Expression of Toll-like receptor 9 (TLR9) in the lungs and lymphoid tissue of pigs

The pattern of distribution of Toll-like receptor 9 (TLR9) in different tissues varies between species. The aim of the present study was to describe the distribution of TLR9 expression in selected tissues and organs of healthy pigs at 3 weeks and 3 months of age. Representative formalin-fixed samples of lung, thymus and secondary lymphoid tissues were evaluated by immunohistochemistry. TLR9 positive staining was observed in epithelial cells, vascular endothelium and myoepithelial-like cells, as well as in cells of the alveolar septa of the lung. Antigen presenting cells of perifollicular zones (interdigitating, macrophage and dendritic-like cells) of the Peyer's patches, lymph nodes, spleen and thymus were also immunoreactive for TLR9. No differences were seen in TLR9 protein expression in tissues from the two age groups.     

Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendritic cells (cMo-DC)

Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs.     

Needle-free delivery of an inactivated avian influenza H5N3 virus vaccine elicits potent antibody responses in chickens

A needle-free delivery system was assessed as a route for providing quick, safe, and effective vaccination against avian influenza (AI). Two groups of chickens were vaccinated with a commercially available inactivated H5N3 virus vaccine delivered either with a needle-free device or with the conventional syringe-and-needle method recommended by the vaccine manufacturer. The kinetic aspects of seroconversion, peak antibody levels, and antibody titers were measured by a combination of an indirect enzyme-linked immunosorbent assay and the hemagglutination-inhibition test and were all found to be similar in the 2 groups of chickens. We conclude that the needle-free delivery system could result in effective immunization against H5N1 AI epidemics and pandemics in chickens.     

Molecular identification and functional expression of porcine Toll-like receptor (TLR) 3 and TLR7

To investigate porcine Toll-like receptors (TLR) responding to viral pathogen associated molecular patterns, the full-length cDNA of porcine TLR3 and TLR7 were identified and characterized. Porcine TLR3 and TLR7 cDNA encode 904- and 1050-amnio-acid polypeptides, respectively. Both porcine TLR3 and TLR7 contain typical functional TLR domains and share about 80% sequence identity to other mammalian orthologues. Tissue expression profiles showed that TLR3 was highly expressed in kidney, duodenum, spleen and liver, and moderately expressed in bone marrow, lung, and skin. Conversely, TLR7 was moderately and constitutively expressed in all tissues evaluated. Stimulation of mammalian cells transfected with porcine TLR3 and TLR7 constructs with TLR3 and TLR7 agonists [poly (I:C) and imiquimod (R837), respectively], and adenovirus elicited activation of interferon regulatory factors (IRFs). These data provide molecular and functional information for porcine TLR3 and TLR7, and implicate their role in mediating immune protection against porcine viral diseases.     

A role for Toll-like receptor 4 in the host response to the lung infection of Yersinia pseudotuberculosis in mice

Although a Yersinia pseudotuberculosis (Yptb) lung infection model has been developed to study Y. pestis pathogenesis, it is still necessary to establish a new animal model to mimic the pathophysiological features induced by Y. pestis infection. Here, we provide a new lung infection model using the Yptb strain, IP2777, which displayed rapid spread of bacteria to the liver, spleen, and blood. In addition, we examined whether TLR4 is involved in Yptb-induced pathogenesis in the lung infection model of mice we generated. Following lung infection of WT and TLR4-deficient mice with the Yptb strain IP2777, the survival rate, bacterial colonization, histopathology, and level of cytokines and chemokines in the lung, spleen, liver, and blood were analyzed. TLR4-deficient mice had a lower survival rate than WT mice in response to Yptb lung infection. Although the bacterial colonization and pathology of the lung were comparable between WT and TLR4-deficient mice, those of the spleen and liver were more severe in TLR4-deficient mice. In addition, the levels of TNF-α and CXCL2 in the liver and IL-6 and CXCL2 in the blood were higher in TLR4-deficient mice than in WT mice. Our results demonstrate that TLR4 is necessary for optimal host protection against Yptb lung infection and TLR4-deficient mice may serve as a better genetic model of Yptb infection for mimicking Y. pestis infection.     

