In order to determine the changes of bacterial community structure and function in the early, middle and late stage of aerobic composting of chicken manure, high-throughput sequencing and bioinformatics methods were used to determine and analyze the 16S rRNA sequence of samples at different stages of composting. Wayne analysis showed that most of the bacterial OTUs in the three composting stages were the same, and only about 10% of the operational taxonomic units (OTUs) showed stage specificity. The diversity indexes including Ace, Chao1 and Simpson showed a trend of increasing at first, followed by decreasing. However, there was no significant difference among different composting stages (P < 0.05). The dominant bacteria groups in three composting stages were analyzed at the phylum and genus levels. The dominant bacteria phyla at three composting stages were the same, but the abundances were different. LEfSe (line discriminant analysis (LDA) effect size) method was used to analyze the bacterial biological markers with statistical differences among three stages of composting. From the phylum to genus level, there were 49 markers with significant differences among different groups. The markers included 12 species, 13 genera, 12 families, 8 orders, 1 boundary, and 1 phylum. The most biomarkers were detected at early stage while the least biomarkers were detected at late stage. The microbial diversity was analyzed at the functional pathway level. The function diversity was the highest in the early stage of composting. Following the composting, the microbial function was enriched relatively while the diversity decreased. This study provides theoretical support and technical guidance for the regulation of livestock manure aerobic composting process.
Animals ; Manure/microbiology* ; Chickens/genetics* ; Composting ; RNA, Ribosomal, 16S/genetics* ; Soil ; Bacteria/genetics*

Overexpression of Krüppel like factor 2 (Klf2) or Klf7 inhibits adipocyte formation. However, it remains unclear whether Klf2 regulates klf7 expression in adipose tissue. In this study, oil red O staining and Western blotting were employed to study the effect of Klf2 overexpression on the differentiation of chicken preadipocytes. The results showed that Klf2 overexpression inhibited the differentiation of chicken preadipocytes induced by oleate and the expression of pparγ, while promoted klf7 expression in chicken preadipocytes. Spearman correlation analysis was used to study the correlation between the expression data of klf2 and klf7 in the adipose tissue of both human and chicken. The results showed that there was a significantly positive correlation between the expression of klf2 and klf7 in adipose tissues (r > 0.1). Luciferase reporter assay showed that overexpression of Klf2 significantly promoted the activity of chicken klf7 promoter (-241/-91, -521/-91, -1 845/-91, -2 286/-91, -1 215/-91; P < 0.05). In addition, the activity of klf7 promoter (-241/-91) reporter in chicken preadipocytes was significantly positively correlated with the amount of klf2 overexpression plasmid transfected (T_au=0.917 66, P=1.074×10^-7). Moreover, Klf2 overexpression significantly promoted the mRNA expression of klf7 in chicken preadipocytes (P < 0.05). In conclusion, upregulation of klf7 expression might be one of the pathways that Klf2 inhibits chicken adipocyte differentiation, and the sequence from -241 bp to -91 bp upstream chicken klf7 translation start site might mediate the regulation of Klf2 on klf7 transcription.
Animals ; Humans ; Chickens/genetics* ; Kruppel-Like Transcription Factors/metabolism* ; Transcription Factors/metabolism* ; Adipocytes/metabolism* ; Adipose Tissue/metabolism*

The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.
Female ; Animals ; Zona Pellucida Glycoproteins ; Membrane Glycoproteins/metabolism* ; Chickens/genetics* ; Egg Proteins/metabolism* ; Receptors, Cell Surface ; Estrogens

Black-bone silky fowl, sweet, pungent, and hot-natured, is one of the valuable domesticated birds with special economic value in China's genebank of poultry breed, which has a long history of medicinal and edible uses. It has the effects of tonifying liver and kidney, replenishing Qi and blood, nourishing yin, clearing heat, regulating menstruation, invigorating spleen, and securing essence. Therefore, it has remarkable efficacy of enhancing physical strength, tonifying blood, and treating diabetes and gynecological diseases. Various local black-bone silky fowl breeds have been generated due to the differences in environmental conditions, breed selection, and rearing conditions in different areas of China, which are mainly concentrated in Taihe, Wan'an, and Ji'an in Jiangxi province and Putian, Jinjiang, and Yongchun in Fujian province. The indigenous chicken breeds in China have different body sizes, appearance, coat colors, etc. The complex lineages lead to extremely unstable genetic traits. The diverse breeds similar in appearance result in the confusion in the market of silky fowl breeds. With the rapid development of molecular biological technology, the genetics of black-bone silky fowls has been intensively studied. This article reviews the research progress of the germplasm resources, genetic diversity, and breed identification of black-bone silky fowl in China at the morphology, chromosome, protein, and DNA levels. Further, it introduces the principles, application status, and limitations of DNA markers such as mitochondrial DNA, microsatellite markers, and SNPs. This review provides a theoretical basis for the mining of elite trait genes and the protection and utilization of local black-bone silky fowl germplasm resources in China.
Animals ; Chickens/genetics* ; DNA, Mitochondrial ; Female ; Genetic Variation ; Microsatellite Repeats ; Polymorphism, Single Nucleotide ; Silk/genetics*

