Reassignment of the land tortoise haemogregarine Haemogregarina fitzsimonsi Dias 1953 (Adeleorina: Haemogregarinidae) to the genus Hepatozoon Miller 1908 (Adeleorina: Hepatozoidae) based on parasite morphology, life cycle and phylogenetic analysis of 18S rDNA sequence fragments

Abstract

SUMMARY Research was undertaken to clarify the true taxonomic position of the terrestrial tortoise apicomplexan, Haemogregarina fitzsimonsi (Dias, 1953). Thin blood films were screened from 275 wild and captive South African tortoises of 6 genera and 10 species between 2009-2011. Apicomplexan parasites within films were identified, with a focus on H. fitzsimonsi. Ticks from wild tortoises, especially Amblyomma sylvaticum and Amblyomma marmoreum were also screened, and sporogonic stages were identified on dissection of adult ticks of both species taken from H. fitzsimonsi infected and apparently non-infected tortoises. Parasite DNA was extracted from fixed, Giemsa-stained tortoise blood films and from both fresh and fixed ticks, and PCR was undertaken with two primer sets, HEMO1/HEMO2, and HepF300/HepR900, to amplify parasite 18S rDNA. Results indicated that apicomplexan DNA extracted from tortoise blood films and both species of tick had been amplified by one or both primer sets. Haemogregarina fitzsimonsi 18S rDNA sequences from tortoise blood aligned with those of species of Hepatozoon, rather than those of species of Haemogregarina or Hemolivia. It is recommended therefore that this haemogregarine be re-assigned to the genus Hepatozoon, making Hepatozoon fitzsimonsi (Dias, 1953) the only Hepatozoon known currently from any terrestrial chelonian. Ticks are its likely vectors.

FirstHemoliviafrom southern Africa: reassigning chelonianHaemogregarina parvulaDias, 1953 (Adeleorina: Haemogregarinidae) toHemolivia(Adeleorina: Karyolysidae)

“…Kinixys belliana zuluensis, Kinixys belliana belliana) (see Dias 1953;Cook et al 2014aCook et al , 2014b. Haemogregarina parvula has only once been reported to parasitise an additional host species, an individual of S. pardalis (see Cook et al 2009), whilst H. fitzsimonsi in comparison has been reported from an additional four tortoise species (see Cook et al 2009Cook et al , 2014a. Given this repeatedly concurrent infection Siddall (1995) synonymised this species with H. fitzsimonsi possibly due to the concern that H. parvula may be an encapsulated form or stage of H. fitzsimonsi.…”

“…graeca and T. marginata, which inhabit temperate to desert regions of northern Africa to the western Palearctic realm (Široký et al 2009). This is in stark contrast to H. parvula, which has only ever been found, during surveys of most of South Africa's tortoise species, including arid to tropical biomes, in tropical KwaZulu-Natal and Mozambique (see Dias 1953;Cook et al 2009Cook et al , 2014aCook et al , 2014b. In addition, H. parvula has only, with one exception as mentioned above, ever been reported to parasitise K. zombensis.…”

“…KR069084) from an additional host (K. zombensis) was produced during this study, and was identical to the isolate (GenBank accession no. KJ702453) described in Cook et al (2014a). In addition, H. parvula and H. fitzsimonsi, which are known to occur concurrently, belonged to separate clades.…”

“…Tortoises were processed and identified to species level at the site of capture using field guides (Branch 2008;Bates et al 2014). Any possible haematophage vectors attached to tortoises, such as ticks, were collected, fixed in 70% ethanol and identified, at a later stage, to species level as in Cook et al (2014a). Blood, 0.2-1 ml depending on the size of the tortoise, was collected from the subcarapacial sinuses using sterile 21G 1½ inch needles fitted to 1 ml syringes (see McArthur et al 2004).…”

“…Haemogregarines were identified to species level by comparing morphometric characteristics to previous data on these parasites (see Cook et al 2009Cook et al , 2014aCook et al , 2014b. Parasitaemia was calculated per 100 erythrocytes, with c. 10 4 erythrocytes examined per blood smear (see Cook et al 2009Cook et al , 2010Netherlands et al 2014a).…”