Joao P. M. C. Maia

MOLECULAR CHARACTERIZATION OF HEPATOZOON SPECIES IN REPTILES FROM THE SEYCHELLES

Abstract Hepatozoon parasites were examined for the first time in reptiles from the Seychelles Islands. Although both prevalence and intensity were low, Hepatozoon species were detected in individuals from 2 endemic species, the lizard Mabuya wrightii and the snake Lycognathophis seychellensis. This was confirmed using visual identification and through sequencing part of the 18s rRNA gene. Phylogenetic analysis indicates that the Hepatozoon on the Seychelles form a monophyletic lineage, although more data are clearly needed to stabilize estimates of relationships based on this marker.

Molecular survey of Hepatozoon Species in Lizards From North Africa

Abstract The prevalence of Hepatozoon parasites in 460 lizards from North Africa was studied by amplification and sequencing of the 18S rRNA gene. The phylogenetic analysis of the 18S rRNA gene provides new insights into the phylogeny of these parasites with multiple genetically distinct lineages recovered. Parasite prevalence differed significantly between lacertid lizards and geckos. Our results show that there is limited host specificity and no clear relation to the geographical distribution of Hepatozoon parasites.

Molecular Survey of Apicomplexa In Podarcis Wall Lizards Detects Hepatozoon, Sarcocystis, and Eimeria Species

Abstract: The occurrence of apicomplexan parasites in Podarcis sp. wall lizards from the Iberian Peninsula and Balearic islands was studied by amplification and sequencing of the 18S rRNA gene. Species from 3 genera, Hepatozoon, Sarcocystis, and Eimeria, were found. The phylogenetic analysis of the 18S rRNA gene provides unexpected insights into the evolutionary history of these parasites. All Hepatozoon spp. specimens were recovered as part of a clade already identified in lizards from North Africa. The Sarcocystis species, detected in Podarcis lilfordi from Cabrera Island in the Balearic Islands, appears related to Sarcocystis gallotiae, known only from endemic Gallotia sp. lizards from the Canary Islands. Based on the lack of snake predators on this island, this parasite presumably presents an atypical transmission cycle that uses the same host species as both intermediate and final host through cannibalism, like S. gallotiae. Eimeria sp. is reported for the first time from Podarcis spp. lizards. This study shows the power of detecting multiple different apicomplexan parasites through screening of tail tissue samples and blood drops that are often collected in reptiles for other purposes.

MOLECULAR ASSESSMENT OF HEPATOZOON (APICOMPLEXA: ADELEORINA) INFECTIONS IN WILD CANIDS AND RODENTS FROM NORTH AFRICA, WITH IMPLICATIONS FOR TRANSMISSION DYNAMICS ACROSS TAXONOMIC GROUPS

Abstract Parasites play a major role in ecosystems, and understanding of host–parasite interactions is important for predicting parasite transmission dynamics and epidemiology. However, there is still a lack of knowledge about the distribution, diversity, and impact of parasites in wildlife, especially from remote areas. Hepatozoon is a genus of apicomplexan parasites that is transmitted by ingestion of infected arthropod vectors. However, alternative modes of transmission have been identified such as trophic transmission. Using the 18S rRNA gene as a marker, we provide an assessment of Hepatozoon prevalence in six wild canid and two rodent species collected between 2003 and 2012 from remote areas in North Africa. By combining this with other predator–prey systems in a phylogenetic framework, we investigate Hepatozoon transmission dynamics in distinct host taxa. Prevalence was high overall among host species (African jerboa Jaculus jaculus [17/47, 36%], greater Egyptian jerboa Jaculus orientalis [5/7, 71%], side-striped jackal Canis adustus [1/2, 50%], golden jackal Canis aureus [6/32, 18%], pale fox Vulpes pallida [14/28, 50%], Rüppells fox Vulpes rueppellii [6/11, 55%], red fox Vulpes vulpes [8/16, 50%], and fennec fox Vulpes zerda [7/11, 42%]). Phylogenetic analysis showed further evidence of occasional transmission of Hepatozoon lineages from prey to canid predators, which seems to occur less frequently than in other predator–prey systems such as between snakes and lizards. Due to the complex nature of the Hepatozoon lifecycle (heteroxenous and vector-borne), future studies on these wild host species need to clarify the dynamics of alternative modes of Hepatozoon transmission and identify reservoir and definitive hosts in natural populations. We also detected putative Babesia spp. (Apicomplexa: Piroplasmida) infections in two canid species from this region, V. pallida (1/28) and V. zerda (1/11).

