The Australian National Quality

Assurance Program

The National Footrot reference Laboratory (NFRL) is situated in the Albany Animal Health laboratories, its primary function is to provide a diagnostic service to the States Footrot Eradication Program.  In addition, associated research on footrot and DNA typing of Dichelobacter nodosus (D.nodosus) is conducted by officers at South Perth office.

The NFRL runs the quality assurance program for the “gelatin gel” test, the primary virulence test for D.nodosus on behalf of ANQAP.

The Albany diagnostic laboratory is staffed by Mike Palmer (supervisor) and Eckard Klien (technologist).

Two forms of Ovine footrot are recognised benign and virulent, flocks infected with the virulent type may experience significant production losses if the disease is uncontrolled.  Both forms of the disease are caused by the bacterium Dichelobacter nodosus (D. nodosus), however some strains posses virulence factors which allow them to produce the more severe virulent disease. All strains of D. nodosus are strongly proteolytic, however the protease enzymes produced by the virulent strains are considerably more resistant to heating than those of the benign types. The “gelatin gel” test was developed to measure the heat stability of protease enzymes, and is accepted as the most reliable laboratory test presently available to identify virulent strains of D. nodosus.

 

D. nodosus strains are grown for 2 days in a broth culture to produce the protease enzymes used in the test, these enzymes are particularly active and capable of digesting a wide range of proteins. In the gelatin gel test, protease activity is measured by inoculating the broths into wells in an agarose gel containing the protein gelatin, after incubation at 370C the gel is treated to precipitate the remaining protein. The diameter of the resulting clear zones of proteolysis is used to measure the protease activity. Using this gel diffusion method we compare the protease activity of the unheated (protease containing) broth, with the same broth which has been heated at 680C. Isolates are classified as positive if 10% or more of the protease remains active after heating for 8 minutes, negative if less than 4% remains, and equivocal if >4% and <10%.

 

Gelatin gel positive isolates are considered potentially Virulent, and in Western Australia, where a footrot eradication scheme is in place, farms with sheep infected with this type are quarantined. Gelatin gel negative isolates are generally considered benign.

Isolates of D. nodosus, which give Equivocal results in the Gelatin gel test, are found occasionally and are considered virulent

In Western Australia the gelatin gel test successfully classifies the majority of the D. nodosus isolated, however occasional anomalies occur. For this reason, the NFRL performs a second test on all isolates, in which the protease enzymes they produce are separated, using polyacrylamide gel electrophoresis, to produce a zymogram. Gelatin gel positive isolates may be divided into 3 zymogram types, S1, S2 and S4, there are eight types, U1 to U8, which are gelatin gel negative, only type S3 is gelatin gel equivocal.

Some gelatin gel negative D. nodosus, classified as zymogram type U5, have been found to cause quite severe lesions, knowing the zymogram alerts us to that possibility, and the flock concerned can be investigated further. Similarly, an isolate giving an equivocal gelatin gel test although strongly suggestive of an S3 type D. nodosus requires a zymogram test to confirm this.

The NFRL provides a zymogram testing service to laboratories outside Western Australia

The NFRL conducts two rounds of Gelatin Gel QA tests each year, in October, and in March, laboratories in New South Wales, Victoria, South Australia, Tasmania, and the NFRL itself participate. Five D. nodosus isolates selected from the National Footrot culture collection are sent to each of the laboratories, which are requested to culture and gelatin gel test them. Laboratories then report the percent remaining protease after heating for each of the unknown isolates and the positive and negative controls, to the NFRL. 

The mean of the “percent remaining protease” results for each isolate is calculated, and laboratories reporting results outside the “acceptable variation range” are required to do re-tests.There is generally a high level of agreement between laboratories for tests on the common benign and virulent strains of D. nodosus.

Content by: ANQAP
Updated: 18th November 2009
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