CASE NOTES

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Evaluation of the new rapid RNA test for Dichelobacter nodosus virulence: the promising and disappointing

Alison Collins, NSW DPI, EMAI

Posted Flock and Herd August 2025

INTRODUCTION

Virulent footrot is a notifiable disease in NSW due to its infectious nature, rapid transmission under optimal conditions and the associated animal welfare issues and production losses. Diagnosing virulent footrot is primarily based on clinical signs, scoring the prevalence and severity of hoof lesions (underrunning) in flock inspections. However, disease expression may be masked by recent treatments, sheep genetics or hot and dry environmental conditions, so quantifying the virulence of the causative agent, Dichelobacter nodosus, has been used to show the potential for the bacteria to cause virulent footrot under optimal conditions. The virulence of D. nodosus is currently quantified by the elastase test, which measures the production of the thermostable acidic protease enzyme responsible for severe lesions and degrading the hoof. Several Australian studies have reported the predictive value of the elastase test in diagnosing virulent footrot at the flock level (Collins et al., 2023; Dhungyel et al., 2013; McPherson et al., 2017). These studies demonstrated that elastase activity in D. nodosus before 12 days was a good predictor of the ability of D. nodosus to cause virulent footrot in NSW flocks, given susceptible sheep and good spread conditions.

A more rapid method to quantify D. nodosus virulence would be to measure the concentration of messenger RNA (mRNA) that translates or synthesises the acidic protease enzyme from an mRNA template (aprV2 RNA). Total RNA is extracted from D. nodosus, then the aprV2 RNA is reverse transcribed to DNA, which can be quantified in an PCR assay. The relative expression of aprV2 RNA copies in each sample is normalised against reference RNAs whose expression isn't affected by environmental conditions (Ganger et al., 2017).

Accurate quantification of virulent RNA transcripts in pathogens depends upon good preservation of RNA in samples, efficient RNA extraction and on the selection of appropriate bacterial reference genes for data normalisation between samples. Once optimised, virulence gene expression and regulation can be studied under varying stages of clinical disease to estimate the impact of virulence gene expression on clinical disease outcomes.

The first objective of this project was therefore to develop a test to quantify the expression of D. nodosus virulence (aprV2 RNA) from purified cultures or directly from foot swabs collected from footrot-affected sheep. The second objective was to validate the aprV2 RNA test against the elastase test and finally to evaluate the predictive value of the aprV2 RNA test in diagnosing virulent footrot.

METHODS

1. Correlation of elastase activity and the concentration of aprV2 RNA in cultured Dichelobacter nodosus isolates

In sample set #1, a total of 206 archived D. nodosus isolates with known elastase activity from 60 NSW sheep flocks were revived on hoof agar and tested for aprV2 RNA concentration. Flocks were sampled from the Central Tablelands LLS (n = 10), Central West LLS (n = 10), Murray LLS (n = 9), Northern Tablelands LLS (n = 1), Riverina LLS (n = 15) and the South East LLS (n = 15). Thirty-one flocks were diagnosed with clinically virulent footrot and 29 with benign footrot.

The significance of correlations between elastase activity and aprV2 RNA concentration were tested by Spearman's rank correlation coefficients in Genstat V23.

2. Correlation of elastase activity and the concentration of aprV2 RNA in foot swabs

In sample set #2, a total of 268 duplicate foot swabs from 65 flocks (53 in NSW and 12 in Victoria) were collected for quantification of elastase activity and aprV2 RNA. Flocks were sampled in NSW between December 2022 and February 2025 from the Central Tablelands LLS (n = 7), Central West LLS (n = 20), Murray LLS (n = 12), Northern Tablelands LLS (n = 2), Riverina LLS (n = 7), North West LLS (n = 3) and the South East LLS (n = 2). Twenty-four of the NSW flocks were diagnosed with clinically virulent footrot and 25 with benign footrot, while an open diagnosis remained for four flocks. Victorian flocks were predominantly diagnosed with benign (n = 6) or intermediate (n = 4) footrot, with only two flocks diagnosed with virulent footrot. Correlations between elastase activity and aprV2 RNA concentration were determined by Spearman's rank correlation coefficients at the individual swab level.

3. Correlation of mean elastase activity and mean concentration of aprV2 RNA at the flock level and modelling both virulence tests as predictors for virulent footrot diagnosis

Using the same swabs from the above 53 NSW flocks in sample set #2, the significance of flock-level correlations between mean aprV2 RNA concentration and the proportion of D. nodosus isolates elastase positive by 12 days was determined by Spearman's rank correlation.

