蒙古马不同组织器官TLR1、TLR2、TLR4和TLR6 mRNA转录水平研究

根据GenBank中马Toll样受体基因序列设计特异性引物,建立检测马Toll样受体（TLRs）mRNA转录水平的SYBR GreenⅠ实时荧光定量PCR方法,检测TLR4、TLR2、TLR1和TLR6在蒙古马不同组织器官中的转录水平。4种TLRs在心脏、肝脏、脾脏、肺脏、肾脏、胃、十二指肠、空肠、盲肠和骨髓中均有转录。其中,TLR4mRNA除在空肠和肝脏外,在其他组织器官中的表达水平均高于TLR2、TLR1、TLR6。免疫器官中,TLR4、TLR1mRNA在骨髓中表达量高于脾脏,而TLR2、TLR6mRNA在脾脏中表达量高于骨髓。各肠段,TLR4、TLR2、TLR1、TLR6mRNA表达水平之间在空肠的差异不是很大,而在十二指肠和盲肠中差异很大。结果表明,TLRs mRNA在马各组织器官转录水平差异较大,可能与其对病原体的识别和抵抗能力有关。     

Toll-like receptor expression in feline lymphoid tissues

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.     

Characterisation of antibodies to bovine Toll-like receptor (TLR)-2 and cross-reactivity with ovine TLR2

Host recognition of conserved pathogen-associated molecular patterns (PAMPs) and their interactions with pattern-recognition receptors, including the Toll-like receptors (TLR) is essential for innate immune response induction. The TLR1 family (TLR1, 2, 6 and 10) is involved in the recognition of gram-positive and gram-negative bacteria and heterodimers of TLR1 or TLR6 with TLR2 are crucial for the identification of several PAMPs. Studies on cell surface expression of TLR in ruminants are hampered by the lack of specific antibodies and no convincingly cross-reactive anti-human antibodies have been described so far. We describe herein four antibodies which recognise bovine TLR2. Differences in TLR2 expression were evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte-derived macrophages. Lower levels of expression were evident on dendritic cell populations derived in vitro and ex vivo, and on alveolar macrophages. One of the antibodies recognised TLR2 expression on ovine peripheral blood monocytes. The identification of antibodies specific for bovine and ovine TLR2 will facilitate studies of the role of this important PRR in the initiation of immune responses to important pathogens.     

Toll-like receptor activation and expression in bovine alpha-herpesvirus infections

The involvement of Toll-like receptors (TLRs) in bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) infections has not been analyzed. In this study, the role of TLR signaling on virus replication was investigated. Blood leukocytes consistently express TLRs. Thus, our approach was to study in vitro the effects of agonist stimulation of TLRs expressed by peripheral blood leukocytes on BoHV-1 and BoHV-5 replication. Furthermore, the patterns of TLRs 3, 7–9 expression on virus-infected-bovine leukocytes were analyzed. Only Imiquimod (TLR7/8 agonist) showed anti-viral activity on infected MDBK cells. This is the first evidence that the timely activation of TLR7/8 signaling is effective in impairing BoHV-1 and 5 replication, thereby providing an experimental indication that Imiquimod may be a promising immune modulator. This work describes, for the first time, the expression patterns of TLRs in BoHV-1- or BoHV-5-infected-bovine leukocytes, suggesting the involvement of TLR7 and TLR9 in the recognition of these viruses.     

Toll-like receptor expression in the nervous system of bovine alpha-herpesvirus-infected calves

In this study, the expression levels of viral Toll-like receptors (TLRs) in the nervous system of bovine herpesvirus type 5 (BoHV-5)-infected calves were investigated. A significant increase in the expression of TLRs 3 and 7–9 was found in the anterior cerebral cortex during acute infection and viral reactivation. In the trigeminal ganglia, only TLR9 expression was significantly affected. The magnitude of the increase was lower in BoHV-1-infected calves, suggesting that a restricted immune response might protect against exacerbated inflammatory responses in the brain. This work describes, for the first time, the involvement of TLRs 3 and 7–9 in the recognition of BoHV in the bovine nervous system, indicating that the expression of these receptors might be associated with the development of neurological disease. Modulation of the signalling pathways mediated by TLRs might provide an effective approach to control the neuro-immune response to BoHV-5, which may be responsible for neurological lesions.     

