Effects Of Orally Administered Recombinant Lactobacillus Casei Expressing HN Protein On Early Growth Performance, Intestinal Health And Protection Against NDV Challenge In Chickens


 Backgroud: Newcastle disease virus (NDV) is considered one of the most important diseases among chickens. In this study, we generated recombinant surface-displayed Lactobacillus casei (L.casei) expressing the hemagglutinin-neuraminidase (HN) of NDV and a live vector pPG alone (named Lc-pPG-HN and Lc-pPG), and evaluated their effects on early growth development, intestinal health and protection against NDV challenge in chickens. 270 chickens were randomly divided into three groups: Lc-pPG-HN, Lc-pPG and physiological saline (control) group, and chickens from each group were respectively immunized with Lc-pPG-HN, Lc-pPG and physiological saline on 1 and 10 days. Results: Recombinant L.casei expressing the HN protein of NDV (Lc-pPG-HN) was successfully constructed. Orally immunized with Lc-pPG-HN could significantly increase body weight (BW) and immune organs index. Moreover, Lc-pPG-HN improved secretory immunoglobulin A (SIgA) in jejunum, the relative abundance of flora in cecum, histomorphological development of small intestine. In addition, the similar enhancement effects were also observed with hemaggluti-nation inhibition (HI) antibody titer and the expression of cytokines in the serum. The oral administration of Lc-pPG-HN also provided effective protection and alleviated the symptoms of NDV challenge.Conclusions: Thus, a recombinant L.casei vaccine expressing HN may be a potential therapeutic candidate against NDV and improve chickens growth and development.


Introduction
Newcastle disease (ND) is considered one of the most devastating infectious diseases among domestic birds and considerably threatens commercial poultry production [1]. Current live attenuated ND virus (NDV) vaccines are unsatisfactory, and a viral vector vaccine that infects chickens but does not cause disease is needed [2,3]. The NDV surface envelope is mainly surrounded by the viral hemagglutininneuraminidase (HN) glycoprotein, which plays a critical role in viral infectivity and pathogenicity, they produce virus-neutralizing antibody responses and are protective antigens [4]. One study showed that HN also protected chickens during a virulent NDV challenge, although the chickens showed lower neutralizing antibody titers. Thus, live vector vaccines centering on the NDV HN may protect against heterologous pathogens [5]. However, the individual contributions of the HN protein to the growth performance and intestinal health was not clearly known.
To date, many probiotic bacteria have been screened for their potential as animal feed additives to increase the nutritional value and provide additional health bene ts. More recently, probiotics have been developed for human use; however, these products must be rigorously tested to ensure their safety and e cacy [6]. Studies have con rmed that probiotics can increase immunity, maintain intestinal health, and reduce pathogen proliferation, thereby enhancing animal and human health [7]. Thus, oral probiotics have signi cant health and economic bene ts. Oral recombinant vaccines using lactic acid bacteria (LAB) as carriers are being extensively researched. As normal inhabitants of the intestines, LAB readily colonize the intestinal mucosa, making LAB vaccines simple to administer while stimulating a good immune response [8]. Previous studies have developed many Lactobacillus-associated model vaccines, such as L. acidophilus expressing Helicobacter pylori adhesin [9], L.casei producing porcine epidermal growth factor (pEGF) [10] and expressing muramyl dipeptide and tuftsin [11]. Studies have shown that intranasally or orally administering these vaccines can directly reduce antigen levels [12]. However, while genetic manipulation can enhance the immunogenicity of the proteins used in a vaccine, intestinal digestive enzymes rapidly degrade the vaccine components. Thus, a major focus of oral vaccine research is to optimize the environmental stress tolerance of antigen-presenting vectors and enhance antigen expression in the gut. Interestingly, LAB have immunoadjuvant properties and readily express foreign antigens, making them excellent candidates as vaccine vectors. Recent studies of genetically engineered LAB vaccines show that they have great potential for oral vaccine use [13,14].
Recently, different type of recombinant L.casei have been studied as a novel vaccine in poultry. In particular, some studies showed that NDV HN-expressing L.casei via oral administration increased immune responses and did not impair the growth performances [15]. There is still little knowledge on how the dosage and the type of vectored vaccines may affect the growth performances, and intestine health of chickens. Therefore, the aim of this study was to evaluate the effects of recombinant L.casei inclusion on growth performance, intestinal health and immune system of chickens. The results suggested that the oral L.casei vaccine may provide a promising strategy for controlling viral infections in poultry.

