Construction of Lactococcus lactis expressing secreted and anchored Eimeria tenella 3-1E protein and comparison of protective immunity against homologous challenge

Experimental Parasitology 178 (2017) 14e20

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Construction of Lactococcus lactis expressing secreted and anchored Eimeria tenella 3-1E protein and comparison of protective immunity against homologous challenge Chunli Ma a, Lili Zhang b, c, Mingyang Gao b, c, Dexing Ma b, c, * a b c

College of Food Science, Northeast Agricultural University, NO. 59 Mucai Street, Harbin 150030, China College of Veterinary Medicine, Northeast Agricultural University, NO. 59 Mucai Street, Harbin 150030, China Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, NO. 59 Mucai Street, Harbin 150030, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Two novel plasmids pTX8048-SP-D31E and pTX8048-SP-NAD3-1E-CWA were constructed.
Two recombinant bacteria L. lactis/ pTX8048-SP-D3-1E and L. lactis/ were pTX8048-SP-NAD3-1E-CWA constructed.
Oral immunization to chickens with live bacteria displaying anchored 31E protein provided more protections.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 November 2016 Received in revised form 8 May 2017 Accepted 16 May 2017 Available online 17 May 2017

Two novel plasmids pTX8048-SP-D3-1E and pTX8048-SP-NAD3-1E-CWA were constructed. The plasmids were respectively electrotransformed into L. lactis NZ9000 to generate strain of L. lactis/pTX8048-SP-D31E in which 3-1E protein was expressed in secretion, and L. lactis/pTX8048-SP-NAD3-1E-CWA on which 3-1E protein was covalently anchored to the surface of bacteria cells. The expression of target proteins were examined by Western blot. The live lactococci expressing secreted 3-1E protein, anchored 3-1E protein, and cytoplasmic 3-1E protein was administered orally to chickens respectively, and the protective immunity and efficacy were compared by animal experiment. The results showed oral immunization to chickens with recombinant lactococci expressing anchored 3-1E protein elicited high 3-1Especific serum IgG, increased high proportion of CD4þ and CD8aþ cells in spleen, alleviated average lesion score in cecum, decreased the oocyst output per chicken compared to lactococci expressing cytoplasmic or secreted 3-1E protein. Taken together, these findings indicated the surface anchored Eimeria protein displayed by L. lacits can induce protective immunity and partial protection against homologous infection. © 2017 Elsevier Inc. All rights reserved.

Keywords: Lactococcus lactis E. tenella Anchored protein Protective immunity

1. Introduction

* Corresponding author. College of Veterinary Medicine, Northeast Agricultural University, NO. 59 Mucai Street, Harbin 150030, China. E-mail address: [email protected] (D. Ma). http://dx.doi.org/10.1016/j.exppara.2017.05.001 0014-4894/© 2017 Elsevier Inc. All rights reserved.

Avian coccidiosis is one of the most common enteric disease caused by protozoan parasite of the Eimeria genus. Eimeria tenella inflicts more severe economic losses on the worldwide poultry industry due to the high morbidity and mortality (Dalloul and

