Introducing new animals to an existing group brings a risk of disease; however there are few validated screening tests for many exotic species. A new study in endangered Asiatic lions shows the benefit of using Actiphage®, a new diagnostic that detects live mycobacteria at a very early stage of infection, as part of a panel of TB screening tools.
Screening lions pre-movement
The study, reported in the Journal of Zoo and Wildlife Medicine, screened two imported Asiatic lions (Panthera leo persica) for Mycobacterium tuberculosis complex (MTBC), a genetically related group of Mycobacterium species that can cause tuberculosis in humans and other animals. Current knowledge of TB testing of big cats is mostly based on experiences from Africa where lions have eaten Cape buffalo infected by bovine TB.
Co-author Dr Ben Swift, Research Fellow in Antimicrobial Resistance and Director of Research and Development at PBD Biotech, has previous experience of mycobacterial infections in wild populations including bison in Canada and in support for zoos, such as Paignton, to help develop a rapid screening test for TB.
He says: “TB screening tools based on the immune system are difficult to deploy for many species, due to fundamental differences in species’ immune systems.
“Sharing all experiences when using experimental tests in a new species is an essential part of data gathering.”
Number of diagnostics used to elicit information
The study of Asiatic lions used the Comparative Intradermal Tuberculosis (TB) test, the feline interferon-c blood test (IGRA) and Actiphage, a novel bacteriophage assay.
Ben continues: “Tests that depend on an immune response such as the Comparative Intradermal TB test and the interferon-y assay can be sensitive when screening recently exposed animals. Using a control group of animals that are highly unlikely to have been exposed to M.bovis or M.tuberculosisincreases confidence in interpreting positive antemortem test results in animals of unknown status.
“However, it’s not known what the sensitivity level is for lions or how the intradermal tuberculin injection impacts the interferon response of this species. So, using experimental tests, such as Actiphage, can help substantiate initial test results from other widely used domestic animal-validated tests and potential identify the presence of a live pathogen.
“If novel tests are used more extensively, this would increase our knowledge of the value of Actiphage and hopefully contribute to the development of more robust tests for difficult to diagnose infections such as tuberculosis. This exploration is a good example of such an evaluation.”
As the skin test has not been fully validated for lions and would require two anaesthetic procedures, it was opted to inject into the eyelids, where inflammation could be easily observed, and to train the lions to allow further blood samples to be taken.
Eyelid used to confirm inflammation
Using the chart for interpreting inflammation of eyelids of non-human primates, the male lion was interpreted as ‘positive’ and the female ‘suspect’ to the Comparative Intradermal TB test. This was followed by the IGRA test, which showed a significant response over the negative control in both.
The blood was also tested with Actiphage, which showed the presence of live bacteria belonging to the MTBC in samples from both animals. Actiphage, the phage-PCR test commercialised by PBD Biotech, has been used to identify mycobacteria in over 30 species and is also used as an un-validated test within a disease management programme to successfully eradicate Bovine TB from cattle.
Actiphage uses bacteriophage to break open viable mycobacterial cells. The DNA released from these cells is then detected by PCR, thus only DNA from a live cell is detected. Bacteriophage have evolved to efficiently break open mycobacteria cells from within, making the phage-PCR assay extremely efficient at releasing mycobacterial DNA for molecular detection.
There is some evidence to demonstrate that mycobacteria can be found in the blood in animals and humans before the onset of clinical signs of infection. Actiphage has the potential to detect the early stage of infection before an immune response can be triggered.
Conclusions - exposure and confirmation of current infection
Combining the evidence, it was concluded that the animals had previously been exposed to the MTBC as shown by the Comparative Intradermal TB test and the IGRA; further testing with IGRA would be required to show an active infection.
However, the rapid Actiphage test revealed in one sample that the animals were currently carrying an infection.
TB is very difficult to treat and represents a risk in terms of cross-infection of other animals and potentially humans. By collating all the evidence available, together with a disease risk analysis that took into account the history of the animals and the likelihood of infection, the decision was made to exclude these animals from the collection.
This study also shows the animal welfare benefit of reducing repeat anaesthetic interventions by placing the Comparative Intradermal TB test in the upper eyelids of lions, after collection of the required blood samples. This plus animal training to allow further collection of blood samples from a conscious animal would greatly assist future screening and disease management.
Reference: Conservation challenges: the limitations of ante mortem tuberculosis testing in captive Asiatic lions (panthera leo persica) Authors: Swift, Benjamin M.C et al Journal of Zoo and Wildlife Medicine, 51(2): 426-432 URL: https://doi.org/10.1638/2019-0084