Materials And Methods
Animal ethics statement 270 healthy chickens (Hy-line Variety White) were obtained from a local chickens factory (Runcheng Chickens Breeding Factory, Changchun, China). We followed all animal care protocols regarding the use of live chickens in our experiments, and all chickens had access to food, water, and necessary veterinary care add libitum.
Bacterial strains, plasmids and growth conditions Lactobacillus casei ATCC 393 was isolated from cheese, a plasmid-free strain grown in De Man, Rogosa and Sharpe grown in De Man, Rogosa and Sharpe (MRS) medium (Oxoid, UK) at 30 • C, without shaking. The Escherichia coli-Lactobacillus shuttle vector pPG, a type of cell-surface expression plasmid containing an anchoring matrix-encoding pgsA gene derived from Bacillus subtilis behind the target gene. The pPG has the ssUSP secretion signal before the target gene to ensure the target protein secretion.
Escherichia coli (E.coli) competent cells MC1061 was grown in Luria-Bertani (LB) medium at 37 • C with shaking for the cloning of plasmid. Chloramphenicol (Cm) (Sigma, USA) was utilized at nal concentration of 10 μg/mL. Newcastle disease virus isolated from the allantoic uid.
Generation of recombinantL. caseiexpressing HN gene The HN gene (1617 bp) of NDV strain (GenBank: ABG56153.1) was ampli ed by PCR using the following primers with Nco I or EcoR V site underlined (forward primer: 5' CCCATGGATGGACCGCGC GGTTAACAGAGTC 3', Reverse primer: 5' AAGATATCTTAAACTCTATCATCCTTGAGGATC 3'); The PCR product of HN gene was cleaved with Nco I/EcoR V restriction endonuclease and inserted into the corresponding sites of pPG to give rise to pPG-HN. Finally, the recombinant pPG-HN and pPG plasmids were transformed into L.casei ATCC 393 by electroporation as previously described [16].

Western blot analysis
The expression of recombinant HN gene in L.casei was detected by Western blot analysis as described previously [17]. Brie y, Lc-pPG-HN and Lc-pPG were grown in basal MRS (MRS Broth) medium supplemented with 10 μg/mL of chloramphenicol Xylose was added to the culture medium to a nal concentration of 10 g/L to induce antigen expression. After induction at 30 • C for 10 h, approximately 1×10 8 cells pellets or supernate (a 10-fold concentration) were examined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk, and then incubated with Rabbit anti-HN serum at dilution of 1:500 with phosphate buffered saline (Bioss, Beijing, China), overnight at 4 • C. A nity-puri ed horseradish peroxidase (HRP)-conjugated goat anti-Rabbit immunoglobulin G (IgG) (Bioss, Beijing, China) was used as second antibody. Finally, the blot was visualized by chemiluminescence detection with Western ECL substrate (Termo Scienti c) in an Amersham Imager 600 (GE Healthcare, UK).

