Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2018, 65: 015 doi: 10.14411/fp.2018.015 http://folia.paru.cas.cz

Research Article Diversity of haemoprotozoan parasites infecting the wildlife of

D. James Harris1,2, Ali Halajian3, Joana L. Santos1, Lourens H. Swanepoel4, Peter John Taylor5,6,7 and Raquel Xavier1

1 CIBIO/InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, Vairão, Portugal; 2 Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal; 3 Department of Biodiversity, University of , South Africa; 4 Department of Zoology, School of Mathematical and Natural Sciences, University of Venda, Thohoyandou, South Africa; 5 NRF/DET SARCHI Chair on Biodiversity Value and Change, School of Mathematical and Natural Sciences, University of Venda, Thohoyandou, South Africa; 6 Core Team Member, Centre for Invasion Biology, Stellenbosch University, Stellenbosch, Republic of South Africa; 7 Honorary Research Associate, University of KwaZulu Natal, Durban, Republic of South Africa

Abstract: Tissue samples from wildlife from South Africa were opportunistically collected and screened for haemoprotozoan parasites using nonspecific PCR primers. Samples of 127 individuals were tested, comprising over 50 different . Haemogregarines were the most commonly identified parasites, but sarcocystids and piroplasmids were also detected. Phylogenetic analyses estimated from the 18S rDNA marker highlighted the occurrence of several novel parasite forms and the detection of parasites in novel hosts. Phy- logenetic relationships, which have been recently reviewed, appear to be much more complex than previously considered. Our study highlights the high diversity of parasites circulating in wildlife in this biodiverse region, and the need for further studies to resolve taxonomic issues. Keywords: , , , , , phylogeny, 18S rDNA

South Africa is the third most biologically diverse coun- Models suggest that co- may be the most try in the world for terrestrial species. It includes three of common form of biodiversity loss (Dunn et al. 2009), yet the 35 recognised biodiversity hotspots, the Cape Floristic paradoxically lack of basic knowledge of most parasite region, the Succulent and the Maputoland-Floris- groups prevents risk assessments for example – Dobson tic region (Mittermeier et al. 2004). Yet despite this well- et al. (2008) state “We have no credible way of estimating known biodiversity richness, parasite diversity remains how many parasitic protozoa … exist”. Part of the problem poorly known. One of the few known patterns of parasite in estimating parasite diversity has classically been that, diversity is a link with overall biodiversity, leading to the especially for endoparasites, often only some life stages ʻhost diversity’ begets parasite diversity” principle, and in- can be identified in host blood or tissue samples, making dicating that parasite diversity should also be very high in identification based on morphological characters difficult. the region. Molecular tools, particularly DNA sequencing approaches, Parasites have dual interests for conservation biologists. should help overcome these problems, but molecular stud- Firstly parasite-driven declines in wildlife have become ies have lagged behind those of free-ranging organisms increasingly common (Pedersen and Fenton 2007), illus- (Criscione et al. 2005). trated by multiple pathogens being implicated in the glob- Currently five groups of haemoprotozoan parasites are al decline of populations (e.g. Hoverman et al. recognised, one group of euglenozoan flagellates and four 2012). Second, parasites also represent a major component groups of apicomplexan parasites (reviewed in O’Dono- of biodiversity. Because of their obligate relationships with ghue 2017). These infect all terrestrial groups their hosts, parasites are particularly at risk through co-ex- and use a variety of including Diptera, ticks tinctions, where the loss of a species leads to the loss of de- and leeches as vectors. Despite their medical and veteri- pendent species so that a cascading extinction effect occurs. nary importance, only a few studies have screened verte-

Address for correspondence: D. James Harris, CIBIO/InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal. Phone: +351252660411; E-mail: [email protected]

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.14411/fp.2018.015 Harris et al.: Haemoprotozoan parasites in South Africa