C. Ma et al. / Experimental Parasitology 178 (2017) 14e20

Lillehoj, 2006; Shirley et al., 2004). The conventional control methods against the disease is still dominated by medication with anticoccidials and immunoprophylaxis with live vaccines, but the new effective and safer alternatives are urgently needed because of the rise of drug-resistant Eimeria species, the drug residues in poultry products, the reversion to virulence for attenuated vaccines (Chapman, 2014). With the aim of exploring new vaccine strategies, several delivery carriers have been tried, including fowlpox virus (Yang et al., 2008), attenuated Salmonella enterica serovar Typhimurium (Konjufca et al., 2008), tobacco (Sathish et al., 2011), ginsenoside-based nanoparticles (Zhang et al., 2012), Lactococcus lactis (Ma et al., 2013), Mycobacterium bovis Bacillus CalmetteGuerin (Wang et al., 2014), Bacillus subtilis (Lin et al., 2015), Pichia pastoris (Chen et al., 2015), Escherichia coli (Yin et al., 2015) and pcDNA (Ma et al., 2011; Hoan et al., 2014). Although vaccines based on the above delivery vectors have already been evaluated to be immunogenic, there is no commercial vaccine available till now. In recent years, lactic acid bacterium (LAB) used as vaccine delivery vector becomes more attractive than many other vehicles (BaheyEl-Din et al., 2010). Lactococcus lactis is generally regarded as an ideal vaccine delivery vector to express heterologous protein and has been used to express antigens of enteric pathogens (Kobierecka et al., 2016; Gao et al., 2015). We previously reported oral immunization with recombinant L. lactis expressing cytoplasmic E. tenella 3-1E protein induced partial protection against homologous challenge (Ma et al., 2013). However, will the protective efficacy induced by secreted or cell wall-anchored 3-1E protein be difference from that induced by cytoplasmic 3-1E protein? The main purpose of the present study was to construct the strain of L. lactis/ pTX8048-SP-D3-1E expressing secreted 3-1E protein, and L. lactis/ pTX8048-SP-NAD3-1E-CWA expressing covalently anchored 3-1E protein, and compare the protective immunity against homologous challenge induced by the three 3-1E-expressing L. lactis. 2. Materials and methods

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amplified with primers pair 3-1E-F1 and 3-1E-R1 (Table 2) using plasmid pTX8048-3-1E as template. In the D3-1E fragment, start codon ATG was not contained with the aim to eliminate the restriction enzyme sites Nco I that would interfere the following DNA manipulations. The purified D3-1E fragment was cloned into the BamH I/Xba I sites of pTX8048 vector to generate plasmid pTX8048D3-1E. Then the forward primer SPtrxA-F2 (Table 2) was designed in which the signal peptide of secretion protein Usp45 (SP) from L. lactis MG1363 and the initial 13 bp of E. coli thioredoxin (trxA) were contained. The Splicing by Overlap Extension (SOE) technique was used to amplify the fusion protein gene SP-trxA-His6-D3-1E (named with SP-D3-1E) with primers pair SPtrxA-F2 and 3-1E-R2 (Table 2) using plasmid pTX8048-D3-1E as template. The above fusion gene fragment was cloned into the Nco I/Xba I sites of pTX8048 to generate plasmid pTX8048-SP-D3-1E. For construction of plasmid pTX8048-SP-NAD3-1E-CWA, primers pair 3-1E-F3 and 3-1E-R3 (Table 2) were utilized to amplify the modified D3-1E fragment without terminator codon TAA (named with NAD3-1E) using plasmid pTX8048-SP-D3-1E as template. In 3-1E-R3 primer sequence, the restriction enzyme site Kpn I was introduced for the following DNA manipulation, and the terminator codon TAA of D3-1E gene was deleted with aim to fuse the cell wall-anchor (CWA) sequence containing LPXTG-type anchoring motif of Streptococcus pyogenes M6 protein. The amplified fragment NAD3-1E was cloned into the BamH I/Xba I sites of plasmid pTX8048-SP-D3-1E to generate pTX8048-SP-NAD3-1E. Then the CWA fragment amplified with primers pair CWAF1 and CWAR1 (Table 2) using pLB113 (Cortes-Perez et al., 2005) as template was inserted into the Kpn I/Xba I sites of pTX8048-SP-NAD31E to generate plasmid pTX8048-SP-NAD3-1E-CWA. The above two recombinant plasmids construction was shown in Fig. 1. Both of the two plasmids were verified by PCR, restriction enzyme digestion and sequencing.

