Differential Toll-Like Receptor (TLR) mRNA Expression Patterns during Chicken Embryological Development

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.     

Dietary l-glutamine supplementation increases Pasteurella multocida burden and the expression of its major virulence factors in mice

This study was conducted to determine the effects of graded doses of l-glutamine supplementation on the replication and distribution of Pasteurella multocida, and the expression of its major virulence factors in mouse model. Mice were randomly assigned to the basal diet supplemented with 0, 0.5, 1.0 or 2.0 % glutamine. Pasteurella multocida burden was detected in the heart, liver, spleen, lung and kidney after 12 h of P. multocida infection. The expression of major virulence factors, toll-like receptors (TLRs), proinflammatory cytokines (interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha) and anti-oxidative factors (GPX1 and CuZnSOD) was analyzed in the lung and spleen. Dietary 0.5 % glutamine supplementation has little significant effect on these parameters, compared to those with basal diet. However, results showed that a high dose of glutamine supplementation increased the P. multocida burden (P < 0.001) and the expression of its major virulence factors (P < 0.05) as compared to those with a lower dose of supplementation. In the lung, high dose of glutamine supplementation inhibited the proinflammatory responses (P < 0.05) and TLRs signaling (P < 0.05). In the spleen, the effect of glutamine supplementation on different components in TLR signaling depends on glutamine concentration, and high dose of glutamine supplementation activated the proinflammatory response. In conclusion, glutamine supplementation increased P. multocida burden and the expression of its major virulence factors, while affecting the functions of the lung and spleen.     

Downregulation of TLR4 and 7 mRNA Expression Levels in Broiler’s Spleen Caused by Diets Supplemented with Nickel Chloride

Toll-like receptors (TLRs) are important immune receptors in discriminating self from nonself and in initiating the innate and adaptive immune response. TLR4 and TLR7 have been proven to be highly expressed in chicken’s spleen. Thus, this study was to evaluate the TLR4 and TLR7 messenger RNA (mRNA) expression levels in the spleen of broilers fed diets supplemented with nickel chloride (NiCl₂) using the methods of quantitative real-time PCR (qRT-PCR). Two hundred forty-one-day-old avian broilers were equally divided into 4 groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600, and 900 mg/kg of NiCl₂ for 42 days. Results showed that TLR4 and TLR7 mRNA expression levels in the spleen were lower (P?<?0.05 or P?<?0.01) in the 300, 600, and 900 mg/kg groups than those in the control group. It was concluded that dietary NiCl₂ in excess of 300 mg/kg could lower TLR4 and TLR7 mRNA expression levels in the spleen of broilers, implying that NiCl₂ could impair the innate and adaptive immunity in spleen by injuring immunocytes and/or decreasing the content of cytokines through TLRs.     

Detection of Polymorphism and Sequence Characterization of Toll-Like Receptor 7 Gene of Indian Goat Revealing Close Relationship Between Ruminant Species

In this study, approximately 3.4 kb nucleotide sequence of caprine TLR7 (Toll-like receptor 7) gene was generated from twelve different Indian goat breeds belonging to different geographical regions. Goat TLR7 gene ORF (Open Reading Frame) was found to be 3141 nucleotides long coding for 1046 amino acids similar to sheep. The sequence analysis at nucleotide level revealed goat TLR7 having 99.5% homology with sheep, followed by other livestock species. Simple Modular Architecture Research Tool (SMART) was used for the structural analysis of goat TLR7 that showed the presence of 22 leucine rich repeats (LRRs) along with single Toll/interleukin-1 receptor (TIR) domains. TIR domain, when compared, was found to be similar in ruminant species, goat, sheep, cattle, and buffalo. The phylogenetic analysis also revealed grouping of all ruminant species together, goat being closer to sheep followed by cattle and buffalo. A total of 22 polymorphic sites were observed in TLR7 gene of 24 goats representing 12 different breeds, out of which 19 were present within the coding region and three in 3'UTR. Out of the seven nonsynonymous SNPs, two were in ectodomains and one in TIR domain. Overall our results indicate substantial variation within goat TLR7 gene, which could be exploited for association with disease susceptibility.     