Table 1. List of positive samples, host identification and locality

Code Species Tissue Tissue Locality Bush915 Aepyceros melampus (Lichtenstein) M/L Pullen Farm, Nelspruit, Mpumalanga province RLMar152 Aethomys chrysophilus (de Winton) M/L Louis Trichardt, Limpopo province RLMar162 Aethomys chrysophilus (de Winton) M/L Louis Trichardt, Limpopo province RVenJa151 Aethomys chrysophilus (de Winton) M/L Vyeboom village, Limpopo province RLMar1517 Aethomys ineptus Thomas et Wroughton M/L Louis Trichardt, Limpopo province MonN1Oc14 Ichneumia albicauda (Cuvier) M/L N1, Close to Louis Trichardt, Limpopo province Mon1015 Ichneumia albicauda (Cuvier) M/L Groothoek, Dendron, R521, Limpopo province RKR37416 Ichneumia albicauda (Cuvier) M R37, Limpopo province BatL152 Neoromicia zuluensis (Roberts) M/L Louis Trichardt, Limpopo province SqVenF15 Paraxerus cepapi (Smith) M/L Vyeboom village, Limpopo province RhaTF142 Rhabdomys sp. M/L Tweefontein, Polokwane, Limpopo province RLMar155 Unidentified M/L Louis Trichardt, Limpopo province GSqR34D14 Xerus inauris (Zimmermann) M/L R34, Wesselsbron, Free State province Sn-Najann4 Naja annulifera Peters M/L Louis Trichardt, Limpopo province Sn-CobO14 Naja mossambica Peters M/L Claudius Hoop 106L, Limpopo province Terr316 Pelomedusa subrufa (Lacépède) M/L R71, Onverwacht junction, Limpopo province Terr516 Pelusios sinuatus (Smith) M/L Flag Boshielo dam, Schuinsdrai nature reserve, Limpopo province M – muscle tissue; L – liver. Tissues shown in bold were positive for the respective hosts. brates from southern Africa for these parasites (e.g. Bos- sequences on GenBank using BLAST. All new sequences were man et al. 2010, Van As et al., 2013, Harris et al. 2017). submitted to GenBank numbers MH924604 - MH924623. Most other assessments of apicomplexan parasites in Since various diverse parasites were detected, separate phy- southern Africa have targeted specific species and par- logenetic analyses were performed for each of the parasite groups asites (e.g. Dubey et al. 2014, Netherlands et al. 2014, recovered. For each data set new sequences produced herein were Cook et al. 2016), which means that presence of parasites aligned with related sequences from GenBank, identified through in many species remains either completely unknown or is the BLAST search. Sequences were aligned using the ClustalW based only on older microscopic screening surveys (e.g. software implemented in Geneious 4.8.5 (Biomatters Ld.). This Basson et al. 1967). This motivated the current study, in led to the production of three data sets, containing 110, 28 and 38 which we screened various vertebrate species for infection taxa, which were 577, 618 and 625 base pairs long, respectively. by these parasites, using primers that are known to ampli- Phylogenetic relationships were estimated using maximum fy across the different groups (e.g. Harris et al. 2013) and likelihood (ML) and Bayesian inference. We implemented the from different sources including muscle and blood (Maia models of sequence evolution selected by PartitionFinder 1.1.1 et al. 2014). (Lanfear et al. 2012). ML analyses were performed with RAxML v 7.4.2 (Stamatakis 2006) with 10 random addition replicates, MATERIALS AND METHODS and node support estimated with 1,000 nonparametric bootstrap Sampling was opportunistic, consisting of either samples col- replicates. Bayesian inference was implemented using MrBayes lected as road kills, or from tissue banks from various collections v3.2 (Huelsenbeck and Ronquist 2001), and run for 1 million (Table 1). A few of the road-killed specimens could not be iden- generations. After 25% burn in, remaining trees were combined tified to the species level due to the poor state of the sample, but in a 50% majority rule consensus. were still tested. When available, both liver and muscle tissues were sampled from the same individual. The molecular approach RESULTS followed standard procedures used in other screening studies (e.g. Of the 236 tissue samples analysed, from 127 individu- Harris et al. 2012). DNA was extracted from the different tissues als, 20 produced amplified PCR products that led to identi- using high salt procedures, which have been shown to be as ef- fiable parasite sequences. Haemogregarines were the most fective as more expensive kits for extracting parasite DNA from commonly identified parasites (Fig. 1). In reptiles, four blood and muscle tissues (Maia et al. 2014). hosts, two freshwater turtles Pelomedusa subrufa (Lacépè- Polymerase Chain Reaction (PCR) amplification was per- de) and Pelusios sinuatus (Smith), and two snakes (Naja formed using the primers HepF300 and HepR900 (Ujvari et al. annulifera Peters and Naja mossambica Peters) were in- 2004) with 35 cycles consisting of 94 °C (30 s), 60 °C (30 s) and fected with haemoprotozoans. The haplotypes recovered 72 °C (1 min). These primers were chosen as they have been from the freshwater turtles were sister taxa and strongly demonstrated to amplify a wide taxonomic range of apicompl- supported as a member of the monophyletic Haemogre- exan parasites from reptilian, amphibian and mammalian hosts garinidae clade that has been identified in previous molec- (e.g. Harris et al. 2013, Maia et al. 2014). Negative and positive ular assessments (e.g. Karadjian et al. 2015). controls were run with each reaction and products sequenced by within haemogregarines is complex (see dis- a commercial company (Genewiz, Takeley, UK). Electrophero- cussion) but remaining haemogregarines were assigned to grams were checked by eye and compared against published a paraphyletic Hepatozoon Miller, 1908 (see Maia et al.