Immuno uorescence assay
Immuno uorescence was performed to determine the surface-displayed localization of the HN protein from Lc-pPG-HN as previously described [17]. In brief, 1x10 5 cells of Lc-pPG-HN were resuspended in 50 μL PBS containing 1% bovine serum albumin (BSA) and rabbit anti-HN antiserum (1:1000 dilution) at 37 • C for 1 h Subsequently, cells were incubated with uorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (Bioss, Beijing, China) containing 1% Evans blue at 37 • C for 2 h. The cells were visualized using a uorescence microscopy (Zeiss LSM710). The Lc-pPG was used as negative control.
Vaccine preparation,animal treatment and sample collection For oral administration, overnight cultures of recombinant L. casei grown in De Man, Rogosa and Sharpe medium supplemented with chloramphenicol and xylose were thoroughly mixed with commercial basal diet feed, then oven-dried at 37 °C for 8 h. Recombinant L.casei counts were expressed as 1×10 9 CFU/mL and 2×10 9 CFU/mL of sample. Three groups of with 3 replicates tray levels of 30 chickens each orally immunized with Lc-pPG-HN, Lc-pPG or the 0.9% physiological saline feed. Oral vaccinations were conducted on days 1 (1×10 9 CFU/g, 1 mL per chick) and 10 (2×10 9 CFU/g, 1 mL per chick), following the immune protocol administered on 3 consecutive days (Fig. 1). All samples were chosen, and the organs weight and body weight (BW) were measured on 1, 7, 14 and 21 days-old. Hematoxylin and eosin (H&E) staining was used to observe the changes in intestinal morphological structure (duodenum, jejunum and ileum) and organ pathologies (spleen, bursa of Fabricius, thymus, lung and liver). Serum was stored at −80 °C until further analysis. Pieces of the cecum and jejunum were rapidly excised, frozen in liquid nitrogen, and stored at −80 °C until DNA extraction.

Immune organ measurement
The bursa of Fabricius, thymus and spleen were the immune organs studied. The chickens were weighed, and the immune organs were excised and weighed on 1, 7, 14, and 21 days-old. The data are presented as the relative weight (index) of the given immune organ weight from the total BW. The indices were calculated as immune organ weight/BW × 100%.

Histomorphological investigations
The collected segments of intestine were the loop of duodenum, the tract before Meckel's diverticulum (jejunum) and the tract before the ileocolic junction (ileum). Gut segments were xed in 10% buffered formalin solution for morphometric analysis. Tissues were routinely embedded in para n wax blocks, sectioned at 5 µm thickness, mounted on glass slides and stained with HE. The villus height (VH) was measured from the top of the villus to the crypt mouth; the invagination depth between adjacent crypt mouths was measured and de ned as the crypt depth (CD), and the VH/CD ratio was calculated. Morphometric analyses were performed on 5 well-oriented and intact villi and 5 crypts chosen from duodenum, jejunum and ileum [18,19]. Histopathological lesions were assessed descriptively according to the presence of degeneration, hemorrhaging, congestion, and edema. Organs were graded based on the focal lesion distribution (low severity) and multifocal lesion distribution (moderate severity).
Observation was conducted using 200× magni cation and 3 view-eld repetitions.
Hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) Blood samples from the immunized chickens were clotted at 37 °C and centrifuged at 3000 rpm for 10 min to collect the serum. Two-fold serial dilutions of the serum were prepared in 96-well plates. Next, equal volumes (25 µL) of 4 hemagglutination units (4 HAU) of NDV antigen were added to all rows except the last row. Thirty minutes later, 25 µL of 1% chickens erythrocytes were added and incubated for 30 min. The agglutination was monitored, and the HI titers were determined as the highest serum dilution that completely inhibited NDV agglutination [20]. The jejunum were infused with 0.5 mL phosphatebuffered saline (PBS); pH 7.4, containing 0.1% bovine serum albumin and aprotinin), washed three times, and centrifuged at 2000 rpm at 4 °C for 15 min to collect the supernatant. Commercial ELISA kits for determining secretory immunoglobulin A (SIgA) from jejunal lavage uid, serum interleukin (IL-2) and interferon (IFN-γ) from chickens (Senbeijia, Nanjing, China) were used to determine the NDV antibody levels and cytokine production [21].

DNA isolation and bacterial quanti cation with qPCR
The cecum were removed, and 20-30 mg aliquots were weighed. Total cecum DNA was extracted according to the instructions with the DNA ΔSimple Total DNA kit-DP431 (Tiangen, Beijing, China). A 10 μL total reaction volume in a 96-well plate contained 5 μL Maxima SYBR Green qPCR Master Mix (Thermo Scienti c/Fermentas, Vilnius, Lithuania), 0.2 μM of each primer speci c to the 16S rDNA of Bi dobacterium spp. [22], Lactobacillus spp. [23], E.coli [24] or universal bacteria [25], and 2 ng of bacterial DNA template. Table 1

Viral challenge
After two oral vaccinations, the chickens in the Lc-pPG-HN and control groups were orally challenged with a 2×10 9 mean chickens infection dose (EID 50 ) of the NDV strain in 0.2 mL viral suspensions per chickens at 21-day-old. The chickens were monitored daily for 10 days after the challenge, and the numbers of dead chickens were recorded.

