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.