Folia Parasitologica 2018, 65: 015 Page 2 of 8 doi: 10.14411/fp.2018.015 Harris et al.: Haemoprotozoan parasites in South Africa

Fig. 1.

MH924604 Hepatozoon sp. ex Aethomys chrysophilus 0.98 MH924605 Hepatozoon sp. ex Unidentified 95 MH924606 Hepatozoon sp. ex Aethomys chrysophilus MH924607 Hepatozoon sp. ex Aethomys ineptus MH924608 Hepatozoon sp. ex Naja mossambica 0.99 EF157822 Hepatozoon ayorgbor 88 KX453597 Hepatozoon sp. JX244267 Hepatozoon sp. KJ408512 Hepatozoon sp. KX453637 Hepatozoon sp. 1 MH924609 Hepatozoon sp. ex Aethomys chrysophilus 97 MH924610 Hepatozoon sp. ex Aethomys chrysophilus KM234650 Hepatozoon sp. FJ719817 Hepatozoon sp. MH924611 Hepatozoon sp. ex Paraxerus cepapi KM234617 Hepatozoon sp. KM234616 Hepatozoon sp. 0.97 KX453613 Hepatozoon sp. KJ408523 Hepatozoon sp. EF222259 Hepatozoon sp. AB181504 Hepatozoon sp. AY600625 Hepatozoon sp. KU680460 Hepatozoon sp. KF939621 Hepatozoon sp. KM234647 Hepatozoon sp. KF939620 Hepatozoon sp. KX453636 Hepatozoon sp. H KR653312 Hepatozoon sp. 1 KX453589 Hepatozoon sp. 88 KX453599 Hepatozoon sp. 1 0.99 KU680444 Hepatozoon sp. 95 KU680464 Hepatozoon sp. 86 HQ292771 Hepatozoon sp. 1 MH924612 Hepatozoon sp. ex Neoromicia zuluensis HQ292774 Hepatozoon sp. KM234648 Hepatozoon domerguei JQ670908 Hepatozoon sp. KJ408521 Hepatozoon sp. KC696564 Hepatozoon sp. 0.93 KJ408522 Hepatozoon sp. KJ408529 Hepatozoon sp. 1 1 HQ734807 Hepatozoon sp.

