the commonly used probiotics, is beneficial in regulating the balance of intestinal flora, promoting the absorption of nutrients, and improving the immunity of the organism (Harding et al., 2008). In the present study on the test of immunoprotective effect against coccidia, the average daily weight gain of both SAG22-DH61 and GJ148-DH61 groups was higher than that of the infected control, which played a mitigating effect on coccidia inhibition of growth and development.

Fecal oocyst counts are a common method to measure the severity of coccidiosis infection in broiler chickens, and the rate of oocyst reduction can, to some extent, reflect the protective effect of coccidiosis vaccines or coccidiostats on chickens. From the results of this study, there were slight differences in the protective effects of different test groups on chickens at different days of infected, with higher oocyst reduction in the d 14 test and lower oocyst reduction in the d 28 test. This indicates that the recombinant protein has an inhibitory effect on the proliferation of oocysts.

The cecum lesion score reflects the severity of intestinal damage in chickens and can evaluate the degree of alleviation of intestinal damage caused by coccidia infection in each test group. Our test showed that the cecum lesion count score of SAG22-DH61 group was significantly lower than that of GJ148-DH61 group in both the d 14 infected test and d 21 infected test, implying that the recombinant protein had a positive effect in alleviating intestinal damage.

The ACI values of the SAG22-DH61 group could reach the moderate anticoccidial level in each of the three tests, and even the high anticoccidial level in the d 14 infected test, which indicated that the recombinant protein had a good protective effect on chickens. It is worth noting that the GJ148-DH61 group had moderate anticoccidial level in the d 14 infected test and low anticoccidial level in the rest of the tests, indicating that B. subtilis coccidioides infected chickens also had a certain degree of protection, which also laid the foundation for future research on B. subtilis as a live bacterial expression host.

Therefore, comprehensive analysis above, recombinant SAG22 B. subtilis SAG22-DH61 was successfully constructed and expressed, and the SAG22-DH61 group had certain immune protection effect on chickens in the immune protection test of E. tenella infection, and the best anticoccidial effect under the infecteding condition at d 14. This may be related to the chicken's own immunity, but the specific influencing factors need to be further investigated, and we will continue to explore the colonization of the recombinant strain in the intestine and the related aspects such as the immune mechanism.

CONCLUSION

The SAG22 gene was synthesized in vitro and cloned into the GJ148 plasmid, and the recombinant plasmid SAG22-GJ148 was introduced into B. subtilis DH61 by electro-transformation techniques to screen out a recombinant SAG22-DH61 B. subtilis strain capable of expressing the SAG22 protein in vitro, and it provided good immune protection against E. tenella infection in chicks by continuous water immunization, with the best anticoccidial level was achieved in the d 14 infection test. In summary, recombinant SAG22 B. subtilis has the potential to become one of the technological reserves in the prevention and control of coccidiosis in chickens in production.

ACKNOWLEDGMENTS

This work was supported by the grants from Guangdong Province Key Construction Discipline Scientific Research Capacity Improvement Project (2022ZDJS040), the Guangdong Province Modern Agriculture Poultry Industry Technology System Innovation Team Construction Project (2023KJ128) and the Special Projects in Key Fields of Guangdong Provincial Department of Education (2019KZDZX2006).

Ethics Statement: The experimental proposals and procedures for the care and treatment of the chicken were approved by the Animal Care and Use Committee of Foshan University, which were in accordance with ethical standards in Laboratory Animal—Guideline for ethical review of animal welfare (The National Standard of the People's Republic of China GB/T 35892-2018).

DISCLOSURES

The 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.

