Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense

Background: Since Trypanosorna spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life -cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T brucei Hp-Hb complex receptor (TbHpHbR). Methods: Trypanosorna congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. Results: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K,A) =2.1x10(-8) M) but free-Hp with low affinity (Kd = 2.2x10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). Conclusion: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T congolense EMF-specific Hb receptor (TcEpHbR)

Karakterizacija podjedinice β (rpoB) RNA polimeraze vrste Ehrlichia canis izdvojene iz pasa i krpelja Rhipicephalus sanguineus u području Cebu na Filipinima.

Ehrlichia canis, a canine tick-borne pathogen with wide geographic distribution, has been serologically and molecularly detected in the Philippines. The present study aimed to characterize E. canis detected from Rhipicephalus sanguineus ticks and dogs in Cebu, Philippines, using the RNA polymerase sub-unit Beta (rpoB), a gene that has been used for disease diagnosis and resolution of phylogenetic relationships between closelyrelated species. Using a 16S rRNA gene-based PCR that screens Ehrlichia spp., DNA samples obtained from the blood of 10 dogs, confirmed to be serologically positive for E. canis, were tested and found positive for E. canis after subsequent DNA sequencing. DNA from infected ticks and the 16S rRNA-E. canis-positive canine blood samples from the present study were further analyzed using the rpoB gene. All registered Ehrlichia spp. rpoB gene sequences were aligned to design specific primers that can amplify a partial 1572-bp length sequence of E. canis. The obtained sequences revealed 99.8-100 % identities with each other, and 99.8-100 % and 87.8-89.1 % identities with registered E. canis and E. chaffeensis sequences from the USA, respectively. Phylogenetic analysis revealed that the obtained partial rpOB sequences formed a clade with E. canis strains from the USA. The present study is the first rpoB characterization of E. canis in the Philippines, and apparently in Asia, and provides additional evidence of the presence of the pathogen in the country. It also adds information on the high conservation of the rpoB gene in E. canis.Ehrlichia canis, geografski vrlo proširena rikecija u pasa, dokazana je serološki i molekularno na Filipinima. Određena su obilježja E. canis dokazane u krpelja Rhipicephalus sanguineus i pasa u pokrajini Cebu na Filipinima. To je učinjeno analizom podjedinice β (rpoB) RNA polimeraze, gena koji je rabljen za dijagnosticiranje bolesti i otkrivanje filogenetske srodnosti između usko srodnih vrsta. Upotrebom lančane reakcije polimerazom temeljene na genu 16S rRNA pomoću kojeg se razlikuju vrste roda Ehrlichia, u krvi 10 pasa serološki pozitivnih na E. canis dokazana je DNA specifična za E. canis. DNA iz zaraženih krpelja i uzorci krvi pasa pozitivnih na 16S rRNA-E. canis bili su dalje analizirani na osnovi gena rpoB. Sve dokazane sekvencije gena rpoB rikecija roda Ehrlichia bile su poravnate radi sinteze specifičnih početnica s kojima se može umnožiti slijed specifičan za E. canis dužine 1572 bp. Umnožene sekvencije pokazivale su 99,8-100 % identičnosti međusobno, 99,8-100 % identičnosti s vrstom E. canis iz SAD-a i 87,8-89,1 % identičnosti sa slijedovima E. chaffeensis iz SAD-a. Filogenetska analiza je pokazala da se sekvencije rpoB nalaze u skupini sa sojevima E. canis iz SAD-a. U istraživanju je prvi put analiziran rpoB vrste E. canis na Filipinima i u Aziji. Ono pruža dodatni dokaz prisutnosti te vrste na tom području. Također pruža informaciju o visokoj konzerviranosti gena rpoB vrste E. canis

Gliding Motility of Babesia bovis Merozoites Visualized by Time-Lapse Video Microscopy

BACKGROUND: Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed "gliding motility". However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite. METHODOLOGY/PRINCIPAL FINDINGS: Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. CONCLUSIONS/SIGNIFICANCE: This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding

Recent Results from LHD Experiment with Emphasis on Relation to Theory from Experimentalist’s View

he Large Helical Device (LHD) has been extending an operational regime of net-current free plasmas towardsthe fusion relevant condition with taking advantage of a net current-free heliotron concept and employing a superconducting coil system. Heating capability has exceeded 10 MW and the central ion and electron temperatureshave reached 7 and 10 keV, respectively. The maximum value of β and pulse length have been extended to 3.2% and 150 s, respectively. Many encouraging physical findings have been obtained. Topics from recent experiments, which should be emphasized from the aspect of theoretical approaches, are reviewed. Those are (1) Prominent features in the inward shifted configuration, i.e., mitigation of an ideal interchange mode in the configuration with magnetic hill, and confinement improvement due to suppression of both anomalous and neoclassical transport, (2) Demonstration ofbifurcation of radial electric field and associated formation of an internal transport barrier, and (3) Dynamics of magnetic islands and clarification of the role of separatrix

Prevalence and Molecular Analyses of Hemotrophic Mycoplasma spp. (Hemoplasmas) Detected in Sika Deer (Cervus nippon yesoensis) in Japan

Hemotropic mycoplasmas (hemoplasmas) are cell-wall deficient, epierythrocytic bacteria that cause infectious anemia in several mammalian species. The prevalence of hemoplasma species was examined by screening and species-specific PCR using blood samples collected from 51 sika deer in Hokkaido, Japan. Molecular analyses were performed for the 16S rRNA, 23S rRNA and RNase P RNA (rnpB) gene sequences. A total of 23/51 (45%) deer DNA samples were positive for hemoplasmas in the screening PCR. Using species-specific PCR, 12 and 17 samples were positive for ‘Candidatus Mycoplasma haemocervae’ and ‘Candidatus M. erythrocervae’, respectively. Sequencing and phylogenetic trees of those three genes indicate that the ‘Candidatus M. haemocervae’ and ‘Candidatus M. erythrocervae’ detected in Japanese deer are potentially different species from the cervine hemoplasma found in deer from America and Brazil