Effects of Combined Application of NRTUAs and ABP on Growth and Humoral Immunity of Chicks

Chao REN, Ling GUI

College of Animal Science and Veterinary Medicine, Tianjin Agricultural University/Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, Tianjin 300384, China

Abstract [Objectives]The paper was to study the effects of combined application of nonreplicating Toxoplasma uracil auxotrophs(NRTUAs)and Agaricus blazei Murill polysaccharide(ABP)on growth and humoral immunity of chicks.[Methods]A total of 120 one-day old female Hyline brown laying hens were randomly divided into 4 groups, 30 hens for each group.The chicks in group 1 were subcutaneously injected with NRTUAs and fed on the diet containing with ABP; the chicks in group 2 were subcutaneously injected with NRTUAs; the chicks in group 3 were subcutaneously injected with equal volume of PBS, and fed on the diet containing with ABP; the chicks in group 4 were subcutaneously injected with equal volume of PBS.The body weight of chicks in each group was counted at the 21st, 42nd, 84th and 112th week.During this period, blood samples were collected from chicks in each group at 0, 7, 14, 21, 28 and 35 d post immunization against Newcastle disease(ND), and serum was separated to detect the antibody titer of ND.[Results]The combined application of NRTUAs and ABP had no effect on growth of chicks, but promoted the humoral immune response of chicks, significantly improved the ND antibody level of chicks, and could maintain high levels of antibodies in the body for a long time.[Conclusions]The study lays a theoretical foundation for further developing the clinical application of NRTUAs and ABP.

Key words NRTUAs, ABP, Chick, Growth, Humoral immunity

1 Introduction

AgaricusblazeiMurill, native to Brazil, has delicious taste and has the functions of enhancing immunoregulatory function, antiviral and antitumor, being an edible and medicinal fungus with health care function[1-3].In animal husbandry and veterinary studies, it has been found that addingA.blazeiMurill polysaccharide(ABP)to diets in the early growth stage of AA broilers can improve the daily gain and serum antioxidant capacity of chicks, thereby improving the body’s immune capacity and reducing the occurrence of disease[4-5].Duan Xianpingetal[6]showed that oral administration of ABP enhanced the humoral immune response of chicks, improved the protection rate of vaccines, and made immunity last longer.At the same time, appropriate doses of ABP increased the C3b receptor chaplet rate of red blood cells and enhanced the immune function of red blood cells at the initial stage of vaccine immunization.A.blazeiextract also has inhibitory effects on bacteria and fungi[7].Therefore, ABP plays an important role in immune regulation by helping the body resist the invasion of bacteria and viruses effectively, and promotes the growth and development of poultry.

NonreplicatingToxoplasmauracil auxotrophs(NRTUAs)is an uracil nutrition-deficient strain ofToxoplasmagondiithat blocks the pyrimidine synthesis pathway by double-knockout of the genes of orotidine-5’-monophosphate decarboxylase(ompdc)and uridine phosphorylase(up)[8].Previous studies show that NRTUAs can invade mammalian cells and form parasitophorous vacuole in uracil-deficient medium, but can not proliferate; NRTUAs can not only invade mammalian cells, but also grow well within mammalian cells in uracil-containing medium; NRTUAs can not proliferate in mammals due to the absence of essential enzymes, but can invade body cells[8-9].

NRTUAs are non-toxic to mice, and can be quickly cleared by the body’s immune system at about 5 d post infection[10-11].Even in immunodeficient animals infected with NRTUAs, such as mice that do not produce IFN-γ or lack T cells, B cells and NK cells(NOD/scid/gamma mice), NRTUAs are not pathogenic to the organism and can be safely tolerated[8-9, 12].NRTUAs can regulate the host’s immune system and improve the immunosuppressive microenvironment of tumors.Similar to wild-typeT.gondii, NRTUAs are more likely to enter the host’s myeloid cells, such as dendritic cells and mononuclear/macrophages, and can activate the immune activity of myeloid cells to significantly improve the expression of costimulatory molecules CD80 and CD86 as well as MHCI molecule in myeloid cells, and increase the production of IL-12[10, 13-14].NRTUAs can induce a strong acquired immune response, stimulate the maturation of CD4+and CD8+T cells, and increase the production of IL-12 and IFN-γ by activating CD8α+dendritic cells and further enhancing antigen presentation, thereby maintaining a high level of tumor-specific antibodies[11, 14].

