Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences

Eimeriatenella and Toxoplasmagondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesised that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites. Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the “housekeeping” gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5′ regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp.     

Cryptosporidium in wild placental mammals

Cryptosporidium species are common parasites of wild placental mammals. Recent parasitological studies combined with molecular genotyping techniques have been providing valuable new insight into the host specificity and potential transmission of various Cryptosporidium species/genotypes among animals and between these animals and humans. Although Cryptosporidium in wild animals may possess a potential public health problem due to oocyst contamination in the environment, studies at various regions of the world have indicated a strong host-adaptation by these parasites and a limited potential of cross-species transmission of cryptosporidiosis among placental mammals, suggesting that these animals are probably not a major reservoir for human infection. However, Cryptosporidium species/genotypes in placental animals have been reported occasionally in humans. Therefore, public health significance of some Cryptosporidium species in wild placental mammals, such as the cervine genotype, should not be overlooked and should be further studied.     

Case study: Pathological and phylogenetic analysis of coccidiosis in two goats with heavy infection of unrecorded Eimeria sp.

Two 3-month-old goats (Capra aegagrus hircus and C. hircus coreanae) died after ataxia. In both goats, white nodules 3 mm in diameter were scattered from the duodenum to the ileum and well-raised white nodules 2–3 mm–diameter in the mucosa of the small intestine. Histopathologically, numerous mucosal polyps with coccidial oocysts were observed in the small intestine and several schizonts, macrogametocytes, microgametocytes, and macrogametes were observed in mucosal polyps in the jejunum. Based on fecal flotation tests, the oocysts morphologically resembled those of Eimeria christenseni and E. sundarbanensis; however, their sizes were different. The 18S rRNA gene and COI were phylogenetically analyzed for the molecular identification and characterization of Eimeria sp. Based on 18S rRNA gene similarity, the isolates formed an independent cluster within the related goat Eimeria clade, and the closest species were E. christenseni C2_42, E. hirci C2_99, and E. arloingi C2_119. Furthermore, these were also distinguished from other related goat Eimeria spp. in the phylogenetic tree based on the COI gene. Considering all histopathological, morphological, and phylogenetic analyses, the current study was diagnosed as fatal coccidiosis due to heavy infection with an unrecorded Eimeria species. Thus, we report in this study with caution regarding coccidiosis caused by an unrecorded Eimeria.     

Stable transfection of Eimeria tenella: constitutive expression of the YFP-YFP molecule throughout the life cycle

The obligate intracellular apicomplexan parasite Eimeria tenella, one of seven species of Eimeria that infect chickens, elicits protective cell-mediated immunity against challenge infection. For this reason, recombinant E. tenella parasites could be utilised as an effective vaccine vehicle for expressing foreign antigens and inducing immunity against heterologous intracellular microbes. A stable line of E. tenella expressing foreign genes is a prerequisite, and in this work an in vivo stable transfection system has been developed for this parasite using restriction enzyme-mediated integration (REMI). Two transgenic populations of E. tenella have been obtained that express YFP-YFP constitutively throughout the parasite life cycle. Southern blotting and plasmid rescue analyses show that the introduced exogenous DNA was integrated at random into the parasite genome. Although the life cycle of the transgenic populations was delayed by at least 12 h and the output of oocysts was reduced 4-fold relative to the parental BJ strain of E. tenella, the transgenic parasites were sufficiently immunogenic to protect chickens against challenge with either transgenic or parental parasites. These results are encouraging for the development of transgenic E. tenella as a vaccine vector and for more detailed investigation of the biology of the genus Eimeria.     

