Parasites of the African painted dog (Lycaon pictus) …...i Parasites of the African painted dog (Lycaon pictus) in captive and wild populations: Implications for conservation Amanda-Lee

i

Parasites of the African painted dog (Lycaon pictus) in

captive and wild populations: Implications for conservation

Amanda-Lee Ash

Bachelor of Animal and Veterinary Biosciences (Hons)

La Trobe University, Victoria

Faculty of Health Sciences

School of Veterinary and Biomedical Sciences

Murdoch University

Western Australia

This thesis is presented for the degree of Doctor of Philosophy of Murdoch University 2011

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I declare that this thesis is my own account of my research and contains as its main

content work which has not previously been submitted for a degree at any other tertiary

educational institution.

……………………………..

Amanda-Lee Ash

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Abstract

The African painted dog (Lycaon pictus) is a highly endangered carnivore of sub-

Saharan Africa, which in the last century has suffered a population decline of almost

99%. With only 3,000-5,500 animals remaining in the wild it is imperative to

understand all threatening processes to which these animals may be exposed. The

impact that parasites and other infectious agents have on wildlife has been increasingly

recognized within conservation programs. Stressors such as human encroachment and

habitat destruction are altering the incidence and effect that these pathogens have on

wildlife populations, especially those endangered and under stress.

A parasitological study was conducted on captive and wild populations of the African

painted dog over a three year period. Collaborations with three captive animal facilities

and three in situ conservation groups within Africa allowed for a broad sample base

from which variation in parasite prevalence and diversity could be identified. A

combination of traditional microscopy techniques and molecular characterisation of

parasite species were employed to obtain comprehensive data on the prevalence and

diversity of gastrointestinal parasites observed in faecal samples collected from painted

dogs.

Parasite prevalence within wild populations was 99% with a similar parasite

community composition observed among all three wild populations. Five of the seven

parasite genera observed in this study have not been reported before in this host.

Additionally, molecular characterisations identified the potentially zoonotic species

Giardia duodenalis, Ancylostoma braziliense and an ambiguous species of taeniid, all

of which have also not been previously reported in this host.

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The prevalence of parasites within captive populations was 15% with Giardia

duodenalis being the dominant of the only two parasite species observed. The overall

lack of prevalence and diversity of parasites observed in captive populations could be

of significance for facilities involved in reintroduction programs. Particularly as

immunologically naïve captive animals may be unable to cope with exposure to a

‘natural’ parasite load in the wild environment, leading to an ultimate decrease in

reintroduction success.

Gastrointestinal parasites detected in faecal samples from wild and captive populations

of the African painted dog during this study

Wild Captive

Parasite Taxon

observed

Taeniid Giardia

Ancylostoma Spirometra

Spirometra

Giardia

Coccidia

Sarcocystis

Filaroides

This study has obtained detailed baseline data of parasitism within populations of the

African painted dog in captive and wild environments. The large proportion of new

discoveries in this study demonstrates the paucity of information currently available on

parasitism within this host species. It is hoped this information will assist in

conservation efforts by a) recognising the challenges of parasite control in captive

populations, particularly those involved in reintroduction and/or translocation

programs, and b) being able to identify deviations from baseline parasite levels in wild

populations which could be indications for emerging exotic and/or zoonotic disease.

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Publications

Refereed journal articles:

Ash, A., Lymbery, A., Lemon, J., Vitali, S., Thompson, R.C.A. (2010). Molecular

epidemiology of Giardia duodenalis in an endangered carnivore – the African painted

dog. Veterinary Parasitology, 174, 206-212.

Conferences

Ash, A., Lymbery, A., Lemon, J., Thompson, R.C.A. (2010). Significance of parasitism

in an endangered carnivore - the African painted dog. Conference Proceedings: XX11

International Congress of Parasitology, Melbourne, Australia. (Abstract)

Ash, A., Lymbery, A., Lemon, J., Vitali, S., Thompson, R.C.A. (2009). Giardia

duodenalis isolates from captive and wild populations of the African painted dog

(Lycaon pictus). Conference Proceedings: ASP & ARC/NHMRC Research Network

for Parasitology Annual Conference. Sydney, Australia, pp 54. (Abstract)

Ash, A., Lymbery, A., Lemon, J., Thompson, R.C.A. (2009). Parasites of the African

Painted dog (Lycaon pictus) in wild and captive populations: potential conservation

impacts. Conference Proceedings: 22nd Conference of the World Association for the

Advancement of Veterinary Parasitology (WAAVP), Calgary Canada, pp 86.

(Abstract)

Ash, A., Lymbery, A., Lemon, J., Thompson, R.C.A. (2008) Parasites of the African

painted dog (Lycaon pictus) in wild and captive populations: potential conservation

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impacts. Conference Proceedings: 21st Australasian Wildlife Management Society

Conference. Fremantle, Australia, pp 44. (Abstract)

Ash, A., Lymbery, A., Lemon, J., Thompson, R.C.A. (2007). Parasites of the African

painted dog (Lycaon pictus) in wild and captive populations: potential conservation

impacts. Conference Proceedings: ASP & ARC/NHMRC Research Network for

Parasitology Annual Conference. Canberra, Australia, pp 95. (Abstract)

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Acknowledgements

I would firstly like to thank my three supervisors, Andy Thompson, Alan Lymbery and

John Lemon for their invaluable guidance throughout this project.

Secondly, the fabulous field assistance given by the staff of the Zambian Carnivore

Project (Matt Becker, Egil Droge and Claire Harrision) and the Wild Dog Project

(Robin Lines and Anna) for making my forays into deepest Africa a little less daunting.

I now have the ability to navigate rough muddy roads in various 4WD vehicles and deal

with angry elephants, hungry hyaena, thieving baboons and the ever present lion all in

the quest for African painted dog poo. Additionally the staff at DeWildt wildlife trust

(Alan and Gabby) for assistance and protection in roaming occupied enclosures for

samples, whilst also chasing cheetah out of trees. And of course the keepers and vets at

Perth and Monarto zoo.

Thirdly, the assistance I was given in the lab was lifesaving. I was fortunate in having

access to expertise from various people including Cath Covacin, Aileen Elliot, Russ

Hobbs and Louise Pallant, all of which made my task that much easier. Additionally,

the post-grad office crowd were always available for a little light comic relief.

And finally, to all my family and friends who tried not to think I was completely mad

for taking on a doctorate and for R who was a calming influence during the rough and

rocky patches of field work, lab work and write up.

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Table of Contents

Abstract ............................................................................................................................... iii

Publications .......................................................................................................................... v

Conferences .......................................................................................................................... v

Acknowledgements ............................................................................................................ vii

Table of Contents ............................................................................................................. viii

List of Tables ..................................................................................................................... xiv

List of Figures ................................................................................................................. xviii

Chapter 1 Introduction ................................................................................... 1

1.1 Parasites in wildlife ........................................................................................................ 2

1.2 Importance of parasites in conservation ...................................................................... 2

1.2.1 Direct impacts ..................................................................................................................4

1.2.2 Indirect impacts ................................................................................................................8

1.2.3 Co-extinction events/biodiversity considerations .......................................................... 11

1.2.4 Assessing effects of human influences .......................................................................... 12

1.3 The host, the African painted dog (Lycaon pictus).................................................... 14

1.3.1 Ecology of the African painted dog ............................................................................... 15

1.3.2 Conservation issues for the African painted dog ........................................................... 16

1.4 Parasites and the African painted dog ....................................................................... 17

1.4.1 Host ecology and parasites ............................................................................................. 17

1.4.2 Parasites recorded from the African painted dog ........................................................... 18

1.4.3 Gaps in current knowledge of parasitism in the African painted dog ............................ 20

1.5 Management of wild species ........................................................................................ 21

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1.5.1 Captive environments .................................................................................................... 21

1.5.2 Reintroduction programs ............................................................................................... 23

1.5.3 Translocation programs ................................................................................................. 23

1.6 Goals for this study ...................................................................................................... 24

Chapter 2 General materials and methods ................................................. 27

2.1. Study design ................................................................................................................. 28

2.2 Captive populations ..................................................................................................... 29

2.2.2 Perth Zoo ........................................................................................................................ 29

2.2.3 Monarto Zoo .................................................................................................................. 31

2.2.3 DeWildt Wildlife Trust .................................................................................................. 32

2.3 Wild populations .......................................................................................................... 33

2.3.1 South Luangwa National Park – Zambia ....................................................................... 33

2.3.2 Nyae Nyae Conservancy – Namibia .............................................................................. 35

2.3.3 Hwange National Park – Zimbabwe .............................................................................. 36

2.4 Sampling methodology ................................................................................................ 37

2.4.1 Ethics and permits .......................................................................................................... 38

2.4.2 Locating packs ............................................................................................................... 39

2.4.3 Sample collection ........................................................................................................... 40

2.4.4 Sample movement .......................................................................................................... 41

2.5 Parasitological techniques ........................................................................................... 42

2.5.1 Microscopy; qualitative analysis .................................................................................... 42

2.5.2 Microscopy; quantitative analysis .................................................................................. 44

2.5.3 Molecular characterisation ............................................................................................. 45

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Chapter 3 Diversity and prevalence of gastrointestinal parasite fauna

within captive and wild populations of the African painted dog .............. 46

3.1 Introduction .................................................................................................................. 47

3.2 Materials and methods ................................................................................................ 49

3.2.1 Sampling protocol .......................................................................................................... 49

3.2.2 Parasite identification and quantification ....................................................................... 51

3.2.3 Host and environmental variables .................................................................................. 52

3.2.4 Data analysis .................................................................................................................. 52

3.2.4.1 Descriptors used ..................................................................................................... 52

3.2.4.2 Levels of testing ....................................................................................................... 53

3.2.4.3 Statistical analysis .................................................................................................. 55

3.3 Results ........................................................................................................................... 56

3.3.1 Variation between captive and wild populations ........................................................... 56

3.3.2 Variation between packs ................................................................................................ 58

3.3.3 Variation among different geographic locations ............................................................ 60

3.3.4 Variation between different seasons .............................................................................. 62

3.3.5 Variation between host age and sex ............................................................................... 64

3.3.6 Parasite aggregation within hosts ................................................................................... 64

3.4. Discussion..................................................................................................................... 65

3.4.1 Parasite diversity ............................................................................................................ 65

3.4.2 Influence of geographic, seasonal and demographic factors ......................................... 68

3.4.3 Parasites of wild and captive populations ...................................................................... 70

3.4.4 Conclusions .................................................................................................................... 71

Chapter 4 Molecular epidemiology of Giardia duodenalis in an

endangered carnivore – the African painted dog ....................................... 72

4.1 Introduction .................................................................................................................. 73

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4.2.2 Parasitological techniques .............................................................................................. 75

4.2.3 DNA extraction .............................................................................................................. 76

4.2.4 Amplification of 18S rRNA locus ................................................................................. 76

4.2.5 Amplification of β-giardin locus .................................................................................... 77

4.2.6 Amplification of the glutamate dehydrogenase locus .................................................... 78

4.2.7 Data analysis .................................................................................................................. 79

4.3. Results .......................................................................................................................... 80

4.3.1 Prevalence of infection .................................................................................................. 80

4.3.2 Molecular characterization of Giardia duodenalis isolates ........................................... 81

4.4. Discussion..................................................................................................................... 84

4.4.1 Conclusions .................................................................................................................... 89

Chapter 5 Hookworm species of the African painted dog and the

significance of infection ................................................................................. 90

5.1 Introduction .................................................................................................................. 91

5.1.1 Hookworm in the canid host .......................................................................................... 92

5.1.2 The zoonotic potential of canid hookworm species ....................................................... 93

5.1.3 Hookworm in the African painted dog and other carnivores in Africa .......................... 94

5.1.4 Diagnosing hookworm infections .................................................................................. 95



5.2.2 Parasitological techniques .............................................................................................. 96

5.2.3 DNA extraction .............................................................................................................. 97

5.2.4 Amplification of Ancylostoma spp. isolates at the ITS1 and ITS2 regions .................... 97

5.2.5 Data analysis .................................................................................................................. 98

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5.3 Results ........................................................................................................................... 98

5.3.1 Prevalence of infection .................................................................................................. 98

5.3.2 Molecular characterisation of Ancylostoma spp. isolates ............................................ 100

5.4 Discussion.................................................................................................................... 101

5.4.1 Conclusions .................................................................................................................. 106

Chapter 6 Detecting Taenia species of the African painted dog ............. 108

6.1 Introduction ................................................................................................................ 109

6.2 Materials and methods .............................................................................................. 114

6.2.1 Sample collection ......................................................................................................... 114

6.2.2 Parasitological techniques ............................................................................................ 115

6.2.3 Identification of proglottids ......................................................................................... 115

6.2.4 DNA extraction ............................................................................................................ 116

6.2.5 Amplification of Taeniid isolates at the 12S rRNA locus ........................................... 116

6.2.6 Data analysis ................................................................................................................ 117

6.3 Results ......................................................................................................................... 118

6.3.1 Prevalence of Infection ................................................................................................ 118

6.3.2 Identification of taeniid spp. proglottids ...................................................................... 120

6.3.3 Molecular characterisation of taeniid sp. isolates ........................................................ 121

6.3.4 Phylogenetic analysis of Taenia spp. isolates .............................................................. 124

6.4 Discussion.................................................................................................................... 125

6.4.1 Conclusions .................................................................................................................. 129

Chapter 7 General discussion ..................................................................... 130

7.1 Conservation status of the African painted dog ...................................................... 131

7.2 Increasing importance of studies of parasites ......................................................... 131

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7.3 Parasitological techniques ......................................................................................... 132

7.4 Implications for biodiversity conservation .............................................................. 133

7.5 Parasitism in natural populations ............................................................................ 134

7.5.1 Baseline data ................................................................................................................ 134

7.5.2 Parasite threats to painted dogs .................................................................................... 135

7.5.3 Zoonoses/anthropozoonoses ........................................................................................ 136

7.6 Parasitism in captive populations ............................................................................. 137

7.7 Project limitations and future research ................................................................... 138

7.7.1 Sample collection and analysis .................................................................................... 138

7.7.2 Continued surveillance ................................................................................................. 139

7.7.3 Threat of emerging disease .......................................................................................... 140

7.7.4 Captive management .................................................................................................... 140

Appendix 1. Taenia spp. proglottid key. From Verster (1969) .................................. 142

References ......................................................................................................................... 143

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List of Tables

Table 1.1 An overview of the major factors of the host/parasite relationship.

Positive effects are those that are assumed to be beneficial to an

individual or population of hosts; negative effects are assumed to be

harmful to an individual or population of

hosts………………………………………………………..……………4

Table 1.2 A brief natural history of parasites observed in the African painted dog…………………………………………………………………19-20

Table 3.1 Number of faecal samples obtained from all captive and wild

populations during the study period….……………………………….50

Table 3.2 A hierarchical list of comparisons made between host groups with

descriptions of the data used at each level……………………….….…54

Table 3.3 The prevalence (with 95% confidence intervals in parentheses) and

diversity of GI parasites detected in faecal samples from all captive and

wild populations of the African painted dog……………………..……57

Table 3.4 Prevalence (with 95% confidence intervals in parentheses) of GI

parasites detected in faecal samples from two packs within the Zambian

study population………………………………………………….…….58

Table 3.5 The overall prevalence (with 95% confidence intervals in parentheses)

and diversity of GI parasites detected in faecal samples from three wild

populations - Zambia, Zimbabwe and Namibia…………………..…...61

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Table 3.6 Prevalence of the main GI parasites (with 95% confidence intervals in

parentheses) detected in faecal samples from all wild populations in wet

and dry

seasons………………………………………………………………....62

Table 3.7 Aggregation of parasite species within host populations in Zambia,

Namibia and Zimbabwe using the Index of Discrepancy values (D).…65

Table 3.8 GI parasite genera observed in this study and in the published

literature………………………………………………………………..65

Table 4.1 Prevalence of Giardia duodenalis (with 95% confidence intervals in

parentheses) detected in faecal samples from captive and wild

populations of the African painted dog……………………..…………80

Table 4.2 Genotypic characterisation of Giardia duodenalis isolates from

individual African painted dogs at the18S rRNA, β -giardin and gdh

loci. * Wild animals over 2yrs of age were allocated as Adult……...…82

Table 4.3 Prevalence (with 95% confidence intervals in parentheses) and

genotypic characterisation of Giardia isolates obtained in a captive

population of African painted dogs over a three year period………….83

Table 4.4 Assemblages of Giardia duodenalis observed in both wild canids and

domestic dogs noted in the literature and this study. Number of times

reported in literature is presented as (r=)…………………………….86

Table 5.1 Prevalence of Ancylostoma spp. (with 95% confidence intervals in

parentheses) detected in faecal samples from captive and wild

populations of the African painted dog………………………………99

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Table 5.2 Prevalence of Ancylostoma spp. observed in the Zambian and Namibian

populations of the African painted dog over a three year period. Sample

size (n) and 95% confidence intervals in parentheses……….…….....99

Table 5.3 Genotypic characterisation of Ancylostoma spp. isolates obtained from

wild populations of the African painted dog within Zambia and

Namibia. Percentages for matched identities with published sequences in

parentheses…………………………………………...……………….100

Table 5.4 Summary of the nucleotide variations detected between the painted dog

isolate from Zambia (Z-Ka01) and reference sequences for A. caninum

and A. duodenale. Alignment was conducted in Sequencher™ 4.8 and

dots indicate identity with the first sequence…………...…………….101

Table 6.1 Definitive and intermediate hosts of Taenia spp. infecting African

carnivores………………………………………………...…………...112

Table 6.2 Prevalence of taeniids (with 95% confidence intervals in parentheses)

detected in faecal samples from wild populations of the African painted

dog………………………………………………………..…………..119

Table 6.3 Prevalence of taeniids observed in the Zambian and Namibian

populations of the African painted dog over a three year period. Sample

size (n) and 95% confidence intervals in parentheses……..…………119

Table 6.4 Species identification of taeniid proglottids obtained from painted dog

populations……………………………………………...…………….120

Table 6.5 Genotypic characterisation of taeniid spp. species isolates obtained from

wild populations of the African painted dog within Zambia, Namibia

xvii

and Zimbabwe. Percentages for matched identities with published

sequences on GenBank in parentheses……………………………….122

Table 6.6 Summary of the nucleotide variations detected between taeniid isolates

from Zambian and Namibian populations of the African painted dog and

reference sequences for T. multiceps and T. serialis (GenBank accession

numbers GQ228818, DQ408418 and EU219546, DQ104238).

Alignment was conducted in Sequencher™ 4.8 and dots indicate identity

with the first

sequence……………………………………..………………………..123

Table 6.7 Pairwise comparison of sequence variation (%) within the 12S rRNA

loci of T. multiceps and T. serialis reference sequences and taeniid

isolates obtained from Namibian and Zambian populations of the

African painted

dog…………………………………..………………………………..124

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List of Figures

Figure 2.1 Location of painted dog study sites within Australia………………....29

Figure 2.2 Painted dog exhibit at Perth Zoo showing the water feature and animals

resting in the shade……………………………………………………30

Figure 2.3 Painted dog exhibit at Monarto zoo showing the animals in the large

fenced

enclosure……………………………………………………………....31

Figure 2.4 Painted dog enclosure at DeWildt Wildlife Trust showing grassy and

shady environment……………………………………………………32

Figure 2.5 Locations of the painted dog study populations within Africa……….33

Figure 2.6 South Luangwa National Park, Zambia. Open grassland area with acacia

and mopane woodland in the background, which is typical of the habitat

frequented by painted dog populations……………………………….34

Figure 2.7 Nyae Nyae Conservancy, Namibia. One of the few sandy roads

available to use, surrounded by thick grass and acacia trees………….35

Figure 2.8 Game managed area of Hwange National Park, Zimbabwe. Open area

with miombo woodland and acacia in the background, typical for

painted dog

habitat………………………………………………………………...37

Figure 2.9 The left and right side views of an individual painted dog from the

Katete pack in Zambia. ID number P3 0308………………………….40

Figure 2.10 Parasite ova of taeniid spp. and Giardia spp. cysts observed during

microscopy analysis of painted dog faecal samples. Morphology and

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size are substantially different between the two genera, allowing for

unambiguous identification……………………………………………43

Figure 3.1 Parasite load (N), species richness (S) and species evenness (H) in

captive and wild populations of the African painted dog……………..56

Figure 3.2 Parasite load (N), species richness (S) and species evenness (H) in two

packs within the Zambian study population…………………………..59

Figure 3.3 A multidimensional scaling plot of data from two African painted dog

packs within the Zambian population based on Bray-Curtis similarity

coefficients of parasite infracommunity composition…………………59

Figure 3.4 Parasite load (N), species richness (S) and species evenness (H) in three

wild populations of the African painted dog…………………………60

Figure 3.5 Parasite load (N), species richness (S) and species evenness (H) between

wet and dry seasons in all wild populations of the painted dog………63

Figure 3.6 A multidimensional scaling plot of data from wet and dry environments

of three wild populations based on Bray-Curtis similarity coefficients of

parasite infracommunity……………………………………………….63

Figure 5.1 Depiction of the lifecycle of Ancylostoma caninum in domestic dogs

adapted from www.Apaws.org...............................................................92

Figure 6.1. The lifecycle of a taeniid species depicting the transmission cycle

between predator (definitive host) and prey (intermediate host)……111

Figure 6.2 A depiction of the genital atrium of T. crocutae as an example of the

morphological features used in identification of proglottids. From

Verster (1969). Appendix 1 displays the proglottid key used for

identifying samples obtained in this study……………………………116

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Figure 6.3 Multiplex PCR of taeniid isolates from wild painted dog populations at

the 12S rRNA locus. Lanes 2-11 are painted dog samples from Zambia

and Namibia. Lane 13 is the negative control; 14-16 are the positive

controls for Taenia sp, E. granulosis and E. multilocularis…………121

Figure 6.4 Phylogenetic relationships, inferred using the Neighbour-joining method

between taeniid isolates from African painted dogs and reference

isolates of T. multiceps and T. serialis based on sequence data of the 12S

rRNA locus. Representatives of Taenia sp. used for comparison are

labelled with accession numbers as published on GenBank, with E.

granulosus as the out-group. Numbers at nodes refer to bootstrap values

(1500 replicates) > 20%.

………………………………………………………………………125

Chapter 1 - Introduction

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Chapter 1

Introduction


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1.1 Parasites in wildlife

Comprehensive research on parasitism within wildlife populations has, to date, been

minimal (De Leo and Dobson, 2002; Altizer et al., 2003a; Morand et al., 2006). Of these

studies most have been focused on the threat disease may pose to livestock or human

populations, rather than gaining insight into the biodiversity and role these host/parasite

interactions have within the ecosystem (Hudson et al., 2006; Thompson et al., 2010).

Parasitism is the commonest life style on earth (Poulin and Morand, 2000) and parasites

are therefore a major component of ecosystems. An increasing number of studies are

showing that parasites are important in maintaining ecosystem function through their effect

on regulating host population sizes, mediating predatory and competitive interactions

among free living organisms and acting as ecosystem engineers (Anderson, 1972; Temple,

1987; Jones et al., 1994; Hudson et al., 1998). Therefore, understanding parasitism within

wild populations should be an integral part of any wildlife monitoring program and a key

component of wildlife conservation efforts.

1.2 Importance of parasites in conservation

As wildlife populations continue to decline, the need for active conservation of many

species becomes vital. For this to be successful it is essential that there is a thorough

understanding of all threatening processes within the wild environment (Peterson, 1991).

Until recently, there has been a very one dimensional view of the role played by parasites

in wildlife conservation programs. Parasite infection has usually been considered simply as

another threatening process which may require intervention (Daszak et al., 2000; Applebee

et al., 2005; Deredec and Courchamp, 2006; Penzhorn, 2006; Thompson et al., 2010). The


3

reality however, is much more complex. An early study by Sachs and Sachs (1968), for

example, noted that wild animals were often heavily parasitised, when compared with

domestic animals, but showed apparent health. This display of health may be the result of

acquired immunity by the host or, as has been previously noted, wild animals tend to

manifest few recognizable signs of disease (Sachs and Sachs, 1968; Young, 1969). For

example, wild populations of zebra are known to harbour a variety of Cyathostomum spp.,

of which several species are pathogenic to domestic equines, but the zebra host appears to

display no clinical effects (Roberts et al., 2002). Sachs and Sachs (1968) qualified that this

display of health should not be confused with non-pathogenicity of parasites in wild

animals but rather a favourable balance between the host and its parasite community

established through a long evolutionary process. This evolutionary process has often been

referred to as an ‘arms race’ in reference to the constant battle between host and parasite to

gain the upper hand (Dawkins and Krebs, 1979). The outcomes of this are complex

host/parasite relationships which can be stable, cyclical or chaotic with equally complex

effects (May and Anderson, 1983). The complexity of the co-evolutionary relationship

between host and parasites means that parasitism can have both positive and negative

effects for individual hosts, for host populations and for communities of free living

organisms (Table 1.1). These effects arise because of both the direct impact of parasites in

causing disease and indirect impacts of parasites in mediating competitive and predatory

interactions among free-living organisms.


4

Table 1.1 An overview of the major factors of the host/parasite relationship. Positive

effects are those that are assumed to be beneficial to an individual or population of hosts;

negative effects are assumed to be harmful to an individual or population of hosts.

Factor Effects at individual level Effects at population/community level

Positive Negative Positive Negative

Direct impacts of parasitism

Clinical &

Sub-clinical

disease

Development of

host immune

system

Morbidity and

death of susceptible

individuals

Avoiding

overpopulation

Maintenance of

genetic diversity

Local extinction

Regulating host

population size

Development of

host immune

system

Reduced fecundity

and survival of host

Increased host

population size

Decreased host

population size

Indirect impacts of parasitism

Interspecific

competition

Increased

competitive success

Decreased

competitive success

Increased resources

for successful

competitor species

Local extinction of

unsuccessful

competitor species

Manipulation of

prey behaviour

Increased prey

capture

Increased predation

rate

Increased

population size of

predator species

Reduced population

size of prey species

1.2.1 Direct impacts

The direct impacts of clinical disease from parasitism on the host are largely negative at

both the individual and population level as a significant parasite burden can lead to severe

morbidity or death. For example, at the individual level a heavy infection of hookworm in

young or immunocompromised animals can lead to severe anaemia and death of those

individuals (Bowman et al., 2003). At the population level gastro intestinal (GI) parasites


5

such as Trichostrongylus sp. are capable of inducing large population crashes in wild and

domestic species. Hudson et al. (1992) observed this in wild populations of red grouse,

whilst livestock farmers have had long standing battles to reduce heavy stock losses due to

strongyle infections (Herlich, 1978; Fabiyi, 1987). Less obvious are the effects of

subclinical disease on the host however, there is growing evidence to suggest the effects

can be severe. One example is the protozoan parasite Giardia spp. Infection with this

parasite is often asymptomatic but studies have shown that long term infections can lead to

growth retardation in the host, particularly when suffering from poor nutrition (Astiazaran-

Garcia et al., 2000; Berkman et al., 2002). Whilst studies on Giardia spp. have been

focused on human infections this parasite is becoming increasingly recognised in wildlife

populations and therefore may be of similar significance, especially to endangered

populations with declining food resources. Evidence for this has been seen in a study on

red colobus monkey (Piliocolobus tephrosceles) populations living in fragmented forests

of Uganda (Chapman et al., 2006). This study found that populations of red colobus

monkeys, which were experiencing a decline in food availability, were at greater risk of the

detrimental effects of gastrointestinal parasites.

To further complicate matters, wild hosts are often infected with more than one parasite

(polyparasitism) which can sometimes alter or amplify the effects of any subclinical

disease. A recent example of this was illustrated in a study by Munson et al. (2008) who

conducted an analysis of historical data of canine distemper virus (CDV) epidemics within

populations of the African lion. They discovered that these epidemics coincided with

unusually high levels of the blood parasite Babesia, which were in turn the result of high

exposure to the tick vector after periods of drought. Babesia infections in wild hosts are


6

often stable but this study suggests that the subclinical effects of this parasitic infection can

leave the host susceptible to other pathogens (Penzhorn, 2006; Munson et al., 2008). These

complex interactions make the ability to detect the causative agent of disease within hosts

harbouring multiple parasites extremely difficult. Compiling comprehensive baseline data

and conducting continual surveillance on the parasite fauna within different wild host

populations may increase the chances of detecting and predicting these pathogenic

outbreaks.

Consequences of clinical and subclinical disease may include the regulation of host

populations. Mathematical modelling has shown that parasites can provide density

dependent regulation of their host populations through effects on both host mortality rates

and host fecundity rates (May and Anderson, 1978; McCallum and Dobson, 1995). This

has been successfully demonstrated in laboratory studies of helminth infections in snails,

fish and mice (Scott and Anderson, 1984; Anderson and Crombie, 1985; Scott, 1988). For

example, the nematode Heligomosomiides polygyrus introduced into mice populations

resulted in dramatically reduced host densities, which subsequently increased on removal

of the nematode (Scott, 1987). This has also been demonstrated in a wild population of red

grouse (Lagopus lagopus scotius) and the nematode Trichostrongylus tenuis (Hudson et

al., 1992; 1998). Experimental removal of this parasite from populations of red grouse led

to greater clutches, higher hatching success and chick survival, however the control

population continued to display reduced fecundity and cyclical population crashes.

A lesser known aspect of parasitism is the beneficial effects which may be gained by the

host. As a result of the evolutionary arms race between host and parasite it has been


7

proposed that parasites actively promote host immune systems and drive genetic diversity,

and consequently act as a buffer against disease (Windsor, 1998; Altizer et al., 2003a;

2003b; Lindstrom et al., 2004; Maillard and Gonzalez, 2006). Early exposure to parasites

can allow the host to build up effective immunity to withstand later heavy challenges. For

example, Carroll and Grove (1985) demonstrated that dogs currently infected with small

numbers of hookworms developed a functional protective immunity when challenged with

a large inoculum of infective hookworm larvae, whereas dogs previously uninfected with

hookworm developed marked anaemia when challenged with the same large inoculum. A

similar benefit observed is con-concomitant immunity which is the ability of the host to

mount an effective defence against parasite larval stages whilst being unable to clear a

burden of adult worms (Smithers and Terry, 1969). This can create an immunological

barrier against continual infection and restrict burden size within the host. The most

common examples of this are the schistosomes whereby immunologically invisible adult

worms established within the host are responsible for generating an anti larval immune

response which prevents any subsequent infection (Smithers and Terry, 1969).

