Advances in Parasitology, Volume 117

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Advances in Parasitology, 117

FIRST EDITION

David Rollinson
Life Sciences Department The Natural History Museum, London, United
Kingdom

Russell Stothard
Department of Tropical Disease Biology Liverpool School of Tropical
Medicine, Liverpool, United Kingdom
Table of Contents

Cover image

Title page

Copyright

Contributors

Chapter One: The microscopic five of the big five: Managing

zoonotic diseases within and beyond African wildlife protected
areas

Abstract

1: Introduction

2: The ‘Microscopic Five’

3: Challenges of BTb control at the wildlife-livestock interface:

The South African case study

4: Drivers of disease: The Kruger National Park case study

5: Disease knowledge gaps and lessons learnt from African

protected areas
 6: Communities and conservation

7: Conclusions

Acknowledgements

References

Chapter Two: Improving translational power in antischistosomal

drug discovery

Abstract

1: Filling the drug pipeline for schistosomiasis

2: Evaluating the importance of S. mansoni isolate origin for

early antischistosomal drug discovery

3: The S. mansoni mouse model for drug efficacy testing

4: Infection intensity of the patent S. mansoni mouse model

5: Pharmacokinetic/pharmacodynamic (PK/PD) relationship of

selected drugs

6: Concluding remarks

Acknowledgements and funding

References

Chapter Three: Unique thiol metabolism in trypanosomatids: Redox

homeostasis and drug resistance

Abstract

1: Introduction
2: Trypanothione metabolism

3: Effector proteins of the antioxidant defence: Old and new

actors

4: Trypanothione metabolism is linked to cysteine, polyamine,

and pentose phosphate pathway

5: The role of redox active compounds and their mechanism in

parasites survival

6: Role of thiol metabolism in drug resistance

7: Anti-parasitic potential of molecules targeted against redox

metabolism

8: Unsolved questions and future prospects

References
Copyright
Contributors
Chapter One: The microscopic five of the big five:
Managing zoonotic diseases within and beyond
African wildlife protected areas
Anya V. Tobera,⁎; Danny Govenderb,c; Isa-Rita M. Russoa,†; Jo Cablea,† a School of Biosciences, Cardiff University, Cardiff,
Wales, United Kingdom
b SANParks, Scientific Services, Savanna and Grassland Research Unit, Pretoria, South Africa
c Department of Paraclinical Sciences, University of Pretoria, Onderstepoort, South Africa
† Authors contributed equally to this work
⁎ Corresponding author: email address: tobera@cardiff.ac.uk.

Abstract
African protected areas strive to conserve the continent's great biodiversity with a targeted focus on
the flagship ‘Big Five’ megafauna. Though often not considered, this biodiversity protection also
extends to the lesser-known microbes and parasites that are maintained in these diverse
ecosystems, often in a silent and endemically stable state. Climate and anthropogenic change, and
associated diversity loss, however, are altering these dynamics leading to shifts in ecological
interactions and pathogen spill over into new niches and hosts. As many African protected areas
are bordered by game and livestock farms, as well as villages, they provide an ideal study system to
assess infection dynamics at the human-livestock-wildlife interface. Here we review five zoonotic,
multi-host diseases (bovine tuberculosis, brucellosis, Rift Valley fever, schistosomiasis and
cryptosporidiosis)—the ‘Microscopic Five’—and discuss the biotic and abiotic drivers of parasite
transmission using the iconic Kruger National Park, South Africa, as a case study. We identify
knowledge gaps regarding the impact of the ‘Microscopic Five’ on wildlife within parks and
highlight the need for more empirical data, particularly for neglected (schistosomiasis) and newly
emerging (cryptosporidiosis) diseases, as well as zoonotic disease risk from the rising bush meat
trade and game farm industry. As protected areas strive to become further embedded in the socio-
economic systems that surround them, providing benefits to local communities, One Health
approaches can help maintain the ecological integrity of ecosystems, while protecting local
communities and economies from the negative impacts of disease.

Keywords
Emerging Infectious Diseases; One Health; Zoonoses; Spill over; Spill Back; Kruger National Park

1: Introduction
As we enter the sixth mass extinction, protecting the world's biodiversity has never been more critical.
Protected areas, including national parks, cover over 18.8 million km2 and are at the forefront of a global
effort to safeguard biodiversity (Chape et al., 2003). Managers of these protected areas must strike a
balance between protecting the ecological integrity of ecosystems and preventing exploitation of local
resources while promoting their use in education and recreation (Chape et al., 2003). If managed
correctly, protected areas can be beneficial to wildlife conservation and the country's economy through
promoting ecotourism and creating local employment opportunities (Cheung, 2012; Spies et al., 2018).
However, the management of protected areas is challenging, particularly in the Anthropocene era of
human mediated global change, and increased emergence and re-emergence of infectious diseases
(reviewed by Cable et al., 2017). These diseases can reduce fitness, alter wildlife population
structure/size and even alter ecosystem function (Holdo et al., 2009; Prins and Weyerhaeuser, 1987;
Scott, 1988). Therefore, to effectively manage wildlife populations and ecosystems, it is essential to
understand the threats posed by pathogens and the diseases they cause.
Of the 3881 terrestrial and marine national parks in the world, almost half are in sub-Saharan Africa,
with terrestrial parks here covering 1 million km2 (4% of the total land area; Chape et al., 2003;
Muhumuza and Balkwill, 2013). These parks aim to conserve Africa's unique and iconic ecosystems
ranging from open savannas and grasslands to dense forest. This variety of habitats supports high levels
of biodiversity, drawing numerous tourists who aspire to spot the ‘Big Five’ megafauna: African buffalo
(hereafter referred to as buffalo), lion, African elephant (hereafter referred to as elephant), rhinoceros
and leopard (Dube and Nhamo, 2019). However, hidden and often forgotten biodiversity within
protected areas includes pathogens, which modulate animal abundance, fitness and behaviour (Gómez
and Nichols, 2013). It is crucial to better understand drivers for past and current wildlife disease
outbreaks within protected areas, to find new approaches to predict and prevent future outbreaks. A
review of all infectious wildlife diseases within protected areas would be too large a task. Instead, we
focus on five diseases referred to here as the ‘Microscopic Five’, which are important at the human-
livestock-wildlife interface due to their broad host range and zoonotic potential. These interface diseases
would all benefit from a ‘One Health’ approach to management (Fawzy and Helmy, 2019; Innes et al.,
2020; Webster et al., 2016). We therefore purposefully included high profile diseases (bovine tuberculosis
(BTb), Rift Valley fever and brucellosis) as well as neglected diseases (cryptosporidiosis and
schistosomiasis) for study. Using Kruger National Park, one of the most researched parks in Africa (van
Wilgen et al., 2016), we will review the key factors that can influence outbreaks and transmission of the
‘Microscopic Five’ within and around protected areas (Fig. 1). By focusing on a select group of
pathogens within a specific park our intention is to highlight drivers of disease common among many
protected areas and the importance of considering all infectious diseases in wildlife management plans.
We will first give a brief introduction to the ‘Microscopic Five’ and then give examples of the
environmental and anthropogenic factors driving the dynamics of these diseases within and around
Kruger National Park. We will then discuss the key knowledge gaps and future challenges for managing
the ‘Microscopic Five’ and other important diseases and touch on different management approaches
followed in various parks.
 FIG. 1 The ‘Big Five’ and ‘Microscopic Five’, and the drivers of disease at the wildlife-
livestock-human interface. Arrows represent anthropogenic drivers from beyond
Kruger National Park. Created with Microsoft PowerPoint (version 2109) and Adobe Photoshop (2021).

2: The ‘Microscopic Five’

The ‘Big Five’ are undoubtedly one of the biggest attractions for tourists visiting South Africa's
protected areas (Dube and Nhamo, 2019). To conserve these and other wildlife, and to reduce
transmission of infectious diseases among wildlife, domestic animals and humans, we focus on the
lesser known ‘Microscopic Five’. These comprise zoonotic diseases caused by pathogens that have
multiple hosts, including humans, and are of particular importance at the human-livestock-wildlife
interface. Although we focus on five specific diseases, there are many more of importance within
protected areas (Table 1) but by highlighting a distinct few we aim to raise the profile of all infectious
diseases and possible drivers. The first three of the ‘Microscopic Five’ (bovine tuberculosis, brucellosis
and Rift Valley fever) are high profile or state-controlled diseases in South Africa and any outbreaks
must be reported to the World Organisation of Animal Health (OIE). All three of these diseases are
trade-sensitive diseases and may change the trading status of a country and its ability to trade on the
global market. The remaining two (schistosomiasis and cryptosporidiosis) are neglected in comparison,
particularly in wildlife. By including these in the ‘Microscopic Five’, we aim to bring greater attention to
overlooked yet highly important diseases (see WHO, 2020). In the following account, we briefly cover
each of the ‘Microscopic Five’ discussing their host specificity, transmission pathways and known
impacts on wildlife, livestock and humans.
Table 1

Some diseases of large herbivores within Kruger National Park which may pose a threat to livesto
Spatial
Transmission Transmission
Disease. Pathogen Drivers distribution
modes routes
in KNP
Bacteria
Anthrax Bacillus Vector, Ingestion of Calcium soil Northern
anthracis environme contamina content, and
ntal, direct ted drought central
contact, vegetation regions
fomites or
carcasses
Bovine Tb Micobacteria Aerosol Respiratory Wildlife/livestock South,
bovis tract interface, host central,
density moving
north
Virus
Foot and mouth Aphtovirus Aerosol, Respiratory Wildlife/livestock North,
fomites tract interface, host central,
density south
African horse Orbivirus Mosquito Cutaneous Introduced Central
sickness vector, penetratio horses,
direct n season
contact
Rift Valley fever Phlebovirus Mosquito Cutaneous Climate change, Higher in
vector, penetratio drought, south
direct n rainfall and
contact central
regions
Protozoa
Bovine Brucella abortus Direct contact Ingestion of Host density North,
brucellosis infected central,
discharges south
during
birth,
milk,
mucus
membrane
s
Cryptosporidiosis Cryptosporidium Environmental Faecal-oral Dependant on Unknown
spp. via species and
contamina host range
tion of
food and
water
 Spatial
Transmission Transmission
Disease. Pathogen Drivers distribution
modes routes
in KNP

Piroplasma
Corridor disease Theileria parva Tick vector Cutaneous Wildlife/livestock North,
penetratio interface central,
n south
Babesiosis Babesia spp. Tick vector Cutaneous Unknown
penetratio
n
Digenea
Fascioliasis Fasciola spp. Environmental Contact with Dependant on Unknown
infected species and
water host range
Schistosomiasis Schistsoma spp. Environmental Contact with Dependant on Unknown
infected species and
water host range

2.1: Bovine tuberculosis

Bovine tuberculosis (BTb) is caused by the bacterium Mycobacterium bovis and predominantly infects
bovines, such as African buffalo (Syncerus caffer caffer) and cattle (Bos taurus), yet most warm-blooded
animals including humans can be infected (Ayele et al., 2004). Transmission mainly occurs through
inhalation of infectious particles, which is particularly problematic when livestock are kept at high
densities (Ayele et al., 2004). Though thought to have spilled over from cattle to buffalo in the early
1960s in South Africa (Bengis et al., 1996), buffalo now serve as the primary maintenance host for BTb
within Kruger National Park and Hluhluwe-iMfolozi Park, spilling over into various species of wildlife
and livestock (Michel et al., 2006). Although BTb is a controlled disease within South Africa, its control
is becoming increasingly challenging due to the presence of wildlife reservoirs, difficulty in controlling
disease in communal herds and lack of practical control options in wildlife (see Section 3). The WHO
estimated 147,000 new cases of zoonotic Tb in humans in 2016 with 12,500 deaths globally but mostly in
Africa (Sichewo et al., 2019b). Humans can become infected through drinking unpasteurised milk,
eating undercooked meat and via aerosols inhaled from infected cattle (DAFF, 2016; Sichewo et al.,
2019a).

2.2: Rift Valley fever

Rift Valley fever (RVF) is caused by a zoonotic, vector borne virus predominantly spread by Aedes
mosquitoes (Clark et al., 2018). The virus was first reported in South Africa in 1950 and subsequent
outbreaks have occurred sporadically every 7–11 years infecting mainly domestic livestock but also a
range of wild mammals and humans (Beechler et al., 2015a; Métras et al., 2015). Human infection occurs
mainly through direct contact with blood or tissue from infected animals or through consuming
unpasteurised milk but can also result from an infected mosquito bite. Symptoms vary from mild, flu-
like to severe haemorrhagic fever that can be fatal (Clark et al., 2018). Over 4000 human cases and
around 1000 deaths have been reported in the last 20 years, predominantly in Africa and Saudi Arabia
(Petrova et al., 2020). Little is known about how the pathogen is maintained during inter-epidemic
periods. One suggestion is vertical transmission from mosquitoes to their ova, which has been
demonstrated with Aedes mosquitoes under laboratory-controlled conditions (Romoser et al., 2011).
Another possibility is that it is maintained in wild animal populations (Beechler et al., 2015a; see Section
4.3.4). Commercial vaccines are available for livestock but there is currently no licensed human vaccine
(Petrova et al., 2020).

2.3: Brucellosis
Brucellosis, caused by bacteria of the Brucella genus, is ranked among the most economically important
zoonotic diseases globally. Although it is an OIE notifiable disease, outbreaks are thought to be greatly
under-reported in Africa (McDermott et al., 2013). The species of medical and veterinary importance are
Brucella abortus, Brucella melitensis and B. suis (see Ducrotoy et al., 2017). Infection in humans can lead to
a debilitating illness known as ‘Mediterranean’ or ‘undulant’ fever and is commonly misdiagnosed as
malaria (Ducrotoy et al., 2017; Godfroid et al., 2011). Human infection occurs through direct contact
with or consumption of an infected animal. Consumption of un-pasteurised milk causes most human
infections, while human to human transmission is rare (Godfroid, 2018). Several wildlife species have
been reported as seropositive for this disease and African buffalo are thought to be a reservoir for B.
abortus (see Godfroid et al., 2013). Infection can cause abortions in livestock reducing farm productivity,
however the effects of the disease on wildlife are largely unknown and may differ between species
(Gorsich et al., 2015). Vaccines are available for livestock and small ruminants but not yet for humans
(Ducrotoy et al., 2017).

2.4: Cryptosporidiosis
Cryptosporidiosis, caused by several species of the protozoan Cryptosporidium genus, can lead to severe
diarrhoea in humans and animals globally. Infectious diarrhoea is a major cause of death in children
under five in Africa and Cryptosporidium is second only to rotavirus as a contributor to this disease
(Kotloff et al., 2013; Squire and Ryan, 2017). Transmission occurs through the faecal oral route via close
contact with infected humans, animals or contaminated food and water (Innes et al., 2020). Currently
there are at least 40 recognised species with varying host specificities but the most important two
species infecting humans and livestock are C. hominus and C. parvum. The latter is the predominant
cause of diarrhoea in young calves and is the most important zoonotic species. Cryptosporidium parvum
is more genetically diverse than C. hominus with several subtypes with differing host specificities,
therefore an integrated genotyping approach has been advocated to differentiate these subtypes (Innes
et al., 2020). Cryptosporidium species have been identified in a range of wildlife, yet most studies focus on
humans and livestock (Zahedi et al., 2016). C. parvum, C. ubiquitum and C. bovis were recently identified
in wildlife within Kruger National Park in elephant (Loxodonta africana), buffalo (Syncerus caffer) and
impala (Aepyceros melampus; see Samra et al., 2011). Oocysts of Cryptosporidium spp. have also been
detected in zebra (Equus zebra), buffalo and wildebeest (Connochaetes gnou) faeces in Mikumi National
Park, Tanzania (Mtambo et al., 1997). There is currently no available vaccine for cryptosporidiosis yet
there is potential to develop one for cattle (Innes et al., 2020).

