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Toll-like receptors mediate immune responses via recognition of pathogen-associated molecular patterns. Polymorphisms of toll-like receptors may affect their recognition of pathogen-associated molecular patterns, leading to varied host resistance to pathogenic infections. However, little is known about the polymorphisms of chicken toll-like receptors (ChTLR) among breeds. In this study, we cloned ChTLR2 type 1 and type 2 genes from 7 chicken breeds and analyzed their sequences. It was found that there were 10 amino acid polymorphism sites in ChTLR2 type 1 and type 2, among which 6 sites were in type 1 (5 sites in the extracellular domain and 1 site in the cytoplasmic domain) and 4 sites were in type 2 (all 4 sites in the extracellular domain). These results demonstrate that ChTLR2 type 1 and type 2 genes are polymorphic among chicken breeds, suggesting a varied resistance among breeds of chicken. We found that Laiwu Black chicken breeds have distinctive polymorphic sites at I699T in the type 1 and H561R in the type 2 ChTLR2 gene. Beijing Fatty chickens have distinctive sites at Q45R in type 1 and V66L in type 2. Nongda No.3 Chickens have distinctive sites at L115P, H232Y, and T494A in type 1. Hy-Line variety brown chickens have distinctive sites at I311V in type 2. Beijing White 939 chickens have distinctive sites at E284G in type 1 and A22V in type 2. These findings may provide some clues toward understanding the resistance of lighter chickens to salmonellosis.

Immunostimulating complexes (ISCOMs), a kind of novel antigen presenting system, could enhance immune protection by antigen presentation. AbISCO®-300 comprising purified saponin, cholesterol and phosphatidyl choline is an effective ISCOM adjuvant. To evaluate the immune protection of recombinant 3-1E protein against Eimeria acervulina infection, chickens were immunized with recombinant 3-1E protein in combination with AbISCO®-300 or recombinant 3-1E protein alone in this study. The protective immunity was assessed with body weight gain, fecal oocyst output, detection of intestinal IgA positive cells and percentages of CD3(+), CD4(+) or CD8(+) intestinal intraepithelial lymphocytes (IELs). Chickens vaccinated with different doses of recombinant 3-1E protein plus AbISCO®-300 showed higher percentages of CD3(+), CD4(+), and CD8(+) intestinal IELs, increased positive expression rate of intestinal IgA, increased body weight gains and decreased oocyst shedding compared with recombinant 3-1E protein-only vaccinated groups. The results showed that immunization with various doses of the recombinant 3-1E protein in AbISCO®-300 adjuvant enhanced immune protection against avian coccidiosis.

Toll-like receptors (TLR) mediate immune responses via the recognition of pathogen-associated molecular patterns, thus playing important roles in host defense. Among the chicken (Ch) TLR family, ChTLR1, 2, 4, 15, and 21 were shown to recognize bacterial components. In our earlier study, we reported polymorphisms of TLR1, 2, and 5. In the present study, we cloned TLR4, 15, and 21 genes from different chicken breeds and analyzed their sequences. We identified 9 amino acid polymorphism sites in ChTLR4 with 8 extracellular domain sites and 1 site in the cytoplasmic domain, 15 amino acid polymorphism sites in ChTLR15 with 14 extracellular domain sites and 1 site in the cytoplasmic domain, and 7 amino acid polymorphism sites in ChTLR21 with 5 extracellular domain sites and 2 sites in the cytoplasmic domain. These results demonstrate that ChTLR genes are polymorphic among different avian breeds, suggesting a varied resistance across numerous chicken breeds. This information might help improve chicken health by breeding and vaccination.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is an effective nucleic acid adjuvant, which can enhance the body immunity, especially in the immune response to the DNA vaccine. In this study, we investigated the function of recombinant chicken GM-CSF (rchGM-CSF). Chicken GM-CSF (chGM-CSF) gene sequence was cloned, the signal peptide in the N terminal sequence was cut, and 3’ end of the stop codon was changed. The rchGM-CSF was labeled with a C-terminal c-myc and His antigen. The recombinant Pichia pastoris expression vector pPICZαA-rchGM-CSF was constructed by inserting the reformed cytokines genes in pPICZαA. The pPICZαA-rchGM-CSF was expressed in GS115 cells. After being screened by yeast peptone dextrose (YPD) containing high concentrations of Zeocin and direct PCR, the positive clone was cultured in flask with buffered minimal methanol (BMMY) and expression induced by methanol. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that rchGM-CSF was expressed and secreted into BMMY and the secreted peak was generally at 72 h in BMMY medium. After large-scale induced expression, the supernatant was collected by centrifuge, concentrated by centrifugal ultrafiltration and purified by His Bind Ni-NTA chromatography. The MTT (3-(4, 5-dimethylthiazolyl-2) -2, 5-diphenyltetrazoliumbromide) assay results showed that rchGM-CSF could stimulate lymphocyte proliferation. The experiment laid the foundation for further study of immune adjuvant. Key words : Granulocyte-macrophage colony stimulating factor (GM-CSF), Pichia pastoris, secretory expression, biological activity.

