Effect Of In Ovo Injection With Recombinant Lactobacillus Casei Expressing HN Of Newcastle Disease Virus On The Growth And Immune Function Of Chicks

Immunity status directly affects the survival and the entire life of hatched chicks.Several studies have demonstrated that in ovo injection vaccine is capable of effectively inducing embryos generating immune responses to enhance their anti-viral infection ability.Newcastle Disease Virus(NDV)hemagglutinin-neuraminidase protein(HN),a novel recombinant vaccine candidate antigen,is capable of inducing humoral immunity in poultry,thereby generating protective antibodies.Therefore,the administration of HN antigen-containing recombinant vaccines via in ovo injection may improve the efficiency of poultry.In the present study,a Lactobacillus casei-pPG612-Newcastle disease virus(NDV)-HN construct was developed,and subsequently,the mucosal immunity and the systemic immune response induced by this construct were evaluated.First,the HN gene of the NDV Lasto strain was cloned and the cell surface expression-type Newcastle disease virus HN gene-containing recombinant lactic acid bacteria(Lactobacillus casei-pPG612-Newcastle disease virus,Lc-pPG-HN)were developed using the Lactobacillus casei-pPG612 vector.A 1617 bp HN gene fragment was obtained by PCR amplification.Western blotting has a clear band and a protein size of 63 k Da.Obvious fluorescence signals can be seen through a fluorescence microscope.The genetic stability of the recombinant lactic acid bacteria was detected,indicating that the HN gene fragment was successfully cloned and recombinant lactic acid bacteria can stably express HN protein;90 healtly chicks were divided into three treatment groups.For every treatment,five replicates were used while six chicks were used per replicate.The three treatment groups were as follows:Physiological saline group(Control),Lc-pPG group and recombinant vaccine group(Lc-pPG-HN).In addition,the growth and development-related indicators,inflammatory cytokines(IL-2 and IFN-?)and HI titers of serum,and SIgA in the intestinal washings were analyzed on Day 1,7,14 and 21,it has good immunogenicity and it can improve the growth performance and immunogenicity.After achieving a stable expression of the HN protein,the following in ovo injection studies were performed:Nine hundred eggs with fertile embryos were divided into six treatment groups.For every treatment,five replicates were used while thirty eggs were used per replicate.The six treatment groups were as follows:1):uninjected group;2):PBS injection group(PBS),3):Newcastle disease vaccine group(NDV,Dilution by instruction),4)Newcastle disease vaccine addition Lc-pPG(NDV+Lc),5):1×10⁹CFU/m L recombinant L.casei vaccine low-dose group(Lc-pPG1-HN),6):1×10¹¹CFU/m L recombinant L.casei vaccine high-dose group(Lc-pPG2-HN).According to normal incubation procedures,incubate to 18.5 embryonic age.The injection dose per egg was 0.2 m L.The recombinant L.casei were induced with lactose and other solutions(for example,PBS),and then injected into the amniotic cavity.In order to select the appropriate concentrations of recombinant L.casei for the in ovo injection,50 healthy chicks from the injection group were randomly selected and orally immunized with the Newcastle disease vaccine,following which they were raised individually(1DPI)and the data regarding their statistics of hatchability and the initial weights were recorded.In addition,the growth and development-related indicators,the alterations in their immune organs and mucosal morphology,and the changes in the distribution and numbers of IgA⁺and IgG in their intestinal mucosa were analyzed duiring Day 1-21.Different doses of recombinant L.casei could improve the hatchability and the initial egg weights of chicks,although the differences were not significant(P>0.05).The Lc-pPG2-HN high-dose group exhibited an increase in the body weight gain(BWG)of the SPF chicks and a reduction in the feed to meat ratio(FCR),both of which were significant(P<0.05).Different doses of recombinant L.casei and the NDV+Lc injection groups promoted the development of immune organs and significantly increased the immune organ index calculated on Day 14–21(P<0.05).In addition,the morphology of the small