Advances in Parasitology, Volume 117 0323989497, 9780323989497

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VOLUME ONE HUNDRED AND SEVENTEEN

ADVANCES IN PARASITOLOGY

SERIES EDITOR D. ROLLINSON

J. R. STOTHARD

Life Sciences Department The Natural History Museum, London, United Kingdom [email protected]

Department of Tropical Disease Biology Liverpool School of Tropical Medicine, Liverpool, United Kingdom [email protected]

EDITORIAL BOARD T. J. C. ANDERSON

K. KING

Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX, United States

Department of Zoology, University of Oxford, Oxford, United Kingdom

M. G. BASÁÑEZ

M. G. ORTEGA-PIERRES

Professor of Neglected Tropical Diseases, Department of Infectious Disease Epidemiology, Faculty of Medicine (St Mary’s Campus), Imperial College London, London, United Kingdom

D. D. BOWMAN Director Cornell CVM MPS—Veterinary Parasitology, Professor of Parasitology, C4-119 VMC, Dept Micro & Immunol, CVM Cornell University, Ithaca, NY, United States

R. B. GASSER Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia

A. L. GRAHAM Professor of Ecology & Evolutionary Biology, Co-Director of the Global Health Program, Princeton University, Princeton, NJ, United States

J. KEISER Head, Helminth Drug Development Unit, Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland

Professor of the Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados IPN, Mexico City, Mexico

D. L. SMITH Johns Hopkins Malaria Research Institute & Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States

R. C. A. THOMPSON Head, WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, Principal Investigator, Environmental Biotechnology CRC (EBCRC), School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA, Australia

X.-N. ZHOU Professor, Director, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People’s Republic of China

VOLUME ONE HUNDRED AND SEVENTEEN

ADVANCES IN PARASITOLOGY Edited by

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

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States 525 B Street, Suite 1650, San Diego, CA 92101, United States First edition 2022 Copyright © 2022 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-323-98949-7 ISSN: 0065-308X For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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Contents Contributors

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1. The microscopic five of the big five: Managing zoonotic diseases within and beyond African wildlife protected areas

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Anya V. Tober, Danny Govender, Isa-Rita M. Russo, and Jo Cable 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

2. Improving translational power in antischistosomal drug discovery

2 4 10 13 28 33 35 37 37

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Alexandra Probst, Stefan Biendl, and Jennifer Keiser 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

3. Unique thiol metabolism in trypanosomatids: Redox homeostasis and drug resistance

48 49 51 54 56 68 69 69

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Vahab Ali, Sachidananda Behera, Afreen Nawaz, Asif Equbal, and Krishna Pandey 1. Introduction 2. Trypanothione metabolism

77 80 v

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Contents

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 Acknowledgements References

90 103 110 118 123 131 132 132

Contributors Vahab Ali Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna, Bihar, India Sachidananda Behera Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna, Bihar, India Stefan Biendl Swiss Tropical and Public Health Institute, Department of Medical Parasitology and Infection Biology; University of Basel, Basel, Switzerland Jo Cable School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom Asif Equbal Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna; Department of Botany, Araria College, Purnea University, Purnia, Bihar, India Danny Govender SANParks, Scientific Services, Savanna and Grassland Research Unit, Pretoria; Department of Paraclinical Sciences, University of Pretoria, Onderstepoort, South Africa Jennifer Keiser Swiss Tropical and Public Health Institute, Department of Medical Parasitology and Infection Biology; University of Basel, Basel, Switzerland Afreen Nawaz Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna, Bihar, India Krishna Pandey Department of Clinical Medicine, ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna, Bihar, India Alexandra Probst Swiss Tropical and Public Health Institute, Department of Medical Parasitology and Infection Biology; University of Basel, Basel, Switzerland Isa-Rita M. Russo School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom Anya V. Tober School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom

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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,†, and Jo Cablea,† a

School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom SANParks, Scientific Services, Savanna and Grassland Research Unit, Pretoria, South Africa c Department of Paraclinical Sciences, University of Pretoria, Onderstepoort, South Africa ∗ Corresponding author: e-mail address: [email protected] b

Contents 1. Introduction 2. The ‘Microscopic Five’ 2.1 Bovine tuberculosis 2.2 Rift Valley fever 2.3 Brucellosis 2.4 Cryptosporidiosis 2.5 Schistosomiasis 3. Challenges of BTb control at the wildlife-livestock interface: The South African case study 3.1 Control in livestock 3.2 Control in wildlife 4. Drivers of disease: The Kruger National Park case study 4.1 Past and present disease management 4.2 Kruger National Park’s current adaptive management approach 4.3 Environmental drivers of disease transmission 4.4 Anthropogenic drivers of disease transmission: Wildlife-livestock-human interface 5. Disease knowledge gaps and lessons learnt from African protected areas 6. Communities and conservation 7. Conclusions Acknowledgements References

†

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Authors contributed equally to this work.

