Ruiping She

Prevalence of hepatitis E virus in swine under different breeding environment and abattoir in Beijing, China

This study was to investigate the prevalence of HEV in pig herds under different breeding environment and in abattoirs located in Beijing, China. In total 638 sera samples and 114 liver samples were collected for a serological survey and a RT-PCR assay, respectively. The average prevalence rate of HEV in pig herds in Beijing suburb were 47.5-100%. Seropositive rate was 76.6% for pig herds of large-scale and 90% for family-scale farms. For sera samples collected from abattoir, 127 samples (78.4%) were found to be positive. Among four liver samples (3.5%) that positive for HEV RNA detection, two strains of HEV has been identified. the two detected HEV sequences shared 99.1% nucleotide sequence identity with each other, and 76.4-81.0%, 76.5-76.8%, 75.7-79.0% and 85.2-96.4% with related strains representing genotype 1, 2, 3, and 4, respectively. Further phylogenetic analysis revealed two HEV sequences belonged to genotype 4 and exhibited a high identity with strain JKO-ChiSai98C (95.4 and 95.7%), and with strains 87, 277 and 292 (96.1-96.4%) isolated from patients with sporadic acute hepatitis E in Beijing, China. The results of this study indicated that the prevalence of HEV in the pig herds were quite high. Additional public-health concerns might be placed on pork safety.

Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein

The use of avian herpesviruses (Mareks disease virus, MDV) as vectors to express the capsid protein of infectious bursal disease virus (IBDV) was well established, and its protection against IBDV challenge has been evaluated previously. However, there is little data about rMDV1 expressing the VP2 protein of IBDV protecting SPF and commercial chickens against virulent IBDV (vIBDV) challenge. In this study, we constructed a stable rMDV1 expressing the VP2 protein of IBDV by inserting the coding sequence within the US10 gene of MDVl by homologous recombination and designated this as rMDVl-US10L, and evaluated effectiveness of the recombinant VP2 protein with SPF chickens and commercial chickens with maternal antibodies in vIBDV challenge. The results can be summarized as follows: (1) We constructed a rMDV1 expressing IBDV-VP2 under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10L]. (2) rMDV-VP2 protein was readily expressed and induced 53% protection against a vIBDV challenge in SPF chickens with 10(3)PFU/chicken, whereas 10(4)PFU induced 73% protection. (3) Vaccination of commercial chickens having maternal antibodies to rMDV1-VP2 induced 87% protection in vIBDV challenge, which was similar to results using the live vaccine, BJ87 IBDV strain, in commercial chickens. These results demonstrate that the VP2 antigen expressed in the MDV vector was an effective and stable vaccine in correlation with the vaccine efficacy against lethal IBDV challenge, and can provide a better protective effect that is likely to persist for the life of the chickens.

Mast cell mediated inflammatory response in chickens after infection with very virulent infectious bursal disease virus.

The potential role of the mast cells in the invasion of very virulent infectious bursal disease virus (vvIBDV) is unknown. We evaluated mast cell activity and tryptase production after vvIBDV infection in special pathogen-free (SPF) chickens using cytochemistry and immunohistochemistry analyses. The results were as follows: (1) severe histologic lesions were observed in the thymus, spleen, cloacal bursa, liver, kidney and other tissues. vvIBDV viral antigens were detected and presented extensively in the parenchymatous organs, in particular, the cloacal bursa, liver, kidney, thymus, spleen and pancreas. (2) In the vvIBDV-infected group, the mast cell population increased markedly in the liver, kidney, thymus, glandular stomach, spleen and cloacal bursa on days 1, 2 and 3 after vvIBDV infection (p<0.05). However, very few mast cells were observed in those same tissues in the controls, especially in the bursa of Fabricius. (3) Tryptase, a marker for activated mast cells, has a positive correlation with mast cell distribution. The mast cells identified in the tissues were likely to be activated since they were associated with cell degranulation and the presence of tryptase. Furthermore, the co-localization of mast cells, and presence of vvIBDV antigens suggests that the mast cells were activated by vvIBDV infection. Our results also suggest that tryptase may contribute to the inflammation of acute IBD induced by vvIBDV infection. Our research contributes to the further understanding of inflammatory response mechanisms and the contribution of mast cell activity to this process.