97 90 JX531921 Hepatozoon sp. / Amphibia / ex Mammalia Reptilia MH924613 Hepatozoon sp. ex Naja annulifera 0.94 1 KC342524 Hepatozoon cuestensis 0.98 100 KC342527 Hepatozoon cuestensis 0.99 KM234612 Hepatozoon sp. et al. 2015) Proposed as Bartozoon (Karadjian 100 KM234615 Hepatozoon sp. AY252108 Hepatozoon sp. 1 KF246565 Hepatozoon seychellensis 100 KF246566 Hepatozoon seychellensis AF297085 Hepatozoon sp. KX453565 Hepatozoon sp. 0.98 KX453566 Hepatozoon sp. 0.94 KJ599676 Hepatozoon theileri 1 1 AF176837 Hepatozoon catesbianae G 100 100 HQ224962 Hepatozoon cf. clamatae HQ224960 Hepatozoon magna KC342526 Hepatozoon cevapii KM234613 Hepatozoon sp. F KM234614 Hepatozoon sp. KM234618 Hepatozoon sp. 0.99 EU908289 Hepatozoon sp. KJ461941 sp. 86 KJ189397 Hepatozoon sp. HQ734793 Hepatozoon sp. KJ461944 Karyolysus sp. HQ734799 Hepatozoon sp. KJ189404 Hepatozoon sp. K Karyolysus JX531917 Hepatozoon sp. 0.95 HQ734791 Hepatozoon sp. JX531910 Hepatozoon sp. 1 KU680457 Hepatozoon sp. 0.99 HQ734795 Hepatozoon sp. 90 HQ734796 Hepatozoon sp. 85 KX453642 Hepatozoon sp. KX011040 Karyolysus paradoxa AY731062 Hepatozoon canis 1 DQ111754 Hepatozoon canis P Hepatozoon 100 AY461375 Hepatozoon canis O 0.99 MH924614 Hepatozoon sp. ex Ichneumia albicauda mostly 0.92 0.99 AF176836 Hepatozoon americanum Mammalia 89 0.99 AY628681 Hepatozoon felis M Carnivora HQ829446 Hepatozoon felis 1 N EF222257 Hepatozoon sp. KU680455 unidentified haemogregarine 91 KU680438 unidentified haemogregarine 0.96 KU680435 unidentified haemogregarine J Unidentified 1 KU680448 unidentified haemogregarine haemogregarines 1 99 KX453595 unidentified haemogregarine 0.97 Reptilia 100 KX453647 unidentified haemogregarine 1 KX453600 unidentified haemogregarine Gekkota 100 I 0.99 KF992706 mauritanica KX453590 unidentified haemogregarine 0.98 KC512766 Hemolivia sp. 0.99 87 1 KR069083 Hemolivia parvula 1 EU430231 Hepatozoon sp. C Hemolivia 100 0.99 97 EU430236 Hepatozoon sp. KF992713 Hemolivia sp. KF992714 Hemolivia sp. JQ080303 Hepatozoon sp. 1 FJ719813 Hepatozoon sp. E Hepatozoon 90 ex Marsupialia FJ719814 Hepatozoon sp. 1 KM887509 pellegrini KM887507 Haemogregarina sacaliae 1 92 KF257926 Haemogregarina stepanowi Haemogregarina 100 0.90 HQ224959 Haemogregarina balli 1 MH924615 unidentified haemogregarine ex Pelusios sinuatus 100 MH924616 unidentified haemogregarine ex Pelomedusa subrufa AF494059 bambaroonidae HQ224958 ranarum AF494058 Adelina bambaroonidae

0.02

Fig. 1. Estimate of relationships of haemogregarines derived from partial 18S rRNA gene sequences using a Bayesian approach. Values above nodes correspond to bootstrap values from an ML approach, and those below nodes correspond to posterior probabilities. New sequences generated for this study are shown in bold. Right-hand side lettres correspond to the major groups identified by Maia et al. (2016).

