WANTED: A Rapid, Sensitive and Accurate Diagnostic Test to Aid Control of Johne’s Disease in Dairy Herds

WANTED: A Rapid, Sensitive and Accurate Diagnostic Test to Aid Control of Johne’s Disease in Dairy Herds

Irene R. Grant, Institute for Global Food Security and School of Biological Sciences, Queen’s University Belfast.

Johne’s disease (or Paratuberculosis) is an endemic disease of domesticated ruminant animals such as cattle, sheep and goats that occurs worldwide. The disease is caused by a bacterium called Mycobacterium avium subsp. paratuberculosis (Map), which is a relative of the bacterium that causes bovine Tuberculosis, but it is even slower growing. Young calves become infected by Map early in life, but it typically takes 2-5 years for the first clinical signs of Johne’s disease to appear and hence for a farmer to become aware of Map infection in their herd. However, whilst animals are asymptomatic, Map infected animals will be excreting live bacteria in their faeces and milk, and spreading infection around the farm environment, leading to transmission of the disease to herdmates, or even beyond the infected farm if cattle are sold into other herds.

Many countries have national Johne’s disease control programmes in place. Generally these entail a risk assessment being carried out at farm level by a veterinarian in order to highlight risky farm management practices that could potentially aid Map transmission. Alongside this, all or a sub-set of animals within a herd are tested for Map, to identify infected animals which may need to be removed from the herd. The diagnostic tests employed are Enzyme-linked immunosorbent assays (ELISA) for screening blood or milk for evidence of antibodies against Map, and then usually a follow-up faecal qPCR to detect Map DNA or culture to grow live Map organisms from bovine faeces on ELISA positive animals to confirm infection. Whilst the specificity of these diagnostic tests (i.e. their ability to detect only Map) is high, the sensitivity of the tests (i.e. their ability to detect Map infection if present) is rather variable, and actually lacking in the case of ELISA tests which are widely relied upon. What this means is that only the most Map infected animals are being picked up by control programmes, rather than all Map infected animals within a herd.

For many years, my research group at Queen’s University Belfast has been endeavoring to develop novel detection methods for Map, specifically living/viable Map cells, which could offer an improvement over the existing tests employed for the diagnosis of Johne’s disease in farm animals. Our latest test for viable Map, termed the phagomagnetic separation (PhMS)-qPCR assay, is a rapid, simple, specific and sensitive test for viable Map1. It uses Mycobacterium-specific phages coated onto small magnetic beads to capture and concentrate Map cells from a veterinary specimen.

Then the phages proceed to infect and reproduce within viable Map cells to the point of bursting them after, around 3-4 hours incubation, to release Map DNA that is detected by a Map-specific qPCR. Our PhMS-qPCR assay provides results for viable Map within one working day (6-8 h), in contrast to culture, the only other test capable of differentiating between viable and dead Map cells, which takes several weeks to yield results. The new PhMS-qPCR assay is applicable for milk, faeces or blood testing, although the bulk of development and validation of the test has been done with milk.

We recently demonstrated the new test’s utility as a screening test to detect viable Map in bulk tank milk at farm level, and subsequently, to test milk from individual animals on four farms that had the highest levels of Map in their bulk tank milk in order to highlight which particular animals were shedding viable Map in their milk2. When PhMS-qPCR and milk-ELISA results for the same milk samples were compared, it was clear that the PhMS-qPCR assay is a more sensitive diagnostic test that would be able to detect more Map-infected animals within a dairy herd and so aid achievement of control of Johne’s disease faster. Queen’s University Belfast is currently seeking to commercialise the new phage-based assay for viable Map, in order to make it available to Johne’s diagnostic laboratories as soon as possible.

References

Foddai, A.C.G. and Grant, I.R. (2020) A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk. Applied Microbiology and Biotechnology. 104, 21, p. 9399-9412
Foddai, A., Watson, G., McAloon, C. and Grant, I. R. (2021) Phagomagnetic separation-qPCR: a rapid, sensitive and specific surveillance tool for viable Mycobacterium avium subsp. paratuberculosis in bulk tank and individual cows’ milk. Journal of Dairy Science, in press, accepted 30 December 2020