Another perspective is the well documented theory of the ‘hygiene hypotheses’ (Cookson

and Moffatt, 1997; Yazdanbakhsh et al., 2002; Cooke et al., 2004). This has been referred

to principally within human hosts and proposes that completely eradicating and/or limiting

exposure of parasites from the host in a short evolutionary time span can lead to a

disruption in the host immune system resulting in autoimmune diseases such as ulcerative

colitis, asthma and other allergies (Yazdanbakhsh et al., 2001; Lakatos et al., 2004; Whary

and Fox, 2004). Evidence for this was documented in a human trial with long-term

Crohn’s disease patients who were seen to have a marked decrease in symptoms when


8

infected with a nematode known to be non-pathogenic to humans Trichuris suis (Summers

et al., 2005). This study proposed that the introduction of a parasite into the host reinstated

normal functionality of the immune system.

Host genetic diversity is known to play an important role in enabling natural populations to

withstand widespread epidemics and is especially important in declining populations

(Frankham, 2005). Parasites represent powerful selective agents in natural populations and

are consequently a driving force behind species and genetic diversity (Altizer et al.,

2003b). This was demonstrated in a study of wild populations of Soay sheep (Ovis aries)

infected with intestinal nematodes (Paterson et al., 1998). Allelic variation within the

major histocompatibility complex (MHC) of Soay sheep was significantly associated with

juvenile survival and parasite resistance and it was concluded that parasites played a large

role in maintaining genetic diversity within this host population.

1.2.2 Indirect impacts

The indirect impacts of parasitism are comparatively less well understood than are the

direct effects, but studies are demonstrating the significance these interactions have within

ecosystems (Loreau et al., 2005). Parasites have been observed to be capable of mediating

interspecific competition between host populations and manipulating predator/prey

dynamics (Anderson, 1972; May and Anderson, 1978; Hudson et al., 1992; McCallum and

Dobson, 1995; Hudson et al., 1998; Jog et al., 2005; Barnes et al., 2007).


9

Interspecific competition mediated by parasites has the potential to influence the structure

of ecological communities, the viability of each species within a community and the

potential for new species to invade a community (Thomas et al., 2005). An example of

parasite mediated competition has been observed in populations of white tailed deer

(Odacoileus virginiatus) infected with the parasite Parelaphostrongylus tenuis. White

tailed deer show minimal effects to an infection with this parasite but other cervids suffer

severe neurological effects and death (Anderson, 1972). This prevents invasion by exotic

cervids, leaving the white tailed deer with substantially less competition for food resources

in the area (Anderson, 1972). The inability to introduce domestic cattle into tsetse fly

zones of Africa is another example of parasite mediated competition. Tsetse flies are the

vector for Trypanosoma brucei and T. congolense which cause fatal nagana disease in

domestic ruminants, but are relatively non-pathogenic to the native wild ruminant

population, thus hindering the introduction of exotic species to this habitat (Bowman et al.,

2003).

Another parasite mediated effect within ecosystems is that of manipulation of prey species.

It is perhaps well recognised that predators regulate prey population size but what is not

acknowledged is the role parasites potentially play in this interaction. Parasites with

complex indirect life cycles often rely on their intermediate host being consumed by a

predator in order to complete their life cycle (Choisy et al., 2003; Poulin, 2007). As a

means to increase the chances of reproduction some parasites appear to have adopted

methods of manipulating the behaviour of the intermediate host enabling easier capture by

predators. Temple (1987) conducted a study comparing levels of parasitism in prey items

caught by a tame hawk and those he shot with a gun. He found that prey captured by the


10

hawk had higher intensity and prevalence of trematodes, nematodes and ectoparasites, than

those shot and suggested parasitism was involved in mediating easier capture by the hawk.

Several studies have since attempted to identify the mechanisms by which parasites are

able to achieve this. One example of this involves the cestode, Echinococcus. The

metacestodes of Echinococcus spp. cause hydatid disease in the intermediate host, usually

a herbivore, through the formation of cysts in the hosts organs (Thompson and Lymbery,

1988). These cysts can often form in the lungs, greatly reducing the animals’ ability to

evade capture by a predator which is the definitive host for this parasite (Fenstermacher,

1937; Cowman, 1947; Joly and Messier, 2004; Barnes et al., 2007). Similar observations

have been made with the coccidian Sarcocystis spp. (Jog et al., 2005; Jog and Watve,

2005). These studies observed that sarcocyst-infected prey (chital deer, Axis axis) were

preferentially taken by the predator (dhole, Cuon alpinus) and although there was no

evidence of clinical signs in the infected prey it was suggested that the large number of

cysts in the heart would compete for oxygen and reduce the ability to evade capture.

Although these examples of parasite mediated increases in predation are often assumed to

be adaptations by the parasite to manipulate prey behaviour, they may simply be by-

products of parasitism. In some cases, however, the adaptive nature of parasite

manipulations has been confirmed. For instance, studies have shown that rats infected with

the protozoan Toxoplasma gondii, display specific behavioural changes that increase the

parasites chances of transmission (Berdoy et al., 2000). Infected rats were found to display

a preference for areas frequented by cats and were therefore vulnerable for predation. The

cat is the definitive host for T. gondii and hence transmission for this parasite was

increased.


11

1.2.3 Co-extinction events/biodiversity considerations

In addition to the positive and negative effects of parasites on individual hosts and host

populations, there is increasing recognition that parasites are important components of

biodiversity in their own right. Parasites are the single largest component of biodiversity

yet are often ignored by conservation biologists (Windsor, 1998; Poulin and Morand,

2000). Eradication and co-extinction events are severely affecting this biodiversity leading

to possible adverse flow on effects (Koh et al., 2004; Dobson et al., 2008; Colwell et al.,

2009; Dunn et al., 2009). Parasites that are host specific run the same risk of extinction as

their host and this is gaining recognition with studies advocating the need for the

conservation of parasites along with their hosts (Dobson, 1995; Gompper and Williams,

1998; Daszak et al., 2000; Altizer et al., 2003a; Colwell et al., 2009). Dobson et al. (2008)

estimated that the extinction of all species currently listed as threatened by the IUCN Red

List (IUCN, 2010) would lead to the co-extinction of c. 2000 species of helminth parasites.

Given the complexities of host/parasite interactions the loss of any parasite species may

well result in detrimental effects to the host and the ecosystem (Gompper and Williams,

1998).

Besides co-extinction events another risk to host specific-parasites is their eradication in

captive populations. Management of captive animals has a heavy focus on de-worming

programs to ensure maximum health but has the adverse effect of causing possible

extinction of host specific parasites. An example of this is the recovery program for the

black footed ferret (Mustela nigripes). To prevent total extinction of the remaining wild

population, all animals were captured and placed into captivity for later release. During

this time efforts to minimize the threat of disease included the eradication of ectoparasites


12

within the population. It has since been proposed that a possible species of Neotrichodectes

louse, which was unique to the black footed ferret may now be extinct (Gompper and

Williams, 1998). However, as there was limited baseline information on the parasite fauna

of this host in the wild, positive confirmation of extinction was not possible. This provides

another example highlighting the need for comprehensive data on the parasite fauna of

wildlife hosts, particularly those endangered and being managed through conservation

programs. Criteria for conserving these host-specific parasites may need to be incorporated

into captive breeding programs to maintain the host parasite fauna and levels of immunity

which are present in natural populations (Daszak et al., 2000; Gompper et al., 2003;

Penzhorn, 2006).

1.2.4 Assessing effects of human influences

All of these host/parasite interactions may be further complicated by the effects of habitat

destruction/fragmentation and human encroachment (McCallum and Dobson, 1995; 2002;

Smith et al., 2009a; 2009b). These factors can lead to a disruption of the ‘normal’

host/parasite interactions, with possible detrimental effects to the host (May and Anderson,

1978).

Habitat destruction results in reduced species ranges and increased interactions between

populations, which in turn raises the risk of disease transmission between these populations

(Scott, 1988; Lyles and Dobson, 1993). It can also result in limited resources for viable

populations. The poor planes of nutrition and stress placed on these declining populations

may then result in a rise in parasitic disease. For example, Penzhorn (2006) identified an


13

endemic stability of the haemoparasite, Babesia spp. in wild populations of lion and

rhinoceros but when individuals of this species were placed under stress, clinical

babesiosis was observed resulting in death of the host.

Perhaps of greater significance is human encroachment and the resultant increased

interaction of humans and their domestic animals with co-habiting wildlife. Approximately

80% of domestic animal pathogens can infect wildlife so the risk of disease spillover into

wild populations is potentially great (Kat et al., 1995; Cleaveland et al., 2001; Applebee et

al., 2005). Examples of this have been documented in populations of the African painted

dog and African lion which have suffered huge population declines due to spillover of

rabies and canine distemper virus (CDV) from domestic dog populations (Kat et al., 1995;

Roelke-Parker et al., 1996). In these cases immunity had not been developed in the wild

population, leaving them open to clinical disease. Another example highlighted by

Gillespie et al. (2005) illustrated the effects of human encroachment in a study on several

primate species living in logged forests compared with those living in unlogged forest.

They identified higher parasite prevalence and richness in populations of the red-tailed

guenon (Cercopithecus ascanius) living in logged forests compared to populations in

unlogged forests. Along with a higher prevalence of parasites species including Trichuris,

Strongyloides and Entamoeba, three additional species were observed in the logged forest

population which were Giardia sp., a liver fluke and Chilomastix mesnili. This study

suggested that the human activity of logging had induced a change in the red-tailed

guenon’s foraging behaviour which in turn increased their exposure to parasites.


14

In summary, it would appear that parasites play a major role in the natural environment and

are an essential component of any healthy ecosystem (Hudson et al., 2006). Additionally,

these examples provide evidence of the need to increase surveillance of wildlife and build

an extensive database of natural parasitism within wild populations from which a baseline

can be formed. This will assist in the ability to identify potential emerging disease

processes which may pose a threat to wildlife and should become an essential component

in any wildlife management program.

1.3 The host, the African painted dog (Lycaon pictus)

Understanding the ecology of the host is important when attempting to understand

parasitism within that host. Factors such as diet, behaviour and habitat provide insight into

the spectrum of parasite fauna likely to exist within these populations.

The African painted dog (Lycaon pictus), also known as the African wild dog and painted

hunting dog, is an endangered carnivore of sub-Saharan Africa which represents a unique

genus of the canid family, having diverged from the rest of the dog family 3 million years

ago (Girman et al., 1993; IUCN, 2010). Lycaon pictus translates to ‘painted wolf’ in

reference to the mosaic of colours and patterns of the animals’ coat which are unique to

each dog. The last century has seen the population decline by almost 99% from

approximately 300,000 individuals to 3,000-5,500 with viable populations existing in only

a few countries (Fanshawe et al., 1991; Woodroffe et al., 1997; IUCN, 2010). The cause of

their decline has been a combination of human persecution, habitat loss, interspecific


15

competition and disease (Fanshawe et al., 1991; Woodroffe et al., 1997; Creel and Creel,

1998; Sillero-Zubiri et al., 2004).

1.3.1 Ecology of the African painted dog

African painted dogs are intensely social pack animals spending much of their time in

close association with each other (Woodroffe et al., 1997). They are co-operative breeders

and hunters which make them almost obligate pack animals in that survival outside the

pack is minimal (Jennions and Macdonald, 1994; Creel and Creel, 1995). Painted dogs are

extremely successful hunters and cooperative hunting allows them to bring down large

prey, usually averaging 50 kg but sometimes as large as 200 kg (Malcolm and van Lawick,

1975; Creel and Creel, 1995). Their diet consists mainly of antelope such as impala, kudu

and gazelle but may also include buffalo, hares and lizards (Creel and Creel, 2002; Pole et

al., 2004; Hayward et al., 2006). Packs are based on a matriarchal society, with the alpha

female governing pack movements. The alpha female and her chosen alpha male are

usually the only pair to breed, whilst the rest of the pack assist in rearing the pups through

hunting and pup-guarding (Malcolm and Marten, 1982; Creel and Creel, 1995). Studies

suggest that in order to breed successfully packs need to consist of a minimum of five

animals which allows for both hunting and pup-guarding to take place (Courchamp et al.,

2002; Sillero-Zubiri et al., 2004). This requirement indicates that maintaining pack

numbers, rather than individuals, is vital to ensure continuation of the species (Creel and

Creel, 1995).


16

The African painted dog is a nomadic species with home ranges greater than 2,000 km2.

The only time packs are stationary is during the denning season which is usually for three

months or until the pups are old enough to follow the pack. During the denning season the

pack, hunting diurnally, will return each time to the den to feed the pups by way of

regurgitation of meat that they have consumed.

1.3.2 Conservation issues for the African painted dog

The predominant threat to the African painted dog is conflict with expanding human

populations, specifically through habitat loss, human persecution and introduced disease

(Creel and Creel, 2002; Sillero-Zubiri et al., 2004). The lack of large protected areas

(greater than 10,000 km2) results in packs roaming outside national parks and into conflict

with humans. Subsequently the risk of death and injury is markedly increased due to roads,

where they are commonly killed, being caught in snare lines, which are set by poachers for

bushmeat, and being shot or poisoned by livestock farmers (Fanshawe et al., 1991; van

Heerden et al., 1995; Creel and Creel, 1998; Rasmussen, 1999). Another human/wildlife

conflict issue is that of domestic dogs. Painted dogs are vulnerable to diseases of domestic

dogs such as rabies and canine distemper virus (CDV). Several painted dog populations

have been adversely affected by epidemic outbreaks of these diseases with a possible

extirpation occurring in the Serengeti ecosystem of Tanzania due to the rabies virus

(Alexander and Appel, 1994; Kat et al., 1996; Woodroffe, 1999). As these animals

naturally live at low population densities catastrophic events such as these pose real threats

to their continued conservation (Creel and Creel, 2002).


17

Measures taken to counteract the dramatic decline in species numbers have included

reintroduction and relocation programs. Currently there are approximately 400 animals in

captivity according to the International Species Information System (ISIS). Many of these

animals have had success in breeding, allowing for possible reintroductions. Few

reintroduction programs, however, have taken place and most have had limited success

(Scheepers and Venzke, 1995; Woodroffe et al., 1997). Some wild populations in South

Africa are being actively managed as metapopulations, which involves translocation of

animals between fenced wild populations to mimic natural immigration and emigration

(Davies-Mosert et al., 2009). Translocation programs are also used to move animals away

from livestock farms, where conflict with farmers is occurring, and back into the relative

safety of national parks.

1.4 Parasites and the African painted dog

As with most wildlife species, there has been only limited research on the parasites of the

African painted dog. In order to obtain a better understanding of the host/parasite

relationship it is beneficial to understand the ecology of the host, obtain data on similar

species and most importantly conduct longitudinal studies over several populations.

1.4.1 Host ecology and parasites

Parasitism is often heavily influenced by the ecology of the host, as the parasite will have

adapted its life cycle according to host diet, behaviour and habitat. The African painted dog

is a carnivore and therefore exposure to parasites will often be through ingested prey


18

species. These can act as intermediate hosts for parasites such as members of the cestode

family Taeniidae, and the protozoans Toxoplasma gondii and Sarcocystis spp. Hunting in

packs will see all individuals exposed to the same infected prey species. Additionally, as

they are extremely social animals, other parasites requiring direct transmission between

hosts such as Ancylostoma spp., Giardia spp. and ectoparasites will be easily transmitted

between pack members (Altizer et al., 2003a). This would be especially important during

the denning period when environmental contamination with parasite stages increase and

subsequently transmission rates of these parasites between individuals would also increase

(Altizer et al., 2003b). Evidence for this has been seen with rabies virus epidemics which

have wiped out entire packs (Kat et al., 1996). Finally, as these animals have large home

ranges, packs will often move outside the park borders and through human communities,

thereby raising the risk of them contracting exotic pathogens from domestic dogs (Creel

and Creel, 2002).

1.4.2 Parasites recorded from the African painted dog

The dramatic decline of the African painted dog has lead researchers to focus on ecological

factors such as pack dynamics, the effects of human/wildlife conflict and habitat

fragmentation. The majority of information regarding the parasite fauna of the African

painted dog comes from one study conducted in the early 90’s (van Heerden et al., 1994;

van Heerden et al., 1995). This involved the sampling of 46 animals in Kruger National

Park over three one-week periods and information recorded on the presence of parasites

from blood and faecal samples. The GI parasites that were reported consisted of Taenia sp,

Ancylostoma caninum and an unknown ascarid species. Toxascaris canis was also reported


19

but this species name is incorrect and was most likely Toxocara canis or Toxascaris

leonina. The haemoparasites reported included Dipetalonema reconditum (microfilariae),

Hepatozoon sp. and Babesia sp. Apart from these studies, there have been incidental

reports resulting from necropsy or parasite specific studies. Young (1975) observed E.

granulosus from one dog in Kruger National Park, Bwangamoi et al. (1993) found

Sarcocystis-like organisms in three animals which had died from rabies. Colly and Nesbit

(1992) reported the death of a painted dog pup due to acute babesiosis. Babesia sp. and

Hepatazoon sp. were also reported by Nietz (1965), Peirce et al. (1995) and more recently

Matjilla et al. (2008). The natural history and importance of these parasites is described in

more detail in Table 1.2.

Table 1.2 A brief natural history of parasites observed in the African painted dog.

Parasite species Transmission Description/lifecycle Importance

Ancylostoma sp. Direct ingestion or skin penetration of infective larvae

Small nematode in the small intestine of the host, which hooks onto the host and sucks blood

Severe anaemia may result in death of host. Possible zoonosis

Taenia sp. Indirect. Intermediate host, usually a herbivore

Large tapeworms in the gut of the definitive host. Gravid proglottids expelled onto pasture and ingested by the intermediate host

Possible intestinal obstruction of definitive host. Intermediate host may be compromised due to cysts forming in organs. Possible zoonoses

Echinococcus sp. Indirect. Intermediate host, usually a herbivore

Small tapeworms in the gut of the definitive host. Gravid proglottids expelled onto pasture and ingested by the intermediate host

Non pathogenic to the definitive host. Causes hydatid disease in the intermediate host. Zoonosis

Toxocara sp. Direct. Ingestion of infective stage

Large roundworms in the intestine of the host

Larval migrans may cause liver and lung damage. Possible zoonoses

Babesia sp. Indirect. Transfer from tick species during a bloodmeal

Protozoan found in blood cells of the host

Anaemia and/or neurological disease causing death


20

Hepatozoan sp. Indirect. Transfer through ingestion of infected tick

Protozoan in various tissues and white blood cells of the host

May cause sub-clinical disease in the host

Sarcocystis sp. Indirect. Intermediate host, usually a herbivore

A protozoan in the gut of the definitive host

Non-pathogenic to the definitive host. May cause disease in the intermediate host

Dipetalonema reconditum

Indirect. Transfer from flea or louse

A small nematode in the connective tissue of the host. Also microfilariae in the blood stream which are taken up by vector during a blood meal

Non-pathogenic to the definitive host

1.4.3 Gaps in current knowledge of parasitism in the African painted dog

The studies referred to above, whilst beneficial, only provide a ‘snap shot’ of parasitism

within this endangered carnivore. Perhaps the most significant gap in knowledge is that of

comprehensive baseline data which document the prevalence and intensity over time of

parasitism within wild populations (Bjork et al., 2000; Mathews et al., 2006; Smith et al.,

2009a). The single random sampling studies commonly undertaken are not able to identify

temporal or spatial variation of parasitic infection within the natural environment (Bjork et

al., 2000). Longitudinal studies are required to obtain this information, which would then

help to determine which potentially pathogenic agents to monitor within natural

populations (Mathews et al., 2006; Smith et al., 2009b). Increasingly, there is recognition

of the need for good surveillance of native fauna, which draws upon data from related

species and uses longitudinal monitoring to identify and predict the parasites and

pathogens of most importance (De Leo and Dobson, 2002; Smith et al., 2009b). Few

studies have achieved this and to date none have been conducted on the African painted

dog.


21

1.5 Management of wild species

Human involvement has become pervasive in the management of nearly all wild species.

Most common is the management of wild animals in captive environments where the focus

is on maintaining breeding populations and genetic diversity with the view to eventually

reintroducing these animals to the wild. Management of wild populations has also

increased as suitable habitat continues to decline and the use of translocation programs is

required to move animals away from human conflict areas. Within both these management

environments detailed knowledge of the ‘natural’ parasite fauna of a particular host may

assist conservation efforts by monitoring for disease outbreaks, identifying exotic

pathogens and perhaps allowing for natural immunity against some parasites (Phillips and

Scheck, 1991; Gompper and Williams, 1998; De Leo and Dobson, 2002).

1.5.1 Captive environments

In wildlife facilities with good veterinary practices, animals are more likely to be exposed

to fewer parasites than their wild relatives. However, relatively low levels of parasitism

may still result in clinical disease, as captivity places stress on wild species, which may

affect the functioning of immune systems. Contrary to this, whilst complete eradication of

parasites in captive populations is beneficial to the animal in the short term, this may later

lead to under developed immune systems and a subsequent greater risk of clinical disease

when exposed to pathogens (Windsor, 1997; Gompper and Williams, 1998; Penzhorn,

2006). Additionally, intensive housing, modern, grassy enclosures and mixed species

enclosures may increase the opportunity for parasite transmission between species. On

occasion, the parasites causing clinical disease are not a natural parasite of the species but


22

result from co-habiting with other species, cross-contamination from other enclosures, and

modified behaviours commonly displayed in captive animals. For example, the tree

browsing giraffe (Giraffa camelopardalis) when kept in modern, grassy enclosures will sit

down and graze (Boomker et al., 1986; Garijo et al., 2004); this modified behaviour

exposes them to parasites from the pasture that they would not normally encounter.

Fukumoto et al. (2004) reported the death of a giraffe due to a heavy infection of the

nematode Camelostrongylus mentulatus, which is only obtained during grazing. This was

compounded by the fact that the giraffe were housed together with camel and antelope, the

natural hosts for this parasite.

Reports such as these, investigating unexpected deaths, are generally how parasitism is

identified in captive environments. There are very few studies directly comparing

parasitism among captive and wild populations. Muller-Graf et al. (1999) conducted a

study on wild populations of lions and then made comparisons with retrospective reports of

parasites in captive populations. Mas Bakal et al.(1980) investigating Toxoplasma gondii,

made direct comparisons between captive and wild animals in Kenya, and Phillips and

Scheck (2001) compared captive and wild populations of red wolves during a

reintroduction program. Whilst it is intuitive that there will be differences between the two

environments, studies documenting specific differences in parasite prevalence and

diversity remain elusive. Data such as these may greatly benefit facilities which use captive

breeding and reintroduction programs as a conservation tool.


23

1.5.2 Reintroduction programs

If when reintroducing captive bred animals into their wild environment, the type of parasite

pressure to which the host is likely to be exposed is known, then measures can be

undertaken to reduce this impact. A simple example of this was documented in a study on

reintroducing captive bred red wolves (Canis rufus). Phillips and Scheck (1991) noted that

it was beneficial to conduct reintroduction programs during winter when the tick burden

was not as high, as in summer animals appeared to suffer from high infestations and this

apparently reduced the success of the program. Pre-exposure to parasites or maintaining

low levels of ‘natural’ parasite fauna within captive animals may limit the effect of the

substantial parasite challenge they will face in wild environments (Viggers et al., 1993).

For example, when the last remaining population of black footed ferrets (Mustela nigripes)

were taken into captivity, infections with the protozoan parasite Eimeria sp. were allowed

to persist in order for the host population to maintain immunity for eventual release into

the wild (Williams et al., 1992). Additionally, there is a risk of introducing exotic parasites,

which animals have acquired from their captive facilities, into naïve wild populations

(Viggers et al., 1993; Cunningham, 1996). However, without detailed knowledge of what

parasites are naturally found in the wild environment this would be difficult to predict and

prevent.

1.5.3 Translocation programs

As with reintroduction programs the translocation of wild animals from one area to another

may pose the risk of introducing exotic disease to either the existing population or the

translocated animals (Davidson et al., 1992; Cunningham, 1996; Wyatt et al., 2008).


24

Translocation is quite often a necessary tool for moving animals away from high

human/wildlife conflict areas, stocking depleted populations or in the management of

metapopulations (Davidson et al., 1992; Davies-Mosert et al., 2009). Again, having

extensive baseline data on the parasite fauna of hosts in their natural environment in

different geographic regions will help identify any risk of introducing possible pathogens

to either population during these events (Cunningham, 1996). Cases of introducing exotic

disease from translocation have often had devastating effects. Examples include the co-

introduction of rinderpest with domestic cattle into East Africa which devastated the native

ungulate population, and the protozoan (Myxobolus cerebralis) which causes whirling

disease in trout and is believed to have been introduced from stocked European trout into

native North American rainbow trout (Oncorhynchus mykissi) (Dobson, 1995; Bergersen

and Anderson, 1997).

These examples provide evidence for the need to increase surveillance of wildlife and to

build an extensive database on natural parasitism within wild populations from which a

baseline can be formed. This will assist in the ability to identify potential emerging disease

processes which may pose a threat to wildlife.

1.6 Goals for this study

Using the African painted dog as a model, this study aims to highlight the importance of

obtaining comprehensive data on parasite biodiversity within an endangered species.

Longitudinal data obtained from several wild populations will provide a baseline on which

further studies can build. Individual animal data such as sex, age and social status, when


25

available, is used to identify susceptibility to parasitism at the individual and population

level. Additionally, environmental factors such as season or geography are incorporated

into the analysis to determine if these are influential in the overall dynamics of parasitism

within the natural environment.

To complement these data, the use of molecular tools will enable species identification,

which has not been possible with traditional microscopy techniques. In many cases,

molecular characterisation of parasites from faecal samples can eliminate the need for

species identification from adult worms, which are usually only obtained during necropsy

of animals. As the host involved in this study is endangered and obtaining animals for

necropsy is almost impossible, these non-invasive methods provide the only way to gain

further insight into the parasite fauna they harbour. Identification of parasite species will

help to ascertain which are host specific, pathogenic, zoonotic or exotic to this species.

It is hoped that this information will assist in the conservation of the African painted dog in

several ways:

providing a comprehensive database on the natural parasitism of Lycaon pictus

which will help identify exotic, zoonotic and emerging diseases within wild

populations.

recognising challenges of parasite control and management within captive

populations

understanding the risks associated with reintroducing parasite-naïve animals into

their natural environment


26

understanding the risks of introducing exotic parasites between populations during

translocation and reintroduction programs

Identifying parasite fauna existing in the natural environment which may become

pathogenic within the host if/when exposed to environmental stressors.

These goals are addressed within the thesis in the following ways:

Chapter 2 details the general research methods utilized throughout the project.

Chapter 3 identifies the prevalence and diversity of parasites within wild and captive

populations, whilst ascertaining significant effects of host sex and age, and environmental

conditions of season and geographical region.

Chapters 4, 5 and 6 address specific parasite threats. Identification through molecular

characterisation will allow species identification and an understanding of species

distribution among hosts and populations. These methods also assist in identifying any

health risks posed to either the host or surrounding human communities within both

captive and wild environments.

Chapter 7 discusses the main findings with reference to the goals identified in Chapter 1.

Chapter 2 – General materials and methods

27

Chapter 2

General materials and methods


28

2.1. Study design

A parasitological study was conducted on the African painted dog (Lycaon pictus) from

both captive and wild populations. Collaborations were established with three captive

animal facilities and three in situ conservation groups within Africa. The captive

populations were housed in a range of environments including a free range savannah, zoo

exhibit style and a rehabilitation/breeding centre. The wild populations were situated in a

range of geographic locations within Africa. This range of facilities and locations was

chosen in order to get a broad sample base from which variations in parasite prevalence

and diversity may be identified.

These collaborations were crucial to the success of this project, as they allowed for

continual sampling to occur from a range of environments. These associations also

provided the opportunity for additional sampling during radio collaring and/or non-related

veterinary events, whereby skin and blood samples could be obtained along with faecal

samples. The majority of samples were collected during project field trips, however, some

samples were collected by collaborators in the field during their own monitoring season.

Sampling was conducted over the three years of the project (2007-2009) with the exception

of samples from Zimbabwe which had been collected previously in 2006. Most

populations were sampled on multiple occasions during the study period. This sampling

protocol was aimed at achieving longitudinal data from a range of environments in order to

obtain a detailed baseline from which comparisons could be made.


29

2.2 Captive populations

The captive populations were located at Monarto Zoo in South Australia, Perth Zoo in

Western Australia (Figure 2.1) and DeWildt Wildlife Trust in South Africa (Figure 2.5).

Figure 2.1 Location of painted dog study sites within Australia.

2.2.2 Perth Zoo

Perth Zoo is a traditional zoo exhibiting animals in enclosures designed to be similar to the

wild environment. The facility had a painted dog population of 17 animals: 12 males which

were in the on-display exhibit and five females which were kept separately off-exhibit to

prevent unplanned breeding. The on-display exhibit was a large sandy area (1550 m2) with

large trees for shade and a small pond water feature (Figure 2.2). The off-exhibit area was

smaller (480 m2) but also sandy with large trees for shade. All animals were given

= Perth Zoo, Western Australia

= Monarto zoo, South Australia


30

ivermectin monthly as a heartworm prophylaxis. Praziquantel had previously been given as

a preventative treatment for Taeniidae infections but medication with this drug had been

halted a year before this project began. The painted dog diet consisted mainly of horse

meat and the occasional sheep carcass which had been previously frozen. The three eldest

animals were captive born and originated from DeWildt Wildlife Trust in South Africa.

The remaining 14 animals were the result of two litters born at Perth zoo. Samples were

collected from both male and female exhibits annually over the three years prior to the

monthly ivermectin treatment in November 2007, 2008 and 2009. A total of 43 faecal

samples were collected and analyzed from the 17 animals over the three year period. Data

collected annually were used to make comparisons within that population, however, data

from 2007 only were used when making comparisons between populations to avoid

including multiple samples from the same animal.

Figure 2.2 Painted dog exhibit at Perth Zoo showing the water feature and animals resting

in the shade.


31

2.2.3 Monarto Zoo

Monarto zoo is an open range safari style zoo located in South Australia. The facility had a

total of 33 animals: 20 males which were in the on-display exhibit and 13 females which

were kept separately off-exhibit to prevent unplanned breeding. The on-display exhibit

area was a large (8 km2) open savannah style which visitors drove through on zoo buses

(Figure 2.3). The off-exhibit was a much smaller sandy area of approximately 500 m2.

Regular anthelmintic treatments were given to prevent parasitic infections however actual

the anthelmintics used is unknown. The painted dog diet consisted mainly of kangaroo and

horse meat which had been previously frozen. The three eldest animals originated from

DeWildt Wildlife Trust with the remaining animals being the result of litters born at the

zoo. Faecal samples were collected from this facility once in May 2008. It is not known

how recently anthelmintic treatment had occurred prior to sample collection. A total of 34

faecal samples were collected randomly by keepers during routine cleaning of enclosures.

Figure 2.3 Painted dog exhibit at Monarto zoo showing the animals in the large fenced

enclosure.


