Herpesvirus associated dermal papillomatosis in Williams' mud turtle Pelusios williamsi with effects of autogenous vaccine therapy.

An adult female of Williams' mud turtle, Pelusios williamsi long-term captive, that was allegedly caught wild in Kenya was found to have developed papilloma-like skin lesions. Excised tumors were examined histologically after routine processing with hematoxylin and eosin (H & E) stained slides, examined for the presence of viral particles by electron microscopy employing negative staining, and examined for the presence of viral DNA by PCR. Microscopic features in pre-treatment biopsies were fully diagnostic and consistent with multifocal squamous cell papilloma. Viral-type inclusion bodies were not identified. Turtle was found to be infected by reptilian herpesvirus. Association with herpesvirus and vast multiplicity of tumors thwarted surgical solution. An autogenous vaccine was prepared using 5 g of excised fresh tissue, aseptically ground, treated with diluted formalin, centrifuged to obtain a supernatant, and subsequently exposed to UV light. Autogenous vaccine induced substantial areas of necrosis of the papillomatous lesions noted by the loss of cytological architecture, nuclear loss, and by edema. The outer edges of the healing biopsies appeared to be regenerating. Therefore, our vaccine application could be considered as effective. It is difficult to treat and eliminate herpesvirus infection because of its cryptic presence and sudden onset of disease. Successful application of autogenous vaccine could be a potentially promising strategy, which deserves further testing.

Phylogeny and Morphological Variability of Trypanosomes from African Pelomedusid Turtles with Redescription of Trypanosoma mocambicum Pienaar, 1962.

Little is known about host specificity, genetic diversity and phylogenetic relationships of African turtle trypanosomes. Using PCR targeting the SSU rRNA gene, we detected trypanosomes in 24 of 134 (17.9%) wild caught African pelomedusid turtles: Pelusios upembae (n=14), P. bechuanicus (n=1), P. rhodesianus (n=3) and P. subniger (n=6). Mixed infection of Trypanosoma species was confirmed by PCR in three specimens of P. upembae, and in one specimen each of P. bechuanicus, P. rhodesianus, and P. subniger. Microscopic examination of stained blood smears revealed two distinct forms (broad and slender) of trypomastigotes. The broad form coincided in morphology with T. mocambicumPienaar, 1962. Accordingly, we have designated this form as the neotype of T. mocambicum. In phylogenetic analysis of the SSU rRNA gene, all the new turtle trypanosome sequences grouped in a single clade within the strongly supported "aquatic" clade of Trypanosoma species. The turtle trypanosome clade was further subdivided into two subclades, which did not correlate with host turtle species or trypanosome morphology. This study provides the first sequence data of Trypanosoma species isolated from freshwater turtles from tropical Africa and extends knowledge on diversity of trypanosomes in the Afrotropical zoogeographical realm.

Phylogeny, Diversity, Distribution, and Host Specificity of Haemoproteus spp. (Apicomplexa: Haemosporida: Haemoproteidae) of Palaearctic Tortoises.

A complex wide-range study on the haemoproteid parasites of chelonians was carried out for the first time. Altogether, 811 samples from four tortoise species from an extensive area between western Morocco and eastern Afghanistan and between Romania and southern Syria were studied by a combination of microscopic and molecular-genetic methods. Altogether 160 Haemoproteus-positive samples were gathered in the area between central Anatolia and eastern Afghanistan. According to variability in the cytochrome b gene, two monophyletic evolutionary lineages were distinguished; by means of microscopic analysis it was revealed that they corresponded to two previously described species-Haemoproteus anatolicum and Haemoproteus caucasica. Their distribution areas overlap only in a narrow strip along the Zagros Mts. range in Iran. This fact suggests the involvement of two different vector species with separated distribution. Nevertheless, no vectors were confirmed. According to phylogenetic analyses, H. caucasica represented a sister group to H. anatolicum, and both of them were most closely related to H. pacayae and H. peltocephali, described from South American river turtles. Four unique haplotypes were revealed in the population of H. caucasica, compared with seven haplotypes in H. anatolicum. Furthermore, H. caucasica was detected in two tortoise species, Testudo graeca and Testudo horsfieldii, providing evidence that Haemoproteus is not strictly host-specific to the tortoise host species.