2.3. Preparation for secreted and cell wall-anchored 3-1E protein samples

2.1. Strain and plasmids The strains and plasmids used are listed in Table 1. Lactococcus lactis cells were grown at 30  C without shaking in M17 medium (Luqiao, Beijing) supplemented with 5 g/l glucose (GM17) and 5 mg/ ml chloramphenicol. 2.2. Plasmids and recombinant strain construction The original plasmid used in the present study was pTX8048 (Douillard et al., 2011). For construction of plasmid pTX8048-SPD3-1E, 3-1E fragment without start codon ATG (D3-1E) was firstly

The plasmid pTX8048-SP-D3-1E and pTX8048-SP-NAD3-1ECWA was respectively electrotransformed into host bacteria L. lactis NZ9000 using gene pulser apparatus (Bio-Rad, Hercules, CA, USA) to screen recombinant strains of L. lactis/pTX8048-SP-D3-1E and L. lactis/pTX8048-SP-NAD3-1E-CWA. The electroporation was carried out using 100 ml of competent cells and 10 ml of plasmid DNA in 0.2 cm-wide electroporation cuvettes with parameter setting of 2000 V and 25 mF. The identified strains were cultured in GM17 medium with chloramphenicol (5 mg/ml) at 30  C for 48 h. An overnight cultures of bacteria were used to inoculate fresh GM17 medium (1:20) with chloramphenicol (5 mg/ml). The cultured

Table 1 Strains and plasmids used in this study. Bacterial strain or plasmids Strain E. coli DH5a L. lactis subsp. cremoris NZ9000 L. lactis/pTX8048 L. lactis/pTX8048-3-1E L. lactis/pTX8048-SP-D3-1E L. lactis/pTX8048-SP-D3-1E-CWA Plasmid pTX8048 pLB113 pTX8048-3-1E pTX8048-SP-D3-1E pTX8048-SP-D3-1E-CWA

Relevant characteristics

Source or References

SupE44DlacU169(480 lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1, plasmid-free derivate strain of MG1363, with nisR and nisK genes for nisin induction, plasmid-free strain with plasmid pTX8048 in L. lactis NZ9000 with plasmid pTX8048-3-1E in L. lactis NZ9000 with plasmid pTX8048-SP-D3-1E in L. lactis NZ9000 with plasmid pTX8048-SP-D3-1E-CWA in L. lactis NZ9000

TaKaRa NIZO Ma et al. (2013) Ma et al. (2013) This study This study

pNZ8048 carrying 6  his-tagged (His6) and thioredoxin (trxA) gene of E. coli; Cmr with fragment cell-wall anchor (CWA) of the Streptococcus pyogenes M6 protein with fragment encoding 3-1E protein in pTX8048 with fragment encoding signal peptide of secretion protein Usp45 (SP) and D3-1E protein in pTX8048 with fragment encoding signal peptide of secretion protein Usp45 (SP), D3-1E protein and cell-wall anchor (CWA) region with LPXTG-type anchoring motif in pTX8048

Douillard et al. (2011) Cortes-Perez et al. (2005) Ma et al. (2013) This study This study

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Table 2 Primer sequences with their corresponding PCR product size.

bacteria were induced with 5 ng/ml nisin (Sigma-Aldrich), and cultured till OD600 was around 0.5. The cytoplasmic 3-1E protein was prepared as previously described (Ma et al., 2013). The secreted 3-1E protein in culture supernatant and the cell wall-anchored 3-1E protein was respectively prepared according to the reported procedure (Piard et al., 1997) with modifications. Briefly, the secreted proteins existed in culture supernatant were firstly filtered by 0.2-mm-pore-size filters (Millipore, USA), and were then precipitated with trichloroacetic acid (TCA) at final concentration of 16%. The above mixture was cooled for 30 min, and then centrifuged at 12,000g for 30 min at 4  C. The pellet was washed twice with cold acetone, dried, and resuspended in 50 mM NaOH. For cell wall-anchored 3-1E protein, cell pellets were washed twice and resuspended in TES (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 25% sucrose). The buffer TES-LMR (TES containing 1 mg/ml lysozyme, 0.1 mg/ml mutanolysin, 0.1 mg/ml RNase) was used to digest cell walls. After digestion for 30 min at 37  C, the cell walls were pelleted by centrifugation at 2500g for 10 min. The objective cell wall-anchored proteins existed in the supernatant were precipitated with 16% TCA. The harvested cell wall-anchored 3-1E proteins was washed, dried, and resuspended in 50 mM NaOH. 2.4. Westernblot detection of secreted and cell wall-anchored 3-1E protein The prepared protein samples were separated by 12% SDS-PAGE, and then electrophoretically transferred to nitrocellulose

