Similar spontaneous release of IP-10 has previously been seen in cattle [17], although the mechanism for this phenomenon is not currently understood

Similar spontaneous release of IP-10 has previously been seen in cattle [17], although the mechanism for this phenomenon is not currently understood. (Fig.?1). Clinical disease often progresses to mortality resulting in changes in population ecology through group-level extinctions [3]. Open in a separate window Fig. 1 Meerkats with pathology typical of tuberculosis caused by and they are therefore unlikely to be suitable diagnostic antigens for this pathogen [2]. As an alternative, the commercially available Bovigam? PC-HP peptide pool, which contains ESAT-6 and CFP-10 peptides, includes antigens derived from the gene and an additional 3 genes, and could be useful for the detection of infection [11]. The purposes of this study were, therefore, to develop enzyme-linked immunosorbent assays (ELISAs) for the measurement of IFN- and IP-10 in meerkat plasma, evaluate the diagnostic utility of the PC-HP peptide pool in this species, and assess the test performance of an optimised diagnostic assay for infection in a population of free-living meerkats. Materials and methods Animals Captive meerkats with no known history of exposure served as an uninfected control group for Demethoxycurcumin the development of the cytokine release assay. These animals (were opportunistically sampled from a population of free-living meerkats, which have been habituated to the presence of researchers [12], from the Kuruman River Reserve, Northern Cape, South Africa (2658S, 2149E). Between September 2014 and February 2015, animals from this population were sampled from 12 social groups. Hereafter, animals were classified according to their presumed infection risk: low risk (category 1), comprising animals from social groups with no known history of TB; intermediate risk (category 2), comprising animals from social groups with either one or two known deaths due to TB in the preceding two years; and high risk (category 3), comprising meerkats from social groups with more than two deaths due to TB in the preceding two years. Permission to perform the study was obtained from the University of Stellenbosch Animal Ethics committee (Reference no. SU-ACUM14-00042). Permits to conduct animal research were obtained from the Northern Cape Department of Environment and Nature Conservation (Permit no. FAUNA 194/2014) and the National Department of Agriculture, Forestry and Fisheries (Reference no. 12/11/1/7/3). Blood collection and processing Meerkats were captured by hand and placed in cotton bags or caught in nets and physically restrained with towels prior to induction and maintenance of anaesthesia with isoflurane (Safeline Pharmaceuticals Rabbit Polyclonal to EGFR (phospho-Ser1071) Ltd, Roodepoort, SA) via facemask. Using a 25G needle and syringe, 2?ml blood was collected from the jugular vein and transferred to a heparinised blood tube (Greiner Bio-one, Kremsmnster, Austria). Animals were monitored after completion of the procedure and returned to their natural environment once fully recovered. Aliquots of whole blood (150?l) were transferred to each of three 1.5?ml microcentrifuge tubes containing, respectively, 15?l phosphate buffered saline (PBS); Demethoxycurcumin 15?l PC-HP peptide solution (Prionics AG, Schlieren, Switzerland), prepared according to the manufacturers instructions; and 15?l pokeweed mitogen (PWM) solution in PBS (final concentration 50?g/ml). Tubes were thoroughly mixed, incubated at 37?C for 24?hours and centrifuged at 1300 g for 6?minutes, after which the plasma was harvested and stored at – 80?C. ELISA protocol All ELISAs described below were performed according to the following protocol. Capture antibody (Table?1) in PBS (50?l) was used to coat wells of 96-well MaxiSorp polystyrene ELISA plates (Thermo Fisher Scientific, Massachusetts, USA) which were incubated at 4?C overnight. All subsequent steps were performed at room temperature. Plates were washed three times with wash buffer (WB) consisting of 0.05% Tween-20 (Sigma-Aldrich, Missouri, USA) in PBS and blocked with 200?l/well blocking buffer (BB) comprising Demethoxycurcumin WB with 0.1% bovine serum albumin (Roche, Basel, Switzerland). After 1?h incubation, plates were washed again. For IFN-? assays, 25?l of each plasma sample was incubated with 25?l BB and for IP-10 assays, 12.5?l of each plasma sample with 37.5?l BB. After 2?h, plates were washed and incubated for 1?h with 50?l/well biotinylated detection antibody (Table?1) diluted in BB..