Courtney A Cook

Hepatozoon species (Adeleorina: Hepatozoidae) of African bufonids, with morphological description and molecular diagnosis of Hepatozoon ixoxo sp. nov. parasitising three Amietophrynus species (Anura: Bufonidae)

BackgroundHaemogregarines comprise a large group of apicomplexan blood parasites. In 1996 all anuran haemogregarines still in the genus Haemogregarina Danilewsky, 1885 were reassigned to the genus Hepatozoon Miller, 1908. Most (11/15, 73%) African anuran Hepatozoon species have been recorded from the family Bufonidae, however, all these are recorded from only two host species, Amietophrynus mauritanicus (Schlegel, 1841) and Amietophrynus regularis (Reuss, 1833) from Northern and central Africa. To the authors’ knowledge the only description of an anuran haemogregarine from South Africa is Hepatozoon theileri (Laveran, 1905), parasitising Amietia quecketti (Boulenger, 1895).MethodsThin blood smears for morphometrics and whole blood for molecular work, were collected from 32 Amietophrynus garmani (Meek, 1897), 12 Amietophrynus gutturalis (Power, 1927), and nine Amietophrynus maculatus (Hallowell, 1854), in Ndumo Game Reserve and Kwa Nyamazane Conservancy, KwaZulu-Natal, South Africa. Smears were Giemsa-stained, screened for haemogregarines, parasite stages measured, compared to each other and to other described African bufonid haemogregarines. Parasite 18S rDNA was amplified using two apicomplexan-specific primer sets, HepF300/HepR900 and 4558/2733. Resulting sequences of the haemogregarine isolates from the three Amietophrynus species were compared with each other and to comparative haemogregarine sequences selected from GenBank.ResultsMorphological characteristics of parasite stages, in particular characteristically capped mature gamont stages, and molecular findings, supported all three haemogregarine isolates from all three Amietophrynus species to be the same, a species of Hepatozoon, and furthermore different morphologically from other previously recorded bufonid Hepatozoon species. The haemogregarine fell within a clade comprising other anuran Hepatozoon species and furthermore, within a monophyletic sub-clade along with H. theileri and are described as Hepatozoon ixoxo sp. nov.ConclusionsThis is the first morphological and molecular account of Hepatozoon species within the family Bufonidae from South Africa, a study hoped to encourage the redescription and molecular analysis of those Hepatozoon species described in the past from Amietophrynus species, as well as to promote the use of both morphological and molecular characteristics in Hepatozoon species descriptions. This will aid in comprehensive Hepatozoon descriptions, which along with the use of phylogenetic analysis will give a better indication of these parasites possible vectors and life cycle dynamics.

Biodiversity of frog haemoparasites from sub-tropical northern KwaZulu-Natal, South Africa.

Highlights • First frog (11 families, 6 genera, 29 spp.) haemoparasite survey in South Africa.• Showing a higher biodiversity as compared with similar research in Africa.• Intraerythrocytic and extracellular haemoparasites of five groups found.• Hepatozoon and Trypanosoma spp. were the most prevalent.• Protected areas boast higher parasite diversity compared with human impacted sites.

First Hemolivia from Southern Africa: Reassigning Chelonian Haemogregarina parvula Dias, 1953 (Adeleorina: Haemogregarinidae) to Hemolivia (Adeleorina: Karyolysidae)

To date, only a single species of Hemolivia, Hemolivia mauritanica (Sergent & Sergent, 1904), has been described from African terrestrial tortoises. Although various haemogregarines have been described from southern African terrapins and tortoises, including species from the genus Haemogregarina and one from the genus Hepatozoon, no species of Hemolivia have been identified previously from southern Africa. Since its morphological redescription, the taxonomic placement of one of these species, Haemogregarina parvula Dias, 1953, was in doubt. Hence, research was undertaken to resolve the true taxonomic position of this haemogregarine. Blood smears from nine wild tortoises of two species, Stigmochelys pardalis and Kinixys zombensis, from South Africa were screened, with the focus on H. parvula. Parasite DNA was extracted from ethanol-preserved blood samples, and fragments of the 18S rDNA gene were amplified by PCR using the primer sets HepF300/HepR900 and 4558/2733. Maximum likelihood and maximum parsimony analyses of 18S rDNA sequences showed that Haemogregarina parvula formed a monophyletic clade with species of Hemolivia within the Hepatozoon clade, not with species of Haemogregarina. It is thus recommended that this haemogregarine be reassigned to the genus Hemolivia, rendering Hemolivia parvula (Dias, 1953) the first species of the genus recorded from southern African tortoises.

Developing an Apicomplexan DNA Barcoding System to Detect Blood Parasites of Small Coral Reef Fishes

Abstract Apicomplexan parasites are obligate parasites of many species of vertebrates. To date, there is very limited understanding of these parasites in the most-diverse group of vertebrates, actinopterygian fishes. While DNA barcoding targeting the eukaryotic 18S small subunit rRNA gene sequence has been useful in identifying apicomplexans in tetrapods, identification of apicomplexans infecting fishes has relied solely on morphological identification by microscopy. In this study, a DNA barcoding method was developed that targets the 18S rRNA gene primers for identifying apicomplexans parasitizing certain actinopterygian fishes. A lead primer set was selected showing no cross-reactivity to the overwhelming abundant host DNA and successfully confirmed 37 of the 41 (90.2%) microscopically verified parasitized fish blood samples analyzed in this study. Furthermore, this DNA barcoding method identified 4 additional samples that screened negative for parasitemia, suggesting this molecular method may provide improved sensitivity over morphological characterization by microscopy. In addition, this PCR screening method for fish apicomplexans, using Whatman FTA preserved DNA, was tested in efforts leading to a more simplified field collection, transport, and sample storage method as well as a streamlining sample processing important for DNA barcoding of large sample sets.