2.5: Schistosomiasis
Schistosomiasis is a waterborne, zoonotic disease of veterinary and medical importance, caused by
digenean parasites of the genus Schistosoma. Schistosomiasis is a major public health threat with an
estimated 207 million people infected and 779 million people at risk globally, with 90% of these
infections in Africa (Steinmann et al., 2006). Like all digeneans, schistosomes have an indirect lifecycle.
They require an intermediate freshwater snail host within which they reproduce asexually ultimately
producing cercariae, which are free-swimming larval stages that subsequently infect a definitive
mammalian host (Cribb et al., 2003). Definitive animal or human hosts can become infected with
schistosomiasis by entering infested waters—the water-borne larvae burrow through the skin of the new
host (Cribb et al., 2003). There are at least 12 known schistosome species in Africa of which 5 are known
to infect humans (S. haematobium, Schistosoma mansoni, S. intercalatum, S. guineensis and S. mattheei).
Schistosoma mattheei is of note as although predominantly a parasite of cattle, it has also been found in
wildlife and humans where it is known to hybridise with S. haematobium (see Pitchford, 1961). The other
species infect a wide range of domestic and wild animals including cattle, horses, buffalo, baboons,
zebra, hippopotamus and rodents (Standley et al., 2012). Traditionally, malacological monitoring
programmes have only targeted snail species known to harbour human infecting schistosomes, but a
wider approach is clearly needed as we become aware of wider host ranges (Pennance et al., 2021) that
are likely to shift with increasing environmental stressors. There is currently no vaccine for
schistosomiasis and the main control strategy for humans is preventative chemotherapy, improved
water, sanitation and hygiene and snail control (WHO, 2022).

3: Challenges of BTb control at the wildlife-livestock interface: The South

African case study
South Africa has been challenged with the control of BTb since the disease was first reported in the
country in 1880, initially focusing on livestock, and now including control in wildlife (DAFF, 2016).

3.1: Control in livestock

Early BTb surveillance included the introduction of tuberculin skin testing in cattle in 1905, followed by
its declaration as a notifiable disease in 1911 and the initiation of the Division of Veterinary Services BTb
scheme in 1969 (DAFF, 2016; Michel et al., 2019). This scheme focused on compulsory testing of
commercial cattle herds suspected to be infected, with slaughter of positive individuals, quarantine and
disinfection of farms. Initially, great progress was made, reducing prevalence to 0.04% by 1991 (1.1
million cattle tested); however, the number of tests have since declined due to budget cuts and a
decreased workforce (DAFF, 2016; Michel et al., 2019). Current prevalence in communal livestock is
variable (< 0.5% to > 15%) (Musoke et al., 2015; Sichewo et al., 2019b).
In 2021 the national cattle herd was estimated at 12 million, consisting of commercial dairy herds
(20%) and beef and dual-purpose herds (80%) (DAFF, 2021). Testing of cattle is no longer compulsory
and current control of BTb is guided by the Interim BTb Manual from the Department of Agriculture,
Forestry and Fisheries (DAFF), South Africa, which proposes the use of four testing programmes (Table
2; DAFF, 2016). All programmes are voluntary apart from the infected herd program, which can be
enforced by the Animal Diseases Act, 1984 (Act No. 35 of 1984) (DAFF, 2016). The approved test is the
cervical intradermal tuberculin (CIT) test (DAFF, 2016).

Table 2

Four levels of Bovine Tb surveillance programmes in South Africa.

Surveillance herd One off survey used by state officials to determine the prevalence of BTb within an
programme area or by a stock owner conducting a self-assessment
Maintenance To join this programme, herds are required to undergo two consecutive tests with
herd 100% negative results at least 3 months apart. These BTb free herds are then
programme tested every 2 years. If an individual tests positive, then the entire herd is
moved to the infected herd programme
Infected herd Compulsory programme for herds that have tested positive with the CIT test, as
programme well as those detected from meat and milk inspection, post-mortems or clinical
cases. These herds are placed under quarantine and kept under supervision of
a state veterinarian, who will order the slaughter of infected animals. The rest
of the herd is tested every 3 months and is only let out of quarantine once the
herd has undergone two consecutive negative tests
Diagnostic Individual cattle destined to be imported or exported. Imported cattle are kept in
testing quarantine and must undergo a compulsory CIT test. Before export, cattle
programme must also receive a comparative CIT test—a requirement for many importing
(individuals) countries
3.2: Control in wildlife
The control of BTb in wildlife is becoming increasingly important as many farms switch from livestock
to game farming, and wild buffalo reservoirs hinder control efforts in cattle (Michel et al., 2019). Bovine
Tb has been identified in 21 different wildlife species in South Africa, including most recently giraffe
(Hlokwe et al., 2019). The current control scheme is focused on domestic cattle and although some tests
have been adjusted for use in buffalo, this is not the case for other wildlife species. The Buffalo
Veterinary Procedural Notice (VPN) was published in 2017 outlining the procedures for disease testing,
movement and contingency planning for disease outbreaks in buffalo (DAFF, 2017). The buffalo VPN
states that for movement purposes, buffalo must have a negative CIT test as outlined in the manual for
cattle. Importantly, the interpretation of CIT has been based on cattle thresholds due to the lack of
species-specific cut-off values for African buffaloes. The gamma interferon test is also an effective
diagnostic tool for buffalo but is not approved by DAFF for movement purposes. There is currently no
guidance on control of BTb in other wildlife species and there are limited verified diagnostic tests in
these species (DAFF, 2017).
Kruger National Park and Hluhluwe-iMfolozi Park are the only two parks within South Africa that
contain buffalo herds maintaining BTb yet they have adopted different control approaches. Bovine Tb
was first detected in Hluhluwe-iMfolozi Park in 1986 and a test and cull disease programme was
initiated in 1999. This programme involved a mobile capture unit to corral buffalo in different areas of
the park, test them by means of the CIT test and culling positive individuals. Between 1991 and 2006,
4733 buffalo were tested, with herd prevalence ranging from 2.3% to 54.7%. Subsequent, data analysis
suggested that the programme was effective at reducing BTb prevalence, particularly in areas with
intensive test and culling operations (Le Roex et al., 2016). Kruger National Park took a different
approach to managing BTb in its buffalo population after the disease was detected in this host species in
1990. They aimed to breed disease free buffalo from Foot and Mouth Disease infected parents within the
park in order to conserve the genetic pool of Kruger buffalo in an ex-situ population (Laubscher and
Hoffman, 2012). This approach, which used dairy cows as foster parents for buffalo calves initially, and
later switched to having the buffalo mothers rear their young, was highly successful and also popular
with farmers, eventually shifting from a few government funded projects to hundreds of private buffalo
breeding farms (Laubscher and Hoffman, 2012). Additionally, Kruger National Park did extensive BTb
monitoring surveys between 1993 and 2007, to assess the spread and impact of BTb in herds, and
determine if the disease was having population level effects. Since it entered the park, BTb has been
detected in 12 spill-over species (Michel et al., 2006) and remains a concern in low density species, such
as wild dog and black rhinoceros (Higgitt et al., 2019).
With the disease currently not shown to be affecting population recruitment or growth in buffalo, the
real concern becomes spill-over to other hosts and therefore finding an effective vaccine that limits
disease severity and spill-over is a priority. Currently there is only one registered vaccine for BTb
control. The BCG vaccine is predominantly used in humans but has yielded promising results for use in
domestic cattle (Arnot and Michel, 2020). However, when trialled in wild buffalo within the Kruger
National Park, the BCG vaccine protection was insufficient and did not limit bacterial shedding (De
Klerk et al., 2010). This was thought to have resulted from priming with environmental non-TB
mycobacteria, which has been shown to reduce the protective efficacy of the BCG vaccine (Brandt et al.,
2002; De Klerk et al., 2010). Importantly similar studies in badgers in the UK found the BCG vaccine to
be effective in limiting disease severity (and therefore bacterial load; Chambers et al., 2011), meaning
that defining the clinical end point for vaccine efficacy trials is important. Another vaccination trial in
buffalo is currently underway, testing both BCG and DNA-sub-unit vaccines.

4: Drivers of disease: The Kruger National Park case study

4.1: Past and present disease management
Kruger National Park first opened as the Sabi Game Reserve in 1898 (10,364 km2) as a response to
campaigns for the conservation of wild animals subjected to uncontrolled hunting and to the 1896
rinderpest epidemic (Mabunda et al., 2003). In 1926, the Sabi Game Reserve was combined with the
Singwitsi Reserve (5000 km2 region named after the Shingwedzi River) and later renamed Kruger
National Park. James Stevenson-Hamilton, who was appointed warden in 1902, was tasked with
managing the aftermath of the rinderpest epidemic which, along with previous hunting activities,
decimated the game population, leaving elephant and white rhinoceros (Ceratotherium simum) locally
extinct (Mabunda et al., 2003). The rinderpest epidemic also severely affected buffalo, eland (Tragelaphus
oryx) and greater kudu (Tragelaphus strepsiceros; hereafter referred to as kudu), whereas wildebeest and
zebra were unaffected (Stevenson-Hamilton, 1957). The first 60 years of park management (1900–1960)
focused on protecting, preserving, and propagating, aiming to increase game numbers through
introductions of large herbivores, provision of water sources and culling of predators (Venter et al.,
2008).
Colonel J.A.B Sanderberg took over from Stevenson-Hamilton as Warden in 1946 and 8 years later the
first case of anthrax was confirmed in the north of the park (Mabunda et al., 2003). This was followed by
repeated outbreaks in 1959–60, 1970 and 1990–91, and outbreaks in the central part of the park in 1993
and 1999 (Bengis et al., 2003; De Vos and Bryden, 1996). The 1959–60 outbreak lasted just 4 months and
yet within this time over 1000 mammals died: kudu, waterbuck (Kobus ellipsiprymnus) and roan
(Hippotragus equinus) being the most affected (Pienaar, 1961). Simultaneously, BTb likely entered the
park, transmitted from cattle to buffalo on the southern border, although it was not detected in the park
until 30 years later (Bengis et al., 1996). At this time, park management shifted to a ‘management by
intervention’ approach and the next 30 years (1960–1990) focused on measuring, monitoring and
manipulation (Mabunda et al., 2003). Fencing of the park was ordered by the National Department of
Agriculture in order to prevent the spread of disease to surrounding livestock, such as foot and mouth
(FMD) endemic in buffalo (Bengis et al., 2003). Fence construction started in the early 1960s with the
western boundary followed by the eastern boundary in the late 1960s, by 1980 all boundaries of the park
were enclosed. The fences (over 360 km in length and 65 km in width) restricted movement of wildlife
leading to increased numbers of large herbivores, such as elephant and buffalo, which were
subsequently controlled by culling operations and in the early 1970s, a certified abattoir was built within
the park to optimise use of the culled meat (Mabunda et al., 2003). From 1990 to 2010 management
shifted again to focus on integration, innovation and internationalisation. The severe drought of 1992–93
followed by the February floods in 2000 as well as the catastrophic wildfire in September 2001, which
killed both people and animals within the park, were indicative of the need for management to become
more adaptive to the increasingly unpredictable environment (Mabunda et al., 2003). Since 1995, Kruger
has used a strategic adaptive management approach, which involves management decisions and actions
guided by research and monitoring while learning from unexpected events or outcomes. This approach
also aims to maximise heterogeneity of the park and led to its expansion across national boundaries
creating the Greater Limpopo transfrontier conservation area (GLTFCA) spanning the Limpopo
(Mozambique), Kruger (South Africa) and Gonarezhou (Zimbabwe) National Parks. A portion of fences
of approximately 45 km was removed between Limpopo and Kruger in 2002 (Caron et al., 2016; Venter
et al., 2008).

4.2: Kruger National Park's current adaptive management approach

In the past, most management issues in Kruger National Park were focused within the park boundaries;
however, since the recognition that threats and drivers to biodiversity conservation often occur outside
of the footprint of the National Parks, management issues are extending beyond the park boundaries
and becoming more socio-economic in nature (Venter et al., 2008). The creation of the Greater Limpopo
transfrontier conservation area shifted the park from being single use for wildlife to a multi-use park,
sharing its land with communities and their livestock. The park's current strategic adaptive
management aims to increase understanding of complex ecosystems and broader societal needs of local
communities. This process is guided by setting appropriate thresholds of potential concern (TPC), a set
of adaptive management goals and endpoints that define upper and lower levels of acceptable change,
enabling management to determine how much a system can be allowed to fluctuate before it becomes a
concern and requires management action. Although TPCs prove useful for simple metrics like invasive
plants and river flows, they have proven more challenging for complex systems such as disease where
drivers and responders are not always known (Gaylard and Ferreira, 2011; Venter et al., 2008).
 Kruger National Park's 2018–28 management plan includes a disease management programme as a
supporting objective to the higher-level objective of biodiversity conservation. This programme
acknowledges endemic wildlife diseases within the park as a key component of biodiversity yet
highlights the need to prevent and mitigate the spread of disease at the wildlife-livestock-human
interface and limit the introduction or impact of novel infectious diseases (Spies et al., 2018).

4.3: Environmental drivers of disease transmission

4.3.1: Spatial heterogeneity and the north/south divide
Topography, climate, geology and the associated soil and vegetation patterns can exert a bottom-up
control on ecosystems. The combination of these abiotic factors can influence fire patterns and animal
behaviours, as well as disease dynamics (Venter et al., 2003).
Kruger National Park lies within part of the north-eastern South African lowveld, which generally has
plains of low to moderate relief with some low mountains and hills. The geology of the park can be
crudely divided into granite plains on the west and basalt plains on the east, separated by a north-south
strip of sedimentary rock (Venter et al., 2003). Rainfall in the park increases along a north to south
gradient with annual mean rainfall of 350 mm in the northeast to 750 mm in the southwest. Geology and
rainfall have influenced the difference in soil and vegetation types between the north and the south of
the park. The south generally consists of deeper and more diverse soil types with predominantly open
canopy acacia tree bushveld and savannah with a well wooded area in the southeast. In contrast, the
north tends to have less diverse, thinner soils with a higher calcium content. Vegetation is dominated by
mopane trees with rare lowveld riverine forest occurring along the rivers in the northeast and sandveld
vegetation type in the northwest (Gertenbach, 1983; Spies et al., 2018). The northern most section of the
park is unique as it contains a varied assemblage of rock formations with associated soil and vegetation
types. It also contains the only true floodplain in Kruger (Venter et al., 2003). For management purposes,
Kruger National Park has been partitioned into 35 landscapes depending on geomorphology,
vegetation, soil, climate types and associated fauna (Gertenbach, 1983; Venter et al., 2003). A social-
economic gradient exists along the northern and southern boundaries of the park. Dense peri-urban to
urban developments lie along the southwestern border, including sugarcane plantations, forestry and
the nearby city of Mbombela (previously known as Nelspruit; Fig. 2). The central and north-western
boundaries are buffered by private nature reserves and community subsistence farming, and further
north becomes more rural with large agricultural areas and poor villages with limited economic
opportunities (Spies et al., 2018). Wildlife densities also differ across the park with megaherbivore
(elephant and buffalo) densities higher in the north than the south (Fig. 2).
 FIG. 2Megaherbivore (African buffalo and elephant) density across Kruger National
Park and fence breakages (red cross) from damage causing animals (DCAs).
Elephant cause most breakages enabling diseased buffalo to escape. Foot and
mouth (FMD) veterinary control zones and nearby villages are also shown. Map
produced by the Skukuza GIS Office.