Advances in Parasitology, Volume 117 ISSN 0065-308X https://doi.org/10.1016/bs.apar.2022.05.001

Copyright

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2022 Elsevier Ltd All rights reserved.

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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.

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

Managing zoonoses in African protected areas

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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 (Go´mez 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.

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Big Five

Microscopic Five

Extreme weather events Co-infections Reservoir hosts Landscape heterogeneity Mixing at waterholes

Translocation Permeable fences Illegal wildlife trade

Fig. 1 The ‘Big Five’ and ‘Microscopic Five’, and the drivers of disease at the wildlifelivestock-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

Table 1 Some diseases of large herbivores within Kruger National Park which may pose a threat to livestock and/or humans. Spatial Known Transmission Transmission distribution in susceptible Disease. Pathogen modes routes Drivers KNP hosts

Reservoir hosts

Bacteria Anthrax

Bacillus anthracis Vector, environmental, direct contact, fomites

Ingestion of contaminated vegetation or carcasses

Calcium soil content, drought

Bovine Tb

Micobacteria bovis Aerosol

Respiratory tract

Wildlife/livestock South, central, Cattle, interface, host moving north buffalo, density humans

Cape buffalo

Aerosol, fomites

Respiratory tract

Wildlife/livestock North, central, Cloven interface, host south hooved density animals

Cape buffalo

Mosquito vector, direct contact

Cutaneous penetration

Introduced horses, Central season

Culicoides mosquito, possibly zebra

Mosquito vector, direct contact

Cutaneous penetration

Climate change, drought, rainfall

Northern and Most central regions mammals including humans

Maintained in environment

Virus Foot and mouth Aphtovirus

African horse sickness

Orbivirus

Rift Valley fever Phlebovirus

Zebra, domestic horses

Higher in Cattle, south and buffalo central regions

Aedes mosquito Cape buffalo Continued

Table 1 Some diseases of large herbivores within Kruger National Park which may pose a threat to livestock and/or humans.—cont’d Spatial Known Transmission Transmission distribution in susceptible Disease. Pathogen modes routes Drivers KNP hosts Reservoir hosts

Protozoa Bovine brucellosis

Brucella abortus

Direct contact

Cryptosporidiosis Cryptosporidium Environmental spp.

Ingestion of Host density infected discharges during birth, milk, mucus membranes

North, central, Cattle, Cape buffalo south buffalo, wild animals, humans

Faecal-oral via Dependant on contamination of species and host food and water range

Unknown

Wild animals, humans, domestic animals

Unknown

Piroplasma Corridor disease

Theileria parva

Tick vector

Cutaneous penetration

Babesiosis

Babesia spp.

Tick vector

Cutaneous penetration

Fascioliasis

Fasciola spp.

Environmental

Contact with infected water

Schistosomiasis

Schistsoma spp.

Environmental

Contact with infected water

Wildlife/livestock North, central, Cattle interface south

Cape Buffalo

Unknown

Rhinoceros, Unknown lions

Dependant on species and host range

Unknown

Ruminants

Unknown

Dependant on species and host range

Unknown

Wild animals, humans

Unknown

Digenea

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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.

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; M
etras 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

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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

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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)

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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 (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).

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

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Table 2 Four levels of Bovine Tb surveillance programmes in South Africa.

Surveillance herd programme

One off survey used by state officials to determine the prevalence of BTb within an area or by a stock owner conducting a self-assessment

Maintenance herd programme

To join this programme, herds are required to undergo two consecutive tests with 100% negative results at least 3 months apart. These BTb free herds are then tested every 2 years. If an individual tests positive, then the entire herd is moved to the infected herd programme

Infected herd programme

Compulsory programme for herds that have tested positive with the CIT test, as 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 testing programme (individuals)

Individual cattle destined to be imported or exported. Imported cattle are kept in quarantine and must undergo a compulsory CIT test. Before export, cattle must also receive a comparative CIT test—a requirement for many importing countries

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).

