Yuncai Xiao

Development of Indirect Enzyme-linked Immunosorbent Assay with Nucleoprotein as Antigen for Detection and Quantification of Antibodies against Avian Influenza Virus

Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. In the present study, we developed an indirect enzyme-linked immunosorbent assay (ELISA) employing E. coli-expressed full-length nucleoprotein (NP) of H9N2 avian influenza virus for the detection and quantification of antibodies against AIV nucleoprotein. The NP-ELISA was compared with the AI agar gel propagation (AGP) test, haemagglutination inhibition (HI) test, and IDEXX-FlockChek ELISA using 263 sera. The NP-ELISA was significantly more sensitive than the AGP and HI tests, and showed 96.2% agreement ratio with IDEXX-FlockChek ELISA. With results obtained using the NP-ELISA, an ELISA titre (ET) prediction equation, with which the ELISA titres of a flock or individual chickens can be determined, was derived from a positive/negative (P/N) ratio standard curve. The NP-ELISA enables an alternative rapid serological diagnosis and is suitable for influenza A antibody screening, especially in species that harbour several influenza subtypes.

Multiple amino acid substitutions involved in enhanced pathogenicity of LPAI H9N2 in mice.

Human infection of avian influenza H9N2 virus highlighted the need to better understand the mechanism of interspecies transmission. In this study, we generated mouse-adapted influenza virus (ma01) through serial lung-to-lung passages of a wild-type H9N2 (A/chicken/Hubei/01/1999). Ma01 caused highly lethal infection in mice with severe lung pathology and extended tissue tropism. Nine amino acid substitutions of ma01 were observed in five viral genes (those for PB2, PA, NA, M1, and NS1). Of these mutations, substitutes of PB2(627), PA(349), PA(605), NA(88), and NA(356) were absent in influenza H9N2. Furthermore, the targets of wild-type virus responding to mouse microRNA mmu-mir-1940 and mmu-mir-1904 were eliminated in ma01. The mutation PB2(627) of ma01 confirmed as a key virulence determinant of influenza H5N1 was responsible for the altered recognition of mmu-mir-1904. In addition, induction of IL-1β, IL-6, TNF-α, and IFN-β was found in significantly higher levels in ma01 infected mouse peripheral blood than parental strain. These results demonstrate that multiple amino acid substitutions and avoidance of microRNA recognitions may be essential for lethal infection and high speed of virus growth can outcompete the antiviral response of infected host.

Genome Sequence of Poultry Pathogen Riemerella anatipestifer Strain RA-YM

Riemerella anatipestifer is a Gram-negative, rod-shaped bacterium associated with epizootic infections in poultry. R. anatipestifer strain RA-YM, belonging to the serotype 1 prevalent in China, is a clinically isolated strain with high-level virulence. Here, we report the first genome sequence of this species.

Haemophilus parasuis infection activates the NF-κB pathway in PK-15 cells through IκB degradation.

Haemophilus parasuis is the causative agent inducing a severe inflammation of the serous membranes in pigs, which contribute to the great economic losses in the pig industry in China in the recent years. In this study, it was demonstrated that H. parasuis could activate the inflammatory transcription factor nuclear factor-kappaB (NF-κB) in a bacteria time- and dose-dependent manner in PK-15 cells, and inactivated H. parasuis significantly reduced the level of NF-κB activation in PK-15 cells compared with the counterpart especially in the later stage. After H. parasuis infection, the degradation of IκBα and phosphorylation of p65 was detected in PK-15 cells. Furthermore, the subcellular localization analyzed using confocal laser microscopy showed that p65-GFP rapidly translocated to the nucleus when PK-15 cells were stimulated with H. parasuis. In addition, real-time RT-PCR showed that the key inflammatory mediators including IL-8, CCL4 and CCL5, regulated by nuclear factor-kappaB (NF-κB) were up-regulated dramatically by the infection of H. parasuis in PK-15 cells. This was the first time to report that H. parasuis infection activated the NF-κB pathway in vitro through IκB degradation.