32

2.2.3 DeWildt Wildlife Trust

DeWildt Wildlife Trust is a breeding and rehabilitation centre for cheetah and African

painted dogs located in Pretoria, South Africa. The facility had approximately 80 African

painted dogs which were housed in up to 25 different enclosures. Enclosures held between

one and six animals made up of various mixes depending on compatibility of animals and

breeding requirements. Enclosures were of varying sizes, ranging from approximately 500

m2 to 1,500 m2, mostly grass covered and with trees for shade (Figure 2.4). Animals were

treated with fenbendazole every four months as a preventative treatment for roundworm,

hookworm and tapeworm. Diet consisted mainly of previously frozen whole chickens and

horse meat with Iams dry dog food. Some animals originated from the wild having been

translocated from livestock farms where they were at risk of being shot. The remaining

population was the result of litters born at the facility. Samples were collected from this

facility once in December 2007, which unfortunately was just after the quarterly

anthelmintic treatment with fenbendazole.

Figure 2.4 Painted dog enclosure at DeWildt Wildlife Trust showing grassy and shady

environment.


33

2.3 Wild populations

The wild populations were located in South Luangwa National Park, Zambia, Nyae Nyae

Conservancy, Namibia and Hwange National Park, Zimbabwe (Figure 2.5).

Figure 2.5 Locations of the painted dog study populations within Africa.

2.3.1 South Luangwa National Park – Zambia

Zambia is a land-locked country situated in central sub-Saharan Africa (Figure 2.5). South

Luangwa National Park is situated at the tail end of the Great Rift Valley and has an area

of 9,050 km2. It is the second largest park in Zambia having been officially proclaimed in

1972 mainly in order to conserve the endemic Thornicroft’s giraffe (Giraffa

camelopardalis thornicroftii). The Luangwa River acts as the eastern border of the park

and is home to large populations of hippopotamus and crocodiles. The park has

= South Luangwa National Park, Zambia

= Hwange National park, Zimbabwe

= Nyae Nyae Conservancy, Namibia

DeWildt Wildlife Trust, South Africa


34

populations of lion, spotted hyaena, leopard and various game animals. The vegetation of

the park is a mosaic of mopane, miombo woodland, acacia and grassland (Figure 2.6).

There are two main seasons within Zambia, the dry season from May to November and the

wet season from December to April.

Figure 2.6 South Luangwa National Park, Zambia. Open grassland area with acacia and

mopane woodland in the background, which is typical of the habitat frequented by painted

dog populations.

The population of African painted dogs within South Luangwa National Park is estimated

at 200 (pers comm. E Droge, Zambian Carnivore Project). Field trips were conducted in

collaboration with the Zambian Carnivore Project, a conservation group based in South

Luangwa National Park. Field agents from this project continually monitor populations of

the African painted dog and other carnivores in order to conserve these species and their

habitats. Samples were collected during field trips in October 2007, June 2008 and May

2009 with occasional sampling between field trips by Zambian Carnivore Project staff


35

during their monitoring periods of painted dog packs in and around the national park.

Where possible a GPS radio collar was placed on an individual within a pack in order to

follow pack movements and ascertain roaming areas. This greatly increased the chances of

locating packs and collecting faecal samples for this project.

2.3.2 Nyae Nyae Conservancy – Namibia

The Nyae Nyae Conservancy is situated in the north eastern corner of Namibia east of

Tsumkwe district (Figure 2.5). It is not a national park but a conservancy area for some of

the last remaining San Bushmen, of the Ju’\Hoansi group. The area is approximately 8,900

km2 and contains a population c.3,000 of predominantly San Bushmen who are traditional

hunter gatherers. The area has populations of hyaena, leopard and a small population of

lion, along with various game animals. The habitat is semi-arid savannah with no perennial

surface water (Figure 2.7), therefore wildlife are reliant on the few water holes existing

throughout the dry season of April – October. A wet season exists from November to

March.

Figure 2.7 Nyae Nyae Conservancy, Namibia. One of the few sandy roads available to

use, surrounded by thick grass and acacia trees.


36

Apparently 75 painted dogs are found in the Tsumkwe area (Lines, 2009). Field trips were

conducted in collaboration with the Wild Dog Project a conservation group conceived in

2002 by the Namibian Nature Foundation in response to a growth in local human/painted

dog conflict. The main aim of the Wild Dog Project is to increase the understanding of

interactions between African wild dogs and humans and finding ways of mitigating

conflict and persecution (www.nnf.org.na/wilddogproject.htm). Samples were collected

during a field trip in July 2008 in conjunction with the Wild Dog Projects own monitoring

period of painted dog packs in the area. Additional sampling was conducted throughout the

monitoring seasons of 2007 and 2008 by Wild Dog Project staff. Some packs had an

individual fitted with a GPS collar, therefore radio-tracking was possible. However,

locating packs within this area was difficult even using telemetry as the flat terrain greatly

reduced signal range and there were minimal roads available to use.

2.3.3 Hwange National Park – Zimbabwe

Zimbabwe is located just below Zambia and is also a land-locked country (Figure 2.5).

Hwange National Park is Zimbabwe’s premier park and is situated in the north east corner

of Zimbabwe, bordering Botswana. The park has populations of lion, spotted hyaena,

leopard and various game animals. The vegetation of the area is a mosaic of miombo

woodland, acacia and open vlei areas (Figure 2.8). Seasons are similar to Zambia with a

dry season from May to November and a wet season from December to April.


37

Figure 2.8 Game managed area of Hwange National Park, Zimbabwe. Open area with

miombo woodland and acacia in the background, typical for painted dog habitat.

A field trip was conducted in May 2009, in collaboration with the Painted Dog

Conservation group which continually monitors populations of painted dogs in and around

adjacent game managed areas of Hwange National Park. Some packs had an individual

with a GPS collar, therefore radio-tracking was possible, however during this field trip no

painted dog packs were located. Although no samples were collected during this time

faecal samples had been stored from previous collections of which a portion collected in

April 2006 were made available for this study.

2.4 Sampling methodology

The majority of samples obtained for this project were faecal samples. This is a non-

invasive method of obtaining biological data from wild animals and has been used for

several previous parasitological studies on wildlife (Muller-Graf, 1995; Marathe et al.,


38

2002; Engh et al., 2003; Smith and Kok, 2006). Non-invasive methods are particularly

useful when dealing with endangered animals. Faecal samples can provide information on

many parasites inhabiting the gastro-intestinal tract including helminths and protozoans.

Additionally, the application of molecular tools increases the information that can be

obtained from these samples.

2.4.1 Ethics and permits

Animal ethics approval was obtained from Murdoch University Animal Ethics Committee

(permit number R2087/07) for the collection of samples including faeces, blood and

ectoparasites from all captive and wild populations of the African painted dog involved in

this study. Additionally the project obtained separate ethics approval from Perth

Zoological Parks Authority Animal Research and Ethics Committee (Project number,

2007-18; reference ZA/3149025776) for the collection of samples from the painted dog

population at that facility.

Permits were obtained from the Zambian Wildlife Authority to conduct research on

parasites of African wild dogs within South Luangwa National Park and adjacent Game

Management Areas. Permits were also obtained from the Ministry of Environment and

Tourism to conduct research and collect samples from the African painted dog populations

in the Kavango and Caprivi regions of Namibia (Permit number 1297/2008). Government

permits were not needed for Zimbabwe as faecal samples obtained had been collected

previously by other researchers with the required documentation.


39

2.4.2 Locating packs

Conducting studies on wildlife can be logistically difficult as finding and tracking animals

within the wild environment is often challenging. The African painted dog has a large

roaming area (>2,000 km2) and is particularly hard to visually locate due to the colour and

pattern of each animals coat, which act as excellent camouflage. During the day, packs are

inactive and are usually resting under trees or scrub and are difficult to locate. Radio

tracking was therefore crucial to locate packs on any given day. Most packs within the

study population had at least one animal radio-collared, which was usually sufficient to

locate the rest of the pack. Radio-tracking was most successful when the packs were on the

move as there was more chance they would move into the main game area and within

telemetry range. This was usually when the packs were hunting which occurs in the

morning and evening. Tracking in the morning was usually from 5am to 11am and in the

afternoon from 3pm to 8pm, however, if it appeared the packs had moved from the vicinity

of base-camp it was necessary to conduct longer searches, whereby the entire day was used

for tracking. Occasionally this also required several days camping in the bush to continue

the search in that area. On occasion a light aircraft was used to radio-track from the air

which enabled the search grid to be greatly expanded.

When the packs were located, they were identified by compiled indentikits. Coat patterns

are unique for each animal and are used as a means of identification. Animals therefore had

both left and right sides of their torso photographed. Identification numbers were allocated

to each animal within each pack and used for later identification in the field (Figure 2.9).


40

Figure 2.9 The left and right side views of an individual painted dog from the Katete pack

in Zambia. ID number P3 0308.

2.4.3 Sample collection

Sample collection in the wild environment involved locating and following packs and

picking up faecal samples as dogs defecated. Faecal samples were placed in a ziplock bag

and kept in a cooler box until return to camp. On return samples were placed into 2 x 10 ml

centrifuge tubes and fixed in 70% ethanol and 10% formalin. Tubes were labelled with

date, place collected, pack name and where possible individual animal identification. Many

times it was not possible to identify individual animals due to the fast movements of

animals as they rouse for a hunt or to interference from safari vehicles. Pack identification

however, was usually obtained. During field trips three animals were anaesthetised in order

to place a radio-collar, and in this case it was possible to obtain blood samples, check for

ectoparasites and obtain faecal samples direct from the animal. Samples of blood were

stored on FTA cards and a smear placed on a microscope slide, while ectoparasites were

placed in 70% ethanol.


41

Sample collection within the captive environment involved walking through enclosures

and collecting faecal samples from the ground. Individual animal information was

therefore not possible in most cases. At Perth zoo however, it was possible to get

individual samples with the use of non-toxic plastic beads to act as faecal markers for

individual dogs. With the help of keepers all animals were fed meat parcels (horsemeat)

which had had coloured plastic markers (1 tsp) inserted into a pocket cut into the meat.

Animals were brought into the night quarters where they could be separated, identified and

handfed the parcel of meat. Colours given to each animal was recorded in order to match

scat samples collected in the next 24-48 hours. This method has been used with success in

several studies on both wild and domestic animals (Delahay et al., 2000; Rolfe et al., 2002;

Staniland, 2002; Hernot et al., 2005).

2.4.4 Sample movement

The appropriate permits were obtained to export samples from Africa to Australia. A

permit (number 73453) was obtained from Ministry of Environment and Tourism for

samples sent from Namibia. Permits (numbers not given) were obtained from the

Department of Veterinary and Livestock Development for each shipment of samples sent

from Zambia.

AQIS permits to import fixed faecal samples into Australia were not required providing

specimens were preserved in either 70% ethanol or 10% formalin and containers were

sealed and packaged correctly. This ensured all pathogens were killed and the risk of

unforseen leakage was contained. Samples were stored in 10 ml centrifuge tubes fixed in


42

the appropriate preservative (70% ethanol or 10% formalin). These tubes were then

wrapped in absorbent cotton material and placed inside two plastic coverings. These were

then boxed and sent with copies of the appropriate export and import documentation for

each country. All packages were sent through international shipping companies as

‘dangerous goods in accepted quantities’ packages to Murdoch University.

2.5 Parasitological techniques

Analysis of fresh faecal samples was preferred as parasite ova are more readily observed

under microscopy, however, for quarantine reasons samples had to be preserved in a

fixative. Faecal samples fixed in 10% formalin (2 parts faeces, 8 parts formalin) were

deemed the preferred fixative for microscopy (Foreyt, 2001). Samples fixed in 70%

ethanol were used for molecular characterisation.

2.5.1 Microscopy; qualitative analysis

Identification of parasite ova from faecal samples was made on size and morphology with

reference to Foreyt (2001) (Figure 2.10). The coccidians observed were unable to be

differentiated on size alone and as samples were preserved in 10% formalin oocyts, were

not able to be sporulated for additional morphological information. Additionally,

carnivores are commonly known to exhibit spurious coccidian infections originating from

ingested prey items further complicating identification (Bowman et al., 2003). However,

whilst it was recognized that some observations of coccidia may not be true infections, it

was impossible to ascertain which were not spurious, therefore all positive samples were


43

included in the prevalence data. In an attempt to observe all parasites existing within each

sample three techniques were employed: Malachite green stain, zinc sulphate flotation and

sodium nitrate flotation.

Figure 2.10 Parasite ova of taeniid spp. and Giardia spp. cysts observed during

microscopy analysis of painted dog faecal samples. Morphology and size are substantially

different between the two genera, allowing for unambiguous identification.

The malachite green stain was based on a simple faecal smear but with a drop of 5%

malachite green added which enabled parasite ova, particularly Cryptosporidium oocysts,

to be more readily observed (Elliott et al., 1999). Flotation techniques are commonly used

to recover parasite eggs and oocysts from faeces and are designed around differences in

specific gravity of parasite ova and faecal debris within specific flotation media (Bowman

et al., 2003). Most parasitic stages float at a specific gravity of 1.2 to 1.3 whilst faecal

debris will sink (Foreyt, 2001). As some parasite ova are heavier (eg. Trichuris sp. ova)

and formalin fixing can alter the normal buoyancy of eggs, two flotation media were used,

sodium nitrate with a specific gravity of 1.27 and zinc sulphate with a specific gravity of

1.18 (pers comm. A. Elliott). Sodium nitrate was used with the commercially available

Faecalyzer® tubes as gravitational force alone was sufficient for eggs to float within this


44

medium (Bowman et al., 2003). A small amount of faecal sample was placed into the tube

which was then filled with sodium nitrate and left for 10 minutes. A coverslip placed on

top of the tube collected the parasite ova which had floated to the top and was then used for

microscopy. Zinc sulphate was used for the remaining sample with 2 g of faeces placed

into a 10 ml centrifuge tube, emulsified in the flotation medium and centrifuged at 2,000

rpm for five minutes. This method concentrated all parasite ova into the top layer of the

tube where a wire loop was used to collect 5 drops of fluid from the meniscus and placed

onto a microscope slide for analysis. Three slides containing blood smears were to be

analysed for the presence of haemoparasites such as Babesia spp. using microscopy,

however, before this could be done slides were lost. Results for ectoparasites obtained

during the study period were not included in this thesis due to the very small sample size.

2.5.2 Microscopy; quantitative analysis

Quantitative analysis was undertaken by calculating eggs per gram (EPG) of faeces.

Calculating EPG is generally done using a McMaster counting chamber (Foreyt, 2001;

Bowman et al., 2003). The zinc sulphate technique used in this project involved a known

amount of faeces and fluid for each test but the wire loop collection method inhibited use

of a McMaster chamber, therefore alternate calculation methods were devised (pers comm.

R. Dobson). As outlined above, five wire loop drops were placed onto a slide and all

parasite ova and cysts under the coverslip were counted. The volume of the fluid placed

onto the slide was weighed and found to be 0.035 g, coming from the meniscus of the 10

ml tube which was estimated at 0.1 ml. Therefore 1/3 of floated ova and cysts were


45

counted. To obtain EPG the total was multiplied by three (for total meniscus count) and

then divided by 2 (as 2 g of faeces was in each 10ml tube).

2.5.3 Molecular characterisation

As species identification for many parasites is not possible from parasite ova, molecular

techniques were employed to characterise these to species level. As these techniques are

time consuming and expensive only parasite genera considered important from a zoonotic

or pathogenic perspective were chosen. Molecular characterisation was therefore

conducted on faecal samples containing taeniid ova, hookworm ova and Giardia cysts.

Full methodologies for these are found in Chapters 4, 5 and 6. Five blood samples on FTA

cards were sent to a laboratory in South Africa for molecular studies on haemoparasites

such as Babesia spp. but results have not yet been received. These same FTA card blood

samples were tested for the presence of Trypanosoma spp. using molecular methods. All

samples were negative and as there were so few samples, results were not included in the

main body of this thesis.

Chapter 3 – Diversity and prevalence of…..

46

Chapter 3

Diversity and prevalence of gastrointestinal parasite fauna within captive and wild populations

of the African painted dog


47

3.1 Introduction

There are at least as many species of parasites as there are free-living organisms, yet except

for humans and a small number of domesticated plants and animals, little is known about

the diversity and prevalence of parasites existing within host populations (Altizer et al.,

2003a; Mathews et al., 2006; Smith et al., 2009a). It is widely acknowledged that parasites

have the ability to regulate host populations through their ability to influence host

mortality, morbidity and fecundity (May and Anderson, 1978; Scott, 1988; McCallum and

Dobson, 1995; Hudson et al., 1998). Parasites may also mediate predatory and competitive

interactions, host immunity, host genetic diversity and host behaviour (Anderson, 1972;

Carroll and Grove, 1985; Temple, 1987; Altizer et al., 2003b; Maillard and Gonzalez,

2006; Barnes et al., 2007). These factors are important processes within ecosystems and

highlight the significance of parasitism within host populations. To add to these complex

interactions, the effects of outside stressors such as habitat destruction and human

encroachment are important influences which can easily disrupt the natural balance of

many host/parasite relationships (McCallum and Dobson, 1995; McCallum and Dobson,

2002; Gillespie et al., 2005).

The increasing recognition of parasites and their interactions within wildlife hosts has

resulted in additional research, yet comprehensive baseline data are still lacking for many

species of wildlife (Daszak et al., 2000; Applebee et al., 2005; Deredec and Courchamp,

2006; Penzhorn, 2006; Thompson et al., 2010). Most studies on parasitism in wildlife

focus either on understanding the life cycle and pathogenesis of a single parasite species or

taking a ‘one-off’ snapshot of the parasite community existing within a particular host

population. While such studies are useful starting points, of greater benefit would be


48

longitudinal studies of several populations in various geographical regions. This detailing

of temporal and spatial variation of parasitic infections within the natural environment

would provide a baseline from which potential pathogenic changes could be identified

(Bjork et al., 2000). Pathogenic effects may originate from introduced exotic parasites for

which host populations have not developed any level of immunity, for example, the

introduction of rabies and canine distemper virus from domestic dog populations into

African lion (Panthera leo) and African painted dog (Lycaon pictus) populations

(Alexander and Appel, 1994; Kat et al., 1995; Cunningham, 1996; Daszak et al., 2000).

Likewise, enzootic parasites which within a healthy population cause little pathogenicity

could give rise to clinical disease in populations experiencing stressors such as drought,

overcrowding due to habitat destruction or human encroachment (McCallum and Dobson,

2002; Penzhorn, 2006; Munson et al., 2008). Without good baseline data of parasitism

within natural environments these events would be difficult to identify or predict. The lack

of good baseline data is in large part due to the difficulties involved in sampling wild

populations. Locating study animals, obtaining individual identifications and obtaining

samples is time consuming and logistically difficult.

The African painted dog is a highly endangered carnivore with approximately 3,000

animals remaining in the wild (IUCN, 2010). These individuals exist in small fragmented

populations which are vulnerable to stochastic disease outbreaks, leading to local

extinctions as already observed in the Serengeti ecosystem (Kat et al., 1996; Sillero-Zubiri

et al., 2004). It is therefore essential for the future conservation of this animal to gain a

broad understanding of threatening processes, such as parasitism, to which this animal is

routinely exposed.


49

This is the first longitudinal parasite survey of the African painted dog and one of few

existing for any wild species. This project was fortunate in being able to collaborate with

three in situ conservation groups in Zambia, Namibia and Zimbabwe, which were

constantly monitoring painted dog packs within their study area. This enabled

comprehensive sample collection from known individuals and packs over various

geographic regions. By sampling over a period of time in various geographic regions it was

hoped to gain insight into the population dynamics of parasitism within a wild and

endangered carnivore.

The aim of this study was to determine the prevalence and diversity of gastrointestinal (GI)

parasites within captive and wild populations of the African painted dog and relate

measures of parasitism to intrinsic factors such as host age and sex, and extrinsic factors

such as location and season. The following hypotheses were tested: 1) levels of parasitism

are markedly different between captive and wild populations; 2) levels of parasitism are

greater in the wet season than in the dry season in the wild; 3) levels of parasitism are

greater in younger animals and 4) there are geographical differences in levels of parasitism

between wild populations.

3.2 Materials and methods

3.2.1 Sampling protocol

Faecal samples were obtained from three wild populations and three captive populations of

the African painted dog over a four year period, 2006 – 2009. Chapter 2 gives detailed

descriptions of populations sampled. A total of 128 samples were collected from a possible


50

83 individual animals within the wild populations and 104 samples from 130 individual

animals within the captive populations (Table 3.1)

Table 3.1 Number of faecal samples obtained from all captive and wild populations during

the study period.

Population Sampling year Total

samples

Individual

animals

2006 2007 2008 2009

Wild populations

Zambia - 11 44 18 73 43

Namibia - 19 10 - 29 28

Zimbabwe 26 - - - 26 12

Total 26 30 54 18 128 83

Captive populations

Perth - 16 14 13 43 17

Monarto - - 34 - 34 33

DeWildt - 27 - - 27 80

Total 0 43 48 13 104 130

Collection of samples from wild populations was conducted during field monitoring of

packs within the study area. Faecal samples were collected soon after being deposited and

individual animal information was obtained where possible. Within Zambia, most samples

were collected during the periods of November to December 2007, May to June 2008 and


51

May to June 2009, however, a small proportion were collected throughout the year by field

collaborators. Within Namibia, most samples were collected during August to November

2007 and May to November 2008 by field collaborators. Within Zimbabwe, samples were

collected in April 2006 and stored for later analysis.

Collection of samples from captive populations was conducted during cleaning of

enclosures by zoo keepers. A single sampling episode was undertaken in December 2007

for the South African population and May 2008 for the South Australian population. These

samples were obtained randomly from group enclosures. Sampling in Western Australia

was conducted each year in November of 2007, 2008 and 2009. In Western Australia,

sampling of individual hosts was made possible by spiking the animals’ meat with non-

toxic plastic beads which allowed for identification on defecation (see Chapter 2).

Two grams of faecal matter from each sample were placed into two 10 ml centrifuge tubes

containing 8 ml of either 10% formalin for subsequent microscopy analysis or 70% ethanol

for molecular characterisation.

3.2.2 Parasite identification and quantification

See Chapter 2 for a full description of microscopy methods used. All parasite ova, cysts

and larvae were recorded. Eggs and/or oocysts per gram (EPG) of faeces were calculated

as described in Chapter 2 and results used for quantitative analyses. EPG values were not

obtained for Spirometra spp. or Filaroides spp. as these were rare observations. Similarly,

EPG values were not obtained for Giardia spp. as it was found that cysts were especially


52

hard to observe in formalin fixed samples due to desiccation, leading to an under

estimation of intensity.

3.2.3 Host and environmental variables

When age and sex of the host was known, individual data were used for comparisons. Ages

of two years and under were defined as juvenile (n=9) and above 2 years were defined as

adult (n=20). Nineteen samples were obtained from identified males and 13 from females.

All wild sample sites had distinctive wet and dry seasons which were also used for

comparisons. Wet season samples (n=28) were from December through to March and dry

season samples (n=62) from April through to November.

3.2.4 Data analysis

This study examined the differences in parasite prevalence and diversity, intensity of

parasite infection and parasite infracommunity composition within and between captive

and wild populations of the African painted dog.

3.2.4.1 Descriptors used

Descriptors used to quantify parasitism within this study followed Bush et al. (1997) and

Poulin (1993) as follows:

Parasite load (N) is the total number of parasites, of all species, in a single infected

host, i.e. the number of individual parasites in an infracommunity. Parasite load

was measured by the total EPG.


53

Prevalence is the percentage or proportion of hosts infected with one or more

individuals of a particular parasite species or taxonomic group.

Diversity is the number of species of parasites present within specific individuals

and/or groups of hosts. Diversity of parasites was measured by species richness (S)

and the Shannon-Wiener index of species evenness (H).

Infracommunity composition is the number and intensity of species in an individual

host.

Parasite aggregation within host populations is defined as when most hosts harbour

few or no parasites, while only few are heavily infected. Aggregation was measured

by the index of discrepancy (D) using EPG data for a particular species.

3.2.4.2 Levels of testing

Levels of parasitism were firstly tested between wild and captive groups for significant

differences. The data from wild populations were then analysed at the pack and population

(all packs within a national park) level and related to intrinsic factors such as host age and

sex, and extrinsic factors such as geographic location and season (see Table 3.2). For many

comparisons sufficiently large sample sizes could only be obtained by pooling data over

age, sex and seasonal classes, or over different packs or populations. Data from year 1

(2007) of the Perth Zoo captive population were pooled with the other two captive

populations for comparison with wild populations. In wild populations, data were pooled

over time when making comparisons as it was found that the composition of packs varied

significantly from season to season with many deaths recorded, consequently duplicate

sampling of individuals was extremely rare. Known duplicates obtained within the same

sampling period were discarded before performing statistical analysis.


54

Table 3.2 A hierarchical list of comparisons made between host groups with descriptions

of the data used at each level.

Comparison Data used for comparison

Between wild and captive populations Data from the three captive populations

were pooled and compared against pooled

data obtained from the three wild

populations.

Among packs Data from two packs within the Zambian

population were pooled over all age, sex

and season classes.

Among populations in different geographic

regions in the wild

Data from Zambia, Namibia and Zimbabwe

were pooled over all age, sex and seasonal

classes in each population.

Among wet and dry seasons in the wild Data were pooled over geographic regions,

age and sex and compared between wet and

dry season.

Among host sex classes in the wild Data were pooled over geographic regions,

season and age and compared between

males and females.

Among host age classes in the wild Data were pooled over all geographic

regions, season and sex and compared

between juveniles and adults.


55

3.2.4.3 Statistical analysis

Prevalences of infection were expressed as a percentage of positive samples found with

95% confidence intervals calculated assuming a binomial distribution, using the software

Quantitative Parasitology 3.0 (Rozsa et al., 2000). Differences in prevalences between

groups were tested using the Fisher’s exact test.

One way analysis of variances (ANOVA’s) were used to compare species richness (S),

species diversity (H) and parasite load (N) among groups of hosts with the software JMP

4.0 (SAS Institute Inc; www.sas.com).

Similarities in parasite species infracommunity composition among individual hosts were

estimated from EPG data using the Bray-Curtis coefficient. Multidimensional scaling plots

were used to represent these data where a stress value of less than 0.2 was taken to indicate

reasonable representation of rank similarities by the ordination plot (Clarke, 1993). The

significance of differences in parasite species composition between groups of hosts was

tested by a permutation procedure applied to the pair-wise similarity matrix (one-way

ANOSIM) using the computer package PRIMER 6.0 (Clarke and Gorley, 2006). The

contribution of individual parasite species to dissimilarities between variables was assessed

by averaging the Bray-Curtis similarity term for each species over all pair-wise variable

combinations using SIMPER procedure in PRIMER 6.0. Parasite aggregation within hosts

was tested using the index of discrepancy, (D) as per Poulin, (1993) with the software

Quantitative Parasitology 3.0 (Rozsa et al., 2000).


56

3.3 Results

3.3.1 Variation between captive and wild populations

Table 3.3 displays the diversity of GI parasites within all captive and wild populations

sampled. Overall prevalence of infection (i.e. infection with any species of parasite) was

significantly greater in wild than in captive populations (Fisher’s exact test, P<0.0001).

The wild population also had a greater parasite load (N; F=503.1, P<0.0001), species

richness (S; F=213.3, P<0.0001)) and species evenness (H; F=80.9, P<0.0001), (Figure

3.1)

-1

0

1

2

3

4

5

6

7

8

9

N S H

Measures of parasitism

Mea

n v

alu

e

Captive

Wild

Figure 3.1 Parasite load (N), species richness (S) and species evenness (H) in captive and

wild populations of the African painted dog.


57

Table 3.3 The prevalence (with 95% confidence intervals in parentheses) and diversity of GI parasites detected in faecal samples

from all captive and wild populations of the African painted dog.

Population Total parasite

prevalence

(%)

Taxon prevalence (%)

Taeniid Ancylostoma Spirometra Giardia Coccidia Sarcocystis Filaroides

Captive

n=130

15

0 0 0.8

(0.04 - 4)

14

(6 – 16)

0 0 0

Wild

n=83

99 32

(22 – 42)

32

(23 – 43)

2

(0.07 – 6)

26

(18 – 37)

13

(8 – 24)

99

(94 – 100)

2.4

(0.4 – 8)


58

3.3.2 Variation between packs

Two packs (Katete and Kaingo) within the Zambian study population were sampled

sufficiently to enable comparisons to be made on parasite prevalence, parasite load,

diversity and infracommunity composition. Table 3.4 displays the prevalences of all GI

parasites observed in the Katete and Kaingo packs of South Luangwa National Park,

Zambia. The Katete pack had a significantly greater prevalence of taeniid spp. (Fisher’s

exact test, P=0.005), Ancylostoma spp. (Fisher’s exact test, P=0.002) and Coccidia

(Fisher’s exact test, P=0.005) than that observed in the Kaingo pack. The Katete pack also

had a greater parasite load (N; F=28.4, P<0.0001), species richness (S; F=34.8, P<0.0001)

and species evenness (H; F=41.9, P<0.0001) (Figure 3.2).

Table 3.4 Prevalence (with 95% confidence intervals in parentheses) of GI parasites

detected in faecal samples from two packs within the Zambian study population.

Pack ID Taxon prevalence (%)


Katete

(n=12)

50

(24-76)

58

(29-82)

8

(0.4-37)

8

(0.4-37)

50

(24-76)

100

(76-100)

16

(3-46)

Kaingo

(n=13)

0 0 0 46

(22-74)

0 100

(77-100)

0

P value 0.005 0.002 0.5 0.07 0.005 1.00 0.2


59

-2

0

2

4

6

8

10

12

14

N S H


Mea

n va

lue

Katete

Kaingo

Figure 3.2 Parasite load (N), species richness (S) and species evenness (H) in two packs

within the Zambian study population.

Infracommunity composition was also found to be significantly different between painted

dogs in the two packs (ANOSIM, R=0.42, P=0.001). This can be clearly seen in the two-

dimensional ordination plot of Bray-Curtis similarity coefficients of parasite

infracommunity composition (Figure 3.3).

Figure 3.3 A multidimensional scaling plot of data from two African painted dog packs

within the Zambian population based on Bray-Curtis similarity coefficients of parasite

infracommunity composition.


60

3.3.3 Variation among different geographic locations

Sampling was conducted in three geographic locations - Zambia, Namibia and Zimbabwe.

Populations in these locations were tested for differences between parasite prevalence,

parasite load, diversity and infracommunity composition. Coccidia were significantly less

prevalent in Namibia when compared with Zambia (Fisher’s exact test, P=0.009) and

Zimbabwe (Fisher’s exact test, P=0.02), however, no significant differences were found in

the prevalence of other parasite species or in the overall prevalence of GI parasite

infections (Table 3.5).