This ecological heterogeneity within the park can create spatial heterogeneity in disease dynamics. A
park wide survey of RVF in buffalo in 1998 showed significantly higher seroprevalence of buffalo herds
in the south and central regions of the park compared to the north (Beechler et al., 2015a). This was
attributed to lower rainfall and different vegetation in the north leading to less suitable breeding
habitats for mosquito vectors (Beechler et al., 2015a). Brucellosis prevalence in buffalo was significantly
associated with park section and soil type (Gorsich et al., 2015). Buffalo captured on the resource poor
granitic soils were twice as likely to be seropositive for brucellosis compared to those on the resource
rich basaltic soils (Gorsich et al., 2015). Moreover, buffalo on granitic soils had higher prevalence in the
southern section of the park compared to the central section (Gorsich et al., 2015). This was attributed to
nutrient poor vegetation in the southwestern granitic soils and general lower body condition of buffalo
in the south of Kruger National Park (Caron et al., 2003; Gorsich et al., 2015). The effect of brucellosis
infection was also dependant on the seasonal heterogeneity of the park, brucellosis infection was
significantly associated with lower body condition but only in the dry season (Gorsich et al., 2015).
Knowledge of this heterogeneity of different disease dynamics and how the landscape and environment
affect this is of great importance and can help target monitoring and management of diseases within the
park.

4.3.2: Climate change and severe weather events

Africa is considered one of the most vulnerable areas to global climate change (Serdeczny et al., 2017).
Average temperature readings from the Skukuza weather station in Kruger National Park have shown a
2 °C increase from 1977 to 2018 with a maximum temperature increase of 0.5 °C per decade (Dube and
Nhamo, 2019). Kruger National Park has suffered numerous droughts in 1966–67, 1982–83, 1991–92,
1995–96 and most recently in 2015–17 (Staver et al., 2019). The most severe drought on record in 1991–92
had a mean total rainfall of 235.6 mm compared to the 534 mm long-term mean annual rainfall and the
number of days with rain within this period (24.2) was significantly less than the mean annual total
(48.3; Zambatis and Biggs, 1995). This was followed by a severe flood event in 1996. Certain disease
outbreaks within the Kruger National Park, such as anthrax and foot and mouth disease (FMD) have
occurred after a dry period (Pienaar, 1961).
Drought not only affects resource availability and body condition, but also the behaviour and
movement of animals, including buffalo, which in turn can alter disease transmission (Cross et al., 2004;
Staver et al., 2019). Combining buffalo behavioural association data with disease models predicted that
dry conditions facilitate increased spread of BTb within buffalo populations due to increased herd
switching (Cross et al., 2004). The 2015–17 drought forced buffalo to move north to areas where the
drought was less severe (Staver et al., 2019). Movements like this could lead to the transmission of
diseases into new areas of the park and is likely to have played a role in the spread of BTb northwards
through the park (Michel et al., 2006).
Drought particularly affects the dynamics of water borne diseases such as schistosomiasis and RVF
carried by vectors (snails and mosquitoes respectively) reliant on water or moisture with populations
that fluctuate depending on climate variations (Cribb et al., 2003; Romoser et al., 2011). Floods and
prolonged wet periods have been associated with outbreaks of RVF. South Africa has experienced three
major RVF epidemics (1950–51, 1973–75 and 2008–11). Epidemiological data from the 2008 to 2011
epidemic was modelled to quantify spatial and temporal environmental factors associated with disease
incidence (Métras et al., 2015). Initial years saw the incidence of RVF increase with increased vegetation
density and presence of wetlands. However, for 2010 and 2011, of which 2010 was the longest lasting
outbreak, the strongest risk factor was temperature. For 2010, the risk of RVF increased by a hazard ratio
of 15.7 in areas between 25 and 32 °C and by 44.35 in areas over 35 °C compared with those below 25 °C
(Métras et al., 2015). This is important for Kruger National Park as the average annual temperature in
the south of the park was 32.3 °C and is set to increase (Dube and Nhamo, 2019).
Modelling the RVF outbreaks in Kruger National Park from 2008 to 2011 suggested that soil
saturation index anomalies exceeding the long-term mean by 20%, followed by a sudden rainfall event
could be a reliable predictor for outbreaks. When tested on previous outbreaks, the model successfully
predicted 90% of outbreaks more than 1 month before they occurred (Williams et al., 2016). Other
factors such as vegetation density and increased temperature are also important risk factors (Métras et
al., 2015).

4.3.3: Water sources

Water is often a focal point for wildlife-livestock interaction, particularly rivers which run between
protected areas and communal farmlands (Kock et al., 2014). Such interaction can enable spread of
diseases between wildlife within the park and livestock on the borders (Miguel et al., 2013; Pienaar,
1961). Within Kruger National Park, five perennial rivers (Sabi-Sands, Crocodile, Olifants, Letaba and
Luhuvu) run through the park as well as several seasonal rivers, natural pans and wetlands (Mabunda
et al., 2003; Pienaar et al., 1997). The Sabi River runs parallel to the fenced south-western border between
Kruger National Park and the adjacent communal lands of Bushbuckridge. Landscape resistance maps
for cattle and buffalo resource utilisation were used to model dispersal of these animals within these
two areas. Contact risk between buffalo and cattle was significantly higher in the dry season and was
concentrated along the Sabi River at the weaker parts of the fence. Contact risk was more widespread
and closer to villages in the wet season, yet still highest along the river (Kaszta et al., 2018). Water
sources can also increase the permeability of nearby fences to wildlife movements. Interviews with
fence maintenance workers in Kruger National Park reported that fences damaged by flooding and
predation were higher in areas with rivers compared to those without. Furthermore, reports of kudu
crossing the fences were significantly higher in areas with rivers, although this was not observed for
other wildlife (elephant, buffalo, warthog (Phacochoerus africanus) and impala) in the park (Jori et al.,
2011).
Contact at water sources has been attributed to disease outbreaks, such as FMD and anthrax (Miguel
et al., 2013; Pienaar, 1961). The Limpopo River runs between the northern edge of Kruger National Park
and adjacent communal land of Pezvi in Zimbabwe, both within the GLTFCA. Satellite data from
collared individual cattle from Pezvi and buffalo from Kruger National Park showed that the two
species shared 16.9% of habitat with most contacts occurring less than 500 m from the riverbed. These
contacts increased during the dry season suggesting that contact is driven by resource availability
(Miguel et al., 2013). Incidence of FMD antibodies in cattle was higher in sites with high buffalo contact
suggesting spread of the infection from buffalo to cattle (Miguel et al., 2013). Water points within
protected areas are also a source for interaction between different wildlife species, particularly in dry
seasons when water sources are limited. The anthrax outbreaks which occurred in the northern section
of Kruger National Park between 5 June and 11 October 1960 were all associated with natural and
artificial water points where large numbers of animals aggregated around the remaining available water
sources during the dry season (Pienaar, 1961).
Water sources also provide habitats for parasite-harbouring vectors such as freshwater snails and
mosquitoes. Freshwater snails harbour a huge number of digenean parasites some of which can cause
diseases of veterinary and medical importance, such as schistosomiasis and fascioliasis. Although
freshwater snails within Kruger National Park have been surveyed several times in the past (De Kock
and Wolmarans, 1998; De Kock et al., 2002; Wolmarans and De Kock, 2006), the diversity and
distribution of digenean parasites hosted by these snails has not yet been studied. The original surveys
identified the intermediate host snails for both schistosomes (Bulinus africanus, B. globosus, Bradyidius
tropicus, B. forskali, Biompharia pfeifferi) and fasciolids (Lymnea columella, L. natalensis; see (De Kock and
Wolmarans, 1998; De Kock et al., 2002), therefore it is important to explore the hidden digenean
diversity within these snails. Within Kruger National Park, schistosome species have been detected in
several animals including baboons, zebra, warthog, giraffe, kudu, wildebeest, buffalo (S. mattheii; see
Beechler et al., 2017; Pitchford et al., 1974) and hippopotamus (S. hippopotami and S.
edwardiense; see Pitchford and Visser, 1981). Animals sampled near man made dams had higher S.
mattheei infection rates and egg outputs than those at natural water sources, suggesting perennial
exposure and transmission at these sites (Pitchford et al., 1974).

4.3.4: Reservoir hosts

As seen with the ‘Microscopic Five’, most pathogens can infect more than one host (Cleaveland et al.,
2001). This is true for 77% of known livestock pathogens and 60% of known human pathogens
(Cleaveland et al., 2001; Haydon et al., 2002). Some hosts can act as reservoir hosts, also known as
maintenance hosts (Ashford, 1997; Haydon et al., 2002; Swinton et al., 2002). Essentially, reservoir hosts
can maintain the pathogen in the absence of cases in other species, and with a high enough prevalence
that parasites can spill over into another host species. Identifying reservoir hosts is crucial to
appropriately manage a disease. The failure to identify the importance of domestic dogs as a reservoir
host for guinea worm in humans has led to the re-emergence of a disease on the brink of eradication
(Durrant et al., 2020; Galán-Puchades, 2017).
In Africa, buffalo are well-known reservoir hosts for diseases such as BTb, FMD and corridor disease
(CD; Michel and Bengis, 2012). Buffalo are a keystone species in the African savanna ecosystem and
have a high economic value due to their importance for wildlife ecotourism, live game trade and
hunting industries (Glanzmann et al., 2016). Buffalo's gregarious nature, tendency to form large herds,
roam long distances, cross park boundaries and undergo regular fission fusion events make them ideal
hosts to maintain and transmit diseases (Caron et al., 2016; Cross et al., 2005; Wielgus et al., 2021). The
population of buffalo in South Africa in 1998 was estimated at over 31,000, of which only 7.7% were
disease-free (Winterbach, 1998). The two largest populations of buffalo in South Africa are found in
Kruger National Park and Hluhluwe-iMfolozi Park both of which are infected with BTb and CD, with
the Kruger population additionally being infected with FMD (Winterbach, 1998).
Within Kruger National Park, buffalo appear to have spread BTb to numerous wildlife species
including warthogs, baboons (Papio ursinus) and lions (Panthera leo). Bovine Tb isolates from all three of
these species were genetically highly similar and, in some cases, identical to buffalo strains (Keet et al.,
2000; Michel et al., 2009). More recently, high sero-prevalence (83%) has been detected in endangered
wild dogs (Lycaon pictus), thought to be eating infected prey such as warthogs (Higgitt et al., 2019).
Although these dogs appeared to be healthy at the time of the study, little is known about how the
disease may progress in this species (Higgitt et al., 2019). Another accepted reservoir for BTb is kudu
(Michel and Mare, 2000; Renwick et al., 2007). Clinical manifestations of BTb in kudu include abscesses
in the cranial lymph nodes from which infectious discharge is secreted onto thorns and leaves while the
animal browses on vegetation (Palmer, 2013; Renwick et al., 2007).
Buffalo are also thought to play a part in the maintenance of RVF during the inter-epidemic periods.
Between 2005 and 2008, a total of 227 buffalo seronegative for RVF were monitored within the park.
During the 4 years, five of these buffalo became seropositive despite no outbreaks being detected in
other species, which suggests circulation of the virus within the buffalo population (Beechler et al.,
2015a).

4.3.5: Co-infections
Since over 80% of all known species are parasitic, co-occurrence of different parasites is the norm
(Vaumourin et al., 2015). Such co-infections can be synergistic, by which one parasite facilitates the
infection of other parasites, antagonistic where one parasite inhibits infection of other parasites, or can
have no effect on each other (Hoarau et al., 2020; Vaumourin et al., 2015). A pathogen can alter the host's
immune response making it more susceptible to others (Ezenwa et al., 2010). Co-infections of closely
related parasite species or strains can lead to hybridisation, potentially creating more virulent pathogens
as seen with certain schistosome species (Huyse et al., 2009). In South Africa the predominantly animal
schistosome species S. mattheei has become increasingly prevalent in humans, thought to be due to
hybridisation with the human species S. haematobium (see Pitchford, 1961). This was confirmed
experimentally, resulting in fertile first generation (F1) hybrids which were more infective and
developed more quickly than the parents (Pitchford, 1961; Taylor, 1970; Wright and Ross, 1980).
However, prior infection with S. haematobium or S. mansoni seems necessary for S. mattheei to become
established in humans (Pitchford, 1961).
Over the last decade a body of research has been conducted assessing the impact of co-infections on
disease dynamics within African parks and the findings are concerning (Beechler et al., 2015b, 2019;
Broughton et al., 2021; Budischak et al., 2012; Ezenwa et al., 2010; Sylvester et al., 2017). In the
Hluhluwe-iMfolozi Park, helminth infections have been shown to alter the immune response of wild
buffalo, which could make them more susceptible to BTb infection (Ezenwa et al., 2010). Nematode
infected individuals had a depressed Th1 immune response, which is important in controlling BTb
infection and other intracellular microparasite infections, whereas Th1 responses were enhanced in
hosts that were nematode resistant. Disease modelling predicted that without these nematodes, BTb
would not have established infection in the buffalo population (Ezenwa et al., 2010). In 2008, an
outbreak of RVF occurred in a buffalo breeding facility close to the southern section of Kruger National
Park. To determine the effect of existing BTb infection on the dynamics of RVF outbreak, BTb positive
and BTb negative individuals were monitored for RVF before and during an outbreak. Bovine Tb
positive individuals had a twofold greater risk of RVF infection than BTb negative individuals. Bovine
Tb infection also worsened the clinical effects of RVF with pregnant co-infected individuals six times
more likely to abort than those with just RVF infections (Beechler et al., 2015b). Scaled-up models of
these data also showed that the presence of BTb increases the risk of RVF infection for the entire herd,
not just those infected with BTb. These findings were mirrored in free-ranging buffalo within the park
(Beechler et al., 2015b). Bovine Tb can also alter the composition of parasites within a host. Buffalo
within Kruger National Park that acquired BTb infections showed significant increases in both
taxonomic and functional parasite richness, as well as shifts in composition associated with the loss of
nematodes and gain of schistosomes (Beechler et al., 2019). Co-infections may also reduce the efficacy of
diagnostic tests. British calves experimentally infected with F. hepatica and M. bovis reacted less strongly
to the single intradermal comparative cervical tuberculin test (SICCT) than those infected with M. bovis
alone (Claridge et al., 2012).
Another important co-infection that could threaten the conservation of another ‘Big Five’ species is
BTb and Feline Immunodeficiency Virus (FIV) in lions (Sylvester et al., 2017). Feline Immunodeficiency
Virus is endemic to these keystone predators, yet BTb was not reported in Kruger National Park's lions
until 1996 (Keet et al., 2010). As FIV can cause lymphocyte deficiencies, infected lions may be
predisposed to infection with BTb. Within Kruger National Park, lions positive for FIV were more likely
(although this was not significant with a sample size of 56) to be infected with M. bovis than those
negative for FIV (Sylvester et al., 2017). A more recent study concluded that total gastrointestinal
parasite burden and richness was significantly higher in FIV positive lions (Broughton et al., 2021). Co-
infections of the ‘Microscopic Five’ and other diseases in Kruger's wildlife need greater attention as this
could greatly alter the dynamics of diseases previously thought to be benign. Co-infections leading to
hybridisations of human and animal specific schistosome species could also hinder control efforts as the
WHO currently focuses on treating human infection by mass drug administration to school age children
and little is known about whether hybrids could be more resistant to preventative chemotherapy or
whether it could change the age profile of infection. This could delay the WHO's target to eliminate
schistosomiasis as a public health problem by 2030 (Stothard et al., 2020; WHO, 2022).