Infectious bursa disease virus causes an acute infection in bursal B cells. The Toll-like receptor (TLR) signaling pathway plays a key role in innate immunity during virus infection. In this study, an Agilent microarray was used to investigate different transcriptional profiles of the TLR pathway and related genes of chicken bursa at 48 h after infection with IBDV, compared with simulated infection. Expression of >58 genes changed significantly. Forty-six genes associated with chicken bursa proinflammatory effects, chemotactic effects, and T-cell stimulation were upregulated, which meant enhancement of these features. Twelve genes that are related to proliferation and differentiation of bursal cells were downregulated, implying suppression of these features. These results revealed that genes of the TLR pathway play an important role in the pathogenicity of IBDV infection. The findings are helpful for understanding the molecular basis of viral pathogenesis and the underlying mechanism of the host antiviral response.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, thus playing important roles in host defense. This study determined the first sequence of a TLR1 type 1 in the guinea fowl (GFTLR1). The open reading frame of GFTLR1 type 1 contains 2,115 nucleotides and encodes 705 amino acids. Amino acid analysis indicated that GFTLR1 type 1 shares 92.3 % homology with the green jungle fowl, 92.1 % with the chicken, 90.4 % with the turkey, and 84.4 % with Cooper’s hawk. Genetic patterns were identified within the TLR1 type 1 of the chicken and the guinea fowl. GFTLR1 type 1 was found to have 92 polymorphic amino acid sites, of which 16 were in the leucine-rich repeat (LRR) domain, 3 in a C-terminal LRR domain, and 6 in a Toll/interleukin-1 receptor domain. The data showed that avian TLR1 type 1 genes are under purifying selection and highly conserved, because dN/dS was less than 1.

Toll-like receptors (TLR) mediate immune responses via recognition of pathogen-associated molecular patterns, thus playing important roles in the host defense. Polymorphisms of TLR may affect their recognition of pathogen-associated molecular patterns, leading to varied host resistance to pathogenic infections. However, little is known regarding the polymorphisms of chicken TLR (ChTLR) among breeds. In this study, we cloned ChTLR1 type 1 and type 2 genes from 9 chicken breeds (2 commercial breeds and 7 Chinese native breeds) and analyzed their sequences. We found 27 amino acid polymorphism sites in ChTLR: 14 sites in type 1 (8 sites in the extracellular domain, 5 sites in the cytoplasmic domain, and 1 site in the transmembrane domain) and 13 sites in type 2 (11 sites in the extracellular domain, 1 site in the cytoplasmic domain, and 1 site in the transmembrane domain). These results demonstrate that ChTLR1 type 1 and type 2 genes are polymorphic among chicken breeds, suggesting a varied resistance among breeds of chicken. This information provides new consideration for future poultry breeding and breed improvement.