Polymorphism of Toll-like receptor 9 (TLR9) gene in sheep

Bacterial and synthetic DNA containing unmethylated CpG dinucleotides in particular sequence contexts, activates the vertebrate immune system through Toll-like receptor 9 (TLR9). In this study, we use PCR-single-strand conformational polymorphism (PCR-SSCP) analysis to investigate genetic variation in a key region of the ovine TLR9 gene. Three novel SSCP patterns, representing three different sequences, were identified. Either one or two different sequences were detected in individual sheep and all the sequences identified shared high homology to the TLR9 sequences from a variety of species, suggesting that these sequences represent allelic variants of the ovine TLR9 gene. Four single nucleotide polymorphisms (SNPs) were detected in the region amplified and two of them were non-synonymous substitutions that would result in amino acid changes. Variation detected here might have an impact on the structure and/or function of TLR9 and hence affect the immune response to pathogens.     

黄芪多糖分级组分对鸡脾淋巴细胞增殖影响

目的：探讨黄芪多糖分级组分对鸡脾淋巴细胞增殖的影响。方法：将黄芪多糖经过不同浓度的乙醇进行分级沉淀,得到3个分级组分：A1、A2、A3;将320只鸡随机分为8组,分别皮下注射A1、A2、A3、A1＋A2、A1＋A3、A2＋A3、A1＋A2＋A3和蒸馏水,每隔7d各组随机捕杀鸡4只,培养脾淋巴细胞,NTT法测定黄芪多糖分级组分对鸡脾淋巴细胞增殖的影响。结果：A2、A3、A2＋A3、A1＋A2＋A3能显著促进淋巴细胞增殖。结论：黄芪多糖免疫功能的有效部位为A2、A3,尤以A2作用显著。     

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.     

Pulmonary congestion and edema (marble spleen disease) of chickens produced by group II avian adenovirus

"Marble spleen disease" of chickens was diagnosed in 22-week-old chickens. Total mortality was 8.9%. Deaths occurred over a period of 2 months. Gross lesions included pulmonary congestion, splenomegaly, hepatomegaly, and congestion of egg follicles. Microscopic lesions included pulmonary congestion and edema, and reticuloendothelial cell hyperplasia of the spleen with concurrent white-pulp necrosis and lymphocyte depletion. The pulmonary lesions were of sufficient intensity to have caused the death of fatally affected birds. Many of the hyperplastic reticuloendothelial cells contained basophilic intranuclear inclusions similar to those that characterize hemorrhagic enteritis of turkeys, marble spleen disease of pheasants, and adenovirus group II splenomegaly of chickens. These characteristic lesions, plus serologic identification of the causal virus, indicate that "marble spleen disease" caused by avian group II adenovirus was affecting the flock under study. This appears to be the first report of death of chickens due to pulmonary congestion and edema caused by spontaneous infection with avian group II adenovirus.     

Characteristics of systemic infection and host responses in chickens experimentally infected with Salmonella enterica serovar Gallinarum biovar Gallinarum

Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing systemic infection in poultry, which leads to significant economic losses due to high mortality. However, little is known about the dynamic process of systemic infection and pathogenic characteristics of S. Gallinarum in chickens. In the present study, we developed an oral infection model that reproduces the pathology of S. Gallinarum and clarified the host immune response of the infected chickens. Chickens at 20 days of age orally inoculated at a dose of 10⁸ colony forming unit (CFU) showed typical clinical signs of fowl typhoid and died between 6 and 10 days post infection. The inoculated S. Gallinarum rapidly disseminated to multple organs and the bacterial counts increased in the liver and spleen at 3 days post infection. Pathological changes associated wirh inflammation in the liver and spleen became apparent at 4 days post infection, and increased expression of interferon (IFN)-γ and interleuikin (IL)-12 in the liver and spleen did not observed until 3 days post infection. These results indicate that S. Gallinarum rapidly spread to entire body through intestine, and the low-level of inflammatory responses in the liver during the early stage of infection may contribute to rapid, systemic dissemination of the bacteria. Our infection model and findings will contribute to the better understanding of the pathogenic mechanism of S. Gallinarum, and provide new insights into the prevention and control of fowl typhoid.     

