Babesia odocoilei is an intraerythrocytic apicomplexan parasite which infects cervidae, sometimes causing babesiosis. It is vectored by the tick Ixodes scapularis and is distributed throughout the southeastern United States. The geographic and host range continue to extend as new incidence of infection is detected. A genomic DNA region spanning the internal transcribed spacer 1 (ITS1), 5.8S rRNA gene, and ITS2 of ribosomal RNA (rRNA) from 18 B. odocoilei isolates (speciation confirmed by small subunit rRNA analysis) was amplified using the polymerase chain reaction, cloned and sequenced. The isolates originated from 6 different cervidae or bovidae hosts in various U.S. geographic areas. Included in the analysis was a previously described reindeer B. odocoilei-like isolate, RD61, which showed only 99.0% identity in SSU rRNA analysis to B. odocoilei. Percent identity pairwise comparisons among the samples were calculated for both the full ITS1-5.8S-ITS2 and individual genomic regions. Identity values for all comparisons ranged from 90% to 100%, with the exception of RD61, which showed no higher than 88% identity for all gene regions. An analysis of fixed differences identified in the ITS1 and ITS2 gene regions of all clones revealed 21 fixed differences in ITS1, and only 11 in ITS2. Most isolates were found to have 2 overall patterns of fixed differences, although some had 1 or 3. Phylogenetic analysis of all sequences for the entire ITS1-5.8S-ITS2 gene region placed most isolates into 2 distinct groups corresponding to those observed in the analysis of fixed differences. This suggested the presence of at least 2 rRNA transcription units in B. odocoilei. ITS analysis failed to demonstrate host or geographic differences that might serve to pinpoint the source of outbreaks of B. odocoilei in farmed and managed host animals. This failure might result from genetic recombination of ITS genomic regions during the tick vector stage. Lack of conspecificity between the RD61 isolate and B. odocoilei was supported by this study; however, more data are needed to clarify the taxonomic status of this B. odocoilei-like isolate.

Ribosomal RNAs (rRNA) undergo several post-transcriptional modifications inside the cell. These modifications can be (1) RNA- independent (enzyme only) and (2) guide RNA-mediated. In the latter mechanism, a group of small, metabolically stable, non-coding RNAs, present as ribonucleoprotein (RNP) particles, modify ribosomal RNAs inside the cell. One of the highly abundant rRNA modifications is pseudouridine (Y) formation. In Archaea and Eukarya, pseudouridine synthases, with the help of small RNAs, form pseudouridines at functionally important regions in rRNA. Cbf5, the pseudouridine synthase, three other core proteins, and a box H/ACA RNA form the ribonucleoprotein complex in sRNP-mediated rRNA pseudouridylation. Certain Ys in rRNAs are evolutionarily conserved from Bacteria to human. Among those, two Ys are present in helix 69 of rRNA and one in helix 90. We successfully deleted Cbf5 in Haloferax volcanii, a haloarchaeon, and showed that the deleted strain was viable. It was the first report where Cbf5 deletion was achieved, because deletion or mutation of cbf5 or of its homologs is lethal in eukaryotes. We also found that the cbf5 deleted strain was unable to produce the three highly conserved Ys in rRNA of H. volcanii (position 1940, 1942 in helix 69, and 2605 in helix 90), whereas the tRNA Ys were intact. To identify the specific structural features of Cbf5 involved in rRNA Ψ formation, we used a cbf5 deleted strain which was complemented with a plasmid borne copy of the gene. Using the crystal structure of Pyrococcous furiosus Cbf5 as template, we created a homology model of H. volcanii Cbf5 (HvCbf5) and identified several residues and motifs/domains of HvCbf5 that might be important to the protein's enzymatic activity. By using an in vivo mutational approach, we confirmed some previously predicted and certain unidentified residues/motifs/domains that serve as positive determinants of rRNA Ys1940, 1942, and 2605 formation inside the cell. A box H/ACA RNA, sR-h45, was bioinformatically predicted before. We confirmed its presence as a double hairpin RNA inside the cell whose level goes down in the absence of Cbf5. We identified that sR-h45 is the guide RNA for sRNP-mediated Ys at the three above mentioned rRNA positions in H. volcanii. Each hairpin of this RNA can independently modify the substrate, both in vivo and in vitro. To characterize the structure of sR-h45, we have used a sR-h45 deleted strain where the function of sR-h45 was complemented with a plasmid-borne copy of the gene. By a combination of in vivo and in vitro mutagenic approaches, we determined specific nucleotides/structures of this RNA, involved in binding to the core proteins and also to the substrate RNA. We also identified that one hairpin of sR-h45 can modify two successive positions (1940 and 1942) in rRNA.

No description available.

No description available.

Background The lack of an effective diagnostic tool for Carrion’s disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. Methodology/Principal Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. Methodology/Principal Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. Conclusions/Significance From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion’s disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.