membranes. The membranes were probed with rabbit anti-3-1E specific antibodies (Ma et al., 2011) for 1.5 h. After washing twice with TTBS (Tris-HCl 20 mmol/l, pH 7.5, NaCl 100 mmol/l, 0.1% Tween-20), horseradish peroxidase (HRP)-conjugated goat antirabbit IgG antibodies (Sigma, USA) were added as the secondary antibody, and incubated for 1.5 h. After washing, the bound antibody complexes were examined with 3, 30 diaminobenzidine (DAB). 2.5. Experimental design and immunizing procedure As shown in Table 3, a total of 150 one-day-old specific pathogen-free (SPF) chickens were randomly assigned to six experimental groups and fed with feed free of anticoccidials. Four groups of 25 chickens each were orally inoculated with 1.5  1010 CFU (100 ml) of induced L. lactis/pTX8048, L. lactis/ pTX8048-3-1E, L. lactis/pTX8048-SP-D3-1E, and L. lactis/pTX8048SP-NAD3-1E-CWA respectively at day 5, 6 and 7. Booster immunizations were administrated at day 15, 16 and 17, and 26, 27 and 28. Chickens in the unchallenged control group and challenged control group were orally inoculated with 100 ml PBS. At 38 days of age, all chickens except in the unchallenged control group were orally challenged with 4  104 sporulated oocysts of E. tenella. The strain of E. tenella used in this study was originally developed and maintained in our lab. Specific pathogen-free (SPF) White Leghorn chickens were obtained on the day of hatch from Harbin Veterinary Research Institute, Harbin, China. Animal experiments were carried out based on the regulations of the Animal Experiment Ethic Committee of Northeast Agricultural University, China.

Fig. 1. The schematic illustration of plasmids pTX8048-SP-D3-1E and pTX8048-SP-NAD3-1E-CWA harboring fusion gene SP-TrxA-His6-D3-1E and SP-TrxA-His6-NAD3-1E-CWA, respectively. Plasmid pTX8048-3-1E (A) was constructed in our previous work (Ma et al., 2013). The amplified 3-1E fragment without start codon ATG (D3-1E) was firstly cloned into pTX8048 to generate pTX8048-D3-1E. The fusion gene fragment SP-trxA-His6-D3-1E (SP-D3-1E) was amplified by SOE (Splicing by Overlap Extension) PCR using pTX8048-D3-1E as template, and was then cloned into pTX8048-D3-1E to substitute D3-1E fragment to generate plasmid pTX8048-SP-D3-1E (B). The modified D3-1E fragment without terminator codon TAA (NAD3-1E) was cloned into pTX8048-SP-D3-1E to generate pTX8048-SP-NAD3-1E. Then cell wall anchor (CWA) gene fragment was inserted into pTX8048-SP-NAD3-1E to generate plasmid pTX8048-SP-NAD3-1E-CWA (C).

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Table 3 Experimental groups used for immunization and challenge experiment. Groups

Number of chickens

Immunization

Challenge

1 2 3 4 5 6

25 25 25 25 25 25

L. lactis/pTX8048 L. lactis/pTX8048-3-1E L. lactis/pTX8048-SP-D3-1E L. lactis/pTX8048-SP-NAD3-1E-CWA 100 ml PBS 100 ml PBS

Challenge Challenge Challenge Challenge Unchallenge Challenge

2.6. Serum antibody levels

significant at P < 0.01.

