Reptiles of South Africa as hosts for tickborne pathogens and haemoparasites

Doctor of Philosophy in Science with Environmental Sciences, North-West University, Vanderbijlpark Campus

Haemoparasites, ticks, and tickborne bacterial microorganisms have been reported in a variety of vertebrate and invertebrate hosts. Reptiles have been documented as hosts for ticks, tickassociated microorganisms, as well as haemoparasites. However, in South Africa, the role of reptiles and their associated invertebrate vectors in the transmission of haemoparasites and tickborne bacterial microorganisms is lacking, in part, due to limited information on the presence, diversity, and molecular evidence of ticks and their associated pathogens. To increase our knowledge on reptilian haemoparasites, their associated tick species and tick-borne bacterial microorganisms, a combined approach (use of microscopy and polymerase chain reaction) study focusing on the diversity and occurrence of haemoparasites, ticks and tick-borne pathogens was undertaken. Most of the fieldwork took place in Ndumo Game Reserve. However archived samples collected from 2014-2017 in and from the region near the village Paternoster and Bonamanzi Game Reserve were also included. The study was divided into five main sections for the collection, analysis and reporting of data. The first section was to determine the infestation, morphology and diversity of reptile associated ticks, as well as their potential as vectors of Hepatozoon, the second to elucidate the life cycle of Hepatozoon fitzsimonsi (Dias 1953) and in conjunction with the former section, third and fourth to determine the morphology, morphometric and phylogenetic analysis of Hepatozoon species and haemosporidian species, respectively. Lastly the occurrence and diversity of tick-borne bacterial microorganisms. Ticks present on the reptiles at the time of blood collection were carefully removed using forceps as to ensure that the hypostome remained intact with minimal damage to the tick. Blood was drawn from the subcarapacial sinuses of tortoises, as well as from the caudal vein of squamate reptiles and thin blood smears prepared for screening and morphometrics; the remaining blood fixed in 70% molecular grade ethanol for later molecular analysis. Giemsastained blood smears were screened microscopically for the presence of any haemoparsasites. All samples were then further analyzed according to the objectives of the respective chapters. Analyses included both morphological and molecular aspects. Morphology was used for the description and identification of species, and molecular analyses were used to assist with the morphology-based descriptions, as well as to allow for phylogenetic relationship comparisons of the ticks, tick-borne pathogens and haemoparasites. For chapter 3 of the thesis, a total of 243 collected ticks from these reptiles were used and morphologically identified as Amblyomma latum (Koch 1844) (n=43), Amblyomma exornatum (Koch 1844) (n=37), Amblyomma marmoreum (Koch 1844) (n=113) and Amblyomma sylvaticum (de Geer 1778) (n=60), morphological identification was then confirmed molecularly using 12S vi rRNA, 16S rRNA, 18S rRNA, ITS2, and CO1 markers. Upon morphological identification, a total number of seven ticks displayed at least one abnormality, such as ectromely and body deformities. These were observed from one of the four species of Amblyomma ticks. Phylogenetic relationships of the Amblyomma ticks from the current study show that the Amblyomma ticks formed a monophyletic clade with closely related Amblyomma species, even though amplification of DNA for some of the genes were unsuccessful. A species of Hepatozoon, H. fitzsimonsi was identified from A. marmoreum and A. sylvaticum ticks, with an overall prevalence of 4.7%. This study provides the first report of anomalous ticks associated with reptiles in South Africa and represents the second study to describe morphological abnormalities in hard ticks in South Africa. Furthermore, the taxonomic position of A. sylvaticum within Amblyomminae have been evaluated for the first time with molecular-phylogenetic methods. Even though ticks have been suggested to be the likely vector of H. fitzsimonsi, with this supported by the findings of Hepatozoon-like developmental stages in ticks collected off infected tortoises, the present study provides further support for ticks acting as the potential vectors of H. fitzsimonsi. A total of 14 tortoises were collected from Bonamanzi Game Reserve for the purpose of the developmental stages of H. fitzsimonsi, one Kinixys natalensis and Kinixys spekii, seven Kinixys zombensis and five Stigmochelys pardalis. Three species of Amblyomma ticks (Order Acari; Family Ixodidae) were collected from the three S. pardalis; Amblyomma herbraeum, A. marmoreum and Amblyomma nuttalli. The sporogonic stages of H. fitzsimonsi were identified in the hemocoel of these Amblyomma ticks, whilst merogonic stages were observed in the liver of one S. pardalis with no observable stages identified in other organs. The phylogenetic analysis show that the species of Hepatozoon from the present study clustered primarily with 18S rDNA sequences of known H. fitzsimonsi and within a larger clade containing sequences of Hepatozoon of other chelonians. Two species of varanid lizards and one species of elapid snake were parasitized with Hepatozoon species. Two species of Hepatozoon, Hepatozoon sp. A and Hepatozoon sp. B that were morphologically, morphometrically, and molecularly distinct were observed from Naja mossambica. The mature gamonts of Hepatozoon sp. A are smaller and wider (13.75 × 4.61) than that of Hepatozoon sp. B (16.52 × 3.36). According to the phylogenetic analysis, 18S rDNA sequences of Hepatozoon sp. A was most closely related to H. ayorgbor Sloboda et al. (2007) described from Python regius in Ghana, while the sequence of Hepatozoon sp. B was most closely related to