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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

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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

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(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

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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

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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). 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

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Fig. 2 Megaherbivore (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.

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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 (M
etras 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 (M
etras 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).

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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 (M
etras 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

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(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

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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; Gala´n-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).

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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

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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)

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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

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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 (Suza´n 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

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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.

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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).

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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 diseaserelated 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).

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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

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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

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(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.

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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 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

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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

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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 Pyh€al€a, 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 Pyh€al€a, 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

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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 Pyh€al€a, 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 Pyh€al€a, 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 wildlifelivestock-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

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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-livestockwildlife 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

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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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Storfer, A., Patton, A., Fraik, A.K., 2018. Navigating the interface between landscape genetics and landscape genomics. Front. Genet. 9, 68. Stothard, J.R., Kayuni, S.A., Al-Harbi, M.H., Musaya, J., Webster, B.L., 2020. Future schistosome hybridizations: will all Schistosoma haematobium hybrids please stand-up! PLoS Negl. Trop. Dis. 14, e0008201. Suza´n, G., Esponda, F., Carrasco-Herna´ndez, R., Aguirre, A.A., Ostfeld, R.S., 2012. Habitat fragmentation and infectious disease ecology. In: Aguirre, A.A., Ostfeld, R., Daszak, P. (Eds.), New Directions in Conservation Medicine: Applied Cases of Ecological Health. Oxford University Press, USA. Suzman, J., 2001. An Introduction to the Regional Assessment of the Status of the San in Southern Africa. Legal Assistance Centre, Windhoek, p. 4. Swanson, A., Kosmala, M., Lintott, C., Packer, C., 2016. A generalized approach for producing, quantifying, and validating citizen science data from wildlife images. Conserv. Biol. 30, 520–531. Swanson, A., Kosmala, M., Lintott, C., Simpson, R., Smith, A., Packer, C., 2015. Snapshot Serengeti, high-frequency annotated camera trap images of 40 mammalian species in an African savanna. Scientific Data 2, 1–14. Swemmer, L., Mmethi, H., Twine, W., 2017. Tracing the cost/benefit pathway of protected areas: a case study of the Kruger National Park, South Africa. Ecosyst. Serv. 28, 162–172. Swinton, J.M.E.J., Woolhouse, M.E.J., Begon, M.E., Dobson, A.P., Ferroglio, E., Grenfell, B.T., Guberti, V., Hails, R.S., Heesterbeek, J.A.P., Lavazza, A., Roberts, M.G., 2002. Microparasite transmission and persistence. In: The Ecology of Wildlife Diseases. Oxford University Press, Oxford. Sylvester, T.T., Martin, L.E.R., Buss, P., Loxton, A.G., Hausler, G.A., Rossouw, L., van Helden, P., Parsons, S.D.C., Olea-Popelka, F., Miller, M.A., 2017. Prevalence and risk factors for Mycobacterium bovis infection in African lions (Panthera leo) in the Kruger National Park. J. Wildl. Dis. 53, 372–376. Taylor, M.G., 1970. Hybridisation experiments on five species of African schistosomes. J. Helminthol. 44, 253–314. Taylor, J.J., 2012. Identity, authority and CBNRM. In: Naming the Land: San Identity and Community Conservation in Namibia’s West Caprivi. vol. 12. Basler Afrika Bibliographien, pp. 28–50. Ung, L., Stothard, J.R., Phalkey, R., Azman, A.S., Chodosh, J., Hanage, W.P., Standley, C.J., 2021. Towards global control of parasitic diseases in the Covid-19 era: one health and the future of multisectoral global health governance. Adv. Parasitol. 114, 1–26. United Nations Environmental Programme (2022). Nairobi, Kenya. https://www.unep. org/news-and-stories/speech/one-health-global-map. Accessed March 12, 2022. van Wilgen, B.W., Boshoff, N., Smit, I.P., Solano-Fernandez, S., Van der Walt, L., 2016. A bibliometric analysis to illustrate the role of an embedded research capability in south African national parks. Scientometrics 107, 185–212. Vaumourin, E., Vourc’h, G., Gasqui, P., Vayssier-Taussat, M., 2015. The importance of multiparasitism: examining the consequences of co-infections for human and animal health. Parasit. Vectors 8, 1–13. Venter, F.J., Scholes, R.J., Eckhardt, H.C., 2003. The abiotic template and its associated vegetation pattern. In: Du Toit, J.T., Rogersand, K.H., Biggs, H.C. (Eds.), The Kruger Experience: Ecology and Management of Savanna Heterogeneity. Island Press, Washington, DC, pp. 83–129. Venter, F.J., Naiman, R.J., Biggs, H.C., Pienaar, D.J., 2008. The evolution of conservation management philosophy: science, environmental change and social adjustments in Kruger National Park. Ecosystems 11, 173–192.

