Effects of subchronic exposure of mercuric chloride on intestinal histology and microbiota in the cecum of chicken.

This study aimed to investigate the influences of mercuric chloride (HgCl2, 250 ppm, drink water) on the growth performance, cecal morphology and microbiota of chickens (n = 60) after 30, 60, and 90 days of exposure. A control group of sixty chickens received water free of HgCl2. Our results suggested that mercury exposure reduced the body weight and changed the cecal morphology of chickens after the 90-day treatment. Furthermore, sequence analysis of 16 S rRNA gene revealed that the diversity and composition of cecal microbiota in chickens differed between the control and exposure group. At the phylum level, Proteobacteria and Tenericutes phyla both significantly increased in mercury exposure groups on day 30 while only Tenericutes phyla significantly increased on day 60. At the genus level, we observed that the change in microbial populations are most dramatic on day 30. Besides, compared with the control group, the genus Prevotellaceae_UCG-001 significantly increased in exposure group on day 30 but showed no significant difference on day 60, whereas there was a significant decrease on day 90. PICRUSt analysis revealed potential metabolic changes, such as Bacterial invasion of epithelial cells and Metabolism of xenobiotics, associated with mercury exposure in chickens. Taken together, the data show that subchronic exposure to mercury not only affected the growth and development but also caused the dysbiosis of gut microbiota, which may further induced metabolic disorders in chickens.

Cloning and prokaryotic expression of the chicken liver kinase B1 (LKB1) and its localization in liver, heart and hypothalamus.

Liver kinase B1 (LKB1) is a member of the serine/threonine kinase family, which plays an indispensable role in the organism of animals. In the current study, the chicken LKB1 protein gene was amplified by PCR based on the primers and cDNA templates. Then, the cloning vector was constructed and the target gene was cloned. After that, the target gene was inserted into the expression vector to construct the recombinant plasmid. The recombinant plasmid was transformed into BL21 (DE3) host cells and the LKB1 recombinant proteins were successfully expressed by using Isopropyl-ß-D-thiogalactopyranoside (IPTG). Finally, purified LKB1 proteins were used as antigen and the rabbit-derived antiserums were collected. The antiserum titer determined by ELISA was not less than 1:128000. The results of Western blot suggested that the polyclonal antibody is highly specific to chicken LKB1 protein. Immunofluorescence indicated that the LKB1 protein is mainly expressed in the cytoplasm of liver, heart and hypothalamus cells of chicken. Our study showed that the LKB1 polyclonal antibodies produced by this method are effective and can be used to further study the role of LKB1 in the pathogenesis of chicken disease.

Activation of AMP-activated protein kinase signaling pathway ameliorates steatosis in laying hen hepatocytes.

The fatty liver hemorrhage syndrome in laying hens is a disease of lipid metabolism disorders. Importantly, energy sensor AMP-activated protein kinase (AMPK) plays an essential role in homeostasis regulation of liver lipid. The current research aims to investigate the relationship between AMPK signaling pathway and lipid metabolism in laying hen hepatocytes and explore the underlying mechanisms. The steatotic hepatocytes model of laying hen was established and treated with AMPK agonist AICAR and inhibitor compound C. The results showed that the levels of triglyceride, total cholesterol, and low-density lipoprotein cholesterol significantly declined while high-density lipoprotein cholesterol level increased in the AICAR-treated steatosis group compared with the steatosis group. Furthermore, the mRNA levels of liver kinase B1 and AMP-activated protein kinase α1 declined significantly in the steatosis group compared with those in the normal group. However, AMPK activation significantly upregulated the mRNA levels of peroxisome proliferator-activated receptor α and carnitine palmitoyl transferase-1 while downregulated the mRNA levels of acetyl CoA carboxylase, fatty acid synthase, 3-hydroxy-3-methyl glutaryl coenzyme A reductase, Sn-glycerol-3-phosphate acyltransferase, and hepatocyte nuclear factor 4α. These results suggest that activated AMPK signaling pathway increases fatty acid oxidation and reduces lipid synthesis in laying hen hepatocytes, thereby ameliorating liver steatosis.

Serum Metabolomic Profiling to Reveal Potential Biomarkers for the Diagnosis of Fatty Liver Hemorrhagic Syndrome in Laying Hens.

