Culture medium

Culture of the bacterium is one of the key tests used in diagnosis of Johne’s disease. It can also be used to confirm an index case of the disease on a property.

In Australia, New Zealand, the USA and some other countries the BACTEC 460 system with modified BACTEC 12B medium (Becton Dickinson) has been the most commonly used liquid culture system. Therefore, the discontinuation of this culture medium in 2012 was a serious global issue. To overcome this problem we have successfully developed a new culture medium and now supply it to animal health diagnostic laboratories in Australia.

Culture bottles
The new culture medium

This medium can be used to culture both cattle and sheep strains of the organism and has several advantages over the commercial medium it replaces: it is safer for the operator and the environment as it is not radioactive, it is  half the cost of the commercial medium and requires less laboratory technician time as the cultures do not have to be monitored as frequently.

With this new culture medium, confirming the growth of the bug is required at the end of the standard incubation period of 12 weeks. This involved a time-consuming protocol that was not well-suited to processing large numbers of samples. We have recently been able to overcome this problem a new technology which allows for rapid processing of multiple samples. It is based on magnetic particles which bind to the DNA of the bug and a semi-automated procedure that prepares the DNA for PCR. A plate of 96 samples (including controls) can be processed at one time using this method.

Processing samples to detect growth of the mycobacterium
Processing samples to detect growth of the mycobacterium

This enhanced protocol follows on from collaborative work we conducted with researchers at NSW DPI (Elizabeth McArthur Agricultural Institute) on the HT-J rapid faecal DNA test, and demonstrates how our research is continuing to produce outcomes that are directly relevant to the Johne’s disease control program in Australia.

This paper describes development of the new culture medium:

Whittington RJ, Whittington AM, Waldron A, Begg DJ, de Silva K, Purdie AC and Plain KM (2013). Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium. Journal of Clinical Microbiology 51, 3993-4000.

October 2014