Vaccination of livestock against Johne’s Disease has been hindered by the lack of a diagnostic that can differentiate vaccinated from infected animals (DIVA test). Now a study presented at ICP 2022 has shown that the blood test Actiphage is able to distinguish between goats that are naturally infected and those vaccinated against Mycobacterium avium subspecies paratuberculosis (MAP), the pathogen that causes Johne’s disease, supporting the potential for a vaccination programme.
Most existing Johne’s tests rely on an animal’s immune response, which develops only as the disease progresses. These tests cannot be used to detect infection in vaccinated animals because if the vaccine has worked, they will automatically give a positive test result. In contrast, Actiphage detects live bacteria in a milk or blood sample and so is not affected by the vaccination status of the animal.
However, Actiphage was originally developed to detect MAP inside the white blood cells of cattle, so it requires optimising for goats, as the properties of blood from other species of animals can be quite different.
Professor Cath Rees of the University of Nottingham has been experimenting with three methods of sample preparation in order to optimise the technique for different host species and she presented the findings in a poster at ICP 2022 Dublin.
Three different techniques of sample preparation were used to separate out the white blood cells: Ficoll density gradients, which takes advantage of the density differences between white blood cells and other types of blood cell; differential lysis using Ammonium Chloride-Potassium (ACK), which lyses the red blood cells, leaving white blood cells intact; and enhanced sedimentation, which causes the red blood cells to separate out under gravity, leaving the white blood cells behind.
Professor Rees, who is also CSO of PBD Biotech, said: “We have now shown that Actiphage is compatible with a range of different methods for purifying white blood cells, demonstrating the versatility of our test and opening up a number of new applications.”
The study found that it was important to select the right method for the host species to optimise the process. Blood samples from domestic goats (caprine) were first tested using Ficoll, the standard Actiphage method used for cattle, which was optimised for caprine white blood recovery by using the ACK lysis method.
Once the blood cells are isolated, Actiphage is used to extract the DNA from MAP cells for identification using PCR. In cattle the primers P90 and P91 are used to bookend the target region on the DNA that needs to be copied. However, analysis showed the genomes of strains of MAP found in goats were different, so the primers were redesigned to improve sensitivity for MAP detection in goat blood.
The optimised Actiphage assay was 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.
Interestingly, in one set of vaccinated goats, Actiphage also 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; this will support the use of vaccines as part of MAP control programs.”
