Screening of Proliferation-Related Genes and Pathological Changes in Thiram-Induced Tibial Dyschondroplasia.

Materials and Methods: Three hundred sixty (n = 360) broiler chickens were equally divided into control (C) and thiram (T) groups. Furthermore, the C and T groups were dividedinto 8-, 9-, 11-, and 13-day-old chickens. Results: Clinically, it was observed that broiler chickens of group T had abnormal posture, gait, and lameness, and histopathological results revealed dead and abnormal chondrocytes of T group on day 6. Real-time qPCR results showed that HDAC1, MTA1, H4, and PCNA genes were significantly expressed (P < 0.05). HDAC1 was upregulated on days 1, 2, 4, and 6 (P < 0.01); MTA1 was upregulated on days 1 and 2 (P < 0.01); H4 was upregulated on days 2 and 4 (P < 0.01), and PCNA was downregulated on days 1, 2, and 4 (P < 0.01). Furthermore, IHC results of HDAC1 protein were significantly (P < 0.01) expressed in proliferative zone of day 1 and hypertrophic zone of day 6. MTA1 protein was significantly (P < 0.01) expressed on days 1, 2, and 6 in all zones, except prehypertrophic zone of day 2. Conclusion: In conclusion, the mRNA expressions of HDAC1, MTA1, H4, and PCNA were differentially expressed in the chondrocytes of thiram-induced TD chickens. HDAC1 and MTA1 protein expression found involved and responsible in the abnormal chondrocytes' proliferation of broiler chicken.

Janus kinase/signal transducer and activator of transcription signaling pathway-related genes STAT3, SOCS3 and their role in thiram induced tibial dyschondroplasia chickens.

Pathogenicity of tibial dyschondroplasia (TD) in broiler chickens is not detected yet. Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway-related genes were investigated in thiram induced TD chickens. Real-time qPCR and immunohistochemical (IHC) technique were used to observe the expression changes of STAT3 and SOSC3 gene on days 1, 2, 4, 6 after feeding 100 mg·kg-1 thiram. Morphological, pathological, and histological results of this study suggested that chondrocyte cells were observed more damaged on day 6 than day 1, 2, and 4. Therefore, Lameness and damaged chondrocytes gradually increased from day 1 to 6. The mRNA expression level of STAT3 was observed insignificant (P > 0.05) in thiram induced TD chickens' group of day 1. However, on days 2, 4, and 6, the expression was significant (P < 0.05). SOCS3 increased in thiram group on days 1, 2 and 6, decreased on day 4 (P < 0.05). The p-STAT3 and SOCS3 protein's protein localization was evaluated in the control and thiram-induced TD broiler chickens through IHC, suggesting that SOSC3 protein was observed significantly higher on days 1, 2, and 6 and down-regulated on day 4. p-STAT3 protein on thiram induced group was observed significantly upregulated on days 4 and 6. In conclusion, the differential expression of STAT3 and SOCS3 showed that the JAK-STAT signaling pathway might play an important role in regulating an abnormal proliferation, differentiation, or apoptosis of chondrocytes in TD at an early stage.

Screening of toll-like receptor signaling pathway-related genes and the response of recombinant glutathione S-transferase A3 protein to thiram induced apoptosis in chicken erythrocytes.

The expression of genes related to the Toll-like receptors (TLRs) signaling pathway were determined. Group A, B and C fed with basal diet and group D, E and F induced TD by feeding a basal diet containing 100 mg·kg-1 thiram. rGSTA3 protein was injected at 20 µg·kg-1 in group B, E and at 50 µg·kg-1 in C, F. Results suggested that lameness and death of chondrocytes were significant on day 14. TLRs signaling pathway related genes were screened based on the transcriptome enrichment, and validated on qPCR. IL-7, TLR2, 3, 4, 5, 7, 15, MyD88, MHC-II, MDA5 and TRAF6 were significantly (p < 0.05) expressed in group E and F as compared to group D on day 14 and 23. IL-7, MHCII, TRAF6, TLR3, TLR5, TLR7, and TLR15 determined insignificant in group D compared to group A on day 23. TD occur in an early phase and alleviated in the later period. rGSTA3 protein can prevent apoptosis and repair degraded chondrocytes.

Antagonistic effect of N-acetyl-L-cysteine against cadmium-induced cytotoxicity and abnormal immune response on chicken peritoneal macrophages.