Orange-spotted grouper (Epinephelus coioides) toll-like receptor 22: molecular characterization, expression pattern and pertinent signaling pathways

The toll-like receptors (TLRs) are an important gene family in host innate immunologic surveillance. The TLR22 gene is an essential member of the TLRs that is only found in aquatic animals and has been detected in some bony fish. Here, a TLR22 homolog, EcTLR22, was characterized in the orange-spotted grouper (Epinephelus coioides) via homology cloning. The 3321 bp full-length cDNA sequence of EcTLR22 was obtained, which included an open reading frame of 2880 bp encoding a putative peptide of 960 amino acids containing three highly typical domains with the characteristics of TLR family members. The deduced amino acid sequence of EcTLR22 showed a relatively high similarity to flounder TLR22. Phylogenetic analysis showed that the orange-spotted grouper TLR22 sequence was clustered with those of Perciforme, such as flounder and croaker. Real-time quantitative PCR analysis revealed broad expression of EcTLR22, with relatively high expression detected in the head kidney, trunk kidney, spleen, peripheral blood leukocytes (PBLs) and heart of orange-spotted grouper. After injection with Vibrio alginolyticus, there was significant up-regulation of the expression of EcTLR22 in the spleen. In evaluating unstimulated/stimulated head kidney leukocytes and spleen leukocytes, a significant increase in EcTLR22 mRNA expression was detected, which implied a sensitive immune response. Furthermore, four important molecules for signal transduction, MyD88, TRIF, TNF-α and IRF3, were chosen to analyze the role of the EcTLR22 signaling pathway in anti-pathogen responses. Upon LPS or Poly I:C challenge, expression of the four genes was induced, with an increasing tendency detected in head kidney leukocytes, suggesting that the four genes might work with EcTLR22 in host defense against pathogenic microbes.     

日本鳗鲡TLR21基因的鉴定、免疫应答与启动子分析

为研究TLR21（Toll like receptor 21）在低等脊椎动物中的功能及表达调控机制,我们扩增获得了日本鳗鲡TLR21（AjTLR21）cDNA序列,其编码的蛋白具有TLR家族的共同特征。AjTLR21基因结构与其他鱼类和两栖类TLR21相同,由单个外显子编码。荧光定量结果显示,AjTLR21在血液、鳃、脾脏、中肾等11个组织/器官中转录表达,其中在血液中表达量最高。经Poly I:C诱导后8h,AjTLR21在脾脏和中肾中的表达量显著性上调;诱导后16h,AjTLR21在血液、鳃、肠和脾脏中的表达量显著性上调（P < 0.05）。双荧光素酶报告基因结果显示,在AjTLR21 5'上游调控序列-1179 bp到+117 bp存在Poly I:C调节的正调控元件。经Edwardsiella tarda诱导后16h和72h,AjTLR21分别在血液和中肾组织的表达量显著性上调,表明AjTLR21同时也参与了抗细菌免疫应答,其在机体免疫系统中的功能具有多样性。研究对于理解日本鳗鲡AjTLR21的免疫学功能具有重要的理论意义和应用价值。     

Expression analysis of turkey (Meleagris gallopavo) toll-like receptors and molecular characterization of avian specific TLR15

Toll-like receptors (TLRs) constitute a multi-gene family, which plays a pivotal role in sensing invading pathogens by virtue of conserved microbial patterns. TLR repertoire of chicken and zebra finch has been well studied. However TLR family of other avian species is yet to be characterized. In the present study, we identified TLR repertoire of turkey, characterized avian specific receptor TLR15 in turkey and profiled the TLRs expressions in a range of tissues of turkey poults. All ten TLR genes orthologous to chicken TLR repertoire were found in turkey. Turkey TLR genes showed 81-93 % similarity at amino acid level to their chicken counter parts. Phylogenetic analysis confirmed the orthologous relationship of turkey TLRs with chicken and zebra finch TLRs. Open reading frame of turkey TLR15 was 2,607 bp long encoding 868 amino acids similar to that of broiler chicken and showed 92.4, 91.1 and 69.5 % identity at amino acid levels with chicken, Japanese quail and zebra finch TLR15 sequences respectively. Overall TLR expression was highest for TLR4 and lowest for TLR21. TLR1A, 2A, 2B and 21 were significantly higher in liver than other tissues investigated (P < 0.01). TLR3 expression was significantly higher in bone marrow (BM) and spleen in comparison to other tissues studied (P < 0.01). Furthermore, no significant differences in the expression levels of TLR1B, 4, 5, 7 and 15 genes were detected among the tissues studied. Our findings contribute to the characterization of innate immune system of birds and show the innate preparedness of young turkey poults to a range of pathogens.     