Ultrastructural Comparison of Hepatozoon ixoxo and Hepatozoon theileri (Adeleorina: Hepatozoidae), Parasitising South African Anurans.

To date, only two haemogregarine parasite species have been described from South African anurans: Hepatozoon ixoxo, infecting toads of the genus Sclerophrys (syn. Amietophrynus); and Hepatozoon theileri, parasitising the common river frog, Amietia quecketti. Both species have been characterised using limited morphology, and molecular data from PCR amplified fragments of the 18S rRNA gene. However, no ultrastructural work has been performed thus far. The aim of this study was to add descriptive information on the two species by studying their ultrastructural morphology. Mature gamont stages, common in the peripheral blood of infected frogs, were examined by transmission electron microscopy. Results indicate that H. ixoxo and H. theileri share typical apicomplexan characteristics, but differ markedly in their external cellular structure. Hepatozoon ixoxo is an encapsulated parasite presenting a prominent cap at the truncate pole, and shows no visible modifications to the host cell membrane. In comparison, H. theileri does not present a capsule or cap, and produces marked morphological changes to its host cell. Scanning electron microscopy was performed to further examine the cytopathological effects of H. theileri, and results revealed small, knob‐like protrusions on the erythrocyte surface, as well as notable distortion of the overall shape of the host cell.

A new lizard malaria parasite Plasmodium intabazwe n. sp (Apicomplexa: Haemospororida: Plasmodiidae) in the Afromontane Pseudocordylus melanotus (Sauria: Cordylidae) with a review of African saurian malaria parasites

BackgroundSaurian malaria parasites are diverse apicomplexan blood parasites including the family Plasmodiidae Mesnil, 1903, and have been studied since the early 1900s. Currently, at least 27 species of Plasmodium are recorded in African lizards, and to date only two species, Plasmodium zonuriae (Pienaar, 1962) and Plasmodium cordyli Telford, 1987, have been reported from the African endemic family Cordylidae. This paper presents a description of a new malaria parasite in a cordylid lizard and provides a phylogenetic hypothesis for saurian Plasmodium species from South Africa. Furthermore, it provides a tabular review of the Plasmodium species that to date have been formally described infecting species of African lizards.MethodsBlood samples were collected from 77 specimens of Pseudocordylus melanotus (A. Smith, 1838) from Platberg reserve in the Eastern Free State, and two specimens of Cordylus vittifer (Reichenow, 1887) from the Roodewalshoek conservancy in Mpumalanga (South Africa). Blood smears were Giemsa-stained, screened for haematozoa, specifically saurian malaria parasites, parasite stages were photographed and measured. A small volume was also preserved for TEM studies. Plasmodium and Haemoproteus primer sets, with a nested-polymerase chain reaction (PCR) protocol, were employed to target a fragment of the cytochrome-b (cyt-b) gene region. Resulting sequences of the saurian Plasmodium species’ isolates were compared with each other and to other known Plasmodium spp. sequences in the GenBank database.ResultsThe presence of P. zonuriae in both specimens of the type lizard host C. vittifer was confirmed using morphological characteristics, which subsequently allowed for the species’ molecular characterisation. Of the 77 P. melanotus, 44 were parasitised by a Plasmodium species, which when compared morphologically to other African saurian Plasmodium spp. and molecularly to P. zonuriae, supported its description as a new species Plasmodium intabazwe n. sp.ConclusionsThis is the first morphological and molecular account of Plasmodium species within the African endemic family Cordylidae from South Africa. The study highlights the need for molecular analysis of other cordylid Plasmodium species within Africa. Future studies should also include elucidating of the life-cycles of these species, thus promoting the use of both morphological and molecular characteristics in species descriptions of saurian malaria parasites.

The distribution and host-association of a haemoparasite of damselfishes (Pomacentridae) from the eastern Caribbean based on a combination of morphology and 18S rDNA sequences

Coral reefs harbor the greatest biodiversity per unit area of any ecosystem on earth. While parasites constitute the majority of this biodiversity, they remain poorly studied due to the cryptic nature of many parasites and the lack of appropriate training among coral reef ecologists. Damselfishes (Pomacentridae) are among the most abundant and diverse fishes on coral reefs. In a recent study of blood parasites of Caribbean reef fishes, the first ever apicomplexan blood parasites discovered in damselfishes were reported for members of the genus Stegastes. While these blood parasites were characterized as “Haemohormidium-like”, they appear to be distinct from any other known apicomplexan. In this study, we examined host associations, geographic distributions, and provide further insights on the phylogenetic affiliation of this parasite. A combination of morphological characteristics and 18S rDNA sequences suggest that this parasite may be the same species at multiple sites and occurs from the southern to the northern extreme of the eastern Caribbean, although it appears rare in the north. At present it appears to be limited to members of the genus Stegastes and infects all life history stages. It is most common in benthophagous species that occur in high population densities and appears basal to a major monophyletic clade containing species of coccidia, distinct from the Piroplasmida, the order to which Haemohormidium spp. have been assigned. These findings suggest a possible fecal-oral mode of transmission.