Cadmium is a highly toxic metal threatening human and animal health. N-acetyl-L-cysteine (NAC) was reported to play a positive role in disease treatment and immune regulation. The present study aimed to explore the effect of NAC administration on Cd-induced cytotoxicity and abnormal immune response on chicken peritoneal macrophages. Peritoneal macrophages isolated from Isa Brown male chickens were exposed to CdCl2 (20 or 50 µM) and/or NAC (500 µM) for different time periods. Results showed that Cd caused dose-dependent damage on chicken peritoneal macrophages characterized by morphologic and ultrastructural alterations, increased cell apoptosis, reactive oxygen species accumulation and mitochondrial injury. Cd exposure inhibited phagocytic activity of chicken peritoneal macrophages, and promoted transcriptional status of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) in both unactivated macrophages and cells in response to lipopolysaccharide (LPS) stimuli. Pretreatment with 500 µM NAC did not affect growth of normal chicken peritoneal macrophages, while remarkably inhibiting Cd-caused cell death, oxidative stress, and mitochondrial membrane depolarization. NAC pretreatment significantly prevented intracellular Cd2+ accumulation in the Cd-exposed macrophages. Inhibitory effects of NAC on Cd-induced ROS accumulation and mitochondrial injury on chicken macrophages were confirmed in HD-11 macrophage cell line. In addition, NAC pretreatment promoted the phagocytic activity of Cd-exposed chicken peritoneal macrophages, and significantly inhibited expression of pro-inflammatory factors (IL-1ß, IL-6 and TNF-α) in both Cd-exposed macrophages and Cd-treated cells in response to LPS stimuli. In conclusion, the present study firstly demonstrated the antagonistic effect of NAC against Cd-caused damage and abnormal immune response on chicken peritoneal macrophages. Protective effect of NAC on chicken macrophages was highly related to its suppression on Cd-induced ROS overproduction, pro-inflammatory reaction and intracellular Cd2+ accumulation.

Integration of gene expression profile data to screen and verify immune-related genes of chicken erythrocytes involved in Marek's disease virus.

Chicken erythrocytes participated in immunity, but the role of erythrocytes in the immunity of Marek's disease virus (MDV) has not been reported related to the immunity genes. The purpose of this study was to screen and verify the immune-related genes of chicken erythrocytes which could be proven as a biomarker in MDV. The datasets (GPL8764-Chicken Gene Expression Microarray) were downloaded from the GEO profile database for control and MDV infected chickens to obtain differentially expressed genes (DEGs) through bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to find enriched pathways, including Gene Ontology (GO). Based on enriched pathways, the top 19 immune-related genes were screened-out and process further to construct the protein-protein interaction (PPI) networks. The screened genes were validated on RT-PCR and qPCR. Results suggested that the mRNA transcription of Toll-like receptors 2, 3, 4, 6 (TLR2, TLR3, TLR4, TLR6), major histocompatibility complex-II (MHCII), interleukin-7 (IL-7), interferon-ßeta (IFN-ß), chicken myelomonocytic growth factor (cMGF) and myeloid differentiation primary response 88 (MyD88) were significantly up-regulated. The expression of toll-like receptor 5, 7 (TLR5, TLR7) interleukin-12 (IL-12 p40), interleukin-13 (IL-13), and interferon-αlpha (IFN-α) were significantly down-regulated in the erythrocytes of the infected group (P < 0.05). In contrast, the expression of toll-like receptor-1, 15, 21 (TLR1, TLR15, TLR21), major histocompatibility complex I (MHCI) and Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) were not significant. In conclusion, it has been verified on qRT-PCR results that 19 immune-related genes, which included TLRs, cytokines and MHC have immune functions in MDV infected chickens.

Transcriptome analysis of MAPK signaling pathway and associated genes to angiogenesis in chicken erythrocytes on response to thiram-induced tibial lesions.