Peripheral blood was collected from chickens in each group (n ¼ 5) at day 38 (on days 10 after third immunity). Sera were prepared and the serum IgG antibody responses were measured by ELISA. Briefly, microtiter plates wells were coated with E. coliexpressed E. tenella 3-1E protein (1 mg/ml) and incubated overnight at 4  C. After three washes with PBS containing 0.05% Tween20 (PBST), the plates were blocked for 1.5 h with 5% skim milk in PBST, washed with PBST and incubated for 1.5 h at 37  C with the diluted sera (1:128). The plates were washed, and the secondary antibody HRP-conjugated goat anti-chicken IgG (Sigma-Aldrich, USA) diluted 1:1500 was added and incubated for 1.5 h at 37  C. The substrate solution containing 0.01% H2O2 and 1 mg/ml o-phenylenediamine was added, and the reaction was stopped with 2M sulfuric acid. Optical density at 450 nm (OD450) was determined with an ELISA reader (Bio-Rad, USA). Each sample was tested in triplicate.

3. Results

2.7. Flow cytometric analysis Spleen samples from chickens in each group (n ¼ 5) were collected in ice-cold PBS and used to prepare single splenocyte suspensions on days 10 post third immunity. The splenocytes were prepared by passage through nylon mesh in PBS with 2% fetal calf serum (FCS), and were isolated on lymphocyte separation medium with a density of 1.007 g/ml (Tianjin Haoyang Biological Manufacture, China) according to the suggested steps. The lymphocytes were collected and washed twice, and finally resuspended in PBS. 1  106 cells in 100 ml PBS were incubated with fluoresceinisothiocyanate (FITC)-conjugated mouse anti-chicken CD4 antibody (0.5 mg/ml) and FITC-conjugated mouse anti-chicken CD8a antibody (0.5 mg/ml) (Southern Biotech Associates, Inc., City, State, Country) for 30 min. Following incubation, the cells were washed with PBS (pH 7.2), and the proportions of CD4þ and CD8aþ cells were measured by flow cytometry (Beckman Coulter). 2.8. Evaluation of immune protection Chickens in each group (n ¼ 10) were weighed both prior to challenge and on day 6 post infection (PI) to record body weight gain (BWG). Five chickens in each group were chosen to assess lesions in cecum on day 7 PI according to the method of Johnson and Reid (1970). Feces from chickens in each group (n ¼ 10) were respectively collected between days 7 and 11 PI and was weighed. Oocyst counting was done as described earlier (Ma et al., 2011), and oocyst decrease ratio was calculated. 2.9. Statistical analyses Data analysis was done by SPSS for Windows version 15.0 (SPSS, Chicago, IL, USA) and was all expressed as means ± SD. Means were evaluated by one-way analysis of variance (ANOVA) Duncan test. Results were considered significant at P < 0.05, and highly

