Isolation and characterization of chicken bile matrix metalloproteinase.

Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies, but the significance of their expression in normal, healthy bile is not understood. We hypothesized that the MMP in bile may aid the digestion of native collagens that are resistant to conventional gastric proteases. Hence, the objective of this study was to characterize the bile MMP and check its regulation in association with dietary factors. We used substrate zymography, azocoll protease assay, and gelatin affinity chromatography to identify and purify the MMP from chicken bile. Using zymography and SDS PAGE, 5 bands at 70, 64, 58, 50, and 42 kDa were detected. The bands corresponding to 64, 50, and 42 kDa were identified as MMP2 using trypsin in-gel digestion and matrix-assisted laser desorption time-of-flight mass spectrometry and peptide mass fingerprinting. Chickens fed diets containing gelatin supplements showed higher levels of MMP expression in the bile by both azocoll assay and zymography. We conclude that the bile MMP may be associated with the digestion of collagens and other extracellular matrix proteins in avian diets.

Tibial dyschondroplasia-associated proteomic changes in chicken growth plate cartilage.

Tibial dyschondroplasia (TD) is a poultry leg problem that affects the proximal growth plate of the tibia, preventing its transition to bone. To understand the disease-induced proteomic changes, we compared the protein extracts of cartilage from normal and TD-affected growth plates. TD was induced by feeding thiram to chickens 2 wk before tissue harvest. Proteins were extracted from whole tissues and from conditioned media (CM) prepared by incubating appropriate growth plate tissues in serum-free culture medium for 48 hr. The extracts were prefractionated to contain proteins ranging between 10 and 100 kD. Equal amounts of proteins were subjected to 2D gel electrophoresis with three individual samples per group. The gels were silver stained, and digital images were compared and analyzed with Melanie software to determine differentially expressed protein spots. On comparison of two sets of gels, 47 matching spots were detected in tissue extracts and 27 in CM extracts. Among the matching spots, 12 were determined to be down-regulated in tissue extracts (P < or = 0.05) and two in CM extracts (P < or = 0.05) of TD-affected growth plates. Altogether, 32 protein spots could be identified in both tissue and CM extracts by in-gel trypsin digestion, followed by peptide mass fingerprinting and mass spectrometry (MS)/MS fragmentation. The down-regulated proteins included alpha-enolase, G protein, origin recognition complex, peptidyl prolyl isomerase, calumenin, type II collagen precursor, and the expressed sequence tag pgm2n.pk014.f20, a protein with homology to human reticulocalbin-3 (RCN3). Most of the downregulated proteins are associated with signal transduction, energy metabolism, and secretory functions that are integral to cell viability. Consistent with our earlier findings that the TD chondrocytes are nonviable, the current results suggest that thiram very likely interferes with basic metabolic functions of chondrocytes, leading to their death and, consequently, to the pathogenesis of TD.

Histopathology and serum clinical chemistry evaluation of broilers with femoral head separation disorder.

Femoral head separation (FHS) and necrosis is a sporadic leg problem of unknown etiology in broiler breeders. To determine the underlying physiology of FHS, the blood chemistry and histopathology of the femoral growth plates of the affected chickens were compared with their age-matched controls and with birds having tibial dyschondroplasia. Femoral problems were categorized on the basis of 1) femoral head separating from articular cartilage without any visible damage to the growth plate (FHS) and 2) FHS with significant tearing and lesions in the growth plate (FHSL). Tibial dyschondroplasia was identified by a widening of the growth plate with an unresorbed plug of cartilage at the proximal end of the tibia. Control birds were without any femoral or tibial problems. The histopathology of FHSL growth plates revealed occasional chondrocyte death, hypocellularity, dysplasia in the prehypertrophic zones, and the absence of inflammatory infiltrates in the lesion areas. Hematoxylin and eosin staining showed brown chromogenic deposits in the metaphyseal bone marrow areas. Blood chemistry of chickens with FHSL showed a modest but significant elevation of cholesterol, triglycerides, and low-density lipoproteins. Only cholesterol and low-density lipoproteins were moderately elevated in FHS-affected chickens. Other blood parameters, such as protein, magnesium, and iron levels, showed differential changes in birds with leg problems, but there were no specific trends. Neither blood ovotransferrin, a marker of chronic inflammation, nor corticosterone, a marker of stress, showed any significant differences from the controls. These results indicate that FHS may be a metabolic problem in poultry, one that is related to fat metabolism disorders, possibly contributing to an unbalanced growth in the articular-epiphyseal complex that leads to its separation under sheer stress.