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Vosloo, W., Thompson, P.N., Botha, B., Bengis, R.G., Thomson, G.R., 2009. Longitudinal study to investigate the role of impala (Aepyceros melampus) in foot-andmouth disease maintenance in the Kruger National Park, South Africa. Transbound. Emerg. Dis. 56, 18–30. Warchol, G., Johnson, B., 2009. Wildlife crime in the game reserves of South Africa: a research note. Int. J. Comp. Appl. Crim. Just. 33, 143–154. Wasserman, T.N., Cushman, S.A., Littell, J.S., Shirk, A.J., Landguth, E.L., 2013. Population connectivity and genetic diversity of American marten (Martes Americana) in the United States northern Rocky Mountains in a climate change context. Conserv. Genet. 14, 529–541. Webster, J.P., Gower, C.M., Knowles, S.C., Molyneux, D.H., Fenton, A., 2016. One health–an ecological and evolutionary framework for tackling neglected zoonotic diseases. Evol. Appl. 9, 313–333. Wielgus, E., Caron, A., Bennitt, E., De Garine-Wichatitsky, M., Cain, B., Fritz, H., Miguel, E., Corn
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e-Jammes, S., 2021. Inter-group social behavior, contact patterns and risk for pathogen transmission in cape buffalo populations. J. Wildl. Manag. 85, 1574–1590. Williams, R., Malherbe, J., Weepener, H., Majiwa, P., Swanepoel, R., 2016. Anomalous high rainfall and soil saturation as combined risk indicator of Rift Valley fever outbreaks, South Africa, 2008–2011. Emerg. Infect. Dis. 22, 2054. Winterbach, H.E.K., 1998. Research review: the status and distribution of cape buffalo Syncerus caffer caffer in southern Africa. S. Afr. J. Wildl. Res. 28, 82–88. Wolmarans, C.T., De Kock, K.N., 2006. The current status of freshwater molluscs in the Kruger National Park. Koedoe 49, 39–44. World Health Organisation, 2017. The Tripartite’s Commitment: Providing Multisectoral, Collaborative Leadership in Addressing Health Challenges. Geneva, Switzerland. https://www.who.int/zoonoses/tripartite_oct2017.pdf Retrieved March 23, 2022. World Health Organization, 2022. WHO Guideline on Control and Elimination of Human Schistosomiasis. Switzerland, Geneva. https://www.who.int/publications/i/item/ 9789240041608. Retrieved March 23, 2022. Wright, C.A., Ross, G.C., 1980. Hybrids between Schistosoma haematobium and S. mattheei and their identification by isoelectric focusing of enzymes. Trans. R. Soc. Trop. Med. Hyg. 74, 326–332. Zahedi, A., Paparini, A., Jian, F., Robertson, I., Ryan, U., 2016. Public health significance of zoonotic Cryptosporidium species in wildlife: critical insights into better drinking water management. Int. J. Parasitol. Parasites Wildl. 5, 88–109. Zambatis, N., Biggs, H.C., 1995. Rainfall and temperatures during the 1991/92 drought in the Kruger National Park. Koedoe 38, 1–16.

CHAPTER TWO

Improving translational power in antischistosomal drug discovery Alexandra Probsta,b,† , Stefan Biendla,b,† and Jennifer Keisera,b,*

,

a

Swiss Tropical and Public Health Institute, Department of Medical Parasitology and Infection Biology, Basel, Switzerland University of Basel, Basel, Switzerland *Corresponding author: e-mail address: [email protected] b

Contents 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 5.1 No correlation between praziquantel exposure and in vivo efficacy 5.2 Chronic S. mansoni infection influences exposure parameters of drugs 5.3 Prolonged drug exposure well above in vitro potency not always correlates with good in vivo efficacy 5.4 Special cases 6. Concluding remarks Acknowledgements and funding References

48 49 51 54 56 56 62 65 67 68 69 69

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 †

Contributed equally (joint first authorship).