A significant difference in parasite load (N; F=3.2, P=0.04), but not species richness (S;

F=1.1, P=0.3) or species evenness (H; F=0.9, P=0.5), was observed among populations

(Figure 3.4). There were, however, no significant differences in parasite infracommunity

composition among populations (ANOSIM R=0.06, P=0.08).

0

1

23

4

5

6

78

9

10

N S H


Mea

n va

lues

Zambia

Namibia

Zimbabwe

Figure 3.4 Parasite load (N), species richness (S) and species evenness (H) in three wild

populations of the African painted dog.


61

Table 3.5 The overall prevalence (with 95% confidence intervals in parentheses) and diversity of GI parasites detected in faecal

samples from three wild populations - Zambia, Zimbabwe and Namibia.

Population Total

prevalence

(%)

Taxon prevalence (%)


Zambia n=43 100

(91-100)

35

(22-50)

33

(19-48)

2

(0.1-13)

30

(18-45)

21

(11-36)

100

(91-100)

5

(0.8-16)

Namibia n=28 96

(83-100)

25

(12-45)

43

(26-62)

0 29

(14-48)

0 96

(83-100)

0

Zimbabwe n=12 100

(76-100)

33

(12-63)

8

(0.4-37)

0 8

(0.4-37)

25

(7-54)

100

(76-100)

0


62

3.3.4 Variation between different seasons

All study sites have definite seasonal variation with a wet and dry season. Table 3.6

displays the prevalence of GI parasites obtained from hosts during the wet and dry seasons

pooled over populations from all geographic locations. A significantly greater prevalence

was observed for Ancylostoma spp. (Fisher’s exact test, P=0.03) and taeniid spp. (Fisher’s

exact test, P=0.005) during the wet season.

Table 3.6 Prevalence of the main GI parasites (with 95% confidence intervals in

parentheses) detected in faecal samples from all wild populations in wet and dry seasons.

Season Taxon prevalence (%)

Taeniid Ancylostoma Giardia Coccidia Sarcocystis

Wet (n=28) 46

(28-65)

61

(41-71)

18

(7-36)

18

(7-36)

100

(88-100)

Dry (n=62) 23

(13-35)

27

(18-40)

26

(16-38)

19

(11-31)

98

(91-100

P-value 0.03 0.005 0.6 1.00 1.00

Parasite load (N; F=0.5, P=0.4) was not significantly different between seasons but a

greater level of species richness (S; F=4.7, P=0.03) and species evenness (H; F=5.2,

P=0.05) was observed in the wet season (Figure 3.5).


63

0

1

23

4

5

6

78

9

10

N S H


Mea

n va

lues

Wet

Dry

Figure 3.5 Parasite load (N), species richness (S) and species evenness (H) between wet

and dry seasons in all wild populations of the painted dog.

Community composition was also found to be significantly different between seasons

(ANOSIM R=0.13, P=0.007) as displayed in Figure 3.6. This is shown in the two-

dimensional ordination plot of Bray-Curtis similarity coefficients of parasite

infracommunity composition among populations (Figure 3.6).

Figure 3.6 A multidimensional scaling plot of data from wet and dry environments of

three wild populations based on Bray-Curtis similarity coefficients of parasite

infracommunity.


64

3.3.5 Variation between host age and sex

No significant relationships were identified between host sex and levels of parasite

prevalence (Fisher’s exact test, P=0.3), parasite load (N; F=0.002, P=0.9), species richness

(S; F=1.6, P=0.2), species evenness (H; F= 0.9, P=0.3) and parasite infracommunity

composition (ANOSIM R=0.16, P=0.09).

No significant relationships were identified between host age and levels of parasite

prevalence (Fisher’s exact test, P=1.00), parasite load (N; F=1.2, P=0.3), species richness

(S; F=0.4, P=0.5), species evenness (H; F=1.1, P=0.9) and parasite infracommunity

composition (ANOSIM R=0.01, P=0.5).

3.3.6 Parasite aggregation within hosts

Parasite species from which sufficient EPG data were obtained (Ancylostoma spp., taeniid

spp., coccidia and Sarcocystis spp.) were tested for aggregation within the host populations

of Zambia, Namibia and Zimbabwe. Aggregation of these parasites was observed within

each population by the Index of Discrepancy (D) values. A value of 0 indicates an even

distribution of counts across all hosts and a value of 1 indicates all parasites aggregated in

a single host (Table 3.7).


65

Table 3.7 Aggregation of parasite species within host populations in Zambia, Namibia and

Zimbabwe using the Index of Discrepancy values (D).

Ancylostoma Taeniid Coccidia Sarcocystis

Zambia 0.83 0.81 0.85 0.7

Namibia 0.8 0.84 - 0.72

Zimbabwe 0.85 0.7 0.8 0.51

3.4. Discussion

3.4.1 Parasite diversity

Overall, a total of seven GI parasite genera were observed in the African painted dog. Five

of these genera have not been reported before in this host: Giardia, Spirometra,

Sarcocystis, Isospora and Filaroides.

Table 3.8 GI parasite genera observed in this study and in published literature.

GI parasite genera detected in this study GI parasite genera reported in published

literature*

Taeniid

Ancylostoma

Spirometra

Giardia

Isospora

Sarcocystis

Filaroides

Taeniid

Ancylostoma

Ascarid (species undetermined)

*Young et al., 1975; van Heerden et al., 1994, 1995.


66

The most prevalent parasite genus observed in wild populations of dogs was Sarcocystis

(99%), followed by taeniids (32%), Ancylostoma (32%), Giardia (26.5%), Coccidia (13%),

Spirometra (2.4%) and Filaroides (2.4%). In contrast, only Giardia (14.5%) and

Spirometra (0.8%) were observed in the captive populations.

The high prevalence of Sarcocystis spp. sporocyst in faecal samples suggests that the

painted dog is a natural definitive host for this organism. This finding is perhaps not

surprising considering carnivores are known definitive hosts for species of parasite with

transmission occurring through ingestion of infected prey. Species identification within the

genus Sarcocystis was not made in this study but based on the prey items of the painted

dog and known intermediate hosts, likely species can be suggested. Cosmopolitan species

which have canids as the definitive host include S. arieticanis (sheep), S. capricanis (goat)

and S. cruzi (cattle) and could all be potential sources of infection for the painted dog.

Species with intermediate hosts specific to Africa include S. nelsoni (puku and waterbuck),

S. melampi (impala), S. woodhousi (springbok, dik dik) and S. phacochoeri (warthog)

(Levine, 1986; Odening, 1986). While definitive hosts for these Sarcocystis species are

unknown, the intermediate hosts are known prey items of the painted dog which could

therefore be considered as a possible definitive host (Estes, 1967; Creel and Creel, 1995;

Hayward et al., 2006).

The only previous study reporting this parasite in the painted dog observed Sarcocystis-

like organisms in the musculature and suggested that the painted dog was a natural

intermediate host (Bwangamoi et al., 1993). Whilst carnivores acting as intermediate hosts

for Sarcocystis is rare, schizonts of S. canis, which are normally only present in the


67

intermediate host have been identified in the tissues of domestic dogs (Dubey and Speer,

1991). Sarcocystis is rarely pathogenic to the definitive host, but acute infection in the

intermediate host may cause extensive tissue damage and subsequent increased mortality

and morbidity (Dubey et al., 1989; Bowman et al., 2003).

Taeniids and Ancylostoma spp. were found at the same prevalence of 32% within the wild

populations, which is similar to a previous report of a wild population of the painted dog

(van Heerden et al., 1995). Taeniids are generally non-pathogenic to the definitive host but

heavy infections may lead to possible intestinal obstruction (Foreyt, 2001). Infections with

Ancylostoma can be pathogenic, particularly to young and/or immunocompromised

animals, and deaths due to ancylostomiasis have been recorded in a captive population of

the painted dog (van Heerden et al., 1994; 1996).

Species of Giardia had not been observed in the African painted dog prior to this study,

but this parasite genus is becoming increasingly recognised in populations of several wild

host species (Graczyk et al., 2002; Gompper et al., 2003; Gillespie et al., 2005; Trout et al.,

2006; Thompson et al., 2009). The prevalence observed in this study indicates a substantial

portion of animals in the wild are infected, with unknown clinical effects. This parasite

does have the ability to cause sub-clinical disease, such as growth retardation in hosts

suffering from poor nutrition, which may be of consequence to declining painted dog

packs experiencing lowered hunting success (Astiazaran-Garcia et al., 2000; Courchamp et

al., 2002).


68

Whilst differentiation among the coccidians was not possible for most observations,

Isospora canis was identified on the basis of the large size of the oocysts, separating it

from most other coccidians. This parasite is host specific to canids, indicating a true

infection rather than a spurious observation of parasites from ingested prey species. Heavy

infections can cause severe enteritis, particularly in pups (Bowman et al., 2003). Filaroides

spp. and Spirometra spp. were only observed rarely in this study but these are the first

reports for the painted dog. Spirometra was observed in both captive and wild populations,

while Filaroides was observed only in wild populations.

3.4.2 Influence of geographic, seasonal and demographic factors

Within wild painted dogs there were some differences observed in prevalence and diversity

of parasite species between packs, geographic populations and seasons, but overall a fairly

consistent parasite community composition existed. Significant differences in parasite

prevalence, diversity and infracommunity composition were observed between packs in

Zambia. This difference is likely to be a consequence of the social aspects of packs, such

as close physical contact and ingestion of the same prey items which will result in minimal

variation of parasite fauna within packs. Differences between packs however, will be

maximised even if these variations arise from chance (stochastic) events, rather than

underlying differences in animals or environment. At the population level the composition

of parasite infracommunity did not differ significantly between dogs from Zambia,

Namibia and Zimbabwe. That is, the same types of parasite species were observed in all

wild populations. There was however, a greater parasite load found in populations from

Zambia and Zimbabwe than in the Namibian population. This may be a reflection of


69

climate differences among geographic areas as Zambia and Zimbabwe have similar

climates, whilst the study site located in Namibia is dryer with a desert-like environment

outside the wet season.

Seasonal factors did appear to have an effect on parasite composition and load. The

parasite composition of populations in the wet season displayed a greater abundance of

Ancylostoma spp. and taeniid spp. whilst populations in the dry season had a greater

abundance of Sarcocystis spp. and coccidians. Moist conditions are favourable for the

transmission of Ancylostoma spp. as it is beneficial for the survival of the infective larval

stage hence, transmission rates between hosts are increased, explaining the higher

abundance observed in the wet season. Whilst taeniid ova are extremely resilient to the

environment, prolonged high temperature can reduce survivability and therefore

transmission rates would be reduced in a hot, dry environment and increased in a cool,

moist environment (Lawson and Gemmell, 1983).

Sex or age of the animal appeared not to have any effect on parasite load, parasite

composition or general prevalence of any parasite species. The result for age was

surprising as young animals are often more susceptible and therefore exhibit higher

parasite loads (Bowman et al., 2003). This result may be a reflection of insufficient

sampling of young animals, as samples were difficult to obtain from painted dog pups

which remain at the den site for the first three months. Similar results have been reported,

however, in prevalence studies on other species of wildlife (Muller-Graf et al., 1999; Bjork

et al., 2000; Feliu et al., 2001). In particular, a study on lions in Tanzania infected with

Spirometra spp. found no relationship between sex or age and the mean number of parasite


70

species, although there was geographic variation (Muller-Graf et al., 1999). Additionally,

studies on trematodes and their hedgehog host (Erinaceus europaeus) found no significant

differences in intensities between host sex or age, but again environmental factors such as

season have been shown to be significant (Feliu et al., 2001).

Aggregation of parasites within hosts was evident for all parasite species tested within this

study. Aggregation has been commonly seen in wildlife populations, including painted dog

competitor species such as African lion and spotted hyaena (Muller-Graf, 1995; Shaw et

al., 1995; Engh et al., 2003). The aggregation of parasites within individual hosts can be

due to several factors such as difference in prior history of exposure (young animals with

undeveloped immune systems); difference in susceptibilities to pathogens,

(immunocompromised animal); or simply chance events of infection (Holt and Boulinier,

2005). Aggregation may well be beneficial to the host population as it is thought to

stabilize the host-parasite dynamics (Holt and Boulinier, 2005).

3.4.3 Parasites of wild and captive populations

In all captive populations the diversity and prevalence of parasites was much reduced

compared to wild populations. This was perhaps expected as captive facilities actively

reduce parasitism through modification of diet (a lack of wild prey) and regular treatment

with anthelmintics. The level of difference, however, was quite marked, with animals in

captive facilities virtually parasite-free. This could be of significant consequence for

animals marked for reintroduction programs as they may have insufficient immunity to

withstand the challenge of a ‘natural’ parasite load in the wild. For example, dogs with


71

prior exposure to Ancylostoma spp. are able to build a protective immunity against later

potentially pathogenic burdens, whereas immunologically naïve animals are highly

susceptible to clinical disease (Carroll and Grove, 1985). In the long term, populations

which remain parasite-free over several generations may exhibit an overall reduction in

genetic diversity, in particular in the major histocompatibility complex (MHC) which plays

an important role within the immune system. Studies have found parasite infections have a

selective force on MHC diversity within the host, subsequently increasing diversity of this

important region of the genome (Paterson et al., 1998; Wegner et al., 2003; Gouy de

Bellocq et al., 2008). An increased polymorphism of the MHC may ultimately increase the

ability of the host to mount adequate immune responses to future parasitic infections and is

therefore an important consideration in captive breeding populations.

3.4.4 Conclusions

In summary, this study has obtained comprehensive data on the prevalence and diversity of

gastrointestinal parasites within the painted dog. Several parasites have been reported for

the first time which demonstrates the paucity of information available for this endangered

carnivore. Studies such as this are important to provide a detailed baseline from which

future conservation programs may benefit. Identifying any departures from the ‘normal’

levels of parasitism may indicate threatening processes at play. This could then allow

predictions and subsequent preventions for emerging diseases, as well as providing

guidance for parasite control in captive breeding populations.

Chapter 4 – Molecular epidemiology of…..

72

Chapter 4

Molecular epidemiology of Giardia duodenalis in an endangered carnivore – the African painted dog

A form of this chapter has been published as:

A. Ash, A. Lymbery, J. Lemon, S. Vitali and R.C.A. Thompson (2010), Molecular

epidemiology of Giardia duodenalis in an endangered carnivore – The African painted

dog. Veterinary Parasitology, 174, 206-212.


73

4.1 Introduction

The impact that parasites and other infectious agents have on wildlife has been

increasingly recognized within conservation programs. Stressors such as human

encroachment and habitat destruction are altering the incidence and effect that these

pathogens have on wildlife populations, especially those endangered and under stress

(McCallum and Dobson, 1995; McCallum and Dobson, 2002; Wyatt et al., 2008; Smith et

al., 2009a; Smith et al., 2009b). Habitat destruction results in reduced species ranges and

increased interactions between populations, which in turn raises the risk of disease

transmission between these populations (Scott, 1988; Lyles and Dobson, 1993). Perhaps of

greater significance is human encroachment and the resultant increased interaction of

humans and their domestic animals with co-habiting wildlife. Approximately 80% of

domestic animal pathogens can infect wildlife (Cleaveland et al., 2001) so the risk of

disease spillover into wild populations is potentially great.

Parasite species previously observed in the African painted dog have included the

macroparasites Taenia sp., Ancylostoma caninum, Dipetalonema reconditum and Toxocara

sp. (van Heerden, 1986; van Heerden et al., 1995), and protozoan parasites Babesia canis,

Hepatozoon sp. and Sarcocystis sp. (Colly and Nesbit, 1992; Bwangamoi et al., 1993;

Peirce et al., 1995; Penzhorn, 2006). To date, Giardia have not been recorded in the

African painted dog.

Giardia duodenalis (syn G. intestinalis; G. enterica) is a ubiquitous protozoan parasite

found in a wide range of animals which can cause diarrhoea and ill thrift in a wide range of

host species (Farthing et al., 1986; Thompson, 2004). Molecular characterisation of


74

Giardia duodenalis isolates has identified seven genetically distinct genotypes which

appear to be host specific (Thompson, 2000; Monis et al., 2009). These genotypes have

been named assemblages A through to G. Of interest to this study are Assemblages C and

D which are specific to dogs and Assemblages A and B which affect humans, but are also

found in a variety of other mammals. Assemblages A and B are thus of particular

significance as they are considered potential zoonotic agents. For this reason, there is an

emerging interest in domestic animals and wildlife as possible reservoirs for this parasite.

There have been many published studies of Giardia duodenalis in domestic dogs but

relatively few for wild canids, reflecting the paucity of parasitological studies in wildlife

(Applebee et al., 2005; Thompson et al., 2010).

Domestic dogs are often infected with G. duodenalis assemblages C and D, but

assemblages A and B are also common (van Keulen et al., 2002; Traub et al., 2004a;

Ipankaew et al., 2007; Leonhard et al., 2007; Covacin et al., 2010). Studies of wildlife

species have also observed infections with assemblages A and B and these have raised

questions on the transmission dynamics of this parasite (Graczyk et al., 2002; Fayer et al.,

2006; Thompson et al., 2009). Zoonotic transfer has been suggested between dogs and

humans living in close conditions (Traub et al., 2004a; Lalle et al., 2005; Ipankaew et al.,

2007; Pelayo et al., 2008; Winkorth et al., 2008). However, the total risk appears to be low

(Hopkins et al., 1997; Berrilli et al., 2004). Alternatively, anthropozoonotic transfer has

been suggested, whereby humans may infect other animals, particularly wildlife (Graczyk

et al., 2002; Applebee et al., 2005; Caccio et al., 2005). Overall the transmission dynamics

and subsequent risks to human health or wildlife are not well understood (Caccio et al.,

2005).


75

The aim of this study was to examine wild and captive populations of the African painted

dog for the presence of Giardia species, and to genetically characterise any isolates

obtained from these populations using three loci, 18S rRNA, β-giardin and the glutamate

dehydrogenase gene. This will enable identification of Giardia duodenalis at the

assemblage and sub-assemblage levels and subsequently any health risk this parasite may

pose within wild and captive environments of this endangered carnivore.



Faecal samples were collected whilst tracking wild populations in Zambia and Namibia

during three field trips conducted in 2007, 2008 and 2009. In most cases these samples

were collected within moments of being deposited. The captive samples came from a

population in a zoo in Australia (n = 17) which was also sampled in 2007, 2008 and 2009.

Additionally, two human faecal samples were obtained from zoo keepers working with the

captive population, which had been approved by Murdoch University’s Human Research

Ethics Committee. All samples were given a consistency score of 1 to 5 with 1 being firm

and 5 being liquid. See Chapter 2 for a full description of sample collection methods.

4.2.2 Parasitological techniques

Refer to Chapter 2 for a full description of microscopy methods used. All parasite ova and

cysts observed were recorded, although only data for Giardia are reported here. All

samples were then analysed using PCR protocols specific for Giardia, as described below.


76

See Table 4.1 for samples which tested positive for Giardia through microscopy and/or

with PCR amplification.

4.2.3 DNA extraction

A 2 ml aliquot of each ethanol-preserved sample was centrifuged and ethanol eluted. The

remaining solid matter was used for DNA extraction using the Maxwell® 16 Instrument

(Promega, Madison, USA) as per manufacturer’s instruction. In an attempt to reduce the

impact of inhibitors in carnivore faecal matter, the final elution was diluted to a 5:1 ratio

with nuclease free water.

4.2.4 Amplification of 18S rRNA locus

A nested PCR was used for amplification of a 130 bp product of the 18S rRNA locus. The

primary reaction utilized the primers RH11 and RH4 (Hopkins et al., 1997) and in the

nested reaction primers GiarF and GiarR (Read et al., 2004) were used. The PCR reaction

was performed in 25 μl volumes consisting of 1 µl of extracted DNA, 2.0 mM MgCl2, 1 x

reaction buffer (20 mM Tris-HCL, pH 8.5 at 25 C, 50 mM KCl), 200 µM of each dNTP,

10 ρmol of each primer, 0.5 units of TAQ-Ti DNA polymerase (Fisher Biotec, Perth

Australia), and H2O. DMSO 5% was added to the primary round and 0.5 µl BSA (10

mg/mL) was added to the nested round. Amplification conditions were modified from

Hopkins (1997) and are as follows: the primary reaction was initiated with a denaturing

step of 96 °C for 5 min, then 35 cycles of 96 °C for 45 s, 58 °C for 30 s and 72 °C for 45 s,


77

followed by a final extension of 72 °C for 7 min. The nested reaction was altered by a

decrease in annealing temperature to 55 °C.

The PCR product was purified using a Wizard SV gel and PCR Clean-up system

(Promega, Madison, USA) after the manufacturer’s instructions, except the final elution

was reduced to 20 µl from 50 µl. Sequence reactions were performed using the Big Dye

Terminator Version 3.1 cycle sequencing kit (Applied Biosystems) according to the

manufacturer’s instructions. PCR products were sequenced in the reverse direction only

(GiarR), as initial sequencing of forward reactions was unsuccessful in this study as also

seen in others (Leonard et al., 2007, Palmer et al., 2008). Reactions were electrophoresed

on an ABI 3730 48 capillary machine. Assemblages were identified via single nucleotide

polymorphisms (SNP’s) within the 130 bp sequence as previously described by Hopkins et

al. (1997).

4.2.5 Amplification of β-giardin locus

A nested PCR was used for amplification of a 492 bp product of the β-giardin gene. The

primary reaction utilized the primers G7 and G759 (Caccio et al., 2002) and in the nested

reaction primers as described by Lalle (2005) were used. The PCR reaction was performed

in 25 μl volumes consisting of 1 µl of extracted DNA, 2.0 mM MgCl2, 1 x reaction buffer

(67 mM Tris-HCl, 16.6 mM (NH4)2SO4, 0.45% Triton X-100, 0.2 mg/ml gelatin), 200 µM

of each dNTP, 10 ρmol of each primer, 0.5 units of Tth Plus DNA polymerase (Fisher

Biotec, Perth Australia), and H2O. The primary reaction was initiated with a denaturing

step of 95 °C for 5 min, then 40 cycles of 95 °C for 30 s, 65 °C for 30 s and 72 °C for 60 s,


78

followed by a final extension of 72 °C for 7 min. The nested reaction was altered by a

decrease in annealing temperature to 55 °C and 35 cycles. The PCR product was purified

and sequenced as described above for the 18S rRNA locus. Assemblages were identified

via SNP’s within the 292 bp sequence when compared with reference sequences

AY072723, AY072727, AY072726, AY072725 and AY45646 (Caccio et al., 2002, Lalle,

2005).

4.2.6 Amplification of the glutamate dehydrogenase locus

A semi-nested PCR was used for amplification of a 432 bp product of the glutamate

dehydrogenase (gdh) gene. The primary reaction utilized the primers GDHeF and GDHiR

with GDHeR and GDHiR in the semi-nested reaction as described by Read et al. (2004).

The PCR reaction was performed in 25 μl volumes consisting of 1 µl of extracted DNA,

3.0 mM MgCl2, 1 x reaction buffer (67 mM Tris-HCl, 16.6 mM (NH4)2SO4, 0.45% Triton

X-100, 0.2 mg/ml gelatin), 200 µM of each dNTP, 10 ρmol of each primer, 0.5 units of Tth

Plus DNA polymerase (Fisher Biotec, Perth Australia), and H2O. DMSO 5% was added to

the primary reaction only. Cycling conditions for both reactions were denatured at 94 °C

for 5 min, then 45 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, followed by

a final extension of 72 °C for 4 min. The PCR product was purified, sequenced and

analysed as previously described for the 18S rRNA locus. Assemblages were identified via

SNP’s within the 432 bp sequence when compared with reference sequences AY178735,

AY178737, AF069059, AY178739, L40508 and AY178738 (Monis et al., 1995; Monis et

al., 1999).


79

4.2.7 Data analysis

Of the 17 samples found positive with microscopy 12 were amplified with PCR along with

27 found negative with microscopy. Prevalence of infection was expressed as percentage

of positive samples found either by microscopy or PCR, with 95% confidence intervals

calculated assuming a binomial distribution, using the software Quantitative Parasitology

3.0 (Rozsa et al., 2000). Faecal consistency scores were compared between dogs testing

positive or negative using a Wilcoxon rank sum test. Differences in prevalence between

populations were tested using the Fisher’s exact test. To compare prevalences between

wild and captive populations, data from year one (2007) of the captive population was

tested against the total individual animals sampled from the wild. Sampling of the captive

population was duplicated each year, but within wild populations animals were sampled

only once as it was found that the composition of packs altered significantly from season to

season, with many deaths recorded and therefore duplicate sampling was extremely rare.

Differences in prevalence of infection among years in the captive population were tested

by a Cochran 2 test, with individual dogs as the blocking factor.

Genotypic information was obtained using the 18S rRNA locus. The multicopy nature and

strong sequence conservation of 18S rRNA provide the sensitivity and specificity required

to determine basic assemblage information (Weilinga and Thompson, 2007). However, to

obtain sub-assemblage information the loci β-giardin and gdh loci were used, as has been

done in many other studies (Abe et al., 2003; Read et al., 2004; Traub et al., 2004a; Lalle

et al., 2005; Leonhard et al., 2007; Palmer et al., 2008). Resultant nucleotide sequences

were compared with published sequences on NCBI. Sequences were aligned against

reference sequences for β-giardin and gdh loci using the sequence alignment program


80

Sequencher™ 4.8 (Gene Codes Corporation, Ann Arbor, USA), allowing assemblage and

sub-assemblage assignment.

4.3. Results

4.3.1 Prevalence of infection

Table 4.1 shows the prevalence of infection with Giardia spp. within the two wild

populations and one captive population of painted dogs. Prevalence of infection with

Giardia did not differ significantly between the two wild populations (P=1.00), but was

significantly greater in the captive population than in either wild population (P=0.03 for

comparison of captive and Zambian populations; P=0.02 for comparison of captive and

Namibian populations). Over all populations, there was no significant difference in faecal

consistency scores between infected (mean score = 2.4 ± SE 0.19) and uninfected (2.3 ±

0.13) dogs (P = 0.78), with only three (n=87) samples presenting with diarrhoea (i.e.

consistency score of 4 or 5).

Table 4.1 Prevalence of Giardia duodenalis (with 95% confidence intervals in

parentheses) detected in faecal samples from captive and wild populations of the African

painted dog.

Captive

(n=16)

Wild - Zambia

(n=43)

Wild - Namibia

(n=28)

Wild-Total

(n=71)

Cap vs Wild

P value

62%

(37-82%)

28%

(16-43%)

25%

(12-44%)

27%

(17-38%)

0.009


81

4.3.2 Molecular characterization of Giardia duodenalis isolates

All samples were initially screened at the 18S rRNA locus and identified as Giardia

duodenalis. Samples successfully amplified at the 18S locus and/or found to be positive

with microscopy were also amplified at the β-giardin and gdh loci. Within the captive

population, isolates from 16 individual animals were successfully amplified at one, two or

three loci. Amplification was successful for isolates from 14 animals at the 18S rRNA

locus, three at β-giardin and six at gdh. Within the wild population, isolates from 14

individual animals were amplified: 13 at 18S rDNA, seven at β-giardin and one at gdh

(Table 4.2). Isolates from both captive and wild populations displayed variation in

assemblage and sub-assemblage type depending on the locus used (Table 4.2).


82

Table 4.2 Genotypic characterisation of Giardia duodenalis isolates from individual

African painted dogs at the18S rRNA, β -giardin and gdh loci. * Wild animals over 2yrs of

age were allocated as Adult (A).

Captive population (n=16) Wild population (n=14)

ID No Sex Age 18S β-g gdh ID No Sex Age* 18S β-g gdh

C1 M 1 A/B - - W1 F A A - -

C2 M 1 A/B - - W2 M 1 B A

C3 M 3 B - A2 W3 ? ? B BIII -

C4 M 1 B BIII/BIV - W4 F A B - -

C5 M 2 B - BIV W5 M A A/B A2 -

C6 M 2 B - - W6 F A A/D A2 -

C7 M 2 A/B - - W7 M A B A2 -

C8 M 3 B/C - - W8 M A A/B/C - -

C9 M 7 B - - W9 ? A B - -

C10 M 9 B - - W10 ? ? A/B - -

C11 M 3 - - BIV W11 ? ? A/B - -

C12 F 2 - BIII/BIV BIV W12 ? A - A2 -

C13 F 1 B - - W13 M A C/D - -

C14 F 4 B BIII/BIV A2 W14 F A A/B/C BIII BIV

C15 F 4 B - D

C16 F 8 B - -

Assemblage information obtained from the 18S rRNA gene showed a dominant proportion

of samples in the captive population to be assemblage B (86%) with mixed assemblages

A/B and B/C/D both being 7%. Within the wild population assemblage B was also

dominant with 38% of isolates however, greater variability was seen in the remaining

isolates with several mixed assemblages.


83

Longitudinal data were obtained from the captive population as sampling was conducted

every year for three years (Table 4.3). Giardia duodenalis was observed in each year, with

prevalences ranging between 50% and 69%. All animals tested positive at least once

throughout the sampling period and there was no significant difference in prevalence

among years (2 = 0.19, P = 0.16). Assemblages A and B were seen in high proportions in

each year.

Table 4.3 Prevalence (with 95% confidence intervals in parentheses) and genotypic

characterisation of Giardia isolates obtained in a captive population of African painted

dogs over a three year period.

Sample year Prevalence 18S rRNA β-giardin gdh

2007

n=16

62%

(37-82%)

B=5

A/B=3

BIII/BIV=1 BIV=1

2008

n=14

50%

(24-76%)

B=5

B/C/D=1

BIII=1

BIII/BIV=1

2009

n=13

69%

(41-88%)

B=6 BIV=1 AII=2

BIII=1 BIV=1

A/B/D=1

Isolates obtained from the two zookeepers were also amplified at the 18S rDNA and β-

giardin loci. Sequencing of the 18S rRNA gene revealed assemblage B and BIII/BIV at the

β-giardin locus for both isolates.


84

4.4. Discussion

This study is the first to identify Giardia duodenalis in the African painted dog. Previous

parasitological studies on this animal have not found this protozoan (van Heerden, 1986;

Colly and Nesbit, 1992; Peirce et al., 1995; van Heerden et al., 1995; Penzhorn, 2006).

The prevalence of G. duodenalis in the wild, 28% in the Zambian population and 25% in

the Namibian population, was similar to other reports in wild canids, e.g. 12.5-32% in

coyotes (Gompper and Williams, 1998; Trout et al., 2006; Thompson et al., 2009), and

46% in wolves (Klock et al., 2005). The greater prevalence found in this study in the

captive population (62.5%) is most likely indicative of their captive environment. Animals

are housed over long periods in the same area, allowing environmental build up and

continued cycling of this protozoan within the population. This effect is reduced in wild

populations as they are extremely nomadic.