4.4: Anthropogenic drivers of disease transmission: Wildlife-livestock-

human interface
Interactions between wildlife and livestock can drive the spread of diseases (Kock et al., 2014). Such
interaction can be linear, across a fence, or focal, at a shared water hole, where pathogens from an
infected animal or population can spill over into a vulnerable population via direct or indirect (vector)
contact. This spill-over can be bi-directional from livestock to wildlife or vice versa (Bengis et al., 2002).
Humans can also be involved with pathogens spilling over from animals to humans known as sylvatic
(from wildlife) or urban (domestic animals) zoonoses (Figueiredo, 2019). Bovine Tb likely first entered
Kruger National Park via transmission from cattle to buffalo in the south-western corner of the park
(Bengis et al., 1996). An outbreak of BTb was then recorded in cattle in the communal rangeland in
Mpumalanga Province on the western border of the park in 2012, suggesting spill back of the disease
from buffalo to cattle (Musoke et al., 2015).

4.4.1: Permeability of wildlife fences

Wildlife fences are commonly used in protected areas to prevent the spread of disease, such as foot and
mouth (FMD), between wildlife and livestock. A veterinary fence of note is the ‘Red Line’ in Namibia,
erected in 1960 with the purpose of separating the FMD endemic north from the rest of the country. This
1250 km fence extends from east to west bisecting the entire country (Miescher, 2012) with unintended
impacts on animal migration (Gadd, 2012) and socio-economic factors in the country. South Africa has
used a combination of fencing and zoning to improve FMD control, splitting the country into an
infected zone, buffer zone and FMD free zone (Fig. 2). This involved erecting a 750 km fence separating
the western and southern boundary of Kruger from neighbouring communal land, private farms and
private game reserves (Jori et al., 2011). The fences between the park and private reserves have since
been removed to allow more space for wildlife and the combined area makes up the infected zone.
Adjacent to this is a 10–20 km wide buffer zone which is split into two sections, one with vaccination
and one without vaccination but increased surveillance (Jori et al., 2009; Kaszta et al., 2018).
Although Kruger National Park's fences were placed as a barrier between infected and buffer zones,
they are highly permeable and, between 1996 and 2006, 1676 buffalo escaped across the park fence
bordering the Mpumalanga Province (Jori et al., 2009). Semi-structured interviews of fence workers
along 357 km of the western and southern border fence reported that higher numbers of kudu and
impala were seen crossing over into communal land than buffalo (Jori et al., 2011). Impala are also
capable of transmitting the FMD virus, they can experience both clinical and sub-clinical infections and
may maintain the South African Territory (SAT) serotype within local populations (Vosloo et al., 2009).
The same workers also reported that the main causes of fence damage were elephant and humans,
followed by predator and prey conflict, flooding and animals digging under the fence (Jori et al., 2011).
Elephant will break fences to access water, desirable trees, notably the Marula (Sclerocarya birrea) or
agricultural crops. Such fence breakages occur more in the northwest of the park, likely due to greater
elephant densities (Fig. 2). There are also more rivers traversing from west to east, through the fences
creating weak spots. These areas are also where livestock and wildlife may share water resources,
particularly in the dry winter months which show peaks of fence breaking activity.
Between 2000 and 2007, five outbreaks of FMD occurred in cattle near the western boundary of the
park (Jori et al., 2009). The most recent outbreak of FMD in Kruger occurred in 2013/14 in cattle in the
Mpumalanga Province, adjacent to the park's western border within the inspection zone with
vaccination. Most of these outbreaks were attributed to contact between wildlife and livestock, mainly
buffalo and cattle (Blignaut et al., 2020). The reliance on fences to act as a barrier against diseases may
exacerbate the problem as this may encourage farmers to relax vaccinating their livestock, or state
officials to not conduct regular animal inspections for early detection of outbreaks.

4.4.2: Edge effects

Edge effects describe the impacts of interactions between habitat patches or fragments and the
surrounding matrix (Suzán et al., 2012). In the case of Kruger National Park, the wildlife reserve is the
habitat patch surrounded by a matrix of communal farms and villages. Such edges can facilitate the
emergence of infectious diseases into new areas.
C. parvum was first detected in Kruger in 2008 with low prevalence in elephant, buffalo and impala
(4.2%, 1.4% and 1.9% respectively; Samra et al., 2011). For all three host animals, prevalence was
significantly higher in areas close to the western park boundary than in the park centre (Samra et al.,
2011). This suggests transmission from livestock or humans inhabiting the bordering farms and villages
to wildlife within the park. A subsequent molecular study within the same area detected a 2.8%
prevalence of C. ubiquitum in impala and C. bovis in buffalo again near the western park boundary
(Samra et al., 2013). Calves from the bordering communal farmlands of Bushbuckridge were also tested,
revealing a prevalence of 4% for C. andersoni and 4% for C. bovis. Farmers from the area reported buffalo
and impala as the most seen wild species outside the park boundary and 6.2% of these farmers reported
bringing their cattle into the park to drink (Samra et al., 2013). This may suggest spill over from either
livestock and or humans to wildlife or vice versa. Cryptosporidium was later confirmed in children from
the same communal grazing area with a 5.6% prevalence, but predominantly infected with the
anthroponotic species C. hominus (see Samra et al., 2016).

4.4.3: Transfrontier conservation areas

Many protected areas across the globe are clustered along international borders that are usually fenced
preventing the natural migration of large mammals. A management decision was taken to remove a
number of these fences within southern Africa to create one large, protected area known as
Transfrontier Conservation Areas (TFCA) and Transfrontier Parks (TFP) with the aim of conserving
biodiversity and enabling the movement of large mammals, aiding socio-economic development, and
promoting a culture of peace (Hanks, 2003). There are at least 13 TFCAs and TFPs within southern
Africa, six of which include South Africa (Lunstrum, 2011). The Greater Limpopo Transfrontier
Conservation Area (GLTFCA), created in 2002, spans the Limpopo (Mozambique), Kruger (South Africa)
and Gonarezhou (Zimbabwe) National Parks and includes conservancies, wildlife ranches and
communal farmland, covering a total area of 85,000 km2 (Caron et al., 2016; Ferreira, 2004). These areas
have many conservation benefits particularly for animals with large home ranges, such as elephant,
buffalo, and wild dog, that need more space to disperse or hunt (Caron et al., 2016; Cook et al., 2015;
Davies-Mostert et al., 2012). However, there is also now increased potential for pathogen spread
between wildlife, livestock and humans within these areas.
In 2009, a strain of BTb related to buffalo in Kruger National Park was identified in buffalo in
Zimbabwe. Telemetry studies revealed that sub-adult female buffalo were moving long distances
between the national parks and even out of the GLTFCA (Caron et al., 2016). One 2.5-year-old collared
female walked 95 km over 6 days during which she crossed into Zimbabwe and Mozambique and
visited a buffalo herd in the Limpopo National Park (Mozambique) as well as a commercial cattle
ranching area (Caron et al., 2016).

4.4.4: Neighbouring game farms and private reserves

South Africa's economy is rapidly transitioning from livestock-based to wildlife-based agriculture and
ecotourism, with the wildlife industry becoming the fastest growing agricultural sector (Saayman et al.,
2018). Kruger National Park is bordered by several private game reserves and farms. Certain private
wildlife reserves on the western border of the park have removed their fences to allow free movement of
animals, creating the Greater Kruger National Park Complex (GKNPC; Hlokwe et al., 2019). These
reserves are under the jurisdiction of the state veterinary office Bushbuckridge East (Orpen) of the
Mpumulanga veterinary services (Hlokwe et al., 2019). Although this creates larger areas for the wildlife
to roam it also increases the likelihood of interspecific and intraspecific interaction, which can be a
driver for disease transmission, particularly problematic when the disease status of some animals is
unknown. In 2013 a novel M. bovis strain was identified in a blue wildebeest (Connochaetes taurinus) that
had been culled after escaping a private game reserve in the GKNPC (Hlokwe et al., 2014). One or more
translocations of untested blue wildebeest likely brought in this new strain (Hlokwe et al., 2014). In 2014
a different M. bovis strain, not previously detected in South Africa, was found in a female giraffe (Giraffa
camelopardalis) within the same nature reserve (Hlokwe et al., 2019). A kudu on a game farm just south
of Kruger National Park tested positive for an M. bovis strain dissimilar to strains previously isolated
within Kruger wildlife or cattle in that region (Bengis et al., 2001). As kudu can easily cross fences and
are a good candidate reservoir host, they could pose a threat of introducing new strains to the park. The
risk of transmission of M. bovis and other infectious diseases from wildlife is increased by the fact that
testing, or even disease risk assessment, before translocation is only mandatory for buffalo and no other
wildlife species or cattle (Hlokwe et al., 2014, 2019). Thus, the translocation of untested animals (both
wild and domestic) from these private farms and reserves could pose a threat to wildlife in Kruger
National Park and surrounding livestock and humans.

4.4.5: Human-wildlife conflicts and illegal wildlife trade

Protected areas may be thought of as a haven for wildlife with tourists and staff often the only human
inhabitants, yet Kruger National Park, for instance, is bordered by seven municipalities in which
approximately two million people reside (Swemmer et al., 2017). The Limpopo Province surrounding
the north of the park is one of the poorest in the nation with a 67% poverty rate (Warchol and Johnson,
2009). It is therefore important to understand how people and their activities can influence disease
dynamics within and around parks and this must be factored into management decisions. A major
threat to the biodiversity and conservation is the illegal wildlife trade, driven by inequality, poverty,
food insecurity and increasing human populations (Bezerra-Santos et al., 2021; Lindsey et al., 2013). The
illegal wildlife trade is also a significant risk factor for the spread of zoonotic pathogens (Bezerra-Santos
et al., 2021). The trade directly threatens one of Kruger National Park's ‘Big Five’, the white rhinoceros,
poached for their horns to be sold on the Asian market (Annecke and Masubelele, 2016).
Hunting of other game including buffalo and kudu for bush meat is also on the rise in Kruger. What
was once a local subsistence practice, the bush meat trade is now a commercialised, global enterprise
and is no longer sustainable (Warchol and Johnson, 2009). Interviews with communities neighbouring
Kruger National Park suggested that bush meat was poached from the park and readily marketed by
local merchants (Warchol and Johnson, 2009). Several social and cultural factors influenced this
including the affordability of bush meat and the use in traditional weddings, as well as the perception
that parks were not benefiting local communities. There were also reports of game rangers in the park
being complicit with poachers (Warchol and Johnson, 2009). Although there are no studies in Kruger
National Park assessing the role of poaching and bush meat in the spread of zoonotic diseases to
humans or their livestock, these activities have been linked to parasitic outbreaks in humans across the
globe (Bezerra-Santos et al., 2021). Risk assessments are needed to determine the threat of zoonotic
disease spread through the trade and consumption of bush meat from Kruger National Park. Poaching
can also exacerbate the problem of permeable fences as fences are cut or damaged by poachers entering
the park which can lead to animals escaping from the park.

