Heterorhabditidoides rugaoensis n. sp. (Rhabditida: Rhabditidae), a Novel Highly Pathogenic Entomopathogenic Nematode Member of Rhabditidae.

A novel entomopathogenic nematode species, Heterorhabditidoides rugaoensis n. sp. RG081015, collected from Rugao, China, is described. The new species is morphologically very similar to H. chongmingensis but can be distinguished from it on the basis of some morphological characteristics, combined with molecular data and a cross-hybridization test. Males of the new species can be recognized on the basis of body length averaging 1396.2 µm; lateral field with one ridge; metastome isoglottoid with one hemispherical swellings comprised of two to three well-developed warts; asymmetric spicules; peloderan bursa. In IJs, EP = 134.5 µm; ES = 149.3 µm; tail length = 82.5 µm; and a = 20.5. Hermaphroditic females have four to five lateral ridges. The 18S rDNA and ITS sequences of the two nematodes share 99% and 98% identity, respectively. Phylogenetic trees of 18S rDNA and ITS indicate that the new species is most closely related to H. chongmingensis; thus, the two nematodes belong to the same genus. Failure of cross-hybridization between them indicates that nematode strain RG081015 is a novel species and is described herein as H. rugaoensis n. sp. The LC50 of the novel species against Galleria mellonella were 24.35 IJs / ml within 48 hours of infection. Morphological characteristics, genetic similarity analyses, and phylogenetic relationships provide strong evidence that some species of Oscheius/Insectivora-group should be reassigned to the genus Heterorhabditidoides.

H9N2 avian influenza virus enhances the immune responses of BMDCs by down-regulating miR29c.

Avian influenza virus (AIV) of the subtypes H9 and N2 is well recognised and caused outbreaks-due to its high genetic variability and high rate of recombination with other influenza virus subtypes. The pathogenicity of H9N2 AIV depends on the host immune response. Dendritic cells (DCs) are major antigen presenting cells that can significantly inhibit H9N2 AIV replication. MicroRNAs (miRNAs) influence the ability of DCs to present antigens, as well as the ability of AIVs to infect host cells and replicate. Here, we studied the molecular mechanism underlying the miRNA-mediated regulation of immune function of mouse DCs. We first screened for and verified the induction of miRNAs in DCs after H9N2 AIVstimulation. We also constructed miR29c, miR339 and miR222 over-expression vector and showed that only the induction of miR29c lead to a hugely increased expression of surface marker MHCII and CD40. Whilst the inhibition of miR29c, miR339 and miR222 in mouse DCs would repressed the expression of DCs surface markers. Moreover, we found that miR29c stimulation not only up-regulate MHCII and CD40, but also enhance the ability of DCs to activate lymphocytes and secrete cytokines IL-6 or TNF-a. Furthermore, we found that Tarbp1 and Rfx7 were targeted and repressed by miR29c. Finally, we revealed that the inhibition of miR29c marvelously accelerated virus replication. Together, our data shed new light on the roles and mechanisms of miR29c in regulating DC function and suggest new strategies for combating AIVs.

H9N2 Avian Influenza Virus Protein PB1 Enhances the Immune Responses of Bone Marrow-Derived Dendritic Cells by Down-Regulating miR375.

Polymerase basic protein 1 (PB1), the catalytic core of the influenza A virus RNA polymerase complex, is essential for viral transcription and replication. Dendritic cells (DCs) possess important antigen presenting ability and a crucial role in recognizing and clearing virus. MicroRNA (miRNA) influence the development of DCs and their ability to present antigens as well as the ability of avian influenza virus (AIV) to infect host cells and replicate. Here, we studied the molecular mechanism underlying the miRNA-mediated regulation of immune function in mouse DCs. We first screened for and verified the induction of miRNAs in DCs after PB1 transfection. Results showed that the viral protein PB1 down-regulated the expression of miR375, miR146, miR339, and miR679 in DCs, consistent with the results of H9N2 virus treatment; however, the expression of miR222 and miR499, also reduced in the presence of PB1, was in contrast to the results of H9N2 virus treatment. Our results suggest that PB1 enhanced the ability of DCs to present antigens, activate lymphocytes, and secrete cytokines, while miR375 over-expression repressed activation of DC maturation. Nevertheless, PB1 could not promote DC maturation once miR375 was inhibited. Finally, we revealed that PB1 inhibited the P-Jnk/Jnk signaling pathway, but activated the p-Erk/Erk signaling pathway. While inhibition of miR375 -activated the p-Erk/Erk and p-p38/p38 signaling pathway, but repressed the P-Jnk/Jnk signaling pathway. Taken together, results of our studies shed new light on the roles and mechanisms of PB1 and miR375 in regulating DC function and suggest new strategies for combating AIV.

