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Spirochaetes of digital dermatitis and super-virulent ovine foot rot - OD0213

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).

Project Documents
• Final Report : Spirochaetes of digital dermatitis and super-virulent ovine foot rot   (163k)
Time-Scale and Cost
From: 2000

To: 2003

Cost: £168,427
Contractor / Funded Organisations
Veterinary Laboratories Agency
Keywords
Animal Diseases              
Animal Health              
Bacteria              
Biotechnology              
Bovine              
Cattle              
Ovine              
Plants and Animals              
Sheep              
Fields of Study
Animal Health