Statistical analysis
Statistical analysis was performed using SPSS 22.0 software and GraphPad PRISM 7.0. For multiple comparisons one-way ANOVA were preformed, followed by Tukey's test. Data are presented as the mean ± SEM. Differences were considered statistically signi cant at P > 0.05, P < 0.05, P < 0.01.

Construction of L.casei vaccine and expression of HN protein
Extract the total RNA of NDV, and amplify the NDV HN gene by RT-PCR according to primers, the gene fragment is 1617 bp (Fig. 1a). The recombinant plasmids pPG-HN was constructed and transferred into L.casei by electroporation. The recombinant plasmids were con rmed by restriction enzyme digestion and sequence analysis (Fig. 1b). To examine whether the properties of the recombinant L.casei changed, growth and hereditary stability were analyzed after the recombinant L.casei were screened by PCR and DNA sequencing. PCR and sequencing analysis (Fig. 1c) showed that recombinant plasmids were stably inherited over 50 generations.

Expression of HN and immuno uorescence assays
To investigate whether the recombinant L.casei is capable of producing HN, cell pellets of Lc-pPG-HN, and Lc-pPG were con rmed by Western blot analyses. The results showed that there was immunoreactive band to 63 kDa were detected in the cell lysates of Lc-pPG-HN (Fig. 2a, lane 2), whereas no band was observed in the Lc-pPG (Fig. 2a, lane 1). These observations suggested that the HN protein is produced and secreted by recombinant L.casei. Clear uorescence signals were observed in Lc-pPG-HN (Fig. 2b,  left), making the morphology of L.casei faintly visible. There was no signal in cells containing the empty vector (Fig. 2b, right). Bacteria could be seen clearly under oil immersion. This nding suggested that Lc-pPG-HN cells could react with the polyclonal Rabbit anti-HN antibody.
Effects of oral immunization with recombinantL.caseion chickens immune organ index Oral immunization with Lc-pPG-HN group had signi cant effect on BW and immune organ index of chickens compared with control and Lc-pPG groups (P < 0.05). However, on 14-21 days, the BW in Lc-pPG-HN group was signi cantly heavier than those in the other two groups (P < 0.01, Table 2).

Intestinal histomorphological analyses
The VH, CD and the ratio of VH/CD of duodenum, jejunum and ileum are shown in Table 3. However, the VH and ratio of VH to CD of chickens in the Lc-pPG and Lc-pPG-HN groups during the 14-21 days periods were signi cantly increased when compared with the control group (P < 0.05). Moreover, when compared with the control group, the CD of chickens in the Lc-pPG group during the 14-21 days periods was also signi cantly decreased (P < 0.05).
SIgA expression levels in the jejunal lavage uid Jejunal lavage uid SIgA was measured to evaluate the systemic immune response. SIgA production was dramatically increased in the jejunal lavage uid of the chickens immunized with Lc-pPG-HN compared with that of the Lc-pPG and control groups on 14 (P < 0.05) and 21-days-old (P < 0.01) (Fig. 3).

Effects of the intestinal ora
The effects of recombinant L.casei on the relative abundance of immune related Lactobacillus spp., E.coli and Bi dobacterium spp. were examined by qRT-PCR analysis (Fig. 4a, b or c). In Lc-pPG-HN group, signi cant changes in Lactobacillus spp. relative abundance observed after booster immunization ( P < 0.05) (Fig. 4a). The relative abundance of E.coli rapid decreased was detected at 7-day-old compared with the control and Lc-pPG groups, and Bi dobacterium spp. reached a fourfold upregulation in caecum at 21-day-old (Fig. 4b or c). Of note, almost no differences in relative abundance were observed between treatments and control groups after prime immunization (P > 0.05).

