Novel inflammatory cell infiltration scoring system to investigate healthy and footrot affected ovine interdigital skin

Ovine footrot is a degenerative disease of sheep feet leading to the separation of hoofhorn from the underlying skin and lameness. This study quantitatively examined histological features of the ovine interdigital skin as well as their relationship with pro-inflammatory cytokine (IL-1_) and virulent Dichelobacter nodosus in footrot. From 55 healthy and 30 footrot ovine feet, parallel biopsies (one fixed for histology) were collected post-slaughter and analysed for lesions and histopathological analysis using haematoxylin and eosin and Periodic Acid-Schiff. Histological lesions were similar in both conditions while inflammatory scores mirror IL-1_ expression levels. Increased inflammatory score corresponded with high virulent D. nodosus load and was significant (

Prediction of Pharmacokinetic Clearance and Potential Drug-Drug Interactions for Omeprazole in the Horse using in vitro Systems

Horses are exposed to various kinds of medication, however, there are limited determinations of plasma clearance (CLp) for the drugs used due to the high cost of equine in vivo studies. Many of the CLp values generated come from the equine sports industry for determining drug plasma screening limits in the control of medications at the time of competition.The kinetics of omeprazole metabolism were investigated in freshly isolated and cryopreserved equine hepatocytes and hepatic microsomes (n = 3 horses). The Vmax, Km and intrinsic clearance (CLint) of omeprazole were determined via the substrate depletion method as well as Km values for the formation of three metabolites. The CLint values were extrapolated to in vivo hepatic plasma clearance (CLH) using the well stirred and parallel tube models.Clp for omeprazole was successfully predicted using freshly isolated or cryopreserved equine hepatocytes, while microsomes under-predicted. Equine microsomes were used to perform a drug-drug interaction (DDI) study between omeprazole and chloramphenicol. The average inhibitor constant Ki, assuming competitive inhibition, was 15.4 ± 5 µM.To the authors’ knowledge, this is the first report showing the successful extrapolation of drug CLp in the horse using equine hepatocytes and the prediction of a DDI using microsomes

The impact of glutaraldehyde based footbaths on Dichelobacter nodosus prevalence and the antimicrobial resistant community of the ovine interdigital skin

Ovine footrot, is a highly contagious polymicrobial bacterial infection, primarily caused by Dichelobacter nodosus. Preventative bactericidal footbaths are commonly used in the sheep industry to reduce the spread of bacteria. However, their effect on the bacterial community is poorly understood. This is the first study to investigate the impact of 2% Digicur (ProGiene,UK) footbath on the bacterial community of the ovine interdigital skin following a common UK footbathing routine. Swab samples were analysed by qPCR to determine prevalence and load of D. nodosus and numerated on MacConkey agar in the presence or absence of tetracycline and ampicillin to determine phenotypic antimicrobial resistance. Metagenomics were used to determine the impact of a single footbath on the bacterial community and genotypic antimicrobial resistance. The results suggest 2% Digicur is ineffective at reducing the load of D. nodosus when applied as a one off or weekly footbath, however sheep may act as a reservoir for multi-drug resistant bacteria creating opportunities to spread antimicrobial resistance to other sheep and their environment

Differential modulatory effects of Annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-α (TNF-α) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-α production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1