This study was not successful in amplifying all isolates at all loci. Previous studies have

also reported lower than expected amplification of Giardia DNA in dogs (Read et al.,

2004; Traub et al., 2004a; Robertson et al., 2006; Leonhard et al., 2007; Caccio and Ryan,

2008; Palmer et al., 2008). The inability to amplify at all loci may be attributed to several

factors. First, β-giardin and gdh loci require high cyst numbers for amplification success

(Castro-Hermida et al., 2007). In addition, faecal samples from carnivores in general have

low PCR success, due to the presence of inhibitors (Kohn and Wayne, 1997; Piggott and

Taylor, 2003; Covacin et al., 2010). Finally, the majority of samples in this study were not

fresh but placed in a fixative, which was necessary to preserve DNA and to comply with

quarantine restrictions for sample importation, but probably reduced the success of DNA


85

amplification due to degradation. Even when amplification was successful, some isolates

showed differences in assemblage and sub-assemblage typing depending on the locus used.

This has also been found in previous studies and may be due to meiotic recombination or

preferential amplification of one assemblage over another in mixed infections (Caccio et

al., 2005; Cooper et al., 2007; Teodorovic et al., 2007; Caccio and Ryan, 2008).

Despite the difficulties mentioned above, it was possible to obtain some assemblage and

sub-assemblage information for both wild and captive populations of the African painted

dog. Genotypic information did not reveal any novel genotypes, but the zoonotic

assemblages B and to a lesser degree A were prevalent within both populations. The

dominance of these zoonotic assemblages was surprising especially as the dog assemblages

C and D were rarely seen. Assemblages A and B have been reported in studies on domestic

dogs, coyotes and wolves but the dog assemblages have also been reported at similar

prevalences (Table 4.4). The lack of assemblages C and D seen in both wild and captive

populations of this study perhaps indicate that the African painted dog is not as susceptible

for this genotype as members of the genus Canis. Interestingly, assemblage A has been the

dominant zoonotic genotype reported in the literature in studies of both wild canids and

domestic dogs, as opposed to this study, where assemblage B was the dominant genotype

(Table 4.4).


86

Table 4.4 Assemblages of Giardia duodenalis observed in both wild canids and domestic

dogs noted in the literature and this study. Number of times reported in literature is

presented as (r=).

Host Assemblage Reference

Canis

familiaris

A (r=11)

B (r=3)

(van Keulen et al., 2002; Berrilli et al., 2004; Read et al., 2004; Traub et al.,

2004a; Itagaki et al., 2005; Lalle et al., 2005; Ipankaew et al., 2007; Leonhard

et al., 2007; Palmer et al., 2008; Covacin et al., 2010; Marangi et al., 2010)

C/ D (r=11) (Monis et al., 1998; Berrilli et al., 2004; Itagaki et al., 2005; Lalle et al., 2005;

Ipankaew et al., 2007; Leonhard et al., 2007; Souza et al., 2007; Palmer et al.,

2008)

Canis latrans

and

Canis lupus

A (r=3)

B (r=1)

C /D (r=3)

(Gompper et al., 2003; Klock et al., 2005; Trout et al., 2006; Thompson et al.,

2009)

Lycaon pictus A

B

Present study

As noted in other wildlife studies, the presence of the zoonotic assemblages found in the

African painted dog raises the question of their potential to act as reservoirs for human

infection (Graczyk et al., 2002; Trout et al., 2006; Thompson et al., 2009). The answer can

perhaps be explained by their ecology and environment. Close interaction between dogs

and humans living in rural communities in India has shown evidence of zoonotic

transmission of Giardia (Traub et al., 2004a) and indeed this kind of transmission could

well be possible within captive populations of the African painted dogs. The zoo keepers in

our study had direct contact with the African painted dogs’ confined environment and were

at risk of exposure through regular cleaning of water features and removing excrement


87

from the enclosure. Isolates from the two keepers typed to the same sub-assemblage as one

of the captive animals in their care, suggesting that zoonotic transmission may have

occurred. Evidence for zoonotic transmission within the zoo environment has also been

observed for another protozoan, Blastocystis sp. whereby keepers and animals were found

to harbour the same genetic sub-type of this parasite (Parkar et al., 2010).

In the wild, however, human contact with the African painted dog would be minimal. They

are not companion animals and have never been domesticated. Due to their nomadic nature

they will roam outside national parks and move through human settlements, although they

actively avoid human contact, making zoonotic transfer unlikely (Creel and Creel, 2002).

However, there is a constant stream of tourists and locals into the national parks and as

suggested by Graczyk et al. (2002) studying gorillas in Uganda, indiscriminate defecation

by humans could be a source of Giardia infection. Other evidence for anthropozoonotic

transmission has been seen between humans and beavers, where humans have been

implicated in introducing Giardia to beavers through sewage runoff upstream from the

beaver colony (Bemrick and Erlandsen, 1988; Fayer et al., 2006).

Whilst the transmission of Giardia duodenalis from humans to African painted dogs

remains speculative, it appears that once established within these packs infection is

maintained by dog to dog transmission. They are very social animals and physical contact

is common in all aspects of pack life, providing ample opportunity for transmission to

occur (Woodroffe et al., 1997; Creel and Creel, 2002). This was evident in the captive

population as each individual animal displayed an infection with G. duodenalis at some

point over the three year study period. As previously mentioned, prevalence was far lower


88

in the wild populations and distribution among packs was not even. G. duodenalis was

observed in several individuals in the same pack and in none in other packs, again

suggesting that once the parasite is introduced into a pack, animal to animal transmission

occurs.

The pathogenesis of Giardia spp. to the host is complicated as many infected hosts can be

asymptomatic while others display severe diarrhoea (Eckmann, 2003). In this study, only

three of the 85 faecal samples positive for G. duodenalis showed signs of diarrhoea,

suggesting that parasitic infection in these animals was generally asymptomatic. Perhaps of

more importance than clinical symptoms are the potential subclinical effects. Infections

with Giardia spp. have been related to growth retardation and malabsorption in children,

particularly those with poor nutrition (Astiazaran-Garcia et al., 2000; Berkman et al.,

2002). With respect to African painted dogs, which often have concurrent enteric

infections (see Chapter 3), subclinical impacts of G. duodenalis could be of great

importance, particularly as pack sizes decrease and hunting efficacy declines (Creel and

Creel, 1995; Courchamp et al., 2002).

Ultimately, the transmission dynamics of G. duodenalis within the African painted dog

remains unclear. While this species could act as a reservoir for zoonotic infections, the lack

of direct human contact in the wild make this unlikely. However, increased human activity

into their natural environment clearly has the potential to increase the prevalence of G.

duodenalis infections in the African painted dog. With other pressures this could

potentially be an emerging disease, which will require monitoring in the future. This

dynamic, however, is altered within captive populations. Due to the necessary interaction


89

between humans and their captive charges in this environment, the risk of zoonotic transfer

is high and should be a consideration in future management protocols.

4.4.1 Conclusions

In summary, the prevalence of G. duodenalis observed in this study indicates that this

parasite is an important part of the parasite fauna of both wild and captive populations of

the African painted dog. Molecular techniques have demonstrated that both wild and

captive populations are predominantly infected with the zoonotic assemblages of G.

duodenalis, and therefore pose a potential zoonotic risk for zoo keepers and surrounding

communities in the natural environment. The subclinical effects of infection are unknown,

but are potentially detrimental to small packs with limited prey capture success and

subsequent poor nutrition. This is the first report of G. duodenalis within the painted dog

and may indicate an emerging disease with possible zoonotic/anthropozoonotic

transmission modes. This highlights the need for continued research to be conducted on

wildlife and their surrounding influences to ascertain more precisely the nature of any

threats to the charismatic but endangered African painted dog.

Chapter 5 – Hookworm species of…..

90

Chapter 5

Hookworm species of the African painted dog and the significance of infection


91

5.1 Introduction

The family Ancylostomatidae contains parasitic nematodes which infect the small

intestine of a wide range of mammalian hosts. These nematodes have a large buccal

cavity, usually armed with teeth, at the anterior end which assists in attachment to the

hosts’ intestinal wall. The anterior end is often ‘hooked’ in appearance, giving rise to

the nomenclature of Ancylostoma with ancyl being Greek for ‘hook’ and stoma for

‘mouth’ (Dubini, 1843).

Infection of the host can occur either through direct ingestion or skin penetration of

infective larvae (L3) (Looss, 1898; Fulleborn, 1914) (Figure 5.1). Larvae which

penetrate the skin usually undergo a tracheal migration where larvae migrate to the

lungs, are ‘coughed’ into the trachea, swallowed and taken through the digestive system

where eventually they reach the small intestine to then attach, feed and reproduce

(Looss, 1898; Bowman et al., 2003). Alternatively L3 may undergo somatic migration

whereby larvae move through the host tissue and localize in the skeletal muscle and

enter into a hypobiotic (dormant) state. These dormant larvae are able to reactivate,

move into the gut and develop into reproducing adults when optimal transmission

conditions occur (Loukas and Provic, 2001). An example of this is when a canid host

gives birth. Encysted larvae are reactivated towards the end of the pregnancy and are

ready to produce large quantities of eggs for quick transmission to the newly born pups

(Bowman et al., 2003). Pups can also be infected through transmammary transmission,

which occurs when larvae emerging from hypobiosis migrate to the mammary glands

and pass through to nursing pups in the dam’s milk (Figure 5.1). Both routes of

infection result in large parasite loads within these young hosts often leading to severe

anaemia as a direct result of the feeding activities of these parasites (Kalkofen, 1970).

Canid hookworm species are important concerns for both animal and human health as


92

besides the ability to cause clinical disease in canids, these hookworms also have the

potential to infect humans.

Figure 5.1 Depiction of the lifecycle of Ancylostoma caninum in domestic dogs

adapted from www.Apaws.org.

.

5.1.1 Hookworm in the canid host

The most widespread species of hookworm infecting canids is Ancylostoma caninum

which has a cosmopolitan distribution (Miller, 1971). Other hookworm species

commonly infecting canids include A. braziliense, A. ceylanicum and Uncinaria

stenocephala all of which have a more restricted distribution. U. stenocephala is

commonly observed in the cooler climates of Europe, North America and Australia,

while A. braziliense and A. ceylanicum are observed in warmer tropical and sub-

tropical regions (Levine, 1968; Provic, 1998; Beveridge, 2002). A. braziliense is quite

widely distributed in these regions but to date A. ceylanicum has only been reported in


93

Southeast Asia, India and more recently Australia (Yoshida et al., 1973; Choo et al.,

2000; Loukas and Provic, 2001; Traub et al., 2004b; Palmer et al., 2007).

Within the canid host, hookworm infections can range from being largely

asymptomatic to severely pathogenic depending on the species of hookworm,

magnitude of infection and the susceptibility of the host (Bowman et al., 2003). A.

caninum infections are often more pathogenic to the canid host due to a greater rate of

blood loss than seen with A. ceylanicum, A. braziliense or U. stenocephala infections

(Miller, 1966; Areekul et al., 1975; Bowman et al., 2003). However, heavy infections

of most hookworm species can lead to clinical disease (Carroll and Grove, 1984). The

magnitude of infection depends on the level of exposure to infective larvae in the

environment, which in turn is dependent on proximity of other infected hosts and

suitability of the environment for the development and survival of the L3.

Host susceptibility is often dependent on age and immune function with young animals

and older immunocompromised animals most at risk, with even light infections capable

of causing illness and death. However, hosts with prior exposure or premunition are

often asymptomatic. Carroll and Grove (1985) demonstrated this ability for acquired

resistance to A. ceylanicum in experimental trials on dogs. They found that dogs

currently infected with small numbers of hookworms were considerably resistant to a

challenge infection of a large inoculum of infective larvae, while dogs previously

uninfected with hookworm developed marked anaemia when inoculated.

5.1.2 The zoonotic potential of canid hookworm species

Canid hookworm species have zoonotic potential and are associated with a variety of

diseases in humans (Carroll and Grove, 1986; Chaudhry and Longworth, 1989; Croese


94

et al., 1994). All canid hookworm species are capable of causing cutaneous larval

migrans (CLM), also known as ‘creeping eruptions’ (Kirby-Smith, 1926; Carroll and

Grove, 1986; Jelinek et al., 1994). CLM is a pruritic skin disease which is the result of

infective hookworm larvae penetrating and migrating through the skin (Jelinek et al.,

1994). Cases of CLM are most often described in hot climates including SE Asia,

Africa and Latin America with A. braziliense the most common etiological agent

involved (Chaudhry and Longworth, 1989). A. caninum has been shown to sometimes

migrate further than the skin reaching the human gut where it can cause eosinophilic

enteritis; however evidence of patency has not yet been observed (Croese et al., 1994).

A. ceylanicum is the only hookworm of dogs that is capable of producing patent

infections in the human host, with patients experiencing intermittent abdominal

discomfort throughout the period of infection (Carroll and Grove, 1986).

5.1.3 Hookworm in the African painted dog and other carnivores in Africa

Reports of hookworm within African animals have mainly been from domestic dogs

and cats with A. caninum most common in dogs and A. tubaeforme in cats (Baker et al.,

1989; Minnaar and Krecek, 2001; Minnaar et al., 2002). A. braziliense was also

reported in these studies but at slightly lower prevalences. Baker et al. (1989) reported

parasitological studies during autopsies on 1,500 stray cats and identified A. ceylanicum

at a prevalence of 1.4%. To date this is the only report of this Ancylostoma species in

Africa in either human or animal populations. Reports of hookworm species in native

African carnivores are less common, however A. caninum and A. braziliense have been

identified in cheetah (Acinonyx jubatus), leopard (Panthera pardus) and jackal (Canis

spp.) (Round, 1968). A. ceylanicum was also reported in an African civet (Viverra

civetta), although during this time A. ceylanicum and A. braziliense were considered


95

synonymous and were not officially differentiated until 1951 (Bioca, 1951; Round,

1968). With this redescription it has been suggested that many old records are in doubt,

therefore this identification is also questionable (Dunn, 1969). The only reports of

hookworm in the African painted dog have been from van Heerden et al. (1994, 1996)

who found A. caninum in a captive and wild population in South Africa.

5.1.4 Diagnosing hookworm infections

In the past, studies using microscopy to identify parasite ova have had to presume or

suggest which species of Ancylostoma was the infecting agent based on previous

observations from that host and the known distribution of hookworm species (Engh et

al., 2003; Smith and Kok, 2006). This may have led to erroneous reports as the

identification of Ancylostoma species from parasite ova alone is not possible.

Traditional methods of species identification have involved obtaining adult worms,

usually during necropsy, which in turn required considerable expertise to differentiate.

Historically, misidentifications have been made, particularly between A. ceylanicum

and A. braziliense due to the morphological similarities between the two, creating doubt

on known distributions (Dunn 1969). Recent development of molecular tools to

differentiate hookworm species from faecal samples has been able to alleviate these

diagnostic problems and allow for more accurate species identification and

subsequently more accurate distribution records for parasites (Palmer et al., 2007;

Traub et al., 2008). For example, the distribution of A. ceylanicum was only recently

found to include Australia (Palmer et al., 2007). This range extension was demonstrated

with the use of molecular tools which were able to differentiate between eggs from

different Ancylostoma species from canine faeces (Traub et al., 2004b).


96

The primary aim of this study was firstly, to establish the prevalence of Ancylostoma

spp. in captive and wild populations of the African pained dog and secondly, to identify

if species of pathogenic and/or zoonotic importance occur within these populations.

These aims were achieved by obtaining data from three wild and three captive

populations for comparative analysis. Molecular characterisation was conducted to

identify species of Ancylostoma.




during three field trips conducted between 2007 and 2009. The population within

Zambia was sampled in each of the three years while the population in Namibia was

sampled in two of these years (2008 and 2009). Samples in Zimbabwe had been

collected in 2006 and stored for later analysis. The samples from captive animals were

collected from two zoo populations in Australia (Perth Zoo, Monarto Zoo) and a

rehabilitation facility in South Africa. See Chapter 2 for a full description of sample

collection methods.


Refer to Chapter 2 for a detailed description of microscopy techniques. All parasite ova

and cysts observed were recorded, although only data for Ancylostoma spp. are reported

here. All samples were then analysed using PCR protocols specific for Ancylostoma

spp., as described below.


97


A 2 ml aliquot of each ethanol-preserved sample was centrifuged and ethanol eluted.

The remaining solid matter was used for DNA extraction using the Maxwell® 16

Instrument (Promega, Madison, USA) as per the manufacturer’s instruction. In an

attempt to reduce the impact of inhibitors in carnivore faecal matter, the final elution

was diluted to a 5:1 ratio with nuclease free water.

5.2.4 Amplification of Ancylostoma spp. isolates at the ITS1 and ITS2 regions

Modifications from Traub et al. (2008) were used for amplification of a 380 bp product

of the ITS1, 58S and ITS2 regions using primers RTHW1F and RTHW1R. The PCR

reaction was performed in 25 μl volumes consisting of 1 µl of extracted DNA, 2.5 mM

MgCl2, 1 x reaction buffer (20 mM Tris-HCl, pH 8.5 at 25 °C, 50 mM KCl), 100 µM of

each dNTP, 10 ρmol of each primer, 0.5 units of Tth plus DNA polymerase (Fisher

Biotec, Perth Australia), and H2O. Amplification conditions were as follows: the

primary reaction was initiated with a denaturing step of 95 °C for 5 min, then 40 cycles

of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, followed by a final extension of 72

°C for 7 min. The PCR product was purified using an Agencourt AMPure XP system

(Beckman Coulter Inc, Brea USA) according to the manufacturer’s instruction.

Sequence reactions were performed using the Big Dye Terminator Version 3.1 cycle

sequencing kit (Applied Biosystems), according to the manufacturer’s instructions.

PCR products were sequenced in both directions. Reactions were electrophoresed on an

ABI 3730 48 capillary machine.


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5.2.5 Data analysis

Prevalences of infection were expressed as a percentage of positive samples found by

microscopy with 95% confidence intervals calculated assuming a binomial distribution,

using the software Quantitative Parasitology 3.0 (Rozsa et al., 2000). Differences in

prevalence between populations and years were tested using the Fisher’s exact test or

chi-square test. Resultant nucleotide sequences were compared with published

sequences on NCBI GenBank using the basic alignment search tool (BLAST). Further

sequence analysis was conducted using the sequence alignment program Sequencher™

4.8 (Gene Codes Corporation, Ann Arbor, USA). Reference sequences for A. caninum,

A. duodenale and A. braziliense (accession numbers A. caninum DQ438079 and

EU159416, A. duodenale EU344797 and AB501351, A. braziliense DQ438054) were

used in the alignments. The levels of sequence differences (D), based on pairwise

comparisons, were calculated using the formula D = 1 – (M/L), where M is the number

of alignment positions at which the two sequences have a base in common and L is the

total number of positions over which the sequences are compared (Chilton et al., 1997).

The level of sequence difference was expressed as a percentage.

5.3 Results

5.3.1 Prevalence of infection

Table 5.1 shows the prevalence of infection with Ancylostoma spp. within captive and

wild populations. Prevalence of infections did not differ significantly between the three

wild populations (Fisher’s exact tests, Zambia vs. Namibia P=0.4; Zambia vs.

Zimbabwe P=0.14; Namibia vs. Zimbabwe P=0.6). There was a significant difference

in prevalence between wild and captive populations, as Ancylostoma spp. were not

observed in any of the captive populations sampled (Fisher’s exact test, P< 0.0001).


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Table 5.1 Prevalence of Ancylostoma spp. (with 95% confidence intervals in

parentheses) detected in faecal samples from captive and wild populations of the

African painted dog.

Zimbabwe

(n=12)

Zambia

(n=43)

Namibia

(n=28)

Total wild

(n=83)

Total captive

(n=130)

8 % 32.6% 43% 32.5% 0

(0.4 – 37%) (19 – 48%) (26 – 62%) (23 – 43%)

Data was obtained over three years for the Zambian population and two years for the

Namibian populations. Ancylostoma spp. were observed in each year with prevalences

ranging from 20% to 66.7% (Table 5.2). There were significant differences in

prevalences among years within the Zambian population (χ2 = 6.306, P=0.04) but not

between years in the Namibian population (Fisher’s exact test, P=1.000).

Table 5.2 Prevalence of Ancylostoma spp. observed in the Zambian and Namibian

populations of the African painted dog over a three year period. Sample size (n) and

95% confidence intervals in parentheses.

Population Year

2007 2008 2009

Zambia

66.7% (n=9)

(32 – 90%)

20% (n=20)

(71 – 42%)

28.6% (n=14)

(10 – 57%)

Namibia

42% (n=19)

(22 – 66%)

44% (n=9)

(17 -75%)

-


100

5.3.2 Molecular characterisation of Ancylostoma spp. isolates

Of the 27 samples identified positive for Ancylostoma spp. ova by microscopy, a subset

were chosen for molecular characterisation. Four samples were successfully amplified

at the ITS1 and ITS2 regions. A BLAST search on GenBank revealed three isolates

from the Namibian population (N-B08, N-K17 and N-U12) to be Ancylostoma

braziliense and one isolate from the Zambian population (Z-Ka01) was found to have a

similar identity to both Ancylostoma caninum and Ancylostoma duodenale (Table 5.3).

Table 5.3 Genotypic characterisation of Ancylostoma spp. isolates obtained from wild

populations of the African painted dog within Zambia and Namibia. Percentages shown

for matched identities with published sequences in parentheses.

Population Isolate Host Sex Host Age Ancylostoma species

Namibia N-B08 ? ? A. braziliense (100)

Namibia N-K17 ? Adult A. braziliense (100)

Namibia N-U12 ? ? A. braziliense (100)

Zambia Z-Ka01 M Yearling A. caninum (99) A. duodenale

(99)

The isolate Z-Ka01 was aligned against reference sequences of A. caninum (accession

numbers DQ438079 and EU159416) and A. duodenale (accession numbers

AB501351and EU344797). Over a 301 bp region there was one different nucleotide


101

difference with A. duodenale and two nucleotide differences with A. caninum (Table

5.4).

Table 5.4 Summary of the nucleotide variations detected between the painted dog

isolate from Zambia (Z-Ka01) and reference sequences for A. caninum and A.

duodenale. Alignment was conducted in Sequencher™ 4.8 and dots indicate identity

with the first sequence.

Ancylostoma isolates Alignment position

324 353 355

A. caninum EU159416 T C C

A. caninum DQ438079 . T .

A. duodenale EU344797 A T T

A. duodenale AB501351 . T T

Z-Ka01 . T T

5.4 Discussion

Ancylostoma spp. were not detected within any of the captive populations of the

African painted dog. This was most likely due to the regular use of anthelmintics in the

captive facilities (see Chapter 2). In comparison, the total prevalence of Ancylostoma

spp. observed in the wild populations of painted dogs in this study was 32.5%. This was

slightly higher than previously reported by van Heerden et al. (1995), (22.4%) but


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varies from studies on competitor species such as hyaena, which reported a prevalence

of 56%, and lion (90%) (Muller-Graf, 1995; Engh et al., 2003). It is not clear whether

the higher prevalences observed in studies of hyaena and lion were due to

environmental conditions or differences among hosts in exposure or resistance to

hookworm infection.

In this study prevalence of infection was not significantly different between African

painted dog populations in different geographic regions, however, within the Zambian

population a significant difference was observed over time. This variation was mainly

due to the higher prevalence observed in 2007, which also had the majority of samples

collected in the wet season. The wet season in these regions provide a moist and warm

environment which are optimal conditions for the transmission and survival of

hookworm larvae and this is the most likely explanation for the observed increased

prevalence in 2007.

The use of molecular tools in this study allowed for species identification of a small

subset of isolates. Using the ITS region of hookworm rDNA it was possible to

distinguish between the main species of canid hookworms (Traub et al., 2004b; Clare e

Silva et al., 2006). A major benefit of using molecular tools is that it avoids the need for

adult worms which are usually only obtained during a necropsy and require substantial

skill to confidently identify (Traub et al., 2004b). This is particularly important when

dealing with endangered wild animals, where the chances of obtaining carcasses for

necropsy is rarely possible. Faecal sampling provides a non-invasive method to obtain

parasitological data, from which molecular tools can then assist in providing species

information.


103

The ability to identify the species of hookworm infecting a host population is of

particular significance as pathogenicity and zoonotic potential vary between the

different species. For example, A. caninum poses a greater health risk to the host as it

causes greater blood loss than the other species, whilst A. braziliense commonly causes

CLM in humans and A. ceylanicum can lead to patent infections in man (Miller, 1966;

Carroll and Grove, 1986). Additionally, as correct species identification has been

impossible through egg morphology, the exact geographic distribution for these

hookworm species is not completely mapped, making molecular characterisation an

important tool in achieving this.

Genetic characterisations conducted in this study identified three isolates from the

Namibian population of painted dogs to be A. braziliense. This is the first identification

of this species of hookworm in the painted dog and potentially represents a zoonotic

risk to the surrounding human communities. A previous study on communities situated

near this study population has implicated A. braziliense as a major cause of CLM

commonly seen in hospital patients (Evans et al., 1990). Theoretically, these animals

could be reservoirs for CLM in human communities but the lack of human contact in

the wild would reduce the chance of transmission (see Chapter 4).

A more likely explanation for the CLM seen in human populations would be zoonotic

infection from the large number of domestic dogs living in close contact with these

communities. A. braziliense has been reported in domestic dogs in Africa with

prevalences ranging from 19% to 79% (Pangui and Kaboret, 1993; Minnaar and

Krecek, 2001; Minnaar et al., 2002). Two concurrent studies conducted in different

regions of South Africa found that the prevalence of A. caninum was significantly less

in the study site with the drier climate (27% vs. 88%) whilst A. braziliense had similar


104

prevalences in both climates (19%, 20%). The authors therefore suggested that A.

braziliense were more resistant to dry environment than A. caninum (Minnaar and

Krecek, 2001; Minnaar et al., 2002). Conclusive evidence for this is lacking in the

literature but another study on domestic dogs in Dakar, Senegal, which has a semi-arid

climate found A. braziliense at much higher prevalences than A. caninum (79% vs.

17%) (Pangui and Kaboret, 1993). A. braziliense was the only species identified from

Namibia in this study, which is a region that experiences a desert dry environment for

most of the year and a shorter wet season than the other study sites of Zambia and

Zimbabwe.

The single isolate which was sequenced from the Zambian population could not be

conclusively identified as either A. caninum or A. duodenale due to the lack of

polymorphisms within the sequenced region. A. duodenale is known as a human

hookworm but is also found in cats and dogs. This species has not been previously

reported in the African painted dog although it has been reported in the spotted hyaena

in Ethiopia (Graber and Blanc, 1979). A. caninum has been reported in both a captive

and wild population of the African painted dog (van Heerden et al., 1994). However, it

must be noted that the study on the wild population within Kruger National Park

identified A. caninum from one adult worm obtained from one painted dog during

necropsy. It appears that it was then assumed that all Ancylostoma spp. ova observed in

faecal samples of that study were of the same species, which may well have been

inaccurate. Studies have since identified A. braziliense within domestic dog populations

of South Africa, providing evidence that the species exists within the region and is

therefore a possible suspect (Minnaar and Krecek, 2001; Minnaar et al., 2002). As

molecular tools become more widely used these issues may be resolved.


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Pathogenicity of hookworm infection within wild populations of the African painted

dog is unknown (van Heerden et al., 1995). It is likely that some mortality events occur

but identifying these in the wild is hindered by the inability to find dead animals in

what is often inaccessible terrain, combined with the efficiency of scavengers in

cleaning up carcases. Nonetheless, the painted dogs are extremely social animals so the

transmission of hookworm between individuals would be greatly enhanced.

Transmission would be significantly increased during the denning period when

reactivated larvae in the dam increase ova output and pups and pack members are

confined to the den and surrounding areas. It appears the prevalence of hookworm

observed in these wild populations was sustainable as study animals appeared robust

and healthy. This display of health may be the result of acquired immunity by the host

or, as has been previously noted, wild animals tend to manifest few recognizable signs

of disease (Sachs and Sachs, 1968; Young, 1969). This apparent health has been

observed in other wild populations who are host to potentially pathogenic parasites.

Wild populations of zebra are known to harbour a variety of Cyathostomum spp., of

which several species are pathogenic to domestic equines, but the zebra host appears to

display no clinical effects (Roberts et al., 2002). Similarly, populations of wild

ruminants within north America are host to many gastrointestinal strongyle parasites,

many of which are pathogenic to domestic livestock, but again negligible clinical

effects have been in observed in these wild hosts (Hoberg et al., 2001).

The ability for the canid host to acquire a level of immunity to hookworm infections is

an important consideration for captive animal management, especially those involved

in reintroduction programs. The prevalences observed in this study indicate that

hookworm infections are common within wild populations while captive animals have

no or minimal exposure. Therefore, facilities with animals marked for reintroduction


106

programs may benefit from maintaining a low level of infection in order for animals to

acquire a level of immunity against species such as hookworm. In addition, exposure to

A. caninum can lead to cross immunity for other hookworm species such as A.

braziliense (Miller, 1967). These factors would then reduce the impact of the

substantial parasite challenge animals will be faced with on release to the natural

environment (Williams et al., 1992; Viggers et al., 1993).

There is a risk involved in maintaining infections with naturally occurring parasites in a

captive environment as the confined conditions can lead to a rapid increase in parasite

numbers. Therefore, adequate monitoring would be required to ensure infection levels

remained low to avoid clinical disease, but the benefits may be worth the extra labour

involved. It should be noted that ancylostomiasis has been implicated in the death of

two captive painted dogs (van Heerden et al., 1994). However, one animal was a pup

and the other was an immunocompromised adult, both of which were particularly

susceptible to hookworm infections. Depending on individual facilities the risk of some

mortality may be acceptable in view of the benefits potentially gained.

5.4.1 Conclusions

In summary, these prevalence data suggest that Ancylostoma species make up an

important part of the parasite fauna of wild populations of the painted dog. Molecular

techniques have been successful in identifying A. braziliense for the first time in this

endangered carnivore and indicate a possible zoonotic risk for the surrounding human

communities. As DNA from only a small subset of samples was successfully amplified

and sequenced, limited information on geographic distribution was obtained. Future


107

studies are required to achieve detailed species distribution within endangered

carnivores such as the African painted dog.