REFERENCES

  1. Ajioka J.W., Boothroyd J.C., Brunk B.P., Hehl A., Hillier L., Manger I.D., Marra M., Overton G.C., Roos D.S., Wan K.L., Waterston R., Sibley L.D. Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the Apicomplexa. Genome Res. 1998;8:18–28. doi: 10.1101/gr.8.1.18. [DOI] [PubMed] [Google Scholar]
  2. Chapman H.D. Evaluation of the efficacy of anticoccidial drugs against Eimeria species in the fowl. Int. J. Parasitol. 1998;28:1141–1144. doi: 10.1016/s0020-7519(98)00024-1. [DOI] [PubMed] [Google Scholar]
  3. Chapman H.D. Milestones in avian coccidiosis research: a review. Poult. Sci. 2014;93:501–511. doi: 10.3382/ps.2013-03634. [DOI] [PubMed] [Google Scholar]
  4. Chen W., Ma C., Wang D., Li G., Ma D. Immune response and protective efficacy of recombinant Enterococcus faecalis displaying dendritic cell-targeting peptide fused with Eimeria tenella 3-1E protein. Poult. Sci. 2020;99:2967–2975. doi: 10.1016/j.psj.2020.03.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chow Y.P., Wan K.L., Blake D.P., Tomley F., Nathan S. Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs) induce inflammatory responses in avian macrophages. PLoS One. 2011;6:e25233. doi: 10.1371/journal.pone.0025233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clark E.L., Macdonald S.E., Thenmozhi V., Kundu K., Garg R., Kumar S., Ayoade S., Fornace K.M., Jatau I.D., Moftah A., Nolan M.J., Sudhakar N.R., Adebambo A.O., Lawal I.A., Alvarez Zapata R., Awuni J.A., Chapman H.D., Karimuribo E., Mugasa C.M., Namangala B., Rushton J., Suo X., Thangaraj K., Srinivasa Rao A.S., Tewari A.K., Banerjee P.S., Dhinakar Raj G., Raman M., Tomley F.M., Blake D.P. Cryptic Eimeria genotypes are common across the southern but not northern hemisphere. Int. J. Parasitol. 2016;46:537–544. doi: 10.1016/j.ijpara.2016.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clarke L., Fodey T.L., Crooks S.R., Moloney M., O'Mahony J., Delahaut P., O'Kennedy R., Danaher M. A review of coccidiostats and the analysis of their residues in meat and other food. Meat. Sci. 2014;97:358–374. doi: 10.1016/j.meatsci.2014.01.004. [DOI] [PubMed] [Google Scholar]
  8. Constantinoiu C.C., Molloy J.B., Jorgensen W.K., Coleman G.T. Antibody response against endogenous stages of an attenuated strain of Eimeria tenella. Vet. Parasitol. 2008;154:193–204. doi: 10.1016/j.vetpar.2008.03.029. [DOI] [PubMed] [Google Scholar]
  9. Dalloul R.A., Lillehoj H.S., Tamim N.M., Shellem T.A., Doerr J.A. Induction of local protective immunity to Eimeria acervulina by a Lactobacillus-based probiotic. Compar. Immunol. Microbiol. Infect. Dis. 2005;28:351–361. doi: 10.1016/j.cimid.2005.09.001. [DOI] [PubMed] [Google Scholar]
  10. Fernando M.A., Lawn A.M., Rose M.E., Al-Attar M.A. Invasion of chicken caecal and intestinal lamina propria by crypt epithelial cells infected with coccidia. Parasitology. 1983;86(Pt 3):391–398. doi: 10.1017/s0031182000050587. [DOI] [PubMed] [Google Scholar]
  11. Geng T., Luo L., Wang Y., Shen B., Fang R., Hu M., Zhao J., Zhou Y. Evaluation of immunoprotective effects of recombinant proteins and DNA vaccines derived from Eimeria tenella surface antigen 6 and 15 in vivo. Parasitol. Res. 2022;121:235–243. doi: 10.1007/s00436-021-07364-9. [DOI] [PubMed] [Google Scholar]
  12. Harding S.V., Fraser K.G., Wykes L.J. Probiotics stimulate liver and plasma protein synthesis in piglets with dextran sulfate-induced colitis and macronutrient restriction. J. Nutr. 2008;138:2129–2135. doi: 10.3945/jn.108.090019. [DOI] [PubMed] [Google Scholar]
