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Spirochaetes of digital dermatitis and super-virulent ovine foot rot - OD0213
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Description
Policy relevance: Lameness in cattle and sheep, particularly bovine digital dermatitis and a novel foot-rot in sheep, “super-virulent ovine foot-rot”, is of increasing welfare and economic concern. Treponemes isolated from lesions from both syndromes share genetic identity with each other and the oral pathogen Treponema denticola. MAFF seeks advice on the role of the Treponemes in foot-rot with a longer-term aim of devising interventions to prevent these diseases.
Objectives: The key aims of the proposed research are: (a) to develop an immunocapture technique with antibodies specific to ovine and bovine Treponemes; (b) to develop a robust method for the sampling of affected animals on-site to include vital biopsy and culture of Treponemes; (c) to characterise fully the Treponemes by: (i) genetic analysis of 16S rDNA gene sequences and other conserved genes such as the flagellin and the major surface antigen (MSP); (ii) analysis of the major immunogens by western blot and ELISA; and (iii) assay pathogenic potential of isolates in the “tissue destructive” mouse model.
Use of results Outputs include: (a) maintaining expertise in these generic technologies for MAFF; (b) to advise MAFF on the significance of Treponemes in foot-rots in the UK through the provision and application of appropriate detection and identification tools; and (c) to support existing MAFF-funded epidemiological analysis of foot-rots (AW1007) toward intervention. |
Objective
Obj. No. Completed by Description Date 01 30/12/2000 Biochemical characterisation of culture requirements for foot- rot Treponemes 01/01 To define nutritional requirements of the foot rot isolates and devise an enhanced medium which better supports the growth of the isolates currently in pure culture. 01/02 To assess the response of these bacteria to antimicrobials and to enable the pragmatic development of transport media (see below) for initial selective cultivation. 01/03 To grow sufficient bacteria for further detailed immunological analysis.
02 30/03/2001 Preparation of polyclonal antisera to ovine and bovine Treponemes
02/01 To raise a large stock of high-titre rabbit polyclonal antisera against ovine and bovine Treponemes. 02/02 To describe the immunogens of the ovine and bovine Treponemes by comparison with those antigens of defined Treponemes such as T. denticola and T. phagedenis, for example. This will identify clinically relevant antigens and, by absorption of the polyclonal rabbit sera against the control antigens, will enable identification of antigens and or epitopes unique to the bovine and ovine isolates.
03 30/03/2002 Development and application of latex immunocapture for foot-rot Treponemes
03/01 To utilise ovine-specific and bovine-specific immune reagents prepared above to prepare antibody-coated magnetic latex beads.
03/02 To apply immunomagnetic separation techniques for the recovery of Treponemes form biopsy material of clinically affected flocks and herds. Matched blood (serum) samples will be taken for serodiagnosis and PCR will be applied to biopsy material also. 04 30/03/2003 Development of PCR specific for foot-rot Treponemes
04/01 To apply the current 16S rDNA PCR strategy developed by this group Rijpkema et al., 1997; Collighan and Woodward, 1997) to all biopsy material. This will provide a survey of similarity of sequences and determine whether the Treponemes are always present in clinical disease. 04/02 To extend the available sequence data and this will be achieved by using universal primers to PCR amplify the spacer regions and the 23S rDNA gene which is adjacent to the 16S rDNA gene.
04/03 To apply new sequence data to the development and application of PCR in order to improve sensitivity and specificity of the existing PCR.
04/04 To enhance the sensitivity of detection of Treponemes by combining immunocapture and PCR. 04/05 To clone genes encoding immunodominant antigens identified by Western blot (objective 01) by “degenerate oligonucleotide primers PCR” (DOP.PCR) approaches.
05 30/03/2003 Definitive identification of foot-rot Treponemes as pathogenic determinants in lesions.
05/01 To examine biopsy samples by histological and electron microscopical analysis.
05/02 To test Treponeme isolates for the secretion of specific enzymes which act upon host tissues (proteases, collagenases, elastases, esterases, etc.).
05/03 To test the virulence of foot-rot Treponemes in the mouse model developed to demonstrate the virulence of the human oral pathogen Treponema denticola (Kesavalu et al., 1997).
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Project Documents
Final Report : Spirochaetes of digital dermatitis and super-virulent ovine foot rot
(163k)
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Time-Scale and Cost
From:
2000
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To:
2003
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Cost: £168,427 |
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Contractor / Funded Organisations
Veterinary Laboratories Agency |
Keywords
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Fields of Study
Animal Health |