Effects of Rabbit Sacculus Rotundus Antimicrobial Peptides on the Intestinal Mucosal Immunity in Chickens

Ninety chickens were randomly divided into 2 groups (45 chickens in each group) to determine the effect of oral administration of rabbit sacculus rotundus antimicrobial peptides (RSRP) on the intestinal mucosal immune responses in chicken. On d 7, 14, 21, and 28, the animals received 0.1 mg of RSRP dissolved in 0.5 mL of physiological saline. The control groups received the same dose of physiological salt solution on the same day. The results showed that RSRP increased the villus height of the duodenum (P < 0.01) and jejunum (P < 0.05) at the ages of 28, 42, and 56 d. The numbers of intestinal intraepithelial lymphocytes in different parts of intestine of the RSRP group were increased significantly more than that of the control (P < 0.01 or P < 0.05) at the ages of 28, 42, and 56 d. The RSRP increased the area of IgA-secreting cells of each fragment of intestine at all 3 time points. These results indicated that the presence of RSRP affected and considerably modified the structure of the intestine and mucosal immune parameters in healthy chickens when compared with controls.

Experimental infection of Mongolian gerbils by a genotype 4 strain of swine hepatitis E virus.

An ideal animal model for hepatitis E virus (HEV) research is still unavailable. To assess the possibility of using Mongolian gerbils as animal model, 28 gerbils were randomly assigned into two groups, 14 for each group. Gerbils in Group 1 were inoculated with a genotype 4 HEV recovered from swine via the intraperitoneal route. Group 2 was used as a negative control and inoculated with normal suspension of swine liver. Sera and feces samples were collected once a week for 7 weeks. Two gerbils from both groups were necropsied weekly, pathological changes were recorded and tissue samples collected for further investigation. Distribution of the virus antigens was determined by immunohistochemical staining. Nested RT‐PCR and a commercial ELISA kit were used to confirm the infection. Research results demonstrated that Mongolian gerbils in Group 1 were successfully infected with HEV. Viremia and fecal virus shedding lasted nearly 4 weeks, while the virus could be detected constantly in the liver, and occasionally in the kidneys and spleen as well as the small intestine. Histopathological changes in the liver were present with slight, multifocal, lymphohistiocytic infiltrates in the portal tracts or distributed irregularly throughout the liver. HEV antigens could be detected in the liver and intestine, and were mainly distributed in the nuclei. The results indicate that Mongolian gerbils could be used as an ideal animal model for the study of HEV. J. Med. Virol. 81:1591–1596, 2009.

Effects of pig antibacterial peptides on growth performance and intestine mucosal immune of broiler chickens

Currently, substitutions for antibiotic growth promoters in animals are attracting interest. This study investigated the effects of pig antibacterial peptides (PABP) on growth performance and small intestine mucosal immune responses in broilers. Three hundred 1-d-old Arbor Acre male broiler chickens were randomly allocated to 5 groups with 60 birds per group. The groups were control group; PABP administered in drinking water at 20 and 30 mg/L of water; or PABP supplemented in feed at 150 and 200 mg/kg of diet. The birds were fed a corn-soybean based diet for 6 wk. Chickens were weighed weekly and killed after 42 d of feeding, and growth performance was measured. Samples of the duodenum and jejunum were collected. The villus height, mucosa thickness, alkaline phosphatase activity, and numbers of secreting IgA and goblet cells were evaluated. The PABP-treated groups had greater BW and average daily gain, greater height of villus and thickness of gut mucosa, greater activity of alkaline phosphatase, higher ratio of secreting IgA, and a greater number of goblet cells compared with the control group (P<0.05). In conclusion, PABP can improve the growth performance, increase the intestinal ability to absorb nutrients, and improve the mucosal immunity of the intestine.

Induction of Adaptive T Regulatory Cells That Suppress the Allergic Response by Coimmunization of DNA and Protein Vaccines

Allergen-induced immediate hypersensitivity (AIH) is a health issue of significant concern. This robust inflammatory reaction is initiated by the allergen-specific T cell responsiveness. Severe lesion reactions on skin are consequential problem requiring medical treatment. Effective Ag-specific treatments or preventions are lacking. Using a rodent model of AIH induced by flea allergens, we first report that coimmunization of DNA and protein vaccines encoding the flea salivary specific Ag-1 ameliorated experimental AIH, including Ag-induced wheal formation, elevated T cell proliferation, and infiltration of lymphocytes and mast cells to the site of allergen challenge. The amelioration of AIH was directly related to the induction of a specific population of flea antigenic specific T cells exhibiting a CD4+CD25−FoxP3+ phenotype, a characteristic of regulatory T (TREG) cells. These TREG cells expressing IL-10, IFN-γ, and the transcriptional factor T-bet after Ag stimulation were driven by a tolerogenic MHC class II+/CD40low dendritic cell population that was induced by the coimmunization of DNA and protein vaccines. The tolerogenic dendritic cell could educate the naive T cells into CD4+CD25−FoxP3+ TREG cells both in vitro and in vivo. The study identified phenomenon to induce an Ag-specific tolerance via a defined Ag vaccinations and lead to the control of AIH. Exploitation of these cellular regulators and understanding their induction provides a basis for the possible development of novel therapies against allergic and related disorders in humans and animals.

