Characterization and biological activity of Taishan Pinus massoniana pollen polysaccharide in vitro.

Taishan Pinus massoniana pollen polysaccharide (TPPPS) improves cellular and humoral immune responses of animals and is a novel potential immunomodulator. However, the components of TPPPS have not been recognized. To investigate the composition of TPPPS, crude polysaccharide was obtained from Taishan P. massoniana pollen through water extraction and ethanol precipitation. Three homogeneous polysaccharide fractions (TPPPS1, TPPPS2, and TPPPS3) were purified from TPPPS by DEAE-cellulose column chromatography. The average molecular weights of the three polysaccharides were 56, 25, and 128 kDa, respectively. Results of high-performance liquid chromatography (HPLC) showed that TPPPS comprised mannose, ribose, xylose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The biological activity assays showed that TPPPS2 and TPPPS3 significantly promoted spleen lymphocyte proliferation, and that TPPPS3 showed better effect than TPPPS2. TPPPS3 enhanced the secretion of cytokine IL-2 and TNF, whereas TPPPS2 mainly elevated IL-2 secretion. By contrast, TPPPS1 exhibited other effects, and it induced the highest amount of NO production, thereby indicating that TPPPS1 had the best antioxidant activity. TPPPS3 at 50 µg/mL significantly inhibited the proliferation of subgroup B Avian Leukosis virus (ALV-B) through virus adsorption interference in vitro. Results indicated that TPPPS comprised three main components, among which, TPPPS1 mainly showed antioxidant effects, whereas TPPPS2 and TPPPS3 played key roles in immunomodulation, especially TPPPS3. Further studies on the use of a reasonable proportion of TPPPS1-3 may facilitate the development of an effective immunomodulator.

Effects of polysaccharide on chicks co-infected with Bordetella avium and Avian leukosis virus.

Chicks' co-infection with immunosuppressive virus and bacteria seriously threaten the development of the poultry industry. In this study, a model was established in which chicks were injected with either subgroup B ALV (ALV-B)+Bordetella avium (B. avium), or ALV-B+B. avium+Taishan Pinus massoniana pollen polysaccharide (TPPPS), or B. avium only, or B. avium+TPPPS. The data showed that the group injected with ALV-B and B. avium exhibited significant inhibition of the immune function and therefore increased pathogenicity compared with the group injected with B. avium-only. Application of TPPPS effectively alleviated immunosuppression, and body weights increased sharply in the TPPPS groups compared with non-TPPPS groups. To some extent, TPPPS may reduce the proliferation of ALV-B. These results suggest that Pinus pollen polysaccharides are beneficial treating co-infections with immunosuppressive virus and bacteria and therefore have potential for development into safe and effective immunoregulator.

Immunoregulatory effects of Taishan Pinus massoniana pollen polysaccharide on chicks co-infected with avian leukosis virus and Bordetella avium early in ovo.

In recent years, co-infection of chicken embryos with immunosuppressive viruses and bacteria occurs with an annually increasing frequency. Consequently, studies on new and safe immunoregulators, especially plant polysaccharides, have become a popular topic in the poultry industry. In the present study, we selected 300 specific pathogen free embryonated eggs, which were injected with subgroup B avian leukosis virus (ALV-B) and Bordetella avium (B. avium) to establish an artificial co-infection model. The chicks that hatched from these co-infected embryonated eggs were treated with Taishan Pinus massoniana pollen polysaccharide (TPPPS). Results indicated that relevant indices in the co-infection group were significantly lower than that in B. avium-only group. Furthermore, pathogenicity of B. avium was exacerbated, with the chicks exhibiting decreased body weights. The TPPPS groups exhibited gradual improvements in immune function and developmental status. Therefore, in terms of improving immunologic function and production performance, TPPPS could be used as immunoregulator for immune responses.

Taishan Pinus massoniana pollen polysaccharides promote immune responses of recombinant Bordetella avium ompA in BALB/c mice.

To study the effects of Taishan Pinus massoniana pollen polysaccharides (TPPPS) on Bordetella avium outer membrane protein A (ompA) recombinant protein vaccine, ompA was expressed, confirmed by Western blotting and mixed with TPPPS. Female BALB/c mice were randomly divided into six groups (I-VI). Groups I, II, and III were treated with TPPPS-ompA at doses of 200, 400, and 800 mg/ml, respectively. Groups IV, V, and VI were treated with Freund's adjuvant-ompA, pure ompA, and physiological saline, respectively. On days 3, 7, 14, 21, 28, 35, 42, and 49 after the first vaccination, antibody titers, interleukin-2 (IL-2) levels, peripheral blood CD4+ and CD8+ levels, and T lymphocyte proliferation rates in peripheral blood, as well as secreting-type immunoglobulin A (SIgA) levels in the duodenum, were measured. The antibody titers against ompA, IL-2, T lymphocyte proliferation rate, CD4+, and CD8+ in Group II were significantly (P<0.05) higher than those in other groups. However, little difference in SIgA content was observed among Groups I, II, and IV. These results indicated that TPPPS strengthened humoral and cellular immune response against recombinant ompA vaccine and 400 mg/ml TPPPS showed significance (P<0.05) compared with Freund's adjuvant. Therefore, TPPPS can be developed into an adjuvant for recombinant protein vaccines or plant-derived medicine for animal husbandry.

Monoclonal antibodies directed against the outer membrane protein of Bordetella avium.

Bordetella avium is the etiologic agent of coryza and rhinotracheitis in poultry. This respiratory disease is responsible for substantial economic losses in the poultry industry. Monoclonal antibodies (MAbs) were produced against the outer membrane proteins (OMPs) of B. avium isolated from diseased chickens. BALB/c mice were immunized with the extracted B. avium OMPs. Then the splenocytes from immunized mice and SP2/0 myeloma cells were fused using PEG 4000. Three stable hybridoma clones (designated as 3G10, 4A3, and 4E8) were produced via indirect ELISA and three rounds of subcloning. The MAbs were classified as IgG1, and can recognize the 58 kDa OMP band by Western blot assays. No MAb cross-reactivity with chicken Proteus mirabilis, Escherichia coli, and Salmonella was observed. A double antibody sandwich ELISA (DAS-ELISA) was developed using the rabbit polyclonal antibodies as the capture antibody and MAb 4A3 as the detection antibody. Under the DAS-ELISA, the minimum detectable concentration of B. avium was 1 × 10(4) CFU/mL, and no cross-reactivity occurred with chicken Proteus mirabilis, Escherichia coli, and Salmonella. Results showed that the DAS-ELISA has good sensitivity and specificity. Clinical application showed the DAS-ELISA was more sensitive than the plate agglutination test. This study may be used to develop a quick and specific diagnostic kit, analyze epitopes, and establish systems for typing B. avium.

Effects of Taishan Robinia pseudoacacia Polysaccharides on immune function in chickens.

To determine the immune function of Taishan Robinia pseudoacacia Polysaccharide (TRPPS) on chickens, 240 chickens were selected as experimental animals and treated with various doses of TRPPS by hypodermic injection before immunized NDV inactivated vaccine. The results indicated that any dose of TRPPS could significantly promote the development of the immune organs, increase the quantity of leukocyte and the ratio of lymphocyte, and improve the antibody titers against Newcastle disease. Meanwhile, it also increased the magnitude of SIgA in duodenum. However, the dose of 200 mg/ml showed to be the most effective. Therefore, in terms of improving immunologic function and production performance, TRPPS could be used as a vaccine immunopotentiator for immune responses.