2016). In N. mossambica the parasitic lineage found was Hepatozoon spp. recovered from various snakes and liz- most closely related to Hepatozoon ayorgbor Sloboda, ards from North Africa (Maia et al. 2011). A white-tailed Kamler, Bulantová, Votýpka et Modrý, 2007 described Ichneumia albicauda (Cuvier) was infected by from a snake in Ghana (Sloboda et al. 2007). The lineage a species clearly sister to Hepatozoon felis Patton, 1908, found infecting N. annulifera was closely related to other known from a wide range of felids. One sample of the Zulu

Folia Parasitologica 2018, 65: 015 Page 3 of 8 Fig.2. doi: 10.14411/fp.2018.015 TGTKGTTLÇHarris et al.: Haemoprotozoan parasites in South Africa MH924617 unidentified ex Xerus inauris AB668373 Theileria orientalis 0.99 AY735134 Theileria cervi AY508455 Theileria ovis 80 L02366 MH924618 Piroplasmida unidentified ex Ichneumia albicauda L19080 Cytauxzoon felis AY485690 Cytauxzoon manul

0.97 AF419313 Babesia bicornis 81 AY150062 Theileria penicillata GQ411405 Babesia lengau AF231350 Babesia gibsoni AY027815 Babesia sp. 98 AF158703 Piroplasmida 85 DQ200887 Babesia poelea

0.99 DQ437686 Theileria fuliginosus DQ437687Theileria penicillata KT937391 Theileria ornithorhynchi MH924619 Piroplasmida unidentified exRhabdomys sp. 1 FJ645725 like 1 100 JX454779 Babesia microti like 93 U09833 Babesia microti M87565 Babesia rodhaini 0.99 AY452708 Babesia leo

1 0.95 MH924620 Piroplasmida unidentified ex Chlorocebus pygerythrus 0.99 GQ225744 Babesia sp. 1 98 AF244913 Babesia sp. AF244912 Babesia felis DQ402155 Babesia bennetti FJ213579 Babesia bennetti 96 AY596279 Theileria fuliginousus X59604 Babesia bigemina AY260176 Babesia crassa 96 EF551335 Babesia kiwiensis U16370 Babesia divergens 81 AY072926 Babesia canis EF222255 Babesia sp. ovale

0.07

Fig. 2. Estimate of relationships of piroplasmids derived from partial 18S rRNA gene sequences using a Bayesian approach. Values above nodes correspond to bootstrap values from an ML approach, and those below nodes correspond to posterior probabilities. New sequences generated for this study are shown in bold. pipistrelle bat, Neoromicia zuluensis (Roberts), gave a pos- complex, with six broad clades identified (reviewed in itive result, with a lineage similar to one obtained from a O’Donoghue 2017). One of these consists of species of snake from the Seychelles. All other positive samples were Theileria Bettencourt, França et Borges, 1907 or Cytaux- from rodents and fell within a clade predominantly consist- zoon Kier, 1979 from carnivores, and one sample from ing of isolates of Hepatozoon identified from other rodents the white-tailed mongoose fell within this group, sister to and snakes. Cytauxzoon felis Kier, 1979. Another sample from a vervet Four different piroplasmids were identified, belonging monkey, Chlorocebus pygerythrus (Cuvier), was identical to the families and Theileridae (Fig. 2). Re- to a sample of Babesia sp. from an olive baboon Papio lationships of accepted genera within these families are anubis (Lesson). Two other samples from a grass mouse,

Folia Parasitologica 2018, 65: 015 Page 4 of 8 doi: 10.14411/fp.2018.015 Harris et al.: Haemoprotozoan parasites in South Africa

L76472 Sarcocystis capracanis Fig.3. 0.99 98 0.99 FJ196261 Sarcocystis gracilis 96 AF176935 Sarcocystis cruzi

AF176930 Sarcocystis sinensis

EF056017 Sarcocystis tarandi

GQ251026 Sarcocystis rangiferi

JN226124 Sarcocystis silva

1 AF176945 Sarcocystis hominis 95 MH924621 Sarcocystis sp. exAepyceros melampus

EU282026 Sarcocystis scandinavica

HM021724 Sarcocystis nesbitti

AF120114 Sarcocystis atheridis 0.99 99 MH924622 Sarcocystis sp. ex Ichneumia albicauda

MH924623Sarcocystis sp. exIchneumia albicauda

JQ762307 Sarcocystis sp. 1 100 AY015112 Sarcocystis gallotae 1 99 AY015113 Sarcocystis lacertae

0.97 KC696570 Sarcocystis sp. 97 AY015111 Sarcocystis rodentifelis

GU253883 Sarcocystis columbae

KC696571 Sarcocystis sp.

