Biodiversity and systematics of apicomplexan parasites infecting South African leopard and hinged tortoises

M.Sc.Research into blood protozoans (haematozoans) infecting African tortoises is scanty with only a few records published, many during the early part of the last century. Little research had been done on the blood parasites of tortoises examined in this study namely, Kinixys lobatsiana, K. belliana belliana, K. natalensis, Geochelone pardalis pardalis, G. pardalis babcocki and Chersina angulata. The study therefore aimed to: 1) examine apicomplexan haematozoan parasites infecting several of South Africa’s indigenous tortoises and compare them with published species descriptions, especially from neighbouring Mozambique; 2) provide host details (identity, ectoparasites, host weight and gender, effects of blood parasites on host cells) and locality records in different seasons for described and new apicomplexan species; 3) describe new and recorded parasites using morphometrics and, if possible, ultrastructural characteristics 4) attempt apicomplexan DNA extraction, amplification and, if feasible, purification; and 5) establish a basis for future research as a result of the acquired knowledge. During the current study, 154 tortoises of six species in three genera, both captive and wild, and from four South African provinces (Gauteng, North West, Kwazulu-Natal and Western Cape) were sampled. Giemsa stained blood smears and use of image analysis enabled morphometric analysis of the apicomplexans and their effects on host cells, while some blood preserved in Karnovsky’s and Todd’s fixatives received detailed examination by transmission electron microscopy. Lastly, blood preserved in lysis buffer during collection, and with the highest parasitaemias, was subjected to parasite DNA extraction and amplification. Comparisons between a published account of apicomplexans recorded from K. b. belliana in Mozambique, and those found in the current study, identified two haemogregarine species. In the present research, Haemogregarina fitzsimonsi Dias, 1953 infected 2/27 (7%) wild North West K. lobatsiana, 2/3 (66%) captive Kwazulu-Natal K. natalensis, 7/14 (50%) captive Kwazulu- Natal K. b. belliana, 3/6 (50%) captive Kwazulu-Natal G. p. pardalis, 2/41 (5%) wild G. p. babcocki and 13/37 (35%) captive Gauteng G. pardalis. In addition, Haemogregarina parvula Dias, 1953, infected 2/14 (14%) captive K. b. belliana and 1/10 (10%) captive G. p. pardalis. An unknown species of haemogregarine, possibly also H. fitzsimonsi occurred in 6/16 (38%) Chersina angulata from the Western Cape. As well as haemogregarines, two haemoproteids were identified: Haemoproteus balazuci Dias, 1953 infected 2/27 (7%) wild North West K. lobatsiana, 2/2 (100%) captive Gauteng K. lobatsiana and 1/41 (2%) wild North West G. p. babcocki; Haemoproteus sp., a likely new species, was found in 1/3 (33%) captive K. natalensis. Infections with Haemogregarina and Haemoproteus were not concurrent in this study, but were found to occur concurrently in Dias (1953) findings, and only the two Haemogregarina spp. occurred together in captive Kwazulu-Natal G. p. pardalis tortoises, which do not occur naturally in the region. Haemogregarina fitzsimonsi did not appear region or host specific, since it infected 5/6 species of tortoises from all provinces sampled. Haemogregarina parvula apparently existed only in tortoises from Kwazulu-Natal. Furthermore, captive Gauteng female tortoises were found to have a higher rate of infection than males and heavier tortoises showed a lower intensity infection than lighter and younger tortoises. On average season appeared to have a slight affect on parasite prevalence, with a higher prevalence during the summer rather than the winter, possibly a result of the activity of the assumed vector, which may be the tick species Amblyomma marmoreum (found on G. pardalis) and/or Amblyomma hebraeum (found on C. angulata). For the new Haemoproteus sp., the small sample size meant that meaningful data on host-specificity and range was not gathered, but Hp. balazuci occurred in K. lobatsiana in the drier regions of the North West and Gauteng. Although DNA extraction was possible for H. fitzsimonsi, the technique requires further refinement and samples with greater parasitemias before it can be used with additional material, and sequencing can be attempted. Thus, new localities, hosts, host data and possible vectors (ticks) were recorded for the apicomplexan species identified by Dias (1953) and they were re-described using modern techniques. Also, possibly new Haemogregarina and Haemoproteus spp. were recorded, but their identity requires confirmation by DNA analysis. It is anticipated that these, and future results, will increase the knowledge of the ecology and biodiversity of apicomplexan haematozoans parasitising chelonian hosts in South Africa, with possible application to the conservation of these and other tortoise species around the world