Field strains of the footrot bacterium Dichelobacter nodosus range from highly virulent through to benign. Within this range there is a point above which a strain causes virulent footrot (which is subject to regulatory quarantine and control measures), and below which strains cause benign footrot (with no regulatory consequences). Highly virulent footrot is easy to recognise clinically and relatively easy to eradicate because infected sheep can be readily identified. Lower-virulent footrot is more difficult to recognise if the environmental conditions are not ideal and if the disease has not been fully expressed.
In NSW the diagnosis of footrot is based on clinical signs, but laboratory bacterial virulence tests can be used to support diagnosis in cases where disease expression is not clear cut. NSW DPI currently uses the elastase virulence test which measures the production of the thermostable acidic protease enzyme responsible for severe lesions by degrading the hoof matrix. While this test has been validated with field studies in South Eastern Australia and Western Australia (Dhungyel et al., 2013; McPherson et al., 2017), it is yet to be validated against NSW DPI clinical diagnosis guidelines.
This study measured the rate of elastase clearing (digestion of elastin particles) in D. nodosus isolate cultures submitted from 184 sheep flocks in NSW between August 2020 and December 2021 to better understand how useful the elastase test was in supporting footrot diagnosis. The elastase activity was combined with other data collected from these flocks including the incidence and severity of foot lesions, the breed and age of affected sheep, environmental conditions on-farm and the number of weeks since foot bathing or antibiotic treatments were given to identify factors that aid in the diagnosis of footrot.
Between August 2020 and December 2021, the NSW LLS performed 438 footrot investigations on 233 NSW sheep and goat flocks, with 46.8% of investigations being repeat clinical disease inspections on the same 233 properties. Laboratory diagnostics (elastase test) were only submitted for 223 of the 438 investigations (51%), with all submissions from unique mobs except for two mobs which were tested twice. However, only 184 of these submissions included sufficient case history information to include in this study. The majority of laboratory accessions were submitted from the Riverina, South East, Central West, Murray and Central Tablelands Local Land Services (LLS) regions, coinciding with the same five LLS regions that produce more than 75% of the annual NSW wool clip by weight.
Bacterial virulence was measured by growing up to 10 pure isolates of D. nodosus on agar media containing elastin particles and recording the clearing of elastin by the bacterial protease every 3-4 days over a 28-day period. Bacterial virulence was measured as the mean rate of elastase activity (diameter of elastin cleared between day 7 and 28); the percentage of D. nodosus isolates that cleared elastin before 12 days; and the minimum number of days before at least one D. nodosus isolate cleared elastin (Figure 1). Correlations between these bacterial virulence measures and clinical disease (% lame sheep and % score 4 and 5 foot lesions per flock) were investigated. Logistic regression models were fitted to the data to identify the minimum number of disease predictors that best discriminate between benign and virulent footrot diagnosis.
Of the 184 flocks investigated, 42.9% were diagnosed with virulent footrot, 46.7% were diagnosed with benign footrot, 8.2% had an open diagnosis and 2.2% showed no disease. Diagnoses were broken down by LLS region (Figure 2).
The majority of samples submitted for elastase testing were from Merino flocks (68.5%), and there was no difference in the proportion of Merino flocks diagnosed with either benign or virulent footrot. Flock numbers for other breeds were too small to analyse. The mob age did not have a significant effect on the proportion of flocks with virulent footrot. Likewise, recent treatment of flocks did not significantly affect the probability of virulent footrot diagnosis.
Virulent flock diagnosis appears to be associated with a higher proportion of score 4 and 5 foot lesions per mob (Figure 3A). However, no clear relationship between higher environmental scores and more severe disease was observed (Figure 3B).
Logistic regression models were fitted to the data to identify the minimum number of disease predictors that best discriminate between benign and virulent footrot diagnosis. The probability of virulent diagnosis increased significantly with an increasing percentage of score 4 and 5 foot lesions per flock, an increasing percentage of D. nodosus isolates elastase positive before 12 days, and an increased mean elastase rate. In contrast, the predictors environment score and the first day of elastase activity were positively associated with benign diagnosis, i.e. as the number of days increased before elastase activity was detected, the probability of benign footrot diagnosis increased. Increasing environmental scores (wet and warm conditions with good pasture growth) aid in the expression and transmission of virulent footrot, but by themselves can't cause virulent footrot. The virulent strain of D. nodosus needs to be present and active in the hoof to cause virulent footrot.
Virulent diagnosis was best predicted by combining the following 5 predictors into a model; % score 4 and 5 lesions (Log10), environmental score, mean elastase rate (Log10), the percentage of D. nodosus isolates elastase positive at 12 days and the first day that elastase activity was detected.
Many attempts have been made to develop a laboratory test that supports virulent footrot diagnosis by measuring the presence or expression of bacterial virulence genes. The elastase test measures the protease activity of D. nodosus in a specialised agar containing elastin particles. In this study, we evaluated three different ways to measure elastase activity from D. nodosus isolates; the mean elastase rate, the first day that any D. nodosus isolate from the mob cleared elastin, and the percentage of isolates that clear elastin before 12 days.
Each of these three elastase measures were good predictors of virulent diagnosis, but not perfect. However, combining these elastase activity measures with clinical disease (the % score 4 and 5 lesions) increased the probability of correctly predicting a virulent footrot diagnosis. It is possible that studies performed under different conditions (different sheep breeds, ages and environment) might highlight other factors important in the diagnosis of virulent footrot, and to that end we plan to continue monitoring the relationship between bacterial virulence (elastase activity) and virulent diagnosis in diagnostic submissions to the EMAI laboratory.
Future work is aimed at developing a more rapid and sensitive molecular test to measure the rate of elastase activity by quantifying the messenger ribonucleic acid concentration (mRNA) that dictates the production of the hoof degrading protease of D. nodosus. This molecular test could reduce testing time from six to approximately two weeks. The ultimate aim is to extract and quantify mRNA for the acidic protease gene aprV2 directly from hoof swabs to calculate the virulence profile of all D. nodosus isolates in an affected hoof, including virulent and lower-virulent isolates. In the meantime, the elastase enzyme activity test can continue to be used to support clinical diagnosis of footrot, but additional measures identified in this study will help in the interpretation of bacterial virulence.