Discussion
The anthrax PA, in addition to the oedema and lethal factors (EF and
LF), make up the tripartite anthrax toxin complex (Schwartz 2009). These
anthrax toxins are encoded by the anthrax-specific virulence plasmid,
pXO1, with PA combining with LF to form the lethal toxin (LT). To date,
no cross reacting antigens to anthrax PA are known and available data
indicate anti-PA antibodies as being the most diagnostically stable
(Leppla, Robbins, Schneerson, & Shiloach, 2002; Turnbull et al., 1992).
However, PA is antigenic complex and can elicit protective and
non-protective antibodies. Abboud & Casadevall (2008) investigated the
relationship between PA structure and antigenicity but could only
conclude the genetic background of the host determines the relative
efficacy of the antitoxin response (Abboud & Casadevall, 2008) and ,
thus the toxin reutilizing antibodies were determine on a random sample
to verify the use of anti-PA antibodies in this study.
Both ELISA and toxin neutralization (TNA) assays can be used to detect
antibodies to PA antigen in sera of animals exposed to B.
anthracis . We report for the first time the dual existence of anthrax
PA-specific and lethal toxin neutralizing antibodies in naturally
exposed African lions. The TNA is a functional assay that measures the
neutralizing activity of sub-sets of PA-specific antibodies against the
cytotoxic effect of the lethal toxin complex formed by PA in association
with LF. Though technically demanding, TNA confirms previous exposure toB. anthracis in animals and is species-independent (Ngundi,
Meade, Lin, Tang, & Burns, 2010; Omland et al., 2008; Savransky et al.,
2017). On the other hand the ELISA assay detects antibodies to PA
antigen in sera of infected individuals and is less demanding but well
correlated with the TNA (Arciniega & DomĂnguez-Castillo, 2011;
Ndumnego, Crafford, Beyer, & van Heerden, 2013; Parreiras, Sirota,
Wagner, Menzies, & Arciniega, 2009). Consequently, the use of ELISA
immunoassays with PA as the sole antigen in diagnostic tests has been
validated in humans (Ghosh et al., 2015; Quinn et al ., 2002;
Semenova et al ., 2012), horses (Caldwell, Hathcock, & Brock,
2017) and goats (Ndumnego et al ., 2013). In this study, we
validated the use of the ELISA assay for detecting anthrax anti-PA
antibodies in exposed African lions by using the TNA assay. Indeed,
earlier researchers (Bagamian, Alexander, Hadfield, & Blackburn, 2013;
Hampson et al ., 2011; Switzer et al ., 2016; Turnbullet al ., 1992), have used the ELISA-PA assay to monitor B.
anthracis exposure in lions among other carnivore species. Hence, the
ELISA-PA immunoassay for the serological survey of B. anthracisexposure in African lions was adopted for use in this study.
Further, on the use of ELISA-PA assay in goats, Ndumnego et al .
(2013) compared and observed that the use of skimmed milk powder gives a
lower background reading compared to the use of foetal calf serum. While
there may be the risk of lion IgG detecting milk or any other ruminant
proteins leading to false positives, the presence of lethal toxin
neutralizing antibodies in sero-positive sera in toxin neutralization
test negates this. The presence of anti-PA antibodies in animals
indicates that non-lethal systemic infection may have taken place
(Hugh-Jones & Blackburn, 2009; Ndumnego et al ., 2013; Turnbullet al ., 1992). Therefore based on anti-PA antigen antibodies
serological assay in African lions, there were indications that B.
anthracis pathogen was present with a wide distribution in Zimbabwe.
In the case of HNP where anthrax outbreaks have not been reported in
over 20 years to 2018, this represents the first confirmation of the
presence and wide exposure of B. anthracis to lions in the park.
There is a possibility that some lions from HNP could have been exposed
to anthrax from consumption of livestock carcasses as outbreaks of
anthrax were reported in adjacent communal lands of Tsholotsho
southeastern to HNP (Fig. 2) (Mukarati et al ., 2018).Indeed,
predation of livestock by lions from the park is an established
human-wildlife conflict issue in the area (Loveridge et al .,
2017). However, other seropositive lions were far removed from this
community at more than 90 km from the nearest park boundary. This
distance was well outside the lions’ home range of about 20 km radius
(Loveridge et al ., 2009) indicating unlikely influence of
diseases between the lions and livestock. It is most likely that such
seropositive lions were exposed to other sources of B. anthracisin HNP. Due to the limitation on samples available, the distribution of
the positive lions was biased towards the northern half of the park. A
uniform distribution of positive lion samples would have provided more
insightful trends.