Effect of dietary nonphytate phosphorus content on ileal lymphocyte subpopulations and cytokine expression in the cecal tonsils and spleen of laying hens that were or were not orally inoculated with Salmonella Typhimurium.

OBJECTIVE: To evaluate the effects of dietary nonphytate phosphorus (NPP) content on ileal lymphocyte subpopulations and cytokine expression in the cecal tonsils and spleen of hens that were or were not inoculated with Salmonella Typhimurium. ANIMALS: 64 Salmonella-free hens. PROCEDURES: Hens were fed a diet with 0.22% (control; n = 32) or 0.42% (high-P; 32) NPP for 6 weeks and then orally inoculated with S Typhimurium (5 × 10(7) CFUs) or PBSS. Tissues were obtained from 8 S Typhimurium-inoculated and 8 PBSS-inoculated hens from each group at 2 and 7 days postinoculation (DPI). Percentages of ileal CD4+ and CD8+ lymphocytes were determined by flow cytometry. Cytokine mRNA expression was determined by quantitative real-time PCR assays. RESULTS: For S Typhimurium-inoculated hens, plasma parathyroid hormone concentration was significantly increased and 1,25-dihydroxyvitamin D3 concentration was decreased in hens fed the high-P diet, compared with values in hens fed the control diet. Salmonella Typhimurium inoculation caused an increase in the percentage of ileal CD8+ lymphocytes and the expression of interleukin (IL)-1ß, IL-6, IL-8, interferon-γ, IL-12, and IL-18 in the cecal tonsils and spleen and a decrease in the expression of IL-4 and IL-10 in the cecal tonsils. Hens fed the high-P diet had significantly increased splenic expression of interferon-γ at 2 DPI and IL-1ß, IL-6, IL-12, and IL-18 at 7 DPI, compared with hens fed the control diet. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested there was a T-helper 1 cytokine reaction in the cecal tonsils and spleen of S Typhimurium-inoculated hens, and dietary NPP content altered calcium regulation hormone concentrations and affected splenic cytokine expression.

Alteration in lymphocytes responses, cytokine and chemokine profiles in laying hens infected with Salmonella Typhimurium.

Salmonella Typhimurium has been reported to contaminate egg production across the world, but the exact nature of the immune mechanisms protective against Salmonella infection in laying hens has not been characterized at the molecular level. The experiment was conducted to determine Salmonella colonization and lymphocytes subpopulation in the ileum and spleen, and the mRNA expression of pro-inflammatory cytokines [interleukin (IL)-1ß and IL-6], chemokine IL-8, and T helper (Th)1/Th2 cytokines [Interferon (IFN)-γ, IL-12 and IL-18; IL-4 and IL-10 respectively] in the cecal tonsil and spleen of Salmonella challenged hens. Forty Salmonella-free laying hens were challenged orally with Salmonella Typhimurium or phosphate-buffered saline (PBS; control). The Salmonella challenged or non-challenged hens (n=10) were sacrificed at 2 and 7 days post-infection (DPI). The lymphocyte subpopulation was determined via flow cytometric analysis in the ileum and spleen. The cecal tonsil and spleen samples were collected for mRNA expression through quantitative-RT-PCR. The Salmonella counts were higher (P<0.05) in the ileum than that in the spleen at 2 and 7DPI, and were higher (P<0.05) at 7DPI than that at 2DPI in the spleen. Salmonella challenge increased (P<0.05) ileal CD4+ and CD8α+ cells ratios at 2 and 7DPI, whereas it increased (P<0.05) splenic CD8α+ cells ratio only at 7DPI. The magnitude of increase in ileal CD8α+ cells ratio was higher (P<0.05) than that in CD4+ cells ratio. The mRNA expression of IL-1ß, IL-6, IL-8, IFN-γ, IL-12 and IL-18 were significantly up-regulated in the cecal tonsil of Salmonella challenged hens, and the magnitude of increases in IL-6, IL-8 and IL-12 were significantly higher at 7DPI than that at 2DPI. However, Salmonella challenge increased (P<0.05) the mRNA expression of IL-1ß, IL-10 and IL-18 at 2 and 7DPI, and IL-8 and IFN-γ mRNA only at 7DPI in the spleen. These findings demonstrated that there appeared the induction of cellular immune responses, and a Th1-cytokines reaction in the intestine and spleen of laying hens infected with Salmonella Typhimurium.