Chapter 6 – Detecting Taenia species in…

108

Chapter 6

Detecting Taenia species of the African painted dog


109

6.1 Introduction

The cestode family Taeniidae consists of two genera, Taenia Linnaeus, 1758 and

Echinococcus Rudolphi 1801¸ both of which include species of medical and veterinary

significance (Eckert et al., 2001; Hoberg, 2002). These taeniids have a two host lifecycle

based around a carnivorous definitive host and an omnivorous or herbivorous intermediate

host. The adult worm lives in the gut of the definitive host where it produces proglottids

which once mature contain multiple ova. These proglottids and ova are passed out in the

hosts’ faeces and are subsequently ingested by grazing herbivores and omnivores. Once

ingested by the intermediate host the ova develop into a larval stage, or metacestode, which

encysts within the tissues and internal organs of the intermediate host. These metacestodes

are fluid-filled cystic structures which depending on the species develop into cysticerci,

strobilocerci, coenuri or hydatid cysts (Bowman et al., 2003). Cysticerci and strobilocerci

contain one scolex from which one mature tapeworm can develop. Coenuri contain

multiple scolices and hydatid cysts can contain thousands of scolices (protoscolices). The

latter two are usually associated with pathogenic disease in the intermediate host due to the

invasive or space occupying nature of the cysts. The taeniid lifecycle is completed when

these metacestodes are ingested by a carnivorous definitive host (Figure 6.1).


110

Figure 6.1.The lifecycle of a taeniid species depicting the transmission cycle between

predator (definitive host) and prey (intermediate host).

There are multiple species within both genera of taeniids and there has been much

discussion and redescription in the literature in an attempt to discern the epidemiology of

each species (Verster, 1969; Rausch, 1994; Hoberg et al., 2000; Thompson and McManus,

2002; Hoberg, 2006). Currently within the genus Taenia there are approximately 42

described species and 3 subspecies, while Echinococcus has a possible 9 species and

several strains of E. granulosus (Loos-Frank, 2000; Thompson and McManus, 2002;

Huttner et al., 2008; Huttner and Romig, 2009). However, it should be noted that many

‘strains’ of E. granulosus have recently been awarded species status (Thompson and

McManus, 2002; Huttner et al., 2008). While complete lifecycles are not known for all

species of taeniids it has been found that in general these parasites have relatively high

definitive host specificity and relatively low intermediate host specificity (Verster, 1969).

There are some exceptions to this, such as E. granulosus and T. hydatigena being found in

Intermediate hosts

Definitive host


111

several different species of definitive hosts (Verster, 1965, 1969). Within African wildlife,

top level carnivores such as lion (Panthera leo), hyaena (Crocuta crocuta, Hyaena brauni,

and Hyaena hyaena), jackal (Canis mesomelas and C.adustus) and African painted dog

(Lycaon pictus) are commonly definitive hosts for taeniids (Dinnik and Sachs, 1969;

Verster, 1969; Jones and Khalil, 1984). The African lion has been found to be a definitive

host for T. regis and T. simbae, hyaena species are definitive hosts for T. dinniki, T.

hyaenae, T. crocutae and T. olngojinei and the silver-backed jackal is host to T. madoquae

(Pellegrini, 1950; Mettrick and Beverley-Burton, 1961; Dinnik and Sachs, 1972; Jones and

Khalil, 1984). The transmission cycles of these Taenia species invariably involves several

wild ungulates, particularly antelopes, as the intermediate host. For example, species of

antelope such as impala (Aepyceros melampus), gazelle (Gazella spp.), kudu (Tragelaphus

spp.) and wildebeest (Connochaetes spp.) have been found to harbour the metacestodes of

T. crocutae, T. hyaenae, T. hydatigena, T. madoquae, T. olngojinei and T. simbae (Table

6.1) (Nelson and Rausch, 1963; Dinnik and Sachs, 1969; Verster, 1969; Dinnik and Sachs,

1972; Jones and Khalil, 1984).

Reports of taeniid species within the African painted dog have been few with the majority

being made in the first half of the 20th century. These early observations included T.

lycaontis, T. brauni and E. lycaontis (Ortlepp, 1934; Mahon, 1954; Baer and Fain, 1955)

all of which were later revised and found to be synonymous with T. hyaenae, T. serialis

brauni and E. granulosus respectively (Verster, 1969). Less disputable are observations


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Table 6.1 Definitive and intermediate hosts of Taenia spp. infecting African carnivores.

Taenia species Distribution Definitive host Intermediate host Metacestode location

T. crocutae Africa Hyaena sp. Antelope: eg. impala, kudu and duiker

Cysticerci – skeletal muscle

T. dinniki Africa Hyaena sp. Unknown Unknown

T. hyaenae

(syn T. lycaontis)

Africa Hyaena sp.

African painted dog

Antelope: eg. impala and sable Cysticerci – muscles, heart, tongue, diaphragm

T. hydatigena Cosmopolitan Dog and other canids Ruminants Cysticerci – pelvic regions, liver, lungs

T. multiceps Cosmopolitan Dog and other canids Sheep and goat Coenurus – CNS, brain

T. serialis Cosmopolitan Dog and other canids Lagomorphs Coenurus – intermuscular tissue, heart

T. s. brauni Africa Dog and other canids

African painted dog

Rodents and primates Coenurus – intermuscular tissue heart

T. madoquae Africa Silver-backed jackal Antelope (Madoqua sp.) Cysticerci – skeletal muscle

T. ovis Cosmopolitan Dog and other canids Sheep and other ruminants Cysticerci – skeletal muscle, heart, diaphragm

T. olngojinei Africa Spotted hyaena Antelope eg. Grants gazelle Cysticerci – intrasacral muscle

T. regis Africa African lion Antelope Cysticerci – skeletal muscle, liver, lung

T. simbae Africa African lion Antelope Unknown


113

of T. pisiformis and E. granulosus infections within the painted dog (Ortlepp, 1934, 1938;

Nelson and Rausch, 1963; Young, 1975). Besides these individual findings, one

parasitological prevalence study has been conducted on painted dog packs within Kruger

National Park which identified the prevalence of taeniid species to be 30% (van Heerden et

al., 1995).

There have been relatively few other prevalence studies of taeniids in African carnivores

but two studies on lions observed a prevalence of 58% and 15% (Muller-Graf, 1995; Bjork

et al., 2000) and one study on hyaena reported 12.9% (Engh et al., 2003). Due to data for

these studies being obtained from faecal samples and the subsequent lack of adult worms,

identification of species was not made.

The presence of taeniids within wildlife does not pose an immediate threat to the definitive

host as most adult tapeworms do not invade or feed off host tissue and are therefore

considered non-pathogenic (Bowman et al., 2003). However, there is a risk of zoonotic

disease as humans can act as intermediate hosts for many of these taeniid species. Of the

two genera, species of Echinococcus are considered more pathogenic as these parasites

form large hydatid cysts in the organs of the intermediate host, resulting in hydatid disease

in humans (Eckert et al., 2001). Among the species of Taenia, which utilize canids as the

definitive host, are members that are also potentially pathogenic to people. Examples

include T. multiceps, T. serialis and T. s. brauni which have been known to cause cerebral

coenurosis and ocular coenurosis in humans (Vanderwick et al., 1964; Templeton, 1968;

Williams and Templeton, 1971; Ibechukwu and Onwukeme, 1991). Humans most at risk to

possible zoonoses in wildlife are those in communities situated near or in national parks,


114

where human/wildlife interactions are more common. Whilst cerebral and/or ocular

coenurosis is relatively rare, hydatid disease has been found to be highly endemic in sub-

Saharan Africa with studies on nomadic pastoralists reporting a prevalence of between 0.1

and 5.6% (Macpherson et al., 1989; Magambo et al., 2006).

The aim of this study was to firstly determine the prevalence of taeniid species within wild

and captive populations of the African painted dog, secondly to identify the species

involved, which may assist in understanding sylvatic predator/prey transmission cycles and

thirdly, to identify any species which may pose a public health risk. These aims were

achieved by obtaining data from three wild and three captive populations for comparative

analysis. Identification of species was achieved by staining proglottids and molecular

characterisations, while the known predator/prey relationships within each study

population were used to infer sylvatic lifecycles.




during three field trips conducted between 2007 and 2009. The population within Zambia

was sampled in each of the three years while the population in Namibia was sampled in

two of these years (2008 and 2009). Samples in Zimbabwe had been collected in 2006 and

stored for later analysis. The samples from captive animals were collected from two zoo

populations in Australia (Perth Zoo, Monarto Zoo) and a rehabilitation facility in South

Africa. See Chapter 2 for a full description of sample collection methods.


115


Refer to Chapter 2 for a detailed description of microscopy methods. All parasite ova and

cysts observed were recorded, although only data for taeniid spp. are reported here. All

samples were then analysed using PCR protocols specific for taeniid species as described

below.

6.2.3 Identification of proglottids

Taeniid proglottids were fixed in 10% formalin and stained using two different methods.

For Semichon’s aceto-carmine stain (Semichon, 1924), proglottids were placed into

ethanol for 24 hours, 1 drop of Semichon’s stain was added and after 24 hours samples

were dehydrated in a series of ethanol dilutions, starting with 70% ethanol and finishing

with 100% ethanol. These were then cleared in methyl salicytate for 20 minutes and

mounted onto microscope slides in Canida balsam. For Harris’s haematoxylin stain

(Harris, 1900), formalin fixed proglottids were placed in a dilution (water and 1.5 drops) of

Harris’s haematoxylin overnight, then washed in 70% ethanol before being destained in

acid alcohol (2% HCl in 70% ethanol) for several minutes until specimens were pale pink

in colour. Destaining was halted in basic alcohol (ammonium hydroxide in 10 ml 70%

ethanol) for up to 15 minutes or until samples turned blue. Samples were then washed in

70% ethanol, dehydrated, cleared and mounted on slides as for Semichon’s stain.

Identification of proglottids was made on the morphology of the female genitalia of each

proglottid as described by Verster (1969) (Figure 6.2).


116

Figure 6.2 A depiction of the genital atrium of T. crocutae as an example of the

morphological features used in identification of proglottids. From Verster (1969).

Appendix 1 displays the proglottid key used for identifying samples obtained in this study.


A 2 ml aliquot of each ethanol-preserved sample was centrifuged and ethanol eluted. The

remaining solid matter was used for DNA extraction using the Maxwell® 16 Instrument

(Promega, Madison, USA) as per the manufacturer’s instruction. In an attempt to reduce

the impact of inhibitors in carnivore faecal matter, the final elution was diluted to a 5:1

ratio with nuclease free water.

6.2.5 Amplification of Taeniid isolates at the 12S rRNA locus

A multiplex PCR at the 12S rRNA locus was used for differentiation of Taenia spp.,

Echinoccocus granulosus and E. multilocularis as per Traschel et al. (2007). Briefly, 5

primers (Cest1, Cest2, Cest3, Cest4 and Cest5) were used for amplification yielding a 267

bp product for Taenia sp, 117 bp for E. granulosus and 395 bp for E. multilocularis. The

Vaginal loops Vaginal sphincter muscle

Vas deferens loops

Cirrus pouch


117

PCR reaction was performed in 50 μl volumes consisting of 2µl of extracted DNA, 10

ρmol of each primer, 25 μl of mastermix (Qiagen multiplex kit, Qiagen, Hilden, Germany)

and 18 μl H2O. The 267 bp amplicons representing Taenia sp. were sequenced using the

internal sequencing primer Cest 5seq, as described by Traschel et al. (2007). The PCR

product was purified using a Wizard SV gel and PCR Clean-up system (Promega,

Madison, USA) following the manufacturer’s instructions. Sequence reactions were

performed using the Big Dye Terminator Version 3.1 cycle sequencing kit (Applied

Biosystems) according to the manufacturer’s instructions. Reactions were electrophoresed

on an ABI 3730 48 capillary machine.

6.2.6 Data analysis

Prevalences of infection were expressed as a percentage of positive samples found by

microscopy with 95% confidence intervals calculated assuming a binomial distribution,

using the software Quantitative Parasitology 3.0 (Rozsa et al., 2000). Differences in

prevalence between populations and years were tested using the Fisher’s exact test or Chi-

square test.

Resultant nucleotide sequences were compared with published sequences on NCBI

GenBank using the basic alignment search tool (BLAST). Further sequence analysis

conducted using the sequence alignment program Sequencher 4.8™ (Gene Codes

Corporation, Ann Arbor, USA). The levels of sequence differences (D), based on pairwise

comparisons, were calculated using the formula D = 1- (M/L), where M is the number of

alignment positions at which the two sequences have a base in common and L is the total


118

number of positions over which the sequences are compared (Chilton et al., 1997). The

level of sequence difference was expressed as a percentage.

Phylogenetic analysis was conducted in MEGA 4 (Tamura et al., 2007) using reference

sequences for T. multiceps, T. serialis, and E. granulosus (accession numbers GQ228819,

DQ408418, EU219546, DQ104238 and GQ161844 respectively). The neighbour-joining

method was used to infer phylogenetic relationships between sequenced isolates and

reference sequences, with support for nodes in the phylogeny assessed using a bootstrap

test with 1,500 replicates (Saitou and Nei, 1987).

6.3 Results

6.3.1 Prevalence of Infection

Table 6.2 shows the prevalence of infection with taeniid spp. within captive and wild

populations. Prevalence of infections with species of taeniid did not differ significantly

between the three wild populations (Fisher’s exact tests; Zambia vs. Namibia P=0.4;

Zambia vs. Zimbabwe P=1.00; Namibia vs. Zimbabwe P=0.7). There was a significant

difference between pooled data of wild and captive populations as taeniids were not

observed in any of the captive populations sampled (Fisher’s exact test, P < 0.0001).


119

Table 6.2 Prevalence of taeniids (with 95% confidence intervals in parentheses) detected

in faecal samples from wild populations of the African painted dog.

Zimbabwe

(n=12)

Zambia

(n=43)

Namibia

(n=28)

Total wild

(n=83)

Total captive

(n=130)

33% 35% 25% 31.3% 0

(12 – 63%) (22 – 50%) (12 – 44%) (22 – 42%)

Data was obtained over three years for the Zambian population and two years for the

Namibian population. Taeniids were observed in each year with prevalences ranging from

14% to 89% (Table 6.3). There were significant differences in prevalences among years

within the Zambian population (χ2 = 15.03, P=0.001) but not in the Namibian population

(Fisher’s exact test, P=0.9).

Table 6.3 Prevalence of taeniids observed in the Zambian and Namibian populations of the

African painted dog over a three year period. Sample size (n) and 95% confidence intervals

in parentheses.

Population Year

2007 2008 2009

Zambia

89% (n=9)

(56 – 99%)

25% (n=20)

(10 – 47%)

14% (n=14)

(26 – 43%)

Namibia

26.3% (n=19)

(11 – 50%)

22% (n=9)

(41 – 56%)

-


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6.3.2 Identification of taeniid spp. proglottids

Five of the nine proglottids obtained from two packs within Zambia were tentatively

identified using the morphological features of the female genital atrium, including the

presence/absence of a vaginal sphincter muscle and the number of loops in the vas deferens

and vagina (Figure 6.2). Table 6.4 lists the morphological data for each of the five

proglottids. Using the criteria of Verster (1969) and Jones and Khalil (1984), one

proglottid resembled that of T. hydatigena, two resembled T. crocutae, one resembled T.

ovis and one resembled T. dinniki.

Table 6.4 Species identification of taeniid proglottids obtained from painted dog

populations.

Population Isolate Host sex

Host age

Morphological characters Probable species

Zambia ZKa01 M Yearling Vaginal sphincter = full Vaginal loops = 2 Vas deferens loop = 2/3 bunched towards end of cirrus pouch

T. dinniki

Zambia ZKa06 F Yearling Vaginal sphincter = full Vaginal loops =0 Vas deferens loops = 2

T. ovis

Zambia ZKa08 ? ? Vaginal sphincter = full Vaginal loops = 1 Vas deferens loops = 3

T. crocutae

Zambia ZRh18 M Adult Vaginal sphincter = 0 Vaginal loops = 1 Vas deferens loops = 2

T. hydatigena

Zambia ZRh26 ? ? Vaginal sphincter = full Vaginal loops = 1 Vas deferens loops = 3

T. crocutae


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6.3.3 Molecular characterisation of taeniid sp. isolates

The multiplex PCR used in this study was able to differentiate between Taenia spp., E.

granulosus and E. multilocularis at the 12S rRNA locus (Figure 6.3).

Figure 6.3 Multiplex PCR of taeniid isolates from wild painted dog populations at the 12S

rRNA locus. Lanes 2-11 are painted dog samples from Zambia and Namibia. Lane 13 is

the negative control; 14-16 are the positive controls for Taenia sp, E. granulosis and E.

multilocularis.

Of the 26 samples identified positive for taeniid ova by microscopy, 22 were successfully

amplified using the multiplex PCR. All of these were species of Taenia. Of the 22

amplicons, 16 were sequenced. Comparisons to published sequences on NCBI GenBank

revealed 3 sequences to be similar to T. hydatigena and 13 to have a similar identity to

both T.multiceps and T.serialis (Table 6.5).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16


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Table 6.5 Genotypic characterisation of taeniid spp. species isolates obtained from wild

populations of the African painted dog within Zambia, Namibia and Zimbabwe.

Percentages shown for matched identities with published sequences on GenBank in

parentheses.

Population Isolate No Host sex Host age Taenia species

Zambia Z-Ka01 M Yearling T. serialis (96) T. multiceps (96)

Zambia Z-Ka02 F Adult T. serialis (96) T. multiceps (96)

Zambia Z-Ka05 F Adult T.serialis (96) T. multiceps (96)

Zambia Z-Ka06 F Yearling T. serialis (96) T. multiceps (96)

Zambia Z-L01 F Adult T. serialis (96) T. multiceps (96)

Zambia Z-G01 F Adult T. serialis (89) T. multiceps (89)

Zambia Z-Rh01 M Adult T. hydatigena (99)

Zambia Z-Rh14 M Adult T. serialis (96) T. multiceps (96)

Zambia Z-Rh18 M Adult T. hydatigena (96)

Zambia Z-Rh29 F Adult T. hydatigena (89)

Namibia N-K17 ? Adult T. serialis (95) T. multiceps (97)

Namibia N-B08 ? ? T. serialis (95) T. multiceps (97)

Namibia N-T15 ? ? T. serialis (95) T. multiceps (97)

Zimbabwe Zw-Mo14 F pup T. serialis (95) T. multiceps (95)

Zimbabwe Zw-Mo12 ? ? T. serialis (89) T. multiceps (90)

Zimbabwe Zw-Ui01 M ? T. serialis (95) T. multiceps (96)


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To clarify these BLAST results, nine sequences which had minimal ambiguities and

maximum bp regions were aligned against reference sequences for T. serialis and T.

multiceps, using the alignment program Sequencher 4.8 ™. Single nucleotide

polymorphisms (SNP’s) were identified between the two geographic regions and also

between the painted dog isolates and the reference sequences (Table 6.6).

Table 6.6 Summary of the nucleotide variations detected between taeniid isolates from

Zambian and Namibian populations of the African painted dog and reference sequences for

T. multiceps and T. serialis (GenBank accession numbers GQ228818, DQ408418 and

EU219546, DQ104238). Alignment was conducted in Sequencher™ 4.8 and dots indicate

identity with the first sequence.

Taenia isolates Alignment position

487 492 493 496 524 577 594 612

T. multiceps A A T G G C G T

T. serialis . . . . . T . .

Z-Rh14 – Zambia T G T A A T . C

Z-Ka06 – Zambia T G T A A T . C


Z-L01 – Zambia T G T A A T . C



N-B08 – Namibia T G C A A C A T

N-K17 – Namibia T G C A A C A T

N-T15 – Namibia T G C A A C A T


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Intraspecific variation was calculated using pairwise comparisons (Table 6.7). Low

intraspecific variation was detected within T.multiceps and T.serialis reference

sequences (0 – 0.6%) and within Zambian and Namibian isolates (0.5 – 0.9%).

Pairwise comparisons between the painted dog isolates from both populations and the

reference sequences were somewhat higher (3.0 – 4.9%).

Table 6.7 Pairwise comparison of sequence variation (%) within the 12S rRNA loci of

T. multiceps and T. serialis reference sequences and taeniid isolates obtained from

Namibian and Zambian populations of the African painted dog.

T.multiceps T.serialis Zambian

isolates

Namibian

isolates

T. multiceps (n=2) 0 4.1 3 – 3.5 2.9 – 3.9

T. serialis (n=2) - 0.6 3 – 3.5 3.9 - 4.9

Zambian isolates (n=6) - - 0 – 0.5 2.5 - 4

Namibian isolates (n=3) - - - 0 – 0.9

T. multiceps (accession numbers GQ228819, DQ408418), T. serialis (accession

numbers EU219546, DQ104238), Zambian isolates (Z-Ka01, Z-Ka02, Z-Ka05, Z-

Ka06, Z-L01, Z-Rh14), Namibian isolates (N-T15, N-K17, N-B08).

6.3.4 Phylogenetic analysis of Taenia spp. isolates

Figure 6.4 represents the phylogenetic relationship between the sequenced isolates from

this study and reference sequences obtained from GenBank for several species of

Taenia. Geographic grouping can be observed within the painted dog isolates. Isolates


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from the painted dogs are not completely aligned with T. multiceps or T. serialis

suggesting significant variation exists between them.

Figure 6.4 Phylogenetic relationships, inferred using the Neighbour-joining method

between taeniid isolates from African painted dogs and reference isolates of T.

multiceps and T. serialis based on sequence data of the 12S rRNA locus.

Representatives of Taenia sp. used for comparison are labelled with accession numbers

as published on GenBank, with E. granulosus as the out-group. Numbers at nodes refer

to bootstrap values (1,500 replicates) > 20%.

6.4 Discussion

Taeniid species were not detected within any of the captive populations of the African

painted dog. This was most likely a reflection of the captive environment which

involves a modified diet of pre-frozen domestic livestock meat and regular anthelmintic


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treatment inhibiting parasite transmission (see Chapter 2). In comparison, the total

prevalence of taeniid spp. observed in the wild study populations was 31.3%. This was

similar to the prevalence reported by van Heerden et al. (1995) in painted dogs (30%).

Studies on competitor species have reported prevalences of 13% in hyaena and 15%

and 58% in lions (Muller-Graf, 1995; Bjork et al., 2000; Engh et al., 2003).

Prevalence of infection in this study was not significantly different between geographic

regions, although a significant difference was observed between years in the Zambian

population. This was mainly due to the high prevalence observed in the first year of

sampling (2007), which also had the majority of samples collected in the wet season.

Taeniid ova are resilient, but prolonged high temperatures can reduce survivability and

subsequently transmission rates are decreased (Lawson and Gemmell, 1983). The

higher prevalence observed in the wet season may also be a reflection of increased

predation, leading to heightened transmission rates and subsequent improved host

condition, allowing for an increase in proglottid output (Mettrick and Munro, 1965).

Conclusive identification of Taenia species was not possible for all samples but with

the use of molecular characterisation, morphological data and knowledge of possible

intermediate hosts, some tentative identifications can be made. The molecular

techniques utilized were successful in determining that the taeniid ova identified

through microscopy were not the pathogenic zoonotic genera of Echinococcus but were

all Taenia species. Three isolates identified closely with T. hydatigena, which

correlated with the identification of a proglottid obtained from one of these samples.

Identification of the remaining 13 isolates was undetermined as a BLAST on GenBank

revealed isolates had similar a identity to both T. serialis and T. multiceps. Pairwise

comparisons of intraspecific variation within the reference sequences were low at 0.6%


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for T. serialis and 0% for T. multiceps. In comparison, a much higher variation was

observed between T. multiceps and T. serialis and the Zambian isolates (3.4% and

3.5%) and the Namibian isolates (2.9% and 4.9%). This suggests that the painted dog

isolates are genetically distinct from the reference sequences. Additionally, the

variation between sequences from each geographic region was equally high (2.5% -

4%). This was also implied in the phylogenetic tree, where the isolates from Zambia

and Namibia grouped separately and apart from T. multiceps and T. serialis. These

results indicate that the taeniid species found in African painted dogs in this study are

not T. multiceps or T. serialis and are most likely an as yet unsequenced African Taenia

species. It would also appear that there are different species infecting the different

geographic regions. The inability to conclusively identify these isolates is most likely

due to the lack of genetic information available for the rarer African Taenia species

such as T. crocutae, T. hyaenae and T. dinniki.

The probability that the infecting species were of African origin was supported with the

identification of proglottids. Whilst the use of only proglottids for the purpose of

species identification is not conclusive the morphology of reproductive organs can be

characteristic enough to discount some species (Verster, 1969). For example, the

presence of a full vaginal sphincter was able to discount T. multiceps as a possible

species as a ½ sphincter (pad) is specific to this species. As all proglottids had either a

full vaginal sphincter or a complete absence of one, this decreased the likelihood that T.

multiceps was the predominant species present as suggested by the molecular data.

Similarly, T. serialis was discounted due to the absence of an extended cirrus pouch

and variation between vaginal sphincters and number of loops in the vagina and vas

deferens. Additionally, a proglottid identified as T. dinniki had come from a faecal

sample from which the sequenced isolate matched with T. multiceps/T. serialis.


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Without adult worms and/or the addition of molecular data of the rarer African species

of Taenia to public databases, the identification of the predominant Taenia species

infecting these populations remains inconclusive. To date the only species of Taenia

reported in the painted dog have been T. pisiformis, T. s. brauni and T. hyaena

(Ortlepp, 1938; Mahon, 1954; Baer and Fain, 1955). While T. crocutae and T. dinniki

are both species previously found in hyaena, they may be shared with painted dogs as is

T. lycaontis (syn. T. hyaenae). The intermediate hosts for T. dinniki are presently

unknown, however, for T. crocutae they include antelope such as impala, lechwe, kudu

and duiker. These intermediate hosts are major food sources for the painted dog with

impala the dominant prey item within the Zambian population and kudu and duiker the

dominant prey item within Namibia (Lines, 2009).

Whilst Echinococcus species were not identified in this study E. granulosus has been

previously reported in the painted dog (Ortlepp, 1934; Nelson and Rausch, 1963;

Verster, 1965; Young, 1975). In particular, experimental infections found painted dogs

to be very susceptible to infection with Echinococcus protoscolices (Verster, 1961).

However, while the painted dog appears to be a suitable host, only a few observations

have been made. This low incidence may be due to the inability to differentiate taeniid

ova or perhaps more likely due to the lack of suitable intermediate hosts acting as prey.

Two studies in Kenya which conducted numerous necropsies on wild herbivores found

very few hydatid cysts within these animals (Nelson and Rausch, 1963; MacPherson et

al., 1983). These same studies presented evidence that the parasite was being

maintained between dogs and domestic livestock rather than sylvatic transmission. A

recent review on known intermediate hosts for hydatid cysts also noted that antelope

appear to play a minor role with very few observations from large sample sizes


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(Huttner et al., 2009). Antelope are a major food source for the painted dogs in this

study and this may provide a reason for the absences of Echinococcus spp.

6.4.1 Conclusions

The prevalence of Taenia species observed in this study indicates that the painted dog

is an important definitive host for taeniid in all geographic regions sampled. Molecular

tools demonstrated that Echinococcus species were not cycling in any of the sampled

wild populations and therefore painted dogs should not be implicated in the spread of

hydatid disease in these regions. The most prevalent cestodes within all sampled

populations are perhaps one or more genetically uncharacterised Taenia species.

Molecularly these appear to be closely related to T. multiceps, a potential zoonotic

threat, but morphological data suggests other species such as T. crocutae and T. dinniki

are possible, both of which have unknown zoonotic potential. Additionally, there

appears to be geographic variation between the Zambian and Namibian isolates. This

was the first observation of Taenia hydatigena within the African painted dog and

possibly the first observations of T. multiceps and T. dinniki.

Molecular tools are allowing substantial advancements in the efforts to unravel the

phylogeny of Taeniidae but further work needs to be done on the lesser known Taenia

species of African carnivores. Increased surveillance and sample collection from wild

populations, combined with molecular characterisations will assist in understanding the

epidemiology of Taenia infection within this endangered carnivore.

Chapter 7 – General discussion

130

Chapter 7

General discussion


131

7.1 Conservation status of the African painted dog

As habitat destruction and human encroachment continue, there is an ever increasing

need for conservation of wild species. The African painted dog has suffered a 99%

decline in population size in the last century, with approximately 3,000 animals left in

the wild environment today (IUCN, 2010). As an endangered species, African painted

dogs are more vulnerable to stochastic events such as disease outbreaks which can lead

to extirpations. It is therefore imperative to understand all threatening processes to

which these animals may be exposed. Parasitism is considered one such threatening

process for which a thorough understanding is still lacking.

The main aim of this study was to obtain detailed baseline data of parasitism within

populations of the African painted dog in both captive and wild environments. With

this information it was hoped to assist in the conservation of the painted dog by a)

recognising the challenges of parasite control in captive populations, particularly those

involved in reintroduction and/or translocation programs, and b) being able to identify

deviations from ‘baseline’ parasite levels in wild populations which could be

indications for emerging exotic and/or zoonotic diseases.

7.2 Increasing importance of studies of parasites

Parasite/host interactions within the natural environment generally involve

polyparasitism with variable host and environmental factors leading to complex and

dynamic systems. Parasitism within individual hosts and host populations can have

both negative and positive effects. Direct impacts cause disease whilst also develop

host immune systems, and indirect impacts mediate competitive and predatory


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interactions, whilst also drive genetic diversity. To add to these complex interactions,

the effects of outside stressors such as habitat destruction and human encroachment are

important influences which can easily disrupt the natural balance of many host/parasite

relationships. To attempt to understand the effects on any particular host species,

comprehensive data must first be obtained on the parasites that are present in natural

host populations, their prevalence and their relationship to host and environmental

variables. This study was successful in obtaining parasitological data on several wild

and captive populations over a three year period and currently represents the largest

parasitological study yet undertaken of the African painted dog.

7.3 Parasitological techniques

The use of traditional microscopy techniques in conjunction with molecular tools to

genetically characterise parasite species was found to be a successful combination for

this project. Microscopy techniques allowed for the identification of the majority of

parasites existing within a specific host along with an estimate of the intensity of

infection for each animal. However, many parasites could not be identified to species

level based on ova/cyst morphology and molecular characterisation was required in

order to obtain this information. Other studies using these techniques include those

which have successfully identified species of hookworm and Giardia in faecal samples

obtained from dogs and coyotes (Traub et al., 2004a; Palmer et al., 2008; Traub et al.,

2008; Thompson et al., 2009). Prior to use of this technology, the most common way to

identify parasite species was by obtaining adult specimens during necropsy of the host,

which still required considerable expertise in order to correctly identify specimens.

Euthanasia and subsequent necropsy is not an option when dealing with endangered

wildlife and finding animals which have died of natural causes in the field is extremely


133

rare due to the efficiency of scavengers. Molecular techniques are therefore vital to

conduct complete parasitological diagnostics within endangered, wild populations. The

combination of microscopy and molecular methods have allowed this project to provide

significant information on parasite infracommunity composition, intensity of infection

within populations and also the pathogenic and zoonotic risks posed by species such as

Giardia duodenalis, Taenia spp. and Ancylostoma spp.

7.4 Implications for biodiversity conservation

As discussed in Chapter 1, parasites make up a large part of the biodiversity in natural

ecosystems and have co-evolved to form complex relationships with their host and

surrounding environment (May and Anderson, 1983; Poulin and Morand, 2000).