Hepatozoon colubri Börner (1901) that Zechmeisterová et al. (2021) recovered from Zamenis langissumus in Iran. Comparison of Hepatozoon from this study’s monitor lizards with other described species of Hepatozoon infecting African varanids show that the Hepatozoon described in the present study from South African Varanus niloticus and Varanus albigularis was vii found to be morphologically similar to Hepatozoon camarai described by Dias (1954) from a specimen of V. a. albigularis in neighbouring Mozambique and Hepatozoon varani originally described from V. niloticus in South Africa by Laveran in 1905, and later in other up African countries. The sequence of the Hepatozoon sp. of monitor lizards from the present study formed a monophyletic clade and clustered with sequences of Hepatozoon species of other Varanus species and the snake Liasis fuscus. This study presents the first formal description of Hepatozoon species parasitizing N. mossambica, and one more Hepatozoon species from V. albigularis and V. niloticus in South Africa based on morphological description and molecular characterization. Furthermore, the present study represents the first morphological and molecular report of Hepatozoon within South African varanids and elapid snakes. Two tortoises (both K. natalensis) were parasitized by a species of Haemoproteus (Haemocystidiumi). This species did not compare morphologically to the previously described species of Haemoproteus (Haemocystidium) from South African nor other African tortoises. Four snakes (N. mossambica and Dendroaspis spp.) were found infected with a species morphologically comparable to Haemoproteus mesnili (Bouet 1909) Wenyon 1926. One tick (A. latum) was found positive with DNA comparable to the latter when molecularly screened targeting a fragment of the cyt-b gene; this most likely due to an incompletely digested bloodmeal. In the phylogenetic analysis, the two sequences of Haemoproteus (Haemocystidium) from two individuals of K. natalensis formed a monophyletic clade with 100% bootstrap support. Furthermore, these sequences clustered in a larger clade with Haemocystidium of both terrestrial and aquatic chelonians. Regarding the sequences of Haemoproteus (Haemocystidium) from snakes and a single tick, these clustered together in a monophyletic clade. With high bootstrap support (99%), this clade formed a sister clade to Haemoproteus mesnili, which in turn clustered within a larger monophyletic clade consisting of Haemocystidium of snakes and lizards. As such this study provides the first available molecular data for a species of Haemocystidium of tortoises in South Africa, and the first combined descriptive approach of Haemocystidium species for chelonians. These Amblyomma ticks (n = 253), as well as their reptiles (n=71) were also screened for the presence of various bacterial microorganisms, including Coxiella, Ehrlichia, Anaplasma, Rickettsia, Borrelia, as well as haemoparasites such as haemogregarines and haemosporidians by amplification, sequencing and phylogenetic analysis of the 16S rRNA, 23S rRNA, gltA, OmpA, Flagellin, and 18S rRNA genes, respectively. This study recorded the presence of reptile associated Borrelia species and Coxiella-like endosymbiont in South Africa for the first time. Furthermore, a spotted fever group Rickettsia species was molecularly detected in seven A. marmoreum and 14 A. sylvaticum from tortoises of genera Kinixys and Chersina. Francisella-like viii endosymbiont was molecularly detected in two A. latum collected from N. mossambica. Moreover, an environmental bacterium genus (Brevibacterium) was observed from A. sylvaticum. Coxiella burnetii, Ehrlichia spp. and Anaplasma spp., were not detected from these ticks. None of the reptilian blood was positive for any of the tick-borne microorganisms. Observations from this study provide indications that reptilian ticks may play a role in the transmission of pathogenic bacteria to homothermic animals. Furthermore, the absence of Ehrlichia, Anaplasma spp., and C. burnetii does not mean that these pathogens should be completely neglected and not screened for in future studies. In summary, this study explored the occurrence and diversity of reptilian haemoparasites, ticks and tick-borne pathogens infecting reptiles from two provinces (KwaZulu-Natal and Western Cape) of South Africa. Based on the results of the study, four species of ticks were recorded infesting chelonians and squamate reptiles, and the phylogenetic position of A. sylvaticum with Amblyomminae was determined for the first time, contributing to studies focusing on the phylogenetic analysis of genus Amblyomma, as well as extending the scope of previous studies on ticks infesting squamates in South Africa. Furthermore, in the present study, sporogonic stages in three species of Amblyomma ticks were molecularly confirmed to be that of H. fitzsimonsi, substantiating that these parasites do act as definitive hosts and strongly suggesting that they are a vector. Moreover, the current study presents the first combined morphological and molecular description of Hepatozoon species parasitizing N. mossambica, and one more Hepatozoon species from V. albigularis and both V. niloticus in South Africa, respectively. The phylogenetic relationships of species of Hepatozoon, Haemoproteus (Haemocystidium) were also characterized based on comparisons to other available molecular data. The presence of different bacterial microorganisms from the study provides indications that reptilian ticks may play a role in the transmission of pathogenic bacteria to homothermic animals. Furthermore, results of the current study provide a baseline for future studies on taxonomy, diversity and phylogenetic relationships of reptilian haemoparasites, ticks, as well as tick-borne bacterial microorganisms in South Africa and worldwide.

-National Research Foundation-Foundational Biodiversity Information Programme (NRF-FBIP)

Doctoral