3.1. Construction of L. lactis/pTX8048-SP-D3-1E and L. lactis/ pTX8048-SP-NAD3-1E-CWA The expected 1006 bp fragment which is approximately equal to signal peptide (SP) of Usp45, trxA, His6 and D3-1E was released from plasmid pTX8048-SP-D3-1E upon digestion with Nco I and Xba I. The 435 bp CWA fragment was observed from plasmid pTX8048-SP-NAD3-1E-CWA digested by Kpn I and Xba I. Both the two plasmids were further confirmed by nucleotide sequence analysis. The plasmids pTX8048-SP-D3-1E and pTX8048-SP-NAD31E-CWA was respectively electroporated into L. lactis NZ9000, and the positive bacteria were screened and identified. 3.2. Protein expression assay and immunoblotting analysis The secreted 3-1E protein in culture supernatant and the cell wall-anchored 3-1E protein was respectively separated by 12% SDSPAGE, transferred to nitrocellulose membranes, and the expression of target protein was detected by Western blot. The band corresponding to secreted 3-1E protein (33 kDa) (Fig. 2A) and cell wallanchored 3-1E protein (47 kDa) (Fig. 2B) was respectively observed. 3.3. Immunization with lactococci expressing anchored 3-1E protein induce more IgG and cellular immune responses On days 10 after third immunity, serum IgG titers to 3-1E protein, and the proportions of CD4þ and CD8aþ splenocyte subpopulation from chickens in each group were shown in Fig. 3. The serum IgG level of chickens immunized with lactococci expressing anchored 3-1E protein increased significantly (p < 0.05) compared to chickens in the other groups. The above data demonstrate chickens immunized with lactococci expressing surface anchored 3-1E protein can induce significant high IgG antibody response which contribute to protective effects against challenge. The percentages of CD4þ and CD8aþ cells in splenocyte of chickens immunized with lactococci expressing anchored 3-1E protein was significantly higher than that immunized with lactococci expressing secreted or cytoplasmic 3-1E protein (p < 0.05), and highly significantly higher than that immunized with lactococci harboring empty vector (p < 0.01). 3.4. Immunization with live lactococci expressing anchored 3-1E protein offer more protection The immunization efficacies of 3-1E-expressing lactococci were described in Fig. 4. On days 7 PI, average cecal lesion score from chickens immunized with lactococci diaplaying anchored 3-1E protein was highly significantly lower than chickens in challenged control group (p < 0.01), significantly lower than chickens immunized with lactococci expressing cytoplasmic 3-1E protein

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Fig. 2. Westernblot detection of secreted and cell wall-anchored 3-1E protein. Target protein was separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with rabbit anti-3-1E polyclonal antibodies (Ma et al., 2011). The expected band 33 kDa for secreted 3-1E protein (A) and 47 kDa for cell wall-anchored 3-1E protein (B) was observed. (A) Lane 1, Secreted protein from induced L. lactis/pTX8048 (negative control). Lane 2, Secreted 3-1E protein from induced L. lactis/pTX8048-SP-D3-1E (slightly less than cytoplasmic 31E protein due to cutting of signal peptide during secretion). Lane 3, Cytoplasmic 3-1E protein from induced L. lactis/pTX8048-SP-D3-1E. Lane 4, Secreted 3-1E protein from L. lactis/ pTX8048-SP-D3-1E without induction by nisin (negative control). Lane 5, pre-stained protein molecular weight marker. (B) Lane 1, Secreted 3-1E protein from induced L. lactis/ pTX8048-SP-NAD3-1E-CWA. Lane 2, Cell-wall anchored 3-1E protein from induced L. lactis/pTX8048-SP-NAD3-1E-CWA. Lane 3, Cytoplasmic 3-1E protein from induced L. lactis/ pTX8048-SP-NAD3-1E-CWA. Lane 4, Cell-wall anchored 3-1E protein from L. lactis/pTX8048-SP-NAD3-1E-CWA without induction by nisin (negative control). Lane 5, Cell-wall anchored 3-1E protein from L. lactis/pTX8048 (negative control). Lane 6, pre-stained protein molecular weight marker.

Fig. 3. The serum IgG antibody levels (A), percentage of CD4þ and CD8aþ T cells in spleenocytes (B, C) from chickens in each group after three immunities by 3-1E-expressing L. lactis. On days 5e7, 15 to 17, and 26 to 28, chickens in each group were orally immunized with 1.5  1010 CFU live bacteria of L. lactis/pTX8048. L. lactis/pTX8048-3-1E, L. lactis/ pTX8048-SP-D3-1E and L. lactis/pTX8048-SP-NAD3-1E-CWA, respectively. Chickens in PBS control group were mock-immunized orally with 100 ml PBS alone. The serum IgG antibody levels, the percentage of CD4þ and CD8aþ T cells in the spleenocytes were collected at 38 days of age (that is on days 10 post third immunity). The values represents mean ± SD (n ¼ 5). *p < 0.05, **p < 0.01.