Displaying the protein of Mycoplasma gallisepticum agglutinin on the cell surface of Bacillus thuringiensis with the S-layer protein.

The S-layer protein CTC surface-display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying the protein of Mycoplasma gallisepticum (MG) agglutinin (pMGA) on the Bt cell surface. By fusing part of pmga1.2 (pmga1.2p) with the surface-anchoring motif of ctc, two recombinant plasmids, pCTC-PMGA1.2P and pCSPMGA1.2P, were constructed. They harboured the fusion genes ctc-pmga1.2p and csa-ctc-pmga1.2p (csa represents csaAB operon, important in anchoring the S-layer protein on the cell surface), respectively. Two recombinant Bt strains were constructed by electro-transferring recombinant plasmids to a Bt plasmid-free derivative strain BMB171. Strains obtained were BCCG (bearing pCTC-PMGA1.2P and the csaAB operon-carrying plasmid pMIL-CSA) and CG (pCSPMGA1.2P). The vegetative cells of both strains were used as antigens for haemagglutination (HA) and haemagglutination inhibition (HI) assays. HA and HI assays showed that recombinant PMGA1.2P proteins were not only displayed on the cell surface of BCCG and CG, but also specific to MG-positive serum. After oral immunization of chickens with spores, both BCCG and CG elicited a humoral response to PMGA1.2P and exhibited immunogenicity, as indicated by serum plate agglutination (SPA) assays. This study suggests the possibility of generating heat-stable and oral vaccines against infectious diseases of fowl with Bt surface-display system.

Gene expression responses to Riemerella anatipestifer infection in the liver of ducks.

Riemerella anatipestifer is one of the most economically important pathogens of farm ducks worldwide. The molecular mechanisms that underlie its pathogenesis, particularly the host response to R. anatipestifer infection, are poorly understood. The differentially expressed gene profile of duck livers at 24 h following R. anatipestifer infection was therefore investigated using suppression subtractive hybridizaton analysis. A total of 45 differentially expressed genes were identified, which primarily included genes for proteins involved in acute-phase response, inflammatory response, immune response, wound healing and iron homeostasis. For the expression level of 20 genes from those 45 analysed by quantitative reverse transcriptase-polymerase chain reaction at 8, 24 and 48 h post infection, significant differences were observed among the three time points of measurements. The result from this study revealed a gene expression profile of duck liver during R. anatipestifer infection, and those genes with a role in the immune response and wound healing deserving further investigation to elucidate their respective roles during infection.

Enterococcus faecium HDRsEf1 Protects the Intestinal Epithelium and Attenuates ETEC-Induced IL-8 Secretion in Enterocytes

The probiotic Enterococcus faecium HDRsEf1 (Ef1) has been shown to have positive effects on piglet diarrhoea, but the mechanism has not yet been elucidated. In this study, using the IPEC-J2 cell line to mimic intestinal epithelial cells and enterotoxigenic Escherichia coli (ETEC) K88ac as a representative intestinal pathogen, the mechanism underlying Ef1 protection against an enteropathogen was investigated. The results demonstrated that Ef1 was effective in displacing K88ac from the IPEC-J2 cell layer. Moreover, Ef1 and its cell-free supernatant (S-Ef1) modulate IL-8 released by IPEC-J2 cells. Ef1 and its cell-free supernatant showed the potential to protect enterocytes from an acute inflammatory response. In addition, Ef1 and its cell-free supernatant increased the transepithelial electrical resistance (TEER) of the enterocyte monolayer, thus strengthening the intestinal barrier against ETEC. These results may contribute to the development of therapeutic interventions using Ef1 in intestinal disorders of piglets.

Pregnane X receptor is required for IFN-α-mediated CYP3A29 expression in pigs.