传染性腔上囊炎疫苗免疫鸡TLR7基因表达的动态变化

给11日龄雏鸡分别口服接种鸡传染性腔上囊炎（IBD）弱毒疫苗（法贝灵，IBD-Blen）1和3头份后，于不同时间采集脾和腔上囊组织，分离纯化淋巴细胞，常规方法抽提总RNA，以鸡伊actin基因为内参，采用半定量RT—PCR法检测两种组织中鸡Toll样受体7（ChTLR7）基因的表达动态。结果显示，接种IBD弱毒疫苗前，试验鸡脾和腔上囊组织中均有ChTLR7的微量表达，疫苗接种后第7h，脾中ChTLR7 mRNA的表达开始显著上调，至接种后第12h达峰值，此后迅速下降，至第130h时降至疫苗接种前的水平；而腔上囊组织中ChTLR7 mRNA的表达自接种后第12h开始增加，第24h时达峰值，随后迅速下降，约72h后恢复至疫苗接种前水平。表明ChTLR7可能参与了IBDV感染早期的天然免疫反应过程。     

Polymorphisms in the canine glucocorticoid receptor alpha gene (NR3C1α)

Corticosteroids are one of the most extensively used class of therapeutic agents in dogs. In human patients, response to corticosteroid therapy has been correlated with the presence of certain polymorphisms of the glucocorticoid receptor gene (NR3C1). Depending on the polymorphism present, patients may show either increased sensitivity to glucocorticoid‐induced adverse effects or resistance to their therapeutic effects. Because response to corticosteroid therapy in dogs can also be variable and unpredictable, we hypothesized that genetic variability exists in the canine NR3C1 gene. The aim of this study was to sequence the coding regions of the canine NR3C1 gene in a representative sample of dogs. Samples from 97 dogs from four previously identified genetic groupings of domestic breeds (Asian/Ancient, Herding, Hunting, and Mastiff) were sequenced and evaluated. Four exons contained polymorphisms and four exons showed no variation from the reference sequence. A total of six single nucleotide polymorphisms (SNPs) were identified including four synonymous SNPs and two nonsynonymous SNPs (c.811A>T and c.2111T>C). No dogs were homozygous for either variant allele, while 23 dogs were heterozygous for the c.811A>T allele and 2 were heterozygous for c.2111T>C allele. The amino acid changes caused by c.811A>T (serine to cysteine) and c.2111T>C (isoleucine to threonine) were both predicted by in silico analysis to be ‘probably damaging’ to structure and function of the resulting protein. We conclude that NR3C1 polymorphisms occur in dogs and may cause individual variation in response to corticosteroid therapy.     

Hypophagic and dipsogenic effect of the 5‐HT1A receptor agonist 8‐OH‐DPAT in broiler chickens

The effects of the 5‐HT_1A receptor agonist 8‐OH‐DPAT on food and water intake in male broiler chickens were investigated. The injection of 25 or 50 μg/kg of 8‐OH‐DPAT 15 min before refeeding in fasted animals produced a decrease in food intake. No effect was observed in drinking. The injection of 25 or 50 μg/kg of the 8‐OH‐DPAT 60 min after the start of refeeding did not produce any significant modification in food intake. No effect on drinking was recorded. The agonist 8‐OH‐DPAT injected 15 min before water presentation in water‐deprived chickens, produced an increased drinking 60 min after the presentation of water. No effect on food intake was observed. The results show that the effect on food intake of the agonist 8‐OH‐DPAT in fasted–refed broiler chickens was similar to those observed in mammals and layer‐strain chickens. However, the agonist did not alter significantly the food intake when the broilers were fed 60 min before the injection. These results are contrary to the observed effects in mammals and in layer‐strain chickens. Probably, the selection for rapid growth rate in broilers causes modifications in the feeding control pattern. The comparison between broilers and layers strain may be a useful tool to elucidate the complex mechanisms involved in food and water intake regulation in chickens.     

In silico identification of components of the Toll-like receptor (TLR) signaling pathway in clustered chicken expressed sequence tags (ESTs)

We have described a bioinformatic approach that involves the clustering of expressed sequence tags (ESTs) to reveal homologs of the Toll-like receptor (TLR) pathway in the chicken. Homology searching of proteins, predicted to be encoded by these EST clusters, resulted in the in silico identification of full-length sequences for Toll-interacting protein (Tollip), IL-1 receptor-associated kinase 4 (IRAK-4), myeloid differentiation factor 88 adapter-like (Mal), TGF beta-activated kinase 1 binding protein 1 (TAB1). We also determined partial sequence information for myeloid differentiation factor 88 (MyD88), two novel TLRs, TNF receptor-associated factor 6 (TRAF6), TGF beta-activated kinase 1 (TAK1), TAB2, inhibitor of nuclear factor kappa B kinase alpha (IKK alpha) and IKK beta. This bioinformatics study has confirmed the evolutionary conservation of the TLR pathway in chicken and demonstrated its essential homology to the TLR pathway in mammals. We have identified in silico the full-length sequence for liver-expressed antimicrobial peptide 2 (LEAP-2). This is the first time a non-mammalian LEAP-2 has been described.