Identification of single nucleotide polymorphisms in the bovine Toll-like receptor 1 gene and association with health traits in cattle

ABSTRACT: Bovine mastitis remains the most common and costly disease of dairy cattle worldwide. A complementary control measure to herd hygiene and vaccine development would be to selectively breed cattle with greater resistance to mammary infection. Toll-like receptor 1 (TLR1) has an integral role for the initiation and regulation of the immune response to microbial pathogens, and has been linked to numerous inflammatory diseases. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) within the bovine TLR1 gene (boTLR1) are associated with clinical mastitis (CM).Selected boTLR1 SNPs were analysed within a Holstein Friesian herd. Significant associations were found for the tagging SNP -79 T > G and the 3'UTR SNP +2463 C > T. We observed favourable linkage of reduced CM with increased milk fat and protein, indicating selection for these markers would not be detrimental to milk quality. Furthermore, we present evidence that some of these boTLR1 SNPs underpin functional variation in bovine TLR1. Animals with the GG genotype (from the tag SNP -79 T > G) had significantly lower boTLR1 expression in milk somatic cells when compared with TT or TG animals. In addition, stimulation of leucocytes from GG animals with the TLR1-ligand Pam3csk4 resulted in significantly lower levels of CXCL8 mRNA and protein.SNPs in boTLR1 were significantly associated with CM. In addition we have identified a bovine population with impaired boTLR1 expression and function. This may have additional implications for animal health and warrants further investigation to determine the suitability of identified SNPs as markers for disease susceptibility.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Identification standard on bovine and ovine derived materials in animal feed passed the examination

The agricultural standard, Identification standard on bovine and ovine derived materials in animal feed - Real-time Fluorescence Polymerase Chain Reaction （PCR） Method, prepared by Shanghai Veterinary Medicine ＆ Feed Institution, passed the examination at 2009 National Feed Industrial Standardization Technical Committee Meeting on Aug. 11, 2009.     

Examination of bovine and ovine fetuses

Diagnosis of the causes of bovine and ovine abortion is difficult and frustrating and requires a systematic and thorough approach. Laboratory assistance is required in all cases. Herd and individual histories occasionally help, as does knowledge of the gestational age and autolytic condition of the fetus when aborted. Tissues from mummified fetuses should be cultured and examined by fluorescent antibody techniques for viruses. The placental tissue from mummified fetuses should be examined for fungi and lesions. Lung, liver, kidney, spleen, abomasal content, body cavity fluid or serum, and fetal placenta from all but completely mummified fetuses should be submitted to a diagnostic laboratory.     

Sequence analysis of Toll-like receptor genes 1-10 of goat (Capra hircus)

This study involved cloning and sequencing of the coding regions of all 10 Toll-like receptor (TLR) genes of goat. Goat TLR 1-10 gene sequences revealed a high degree of nucleotide identity with sheep and cattle sequences (>90%) and 75-85% with pig, mouse and human sequences. At the amino acid level, 85-99% similarity was observed with sheep and cattle and 60-85% with pig, mouse and human. TLR9c DNA of goat showed the highest amino acid identity to that of sheep (99%) while TLR8 cDNA showed the lowest identity of 88.7% to that of sheep. Variations were seen in the number of leucine rich repeats (LRRs) of goat TLRs as compared to other ruminant species with maximum differences in the TLR3 gene. Phylogenetic analysis through molecular evolution and genetic analysis (MEGA) software and multi dimensional scaling revealed a high degree of conservation of goat TLRs with those from other species. However when the TIR domain of all the TLRs were compared, goat TLR7 TIR alone showed a high divergence of 19.3 as compared to sheep sequences. This is the first report of the full-length cDNA sequences of all the 10 TLR genes of goats which would be a useful tool for the study of evolutionary lineages and for phylogenetic analysis.     

Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis

In the present work, evidence of Listeria monocytogenes antigens based on the avidin-biotin complex (ABC) immunoperoxidase technique was performed on formalin-fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine). Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens. Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination. Positive L. monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases. Paraffin-embedded tissues assayed were stored up for 17 years. Morbidity of the outbreaks was between 0.3-3% and 0.1-1% for ovine and bovine cases, respectively. In all the ovine cases, flocks involved were under extensive grazing conditions. In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used. The ABC test can help as a practical tool for the diagnosis of natural cases of L. monocytogenes encephalitis on formalin-fixed specimens from ovine and bovine.     

Cloning and expression of Toll-like receptors 1 and 2 from a teleost fish, the orange-spotted grouper Epinephelus coioides

The two Toll-like receptors, TLR1 and TLR2 were cloned from orange-spotted grouper, Epinephelus coioides, an important teleost fish in mariculture of Asia. The cDNA sequences of TLR1 and TLR2 are 3195 and 3439 bp long, with an open reading frame (ORF) of 2406 and 2466 bp, encoding 801 and 821 amino acids, respectively. The TLR family motifs, i.e. leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains are conserved in the TLR1 and TLR2, with nine and ten leucine-rich repeat (LRR) domains, and with one TIR domain, respectively. The TLR1 and TLR2 had a constitutive expression in examined organs/tissues of na?ve orange-spotted grouper, and an increased expression of TLR1 and TLR2 at mRNA level was observed in immune organs such as in spleen of LPS and Poly(I:C) stimulated fish. An increased expression of TLR1 and TLR2 was also recorded in immune organs of the fish injected with the bacterial pathogen, Vibrio alginolyticus. Similarly, a significant rise in the expression of MyD88, an adaptor molecule which forms signalling complex with intracellular TIR domain, thus leading to the production of pro-inflammatory cytokines, such as IL-1β, was also observed in the LPS- and Poly(I:C)-stimulated, and V. alginolyticus-infected fish, indicating the possible role of TLR1 and TLR2 in the MyD88 signalling pathway. However, the mechanism involved in the increased expression of TLR1 and TLR2 following LPS and Poly(I:C) stimulation is at present unknown in fish, and further research should be carried out to identify ligands of fish TLR1 and TLR2 in order to understand the function of these receptors.     

Extra hepatic expression of the acute phase protein alpha 1-acid glycoprotein in normal bovine tissues

Combined quantitative (real-time) polymerase chain reaction and immunohistochemistry were used to evaluate the expression of the minor acute phase protein alpha 1-acid glycoprotein (AGP, orosomucoid) in bovine extrahepatic tissues. AGP was produced mainly in the salivary glands and spleen, whereas minor expression was detected in all other tissues sampled, including lung, lymph nodes, uterus, ovary, kidney and tongue. The findings were consistent with immunohistochemical results. In view of the immunomodulatory and direct antibacterial activity of AGP, its expression in the salivary glands may signal an involvement in the regulation of the local immunity, even in non-pathological conditions.     

驯鹿β-防御素-1(reBD-1)mRNA在驯鹿组织器官中的表达检测

为了检测reBD-1 mRNA在驯鹿体内可能的表达器官,研究根据已知的reBD-1 cDNA序列设计了一对预计扩增产物为121 bp的引物,以驯鹿舌、食管、瘤胃、网胃、皱胃、十二指肠、回肠、结肠、肝、肺、气管、肾、膀胱、睾丸、附睾、心脏、脾脏中提取的总RNA为模板,采用反转录PCR(RT-PCR)技术检测驯鹿的上述器官内reBD-1 mRNA的表达情况.结果显示:reBD-1 mRNA在上述组织器官中均有表达,其中在舌、瘤胃、睾丸中表达最强;在食管、十二指肠、结肠、气管、脾脏中有中等量的表达;在网胃、皱胃、回肠、肝、肺、肾、膀胱、附睾、心脏内的表达较弱.β-防御素reBD-1在驯鹿体内的广泛表达提示reBD-1有助于驯鹿的先天性宿主防御.