Patterns of genetic diversity in Hepatozoon spp. infecting snakes from North Africa and the Mediterranean Basin

Species of Hepatozoon Miller, 1908 are blood parasites most commonly found in snakes but some have been described from all tetrapod groups and a wide variety of hematophagous invertebrates. Previous studies have suggested possible associations between Hepatozoon spp. found in predators and prey. Particularly, some saurophagous snakes from North Africa and the Mediterranean region have been found to be infected with Hepatozoon spp. similar to those of various sympatric lizard hosts. In this study, we have screened tissue samples of 111 North African and Mediterranean snakes, using specific primers for the 18S rRNA gene. In the phylogenetic analysis, the newly-generated Hepatozoon spp. sequences grouped separately into five main clusters. Three of these clusters were composed by Hepatozoon spp. also found in snakes and other reptiles from the Mediterranean Basin and North Africa. In the other two clusters, the new sequences were not closely related to geographically proximate known sequences. The phylogeny of Hepatozoon spp. inferred here was not associated with intermediate host taxonomy or geographical distribution. From the other factors that could explain these evolutionary patterns, the most likely seems series of intermediate hosts providing similar ribotypes of Hepatozoon and a high prevalence of host shifts for Hepatozoon spp. This is indicated by ribotypes of high similarity found in different reptile families, as well as by divergent ribotypes found in the same host species. This potentially low host specificity has profound implications for the systematics of Hepatozoon spp.

Molecular Assessment of Apicomplexan Parasites in the Snake Psammophis from North Africa: Do Multiple Parasite Lineages Reflect the Final Vertebrate Host Diet?

Abstract:  The Apicomplexa are intracellular pathogens of animals, with the Coccidia being the largest group. Among these are the hemogregarines, which include some of the most common hemoparasites found in reptiles. Several studies have reported a possible pattern of prey–predator transmission for some of these parasites. Snakes from the Mediterranean region have been found to be parasitized with Hepatozoon spp. similar to those in lacertids and gekkonids, supporting the prey–predator transmission hypothesis. Here we analyzed specimens of the saurophagous genus Psammophis from North Africa, an ecologically different region. Through molecular analysis of tissue samples we detected 3 different apicomplexan parasites: Caryospora, Sarcocystis, and Hepatozoon. Caryospora was detected in a Forskåls sand snake Psammophis schokari from Algeria, constituting the first time these parasites have been detected from a tissue sample through molecular screening. The obtained Sarcocystis phylogeny does not reflect the relationships of their final hosts, with the parasites identified from snakes forming at least 3 unrelated groups, indicating that it is still premature to predict definitive host based on the phylogeny of these parasites. Three unrelated lineages of Hepatozoon parasites were identified in Psammophis, each closely related to lineages previously identified from different lizard groups, on which these snakes feed. This once again indicates that diet might be a key element in transmission, at least for Hepatozoon species of saurophagous snakes.

Molecular Detection of Hemolivia (Apicomplexa: Haemogregarinidae) from Ticks of North African Testudo graeca (Testudines: Testudinidae) and an Estimation of Their Phylogenetic Relationships Using 18S rRNA Sequences

ABSTRACT: Hemolivia mauritanica is an apicomplexan parasite infecting the tortoises Testudo graeca and Testudo marginata as the intermediate vertebrate host, and the tick Hyalomma aegyptium as the definitive invertebrate host. Here, we used molecular techniques to detect an apparent H. mauritanica in the ticks H. aegyptium, attached to Testudo graeca from North Africa. Sequences from 18S rRNA were used to place the new sequences, and others retrieved from GenBank, in a phylogenetic framework. A single Hemolivia haplotype was recovered from 16 ticks. Prevalence was estimated at 22.6% of hosts having ticks infected with H. mauritanica.