Therefore, parasites warrant consideration for conservation programs in their own right.

Of particular concern would be species of parasites that are host specific and run the

risk of becoming extinct with the host. With reference to this study some parasite

species could not be completely identified, leaving the possibility that host specific

parasites exist within painted dog populations. For example, the majority of sequenced

isolates of Taenia spp. obtained from wild populations were not completely matched

with the available genetic information. It is possible that the species of Taenia infecting

these animals are known species which are not yet genetically characterized or they

could be previously unidentified species unique to the painted dog. As the painted dog

is an endangered carnivore, the risks of co-extinction events occurring with this host

are significant. It is possible that there is the risk of losing species before science has

even been introduced to them.


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7.5 Parasitism in natural populations

7.5.1 Baseline data

This study has gained an insight into the natural abundance, prevalence and intensity of

parasitism within wild populations of the painted dogs over several geographic regions.

The similar parasite community compositions observed among all three wild

populations during the three year study suggest that this represents the ‘natural’ parasite

fauna of the African painted dog. These data can therefore be confidently used as a

baseline for the GI parasites infecting wild populations of the African painted dog, and

can be used for comparisons with future results. Few studies have been able to obtain

this information with most conducting single random sampling studies which are

unable to provide comprehensive data on the host/parasite systems. The need for this

data has been noted by several authors of similar studies who have only been able to

conduct single studies on small population, such as those conducted on the African lion

and spotted hyaena (Bjork et al., 2000; Engh et al., 2003)

Of the seven GI parasite genera observed in populations of the African painted dog,

five have not been previously reported. Within the two genera previously reported

(Taenia and Ancylostoma), three species are reported here for the first time

(Ancylostoma braziliense, Taenia hydatigena and T. dinniki). This large proportion of

new discoveries demonstrates the paucity of information currently available on

parasitism within this host species and is a reflection of the limited knowledge of

parasitism in wildlife in general.


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7.5.2 Parasite threats to painted dogs

The ecology of the African painted dog plays a pivotal role in its exposure to parasitic

disease and potential disease outbreak within populations. Social aspects of the pack

such as the maintenance of close contact by all animals can lead to the rapid spread of

directly transmitted parasites such as hookworm and Giardia spp. Likewise, feeding on

the same prey items results in the same exposure to indirectly transmitted parasites such

as Sarcocystis spp. and Taenia spp. Painted dog packs are renowned for having large

roaming areas (<2,000 km2) which raises the likelihood of packs moving through

human communities and contracting disease from domestic animals, particularly

domestic dogs. The exposure of one member of the pack to disease will then be likely

to result in all members becoming infected. Finally, as populations of painted dogs

appear to exist at natural low densities they are particularly vulnerable to catastrophic

events such as disease which can result in local extinction.

It was difficult to ascertain clinical effects of parasitism within wild populations as

visually all animals appeared to be strong and robust. The only obvious clinical signs

were from those animals carrying injuries i.e. broken legs or snare line injuries. This

appearance of apparent health is common within the natural environment as most wild

animals tend to manifest few recognizable signs of disease (Sachs and Sachs, 1968;

Young, 1969; Roberts et al., 2002). The effects of parasitism on the African painted

dog are therefore most likely to be at a subclinical level. With reference to the findings

in this project, parasites such as Giardia duodenalis could result in sub-clinical disease,

particularly on populations with a low nutritional status or during periods of stress.

Likewise, the intensity of hookworm infection may cause clinical disease in

immunocompromised animals, such as old, injured or stressed individuals. Evidence

for this has been seen in the death of an immunocompromised captive painted dog due


136

to a hookworm infection, and stress has been suggested as a cause for clinical

babesiosis observed in wild rhinoceros and lion (van Heerden, 1986; Penzhorn, 2006).

7.5.3 Zoonoses/anthropozoonoses

Wildlife are often implicated as reservoirs for zoonotic disease and are therefore

considered a threat to surrounding human communities and livestock. The results from

this study suggest the painted dog is not a major threat for zoonotic disease. Of the

parasites observed, the taeniids, Giardia duodenalis and hookworm are potential

zoonotic threats. Within the taeniids, the genus Echinococcus is considered the more

pathogenic as it can cause hydatid disease in humans. Although there was no evidence

of this genus in any of the wild populations of painted dogs which were sampled, the

unidentified Taenia spp. cannot be discounted as potential zoonoses.

The observation of the zoonotic assemblages of G. duodenalis in wild populations

raised the question of whether painted dogs may transmit this parasite to people, but

given the lack of direct contact between these animals and humans, transmission from

painted dogs is unlikely. However, increased human activity into areas such as national

parks has the potential to increase the prevalence of G. duodenalis infection in the

painted dog. Human to animal transmission (anthropozoonotic) is not well understood

or recognised but there has been some evidence for this occurring. Examples include

the transmission of Giardia spp. from human faecal contamination into populations of

gorillas in Uganda and beavers in North America (Bemrick and Erlandsen, 1988;

Graczyk et al., 2002; Fayer et al., 2006). Similarly, the observation of Ancylostoma

braziliense in this study indicates a zoonotic risk but again the lack of direct human

contact would indicate the risk of transmission from painted dogs in the wild

environment would be minimal.


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7.6 Parasitism in captive populations

This is one of few studies to directly compare levels of parasitism between captive and

wild environments in a specific host. A significant difference was observed between the

two environments with 15% of captive and 99% of wild animals found to be host to

one or more parasite species. The implications of the lower exposure to parasites

observed in captive populations may be significant, particularly for those involved in

reintroduction programs, as immunologically naïve animals will be more susceptible to

clinical disease (Viggers et al., 1993; Kock et al., 2010).

The management of captive animals is governed by the need for healthy and

reproductively sound animals and therefore parasite control is a major consideration.

Often the objective is to completely eradicate all parasites from the resident populations

but this may also result in extinction of host-specific parasites (Gompper and Williams,

1998). Additionally, as captive breeding facilities move toward conducting

reintroduction programs, management of captive animals may need to alter the focus

from complete parasite eradication to allowing a low level of parasitism to exist within

the population. In the short term this may assist in the development and maintenance of

host immunity which will be required when animals are reintroduced back into the wild

environment. The most pertinent example of this that is relevant to this study is the

observed prevalence of hookworm in the wild environment (32.5%) compared to the

complete absence in the captive environment. Exposure to a large burden of hookworm

by immunologically naïve painted dogs would most likely result in severe anaemia and

possible death as seen in domestic dogs (Miller, 1971; Carroll and Grove, 1985).

However, if captive animals had prior exposure to this parasite and had developed low

levels of immunity this effect would be minimized (Carroll and Grove, 1985). In the

long term, parasite exposure has been shown to assist in maintaining genetic diversity,


138

particularly of the major histocompatibility complex (MHC) which is largely involved

in immune function (Paterson et al., 1998). As a major concern of captive breeding is

loss of genetic diversity this beneficial effect of parasitism may outweigh the

occasional loss of an animal due to the direct effects of parasitism.

7.7 Project limitations and future research

7.7.1 Sample collection and analysis

The logistics of field work prevented sufficient sampling of all wild populations in

order to answer all hypotheses. For example, the lack of samples from pups did not

allow for the significance of host age to be sufficiently tested. In addition, sampling

was predominantly restricted to faecal samples and it was therefore not possible to

detect parasites such as Toxoplasma spp., Sarcocystis spp., Babesia spp, and

Dirofilaria immitis (heartworm) existing in host blood or tissues.

The use of fresh samples is preferred for accurate detection of parasite stages but the

lack of facilities in the field meant that samples had to be preserved in 10% formalin

and 70% ethanol for shipping and later analysis. Several microscopy techniques were

employed in an effort to observe all parasite species, however, the most accurate

method for prevalence data is usually obtained during necropsy. This method was not

an option for studies on endangered wildlife and finding carcasses in the field did not

occur during the course of the project.

The molecular techniques employed were of great value in identifying several parasite

species, however not all positive samples could be amplified by PCR. In the case of


139

hookworm only a small subset of samples was successfully amplified and sequenced.

Additional sequences would have been beneficial in providing detailed information on

the prevalence of species infecting packs and also in obtaining accurate geographic

distributions for the hookworm species observed. Finally, as the use of molecular

techniques is relatively new not all species of parasites have been genetically

characterised and sequences are therefore not available for comparison in databases.

This was the case with the predominant Taenia spp. observed in this study which

remains unknown despite the successful amplification and sequencing of many

samples.

7.7.2 Continued surveillance

The data obtained from this study has provided a baseline for the ‘normal’ parasite

diversity and prevalence of GI parasites to which painted dogs are exposed within the

natural environment. This information can now be used to identify future deviations

from this baseline which may then assist to identify potential emerging disease. In

order for this to be of use, continued surveillance of parasitism within painted dog

populations needs to be conducted. The data from this study, used in conjunction with

results from future surveillance, may also help to predict how continued pressure on the

already small populations existing in the wild may change the dynamics of the

host/parasite systems currently in play. The host/parasite relationship is often a careful

balance which can be tipped in favour of the parasite if the host is immunologically

compromised. For instance, the decreased hunting success in small painted dog packs

may lead to a lowered nutrition plane which could in turn lead to immunocompromised

animals. Other factors such as habitat destruction, human encroachment and disease

may also result in an imbalance in the host/parasite relationship leading to an increase

in clinical disease.


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7.7.3 Threat of emerging disease

As outlined throughout this thesis the host/parasite interaction is complex and dynamic.

These interactions will be further complicated by human influences such as habitat

destruction and human encroachment. Habitat destruction disturbs ecosystems and can

subsequently alter host/parasite interactions. Evidence for this was seen in a study by

Gillespie et al. (2005) which identified altered patterns of parasite infection among red

tailed guenon populations living in unlogged and logged habitats. Likewise, human

encroachment brings the increased risk of disease spillover from co-habiting domestic

animals. As approximately 80% of domestic animal pathogens can infect wildlife the

threat is significant (Cleaveland et al., 2001). The painted dog has already been seen to

be vulnerable to domestic dog diseases such as rabies and canine distemper virus

suggesting they could be vulnerable to additional disease spillover (Kat et al., 1995;

Roelke-Parker et al., 1996). The identification in this study of predominantly zoonotic

assemblages of Giardia duodenalis in the painted dog may be indicative of human

encroachment and consequently should be considered an emerging disease within this

species. Future parasitological studies comparing results to this newly obtained baseline

may be able to predict and identify future emerging diseases.

7.7.4 Captive management

This project was only able to conduct a captive versus wild comparison for a single

host species but given the significance of results obtained, could be used as a model for

other host species. As reintroduction programs become a main aim for many captive

facilities a host specific knowledge of parasitism in the natural environment may

provide important information. The ability to predict the type of parasitic challenge that

reintroduced hosts may face could lead to vaccination programs or deliberate exposure

to specific parasites in order to increase the chance of successful reintroduction.


141

Additionally, these parasitological studies may prevent future introductions of exotic

pathogens from reintroduced captive stock into naïve wild populations (Viggers et al.,

1993; Kock et al., 2010). Similar studies on other endangered captive host species

could, therefore, be of benefit to many reintroduction and/or translocation programs.

142

Appendix 1. Taenia spp. proglottid key. From Verster (1969)

143

References

Abe, N., Kimata, I., Iseki, M., 2003. Identification of genotypes of Giardia intestinalis isolates from dogs in Japan by direct sequencing of the PCR amplified glutamate dehydrogenase gene. Journal of Veterinary Medical Science 65, 29-33.

Alexander, K.A., Appel, M.J., 1994. African wild dogs (Lycaon pictus) endangered by a canine distemper epizootic among domestic dogs near the Masai Mara National Reserve, Kenya. Journal of Wildlife Diseases 30, 481-485.

Altizer, S., Harvell, C.D., Friedle, E., 2003a. Rapid evolutionary dynamics and disease threats to biodiversity. Trends in Ecology and Evolution 18, 589-596.

Altizer, S., Nunn, C.L., Thrall, P.H., Gittleman, J.L., Antonovics, J., Cunningham, A.A., Dobson, A., Ezenwa, V.O., Jones, K.E., Pedersen, A.B., Poss, M., Pullimam, J.R.C., 2003b. Social organization and parasite risk in mammals: Integrating theory and empirical studies. Annual Review of Ecology, Evolution and Systematics 34, 517-547.

Anderson, R.C., 1972. The ecological relationships of meningeal worm and native cervids in North America. Journal of Wildlife Diseases 8, 304-310.

Anderson, R.M., Crombie, J.A., 1985. Population dynamics of Schistosoma mansoni in mice repeatedly exposed to infection. Nature 315, 491-493.

Applebee, A.J., Thompson, R.C.A., Olson, M.E., 2005. Giardia and Cryptosporidium in mammalian wildlife - current status and future needs. Trends in Parasitology 21, 370-376.

Areekul, S., Saenghirun, C., Ukoskit, K., 1975. Studies on the pathogenicity of Ancylostoma ceylanicum. I. Blood loss in experimental dogs. Southeast Asian Journal of Tropical Medicine and Public Health 6, 234-240.

Astiazaran-Garcia, H., Espinosa-Cantellano, M., Castanon, G., Chavez-Munguia, B., Martinez-Palomo, A., 2000. Giardia lamblia: Effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus). Experimental Parasitology 95, 128-135.

Baer, J.G., Fain, A., 1955. Cestodes. Exploration du Parc National de Upemba. Mission G.F.de Witte 36, 1-38.

Baker, M.K., Lange, L., Verster, A., Van der Plaat, S., 1989. A survey of helminths in domestic cats in the Pretoria area of Transvaal, Republic of South Africa. Part 1. Journal of South African Veterinary Association 60, 139-142.

Barnes, T.S., Hinds, L.A., Jenkins, D.J., Coleman, G.T., 2007. Precocious development of hydatid cysts in a macropodid host. International Journal for Parasitology 37, 1379-1389.

Bemrick, W.J., Erlandsen, S.L., 1988. Giardiasis - is it really a zoonosis? Parasitology Today 4, 69-71.

144

Berdoy, M., Webster, J.P., Macdonald, D.W., 2000. Fatal attraction in Toxoplasma-infected rats: a case of parasite manipulation of its mammalian host. Proceedings of the Royal Society of London B 267, 1591-1594.

Bergersen, E.P., Anderson, D.E., 1997. The distribution and spread of Myxobolus cerebralis in the United States. Fisheries 22, 6-7.

Berkman, D.S., Lescano, A.G., Gilman, R.H., Lopez, S.L., Black, M.M., 2002. Effects of stunting, diarrhoeal disease, and parasitic infection during infancy on cognition in late childhood: a follow-up study. The Lancet 359, 564-571.

Berrilli, F., Di Cave, D., De Liberato, C., Franco, A., Scaramozzino, P., Orecchia, P., 2004. Genotype characterisation of Giardia duodenalis isolates from domestic and farm animals by SSU-rRNA gene sequencing. Veterinary Parasitology 122, 193-199.

Beveridge, I., 2002. Australian hookworms (Ancylostomatoidea): a review of the species present, their distributions and biogeographical origins. Parasitologia 44, 83-88.

Bioca, E., 1951. On Ancylostoma braziliense (de-Faria, 1910) and its morphological differentiation from A. ceylanicum (Looss, 1911). Journal of Helminthology 15, 1-10.

Bjork, K.E., Averbeck, G.A., Stromberg, B.E., 2000. Parasites and parasite stages of free-ranging wild lions (Panthera leo) of northern Tanzania. Journal of Zoo and Wildlife Medicine 31, 56-61.

Boomker, J., Horak, I.G., de Vos, V., 1986. The helminth parasites of various artiodactylids from some African nature reserves. Onderstepoort Journal of Veterinary Research, 53, 93-102.

Bowman, D.D., Lynn, R.C., Eberhard, M.L., 2003. Georgis' parasitology for veterinarians. Saunders, St Louis.

Bwangamoi, O., Ngatia, T.A., Richardson, J.D., 1993. Sarcocystis-like organisms in musculature of a domestic dog (Canis familiaris) and wild dogs (Lycaon pictus) in Kenya. Veterinary Parasitology 49, 201-205.

Caccio, S.M., De Giacomo, M., Pozio, E., 2002. Sequence analysis of the giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. International Journal for Parasitology 32, 1023-1030.

Caccio, S.M., Thompson, R.C.A., McLauchin, J., Smith, H.V., 2005. Unravelling Cryptosporidium and Giardia epidemiology. Trends in Parasitology 21, 430-437.

Caccio, S.M., Ryan, U., 2008. Molecular epidemiology of giardiasis. Molecular and Biochemical Parasitology 160, 75-80.

Carroll, S.M., Grove, D.I., 1984. Parasitological, hematologic and immunologic responses in acute and chronic infections of dogs with Ancylostoma ceylanicum: A model of human hookworm infection. The Journal of Infectious Diseases 150, 284-294.

145

Carroll, S.M., Grove, D.I., 1985. Response of dogs to challenge with Ancylostoma ceylanicum during the tenure of a primary hookworm infection. Transactions of the Royal Society of Tropical Medicine and Hygiene 80, 406-411.

Carroll, S.M., Grove, D.I., 1986. Experimental infection of humans with Ancylostoma ceylanicum: clinical, parasitological, haematological and immunological findings. Tropical and Geographical Medicine 38, 38-45.

Castro-Hermida, J., Almeida, A., Gonzalez-Warleta, M., Correia da Costa, J., Rumbo-Lorenzo, C., Mezo, M., 2007. Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants. Parasitology Research 101, 1143-1148.

Chapman, C.A., Wasserman, M.D., Gillespie, T.R., Speirs, M.L., Lawes, M.J., Saj, T.L., Ziegler, T.E., 2006. Do food availability, parasitism, and stress have synergistic effects on red colobus populations living in forest fragments? American Journal of Physical Anthropology 131, 525-534.

Chaudhry, A.Z., Longworth, D.L., 1989. Cutaneous manifestations of intestinal helminthic infections. Dermatologic Clinics 7, 275-290.

Chilton, N.B., Gasser, R.B., Beveridge, I., 1997. Phylogenetic relationships of Australian strongyloid nematodes inferred from ribosomal DNA sequence data. International Journal for Parasitology 27, 1481-1494.

Choisy, M., Brown, S.P., Lafferty, K., D., Thomas, F., 2003. Evolution of trophic transmission in parasites: why add intermediate hosts? American Naturalist 162, 172-181.

Choo, J., Pang, E., Provic, P., 2000. Hookworms in dogs in Kuching, Sarawak (North Borneo). Transactions of the Royal Society of Tropical Medicine and Hygiene 21, 300-301.

Clare e Silva, L.M., Miranda, R.C., Santos, H.A., Rabelo, E.M.L., 2006. Differential diagnosis of dog hookworms based on PCR-RFLP from the ITS region of the rDNA. Veterinary Parasitology 14, 373-377.

Clarke, K.R., 1993. Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology 18, 117-143.

Clarke, K.R., Gorley, R.N., 2006. PRIMER v6: User Manual/Tutorial. PRIMER, Plymouth.

Cleaveland, S., Laurenson, M.K., Taylor, L.H., 2001. Diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence. Philosophical Transactions of the Royal Society of London B 356, 991-999.

Colly, L.P., Nesbit, J.W., 1992. Fatal acute babesiosis in a juvenile wild dog (Lycaon pictus). Journal of the South African Veterinary Association 63, 36-38.

Colwell, D.D., Otranto, D., Stevens, J.R., 2009. Oestrid flies: eradication and extinction versus biodiversity. Trends in Parasitology 25, 500-504.

146

Cooke, A., Zaccone, P., Raine, T., Phillips, J.M., Dunne, D.W., 2004. Infection and autoimmunity: are we winning the war, only to lose the peace? Trends in Parasitology 20, 316-321.

Cookson, W., Moffatt, M.F., 1997. Asthma: An epidemic in the absence of infection? Science 275, 41-42.

Cooper, M.A., Adam, R.D., Worobey, M., Sterling, C.R., 2007. Population genetics provides evidence of recombination in Giardia. Current Biology 17, 1984-1988.

Courchamp, F., Rasmussen, G., Macdonald, D., 2002. Small pack size imposes a trade-off between hunting and pup-guarding in the painted hunting dog Lycaon pictus. Behavioural Ecology 13, 20-27.

Covacin, C., Aucoin, D.P., Elliot, A., Thompson, R.C.A., 2010. Genotypic characterisation of Giardia from domestic dogs in the USA. Veterinary Parasitology 177, 28-32.

Cowman, I.M., 1947. The timber wolf in the Rocky Mountain national parks of Canada. Canadian Journal of Research 25, 139-174.

Creel, N., Creel, S., 2002. The African wild dog. Behaviour, ecology and conservation. Princeton University Press, Oxford.

Creel, S., Creel, N., 1995. Communal hunting and pack size in African wild dogs, Lycaon pictus. Animal Behaviour 50, 1325-1339.

Creel, S., Creel, N., 1998. Six ecological factors that may limit African wild dogs, Lycaon pictus. Animal Conservation 1, 1-9.

Croese, J., Loukas, A., Opdebeeck, J., Provic, P., 1994. Occult enteric infection by Ancylostoma caninum: a previously unrecognised zoonosis. Gastroenterology 106, 3-12.

Cunningham, A.A., 1996. Disease risks of wildlife translocations. Conservation Biology 10, 349-353.

Daszak, P., Cunningham, A.A., Hyatt, A.D., 2000. Emerging infectious diseases of wildlife - threats to biodiversity and human health. Science 287, 443-449.

Davidson, W., R., Appel, M.J., Doster, G.L., Baker, O.E., Brown, J.F., 1992. Diseases and parasites of red foxes, grey foxes and coyotes from commercial sources selling to fox-chasing enclosures. Journal of Wildlife Diseases 28, 581-589.

Davies-Mosert, H.T., Mills, M.G., Macdonald, D., 2009. A critical assessment of South Africa's managed metapopulation recovery strategy for African wild dogs. In: Hayward, M.W.,Somers, M. (Eds.), Reintroduction of top-order predators, Wiley-Blackwell, Oxford, pp. 10-42.

Dawkins, R., Krebs, J.R., 1979. Arms races between and within species. Proceedings of the Royal Society of London B 205, 489-511.

147

De Leo, G., Dobson, A., 2002. Virulence management in wildlife populations. In: Dieckmann, U., Metz, J.A.J., Sabelis, M.W.,Sigmund, K. (Eds.), Adaptive dynamics of infectious diseases: In pursuit of virulence management, Cambridge University Press, Cambridge, pp. 413-424.

Delahay, R.J., Brown, J.A., Mallinson, P.J., Spyvee, P.D., Handoll, D., Rogers, L.M., Cheeseman, C.L., 2000. The use of marked bait in studies of the territorial organization of the European Badger (Meles meles). Mammal Review 30, 73-87.

Deredec, A., Courchamp, F., 2006. Combined impacts of Allee effects and parasitism. Oikos 112, 667-679.

Dinnik, J.A., Sachs, R., 1969. Cysticercosis of the sacrum bone of antelopes and Taenia olngojinei sp.nov. of the spotted hyena - Article in German. Zeitschrift fur Parasitenkunde 31, 326-329.

Dinnik, J.A., Sachs, R., 1972. Taeniidae of lions in East Africa. Zeitschrift fur Tropenmedizin und Parasitologie 23, 197-210.

Dobson, A., Lafferty, K., D., Kuris, A.M., Hechinger, R.F., Jetz, W., 2008. Homage to Linnaeus: How many parasites? How many hosts? Proceedings of the National Academy of Sciences of the United States of America 105, 11482-11489.

Dobson, A.P., 1995. Rinderpest in the Serengeti ecosystem: the ecology and control of a keystone virus. In: Junge, R.E. (Ed). Proceedings of a joint conference American Association of Zoo Veterinarians, Wildlife Disease Association and American Association of Wildlife Veterinarians, pp. 518-519.

Dubey, J.P., Speer, C.A., Fayer, R., 1989. Sarcocystosis of animals and man. Boca Raton, Florida.

Dubey, J.P., Speer, C.A., 1991. Sarcocystis canis n. Sp. (Apicomplexa: Sarcocystidae), etiological agent of generalized coccidiosis in dogs. Journal of Parasitology 77, 522-527.

Dubini, A., 1843. Nuovo verme intestinal umano (Agchylostoma duodenale) costituente un sesto genere dei nematoidea proprii dell'uomo. Annali Universali di Medicina 106, 5-13.

Dunn, A.M., 1969. Veterinary Helminthology. William Heinemann medical books Ltd, London.

Dunn, R.R., Harris, N., Colwell, R.K., Koh, L.P., Sodhi, N.S., 2009. The sixth mass coextinction: are most endangered species parasites and mutualists? Proceedings of the Royal Society of London B 276, 3037-3045.

Eckert, J., Gemmell, M.A., Meslin, F.X., Pawlowski, Z.S. (eds.), 2001. WHO/OIE Manual on Echinococcosis in humans and animals: a public health problem of global concern. World Organisation for Animal Health and World Health Organisation, Paris, France.

148

Eckmann, L., 2003. Mucosal defences against Giardia. Parasite Immunology 25, 259-270.

Elliott, A., Morgan, U., Thompson, R., C,A, 1999. Improved staining method for detecting Cryptosporidium oocysts in stools using malachite green. Journal of General Applied Microbiology 45, 139-142.

Engh, A.L., Nelson, K.G., Peebles, R., Hernandez, A.D., Hubbard, K.K., Holekamp, K.E., 2003. Coprological survey of parasites of spotted hyenas (Crocuta crocuta) in the Masai Mara National Reserve, Kenya. Journal of Wildlife Diseases 39, 224-227.

Estes, R.D., 1967. Prey selection and hunting behaviour of the African wild dog. The Journal of Wildlife Management 31, 52-70.

Evans, A.C., Markus, M.B., Steyn, E., 1990. A survey of the intestinal nematodes of bushmen in Namibia. American Journal of Tropical Medicine and Hygiene 42, 243-247.

Fabiyi, J.P., 1987. Production losses and control of helminths in ruminants of tropical regions. International Journal for Parasitology 17, 435-442.

Fanshawe, J.H., Frame, L.H., Ginsberg, J., 1991. The wild dog - Africa's vanishing carnivore. Oryx 25, 137-146.

Farthing, M.J., Mata, L., Urrutia, J.J., Kronmal, R.A., 1986. Natural history of Giardia infection of infants and children in rural Guatemala and its impact on physical growth. The American Journal of Clinical Nutrition 43, 395-405.

Fayer, R., Santin, M., Trout, J., DeStefano, S., Koenen, K., Kaur, T., 2006. Prevalence of microsporidia, Cryptosporidium spp., and Giardia spp. in beavers (Castor canadensis) in Massachusetts. Journal of Zoo and Wildlife Medicine 37, 492-497.

Feliu, C., Blasco, S., Torres, J., Miquel, J., Casanova, J.C., 2001. On the helminth fauna of Erinaceus europaeus Linnaeus, 1758 (Insectivora, Erinaceidae) in the Iberian Peninsula. Research and Reviews in Parasitology 61, 31-37.

Fenstermacher, R., 1937. Further studies of diseases affecting moose. II. Cornell Veterinarian 27, 25-37.

Foreyt, W.J., 2001. Veterinary Parasitology. Blackwell publishing, Iowa.

Frankham, R., 2005. Genetics and extinction. Biological Conservation 126, 131-140.

Fulleborn, F., 1914. Untersuchungen uber den Infektionsweg bei Strongyloides und Ankylostomum und die Biologie dieser Parasiten. Archiv fur Schiffs und Tropen-Hygiene 18, 26-80.

Garijo, M., Oritz, J.M., Ruiz de ibanez, M.R., 2004. Helminths in a giraffe (Giraffa camelopardalis giraffa) from a zoo in Spain. Onderstepoort Journal of Veterinary Research 70, 153-156.

149

Gillespie, T.R., Greiner, E.C., Chapman, C.A., 2005. Gastrointestinal parasites of the colobus monkeys of Uganda. Journal of Parasitology 91, 569-573.

Girman, D.J., Kat, P.W., Mills, M.G., Ginsberg, J., Borner, M., Wilson, V., Fanshawe, J.H., Fitzgibbon, C., Lau, L.M., Wayne, R.K., 1993. Molecular genetic and morphological analyses of the African Wild Dog (Lycaon pictus). Journal of Heredity 84, 450-459.

Gompper, M.E., Williams, E.S., 1998. Parasite conservation and the black-footed ferret recovery program. Conservation Biology 12, 730-732.

Gompper, M.E., Goodman, R.M., Kays, R.W., Ray, J.C., Fiorello, C.V., Wade, S.E., 2003. A survey of the parasites of coyotes (Canis latrans) in New York based on fecal analysis. Journal of Wildlife Diseases 39, 712-717.

Gouy de Bellocq, J., Charbonnel, N., Morand, S., 2008. Coevolutionary relationship between helminth diversity and MHC class II polymorphism in rodents. Journal of Evolutionary Biology 21, 1144-1150.

Graber, M., Blanc, J.P., 1979. Ancylostoma duodenale (Dubini, 1843) Creplin 1843 (Nematoda: Ancylostomidae) parasite de l'hyene tachetee Crocuta crocuta (Erxleben), en Ethiopie. Revue d'Elevage et Medecine Veterinaire des Pays Tropicaux 32, 155-160.

Graczyk, T.K., Bosco-Nizey, J., Ssebide, B., Thompson, R.C.A., Read, C., Cranfield, M.R., 2002. Anthropozoonotic Giardia duodenalis genotype (Assemblage A) infections in habitats of free-ranging human-habituated gorillas, Uganda. Journal of Parasitology 88, 905-909.

Harris, H.F., 1900. On the rapid conversion of hematoxylin into haematein in staining reactions. Journal of Applied Microscopic Laboratory Methods 3, 777.

Hayward, M.W., O'Brien, J., Hofmeyr, M., Kerley, G.I., 2006. Prey preferences of the African wild dog Lycaon pictus (Canidae:Carnivora): ecological requirements for conservation. Journal of Mammalogy 87, 1122-1131.

Herlich, H., 1978. The importance of helminth infections in ruminants. World Animal Review 26, 22-26.

Hernot, D.C., Biourge, V.C., Martin, L.J., Dumon, H.J., Nguyen, P.G., 2005. Relationship between total transit time and faecal quality in adult dogs differing in body size. Journal of Animal Physiology and Animal Nutrition 89, 189-193.

Hoberg, E.P., Jones, A., Rausch, R.L., Eom, K.S., Gardner, S.L., 2000. A phylogenetic hypothesis for species of the genus Taenia (Eucestoda: Taeniidae). Journal of Parasitology 86, 89-98.

Hoberg, E.P., Kocan, A.A., Rickard, L.G., 2001. Gastrointestinal strongyles in wild ruminants. In: Samuel, W.M., Pybus, M.J.,Kocan, A.A. (Eds.), Parasitic diseases of wild mammals, Manson publishing Ltd., London.