(p < 0.05). Chickens immunized with lactococci displaying surface anchored 3-1E protein showed highly significantly lower oocyst production than that in challenge control group and empty vector group (p < 0.01), significantly lower than chickens immunized with lactococci expressing cytoplasmic or secreted 3-1E protein (p < 0.05). The above results demenstrate chickens immunized with lactococci expressing anchored 3-1E protein imparted better protection than lactococci expressing secreted or cytoplasmic 3-1E protein. Average body weight gain (BWG) in chickens vaccinated with the three 3-1E-expressing lactococci was highly significant difference from that of chickens in challenge control group and empty vector group (p < 0.01). However, the statistical difference in average BWG was not observed among the groups immunized with three recombinant 3-1E-expressing lactococci.

4. Discussion Nowadays there was no commercial vaccines available for avian coccidiosis, and widespread coccidiosis still causes severe annual economic losses to worldwide poultry industries (Chapman et al., 2013). The development of effective vaccines against coccidiosis is urgently required (Blake and Tomley, 2014). For developing a successful vaccine, a stable and effective antigen carrier system is essential. In recent years, lactococcus lactis is extensively used as vector to delivery heterologous protein. We previously reported oral immunization with lactococci expressing cytoplasmic 3-1E protein offered protection against E. tenella challenge (Ma et al., 2013). To further explore the secretion and cell wall-anchoring of 3-1E protein in L. lactis NZ9000, and compare the protective

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Fig. 4. Protective effects of oral immunization with 3-1E-expressing L. lactis against E. tenella infection. At 38 days of age, all chickens except in the unchallenged control group were orally infected with 4  104 sporulated oocysts of E. tenella. Ten chickens in each group were weighed to record body weight gain (A). Five chickens in each group were chosen to assess lesions in cecum on days 6 post infection (PI) (B). Feces from chickens in each group were respectively collected between days 7 and 11 PI to count oocyst shedding and calculate oocyst output per chicken (C). bar represents mean ± SD of values. *p < 0.05, **p < 0.01.

immunity against Eimeria infection, two novel plasmids and two 31E-expressing lactococci were constructed based on our previous research. Plasmid pTX8048-SP-D3-1E carried 3-1E protein gene and signal peptide of secretion protein Usp45 (SP) from L. lactis MG1363, and the live lactococci expressing secreted 3-1E protein was constructed. Plasmid pTX8048-SP-NAD3-1E-CWA harbored 31E protein gene and cell wall-anchor (CWA) sequence containing LPXTG-type anchoring motif of Streptococcus pyogenes M6 protein, and the live lactococci displaying surface anchored 3-1E protein was identified. Both of the target protein were examined by Western blot showing with the expected protein band of 33 kDa and 47 kDa, respectively. It was generally accepted from early studies that cellular immunity plays a key role in mediating protection against Eimeria infection (Lillehoj and Trout, 1996), and humoral immunity appears to be minor role in protection against infection (Sathish et al., 2012). However, several reports showed Eimeria-specific antibodies inhibited parasite invasion into cells in vitro and in vivo (Wallach, 2010; Jiang et al., 2012), demonstrating the role of antibody in eliminating Eimeria infection. In the present study, the 3-1E specific IgG antibody levels in peripheral blood were examined following oral immunization with 3-1E-expressing lactococci. The IgG antibody levels in chickens immunized with L. lactis expressing anchored 3-1E protein was higher than that of chickens in other groups (p < 0.05) after third immunity. Considering the obvious reduced oocyst output per chicken, lower average lesion scores in the ceca of chickens immunized with bacteria expressing anchored 3-1E protein, we deduce that higher anti-3-1E IgG antibodies might bind to sporozoite and merozoite of Eimeria, and inhibit the invasion into intestinal epitheliums. In addition, the lactococci displaying anchored 3-1E protein induced significantly higher percentages of CD4þ and CD8aþ cells compared with lactococci expressing cytoplasmic or secreted 3-1E protein (p < 0.05). The percentage of CD8aþ cells in spleen may partially reflect the enhanced cellular immune status against Eimeria infection. The above results suggested the lactococci expressing anchored 3-1E

