HIERDIE EKSEMPlAAR MAG 0NDER
GEEN OMSTANDIGHEDE UIT DIE
BIBLIOTEEK VERWYDERWORp NIE
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34300002278970
Universiteit Vrystaat
BLOOD PARASITES OF FREE STATE AND
LESOTHO REPTILES
By
Johann Van As
Dissertation submitted in fulfilment of the requirements for the
degree Magister Scientiae in the Faculty of Natural and
Agricultural ~cif!nces . ,~ .tv • .'!-:.
Department of Zoology and Entomology; Universiiy ;of the Free
State '.:y ,~'.';'~: .'
Supervisor Prof. Angela Davies
Co- supervisor Prof. Linda Basson
July 2003
~n1voroltelt van ~le
orc:nJo-vrvotaat
BL~T~lN
2 7 JUL 2004
uoVS 9AGOl !IDLIOTEEK
CONTENTS
1. INTRODUCTION
1.1 Distribution and evolution of reptiles 1
1.2 Squamata of the Free State 2
1.3 Parasites of'Squamata and aims of the project 6
1.4 Blood parasites of reptiles 7
1.5 Problems oftaxonomy 9
1.6 Reptilian viral and viral-like infections 9
1.7 Reptilian Protozoa or so-called Protozoa 10
PHYLUM APICOMPLEXA LEVINE, 1970 11
CLASS CONOIDASIDA LEVINE, 1988 11
Order Eucoccidiorida Léger & Dubosq, 1910 12
CLASS ACONOIDASIDA MEHLHORN,
PETERS & HABERKORN, 1980 17
Order Haemospororida Danilewsky, 1885 18
Order Piroplasmorida Wenyon, 1926 19
1.8 Reptilian filaria I nematodes 21
Phylum Nematoda Potts, 1932 21
Family Onchocercidae Leiper, 1911 21
J.9 The current study 22
2. MATERIALS AND METHODS 23
2.1 Collections 23
2.2 Blood sampling, light microscopy and image capture 23
2.3 Transmission electron microscopy 24
2.4 Cell infection data 25
2.5 Digital imaging. 25
2.6 Miscellaneous 26
3. RESULTS 31
3.1 HOSTS COLLECTED 31
3.2 VIRUSES AND SUSPECTED VIRAL INFECTIONS 63
3.2.1. PIRHEMOCYTON CHA TTON & BLANC 1914 63
• Pirhemocyton from Agama atra atra Daudin, 1802. 63
• Pirhemocyton from Pseudocordylus melanotus 65
subvindis (A. Smith, 1838) 57
3.2.2. SAVROPLASMA DU TOIT, 1937 66
• Sauroplasma from Cordylus giganteus (A. Smith, 1844) 66
e Sauroplasma from Cordylus polyzonus polyzonus A. Smith, 1838 70
• Sauroplasma from Mabuya sulcata (Peters, 1867) 71
lil Sauroplasma from Nucras intertexta (A. Smith, 1838) 71
• Sauroplasma from Mabuya striata punctatissima (A. Smith, 1849) 72
• Sauroplasma from Acontias gracilicauda gracilicauda Essex, 1925. 75
• Sauroplasma from Varanus exanthematicus albigularus (Daudin, 1802)75
• Sauroplasma from Pseudocordylus melanotus 76
subviridis (A. Smith, 1838) and Pseudocordylus melanotus
melanotus (A. Smith, 1838).
3.2.3. SA VROMELLA PIENAAR, 1954 78
• Sauromella from Pachydactylus capensis (A. Smith, 1845) 78
3.2.4. SERPENTOPLASMA PIENAAR, 1954 80
• Serpentoplasma from Crotaphopeltis hotamboeia (Laurenti, 1768) 80
• Serpentoplasma from Lycophidion capense capense (A. Smith, 1831) 80
• Serpentoplasma from Elapsoidea sundevallii media Broadley, 1971 81
• Serpentoplasma from Hemachmus haemachatus (Lacêpéde, 1788) 82
• Serpentoplasma from Causus rhombeatus (Lichtenstein, 1823) 82
• Serpentoplasma from Bitis arietans (Merrem, 1820). 84
3.3. PROTOZOA 86
3.3.1. HAEMOGREGARINES 86
• Hepatozoon sp. A from Agama atra atra Daudin, 1802 86
• Hepatozoon sp. B from Pseudocordylus melanotus 89
melanotus (A. Smith, 1838)
• Hepatozoon sp. C from Pseudocordylus melanotus 91
subviridis (A. Smith, 1838)
• Hepatozoon sp. D from Pseudocordylus melanotus 94
subviridis (A. Smith, 1838)
e Heputozoon sp. E from Pseudocordylus melanotus 98
subviridis (A. Smith, 1838)
9 Hepatozoon (Haemogregarina) sebue (Laveran and Pettit, 1909) 100
Smith, 1996 from Python sebae natalensis (Gmelin, 1789)
• Hepatozoon sp. F from Psammophylax tritaeniatus (Gunther, 1868) 103
SAURIAN MALARIA 106
3.3.2. PLASMODIDAE 106
• Plasmodium sp. A from Cordylus polyzonus polyzonus A. Smith, 1838 106
• Plasmodium sp. B from Pseudocordylus melanotus 109
melanotus (A. Smith, 1838)
• Plasmodium or Haemoproteus sp. C from 112
Pseudocordylus melanotus subviridis (A. Smith, 1838)
• Plasmodium or Haemoproteus sp. D from 113
Pachydactylus bibronii (A. Smith, 1845)
3.4. MICROFILARIA E 114
3.4.1. NEMATODA 114
• Microfilaria sp. A from Agama atra atra Daudin, 1802 114
• Microfilaria sp. B from Cordylus polyzonus
• polyzonus A. Smith, 1838 115
• Microfilaria sp. C from Pseudocordylus melanotus 115
subviridis (A. Smith, 1838)
• Microfilaria sp. D from Pseudocordylus melanotus 116
melanotus (A. Smith, 1838)
4. DISCUSSION 119
5. REFERENCES 127
ABSTRACT 138
OPSOMMING 139
ACKNOWLEDGEMENTS 140
APPENDIX A 141
CHAPTER 1 INTRODUCTION
INTRODUCTION
1.1Distribution and evolution of reptiles
Evolutionary history and fossil evidence suggest that reptiles have existed for more than
300 million years. According to this evidence this was, and is, a very successful group of
vertebrates. In this time-scale reptiles have successfully radiated onto most of the
continents, with the exception of Antarctica. They are thought to have arisen from
amphibians during the Carboniferous period and the earliest reptile fossils are about 315
million years old. According to Branch (1998), living reptiles are either remnants of this
period, or a recent flowering has taken place since the extinction of the dinosaurs, 65
million years ago. They are so successful in terms of diversity and radiation that there
are more species of reptiles in South Africa than mammals. In the western deserts they
exceed the birds in number, if not diversity (Branch 1998). Furthermore, there are more
endemic reptile species in South Africa than any other vertebrate group and according to
Branch (1998) in the period from 1988-1998, 83 new species were described, that is, one
in every 44 days.
The key to the reptiles' success was the development of the amniotic egg, which is
resistant to desiccation and without the free-living tadpole stage. The absence of this
typical aquatic larval stage was instrumental in freeing reptiles from the aquatic world.
Some have evolved cleiodic eggs, with thick shells and yolk stores. Reptiles also have
particular features that they share with amphibians: scaly skin, the presence of lungs and
of four legs, at least in the primitive forms. In some cases evolution has brought the loss
of two and even four external appendages. According to Low (1978) there are four
orders of reptiles, namely the Crocodylia (crocodiles, caimans and alligators), Chelonia
(tortoises, terrapins and turtles), Squamata (lizards, amphisbaenians and snakes) and
Sphenodonta (the tuataras). The lizard-like tuataras are restricted to a few islands on the
north coast of New Zealand. The other three orders are well represented in South Africa,
comprising 480 species.
CHAPTER 1 INTRODUCTION
1.2 Squamata of the Free State
The first contribution to the knowledge of the Free State reptiles was that of Boettger
(1883), who recorded a few lizard and snake species from Smithfield. FitzSimons added
much more to this subject in his books on the lizards (FitzSimons 1943) and snakes
(FitzSimons 1962) of Southern Africa. Later, De Waal (1978) compiled a list of the Free
State squamates, collecting in 16 habitats, in each degree unit, making this the most
intensive survey to date in Africa. He also sampled in every quarter degree in the Free
State. His results are summarised in Table 1.
A more recent survey by Bates (1996) shed new light on the diversity of reptiles in the
Free State. He added Il lizard and two snake species to the list, bringing the total of
known representatives of Squamata of the Free State to 53 lizard, one amphisbaenian and
38 snake species. The following table (Table 1) is a complete list of known squamates in
the Free State, as well as Bates' newer records indicated with asterisks.
Table 1 A summary of reptiles of the Free State from De Waal (1978) and Bates (1996).
Newer records from Bates (1996) are indicated by asterisks.
Host
Class: Reptilia
Order: Squamata Oppel, 1811
Suborder: Sauria MacCartney, 1802
Family: Gekkonidae Cuvier, 1817
Afroedura anivaria (Boulenger, 1894)
Afroedura karroika halli (Hewitt, 1935)
"Hemidactylus maboeia (Moreau de Jonnes, 1818)
Lygodactylus capensis capensis (A. Smith, 1849)
Pachydactylus bibronii (A. Smith, 1845)
Pachydactylus capensis capensis (A. Smith, 1845)
*Pachydactylus laevigatus laevigatus Fisher, 1888
Pachydactylus maculatus ocelatus (Hewitt, 1927)
Pachydactylus mariquensis mariquensis (A. Smith, 1849)
2
CHAPTER 1 INTRODUCTION
Table 1 (Continued). A summary of reptiles of the Free State from De Waal (1978) and
Bates (1996). Newer records from Bates (1996) are indicated by asterisks.
*Paehydaetylus vansoni FitzSimons, 1933
Ptenopus_garrulus garrulus (A. Smith, 1849)
Family: Agamidae Gray, 1827
Agama atra Daudin, 1802
Agama hispida (Linnaeus, 1758)
Agama makarikarica FitzSimons, 1932
Family: Chameleonidae (Methuen & Hewitt, 1915)
*Bradypodion sp (Qua Kwa & Zastron varieties)
*Bradypodion draeomontanum Raw, 1976
*Bradypodion cf karooicum (Methuen & Hewitt, 1915)
Chameleo dilepis dilepis Leach, 1819
Family: Scincidae
Aeontias graeilieauda graeilicauda Essex, 1925
Afroablepharus wahlbergii (A. Smith, 18491
Mabuya oeeidentalis (Peters, 1867)
Mabuya striatapunetatissima (A. Smith, 1849)
Mabuya sulcata sulcata (Peters, 1867)
Mabuya varia(Peters, 1876)
Mabuya variegata punctulata (Bocage, 1872)
Mabuya variegata variegata (Peters, 1869)
Tetradactylus africanus africanus (Gray, 1838)
"Tetradactylus seps (Linnaeus, 1758)
"Tetradactylus tetradactylus (Lacêpéde, 1803)
Family: Cordylidae Gray, 1837
"Charnaesaura aenea (Wiegmann, 1843)
Cordylus eordy/us cordylus (Linnaeus, 1758)
COl-dylusgiganteus A. Smith, 1844
Cordylus polyzonus polyzonus A. Smith 1838
Cordylus vittifer vittifer (Reichenow, 1887)
3
CHAPTERl INTRODUCTION
Table 1 (Continued). A summary of reptiles of the Free State from De Waal (1978) and
Bates (1996). Newer records from Bates (1996) are indicated by asterisks.
Pseudocordylus melanotus melanotus (A. Smith, 1838)
Pseudocordylus melanotus subviridis (A. Smith, 1838)
Pseudocordylus spinosus FitzSimons, 1947
*Tropidosaura essexi Hewitt, 1927
Family: Lacertidae Bonaparte, 1831
Eremias burchelli Dumeril & Bibron, 1839
Eremias lineoocellata lineoocellata Dumeril & Bibron, 1839
Eremias namaquensis Dumeril & Bibron, 1839
Ichnotropis squamulosa Peters, 1854
Nucras intertexta (A. Smith, 1838)
Nl/eras lalandii (Milne-Edwards, 1829)
Nueras taeniolata ornata (Gray, 1864)
Varanus exanthematicus albigarus (Daudin, 1802)
Varanus niloticus niloticus (Linnaeus, 1766)
Suborder: AMPHISBAENIA
Family: Amphisbaenidae Gray, 1825
Monopeltis capensis capensis A. Smith, 1848
Suborder: SERPENTES Linnaeus, 1758
Family: Typhlopidae Gray, 1825
Rhinotyphlops lalandei (Schlegel, 1844)
Typhlops bibronii ( A. Smith, 1846)
Family: Leptotyphlopidae Stjneger, 1891
*Leptotyphlops conjunctus conjunctus (Jan, ] 861)
Leptotyphlops scutifrons scutifrons (Peters, 1854)
Family: Colubridae Gray, 1825
Aparallactus capensis A. Smith, 1849
Crotahopeltis hotamboeia (Laurenti, 1768)
Dasypeltis scabra (Linnaeus, 1785)
Dispholidus typus typus (A. Smith, 1829)
4
CHAPTER 1 INTRODUCTION
Table 1 (Continued). A summary of reptiles of the Free State from De Waal (1978) and
Bates (1996). Newer records from Bates (1996) are indicated by asterisks.
Duberria lutrix lutrix (Linnaeus, 1785)
Lamprophis aurora (Linnaeus, 1754)
Lamprophisfuliginosusfuliginosus (Baie, 1827)
Lamprophis fuscus Boulenger, 1893
Lamprophis guttatus (A. Smith, 1843)
Lamprophis inoratus Dumeril & Bibron, 1854
"Lycodonomorphus laevissimus (Gunter, 1862)
Lycodonomorphus rufulus (Lichtenstein, 1823)
Lycophidion capense capense (A. Smith, 1831)
Philotamnus natalensis occidentalis Broadley, 1966
Prosymna bivittata Werner, 1903
Prosymna sundevalli sundevalli (A. Smith, 1849)
Psammophis crucifer (Daudin, 1803)
Psammophis leightoni trinasalis Werner, 1902
Psammophis notostictus Peters, 1867
Psammophylax rhombeatus rhomheatus (Linnaeus, 1754)
Psammophylax tritaeniatus (Gunter, 1868)
Pseudaspis cana (Linnaeus, 1785)
Xenoealamus hieolor Ncolor Gunther, 1868
Family: Elapidae Baie, 1827
A!:>pidelapslubneus luhrieus (Laurenti, 1786)
Elaps dorsalis A. Smith, 1849
Elaps lacteus (Linnaeus, 1754)
Hemachatus haemaehatus (Lacépéde, 1788)
Naja nivea A. Smith, 1849
Family: Viperidae Gray, 1825
Atraetaspis hihronii A. Smith, 1849
Bitis arietans arietans (Merrem, 1820)
Bitis artropos (Linnaeus, 1754)
Causus rhombeatus (Lichtenstein, 1823)
5
CHAPTER 1 INTRODUCTION
1.3 Parasites of Squamata and aims of the project
Several groups of parasites are known from most types of reptiles, including the
Squamata (see Mader 1996). Work on parasites of reptiles in general is mostly confined
to Europe and the Americas, although Mackerras (1961) compiled a comprehensive list
of blood parasites of Australian reptiles.
It is impossible in a study like this one to focus on all parasites found in the Squamata.
The aims of this project are therefore to concentrate on the blood parasites, since these
are readily accessible and once sampled, allow reptiles to be returned to the wild shortly
after capture, without harm. It focuses particularly on blood parasites of Squamata of the
Free State, but also includes some work in Lesotho. The project also aims to provide
baseline studies, so that in future more detailed research will be possible. In doing a
survey of reptiles, many of which are protected species, an initial assessment of the
identity and taxonomy of their blood parasites is necessary. This approach can identify
taxonomic problems that merit future, more detailed work. For example, the study makes
preliminary, yet determined attempts to identify the nature of infections in the blood of
CITES listed red data species Cordylus giganteus A. Smith, 1844 in the northeastern
parts of the Free State. This particularly involves the use of transmission electron
microscopy (TEM) techniques.
By surveying the blood parasites of these animals, a deeper understanding of the
population structures and general biology of their parasites can be achieved. This also
provides a greater knowledge of the distribution of these infections and whether they
might be of a pathological nature. Focusing on the biology of blood parasites in reptiles
might just lead to a greater insight into parasites infecting man, like the malarias, which
are common in lizards. A well-known example of such an event using non-human
parasite models was that involving Ronaid Ross, who famously completed his work on
human malaria in 1898 by observing the transmission of bird malaria parasites (see
Roberts & Janovy, 2000).
6
CHAPTER 1 INTRODUCTION
1.4 Blood parasites of reptiles
The blood parasites of reptiles represent a rather unexplored field. These parasites were
first recorded in the late 1800s, and the work of Robertson (1906) and Sambon &
Seligmann (1907) are classical examples of early research on turtle and snake
haemogregarines, blood parasites broadly related to the malarias. Labbé (I 894) proposed
to divide the haemogregarines known at that stage into three distinct groups, on the
grounds of the relative proportions of the parasite to the host blood cell. In Drepanidium
Labbé, 1894, the parasite was no more than three fourths the host cell in length. In
Karyolysus Labbé, 1894, the parasite did not exceed the host erythrocyte in length and
exercised a destructive influence on the cell nucleus. For Danilewskya Labbé, 1894, the
parasite exceeded the host cell and doubled up in it. Sambon & Seligmann (I907) later
noted that except for the substitution of the name LankesterelIa Labbé, 1899 for
Drepanidium and Haemogregarina Danilewsky, 1885 for Danilewskya, Labbé's
classification was followed by the great majority of authors at that time.
During the first half of the last century, work on blood parasites of reptiles tended to be
sporadic. Some enigmatic new genera were reported, including Toddia Franca, 1911,
Pirhemocyton Chatton & Blanc, 1914, Cingula Awerinzew, 1914, TunetelIa Brumpt &
Lavier, 1935, Sauroplasma Du Toit, 1937, Serpentoplasma Pienaar, 1954 and
Sauromella Pienaar, 1954 (see Davies & Johnston, 2000) as well as coccidian genera
such as Schellackia Reichenow, 1919. In the 1960s, Mackerras (1961) reported on the
haematozoa of Australian reptiles and Stehbens & Johnston (1966) proved by TEM that
Pirhemocyton from the same region is a viral infection, now known from the erythrocytes
of Iizards, turtles and snakes. The 1970s revealed evidence that Toddia, like
Pirhemocyton, is a viral infection in the blood of snakes (De Sousa & Weigl 1976).
Furthermore, from the 1970s to the 1990s the work of authors such as Ayala (1977),
Telford (1972, 1973, 1988, 1989, 1993), Lainson & Paperna (1996) and Schall (1990,
1996) on lizard malarias (mainly Garnia Lainson, Landau & Shaw, 1971, Haemoproteus
Kruse, 1890 and Plasmodium Marchiafava & Celli, 1885) was undertaken (see Ayala
1977; Schall, 1996). This period also saw efforts to understand the effects of reptilian
blood parasites on host behavior (see Schall, 1996). The 1990s onwards have also
7
CHAPTER 1 INTRODUCTION
produced descriptions of reptilian blood parasites, including new genera such as
Hemolivia Petit, Landau, Baccam & Lainson, 1990 and Billbraya Paperna & Landau,
1990 and new species of established genera such as Schellackia, Plasmodium and
Haemoproteus (see Paperna & Finkelman, 1996, Lainson & Paperna, 1996 and Paperna
& Landau, 1991). Also during this period, except for those in chelonians, the majority of
reptilian haemogregarines has been transferred to the genus Hepatozoon Miller, 1908 by
Smith (1996). Finally, some of the most complex life cycles of members of this genus
have been elucidated, particularly by Desser (1993) and his eo-workers (see Desser &
Bennett, 1993, Smith, Desser & Martin, 1994, Smith & Desser, 1997a, 1997b) and by
Lainson, Paperna & Naiff (2003).
Literature concermng the blood parasites of reptiles in Africa appears scanty in
comparison with that noted above. Sambon & Seligmann (1907), Fantham (1925) and
Pienaar (1962) have probably made the most significant contributions to knowledge of
these blood parasites in South Africa, although in the last decade there has been some
research done on the blood parasites of reptiles in this region (see Paperna & De Matos,
1993a). Pienaar (1962) described several new species of parasites in South African
reptiles, including a trypanosome (Trypanosoma mocambicum Pienaar, 1962) in the
blood of a Mozambican terrapin, Pelosios sinuatus sinuatus, a species of lizard malaria
(Plasmodium zonurae Pienaar, 1962), a piroplasmid (Sauroplasma zonurum Pienaar,
1962) and infections of a viral nature (Pirhemocyton zonurae Pienaar, 1962) in the
girdled lizard, Cordylus vittifer Reichenow, 1887. A new haemogregarine
(Haemogregarina pelusiensi Pienaar, 1962) from the terrapin, Pelosios sinuatus sinuatus
and another suspected piroplasm (Serpentoplasma najae Pienaar, 1962) were also
recorded from the blood of a black-necked cobra, Naja nigricollis Bogert, 1940 by
Pienaar (1962). Paperna & de Matos (1993a) reported new hosts and geographical
locations of erythrocytic viral infections, of which some records were from South Africa.
However, in the Free State Province, work on blood parasites of reptiles or any other
vertebrate group appears very limited.
8
CHAPTERl INTRODUCTION
1.5 Problems of Taxonomy
Several of the reptilian blood infections in this study are known or suspected to be of
viral origin, although some of these are currently classified with the so-called piroplasms
of the protozoan Phylum Apicomplexa Levine, 1970. Other infections in the study result
from the presence of haemogregarines and the malarias, both groups, like the piroplasms,
belonging to the Apicomplexa. Also observed are nematode stages (microfilariae), which
will be considered last in this section and which are considered only briefly in this study.
1.6 Reptilian viral and viral-like infections
Classification of viral and viral-like infections in the blood of reptiles is probably unwise,
given the current uncertainties concerning their identity, nomenclature and classification
(see Davies & Johnston 2000). Some infections are thought to result from icosahedral
viruses (e.g. Pirhemocyton) related to the iridoviruses, others may be herpesviruses, and
yet more may be oncornaviruses (see Davies & Johnston 2000). Viral-like infections in
reptiles probably include Sauromella, an infection of uncertain status (see Johnston
1975), and Sauroplasma, recently classified with the Protozoa as a piroplasm (see Section
1.7 below). Serpentoplasma may be a similar infection.
Chatton & Blanc (1914) noted an organism resembling a piroplasm within the red blood
cells of the North African gecko (Tarentola mauritanica), which they named
Pirhemocyton tarentolae Chatton & Blanc, 1914. Later, Brumpt (1936) defined the
genus Pirhemocyton Chatton & Blanc, 1914 as "nonpigmented endoglobular parasites of
saurian red cells with diffuse chromatin or central chromatin dot, giving rise, in infected
blood, to albuminoid inclusions in the red corpuscles. Multiplication and replication
unknown". It was Stehbens & Johnston (1966) who discovered the viral nature of
Pirhemocyton by examining its ultrastructure and a recent study of this infection also
confirmed its viral nature (Paperna & de Matos 1993b). Johnston (1975) listed 35 hosts
for Pirhemocyton. All these viruses comprise icosahedral, intracytoplasmic, iridovirus-
like particles (see Paperna & de Matos 1993b).
9
CHAPTER 1 INTRODUCTION
According to Pienaar's (1962) post-mortem observations there is a chance that severe
infections such as these in reptiles may run a fatal course. Heavy invasions of the
erythrocytes with Pirhemocyton invariably lead to extensive ani so- and poikilocystosis,
gross cellular distortion and cytolysis leading to severe anemia. Cellular deformation and
disruption is effected primarily through the association of the parasite with the "curious"
albuminoid bodies that appear in the cytoplasm of the host cells. In this condition,
according to Pienaar (1962), there is nuclear displacement.
Sauromella haemolysus Pienaar, 1954 is the only parasite of its type reported from the
blood of lizards. The parasite was originally noted in a South African lizard
iPachydactylus capensis (A. Smith, 1845)), which is also found in the Free State.
According to Pienaar (1962) this endoglobular parasite was of a doubtful nature and
could possibly be of the Anaplasma type. He described forms as minute, dark, spherical
or rod-like bodies. These bodies could occur singly or in groups, and could be associated
with a Pirhemocyton-type infection since the red cell stroma de-haemoglobinized.
According to Pienaar (1962) multiplication was apparently effected through binary or
multiple fission, and the infection appeared to be of an acutely pathological nature, as it
not only destroyed the haemoglobin pigment of the host cell, but may have also caused
heavy anemia and hyperactive erythropoitic activity.
J. 7Reptilian Protozoa or so-called Protozoa
For the purposes of the study, the classification system of Lee et al. (2000) is employed
for the Protozoa. There have been several attempts to re-define the classification of the
Protozoa in recent years. Notable examples have been firstly by Levine et al. (1980),
then Corliss (1994), who designed a "user friendly", six-kingdom classification of life
and then Cavalier-Smith (1998), who elevated the Protozoa to kingdom status. However,
Patterson (2000) notes in the Society of Protozoologists' publication that Protozoa form
an artificial group of eukaryotes, rather than a natural one. Patterson (2000) also
concludes that although the "bricks (groups with distinctive ultrastructural identities)"
and the "cement (phylogenetic systematics)" for a "systematic edifice" exist, "the plans"
are lacking. Such plans, he believes, will probably come from a "molecular
10
CHAPTER! INTRODUCTION
understanding of evolutionary relationships among taxa". As a result of the current
uncertainties, Lee et al. (2000) divides the Protozoa into "Key Major Groups", many
corresponding to phyla, others to orders. The phylum Apicomplexa, is one such group.
