Tests for protease activity

There is an association between the degree of proteolytic activity expressed by isolates of D. nodosus and the capacity of these isolates to cause underrunning of hard horn in susceptible sheep under appropriate environmental conditions. Tests for protease activity include the elastase test, the gelatin gel test and the zymogram. 

All tests of actual proteolytic activity require the establishment of pure cultures of D. nodosus and thus take a considerable time to complete. The elastase test in particular requires up to 30 days to yield a negative result after inoculation of elastin agar plates with pure cultures of D. nodosus. However, results for the elastase test are provided on a continuous scale (number of culture days required to cause clearing of elastin) and thus allow classification of isolates into several categories (for example, <10 days = virulent, 11-21 days = intermediate, >21 days = benign). 

The literature concerning the correlation between phenotypic characteristics of D. nodosus such as protease activity and virulence is difficult to review because authors have used different methods or have published incomplete materials and methods, abbreviated data and the results of the same experiment in several publications (Whittingtonet al., 2016).  Furthermore, as a number of strains of D. nodosus of varying phenotype may be present in an affected foot it cannot be assumed that any given isolate is representative of those present in lesions or responsible for the lesions observed (Hill et al., 2010). Sample size is very important in diagnosis.

The need to provide laboratory tests in a climate of diminishing financial resources led to replacement of the elastase test with the gelatin gel test and required the re-definition of ovine footrot into two categories, benign and non-benign based on the capacity of the gelatin gel test to provide only a dichotomous classification of isolates. Artificial classification of the disease creates confusion for producers (Whittington et al., 2016). 

This problem was highlighted by discrepancies between field diagnoses and the gelatin gel test. In 2006, Cheetham et al. (2006) reported that 63% (233/370) of isolates collected from outbreaks of benign footrot produced heat-stable (virulent) or equivocal proteases, as determined by the gelatin gel test. There is also evidence of discrepancies between the gelatin gel test and the elastase test. In a comparative study in which 2851 D. nodosus isolates were subjected to both the elastase and gelatin gel tests, the level of agreement between the results of the two tests was slight (Dhungyel et al., 2013). 84% (857/1017) of isolates classified as benign by the elastase test were classified as virulent by the gelatin gel test. Given that both tests evaluate characteristics of the same protease, one or both tests are unreliable (Whittington et al., 2016).

Based on subsequent evaluation of these tests in 40 sheep flocks in Australia, false positive diagnosis of virulent footrot is likely to occur more often with the gelatin gel test than it is with the elastase test (McPherson et al., 2017).

Select a page from the Dropdown menu or click on one of these links to learn more about protease tests:

Protease – elastase test

Protease – gelatin gel test

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