Logistic regression was used to identify the best predictors for virulent footrot diagnosis (binary response: virulent footrot = 0 and benign footrot diagnosis = 1). Predictors included the percent of sheep with severe hoof lesions (score ≥ 4), the percent of lame sheep, the total environmental score (moisture + pasture + minimum overnight temperature), breed of sheep, recent treatment, relative aprV2 RNA concentration, mean elastase rate, percent of isolates elastase positive at 12 days and the first day that any isolate was elastase positive. A range of models were examined to find the smallest number of predictors that best explained the factors important in the flock diagnosis of virulent footrot. The first model included elastase activity and the second included aprV2 RNA concentration. Potentially significant predictors were included in the model in a stepwise process.

RESULTS

1. Correlation of elastase activity and the concentration of aprV2 RNA in cultured Dichelobacter nodosus isolates

A significant negative correlation was observed between the relative aprV2 RNA concentration and the first day that isolates were elastase positive (r = -0.263, p < 0.001), i.e. aprV2 RNA concentration was higher in isolates with earlier elastase activity. Significant positive correlations (p < 0.001) were also observed between aprV2 RNA concentration and elastase clearance rate (r = 0.348), with higher aprV2 RNA concentration in cultures with rapid rates of elastin clearing (diameter of elastin cleared from around the culture over 21 days). Higher aprV2 RNA concentrations were also significantly correlated with elastase activity before 12 days (r =0.469, p < 0.001).

The majority of isolates elastase positive before 12 days were from flocks diagnosed with virulent footrot and conversely most isolates with late elastase activity were from flocks diagnosed with benign footrot (Figure 1).

The relative concentration of aprV2 RNA was highly variable (ranging from 5 to 150) in cultured isolates with early elastase activity (before 12 days). As expected, the highest aprV2 RNA concentration was seen in D. nodosus with early elastase activity and declined with later elastase activity. Benign isolates with elastase activity at about 28 days expressed predominantly lower aprV2 RNA concentrations (below 40) except for one isolate (circled in red in Figure 1). Unexpectedly, relatively low aprV2 concentrations were also observed in D. nodosus with early elastase activity (circled in blue, Figure 1) from flocks diagnosed with virulent footrot.

Figure 1. Negative correlation between normalised (relative) aprV2 RNA concentration and the first day that D. nodosus isolates are elastase positive from flocks diagnosed with benign (red cross) and virulent footrot (black cross). Exceptions to the negative correlation included one isolate from a benign flock with moderately high aprV2 concentration at 28 days (circled in red) and isolates from virulent flocks with early elastase activity (circled in blue), but low aprV2 RNA concentrations.

2. Correlation of elastase activity and the concentration of aprV2 RNA in foot swabs

Contrary to expectations, when aprV2 concentration was quantified directly from foot swabs, a significant positive correlation was observed between aprV2 RNA concentration and the first day that isolates were elastase positive (r = 0.249, p = 0.009), i.e. aprV2 RNA concentration was lower in isolates with earlier elastase activity. Only a small set of swabs with early elastase activity had elevated aprV2 RNA concentrations (circled in blue, Figure 2), as expected. However, a significant number of swabs with late elastase activity (28 and 56 days) also had elevated aprV2 RNA concentrations (red circles in Figure 2), which was not expected.

Figure 2. Association between relative aprV2 RNA concentration and the first day that D. nodosus was elastase positive on 268 swabs from 53 NSW and 12 Victorian flocks, showing that swabs with high aprV2 RNA concentration were associated with D. nodosus isolates with early elastase activity (blue circle), but also with late elastase activity (red circles).

3. Correlation of mean elastase activity and mean concentration of aprV2 RNA at the flock level and modelling of both virulence tests as predictors for virulent footrot diagnosis

Only 42 of the 53 flocks had data for all our predictive parameters and could therefore be included in the correlation and logistic regression analysis. No significant correlations were observed between the relative aprV2 RNA concentration and the percent of isolates per flock with elastase activity before 12 days (p = 0.884), the mean elastase rate (p = 0.480) per flock and the first day that isolates were elastase positive (p = 0.497). However, relative aprV2 RNA concentration was significantly associated with clinical diagnosis in the same flocks (p = 0.022). Flocks with a virulent footrot diagnosis showed higher mean aprV2 RNA concentrations than flocks diagnosed with benign footrot (Figure 3).As demonstrated previously (Figure 1 and 2), some flocks diagnosed with benign footrot have swabs containing D. nodosus that express high levels of aprV2 RNA (Figure 3, flocks circled in red).

Figure 3. Dot histogram of relative aprV2 RNA concentration in 42 NSW flocks diagnosed with either virulent (0) or benign footrot (1)

The first logistic regression model investigated the value of including elastase activity of D. nodosus to other more significant predictors such as clinical signs (% sheep with score 4 and 5 lesions) to increase the predictive model for virulent footrot diagnosis. The model of best fit for predicting virulent footrot diagnosis included "the percentage of sheep with score 4 and 5 lesions", "the percentage of elastase-positive D. nodosus isolates at 12 days" and the total environmental score.

