Direct and indirect markers of cartilage metabolism in synovial fluid obtained from dogs with hip dysplasia and correlation with clinical and radiographic variables

OBJECTIVE: To compare activities of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and matrix metalloproteinase (MMP)-3 and contents of sulfated glycosaminoglycan (S-GAG) in joint fluid obtained from dogs with hip dysplasia (HD) and clinically normal dogs, evaluate correlations among these markers in joint fluid obtained from dogs with HD, and evaluate correlations between each marker and clinical and radiographic variables. Animals-26 dogs with HD (clinical group) and 43 clinically normal Beagles (control group). PROCEDURE: Joint fluid was aseptically collected from the hip joints of all dogs. For each dog in the clinical group, age, duration of lameness, radiographic osteoarthritis (OA) score, and Norberg angle in each affected joint were recorded. Activities of IL-1beta, IL-6, TNF-alpha, and MMP-3 and S-GAG contents were measured. Values were compared between groups by use of Mann-Whitney U tests, and the Spearman rank correlation test was used to evaluate correlations among markers and between each marker and clinical or radiographic variables. RESULTS: Values of all markers were significantly higher for the clinical group, compared with values for the control group. There was a moderate positive correlation between lameness duration and IL-6 activity and a strong negative correlation between the Norberg angle and IL-1beta activity. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of our results indicated that there was a significant increase in markers of OA in dogs with HD. Activities of IL-1beta and IL-6 in joint fluid of dogs with HD may be influenced by the severity of laxity in the hip joint and lameness duration, respectively.     

Establishment of a new canine cell line (CCT) originated from a cutaneous malignant histiocytosis

A new canine cell line, named CCT, was established from the cutaneous malignant histiocytosis in a 4-year-old male Borzoi. CCT proliferated with loose adherence and doubling time was approximately 30 hr. When co-cultured with latex beads, CCT phagocytized beads vigorously. Lysozyme and vimentin were positive by immunostaining, and non-specific esterase and acid phosphatase were positive by cytochemical staining. These features indicated the cells had a histiocytic nature. Furthermore, by subcutaneous injection to nude mice CCT could successfully form tumors with the morphological and immunohistochemical features similar to the original tumor.     

Distribution of TNF receptors and TNF receptor-associated intracellular signaling factors on equine tendinocytes in vitro

Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-R-associated factor 2 (TRAF2) and nuclear factor-kappa B (NF-kappaB), and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTs of thoroughbred horses. The tendinocytes were treated with 10 ng/ml equine TNFalpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF -R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFalpha-treated cells (inflamed condition). Intense TRAF2 and NF-kappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in vitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.     

Growth characteristics of canine distemper virus in a new cell line CCT cells originated from canine malignant histiocytosis

Canine distemper virus (CDV) growth and the morphological characterization were examined in a cell line established from a canine malignant histiocytosis (CCT cell line). The susceptibility of the CCT cells to 3 CDV strains, FXNO, YSA-TC and MD-77 was shown by detection of the antigen in the indirect fluorescent assay. After passaging 4 and 9 times through the CCT cells, only FXNO strain could produce the syncytia where demonstrated the antigens. Titers of 9 passaged viruses through the CCT cells showed slightly higher in the CCT cells than those in Vero cells. Morphological characterization of karyorrhexis and specific DNA ladder by extracted DNA electrophoresis indicated apoptosis in the CDV infected CCT cells.     

Comparative studies of the persistence of animal mycoplasmas under different environmental conditions

A comparison of the persistence of mycoplasmas in animals was carried out. When inoculated into liquid media, strains of Mycoplasma bovis, M. arginini, Acholeplasma laidlawii, and A. axanthum persisted for 59-185 days post-inoculation. The survival periods were not significantly influenced by temperature (4, 30, 37 degrees C, and room temperature). The survival periods for M. bovigenitalium, M. gallisepticum, M. bovirhinis, and M. gateae ranged from <7 to 185 days depending on medium components and temperature. Further, it was determined that strains of M. bovigenitalium, M. bovis, M. bovirhinis, M. arginini, and A. laidlawii persisted in a dry paper disc for at most 28, 126, 154, 56 and over >168 days at 4 degrees C, respectively. At 4 degrees C, strains of M. gallisepticum, M. columborale, M. edwardii, M. felis, and M. gateae survived for at most 28, 21, 42, 28, 28 and 70 days, respectively. At 30 degrees C, strains of M. bovis, M. bovirhinis, M. arginini, A. laidlawii, and M. gallisepticum persisted for at most 28, 84, 56, >168 and 14 days, respectively, but strains of M. gallisepticum, M. columborale, M. edwardii, M. felis, M. gateae, and U. diversum did not survive for more than 14 days. In an outdoor environment, strains of M. bovirhinis and A. laidlawii survived for at most 28 and 14 days, respectively. Finally, it was found that 14 isolates of M. gallisepticum persisted for periods similar to those of the reference strains. The results under dry conditions at a variety of temperatures presented contribute to understanding the epizootiology of mycoplasmal infections in the field.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Use of monoclonal antibodies for analyses of Coxiella burnetii major antigens

Monoclonal antibodies (MAbs) to major antigens of Coxiella burnetii were produced. Some of the MAbs to a 62-kDa protein antigen, peptidoglycan protein complex and lipopolysaccharide (LPS) O-chains reacted with other bacteria whereas none of the MAbs to outer membrane proteins and LPS outer-core did. The LPS outer-core and OMPs may be useful antigens for specifically detecting antibodies to C. burnetii.     

Generation of monoclonal antibody against canine neural-cell adhesion molecule

A monoclonal antibody, K9BYU, was generated using Escherichia coli recombinant extracellular domain of canine neural-cell adhesion molecule (N-CAM) as an antigen. Immunoreactivity of K9BYU to insect cell recombinant canine N-CAM was demonstrated by Western blotting using Sf9 insect cells transfected with the canine N-CAM gene. In Western blotting against canine brain tissue, K9BYU detected three isoforms of N-CAM that correspond to three major isoforms of human and mouse N-CAM (N-CAM-120, -140, and -180). From these results, K9BYU was considered to be a useful tool for research of canine N-CAM.