Animals Testing

Blood samples were collected in heparinized tubes the same day SICT was performed, transported at ambient temperature (22 ± 5°C, avoiding extreme temperatures) to the laboratories and processed within 8 h postcollection. Stimulation of whole blood was done with PPDB and avian purified protein derivative (PPDA) (Prionics AG, Schlieren, Switzerland). PPDB and PPDA were prediluted with phosphate buffer saline (PBS) to achieve a final assay concentration of 20 µg/ml. Pokeweed mitogen (PWM) at 5 µg/ml and NIL antigen PBS were used, respectively, to control the blood cells ability to produce gamma-IFN and to detect a non-specific gamma-IFN production. Whole blood cultures were performed with 24-well plates from 2006 to 2009 (1.5 ml of heparinized blood with 100 µl of antigen solution) and 96-well plates from 2009 to 2012 (250 µl of blood with 25 µl of antigen solution, 2 stimulation wells per antigen). Blood samples were evenly mixed before aliquoting. Plates were incubated at 37°C in a humidified atmosphere for 16–24 h, then centrifuged at 500 g for 10 min at room temperature. After incubation, approximately 100 µl (96-well plates) or 500 µl (24-well plates) of plasma were removed from above the sedimented red cells using a variable-volume pipette. The absence of significant effect of vessel geometry has been demonstrated by Schiller et al. (9). Plasma samples were tested using Bovigam^® (Prionics AG, Schlieren, Switzerland) in duplicate wells. The same analytical protocol was used in Camargue and Dordogne regions. Some animals were tested with the SICT using the official bovine PPD (Synbiotics, France).

PCR and mycobacterial culture were performed on tracheobronchial, retropharyngeal, and mediastinal lymph nodes presenting or not VL at the slaughter house. Lymph nodes were analyzed individually. Culture was performed according to the French ad hoc guideline, using solid Lowenstein and Coletsos agar after decontamination with 4% H2SO4 solution neutralized by adding a 6% NaOH solution. After mechanical lysis of tissue, DNA was extracted by using the QIAamp DNA mini kit (Qiagen) or by Magvet MV384 (Life Technologies) with a King Fisher KF96 automate, following the manufacturer’s instructions. PCR was performed with a commercial kit (LSI VetMAXTM Mycobacterium tuberculosis Complex PCR Kit 2 wells) targeting IS6110, which is present in all species of the M. tuberculosis complex (10): 5 μl of the extracted DNA was mixed with 20 µl of reaction mix and the reaction was carried out at 50°C for 2 min (1 cycle), followed by one cycle of 10 min at 95°C and 40 cycles of 15 s at 95°C and 1 min at 60°C. Results were interpreted following the manufacturer’s recommendations and by comparison with negative and positive controls. Thermolysates of bacterial isolates were confirmed as M. bovis by Luminex spoligotyping as described by Zhang et al. (11).