Although this was not strictly a cross-sectional survey in that the
lions were sampled over 20 years, it nonetheless provides useful
information. That lions sampled over this period were positive forB. anthracis in the absence of reported disease outbreaks
suggests either occurrence of undetected outbreaks of the disease or
sublethal infections in wildlife in HNP (Hugh-Jones and Blackburn, 2009;
Cizauskas et al., 2014). Thus, the hitherto general belief that
HNP was free of anthrax based on no reported cases is uncertain. The
presence of antibodies to anthrax in lions in Mana Pools NP, Save Valley
Conservancy and Malilangwe Wildlife Reserve indicates that B.
anthracis was already circulating in the area long before the disease
outbreaks occurred (Mukarati et al., 2018; Clegg et al.,2007; OIE, 1997). However, there was no correlation between anthrax
outbreaks and seropositivity in lions from this dataset. Improved
surveillance and additional studies on possible environmental and soil
geochemical factors possibly influencing B. anthracisdistribution in protected areas are necessary (Hugh-Jones and Blackburn,
2009; Griffin et al., 2014).
There were no known anthrax outbreaks that could account for exposure
and seroconversion in captive lions in this study. According to de Vos
and Turnbull (2004), anthrax outbreaks affecting wildlife in captivity
are limited to consumption of infected meat. Carnivores on small game
parks are managed essentially as in zoological gardens and thus are
presumably similarly exposed to anthrax. An anthrax outbreak occurred in
lions and cheetahs in 1997 at Lion and Cheetah Park, Harare, when they
were fed infected cattle meat donated by a farm (OIE, 1997) . On
the other hand, wild carnivores in captivity maybe exposed to B.
anthracis from infected meat without necessarily developing clinical
disease either due to their relative resistance or exposure to sublethal
anthrax doses (Beyer and Turnbull, 2009; Hugh-Jones and Blackburn, 2009;
Cizauskas et al ., 2014).
A pertinent question which arises is the specificity of the serological
assay used in this survey, given that there are few reports of atypicalB. cereus strains causing similar disease in humans and wild
primates (Marston et al., 2016). Rare cases of anthrax-like
illness in humans and wild chimpanzees caused by a B. cereusstrain possessing the anthrax toxin genes have been reported in the US
and West Africa (Antonation et al ., 2016; Hoffmaster et
al ., 2006; Klee et al ., 2006). However, to date there are no
documented cases of outbreaks caused by this strain of B. cereusin wild or domestic ruminants, or the carnivores that predate on these
animals. While not ruling out the possible exposure to environmentalB. cereus in grazing animals, the presence of the rare
toxin-producing B. cereus strains have not been reported in
Southern Africa (Romero-alvarez et al., 2020). Thus, the seropositivity
of lions in this study is assumed to be attributable to exposure toB. anthracis strains.
The finding of no significant difference in seropositivity between lions
located in currently recognized high- and low-risk zones for anthrax (p
= 0.852, Table 1) tally with earlier findings in domestic dogs (Mukaratiet al., 2018). A much wider range of anthrax endemic areas in
Zimbabwe can be hypothesized similar to other endemic regions of the
world. This suggests that the categorization of areas in Zimbabwe into
high- and low-risk zones may not represent the true status of anthrax
presence across the country. This needs review based on improved
surveillance and epizootiological investigations. Anthrax serology in
resident wild and/or domestic carnivores could serve as sentinel and
indicator of anthrax circulation in given areas and thus can be useful
epidemiological tools.
The widespread presence of anthrax antibodies in lions in protected
areas, irrespective of absence of reported disease outbreaks in wild or
domestic ungulates or humans, confirms a much larger circulation ofB. anthracis in Zimbabwe. These results raise new questions on
the epidemiology of anthrax in endemic regions. There is need to
investigate local factors that could be associated with anthrax
outbreaks apart from the presence of the pathogen. On the other hand,
there could be smaller outbreaks of anthrax occurring but going
unnoticed in HNP as has been noted elsewhere (Cizauskas, Bellan, Turner,
Vance, & Getz, 2014). Overall, improved surveillance of anthrax in all
animals could shed more light on whether outbreaks were indeed taking
place but being missed because of inadequate surveillance and may also
give pointers on risk factors. Further, the spatial distribution of
lions tested for anthrax anti-PA antibodies in this study in the
northern half of HNP meant that insightful trends in the whole of the
park could not be determined. A more uniform distribution of lion
samples tested for anthrax anti-PA antibodies across the park would be
helpful in establishing likely risk areas for the disease outbreaks.