Jan Šlapeta

A photosynthetic alveolate closely related to apicomplexan parasites

Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functional photosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model with which to study the evolution of parasitism in Apicomplexa.

The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes

Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described.

Australian dingoes are definitive hosts of Neospora caninum.

To provide objective data on the potential role of dingoes (Canis lupus dingo) in the life cycle of Neospora caninum in Australia, the production of N. caninum oocysts by experimentally infected canids was investigated. Three dingo pups raised in captivity and three domestic dogs were fed tissue from calves infected with an Australian isolate of N. caninum, Nc-Nowra. Oocysts of N. caninum, confirmed by species-specific PCR, were shed in low numbers by one dingo pup at 12-14 days p.i. The remaining animals did not shed oocysts. Furthermore, the blood from two out of three dingoes tested positive for DNA of N. caninum using PCR tests at 14 and 28 days p.i. Oocyst shedding from the intestinal tract of a dingo demonstrates that dingoes are definitive hosts of N. caninum and horizontal transmission of N. caninum from dingoes to farm animals and wildlife may occur in Australia.

Dog shedding oocysts of Neospora caninum: PCR diagnosis and molecular phylogenetic approach

Results of molecular determination of a dog isolate of Neospora caninum in the Czech Republic are presented. Colorless bisporocystic oocysts measuring 10-13 micro m x 10-11 micro m were recovered from feces and used for DNA isolation. A diagnostic PCR procedure using previously described molecular methods was performed to determine the species. The N. caninum species-specific primers based on the Nc 5 region produced a positive result, while primers specific for Hammondia heydorni rDNA internal transcribed spacer 1 (ITS1) was negative. Sequencing and phylogenetic comparison of ITS1 rDNA and the D2 domain of the large subunit rDNA (D2 LSU) determined our isolate to be N. caninum. Phylogenetic analysis of closely related genera Toxoplasma, Neospora and Hammondia based on ITS1 and D2 LSU robustly distinguished three clades: (i). Toxoplasma gondii + Hammondia hammondi, (ii). N. caninum + Neospora hughesi, and (iii). H. heydorni. Based on phylogenetic relationships we propose three acceptable suggestions to solve the problem of taxonomy of these genera.

The Phylogeny of Goussia and Choleoeimeria (Apicomplexa; Eimeriorina) and the Evolution of Excystation Structures in Coccidia

The phylogenetic relationships of Goussia janae and Choleoeimeria sp. were analyzed using the small subunit ribosomal RNA gene (SSU rDNA). This is a first attempt to study the molecular phylogeny of coccidian genera parasitizing strictly poikilotherm hosts. The biliary Eimeria-like coccidia of reptiles classified into the genus Choleoeimeria form a sister clade to the family Eimeriidae, which confirms the separate generic status of the genus Choleoeimeria. The position of Goussia is less robustly resolved, since it forms a trichotomy with the Eimeriidae and Sarcocystidae, or alternatively constitutes the earliest branch of the coccidian lineage. Morphological similarities, namely the extracytoplasmic location of the endogenous stages, and the presence of sutures in the sporocyst wall are discussed in the context of the traditional classification of eimeriids. In contrast to the morphology-based systematics, the monophyly of Goussia and Choleoeimeria is not supported by the SSU rDNA data.

A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife.

Highlights • The importance of Neospora caninum-associated disease in wildlife is reviewed.• There are only 12 reports of clinical neosporosis in wildlife species to date.• The best practice guidelines to follow for reporting wildlife cases of neosporosis are presented.

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.

Cryptosporidium parvum Mitochondrial-Type HSP70 Targets Homologous and Heterologous Mitochondria

ABSTRACT A mitochondrial HSP70 gene (Cp-mtHSP70) is described for the apicomplexan Cryptosporidium parvum, an agent of diarrhea in humans and animals. Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont. The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH2-terminal mitochondrion-like targeting presequence. Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondrial clade of HSP70 homologues. Using reverse transcription-PCR, Cp-mtHSP70 mRNA was observed in C. parvum intracellular stages cultured in HCT-8 cells. Polyclonal antibodies to Cp-mtHSP70 recognize a ∼70-kDa protein in Western blot analysis of sporozoite extracts. Both fluorescein- and immunogold-labeled anti-Cp-mtHSP70 localize to a single mitochondrial compartment in close apposition to the nucleus. Furthermore, the NH2-terminal presequence of Cp-mtHSP70 can correctly target green fluorescent protein to the single mitochondrion of the apicomplexan Toxoplasma gondii and the mitochondrial network of the yeast Saccharomyces cerevisiae. When this presequence was truncated, the predicted amphiphilic α-helix was shown to be essential for import into the yeast mitochondrion. These data further support the presence of a secondarily reduced relict mitochondrion in C. parvum.

