Development of recombinant BCG vaccine and complementary diagnostics for TB control in cattle.

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Bovine tuberculosis (TB) caused by Mycobacterium bovis remains a major problem in both the developed world and in developing countries such as India. As well as being a cause of huge economic loss in livestock farming, M. bovis infection can be spread to humans from infected cattle by aerosol or by consumption of contaminated dairy products to cause zoonotic tuberculosis. Control of bovine TB in the west is by detection and subsequent killing of infected animals. Detection of infection is primarily by the tuberculin skin test (PPD) in which a preparation of crude denatured antigen from the tubercle bacillus (tuberculin) is injected under the skin of the animal and is then examined several days later, by which time infected animals have generated a strong visible inflammatory reaction at the site of injection. The test is cheap and effective but it suffers from a major drawback: animals that have been vaccinated with the standard TB vaccine, BCG (an attenuated strain of the bovine TB bacillus Mycobacterium bovis), report positive in the test. This cross-reactivity means that the PPD test cannot be used to diagnose TB in vaccinated animals. So, in order to retain the PPD test, BCG vaccination is not used to control bovine tuberculosis, despite its established efficacy in cattle. An additional problem for any diagnosis and killing control strategy in India is that it is culturally unacceptable to cull infected cattle. The same problem of PPD cross-reactivity with BCG besets programmes for control of TB in humans and is the reason why BCG vaccination is not used in several countries, most notably the USA. However, new diagnostic tests have recently been developed that can distinguish between BCG vaccination and natural infection with the TB bacillus. These tests are based on immunogenic proteins whose genes were spontaneously lost from the genome of M. bovis during the development into the BCG vaccine and so can be used to Differentiate between natural Infection with M. bovis and VAccination with BCG (DIVA antigens). These tests, applied as blood-based interferon-gamma release assays (IGRA), are now widely used to diagnose TB in humans; but they are not as sensitive as PPD for detecting M. bovis infection in cattle. An additional drawback is that the tests are laboratory-based requiring blood samples to be taken from infected animals, transported to the laboratory within a tight timeline and under temperature controlled transport conditions, the test performed and result reported back to the field. This makes current IGRA DIVA tests expensive and inappropriate for countries, such as India, with limited technological and scientific infrastructure. However, over recent years significant progress has been made in applying these DIVA antigens in skin tests. These studies demonstrated that DIVA skin testing constitutes a realistic and practical alternative to IGRA tests with sensitivities approaching, but not yet reaching, those of PPD. Importantly, their specificity, when applied in skin tests, appears to be superior compared to their use in IGRA (>99 %) Therefore, these studies provided proof of principle that a much more cost-effective control strategy for bovine tuberculosis, particularly in India, would be to implement a combination of a cheap and effective vaccine (such as BCG) together with a cheap and effective skin test diagnostic reagent to identify and segregate infected animals from the rest of the herd. It is the aim of this project to develop such a system. The project will have two complementary components. First we will delete genes encoding additional dispensable antigens from the current BCG vaccine, effectively convert non-DIVA antigens present in both in M. bovis and BCG into DIVA antigens present in M. bovis but absent from BCG. In parallel, we will develop a standardized skin test reagent containing a cocktail of both current and new DIVA antigens (those removed from the new BCG vaccine).