protein could induce more effective cellular and humoral immunity in chickens. The protective effects were compared by recording body weight gain (BWG), fecal oocyst output, and average lesions scores in the ceca of infected chickens. The animal experimental results demonstrated that chickens immunized with live lactococci expressing cell wall-anchored 3-1E protein were more protected, as showed by lower oocyst production, lower average lesion scores in ceca compared to chickens in other immunized groups. The above results indicated live lactococci displaying cell wall-anchored 3-1E protein elicited more effective immune responses, which decreased or prevented Eimeria damage to chicken gut tissue. Although the average BWG in the groups immunized with the three recombinant lactococci expressing 3-1E protein was all highly significantly higher than that in the challenge control group and empty vector group, the statistical difference was not observed among the three 3-1E-expressing L. lactis groups, which could be explained that the induced immune response was not strong enough to influence the BWG of experimental chickens. In preliminary experiment, the statistical difference in the immune responses and protective efficacies elicited by the three 3-1Eexpressing lactococci were not observed by inoculating 1  1010 CFU (100 ml) of live bacteria and by challenging 5  104 sporulated oocysts of E. tenella. These previous results could be explained by the fact that the expression levels of target protein in lactococci were generally not high, the degradation of target proteins could not be avoided, and also the host strain L. lactis NZ9000 was lack of long-lasting colonization in chicken intestinal tract, all of which made the existed effective target protein was not difference. In the present study, the dose of 3-1E-expressing lactococci used for immunization was increased, and meanwhile the challenging dose of E. tenella sporulated oocyst was reduced, then the statistical difference among the groups was observed. However, the observed protective efficacy against homologous infection resulting from oral immunization of 3-1E-expressing lactococci was still rather moderate than other reported vaccines based on 3-1E

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antigen (Ma et al., 2011; Lin et al., 2015). We are now conducting the work using the symbiotic bacteria in chicken intestinal as carriers to deliver heterologous antigens, and the related research will be further reported. Acknowledgments We would like to thank Prof. Douwe van Sinderen and Dr. Francois P Douillard (Department of Microbiology, University College Cork), who kindly provided us vector pTX8048, and also thank Prof. Chatel JM and Langella P (INRA, France), who kindly provided us vector pLB113. This work was supported by the National Natural Science Foundation of China (No. 30901061), by the Natural Science Foundation of Heilongjiang Province (No. C201422), by the postdoctoral scientific research developmental fund of Heilongjiang Province (No. LBH-Q14019). References Bahey-El-Din, M., Gahan, C.G., Griffin, B.T., 2010. Lactococcus lactis as a cell factory for delivery of therapeutic proteins. Curr. Gene Ther. 10, 34e45. Blake, D.P., Tomley, F.M., 2014. Securing poultry production from the ever-present Eimeria challenge. Trends Parasitol. 30, 12e19. Chapman, H.D., 2014. Milestones in avian coccidiosis research: a review. Poult. Sci. 93, 501e511. Chapman, H.D., Barta, J.R., Blake, D.P., Gruber, A., Jenkins, M., Smith, N.C., Suo, X., Tomley, F.M., 2013. A selective review of advances in coccidiosis research. Adv. Parasitol. 83, 93e171. Chen, P., Lv, J., Zhang, J., Sun, H., Chen, Z., Li, H., Wang, F., Zhao, X., 2015. Evaluation of immune protective efficacies of Eimeria tenella EtMic1 polypeptides with different domain recombination displayed on yeast surface. Exp. Parasitol. 155, 1e7.
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