AF109679 Sarcocystis mucosa

1 1 AF120115 Sarcocystis dispersa 100 U07818 Sarcocystis neurona

AF009244 Sarcocystis microti

AF009245 Sarcocystis glareoli

1 besnoiti 100 Hyaloklossia lieberkuehni 0.06

Fig. 3. Estimate of relationships of sarcocystids derived from partial 18S rRNA gene sequences using a Bayesian approach. Values above nodes correspond to bootstrap values from an ML approach, and those below nodes correspond to posterior probabilities. New sequences generated for this study are shown in bold.

Rhabdomys sp., and the Cape ground squirrel, Xerus inau- from very different parasite lineage. One lineage was sister ris (Zimmermann), were infected with parasites that were taxon to Sarcocystis atheridis Šlapeta, Modrý et Koudela, distinct from published sequences but related to two differ- 1999 from Nitsche’s bush viper, Atheris nitschei Tornier, ent lineages of Theileria (Fig. 2). whereas the relationships of the other lineage are not clear The remaining positive samples were all phylogeneti- (Fig. 3). cally related to the . One sample from the im- pala Aepyceros melampus (Lichtenstein) was identical to DISCUSSION Sarcocystis truncata Gjerde, 2013 (originally reported as The primers used in this assessment allowed the de- S. rangiferi) from red deer, Cervus elaphus Linnaeus. Two tection of multiple and phylogenetically diverse lineages white-tailed mongoose were infected, each by a species of the . Haemogregarines are composed of