Serum IL-2 and IFN-γ expression levels
The effects of the recombinant L.casei on IL-2 levels of serum were shown in Fig. 5a. Chickens that oral administration of Lc-pPG-HN and Lc-pPG resulted in higher IL-2 activity as compared to the controls (P < 0.01) after booster immunization at 14-day-old. Likewise, the higher serum IFN-γ level (Fig. 5b) was observed in chickens received Lc-pPG-HN at 21-day-old compared to the controls (P < 0.05).

NDV-HN antibody titers by HI
The level of HI anti-HN in the serum of immunized chicken was determined. As the results showing (Fig.  6), signi cant level of HI was elicited in chickens that received Lc-pPG-HN (P < 0.05) as compared to the controls. After booster immunization, the higher antibodies titers in the serum was observed in chickens by oral administration of Lc-pPG-HN compared to the control and Lc-pPG (P < 0.05). By contrast, no signi cant elicitation of antibodies was detected in the control groups. No signi cant difference (P > 0.05) was observed between treatments and controls prior to the prime immunization.

Viral challenge
The NDV strain challenge experiment was performed to evaluate the protective immunity of the recombinant L.casei. Of the chickens immunized with Lc-pPG-HN, 80 % survived after being challenged with a low dose of NDV, whereas the control group exhibited the lower survival rate (Table 4).

Pathological organ changes
Clinical signs appeared in infected chickens and were rst observed 2 days postchallenge, consisting of green-white soft feces, anorexia, diarrhea, crouch, paralysis, and eyelid edema. Histopathological changes in the visceral organs following the virulent viral challenge were studied separately for NDV.
In the control group, the immune organs showed moderately severe lymphoid depletion in the medulla, and the thinned cortical lymphocytes were loosely arranged, appearing as a few bleeding spots in the medullary lymph (Fig. 7A-F). The control group also presented lymphopenia in the smooth muscle wall of the tertiary bronchi and stenosis in the pulmonary atrium. The pulmonary capillary blood vessels had a few divided red blood cells, a messy cloth-like structure, and a lower cell density with low severity (Fig.  7H). The control group also presented central vein dilation in the liver and liver sinusoid expansion (Fig.  7J). The lung and liver in the Lc-pPG-HN group showed no obvious lesions ( Fig. 7G and I). Pathological lesions and histopathological changes were reduced in the vaccinated chickens following the virulent NDV challenge.

