Experiments were conducted to investigate the effect of a fusion gene of porcine IL-4 and IL-6 (PIL4/IL6) packaged with chitosan nanoparticles (CNPs) in terms of the development of a novel effective adjuvant. The IL4/PIL6 fusion gene was constructed and inserted into a eukaryotic expression vector. The plasmid was bound to CNP and then utilized to orally inoculate 21-day-old female Kunming mice that simultaneously received intramuscular injection of inactivated Escherichia coli vaccine. At 35 days post-vaccination, the mice were challenged by oral feeding with virulent O139: K88 strain EPEC E. coli bacteria. Compared with those of control mice, the content of immunoglobulins and specific antibodies to E. coli increased significantly in the sera of mice immunized with VPIL4/IL6-CNP (P<0.05). Furthermore, the levels of IL-2, IL-4 and IL-6 increased remarkably in the sera of immunized mice (P<0.05). After challenge, these immunological markers were elevated to different degrees in the mice immunized with the fusion gene construct (IL4/VPIL6-CNP) or individual plasmids (VPIL4+VPIL6-CNP). The immunized mice all survived the challenge and did not show any symptoms or lesion, whereas the VR1020-CNP control mice manifested obvious clinical symptoms and hemorrhagic lesions in the digestive tracts. These results demonstrated that VPIL4/IL6 entrapped with CNP is a novel promising adjuvant to promote specific immunity and resistance of animals against infectious pathogen.

In order to explore the safe and effective adjuvant for promotion of immunity of animals against infection, the experiment was carried out to shuffle Tibet pig IL-2 cDNAwith other IL-2 cDNAfrom human, yak and mouse, and the effect of shuffled IL-2 (IL-2S) gene in vivowas investigated on immunity of mice to Pasteurella multocida. The IL-2S protein was found to remarkably promote the proliferation of pig lymphoblasts than the native pig IL-2 protein. Then the IL-2S gene was cloned into VR1020 eukaryotic plasmid (VRIL2S) and enwrapped with chitosan nanoparticles (CNP-VRIL2S). Twenty-one day old female Kunming mice were muscularly inoculated respectively with the CNP-VRIL2, CNP-VRIL2S and CNP-VR1020 along with Pasteurella multocida vaccine, and orally challenged with virulent Pasteurella multocida on 28 days post-vaccination. The blood was weekly collected to detect the change of IgG, IgA, IgM, specific antibody, IL-2, IL-4 and IL-6 by ELISA. The immunoglobulins, specific antibody and interleukins significantly increased in CNP-VRIL2S group compared with the control mice after vaccination and challenge, and 9 of 10 immunized mice survived challenge, while the all control mice manifested severe symptoms and lesions, and finally died of infection. These indicated that VRIL2S entrapped with CNP is a novel safe and effective adjuvant to boost the specific immunity and resistance of animal against infectious pathogen, which could facilitate the development of highly promising powerful adjuvant.

Epitope-based vaccines designed to induce cellular immune response and antibody responses specific for infectious bronchitis virus (IBV) are being developed as a means for increasing vaccine potency. In this study, we selected seven epitopes from the spike (S1), spike (S2), and nucleocapsid (N) protein and constructed a multi-epitope DNA vaccine. The 7-day-old chickens were immunized intramuscularly with multi-epitope DNA vaccine encapsulated by liposome and boosted two weeks later, and were challenged by virulent IBV strain five weeks post booster. The results showed that multi-epitope DNA vaccine led to a dramatic augmentation of humoral and cellular responses, and provided up to 80.0% rate of immune protection. The novel immunogenic chimeric multi-epitope DNA vaccine revealed in this study provided a new candidate target for IBV vaccine development.

To study the prevalence of antimicrobial resistance in faecal bacteria from Giant pandas in China, 59 isolates were recovered from faecal pats of 30 Giant pandas. Antimicrobial susceptibility testing of the isolateswas performed by the standardised disk diffusion method (Kirby-Bauer). Of the 59 study isolates, 32.20% were resistant to at least one antimicrobial and 16.95% showed multidrug-resistant phenotypes. Thirteen drug resistance genes [aph(3')-IIa, aac(6')-Ib, ant(3")-Ia, aac(3)-IIa, sul1, sul2, sul3, tetA, tetC, tetM, cat1, floR and cmlA] were analysed using four primer sets by multiplex polymerase chain reaction (PCR). The detection frequency of the aph(3')-IIa gene was the highest (10.17%), followed by cmlA (8.47%). The genes aac(6')-Ib, sul2 and tetAwere not detected. PCR productswere confirmed by DNA sequence analysis. The results revealed that multidrug resistancewas widely present in bacteria isolated from Giant pandas.

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp® DNA Stool Mini Kit.