Eimeria is an apicomplexan parasite causing disease coccidiosis that is most prominent in poultry farming industry. This thesis is aimed to develop new molecular tools and resolve issues that would be a valuable contribution in the field from both research and industry perspective. Because immunity to Eimeria is strictly species- specific, it is important to know and recognize correctly all species that parasitize the host. Traditional diagnostic approaches rely on classical methods such as oocyst morphology determination under the microscope, measurement of prepatent period or in-vivo assessment of lesions caused by this parasite. However, diagnostics of individual species using these methods is very time-consuming and it is often unreliable, especially when mixture of multiple species whose parameters overlap is analyzed. Methods utilizing conventional PCR to distinguish species already exist, however, they lack advantages offered by quantitative real-time PCR (qPCR). The first aim of this thesis was to develop qPCR assays for detection and quantification of seven Eimeria species which infect chicken utilizing single-copy non-polymorphic targets in order to ensure maximal specifity and coverage of all strains of each species. Usefulness of this method was demonstrated by analysis of field...

O estudo sobre a cinética de incorporação de uridina em ácidos nucleicos permitiu estabelecer que o tamanho do \"pool\" de precursores permanece constante na presença de concentrações de uridina exógena acima de aproximadamente1 µM. Concluiu-se ainda que todo o sistema de tomada de uridina, medido pela sua incorporação em ácidos nucleicos, apresenta um Km de 5,1 µM e opera a uma velocidade máxima de 11 pmoles/min/l,3 x 107 células. Um estudo análogo, em presença de rifampicina, possibilitou calcular que a meia vida de RNAs mensa - geiros em B. subtilis é de 2 minutos e que 44% da radiatividade incorporada em RNA num determinado instante se encontra na fração de RNA estável (ribossômico e de transferência). A análise do mecanismo de transcrição dos genes para RNA ribossômico foi abordada por meio do estudo do alongamento de cadeias de rRNA já iniciadas, em presença de rifampicina. As relações iniciais de radiatividade incorporada em rRNA 16S e 23S, quando rifampicina e uridina tritiada são adicionadas concomitantemente, bem corno a cinética de decaimento de marcação presente em ambas as espécies de rRNA sugerem que os genes para RNA ribossômico 16S e 23S são cotranscritos nessa ordem, em B. subtilis. Levando-se em consideração essas e outras evidências, propõe-se o seguinte relacionamento estrutural para a unidade de transcrição: [Obs.: Ver no arquivo em PDF] Os resultados experimentais foram comparados com modelos teóricos descritos no Apêndice. A existência de um mecanismo de cotranscrição para os genes responsáveis pela síntese de rRNA em procariotos e eucariotos parece sugerir que esse mecanismo, de fundamental importância, instalou-se precocemente e foi mantido durante a evolução. / The kinetics of uridine incorporation into nucleic acids has shown that the precursor pool size remains constant in the presence of exogenous uridine concentrations above 1 µM, approximately. It has also been shown that the whole system of uridine uptake, as measured by the incorporation of uridine into nucleic acids, has an apparent Km of 5.1 µM and operates at a maximal rate of 11 pmoles/min/ 1.3 x 107 cells. The incorporation, in the presence of rifampicin, made it possible to calculate the half life of the messenger RNA\'s in B.subtilis as being 2 minutes. In any given instant, 44% of the radioactivity incorporated into RNA belongs to the stable RNA fraction (ribosomal and transfer). The analysis of the ribosomal RNA genes transcription mechanism was undertaken by a study of the rRNA chain elongation in the presence of rifampicin. The initial ratios of radioactivity incorporated in 16S and 23S rRNA\'s, when rifampicin and tritiated uridine are concomitantly added, and the decay kinetics of the radioactivity present in both rRNA species, suggest that the 16S and 23S rRNA genes are cotranscribed, in that order, in B.subtilis. Taking into consideration these and other evidences, the following structural relationships are proposed for the transcriptional unit: The experimental results were compared with theoretical models described in the Appendix. The existence of a cotranscription mechanism for the rRNA genes in prokaryotes and eukaryotes seems to suggest that such mechanism, being of fundamental importance, established itself very early and was maintained through evolution.