This study was planned to investigate TD (Tibial dyschondroplasia) on the potential MAPK signaling pathway and angiogenesis related genes. Forty-eight broilers were allotted into control (C) and treatment (T) groups of 2, 6 and 15 days as C1, C2, C3, T1, T2 and T3. The histopathology results revealed that tibiotarsus bone of chickens had more lesions on day 6 (T2 group). The chondrocytes were disordered, and the size, shape and proliferation were affected. Transcriptome results revealed that differentially expressed genes (DEGs) identified were 63, 1026, 623, 130, 141 and 146 in C1 (2 days control vs 6 days control); C2 (2 days control vs 15 days control); C3 (6 days control vs 15 days control); T1 (2 days treatment vs 6 days treatment); T2 (2 days treatment vs 15 days treatment) and T3 (6 days treatment vs 15 days treatment) groups respectively. Whereas, 10 angiogenesis related-genes RHOC, MEIS2, BAIAP2, TGFBI, KLF2, CYR61, PTPN11, PLXNC1, HSPH1 and NRP2 were downregulated on day 6 in the treatment group. The pathway which was found enriched in the control and treatment groups was MAPK signaling pathway. Therefore selected 10 MAPK signaling pathway-related genes RAC2, MAP3K1, PRKCB, FLNB, IL1R1, PTPN7, RPS6KA, MAP3K6, GNA12 and HSPA8 which were found significantly downregulated in the treatment group on day 6. It is concluded that angiogenesis and MAPK signaling pathway related genes has an essential role in TD, as those top screened genes found downregulated in the thiram fed chickens when TD observed severed on day 6.

Transcripts of antibacterial peptides in chicken erythrocytes infected with Marek's disease virus.

BACKGROUND: Chicken erythrocytes are involved in immunity through binding of toll-like receptors (TLRs) with their ligands to activate downstream signaling and lead to cytokine production in erythrocytes. Some avian ß-defensins (AvBDs) are constitutively expressed in tissues and some others can be induced by various bacteria and viruses. However, the expression of AvBDs in erythrocytes has not yet been studied extensively. RESULTS: The transcripts of eight AvBDs (AvBD1 to AvBD7, and AvBD9) and liver-expressed antimicrobial peptide-2 (LEAP-2) were found in normal chicken erythrocytes. The expression levels of AvBD2, 4 and 7 were significantly increased (P < 0.01), whereas the levels of AvBD1, 6 and 9 were significantly decreased (P < 0.01) after Marek's disease virus (MDV) infection. The mRNA expression level of LEAP-2 was not significantly changed after MDV infection. Highest viral nucleic acid (VNA) of MDV in the feather tips among the tested time points was found at 14 days post-infection (d.p.i.). In addition, 35 MD5-related gene segments were detected in the erythrocytes at 14 d.p.i. by transcriptome sequencing. CONCLUSIONS: These results suggest that the AvBDs in chicken erythrocytes may participate in MDV-induced host immune responses.

Screening of differentially expressed genes in the growth plate of broiler chickens with tibial dyschondroplasia by microarray analysis.

BACKGROUND: Tibial dyschondroplasia (TD) is a common skeletal disorder in broiler chickens. It is characterized by the presence of a non-vascularized and unmineralized cartilage in the growth plate. Previous studies have investigated differential expression of genes related to cartilage development during latter stages of TD. The aim of our study was to identify differentially expressed genes (DEGs) in the growth plate of broiler chickens, which were associated with early stage TD. We induced TD using tetramethylthiuram disulfide (thiram) for 1, 2, and 6 days and determined DEGs with chicken Affymetrix GeneChip assays. The identified DEGs were verified by quantitative polymerase chain reaction (qPCR) assays. RESULTS: We identified 1630 DEGs, with 82, 1385, and 429 exhibiting at least 2.0-fold changes (P < 0.05) at days 1, 2, and 6, respectively. These DEGs participate in a variety of biological processes, including cytokine production, oxidation reduction, and cell surface receptor linked signal transduction on day 1; lipid biosynthesis, regulation of growth, cell cycle, positive and negative gene regulation, transcription and transcription regulation, and anti-apoptosis on day 2; and regulation of cell proliferation, transcription, dephosphorylation, catabolism, proteolysis, and immune responses on day 6. The identified DEGs were associated with the following pathways: neuroactive ligand-receptor interaction on day 1; synthesis and degradation of ketone bodies, terpenoid backbone biosynthesis, ether lipid metabolism, JAK-STAT, GnRH signaling pathway, ubiquitin mediated proteolysis, TGF-ß signaling, focal adhesion, and Wnt signaling on day 2; and arachidonic acid metabolism, mitogen-activated protein kinase (MAPK) signaling, JAK-STAT, insulin signaling, and glycolysis on day 6. We validated seven DEGs by qPCR. CONCLUSIONS: Our findings demonstrate previously unrecognized changes in gene transcription associated with early stage TD. The DEGs we identified by microarray analysis will be used in future studies to clarify the molecular pathogenic mechanisms of TD. From these findings, potential pathways involved in early stage TD warrant further investigation.