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.
Congenic PVG.RT1u rats rapidly reject Aa class I-disparate kidney allografts from recombinant PVG R8 donors and we recently demonstrated that anti-class I MHC alloantibody plays a critical role in effecting acute rejection in this experimental model. In this article, we show that PVG.RT1u recipients can be rendered permanently and specifically tolerant to R8 kidney allografts by administration of four weekly donor-specific transfusions (DST) combined with a 7-day course of cyclosporine given with the first DST. Tolerance induction correlated with abrogation of a cytotoxic alloantibody response by thymus-independent, i.e., peripheral mechanisms; IgM and all IgG subclasses of anti-class I alloantibody were abolished. In contrast, nonrejecting kidney allografts in tolerant rats and rejecting grafts from unmodified recipients were similarly infiltrated by mononuclear cells, and intragraft transcripts for interleukin (IL)-2, interferon-gamma, and IL-13 were readily detected by reverse transcriptase polymerase chain reaction with no apparent quantitative difference between the two groups. Messenger RNA for IL-4 and IL-10 was present in rejecting grafts but barely detectable in grafts from tolerant animals. These results suggest that tolerance induction by DST and cyclosporine is, in this experimental model, associated with a selective impairment in humoral alloimmunity.
Bovine tuberculosis (bTB) is a disease of livestock with severe and worldwide economic, animal welfare and zoonotic consequences. Application of test-and-slaughter-based control polices reliant on tuberculin skin testing has been the mainstay of bTB control in cattle. However, little is known about the temporal development of the bovine tuberculin skin test response at the dermal sites of antigen injection. To fill this knowledge gap, we applied minimally-invasive sampling microneedles (SMNs) for intradermal sampling of interstitial fluid at the tuberculin skin test sites in Mycobacterium bovis BCG-vaccinated calves and determined the temporal dynamics of a panel of 15 cytokines and chemokines in situ and in the peripheral blood. The results reveal an orchestrated and coordinated cytokine and local chemokine response, identified IL-1RA as a potential soluble biomarker of a positive tuberculin skin response, and confirmed the utility of IFN-γ and IP-10 for bTB detection in blood-based assays. Together, the results highlight the utility of SMNs to identify novel biomarkers and provide mechanistic insights on the intradermal cytokine and chemokine responses associated with the tuberculin skin test in BCG-sensitized cattle.
Although France is officially declared free of bovine tuberculosis (TB), Mycobacterium bovis infection is still observed in several regions in cattle and wildlife, including badgers (Meles meles). In this context, vaccinating badgers should be considered as a promising strategy for the reduction in M. bovis transmission between badgers and other species, and cattle in particular. An oral vaccine consisting of live Bacille Calmette–Guérin (BCG) contained in bait is currently under assessment for badgers, for which testing bait deployment in the field and assessing bait uptake by badgers are required. This study aimed to evaluate the bait uptake by badgers and determine the main factors influencing uptake in a TB-infected area in Burgundy, north-eastern France. The baits were delivered at 15 different setts located in the vicinity of 13 pastures within a TB-infected area, which has been subject to intense badger culling over the last decade. Pre-baits followed by baits containing a biomarker (Rhodamine B; no BCG vaccine) were delivered down sett entrances in the spring (8 days of pre-baiting and 4 days of baiting) and summer (2 days of pre-baiting and 2 days of baiting) of 2018. The consumption of the marked baits was assessed by detecting fluorescence, produced by Rhodamine B, in hair collected in hair traps positioned at the setts and on the margins of the targeted pastures. Collected hairs were also genotyped to differentiate individuals using 24 microsatellites markers and one sex marker. Bait uptake was estimated as the proportion of badgers consuming baits marked by the biomarker over all the sampled animals (individual level), per badger social group, and per targeted pasture. We found a bait uptake of 52.4% (43 marked individuals of 82 genetically identified) at the individual level and a mean of 48.9 and 50.6% at the social group and pasture levels, respectively. The bait uptake was positively associated with the presence of cubs (social group level) and negatively influenced by the intensity of previous trapping (social group and pasture levels). This study is the first conducted in France on bait deployment in a badger population of intermediate density after several years of intensive culling. The results are expected to provide valuable information toward a realistic deployment of oral vaccine baits to control TB in badger populations.
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.
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