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Animals ; Antibodies, Viral ; Chickens ; HN Protein/metabolism* ; Newcastle Disease/prevention & control* ; Newcastle disease virus/metabolism* ; Oryza/genetics*

In this study, we cloned the complete coding sequence (CDS) of chicken foxp3 (chfoxp3) gene, analyzed its structure, and investigated its expression profile in different chicken tissues. To be specific, chfoxp3 was cloned from the splenic tissue of 50-day-old specific-pathogen-free chickens, and analyzed by using online bioinformatics tools or software. The expression profiles of the chfoxp3 gene in different chicken tissues were detected by quantitative real-time PCR (qRT-PCR). The results indicated that the chfoxp3 gene contains an 882-bp open reading frame, encoding 293 amino acids hydrophilic protein with a molecular weight of 33.44 kDa. The chFoxp3 protein has a forkhead domain and carries a nuclear localization signal, which is typical in the Fox transcription factor family. The secondary structure of chFoxp3 consists of α-helix (29.35%), extended chain (10.92%), β-turn (5.12%) and random coil (54.61%). The expression of chfoxp3 varied in different tissues. The expression levels of chfoxp3 in chicken heart and pancreas were higher than in spleen, bursa of Fabricius, thymus, and other immune organs (P < 0.01), which was quite different from that of mammals. Phylogenetic tree analysis showed that chFoxp3 belonged to the same clade as other wild birds did, but was far different from that of mammals. These results may facilitate further research on the role of chFoxp3 in immune regulation.
Amino Acid Sequence ; Animals ; Chickens/genetics* ; Cloning, Molecular ; Gene Expression Regulation ; Mammals/genetics* ; Phylogeny

This paper aims to explore the effects of chicken interferon-γ (ChIFN-γ) and interleukin-2 (ChIL-2) on type 1 helper (Th1) T lymphocyte differentiation. To be specific, ChIFN-γ and ChIL-2 were first expressed in Escherichia coli competent cells and then purified by Ni-NTA affinity chromatography. Different concentration of ChIFN-γ and ChIL-2 were employed to stimulate the lymphocytes in chicken peripheral blood which had been activated by concanavalin A (Con A), and the mRNA levels of cytokines related to Th1 cell differentiation were detected by real-time quantitative PCR (RT-qPCR). The results showed that both ChIFN-γ and ChIL-2 can significantly up-regulate mRNA levels of cytokines related to Th1 cell differentiation and the optimal concentration was 12.5 μg/mL and 25.0 μg/mL, respectively. In addition, specific-pathogen-free (SPF) chickens were immunized with ChIL-2 or ChIFN-γ together with H9N2 vaccine, or H9N2 vaccine alone by oral administration or intramuscular injection, respectively. The mRNA levels of cytokines related to Th1 cell differentiation were detected after immunization. The results showed that ChIFN-γ and ChIL-2 significantly up-regulated the mRNA levels of cytokines related to Th1 cell differentiation induced by H9N2 vaccine compared with H9N2 vaccine alone, and that the intramuscular injection was better than oral administration. In this study, we verified that ChIFN-γ and ChIL-2 can significantly enhance mRNA levels of cytokines related to Th1 cell differentiation induced by ConA or H9N2 vaccine in vitro and in vivo. The results of this study can lay a theoretical basis for using ChIFN-γ and ChIL-2 as vaccine adjuvants.
Animals ; Cell Differentiation ; Chickens ; Concanavalin A ; Cytokines/genetics* ; Influenza A Virus, H9N2 Subtype/genetics* ; Interferon-gamma/metabolism* ; Interleukin-2/genetics* ; RNA, Messenger

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
Animals ; Chick Embryo ; Chickens ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; Herpesvirus 2, Gallid/genetics* ; Marek Disease

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.
Animals ; Baculoviridae/genetics* ; CD40 Ligand/genetics* ; Chickens ; Cloning, Molecular ; Genetic Vectors/genetics* ; Recombinant Proteins/genetics*

Aged ; Animals ; Arcobacter/genetics* ; Chickens ; Diarrhea/microbiology* ; Drug Resistance, Bacterial/genetics* ; Genes, Bacterial ; Gram-Negative Bacterial Infections/veterinary* ; Humans ; Male ; Meat ; Microbial Sensitivity Tests ; Phylogeny ; Poultry Diseases/microbiology* ; Virulence ; Virulence Factors/genetics*