Hepatozoon Infection Prevalence in Four Snake Genera: Influence of Diet, Prey Parasitemia Levels, or Parasite Type?

Abstract: Hepatozoon spp. (Apicomplexa: Haemogregarinidae) are the most commonly reported hemoparasites from snakes. Of over 300 Hepatozoon species identified, more than 120 were described from snakes. However, recent genetic assessments have found Hepatozoon lineages recovered from both prey and predators, indicating that diet may play an important role in the infection of final vertebrate hosts. Here 4 different snake genera with different diets were assessed. Hepatozoon spp. prevalence varied greatly between the genera, but only lineages already identified from potential prey, i.e., gecko and lacertid lizards, were recovered from the snakes. Interestingly, the Hepatozoon spp. lineage known from geckos was the most common in the snakes, but this does not reflect their diet. Higher parasitemia levels, reported for some geckos relative to lacertid lizards, may play a role. Alternatively, this lineage may be more effective at parasitizing snakes or may occur, despite being unrecorded, in other vertebrate groups consumed by snakes.

A comparison of multiple methods for estimating parasitemia of hemogregarine hemoparasites (apicomplexa: Adeleorina) and its application for studying infection in natural populations

Identifying factors influencing infection patterns among hosts is critical for our understanding of the evolution and impact of parasitism in natural populations. However, the correct estimation of infection parameters depends on the performance of detection and quantification methods. In this study, we designed a quantitative PCR (qPCR) assay targeting the 18 S rRNA gene to estimate prevalence and intensity of Hepatozoon infection and compared its performance with microscopy and PCR. Using qPCR, we also compared various protocols that differ in the biological source and the extraction methods. Our results show that the qPCR approach on DNA extracted from blood samples, regardless of the extraction protocol, provided the most sensitive estimates of Hepatozoon infection parameters; while allowed us to differentiate between mixed infections of Adeleorinid (Hepatozoon) and Eimeriorinid (Schellackia and Lankesterella), based on the analysis of melting curves. We also show that tissue and saline methods can be used as low-cost alternatives in parasitological studies. The next step was to test our qPCR assay in a biological context, and for this purpose we investigated infection patterns between two sympatric lacertid species, which are naturally infected with apicomplexan hemoparasites, such as the genera Schellackia (Eimeriorina) and Hepatozoon (Adeleorina). From a biological standpoint, we found a positive correlation between Hepatozoon intensity of infection and host body size within each host species, being significantly higher in males, and higher in the smaller sized host species. These variations can be associated with a number of host intrinsic factors, like hormonal and immunological traits, that require further investigation. Our findings are relevant as they pinpoint the importance of accounting for methodological issues to better estimate infection in parasitological studies, and illustrate how between-host factors can influence parasite distributions in sympatric natural populations.

Prevalence and Diversity of Hepatozoon in Native and Exotic Geckos from Brazil

Abstract:  Hepatozoon is a genus of hemogregarines constituting the most widespread and common reptile hemoparasite. Although various molecular assessments of these parasites have been conducted in lizards from Africa and Europe, similar studies are needed for South American lizards. Through amplification and sequencing of fragments of the 18S rRNA gene, we assess the prevalence of Hepatozoon parasites in 230 geckos from South America, including the endemic species Hemidactylus agrius, Hemidactylus brasilianus, Lygodactylus klugei, Phyllopezus pollicaris, Phyllopezus periosus, and an exotic species, Hemidactylus mabouia. We found an overall low prevalence of Hepatozoon infection (7/230, 3%) with only 3 of the 6 host species infected with Hepatozoon (Hemidactylus mabouia, P. pollicaris, and P. periosus). Within the 7 infected host samples, 5 genetically distinct lineages of Hepatozoon parasites were identified, only 1 of which was similar to previously published haplotypes. Thus, although prevalence is low, genetically based diversity of Hepatozoon in geckos from South America is very high. Three of these lineages appear basal to 1 of the major clades of Hepatozoon, suggesting that this clade might have originated in South America, and thereby indicating a potential phylogeographic pattern that had not been previously identified. Future studies should assess the distribution and competence of invertebrate hosts in the regions analyzed, and Hepatozoon diversity in other less well-known regions of the world.