Pregnane X receptor (PXR) has been identified as a central mediator for coordinate responses to xenobiotic and drug metabolism, and is the major transcriptional regulator of cytochrome P-450 (CYP). Interferon (IFN)-α is known to induce antiviral mechanisms and exert immune regulatory capacity in various cell types. Here, we used primary porcine hepatocytes and a cultured hepatocyte cell line to identify the metabolic role of PXR in IFN-α-mediated CYP3A29 expression. We found that IFN-α could activate PXR in both time- and dose-dependent manners in pigs. Activation of PXR significantly increased CYP3A29 mRNA and protein expression. Meanwhile, the expression of CYP3A29 induced by IFN-α occurred after the increase of PXR expression in porcine hepatocytes. In addition, the IFN-α-induced CYP3A29 expression was blocked by PXR knockdown. The PXR-overexpressed cells (transfected with porcine PXR) increased CYP3A29 mRNA and protein expression. Furthermore, in animal experiments, we found that IFN-α increased both CYP3A29 mRNA and protein levels. Collectively, our results suggest that PXR plays an important role in IFN-α-mediated CYP3A29 expression in porcine hepatocytes.

Probiotic mixture ameliorates heat stress of laying hens by enhancing intestinal barrier function and improving gut microbiota

Abstract Heat stress (HS) is a major stressor for laying hens. It causes enormous financial losses worldwide annually. The purpose of this study was to investigate the effect and mechanism of a probiotic mixture of Bacillus subtilis and Enterococcus faecium on the performance of laying hens under HS. Eight hundred and fifty-six commercial laying hens (Hy-Line Brown, aged 40 weeks) were randomly allocated to three groups, Group C (26 °C), Group H (33 °C) and Group H + PM (33 °C + probiotic mixture), respectively. The trial lasted for 20 days. Compared with Group H, we demonstrated that treatment with the probiotic mixture significantly increased the egg production rate, average daily feed intake (ADFI), and egg weight of laying hens under HS (p < .05). In addition, the eggshell thickness, eggshell strength and albumen height also improved. We further showed that these probiotics improved gut microbiota as well as enhanced intestinal integrity, which may be responsible for inhibiting the invasion of bacteria and improving the performance indices. The study suggests that this probiotic mixture may be an effective strategy for treating laying hens under HS.

The Role of the Regulator Fur in Gene Regulation and Virulence of Riemerella anatipestifer Assessed Using an Unmarked Gene Deletion System

Riemerella anatipestifer, an avian pathogen, has resulted in enormous economic losses to the duck industry globally. Notwithstanding, little is known regarding the physiological, pathogenic and virulence mechanisms of Riemerella anatipestifer (RA) infection. However, the role of Ferric uptake regulator (Fur) in the virulence of R. anatipestifer has not, to date, been demonstrated. Using a genetic approach, unmarked gene deletion system, we evaluated the function of fur gene in the virulence of R. anatipestifer. For this purpose, we constructed a suicide vector containing pheS as a counter selectable marker for unmarked deletion of fur gene to investigate its role in the virulence. After successful transformation of the newly constructed vector, a mutant strain was characterized for genes regulated by iron and Fur using RNA-sequencing and a comparison was made between wild type and mutant strains in both iron restricted and enriched conditions. RNA-seq analysis of the mutant strain in a restricted iron environment showed the downregulation and upregulation of genes which were involved in either important metabolic pathways, transport processes, growth or cell membrane synthesis. Electrophoretic mobility shift assay was performed to identify the putative sequences recognized by Fur. The putative Fur-box sequence was 5′-GATAATGATAATCATTATC-3′. Lastly, the median lethal dose and histopathological investigations of animal tissues also illustrated mild pathological lesions produced by the mutant strain as compared to the wild type RA strain, hence showing declined virulence. Conclusively, an unmarked gene deletion system was successfully developed for RA and the role of the fur gene in virulence was explored comprehensively.