Rumen prokaryotic communities of ruminants under different feeding paradigms on the Qinghai-Tibetan Plateau

Yak and Tibetan sheep are the major indigenous ruminants on the Qinghai-Tibetan Plateau in China. The aim of this work was to study the differences in ruminal fermentation parameters and rumen prokaryotic community composition between hosts and feeding paradigms. The 16S rRNA genes targeting bacteria and archaea were sequenced using the MiSeq platform. The results showed that the prokaryotic community structure between yak and Tibetan sheep was significantly different (P < 0.01). A significant difference in structure was also found between groups of yaks barn fed with a total mixed ration (TMR) and those naturally grazing (NG) (P = 0.034), as well as for Tibetan sheep of the two groups (P = 0.026). The core prokaryotic populations that existed in the rumen mostly dominated the structure. There was an obvious correlation of the prokaryotic community composition at the phylum and genus levels with the host or the feeding pattern. In addition, Tibetan sheep showed significantly higher yields of volatile fatty acids (VFAs) than yak, as did the NG group compared with the TMR group. In conclusion, both the host and feeding pattern may influence rumen microbial ecology system, with host effects being more important than those of the feeding pattern.     

Gene cloning and expression analysis of IRF1 in half-smooth tongue sole (Cynoglossus semilaevis)

Interferon regulatory factor 1 (IRF1) was known to play key roles in antiviral defense in several species, and some other important biological processes. In this report, full length cDNA of IRF1 from Cynoglossus semilaevis (CsIRF1) was identified. It was of 1,455 bp, containing a 5′ UTR of 104 bp, a 3′ UTR of 541 bp with a poly (A) tail and an ORF of 810 bp encoding a putative protein of 269 amino acids. The putative CsIRF1protein contained one conserved IRF domain (1–113aa), and two low complexity regions (140–158aa and 230–242aa, respectively). Phylogenetic analysis showed that CsIRF1 was conserved in the teleost evolutionary branch, which was independent of mammalian, birds and amphibians. Additionally, CsIRF1 had the 96 % homology with marine fishes, while 66 % with freshwater fishes. The expression profiles of CsIRF1was analyzed by quantitative real-time PCR in healthy tissues and in immune tissues challenged with different pathogens [Vibrio anguillarum and Lymphocystis disease virus (LCDV)], respectively. CsIRF1 was widely expressed in healthy tissues of Cynoglossus semilaevis and with the highest expression in blood, as much as 19 times of that in liver. V. anguillarum and LCDV both induced the CsIRF1 gene expression distinctly in liver, with the peak value reached to 98-fold at 6 h and 25-fold at 24 h, respectively. The bacteria induced CsIRF1 suddenly up-expression in each detected tissues. However, at the initial stage of the challenge of virus LCDV, the CsIRF1 expression in blood and spleen were up regulated; on the contrary, its expression in liver and head kidney were down regulated, 0.3 and 0.4-fold 6 h post virus injection, respectively. These results suggested that CsIRF1 gene might involve in not only antiviral activity but also antibacterial procedure, indicating its vital role in Cynoglossus semilaevis innate defense system.     

Summer season induced heat stress impact on the expression patterns of different toll-like receptor genes in Malabari goats

The study was conducted with the primary objective of establishing the impact of heat stress on the expression pattern of different toll-like-receptors (TLRs) in Malabari goats. The study was conducted for a period of 45 days using twelve Malabari goats. The goats were randomly allocated into two groups: MC (n = 6; Malabari control) and MHS (n = 6; Malabari heat stress). At the end of study, all 12 animals were slaughtered and their mesenteric lymph node (MLN) tissues were collected for gene expression. Heat stress significantly (p < 0.05) down regulated TLR1, TLR4, TLR5 and TLR6 and significantly increased (p < 0.05) TLR2 expression pattern. A negative correlation (p < 0.01) was also established between THI and different TLRs except TLR3, TLR7 and TLR10. The study indicated that TLR2 could serve as ideal immunological marker for establishing the superior thermo-tolerance ability of Malabari breed in terms of maintaining the immune status during heat stress challenges.     

Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines.

Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4(+), CD8(+), CD19(+), monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.     

Complete sequence of the yak (Bos grunniens) mitochondrial genome and its evolutionary relationship with other ruminants

The yak (Bos grunniens) is the most important domesticated species in the Qinhai-Tibetan Plateau. In present study, the complete sequence of the yak mitochondrial genome was determined. Sequence analysis revealed that there are no differences with cattle in the yak mitochondrial genome organization. Interestingly, within the D-loop, the conserved sequence blocks are less conserved than surrounding regions. Neighbor-Joining (NJ) trees based on single genes, gene sets and concatenated genes of mitochondrial genome were constructed. The analysis identified the yak as a sister group of a cattle/zebu clade. Based on substitutions in 22 tRNA genes, 12S rRNA gene and 16S rRNA gene, the dating of divergence between yak and cattle/zebu, and yak and water buffalo, was proposed to have occurred 4.38-5.32 and 10.54-13.85 million years before present, respectively. This is consistent with the paleontologyical data. Yak and sheep/goat divergent dating predicts that their divergence occurred at 13.14-27.99 million years before the present day.     

Sequence Based Structural Characterization and Genetic Diversity Analysis of Full Length TLR4 CDS in Crossbred and Indigenous Cattle

The exploration of candidate genes for immune response in cattle may be vital for improving our understanding regarding the species specific response to pathogens. Toll-like receptor 4 (TLR4) is mostly involved in protection against the deleterious effects of Gram negative pathogens. Approximately 2.6 kb long cDNA sequence of TLR4 gene covering the entire coding region was characterized in two Indian milk cattle (Vrindavani and Tharparkar). The phylogenetic analysis confirmed that the bovine TLR4 was apparently evolved from an ancestral form that predated the appearance of vertebrates, and it is grouped with buffalo, yak, and mithun TLR4s. Sequence analysis revealed a 2526-nucleotide long open reading frame (ORF) encoding 841 amino acids, similar to other cattle breeds. The calculated molecular weight of the translated ORF was 96144 and 96040.9 Da; the isoelectric point was 6.35 and 6.42 in Vrindavani and Tharparkar cattle, respectively. The Simple Modular Architecture Research Tool (SMART) analysis identified 14 leucine rich repeats (LRR) motifs in bovine TLR4 protein. The deduced TLR4 amino acid sequence of Tharparkar had 4 different substitutions as compared to Bos taurus, Sahiwal, and Vrindavani. The signal peptide cleavage site predicted to lie between 16th and 17th amino acid of mature peptide. The transmebrane helix was identified between 635–657 amino acids in the mature peptide.     

Molecular characterization and phylogenetic analysis of a yak (<Emphasis Type="Italic">Bos grunniens</Emphasis>) κ-casein cDNA from lactating mammary gland

κ-Casein is one of the major proteins in the milk of mammals. It plays an important role in determining the size and specific function of milk micelles. We have previously identified and characterized a genetic variant of yak κ-casein by evaluating genomic DNA. Here, we isolate and characterize a yak κ-casein cDNA harboring the full-length open reading frame (ORF) from lactating mammary gland. Total RNA was extracted from mammary tissue of lactating female yak, and the κ-casein cDNA were synthesized by RT-PCR technique, then cloned and sequenced. The obtained cDNA of 660-bp contained an ORF sufficient to encode the entire amino acid sequence of κ-casein precursor protein consisting of 190 amino acids with a signal peptide of 21 amino acids. Yak κ-casein has a predicted molecular mass of 19,006.588 Da with a calculated isoelectric point of 7.245. Compared with the corresponding sequences in GenBank of cattle, buffalo, sheep, goat, Arabian camel, horse, and rabbit, yak κ-casein sequence had identity of 64.76–98.78% in cDNA, and identity of 44.79–98.42% and similarity of 53.65–98.42% in deduced amino acids, revealing a high homology with the other livestock species. Based on κ-casein cDNA sequences, the phylogenetic analysis indicated that yak κ-casein had a close relationship with that of cattle. This work might be useful in the genetic engineering researches for yak κ-casein.