Advances in Parasitology, Volume 117 ISSN 0065-308X https://doi.org/10.1016/bs.apar.2022.05.002

Copyright

#

2022 Elsevier Ltd All rights reserved.

47

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Alexandra Probst et al.

to improve the drug profile to accelerate the transition from the early drug discovery phase to new clinical candidates.

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

Improving translational power in antischistosomal drug discovery

49

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.

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).

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

50

Alexandra Probst et al.

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 in the online version at https://doi.org/10.1016/ bs.apar.2022.05.002). 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 in the online version at https://doi.org/10.1016/bs.apar.2022.05.002). Drug activity did not improve significantly for prolonged exposure times. In summary, we did not observe significant differences of praziquantel or (R)-praziquantel activity against both tested isolates for any evaluation time point (Fig. 1). Therefore, we hypothesize that there is not sufficient reason to expect

Fig. 1 Praziquantel (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.

Improving translational power in antischistosomal drug discovery

51

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.

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). 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

Table 1 The S. mansoni mouse model for drug efficacy testing. Host

Institution

Strain

Age at infection [weeks]

Animals per Isolate origin Sex group

Infection

Intensity [number of Route cercariae]

Evaluation Duration [weeks] ad.

j.

post treatment Worm [weeks] recovery

Egg burden Ref.

Swiss TPH NMRI 4

f

4

Liberia s.c.

100

49

21

2–3

Perfusion No (j.), Picking (ad.)

a)

LSHTM

CD1

f

5

Puerto s.c. Rico

150

42

21

1–2

Perfusion

No

b)

UCSD/ UCSF

Swiss 3–6 webster

f

3–6

Puerto s.c. Rico

140–150


42 21

2

Perfusion

Yes

c)

TBRI

CD1

m

10

Egypt

imm. 80

49

1d-2

Perfusion

Yes

d)

UNG

Balb/c, 3 Swiss

NR 3–10

Brazil (BH)

imm., 70–80 s.c.


42 21

2–3

Perfusion, Picking

Yes

e)

f

Brazil (LE)

s.c.

45

Perfusion

Yes

f)

FIOCRUZ Albino

5

NR

NR

10

100

21

NR 1d-2

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).

Improving translational power in antischistosomal drug discovery

53

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.

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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 for example Methods, 2.6 Data analysis in Supplementary Material in the online version at https://doi.org/10.1016/bs.apar.2022.05.002). 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 in the online version at https://doi.org/10.1016/bs.apar.2022.05.002). 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 cercarium 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 in the online version at https://doi. org/10.1016/bs.apar.2022.05.002). 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). Furthermore, our analysis revealed considerable variability/variation even at the most commonly employed infection dose of 100 cercariae, with yields ranging between 5 and 71 adult worms (interquartile range: 14,

Improving translational power in antischistosomal drug discovery

55

Fig. 2 Infection intensity of the patent S. mansoni mouse model. Boxplot depicting total worm count (worm burden) per mouse extracted from mice (n ¼ 9, 245, 23, 6, 6) infected by s.c. injection of cercariae (50, 100, 200, 300, 400, respectively). Diamond points in light blue represent the mean of the distribution. The median is depicted as dividing white space between the 25th and 75th percentile in black. The whiskers extend to the minimum or maximum value in the data or to a maximal distance from the quartiles of 1.5*interquartile range (IQR) respectively. Dots represent potential outliers. The line represents the linear fit and the grey area shows the 95% confidence area of the fit.

n ¼ 245). Factors acting as underlying causes for biological variability need to be carefully considered during experiment planning to improve comparability between studies. These factors include mouse age at infection linked to immune system maturity as well as mouse stress levels at infection and during worm development. Typically, young mice are infected after at least 1 week of acclimatization and before reaching an age of 6 weeks to improve susceptibility to infection (Lombardo et al., 2019a; Silva et al., 2021; Wolfe et al., 2018). Besides mouse acclimatization, training of the experimenter conducting infection is essential to reduce variation at infection. Finally, based on our experimental experience over the last decade, we suspect batch-to-batch variations in cercarial viability and virulence to be a key driver of inter-experiment variability that can seldom be detected or assessed prior to mouse infection.