Folia Parasitologica 2018, 65: 015 Page 5 of 8 doi: 10.14411/fp.2018.015 Harris et al.: Haemoprotozoan parasites in South Africa the genera Haemogregarina Danilewsky, 1885, proaches. Clearly, the finding of a species of Hepatozoon Siddall, 1995 and Lainson, 1981 (Haemogrega- in two different continents and from two different species rinidae), Hepatozoon (Hepatozoidae), Hemolivia Petit, means that the original finding of Pinto et al. (2013) cannot Landau, Baccam et Lainson, 1990 and Karyolysus Labbé, be disregarded. Rather, it highlights the need for an exten- 1894 (), and Dactylosoma Labbé, 1894 and sive, targeted microscopic approach to confirm, whether Babesiosoma Jakowska et Nigrelli, 1956 (Dactylomatidae) some species of Hepatozoon can use bats as their verte- (see Smith and Desser 1997, Telford 2009). Parasites of the brate hosts. Haemogregarina are known to use chelonians as the The finding of two very different lineages of Hepato- vertebrate host. In this study, however, both samples from zoon in two species of cobras, Naja mossambica and Naja freshwater terrapins, Pelusios sinuatus and Pelomedusa annulifera, is less unexpected. Various studies have shown subrufa, appear to be infected with parasites that formed a that isolated of Hepatozoon identified in snakes reflected distinct lineage that was more closely related to Dactylo- in many cases dietary preferences (e.g. Tomé et al. 2014). soma than to different species of Haemogregarina. Further Similarly, all Hepatozoon from rodents seem to be part of studies on South African terrapins using both microscopy the same clade. Although four different haplotypes were and molecular tools are clearly needed to better identify detected from at least four different host species in the these parasites. present study, they all form part of the same clade, along While the taxonomy within the Haemogregarinidae is with all other published sequences retrieved from rodent not controversial, the situation in the remaining families is and many snake hosts. far from stable. The genus Hepatozoon is the most wide- The detection of isolates of Sarcocystis Lankester, 1882 ly reported and the most speciose, with over 300 species in tissue samples was also not unexpected, since they have (Telford 2009). In a recent review of phylogenetic rela- been detected previously in muscle tissue samples tionships, O’Donoghue (2017) reported that Hepatozoon using these primers (Harris et al. 2012). One sequence re- was paraphyletic, with one group using carnivores as the trieved from an was almost identical to sequences vertebrate host related to Karyolysus, and another us- from other bovids such as Sarcocystis truncata from red ing non-carnivores related to Hemolivia. Karadjian et al. deer, Cervus elaphus. Several studies have proposed that (2015) had previously proposed that the lineage found in the phylogeny of Sarcocystis reflects the relationships of carnivores should remain Hepatozoon, and the other line- the final hosts (e.g. Doležel et al. 1999). However, Tomé et age of parasites considered Hepatozoon should be placed al. (2013) have already shown that Sarcocystis from snakes in a new genus Bartozoon Karadjian, Chavatte et Landau, form multiple, unrelated groups. This is important as some 2015. However, the situation is far more complex, since authors have attempted to predict definitive hosts based on relationships between Hepatozoon and Hemolivia are not phylogenetic analysis of the parasite. For example, Tian et stable, and tend to change depending on which outgroup al. (2012) proposed a snake as the definitive host of Sar- is used (as highlighted in Maia et al. 2016), and an entire cocystis nesbitti Mandour, 2007 based on their estimate of new lineage that use geckos as vertebrate hosts has recent- phylogenetic relationships in which this taxon was sister ly been identified (Tomé et al. 2016), which does not fit taxon to Sarcocystis atheridis which does use a snake as into the newly proposed taxonomic scheme. Therefore, we the definitive host. follow Maia et al. (2016) and continue to refer to these The identification of two distinct lineages from two in- forms as Hepatozoon, pending a complete taxonomic revi- dividuals of white-tailed mongoose shows how complex sion of the group. the situation is and how difficult it is to identify coevo- Similarly controversial is the identification of Hepato- lutionary patterns between these parasites and their verte- zoon parasites in bats. Pinto et al. (2013) provided the first brate hosts. In our estimate of relationships one parasite evidence of Hepatozoon parasites infecting Hipposideros lineage from the mongoose is more closely related to S. cervinus (Gould) from Borneo, with molecular phyloge- atheridis, whereas the relationship of S. nesbitti is poorly netic assessments indicating they formed a clade with para- supported. Relationships of the Sarcocystis detected in the sites from reptiles, rodents and marsupials. However, Kar- second white-tailed mongoose, while clearly distinct from adjian et al. (2015) suggest this result should be interpreted the first lineage from this species and from other published with caution. We have observed on several occasions cysts sequences from other Sarcocystis, was similarly poorly from a haemogregarine in sections of the liver from a spe- supported. Sequencing of additional genes for these par- cies of Miniopterus Bonaparte and considered them to be asites may help to resolve relationships and confirm if the cysts from a Hepatozoon developing in a dipteran ingested phylogeny of some of these parasites does reflect that of by the bat. Haemogregarine gametocytes were never found the final host, or if this only occurs in some groups, such in the blood of any bat. These cysts are probably a dead as the bovids. end. The remaining parasites detected in the present study Here we report only the second evidence for species were piroplasmids belonging to the families Babesiidae of Hepatozoon from a bat, with a sample from the Zulu and Theileridae. Although some species of these parasites pipistrelle being part of a clade with parasites primarily can infect humans, causing malaria-like symptoms, cur- from reptiles and rodents. Screening molecular tissue sam- rent knowledge regarding their taxonomy is surprisingly ples allows far more samples to be included in large-scale confused. O’Donoghue (2017) proposed that molecular assessments than would be available for microscopic ap- phylogenetic assessments of 18S rRNA sequences clear-

