Immunoproteomic characterization of protein derivatives from Mycobacterium bovis, Mycobacterium avium (subspecies avium and paratuberculosis) and Corynebacterium pseudotuberculosis. Application in diagnosis
PhD Thesis defense by Jose Antonio Infantes Lorenzo at the VISAVET Centre of the Complutense University of Madrid
December 14th, 2018
Animal tuberculosis is a zoonotic infection caused by members of the Mycobacterium tuberculosis complex (MTBC), especially M. bovis and M. caprae. It represents an important health problem, in humans, domestic animals and wildlife. For this reason, in the European Union, cattle are subject to an eradication programme, based on " test-and slaughter" strategies, which implies the removal of animals that react positively to diagnostic tests. The official diagnostic tests are based on the cell immune response against the bovine tuberculin or PPDb. This is a purified protein derivative (PPD) prepared from M. bovis which has a complex composition and needs a careful titration. This is why, in this work, by means of studying the composition of this bovine tuberculin, we considered improving the diagnosis of the tuberculosis disease.
Firstly, we carried out the immunoproteomic characterization of PPDb using monoclonal antibodies (mAbs). We obtained four types of mAb that allowed us to identify five immunodominant proteins present in PPDb, which are MPB70, MPB83, HspX and ESAT-6 exclusives in MTBC, and meromycolate acyl carrier protein, also present in the PPDa, the purified protein derivative from M. avium subsp avium. The PPDb exclusive identified proteins are included in a multiprotein complex which is always obtained by immunopurification from the PPDb with the mAb SIM 377-18 and we named P22, because in the western blot analysis shows a major protein with electrophoretic mobility at 22 kDa. This mAb recognizes a common epitope in the MPB70 and MPB83 proteins. The results of this first study also indicate that when mice are inoculated with the PPDb, there is a highly exclusive immune response against MPB70/83. The major reactivity against these proteins could be due to their higher concentration in the P22 complex, their orientation and their possible micellar structure, which would benefit their processing by the immune system.
On a second study, we addressed the specificity-related problems by studying the proteins shared between M. bovis, M. avium subsp. avium, M. avium subsp. paratuberculosis and C. pseudotuberculosis, using mAbs obtained with the aforementioned bacteria or their products. We obtained a total of 21 mAbs that recognize the following common proteins: ModD protein, PPE family protein, Serine protease, Meromycolate acyl carrier protein, Diacylglycerol acyltransferase, GroEL and Enolase.
In addition to the above studies, a mass spectrometry analysis was conducted in order to make a theoretical approximation of the proteins responsible for the cross-reactivity between PPDb, PPDa and P22 complex. This study allowed us to 1.- Identify the constituent proteins of the three preparations as well as the proteins shared between them; 2.- Calculate the concentration of their constituent proteins; 3.- Calculate their antigenicity in a humoral immunity response; and 4.- Propose the use of the P22 protein complex in ELISA assays, allowing the arrest of specific antibodies against M. tuberculosis complex bacteria.
We also developed and evaluated new "multi-species" assays for ante-mortem and post-mortem diagnosis of tuberculosis in animals. As notable results, we found that the mAb SIM 377-18 has a great potential in immunohistochemistry studies testing tissues from TB infected animals, and that the P22 protein complex is more sensitive than the recombinant proteins MPB70 and MPB83 in the detection of antibodies against M. bovis and M. caprae.
Finally, we have developed and validated an indirect ELISA assay based on the protein complex P22, for the diagnosis of tuberculosis in different domestic animals (cattle, goats, sheep, pigs and llamas and alpaca herds) and wild species (deer, wild boar and badgers). This ELISA has a sensitivity between 63% and 100% and a specificity between 75% and 100%, depending on the species and country studied. Due to its excellent specificity in pigs and camelids, and having an acceptable one in cattle, sheep and badgers, this assay is a good option for the diagnosis of tuberculosis in animal herds. We therefore propose the serological testing as a surveillance technique to detect specific antibodies against the MTBC, especially in countries that are officially free of tuberculosis and animals which are not subject to the intradermal reaction test, showing higher specificities in these conditions. This adds to the existing advantages of these techniques which are simple, low costing and require minimum resources.
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