A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne’s Disease

Abstract

Tuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

Utility of the Intradermal Skin Test in a Test-and-Cull Approach to Control Bovine Tuberculosis: A Pilot Study in Ethiopia

“…It is important to acknowledge here that bacteriological culture and confirmation of reactor animals could not be performed and is a limitation of the study. While the number of animals positive per SCT was high, it is also important to note the relative low specificity of this test, in particular in the context of regions with high environmental bacteria such as Ethiopia, further stressing the need for more accurate and specific defined diagnostic reagents ( 8 , 10 , 16 ). The increase in the number of reactor animals due to the interruption in routine testing between June 2018 and May 2021 indicates that expenses involved in the control of bTB are anything but short-term in nature.…”

“…The CCT test was performed following the OIE guidelines ( 3 , 10 ). Bovine PPD (3000IU/dose) and avian PPD (2500IU/dose) were administered intradermally with the bevel edge of needle faced outwards (0.1 ml final volume; Prionics/ThermoFisher Scientific, Lelystad B.V., The Netherlands).…”

Peripheral IFN-ɣ Production after Blood Stimulation with Different Mycobacterial Antigens in Goats Vaccinated against Paratuberculosis

“…These studies have been performed in some experimental kids or calves infected with M. bovis [ 6 , 8 , 12 , 13 , 14 ] and Map [ 15 ], but they have been barely assessed in relation to paratuberculosis vaccination, only being investigated in a limited number of goats or cattle [ 6 , 13 , 14 , 15 , 16 , 17 ] under experimental conditions rather than in field or experimental studies with Map-infected or vaccinated animals for longer time and at different age range.…”

Predicting Antigenic Peptides from Rocio Virus NS1 Protein for Immunodiagnostic Testing Using Immunoinformatics and Molecular Dynamics Simulation

“…However, to our knowledge, no diagnostic tests exist for ROCV. Synthetic peptides have recently emerged as novel targets for efficient serological diagnosis of infectious viral, bacterial, and parasitic diseases [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Such molecules could be applied in rapid diagnostic tests or other immunodiagnostic platforms such as ELISA to diagnose acute ROCV infection, offering lower production costs, higher specificity and reproducibility with no variation between batches, and larger-scale production compared to diagnosis based on whole antigens [ 30 ].…”