Molecular and phylogenetic characterizations of an Eimeria krijgsmanni Yakimoff & Gouseff, 1938 (Apicomplexa: Eimeriidae) mouse intestinal protozoan parasite by partial 18S ribosomal RNA gene sequence analysis

Previously, we characterized an undocumented strain of Eimeria krijgsmanni by morphological and biological features. Here, we present a detailed molecular phylogenetic analysis of this organism. Namely, 18S ribosomal RNA gene (rDNA) sequences of E. krijgsmanni were analyzed to incorporate this species into a comprehensive Eimeria phylogeny. As a result, partial 18S rDNA sequence from E. krijgsmanni was successfully determined, and two different types, Type A and Type B, that differed by 1 base pair were identified. E. krijgsmanni was originally isolated from a single oocyst, and thus the result show that the two types might have allelic sequence heterogeneity in the 18S rDNA. Based on phylogenetic analyses, the two types of E. krijgsmanni 18S rDNA formed one of two clades among murine Eimeria spp.; these Eimeria clades reflected morphological similarity among the Eimeria spp. This is the third molecular phylogenetic characterization of a murine Eimeria spp. in addition to E. falciformis and E. papillata.     

Cross immunity of DNA vaccine pVAX1-cSZ2-IL-2 to Eimeria tenella, E. necatrix and E. maxima

The study describes cross protection experiments with chimeric DNA vaccine pVAX1-cSZ2-IL-2 to determine its efficacy against four important Eimeria species. Seven-day-old chickens were randomly divided into nine groups; group 1 negative control, groups 2, 3, 4, 5 positive controls; and groups 6, 7, 8 and 9 experimental groups. On days 7 and 14, groups 1-5 were injected with TE buffer, and groups 6-9 with the vaccine. At 21 days of age, all chickens were inoculated with 5 × 10⁴ sporulated oocysts except for the negative control. Groups 2 and 6 were inoculated with Eimeria tenella, groups 3 and 7 with Eimerianecatrix, groups 4 and 8 with Eimeria acervulina and groups 5 and 9 with Eimeria maxima. Seven days later, all chickens were weighed and slaughtered to obtain intestinal samples. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and anti-coccidial index. The results indicated that the recombinant plasmid can induce host immune responses by alleviating intestinal lesions, body weight loss and oocyst ratio and imparting good protection against E. tenella and E.acervulina, medium protection against E. necatrix but little effect against E. maxima. It is concluded that the conserved antigen can provide cross protection and should be explored further.     

Phylogenetic relationships in European Ceriporiopsis species inferred from nuclear and mitochondrial ribosomal DNA sequences

The aim of this work was to clarify taxonomy and examine evolutionary relationships within European Ceriporiopsis species using a combined analysis of the large subunit (nLSU) nuclear rRNA and small subunit (mtSSU) mitochondrial rRNA gene sequences. Data from the ITS region were applied to enhance the view of the phylogenetic relationships among different species. The studied samples grouped into four complex clades, suggesting that the genus Ceriporiopsis is polyphyletic. The generic type Ceriporiopsis gilvescens formed a separate group together with Ceriporiopsis guidella and Phlebia spp. in the phlebioid clade. In this clade, the closely related species Ceriporiopsis resinascens and Ceriporiopsis pseudogilvescens grouped together with Ceriporiopsis aneirina. C. resinascens and C. pseudogilvescens have identical LSU and SSU sequences but differ in ITS. Ceriporiopsis pannocincta also fell in the phlebioid clade, but showed closer proximity to Gloeoporus dichrous than to C. gilvescens or C. aneirina–C. pseudogilvescens–C. resinascens group. Another clade was composed of a Ceriporiopsis balaenae–Ceriporiopsis consobrina group and was found to be closely related to Antrodiella and Frantisekia, with the overall clade highly reminiscent of the residual polyporoid clade. The monotypic genus Pouzaroporia, erected in the past for Ceriporiopsis subrufa due to its remarkable morphological differences, also fell within the residual polyporoid clade. Ceriporiopsis subvermispora held an isolated position from the other species of the genus. Therefore, the previously proposed name Gelatoporia subvermispora has been adopted for this species. Physisporinus rivulosus appeared unrelated to two other European Physisporinus species. Moreover, Ceriporiopsis (=Skeletocutis) jelicii grouped in a separate clade, distinct from Ceriporiopsis species. Finally, the ITS data demonstrated the proximity of some Ceriporiopsis species (Ceriporiopsis portcrosensis and Ceriporiopsis subsphaerospora) to Skeletocutis amorpha.     