5: Disease knowledge gaps and lessons learnt from African protected

areas
Research underpins Kruger National Park's Strategic Adaptive Management approach but going
forward we need to plug key knowledge gaps with both traditional and novel research techniques. Most
disease-related research within parks have focused on pathogens of economic importance with simple
(direct) life cycles such as FMD and BTb. Pathogens with complex life cycles, such as digeneans, have
been largely neglected. Increased knowledge of these parasites including their genetic diversity and
distribution is hugely important, especially with mounting evidence on the impact of co-infections
(Ezenwa et al., 2010).
There is a global lack of knowledge about the transmission of Cryptosporidium oocysts within the
environment (Innes et al., 2020). African protected areas would provide a good study system to better
understand this transmission and determine exactly how these parasites enter, and possibly accumulate
in parks. It is also crucial to determine whether the species detected negatively affect the fitness of
infected wildlife and whether infection with Cryptosporidium species could increase the pathogenicity of
existing diseases (Beechler et al., 2015a). There is an apparent lack of empirical data for the more
neglected ‘Microscopic Five’ (schistosomiasis and cryptosporidiosis) and for other similar diseases. The
recent pandemic has further hindered the control efforts of neglected diseases, but also highlighted the
importance of monitoring them (Ung et al., 2021). It is imperative that on the ground surveys and
collection of empirical data is carried out to understand disease epidemiology in parks. This data can be
easily and relatively cheaply collected through traditional epidemiological methods, such as serological
surveys and questionnaires, which can be used to analyse the risk of these diseases to people, their
livestock and wildlife within the park.
For some of the more high-profile diseases such as BTb and brucellosis, much empirical data has
already been published and can be assimilated together in mathematical models, which can aid the
adaptive management process. Where it is difficult to acquire sufficient empirical data needed for
traditional modelling of disease dynamics, a Bayesian approach has been suggested as it allows for
diseases to be modelled in complex systems with limited data (Kosmala et al., 2016). Using this
approach to assess the dynamics of BTb in lions in Kruger National Park has revealed that the pathogen
is primarily transmitted from buffalo to lion, while lion to lion transmission is low (Kosmala et al., 2016).
Longitudinal studies are necessary to determine trends over time. One resource that can help are
biobanks, where samples and corresponding data from a variety of wildlife species have been frozen for
decades, over 20 years in the case of Kruger National Park. Such resources should be used to aid our
understanding of long-term trends in disease, which can then be incorporated into mathematical
models for forecasting. Other protected areas should strive to create their own biobanks from collected
samples and data as this can be a valuable resource for disease management as well as species
conservation.
The increasing switch from livestock farming to game farming poses some important questions
regarding the transmission of zoonotic infectious diseases within and around protected areas. Many
national disease control measures, including those for BTb and brucellosis in South Africa, are focused
on vaccination and monitoring of livestock while wildlife is largely neglected. In South Africa disease
testing for BTb is only mandatory for livestock/buffalo but not for other wildlife (DAFF, 2016; Michel et
al., 2019). The translocation of wildlife between game farms and reserves around Kruger National Park
has already brought new strains of BTb, which could spill over into the park (Hlokwe et al., 2014).
Moreover, this could have health implications as the industry for game meat expands globally,
prompting the need to adopt hazard identification and critical control points protocols for safe food
production.
Landscape characteristics influence dynamics of several diseases within African protected areas (Dion
et al., 2011). Kruger National Park's heterogeneous landscape has led to marked differences in disease
dynamics between the northern and southern regions of the park. A handful of studies have assessed
spatial epidemiology within Kruger National Park (Dion and Lambin, 2012; Dion et al., 2011), but this
cannot be achieved without disease prevalence and host-parasite abundance data (Schwabl et al., 2017).
Landscape genetics/genomics has been strongly advocated as a method to overcome these issues
(Schwabl et al., 2017). The method combines spatial and molecular data to assess correlations between
gene flow or local adaptation and landscape features (Schwabl et al., 2017; Storfer et al., 2018). Previous
applications of landscape genetics/genomics have focused on assessing barriers to gene flow within
vulnerable populations to aid conservation efforts (Corlatti et al., 2009; Wasserman et al., 2013). More
recently it has been applied to track parasite transmission (Biek and Real, 2010). However, its use for
parasites with complex lifecycles and multiple hosts is currently limited (Sprehn et al., 2015). Landscape
genomics would be particularly useful in predicting the spread of diseases maintained by important
reservoir species such as buffalo. Tracking the movement or restriction of buffalo gene flow across the
landscape may help to predict the movement of diseases such as BTb and FMD. This approach will be
more complex for pathogens that require an intermediate host as part of their life cycle, such as
schistosomes. In this case landscape genomics could be applied to determine the effect of intermediate
and definitive hosts on gene flow and local adaptation of parasites (Sprehn et al., 2015).
Preventing emerging infectious diseases from entering the park is part of Kruger National Park's 10-
year management plan. Edges of the park, especially those adjacent to human settlements and
farmlands, are important areas for introduction of diseases as seen for M. bovis and Cryptosporidium spp.
(see Samra et al., 2011). Routine monitoring and surveillance of potential diseases is key to the early
detection and prevention of disease outbreaks (Karimuribo et al., 2012; Webster et al., 2016). Surveillance
and monitoring can be simple and should ensure optimal use of the resources available (Karimuribo et
al., 2012). Some innovative examples have made use of available technology such as mobile phone apps
to collect epidemiological data (Aanensen et al., 2009), this opens the possibility of a more participatory
and citizen science approach where locals and tourists could become a part of disease surveillance
programmes. A successful citizen science project was carried out in the Serengeti National Park in
Tanzania, where over 1 million images from a large-scale camera trapping survey were classified by
over 28,000 volunteers (Swanson et al., 2015). Each image was circulated to multiple volunteers and
responses were aggregated, using an algorithm to produce a quality data set with 97.9% agreement with
a subsample analysed by experts (Swanson et al., 2015, 2016). Similar methods could be useful for
notifying mangers of sick or dead animals which could then be monitored for disease. South African
National Parks (SANParks) are developing a surveillance system and training conservation staff to
identify basic disease syndromes and a system for reporting sick or dead animals (Spies et al., 2018).
Involving local people in conservation and disease management is another important strategy as
many drivers of disease dynamics around Kruger National Park are anthropogenic in nature.
Community Based Natural Resource Management has become popular in Africa and in some protected
areas, yet it is important that both parks and communities benefit equally (see Section 6). Community
participation played a key part in the global eradication of rinderpest in 2011 (reviewed by Roeder et al.,
2013), whereby locals were trained in vaccination and sero-monitoring of cattle which enabled 80% herd
immunity in rural areas of Africa. This was successful as local people benefitted from the vaccination of
their livestock and training empowered local communities (Roeder et al., 2013). Social studies are
important to understand how communities perceive protected areas and why human-wildlife conflicts
may occur.
Integrating the needs of the environment, animals and humans underpins the fast growing ‘One
Health’ concept, which is advocated for the control of zoonotic diseases (Webster et al., 2016). There are
several governmental and non-governmental organisations in sub-Saharan Africa that are using the
‘One Health’ approach to monitor and control zoonotic diseases at the human-livestock-wildlife
interface (reviewed by Rwego et al., 2016). As many protected areas have borders which create such an
interface, it is important that park managers work with existing institutions and agencies working in
this field and share data and management strategies. Vaccine research and development is advocated in
many One Health frameworks and there is potential for their development or improvement in each of
the Microscopic Five, however parasitic diseases with complex life cycles and immune invasive
behaviour as seen in schistosomes pose a greater challenge (Driciru et al., 2021).
Although different authorities are responsible for wildlife, livestock and human health, the
intersectoral impact of zoonotic diseases means these authorities all need to take equal responsibility for
monitoring and preventing outbreaks and spread of such diseases. Lack of communication and
 g p g p
cooperation between these sectors can hinder control efforts and the ‘One Health’ approach advocates
working across disciplines to achieve effective control of zoonoses (Randolph, 2020; Webster et al., 2016).
Nevertheless, new funding is essential to coordinate this joint effort. Zoonotic diseases and their
ecological drivers should be brought into the curriculum for student doctors along with knowledge
sharing and joint training between doctors and veterinarians. The Wits rural facility, a campus of the
University of Witwatersrand located near the southwest border of Kruger National Park, aims to
achieve this through a practical training campus where student veterinarians and medics receive
multidisciplinary training right at the human-livestock-wildlife interface.
This transdisciplinary ‘One Health’ approach is being spearheaded by three intergovernmental
organisations the World Health Organisation (WHO), the Food and Agricultural Organisation (FAO)
and the World Organisation for Animal Health (OIE), which formed a Tripartite alliance in 2010
(recently joined by the UN Environment Program (UNEP) forming a Quadripartite; UNEP, 2022; WHO,
2017). While these organisations are important for coordinating global responses to pandemics and
promoting communication and collaboration between nations, participation is voluntary, and they
cannot legally enforce policy. It is therefore the responsibility of local and national government
authorities to create new policies and enforce them. Ministries of health, agriculture, wildlife,
environment, trade and tourism must collaborate and work towards a joint commitment to long term
monitoring and prevention of zoonotic disease outbreaks (Randolph, 2020). Some African countries
have already set up collaborations between different ministries (Rwego et al., 2016).
Initial disease monitoring and prevention should start at the local level but for this to be successful,
coordination is needed at the regional, national and global level. Local communities, farmers and
consumers should also practice behaviours to prevent the spread of diseases, however community
engagement and training is needed to facilitate this. Improvements in policy and regulation in testing
and movement of animals, farming practices and the wildlife trade are also needed and must come from
local and national governments (Randolph, 2020). It is important to bridge the gap between the
management of diseases within protected areas and the surrounding communities and farms. Public
health officials should work with park managers to assess potential threats to human health as well as
threats to wildlife. This has been demonstrated in Uganda Bwindi Impenetrable National Park (BINP)
where the Ugandan wildlife authority, Bwindi community hospital and Kayonza Government Health
Centre, medical and veterinary officers are working together with an NGO called Conservation Through
Public Health (CTPH) to protect gorillas in the park and surrounding communities from disease (Rwego
et al., 2016).
A major barrier to this global co-operation is the lack of political will and lack of funding particularly
in African countries. Public-private partnerships may help alleviate these issues for example the NGO
African Parks has secured long term contracts with governments of 11 countries to manage 19 parks in
central and southern Africa and has mobilised a large funding base (African parks, 2022). Conflicts
between the local population and park authorities in addition to human wildlife conflict can also hinder
the above actions and efforts must be made to rebuild relationships and perceptions which may have
been damaged throughout history. The aftermath of the recent Covid-19 pandemic also poses
challenges and has led to large declines in funding for national parks which rely heavily on tourism for
income (Lindsey et al., 2020). Yet this creates a good opportunity to reflect and re-assess. Parks should
consider diversifying income streams to not be so reliant on international tourism for income, given its
vulnerabilities to shocks such as pandemics and economic recession. The pandemic has proven that
humans are not just threats to biodiversity, but important custodians of nature, and finding diverse
ways to fund their continued involvement as scientists, conservation managers and rangers is important
to ensure a healthy planet for people and nature (Bates et al., 2021).

6: Communities and conservation

Many areas of southern Africa are still inhabited by indigenous tribes, initially hunter gatherers, who's
knowledge of wildlife and land management has been passed down over centuries (Suzman, 2001). In
the early 1900s many of these indigenous people were cleared from their homelands to make way for
national parks and those who remained within parks had little control over the area's resources, creating
conflicts between parks and local communities (Suzman, 2000). Although many parks are now shifting
policies to involve local communities in conservation through Community Based Natural Resource
Management (CBNRM), they still face many challenges and many schemes have been criticised (Blaikie,
2006). According to a metanalysis in 2013, factors contributing most to the success or failure of African
parks in conserving biodiversity, were socio-economic and cultural in nature, highlighting how
important it is for parks to engage with local communities whether they are living within the park or in
bordering areas (Muhumuza and Balkwill, 2013).
In the early days of Kruger National Park, wildlife conservation was the priority and so local
communities were moved out of the park to relocated to its borders, however since the democratic
elections in 1994, South African National Parks policy shifted to include the needs of local communities,
establishing a social ecology department for the first time in 1995 (Anthony, 2007). As the flagship of
SANParks, Kruger National Park developed its own social ecology programme which enables
participatory communication with the 120 surrounding villages and private game farms (Anthony,
2007). One study assessing the attitudes and perceptions of villagers surrounding the park (Anthony,
2007) found that although most participants had a positive perception of Kruger National Park (88.7%)
and were satisfied with living near to the park (70.8%), a large percentage (77.9%) felt that no one in
their household had benefited from the park directly (Anthony, 2007). Positive attitudes towards the
park were significantly associated with having a household member working in the park. Despite the
parks efforts to include community members, more is needed to improve community knowledge and
engagement in its various programmes.
In contrast to the closed system of the Kruger National Park, Bwabwata National Park in Namibia
represents an open system where wildlife and the indigenous Khwe San tribes and their livestock
coexist (Paksi and Pyhälä, 2018; Taylor, 2012). Usage of the park falls into three zones: the Buffalo Core
Area in the west, the Mahango Core Area in the east, which are designated for wildlife only, and the
large mixed use central area, where human settlements and small-scale agriculture are permitted (Paksi
and Pyhälä, 2018). Namibia is known for using conservancies as a model for CBNRM, which consist of
groups of private landowners who self-govern and manage their land collectively, with trophy hunting
and tourism as their two main sources of income (Mufune, 2015). The Khwe San people residing within
Bwabwata National Park, however, cannot form their own conservancy so an official resident's
organisation known as the Kyaramacan Associaton (KA) was formed as an alternative. As the Ministry
of Environment, Forestry and Tourism (MEFT) states that park residents should be actively involved in
park management, they work closely with the KA, providing opportunities similar to those in
communal conservancies (Paksi and Pyhälä, 2018). Despite these efforts though, a recent social study
highlighted that the CBNRM approach was contributing very little to local income and was unstable
compared to other forms of income. Furthermore, residents still perceived the park to value income
from wildlife and tourism over the welfare of the people (Paksi and Pyhälä, 2018).
Although Kruger and Bwabwata National Parks have different management strategies surrounding
local communities, they both seem to face similar challenges and it seems that local people are not
recognising the benefits from the parks and their CBNRM strategies. In order for this relationship to
work both parties must benefit equally otherwise the partnerships will break down. Having good
communication and cooperation with local communities is crucial to aid in zoonotic disease control as
many drivers of disease around protected areas are anthropogenic in nature.

7: Conclusions

(1) This review focussed on five zoonotic diseases of importance at the wildlife-livestock-human
interface, which pose a threat to the economy, public health and conservation and should be
prioritised for monitoring and management. Using Kruger National Park as a case study we
identified the environmental and anthropogenic drivers of disease dynamics for the
‘Microscopic Five’ within and beyond protected areas. Environmental drivers are important in
within-park, wildlife to wildlife transmission and include climate change, heterogeneity of the
landscape, reservoir/maintenance hosts, mixing at water bodies and co-infections.
Anthropogenic drivers are important at the wildlife-livestock-human interface and include
permeable fences, management of livestock, translocation of game animals, illegal wildlife trade
 and transfrontier conservation areas. Environmental drivers may be difficult to control but can
be monitored and management decisions can help mitigate their effects, whereas the
anthropogenic drivers can be controlled by management decisions, communication and
collaboration with local communities, non-government organisations and government
departments.
(2) The wildlife-livestock interface is a key driver of diseases entering and leaving protected areas.
Areas with water sources adjacent to park edges, particularly in the north where livestock and
wildlife densities are greater and more fence breakages occur, should be prioritised for disease
monitoring. Increased human development such as lifestyle estates on the boundary of protected
areas have the potential to introduce additional infections such as Cryptosporidium species. Many
of these interactions are influenced by management strategies including the decision to instal or
remove fencing, creation of water holes or translocation of species. Currently, testing for BTb
and brucellosis before translocation is only mandatory for buffalo yet there are other wildlife
species (and cattle) that are known reservoir hosts. Testing for these high-profile diseases in any
potential reservoir host being translocated across parks and private reserves should be
mandatory. Additionally, if the purpose of wildlife testing is to prevent infection in cattle, then
cattle should be monitored regularly and thoroughly to establish a national negative herd.
Schemes to test and slaughter positive communal cattle and routine testing at abattoir should
also be implemented to identify hot-spot areas.
(3) More research is needed to understand the impact of the ‘Microscopic Five’ on wildlife within
protected areas and other protected areas and particularly how co-infections alter the dynamics
of diseases. For two of the ‘Microscopic Five’, BTb and brucellosis, there are some national
control programs in place, but these may be hindered by co-infections, reservoir species and
climate change. Neglected and emerging diseases such as schistosomiasis and cryptosporidiosis
must be put on the agenda for disease monitoring and control particularly at the human-
livestock-wildlife interface and empirical data is desperately needed to better understand the
threat of these diseases to wildlife, livestock and humans.
(4) Many African protected areas are striving to involve neighbouring communities in their
management approach. Social science and economic studies could help identify mutually
beneficial approaches for both wildlife and people. This would be particularly helpful in
combatting human wildlife conflict and the bush meat trade. As seen in the surrounding
communities of Kruger National Park, complex interactions between cultural practices,
traditions and/or income likely drive human behaviour. Understanding these interactions and
working in mutually beneficial partnerships can help manage some of the anthropogenic drivers
of disease.
(5) Ultimately, conservation of biodiverse ecosystems and maintaining ecological balance is
fundamental to a healthy planet. Protected areas are integral to maintaining this balance yet to
do this successfully, they must adapt and work with the ever-increasing domestic world that
surrounds them, keeping a ‘One Health’ approach in mind. The key knowledge gaps and lessons
from Kruger National Park can be applied across many protected areas and different wildlife-
livestock-human interfaces. On the tail end of a global pandemic, which may well have had its
origin in wildlife, the lesson is clear, holistic approaches to health and well-being are not just
necessary, but vital for our continued existence.

Acknowledgements
We thank Michael Bruford and Sarah Perkins for their comments on earlier versions of this manuscript.
We thank Chenay Simms from Skukuza GIS Office for creating the map of Kruger National Park. This
study was supported by the Natural Environment Research Council GW4 + DTP [NE/L002434/1
studentship to A.V.T].