PHYLUM APICOMPLEXA LEVINE, 1970
The phylum Apicomplexa comprises unicellular endosymbionts, characterised by having
an apical complex, composed of one or more polar rings, a number of rhoptries and
micronemes, a conoid and sub-pellicular microtubules. The phylum has the following
three classes: Perkinsasida Levine, 1987, Conoidasida Levine, ]988 and Aconoidasida
Melhorn, Peters & Haberkorn, 1980.
In the Society of Protozoologists' system (Lee et al. 2000), the haemogregarines found in
the current study may fall within the class Conoidasida, order Eucoccidiorida Léger &
Duboscq, 1910, suborder Adeleorina Léger, 1911, or in the suborder Eimeriorina, Léger
1911 of the same order (Eucoccidiorida). The malarias are all classified within the class
Aconoidasida, order Haemospororida Danilewsky, 1885 and the so-called piroplasms
within the same class (Aconoidasida), but the order Piroplasmorida Wenyon, 1926.
Details of this classification are given below.
CLASS CONOIDASIDA LEVINE, 1988
Such organisms have organelles of the apical complex, and generally both sexual and
asexual reproduction occur, followed by sporogony. Sporogony results in oocysts with
infective sporozoites. Cellular motility exists, but flagella are found only on the
mierogametes of some taxa. Pseudopods may exist for feeding. Homoxenous and
heteroxenous species are known.
Il
CHAPTER 1 INTRODUCTION
Order Eucoccidiorida Léger & Dubosq, 1910
Members of this order demonstrate merogony, gamogony and sporogony and these occur
in vertebrates and/or invertebrates.
Suborder Adeleorina Léger, 1911
Organisms within this suborder exhibit syzygy, with conjugation and subsequent
sporogony usually in an invertebrate definitive host. Complex life-cycles exist, involving
at least one cycle of merogony, followed by gametogony, syngamy and sporogony. Two
types of meronts may occur. The Adeleorina comprises seven families, four of which
contain genera of reptilian haemogregarines.
Family Hepatozoidae Wenyon, 1926
This family contains only the genus Hepatozoon Miller, 1908. Members of the genus
demonstrate such diversity that the genus may be paraphyletic (see Barta, 2000).
The genus Hepatozoon Miller, 1908
Type species Hepatozoon muris (Balfour, 1905) Wenyon, 1926 in Rattus norvegicus
The majority of species within the genus have been reported on the appearance of their
gamonts in the erythrocytes and/or leucocytes of vertebrate hosts, including reptiles.
Merogony does not usually occur within erythrocytes, but in vascular endothelial cells.
Latent monozoic and dizoic cysts can also exist in vertebrate tissues. In invertebrate hosts
such as mites, ticks, insects and possibly leeches, mierogametes may be flagellated, but
no sporokinetes are formed. Normally in the haemoecel of these same invertebrates, large
polycystic oocysts are produced with sporocysts containing four to 16 or more
sporozoites. Transmission occurs when the vertebrate host ingests the infected
invertebrate, or through predation on another vertebrate containing tissue cysts.
12
CHAPTER 1 INTRODUCTION
More than 121 species of the genus Hepatozoon have been described worldwide (Levine
1988, Smith 1996). The range of blood sucking invertebrates that these parasite utilise
include ixodid and argasid ticks, mites, assassin bugs (Hemiptera: Reduviidae); Diptera
(sandflies, mosquitoes, tsetse flies), Anopleura (sucking lice), Siphonaptera (Fleas) and
the Hirudinea (leeches) (Smith, 1996).
Family Haemogregarinidae (Neveu-Lemaire) Léger, 1911
The numerous species compnsmg this family, particularly those of the genus
Haemogregarina Danilewsky, 1885 have been described as a "taxonomic mess" and the
genus itself, a "taxonomic repository of poorly described forms" (Barta, 2000). In fact,
Mohammed and Mansour (1959) recommended the qualifier "senso lato ,. to include
species whose life cycles have not yet been described or studied and "senso stricto '.' for
those with a known life history. Representatives of the family Haemogregarinidae
comprise three genera, but only one of these, Haemogregarina, is known from reptiles.
The genus Haemogregarina Danilewsky, 1885
Type species: Haemogregarina stepanowi Reichenow, 1885 in Emys orbicularis
More than 300 Haemogregarina species have been described in many groups of
vertebrates (Desser, 1993). Siddall (1995) listed 19 chelonian species infected with
representatives of the genus Haemogregarina (senso stricto). A further 10 chelonian
species were added to this list by Smith (1996). Siddall (1995) also recommended that all
the remaining species that parasitise fish, turtles, snakes, crocodilians, lizards, and birds
that he could not place in the genera Haemogregarina (sensu lato), Cyrilia Lainson, 1981
and Desseria Siddall, 1995 be transferred to the genus Hepatozoon. Smith (1996)
completed this task. Haemogregarina are adeleid coccidia with heteroxenous life cycles.
Generally, the gamont stages that are a product of merogony occur in the erythrocytes of
the vertebrate host and according to Davies & Johnston (2000), the sporozoites, a product
of sporogony, occur in haematophagous invertebrates. Desser (1993) noted that the
13
CHAPTER 1 INTRODUCTION
characteristics of Haemogregarina species are that they have small oocysts with eight
sporozoites, formed from a single germinal centre.
Members of this genus (Haemogregarina) occur therefore in vertebrate hosts such as
chelonians, fishes and possibly other ectotherms. In their vertebrate hosts, vermicular
meronts exist in blood cells and fixed tissue cells, with gamonts mainly in erythrocytes.
In their invertebrate hosts, such as leeches, sporogony occurs in the intestinal epithelium
and oocysts produce eight naked sporozoites. Post-sporogonic merogony also occurs in
the invertebrate host and transmission is by bite, when merozoites are transferred to the
vertebrate host, or perhaps when the invertebrate is ingested.
Family Karyolysidae Wenyon (1926)
Karyolysids may represent a sister taxon to piroplasms of veterinary importance (see
Barta, 2000). They have definitive hosts in common (arachnids) and both initiate
merogony in these invertebrate hosts, or in their progeny. Their vertebrate hosts are
amphibians and reptiles. The family contains two genera, both of which parasitise
reptiles.
The genus Karyolysus Labbé, 1894
Type species: Karyolysus lacertae (Danilewsky, 1886) Reichenow, 1913 in Lacerta
muralis
Members of this genus probably infect only lacertid lizards. Merogony occurs in the
endothelial cells of lizards and gamonts primarily infect erythrocytes. Syzygy and
sporogony occur in the gut of female mites, forming motile sporokinetes (sporozoites,
according to Barta, 2000). These enter mite eggs to form sporocysts with 20-30
sporozoites each (merozoites, according to Barta, 2000). Transmission occurs when the
vertebrate host eats an infected mite of the next generation (mite nymph).
14
CHAPTER 1 INTRODUCTION
The genus Hemolivia Petit, Landau, Baccam & Lainson, 1990
Type species: Hemolivia stellata Petit, Landau, Baccam & Lainson, 1990 in Bufo
marinus and Amblyomma rotondatum
This genus has been reported from a toad from Brazil, a lizard from Australia and an
African tortoise (see Davies & Johnston, 2000), with ticks as invertebrate hosts. In the
vertebrate hosts merogony and cyst formation occur.in endothelial cells and erythrocytes,
while gamonts occur in erythrocytes. In ticks, sporogony exists in cells of the intestine
and a typically star-shaped oocyst is produced. Numerous sporokinetes (possibly
sporozoites) from the oocyst invade the intestinal cells, form sporocysts and then
sporozoites (possibly merozoites). Transmission occurs when the vertebrate host ingests
an invertebrate containing sporocysts/sporozoites and by predation of another vertebrate
with tissue cysts.
Family Dactylosmatidae Jakowska & Nigrelli, 1955
This family comprises two genera, one of which may occur in reptiles. Dactylosomatids
are heteroxenous blood parasites of eetothermie vertebrates that appear to use leeches as
definitive hosts (Barta 1991).
The genus Dactylosoma Labbé, 1894
Type species: Dactylosoma ranarum (Lankester, 1882) Wenyon, 1926 in Rana esculenta
In vertebrate hosts that include fishes, newts, anurans and possibly lizards, merogony and
gamogony occur in the erythrocytes of the peripheral blood. Primary merogony yields
six to 16 merozoites by budding to form a "hand-like" structure. Secondary merogony
may produce six individuals that form the gamonts. In the leech, budding produces 30 or
more sporozoites within cells of the intestinal lining, but transmission has not been
demonstrated.
15
CHAPTER 1 INTRODUCTION
Suborder Eimeriorina Léger, 1911
This suborder contains numerous genera and species, many of uncertain taxonomic
status. Species develop in both vertebrates and invertebrates, some alternating between
them. Syzygy does not occur and microgamonts produce many microgametes.
Sporozoites develop within oocysts (or membranes corresponding to the oocyst wall) and
sporocysts may be present. Zygotes are often motile. Upton (2000) divided the
Eimeriorina into nine families, one of which (Lankesterellidae Noller, 1920) has
members occurring in the blood of reptiles.
Family Lankesterellidae Nëller, 1920
This family shares some characteristics with the representatives of the family
Haemogregarinidae (Neveu-Lemaire) Léger, 1911, having stages in blood cells, and
members with heteroxenous life cycles. The representatives of the family
Lankesterellidae can be distinguished from those of the family Haemogregarinidae in
having sexual and replicative stages in the tissues (gut, connective tissue and/or viscera)
of the vertebrate host. In this family therefore, merogony, gamogony and sporogony
occur in the gut, connective tissue and viscera of the vertebrate host. According to
Desser (1993), the oocysts are asporoblastic and thus a variable number (eight
commonly) of sporozoites are produced, and these enter the red and white blood cells of
the host. The dispersive agents of these parasites are haematophagous invertebrates that
ingest the sporozoites, but development of these is minimal (probably maturation only) in
the intermediate hosts. Few life cycles have been described, and more confusion arises
because the descriptions of lankesterellid sporozoites and haemogregarinid gamonts
resemble each other. Twenty species have been described within the family
Lankesterellidae, many of which that could just as well be members of the
Haemogregarinidae, because relatively little attention was given to the multinucleate
stages.
Representatives of the Lankesterellidae consist of two genera (LankesterelIa Labbé, 1899
and Schellackia Reichenow, 1919), according to Upton (2000). Lankesterella spp. are
suspected to parasitise reptiles, whereas Schellackia spp. are known to do so. The genus
16
CHAPTER 1 INTRODUCTION
Lainsonia Landau, 1973 has also been recognised as a member of the same family
(Lankesterellidae) (see Desser, 1993; Davies & Johnston, 2000), but this genus is not
recorded by Upton (2000), who presumably, like Levine (1988), regarded it as
synonymous with the genus Schellackia.
The genus LankesterelIa Labbé, 1899
Type species: LankesterelIa minima (Chaussat, 1850) Noller, 1920 in Rana esculenta
Members of this genus undergo merogony, gamogany and sporogony in cells of the
reticuloendothelial system and have oocysts with 32 or more sporozoites. Sporozoites
exist in blood cells, but dormant sporozoites may also occur in the vertebrate tissues. In
invertebrates such as mites, mosquitoes or leeches, sporozoites undergo little or no
development. Transmission occurs when the invertebrate is ingested, or by predation
between vertebrates.
The gen us Schellackia Reichenow, 1919
Type species: Schellackia bolivari Reichenow, 1919 in Acanthodactylus vulgaris
Species within this genus undergo merogony, gamogony and sporogony in the intestinal
epithelium or lamina propria, with possible development in the spleen and liver. The
oocyst yields eight sporozoites that enter erythrocytes and Iymphocytes, but dormant
sporozoites can occur in tissues. In invertebrates, such as mites and some Diptera,
sporozoites exist without development. Transmission occurs on ingestion of an infected
invertebrate or by predation between vertebrates.
CLASS ACONOIDASIDA MEHLHORN, PETERS & HABERKORN, 1980
Members of this class are without a conoid, except for the ookinete of some species of
Haemospororida Danilewsky, 1885. There are two orders found in reptiles, the
Haemospororida and the Piroplasmorida Wenyon, 1926.
17
CHAPTERl INTRODUCTION
Order Haemospororida Danilewsky, 1885
This order contains apicomplexans that do not demonstrate syzygy. About eight
flagellated mierogametes are produced and the zygote is motile (ookinete). Sporozoites
are naked and the life cycle is heteroxenous. Blood-sucking insects usually transmit
these parasites. Merogony occurs in the vertebrate host and sporogony in the
invertebrate. Pigment (haemozoin) may be formed from host cell haemoglobin, with the
macro- and mierogametes that develop independently.
Family Plasmodiidae Mesnil, 1903
This family includes Plasmodium Marchiafava and Celli, 1885, Haemoproteus Kruse,
1890, Saurocytozoon Lainson and Shaw, 1969 and seven other genera causing malaria or
similar diseases in vertebrates. Only the first three (Plasmodium, Haemoproteus and
Saurocytozoony occur in reptiles. The genera (and subgenera) are differentiated by: the
morphology of the erythrocytic stages; development in the tissues of the vertebrate host
and the vector.
The genus Plasmodium Marchiafava and Celli, 1885
Type species: Plasmodium malariae (Feletti & Grassi, 1889) Int. Com. Zool. Nomen.,
1954 in Homo sapiens and other primates
Numerous species of this genus have been described in the blood of reptiles, birds and
mammals. The parasites exist as meronts in erythrocytes and other tissues, and
gametocytes in erythrocytes, which characteristically produce pigment. Invertebrate
hosts are mostly anopheline mosquitoes, midges and possibly mites. The oocyst stages of
Plasmodium exist in the stomach wall of the invertebrate, and sporozoites occur in the
salivary glands. The parasites are transmitted and distributed through the bite of the
invertebrate.
Peirce (2000) regards Plasmodium in reptiles as cornpnsmg about 90 species or
subspecies divided into subgenera including: Asiamoeba, Carinamoeba, Fallisia, Garnia,
Lacertamoeha, Ophidiella, Parasplasmodium, Sauramoeba and possibly Billhraya.
Presumably, Progarnia would be another.
18
CHAPTERl INTRODUCTION
The first known saurian Plasmodium species was described by Weynon (1909) from the
rainbow lizard Agama agama in Africa. According to Schall (1990), half of the known
malaria parasites are described from lizards, these comprising seventy-six of the 196
Plasmodium species. OfSchall's (1990) listed Plasmodium species that infect lizards, six
species are present in Africa, and one in South Africa. Lizard malaria has been found on
all the warm continents, except Europe. Schall (1990) stated that most of the well-known
families of lizards are infected with malaria, in temperate woodlands, tropical rain forests
and cool upland tropical habitats. Only a few distributions of such malaria populations
are known, and it is concluded that at least some parasite-host associations are ancient.
The genus Haemoproteus Kruse, 1890
Type species: Haemoproteus columbae Kruse, 1890 in Columba livia
According to Peirce (2000), Haemocystidium Castellani & Willey, 1904 is synonymous
with this genus. Within Haemoproteus, merogony occurs in the endothelial cells of blood
vessels and gamonts exist in erythrocytes. Pigment is formed and vectors are
hippoboscid flies, Culicoides spp. (Ceratopogonidae) or Chrysops spp. (Tabanidae).
The genus Saurocytozoon Lainson & Shaw, 1969
Type species: Saurocytozoon tupinambi Lainson & Shaw, 1969 In Tupinambus
nigopunctatus
Meronts occur in lymphocytes, gamonts in leucocytes and pigment IS not formed.
Oocysts are large and slow to develop, forming hundreds of slender sporozoites. Vectors
are presumed to be culicine mosquitoes.
Order Piroplasmorida Wenyon, 1926
This order contains generally pyriform, round, rod-shaped or amoeboid organisms found
in the erythrocytes of a variety of vertebrates. Oocysts, spores and pseudocysts are
absent, as are flagella. Subpellicular microtubuies may be present, and polar rings and
19
CHAPTER 1 INTRODUCTION
rhoptries occur. Asexual reproduction is present and sexual reproduction may well exist.
Merogony occurs in vertebrates and sporogony in invertebrates such as ticks. They are
therefore heteroxenous parasites. Peirce (2000) names four families with the order, one
of these, the Haemohormidiidae Levine, 1984, having a genus found in reptiles
iSauroplasma Du Toit, 1937).
Family Haemohormidiidae Levine, 1984
Members of this family undergo merogony and binary fission. The nucleus lacks an
endosome or nucleolus and fish, reptiles and birds are hosts. Vectors are unknown.
The genus Sauroplasma Du Toit, 1938
Type species: Sauroplasma thomasi Du Toit, 1938 in Cordylus giganteus A. Smith,
1844.
Binary fission or budding into daughter cells exists in reptiles. Vectors are unknown.
Peirce (2000) makes no mention of the very similar genus Selpentop/asma Pienaar, 1962
and Davies & Johnston (2000) were not convinced that Sauroplasma is of protistan
ongin.
These so-called piroplasms were discovered by Du Toit (1937) in girdled lizards
Cordylus giganteus in Africa. According to Du Toit the degree of infection varied and
the parasites were small in comparison with the host erythrocytes. The smaller forms
were anaplasmoid forms consisting of granules or small ring-shaped bodies. According
to Du Toit (1937), these bodies arose from an anaplasmoid body with the gradual
enlargement of the central vacuole. Multiplication subsequently took place by binary
fission or by a process of budding. In the first, the spherical bodies elongated, and the
"nuclear" material concentrated at the two opposite extremities. A constriction appeared
in the middle of the elongated parasite, this constriction tightening until two separate and
approximately equal daughter cells were formed. The second procedure, a budding
process, achieved the same result. According to Du Toit, (1937), this process was very
similar to that seen in many mammalian piroplasms.
20
CHAPTER! INTRODUCTION
Possibly, these parasites can be transmitted by the bite of ticks and mites. According to
Pienaar (1962) the prostigmatic mites tZonurobia circularis) that infect these giant
girdled lizards may transmit Sauroplasma infections. Pienaar (1962) described a new
piroplasm from a cordylid lizard Cordylus vittifer and named it Sauroplasma zonurum.
He also recorded a piroplasm from a black-necked cobra (Naja nigricollis) and named it
Serpentoplasma najae. He described it in a similar way to Du Toit, referring to these
parasites as sporozoans. Davies & Johnston (2000) suggested that these structures are
unlikely to be of a protistan origin, after examining a specimen Cordylus polyzonus A
Smith, 1838 from the Free State I collected in my undergraduate years.
1,8 Reptilian filaria I nematodes
These are classified below broadly according to Roberts and Janovy (2000).
Phylum Nematoda Potts, 1932
Nematodes are typically multicellular, bilaterally symmetrical, elongated cylindrical
animals and tapered at both ends. They possess a pseudocoele and a complete digestive
system with an anterior mouth and a posterior anus. The body is covered with non-
cellular cuticle and body wall muscles are all longitudinal. Most species are dicecious,
but some are hermaphroditic, others parthenogenetic. Most are oviparous, some
ovoviviparous.
Family Onchocercidae Leiper, 1911
Members of this family live in amphibian, reptilian, avian and mammalian tissues. All
species of filaroids have arthropods as intermediate hosts, most of which deposit third
stage larvae on the vertebrate host when they bite. These juveniles often migrate to areas
such as subcutaneous tissues, intermuscular connective tissue, the body cavity and lymph
nodes, where they develop into adult male and female worms. Adults mate and the
female releases microfilariae that migrate to the blood stream. These are ingested when
the vector bites. Members of about 14 genera of filarial nematodes can be found in
reptiles (Mad er, 1996).
21
CHAPTER! INTRODUCTION
1.9 The current study
The current work includes new records of blood parasites from reptiles collected during
surveys of sites in the Free State and Lesotho. Two hundred and four lizards, one
amphisbaenian and 59 snakes were screened for blood parasites. Pirhemocyton
infections are described from numerous specimens of Agama atra atra Daudin, 1802.
Sauroplasma thomasi Du Toit, 1937 and Sauromella haemolysus are redescribed, and
Sauroplasma thomasi infections from Cordylus giganteus were observed and analysed by
transmission electron microscopy (TEM). Eight new distribution records for
Sauroplasma are reported, involving five families of lizards. In addition, nine new
distribution records for Serpentoplasma are described across three families of snakes,
including preliminary ultrastructural studies by TEM of blood infections of a captive
African rock python (Python sebae natalensis (Gmelin, 1789)) and Serpentoplasma
infections of a puff adder (Bitis arietans (Merrem, 1820)). Previously unreported
haemogregarines are recorded from girdled lizards (Pseudocordylus melanotus melanotus
(A. Smith, 1838)) and Pseudocordylus melanotus subviridis (A. Smith, 1838)), Agamid
lizards (Agama atra atra Daudin, 1802) and a striped skaapsteker (Psammophylax
tritaeniatus (Gunther, 1868)). Five possible new infections of lizard malarias are
described from three families of lizards, and five species of unidentified microfilaria are
noted in the blood of Agamid, Gekkonid and Cordylid lizards.
22
CHAPTER2 MATERIALS ANDMETHODS
MATERIALS AND METHODS
All reptiles were collected in the Free State with permission from the Free State Province
Department of Tourism, Environment and Economic Affairs (Permit nr.
HKfPlI03894/002) (see appendix A). Reptiles collected in Lesotho were collected with
permission from the Ministry of Environment and Tourism, Lesotho. Reptiles were
captured at various localities in the Free State and Lesotho (Fig. 2.1 & 2 2), and then
released back into their habitats at the same collection sites. Table 2.1 provides details of
the reptiles collected in nine main areas.
2.1 Collections
Representatives of the Gekkonidae Cuvier, 1817, Agamidae Gray, 1827, Scincidae,
Cordylidae Gray, 1837, Lacertidae Bonaparte, 1831 and Varanidae Hardwieke & Gray,
1828 were collected by hand where possible. A noose was used to catch specimens
lodged inside rock cracks and a crowbar was employed to lift large rocks. Cordylus
giganteus were collected by inserting a 10cm nail into earth above burrows and a nylon
noose was attached to the nail. Specimens were trapped in the nooses as they left the
burrows. After blood samples had been taken, specimens were released back into the
same burrows.
All snakes were collected by hand and a 1.2m long (9mm diameter) aluminium rod was
used to pin venomous snakes to the ground. These snakes were then put head-first into
Perspex pipes ranging from 10mm to 60mm in diameter for further investigation.
2.2 Blood sampling, light microscopy and image capture
Reptile blood was sampled mainly in the field, but also in the laboratory. Lizard blood
was collected from toe clips or a tail end, using sharp scissors. Snakes were put in
Perspex pipes (as above) to make tail clips. In some cases a micropipette was used to
draw blood from the clip site. Blood samples were smeared on clean glass slides marked
with each reptile's identity, age and sex (if possible), as well as date and site of capture.
23
CHAPTER2 MATERIALS ANDMETHODS
Smears were then air-dried and stored in dust-free slide boxes for transport back to the
laboratory.
In the laboratory, these thin blood films were fixed in absolute methanol for at least 60
seconds. Fixed blood films were then stained wi th 10% Gurr's Giemsa Improved R66
stain solution (lOml in 90ml tap water) for up to one hour. Blood films were then
examined with a Zeiss Axiophot photo microscope using 63X and lOOX oil immersion
objectives. Slides were also examined at Kingston University (UK) with a Zeiss
Axiophot 20 microscope and a 100X oil immersion objective lens. Images from the
Axiophot 20 were captured by Nikon digital camera (DN 100), and stored on computer
discs as JPEG files. These images were then measured using an Eclipse Net (Nikon)
image analysis package calibrated to a stage micrometer.
2.3 Transmission electron microscopy
Two giant girdled lizards (COl-dy/us giganteus), one puff adder (Bitis arietans) and one
captive African rock python (Python sehae natalensis) (permit nr: HK/P19C/03894/002)
were sampled for electron microscopy. Four to five drops of fresh blood from each
reptile were dropped into 2.5% glutaraldehyde (lOml in 90ml 0.2M Sorensen's phosphate
buffer at pH 7.2). Glutaraldehyde-fixed material was then centrifuged at 10000rpm and
the pellet washed in 0.2M Sorensen s phosphate buffer and post-fixed with a 2% solution
of osmium tetroxide in 0.2M phosphate buffer. Post-osmication, the pellet was rinsed in
buffer and then dehydrated in a graded series (30 - 100%) of ethanol solutions. The final
dehydration in ethanol (100%) was dried over a 4Á molecular sieve. Ethanol was
removed by transfer of the sample through three changes of propylene oxide (1,2 epoxy
propane). The pellet was then left for 12 hours in a mixture of one volume propylene
oxide mixed with three volumes of Agar 100 resin. The epoxy resin mixture was made by
mixing the following components: 23 ml Agar 100 resin, 12 ml methyl nadic anhydride
and 15 ml dodecaenyl succinic anhydride, with dropwise addition of l.5 ml benzyl
dimethyl amine during mixing.