Eimeria species and genetic background influence the serum protein profile of broilers with coccidiosis

Background

Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria.

Methodology/Principal Findings

Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05−0.01 (250 spots), P<0.01−0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001.

Conclusions/Significance

Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex biochemical mechanisms involved in host response to Eimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods.     

Molecular identification of Eimeria species in Spanish bats

This is the first study reporting the detection and molecular characterization of Eimeria in bats in Spain, specifically in 12 of 32 chiropteran species described in the Iberian Peninsula. A total of 76 faecal samples were collected from different bat roosting sites across Spanish territory. The DNA was extracted from the samples and sequenced by targeting the Eimeria SSU-rRNA gene. Two Eimeria species were detected in 29 of the 76 faecal samples (38%), and the bat-specific Eimeria rioarribaensis and rodent-specific Eimeria jerfinica were detected in 4 (5%) and 25 (33%) of the samples, respectively. This is the first report of E. rioarribaensis in the bats Rhinolophus euryale, Myotis myotis and Nyctalus lasiopterus, extending the host and geographical ranges for this bat coccidian parasite. The identification of the rodent-specific parasite species E. jerfinica in bats indicates the occurrence of this species in Spain, although its presence has not previously been reported in wild rodents in this country. Considering that most of the Eimeria spp. reported in bats were described only on the basis of morphometric data, molecular studies are required to determined which Eimeria species occur in bats, to complete the identification of these species and to clarify the phylogeny.     

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.     

淫羊藿属植物查耳酮合成酶启动子序列的克隆及比较分析

采用TAIL-PCR技术从箭叶淫羊藿(Epimedium sagittatum)叶片中克隆得到查耳酮合成酶基因(Chalcone synthase)的启动子,命名为ECHSP, 长度为750 bp,其具有TATA BOX、CAAT BOX、MYB结合位点、MYC结合位点等元件。对箭叶淫羊藿、木鱼坪淫羊藿(E. franchetii)、粗毛淫羊藿(E. acuminatum)、直距淫羊藿(E. mikinorii)、黔岭淫羊藿(E. leptorrhizum)等5种植物的CHS启动子序列进行比较分析,结果表明,CHS启动子的核苷酸多态性丰富,表明CHS 的表达可能受到多种因素的灵活调节。ECHSP与其他植物的CHS启动子序列进行比较,其TATA box的位置可能与黄酮类化合物的积累相关。此外,进化分析表明ECHSP的核苷酸序列可应用于淫羊藿属植物的系统进化研究。     

Eimeria tenella,Eimeria necatrix, and Eimeria adenoeides: Peripheral blood leukocyte response of chickens and turkeys to strains adapted to the turkey embryo

The peripheral blood leukocyte responses of chickens and turkeys inoculated with one of three strains of a chicken Eimeria species adapted to the turkey embryo with their respective parent lines, or with E. adenoeides of the turkey were studied. The adapted lines tended to cause hematological changes in chickens and turkeys similar to those caused by E. adenoeides. These parasites caused the most significant increases in large mononuclear white blood cells = (monocytes) in both chickens and turkeys. These results provide further evidence for a monocyte/macrophage effector mechanism in the rejection of heterologous species of Eimeria from a nonspecific host. The results also agree with previous studies that show that increases in mononuclear white blood cells during parent E. tenella and E. necatrix infections in chickens occur during the periods of greatest tissue damage (3–4 days after inoculation). The generally unaffected lymphocyte numbers and increases in mononuclear white blood cells during infections with the adapted lines probably explain the reduced pathogenicity and the lack of immunogenicity seen previously in chickens inoculated with these three lines. Possibly, monocytes/macrophages play a role in the host specificity of the parasites.     