Effects of swine gut antimicrobial peptides on the intestinal mucosal immunity in specific-pathogen-free chickens

Sixty specific-pathogen-free chickens were randomly divided into 2 groups (30 chickens for each group) to determine the effect of swine gut antimicrobial peptides (SGAMP) on intestinal mucosal immunity. All chickens were raised in negative-pressure isolators and fed the same diet. The results were as follows. (1) In the SGAMP group, the number of mast cells was increased markedly in the duodenum from d 21 to 49 (P < 0.05), and similar results were observed in the jejunum and ileum after being treated with SGAMP (P < 0.05). However, fewer mast cells were observed in those same tissues in the control. (2) Intraepithelial lymphocytes in the duodenum, jejunum, and ileum were increased significantly from d 21 to 49 in the SGAMP group compared with the control (P < 0.05). (3) Compared with the control, goblet cells were increased significantly in duodenum and jejunum from d 35 to 49 (P < 0.05) after SGAMP treatment and in the ileum were increased from d 21 to 49 (P < 0.05). (4) Swine gut antimicrobial peptides upgrade the expression of secretory IgA at different sites within the intestinal tract. The results strongly support that SGAMP can enhance the intestinal mucosal immune parameters of specific-pathogen-free chickens. Our research contributes to the further understanding of immunoregulatory mechanisms of intestinal mucosal immunity and the contribution of SGAMP to this process.

Evidence for a role of mast cells in the mucosal injury induced by Newcastle disease virus

We have previously demonstrated that mast cells were significantly increased during Newcastle disease virus (NDV) infection, but their precise role in the process is unknown. In this study, we investigated the role of mast cells in this process by using ketotifen, a mast cell membrane stabilizer. A total of 60 specific-pathogen-free chickens were randomly divided into 3 groups of 20 birds each (NDV-infected group, ketotifen-pretreated group, and the control group). The ketotifen-pretreated group was administered orally with ketotifen before NDV infection. On 12, 24, and 48 h postinfection, 5 chickens from each treatment were killed. Tissues of proventriculus were collected to quantify mast cells, the content of tryptase and histamine by cytochemistry, immunohistochemistry, and fluorescence analysis, respectively. The results showed that the population of mast cells and the content of tryptase and histamine were increased significantly in the proventriculus (P < 0.01) of infected birds compared with the control group. An acute mucosal injury was observed in the infected chickens. In contrast, among chickens pretreated with ketotifen, followed by NDV infection, the mast cells number and the content of tryptase and histamine were decreased significantly (P < 0.01). Likely as a result, the mucosal injury was remitted remarkably. The overall results of this experiment suggest that mast cells are implicated in NDV-induced mucosal injury. Inhibition of mast cell mediator release may represent a novel strategy to modulate this process.

Chicken intestine defensins activated murine peripheral blood mononuclear cells through the TLR4-NF-κB pathway.

Defensins serve as alarm signals in mobilizing the immune system and activating the innate and adaptive immune responses. In order to investigate whether avian defensins could activate monocytes of another species, and whether chicken defensins could modulate or amplify the adaptive immune responses of murine through the TLR-NF-kappaB pathway, the relationship between the chicken intestinal defensin AvBD13 and TLR4 in murine peripheral blood mononuclear cells (PBMCs) was explored in vitro. Monocytes were stimulated with AvBD13 (1 microg/mL). The levels of NF-kappaB p65, CD80, CD86, IL-12 and IFN-alpha were measured by immunohistochemical analysis of the cells or enzyme-linked immunosorbent assay (ELISA), and the TLR4 levels in monocytes were measured by flow-cytometry. We found that AvBD13 can activate NF-kappaB, induce the inflammatory cytokines IL-12 and IFN-alpha, and upregulate costimulatory molecules like CD80 and monocyte proliferation, which was clearly inhibited by the anti-TLR4 antibody. TLR4 expression was rapidly downregulated in the presence of AvBD13. AvBD13 could modulate monocytes directly and serve as an endogenous ligand for TLR4 and upregulate costimulatory molecules and monocyte proliferation. Thus, TLR4 is involved in AvBD13-mediated activation of adaptive immune responses.