Host-derived bacillus spp. as probiotic additives for improved growth performance in broilers.

In recent years, the utilization of antibiotics in animal feed has been restricted, probiotics have been increasingly used to replace antibiotics in maintaining animal health. The aim of this study was to screen and evaluate probiotics with excellent probiotic potential from the gut of healthy goslings for clinical application. Thirteen strains of Bacillus (named AH-G201 to AH-G2013), including 2 strains of Bacillus subtilis (B. subtilis), 6 strains of Bacillus licheniformis (B. licheniformis) and 5 strains of Bacillus amyloliquefaciens (B. amyloliquefaciens), were isolated and identified. Then, acid and bile salts tolerance tests were performed to screen probiotics strains that could survive under different environments. The effects of screened probiotics on the growth of pathogenic Escherichia coli (E. coli) and Salmonella were assessed. Furthermore, we performed the drug resistance tests and safety tests in animals. The results showed that B. Subtilis AH-G201, B. licheniformis AH-G202 and AH-G204 exhibited higher gastrointestinal resistance under in vitro conditions, and showed a moderate level of resistance to the tested antibiotics. Importantly, AH-G201 and AH-G202 showed 24 to 60% inhibition rate against pathogenic E. coli and Salmonella. Moreover, the safety analysis of AH-G201 and AH-G202 suggested that the 2 probiotics strains have no adverse effects on body weight gain and feed intake in the broilers, and in addition, they have significantly improved growth performance. Finally, we analyzed effects of B. Subtilis AH-G201and B. licheniformis AH-G202 on growth performance, immune organ index and the feces microbes of broilers. The results showed that broilers fed with high doses (5 × 109 CFU/mL, for single strain) of a mixture of AH-G201 and AH-G202 exhibited good growth performance, and exhibited the greatest gain in spleen weight and the highest lactic acid bacteria counts. These findings indicate that the combined addition of B. Subtilis AH-G201 and B. licheniformis AH-G202 has the potential to replace antibiotics and to improve the growth performance of broilers.

[Preparation of monoclonal antibody against E domain III (ED3) protein of duck Tembusu virus with neutralizing activity].

Objective To prepare the specific monoclonal antibody (mAb) against E domain III (ED3) of duck Tembusu virus (DTMUV) and explore its neutralization activity. Methods The ED3 gene was amplified by using reverse transcription PCR according to the genome of the DTMUV AH-F10 strain. Then, the recombinant expression vector pET32a-ED3 was constructed and transformed into E.coli Rosetta. The ED3 protein was expressed and purified by nickel column affinity chromatography. After the recombinant ED3 protein was identified by SDS-PAGE and Western blot analysis, the BALB/c mice were immunized subcutaneously three times. The splenocytes of the immunized mice were hybridized with Sp2/0 myeloma cells, and the hybridization was screened by the limiting dilution method. The specificity and sensitivity of the antibody were identified by indirect immuno-fluorescent assay and Western blot analysis. Subsequently, antibody titers were determined by ELISA. Finally, this study titrated the neutralization titers of the antibodies on DTMUV-infected BHK-21 cells. Results The ED3 protein was successfully prepared and purified using the prokaryotic expression system. Three strains of monoclonal antibodies named B9D10C7, B9D7B8G10 and B9D7B8F11 were prepared. Their subtypes were IgG1, IgG2a and IgG2b, respectively. The titers of monoclonal antibody ascites can reach 1:51 200, and they could specifically recognize the E protein of DTMUV. Neutralization test showed that they had a certain neutralizing activities. Conclusion The monoclonal antibodies against ED3 protein of DTMUV are successfully prepared.

[Preparation of mouse polyclonal antibodies against virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin)].

Objective Mice were immunized with purified virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) to prepare polyclonal antibody and identify specificity. Methods BALB/c mice were injected with duck tembusu virus to generate viperin in mouse brain by intracranial injection. Viperin gene, cloned from mouse brain tissue by reverse transcription PCR, was inserted into pGEX-6p-1 prokaryotic expression vector and transformed into E. coli Rosetta. The recombinant viperin protein was induced by isopropyl thiogalactoside (IPTG) and its solubility was analyzed. The protein was purified by potassium chloride (KCl) staining and gel cutting method. Polyclonal antibody was prepared by immunizing mice with purified recombinant viperin protein subcutaneously through abdomen, and the titer of polyclonal antibody was determined by indirect ELISA. Western blot analysis and indirect fluorescence assay (IFA) were used to detect the transient expression of viperin protein in BHK-21 cells to identify the specificity and sensitivity of the prepared polyclonal antibody against viperin protein. Results The mouse viperin gene was successfully cloned and the viperin protein was expressed. The titer of the prepared anti-viperin polyclonal antibody reached 1:25 600. The mouse anti-viperin polyclonal antibody could specifically recognize the transient expression of viperin protein in BHK-21 cells. Conclusion Mouse polyclonal antibody against viperin protein with high specificity and sensitivity was successfully prepared.

Development of a potential diagnostic monoclonal antibody against capsid spike protein VP27 of the novel goose astrovirus.

Goose astrovirus (GAstVs) is an emerging pathogen of goslings that causes fatal gout, kidney hemorrhages, renomegaly, and high mortality. The GAstVs VP27 protein is an important capsid protein and a candidate for the development of diagnostic reagents. The aim of this study was to clone and express the VP27 gene for preparation of a specific monoclonal antibody (mAb). The VP27 protein was expressed and purified in the supernatant of Escherichia coli BL21. Then, the mAb was obtained with the hybridoma technique and named 2AF11. It was differentiated as IgG1 with the help of immunoglobulin subclass tests. This mAb can specifically recognize the VP27 protein in GAstVs-infected cells, as evidenced by western blot analysis and immunofluorescent assay. Furthermore, this mAb could also detect the VP27 protein in GAstVs-infected tissues, as demonstrated by immunohistochemistry. These findings indicate that this mAb has high diagnostic potential. Therefore, the newly produced anti-VP27 mAb, 2AF11, could be a useful tool as a specific diagnostic marker for GAstVs.