Reelin and apolipoprotein E receptor 2 in the embryonic and mature brain: effects of an evolutionary change in the apoER2 gene

In the mature cerebral cortex of higher vertebrates, neurons are arranged in layers, each layer containing neurons of the same functional class. The cortical layering pattern is laid down during development by migration of young post-mitotic neurons along glial fibres to their correct positions in the cortical plate. The mechanics of whole-cell movement are well understood, but there is still uncertainty as to how a migrating neuron is instructed to leave its glial support when it reaches its destination. An intraneuronal signalling pathway initiated by reelin and containing apolipoprotein E receptor 2 (apoER2) is essential for normal cortical layering, and there is strong evidence that detachment of a migrating neuron is brought about by reelin-dependent downregulation of α3 integrin. But there remains the problem of how the reelin signal is switched on at a position in the cortex appropriate for each class of neuron. ApoER2 of placental mammals contains an amino acid sequence that is encoded in a separate exon in the apoER2 gene and is required for normal memory and spatial learning. The separate exon is not present in marsupials, birds or reptiles. The addition of this exon to the evolving apoER2 gene may have contributed to the success of placental mammals.     

Ancient collagen reveals evolutionary history of the endemic South American ‘ungulates’

Since the late eighteenth century, fossils of bizarre extinct creatures have been described from the Americas, revealing a previously unimagined chapter in the history of mammals. The most bizarre of these are the ‘native’ South American ungulates thought to represent a group of mammals that evolved in relative isolation on South America, but with an uncertain affinity to any particular placental lineage. Many authors have considered them descended from Laurasian ‘condylarths’, which also includes the probable ancestors of perissodactyls and artiodactyls, whereas others have placed them either closer to the uniquely South American xenarthrans (anteaters, armadillos and sloths) or the basal afrotherians (e.g. elephants and hyraxes). These hypotheses have been debated owing to conflicting morphological characteristics and the hitherto inability to retrieve molecular information. Of the ‘native’ South American mammals, only the toxodonts and litopterns persisted until the Late Pleistocene–Early Holocene. Owing to known difficulties in retrieving ancient DNA (aDNA) from specimens from warm climates, this research presents a molecular phylogeny for both Macrauchenia patachonica (Litopterna) and Toxodon platensis (Notoungulata) recovered using proteomics-based (liquid chromatography–tandem mass spectrometry) sequencing analyses of bone collagen. The results place both taxa in a clade that is monophyletic with the perissodactyls, which today are represented by horses, rhinoceroses and tapirs.     

Retama species growing in different ecological–climatic areas of northeastern Algeria have a narrow range of rhizobia that form a novel phylogenetic clade within the Bradyrhizobium genus

Sixty-seven isolates were isolated from nodules collected on roots of Mediterranean shrubby legumes Retama raetam and Retama sphaerocarpa growing in seven ecological–climatic areas of northeastern Algeria. Genetic diversity of the Retama isolates was analyzed based on genotyping by restriction fragment length polymorphism of PCR-amplified fragments of the 16S rRNA gene, the intergenic spacer (IGS) region between the 16S and 23S rRNA genes (IGS), and the symbiotic genes nifH and nodC. Eleven haplotypes assigned to the Bradyrhizobium genus were identified. Significant biogeographical differentiation of the rhizobial populations was found, but one haplotype was predominant and conserved across the sites. All isolates were able to cross-nodulate the two Retama species. Accordingly, no significant genetic differentiation of the rhizobial populations was found in relation to the host species of origin. Sequence analysis of the 16S rRNA gene grouped the isolates with Bradyrhizobium elkanii, but sequence analyses of IGS, the housekeeping genes (dnaK, glnII, recA), nifH, and nodC yielded convergent results showing that the Retama nodule isolates from the northeast of Algeria formed a single evolutionary lineage, which was well differentiated from the currently named species or well-delineated unnamed genospecies of bradyrhizobia. Therefore, this study showed that the Retama species native to northeastern Algeria were associated with a specific clade of bradyrhizobia. The Retama isolates formed three sub-groups based on IGS and housekeeping gene phylogenies, which might form three sister species within a novel bradyrhizobial clade.     