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Chapter Two: Improving translational power in
antischistosomal drug discovery
Alexandra Probsta,b,†; Stefan Biendla,b,†; Jennifer Keisera,b,* a Swiss Tropical and Public Health Institute, Department of
Medical Parasitology and Infection Biology, Basel, Switzerland
b University of Basel, Basel, Switzerland
† Contributed equally (joint first authorship)
* Corresponding author: email address: jennifer.keiser@swisstph.ch.

Abstract
Schistosomiasis is a poverty-associated tropical disease caused by blood dwelling trematodes that
threaten approximately 10% of the world population. Praziquantel, the sole drug currently
available for treatment, is insufficient to eliminate the disease and the clinical drug development
pipeline is empty. Here, we review the characteristics of the patent Schistosoma mansoni mouse
model used for in vivo antischistosomal drug discovery, highlighting differences in the
experimental set-up across research groups and their potential influence on experimental results.
We explore the pharmacokinetic/pharmacodynamic relationship of selected drug candidates,
showcasing opportunities to improve the drug profile to accelerate the transition from the early
drug discovery phase to new clinical candidates.

Keywords
Anthelminthics; Drug discovery; Schistosoma mansoni; Schistosomiasis; Mouse model;
Pharmacokinetics/pharmacodynamics

1: Filling the drug pipeline for schistosomiasis

Schistosomiasis, a disease with a tremendous global burden, is caused by the blood dwelling trematodes
of the genus Schistosoma. Associated with poverty, approximately 10% of the world population is at risk
for an infection with one of the three clinically important species, Schistosoma haematobium, Schistosoma
japonicum and Schistosoma mansoni (King, 2010; WHO, 2020). Praziquantel (PZQ) is the sole drug
currently available for the treatment of schistosomiasis (Colley et al., 2014; WHO, 2020). Ever since its
discovery by Bayer AG, Leverkusen and Merck KGaA, Darmstadt decades ago, praziquantel has been
the key contributor to control schistosomiasis (within preventive chemotherapy campaigns) by reducing
the disease morbidity (Spangenberg, 2021). However, praziquantel is far from being a perfect drug. The
fact that it is rarely curative coupled to the main drawback of the drug, which is its inactivity against
developing infections, call for a better understanding of the underlying mechanisms of drug action (only
recently, a transient receptor (TRP) channel in S. mansoni has been discovered (Sm.TRPPZQ) (Park and
Marchant, 2020)), as well as the discovery and development of new drugs in the fight against
schistosomiasis (Panic and Keiser, 2018; Spangenberg, 2021).
With the aim to eliminate schistosomiasis as a public health problem by 2030 (WHO, 2020), the drug
discovery landscape has gained momentum over the past few years, with research efforts mainly driven
by academic institutions. Different research groups all over the globe have brought promising drug
candidates forward, as recently summarized (Dziwornu et al., 2020). Despite the preclinical pipeline
being filled with several drug candidates (repurposed, rescued and new chemotypes), many gaps
remain in the understanding of the profile of a compound required for the treatment of schistosomiasis.
To facilitate the discovery of novel antischistosomal drugs this review aims to increase our
understanding of key elements of the schistosome drug discovery process, including the predictive
pharmacology of lead compounds as well as the experimental setup used. We first summarize
characteristics on the most commonly used in vivo model, the S. mansoni mouse model. Moreover, we
highlight new findings on praziquantel, filling some remaining knowledge gaps of the drug of choice.
Lastly, we aim to shed light on the required pharmacokinetic/pharmacodynamic (PK/PD) relationship
analysing the correlation between kinetic disposition and in vivo drug efficacy of selected candidates. A
better understanding of the profile of a compound required for antischistosomal treatment and the
available tools might help to accelerate the transition from the early drug discovery phase to the
preclinical stage and eventually pave the way for a new clinical candidate (Box 1).

Box 1

Highlights

• Praziquantel activity against ex vivo adult S. mansoni isolates of South American and Western
African origin does not differ significantly.
• Academic research groups distributed over only a few public institutions drive in vivo drug
efficacy research on S. mansoni. Biologic variability of mouse strain and gender as well as parasite
isolates origin offer the advantage of obtaining a broader coverage of naturally occurring
pathogen and host disease variability.
• Infection intensity of the patent S. mansoni mouse model follows a linear correlation to the
number of cercariae s.c. injected per mouse.
• Praziquantel plasma exposure does not correlate with the in vivo efficacy in juvenile S. mansoni-
infected mice.
• Chronic S. mansoni infection influences exposure parameters of drugs.
• No general rules apply regarding the correlation between in vivo drug efficacy and kinetic
disposition; experimental differences must be taken in account when evaluating PK/PD
relationship.

2: Evaluating the importance of S. mansoni isolate origin for early

antischistosomal drug discovery
Phenotypic activity screens of compound libraries against S. mansoni are still the mainstay in
antischistosomal drug discovery to provide small drug-like molecules as starting points for drug
development programs (Biendl et al., 2021; Pasche et al., 2018). Recent multicenter activity screens of the
same drug libraries, however, resulted in only limited overlap in identified hit compounds between
different laboratories and these inconsistencies were proposed to be caused by differences in the
parasite strain among others (Maccesi et al., 2019; Panic et al., 2015). To examine the hypothesis of
differences in S. mansoni strains driving outcomes of antischistosomal drug activity tests, we evaluated
the phenotypic response of adult schistosomes, derived from two different isolate origins, to
praziquantel in the same laboratory. Such an evaluation allows excluding differences in assay
methodology and read-out (and weighing thereof, see below) which are the main drivers of inter-
laboratory inconsistencies. We selected isolates from South America (Brazil) and western Africa
(Liberia), to cover a large spatial distance of isolate origin (see Methods in Supplementary Material).
Following exposure to praziquantel, viability of ex vivo adult S. mansoni of both isolates decreased
rapidly. The fast onset of action resulted in high double-digit to low triple digit nanomolar EC50 values
after 1 h of drug exposure, independent of isolate origin (Fig. 1, Table S1 in Supplementary Material).
Drug activity did not improve significantly for prolonged exposure times. In summary, we did not
observe significant differences of praziquantel or (R)-praziquantel action against both tested isolates for
any evaluation time points (Fig. 1). Therefore, we hypothesize that there is not sufficient reason to
expect differences in the response of new compounds on different isolates. Thus, we believe that for
early antischistosomal drug discovery it is sufficient to initially screen compounds against single isolates
only. However, for compounds that have further advanced the antischistosomal drug discovery
cascade, characterization of in vitro and in vivo drug activity against multiple strains offers additional
value, to potentially identify compound specific effects. Recent studies of different isolates and strains at
the genomic and transcriptomic levels showed remarkable differences (Berger et al., 2021; Doyle and
Cotton, 2019; Gower et al., 2013; Stroehlein et al., 2022)—some of these might have an influence on drug
sensitivity. Hence, further studies evaluating the effect of such differences on drug efficacy are required.

FIG. 1Praziquantel (PZQ) and (R)-Praziquantel (R-PZQ) in vitro activity against adult
S. mansoni from isolates of Brazilian and Liberian origin after 1, 24 and 72 h of drug
exposure. Each point represents the mean EC50 value, and the error bars represent
the 95% confidence interval of the mean.

3: The S. mansoni mouse model for drug efficacy testing

Academic research groups distributed over only a few public institutions drive in vivo drug efficacy
research on S. mansoni. Besides high research activity in Brazil, where S. mansoni still presents a public
health concern, and Egypt, where Theodor Bilharz first described Schistosoma parasites, for example,
groups at Swiss TPH, LSHTM and the University of California continuously publish evaluations of new
or repurposed compounds in the S. mansoni mouse model. We collected and summarized characteristics
of host mice, S. mansoni infection and evaluation methods employed by key institutions driving
antischistosomal drug discovery, to allow appraisal and comparisons of results generated in these
laboratories (Table 1).

Table 1

The S. mansoni mouse model for drug efficacy testing.

Host Infection Eval
Intensity Duration
Age at Animals post
Institution Isolate [number [weeks] Wo
Strain infection Sex per Route treatment
origin of rec
[weeks] group [weeks]
cercariae] ad. j.
Swiss NMRI 4 f 4 Liberia s.c. 100 49 21 2–3 Per
TPH

LSHTM CD1 5 f 5 Puerto s.c. 150 42 21 1–2 Per

Ric
o
UCSD/ Swiss 3–6 f 3–6 Puerto s.c. 140–150 ≥ 42 21 2 Per
UCSF web Ric
ster o
TBRI CD1 NR m 10 Egypt imm. 80 49 21 1d-2 Per
UNG Balb/c, 3 NR 3–10 Brazil imm.,s.c. 70–80 ≥ 42 21 2–3 Per
Swi (B
ss H)
FIOCRUZ Albino NR f 10 Brazil s.c. 100 45 NR 1d-2 Per
(LE
)
The table summarizes characteristics of host mice, S. mansoni infection and evaluation methods employed by key institutions working on
antischistosomal drug discovery and development.
Abbreviations: Swiss TPH: Swiss Tropical and Public Health Institute, Basel, Switzerland; LSHTM: London School of Hygiene and Tropical
Medicine, London, United Kingdom; UCSD: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego,
San Diego, USA; UCSF: University of California San Francisco, San Francisco, USA; TBRI: Theodor Bilharz Research Institute, Giza,
Egypt; UNG: Universidade Guarulhos, Guarulhos, Brazil; FIOCRUZ: Laboratory of Schistosomiasis, Rene Rachou Research Center
Fiocruz, Belo Horizonte, Brazil; BH: Belo Horizonte; imm.: tail (UNG) or body (TBRI) immersion; s.c.: subcutaneous; ad.: adult; j.: juvenile;
Perfusion: reverse perfusion of the hepatic portal system; m: male; f: female; d: day; Ref.: Reference; NR: not reported.
References: (a): (Biendl et al., 2021; Lombardo et al., 2019a; Probst et al., 2020b). (b): (Gardner et al., 2021). (c): (Wolfe et al., 2018). (d):
(Botros et al., 2020). (e): (de Moraes et al., 2014; Silva et al., 2017, 2021; Xavier et al., 2020). (f): (Castro et al., 2018; Katz et al., 2013).

Generally, young animals are infected shortly after purchase and acclimatization to improve
susceptibility to infection. Further, there is good accordance between research groups on infection
duration to achieve juvenile and adult (patent infection) worm life stages reflecting the parasite life cycle
in the host organism.
We identified three main variations influencing mouse model characteristics of the various
laboratories and complicating direct comparability of experimental results. First, utilization of mice of
different strain and gender. All research groups use other but similar general purpose mouse strains
(e.g. NMRI, Swiss Webster), with the exception of researchers at LSHTM and TBRI both employing CD1
mice (Botros et al., 2020; Gardner et al., 2021). However, researchers at TBRI utilize male mice, while
most other groups, including the group at LSHTM, utilize female mice in their studies due to their
increased susceptibility to infection (Eloi-Santos et al., 1992; Nakazawa et al., 1997). The effect of mice
strain on experimental end-points remains largely unknown, but is likely to influence susceptibility to
infection, parasite and disease development as well as drug pharmacokinetics (Bickle et al., 1980;
Cheever et al., 1987), similar to mouse gender. Finally, insufficient reporting of mouse characteristics
occasionally impedes complete appraisal of individual study methodology (Katz et al., 2013; Silva et al.,
2021; Xavier et al., 2020).
Second, utilization of parasites of different isolate origin (described above) as well as infection route
and dose. Although mouse body or tail immersion into a cercariae suspension was predominantly used,
especially by groups from the Middle East and South America (Botros et al., 2020; de Moraes et al., 2014;
Silva et al., 2017), s.c. infection nowadays is most commonly employed, likely due to the ease of
application and higher control over the quantity of introduced parasites (Gardner et al., 2021; Lombardo
et al., 2019a; Silva et al., 2021). Infection intensity varies between 70 and 150 applied cercariae per mouse
rendering an optimized trade-off between worm yield and severity of disease responsibly for disease
burden and stress of the animal subject.
Third, worm recovery methodology and drug effect evaluation. Worm burden reduction (WBR) is
consensually considered as the main read-out for drug effect evaluation. Additionally, most groups,
except the European groups at LSHTM and Swiss TPH, evaluate drug effect on egg reduction rate.
Differential weighing of such additional read-outs in drug effect evaluation can provoke minor
deviations in conclusions arising from consistent experimental results. Further, to accurately determine
worm burden, worms need to be removed exhaustively from sacrificed mice and counted. While reverse
perfusion of the hepatic portal system is the mainstay of adult worm recovery and the only practicable
methodology for the recovery of the smaller juvenile worms, direct picking of adult worms from their
dwelling site after mouse dissection provides equivalent results and offers an alternative procedure that
is less time sensitive and offers the advantage that the hepatic shift can be determined. While most other
of these identified variations are likely to affect experimental results only slightly, differences in mouse
strain and gender are gateways for biologic variability potentially resulting in discrepancies between
laboratories. However, especially the later differences in addition to utilization of parasite isolates of
different origin offer the advantage of obtaining a broader coverage of naturally occurring pathogen and
host disease variability and might thus improve translatability of laboratory examinations to real-world
environments.

4: Infection intensity of the patent S. mansoni mouse model

As mentioned above, WBR is the main efficacy read-out employed in in vivo studies evaluating
antischistosomal drug activity. Calculation of the WBR is based on the absolute worm count in infected
and treated mice compared to infected, but untreated (vehicle control) mice (see Methods, 2.6 Data
analysis in Supplementary Material). Thus, the worm load of experimental mice might impact efficacy
results and their statistical significance.
To better understand the underlying biological variation and statistical distribution of infection
intensities achieved by s.c. infection with S. mansoni cercariae, we reanalysed adult worm yields from
control mice studied in experiments conducted in our group during the last decade of continued
research activity (see Methods in Supplementary Material).
Infection intensity of the patent S. mansoni mouse model follows a linear correlation to the number of
cercariae injected per mouse, where roughly every fourth s.c. injected cercariae develops into an adult
worm (Fig. 2). At the most commonly employed infection dose for many years for drug activity studies
in our laboratory of 100 cercariae per mouse, (Biendl et al., 2021; Probst et al., 2020b) our S. mansoni
mouse model yields 22 adult worms on average. Adult worm infection intensity increases on average by
28 adults for every additional 100 cercariae injected, and this rate remains stable up to the maximally
tested dose of 400 cercariae per mouse (Table S2 in Supplementary Material). Our linear model is in
good accordance with observations from other groups working in the field (see Table 1) (Castro et al.,
2018; Gardner et al., 2021; Wolfe et al., 2018; Xavier et al., 2020). For other infection doses frequently
employed in these laboratories, namely, 80 and 150 cercariae per mouse, our linear model estimates
yields of ca. 17 and 36 adult worms, respectively. Notably, infection by whole body or tail appears to
yield higher worm counts compared to s.c. infection (Botros et al., 2020; Silva et al., 2017).
Another random document with
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of the system was the clan, the members of which, forming a close
brotherhood, were bound to their lord by ties of affection and fidelity
like those which in Europe theoretically bound the retainer to his
193
lord. This system exerted a great influence upon the moral type. It
developed a martial ideal of character known as Bushido, many of
the virtues of which are almost identical with corresponding virtues in
the European ideal of chivalry. Probably this system has had more to
do with creating in Japan a moral consciousness in many respects
like our own than has any other single agency. To the lack in the
Chinese social system of any institution like Japanese feudalism
may be ascribed in part at least the wide difference which exists
between the moral ideals of the two peoples, especially in regard to
the rank assigned the military virtues.