intestinal mucosa was improved,the height of the villi(VH)was increased,the crypt depth(CD)was reduced,and the VH/CD and activity of digestive enzymes(amylase,trypsin,and lipase)were significantly increased on Day 14–21(P<0.05).The indicators in the different doses of the recombinant L.casei injection group were significantly higher than those in the NDV+Lc group(P<0.05).On Day 14 and Day 21,the NDV+Lc and different doses of recombinant L.casei groups exhibited promoted the secretion of SIgA in the jejunum and IgA⁺in the villi propria of the duodenum,jejunum,and ileum,with the cells rapidly distributed at the top of the intestinal villi;However,the differences were not significant(P>0.05).Furthermore,the cytokine(IL-2?IL-4?IL-6?IFN-?)and SIgA contents in serum and the activity of the jejunum digestive enzyme were determined.Quantitative PCR was employed for the detection of jejunum,ileum,and spleen inflammatory cytokines(IL-2,IL-6,IFN-?,and TLR4)and the m RNA expression levels of jejunum and ileum mucosal barrier proteins(mucin-1,ZO-1,and occludin),for which the bacterial DNA was extracted from the cecal samples and the relative flora abundances of Bifidobacterium spp.,Lactobacillus spp.,E.coli,and Enterococcus spp.were determined.MTT proliferation and Flow cytometry were used for determining the proliferation of the lymphocytes derived from blood and spleen samples and the percentage of CD4⁺and CD8⁺T lymphocytes.In comparison to the PBS control group,the NDV,NDV+Lc,and different doses of recombinant L.casei groups exhibited increased relative expressions of the m RNA of the jejunum and ileum mucosal barrier proteins and the levels of inflammatory cytokines were significantly increased at Day 21(P<0.05).On Day 14,the Lc-pPG2-HN high-dose group exhibited significantly increased relative abundances of the three probiotics(P<0.05)and decreased relative abundance of E.coli;In comparison to the NDV+Lc group,the different doses of recombinant L.casei group exhibited significant promotion of the HI and SIgA antibody titers in serum on Day 14 and Day 21(P<0.05),induction of higher production of cytokines(IL-2?IL-4?IL-6?IFN-?)in serum,and stimulation of the proliferation of lymphocytes.The Lc-pPG2-HN high-dose group exhibited a significant increase in the percentage of CD4⁺and CD8⁺T lymphocytes in the blood and spleen of the SPF chicks(P<0.05).On Day 21,the percentage of the CD4⁺and CD8⁺T lymphocytes in the spleen was as high as 60%.In addition,Different doses of recombinant L.casei groups exhibited increased relative expressions of the m RNA of the spleen.The clinical morbidity and the pathological changes occurring in the organs on Day 22after challenge with the NDV strain were observed in the PBS,NDV+Lc,1DPI,and Lc-pPG2-HN high-dose groups.The antibody titers of NDV HI in serum and SIgA in the intestinal washings were determined at different ages,1 day before challenge and at Day 7post-infection,followed by the determination of statistical survival.The obtained results are provided below.The survival rates in the NDV+Lc,1DPI,and Lc-pPG2-HN high-dose groups were 76%,67%,and 89%,respectively,while all the SPF chicks in the PBS group died 7 days after the infection.The pathological analysis revealed that the Lc-pPG2-HN high-dose group did not present the typical clinical symptoms and there were no obvious lesions in the structure of each tissue.The HI titers and SIgA secreted by the serum and the intestinal washings of the chicks in the Lc-pPG2-HN high-dose group were significantly higher than those in the 1DPI group(P<0.05)and not significantly different from those in the NDV+Lc group(P>0.05).In summary,the HN antigen expressed by the lactic acid bacteria vector was injected into the digestive tract of chicks through the amniotic cavity.The vaccine administration could inhibit the colonization of pathogenic microorganisms in the intestine,resulted in the formation of a mucosal barrier for stimulating the body to produce local immunity,and further caused an adaptive immune response.It could effectively alleviate the acute infection caused by NDV and played a protective role.These results would serve as a theoretical basis for the future development of in ovo injection-based recombinant Lactobacillus casei vaccine.