Johne’s Disease is a wasting disease of ruminants that is caused by Mycobacterium avium subspecies paratuberculosis or MAP. MAP has also been linked to Crohn’s Disease in humans but the connection is not conclusive.
Actiphage is a test for MAP that can detect live bacteria in a sample of white blood cells or milk. It was originally developed to detect MAP inside the white blood cells of cattle, but as the properties of blood from other species of animals can be quite different, sample preparation needs to be optimised for the host.
In a poster presented at ICP 2022, Professor Cath Rees of the University of Nottingham described experiments conducted to optimise Actiphage for use with blood samples from different host species.
This is the first time Actiphage has been used to detect MAP in a human blood sample and also to identify a live infection in a vaccinated goat, supporting its use as a test to ‘Differentiate Vaccinated from Infected Animals’ (DIVA) test.
Actiphage extracts DNA from viable MAP cells
Work has now been carried out to show that the Actiphage assay is compatible with a range of different methods that can be used to purify white blood cells (described below).
Ficoll density gradients – the traditional method of purifying peripheral blood mononuclear cells (PBMCs) that takes advantage of the density differences between white blood cells and other elements found in the blood sample. Specifically this method recovers lymphocytes and monocytes as the “buffy coat layer” but granulocytes are removed along with the majority of the red blood cells. This method is the most laborious, but has been used to recover white blood cells for Actiphage testing from a wide range of animal species, including a number of exotic species, and also for humans
Differential lysis using Ammonium Chloride-Potassium (ACK) – this method specifically lyses just erythrocytes in whole blood as a result of osmotic stress caused by the uptake of ammonium chloride added to the sample and therefore the sample contains lymphocytes, monocytes and granulocytes. After the red blood cells have been lysed, the intact white cells are collected by centrifugation. This method has now been successfully used for cattle, deer, goat and human blood samples prior to the Actiphage assay.
Differential sedimentation – this method causes the red blood cells to stack together to form large complexes (“rouleaux”) that then rapidly fall to the bottom of the sample, leaving the nucleated white blood cells in the upper layer of the sample which can then be recovered using a pipette. Hetasep has been specifically developed for human blood and is compatible with the Actiphage assay, but this product will not achieve this separation for blood from all animals.
Professor Rees, who is also CSO of PBD Biotech, said “It has now been shown that all these methods can be successfully used for purifying white blood cells for use with the Actiphage assay, but the method chosen will depend on the species being tested. However these results demonstrate the versatility of our test and opens up a number of new applications”
Actiphage extracts DNA from viable cells
Actiphage uses bacteriophage as the lysing agent to extract DNA from viable mycobacterial cells ahead of identification with PCR. The robust nature of the mycobacteria means that physical or chemical lysis methods are inefficient. In contrast, the phage lysis has evolved to be highly efficient and this allows Actiphage to detect very low (less than 10 per 50ml) numbers of cells.
Actiphage detects MAP in human blood for first time
For human blood, Hetasep was found to be the best method to purify PBMCs as it is simpler than Ficoll and good agreement was found for test results for samples prepared using both methods.
Samples were obtained from patients with atypical symptoms that had been referred to a tuberculosis clinic. After samples were screened for the presence of Mycobacterium tuberculosis complex (MTBC) DNA, any negative samples (=5) were then screened for the presence of Mycobacterium avium complex (MAC) organisms by amplifying IS1311.
One sample gave a positive result for IS1311, and further analysis (PCR-REA) confirmed that the organism detected in the blood of this patient was a sheep strain of MAP. This is the first report of successful detection of MAP in human blood using the Actiphage assay.
Professor Rees says “This is a very exciting result, and opens the door for using Actiphage to screen for a range of non-tuberculosis mycobacterial diseases in human patients”
Blood samples from domestic goats (caprine) were initial tested using the standard Actiphage method (Ficoll) used for cattle. However better results were achieved using the ACK lysis method.
Detection of MAP was attempted using the well characterised P90 and P91 primers which target the IS900 element. Previously we found these to be very effective for detection of MAP in bovine blood samples. However when working with goat blood samples it was noticed that PCR sensitivity was poor, especially using a real-time PCR format.
Comparison with MAP genome sequences found a sequence mismatch at the 3’ end of the P90 primer was common in many of the non-cattle MAP strains. A redesigned P90 was designed to avoid this region and this improved PCR sensitivity for caprine samples
The Actiphage assay was then used to screen caprine blood samples of known ELISA status. 60% of ELISA-positive animals gave a positive Actiphage test result, confirming the infection status of these animals. In a set of animals vaccinated with a sub-unit vaccine, Actiphage detected viable MAP in the blood samples from one animal indicating that this animal was infected despite vaccination.
Professor Rees commented “This study provides evidence that Actiphage can also be used as a DIVA test, allowing us to differentiate between naturally infected and vaccinated animals, which will support the use of vaccines as part of MAP control programs.”