The above studies indicate that NRTUAs have good safety and immunomodulatory functions in mice, and ABP also has important immunomodulatory effects, which can promote the growth and development of poultry.However, few efforts have been dedicated to combined application of NRTUAs as a potential immunomodulator and ABP in poultry.Therefore, the study applied NRTUAs and ABP to chicks in combination to study their effects on growth and humoral immunity of chicks, laying a theoretical foundation for further developing the clinical application of NRTUAs and ABP.

2 Materials and methods

2.1 Materials

2.1.1Animals.A total of 120 one-day old female Hyline Brown laying hens were purchased from Tianjin Jinhua Breeding Farm.521 formula feed for laying chicks was purchased from Shijiazhuang Zhengda Co., Ltd.Experimental diets and water were availableadlibitumduring the entire experimental period.

2.1.2Cells and parasite strain.Human foreskin fibroblasts(HFF)and NRTUAs were preserved by the basic veterinary laboratory of Tianjin Agricultural University.

2.1.3Reagents.ABP(purity 70%)was purchased from Xi’an Ruidi Biotechnology Co., Ltd.Newcastle disease(ND)attenuated vaccine was purchased from Qianyuanhao Biological Co., Ltd.

2.2 Methods

2.2.1Effects of combined application of NRTUAs and ABP on growth of chicks.A total of 120 one-day old female Hyline brown laying hens were randomly divided into 4 groups, 30 hens for each group.100 mg/kg ABP was supplemented to the basal diet, and NRTUAs were subcutaneously injected into chicks at the dose of 106for each.The specific treatment methods were as follows: group 1, NRTUAs+ABP; group 2, NRTUAs+/; group 3, PBS+ABP; group 4, PBS+/.Experimental diets and water were availableadlibitumduring the entire experimental period.Chicks were immunized according to the routine immunization procedure of commercial Hyline brown laying hens.Manure cleaning and disinfection should be done well in time every day.The body weight of chicks in each group was measured at the 21st, 42nd, 84thand 112thweek.

2.2.2Effects of combine application of NRTUAs and ABP on humoral immunity of chicks.According to the immunization procedures of Hyline brown laying hens, chicks in each group were immunized with ND IV vaccine by intraocular-nasal vaccination.At 0, 7, 14, 21, 28 and 35 d post immunization, blood samples were collected from wing vein of chicks in each group.The antibody titers of ND in serum were detected by hemagglutination(HA)test and hemagglutination inhibition(HI)test, so as to evaluate the effects of combined application of NRTUAs and ABP on humoral immunity of laying hens.

2.2.3Statistical analysis.All data were analyzed by the Two-Way ANOVA method, and the data of each group were conducted multiple comparisons by the Turkey method.The data were expressed as "mean±standard error".

3 Results and analysis

3.1 Effects of combined application of NRTUAs and ABP on growth of chicksSingle or combined application of NRTUAs and ABP had no significant effects on the average weight and average daily gain of chicks during brooding, growing and laying periods(Table 1), indicating that the combined application of NRTUAs and ABP had no effect on growth of chicks.