  13. Henriques A.O., Moran C.P., Jr Structure, assembly, and function of the spore surface layers. Annu. Rev. Microbiol. 2007;61:555–588. doi: 10.1146/annurev.micro.61.080706.093224. [DOI] [PubMed] [Google Scholar]
  14. Jeong J.S., Kim I.H. Effect of Bacillus subtilis C-3102 spores as a probiotic feed supplement on growth performance, noxious gas emission, and intestinal microflora in broilers. Poult. Sci. 2014;93:3097–3103. doi: 10.3382/ps.2014-04086. [DOI] [PubMed] [Google Scholar]
  15. Johnson J., Reid W.M. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Exp. Parasitol. 1970;28:30–36. doi: 10.1016/0014-4894(70)90063-9. [DOI] [PubMed] [Google Scholar]
  16. Kundu K., Garg R., Kumar S., Mandal M., Tomley F.M., Blake D.P., Banerjee P.S. Humoral and cytokine response elicited during immunisation with recombinant immune mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella. Vet. Parasitol. 2017;244:44–53. doi: 10.1016/j.vetpar.2017.07.025. [DOI] [PubMed] [Google Scholar]
  17. Liao Y. Nanjing Agricultural University; 2008. Immunogenicity Study of the Surface Antigens SAG21 and SAG23 of Eimeria tenella. Master Thesis. [Google Scholar]
  18. Liu T., Huang J., Li Y., Ehsan M., Wang S., Zhou Z., Song X., Yan R., Xu L., Li X. Molecular characterisation and the protective immunity evaluation of Eimeria maxima surface antigen gene. Parasit. Vectors. 2018;11:325. doi: 10.1186/s13071-018-2906-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lopez-Osorio S., Chaparro-Gutierrez J.J., Gomez-Osorio L.M. Overview of poultry Eimeria life cycle and host-parasite interactions. Front. Vet. Sci. 2020;7:384. doi: 10.3389/fvets.2020.00384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ma C., Li G., Chen W., Jia Z., Yang X., Pan X., Ma D. Eimeria tenella: IMP1 protein delivered by Lactococcus lactis induces immune responses against homologous challenge in chickens. Vet. Parasitol. 2021;289 doi: 10.1016/j.vetpar.2020.109320. [DOI] [PubMed] [Google Scholar]
  21. McDonald V., Wisher M.H., Rose M.E., Jeffers T.K. Eimeria tenella: immunological diversity between asexual generations. Parasite Immunol. 1988;10:649–660. doi: 10.1111/j.1365-3024.1988.tb00251.x. [DOI] [PubMed] [Google Scholar]
  22. McManus E.C., Campbell W.C., Cuckler A.C. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 1968;54:1190–1193. [PubMed] [Google Scholar]
  23. Palmieri N., Shrestha A., Ruttkowski B., Beck T., Vogl C., Tomley F., Blake D.P., Joachim A. The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates. Int. J. Parasitol. 2017;47:189–202. doi: 10.1016/j.ijpara.2016.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Park J.H., Kim I.H. Supplemental effect of probiotic Bacillus subtilis B2A on productivity, organ weight, intestinal Salmonella microflora, and breast meat quality of growing broiler chicks. Poult. Sci. 2014;93:2054–2059. doi: 10.3382/ps.2013-03818. [DOI] [PubMed] [Google Scholar]
  25. Rafiqi S.I., Garg R., R. K K., Ram H., Singh M., Banerjee P.S. Immune response and protective efficacy of Eimeria tenella recombinant refractile body protein, EtSO7, in chickens. Vet. Parasitol. 2018;258:108–113. doi: 10.1016/j.vetpar.2018.06.013. [DOI] [PubMed] [Google Scholar]
  26. Reid A.J., Blake D.P., Ansari H.R., Billington K., Browne H.P., Bryant J., Dunn M., Hung S.S., Kawahara F., Miranda-Saavedra D., Malas T.B., Mourier T., Naghra H., Nair M., Otto T.D., Rawlings N.D., Rivailler P., Sanchez-Flores A., Sanders M., Subramaniam C., Tay Y.L., Woo Y., Wu X., Barrell B., Dear P.H., Doerig C., Gruber A., Ivens A.C., Parkinson J., Rajandream M.A., Shirley M.W., Wan K.L., Berriman M., Tomley F.M., Pain A. Genomic analysis of the causative agents of coccidiosis in domestic chickens. Genome Res. 2014;24:1676–1685. doi: 10.1101/gr.168955.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rochell S.J., Parsons C.M., Dilger R.N. Effects of Eimeria acervulina infection severity on growth performance, apparent ileal amino acid digestibility, and plasma concentrations of amino acids, carotenoids, and α1-acid glycoprotein in broilers. Poult. Sci. 2016;95:1573–1581. doi: 10.3382/ps/pew035. [DOI] [PubMed] [Google Scholar]