24
CHAPTER2 MATERIALS ANDMETHODS
Blood pellets were transferred from the propylene oxide/Agar 100 mixture into freshly
made Agar 100 resin mixture (2 x 24 hour changes), then transferred to fresh Agar 100
resin mixture in silicone rubber embedding moulds. The resin mixture was polymerised
at 60 degrees Celsius for 48 hours. Sections, showing pale gold interference colours were
cut from the blocks of embedded tissue, using glass knives on a Huxley Mark Il
ultramicrotome and collected on copper, 300 hexagonal-mesh grids. The sections on the
grids were stained for 20 minutes with a solution of 10% uranyl acetate in Analar grade
methanol, washed with Analar methanol and allowed to dry. They were then stained for
20 minutes with Reynold's lead citrate solution, washed with 0.02M sodium hydroxide
solution followed by distilled water, before examination with a JEOL JEM- 1010
transmission electron microscope operated at 80-100 kV. Digital images were captured
using a Soft lmaging Systems' Mega View III camera mounted in the microscope
column.
All chemicals used were obtained from Agar Scientific Ltd, 66A Cambridge Road,
Stansted, Essex, England with three exceptions. The Analar methanol and the 4Á
molecular sieve were supplied by BDH Laboratory Supplies, Poole, Dorset, England, and
the Analar ethanol was obtained from Hayman Ltd, East Ways Park, Witham, Essex,
England.
2.4 Cell infection data
Levels of infection (intensity) among blood cells (erythrocytes) were calculated by taking
10random fields on each slide with the 63X objective and 2 x converter and with a Zeiss
Axiophot 20 microscope and a 100X oil immersion objective lens. In each sample field
the number of cells ranged from four to 150 but on average ±50 blood cells were counted
of which some were infected. This was repeated 10 times in different areas of the sIideo
Filarial nematodes were counted in a field using the 10x objective, with an average of
500 host cells counted in the same field.
25
CHAPTER2 MATERIALS AND METHODS
2.5 Digital imaging
Pictures of hosts were taken from field guides of Branch (1998) and Patterson (1987) by
aid of a Nikon Coolpix 990 digital camera. Images were digitally reduced of noise,
resampled, cropped, sharpened and flipped in Corel draw™ 10. These selected images
only serve as illustrations for the thesis and are not intended for publication purposes.
2.6 Miscellaneous
Names of host species collected from South Africa are provided with author names.
Those host localities from other than South African are without authors. The author name
of Scincidae was not found.
26
KALAHARI
BOTSWANA
NAMIBIA
Kalaha,.i
G~msbOH N.P.
<,
Calvinia0 SOUTH AFRICA
Middleburg· Great . 0Umt
cradock~iSh~
Atlantic OREAZT~~~aff_~Lineth.lice. ,Bisho
Ocean Saldanha \ ''kiflg w~m' TOW~ ®E8st London
• Worcest~rLl LE KAROO, '
Paarl0 . _.-tIitenha'"g'e0 00 -ahamstown
CAPE TOVVN Swellenda. 0George ® Sunda_J s In
~tellenbosch ~ossel Bay Port Elizabeth
'-- Cape of
SOkm
Fig.2.1 Map of South Africa, Free State and Lesotho showing collection localities. Collections
on farms and other sites are shown in marked areas. A: Jagersfontein district (The farm
Zuurfontein), B: Bloemfontein and Brandfort districts (The farms Deelhoek and Hopefield), C:
Koppies district (The farm Koffielaagte), D: Clarens district, E: Reitz district, F: Harrismith
district G: Lesotho. Map taken and digitally adapted from Microsoft Encarta™ Scale: 50 Km.
Fig.2.2 Map ofLesotho where collections were made showing the three Catchment areas
(highlighted areas) with the nearby villages indicated in red. Scale 50 km and 5 km
respectively. Map taken and digitally adapted from Microsoft Encarta™
CHAPTER2 MATERIALS AND METHODS
Table 2.1 Main collection sites where specimens were collected. The total number of specimens collected
is indicated in column Number Captured. In South Africa eight different districts are presented where
collections were made. Collected specimens from various parts in Lesotho and endemic species to these
regions are given. The presence of collected specimens in a particular district site is indicated with X
-
Z W
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Zi..c:l U N ~":I: c:l~
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L .capensis
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~A.=gra='cib=·call=:=da=~I~~DDDDDDD~[CJ
:=M.=cape=nsis=~Ic~D=DJ~~DDDDDDDM. striatapunctatissima DDDDDDDDD
~M"~dCO,o==~I~DD~DDDDDDD
Cordylidae Gra1y8,37
:==:::=Cg=ig~aIn~=/DeIuDD.\=DD. DDDDDDDDD
C. polyzonus
~Pl=II.e=;=1'/{=O:I=7l=eo{aw=I=nsOEI=J
D
usL
DJDDDL~JDD[CJ~I~D~DDD~DDDD[DCJLJDDLJ
~p.=111e==lonO=lu.I=='sub=Vir=idis~I~DDDDD
:J~:;rBtoniadparate,e DDDDDDDDDDDD~[DCJD
:=iV=':1i/7(er=/eXlo=~Ic=JDDDDD~DDD[CJ
Vraa1ra8HnIc ~y,VO2
arid8wdieakee&{b~~DUDW1D=D'iDSD~lDuDDDDLJDDDDDDDDD
29
CHAPTER2 MATERIALS AND METHODS
Table 2.1 (continued). Main collection sites where specimens were collected. The total number of
specimens collected is indicated in column Number Captured. In South Africa eight different districts are
presented where collections were made. Collected specimens from various parts in Lesotho and endemic
species to these regions are given. The presence of collected specimens in a particular district site is
indicated with X
"c...".~-e
~..l
~
'o"'
::.::
S1ER7PE5N8TES Linnaeus,
:=:=C=G'u=lur1a=8y=:=:b:,:r;0i:=2d5a0=c=::=0::=:::I0:D' D0DD0DD0DD0DD0D00
.'I. capensis
~Cho~/all1IbDoD=eDiaD=D==D=D=D:ID~DDDDDDDDDDDD
D. seobra
~L.all=roraI=c==~JDIDDDDDDDDDDDDDDDDDDDD
L. capense
I:===:::L.fIl=:===/ig=IinD=OSD=·I;HD1cD=DJDDDDDDDDDDDDDDDD
L. gilt/a/lis
~p.c=ana==~I~I~DDDDDDDDDDDDDDDDDD[]D
I Pi notostictus I~DDD~DDDDDD
I~IP /=ri/aen=ia/lls=~I~c:JDDc:JDDDDDD
IElapidacBuic,1ID82D7DDDDDDDDD
F=::=E. =h.l'IInad:==e=:=mevall:=a==iichme=a:==d/iaus=====:I~DDD~DDDDDDH.
~Vi=PCri~daC~luGnD=IYD,1~D825D~IDcD:DJDDDDDDDDDD~Du
B. arietans
~c.rh=ombea=/lls~I[]DI~D~DDDDDDDDDDDDDDDDD
30
CHAPTERJ RESULTS
RESULTS
A list of the reptiles collected in the Free State and Lesotho for this study is provided
below. The terminologies of De Waal (1978) and Branch (1998) are used in the
descriptions of these reptiles. For each species the following information is provided
synonyms, range and distribution patterns, characteristics, general biology and breeding,
and a short note on the haematological findings for the present study. Most parasitic
infections are new host records from the Free State, except for the viral infection known
as Pirhemocyton, found earlier in the blood of the skink Mabuya capensis (Gray, 1830)
and the agama Agama atra by Paperna & De Matos (1993a). Sauroplasma thomasii from
Cordylus giganteus and Sauromella haemolysus from Pachydactylus capensis have also
been reported previously from the Free State.
3.1 HOSTS COLLECTED
SAURIA: GECKONIDAE
Synonyms: Hemidactylus capensis (A. Smith, 1849)
Lygodactylus strigatus Gray, 1864
Range: Mpumalanga, KwaZulu-Natal, Northern Cape, northwestern Free State, Zaire,
Angola and Botswana (De Waal, 1978).
Characteristics: Nostril bordered by two nasals, anterior nasals separated by one
granule; mental with deep lateral clefts; post-mentals two to three; supra-labials seven to
nine, infra-labials six to seven, males pre-anal pores five; original tail with six or seven
scales above.
31
CHAPTER3 RESULTS
Biology: These geckos have been translocated to various regions such as Bloemfontein,
Port Elizabeth and Grahamstown (Branch 1998). They prefer to forage in low shrubs, but
can also tolerate urban areas. They live for 15-18 months and sexual maturity is reached
in eight months. Hard-shelled eggs are laid in cracks or under loose bark.
Haematological observations: A single specimen was caught in the urban area of
Bloemfontein (Fig. 2.1) and was infested with mites. The peripheral blood showed a
high infection of a hitherto undescribed haemogregarine. It also had a suspected viral
infection (possibly Pirhemocyton).
Synonyms: Tarentola capensis A. Smith, 1845
Pachydactylus elegans Gray, 1845
Pachydactylus leopardinus Sternfeld, 1911
Range: Throughout Plateau areas of Southern Africa (FitzSimons 1943; Loveridge
1947).
Characteristics: Nostrils bordered by two or three nasals, anterior nasals in contact or
separated by one granule; supra-labials six to eight, seldom five or nine; infra-Iabials five
to seven; five transversely enlarged adhesive lamellae under fourth toe; two or three
underdeveloped tubercles on either side of base of tail; original tail verticillate, four scale
rows per vertical above, with second, third or last row enlarged, into keeled tubercles.
Biology: Commonly found under stones, logs, old termitaria and occasionally in cracks
of houses. Nocturnal and prey on insects. Clutches of two eggs are laid in old terrnitaria;
incubation time is 90-110 days.
32
CHAPTER3 RESULTS
Haematological observations: Four specimens were collected and examined from
Bloemfontein and Jagersfontein districts; all four were infested with mites. The blood
smears showed a heavy infection of so-called Sauromella haemolysus.
Synonyms: Tarentola bibronii A. Smith, 1845
Homodactylus tuneri Gray, 1864
Pachydactylus bibronii var. stellatus Werner, 1910
Pachydactylus bibronii pulitzerae Schimdt, 1933
Range: Restricted populations in the Cape Provinces, just extending into adjacent Free
State and Namibia (Branch 1998). Mpumalanga and KwaZulu-Natal, Botswana, Malawi,
Zambia, Angola to Tanzania (Fitzsimons 1943; Loveridge 1947).
Characteristics: Large stout gecko with strongly keeled tubercles separated by granular
scales on back. Middle row of scales below toes and above scansors not enlarged (Branch
1998). Ten to 13 transversely enlarged adhesive lamellae under fourth toe. Original tail
verticillate, four to six rows per scale above, one row consisting of large keeled stellate
tubercles; sub-caudals in periods of two, sometimes divided anteriorly.
Biology: These geckos are found in rock outcrops. Many of them are translocated to
urban areas and can sometimes be found in and around houses. They are gregarious and
can often be found in dense colonies. They feed on a variety of insects, but smaller
geckos can also be taken. Two eggs are laid under bark or in a rock crack.
Haematological observations: Two of these geckos were collected on the farm
Zuurfontein (Fig. 2.1) and both specimens had a malaria infection. Both of these geckos
also had a suspected viral infection.
33
CHAPTER3 RESULTS
SAURIA: AGAMIDAE
Synonyms: Agama mieropholis Matschie, 1890
Agama micropterolepis Boulenger, 1896
Agama holubi Bocage, 1896
Agama atra var. rudis Boulenger & Power, 1921
Range: Throughout the Cape Province, absent from sandy areas, South Namibia and east
to the escarpment KwaZulu-Natal and Maputuland (Branch 1998). Southern Namibia
and southeastern corner of Botswana (De Waal 1978). No specimens recorded in Kruger
National Park (Pienaar 1966).
Characteristics: Mid body scale rows 120-150, seldom as low as 109 and high as 170.
Supra-labials 10-15, mostly 13-14; pre-anal pores in males 10-16; fourth toe longer than
third. Lamellae under fourth toe 16-20, seldom 15 or 22. White vertebral streak running
from nape to base of tail; throat, chest and ventral parts of upper arm greenish blue;
lateral side of body rust-red; tail often yellow with dark cross bands.
Biology: Agamas live in rocky outcrops and in mountain ranges. They are colonial and
can form dense colonies, according to Branch (1998) up to 165 specimens per hectare can
be found. Male territories are approximately 90m. Their diet is mostly insectivorous, but
plant matter can also be taken. Females dig a shallow hole in damp soil and lay eggs
which take 2-3 months to hatch.
Haematological observations: Sixty-five of these lizards were collected from various
parts of the Free State and Lesotho, but the greatest number of these was from a farm
Zuurfontein near Jagersfontein (Fig 2.1). Most lizards (precise numbers not recorded)
were infested with mites and some had engorged ticks behind their legs or in the neck
folds. The blood infections of these lizards showed a great diversity of parasites.
34
CHAPTER3 RESULTS
Infections ranged from viral-like infections (Pirhemocyton), haemogregarines, suspected
malaria and filarial nematodes.
Synonyms: Lacerta hispida Linnaeus, 1758
Agama aculeata Merrem, 1820
Agama armata Peters, 1845
Agama irfralineata Peters, 1877
Agama hrachyura Boulenger, 1885
Agama distanti Boulenger, 1902
Range: Two varieties, Southeastern (Kalahari form) and the Eastern variety. The Eastern
variety occurs in South central and an isolated population in the Northwestern Free State,
(Branch 1998).
Characteristics: Medium-sized agama with broad head and rounded snout. Ear holes
small and tympanums cannot easily be seen. Scales overlap from head towards tail
(Branch 1998). According to de Waal (1978), mid body scale rows 84-112 supra-Iabials
10-14, pre-anal pores in males10-14; fourth toe shorter than third; fifth toe shorter than
equal to first; tail shorter than body and head in females; males dorso-ventrally dark;
females with large dark spots on either side of vertebral band.
Biology: These lizards dig a short tunnel at the base of a bush in sandy areas; their main
diet is ants and beetles. They are solitary and females lay seven to Il eggs in spring.
Haematological observations: A suspected viral infection (Pirhemocyton) was present
in the peripheral blood of one specimen caught on the farm Zuurfontein.
35
CHAPTER3 RESULTS
SAURJA: SCINCIDAE
Synonyms: Scineus trivittatus Cuvier, 1829
Tiliqua capensis Gray, 1838
Tiliqua ascensionis Gray, 1830
Euprepes merremi Dumeril & Bibron, 1839
Range: Zambia, Botswana, Zimbabwe (Broadley 1966), Namibia (Mertens 1955), rest
of South Africa except for arid areas of the Western Cape (FitzSimons 1943). According
to Branch (1998) there are relict populations in the Inyanga Mountains in Zimbabwe and
Luiwa Plain in Zambia.
Characteristics: In De Waal' s (1978) terminology, centre of nostril always posterior to
structure of rostal. Supra-nasals always in contact; pre-frontals usually in contact; supra-
labiais four; 32-38 scale rows around middle of body; lamellae under fourth toe 15-20;
light brown or grey-brown; three pale stripes on back extending to tail.
Biology: Found in numerous habitats, including around houses, termitaria and in open
fields. They dig in loose sand, favour fallen logs and rocky outcrops. The femaie gives
birth to five to 18 young, but in Pretoria and Port Elizabeth females have been known to
lay clutches of eggs.
Haematological observations: Three specimens were collected on the farm Zuurfontein.
Two of these had mite infestations and all three had Sauroplasma -like infections.
36
CHAPTER3 RESULTS
Synonym: Euprepes sulcata Peters, 1867
Range: From Southern Angola, south through Namibia, Northern half of the Cape and
the south Free State (FitzSimons 1943).
Characteristics: Centre of nostril posterior to suture of rostral/ first supra-Iabials; supra-
nasals always in contact (De Waal 1978). Coloration varies between sexes; males
completely jet-black on dorsal and ventral sides. Females and juveniles pale olive to olive
brown with six golden stripes on dorsal surface.
Biology: These are active skinks that live in rocky outcrops and feed on insects. They
shelter in rock cracks where they have three to five young. According to Branch (1998)
there are informal reports that these skinks also lay eggs.
Haematological observations: Six of these skinks were collected on the farm
Zuurfontein near Jagersfontein. The peripheral blood showed an infection of
Sauroplasma-like inclusions in all six specimens studied.
Synonyms: Euprepes punctatissimus A. Smith, 1849
Euprepes sunderallii A. Smith, 1849
Euprepes grutzneri Peters, 1869
Range: Eastern temperate highveld regions of South Africa to southeastern Botswana.
Relict populations exist on the Eastern Highlands of Zimbabwe (Broadley 1966).
37
CHAPTERJ RESULTS
Characteristics: Centre of nostril posterior to suture of rostral or super-labial; supra-
nasals in contact; pre-frontals usually separated; lamellae under fourth toe 16-21; yellow
dorso-Iateral stripe; scales between these stripes are pale spots.
Biology: They feed on invertebrates and are active climbers of rocks and the habitats are
varied. According to Branch (1998) the southern populations give birth to three to nine
young.
Haematological observations: Infections resembling Sauroplasma, were present in the
red blood cells of one specimen collected in Bloemfontein suburbs.
Range: North East Cape, Free State, Southern Mpumalanga and Northern Cape Province
(Broadley 1966). Two isolated populations in Little Namaqualand, Eastern Cape and Free
State (Branch 1998)
Characteristics: According to De Waal (1978), three sub-oculars; second supra-Iabials
usually entering eye, five supra-Iabials, scale rows 18; sub-caudals 30 to 40. According to
Branch (1998), lower eyelids opaque, coloration pale golden olive to olive brown.
Biology: These skinks show a preference for compact, moist soils. Females give birth to
two young in February (Branch 1998).
Hematological observations: Infections resembling Sauroplasma, were present in the
peripheral blood erythrocytes of one specimen captured in Bloemfontein and one
captured in Lesotho.
38
CHAPTER3 RESULTS
SAURIA: CORDYLIDAE
Synonym: Zonurus derhianus Gray, 1845
Range: Northeastern half of the Free State and adjacent areas of southern Gauteng
(Loveridge 1944). Scattered populations in southeast Mpumalanga and extreme western
KwaZulu- Natal (Branch 1998).
Characteristics: Nasals separated by rostral; four large occipital spines; supra-Iabials
five to seven; males with nine to 13 pores and a patch of differentiated femoral scales;
females with 10-13 femoral pores (De Waal 1978). Tails have very large spines. Lateral
sides are golden yellow.
Biology: These vulnerable lizards live in colonies in burrows they dig in soil. These are
usually about 17 meters apart, 50 centimeters under ground and 2 meters long. They are
long lived, up to 20 years and females give birth to one or two young every two-three
years.
Haematological observations: Thirty-one of these lizards were collected on the farm
Koffielaagte near Kroonstad, in the Koppies district (Fig. 2.1). The locality consists of
two disjunct populations, separated by cultivated lands. Light microscopy showed
Sauroplasma thomasi infections in all captured specimens, but TEM studies failed to
reveal the true nature of these infections and further examination is needed to establish
what these enigmatic structures represent.
39
CHAPTER3 RESULTS
Range: Southern half of Free State, Karoo and Western Cape, into Namibia (FitzSimons
1943).
Characteristics: Two nasals; supra-nasals in contact; occipitals four to eight, supra-
labials anterior to sub-ocular four, dorsal scales 39-45 transverse and 32-41 longitudinal
rows; femoral pores in males 10-15, lamellae under fourth toe 13-16, mostly 15 to 16,
dorsal color brown to black, ventral lighter brown below.
Biology: Found on low lying koppies in rocky outcrops. Insectivorous and two or three
large young are born in late summer.
Haematological observations: Forty SIX of these shy lizards were caught in
Bloemfontein, Jagersfontein and Brandfort districts (Fig. 2.1). Most of these were
collected on a farm at Zuurfontein and most of these lizards were infected with malaria.
This type of infection is not mentioned in existing literature for C. polyzonus polyzonus,
and therefore this represents a new host record for this blood infection. All of the
specimens examined also harbored a Sauroplasma-like infection. The specimens
collected in the Brandfort district had viral infections, haemogregarines, malarias and
microfilarial nematodes. These are also new host records.
Synonym: Cordylus (Pseudocordylus) melanotus (A. Smith, 1838)
Range: Eastern Free State (FitzSimons 1943), Gauteng to Mpumalanga escarpment to
northern KwaZulu Natal and North to Northeastern Free State (Branch 1998).
40
CHAPTER3 RESULTS
Characteristics: Pseudocordylus m. melano/us has a divided fronto-nasal and the
femoral pits in females are only shallow pits. Breeding males has one to 17 glandular
femoral scales, a broad brown band around neck and a diffuse grey patch on throat.
Biology: Found in large colonies with a single dominant male. They are ambush
predators and feed on small insects, as well as vegetable matter. One to six young are
born in late summer.
Haematological observations: A total of 12 lizards were collected, four from the
Clarens district and five in the Harrysmith district in Collins Pass (Fig. 2.1). All of these
lizards had heavy infections of Sauroplasma and some of them had malaria and
haemogregarines. None of these parasitic haematozoan infections have been recorded
from this host in previous surveys.
Range: Mount-aux-Sources through Lesotho and Transkei, with an isolated population in
the Amatola Mountains in Eastern Cape (Branch 1998).
Characteristics: According to Branch (1998) this smaller race does not exceed 118cm in
snout-vent length and has undivided fronto-nasal, with large lateral scales.
Biology: Found in large colonies with a single dominant male. They are ambush
predators and feed on small insects, as well as vegetable matter. One to six young are
born in late summer.
Haematological observations: Thirty-five lizards were collected during a survey done in
Lesotho. The infections ranged from viral-like (Pirhemocyton), Sauroplasma-like to three
types of haemogregarines, as well as malaria and filarial nematodes.
41
CHAPTER3 RESULTS
SAURlA: LACERTIDAE
Synonyms: Lacerta intertexta A. Smith, 1838
Nucras tesselata var. ocel/ata Boulenger, 1910
Range: Western Free State, Northern Cape, Botswana, Namibia, Northern Mpumalanga,
southern Mozambique and south eastern Zimbabwe (Broadley 1972).
Characteristics: Parietal foramen present, three nasals, three to six granules between
supra-ciliaries and sub-oculars (De Waal 1978). Eleven to fifteen pre-femoral pores on
each thigh. Belly cream white and lateral scales have distinct cream spots.
Biology: These lizards forage widely in open savanna and search for slow moving food
and even other lizards.
Haematological observations: One of these lizards was collected on Collins Pass in the
Harrismith district (Fig. 2.1), and had a Sauroplasma-like infection.
SAURIA: VARANIDAE
Synonyms: Tupinambis albigularus Daudin, 1802
Varanus gillii A. Smith, 1831
Monitor exanthematicus var. capensis Schlegel, 1844
Varanus exanthematicus ionidesi, Laurent, 1964
42
CHAPTER3 RESULTS
Range: Savannas of southern and eastern Africa (Broadley 1966). Absent from West
Cape (Branch, 1998)
Characteristics: Large stout lizard with sharp claws and stocky limbs. Head with a
bulbous snout. Tail longer than body, cylindrical at base and compressed laterally.
Dorsal dark brown, with five to six pale yellow markings on back (Branch 1998).
Biology: These lizards live in burrows or under fallen logs. They are scavengers,
insectivorous and will kill and eat any animal small enough to swallow (Branch 1998).
Haematological observations: One juvenile was sampled. It was found in a rocky
outcrop on the farm at Zuurfontein (Fig. 2.1). Two mosquitoes were observed taking a
blood meal on one of its eyelids. The blood smears revealed a Sauroplasma-Iike
infection in the erythrocytes, as well as haemogregarines.
SERPENTES: BOIDAE
Synonyms: Coluher speciosus Bonnaterre, 1789
Boa hieroglyphica Schneider, 1801
Python hivittatus Kuhl, 1820
Python natalensis A. Smith. 1833
Hortulia sehae Gray, 1840
Range: Restricted to north and north-eastern parts of Southern Africa, Eastern Cape,
Pondoland and Transkei (FitzSimons 1962).
Characteristics: Rostral as broad as deep; two elongate pits on either side; internasals
one and a half times as long as broad; supraocular broken into two shields; a pair of
frontals broken into smaller shields; eight to 12 scales around eye; 12 to 15 upper labials;
43
CHAPTER3 RESULTS
first two are deeply pitted; smooth scales 71 to 93 at midbody; ventrals 265 to 286; anal
plate divided or undivided; vestiges of hind legs are visible as spurs either side of vent
(FitzSimons 1962).
Biology: Common in well flooded valleys, plantations and bushveld. Seldom far from
permanent water where they may lie submerged for long periods on end. Non-venomous
with needle-sharp solid recurved teeth. Diurnal and feeds on mammals, can swallow
large prey. Female lays 30-50 (exceptionally up to 100 eggs) and incubates and protect
the eggs.
Haematological observations: One captive specimen In Bloemfontein had a
haemogregarine infection (Hepalozoon sebae (Laveran & Pettit, 1909) Smith, 1996) in its
red cells. It also had a Serpentoplasma-ïise infection.
SEPENTES:COLUBRIOAE
Synonym: Coluher aurora Linnaeus, 1754
Range: Eastern Cape, Free State, Lesotho, Southern KwaZulu Natal and Mpumalanga
(FitzSimons 1962).
Characteristics: Pre-ocular one; post-oculars two; supra-labials eight, fourth and fifth
entering orbit; infra-labials eight (De Waal 1978). Scales smooth with 21-23 rows; sub-
caudals 28-34 (Branch 1998).
Biology: This snake is not common and feeds on nestling rodents. It can sometimes be
found in old termitaria.