Genetic characterisation of uninucleated cyst-producing Entamoeba spp. from ruminants

Six ssrRNA gene sequences were obtained by PCR amplification of DNA from uninucleated Entamoeba cysts isolated from fresh faeces of sheep, cows, a roe deer and a reindeer. Phylogenetic analysis using sequences of non-, uni-, quadri- and octonucleate cyst-producing Entamoeba spp. for comparison showed that all six isolates formed a separate clade nested within the clade of quadrinucleate cyst producers. The data indicate that Entamoeba bovis can be isolated from ruminant hosts other than cattle, and we suggest that organisms clustering with the sheep and cattle isolates analysed in the present study be named E. bovis.     

Host-Parasite Incongruences in Rodent Eimeria Suggest Significant Role of Adaptation Rather than Cophylogeny in Maintenance of Host Specificity

The degree of host specificity, its phylogenetic conservativeness and origin are virtually unknown in Eimeria. This situation is largely due to the inadequate sample of eimerian molecular data available for reliable phylogenetic analyses. In this study, we extend the data set by adding 71 new sequences of coccidia infecting 16 small-mammal genera, mostly rodents. According to the respective feasibility of PCR gene amplification, the new samples are represented by one or more of the following genes: nuclear 18S rRNA, plastid ORF 470, and mitochondrial COI. Phylogenetic analyses of these sequences confirm the previous hypothesis that Eimeria, in its current morphology-based delimitation, is not a monophyletic group. Several samples of coccidia corresponding morphologically to other genera are scattered among the Eimeria lineages. More importantly, the distribution of eimerians from different hosts indicates that the clustering of eimerian species is influenced by their host specificity, but does not arise from a cophylogenetic/cospeciation process; while several clusters are specific to a particular host group, inner topologies within these clusters do not reflect host phylogeny. This observation suggests that the host specificity of Eimeria is caused by adaptive rather than cophylogenetic processes.     

Phylogeny of the reptilian Eimeria: are Choleoeimeria and Acroeimeria valid generic names?

Reptiles are the animals with the most described coccidian species among all vertebrates. However, the co‐evolutionary relationships in this host–parasite system have been scarcely studied. Paperna & Landsberg (South African Journal of Zoology, 24, 1989, 345) proposed the independent evolutionary origin of the Eimeria‐like species isolated from reptiles based on morphological and developmental characteristics of their oocysts. Accordingly, they suggested the reclassification of these parasites in two new genera, Choleoeimeria and Acroeimeria. The validity of the genera proposed to classify reptilian Eimeria species remained unresolved due to the lack of species genetically characterized. In this study, we included 18S rRNA gene sequences from seven Eimeria‐like species isolated from five different lizard host families. The phylogenetic analyses confirmed the independent evolutionary origin of the Eimeria‐like species infecting lizards. Within this group, most species were placed into two monophyletic clades. One of them included the species with ellipsoidal oocysts (i.e. Choleoeimeria‐like oocysts), whereas the species with more spheroidal oocysts (i.e. Acroeimeria‐like oocysts) were included in the second one. This result supports the taxonomic validity of the genera Acroeimeria and Choleoeimeria.     

Paleovirology of ‘syncytins’, retroviral env genes exapted for a role in placentation

The development of the emerging field of ‘paleovirology’ allows biologists to reconstruct the evolutionary history of fossil endogenous retroviral sequences integrated within the genome of living organisms and has led to the retrieval of conserved, ancient retroviral genes ‘exapted’ by ancestral hosts to fulfil essential physiological roles, syncytin genes being undoubtedly among the most remarkable examples of such a phenomenon. Indeed, syncytins are ‘new’ genes encoding proteins derived from the envelope protein of endogenous retroviral elements that have been captured and domesticated on multiple occasions and independently in diverse mammalian species, through a process of convergent evolution. Knockout of syncytin genes in mice provided evidence for their absolute requirement for placenta development and embryo survival, via formation by cell–cell fusion of syncytial cell layers at the fetal–maternal interface. These genes of exogenous origin, acquired ‘by chance’ and yet still ‘necessary’ to carry out a basic function in placental mammals, may have been pivotal in the emergence of mammalian ancestors with a placenta from egg-laying animals via the capture of a founding retroviral env gene, subsequently replaced in the diverse mammalian lineages by new env-derived syncytin genes, each providing its host with a positive selective advantage.