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The detection of animal derived DNA in stockfeed
Bovine spongiform encephalopathy (BSE) is a fatal degenerative disease affecting the central nervous system of cattle. The disease was first reported in the UK in 1986, with up to 100,000 cattle affected by 1993. There are several theories as to the origin of BSE and the mechanism by which it is spread. One theory is that it originated from a spontaneous mutation in a cow but a second theory is that it was transmitted from scrapie infected sheep meat in stockfeed. In the past it has been common practice to produce meat and bone meal (MBM) from the waste meat and skeleton of animal tissues that could not be used for human consumption and to incorporate the MBM into animal stockfeed. The practice of feeding MBM to cattle has been implicated in the lateral transmission of BSE through the cattle population (1). More than 80 cases of a variant of the fatal Creutzfeldt-Jakob disease (nvCJD), which affects humans, have been reported in the UK. This human disease has been linked etiologically to BSE (2).
In 1996, the World Health Organisation made a recommendation that ruminant protein should not be included in cattle feeds. This was implemented as a preventative measure to avoid the spread of BSE among cattle population and to minimize transmission of BSE from bovines to humans. Since 1988, a ban on addition of animal derived materials in cattle feeds has led to a decline in the incidence of BSE in Great Britain (3). With the recent increased incidence of BSE in Europe, a ban on the feeding of all animal-derived material is currently in place in Europe. This ban excludes tallow.
Australia currently has a BR Level 1 rating and is placed at very low risk group for BSE in cattle. However control measures, to ensure that bans on feeding ruminant derived materials to cattle and sheep are implemented, are considered a critical element in the maintenance of this status.
The major objective of this study was to develop a test that would detect the presence of vertebrate tissue in stockfeed and, in the first instance, to focus on developing a test that would detect the main animal species of interest, bovine and ovine. The Australian Government Analytical Laboratories (AGAL) has previously developed a universal test to detect DNA extracted from fresh vertebrate tissue using the polymerase chain reaction (PCR) (4). However, preliminary investigations indicated that this method has limited application for stockfeed where the meat material has undergone a rendering process and the DNA has been significantly degraded.
Following these initial studies, a new assay was designed, specifically, to detect and amplify a much shorter fragment of the cytochrome B (cytb) gene. This region of the cytb gene is highly conserved among several animal species including bovine, ovine, caprine and porcine. For this reason, it was predicted that such an assay would be suitable for the detection of several vertebrate species. The method was developed and validated for detection of rendered ovine or bovine material in stockfeed.
The specificity of the test for other animal species was examined by analysis of rendered meat from pig, chicken and fish and by analysis of DNA extracted from goat, horse and human tissue. The test showed strong reactivity with DNA extracted from caprine tissue and weak reactivity with rendered pork samples.
Samples of rendered chicken and fish were not detected nor did the test detect DNA extracted from either horse or human tissue. A range of stockfeed samples were spiked with either 0.5% rendered bovine or ovine tissue (n=17). Based on reporting criteria that were established in the validation process, more than 95% of these spiked samples would be reported as positive.
The PCR method developed and described herein can be used as part of the auditing process to monitor compliance with the Australian Government's legislation banning the addition of animal-derived tissue to stockfeed. This will not only assist in the protection of Australia’s meat industry from the threat of BSE transmission among the cattle population but will also support access of Australia’s beef and mutton to international markets.
Note: The practice of feeding MBM, the cattle feed made from the rendered remains of dead animals, to cattle has been implicated in the spread of BSE through the cattle population. However, a safe level of rendered meat in stockfeed that will exclude transmission of BSE has not been established. Presence of rendered bovine or ovine material in stockfeed below the Reporting Threshold established in this method does not exclude the possibility that BSE could be transmitted via that stockfeed.
This page was last updated on 10/11/2014
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