Confucianism Along with the Chinese classics Confucianism was

introduced into Japan about the middle of the sixth century of our
era, and being in perfect accord with the native system of Shinto and
with the Japanese ways of thinking, this cult of ancestors tended to
reënforce native ethical tendencies and thus contributed essentially
to make the virtues of filial obedience and reverence for superiors
prominent in the growing type of character.

Buddhism Buddhism was introduced into Japan in the sixth

century of our era. Its incoming had deep import for the moral life of
the Japanese people. It inculcated the gentler virtues, exerting here
in this respect, as elsewhere in the Far East,—save in China, where
it too quickly became shockingly degenerate,—an influence like that
exerted by Christianity in the Western world. It helped to make
gentleness, courtesy, and tenderness distinctive traits of the
Japanese character. Through the regard which it instilled for dumb
animals it placed the whole lower world of animal life under the
194
protection of the moral sentiment.

Western A little more than a generation ago the civilization of

civilization
Japan came into vital contact with the civilization of
the West. Almost every element of the old Japanese culture has felt
the modifying effect of this contact. The political, the economic, the
social, the domestic, and the religious institutions have undergone or
are undergoing great changes. These changes in these departments
of life and thought have caused, as such changes always do,
important modifications in ethical sentiments and convictions. Of all
the influences which for more than two thousand years have been at
work shaping and molding the moral ideal of the Japanese nation,
those now entering from the Occidental world will doubtless leave
the deepest impress upon the ethical type.
In a still more direct way is this contact of Japan with Western
civilization resulting in important consequences for Japanese
morality. Christian ethics, like Buddhist ethics, is making a strong
appeal to certain classes of Japanese society. The result is what in
an earlier chapter was designated as a “mingling of moralities” and
the creation of a new composite conscience.

II. The Ideal

Bushido The heart of Japanese morality is to be sought in

195
Bushido, the ethics of the samurai. We shall best
understand this moral code by thinking of it as the Japanese ideal of
chivalry or, perhaps better, as a blending of the Western chivalric,
Spartan, and Stoic ideals of goodness and nobility, since in the list of
virtues making up the Bushido ideal we find several of the cardinal
virtues entering into each of these three distinct types of character.
As we have already intimated, Bushido is an ideal of excellence
which grew up out of the root of Japanese feudalism, just as the
Western ideal of chivalry developed out of European feudalism. It
was essentially an ideal of knighthood, the prime virtue of which was
personal loyalty to one’s superior. Fealty to one’s chief was made so
dominant a virtue that it overshadowed all other virtues. In the
defense or in the service of his lord a samurai might commit, without
offense to his sense of moral right, practically any crime, such as
blackmailing, lying, treachery, or even murder.
Grouped about this cardinal virtue of loyalty were the other
knightly virtues of courage, fidelity to the plighted word, liberality,
self-sacrifice, gratitude, courtesy, and benevolence. Liberality, or
free-handedness, was carried to such an extreme as to become a
defect of character. The true samurai must have no thought of
economy and money-making. “Ignorance of the value of different
196
coins was a token of good breeding.” To handle money was
thought degrading.
In one respect the code of honor of the Japanese knight was
wholly unlike that of the Western knight. It did not include any special
duty to woman. “Neither God nor the ladies inspired any enthusiasm
in the samurai’s breast.”
The Spartan element in the samurai code appears particularly in
the training of the youth. The boy was taught always to act from
motives of duty. He was denied every comfort. His clothing and his
diet were coarse. He was allowed no fire in the winter. “If his feet
were numbed by frost, he would be told to run about in the snow to
make them warm.” To accustom him to the sight of blood, he was
taken to see the execution of criminals; and to banish foolish fear
from his mind, he was forced to visit alone at night the place of
197
execution.
The Stoic element in the ideal appears in the high place assigned
to the virtue of self-control. The samurai, like the Stoic, must
suppress all signs of his emotions. Like the Stoic, too, he must have
courage to live or courage to die, as enjoined by duty. And his code
of honor taught him what true courage is: “It is true courage to live
198
when it is right to live, and to die only when it is right to die.”
This samurai ideal of character constitutes, as we shall see, a
molding force in the moral life of Japan. Bushido, it is true, died with
199
the passing of feudalism, but the spirit of Bushido lived on. The
samurai’s sense of honor and of duty became the inheritance of the
Japanese people. This great bequest of honor and valor and of all
samurai virtues is, in the words of the author of the Soul of Japan,
but “a trust to the nation, and the summons of the present is to guard
this heritage, nor to bate one jot of the ancient spirit; the summons of
the future will be so to widen its scope as to apply it in all walks and
200
relations of life.”

The virtue of The belief in the divine origin of the imperial house
loyalty to the
Emperor, or
of Japan makes loyalty to the Emperor the supreme
patriotism 201
duty. During the ascendancy of feudalism this duty,
in so far as the samurai class was concerned, was, it is true,
overshadowed by the duty of loyalty to one’s immediate feudal
superior. The sentiment due the Emperor was intercepted by the
daimyos. But in theory loyalty to the imperial house has ever been
the paramount virtue of the Japanese. The Emperor’s command is to
his subjects as the command of God to us, and obedience must be
perfect and unquestioning. So dominant is the place assigned this
virtue of loyalty to the head of the nation that the Japanese moralist
seems almost to make morality consist in this single virtue, as if “to
fear the Emperor and to keep his commandments” were the full duty
202
of man.
This sentiment of the people toward the imperial family renders
the government a sort of theocracy. Hence patriotism with the
Japanese is in large measure a religious feeling. Indeed, patriotism
has been called the religion of the Japanese. It is this virtue, exalted
to a degree which the world has never seen surpassed, which has
contributed more than any other quality of the Japanese character to
make Japan a great nation and to give her the victory over a
powerful foe in one of the most gigantic wars of modern times.

Family ethics If the first duty of the Japanese is to his Emperor,

his second is to his parents. In Japanese phrase, the two virtues of
loyalty and filial piety are “the two wheels of the chariot of Japanese
203
ethics.” Shinto and Confucianism, as we have seen, have both
contributed to the fostering in children of the moral sentiments of
grateful love, reverence, and obedience toward parents and all
ancestors living and dead. The Japanese regard the high place
assigned to these filial duties in the standard of character as a mark
204
of the vast superiority of their morality to ours. The sentiments of
filial affection and reverence, coloring as they do the whole moral
life, lend to Japanese society an ethical cast which places it in many
respects in strong contrast to the social order of the Western nations,
and makes it difficult for the Japanese to understand us and for us to
205
understand them.

Woman as wife As respects the position of woman the family ethics

and as mother
of Japan are the family ethics of the East. In a work
from every page of which breathes the spirit of the Orient, a
Japanese writer, dwelling upon the difference between the ethical
sentiments respecting family relationships which have been evoked
by the different social environments of the East and the West, says:
“In the East woman has always been worshiped as the mother, and
all those honors which the Christian knight brought in homage to his
206
ladylove, the samurai laid at his mother’s feet.”
Lafcadio Hearn, touching upon this same feature of the family
ethics of the Japanese, declares that the Bible text, “For this cause
shall a man leave his father and mother and shall cleave unto his
wife,” is, to their way of thinking and feeling, “one of the most
207
immoral sentences ever written.”
In another important respect does the domestic morality of the
Japanese differ essentially from that of the Christian West. The
family is not strictly monogamous, as with us. The moral sense of the
208
Japanese discerns nothing wrong in polygamy or concubinage.
As respects the whole relation of marriage, the Japanese appear to
be in about the same stage of evolution as had been reached by the
Hebrews at the time of Abraham.
 A chief virtue of the Japanese women in all their relations is
obedience to the one—whether father, or husband, or son—to whom
obedience is due. It is the setting of this duty before all other duties
that causes the Japanese women sometimes to do what appears to
us immoral, but which seems to them truest piety and noblest self-
209
sacrifice. In loyalty to duty, as they interpret duty, they maintain a
210
standard rarely surpassed by the women of any land.

Suicide Suicide is infrequent among savage and barbarian

regarded as a
virtuous act
races, but is common among all peoples in an
advanced stage of civilization. It is not so much the
fact itself of self-destruction that claims the attention of the historian
of morals, as the light in which the act is viewed; that is, whether it is
considered a virtuous or a reprehensible deed.
Now the Japanese regard suicide, if prompted by a good motive,
as a justifiable and noble act. The motives with them for the deed are
various, as in the case of other peoples, but among these motives is
one which discloses the existence among the Japanese of a
sentiment unknown or almost unknown among ourselves. The deed
is often committed in the way of making a solemn protest against
something disapproved of in the conduct or acts of others. Thus
when the Japanese government after war with China in 1898
acceded to the demands of Russia, France, and Germany
respecting the recession to China of certain territories on the
Continent, forty men in the Japanese army, by way of protest,
211
committed suicide “in the ancient way.”
Tyrannicide is also looked upon by the Japanese as an heroic and
praiseworthy deed, provided the person committing the act makes
clear the self-sacrificing and patriotic character of his motives by at
once taking his own life.

Low estimation A marked defect of the moral standard of the

of the virtue of
truthfulness
Japanese is the low place assigned to the virtue of
truthfulness. Among the Japanese, to call a person a
liar is not to apply to him a term of reproach, but rather to pay him a
pleasant compliment as a person of tact and shrewdness.
This lack of reverence for truth probably springs in part from the
virtue of politeness as a root. The extreme emphasis laid upon
courtesy as the sign and expression of reverence and loyalty toward
superiors fosters the general habit of saying things which are
pleasant and agreeable whether they are true or not. This
complacent disregard of truth in social intercourse would seem to
have dulled the sense of obligation of truth-speaking in other
relations.

III. Some Significant Facts in the Moral

History of Japan

General The Japanese knightly ideal, which, as we have

influence of the
said, constitutes the heart and core of theoretical
ideal of Bushido
Japanese morality, has a history somewhat like that of
the ideal of European knighthood. It was a lofty ideal very imperfectly
realized, yet realized to such a degree as to make it a chief motive
212
force in the political and social life of Japan for several centuries.
It left a permanent impress upon the moral consciousness of the
Japanese nation, an impress certainly deeper and more enduring
than that left by the ideal of European chivalry upon the moral
consciousness of the peoples of Western Europe. New Japan is
directly or indirectly the creation of Japanese knighthood.
We have seen that loyalty to his chief was the preëminent virtue
of the samurai. Upon the downfall of feudalism this loyalty was
transferred to the Emperor. The spirit of the samurai came to inspire
the Japanese nation. Since the time when the loyalty of Scottish
clansmen to their chief was transferred to Scottish royalty, there has
not been seen a more remarkable example of the absolute devotion
of a people to their sovereign than that exhibited to-day by the
people of Japan.
 The samurai were taught to despise the love of gain, and thus
these knights of Japan were strangers to those vices which spring
from the love of money. To this circumstance may be ascribed the
fact that the statesmen of Japan, who almost invariably are of the
samurai class, have been so notably free from venality and
213
corruption.
Finally, Bushido held aloft a high standard of truthfulness. The
true samurai regarded an oath as a derogation of his honor. It cannot
be affirmed that this Bushido virtue of veracity has yet become the
inheritance of the mercantile and peasant classes of Japan, but it
has at least been retained by the samurai as a class, and is working
to-day like leaven in the mass of Japanese society.

The Bushido There are two remarkable passages in recent

code in action
Japanese history which well illustrate in what way and
to what degree the spirit of the samurai, “the spirit of not living unto
one’s self,” has become an inspiration to the whole Japanese nation.
The first passage has to do with the Russo-Japanese War of 1904–
1905, which on the part of Japan was a struggle for national
existence. It was the samurai morality, a morality of loyalty, of valor,
of selflessness, of fidelity to duty, that formed a chief element of the
strength of Japan in that critical juncture of the nation’s life. The
Bushido code of honor showed itself equal to the Spartan code in
creating a race of invincible warriors. Since the Spartan Leonidas
and his companions died for Greece in the pass of Thermopylæ
there has been no sublimer exhibition of fortitude and self-devotion
in a great cause than that shown by Japanese soldiers in the
trenches before Port Arthur and on the battlefields of Manchuria.
This war for national independence also afforded proof of how the
gentle virtue of Japanese knighthood, courteous generosity to the
vanquished, has passed as a noble legacy to the nation at large; for
as an eminent Japanese statesman affirms, “In the tender care
bestowed upon our stricken adversary of the battlefield will be found
214
the ancient courtesy of the samurai.”
 The second passage shows the morality of the
The moral samurai in competition with the morality of the
standard of the common Japanese shopman. Now the morality of the
samurai in
competition with
plebeian Japanese trader is about on a level with that
that of the 215
plebeian trader
of the ancient Greek shopkeeper. And a chief
cause of his low moral standard is the same, namely,
the general disesteem in which the trader’s business has been held.
This social stigma has resulted in the mercantile business being left
in the hands of the lowest class socially, intellectually, and
216
morally. The great mass of the people have from time immemorial
been engaged in the honorable business of agriculture; while the
samurai class, as we have seen, regarded it as degrading to engage
in trade or even to handle money. In these circumstances it was
inevitable that the mercantile class should evolve a very low code of
business ethics; for, as the author of Bushido very justly observes,
“put a stigma on a calling and its followers adjust their morals to it.”
The strictly class character of this loose commercial morality is
shown by the experience of the samurai after the abolition of
feudalism in 1868. Upon that event many of them engaged in
mercantile business, carrying with them their high moral standard,
with results pathetically depicted by Nitobé in these words: “Those
who had eyes to see could not weep enough, those who had hearts
to feel could not sympathize enough, with the fate of many a noble
and honest samurai who signally and irrevocably failed in his new
and unfamiliar field of trade and industry, through sheer lack of
shrewdness in coping with his artful plebeian rival.... It will be long
before it will be recognized how many fortunes were wrecked in the
attempt to apply Bushido ethics to business methods, but it was
soon apparent to every observing mind that the ways of wealth were
217
not the ways of honor.” About ninety-nine out of every hundred
samurai who ventured into business are said to have failed.
This passage out of the history of New Japan carries with it
various lessons, but particularly does it teach how unjust it is to
218
judge the morality of a people by the morality of a class.
 Notwithstanding the disastrous outcome of their first venture into
the mercantile field, the samurai still remain in business, so that
there is going on to-day in Japan in the commercial domain a
competition between two moral standards. The triumph of the
standard of the samurai over that of the plebeian trader would mean
the development in Japan of a matchless business morality, which,
in the increasing closeness of commercial relations between the
East and the West, might well act cleansingly on our own business
219
ethics.