  28. Song X., Yang X., Zhang T., Liu J., Liu Q. Evaluation of 4 merozoite antigens as candidate vaccines against Eimeria tenella infection. Poult. Sci. 2021;100 doi: 10.1016/j.psj.2020.12.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Song X., Zhao X., Xu L., Yan R., Li X. Immune protection duration and efficacy stability of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 against coccidiosis. Res. Vet. Sci. 2017;111:31–35. doi: 10.1016/j.rvsc.2016.11.012. [DOI] [PubMed] [Google Scholar]
  30. Tabares E., Ferguson D., Clark J., Soon P.E., Wan K.L., Tomley F. Eimeria tenella sporozoites and merozoites differentially express glycosylphosphatidylinositol-anchored variant surface proteins. Mol. Biochem. Parasitol. 2004;135:123–132. doi: 10.1016/j.molbiopara.2004.01.013. [DOI] [PubMed] [Google Scholar]
  31. Tang L. Huazhong Agricultural University; 2018. Study on the Immunoprotective Effect of the Surface Antigens EtSAG4, EtSAG16 and EtSAG22 of Eimeria tenella. PhD Thesis. [Google Scholar]
  32. Vo T.C., Naw H., Flores R.A., Le H.G., Kang J.M., Yoo W.G., Kim W.H., Min W., Na B.K. Genetic diversity of microneme protein 2 and surface antigen 1 of Eimeria tenella. Genes (Basel) 2021;12 doi: 10.3390/genes12091418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang Y. Shandong Agricultural University; 2021. Role of EtCab Protein in Chicken Coccidia Invasion and Its Immunoprotection by Recombinant Lactic Acid Bacteria Vaccine. Master Thesis. [Google Scholar]
  34. Wickramasuriya S.S., Park I., Lee Y., Kim W.H., Przybyszewski C., Gay C.G., van Oosterwijk J.G., Lillehoj H.S. Oral delivery of Bacillus subtilis expressing chicken NK-2 peptide protects against Eimeria acervulina infection in broiler chickens. Front. Vet. Sci. 2021;8 doi: 10.3389/fvets.2021.684818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yang G., Li J., Zhang X., Zhao Q., Liu Q., Gong P. Eimeria tenella: construction of a recombinant fowlpox virus expressing rhomboid gene and its protective efficacy against homologous infection. Exp. Parasitol. 2008;119:30–36. doi: 10.1016/j.exppara.2007.12.009. [DOI] [PubMed] [Google Scholar]
  36. Yang J. Jilin Agricultural University; 2015. Evaluation of the Effect of Probiotics on the Immune Level and Immune Protection of Eimeria flexneri-Infected Chicks. Master Thesis. [Google Scholar]
  37. Zhao N., Lv J., Lu Y., Jiang Y., Li H., Liu Y., Zhang X., Zhao X. Prolonging and enhancing the protective efficacy of the EtMIC3-C-MAR against Eimeria tenella through delivered by attenuated salmonella typhimurium. Vet. Parasitol. 2020;279 doi: 10.1016/j.vetpar.2020.109061. [DOI] [PubMed] [Google Scholar]
  38. Zhao P., Li Y., Zhou Y., Zhao J., Fang R. In vivo immunoprotective comparison between recombinant protein and DNA vaccine of Eimeria tenella surface antigen 4. Vet. Parasitol. 2020;278 doi: 10.1016/j.vetpar.2020.109032. [DOI] [PubMed] [Google Scholar]
  39. Zhao P., Wang C., Ding J., Zhao C., Xia Y., Hu Y., Zhang L., Zhou Y., Zhao J., Fang R. Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo. Parasitol. Res. 2021;120:1861–1871. doi: 10.1007/s00436-021-07105-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Poultry Science are provided here courtesy of Elsevier

RESOURCES