Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Dichelobacter nodosus and Fusobacterium necrophorum in ruminants with lameness and footrot A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by G. N. Bennett Lincoln University 2011 ii Declaration of parts of thesis that have been accepted for publication Chapter 2 Bennett G., Hickford J.G., Sedcole R., Zhou H., 2009(a). Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot. Anaerobe 15, 173-6. Bennett G., van Loenen A., Hickford J.G., Sedcole R., Zhou H., 2009(b). Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot in goats. Anaerobe 15, 177. Chapter 5 Bennett G., Hickford J.G., Zhou H., Laporte J., Gibbs J., 2009(c). Detection of Fusobacterium necrophorum and Dichelobacter nodosus in lame cattle on dairy farms in New Zealand. Res. Vet. Sci. 87, 413-5. Chapter 6 Zhou H., Bennett G., Hickford J.G., 2009(a). Variation in Fusobacterium necrophorum strains present on the hooves of footrot infected sheep, goats and cattle. Vet. Microbiol. 135, 363-7. Zhou H., Bennett G., Kennan R.M., Rood J.I., Hickford J.G., 2009(b). Identification of a leukotoxin sequence from Fusobacterium equinum. Vet. Microbiol. 133, 394-5. Zhou H., Bennett G., Hickford J.G., 2009(c). Detection of Fusobacterium equinum on footrot infected hooves of sheep and cattle. Vet. Microbiol. 134, 400-1. Chapters 5 and 6 Hickford J.G., Bennett G., Zhou H., 2010. The presence of Dichelobacter nodosus and Fusobacterium necrophorum on the claws of lame dairy cattle in New Zealand. Proceedings of the 4th Australasian Dairy Science Symposium. 428-31. iii Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy Dichelobacter nodosus and Fusobacterium necrophorum in ruminants with lameness and footrot By Grant Norman Bennett Footrot is a destructive hoof disease found predominantly in sheep and goats that is caused by Dichelobacter nodosus (D. nodosus), a gram-negative, anaerobic bacterium. It has been shown that D. nodosus requires a second, gram-negative, anaerobic bacterium, Fusobacterium necrophorum (F. necrophorum), to initiate footrot. However, once footrot is established, the role, if any, that F. necrophorum plays is unknown. In this thesis, the roles D. nodosus and F. necrophorum play in footrot were investigated. To facilitate the study of F. necrophorum a new diagnostic Polymerase Chain Reaction (PCR) specific for F. necrophorum was developed and tested. This method was used in combination with an existing D. nodosus-specific PCR to study the prevalence of D. nodosus and F. necrophorum in sheep and goats with footrot in the field. This found D. nodosus and F. necrophorum were associated with under-running footrot and tended to be detected together. This supported the contention that F. necrophorum acts cooperatively with D. nodosus to cause under-running footrot in sheep and goats as well as that ovine and caprine footrot have similar bacteriologies. To ascertain the possible roles D. nodosus and F. necrophorum play during footrot development, two footrot trials were conducted. These trials ran disease-free sheep and iv sheep with footrot together in an experimental group as well as a control group of disease free sheep. Data were collected describing hoof pathologies and bacterial prevalence which was assessed using a combination of case study and statistical analyses The case studies suggested that F. necrophorum may be involved in the most rapidly developing and destructive under-running pathologies, both D. nodosus and F. necrophorum persist together in old cryptic lesions and that both D. nodosus and F. necrophorum can be found in sheep with no clinical signs of footrot. Furthermore, it was observed that before under-running occurred, only F. necrophorum could be detected and D. nodosus only became detectable once under-running began. These case studies not only suggested that F. necrophorum is involved with D. nodosus in initiating footrot, but also supports the contention that it is involved in under-running lesions and persists in cryptic lesions with D. nodosus. The observation that D. nodosus was also detected in sheep with no signs of disease suggested D. nodosus was either transmitted to, or able to persist in these animals, without causing disease. Statistical analyses of the two footrot trials revealed that detection of F. necrophorum on the skin-horn junction and variance in the detection of D. nodosus were strongly correlated with disease. This suggested that F. necrophorum is important in disease and implied D. nodosus numbers “wax and wane” during disease. In turn, this suggests D. nodosus