A curious coincidence: mosquito biodiversity and the limits of the Japanese encephalitis virus in Australasia

BackgroundThe mosquito Culex annulirostris Skuse (Diptera: Culicidae) is the major vector of endemic arboviruses in Australia and is also responsible for the establishment of the Japanese encephalitis virus (JEV) in southern Papua New Guinea (PNG) as well as its incursions into northern Australia. Papua New Guinea and mainland Australia are separated by a small stretch of water, the Torres Strait, and its islands. While there has been regular JEV activity on these islands, JEV has not established on mainland Australia despite an abundance of Cx. annulirostris and porcine amplifying hosts. Despite the public health significance of this mosquito and the fact that its adults show overlapping morphology with close relative Cx. palpalis Taylor, its evolution and genetic structure remain undetermined. We address a hypothesis that there is significant genetic diversity in Cx. annulirostris and that the identification of this diversity will shed light on the paradox that JEV can cycle on an island 70 km from mainland Australia while not establishing in Australia itself.ResultsWe sequenced 538 bp of the mitochondrial DNA cytochrome oxidase I gene from 273 individuals collected from 43 localities in Australia and the southwest Pacific region to describe the phylogeography of Cx. annulirostris and its sister species Cx. palpalis. Maximum Likelihood and Bayesian analyses reveal supporting evidence for multiple divergent lineages that display geographic restriction. Culex palpalis contained three divergent lineages geographically restricted to southern Australia, northern Australia and Papua New Guinea (PNG). Culex annulirostris contained five geographically restricted divergent lineages, with one lineage restricted to the Solomon Islands and two identified mainly within Australia while two other lineages showed distributions in PNG and the Torres Strait Islands with a southern limit at the top of Australias Cape York Peninsula.ConclusionThe existence of divergent mitochondrial lineages within Cx. annulirostris and Cx. palpalis helps explain the difficulty of using adult morphology to identify Cx. annulirostris and its ecological diversity. Notably, the southern limit of the PNG lineages of Cx. annulirostris coincides exactly with the current southern limit of JEV activity in Australasia suggesting that variation in these COI lineages may be the key to why JEV has not yet established yet on mainland Australia.The mosquito Culex annulirostris Skuse (Diptera: Culicidae) is the major vector of endemic arboviruses in Australia and is also responsible for the establishment of the Japanese encephalitis virus (JEV) in southern Papua New Guinea (PNG) as well as its incursions into northern Australia. Papua New Guinea and mainland Australia are separated by a small stretch of water, the Torres Strait, and its islands. While there has been regular JEV activity on these islands, JEV has not established on mainland Australia despite an abundance of Cx. annulirostris and porcine amplifying hosts. Despite the public health significance of this mosquito and the fact that its adults show overlapping morphology with close relative Cx. palpalis Taylor, its evolution and genetic structure remain undetermined. We address a hypothesis that there is significant genetic diversity in Cx. annulirostris and that the identification of this diversity will shed light on the paradox that JEV can cycle on an island 70 km from mainland Australia while not establishing in Australia itself. We sequenced 538 bp of the mitochondrial DNA cytochrome oxidase I gene from 273 individuals collected from 43 localities in Australia and the southwest Pacific region to describe the phylogeography of Cx. annulirostris and its sister species Cx. palpalis. Maximum Likelihood and Bayesian analyses reveal supporting evidence for multiple divergent lineages that display geographic restriction. Culex palpalis contained three divergent lineages geographically restricted to southern Australia, northern Australia and Papua New Guinea (PNG). Culex annulirostris contained five geographically restricted divergent lineages, with one lineage restricted to the Solomon Islands and two identified mainly within Australia while two other lineages showed distributions in PNG and the Torres Strait Islands with a southern limit at the top of Australias Cape York Peninsula. The existence of divergent mitochondrial lineages within Cx. annulirostris and Cx. palpalis helps explain the difficulty of using adult morphology to identify Cx. annulirostris and its ecological diversity. Notably, the southern limit of the PNG lineages of Cx. annulirostris coincides exactly with the current southern limit of JEV activity in Australasia suggesting that variation in these COI lineages may be the key to why JEV has not yet established yet on mainland Australia.

A Suspected Parasite Spill-Back of Two Novel Myxidium spp. (Myxosporea) Causing Disease in Australian Endemic Frogs Found in the Invasive Cane Toad

Infectious diseases are contributing to the decline of endangered amphibians. We identified myxosporean parasites, Myxidium spp. (Myxosporea: Myxozoa), in the brain and liver of declining native frogs, the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis). We unequivocally identified two Myxidium spp. (both generalist) affecting Australian native frogs and the invasive Cane toad (Bufo marinus, syn. Rhinella marina) and demonstrated their association with disease. Our study tested the identity of Myxidium spp. within native frogs and the invasive Cane toad (brought to Australia in 1935, via Hawaii) to resolve the question whether the Cane toad introduced them to Australia. We showed that the Australian brain and liver Myxidium spp. differed 9%, 7%, 34% and 37% at the small subunit rDNA, large subunit rDNA, internal transcribed spacers 1 and 2, but were distinct from Myxidium cf. immersum from Cane toads in Brazil. Plotting minimum within-group distance against maximum intra-group distance confirmed their independent evolutionary trajectory. Transmission electron microscopy revealed that the brain stages localize inside axons. Myxospores were morphologically indistinguishable, therefore genetic characterisation was necessary to recognise these cryptic species. It is unlikely that the Cane toad brought the myxosporean parasites to Australia, because the parasites were not found in 261 Hawaiian Cane toads. Instead, these data support the enemy-release hypothesis predicting that not all parasites are translocated with their hosts and suggest that the Cane toad may have played an important spill-back role in their emergence and facilitated their dissemination. This work emphasizes the importance of accurate species identification of pathogens relevant to wildlife management and disease control. In our case it is paving the road for the spill-back role of the Cane toad and the parasite emergence.