Moral education The rapid transformation in the institutions and

in the schools;
the Imperial
ideas of Old Japan after the revolution of 1868 created
Rescript a crisis in the moral life of the Japanese people. The
old basis of the national morality was destroyed.
Reverence for the Confucian teachings was lost. Respect for
ancestral customs was seriously impaired. Moral anarchy impended.
In this critical juncture some proposed that Buddhism, others that
Christianity, should be made the basis of the moral code.
Especially in the schools was the urgency of the need of some
new sanction for morality felt, because moral instruction and training
have always formed an essential part of the education of the youth of
Japan. The Japanese have ever believed that it is possible to mold
the character of the nation by education. “With us,” says a native
writer, “education has meant moral education more than anything
220
else for centuries.” “The object of teaching,” says the official
regulations for teaching in elementary schools, “is to cultivate the
moral nature of children and to guide them in the practice of
221
virtues.” Because of this central place assigned moral education
in the work of the schools, the necessity for removing all uncertainty
as to what should be inculcated was all the more exigent.
To meet the crisis the following imperial rescript was issued—
certainly one of the most remarkable state papers ever promulgated:

“Know ye, our subjects:

 “Our imperial ancestors have founded our Empire on a basis
broad and everlasting and have deeply and firmly implanted
virtue; our subjects, ever united in loyalty and filial piety, have
from generation to generation illustrated the beauty thereof. This
is the glory and the fundamental character of our Empire, and
herein also lies the source of our education. Ye, our subjects, be
filial to your parents, affectionate to your brothers and sisters; as
husbands and wives be harmonious; as friends true; bear
yourselves in modesty and moderation; extend your
benevolence to all; pursue learning and cultivate arts, and
thereby develop intellectual faculties and perfect moral powers;
furthermore, advance public good and promote common
interests; always respect the Constitution and observe the laws;
should emergency arise, offer yourselves courageously to the
state; and thus guard and maintain the prosperity of our imperial
throne coeval with heaven and earth. So shall ye not only be our
good and faithful subjects, but render illustrious the best
traditions of your forefathers.
“The way here set forth is indeed the teaching bequeathed by
our imperial ancestors, to be observed alike by their
descendants and the subjects, infallible for all ages and true in
all places. It is our wish to lay it to heart in all reverence, in
common with you, our subjects, that we may all thus attain to the
same virtue.
“The 30th day of the 10th month of the 23d year of Meiji”
222
[1890].

It would be almost impossible to exaggerate the influence of this

imperial edict. “Our whole moral education,” affirms Baron Kikuchi,
“consists in instilling into the minds of our children the proper
223
appreciation of the spirit of this rescript.” The children learn it by
heart just as the Roman children committed to memory the Twelve
Tables of the laws.
Japanese believe that the effect of this instruction upon the
national character, reënforcing the ancestral virtues of loyalty and
 224
devotion to duty, was exhibited in the recent war with Russia.
A noteworthy feature of the rescript is that it is simply a
reaffirmation of the teachings of the ancient moralists and the ethical
traditions of the fathers—an inculcation of those virtues of loyalty and
filial piety which the Japanese people have held in esteem and
practiced from generation to generation.
A second feature of the edict which arrests attention is the
universalistic and secular character of the morality inculcated. The
virtues enjoined are universal benevolence, loyalty to duty, and self-
devotion to the common good—a morality of the universal human
heart and conscience, a morality, as the edict declares, good for all
ages and for all places.

Japanese The foregoing anticipates and gives answer to the

morals and
Western
questions: What will be the effect upon Japanese
civilization morality of those changes now going on in the life and
thought of Japan through contact with the civilization
of the West? What will be the effect upon Japanese public morality
when the common belief in the divine descent of the Emperor, which
is the root from which springs the primal duty of loyalty, is
undermined, as modern science is certain to undermine it? What will
be the effect upon Japanese domestic morality when Occidental
conceptions of the family and of woman’s place in it come to modify,
as they seem likely to do, those ideas and sentiments which from
time immemorial have formed the basis of the family ethics of the
East? What will be the ethical consequences when Western science
renders obsolete the Shinto learning and the Confucian classics,
which have hitherto formed the basis of so large a part of Japanese
morality? What will be the effect upon the ancient ideal of character
of the adoption of Christian ideas and teachings in place of those
which have so long nourished the ethical feelings and sentiments of
the Japanese people?
That the intrusion into the ancient culture of Japan of these
various elements of Western civilization has deep import for
Japanese morality cannot be made a matter of doubt. In the new
environment, so different from that in the midst of which the ancient
ideal of goodness was developed, this ideal must inevitably undergo
important changes. Some of those qualities of character which have
so long held high places in the ideal of excellence will cease to
evoke the old-time homage, while other qualities at present assigned
low places in the standard will be exalted. Virtues now practically
unrecognized by the Japanese as virtues, but which among us are
highly esteemed moral qualities, will certainly be incorporated in the
modified ideal, giving it a new cast, yet probably without changing
fundamentally the type; for the moral life of the Japanese people is
too virile and too essentially sound to permit us to think that the new
influences now coming in will produce such radical changes in the
ethical feelings and convictions of the race as to result in a repetition
of what happened upon the entrance of Christianity into the morally
decadent Greco-Roman world—the displacement of the old ideal of
character by a new and essentially different ideal.
 CHAPTER VII
THE ETHICAL IDEALS OF INDIA

PART I. THE ETHICS OF

BRAHMANISM—A CLASS MORALITY

I. Historical and Speculative Basis of the

System

The conception As in Judea so in India the conception formed of

of the First the Supreme Being reacted potently upon morality.
Cause​— ​
Brahma Hence in naming the influences under which the moral
ideal of Brahmanism was molded we must speak first
of the Indian conception of the First Cause.
The Aryan conquerors of India originally held notions of the gods
in general like those held by their kinsmen, the early Greeks and
Romans. When they entered India they were ancestor worshipers
and polytheists. They had earth gods and sky gods. The gods of the
celestial phenomena gradually acquired ascendancy. Then, as in
Egypt, there came a tendency toward unity. The various gods came
to be looked upon by the loftier minds as merely different
225
manifestations of one primal being.
It is right at this point that we find the great antithesis between
Indian modes of thought and those of all or almost all other peoples.
When the thinkers of Egypt, of the Semitic lands, of Persia, of
Greece and Rome, had at last through reflection evolved the lofty
conception of a single great First Cause, they endowed this cause
with conscious personal life. This mode of thought is our heritage
from the past. It is to us almost or quite impossible to conceive of
conscious personal life as springing from an unconscious impersonal
cause. Hence we place behind the manifold phenomena of the
universe a conscious personal being as the origin and source of all
226
things and all life.
It is wholly different with the thinkers of India. They seem to be
able to postulate as the beginning of things an impersonal cause, a
cause without perception, thought, or consciousness. They affirm
that out of unconsciousness consciousness arises. They teach that
out of Brahma, the unconscious, impersonal, passionless One,
emanate all material worlds and sentient beings, gods as well as
men.
How profoundly this conception of the First Cause has reacted on
the ethical speculations of the Hindu sages and on the moral life of
India will appear a little further on.

The god Brahma But this incomprehensible, unconscious,

(Brahman)
passionless Brahma is not the Brahma of the popular
faith. The masses and even the philosophers themselves must have
something more concrete. So this impersonal, neuter Brahma is
conceived as giving existence to the personal, masculine God
Brahma (Brahman), “the progenitor of all worlds, the first-born
227
among beings.”
It is very necessary for the student of Brahmanic ethics to keep in
mind the distinction between the uncreated, unconditioned,
impersonal Brahma and the created, conditioned, personal Brahma,
since there is here laid the foundation of a double goal for rational
moral striving: the goal of the ascetic whose ultimate aim is
deliverance from individual existence and absorption into the
absolute, unchangeable, impersonal Brahma, which means a state
of eternal unconsciousness—dreamless sleep; and the goal of the
multitude, whose hope and aim is blissful, though temporary, union
with the personal Brahma in the heaven of the mortal, conditioned
228
gods.

The system of The ethical evolution in India was also profoundly

castes
influenced by a prehistoric event, namely, the
subjection of the original non-Aryan population of the land by an
intruding Aryan people. As a result of the long and bitter struggle the
two races became separated by a sharp line of race prejudice and
hatred. The dark-skinned natives were reduced to a state of
servitude or dependence upon their conquerors. Intermarriages
between the two races were strictly prohibited, and thus the
population of the conquered districts of the peninsula became
divided into two sharply defined classes. These constituted a model
upon which Indian society was framed. Other classes were formed,
and these gradually hardened into castes, that is, into classes
between which marriages were prohibited. Four great castes arose:
namely, priests or Brahmans, warriors and rulers, peasants and
merchants, and sudras. Below these castes were the pariahs, or
outcasts, made up of the most degraded of the natives. As time
passed, still other divisions were formed, every occupation coming to
constitute the basis of a new caste, till society was stratified like a
geologic deposit.
Religion came in to consecrate this division of the people into
229
privileged and nonprivileged classes. The sacred scriptures
declare that the Brahmans sprang from the mouth of Brahma, the
warriors from his arms, the peasants and traders from his thighs, and
230
the sudras from his feet.
No institution known among men ever exercised a more fateful
and sinister influence upon morality than this caste system has
exercised upon the morality of the peoples of India. The rooted belief
and dogma of the natural inequality of men has made Brahmanic
ethics a thing of grades and classes, and has thus rendered
impossible the evolution of a true morality, which requires for its
basis genuine sentiments of equality and brotherhood.
 We easily realize the importance for morality of a
The doctrine of belief in a life after death. But a belief in preëxistence
transmigration may exert an even greater influence upon the moral
231
code of a people than a belief in post-existence.
Now the morality of the Hindus has been molded by both these
doctrines, for according to the teachings of Brahmanism a man has
lived through many lives before his “birth,” and may wander through
“ten thousand millions of existences” after death has freed him from
232
his present body. The class and the condition into which he is
born here on earth is believed to be determined by the sum total of
his merits or demerits earned in preceding existences. As a result of
sin he may in his next birth be reborn in a lower caste, or may be
imprisoned in some animal or vegetable form. He may pass a
thousand times through the bodies of spiders, snakes, and lizards,
and hundreds of times through the forms of grasses, shrubs, and
creepers. And all this experience may come after the soul has
passed through dreadful and innumerable hells for vast cycles of
233
years.
This transmigration theory was framed by the thinkers of India to
explain among other things the seemingly unjust inequalities of
234
human life. It afforded an explanation why one man should be
born a Brahman and another a sudra, one born in a hovel and
another in a palace, by conceiving the place of every person born
into the world as being determined by the manner of his life in former
235
existences.
It is unnecessary to dwell upon the profound influence which this
doctrine of transmigration, or round of births, has exerted upon the
moral life of India. The tendency of this theory, as soon as
elaborated, was to render still more intolerable the position of the
lower castes, particularly that of the sudras, since it made their low
place and hard lot to be the merited punishment of crimes and
misdoings in previous lives; while at the same time it fed the pride
and enhanced the arrogance of the Brahmans, since their superior
lot was, according to the theory, attributable to merit acquired in
other existences. Thus did the theory tend to give a more sinister
aspect to the baneful caste system, to make it appear a part of the
unchangeable order of things, and to render impossible the growth of
any other than a class morality.

Indian Hardly less important than the doctrine of

pessimism
transmigration for Hindu morality is the Indian
conception of life—of all individual, conscious existence whether
here on earth or in other worlds—as inseparable from misery, pain,
decay, and death.
The Aryan immigrants into India seem to have been, like their
kinsmen the Greeks, a light-hearted folk, filled with a strong joy in
life. But as in their journeyings they pressed southward into the
valleys of the Indus and the Ganges and came under the influences
of the hot, depressing climate, and of an oppressive social and
236
political system, they appeared to have lost their buoyant spirits.
The skies seemed less bright and life less worth living, and, weary of
it all, they at last came to regard eternal death, annihilation, as the
greatest of boons.
This pessimistic view of the world and of life, as we shall see a
little further on, forms the basis of large sections of Indian ethics,
since it makes the ultimate goal of rational or moral effort to be the
getting rid of conscious existence.

The conception Another conception which has exerted a profound

of sacrifice
influence upon the religious ethics of Brahmanism is
that respecting sacrifice. This conception is that the gods need
sustenance, and can only exist through the gifts and offerings made
237
to them by men. “The gods live by sacrifice” say the sacred
scriptures; “the sun would not rise if the priests did not make
sacrifice.”
To understand this teaching we must connect it with the belief of
primitive man that the spirits of the dead have absolute need of meat
and drink offerings at the hands of the living, and remember that in
India there is no sharp distinction drawn between the gods and the
souls of men. The gods, like the spirits of the dead, are dependent
for life and strength upon the offerings laid on their altars. Without
these gifts they would die or pine away, and all the movements of the
238
universe controlled by them would cease.
From this conception of the gods came the emphasis laid by
Brahmanism upon sacrifice, and the prominence given the religious
duty of bringing rich gifts to the priests and keeping the altars of the
239
gods heaped with food.

II. The Various Moral Standards

A class morality The fundamental fact of Brahmanic morality is that

as a result of the caste system it is a class morality;
that is, there is a different moral standard or code for each of the
different castes.
In the account given in the Laws of Manu of the origin of the four
chief castes, the occupation and the duties of each class are
carefully prescribed. To the Brahman was assigned teaching and
offering sacrifice; to the warriors and rulers the protection of the
people; to the peasants and merchants the tilling of the ground and
trading; and to the sudras—“One occupation only,” reads the sacred
law, “is prescribed to the sudra, to serve meekly the other three
240
castes.”
241
The Brahman is by right the lord of the whole creation. His
name must express something auspicious, but the first part of the
sudra’s name must express something contemptible, and the second
242
part must be a word denoting service.
For a man of a lower caste to affect equality with a person of a
higher caste is a crime: “If a man of an inferior caste, proudly
affecting an equality with a man of superior caste, should travel by
his side on the road, or sit or sleep upon the same carpet with him,
the magistrate shall take a fine from the man of inferior caste to the
243
extent of his ability.”
For a Brahman to explain to a sudra the sacred Vedas is a sin:
“Let him [the Brahman] not give to a sudra advice nor the remnants
of his meal ...; nor let him explain the sacred law to such a man; ...
for he who explains the sacred law to such ... will sink together with
244
that man into hell.”
In the matter of punishments for crimes the laws are grossly
unequal, the punishment of a person of inferior caste being always
more severe than that of a person of a superior caste for the same
offense. Thus for a crime punishable with death if committed by a
person of an inferior caste, tonsure only is ordained if committed by
245
a Brahman; for a Brahman must never be slain, “though he have
246
committed all horrible crimes.” There is no crime in all the world
247
as great as that of slaying a Brahman.
A knowledge of the inequality of these sacred laws of the
Brahmans and the burdensomeness of this caste morality as it
pressed upon the lower classes is necessary to an understanding of
the rise and rapid spread of Buddhism, and the fervor with which its
teachings of equality and brotherhood were embraced by the
masses of Brahmanic India.

The highest Of the different standards of morality of the several

moral
excellence
castes that of the Brahman is of course the highest.
attainable in The study of the sacred books is for him the chief duty.
general only by “Let him,” says the sacred law, “without tiring daily
Brahmans
mutter the Veda at the proper time; for that is one’s
248
highest duty; all other observances are secondary duties.”
249
Knowledge of the Veda destroys guilt as fire consumes fuel.
Among the secondary duties are observance of the rules of