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5. Pharmacokinetic/pharmacodynamic (PK/PD) relationship of selected drugs While it is well-known that PK/PD studies are often employed in the drug discovery pipeline for the majority of drugs, it has not been established whether there is a PK/PD correlation in the S. mansoni rodent models. The location of the worms should be kept in mind when discussing pharmacokinetics. While the adult worms are found in the mesenteric veins of the intestine (S. mansoni and S. japonicum) or the urinary plexus surrounding the bladder (S. haematobium), juvenile worms travel through the inferior vena cava to the heart, the left lung (and back to the heart) before entering the abdominal aorta and finally locating to the portal vasculature, where the parasites mature (Nation et al., 2020). In the following section, we analyse and compare different drug candidates that were characterized by us and other research groups with regard to the relationship between pharmacokinetics and in vivo efficacy (Table 2) to better understand PK/PD relationships.

5.1 No correlation between praziquantel exposure and in vivo efficacy As a Class II drug (Biopharmaceutical Classification System, BCS), praziquantel possess high permeability and a limited solubility, it undergoes slow absorption in the gut lumen and is marked by extensive first-pass metabolism in the liver by the cytochrome P450 system (Lindenberg et al., 2004). Praziquantel is rapidly cleared and has a short half-life (Olliaro et al., 2014). In vitro, the drug is fast acting and has an IC50 value of approximately 0.1 μM after 72 h. Several years ago, our research group reported on the PK/PD relationship of praziquantel in the S. mansoni mouse model (Abla et al., 2017). Briefly, racemic praziquantel, given at a single oral dose of 400 mg/kg, resulted in worm burden reductions of 84–100% and the same applies for the levo enantiomer, which is consistent with the R-praziquantel being the pharmacologically active form of the drug (Abla et al., 2017; Lombardo et al., 2019b; Meister et al., 2014). Contrary, the S-praziquantel enantiomer, responsible for most of the bitter taste of the tablet (Meyer et al., 2009), did not reduce the worm burden in infected S. mansoni mice (WBR ¼ 18%) (Meister et al., 2014). Abla and colleagues reported that plasma exposures of the active enantiomer were increased by 10- to 20-fold when 1-aminobenzotriazole (ABT), a pan-CYP inhibitor

Table 2 PK/PD relationship of antischistosomal compounds.

Chemical class

ID

Dose p.o. (mg/kg)

Pyrazino-isoquinoline

PZQ

400

WBR (%)

97

T1/2 (h)

Tmax (h)

84

Cmax (μM)

In vitro AUC0-t IC50 (μM*h) (μM)a

Mouse model: infected (gender, strain)

6.3c

11.4c

Yes (f, NMRI)

11.7

c

12.0

400

R-PZQ

100

S-PZQ

18

R-PZQ

84

a

0.8

8.6

20.2

0.06

4.7

0.8

1.6

5.8

18.9

183

12

19

1501

11

34

12

27

1332

182

24

14

2027

72

48

19

2128

No (f, NMRI)

39

2

459

22,463 66

No (f, NMRI)

96

17e

0.5e

4.6e

127e

100 (i.p.) 99

e

e

e

e

Yes (f, Swiss Webster)

b

72–80

83–93

Ozonides

c

400

9-acridanone hydrazone

d

100

3.4

0

a)

11.8 (L6)

3.7

0

b)

9.7 (L6)

4.4

0

4.8

0

c)

4

0

d)

0.10

3.3

200

violation of Limpinski’s References cLogP rule of 5

94

S-PZQ Aryl-methanols

In vitro cytotox. (μM) (cellline)

c

100 PZQ

0.16

In vitro intr. In vitro met. Cl. (μL/ Stability min/mg (% rem.)b protein)

No (f, NMRI) 6

80

12.5

100

25

99

Yes (f, NMRI)

Yes (f, NMRI)

20

0.5

e

e

5

50

Yes (f, NMRI)

3

6.3

e

21

126

254e

85

100 (U-20S, HEK293, HC-04)

5.8

1

3.3

0

i 20

N,N0 -Diarylureas

Tmax (h)

2.4

20

2 (i.v.) N,N0 -Diarylureas

T1/2 (h)

In vitro AUC0-t IC50 (μM*h) (μM)a

Yes (f, NMRI)

No (f, NMRI)

j

400

0

25

3

171

691

0.1

Yes (f, NMRI)

k

100

40

11

4

31

394

0.2

20

10

4

40

953

0.9

Efficacy: yes (f, NMRI), PK: no (m, Swiss outbread)

l

10 (HML) / 7 (MLM)