150

Hoberg, E.P., 2002. Taenia tapeworms: their biology, evolution and socioeconomic significance. Microbes and Infection 4, 859-866.

Hoberg, E.P., 2006. Phylogeny of Taenia: Species definitions and origins of human parasites. Parasitology International 55, S23-S30.

Holt, R., Boulinier, T., 2005. Ecosystems and parasitism: the spatial dimension. In: Thomas, F., Renaud, F.,Guegan, J.F. (Eds.), Parasitism and ecosystems, Oxford University Press, New York.

Hopkins, R.M., Meloni, B.P., Groth, D.M., Wetherall, J.D., Reynoldson, J.A., Thompson, R.C.A., 1997. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living the same locality. Journal of Parasitology 83, 44-51.

Hudson, P.J., Dobson, A.P., Newborn, D., 1992. Do parasites make prey vulnerable to predation? Red Grouse and parasites. The Journal of Animal Ecology 61, 681-692.

Hudson, P.J., Dobson, A., Newborn, D., 1998. Prevention of population cycles by parasite removal. Science 282, 2256-2258.

Hudson, P.J., Dobson, A., Lafferty, K., D., 2006. Is a healthy ecosystem one that is rich in parasites? Trends in Ecology and Evolution 21, 381-385.

Huttner, M., Nakao, M., Wassermann, T., Siefert, L., Boomker, J., Dinkel, A., Sako, Y., U., M., Romig, T., Ito, A., 2008. Genetic characterization and phylogenetic position of Echinococcus felidis Ortlepp, 1937 (Cestoda:Taeniidae) from the African Lion. International Journal for Parasitology 38, 861-868.

Huttner, M., Romig, T., 2009. Echinococcus species in African wildlife. Parasitology 136, 1089-1095.

Huttner, M., Siefert, L., Mackenstedt, U., Romig, T., 2009. A survey of Echinococcus species in wild carnivores and livestock in East Africa. International Journal for Parasitology 39, 1269-1276.

Ibechukwu, B.I., Onwukeme, K.E., 1991. Intraocular coenurosis: a case report. British Journal of Ophthalmology 75, 430-431.

Ipankaew, T., Traub, R.J., Thompson, R., C,A, Sukthana, Y., 2007. Canine parasitic zoonoses and temple communities in Thailand. South Asian Journal of Tropical Medicine and Public Health 38, 247-255.

Itagaki, T., Kinoshita, S., Aoki, M., Itoh, N., Saeki, H., Sato, N., Uetsuki, J., Izumiyama, S., Yagita, K., Endo, T., 2005. Genotyping of Giardia intestinalis from domestic and wild animals in Japan using glutamate dehydrogenase gene sequencing. Veterinary Parasitology 133, 283-287.

IUCN 2010, posting date. IUCN Red List of Threatened Species. www.iucnredlist.org. [Online.]

151

Jelinek, T., Maiwald, H., Nothdurft, H.D., Loscher, T., 1994. Cutaneous larva migrans in travellers: synopsis of histories, symptoms, and treatment of 98 patients. Clinical Infectious Diseases 19, 1062-1066.

Jennions, M.D., Macdonald, D., 1994. Cooperative breeding mammals. Trends in Ecology and Evolution 9, 89-93.

Jog, M.M., Marathe, R., R., Goel, S.S., Ranade, S.P., Kunte, K.K., Watve, M.G., 2005. Sarcocystosis of chital (Axis axis) and dhole (Cuon alpinus): ecology of a mammalian prey-predator-parasite system in Peninsular India. Journal of Tropical Ecology 21, 479-482.

Jog, M.M., Watve, M.G., 2005. Role of parasites and commensals in shaping host behaviour. Current Science 89, 1184-1191.

Joly, D., O., Messier, F., 2004. The distribution of Echinococcus granulosus in moose: evidence for parasite-induced vulnerability to predation by wolves? Oecologia 140, 586-590.

Jones, A., Khalil, L.F., 1984. Taenia dinniki sp. nov. (Cestoda: Taeniidae) from the striped and the spotted hyaena in East Africa. Journal of Natural History 18, 803-809.

Jones, C.G., Lawton, J.H., Shachak, M., 1994. Organisms as ecosystems engineers. Oikos 69, 373-386.

Kalkofen, V.P., 1970. Attachment and feeding behaviour of Ancylostoma caninum. Parasitology Research 33, 339-354.

Kat, P.W., Alexander, K.A., Smith, J.S., Munson, L., 1995. Rabies and African wild dogs in Kenya. Proceedings of the Royal Society of London B 262, 229-233.

Kat, P.W., Alexander, K.A., Smith, G., Richardson, J.D., Munson, L., 1996. Rabies among African wild dogs (Lycaon pictus) in the Masai Mara, Kenya. Journal of Veterinary Diagnostic Investigation 8, 420-426.

Kirby-Smith, J.L., 1926. Creeping eruption. Archives of Dermatology and Syphilology 13, 137-173.

Klock, A., Bednarska, M., Bajer, A., 2005. Intestinal macro and microparasites of wolves (Canis lupus l.) from north-eastern Poland recovered by coprological study. Annals of Agricultural and Environmental Medicine 12, 237-245.

Kock, R.A., Woodford, M.H., Rossiter, P.B., 2010. Disease risks associated with the translocation of wildlife. Revue scientifique et technique - Office International et Epizootics 29, 329-350.

Koh, L.P., Dunn, R.R., Sodhi, N.S., Colwell, R.K., Proctor, H.C., Smith, V.S., 2004. Species coextinctions and the biodiversity crisis. Science 305, 1632-1634.

152

Kohn, M.H., Wayne, R.K., 1997. Facts from feces revisited. Trends in Ecology and Evolution 12, 223-227.

Lakatos, L., Mester, G., Erdelyi, Z., Balogh, M., Szipocs, I., Kamaras, G., L., L.P., 2004. Striking elevation in incidence and prevalence of inflammatory bowel disease in a province of western Hungary between 1977-2001. World Journal of Gastroenterology 10, 404-409.

Lalle, M., Pozio, E., Capelli, G., Bruschi, F., Crotti, D., Caccio, S.M., 2005. Genetic heterogeneity at thegiardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. International Journal for Parasitology 35, 207-213.

Lawson, J.R., Gemmell, M.A., 1983. Hydatidosis and cysticercosis: the dynamics of transmission. Advances in Parasitology 22, 261-308.

Leonhard, S., Pfister, K., Beelitz, P., Thompson, R.C.A., 2007. The molecular characterisation of Giardia from dogs in southern Germany. Veterinary Parasitology 150, 33-38.

Levine, N.D., 1968. Nematode parasites of domestic animals and of man. Burgess Publishing Company, Minneapolis.

Levine, N.D., 1986. The taxonomy of Sarcocystis (Protozoa, Apicomplexa) species. Journal of Parasitology 72, 372-382.

Lindstrom, K.M., Foufopoulos, J., Parn, H., Wikelski, M., 2004. Immunological investments reflect parasite abundance in island populations of Darwin's finches. Proceedings of the Royal Society of London B 271, 1513-1519.

Lines, R., 2009. Background information and species management guidelines for Namibia's rare and valuable wildlife. Namibian Nature Foundation.

Loos-Frank, B., 2000. An up-date of Verster's (1969) 'Taxonomic revision of the genus Taenia Linnaeus' (Cestoda) in table format. Systematic Parasitology 45, 155-183.

Looss, A., 1898. Zur Lebensgeschichte des Ankylostoma duodenale. . Centralblatt fur Bakteriologie und Parasitenkunde, Abteillung originale 24, 441-449, 483-488.

Loreau, M., Roy, J., Tilman, D., 2005. Linking ecosystem and parasite ecology. In: Thomas, F., Renaud, F.,Guegan, J.-F. (Eds.), Parasitism and ecosystems, Oxford University Press, New York, pp. 13-21.

Loukas, A., Provic, P., 2001. Immune responses in hookworm infections. Clinical Microbiology Reviews 14, 689-703.

Lyles, A.M., Dobson, A., 1993. Infectious disease and intensive management: population dynamics, threatened hosts, and their parasites. Journal of Zoo and Wildlife Medicine 24, 315-326.

153

MacPherson, C.N.L., Karstad, L., Stevenson, P., Arundel, J.J., 1983. Hydatid disease in the Turkana district of Kenya. III. The significance of wild animals in the transmission of Echinococcus granulosus, with particular reference to Turkana and Masailand in Kenya. Annals of Tropical Medicine and Parasitology 77, 61-73.

Macpherson, C.N.L., Spoerry, A., Zeyle, E., Romig, T., Gorfe, M., 1989. Pastoralists and hydatid disease: an ultrasound scanning prevalence survey in East Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 243-247.

Magambo, J., Njoroge, E., Zeyle, E., 2006. Epidemiology and control of echinococcosis in sub-Saharan Africa. Parasitology International 55, S193-S195.

Mahon, J., 1954. Tapeworms from the Belgian Congo. Annales du Musee royal du Congo belge C. Zoologie, 137-264.

Maillard, J.-C., Gonzalez, J.-P., 2006. Biodiversity and emerging diseases. Annals of the New York Academy of Sciences 1081, 1-16.

Malcolm, J.R., van Lawick, H., 1975. Notes on wild dogs (Lycaon pictus) hunting zebras. Mammalia 39, 231-240.

Malcolm, J.R., Marten, K., 1982. Natural selection and the communal rearing of pups in African wild dogs (Lycaon pictus). Behavioural Ecology and Sociobiology 10, 1-13.

Marangi, M., Berrilli, F., Otranto, D., Giangaspero, A., 2010. Genotyping of Giardia duodenalis among children and dogs in closed socially deprived community from Italy. Zoonoses and Public Health 57, e54-e58.

Marathe, R., R., Goel, S.S., Ranade, S.P., Jog, M.M., Watve, M.G., 2002. Patterns in abundance and diversity of faecally dispersed parasites of tiger in Tadoba National Park, central India. BMC Ecology 2.

Mas Bakal, P., Karstad, L., Veld, N.I.T., 1990. Serologic evidence of toxoplasmosis in captive and fee-living wild mammals in Kenya. Journal of Wildlife Diseases 16, 559-564.

Mathews, F., Moro, D., Strachan, R., Gelling, M., Buller, N., 2006. Health surveillance in wildlife reintroductions. Biological Conservation 131, 338-347.

Matjila, P.T., Leisewitz, A.L., Jongejan, F., Bertschinger, H.J., Penzhorn, B.L., 2008. Molecular detection of Babesia rossi and Hepatozoan sp. in African wild dogs (Lycaon pictus) in South Africa. Veterinary Parasitology 157, 123-127

May, R.M., Anderson, R.M., 1978. Regulation and stability of host-parasite population interactions. II. Destabilising processes. Journal of Animal Ecology 47, 249-267.

May, R.M., Anderson, R.M., 1983. Epidemiology and genetics in the coevolution of parasites and hosts. Proceedings of the Royal Society of London B 219, 281-313.

McCallum, H., Dobson, A.P., 1995. Detecting disease and parasite threats to endangered species and ecosystems. Trends in Ecology and Evolution 10, 190-194.

154

McCallum, H., Dobson, A., 2002. Disease, habitat fragmentation and conservation. Proceedings of the Royal Society of London B 269, 2041-2049.

Mettrick, D.F., Beverley-Burton, M., 1961. Some cyclophyllidean cestodes from carnivores in Southern Rhodesia. Parasitology 51, 533-544.

Mettrick, D.F., Munro, H.N., 1965. Studies on the protein metabolism of cestodes. Effects of host dietary constituents on the growth of Hymenolepis diminuta. Parasitology 55, 453-466.

Miller, T.A., 1966. Blood loss during hookworm infection, determined by erythrocyte labelling with radioactive 51chromium. II. Pathogenesis of Ancylostoma braziliense infection in dogs and cats. Journal of Parasitology 52, 856-865.

Miller, T.A., 1967. Immunity of dogs to Ancylostoma braziliense infection following vaccination with x-irradiated A. caninum larvae. Journal of the American Veterinary Medical Association 150, 508-515.

Miller, T.A., 1971. Vaccination against the canine hookworm diseases. Advances in Parasitology 9, 153-180.

Minnaar, W.N., Krecek, R.C., 2001. Helminths in dogs belonging to people in a resource-limited urban community in Gauteng, South Africa. Onderstepoort Journal of Veterinary Research 68, 111-117.

Minnaar, W.N., Krecek, R.C., Fourie, L.J., 2002. Helminths in dogs from a peri-urban resource-limited community in Free State Province, South Africa. Veterinary Parasitology 107, 343-349.

Monis, P.T., Mayrhofer, G., Andrews, R.H., Homan, W.L., Limper, L., Ey, P.L., 1995. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112, 1-12.

Monis, P.T., Andrews, R.H., Mayrhofer, G., Mackrill, J., Kulda, J., Isaac-renton, J.L., Ey, P.L., 1998. Novel analysis of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia. Parasitology 116, 7-19.

Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 1999. Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol 16, 1135-1144.

Monis, P.T., Caccio, S.M., Thompson, R.C.A., 2009. Variation in Giardia: towards a taxonomic revision of the genus. Trends in Parasitology 25, 93-100.

Morand, S., Krasnov, B.R., Poulin, R. (eds.), 2006. Micromammals and macroparasites. From evolutionary ecology to management. Springer-Verlag, Tokyo.

Muller-Graf, C.D.M., 1995. A coprological survey of intestinal parasites of wild lions (Panthera leo) in the Serengeti and Ngorongoro Crater, Tanzania, East Africa. Journal of Parasitology 81, 812-814.

155

Muller-Graf, C.D.M., Woolhouse, M.E.J., Packer, C., 1999. Epidemiology of intestinal parasite (Spirometra spp.) in two populations of African lions (Panthera leo). Parasitology 118, 407-415.

Munson, L., Terio, K.A., Kock, R., Mlengeya, T., Roelke, M.E., Dubovi, E., Summers, B.A., Sinclair, A.R., Packer, C., 2008. Climate extremes promote fatal co-infections during canine distemper epidemics in African lions. PloS One 3, e2545.

Neitz, W.O., 1965. A check-list and host-list of the zoonoses occurring in mammals and birds in South and South West Africa. Onderstepoort Journal of Veterinary Research 32, 189-374.

Nelson, G.S., Rausch, R.L., 1963. Echinococcus infections in man and animals in Kenya. Annals of Tropical Medicine and Parasitology 57, 136-149.

Odening, K., 1986. The present state of species-systematics in Sarcocystis Lankester, 1882 (Protista, Sporozoa, Coccidia). Systematic Parasitology 41, 209-233.

Ortlepp, R.J., 1934. Echinococcus in dogs from Pretoria and vicinity. Onderstepoort Journal of Veterinary Science and Animal Industry 3, 97-108.

Ortlepp, R.J., 1938. South African helminths. Pt. II. Some Taenias from large wild carnivores. Onderstepoort Journal of Veterinary Research 10, 253-278.

Palmer, C.S., Traub, R.J., Robertson, I.D., Hobbs, R.P., Elliott, A., While, L., Rees, R., Thompson, R.C.A., 2007. The veterinary and public health significance of hookworm in dogs and cats in Australia and the status of A. ceylanicum. Veterinary Parasitology 145, 304-313.

Palmer, C.S., Traub, R.J., Robertson, I.D., Devlin, G., Rees, R., Thompson, R.C.A., 2008. Determining the zoonotic significance of Giardia and Cryptosporidium in Australian dogs and cats. Veterinary Parasitology 154, 142-147.

Pangui, L.J., Kaboret, T., 1993. Les helminthes du chien a Dakar, Senegal. Revue de Medecine Veterinaire 144, 791-794.

Parkar, U., Traub, R.J., Vitali, S., Elliot, A., Levecke, B., Robertson, I., Geurden, T., Steele, J., Drake, B., Thompson, R.C.A., 2010. Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Veterinary Parasitology 169, 8-17.

Paterson, S., Wilson, K., Pemberton, J.M., 1998. Major histocompatibility complex variation associated with juvenile survival and parasite resistance in a large unmanaged ungulate population (Ovis aries). Proceedings of the National Academy of Sciences of the United States of America 95, 3714-2719.

Peirce, M.A., Laurenson, M.K., Gascoyne, S.C., 1995. Hepatozoonosis in cheetah and wild dogs in the Serengeti ecosystem. African Journal of Ecology 33, 273-275.

156

Pelayo, L., Nuez, F.A., Rojas, L., Furuseth Hansen, E., Gjerde, B., Wilke, H., Mulder, B., Robertson, L., 2008. Giardia infections in Cuban children: the genotypes circulating in a rural population. Annals of Tropical Medicine and Parasitology 102, 585-595.

Pellegrini, D., 1950. Una nuova speci di larva di tenia (Cysticercus madoquae) nel dig-dig. Rivista di Parassitologia 11, 211-217.

Penzhorn, B.L., 2006. Babesiosis of wild carnivores and ungulates. Veterinary Parasitology 138, 11-21.

Peterson, M.J., 1991. Wildlife parasitism, science, and management policy. Journal of Wildlife Management 55, 782-789.

Phillips, M.K., Scheck, J., 1991. Parasitism in captive and reintroduced red wolves. Journal of Wildlife Diseases 27, 498-501.

Piggott, M.P., Taylor, A.C., 2003. Extensive evaluation of faecal preservation and DNA extraction methods in Australian native and introduced species. Australian Journal of Zoology 51, 341-355.

Pole, A., Gordon, I.J., Gorman, M.L., MacAskill, M., 2004. Prey selection by African wild dogs (Lycaon pictus) in southern Zimbabwe. Journal of Zoology, London 262, 207-215.

Poulin, R., 1993. The disparity between observed and uniform distributions: A new look at parasite aggregation. International Journal for Parasitology 23, 937-944.

Poulin, R., Morand, S., 2000. The diversity of parasites. Quarterly Review of Biology 75, 277-293.

Poulin, R., 2007. Evolutionary ecology of parasites. Princeton University Press, Princeton.

Provic, P. (ed.), 1998. Zoonotic hookworm infections (Ancylostomiasis). Oxford Medical Publications, Oxford.

Rasmussen, G., 1999. Livestock predation by the painted hunting dog Lycaon pictus in a cattle ranching region of Zimbabwe: a case study. Biological Conservation 88, 133-139.

Rausch, R.L., 1994. Family Taeniidae Ludwig, 1886. In: Khail, L.F., Jones, A.,Bray, R. (Eds.), Keys to the cestode parasites of vertebrates, International Institute of Parasitology, Commonwealth Agricultural Bureaux, Wallingford, U.K., pp. 665-672.

Read, C.M., Monis, P.T., Thompson, R.C.A., 2004. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infection, Genetics and Evolution 4, 125-130.

Roberts, M.G., Dobson, A.P., Arneberg, P., de Leo, G.A., Krecek, R.C., Manfredi, M.T., Lanfranchi, P., Zaffaroni, E., 2002. Parasite community ecology and biodiversity. In: Hudson, P.J. (Ed.), The ecology of wildlife diseases, Oxford University Press Inc., New York.

157

Robertson, L.J., Hermansen, L., Gjerde, B., Strand, E., Alvsvag, J.O., Langeland, N., 2006. Application of genotyping during an extensive outbreak of waterborne giardiasis in Bergen, Norway, during autumn and winter 2004. Applied and Environmental Microbiology 72, 2212-2217.

Roelke-Parker, M.E., Munson, L., Packer, C., Kock, R., Cleavelend, S., Carpenter, M., Obrien, S.J., Pospischil, A., Hofmann-Lehmann, R., Lutz, H., Mwamengele, G.L., Mgasa, M.N., Machange, G.A., Summers, B.A., Appel, M.J., 1996. A canine distemper virus epidemic in Serengeti lions (Panthera leo). Nature 379, 441-445.

Rolfe, V.E., Adams, C.A., Butterwick, R.F., Batt, R.M., 2002. Relationship between faecal character and intestinal transit time in normal dogs and diet-sensitive dogs. Journal of Small Animal Practice 43, 290-294.

Round, M.C., 1968. Check list of the helminth parasites of African mammals of the orders Carnivora, Tubulidentata, Proboscidea, Hyracoidea, Artiodactyla and Perissodactyla. Technical Communication No. 38. Commonwealth Agricultural Bureaux.

Rozsa, L., Reiczigel, J., Majoros, G., 2000. Quantifying parasites in samples of hosts. Journal of Parasitology 86, 228-232.

Sachs, R., Sachs, C., 1968. A survey of parasitic infestation of wild herbivores in the serengeti region in northern Tanzania and the Lake Rukwa region in southern Tanzania. Bulletin for Epizootological Disease in Africa 16, 455-472.

Saitou, N., Nei, M., 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406-425.

Scheepers, J.L., Venzke, K.A.E., 1995. Attempts to reintroduce African wild dogs Lycaon pictus into Etosha National Park, Namibia. South African Journal of Wildlife Research 25, 138-140.

Scott, M.E., Anderson, R.M., 1984. The population dynamics of Gryodactylus bullatarudis (Monogenea) on guppies (Poecilia reticulata). Parasitology 89, 159-154.

Scott, M.E., 1987. Regulation of mouse colony abundance by Heligmosomoides polygyrus. Parasitology 95, 111-124.

Scott, M.E., 1988. The impact of infection and disease on animal populations: Implications for conservation biology. Conservation Biology 2, 40-56.

Semichon, L., 1924. Procedee de coloration et de regonflement des parasites animaux. Revue de Pathologie Vegetale et d'Entomologie Agricole 11, 193.

Shaw, D.J., Grenfell, B., Dobson, A., 1995. Patterns of macroparasite abundance and aggregation in wildlife populations: a quantitative review. Parasitology 111, S111-S133.

158

Sillero-Zubiri, C., Hoffmann, M., Macdonald, D. (eds.), 2004. Canids: foxes, wolves, jackals and dogs: Status survey and conservation action plan. IUCN/SSC Canid Specialist Group, Cambridge.

Smith, K.F., Acevedo-Whitehouse, K., Pedersen, A.B., 2009a. The role of infectious diseases in biological conservation. Animal Conservation 12, 1-12.

Smith, K.F., Behrens, M.D., Dov, F.S., 2009b. Local scale effects of disease on biodiversity. EcoHealth 6, 287-295.

Smith, Y., Kok, O.B., 2006. Faecal helminth egg and oocysts counts of a small population of African lion (Panthera leo) in the southwestern Kalahari, Namibia. Onderstepoort Journal of Veterinary Research 73, 71-75.

Smithers, S.R., Terry, R.J., 1969. Immunity in schistosomiasis. Annals New York Academy of Sciences 160, 826-840.

Souza, S.L.P., Gennari, S.M., Richtzenhain, L.J., Pena, H., Funanda, M.R., Cortez, A., Gregori, F., Soares, R.M., 2007. Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of Sao Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Veterinary Parasitology 149, 258-264.

Staniland, I.J., 2002. Investigating the biases in the use of hard prey remains to identify diet composition using Antarctic fur seals (Arctocephalus gazella) in captive feeding trials. Marine Mammal Science 18, 223-243.

Summers, R.W., Elliott, D.E., Urban, J.F., Thompson, R., Weinstock, J.V., 2005. Trichuris suis therapy in Crohn's disease. Gut 54, 87-90.

Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596-1599.

Temple, S.A., 1987. Do predators always capture substandard individuals disproportionately from prey populations? Ecology 68, 669-674.

Templeton, A.C., 1968. Human coenurus infection: a report of 14 cases from Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene 62, 251-255.

Teodorovic, S., Braverman, J.M., Elmendorf, H.G., 2007. Unusually low levels of genetic variation among Giardia lamblia isolates. Eukaryotic Cell 6, 1421-1430.

Thomas, F., Renaud, F., Guegan, J.F. (eds.), 2005. Parasitism and ecosystems. Oxford University Press, Oxford.

Thompson, R.C.A., Lymbery, A.J., 1988. The nature, extent and significance of variation within the genus Echinococcus. Advances in Parasitology 27, 209-258.

159

Thompson, R.C.A., 2000. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitology Today 16, 210-213.

Thompson, R.C.A., McManus, D.P., 2002. Towards a taxonomic revision of the genus Echinococcus. Trends in Parasitology 18, 452-457.

Thompson, R.C.A., 2004. The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Veterinary Parasitology 126, 15-35.

Thompson, R.C.A., Colwell, D.D., Shury, T., Appelbee, A.J., Read, C., Njiru, Z., Olson, M.E., 2009. The molecular epidemiology of Cryptosporidium and Giardia infections in coyotes from Alberta, Canada, and observations on some cohabiting parasites. Veterinary Parasitology 159, 167-170.

Thompson, R.C.A., Lymbery, A.J., Smith, A., 2010. Emerging parasite diseases and wildlife. International Journal for Parasitology 40, 1163-1170.

Trachsel, D., Deplazes, P., Mathis, A., 2007. Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA. Parasitology 134, 911-920.

Traub, R.J., Monis, P.T., Robertson, I.D., Irwin, P.J., Mencke, N., Thompson, R.C.A., 2004a. Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128, 253-262.

Traub, R.J., Robertson, I.D., Irwin, P.J., Mencke, N., Thompson, R.C.A., 2004b. Application of a species-specific PCR-RFLP to identify Ancylostoma eggs directly from canine faeces. Veterinary Parasitology 123, 245-255.

Traub, R.J., Inpankaew, T., Sutthikornchai, C., Sukthana, Y., Thompson, R.C.A., 2008. PCR-based coprodiagnostic tools reveal dogs as reservoirs of zoonotic ancylostomiasis caused by Ancylostoma ceylanicum in temple communities in Bangkok. Veterinary Parasitology 155, 67-73.

Trout, J., Santin, M., Fayer, R., 2006. Giardia and Cryptosporidium species and genotypes in coyotes (Canis latrans). Journal of Zoo and Wildlife Medicine 37, 141-144.

van Heerden, J., 1986. Disease and mortality of captive wild dogs (Lycaon pictus). South African Journal of Wildlife Research 16, 7-11.

van Heerden, J., Boomker, J., Booyse, D.G., Mills, M.G., 1994. The wild dog (Lycaon pictus): A new host for Ancylostoma caninum. Journal of the South African Veterinary Association 65, 18-19.

van Heerden, J., Mills, M.G., van Vuuren, M.J., Kelly, P.J., Dreyer, M.J., 1995. An investigation in the health status and diseases of wild dogs (Lycaon pictus) in the Kruger National Park. Journal of the South African Veterinary Association 66, 18-27.

van Heerden, J., Verster, R., Espie, I., 1996. Disease and mortality in captive wild dogs (Lycaon pictus). Journal of the South African Veterinary Association 67, 141-145.

160

van Keulen, H., Macechko, P.T., Wade, S., Schaaf, S., Wallis, P.M., Erlandsen, S.L., 2002. Presence of human Giardia in domestic, farm and wild animals, and environmental samples suggests a zoonotic potential for giardiasis. Veterinary Parasitology 108, 97-107.

Vanderwick, F., Fain, A., Langi, S., 1964. Two new cases of human coenurosis caused by Taenia brauni in Rwanda, with orbital localization of the coenurus. Annales des Societes Belges de Medecine Tropicale, de Parasitologie, et de Mycologie 44, 1077-1079.

Verster, A., 1961. Helminth research in South Africa. V. Journal of South African Veterinary Medical Association 32, 181-185.

Verster, A., 1965. Review of Echinococcus species in South Africa. Onderstepoort Journal of Veterinary Research 32, 7-118.

Verster, A., 1969. A taxonomic revision of the genus Taenia Linnaeus, 1758 S. Str. Onderstepoort Journal of Veterinary Research 36, 3-58.

Viggers, K.L., Lindenmayer, D.B., Spratt, D.M., 1993. The importance of disease in reintroduction programmes. Wildlife Research 20, 687-698.

Wegner, K.M., Reusch, T.B.H., Kalbe, M., 2003. Multiple parasites are driving major histocompatibility complex polymorphism in the wild. Journal of Evolutionary Biology 16, 224-232.

Weilinga, C., Thompson, R.C.A., 2007. Comparative evaluation of Giardia duodenalis sequence data. Parasitology 134, 1795-1821.

Whary, M.T., Fox, J.G., 2004. Th1-mediated pathology in mouse models of human disease is ameliorated by concurrent Th2 responses to parasite antigens. Current Topics in Medicinal Chemistry 4, 531-538.

Williams, E.S., Thorne, E.T., Kwiatkowski, D.R., Oakleaf, B., 1992. Overcoming disease problems in the black-footed ferret recovery program. Transactions of North American Wildlife and Natural Resources Conference 57, 474-485.

Williams, P.H., Templeton, A.C., 1971. Infection of the eye by tapeworm Coenurus. British Journal of Ophthalmology 55, 766-769.

Windsor, D.A., 1997. Equal rights for parasites. Perspectives in Biology and Medicine 40, 222-229.

Windsor, D.A., 1998. Most of the species on Earth are parasites. International Journal for Parasitology 28, 1939-1941.

Winkorth, C.L., Learmonth, J.J., Matthaei, C.D., Townsend, C.R., 2008. Molecular characterization of Giardia isolates from calves and humans in a region in which dairy farming has recently intensified. Applied and Environmental Microbiology 74, 5100-5105.

161

Woodroffe, R., Ginsberg, J., Macdonald, D., 1997. The African wild dog: status survey and conservation action plan. IUCN/SSC Canid Specialist Group, Cambridge.

Woodroffe, R., 1999. Managing disease threats to wild mammals. Animal Conservation 2, 185-193.

Wyatt, K.B., Campos, P.F., Gilbert, M.T.P., Kolokotronis, S., Hynes, W.H., Delasse, R., Daszak, P., MacPhee, R.D., Greenwood, A.D., 2008. Historical mammal extinction on Christmas Island (Indian Ocean) correlates with introduced infectious disease. PloS One 3, e3602.

Yazdanbakhsh, M., van den Biggelaar, A., Maizels, R.M., 2001. Th2 responses without atopy: immunoregulation in chronic helminth infections and reduced allergic disease. Trends in Immunology 22, 372-377.

Yazdanbakhsh, M., Dremsner, P.G., van Ree, R., 2002. Allergy, parasites and the hygiene hypothesis. Science 296, 490-494.

Yoshida, Y., Okamoto, D., Matsuo, K., Kwo, E.H., Retnasabapathy, A., 1973. The occurrence of Ancylostoma braziliense (de Faria, 1910) and Ancylostoma ceylanicum (Looss, 1911) in Malaysia. Southeast Asian Journal of Tropical Medicine, Public Health 4, 498-503.

Young, E., 1969. The significance of infectious diseases in African game populations. Zoologica Africana 4, 275-281.

Young, E., 1975. Echinococcosis (Hydatidosis) in wild animals of the Kruger National Park. Journal of the South African Veterinary Association 46, 285-286.

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