Dichelobacter nodosus metapopulations and epidemiology of footrot in endemically infected flocks (PhD)

From investigating the load of D. nodosus on the interdigital skin and non-interdigital lesions of ewes' feet, mean load increased with increasing ID score. Both foot trimming plus topical antibiotic foot spray and parenteral antibiotics plus topical antibiotic foot spray yielded a reduction in mean D. nodosus load after treatment. This reduction in mean D. nodosus load was not seen on sheep without footrot after treatment with parenteral antibiotics and no topical treatment. This suggests that the topical antibiotic foot spray reduced D. nodosus load on the interdigital skin. Topical antibiotics are likely to reduce the spread of D. nodosus and so reduce the chances of further disease developing. The action of topical antibiotics are independently effective alongside using parenteral antibiotics - this study shows topical antibiotics to be a key part of the control of footrot.

When assessing footrot lesions, it is recommended that farmers turn sheep over and inspect their feet at gathering times and treat any signs of ID or Severe FR immediately. Almost half of routine samples in this study from ewes showing no signs of lameness had a positive ID and/or SFR score. If farmers purely use visible lameness this may not always be an accurate measure of footrot in their flock.

With regards to laboratory methodologies, mean D. nodosus load of swab DNA where isolates were successfully cultured was higher than where isolates were not obtained. In addition, more than one fifth of samples without isolates could be detected using quantitative PCR but would unlikely be detected through culturing. This shows that detecting D. nodosus from culturing alone gives a qualitative indication which could be sufficient to help in vaccine development for outbreaks of footrot. But does not give as true a representation of D. nodosus presence as direct quantitative DNA analysis for more in-depth analyses of D. nodosus pathogenicity. PCR based laboratory methods should be used in future analyses to ensure a more accurate detection rate. In addition, swab DNA analysis yielded higher detection rates for serogroups H, I and D (H in particular) than isolate DNA analysis. More than three quarters of samples where additional serogroups could be detected in swab DNA than isolate DNA, serogroup H was the additional serogroup. This indicates that serogroup H may be less detectable by culture from this flock. Non-culture-based swab DNA analysis was more sensitive and therefore provides a more accurate representation of D. nodosus present. Furthermore, the coherence of genomic serogroup analysis shown in the studied flock isolates, indicated genomic serogroup identification shows even greater accuracy than laboratory isolate analysis.

The isolates sequenced from the studied flock showed three phylogenetically distinct clades. Temporal signal was identified in clade 1 and the most recent common ancestor identified to a date from 1998 when new stock was brought into the previously closed flock. This gives further evidence to the importance of adequate biosecurity measures in minimising footrot in flocks. Quarantine of new and diseased sheep should be a top priority for farmers, insuring there is no spread of contact to current stock until the quarantine period of 4 weeks (ideally) is completed. All four feet of new sheep should be disinfected before being introduced to the current flock. The two remaining clades could have developed from the introduction of rams from different farms. This is commonly thought to be a source of new strains of D. nodosus but needs further research into the phylogeny of D. nodosus before and after ram introduction.

From genomic serogroup analysis of the studied flock, clade 1, the smallest clade, comprised serogroups D and H and the larger clades, 2 and 3, both comprised serogroups B and I. Yet phylogenetically clades 1 and 2 were more closely linked. Although only two known serogroups were identified in each clade, all three clades exhibited the entire range of ID and SFR scores recorded. So isolates that appear phylogenetically almost identical were identified across the range of disease severities. Moreover, no associations were found between serogroup identification by genomic analysis and ID or SFR score in the studied flock isolates. The same with laboratory identification of serogroups by singleplex PCR. All four serogroups caused footrot.

With regards to the current vaccine, it would be advisable for farmers not already using the vaccine to start using it biannually and monitor its effects. This should be done alongside current recommended management strategies for treatment of footrot. However, further research is needed in vaccine design as the variation in success could be attributed to the lack of association between serogroup (dominant or not) and disease state as indicated by this study.

From global D. nodosus isolate analysis where both aprB2 and aprV2 were identified, acidic protease identification was identical between laboratory and genomic analysis in all but one D. nodosus isolate. Accuracy of acidic protease identification appeared very strong, but these proteases did not correlate with disease presentation on the feet. Both genotypes were identified on healthy feet and feet with a form of footrot. The majority of D. nodosus isolates positive for aprB2 came from Sweden and Norway where aprB2 dominates suggesting the acidic protease genotype simply mirrors the phylogeographical structure of D. nodosus. This indicates that all strains of D. nodosus regardless of acidic protease or serogroup are virulent and able to cause footrot. However, previous studies have shown that identifying serogroups present in a new outbreak of footrot in an area can then be used in targeted vaccine formulation which can help reduce the prevalence of footrot.

In both the studied flock isolate analysis and globally sourced isolate analysis, considerable recombination was identified with more mutation events occurring than recombination events. In both analyses, peaks in recombination were found for the fimbrial and outer membrane protein 1 gene regions which may aid pathogenesis. The outer membrane protein region may shed more light on the virulence of D. nodosus and disease state and so should be investigated further. 

The Challenge

Footrot has an estimated economic impact of between £24 and £80 million per annum to the sheep industry and causes lameness, loss of body condition and reduced productivity. Footrot has two main presentations, interdigital dermatitis (ID) which is an inflammation of the interdigital skin and severe footrot (SFR) where there is separation of the hoof horn from the underlying soft tissue.

Dichelobacter nodosus is the bacterial cause of footrot and the focus of my PhD. There are often several strains of D. nodosus on feet simultaneously. A number of virulence factors have been identified in D. nodosus and the bacterium is broadly categorised as benign or virulent by absence or presence of virulence factors, however, the association between virulence factors and disease severity is not well characterised.

The Problem

The high genetic diversity often found in D. nodosus isolates provides a challenge for identifying virulent strains. Often a select number of strain types can dominate, which highlights the importance of strain detection early on in an outbreak, the strategic use of vaccines, and separating diseased animals. The phylogenetic diversity and virulence factors of D. nodosus are varied and complex and it is important to investigate them further in order to improve our understanding of how D. nodosus establishes infection in sheep feet.

Aims and Objectives 

1. To gain a clear understanding of how the load of D. nodosus changes over the duration of disease and how this reflects the severity of lesions observed
2. To use non-culture-based techniques to detect the presence of key virulence factors of D. nodosus
3. To use whole genome sequencing to gain a clearer understanding of the key virulence factors of D. nodosus, namely aprV2 and aprB2 and fimA defining the serogroups, and how they associate with the severity of disease
4. To compare laboratory and genomic detection of these key virulance factors to understabnd better how genetic elements link with clinical disease
5. To investigate the phylogenetic diversity of D. nodosus strains and associations with disease severity.

Approach

The majority of the work in this study was focused around analysis of swab DNA and isolate DNA taken from the feet of a flock of 91 ewes from one farm studied in detail previously over 10 months. This work brought to light new knowledge around D. nodosus load and the severity of footrot, how D. nodosus load is affected by treatment, and differences in methods of detecting D. nodosus. The key virulence factors previously associated with clinical disease were then investigated both through laboratory and genomic analysis. These factors along with the phylogenetic diversity of D. nodosus were investigated on both a single farm scale and global scale.

Student

Zoë Willis, University of Warwick