Table 1 Effects of combined application of NRTUAs and ABP on growth of chicks g

3.2 Effects of combined application of NRTUAs and ABP on humoral immunity of chicksThe ND antibody levels in various groups increased gradually after immunization and reached the maximum on the 21stday after immunization.At 14 d post immunization, the ND antibody level in group 1 was significantly higher than those in other groups(P<0.05); at 21 d post immunization, the ND antibody level in group 1 was significantly higher than those in groups 3 and 4(P<0.05), and the ND antibody level in group 2 was significantly higher than that in group 4(P<0.05); at 28 d post immunization, the ND antibody levels in groups 1 and 2 were significantly higher than those in groups 3 and 4(P<0.05); at 35 d post immunization, the ND antibody levels in group 1 was significantly higher than those in other groups(P<0.05, Table 2).Therefore, the combined application of NRTUAs and ABP can significantly improve the ND antibody level in chicks, and maintain high levels of antibodies in the body for a long time.

Table 2 Effects of combined application of NRTUAs and ABP on ND vaccine immunization of chicks

4 Discussion and conclusions

ABP can reduce the cadmium content in thymus, spleen and bursa of Fabricius, lower the expression of TLR4, MDA5, IPS-1, IRF-3 and NF-κB, and decrease the tissue contents of inflammatory cytokines IL-1β, IL-6, TNF-α and IFN-β of chickens with cadmium-induced damage; moreover, it can improve the activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px), enhance antioxidant activity and alleviate the inflammatory response to various organs caused by cadmium intoxication, so as to regulate immune response and protect various organs and tissues[15-17].All kinds of mushrooms and their polysaccharides play an important role in poultry production.Lentinan can be used as an immunoenhancer or immunomodulator, and have antibacterial, antiviral and anti-parasitic biological activities.Phenolic compounds in mushrooms can be used as antioxidants and mushrooms in broiler diets can be used as growth promoters instead of antibiotics; dietary addition of mushrooms can improve egg production and egg quality, and all kinds of mushrooms and their polysaccharides can be used as safe and effective antibiotic substitutes to promote molting, enhance poultry immunity, combatSalmonellaandEimeriainfection, and restore egg production[18-19].In this study, the combined application of NRTUAs and ABP(group 1)and single application of ABP(group 3)had no significant effects on the average weight and average daily gain of chicks during brooding, growing and laying periods, indicating that feeding ABP had no impact on growth of chicks.

Because of differences in body temperature, immune organs, physiological and biochemical metabolism between poultry and mammals, poultry becomes an insensitive intermediate host forT.gondii.In this study, the combined application of NRTUAs and ABP(group 1)and single application of NRTUAs(group 2)had no significant effects on the average weight and average daily gain of chicks during brooding, growing and laying periods, indicating that NRTUAs immunity had no impact on growth of chicks.

Studies have shown that active NRTUAs actively invade host cells and can produce and secrete MICs, ROPs and GRAs to induce T cells to produce IFN-γ and activate antitumor immune response[20].NRTUAs can induce a strong adaptive immune response against the body’s immune response to unrelated antigens, which is similar to the heterogenic effect produced by BCG immunization[21].It has been found that after invading macrophages and dendritic cells,T.gondiicarries out designated reprogramming of host cells through corresponding transcriptional mechanisms[22].ROP16 and GRA15 secreted during invasion can reprogram the host cell’s immune.T.gondiiROP16 phosphorylates STAT3 and STAT6, promoting the production of IL-6 and IL-12p40 in infected macrophages and enhancing the inflammatory response of the body[23].During the whole development process ofT.gondiiin the host cells, GRAs are released in the host cells to continuously stimulate the host mononuclear macrophage system and promote the body’s immune response[24-25].Therefore, NRTUAs is essentially a safe and effective Th1 type adjuvant targeting the mononuclear macrophage system[8].In this study, the ND antibody levels in combined application of NRTUAs and ABP(group 1)and single immunization of NRTUAs(group 2)were significantly higher than that in group 4 at 21 and 28 d post immunization, indicating that active NRTUAs can promote the humoral immune response against ND vaccine.And combined application of NRTUAs and ABP can maintain high levels of antibodies from 14 to 35 d after immunization.

In conclusion, the combined application of NRTUAs and ABP does not affect the growth of chicks, but promotes the humoral immune response of chicks, significantly improves the ND antibody level of chicks, and can maintain high levels of antibodies in the body for a long time.