When the relative aprV2 RNA concentration was added to the predictive model for virulent footrot diagnosis, the best predictors included "the percentage of sheep with score 4 and 5 lesions" and the total environmental score. Including the relative aprV2 RNA concentration to the model did not increase the predictive capacity of the model.

DISCUSSION

The concentration of aprV2 RNA was a good measure of D. nodosus virulence in that it correlated significantly with elastase activity. Quantifying the RNA concentration of aprV2 provided a good estimate of the amount of acidic protease enzyme that D. nodosus produces to degrade hooves. The quantitative RT-PCR assay can be performed on 48-96 isolates within a 3-day period, whereas the elastase test requires up to 28 days to get a result. However, in a small number of isolates, high concentrations of aprV2 RNA were observed in D. nodosus with low acidic protease activity (late elastase activity). This finding may suggest that other factors in the host or bacteria are capable of suppressing the translation or synthesis of acidic protease enzyme from the mRNA template of aprV2. Genes often have multiple regulators or inhibitors, so further studies are needed to identify what factors may inhibit protease production in D. nodosus expressing high levels of aprV2 RNA.

The isolation and purification of D. nodosus from foot swabs of the five worst-affected sheep per flock only tests virulence in 10 isolates from the potentially thousands of D. nodosus found in every hoof swab. This study investigated whether the virulence of all D. nodosus in a swab could be measured by reverse transcription PCR of the aprV2 RNA extracted directly from each swab. While it was possible to calculate the relative concentration of aprV2 RNA extracted directly from foot swabs, this value did not correlate with elastase activity in D. nodosus cultured from duplicate swabs. Poor correlations were also observed at the flock level between the mean aprV2 RNA concentration and the mean elastase activity.

This study reinforced the association between early elastase activity (before 12 days) and diagnosing virulent footrot in flocks. The top predictor for diagnosing virulent footrot was the percentage of sheep with severe underrunning (score 4 and 5 lesions), but including early elastase activity and the total environment score led to an increased 'goodness of fit' for the modelled data. Although relative aprV2 RNA concentration in foot swabs was significantly associated with clinical diagnosis of footrot at the flock level, the relative aprV2 RNA concentration was not a significant predictor for virulent footrot diagnosis. The expression of virulent footrot and underrruning of the hoof depends on the presence of virulent D. nodosus, as well as a combination of host susceptibility and optimal environmental conditions for disease spread.

While the results suggest that the relative concentration of aprV2 RNA extracted from hoof swabs is not a good predictor for virulent footrot diagnosis, it seems that the aprV2 RNA concentration from purified D. nodosus isolates is a good correlate for elastase activity and therefore D. nodosus virulence. Isolation and purification of D. nodosus from hoof swabs routinely takes two weeks, but the quantification of virulence from D. nodosus isolates can be determined more rapidly by aprV2 RT-PCR compared with the elastase test.

In the future, using the same D. nodosus isolates for virulence quantification and serogrouping may provide better management tools for the control or even eradication of virulent footrot. Once the serogroups of all virulent D. nodosus are identified in a flock, mono- or divalent vaccines can be used to eliminate the serogroups of D. nodosus causing virulent footrot (Dhungyel et al., 2013; Dhungyel et al., 2014).

ACKNOWLEDGEMENTS

The author would like to acknowledge the financial support of the McGarvie Smith Trust and the field support of many LLS vets in submitting clinical data, case histories and duplicate swabs to enable the comparison of the two D. nodosus virulence tests. Dr Om Dhungyel from the University of Sydney provided invaluable support in both clinical and technical aspects of this study. Technical support for the elastase testing was performed with great skill and patience by Karen Smith and the significant volume of media required was prepared by Amanda Azzopardi at EMAI.

REFERENCES

1. Collins A, Collins D, & Dhungyel O (2023) The virulence of Dichelobacter nodosus, measured by the elastase test, is an important predictor for virulent footrot diagnosis in New South Wales sheep flocks Australian Veterinary Journal 2023;Dec;101(12):522-530 doi.org Epub 2023 Oct 4. PMID: 37794558
2. Dhungyel OP, Hill AE, Dhand NK et al. (2013) Comparative study of the commonly used virulence tests for laboratory diagnosis of ovine footrot caused by Dichelobacter nodosus in Australia Veterinary Microbiology 2013;162:756-760
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4. Dhungyel O, Schiller N, Eppleston J, Lehmann D, Nilon P, Ewers A & Whittington R (2013) Outbreak-specific monovalent/bivalent vaccination to control and eradicate virulent ovine footrot>Vaccine 2013;31(13):1701-6 doi.org Epub 2013 Feb 5. PMID: 23395621
5. Ganger, NP, Dietz GD & Ewing SJ (2017) A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments BMC Bioinformatics 2017;18:1-11
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