44
CHAPTERJ RESULTS
Haematological observations: Two specimens were collected. One on the farm
Deelhoek (Fig. 2.1) 30 kilometers north of Bloemfontein and one in Bloemfontein
suburbs itself. Both of these snakes had what appeared to be a Serpentoplasma infection
in the red blood cells.
Synonyms: Lycodon guttatus A. Smith, 1843
Alopeeion annulifer Dumeril & Bibron, 1854
Range: Southern Cape, Great Namaqualand, KwaZulu-Natal, Eastern Mpumalanga,
Eastern Free State. Inland Mountains of the Cape and Cape Fold Mountains (FitzSimons
1962)
Characteristics: A small slender snake, with 21-25 scale rows and large eyes. Blotches
on dorsal side, varying according to habitat.
Biology: It shelters in rocky areas where it preys on geckos and other lizards. The female
lays three to six elongated eggs in midsummer.
Haematological observations: All five specimens captured in Lesotho had an infection
resembling Serpentoplasma in the erythrocytes of this snake species.
Synonyms: Lycodonfuliginosus Boie, 1827
Lycodon unicolor Schlegel, 1837
Boaedon quadrivittatum Hallowel, 1857
Boaedon quadrilineatum Dumeril, 1859
Alopeeion variegatum Bocage, 1867
45
CHAPTERJ RESULTS
Boodon bipraeocularis Gunhter, 1888
Boodon lineatus var. plutonis Werner, 1902
Range: All over Sub Saharan Africa (Branch 1998).
Characteristics: Posterior sub-linguals usually in contact, separated by prolongation of
anterior sub-linguals; pre-oculars one or two (De Waal 1978). Large head with two pale
yellow streaks along head, small body scales with 27-29 rows (Branch 1998).
Biology: These nocturnal snakes forage for rodents and sometimes bats, they seldom take
lizards. They usually occupy old termitaria and can be found in almost any habitat, as
they usually occur nearby houses.
Haematological observations: Twelve specimens were collected in Bloemfontein
suburbs and on the farm Deelhoek. All the specimens studied had Serpentoplasma-like
blood infections.
Synonyms: Lycodon capensis A. Smith, 1831
Lycodon horstokii Schlegel, 1837
Range: Throughout most of Southern Africa, except the western parts of the Cape and
Namibia (FitzSimons 1962).
Characteristics: Flattened head; post-nasal touches - first upper labial. Ventral scales are
150-205 (Branch 1998). Sub-caudals 32-41 in males; 24-31 in females (De Waal 1978).
Biology: This species prefers well-vegetated areas and the Cape skink tMabuia capensis)
is an important part in their diet.
Haematological observations: A Serpen/oplasma-like infection was observed m the
erythrocytes of two specimens captured in Bloemfontein and the farm Deelhoek.
46
CHAPTERJ RESULTS
Synonyms: Coluber canus Linnaeus, 1758
Coluber elegantissimus Laurenti, 1768
Coluber ocel/atus Gmelin, 1789
Cadmus cuneiformis Theobaid, 1868
Coronella phocarum Gunter, 1872
Ophirhina anchietae Bocage, 1882
Range: Throughout Southern Africa and on Robben Island, elsewhere Angola and
Kenya (Branch 1998).
Characteristics: Ventral scales 186-197 in males, 206-218 in females (De Waal 1978).
A large thick snake with hooked nose. Body scales smooth, but sometimes keeled in
black specimens from Western Cape (Branch 1998) and counts 25-31 rows. Eyes with
round pupils. Young blotched, older specimens darker and more uniform in color.
Biology: They feed on moles, rodents, other snakes, eggs and lizards. They are
constrictors that live underground.
Haematological observations: One specimen was collected from Bloemfontein; the
presence of a Serpentoplasma-like infection was found in the erythrocytes of this snake.
Synonyms: Anodon typus A. Smith, 1929
Rachiodon abyssinus Dumeril & Bibron, 1854
Dasypeltis scaber var. capensis Peters, 1864
Dasypeltis scaber var. mosambicus Peters, 1864
Dasypeltis scaber var. breviceps Peters, 1864
Dasypeltis lineolatus Peters, 1878
47
CHAPTER3 RESULTS
Dasypeltis seahra loveridgei Mertens, 1954
Range: Africa, except high mountain peaks and dense lowland forest, Southern Arabia.
Characteristics: Post-ocular scales two; pre-ocular scales one; dorsal scales 21-28 and
17-22 anterior to vent (De Waal 1978). Lateral scales small and have serrated scales. Tail
short, head rounded.
Biology: This snake is very common and can be found in old termitaria. It is adapted for
a special diet of only eggs.
Haematological observations: A Serpentop/asma-like infection was observed in the red
blood cells of 18 specimens captured from the farm Deelhoek.
Synonyms: Coronella hotamhoeia Laurenti, 1768
Coronella virginia Laurenti, 1768
Coluher bicolor Leach, 1819
Ophis heterurus Duvernoy, 1833
Dipsas hippoerepis Reinardt, 1843
Dipsas inorhatus A. Smith, 1849
Oxyropus me/anoerotaphos Cope, 1860
Range: Tropical Africa south of the Sahara, southwards over eastern Africa to Cape
Town; absent from arid western deserts (FitzSimons 1966).
Characteristics: Ventral scales 146-160 in males, 146-161 in females; anal plate entire.
Dorsal color dark brown with uniform white spots. Distinct black patch from eye to
neck; upper lip orange red.
Biology: These snakes live in marshy areas and feed at night on amphibians. They can
also be found in old termitaria.
48
CHAPTER3 RESULTS
Haematological observations: A Serpentoplasma-like infection was observed 10 the
erythrocytes of six specimens from Bloemfontein.
Synonym: Rhagerrhis tritaeniatus Gunther, 1868
Range: Tanzania to Southern Zaire, Namibia, KwaZulu-Natal, Free State, Eastern Cape
(Broadley 1966).
Characteristics: Ventral scales 151-160 in males, 153-169 in females; anal plate usually
divided. Three black edged dark stripes on back, vertebral line is yellow.
Biology: These snakes forage in grass for small mammals; female lays five to 18 eggs in a
hole.
Haematological observations: Six specimens were collected in the Free State. Two
were collected in Bloemfontein district and four on the farm Deelhoek, 30 Km north of
Bloemfontein. All six of these snakes had a haemogregarine infection as well as
Serpentoplasma-use infections in the blood. These also represent new records.
Synonyms: Psamophis moniliger var. notostictus
Psammophis sibilans var stenocephalus Bocage, 1887
Range: Cape Province to Southwestern Angola (Broadley 1975). Southern Free State to
Gauteng, Northern Province, Botswana and Tanzania (Branch 1998).
Characteristics: Upper labials four and five enter the eye; 17 scale rows; one pre-
ocular; the tail is long: 80-107 sub-caudals; striped or blotched coloration.
Biology: These snakes are fast predators of skinks and lacertids. They live 10 old
termitaria, under rocks and in rock cracks.
49
CHAPTER3 RESULTS
Haematological observations: One specimen was collected on the farm at Deelhoek
(Fig. 2.1). lts erythrocytes contained Serpentoplasma-like inclusions, as well as an
undescribed haemogregarine.
Synonyms: Cercocalamus collaris Gunther, 1863
Aparallactus punctatolineatus Boulenger, 1895
Aparallactus hocagii Boulenger, 1895
Aparallactus luhherti Sternfeld, 1910
Range: Free State, KwaZulu-Natal, Eastern Cape (Broadley 1966).
Characteristics: Supra-Iabials six; ventral scales 141-157 in males, 159-173 in females;
dorsum dark brown (De Waal 1978). Lower labials separated by mental black collar and
brown head (Branch 1998).
Biology: These snakes are abundant in old termitaria, where they shelter and feed.
Haematological observations: A total of five specimens were collected at the farm
Deelhoek near the Soetdoring Nature Reserve in the Bloemfontein district (Fig. 2.1).
These snakes were easily obtained from old termitaria. Their blood contained a
Serpentoplasma-use infection.
50
CHAPTER3 RESULTS
SEPENTES:ELAPIDAE
Range: Highveld areas of Free State and Mpumalanga, North Cape in the Kimberly
region (Broadley 1971).
Characteristics: Ventral scales 161-164 in males; 143-152 in females; sub-caudals 21-22
in males, 14-17 in females, dorsum with alternating black and white cross bands.
Biology: These are slow moving snakes and reluctant to bite. Rarely seen, they live in
old termitaria and under rocks. The venom has not yet been studied.
Haematological observations: Three specimens were collected on the farm Deelhoek 30
km north of Bloemfontein. All of these specimens had a Serpentoplasma-like infection
in the blood.
Synonyms: Vipere haemachate Lacêpéde, 1788
Naja capensis A. Smith, 1826
Range: Eastern parts of the country, south of the 25° South latitutude line (FitzSimons
1966).
Characteristics: Sub-caudals 40-47 in males, 38-45 in females; ventrum black with
white cross bands on ventral part of neck (De WaaI1978). Keeled scales, 17-19 rows,
and broad head, interscaly areas of spread hood sometimes yellow-orange tinged (De
WaaI1978).
Biology: Nocturnal, eats small vertebrates, spread a hood when cornered and can spit
venom up to three meters.
Haematological observations: One specimen was collected near Excelsior in the Reitz
district, Eastern Free State (Fig. 2.1). Its blood contained a Serpentoplasma-like infection
similar to that seen in all the other snakes. The inclusions were, however, much bigger
51
CHAPTER3 RESULTS
than those Sauroplasma infections found in lizards. Another specimen from Lesotho
(Fig. 2.2) also had the same kind of Serpentaplasma-like infection.
SEPENTES: VIPERIDAE
Synonyms: Naja (Coluher) var. nigrum Cuvier (sic) Boie, 1827
Causus rhamheatus var. taeniata Sternfeld, 1912
Range: Sudan and Somalia, Cape, Zaire and Angola (Broadley 1966). Eastern regions
and in Eastern Free State (FitzSimons 1966).
Characteristics: Ventral scales 136, anal plate entire, sub-caudals 23, dorsal scales in 19
rows on nape and mid-body, 13 anterior to vent.
Biology: It rests during the day in undergrowth, termitaria and under logs. At night it
forages for amphibians, the venom glands are large, extending into neck.
Haematological observations: One specimen was collected in the Eastern Free State.
Its blood contained Serpentoplasma -like inclusions.
Synonyms: Vipera (Echidna) arietans Merrem, 1820
Vipera in.flata Burchell, 1822
Vipera hrachyura Cuvier, 1829
Clotho lateristriga Gray, 1842
Vipera irflata Burchell, 1822
Vipera hrachyura Cuvier, 1829
Clotho lateristriga Gray, 1842
52
CHAPTER3 RESULTS
Range: Throughout Africa, absent from rain forests and extreme deserts (FitzSimons
1962).
Characteristics: Ventral scales 135-142 in males, 134-146 in females, anal plate entire,
sub-caudals 26-32 in males, 16-20 in females, yellow or white inter-ocular band (De
Waal 1978). Heavily keeled scales, large triangular head with nostrils pointed upward
(Branch 1998).
Biology: It emerges at dusk and waits and ambushes its prey. This snake relies on its
camouflaging and poison action.
Haematological observations: Two specimens were collected in the Bloemfontein
district. Both of these specimens also had Serpentoplasma-like infections in the
peripheral red blood cells.
53
CHAPTER3 RESULTS
The following table (Table 3.1) provides a summary of infections in reptiles collected. It
includes parasite species, hosts, localities and levels of infection.
Table 3.1 Summary of parasitic infections examined and presented from some of the reptiles collected with
their localities. N indicates the number of specimens examined and (%) gives parasite prevalence.
Intensity was calculated by number of infected erythrocytes in 10000 erythrocytes. In each case the highest
intensity found is given.
Viral and viral-like Host Locality N (%) Intensity
infections (includ-
ing so-called
piroplasms)
Pirhemocyton Agama atra atra Bloemfontein 65/65(100) 2800/10000
(26°04'S,
29°13'E),
Zuurfontein
(29°54'S,
25°22'E),
Lesotho,
Clarens
(28°31 'S,
28°25'E)
Pirhemocyton Cordylus Ha lil (100) 8900/10000
po/yzonus Rapokolana
polysonus (29'22'36"S
28'01 '53E)
Sauroplasma Cordylus Koffielaagte 31131 (lOO) 7400/1 0 000
thornasi giganteus (27'29'S,
27'28'E)
Sauroplasma Cordylus Zuurfontein 38146 6900110000
polyzonus (29'54'S (82.6)
polyzonus 25'22 'E)
Sauroplasma Mabuya su/cala Zuurfontein 6/6 (100) 1100/10000
sulcata (29'54'S
25'22'E)
Sauroplasma Mabuya striata Bloemfontein 3/3 (100) 600110000
punctatissima (26'04'S
29'13'E),
Sauroplasma Nucras intertexta Bloemfontein 1/1 (lOO) 3700/10000
(26'04'S
29'13'E),
Sauroplasma Acontias Bloemfontein III (100) 600/10000
gracilicauda (26'04'S
gracilicauda 29'13'E),
Sauroplasma Varanus Zuurfontein III (100) 2500/10000
exanthematicus (29'54'S
albigularus 25'22'E),
Sauromella Pachydactylus Deelhoek 4/4 (100) 900\10000
haemolysus capensis (28'54'S,
26'09'E)
54
CHAPTER3 RESULTS
Table 3.1 (continued). Summary of parasitic infections examined and presented from some of the reptiles
collected with their localities. N indicates the number of specimens examined and (ly") gives parasite
prevalence. Intensity was calculated by number of infected erythrocytes in 10000 erythrocytes. In each
case the highest intensity found is given.
Viral and viral-like Host Locality N (%) Intensity
infections (include-
ing so-called
piroplasms)
Sauroplasma Pseudocordylus Ha 39/39 6780/10000
melanotus Rapokolana (100)
subviridis (29°22'36"S
28°01' 53E),
Ha Khojane
(28°22 'S,
28°01 'E) and
Ha Thaba
Bosiu
(29°20'15"S
28°11 '58"E),
Lesotho.
Sauroplasma Pseudocordylus Clarens 5/5 (100) 5620/10000
melanotus (28°31 'S,
melanotus 28°25'E)
Serpentoplasma Bitis arietans Bloemfontein 2/2 (100) 800/10000
arietans (26°04'S
29°13'E)
Serpentoplasma Crotaphopeltis Bloemfontein 6/6 (100) 700/10000
hotamboeia (26°04'S
29°13'E)
Serpentoplasma Lycophidion Deelhoek 2/2 (100) 2700/10000
capense capense (28°54'S,
26°09'E
Serpentoplasma Causus Clarens 1/1 (lOO) 100/ 10000
rhombeatus (28°31 'S,
28°25'E),
Serpentoplasma Elapsoidea Deelhoek 3/3 (100) 85/10 000
sundevallii media (28°54'S,
26°09'E),
Serpentoplasma Hemachatus Excelsior 1/1 (100) 2500/10000
haemachatus
Serpentoplasma Hemachatus Ha 1/1 (100) 3540/10000
haemachatus Rapokolana
(29°22'36"S
28°01 '53E)
Serpentoplasma Lamprophis DeeIhoek 2/2 (100) 750/10000
aurora (28°54'S,
26°09'E)
Serpentoplasma Lamprophis Deelhoek 12/12 900/10000
fuliginosus (28°54'S,26°) (100)
55
CHAPTER3 RESULTS
Table 3.1 (continued). Summary of parasitic infections examined and presented from some of the reptiles
collected with their localities. N indicates the number of specimens examined and (0;',) gives parasite
prevalence. Intensity was calculated by number of infected erythrocytes in 10000 erythrocytes. In each
case the highest intensity found is given.
Viral and viral-like Host Locality N (%) Intensity
infections (include-
ing so-called piro-
plasms)
Serpentop/asma Psammophylax Bloemfontein 5/5 30l)/1O 000
tritaeniatus suburbs
Haemogregarines Host Locality N (%) Intensity
Hepatozoon sp.A Agama atra atra Ha Khojane 3/3 (100) 1263/10000
(28'22'S,
28'01 'E)
Zuurfontein 7/62 (3.5) 2200/10000
(29'54'S
25'22'E)
Hepatozoon sp. B Pseudocordy/us Clarens 5/5( 1(0) 6850/10000
melanotus (28'31 'S,
melanotus 28'25'E)
Hepatozoon sp. C Pseudocordylus Ha Thaba 7/8 (87.5) 1000/10000
melanotus Bosiu
subviridis (29'20'15"S
28'11 '58"E
Hepatozoon sp. D. Pseudocordylus Ha 4/4 (lOO) 1850/10000
melanotus Rapokolana
subviridis (29'22'36"S
28'01' 53E),
Hepatozoon sp. E. Pseudocordylus Ha 3/3 (lOO) 550/10000
melanotus Rapokolana
subviridis (29'22'36"S
28'lH ' 53E),
Hepatozoon sp. F Psammophylax Bloemfontein 5/5 (100) 150/10000
tritaeniatus (26'lW S
29'l3'E)
Hepatozoon Python sebae Bloemfontein 1/1 (100) 450/10000
(1-a[emogregari na) natalensis (26°04'S
sebae 29'13'E)
56
CB.APTER3 RESULTS
Table 3.1 (continued). Summary of parasitic infections examined and presented from some of the reptiles
collected with their localities. N indicates the number of specimens examined and (%) gives parasite
prevalence. Intensity was calculated by number of infected erythrocytes in 10000 erythrocytes. In each
case the highest intensity found is given.
Malarias Host Locality N (%) Intensity
Plasmodium sp. A Cordylus Zuurfontein 17/38 3800/10000
polyzonus (29'54'S (44.7)
polyzonus 25'22'E)
Plasmodium sp. B Pseudocordylus Clarens 1/5 (20) 1200/10000
melanotus (28'31 'S,
melanotus 28'25'E)
Plasmodium sp .C Pseudocordylus Ha 3/4 (75) 800/10000
melanotus Rapokolana
subviridis (29'22'36"S
28'0 I' 53"E)
Plasmodium sp. D Pachydactylus Zuurfontein 2/2 (lOO) 4100/10000
bibronii (29'54'S
25'22'E)
Microfilariae Host Localitv N (%) Intensitv
Microfilaria sp. A Agama atra atra Ha Exact Exact numbers not
Rapokolana numbers recorded
(29'22'36"S not
28'0 I' 53 "E) recorded
Ha Khojane
(28'22'S,
28'01 'E)
Zuurfontein
(29'54'S
25'22'E)
Microfilaria sp. B Cordylus Zuurfontein Exact Exact numbers not
polyzonus (29'54'S numbers recorded
polyzonus 25'22'E) not
recorded
Microfilaria sp. C Pseudocordylus Ha Exact Exact numbers not
melanotus Rapokolana numbers recorded
subviridis (29'22'36"S not
28'01 '53"E) recorded
Ha Khojane
(28'22'S,
28'01 'E)
Ha Thaba
Bosiu
(29'20'15"S
28'11'58"E
Microfilaria sp. D Pseudocordylus Clarens Exact Exact numbers not
melanotus (28'31 'S, numbers recorded
melanotus 28'25'E) not
recorded
57
r-----------------------------------------------------------------------~---~~
Figure 3.].. Photographs of reptiles collected in the Free State and Lesotho. A:
Lygodactylus capensis capensis (A. Smith, 1849), B: Pachydactylus capensis capensis
(A. Smith, 1845), C: Pachydactylus bibronii (A. Smith, 1845) ]): Agama atra atra
Daudin, 1802lE: Agama hispida (Linnaeus, 1758).IF:Mabuya capensis (Gray, 1830), G:
Mabuya sulcata sulcata (Male & Female) (peters, 1867) lHI: Mabuya striata
punctatissima (A. Smith, 1849), I: Acontias gracilicauda gracilicauda Essex, 1925 J:
Cordylus giganteus A. Smith, 1844, K: Cordylus polyzonus polyzonus A. Smith 1838, L:
Pseudocordylus melanotus melanotus (A. Smith, 1838). A-C, lE-:n:K, &L are taken from
Branch (1998) and digitally adapted and reproduced. This is only for illustration purposes
for this thesis and not for any publication purposes.

Figure 3.2. Photographs of reptiles collected in the Free State and Lesotho. A:
Pseudocordylus melanotus subviridis (A. Smith, 1838), B: Nucras intertexta (A. Smith,
1838), C: Varanus exanthematicus albigularus (Daudin, 1802), Dl: Lamprophis aurora
(Linnaeus, 1754), lE: Lamprophis guttatus (A. Smith, 1843), lF: Lamprophis fuliginosus
fuliginosus (Boie, 1827), G: Lycophidion capense capense (A. Smith, 1831), lHl:
Pseudaspis cana (Linnaeus, 1785), lI: Dasypeltis scabra (Linnaeus, 1785), .M:
Crotaphopeltis hotamboeia (Laurenti, 1768), K: Psammophylax tritaeniatus (GUnter,
1868), I.:Psammophis notostictus Peters, 1867. B & C, lE,G & H, .M-I. were taken from
Branch (1998), IJ) & F: were taken from Patterson (1987) and digitally adapted and
reproduced. This is only for illustration purposes for this thesis and not for any
publication purposes. A & lI: Taken by author.

Figure 3.3. Photographs of reptiles collected in the Free State and Lesotho. A: Aparallactus
capensis A. Smith, 1849, B: Elapsoidea sundevallii media Broadley, 1971, C: Hemachatus
haemachatus (Lacêpéde, 1788), D: Causus rhombeatus (Lichtenstein, 1823), E: Bitis arietans
arietans (Merrem, 1820), F: Python sebae natalensis (Gmelin, 1789). A & B: were taken
from Branch (1998), E: was taken :fromBuys & Buys (1983) and digitally adapted and
reproduced. This is only for illustration purposes for this thesis and not for any publication
purposes. C & D: Taken with permission :fromProt L.H. du Preez, F: was taken by author.
CHAPTER3 RESULTS
BLOOD PARASITES
As reported above, a variety of blood parasites were seen in the reptiles captured in the
Free State and the border regions of Lesotho, including viral infections (Pirhemocyton)
and suspected viral infections (Sauroplasma. Sauromella, Serpentoplasmas. protozoans
(haemogregarines of the genus Hepatozoon, malaria parasites of the genus Plasmodium
and possibly Haemoproteusi and filarial nematodes (microfilariae). Many infections
probably provide the basis for new parasite species and most yield new host records.
Of those infections noted in the previous section 3.1 (Hosts collected), only host blood
samples that were adequate enough to provide good descriptions are recorded below. For
the remainder, further collections will be required before they can be examined in
sufficient detail to allow description.
3.2 VIRUSES AND SUSPECTED VIRAL INFECTIONS
3.2.1. PIRHEMOCYTON CHATTON & BLANC 1914
Pirhemocyton from Agama atra atra Daudin, 1802
Type host: Tarentola mauriticana
Type localities: Morocco, Spain
Present study:
Localities: Bloemfontein; Zuurfontein (29°54'S, 25°22'E); Clarens (28°31 'S, 28°25'E);
Lesotho
Host: Agama atra atra Oaudin, 1802
Pirhemocyton infections were observed in the lizard Agama atra atra collected in the
Bloemfontein, Clarence and Jagersfontein districts (Fig. 2.1) and in Lesotho (Fig. 2.2).
Such infections have been described from lizards in Australia by Stehbens & Johnston
63
igure 3.4. Light micrographs of Giemsa stained blood films of Pirhemocyton infections of 15 different
pecimens of Agama atra atra collected at different localities. A-C: Pirhemocyton infections from two éS
and one Sf? specimen from the farm at Zuurfontein, D-F: Pirhemocyton infections from three éS specimens
ollected in the Bloemfontein district, with D (white arrow) showing areas of pinkish granules, E & F
showing comet tail effects, G-I: Pirhemocyton infections from two Sf? and one éS specimen collected in
loemfontein, J: Pirhemocyton infections from one éS specimen collected at Ha Thaba-Bosiu, K-M:
Pirhemocyton infections from two Sf? specimens collected at Ha Khojane, N-O: Pirhemocyton infections
from one (S, one Sf? and one juvenile collected at Ha Rapokolana. A-C, E&F, I, K-O (red arrows):
albuminoid bodies. B, D, G-J, N (blue arrows): rounded purple staining bodies, M (yellow arrow): deep red
pigment inside purple staining areas, A-F, L, 0 (white arrows): effects on host cell: rounded nucleus is
distinctively displaced to the lateral side in the cell. Scale: A-O: 10 J..lID.
CHAPTER3 RESULTS
(1966) and Johnston (1975), while Paperna & De Matos (l993b) studied the
ultrastructure of the same type of infection from the same species of lizards from the Free
State.
In the present study, these infections were abundant in all of the captured lizards and
infections varied from low levels among erythrocytes to almost 100% red cells infected.
Infections could be seen as purple bodies (Fig.3.4A-O), sometimes accompanied by an
albuminoid body (vacuole) (Fig.3.4A-C, E&F, I, K-O (red arrows». Albuminoid bodies
measured 2.88 ± 0.55 (0.26-5.5) firn (n=30). The centre of infection itself was largely
stained deep purple with Giemsa, measuring 2.2 ± 0.57 (0.9-3.5) firn (n=60) in diameter.
Although in general, infection centres were seen as purple staining bodies which were
mostly round (Fig.3.4B, D, G-J, N (blue arrows)), sometimes a deep red pigment was
visible within the round body (Fig.3.4M (yellow arrow». In addition, patches of fine
pink granules were sometimes visible scattered within the host erythrocyte cytoplasm
(Fig.3.4D (white arrow», or forming a comet-tail leading from the round body (Fig.3.4E,
F), and associated effects on host cells were marked, these cells appearing rounded with a
distinctive round nucleus, sometimes displaced to one side of the cell. (Fig.3.4A-F, L, 0
(white arrows». Some infected host erythrocytes were pale-stained with Giemsa (Fig.
3.41 (red and blue arrows». Pirhemocyton infections were also found in Agama atra atra
in Lesotho, (Fig.3.1 OB, E-I (blue arrows» where they were accompanied by another
parasite, Hepatozoon sp. A, illustrated in Fig.3 .1OA-I.
Pirhemocyton from Pseudocordylus melanotus subviridis (A. Smith, 1838)
Host: Pseudocordylus melanotus suhviridis (A. Smith, 1838)
Locality: From the village of Ha Rapokolana (29°22'36"S 28°01 '53E), Lesotho (Fig.
2.2)
A similar infection, but apparently producing smaller intraerythrocytic bodies was seen in
this lizard and was accompanied by Hepatozoon sp. E. (Fig.3.14A-F). This possible
Pirhemocyton, was found in almost all erythrocytes (Fig.3.14A-F). Some cells appeared
65
CHAPTER3 RESULTS
to have multiple infection centres (Fig.3.14C&D (red arrows)) and small granular areas
were observed. As previously described for Pirhemocyton, the infection itself had a
rounded, purple-stained centre (Fig.3.14A-F (blue arrows)), measuring 1.29 ± 0.9 (0.25-
2.33) urn (n=60) in diameter. However, no albuminoid bodies like those in infections of
Agama atra atra were observed. Host cells did not seem to be unduly affected by these
infections, in contrast to the infection described above.
Comments
Pirhemocyton zonurae was described from a cordylid lizard (Cordylus viuiferï in
Southern Africa (Pienaar 1962). The current study demonstrates the first record of a
possible Pirhemocyton infection of Pseudocordylus melanotus subviridis (A. Smith,
1838) in South Africa, thus extending the range of distribution of these viral infections
among the Cordylidae. However, this differs from most other known Pirhemocyton
infections, including that seen in Agama atra atra (above), in that it is not accompanied
by an albuminoid body. Morphometrically, the infected areas are also smaller (1.29 ± 0.9
(0.25-2.33) urn (n=60) in diameter) than those (2.2 ± 0.57 (0.9-3.5) urn (n=60) in
diameter) for Pirhemocyton from Agama atra atra and the characteristic red pigment is
also absent from the current infection.
3.2.2. SAUROPLASMA DU TOIT, 1937
Sauroplasma from Cordylus giganteus (A. Smith, 1844)
Type species: Sauroplasma thomasi Du Toit, 1937
Type Host: Cordylus giganteus (A. Smith, 1844)
Type locality: Wesselsbron (2T52'S, 26°21'E) and Odendaalsrust (27°51'S, 26°41'E),
Free State
Present study:
Host: Cordylus giganteus (A. Smith, 1844)
Locality: On the farm Koffielaagte (27°29' S, 2T28'E), near Kroonstad
66
CHAPTER3 RESULTS
Sauroplasma infections, indistinguishable from those of Du Toit (1937) were found in all
of 31 C. giganteus captured on a farm near Kroonstad. The infected lizards were 10
males and 21 females.
Light microscopy
Parasites were intracytoplasmic and found in mature erythrocytes of which 740011 0 000
(74%) cells were infected. The effects on the host cells were seemingly minimal (Fig.
3.5A-F), as they were of normal size and shape and only a small area of the host
cytoplasm was normally affected. In some heavy infections, larger areas within the cell
cytoplasm appeared to be vacuolated, constituting a significant lesion in the infected
cells.
In Giemsa-stained blood films parasites usually occurred singly, but occasionally in pairs,
in a single erythrocyte. They were rounded, ring-like structures, sometimes with several
small buds or protrusions (not shown in light micrographs). The main body of the
structure measured l.51 ± 0.7 (l.35-l.79)!lm by l.58 ± 0.74 (1.46-1.62)!lm (n= 50),
with buds 0.61 ± 0.54 (0.45-0.78) urn by 0.67 ± 0.48 (0.47-0.81) urn (n=25). The centre
of the parasite was largely unstained and vacuole-like, but a narrow rim around most of
its periphery was deeply stained with Giemsa, sometimes bearing purple-stained
granules. Except for budding forms, no evidence of division was observed and no pink-
stained regions similar to those seen in Pirhemocyton infections were observed in host
erythrocytes.
Electron microscopy
Examination of blood samples by transmission electron microscopy (TEM) revealed
something of the curious nature of these structures. Images showed multiple daughter
structures or budding forms (Fig.3.5G & H). These were filled with lightly granular
material embedded in an otherwise largely electron-lucent matrix, surrounded by a
boundary membrane (Fig.3.5G & H (black arrows)). In just one image the faint
impression of a structure resembling a small region of heterochromatin was observed
(Fig. 3.5H (white arrow)), but this was the only image of its type.
67
Figure 3.5. Light micrographs of Sauroplasma thomasi Du Toit, 1937. A-F:
Sauroplasma thornasi from Giemsa stained blood films of six different specimens of
Cordylus giganteus A. Smith, 1844. G-L: Transmission electron micrographs of S.
thornasi in mature erythrocytes of C. giganteus. G & H: Multiple daughter parasites or
budding forms, with boundary membrane (black arrows). H: (white arrow) A small area
of heterochromatin. G & H: Budding: surface of mother structure expands until a bud is
produced. I & J: Membranous whorls and granules within the structure. K & L:
Peripheral membranous whorls with a prominent vacuolated center. Scale: A-F: 10 J.1m,
G, H, J & L: 500 nm, I & K: 1J.1ID.
CRAPTER3 RESULTS
Budding appeared to occur when the surface of the mother structure expanded to produce
a bud that was also filled with granular material (Fig. 3.5G & H.). Presumably, by a
process of constriction, the bud eventually separates from the mother structure and grows
until roughly the same size of the mother. Some structures also seemed to be filled with
membranous whorls and particulate material (Fig. 3.SI & J), while in others, the
membranous material was confined to the perimeter of the structure and the centre was
vacuole-like (Fig. 3.5K & L).
These structures have been described as parasites belonging to the Order Piroplasmorida
Wenyon, 1926 because of their absence of pigment, mode of multiplication, size and
shape (see Davies & Johnston (2000); Peirce (2000)). They were also suspected to be
viral in nature because of their similarity with Pirhemocyton-like infections found in
other members of the family Cordylidae (see Davies & Johnston, 2000). The current
TEM study does not reveal any apicomplexan structures, or provide any clear indication
of the identity of Sauroplasma, including whether or not it might be of host origin. No
viral particles were found in the current electron micrographs, but Sauroplasma appears
very similar to Serpentoplasma and the latter infection might be of viral origin (see
Section 3.2.4 on Serpentoplasma of snakes).
Comments
By light microscopy, the intra-erythrocytic structures found in this study are identical to
those described by Du Toit (1937) and later Pienaar (1962) from C. giganteus and
therefore they are identified as Sauroplasma thomasi. Although leading to no firm
conclusions about its identity, this is the first description of the ultrastructure of S.
thomasi. The infection found in C. giganteus also closely resembles those found in all
other species of lizards collected in this study. This leads to the suspicion that
Sauroplasma-like infections are more widespread than previously thought and that they
may cross specific, generic and even family boundaries. The true identity of
Sauroplasma, the boundaries of its distribution and its mode of transmission between
hosts are currently unknown.
69
CHAPTER3 RESULTS
Additional records of Sauroplasma
The following descriptions of Sauroplasma all represent new host records, as far as is
known. Although they are the first morphometrical descriptions in new host erythrocytes,
these descriptions are not considered sufficient to distinguish between species, especially
since the identity of Sauroplasma is currently unclear and all forms are round, ring-like,
budding structures. However, these data provide useful new information on the
dispersion of these structures and individual variations between them. Furthermore, since
host specimens were collected from areas separated by extensive mountain ranges, rivers
and grasslands, these descriptions may well prove eventually to be those of new species,
if protozoan, or strains, if viral.
Sauroplasma from Cordylus polyzonus polyzonus A. Smith, 1838
Host: Cordylus polyzonus polyzonus A. Smith, 1838.
Locality: On the farm Zuurfontein (29°54'S 25°22'E), Jagersfontein district, Free State.
Sauroplasma infections were found in 38 of 46 C. polysonus captured on a farm near
Jagersfontein (Fig. 2.1). The infected lizards were 21 males and 25 females. The
parasites were intracytoplasmic and found in mature erythrocytes of which an average
6900/10 000 (69%) cells were infected. The effects on the host cells were seemingl y
minimal (Fig. 3.6A-E). In some heavy infections large areas within the cell cytoplasm
appeared to be vacuolated.
In Giemsa-stained blood films the structures occurred singly, or m pairs m a single
erythrocyte (Fig.3.6A-C), although some were multiple (Fig.3.6D & E). They were
irregularly round, ring-like structures. The main body of the structure measured 2.37 ±
0.64 (0.37-2.20) urn by 1.36 ± 0.7 (0.26-2.46) urn (n= 60). The centre was largely
unstained and vacuole like, but a narrow rim around most of its periphery was deep
stained with Giemsa. No evidence of division was observed.
70
CHAPTER3 RESULTS
Sauroplasma from Mabuya sulcata sulcata (Peters, 1867)
Host: Mabuya su/cala su/cata (peters, 1867)
Locality: on the farm Zuurfontein (29°54'S 25°22'E), Jagersfontein district, Free State.
Sauroplasma infections were found in all six M. sulcata (Peters, 1867) captured on a
farm near Jagersfontein (Fig. 2.1) and infected lizards were four males and two females.
The parasites were intracytoplasmic and found in mature erythrocytes of which 1100/1 0
000 (11%) cells were infected. The effects on the host cells were seemingly minimal (Fig.
3.6F & G) (arrows». Only small areas within the cell cytoplasm appeared to be
vacuolated.
In Giemsa-stained blood films the structures occurred singly within erythrocytes
(Fig.3.6F & G). They were round and ring-like with no buds or protrusions. The main
body of the structure measured 0.66 ± 0.67 (0.37-0.87) urn by 0.655 ± 0.77 (0.21-1.10)
urn (n= 24) and its centre was largely unstained and vacuole like. A narrow rim around
most of its periphery, deep stained with Giemsa (Fig3.6F & G). No evidence of division
was observed, but faintly pink-staining regions similar to those seen in Pirhemocyton
infections were seen in some cases.
Sauroplasma from Nucras interte.xta (A. Smith, 1838)
Host: Nueras intertexta (A. Smith, 1838)
Locality: Bloemfontein district, Free State.
Sauroplasma infections were found in one specimen of Nucras intertexta (A. Smith,
1838) captured in the Bloemfontein area (Fig. 2.1). The infected lizard was a male. The
structures were intracytoplasmic and found in mature erythrocytes of which 3700/1 0 000
(37%) cells were infected. The structures themselves were quite large and bullet hole
shaped.
71
CHAPTER3 RESULTS
In Giemsa-stained blood films Sauroplasma occurred singly in erythrocytes (Fig.3.6H &
I). Each was a round, ring-like structure with no buds or protrusions. The main body of
the structure measured 1.35 ± 0.4 (1.20-1.45)!lm by 1.33 ± 0.46 (1.17-1.49)!lm (n= 30).
The centre was unstained and vacuole-like (Fig. 3.6H & I). No evidence of division was
observed and pink-staining regions like those seen in Pirhemocyton infections were
absent.
Sauroplasma from Mabuya striata punctatissima (A. Smith, 1849)
Host: Mabuya striata punctatissima (A. Smith, 1849).
Locality: Bloemfontein district, Free State.
Sauroplasma infections were found in all three Mabuya striata punctatissima (A. Smith,
1849) specimens captured in the Bloemfontein suburbs. The infected lizards were all
males. The structures were intracytoplasmic and found in mature erythrocytes of which
60011 0 000 (6%) cells were infected. The effects on the host cells were seemingly
minimal (Fig. 3.6J & K), although in some heavy infections large areas of host cytoplasm
appeared to be vacuolated (Fig. 3. 6J & K).
In Giemsa-stained blood films Sauroplasma occurred singly (Fig. 3.6J), or in pairs (Fig.
3.6K) in a single erythrocyte. They were irregularly round, ring-like structures with the
main body of the structure measuring 1.44 ± 0.57 (0.61-2.24) urn by 1.54 ± 0.59 (0.91-
1.97) urn (n= 40). No buds were observed. The centre of the parasite was largely
unstained and vacuole-like, without a staining rim at its periphery. No evidence of
division was observed and no pink-staining regions similar to those seen in Pirhemocyton
infections were noted.
72
Figure 3.6. Sauroplasma Du Toit, 1937 infections of various lizards. A-E: Sauroplasma
from Cordylus polyzonus polyzonus A. Smith, 1838, A-C: Single infections, D & E:
multiple infections, F & G: Sauroplasma from Mabuya sulcata sulcala (peters, 1867),
JH[-][: Sauroplasma from Nucras intertexta (A. Smith, 1838). J & K: Sauroplasma from
Mabuya striata punctatissima (A. Smith, 1849) with vacuolated cytoplasm in infected
areas. JL & M: Sauroplasma from Acontias gracilicauda gracilicauda Essex, 1925 with
large vacuoles in infected cells. N & 0: Sauroplasma from Varanus exanthematicus
albigilarus (Daudin, 1802) with vacuolated areas in infected cells.

CHAPTER3 RESULTS
Sauroplasma from Acontias gracilicauda gracilicauda Essex, 1925.
Host: Acontias gracilicauda gracilicauda Essex, 1925.
Locality: Bloemfontein district, Free State.
Sauroplasma infections were found in one specimen of Acontias gracilicauda
gracilicauda Essex, 1925. The sex of this specimen was undetermined. The parasites
were intracytoplasmic and found in mature erythrocytes of which 600/1 0 000 (6%) cells
were infected. These infections involved large areas of host cytoplasm which appeared
vacuolated (Fig. 3.6L & M).
In Giemsa-stained blood films the structures occurred singly, in pairs, or up to three were
present in a single erythrocyte and were irregularly round, ring-like structures. The main
body of the structure measured 2.5 ± 0.9 (1.1-3.8)!lm by 1.76 ± 0.86 (0.98-2.97) urn (n=
20). No buds were observed. The centre of the parasite was largely unstained and
vacuole-like, with no narrow rim around its periphery. No evidence of division was
observed and no pink-staining regions similar to those seen in Pirhemocyton infections
were noted.
Sauroplasma from Varanus exanthematicus alhigularus (Daudin, 1802)
Host: Varanus exanthematicus albigularus (Daudin, 1802).
Locality: On the farm Zuurfontein (29°54'S 25°22'E), Jagersfontein district, Free State.
Sauroplasma infections were found in one specimen of Varanus exanthematicus
albigularus (Daudin, 1802). The sex of this specimen was undetermined. The structures
were intracytoplasmic and found in mature erythrocytes of which 2500/10 000 (25%)
cells were infected. The effects on the host cells were marked (Fig. 3.6N & 0),
sometimes involving large areas of host cytoplasm that appeared to be vacuolated (Fig.
3.6N & 0).
75
CHAPTER3 RESULTS
In Giemsa-stained blood films the structures occurred singly, in pairs, and up to three
were present in a single erythrocyte. As in other infections, they were irregularly round,
ring-like, structures. The main body of the structure measured 1.4 ± 0.81 (1.0-3.8)!lm by
1.8 ± 0.97 (l.12-3.2) urn (n= 25) and no buds were observed. The centre of the parasite
was largely unstained and vacuole-like, without a narrow rim at its periphery. No
evidence of division was observed and no pink-staining regions similar to those seen in
Pirhemocyton infections were noted.
Sauroplasma from Pseudocordylus melanotus subviridis (A. Smith, 1838) and
Pseudocordylus melanotus melanotus (A. Smith, 1838).
Hosts: Pseudocordylus melanotus subviridis (A. Smith, 1838) and Pseudocordylus
melanotus melanotus (A. Smith, 1838).
Localities: Clarens district (28°31 'S, 28°25'£), Eastern Free State and Jorotane river
valley near village of Ha Rapokolana (29°22'36"S 28°01 '53E), Lesotho.
Sauroplasma infections were found in all 44 lizards collected in the different localities
noted above. Infections varied from only a few cells affected to near2'_. all erythrocytes
infected. The parasites were intracytoplasmic and found in mature erythrocytes, often
accompanying Plasmodium, haemogregarine and filarial nematode infections. Effects on
the host cells were apparently minimal (Fig. 3.18A-O (blue arrows»; although in some
cases multiple infections occurred (Fig. 3.18K (blue arrow». In heavy infections,
relatively large areas within the cell cytoplasm appeared to be vacuolated (Fig. 3.18A-D,
F).
In Giemsa-stained blood films, one to five structures were present in a single erythrocyte.
They were irregularly round and ring-like, and the main body of the structure measured
1.6 ± 0.45 (0.30-2.47) urn by 2.45 ± 0.51 (0.91-3.1) urn (n= 50). No buds were observed.
The centre of the parasite was largely unstained and vacuole-like, with no narrow rim
around its periphery. No clear evidence of division was observed, but the presence of
76
CHAPTER3 RESULTS
small forms suggested that multiplication or budding may have occurred. Pink-staining
regions similar to those seen in Pirhemocyton infections were not encountered.
Comments
Sauroplasma and Sauroplasma-like infections, have been previously described from
three species of lizards in South Africa (c. giganteus, Cordylus jonesii and Cordylus
vittifer), as well as from chameleons (Zonosaurus mascareniensisï and geckoes
(Uroplatus .fibriatus) in Madagascar, unidentified reptiles in Uzbekistan, and lizards
(Lacerta agilis) in Scandinavia (Du Toit (1937); Pienaar (1962); Brygoo (1963);
Uilenberg & Blanc (1966); Zakharyan (1970) and Svahn (1976)). Most of these
infections have been grouped with the piroplasms (see Du Toit (1937); Pienaar (1962);
Svahn (1976); Levine (1971, 1988); Johnston (1975); Frye (1991); Barnard & Upton
(1994) and Peirce (2000)), however, similar parasites in lizards (Gehyra variegata) in
Australia were considered to be early trophozoites of the haemoproteid Haemocystidium
Castellani & Willey, 1904 (see Johnston 1975).
Johnston (1975) also reported that some Sauroplasma infections can resemble the viral
infection Pirhemocyton. Davies & Johnston (2000) examined blood samples of Lacerta
agilis from Scandinavia containing both Sauroplasma and a Pirhemocyton-ïw:e infection.
Viral particles were found by TEM, but the authors were unable to state with certainty
that Sauroplasma is viral in nature.
By light microscopy, Sauroplasma thomasi could easily be interpreted as a piroplasm
infection, and is similar to the enigmatic infections of Serpentoplasma (seen in snakes),
Chelonoplasma Frye, 1981 (in tortoises) and Haemohormidium Henry, 1910 and
Haematractidium Henry, 1910 (from fishes). It could also be a viral infection, since the
albuminoid bodies of some Pirhemocyton infections are not unlike Sauroplasma (see
Davies & Johnston (2000)).
Current TEM studies (see Sauroplasma from Cordylus giganteus) have revealed that
Sauroplasma is probably not a typical apicomplexan because of the apparent absence of
an apical complex including rhoptries, mieronemes and other characteristic structures of
77
CHAPTER3 RESULTS
the Apicomplexa. However, when it is compared with Serpentoplasma from Python
sebae natalensis and Serpentoplasma from Bitis arietans by TEM they appear to be of
very similar infections, possibly of viral origin (see electron microscopy section of
Serpentoplasma infections of Bi/is arietans).
3.2.3. SAUROMELLA PIENAAR, 1954
Sauromella from Pachydactylus capensis (A. Smith, 1845)
Type species: Sauromella haemolysus Pienaar, 1954.
Type Host: Pachydactylus capensis (A. Smith, 1845).
Type Locality: On the farm Elandsfontein, Potchefstroom district.
Present study:
Host: Pachydactylus capensis (A. Smith, 1845).
Locality: On a farm Deelhoek, Bloemfontein district.
Sauromella haemolysus was found in the peripheral blood of Pachydactylus capensis
captured in the Bloemfontein district. Two males, one female and one juvenile were
infected. The parasites were intracytoplasmic and found in mature erythrocytes of which
900\10000 (9%) cells were infected. The effects on host cells were mostly minimal (Fig.
3.7A-F), but in some heavy infections large areas within the cell cytoplasm appeared to
be vacuolated (Fig. 3.7B, D-F (arrows)).
Sauromella comprised a small, round or streaked, purple or dark blue-stained area,
sometimes associated with a large vacuole (Fig.3. 7A, C, D, & E). The purple structure
appeared to become larger as it matured and exhibit peripheral granules (Fig.3. 7D & E
(red arrow)), and measured 1.79 to 2.57 urn by 0.94 to 1.48 urn (n= 21). Vacuoles were
of similar size, but sometimes slightly larger (Fig. 3.7B, E, & F). No evidence of division
was observed, but the pink-staining regions and vacuoles were similar to those seen in
viral Pirhemocyton infections. These infections were indistinguishable from those found
by Pienaar (1954) in the same host.
78
Figure 3.7. Sauromella haemolysus Pienaar, 1954 in Giemsa stained blood films from two C), one
~ and one juvenile (A-c), B-c), C-juvenile D-F, ~) Pachydactylus capensis (A. Smith, 1845)
specimens. A, C, D: Pigmented structures, B, E, & F: unstained and vacuolated apparent
structures. Red arrow in E points to stained and streaked structure. Scale: A-F: 10 um,
CHAPTER3 RESULTS
3.2.4. SERPENTOPLASMA PIENAAR, 1954
The following species are new host records of Serpentoplasma found in snakes. Their
morphology appears to be similar to Sauroplasma infections found in lizards, although
some resemble Pirhemocyton.
Serpentoplasma from Crotaphopeltis hotamboeia (Laurenti, 1768)
Host: Crotaphopeltis hotamboeia (Laurenti, 1768)
Locality: On the farm Deelhoek (28°54'S, 26°09'E), Bloemfontein district, Free State.
These infections were similar to those described by Pienaar (1962). They were found in
six (two males and four females) Crotaphopeltis hotamboeia (Laurenti, 1768)specimens,
collected in the Bloemfontein district. The structures were intracytoplasmic and occurred
as prominent round and oval bodies (Fig. 3.8A-C). They were found in mature
erythrocytes of which 700/1 0 000 (7%) cells were infected.
In Giemsa-stained blood films the structures occurred singly, or up to three in mature
erythrocytes. They were irregularly round, vacuole-like structures (Fig. 3.8A-C). The
main body of the structure measured l.8 ± 0.93 (1.4-2.3) urn by l.94 ± 0.7 (l.2-2.10) urn
(n=36). The centre of the parasite was largely unstained and vacuole-like, with no
narrow rim around its periphery. No pink-staining bodies similar to those seen in
Pirhemocyton infections from lizards were noted.
Serpentoplasma from Lycophidion capense capense (A. Smith, 1831)
Host: Lycophidion capense capen.<;e(A. Smith, 1831).
Locality: On the farm Deelhoek (28°54'S, 26°09'E), Bloemfontein district, Free State.
These infections were also similar to those described by Pienaar (1962). They were
found in two male specimens, collected in Bloemfontein and on a farm at Deelhoek in the
Bloemfontein district. The structures were largely vacuole-like intracytoplasmic and
80
CHAPTER3 RESULTS
occurred as small round and oval bodies (Fig. 3.8D-F). They were found in mature
erythrocytes of which 2700110 000 (27%) cells were infected.
In Giemsa-stained blood films parasites occurred singly in mature erythrocytes. The
main body of the structure measured 1.12 ± 0.84 (0.4-1.5)!lm by 0.97 ± 0.71 (0.5-1.64)
urn (n=20). The centre of the parasite was largely unstained and vacuole-like, with a
narrow rim around its periphery. Small pink-staining bodies similar to those seen in
Pirhemocyton infections from lizards were also noted (Fig. 3.8F).
Serpentoplasma from Elapsoidea sundevallii media Broadley, 1971
Host: Elapsoidea sundevallii media Broadley, 1971.
Locality: On the farm Deelhoek (28°54'S, 26°09'E), Bloemfontein district, Free State.
These Serpentoplasma infections also showed some similarities with Pirhemocyton.
They were found in three specimens collected on a farm Deelhoek in the Bloemfontein
district. The structures were intracytoplasmic and occurred as small oval bodies (Fig.
3.8G). They were found in mature erythrocytes of which 85110 000 (0.85%) of the cells
were infected.
In Giernsa-stained blood films the structures occurred singly in mature erythrocytes. They
were irregularly round, dark-purple structures with some dark pigment around the
•
periphery of the organism. The main body of the structure measured 0.98 ± 0.99 (0.68-
1.2) urn by 0.65 ± 1.2 (0.55-l.1) urn (n= 11) and its centre appeared solid.
81
CHAPTER3 RESULTS
Serpentoplasma from Hemachatus haemachatus (Lacêpéde, 1788)
Host: Hemachatus haemachatus (Lacépéde, 1788).
Locality: Near the village of Excelsior and in the village of Ha Rapokolana(29°22'36"S
28°01 '53E), Lesotho.
lntra-erythrocytic infections were found in two male specimens, collected near the village
of Excelsior in eastern Free State and in the village of Ha Rapokolanathe Lesotho
highlands. The structures were intracytoplasmic, measured 0.67 ± 0.57 (0.4-1.2) urn by
1.16 ± 0.6 (0.6-1.67) urn (n=30) and occurred as rounded vacuole-like structures, one in
each infected erythrocyte (Fig. 3.8H). They were found in mature erythrocytes of which
2700/1 0 000 (27%) cells were infected. Bodies with a purple-stained periphery (Fig.
3.8H (red arrow», and vacuole-like bodies (Fig. 3.8H (black arrows», were observed.
Serpentoplasma from Causus rhombeatus (Lichtenstein, 1823)
Host: Causus rhomheatus (Lichtenstein, 1823).
Locality: Clarens district (28°31 'S, 28°25'E), Eastern Free State.
These Serpentoplasma infections also showed great similarities with those of lizard
Pirhemocyton. They were found in one female specimen, collected on a road in the
Clarens district. The structures were intracytoplasmic, occurring as small or prominent
rounded, oval and elongate purple-stained bodies, with or without a vacuole or pale-
stained region of host cytoplasm (Fig. 3.8I-L (arrows». They were found in mature
erythrocytes of which 100/10 000 (1%) of the cells were infected.
In Giemsa-stained blood films the structures occurred singly in mature erythrocytes. The
effects on the host cells (Fig. 3.8I-L) were most evident in cells containing deep-stained
bodies and pale-stained cytoplasm (Fig. 3.81). The main body of the purple-stained
structure measured 2.68 ± 1.4 (1.89-2.90) urn by 3.89 ± 1.1 (2.24-4.2) urn (n=12).
82
Figu re 3.8. Serpentoplasma Pienaar, 1954 infections in Giemsa stained blood films
from different snake species: A-C: from Crotaphopeltis hotamboeia (Laurenti, 1768)
with prominent vacuole-like bodies. D-F: similar vacuole-like structures from
Lycophidion capense capense (A. Smith, 1831). G: from Elapsoidea sundevalli media
Broadley, 1971 with pink-stained bodies similar to Pirhemocyton Chatton & Blanc,
1914 infections. H: from Hemacatus haemachatus (Lacêpéde, 1788), with purple
staining periphery (red arrow). I-L: from Causus rhombeatus (Lichtenstein, 1823) (see
arrow in K) but also similar to Pirhemocyton consisting of purple stained bodies (I
(arrow». Scale: A-L: 10 urn
CHAPTER3 RESULTS
Serpentoplasma from Bitis arietans (Merrem, 1820).
Host: Bitis arietans arietans (Merrem, 1820).
Locality: Bloemfontein district, Free State.
Serpentoplasma infections were found in two specimens of Bitis arietans arietans. One
male and one female were sampled.
Light microscopy
The structures were intracytoplasmic and occurred as tiny round or oval bodies (Fig.
3.9A-C). They were found in mature erythrocytes of which 800110 000 (8%) cells were
infected.
In Giemsa-stained blood films parasites occurred singly in mature erythrocytes. They
were ring-like structures, without any small buds or protrusions. The main body of the
structure measured 1.1 ± 0.99 (0.9-1.4)!lm by 1.4 ± 0.81 (0.91-1.35)!lm (n=30), and was
largely unstained and vacuole-like, with no narrow rim around its periphery. No evidence
of division was seen.
Electron microscopy
Blood samples from the same specimen of Bitis arietans showed by TEM (Fig.3.9D-I)
that these Serpentoplasma infections were largely similar to Sauroplasma infections from
lizards (Fig.3.5G-L). Stages with rather fibrous and whorled contents, and particulate
matter were very close in appearance to those seen in Fig. 3.5G-J of Sauroplasma
infections. Furthermore, stages of Serpentoplasma were present with a vacuole-like
centre and marginal whorled material (Fig. 3.9G & H), also similar to those seen in
Sauroplasma (Fig. 3.5G, H). However, in Serpentoplasma, the forms with marginated
contents also showed small particles both within the body itself, and on the surface of the
host erythrocyte (Fig. 3.9H & I). These particles are reminiscent in position and
appearance to particles observed by Daly, Mayhue, Menna & Calhoun (1980) in the
erythrocytes of the yellowbelly water snake, Nerodia erythrogaster flavigaster. Daly et
al. (1980) interpreted their structures as resembling oncornaviruses.
84
Figure 3.9. A-C: Light micrographs of Serpentoplasma Pienaar, 1945 infections in
Giemsa stained blood films of the Puff adder (Bitis arietans arietans (Merrem, 1820).
D-I: transmission electron micrographs of Serpentoplasma infections in peripheral
blood cells of the puff adder (Bitis arietans) collected in the Bloemfontein district. D-
F: Structures filled with granular and fibrous material, and membranous whorls, G &
H: structures with marginated whorled contents. I: possible viral particles attached
inside vacuole and on surface of host erythrocyte. Scale: A-C: 10 urn, D: I urn, E &
G: 500 nm, F & H: 200 nm, I: 100nm.
CHAPTER3 RESULTS
3.3. PROTOZOA
3.3.1. HAEMOGREGARINES
All haemogregarines noted in these studies have been allocated the genus name
Hepatozoon, in line with the recommendations of Smith (1996). When their life cycles
are elucidated, however, they may prove to be Hemolivia, Schellackia or other genera of
haemogregarines reported from reptiles.
Hepatozoon sp. A from Agama atra atra Daudin, 1802
Host: Agama atra atra Daudin, 1802.
Localities: Bokong River near Ha Khojane (28°22'S, 28°01 'E), Lesotho. On the farm
Zuurfontein (29°54'S 25°22'E) Jagersfontein district, Southern Free State.
Hepatozoon sp. A was found in three of three Agama atra atra captured in three areas in
the Bokong catchment. The first site was a river bank near the village of Ha Khojane
(28°22'S, 28°01 'E), where a male specimen was collected (Fig. 3.10A-I). Another male
was caught roughly halfway up the adjacent mountain on its southwest facing slopes
(29°18'S, 28°19'E) with the same infection. A gravid female was collected above the
snowline on top of the mountain (29°18'S, 28°07'E), and had a similar blood picture.
This parasite was also found in seven other specimens of the same host from the southern
parts of the Free State on the farm Zuurfontein (29°54'S 25°22'E). All these lizard
specimens had morphologically the same infections of Hepatozoon and Pirhemocyton.
The haemogregarines were intracytoplasmic and found in mature erythrocytes. Mature
and immature stages of these parasites were noted.
In Giemsa-stained blood films parasites occurred singly in erythrocytes. As noted above,
these infections were accompanied by Pirhemocyton infections (Fig. 3.10B, E, F, H & I
(blue arrows)), either in the same cells as the haemogregarine or in erythrocytes lacking
86
Figure 3.10 A-I: Light micrographs of Giemsa stained blood films showing
Hepatozoon Miller, 1908 sp. A infecting red blood cells of Agama atra atra Daudin,
1802 collected near the village of Ha Khojane in Lesotho. B, E, F, H & I (Blue
arrows)): Pirhemocyton Chatton & Blanc, 1914 infections accompanying the
Hepatozoon sp. A infection. B & C (black arrows)): slightly elongated immature
stages, A, D & G (black arrows): pale whitish cap at the anterior end of the more
mature parasites. Scale: A-I: 10 urn,
CHAPTER3 RESULTS
the apicomplexan. Only gamont stages of the haemogregarine were recognised in blood
films and these were typically only slightly elongate when immature (Fig. 3.10 B-C
(black arrows)). The cytoplasm of these immature gamonts stained pale blue with
Giemsa, and the nucleus, which lay slightly nearer the posterior end, consisted of
stranded, pale-stained chromatin. Cytoplasm contained dark peripheral granules in the
vicinity of the nucleus. Immature stages measured 4.2 ± 0.7 (3.1-5.3) urn by 3.8 ± 0.85
(2.7-4.9) urn (n= 12).
Mature gamonts were found in 220\10000 (0.2%) of mature erythrocytes and were
elongate. These measured 8.8 ± 2.06 (7.31-10.36) urn long by 3.7 ± 0.93 (3.54-3.9) urn
wide (n = 62). They were generally pale-stained with Giemsa, while the nucleus stained
pale pink and the anterior end of the parasites bore a pale, almost whitish, cap (Fig.
3.10A, D & G (black arrows)). The nucleus measured 3.1 ± 0.9 (2.7-3.5) urn by 3.6 ± 0.8
(3.4-3.9) urn (n = 26). The nucleus was located in the posterior half of the parasite body.
Comments
These infections are the first haemogregarines reported from Agama atra atra in South
Africa. The parasite differs from all other similar species in its overall morphological
dimensions: mature gamonts are much smaller than cordylid haemogregarines and have a
pale stained nucleus. This is the first description of a Hepatozoon species from Agama
atra atra. In comparison with an unnamed haemogregarine described from the agamid
Agama ruppelli by Ball (1967) from East Africa, the gamont stages in A. ruppelli
measured 9.8 by 4.31lm with a lightly stained nucleus and prominent granules in the
cytoplasm, while those of Hepatozoon sp. A gamonts were smaller (8.8 ± 2.06 (7.31-
10.36) urn long by 3.7 ± 0.93 (3.54-3.9) urn wide (n=62)). Hepatozoon sp. A were also
generally pale-stained with Giemsa, while the nucleus stained pale pink and the anterior
end of the parasites bore a pale, almost whitish, cap and did not have the granules
characteristic of the parasite from A. ruppelli. It is interesting that Hepatozoon sp. A was
found in Agama atra atra located in disjunct populations.
88
CHAPTER3 RESULTS
Hepatozoon sp. B from Pseudocordylus melanotus melanotus (A. Smith, 1838)
Host: Pseudocordylus melanotus melanotus (A. Smith, 1838).
Locality: Clarens (28°31 'S, 28°25'E), Eastern Free State.
Hepatozoon sp. B was found in all five Pseudocordylus melanotus melanotus (A. Smith,
1838) captured at an isolated rocky outcrop (28°31 'S, 28°25'E) near Clarens in the
Eastern Free State. The infected lizards were two males and three females (of which one
also had a Plasmodium infection (Fig. 3.11A-C). The haemogregarines were usually
intracytoplasmic, found in mature erythrocytes. Extracellular and immature stages were
noted. Effects on host cells were marked, the mature stages displacing the host nucleus
to the side of the erythrocyte and apparently reducing its width by up to 50 %.
In Giemsa-stained blood films parasites occurred singly in erythrocytes and extracellular
parasites were also present (Fig. 3.11H-I). Only gamont stages were recognised in blood
films and these were typically rather elongate when immature (possibly those in Fig.
3.11A-C, but these could also be malarial parasites). These immature gamonts stained
purple with Giemsa and their nuclei were difficult to discern. Immature stages measured
2.5 ± 0.95 (2.25-4.4)!Jm by 4.2 ± 0.87 (3.1-7.21)!Jm (n=10).
Mature gamonts infected 6850110 000 (69%) of cells and were elongate structures
sometimes with a slightly retlexed posterior region. The main body of the parasite
measured 19.2 ± 0.83 (18.4-20.7) urn by 6.2 ± 0.7 (5.9-6.6) urn (n=23). The mature
gamont stained mostly deep purple to purple with Giemsa and its cytoplasm was
relatively free of granules, while its nucleus stained reddish purple and comprised dense
strands of chromatin. The anterior end of the parasite bore a characteristic pinkish cap
(Fig. 3.11E (arrows». The nucleus measured 5.8 ± 0.9 (5.5-6.4) urn (n=23) and was
located nearer to the posterior end of the parasite body.
89
H 1
Figure 3.11. Light micrographs of Hepatozoon Miller, 1908 sp. B. from
peripheral blood of Pseudocordylus melanotus melanotus (A. Smith, 1838).
collected in Clarens in the eastern Free State. A-C: possible immature stages of
Hepatozoon sp. B or perhaps a species of Plasmodium Marchiafava and Celli,
1885. D-G: mature gamonts inmature erythrocytes. B-1: Extracellular gamont in
capsule (arrow). I: Free gamont. Scale:A-I: 20 urn.
CHAPTER3 RESULTS
The intraerythrocytic mature gamont was surrounded by a purple-stained capsule (see
Fig. 3.11H which shows an extracellular example). This was only slightly bigger than
the gamont. Extracellular mature gamonts appeared longer and narrower than their
intracellular counterparts when free of the capsule and measured 21.4 urn by 3.2 urn
(n=l ). The tail regions of these extracellular forms were slightly curved.
Comments
This investigation led to the first description of this parasite from Pseudocordylus
melanotus melanotus in Clarens (28°31 'S, 28°25'E), Eastern Free State. It compared
with the general morphology of Haemogregarina boskani Catouillard, 1909 from a
Tunisian lizard Acanthodacty/us boskianus. Haemogregarina boskani gamonts were oval
or slightly reniform with sometimes a broader end than the other, while Hepatozoon sp. B
differed in having rounded nuclei and cytoplasm relatively free of granules. It was also
wider than all of those haemogregarines described in the Cordylidae in the current
survey. Hepatozoon sp. B has nuclear dimensions of 5.8 ± 0.9 (5.5-6.4) urn (n=23) and
the nucleus is located nearer to the posterior end of the parasite body. The overall
dimensions of Hepatozoon sp. B were 19.2 ± 0.83 (18.4-20.7) urn by 6.2 ± 0.7 (5.9-6.6)
urn (n=23). Haemogregarina boskani measured 8.5-16.5 urn by 4-16 urn with prominent
chromatophillic granules. Furthermore, Hepatozoon sp. B has characteristically purple to
deep purple-stained capsules with a light purple cytoplasm and a rounded compact
slightly posteriorly situated nucleus that are not common in other haemogregarines found
in lizards.
Hepatozoon sp. C from Pseudocordylus melanotus subviridis (A. Smith, 1838)
Host: Pseudocordylus melanotus subviridis (A. Smith, 1838).
Locality: Ha Thaba Bosiu (29°20'15"S 28°}} '58"E), Lesotho.
Hepatozoon sp. C was found in seven of eight Pseudocordylus melanotus subvindis
captured at Ha- Thaba Bosiu in Lesotho. The infected lizards were five males and three
91
Figure 3.12. A-F: Light micrographs of Hepatozoon Miller, 1908 sp. C collected from the
peripheral blood of Pseudocordylus melanotus subviridis (A. Smith, 1838) in the Syncunjane river
catchment near the village Ha Thaba Bosiu. A-F: Various gamont stages parasitizing mature
erythrocytes. A: immature gamont with dense nucleus near the posterior end, note the polar cap at
the anterior end (arrow). B: slender gamont with dense pigmentation along one extremity (arrow),
note the lateral displacement of the host cell's nucleus. C, D, E: different gamonts with dense
nuclear pigmentation. F: mature gamont in mature erythrocyte with a compact dense nucleus.
The effect on the host cell is marked with a displacement of the host cell nucleus. Scale: A-F: 10
um,
CHAPTER3 RESULTS
females. The parasites were intracytoplasmic and found in mature erythrocytes, of which
1000/10 000 (10%) of cells were infected.
Effects on host cells were marked, the mature stages enlarging the host cell and
displacing the host nucleus to one side or the end of the erythrocyte (Fig 3.12A-F).
Sometimes the host nucleus was reduced in size (Fig 3.12D, F & G).
In Giemsa-stained blood films parasites occurred singly in a single erythrocyte.
Extracellular parasites were not detected and only mature gamont stages were recognised.
Mature gamonts (Fig. 3.12A-F) were elongate structures with a slightly reflexed posterior
region (Fig 3.12B (arrow)). The main body of the parasite measured 19.5 ± 0.89 (17-25)
urn by 2.24 ± 0.99 (2.30-3.52) urn (n=30).
In mature gamonts the cytoplasm stained pale pink or purple with Giemsa, and the
centrally or posteriorly placed nucleus (measuring 3.45 ± 0.5 (3-4)!lm by 1.61 ± 0.44 (2-
4) urn (n=30) consisted of banded chromatin that stained purple. Parasite cytoplasm also
contained purple-stained granules posterior to the nucleus (Fig. 3.12F). The anterior end
of the mature gamont sometimes bore a characteristic cap (Fig. 3.12A (arrow)). A
capsule was not visible
Comments
This is the first description of a haemogregarine in the Cordylidae from Lesotho. This
new parasite seems to be very narrow in comparison to the rest of Hepatozoon
descriptions in this thesis, but broader than Hepatozoon gracilis from an Egyptian skink
(Bashtar, Abdel-Ghaffar & Shazly 1987). Haemogregarina acanthodactylii Ramadan,
1974 described from an Egyptian lizard Acanthodactylus boskianus by Ramadan (1974)
had broad forms with rounded ends, a rounded, centrally placed, nucleus and red staining
granules. These measured 10-11 by 5.5-5.6!lm for the broad forms and 11.5-13.5 by 4-5
urn for the slender ones. However, Hepatozoon sp. C was longer and its morphometrical
dimensions were (19.5 ± 0.89 (17-25) urn by 2.24 ± 0.99 (2.30-3.52) urn (n=30) in
contrast with Haemogregarina acanthodactylii. These slender gamonts of Hepatozoon
93
CHAPTER3 RESULTS
sp. C have a marked effect on host cells when mature, which can mean displacement of
the host cells nucleus to one extremity of the cell, where the nucleus itself is laterally
compacted.
Hepatozoon sp. D from Pseudocordylus melanotus subviridis (A. Smith, 1838)
Host: Pseudocordylus melano/us subviridis (A. Smith, 1838).
Locality: Jorotane river near village of Ha Rapokolana (29°22'36"S 28°01' 53E),
Lesotho.
Hepatozoon sp. 0 was found in all of Pseudocordylus melanotus subviridis captured at
the Jorotane Catchment in Lesotho. The infected lizards were two males and two
females. The parasites were intracytoplasmic and found only in mature erythrocytes and
1850/10000 (19%) of cells were infected. Effects on host cells were marked, the mature
stages displacing the host nucleus to one side and enlarging the host cells (Fig. 3.13A, D,
E, F, G, H, K & L). Enlarged host cells were also sometimes de-haemoglobinised (Fig.
3.13A, 0, F, H, K, L & M).
In Giemsa-stained blood films parasites occurred singly in mostly mature erythrocytes
and extracellular parasites were also present. Only gamont stages were recognised in
blood films and these were typically rather elongated even when very immature (Fig.
3.13A (red arrow)). These very young gamonts stained lightly purple with Giemsa, and a
centrally placed nucleus consisting of granular chromatin stained dark purple with
Giemsa. Cytoplasm contained two distinct pink-stained areas on either side of the host
nucleus. Immature gamonts measured 8.5 ± l.5 (5.2-10.1) urn long and 2.2 ± 5.7 (2.0-
2.9) urn (n=11) wide.
Maturing and mature gamonts were elongate structures often with a retlexed posterior
region when intracellular. The mature gamont stained mostly pale purple with Giemsa
and its cytoplasm contained abundant characteristic pink-stained granules (Fig. 3.13E).
lts nucleus stained pink and comprised large granules of chromatin (Fig. 3.13E (arrow)).
94
CHAPTER3 RESULTS
The anterior end of the parasite bore a characteristic pink-stained cap (Fig. 3.13A, F, G,
L, M (arrows)). The nucleus measured 5.2 ± 0.7 (4.8-6.61) urn by 3.64 ± l.12 (3.1-3.92)
95
Figure 3,]1.3. Light micrographs of Hepatozoon Miller, 1908 sp. D. from peripheral
blood of Pseudocordylus melanotus subviridis (A. Smith, 1838) collected in the Jorotane
River catchment in Lesotho. A-C (red arrows): Immature gamonts. A-M: mature
gamont stages in mature erythrocytes. Black arrows of A, IF, G, L & M: indicate the
polar caps, arrows of lE: indicate nuclear granules. A, 11), lE, IF,G, H, K, & L: mature
stages displacing the host nucleus to one side and causing severe hypertrophy. A:
immature and mature gamonts, B: maturing and mature gamonts. C: possible early
stages of host cell occupation. D: damaging effects of a mature gamont on the host cell.
E: different nuclear positions, note the folding of the tail. IF:Dehaemoglobinisised effect
on the host cell, arrow shows polar cap. G: folding of the haemogregarine with lateral
displacement of host cell nucleus. H, K, L & M: dehaemoglobinisised host cells B, I, &
J: characteristic folding of mature gamonts around host cell nucleus with maturing
gamont in M: (black arrow). N: extracellular gamont. Scale: A-N: l Oum

CHAPTER3 RESULTS
urn (n=12) and was located centrally (Fig. 3.111) or commonly nearer to the anterior end
of the parasite body (Fig. 3.11B). The main body of the parasite measured 25.9 ± 0.8
(25.1-27.05) urn by 5.25 ± 0.98 (4.98-5.56) urn (n=20), with a reflexed tail measuring
5.87 ± 0.94 (5.25-7.84) urn (n=12), that was therefore roughly a third of the length of the
main body of the haemogregarine. Extracellular mature gamonts were rather worm-like
in appearance, appearing much longer and thinner than their intracellular counterparts
(Fig. 3.13N). They measured 351lm by 3.2 urn (n=2). The tail regions of these extra
cellular forms were often, though not invariably, reflexed.
Comments
These are most likely new host and species records. The gamonts differ from those of
other species of Hepatozoon found in such lizards by having a characteristically large,
curved granulated body and a heavily granulated nucleus consisting of both large and
small granules. It is a little similar morphologically to described species like those of
Haemogregarina damiettae Ramadan, Saoud, Mohammed & Fawzi, 1996 in an Egyptian
lizard Acanthodactylus hoskianus (see Ramadan, Saoud, Mohammed & Fawzi, 1996).
One end of this haemogregarine was broader with the nucleus closer to one end than the
other, but it measured 15 Ilm-16.5 urn by 2.5 Ilm-7.5 urn whereas Hepatozoon sp. D
measures 25.9 ± 0.8 (25.1-27.05) urn by 5.25 ± 0.98 (4.98-5.56) urn (n=20) and differs
from Haemogregarina damiettae in the absence of a thick sheath and in its nuclear
arrangements. The current species has a distinct nuclear arrangement with heavily pink
staining chromatin with one or two darker and larger granules. Although the nuclei of
Haemogregarina damiettae are in the same way diffused they consist of discreet strands
whereas Hepatozoon sp. D has a granulated nucleus which measures 5.2 ± 0.7 (4.8-6.61)
urn by 3.64 ± 1.12 (3.1-3.92) urn (n=12) and is located centrally. Infections of
Hepatozoon sp. 0 had a severe effect on the host cells. Infections of mature gamonts
caused dehaemoglobinised areas in some host cells. The gamonts can also occupy all or
most of the host cell cytoplasm and displace the nucleus to one side of the cell (Fig.
3.13A, E, G, H, K, L, & M).
97
CHAPTERJ RESULTS
Hepatozoon sp. E from Pseudocordylus melanotus subviridis (A. Smith, 1838)
Host: Pseudocordylus melanotus subviridis (A. Smith, 1838).
Localities: In the village of Ha Rapokolana (29°22'36"S 28°01 '53E), Lesotho.
Hepatozoon sp. E was found in three of three Pseudocordylus melanotus subviridis
captured in the village of Ha Rapokolana in the Jorotane catchment. Three male
specimens were collected and sampled.
The parasites were intracytoplasmic and found in mature erythrocytes of all three male
specimens and 550110 000 (5.5%) of cells were infected in one of them. Effects on host
cells were marked, both the immature and mature stages displacing the host nucleus to
the side of the erythrocyte (Fig. 3.14A-F). In most cases the host nucleus was laterally
compacted (Fig. 3 .14A-F (black arrows».
In Giemsa-stained blood films parasites occurred singly in an erythrocyte. These
infections were accompanied by Pirhemocyton or (Saurop/asma-like) infections (Fig.
3.14A-F (blue and red arrows), but not in the same cells. Only gamont stages were
recognised in blood films and these were typically rather oval when immature (Fig.
3.14A). Immature gamonts stained purple with Giemsa, the possible posteriorly placed
nucleus consisting of granular chromatin stained dark purple with Giemsa. Their
cytoplasm contained dark granules peripherally and a large vacuole. Immature stages
measured 3.6 ± 0.5 (3-4.4) urn by 4.8 ± 0.71 (4.5-6.2) urn (n=11).
Mature gamonts were thin, elongate structures with rounded extremities, measuring 11.46
± 0.41 (9.24-13.8) urn by 2.4 ± 0.4 (2.2-3.54) urn (n=30). The mature gamont stained
mostly deep blue purple to purple along its sides with Giemsa and its cytoplasm was
finely granulated. Nuclei stained pale reddish purple and comprised finely stranded
chromatin (Fig.3.14A-F (black arrows». The nucleus measured 2.4 ± 0.47 (2.1-3.2) urn
by 3.15 ± 0.66 (2.4-3.7) urn (n=20) and was located towards the anterior half of the
parasite body.
98
Figure 3.14. A-F: Light micrographs of Hepatozoon Miller, 1908 sp. E. from the
peripheral blood of Pseudocordylus melanotus subviridis (A. Smith, 1838) captured in
the village of Ha Rapokolana in the Jorotane catchment. A-F (black arrows): show
lateral compacted host nuclei. A-F (blue arrows): Pirhemocyton Chatton and Blanc
1914 -like infections. C & D (red arrows): multiple infection centres of a possible
Pirhemocyton infection. A: Immature stage of the haemogregarine. Scale: A-F: l Oum.
CHAPTER3 RESULTS
Comments
These infections most likely represent new records for Pseudocordylus me/anotus
subviridis in its locality and new species compared with all others described. Hepatozoon
sp. E differs from all other species in the overall morphological dimensions: the mature
gamont is much slimmer than others described from cordylid lizards with
characteristically pigmented granules in the cytoplasm and nucleus. It shows some
resemblance to the general morphology of Haemogregarina capensis Conor, 1909 from
an Egyptian lizard Acanthodacty/us pardalis, but this has a nucleus in the form of a
rectangular band. Hepatozoon sp. E has a characteristically granulated nucleus
measuring 2.4 ± 0.47 (2.1-3.2) urn by 3.15 ± 0.66 (2.4-3.7) urn (n=20). Haemogregarina
capensis measured Sum by 3.4 urn and slightly affected the host cell with a displaced
host cell nucleus. Hepatozoon sp. E gamonts measured 11.46 ± 0.41 (9.24-13.8) urn by
2.4 ± 0.4 (2.2-3.54) urn (n=30) and displaced the host nucleus and in some infections
caused dehaemoglobinised areas in the host cell.
Hepatozoon (Haemogregarina) sebae (Laveran and Pettit, 1909) Smith, 1996 from
Python sebae natalensis (Gmelin, 1789)
Type Host: Python sebae (Gmelin, 1789)
Type Locality: Senegal.
Present study
Host: Python sebae nata/ensis (Gmelin, 1789).
Locality: Bloemfontein, Free State (snake bred in captivity by a licenced breeder).
Hepatozoon (Haemogregarina) sebae (Laveran and Pettit, 1909) Smith, 1996 was found
in an African rock python, Python sebae (Gmelin, 1789) in Senegal, North Africa. The
same species was found in the present study, but in a different variety (Southern race) of
African rock python (Python sebae natalensist This subspecies has larger shields and a
different colour pattern than the typical race (Branch 1998).
100
CHAPTER3 RESULTS
The current investigation led to the discovery of Hepatozoon sebae in a captive African
rock python (Python sehae natalensis) (permit nr: HKlPI9C/03894/002 (appendix A)) in
the Bloemfontein district. The infected snake was a female. The parasites were
intracytoplasmic and found in mature erythrocytes of which 450/1 0 000 cells were
infected. Effects on host cells were marked, the mature stages displacing the host nucleus
to one side of the erythrocyte.
Light microscopy
In Giemsa-stained blood films parasites occurred singly in erythrocytes. Extracellular
parasites were absent. Only gamont stages were recognised in blood films and these
appeared to be mature (Fig. 3.15A-C). These stages stained bluish purple, with a nucleus
that stained deep purple with Giemsa, positioned near the posterior end of the parasite.
Cytoplasm contained coarse dark granules, especially in the anterior portion of the
parasite (Fig. 3.15A-C).
Mature gamonts were overall elongated and slightly curved structures with rounded ends
(Fig.3.15A-C). The main body of the parasite measured 11.36 ± 1.1 (10.4-12.33)!lm
long and 3.8 ± 1.2 (3.22-4.51)!lm wide (n=10). A small pinkish-red cap occurred on the
anterior end of the parasite, and the entire haemogregarine appeared to be surrounded by
a capsule. The nucleus measured 2.3 ± 2.5 (2.1-2.5) by 4.42 ± 2.7 (4.24-4.46) urn (n=I 0)
and was located nearer to the posterior end of the parasite body. The parasite closely
resembled the type description of Laveran & Pettit (1909).
Electron microscopy
Initial observations on Hepatozoon (Haemogregarina) sehae by TEM showed that the
gamont had a typical apicomplexan structure. The extracellular haemogregarine shown
in Fig. 13D-F is surrounded by a typical plasma-membrane or membranous sheath
(pellicle) (Fig. 3.15F) and it contains abundant mieronemes (Fig. 3.15E & F), a few
rhoptries (Fig. 3.15E & F) and a distinct nucleus (Fig. 3.15D & E). A capsule is not
visible surrounding this extracellular form. Serpentoplasma-like structures similar to
those oflizards (See Fig. 3.5G-L) and of snakes (Fig. See Fig. 3.9D-l) were also seen
101
Figure 3.15. A-C: light micrographs of Hepatozoon (Haemogregarina) sebae (Laveran and
Pettit, 1909) Smith, 1996 in Giemsa stained films of peripheral red blood cells of a captive
African rock python (Python sebae natalensis (Gmelin, 1789) ). D-F: Transmission electron
micrographs of infections from peripheral erythrocytes of the same python (Python sebae
natalensis), D-F: extracellular haemogregarine showing the gamont nucleus (gn), rhoptries
(rh) mieronemes (mn) and pellicle (P) in F. E & F: abundant mieronemes (mn) and rhoptries
(rh). D & E: (gn): gamont nucleus. Scale: A-C: 10 urn, D: 2 urn, E: 1 urn, F : 500 nm.
CHAPTER3 RESULTS
(not shown). Further TEM studies are needed to elucidate the fine structure of these
enigmatic infections and those of the apicomplexan in any detail.
Hepatozoon sp. F from Psammophylax tritaeniatus (Giinther, 1868)
Host: Psammophylax tritaeniatus (Gunther, 1868).
Locality: Bloemfontein, Free State.
Hepatozoon sp. F was found in all five Psammophylax tritaeniatus (Gunther, 1868)
specimens captured in the Bloemfontein district. The infected snakes were two males,
two females and a juvenile. The parasites were intracytoplasmic and found in mature
erythrocytes of which an average of 150/10000 (1.5%) of cells were infected. Effects on
host cells were marked, the mature stages displacing the host nucleus to one side of the
erythrocyte and causing severe dehaemoglobinisation of the host cells. The juvenile male
snake had immature gamonts (Fig. 3.16A-C), with the host erythrocyte nuclei showing
lateral displacement and compaction. The young female snake had a series of
developmental stages in its cells (Fig. 3.16D-F). A gravid female (Fig. 3.16G-I) and two
adult males (Fig. 3.16J-L) and (Fig. 3.16M-O) respectively, had mature gamonts in their
blood and a severe effect on the morphology of the host cells was noted. The host
nucleus was compacted to about half of its original width. The cytoplasm was severely
dehaemoglobinised and the cell perimeter stretched to about twice its original size.
In Giemsa-stained blood films parasites occurred singly in erythrocytes. Extracellular
parasites were absent. Only gamont stages were recognised in blood films and these
were typically elongate when immature (Fig. 3.16A-E). These immature gamonts stained
pale blue with Giemsa, and the nucleus, which was located centrally, or slightly
posteriorly, consisted of banded chromatin and stained a deep purple with Giemsa.
Immature stages were small and measured 7.8 ± 0.96 (7.1-8.5) urn long and 4.1 ± 0.7
(3.5-4.7) urn wide (n=15), with nuclei 3.32 ± 0.7 (2.88-3.76) by 4.56 ± 0.79 (4.30- 4.94)
urn (n=15).
103
B c
Figure 3.16. Á-O: light micrographs of Giemsa-stained blood films showing
Hepatozoon Miller, 1908 sp. F from the peripheral blood of five different specimens
of striped skaapstekers (Psammophylax tritaeniatus (GUnther, 1868)) collected in
the Bloemfontein district. Á-E: immature gamonts. Á-C: immature stages from a
young 0 20cm SVL. D-F: immature stages and mature stage F: ofa young ~ 31cm
SVL. G-I: different maturity stages of gamonts from a gravid ~, 61cm SVL. J-L:
mature gamonts from a mature 0 (62cm SVL.) showing cell degradation. M-O:
mature gamonts from a mature 0 showing severe hypertrophy of the cells and
hemoglobin loss. F: mature gamont showing rounded anterior and posterior regions,
(arrow). Scale: Á-O: 10 um,
CHAPTER3 RESULTS
Mature gamonts were also elongate structures with rounded anterior and posterior regions
(Fig. 3.16F (arrow». The main body of the parasites measured 12.65 ± 0.83 (10.2-15.1)
urn long by 6.02 ± 0.88 (5.4-6.65) urn (n=35). The mature gamonts stained mostly pale
purple with Giemsa and their cytoplasm largely lacked granules, while their nuclei
stained dark purple and comprised strands or granules of dense chromatin. A cap was
detected at the anterior end of some mature gamonts, but was not visible in most
parasites. The nucleus measured 4.5 ± 0.6 (4.14-4.97) by 5.01 ± 0.79 (4.57-5.46) urn
(n=20). In mature gamonts, and as in immature forms was mostly located in the posterior
half of the parasite body.
Comments
This is the first record and description of a haemogregarine from Psammophylax
tritaeniatus in South Africa One distinguishable characteristic of this species is the
damage it causes to the host cell. The host cell cytoplasm is often robbed of
haemoglobin, and may increase in volume (hypertrophy) and its nucleus is distorted.
Infected cells are therefore changed in form and their function may be altered. This
species shows some resemblance to the dimensions of Hepatozoon bitis (Fantham, 1925)
Smith, 1996 found in the puff adder, but now considered a synonym (according to Peirce
& Bengis (1998) of Hepatozoon dogieli Hoare, 1920). The dimensions of H. bitis were
12.5-15.3 urn and for H. dogieli 14 by óurn. Hepatozoon sp. F had dimensions of 12.65
± 0.83 (10.2-15.1) urn long by 6.02 ± 0.88 (5.4-6.65) urn (n=35). This species was also
compared with the descriptions given by Sambon & Seligmann (1907) of Hepatozoon
stattocki Sambon & Seligmann, 1907 in the diamond snake Python (Mm-elia) spilotes. It
had nearly the same morphometrical dimensions, but effects on the red cells were
different. Whereas the current parasites compressed and elongated the nucleus, H.
stattocki had little or no influence on the cell, with the nucleus only slightly displaced and
parasite occupying nearly half the cell.
105
CHAPTER3 RESULTS
SAUR1AN MALARIA
3.3.2. PLASMODIDAE
A number of endoglobular pigmented parasites were found in representatives of the
Cordylidae, Agamidae and Gekkonidae. Apart from previous descriptions by Pienaar
(1962) and Garnham (1966), Ayala (1977) listed nine species of Plasmodium from
African lizards and Mutinga & Dipeolu (1990), noted 10more that they had found in
Kenya (see Dipeolu & Mutinga 1989). Mutinga & Dipeolu (1990), focusing particularly
on Mabuya striata and Agama agama, also reported that these lizards could carry up to
four concurrent infections with different malaria species.
Clearly, Plasmodium species in lizards can be difficult to identify, particularly if they
form mixed infections. For this reason, precise identification of the species (or mixed
species) found in the current work awaits further study, especially as some infections
were found in only one or two lizards. Preliminary searches through the literature, do
suggest that at least some of these infections will eventually prove to be new species.
However, they are designated Plasmodium species A, B, C & D at present.
Plasmodium sp. A from Cordylus polyzonus polyzonus A. Smith, 1838
Host: Cordylus polyzonus polyzonus A. Smith, 1838
Locality: On the farm Zuurfontein (29°54'S 25°22'E) Jagersfontein district, Southern
Free State.
Plasmodium sp. A infections were discovered in the blood of COl-dylus polyzonus
polyzonus A. Smith, 1838 in 17 of 38 specimens captured on a farm Zuurfontein in the
Jagersfontein district, Southern Free State. These infections appeared different from all
descriptions given for malarias occurring in the family Cordylidae in South Africa.
106
Figure 3.17. Light micrographs of Giemsa stained blood films showing Plasmodium
Marchiafava and Celli, 1885 sp. A from Cordylus polyzonus polyzonus A. Smith,
1838 captured on the farm Zuurfontein in the Southern Free State. A-F: (black
arrows): trophozoites with fine malaria pigment, E: two trophozoites located at
opposite poles of the host cell D-F: different maturing meronts (E & F) (blue
arrows). E, F, & H: coarse pigment granules within erythrocyte cytoplasm (arrows).
G-I: gametocytes and meronts, G: micro gametocyte, H: meront probably yielding
16 merozoites, I: macrogametocyte (yellow arrow) with meront (red arrow). Scale:
A-I: 10 urn,
CHAPTER3 RESULTS
Trophozoites
The earliest asexual forms of the parasites were situated mostly singly in a polar position
within mature erythrocytes. Some erythrocytes contained two trophozoites located at
opposite poles of the host cell (Fig. 3.17E). These trophozoites were mostly round to
oval in shape, but sometimes they were rather triangular, or elongate with extensions
(Fig.3.17A-C). They measured 2.21 ± 0.98 (1.12-4.89) urn by 3.1 ± 1.45 (2.15-5.22) urn
(n=10) in diameter. Trophozoites stained dark purple with Giemsa around their
periphery, while the vacuole-like centre was relatively unstained. Fine malaria pigment
granules were found within some trophozoites, and these, and much coarser pigment
granules were found scattered throughout the blood films (Fig 3.17B, E, F & H). Host
cells containing trophozoites and other stages of this parasite (below) were largely of
normal appearance.
Meronts
These stages varied from round to rather triangular in shape (Fig.3.17F (blue arrow), I
(red arrow) & H) and were also polar within erythrocytes. Parasite cytoplasm became
increasingly eosinophilic as development of the meront progressed (compare Fig. 3.17F
(black arrow) with Fig 3.171 (red arrow)), and merozoite nuclei were seen forming a deep
purple band around the perimeter of the maturing meront (Fig 3.171 (red arrow)) these
measured 4.48 ± 3.4 (3.4-5.56) urn across (n=11). As they approached maturity,
triangular or elongate meronts were seen to contain at least 16 merozoites (Fig. 3.17H)
and measured 6.25 ± 4.1 (5.1-7.4) urn (n=13) in diameter.
Gametocytes
Two distinct types of gametoeytes were observed and because of their small size, both
were suspected to represent immature stages (Fig. 3.17G & 1 (yellow arrow)). Both types
lay in a polar position within the host erythrocyte, and often a trophozoite shared the
same host cell (Fig. 3.17D, I). The presumed male gametocyte (microgametocyte)
(Fig.3.17G) was round and stained deep purple with an eccentrically placed red-stained
nucleus. Presumed macrogametocytes were also round in shape (Fig. 3.171 (yellow
108
CHAPTER3 RESULTS
arrow» and measured 8.8 ± 2 (8.2-9.4) by 6.17 ± 1.4 (5.8-6.54) urn (n=12) and stained
light purple, with a peripherally placed nucleus stained deep purple or red.
Comments
When compared with Plasmodium zonurae Pienaar, 1962 the trophozoites of this species
were smaller (2-2.8) urn than Plasmodium sp. A (2.21 ± 0.98 (1.12-4.89) urn (n=10) in
diameter). Plasmodium zonurae trophozoites also had a light blue cytoplasm with
reddish blue chromatin, while Plasmodium sp. A stained a dark purple with Giemsa along
the periphery of the trophozoite with fine pigment in the centre of the parasite. The
occurrence of fine pigment in Plasmodium sp. A was one of the main differences
between these parasites.
Young meronts of Plasmodium sp. A measured 4.48 ± 3.4 (3.4-5.56) urn across (n=l l )
and more mature ones measured 6.25 ± 4.1 (5.1-7.4) urn (n=13) in diameter. Young
segmenters were seen to produce at least 16 merozoites, whereas P. zonurae produced an
average of 18 merozoites. Microgametocytes measured 12.5 ± 4.0 (12.3-12.8) urn by 6.4
± 3.1 (6.2-6.6) urn (n=12) in contrast with 8-8.4 by 4.2 urn for P. zonurae. Plasmodium
sp. A was overall bigger than the Plasmodium described from Cordylus vittifer by
Pienaar (1962) and carried considerably more pigment.
Plasmodium sp. B from Pseudocordylus melanotus melanotus (A. Smith, 1838)
Host: Pseudocordylus melanotus melanotus (A. Smith, 1838).
Locality: Clarens (28°31 'S, 28°25'E), Eastern Free State.
Plasmodium infections were discovered in the blood of in one specimen of
Pseudocordylus melanotus melanotus captured on a rocky outcrop in the Clarens district,
Southern Free State and in the village of Ha Rapokolana(29°22'36"S 28°01 '53E),
Lesotho. The lizard also contained a Sauroplasma-like infection (Fig.3.18 (blue
arrows».
109
Figure 3.18. A-R: light micrographs of lizard malaria and Sauroplasma Pienaar, 1937
infections of three lizards. A-E: light micrographs of Plasmodium Marchiafava and Celli, 1885
sp. B from Pseudocordylus melanotus melanotus (A. Smith, 1838) captured on a rocky outcrop
in the Clarens district. Blue arrows of A-E indicate a Sauroplasma-like infection. A: (black
arrow): oval trophozoite with a purple stained periphery and a pinkish central vacuole. B & D:
trophozoites/ possible early meronts D: (Yellow arrow): amoeboid meront. C & E:
gametocytes. F-M: Plasmodium or Haemoproteus Kruse, 1890 sp. C from Pseudocordylus
melanotus subviridus (A. Smith, 1838) captured near the village Ha Rapokolana. F-M (blue
arrows): Sauroplasma-like infection. F-I: shows fine malaria pigment. K-M: suspected
juvenile gametocytes. N-R: Plasmodium or Haemoproteus sp. D from Pachydactylus bibronii
(A. Smith, 1845) collected on the farm Zuurfontein, Free State. N: (blue arrows)
Sauroplasma-like infection. N-R: gametocytes. Scale: A-R: 10 urn,
CHAPTER3 RESULTS
Trophozoites and possible early meronts
The earliest infecting forms of the parasite seen In blood smears were small, oval
trophozoites with a purple-stained periphery and a pinkish central vacuole (Fig. 3. l8A
(black arrow)) 0.98 ± 0.97 (1.14-2.12) urn to 2.30 ± 0.88 (2.47-3.35) urn (n=15) in
diameter. Larger trophozoites/ possible early meronts (Fig. 3.18B, 0) were similar in
staining properties to young trophozoites, but some were triangular or amoeboid in shape
(Fig. 3.180 (yellow arrow) and measured 3.3 ± 1.1 (3.1-5.8) urn in width by 3.8 ± 1.4
(3.4-6.1) urn in length. Mature meronts were not observed. No marked effects on the host
cell were seen associated with trophozoite and early meront stages, or with presumed
gametocytes (below).
Gametocytes
Stages presumed to be juvenile gametocytes were positioned in the polar regions of the
host erythrocytes, and some host cells contained two such parasites (Fig. 3. 18C). They
were kidney-shaped or somewhat amoeboid (Fig 3.18C & E), stained deep pink with
Giemsa and contained fine granules of malaria pigment. The biggest of these forms
measured 4.0 ± 0.9 (3.84-5.55)!lm in width by 5.8 ± 1.9 (4.47-7.10) urn (n=12) in length.
Comments
The parasites were smaller than Plasmodium sp. A and about the same dimensions (4.0 ±
0.9 (3.84-5.55) urn in width by 5.8 ± 1.9 (4.47-7.10) urn (n=12)) as P. zonurae but had
different staining properties and carried lots of fine pigment in the mainly eosinophilic
cytoplasm. Macrogametocytes also had more pigment than those described from
Cordylidae in South Africa by Pienaar (1962).
III
CHAPTER3 RESULTS
Plasmodium or Haemoproteus sp. C from Pseudocordylus melanotus subviridis (A.
Smith, 1838)
Host Pseudocordylus melanotus subviridis (A. Smith, 1838)
Locality: Village of Ha Rapokolana (29°22'36"S 28°01 '53E), Lesotho.
Plasmodium sp. C infections were discovered in the blood of three of four
Pseudocordylus melanotus suhviridis captured near the village Ha Rapokolana in
Lesotho. These infections were similar to Plasmodium zonurae in the girdled lizard
Cordylus vittifer, and were accompanied by a Sauroplasma-like infection (Fig. 3.18F-M
(blue arrows».
Trophozoites
Trophozoites were round to oval In shape and measured 2.25 ± 0.84 (1.12-5.16) urn
(n=20) in diameter. They stained dark purple with Giemsa peripherally, with some
malaria pigment (Fig.3.18F-I) with a pale central vacuole. No marked effects on the host
cell were seen and no meronts were identified.
Gametocytes
Suspected juvenile gametocytes (Fig. 3 .18K-M) of this malaria were also observed. They
were mostly polar in position within erythrocytes, rounded, rather amoeboid or broadly
crescent-shaped and contained pigment granules. They stained either pinkish or pale lilac
with Giemsa and measured 3.4 ± 0.8 (3.2-4.5) urn in width and 5.54 ± 1.0 (5.18-6.97) urn
(n=14) in length. No effects on host cells were detected.
Comments
These specimens were found only in one developmental stage, therefore it will be
considered as Plasmodium or Haemoproteus sp. C. When compared with Plasmodium
zonurae this Plasmodium or Haemoproteus species appears similar in the general
morphology. Plasmodium or Haemoproteus sp. C has round or oval-shaped trophozoites
with dimensions of2.25 ± 0.84 (1.12-5.16) urn (n=20) in comparison with 2-2.8 urn of P.
zonurae. The gametocytes varied slightly in size, being 3.4 ± 0.8 (3.2-4.5) urn in width
112
CHAPTER3 RESULTS
and 5.54 ± l.0 (5.18-6.97) urn (n=14) in length in Plasmodium or Haemoproteus sp. C,
whilst P. zonurae had gametocytes that measured 8-8.4 by 4.2 urn.
Plasmodium or Haemoproteus sp. D from Pachydactylus bibronii (A. Smith, 1845)
Type Host: Pachydactylus hihronii (A. Smith, 1845).
Type Locality: On the farm Zuurfontein (29°54'S 25°22'E) Jagersfontein district,
Southern Free State.
Plasmodium infections were observed in the blood of two specimens (male and female)
of Pachydactylus hihronii (A. Smith, 1845) captured on the farm Zuurfontein. These
infections were accompanied by a Sauroplasma-Iike infection (Fig.3. 18N (blue arrowsj).
Gametocytes
Only gametocytes were observed in the blood of these geckos (Fig. 3.18N-R) and two
distinct types of gametocytes were observed. Presumed macrogametocytes were broadly
crescentic in form (Fig.3 .18R (black arrow), stained faintly lilac or purple, had fine
pigment granules and measured 8.4 ± 0.7 (8.2-8.7) urn by 9.3 ± 0.44 (8.8-9.4) urn (n=20).
Microgametocytes stained deeper purple, and scattered, slightly coarser, pigment
granules were present (Fig. 3 .I8P (black arrow), Q & R (red arrow), and they measured
9.4 ± 0.51 (8.87-10.2) by 5.6 ± 0.45 (5.5-5.9) urn (n=20). Both types of gametocytes
usually lay at one extremity of the host cell. Large forms (Fig. 3.18R (red arrow)) filled
the cell cytoplasm, causing displacement of the host cell nucleus and general cell
enlargement.
Comments
These infections, like those of the previously described malaria (from Lesotho) could be
Haemoproteus sp. if it can be proved that there is lack of merogony in the red cells.
Certainly some stages of the parasite almost fill the host cell cytoplasm (Fig. 3 .18R) as in
Haemoproteus. Only gametocytes were present in the blood cells and if these prove to be
a Plasmodium infection, then it is possible that it is an old infection. These infections
113
CHAPTER3 RESULTS
correspond closely to the gametocytes described as Plasmodium mexicanum Thompson
& Huff, 1944 from Sceloporis undulates, but the current species differs as it seems to
produce more pigment.
3.4. MICROFILARJAE
3.4.1. NEMATODA
A number of different microfilarial nematodes were observed in the blood of Agama atra
atra, Cordylus polyzonus and Pseudoeordylus melanotus subviridis collected in different
localities in Lesotho and the Free State.
Microfilaria sp A from Agama atra atra Daudin, 1802
Specimens were collected (exact numbers not recorded) in the village of Ha Rapokolane,
Ha Khojane in Lesotho and on the farm Zuurfontein (29°54'S 25°22'E) in the southern
Free State.
These microfilariae (Fig. 3.19A-F) measured 54 ± 0.8 (40-60) urn (n=10) long with a
pale pink or purple-stained, highly inflated, sheath and a slightly pointed anterior. The
tail tip was relatively slender and bluntly rounded, with two single nuclei close to its
terminus. Nuclei in the tail region stained deep purple, the remainder, almost black.
Pink-staining organ primordia could be seen towards the centre of the nematode.
Comments
All of these microfilariae were distinctly stained and had a characteristically inflated
sheath. It seems that these unidentified species are specific to A. atra atra hosts even
over disjunct localities.
114
CHAPTER3 RESULTS
Microfilaria sp B from Cordylus polyzonus polyzonus A. Smith, 1838
Specimens were collected from various sites on the farm Zuurfontein (29°54'S 25°22'E)
in the southern Free State.
Microfilariae from these lizards (Fig. 3.19G-K) measured 82.5 ± l.8 (80-95) urn (n=20)
long with a pale blue-stained, loose fitting, sheath and a rounded anterior, with eight pairs
of nuclei clearly visible. The tail tip was slender and sharply pointed (Fig. 3. 19G-J), with
5-6 elongate, widely spaced single nuclei. Nuclei in the tail region stained deep purple,
the remainder, almost black. As above, pink-staining organ primordia could be seen
towards the centre of the nematode.
Comments
These microfilariae seem distinct if compared to the other described forms in this study.
It has a robust thick body with a characteristically blue-stained body with a sharp pointed
tail.
Microfilaria sp C from Pseudocordylus melanotus subviridis (A. Smith, 1838)
These microfilariae were found in numerous lizards (exact numbers not recorded)
collected in the Bokong, Jorotane and Syncunjane river catchments in Lesotho.
Microfilariae of this specres measured 95 ± 0.955 (40-110) urn (n=20) long with an
almost colourless, loose-fitting, sheath and a broad, bluntly rounded anterior (Fig. 3.19L-
0). The tail tip was rather broad and stumpy and individual nuclei within it were difficult
to count. Nuclei throughout the body stained deep red-purple. Organ primordia could be
seen as colourless and red-stained areas towards the centre and two-thirds down the body
of the nematode.
Comments
These lizards were collected in various localities separated by high mountain ranges. It
seems from their general morphology that these microfilariae are specific to these lizards
115
CHAPTER3 RESULTS
(Pseudocordylus melanotus subviridis) in Lesotho. These are morphometrically the
biggest forms of nematode observed in the blood plasma of all lizards infected examined
in this study.
Microfilaria sp D from Pseudocordylus melanotus melanotus (A. Smith, 1838)
Lizards with this microfilarial nematode were collected in the Clarens district In the
eastern Free State.
Microfilariae in Fig. 3.19P-R measured 55 ± 1.4 (40-77) urn (n=20) long with deep
purple-staining nuclei. The anterior tip of each was pale-stained. The parasites were
irregularly coiled with a rough outline and the sheath that seemed to be tight fit.
Comments
These lizards were collected deep in the Maluti Mountains. They were collected from a
few sites relatively close to each other, and it seems that these species of microfilariae are
specific to these subspecies of lizards. More surveys will clarify this.
116
lFngunre3.19. Light micrographs Giemsa-stained blood films of filarial nematodes found
in the blood plasma of Agama atra atra Daudin, 1802, Pseudocordylus melanotus
melanotus (A. Smith, 1838), Pseudocordylus melanotus subviridis (A. Smith, 1838) and
Cordylus polysonus A. Smith, 1838. A-lf: microfilaria sp A in six different specimens of
Agama atra atra collected on the farm Zuurfontein, G-K: microfilaria sp B in blood
plasma from Cordylus polysonus collected in same locality (Zuurfontein). L-O:
microfilaria sp. C from Pseudocordylus melanotus melanotus collected in Clarens, P-R.:
microfilaria sp. D from Pseudocordylus melanotus subviridis collected in the village of
Ha Khojane, Lesotho. Scale: A-R: 20 urn,

CHAPTER4 DISCUSSION
DISCUSSION
The Free State is an area of high altitude grasslands and deep soils where the summers
are hot with frequent thunderstorms and the winters are dry and cold. It was noted by
Lynch (1983) that 75% of the Free State consists of grassland with 20% karoo in the
South West and the remaining 5% is thornveld in the North West. The Eastern parts are
mountainous with montane grasslands and scattered patches of Afromontane forests.
Temperature increases from east to west while rainfall, elevation and reptile diversity
increase from west to east. The overall climatic conditions have an influence on reptile
diversity, i.e. it increases to the east. Therefore, most of the sample areas in the present
study were selected in the eastern parts of the Free State and in the heavily mountainous
and rocky areas in Lesotho.
Of the 59 ophidian specimens representing 13 snake species, and 204 lizards consisting
of 15 species in six fami lies, examined during this study all apparently revealed at least
some kind of blood infection. Some specimens with wide distribution ranges like the
puff adder (BUis arietans), and others with specific and endemic distribution patterns like
giant girdled lizards (Cordylus giganteusy, showed over dispersed parasite infections.
Others like the Karoo girdled lizards (Cordylus polyzonusy, which are distributed
throughout the South western parts and consist of scattered and isolated populations
living almost exclusively on dolerites, were found to have high and sometimes mixed
infections. These reptiles could serve as manageable models for future studies to
investigate parasite populations, since each isolated outcrop serves as an island for the
host population.
A broad spectrum of blood parasites (summarised in Table 3.1) ranging from viral
infections and apicomplexans to nematodes were found in all locations. No clear patterns
were found in terms of the distribution arrangements of parasites. Apart from parasites
that are over dispersed, all of the reptiles sampled apparently had some sort of an
infection in their peripheral erythrocytes. It is well-known that reptiles are an ancient
group and some of them have remained virtually unchanged, perhaps for many millions
119
CHAPTER4 DISCUSSION
of years. It could thus be assumed that they possibly had time to adapt to their infections,
but the question arises why most reptiles have an infection and why it is that the
infections are so severe in some members of populations. Furthermore, infections like
Sauroplasma, Hepatozoon and Plasmodium undoubtedly have a destructive effect on the
host cells, but how do they affect the hosts overall? Surely if a cell is robbed of its
haemoglobin content (by several infections) the normal metabolic functions would be
changed, most likely to the detriment of the host. Ultimately what effect does this have
on the population dynamics?
One of the reasons that the giant girdled lizards (Cordylus giganteus) are regarded as (SA
RDB) vulnerable is because they have become isolated due to roads and agricultural
events. Apart from their stagnating gene pools, should one be concerned when almost
every blood cell appears infected? Although the nature of this study was not to determine
the physiological state of reptiles, no overall apparent effects on the hosts itself could be
observed. Could these infections be due to human disturbance? Human disturbance has
created island populations instead of the once free areas for the exchange of genes regime
amongst animals. These induced host isolations may have carried on for the last few
hundred years and on a comparative morphological level perhaps no differences could be
seen in their parasites. It would be interesting to see the genetic or molecular changes in
these parasites trapped in these human induced refuges.
Very few habitats these days are in pristine condition and some human influence could
perhaps have an effect on the parasite infections of reptiles. However, lizards from
pristine habitats with almost no human impact, like those from the highlands of Lesotho,
are seemingly healthy, but have a blood picture that is abnormal, with extremely high
levels of (often mixed) infections. From an objective point of view something is wrong.
Is it normal for these undisturbed reptiles to have such high infection levels? Clearly, it
would seem to be so.
Haemogregarines discovered in the blood of some reptiles in this study undoubtedly
constituted new host records and very likely new species, with the exception of
120
CHAPTER4 DISCUSSION
Haemogregarina (Hepatozoon) sebae (Laveran & Pettit, 1909) Smith, 1996 from the
African python. The current investigation led to the discovery of this latter parasite from
a different race (southern race) of python, Python sebae natalensis. This was also the
first description of the ultrastructure of H. sebae natalensis and, although an initial and
rather brief study, it clearly demonstrated the apicomplexan features of this
haemogregarine. Distribution ranges and probably numbers of species for reptilian
haemogregarines are extended during this study to include six likely new species of
haemogregarines present in the blood cells of a snake, cordylid and agamid lizards. They
have been named Hepatozoon sp. A to F for the purpose of this dissertation, because their
life cycles are not known at this stage. When these have been determined it may still be
appropriate that they are named Hepatozoon, or it may be necessary to transfer these new
haemogregarines to different genera, such as Hemolivia, Karyolysus or Schellackia.
Some Hepatozoon spp. found in this study were particularly interesting. Two in
particular deserve further study, namely, the distinctive huge Hepatozoon sp. D from
Lesotho in the lizard Pseudocordylus melanotus subviridus, and Hepatozoon sp. F. from
Bloemfontein which has severe effects on host erythrocytes.
It seems that these Hepatozoon spp. have adapted to infect isolated populations of species
of hosts. The Lesotho highlands provide a number of isolated localities where a spectrum
of new parasites and infections were discovered. The catchment area of Mohale Dam in
Lesotho consists of the Syncunjane, Bokong and Jorotane rivers separated by huge
mountains with peaks sometimes exceeding 2900m in height. These barriers probably
isolate host populations where distinct morphological differences between Hepatozoon
spp. are evident. Future studies will involve in-depth sampling in these and other
locations to map the different host and haemogregarine species distribution.
Furthermore, agamid lizards, which are likely an older group than cordylid lizards in
evolutionary terms, seem more adapted to their parasites, and their parasites to them, to
such an extent that their haemogregarines, filarial nematodes and Pirhemocyton
infections are host (species) specific. Although host specific parasites have probably
evolved in most of the major reptile groups, it seems from the current survey that these
parasites have evolved particularly to Agama atra atra hosts. Although these lizards
121
CHAPTER4 DISCUSSION
have a wide distribution range and were collected in various disjunct localities, infections
in the blood of these lizards seemed identical, hence the tentative conclusion regarding
the host specificity.
Lizard malaria has been found on five continents and in all the major families of lizards
as well as in some snakes. Infections of representatives of the Plasmodidae are so far not
recorded from any representatives of the Testudes and Rhynchocephalia. Ayala (1977)
was of the opinion that lizard malaria is most common in wet tropical forests, is
distributed as far north as Japan and as far south as New Zealand. These distributions
probably reflect, in part, that more surveys were conducted in those areas. The subject of
reptile malaria had been studied, (according to Ayala, ] 977), by about 80 authors who
had contributed significantly in their ] 53 papers in their knowledge of infections in
Africa, Australasia and the Americas. In the African region a total of 16 species have
been described thus far, with one, Haemoproteus mungutti Mutinga and Dipeolu, 1989
from Agama agama and another Plasmodium pitmani Hoare, 1932 from a skink Mabuya
striata, while in South Africa three species have been recorded by Pienaar (1962) and
Garnham (1966) in three families of lizards (Agamidae, Cordylidae and Gerrhosauridae
Fitzinger, 1843). Investigations in the present study of the blood of Pseudocordylus
melanotus subviridis, Pseudocordylus melanotus melanotus, Cordylus polyzonus
polyzonus (Cordylidae) and Pachydactylus bibronii (Gekkonidae) revealed four likely
new species of lizard malaria, namely Plasmodium sp. A-D, although some may prove to
be Haemoproteus sp. These are new host and distribution records for these parasites. In
general, lizards live their entire lifetime in a small habitat range with no migratory
movements as in birds. Therefore, Plasmodium infections in lizards are well suited for
evolutionary and epidemiological studies in natural infections. According to Ayala
(1977), typical saurian infections follow the same general cycles as their bird and
mammal counterparts: a period of initial rise, then a peak followed by a gradual or abrupt
decline. A chronic period is usually interrupted by a relapse. Future studies in South
Africa should involve complete life cycle studies of this prehistoric, but successful group.
122
CHAPTER4 DISCUSSION
Transmission of representatives of the Plasmodidae between South African lizards
remains unresolved. Pienaar (1962) was of the opinion that although Tabanus and
Haematopota spp. (Diptera: Tabanidae) are prevalent it seems inconceivable that these
flies could act as vectors transmitting Plasmodium infections. However, it was noted that
all lizards that harbored a Plasmodium infection, had infestations of prostigmatic mites.
These acarines could be considered potential vectors, but Ayala (1977) considered that
these would not support sporogonic development, although parasites might be retained in
the stomach for several days. Although much of the skin of many reptiles seems resistant
to insect bites, it actually consists of an extensive vascular system where haematophagous
arthropods can easily take a blood meal. Numerous "soft" areas can serve as possible and
accessible sites for attachment of acarines or as a feeding spot for capillary feeders such
as Diptera (Culicidae), or Hemiptera (Reduviidae) which are undoubtedly present in the
Free State. I observed a few culicine mosquitoes feeding on the soft skin beneath the eye
of a monitor lizard lodged in a rock crack. Ayala (1971 & 1973) proved that lizards can
be infected following inoculation with sporozoites from heavily infected sandflies
(Diptera: Phlebotidae). It thus seems that transmission from invertebrate vectors to
reptile hosts may take place by inoculation of salivary fluids, or due to regurgitation
when taking a blood meal, or if the invertebrate is ingested. For specimens collected in
South Africa and Lesotho, it seems unlikely that these lizards prey on minute mites that
are usually engorged. I doubt that lizards can even see these highly camouflaged acarines
safely wedged between their scales. The four likely new Plasmodium species are
described from disjunct localities, and each will have their own characteristic
haematophagous invertebrates. Jupp (1996) reported numerous culicine and
toxorhynchitine mosquitoes from South Africa some of which are found in Free State
(Aedes aegipi, A caballus, A circumluteolus, A dentatus, A durbanensis. A hirsutus, A
luridus, A luteolateralus, A mcintoshi, A mixtus, A natalensis, A natronius, A sudanensis,
A unidentatus, A vittatus, Culex annulioris, C lineate, C pipiens, C poicilipes, C
quinquefasciatus, C salisburiensis, C theileri, C tigripes, C univittatus, Culiseta
longiaeriolata and Mansonia uniformess and in Lesotho (Aedes hirsutus, A juppi, A
unidentatus, Culex andersoni, C. theileri, C salisburiensis, C. univiuatus and Culiseta
longiaeriolata). Plasmodium sp. A, B & D are described from the Free State and
123
CHAPTER4 DISCUSSION
Plasmodium sp. C. was described from Lesotho. Apart from the two subfamilies of
mosquitoes that could likely be responsible for transmission, the only common link
between infections is the presence of prostigmatic mites. Future studies will involve
seeking vectors that are likely to be responsible for Plasmodium infections. These
invertebrates will be prepared for investigation by light microscopy (for example, by
histology), TEM and confocallaser scanning microscopy.
The enigmatic and so-called piroplasms of reptiles, Sauroplasma and Serpentoplasma,
were found in most of the reptiles collected. Morphologically some of these apparent
infections appeared similar to Pirhemocyton infections, which are viral in nature and now
often designated LEV (lizard erythrocytic virus) or SEV (snake erythrocytic virus) (see
Telford & Jacobson 1993; Davies & Johnston 2000). Strangely perhaps, Toddia was not
seen in snakes from South Africa, but Sauromella (from the lizard Pachydactylus
capensis) was found to be particularly close to Pirhemocyton in appearance, suggesting
that this is also a viral infection.
Attempts were made to study the nature of Sauroplasma and Serpentoplasma by means
of transmission electron microscopy (TEM). The threatened and CITES listed giant
girdled lizards (Cordylus giganteus) are endemic and found only in small isolated areas.
They are considered vulnerable South Africa Red Data Species due to massive habitat
destruction by farmers producing monocultures (mostly maize and corn) and the recently
booming (illegal) pet trade. These lizards serve as an excellent model to try and
understand the biology of Sauroplasma infections, because they are colonial, viviparous,
long-lived and reasonably easily obtained. Ultrastructural studies failed to give a firm
indication of the nature of these infections. Their boundary membrane, heterochromatin
and their budding suggested a possible eukaryotic identity, but no structures consistent
with those found in the Apicomplexa were observed. However, when they were
compared with Serpentoplasma, Sauroplasma infections were found to be very similar,
and Serpentoplasma might just be viral in nature (see below). Future studies will involve
attempts to finally resolve the identity of these strange infections, to find their mode of
transmission, and an assessment of their pathogenicity to their host lizards.
124
CHAPTER4 DISCUSSION
A puff adder (Bitis arietans) and a captive African rock python (Python sebae natalensis)
were used to study Serpentoplasma infections by means of TEM. The infections were
similar in morphometrical dimensions and morphological appearance to those
(Sauroplasmay found in lizards. By TEM they were also reminiscent of the viral
infections seen by Daly et al. (1980) in water snake erythrocytes, but again further studies
are needed to confirm this. Future studies will also focus on the mode of transmission of
Serpentoplasma.
Mixed infections were common amongst host species. In general, Sauroplasma was
observed together with haemogregarines and with Plasmodium species. Pirhemocyton
infections and Hepatozoon sp. A. were found to coexist in the erythrocytes of Agama atra
atra. All six likely new species of haemogregarines were accompanied by Sauroplasma
infections. It was not uncommon to find malaria, haemogregarines, nematodes
(microfilariae) and so-called piroplasms in the same specimens of some lizards. The
identity of the microfilariae, the location of their adult stages and their mode of
transmission are further problems that require resolution.
Transmission of Pirhemocyton, Sauroplasma and Serpentoplasma also remains
unresolved. It is possible that prostigmatic mites transmit these infections via their
salivary glands while having a blood meal. On most wild reptilian specimens these
engorged ectoparasites were found and I also observed on several occasions mosquitoes
feeding on geckos and on the eyelids of a monitor lizard tVaranus exanthematicus
albigularusy. This latter lizard's blood revealed a Sauroplasma-like infection, as well as
haemogregarines.
Pathological effects on host cells were apparent. Several infections revealed infected
blood cells showing nuclear displacement hypertrophy and dehaemoglobinisation. These
infections must have an effect on the host itself. Infections like those of Sauroplasma
that infect nearly every cell, possibly reduce the haemoglobin content extensively. The
Hepatozoon sp. F from the striped skaapsteker (Psammophylax tritaeniatusï was another
amazing example of a parasite that dehaemoglobised the host erythrocyte, displaced the
125
CHAPTER4 DISCUSSION
host cell nucleus and reduced it in size. As a result of these infections perhaps less
oxygen is available at a cellular level which might reduce the mobility of the reptile.
These lower energy levels could have an impact on the reproduction abilities. SchaIl
(1996) did some ecological work on malaria parasites affecting lizard behaviour, effects
on showy male traits being a good example.
Even though short in geological time, reptiles have had at least a few hundred million
years to successfully adapt to their parasite infections. The present survey, although
relatively scanty in reptile diversity, showed an awesome diversity of parasites in their
erythrocytes. Some of the infections may prove to be detrimental to their host's health,
and, like any system under pressure, the host may then experience further increase in
parasite loads. The outcomes of this study opened up one main question: what are the
normal levels of parasite loads in reptiles? Is it only in the present study area that such
levels of infection are seen? Are the host reptiles adapted to these seemingly heavy
levels of infection or is there something that we do not see?
Huchzermeyer, Williamson and Swanepoel (1986) briefly examined (reported in a
conference presentation) the blood of a few lizards from the Eastern Cape, where they
found infections ranging from viral to haemogregarines and malarias, but the present
investigation was the first study of its kind in the Free State and in Lesotho. These
remarkable findings of infections of such high frequencies and the presence of mixed
infections, proves that these varied and relatively unknown parasites are indeed
widespread and possibly threatening to their hosts. Are we witnessing gradual extinction
of these successful reptiles, due to parasitic infections? It is regrettable that so few
studies like this one have been conducted. Further studies like these might indicate
whether these infections are increasing, and, if so, are they a cause for concern? Should
future nature conservation efforts for red data species like the endemic giant girdled
lizards be focused on monitoring intraerythrocytic infections in view of the demonstrable
effects of these on the host cells? Certainly, further studies on these fascinating
infections are urgently needed.
126
CHAPTERS REFERENCES
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137
ABSTRACT
The study of blood parasites of reptiles is a relatively new and unexplored field of
research in South Africa. The Free State province and the Lesotho highlands provide a
range of reptiles in which their intraerythrocytic parasite fauna were explored.
Objectives of this study were to set a baseline of blood parasite diversity and to identify
the enigmatic Sauroplasma Du Toit, 1937 and Serpentoplasma Pienaar, 1954 infections
in lizards and snakes, respectively. Surveys were conducted in various localities in the
Free State and Lesotho. Although low in diversity, 204 specimens representing 14
species of lizards, and 59 specimens representing 13 species of snakes were investigated
for the presence of blood parasites. Three known infections were found: Sauroplasma
thornasi Du Toit, 1937, Sauromella haemolysus Pienaar, 1954 and Hepatozoon
(Haemogregarina) sebae (Laveran and Pettit, 1909) Smith, 1996. These were
redescribed and S. thomasii and H. sebae were examined by aid of transmission electron
microscopy. The investigation led to the discovery of six new records and possibly new
species of haemogregarines named Hepatozoon sp. A-F, four new records and possibly
new species of lizard malaria named Plasmodium sp. A-D, and a viral infection possibl y
of the Pirhemocyton type. Furthermore nine new host and distribution records for
Sauroplasma in lizards and nine for Serpentoplasma in snakes are described.
Ultrastructural investigations of S. thomasi in Cordylus giganteus A. Smith, 1844,
Serpentoplasma in Bitis arietans arietans (Merrem, 1820) and H. sebae in Python sebae
natalensis (Gmelin, 1789) were the first to examine the nature of infections in this
manner. This is the first comprehensive survey of the biodiversity of blood parasites in
reptiles in the Free State and Lesotho highlands.
Key words: Reptiles, blood parasites, haemogregarines, lizard malaria, Sauroplasma,
Serpentoplasma, Pirhemocyton, Free State, Lesotho.
138
OPSOMMING
Die studie van bloedparasiete van reptiele is 'n relatiewe nuwe en onbekende veld van
navorsing in Suid Afrika. Die Vrystaat Provinsie en Lesothohooglande het 'n
verskeidenheid van reptiele waarvan hul intraeretrositiese parasietfauna bestudeer was.
Die doel van die studie was om 'n basislyn van bloedparasietdiversiteit op te stel, en orn
die enigmatiese Sauroplasma Du Toit, 1937 en Serpentoplasma Pienaar, 1954 infeksies
in akkedisse en slange onderskeidelik te identifiseer. Opnames was in verskeie lokaliteite
in die Vrystaat en in Lesotho gedoen. Alhoewel laag in biodiversiteit, was 204
eksemplare, verteenwoordigend van ] 4 spesies akkedisse en 59 slange,
verteenwoordigend van 13 spesies, ondersoek vir die teenwoordigheid van
bloedparasiete. Drie bekende infeksies was gevind: Sauroplasma thomasi Du Toit,
1937, Sauromella haemolysus Pienaar, 1954 en Hepatozoon (Haemogregarina) sebae
(Laveran and Pettit, 1909) Smith, 1996. Die material is herbeskryf en S. thomasii en H.
sebae was deur middel van transmissie-elektron mikroskopie ondersoek. Die ondersoek
het tot die ontdekking van ses nuwe rekords en moontlik nuwe haemogregariene spesies,
naamlik Hepatozoon sp. A-F, vier nuwe rekords en moontlike nuwe spesies van akkedis-
malaria, naamlik Plasmodium sp. A-D, en 'n virus-infeksie wat moontlik van die
Pirhemocyton tiepe is, gelei. Verder is nege nuwe gasheerverspreidingsrekords vir
Sauroplasma spp. in akkedisse en nege vir Serpentoplasma spp. in slange beskryf
Ultrastrukturele ondersoeke van S. thornasi in Cordylus giganteus A. Smith, 1844,
Serpentoplasma sp. in Bitis arietans arietans (Merrem, 1820) en H. sebae in Python
sebae natalensis (Gmelin, 1789) was die eerste keer gedoen op hierdie manier orn die
aard van infeksies vas te stel. Hierdie is die eerste samevattende opname van die
biodiversiteit van bloedparasiete van reptiele in die Vrystaat en Lesothohooglande.
Sleutelwoorde: Reptiele, bloedparasiete, haemogregarienes, akkedis-malaria,
Sauroplasma, Serpentoplasma, Pirhemocyton, Vrystaat, Lesotho.
139
ACKNOWLEDGEMENTS
The author acknowledges and expresses his deep and sincere appreciation to the
following persons and institutions:
To my supervisors:
Prof. Angela Davies for sharing her profound knowledge and deep understanding of
blood parasites. Also for making it possible to travel abroad.
Prof. Linda Basson for her guidance and extreme patience during this study.
Prof. Jo. Van As (my dad) for his constant guidance and patience.
All my friends especially Jonie, Vaughn and Charles for their support and help during
field work.
Or. Bill Edwards for his profound interest and help preparing tissue samples for electron
nucroscopy.
TEOL (UK) Ltd, especially Andy Yarwood of their applications laboratory and assistance
in using the TEM, and for hospitality, during my visit to their laboratories.
140
APPENDIX A
141
r' .....~.-.-----.Permit om lewende Wilde en/of Uitheemse Reptiele in gevangenskap aan te hou
I HIERDIE PERMIT IS NIE OORDRAAGBAAR NIEi: -- - - -------- --------- ------- -- ----
j~K.ragtens die Ordonnansie op Natuurbewaring Nr 8 van 1969, word magtiging hiermee verleen aan:1'1
~ PERMITHOUER BESONDERHEDE~-
I~~_ Mnr. van As ID Nommer 7707225055089J. Nauhaus 12
Brandwag
~, Bloemfontein.
it. Suid-Afrika
}.
j Tel No (H) 051-4300691
~t
jl Tel No (W) 051-4012370 Faks No:
~~
{.
in gevangenskap aan te hou
-- - ---------------------
I Handtekening van Permithouer namens die LUR: Omgewingsake en Toerisme
,I
P.O. Box 517 Tel: 051 - 447U4U7Fax: 051 - 4475240
BLOEMFONTEIN
9300 South Africa
-------_ .._- .----------------------_._ .._._----_.- . _-_ .._._- -
PERMITTEE DETAILS
Mnr. van As ID Number 770i225055089
J.
Nauhaus 12
Brandwag
Bloemfontein.
Suid-Afriklll
Tel No (H) 051-4300691 Fax No
Tel No (W) 051-4012370
- _. __ ._ .. ~._ ••• _,_ ~. ••• _, "_0' _ •• . .---.-.------
In terms of l'\ature Conservation Ordinance no 8 of 1969, permission is hereby granted to the holder of this permit to:
Collect reptiles in the Free State for a M.Se. Project.
Subject to the following conditions:
1. This permit: is invalid unless all requirements of any other legislation in respect of the act mentioned are complied wi
2. This permit is invalid if it is not signed by the permittee and is not transferable.
3. All specimens are to be released at the point of collection within 24 hour of capture.
4. All wounds from capture and drawing blood are to be treated appropiately before release.
5. No more than one voucher specimen per site is allowed to be collected for identification. Such specimens must be
fixated in the field and deposited as soon as possible with the National Museum, following each field trip. The
neceessary collection data as required by the National Museum must accompany eachspecimen.
6. Live specimens can be transported backto, or kept in captivity at the laboratory and or University premises for not
longer than two months.
7. A register must be kept of all animals caught and released, with dates and localities.