Haemogregarines of freshwater turtles from Southeast Asia with a description of Haemogregarina sacaliae sp. n. and a redescription of Haemogregarina pellegrini Laveran and Pettit, 1910.

The uniform morphology of the developmental stages of Haemogregarina species and the insufficient information supplied by the simplistic descriptions of previous authors complicates their differential diagnosis and proper species identification. In this study, we detected Haemogregarina spp. in 6 out of 22 (27.2%) examined turtles originating from Southeast Asia, Malayemys subtrijuga (n = 4), Sacalia quadriocellata (n = 1) and Platysternon megacephalum (n = 1), and compared them with the available literature data. Microscopic analysis of our isolates distinguished 2 morphological species, Haemogregarina pellegrini and one new species, being described in this paper as Haemogregarina sacaliae sp. n. Phylogenetic analyses based on 1210 bp long fragment of 18S rDNA sequences placed both haemogregarines firmly within the monophyletic Haemogregarina clade. Isolates of H. pellegrini from 2 distantly related turtle hosts, M. subtrijuga and P. megacephalum, were genetically identical. Despite the fact that numerous Haemogregarina species of turtles have been described, the incompleteness of the morphological data and relatively low host specificity provides the space for large synonymy within this taxon. Therefore, a complex approach combining microscopic analyses together with molecular-genetic methods should represent the basic standard for all taxonomic studies.

Hemolivia and hepatozoon: haemogregarines with tangled evolutionary relationships.

The generic name Hemolivia has been used for haemogregarines characterized by morphological and biological features. The few molecular studies, focused on other haemogregarine genera but involving Hemolivia samples, indicated its close relationship to the genus Hepatozoon. Here we analyze molecular data for Hemolivia from a broad geographic area and host spectrum and provide detailed morphological documentation of the included samples. Based on molecular analyses in context of other haemogregarines, we demonstrate that several sequences deposited in GenBank from isolates described as Hepatozoon belong to the Hemolivia cluster. This illustrates the overall difficulty with recognizing Hemolivia and Hepatozoon without sufficient morphological and molecular information. The close proximity of both genera is also reflected in uncertainty about their precise phylogeny when using 18S rDNA. They cluster with almost identical likelihood either as two sister taxa or as monophyletic Hemolivia within paraphyletic Hepatozoon. However, regardless of these difficulties, the results presented here provide a reliable background for the unequivocal placement of new samples into the Hemolivia/ Hepatozoon complex.

Haemogregarines from western Palaearctic freshwater turtles (genera Emys, Mauremys) are conspecific with Haemogregarina stepanowi Danilewsky, 1885.

The majority of Haemogregarina species have been based on the morphology of their erythrocytic stages and supposed strict host specificity. The quantity of species with a limited number of overlapping diagnostic traits has led to a considerable mess in haemogregarine taxonomy and significant synonymy. We analysed host specificity, intra- and interspecific variability, evolutionary relationships, and the distribution of the type species of the genus Haemogregarina--H. stepanowi. The morphology of blood stages and 18S rDNA sequences of this haemogregarine from four western Palaearctic hard-shelled freshwater turtles (Emys orbicularis, Mauremys caspica, Mauremys leprosa and Mauremys rivulata) were compared with Haemogregarina balli. Additional sequences of 18S rDNA of Haemogregarina-like isolates collected from three species of African hinged terrapins (genus Pelusios) were used to enlarge the dataset for phylogenetic analyses. Thirteen sequences (1085 bp) of Haemogregarina representing all four western Palaearctic turtle species were identical, corresponding to H. stepanowi, which is closely related to the Nearctic species H. balli. In our analyses, Haemogregarina spp. constituted a monophyletic clade sister to the genus Hepatozoon. Haemogregarina stepanowi possesses a wide distribution range from the Maghreb, through Europe, Turkey and the Middle East to Iran. We consider that the genus Haemogregarina has a low host specificity crossing the family level of its vertebrate hosts and that its distribution is likely to be linked to the vector and definitive host--the leech.