Folia Parasitologica 2018, 65: 015 Page 6 of 8 doi: 10.14411/fp.2018.015 Harris et al.: Haemoprotozoan parasites in South Africa ly distinguished between theileriid and babesiid piroplas- found in squirrels (e.g. Tsuji et al. 2006). Indeed, although mids. Within these, six broad clades were identified, of rodents can be infected with very different lineages of which theileriid parasites could be split into three, Thei- Babesia, they are not known to be hosts to species of Thei- leria/Cytauxzoon from felids, Theileria from equids and leria (reviewed in O’Donoghue 2017). However, the lin- rhinoceroses, and Theileria from bovids. In our estimate eage identified in X. inauris is clearly related to Theileria of relationships, separation between theileriid and babesiid orientalis (Yakimoff and Soudatschenkoff 1931) and other piroplasmids is less clear. One possible explanation is that species of Theileria known to infect bovids. The sample forms were misidentified on GenBank, since errors of this from the grass mouse, Rhabdomys sp., is more difficult to type are not uncommon (Harris 2003). Tissue samples can place within a phylogenetic framework, because relation- also be infected with multiple parasites, leading to discrep- ships of this new sequence with others are less well sup- ancies between observed parasites and sequences obtained ported (Fig. 2). from infected tissues. Despite these uncertainties, the four One sample of white-tailed mongoose was infected with samples positive for these parasites in our analysis can be both Hepatozoon and Sarcocystis spp., detected in liver and placed within the overall phylogeny with little ambiguity. muscle tissue samples, respectively. Parasite coinfection One sample, from the white-tailed mongoose, was appar- and interactions can have important influences on the host ently infected with a species of Cytauxzoon, more closely (e.g. Cattadori et al. 2008). This finding highlights both related to Cytauxzoon felis which is widespread in vari- the value of examining different tissue sources from single ous felids, than to Cytauxzoon manul Reichard, Van Den animal carcass and also the need for further assessments of Bussche, Meinkoth, Hoover et Kocan, 2005 from the Pal- apicomplexan parasite interactions within hosts. las , Otocolobus manul (Pallas). Species of Cytauxzo- Even though conclusive taxonomic inferences regarding on are common in many felids and rarely reported from the parasites infecting the wide range of hosts included in other hosts. A similar relationship to the one identified the present study were not provided, it is clear that, molec- here was reported for a parasite from the Burmese ferret ular screening repeatedly identifies new lineages of para- badger Melogale personata Geoffroy Saint-Hilaire, but the sites in new hosts, which have profound implications. This sequence is not available for comparisons (Sukmak et al., highlights the great value of this kind of approach, even if unpublished conference report). South-African it then requires a follow up with additional markers and, were also often identified as infected by species of Cytaux- when possible, microscopy to allow more integrative con- zoon, although these were suggested to be the sister taxon clusions. As a first step, the method clearly can demonstrate to the C.felis/C.manul clade (Leclaire et al. 2015). Further how limited current knowledge still is regarding these im- assessments are clearly needed to identify the range of portant parasites, and can direct further studies to key hosts hosts of Cytauxzoon outside of felids, and the relationships that have been previously overlooked. between the different species. The identification of isolate of Babesia Starcovivi, 1893 Acknowledgements. DJH and RX are supported by the Fundação para a Ciência e a Tecnologia (FCT, Portugal) through IF con- in the was not unexpected, since this has tracts 01627/2014 and 00359/2015, respectively. Thanks are due been previously reported for many African primates (re- to Chris Burger for identification of snakes, Philippe Gaubert and viewed by Cormier and Jolly 2017). The haplotype was Emmanuel Do Linh San for confirming the identification of gen- identical to one recovered from an olive baboon, Papio ets, and to all those who helped AH with roadkills and checking anubis. In contrast, the relationships of the parasites re- them: Wilmien J Luus-Powell, David Kunutu, Michael Rampedi, covered from the ground squirrel Xerus inauris were un- Makubu P. Mokgawa, Sello Matjee (University of Limpopo) and expected. Previously, Babesia microti-like parasites were Jabu Linden (Lajuma Research Centre).

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Received 30 November 2017 Accepted 31 May 2018 Published online 2 October 2018

Cite this article as: Harris D.J., Halajian A., Santos J.L., Swanepoel L.H., Taylor P.J., Xavier R. 2018: Diversity of haemoprotozo- an parasites infecting the wildlife of South Africa. Folia Parasitol. 65: 015

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