Fatty liver hemorrhage syndrome (FLHS), a nutritional and metabolic disease that frequently occurs in laying hens, causes serious losses to the poultry industry. Nowadays, the traditional clinical diagnosis of FLHS still has its limitations. Therefore, searching for some metabolic biomarkers and elucidating the metabolic pathway in vivo are useful for the diagnosis and prevention of FLHS. In the present study, a model of FLHS in laying hens induced by feeding a high-energy, low-protein diet was established. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) was used to analyze the metabolites in serum at days 40 and 80. The result showed that, in total, 40 differential metabolites closely related to the occurrence and development of FLHS were screened and identified, which were mainly associated with lipid metabolism, amino acid metabolism, and energy metabolism pathway disorders. Further investigation of differential metabolites showed 10 potential biomarkers such as 3-hydroxybutyric acid, oleic acid, palmitoleic acid, and glutamate were possessed of high diagnostic values by analyzing receiver operating characteristic (ROC) curves. In conclusion, this study showed that the metabolomic method based on GC-TOF-MS can be used in the clinical diagnosis of FLHS in laying hens and provide potential biomarkers for early risk evaluation of FLHS and further insights into FLHS development.

Nephropathogenic Infectious Bronchitis Virus Infection Altered the Metabolome Profile and Immune Function of the Bursa of Fabricius in Chicken.

Infectious bronchitis is a highly contagious, acute viral respiratory disease of chickens, regardless of the strain, and its infection may lead to considerable economic losses to the poultry industry. New nephropathogenic infectious bronchitis virus (NIBV) strains have increasingly emerged in recent years; hence, evaluating their infection-influenced immune function changes and the alteration of metabolite profiling is important. Initially, chickens were randomly distributed into two groups: the control group (Con) and the disease group (Dis). Here, the partial cytokines were examined, and the metabolome alterations of the bursa of Fabricius (BF) in NIBV infections in chickens were profiled by gas chromatography time-of-flight/mass spectrometry (GC-TOF/MS). The results revealed that the NIBV infection promotes the mRNA expression of inflammatory cytokines. Metabolic profile analysis indicated that clustering differed between the two groups and there were 75 significantly different metabolites detected between the two groups, suggesting that the host metabolism was significantly changed by NIBV infection. Notably, the following 12 metabolites were identified as the potential biomarkers: 3-phenyllactic acid, 2-deoxytetronic acid, aminomalonic acid, malonamide 5, uric acid, arachidonic acid, 2-methylglutaric acid, linoleic acid, ethanolamine, stearic acid, N-alpha-acetyl-l-ornithine, and O-acetylserine. Furthermore, the results of the correlation analysis showed that a strong correlation existed between metabolic biomarkers and inflammatory cytokines. Our results describe an immune and metabolic profile for the BF of chickens when infected with NIBV and provide new biomarkers of NIBV infection as potential targets and indicators of indicating therapeutic efficacy.

16S rRNA gene sequencing reveals an altered composition of the gut microbiota in chickens infected with a nephropathogenic infectious bronchitis virus.

Infectious bronchitis virus (IBV), a member of the Coronaviridae family, causes serious losses to the poultry industry. Intestinal microbiota play an important role in chicken health and contribute to the defence against colonization by invading pathogens. The aim of this study was to investigate the link between the intestinal microbiome and nephropathogenic IBV (NIBV) infection. Initially, chickens were randomly distributed into 2 groups: the normal group (INC) and the infected group (IIBV). The ilea were collected for morphological assessment, and the ileal contents were collected for 16S rRNA gene sequencing analysis. The results of the IIBV group analyses showed a significant decrease in the ratio of villus height to crypt depth (P < 0.05), while the goblet cells increased compared to those in the INC group. Furthermore, the microbial diversity in the ilea decreased and overrepresentation of Enterobacteriaceae and underrepresentation of Chloroplast and Clostridia was found in the NIBV-infected chickens. In conclusion, these results showed that the significant separation of the two groups and the characterization of the gut microbiome profiles of the chickens with NIBV infection may provide valuable information and promising biomarkers for the diagnosis of this disease.

A Multi-Omics Study of Chicken Infected by Nephropathogenic Infectious Bronchitis Virus.

Chicken gout resulting from nephropathogenic infectious bronchitis virus (NIBV) has become a serious kidney disease problem in chicken worldwide with alterations of the metabolic phenotypes in multiple metabolic pathways. To investigate the mechanisms in chicken responding to NIBV infection, we examined the global transcriptomic and metabolomic profiles of the chicken's kidney using RNA-seq and GC-TOF/MS, respectively. Furthermore, we analyzed the alterations in cecal microorganism composition in chickens using 16S rRNA-seq. Integrated analysis of these three phenotypic datasets further managed to create correlations between the altered kidney transcriptomes and metabolome, and between kidney metabolome and gut microbiome. We found that 2868 genes and 160 metabolites were deferentially expressed or accumulated in the kidney during NIBV infection processes. These genes and metabolites were linked to NIBV-infection related processes, including immune response, signal transduction, peroxisome, purine, and amino acid metabolism. In addition, the comprehensive correlations between the kidney metabolome and cecal microbial community showed contributions of gut microbiota in the progression of NIBV-infection. Taken together, our research comprehensively describes the host responses during NIBV infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout.