Materials and methods
African lion (Panthera leo ) serum samples (n = 114) were obtained from serum banks from wild carnivore conservation projects in protected areas and recreational game parks in Zimbabwe. Each sample was allocated to high or low risk areas for anthrax (Figure 1, Table 1) based on a previous classification ( Chikerema, Murwira, Matope, & Pfukenyi, 2013). The samples were collected from lions immobilized for various reasons over a period of 20 years (1996 – 2016) and stored frozen at -20oC at the Wildlife Unit of the Department of Veterinary Services, Ministry of Lands, Agriculture and Rural Resettlement, Zimbabwe. The 114 samples represented 114 different individuals. Demographic and other details of the lions used in this study were not included as the main objective was limited to determination of seropositivity according to low and high-risk anthrax zones.
The sera samples were tested for antibodies to B. anthraciscapsule (PA) antigen as detailed in Mukarati et al. (2018). A conventional PA ELISA was used to analyze samples for specific immunoglobulins according to (Hahn, Alex, Czerny, Böhm, & Beyer, 2004) and modified by Ndumnego et al. (2013). For determination of the cut-off value for positive lion sera in the multi-species ELISA a similar approach depicted previously by Mukarati et al (2018) was adopted. Previously identified negative and positive control sera were sourced from a domestic cat presented for an unrelated condition at the Onderstepoort Veterinary Academic Hospital and from a vaccinated goat (Ndumnego, Koehler, Crafford, Beyer, & van Heerden, 2018) respectively. Each ELISA plate contained duplicate wells of the known negative and positive control sera. Also six blank wells containing only the blocking solution (skimmed milk powder) were provided for each plate and background OD values from these wells were subtracted from the test sera wells. Seropositivity estimates of anthrax with 95% confidence intervals were computed using Stata Version SE/11. The primary sampling strata were the recognized high and low anthrax risk zones. Wildlife management systems (protected areas vs. recreational game parks), represented the secondary strata while the individual animals were the sampling units. Data analysis was done in Stata Version SE/11 for Windows (Stata Corp., College Station, USA) at a 95% confidence interval, and differences between strata noted.
The presence of anthrax-specific antibodies in lion sera using the ELISA-PA protocol was validated using a functional toxin neutralization assay (TNA). For this purpose, 27 randomly selected sera samples comprising 17 positive and 10 negative for anti-PA antibodies on ELISA-PA were further tested with TNA. The in vitro TNA was conducted using J774A.1 mouse macrophage cell line (ECACC cat no 91051511) with modifications as described (Hering et al., 2004; Ndumnego et al., 2018). The cell line was added at 100,000 cells in Dulbecco’s modified eagle medium with 10% foetal bovine serum per well in flat-bottomed 96-well culture plates (Corning ™, Germany) and allowed to incubate at 37oC in 5% CO2 for 24 hours. Test sera were duplicated and diluted two-fold starting with a 1:50 PA 500 ng/mL in lethal factor (LF) 400 ng/mL (List Biological Laboratories Inc., USA). The mixture was incubated for 1 hour at 37oC before transferring to the previously seeded cells and incubated for 3 hours at 37oC. Apart from the test sera added, each 96-well plate had blank wells dedicated to toxin (3 wells) and medium (2 wells) control while three wells were left blank. Each batch also contained a single dilution for the positive control obtained from a lion which was vaccinated with the Sterne live spore vaccine (Onderstepoort Biological Products, South Africa) to assess uniformity and duplicability of the assay. A 25 µL of 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Invitrogen, USA) was then added per well and the chromogenic mixture allowed to develop in darkness in a CO2 incubator at 37oC for 2 hours. Finally, a mixture of 90% isopropyl alcohol, 0.5% SDS and 25 mM HCl was added to all the wells to lyse the cells and the mixture further agitated by gentle pipetting several times before allowing it to rest for 5 minutes. The plates were then read at an absorbance of 570nm in a Biotek power wave XS2 reader. The neutralization was calculated as:
Neutralization titre ( \(NT50)=\frac{\text{Sample}-\text{Toxin}\ \text{control}}{(\text{Medium}\ \text{control}-\text{Toxin}\ \text{control}}\ X\ 100\)
The Gen5 analysis software (Biotek Instruments, USA) was used to obtain the neutralization titres (NT50) which were conveyed as the corresponding value of the maximum dilution of the serum at which the antibodies protected 50% of the macrophages (NT50). Sera that could not protect up to 50% of the cells were recorded as having no anti-toxin neutralizing antibodies.
For the assay validation, the paired results for the 27 sera samples for ELISA and TNA assays were captured in SPSS, cross tabulated in a 2x2 contingency table and analysed for agreement using the McNemar’s χ2 test (Adedokun & Burgess, 2012) at 95% confidence interval.
The geographic location data for sampled lions was used to create spatial distribution maps for seropositive animals across the country and in Hwange National Park. The maps were created in a GIS environment using ArcMap 10.1 (cite ArcMap 10.1) using location points for seropositive lions which were overlaid on shapefiles of administrative districts and wildlife conservation areas of Zimbabwe downloaded from www.diva-gis.org/gdata. Location points of positive cases in HNP were also overlaid on shapefile of the park to show the distribution.