Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology

Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.     

Immunological and pathological investigations in equine experimental uveitis

Background

The pathogenic mechanism of equine recurrent uveitis (ERU) is still poorly defined and many variations between experimental animal models and spontaneous disease exist.

Objectives

The aim of our study was to investigate if Th17 cell-mediated response plays role in the pathogenesis of the used experimental model in horses and to reveal its pathological findings.

Methods

Experimental uveitis was induced in 6 healthy horses. The concentrations of retinal autoantigen CRALBP and IL-17 were measured using ELISA in aqueous humor and vitreous body of the 12 inflamed eyes as well as in 12 control non-inflamed eyes taken from 6 horses in slaughter house. After centrifugation of the two eye media, smears were prepared and cytological investigation was performed. Tissue specimens were taken from all eye globes and were submitted to histopathological investigation.

Results

CRALBP and IL-17 concentrations were significantly elevated in eye media of horses with experimental uveitis in comparison with controls. Cytological and histopathological findings corresponded to the changes characteristic of chronic immune-mediated inflammation with mononuclear cell infiltration of uvea, choroid, retina, and eye media as well as severe retinal destruction.

Conclusions

Our study demonstrated the involvement of the retinal autoantigen CRALBP as well as IL-17 in the pathogenesis of experimental uveitis in horses. These findings suggests that this experimental uveitis in horses may serve as a suitable animal model for investigation of IL-17- mediated immune response during spontaneous autoimmune uveitis in horses as well as in humans.

     

Efficacy of bovine herpesvirus-1 inactivated vaccine against abortion and stillbirth in pregnant heifers

OBJECTIVE: To evaluate the efficacy of an inactivated bovine herpesvirus-1 (BHV-1) vaccine to protect against BHV-1 challenge-induced abortion and stillbirth. DESIGN: Prospective study. ANIMALS: 35 beef heifers. PROCEDURES: Before breeding, heifers were vaccinated with a commercially available BHV-1 inactivated vaccine SC or IM. The estrous cycle was then synchronized, and heifers were artificially inseminated 30 to 60 days after vaccination. Heifers (n = 21) were challenge inoculated IV at approximately 180 days of gestation with virulent BHV-1. Fourteen control heifers were not vaccinated. Clinical signs of BHV-1 infection were monitored for 10 days following challenge; serologic status and occurrence of abortion or stillbirth were evaluated until time of calving. RESULTS: 18 of 21 (85.7%) heifers that received vaccine were protected from abortion following challenge, whereas all 14 control heifers aborted. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that an inactivated BHV-1 vaccine can protect against abortion resulting from a substantial challenge infection, with efficacy similar to that of modified-live BHV-1 vaccines.     

Adrenomedullin (AM) and adrenomedullin binding protein (AM-BP) in the bovine mammary gland and milk: Effects of stage of lactation and experimental intramammary E. coli infection

Adrenomedullin (AM) has been characterized as an endogenous tissue survival factor and modulator of many inflammatory processes. Because of the increased susceptibility of the mammary gland to infection during the time surrounding parturition in the cow, we investigated how milk and tissue content of AM and its binding protein (AM-BP) might be affected by the stage of lactation and the udder health status. Milk and mammary biopsy samples were obtained from Holstein cows 21 days prior to and at various times after calving to represent the dry period and early and mid-stages of lactation. Additional cows received an intramammary challenge with Escherichia coli for immunohistochemical characterization of AM and AM-BP. Milk AM concentrations were relatively constant across the stages of lactation while AM-BP increased two-fold (P<0.04) between early and mid-lactation. Milk AM (P<0.04) and AM-BP (P<0.03) increased as somatic cell counts (SCCs) increased within a given stage of lactation. Tissue content of both (AM and AM-BP) were significantly affected by stage of lactation, lowest in the dry period and progressively increasing to peak at mid-lactation as well as increasing in association with higher levels of SCCs. Following E. coli challenge, AM increased in epithelial cells surrounding mammary alveoli presenting high levels of SCCs. The data suggest that AM and AM-BP are cooperatively regulated in the mammary gland during lactation; changes in localized tissue AM and AM-BP content reflect a dynamic regulation of these tissue factors in the bovine mammary gland consistent with their protective effects within inflamed tissue.     

Broad range 16S rRNA gene PCR compared to bacterial culture to confirm presumed synovial infection in horses

The objectives of the present study were to evaluate the accuracy of broad range 16S rRNA gene PCR compared to bacterial culture for the detection of synovial infection in horses. The study included 57 synovial fluid samples from horses with presumed synovial infection and a control group consisting of 31 synovial fluid samples originating from clinically normal horses and horses with aseptic synovial inflammation. All samples were analysed by 16S PCR with reverse line blot (RLB) hybridisation. Synovial fluid samples were cultured using conventional agar plate methods (APM) and/or blood culture medium (BCM). The results of the study showed a superior detection rate (89.5%) for 16S PCR with RLB. Bacterial culture had lower sensitivity, but highly acceptable detection rates (77.6%) were observed using BCM. APM had very low sensitivity (37.8%) and infection was never detected by plate isolation without positive incubation in BCM. The highest sensitivity (91.8%) for the detection of synovial infection was achieved when the results of incubation in BCM and 16S PCR were combined. For all the tests, the specificity was higher than 90%.     

Association between floor type and behaviour, skin lesions, and claw dimensions in group-housed fattening bulls

Rubber mats covering concrete slatted flooring are a developing market in dairy barns but remain rare in beef cattle facilities. The aim of this study was to investigate the effects of covering slatted concrete floor with perforated rubber mats on behaviour and occurrence of skin and claw lesions in fattening bulls. The groups of six bulls each with a total average age of 9.8 months were kept over 1 year on either slatted concrete (CONCRETE PEN) or on slatted concrete covered completely (RUBBER PEN) or partially (CHOICE PEN) with perforated rubber mats. Every quarter-year, behaviour (preference of flooring, lying, aggression, mounting) was recorded. In two-weekly intervals the incidence of skin lesions was examined. At 12 and 18 months of age the rising time of the bulls was measured. At the beginning of the study and after slaughter claw dimensions were recorded. Bulls in the CHOICE PEN preferred (P<0.01) the rubber coated area throughout the experiment. Animals in the RUBBER and the CHOICE PENS showed more lying periods (P<0.01) and had a lesser incidence of skin lesions (P<0.01) compared to bulls in the CONCRETE PEN. Bulls in the CHOICE PEN needed less time for rising (2.7+/-0.3s) than bulls in the CONCRETE PEN (4.4+/-0.5s, P<0.01). Net claw growth differed significantly between all pens (RUBBER>CHOICE>CONCRETE; P<0.05). In conclusion, the results of this study indicate that rubber coated slatted flooring has a positive influence on the housing conditions of beef cattle.