undergoes a "boom and bust" lifecycle during disease and in combination with the strong correlations between variables; it highlights the complex multi-factorial nature of footrot. v An investigation was undertaken to identify if D. nodosus could be persisting in the gastro-intestinal tract of sheep. This site was studied since it had the potential to act as a subclinical reservoir of D. nodosus as an anaerobic habitat in close proximity to the hoof. PCR was used to test if D. nodosus was detectable in the mouth, rumen, duodenum, caecum or colon of 25 culled sheep; or shed via faeces or the mouth of 36 monitored sheep. D. nodosus was not detected in the mouth, faeces, or gastro-intestinal tract of these sheep. F. necrophorum carries a leukotoxin (lktA) gene that has previously only been described in F. equinum and F. necrophorum. Here, four variants (A, B, C and D) of the lktA gene of F. necrophorum were identified on lame cattle and cattle, sheep and goats with footrot. After sequence comparison, it was found that variant A of the lktA matched the lktA of the type strain of F. necrophorum sub sp. necrophorum (NCTC no.10575) while variants B, C and D have not been described previously. Of the four lktA variants observed, variants A and C were found most often. Furthermore, variant A was predominantly found in lame cattle (87.7% of cattle) while variant C was predominantly found in footrot-infected sheep and goats (83.3% of sheep and goats). During the development of the F. necrophorum specific lktA PCR, a novel variant of the lktA gene was identified in F. equinum and, subsequently, detected in lesion material collected from cattle and sheep. This novel lktA sequence was different from other F. necrophorum type strains (67.8-68.1 % homologous). Moreover, blocks of sequence conservation were observed suggesting the lktA gene may be structurally conserved across species. vi The work described here suggests that F. necrophorum may play an important role in ovine footrot, being found in under-running, cryptic and developing lesions. It was also shown that a distinctive variant of F. necrophorum may be present in footrot lesions and if the genetic variation in this portion of lktA gene is representatives of variation in the genome, it implies these variants represent an un-described species or sub-species of Fusobacteria. While ovine footrot has been well investigated with regard to D. nodosus and its virulence, it is clear that the study of F. necrophorum has been neglected. The work presented here suggests that F. necrophorum plays an important role in ovine footrot and it should be studied further. Key words: Footrot, lameness, Dichelobacter nodosus, Fusobacterium necrophorum, Fusobacterium equinum, leukotoxin, sheep, goats, cattle. vii Publications arising from this thesis Bennett G., Hickford J.G., Zhou H., 2006. Convenient anaerobic techniques, science from the supermarket shelf. Anaerobe 12, 49-51. Bennett G., Hickford J.G., Sedcole R., Zhou H., 2009(a). Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot. Anaerobe 15, 173-6. Bennett G., van Loenen A., Hickford J.G., Sedcole R., Zhou H., 2009(b). Dichelobacter nodosus, Fusobacterium necrophorum and the epidemiology of footrot in goats. Anaerobe 15, 177. Bennett G., Hickford J. G., Zhou H., Laporte J., Gibbs J., 2009(c). Detection of Fusobacterium necrophorum and Dichelobacter nodosus in lame cattle on dairy farms in New Zealand. Res. Vet. Sci. Res. Vet. Sci. 87, 413-5. Zhou H., Bennett G., Hickford J.G., 2009(a). Variation in Fusobacterium necrophorum strains present on the hooves of footrot infected sheep, goats and cattle. Vet. Microbiol. 135, 363-7. Zhou H., Bennett G., Kennan R.M., Rood J.I., Hickford J.G., 2009(b). Identification of a leukotoxin sequence from Fusobacterium equinum. Vet. Microbiol. 133, 394-5. Zhou H., Bennett G., Hickford J.G., 2009(c). Detection of Fusobacterium equinum on footrot infected hooves of sheep and cattle. Vet. Microbiol. 134, 400-1. Bennett G., Zhou H., Hickford J.G., 2010(a). Undetected lktA genes within Fusobacterium necrophorum? J. Med. Microbiol. 59, 499-500. Hickford J.G., Bennett G., Zhou H., 2010(b). The presence of Dichelobacter nodosus and Fusobacterium necrophorum on the claws of lame dairy cattle in New Zealand. Proceedings of the 4th Australasian Dairy Science Symposium. 428-31. viii Bennett G., Hickford J.G., 2011(a). Ovine footrot: New approaches to an old disease. Review, Vet Microbiol. 148, 1-7. Zhou H., Bennett G., Buller N, Hickford J.G, 2011(b). Isolation of two novel Fusobacterium necrophorum variants from sheep in Australia.