Discussion
Newcastle disease (ND) is an economically important disease of poultry industry, and naturally occurring avirulent NDV strains are widely used as live attenuated vaccines all over the world [26]. Several previous studies have suggested that there is a need to develop live-virus-vectored vaccines that do not involve attenuated NDV strains [27]. In poultry, the small intestine is one of the main mucosal surfaces and immune barriers [28]. As a mucosal vaccine vector, L.casei was used very pro ciently whereas excellent result was also found when it was used as a carrier in DNA vaccine through oral route [29,30]. In this study, we engineered and evaluated the recombinant L. casei expressing HN protein of NDV to induce an individual development and immunity response in chicken.
In our study, the HN antigen was stably expressed on L.casei. HN detection in both the supernatant uid and cell pellets indicated that the expression system also enabled the L.casei to surface display the HN antigen. In analyzing the properties of the recombinant L.casei, although overproduction of heterologous proteins often hampers bacterial growth, the HN-expressing strains grew well and were genetically stable. Our results are consistent with those of a previous report that L. plantarum expressing the Hirep2 (H2) antigen of Chlamydia trachomatis had only limited effects on bacterial tness [31]. These data enable hypothesizing that HN immunogenicity based on the L.casei-expression system can be further investigated via oral administration in a chick model.
Immune organ index and intestinal histomorphology had been widely used for assessing growth performance and intestinal development. For instance, both Ding et al. [32] and Bu [33] found similar increasing growth performance and organs index when feeding chickens with L.casei or Lactobacillus vaccine. Also, Sumarsih et al [34]. Previous studies also indicated that increased VH and VH/CD are directly correlated with increased epithelial turnover, and longer villi are linked with activation of cell mitosis, with shortening of villi and deeper crypts leading to poor nutrient absorption, increased secretion in the gastrointestinal tract and reduced performance [35,36]. In our study, chickens oral immunization Lc-pPG-HN consistently increased the VH of jejunum, duodenum and ileum, and the VH/CD signi cantly increased, which indicated the better digestive and absorption capability [37].
We conducted a oral route of vaccination to evaluate the immunogenicity of our developed vaccine candidate. SIgA is the principal antibody type for host mucosal defense system [38]. The detection of speci c IgA from jejunum indicated that recombinant strain Lc-pPG-HN triggered strong mucosal immune response.
Relevant studies have shown that intestinal microbiota of animals plays an important role in the life activities of the host [39]. L.casei also determined a modi cation of the gut microbiota composition in chickens. Furthermore, the results show that Lc-pPG-HN can signi cantly increase intestinal the number of Lactobacillus and Bi dobacteria and reduce the number of Escherichia coli bacteria. Probiotics, such as Lactobacillus spp., Bi dobacterium spp. are living microorganisms that confer health bene ts on the host [40]. Accordingly, recombinant lactic acid bacteria vaccine promising candidates to study intestinal microbiota/immune system modulation in chickens.
For the detection of cellular response, IL-2 and IFN-γ were selected which are produced by Th1 cells and play a critical role triggering in cell-mediated immune response for the clearance of intracellular pathogens. Oral administration group induced signi cant (P < 0.05) rise of IFN-γ and IL-2 from 14-day-old in comparing control and Lc-pPG groups. Although the level of IFN-γ and IL-2 induction is not so high but consistent with other observation [41]. From the cytokine study, it is also observed that serum from Lc-pPG group also produced a considerable amount of IL-12 and IFN-γ comparing control PBS group (Fig. 5) which may be due to the ability of Lactobacillus casei to induce IL-2 and IFN-γ by itself [42].
The results of the HI assay revealed that the Lc-pPG-HN signi cantly elicited higher antibody titers than the groups received Lc-pPG or saline at 21 day-old (P < 0.05). However, no signi cant changes were evident for any treatment when compared to the vaccinated Lc-pPG or control group at any time point (P > 0.05). Interestingly, The survival study revealed that the Lc-pPG-HN could provide strong protection (80%) for chickens against NDV challenge. However, the lesions observed in NDV-infected chickens were severe and manifested multifocal lesions in the immune organs. The most numerous and consistent gross lesions in these organs involved severe lymphoid depletion and necrosis. H&E staining showed obvious lymphocyte degeneration and necrosis in the immune organs, lung and liver 2 days post challenge. Another study showed that the clinical signs and pathological changes in experimentally infected geese were similar to those of ND induced by viscerotropic velogenic NDV strains in chickens [43]. In addition, anti-HN antibody induced by Lc-pPG-HN demonstrated signi cant increase and provided protection for vaccinated chicken, immunogenic of Lc-pPG-HN requires to be further identi ed.

Conclusion
In conclusion, we demonstrated ve major points in this paper: (1) We generated a recombinant L.casei (surface-displayed) expressing the HN of NDV, and the recombinant plasmid in L.casei could be stably inherited over 50 generations; (2) Oral vaccination chickens with the recombinant vaccine candidate induced a higher immune organ index and intestinal development; (3) The recombinant vaccine candidate also stimulated jejunum mucus speci c SIgA and serum HI antibody titers, and induced a higher IL-2, IFN-γ levels, eliciting in ammatory response and cell immune response; (4) Relative abundance of Bi dobacterium spp, Lactobacillus spp, E. coli in the oral immunization group with engineered L.casei was bene cially modulated in chickens cecum, improving intestine health; (5) Our study clearly demonstrates that the engineered L.casei developed in this study provide protection for chickens against infection with NDV. These results suggest that the potential of oral administration as a mucosal vaccine may be an attractive candidate for treating NDV response and improving early growth performance and intestinal health.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.          Route of the HN-expressing L.casei in oral immunization