Rickettsia rickettsii é o agente etiológico da Febre Maculosa das Montanhas Rochosas, que no Brasil é transmitida pelos carrapatos Amblyomma cajennense e A. aureolatum. Para elucidar os mecanismos de virulência sobre seus vetores, construímos bibliotecas subtrativas utilizando RNA de A. aureolatum infectados ou não com o patógeno. Com a análise bioinformática, foram obtidas 56 sequências únicas com expressão induzida e 12 com expressão reprimida pela infecção. Após a validação dos dados por RT-qPCR 3 genes foram caracterizados por RNAi: uma hebraeína, uma proteína dissulfeto isomerase (PDI) e uma proteína com domínio Kunitz-type. Um maior número de carrapatos adquiriu R. rickettsii quando a expressão gênica da hebraeína e da PDI foi silenciada, sugerindo que elas participam na defesa do carrapato contra a infecção. Nenhum efeito foi observado sobre a transmissão da bactéria para o hospedeiro ou sobre o fitness de carrapatos nos três genes analisados. O presente estudo apontou genes importantes que possibilitam uma melhor compreensão da relação carrapato-riquétsia. / Rickettsia rickettsii is the etiological agent of Rocky Mountain Spotted Fever and, in Brazil, it is transmitted by Amblyomma cajennense and A. aureolatum. To elucidate mechanisms of virulence to its vectors, we construct cDNA libraries with RNA of ticks A. aureolatum infected or not with this pathogen. After bioinformatic analysis, 56 unique sequences were obtained representing up-regulated genes and 12 down-regulated by infection. After data validation by RT- qPCR, 3 genes were characterizated by RNAi: a hebraein, a protein disulfide isomerase (PDI), and a protein with Kunitz-type domain. A higher number of ticks acquired R. rickettsii when the gene expression of hebraein and PDI was silenced, suggesting that both proteins participate in the defense of the tick against infection. No effect on the transmission of the bacterium to the host or on the fitness of ticks was observed after knockdown of the 3 analyzed genes. Data obtained by the present study pointed out important genes that provide information to better understand of the tick-rickettsia relationship.

Canine babesiosis is an important worldwide disease caused by protozoan hemoparasites of the genus Babesia, which are primarily transmitted to a dog by the bite of an Ixodid tick, although vertical transmission has recently been reported. The disease is typically characterized by hemolytic anemia, fever, splenomegaly, and thrombocytopenia, with clinical signs ranging from clinically normal to acute anemia. Death may even result in some severe cases. Two species of Babesia, Babesia gibsoni and Babesia canis, have long been known to cause babesiosis in dogs. To date, almost all B. gibsoni infections in the United States have been reported in American Pit Bull Terriers or in dogs associated with the breed through either transfusion or fighting. Dog blood samples received from kennels, shelters, and veterinary clinics throughout Texas were tested for the presence of B. gibsoni and B. canis. A total of 254 samples were tested for B. gibsoni and B. canis by light microscopy and polymerase chain reaction (PCR). Babesia gibsoni was detected in four of the dogs tested and B. canis was detected in one of the dogs tested. The average packed cell volumes (PCVs) of infected dogs were compared with those of uninfected dogs, with the infected, on average, having lower PCVs. Molecular characterization of the small subunit ribosomal RNA gene and the ribosomal RNA internal transcribed spacer regions was performed on all sequences obtained in this study, and results were consistent with those previously reported for B. gibsoni and B. canis. Also, positive samples and additional samples provided by North Carolinia State University were used to initiate in vitro cultures of the parasites. To date, one isolate of a large unknown Babesia sp. from a North Carolina dog was successfully established in vitro. The establishment of Babesia spp. parasites in culture may aid in the development of a vaccine for babesiosis and will also be beneficial in improving diagnostic tests for the parasite.

We describe the use of a tensor mode-1 higher-order singular value decomposition (HOSVD) in the analyses of alignments of 16S and 23S ribosomal RNA (rRNA) sequences, each encoded in a cuboid of frequencies of nucleotides across positions and organisms. This mode-1 HOSVD separates the data cuboids into combinations of patterns of nucleotide frequency variation across the positions and organisms, i.e., "eigenorganisms"' and corresponding nucleotide-specific segments of "eigenpositions," respectively, independent of a-priori knowledge of the taxonomic groups and their relationships, or the rRNA structures. We show that this mode-1 HOSVD provides a mathematical framework for modeling the sequence alignments where the mathematical variables, i.e., the significant eigenpositions and eigenorganisms, are consistent with current biological understanding of the 16S and 23S rRNAs. First, the significant eigenpositions identify multiple relations of similarity and dissimilarity among the taxonomic groups, some known and some previously unknown. Second, the corresponding eigenorganisms identify positions of nucleotides exclusively conserved within the corresponding taxonomic groups, but not among them, that map out entire substructures inserted or deleted within one taxonomic group relative to another. These positions are also enriched in adenosines that are unpaired in the rRNA secondary structure, the majority of which participate in tertiary structure interactions, and some also map to the same substructures. This demonstrates that an organism's evolutionary pathway is correlated and possibly also causally coordinated with insertions or deletions of entire rRNA substructures and unpaired adenosines, i.e., structural motifs which are involved in rRNA folding and function. Third, this mode-1 HOSVD reveals two previously unknown subgenic relationships of convergence and divergence between the Archaea and Microsporidia, that might correspond to two evolutionary pathways, in both the 16S and 23S rRNA alignments. This demonstrates that even on the level of a single rRNA molecule, an organism's evolutionary pathway is composed of different types of changes in structure in reaction to multiple concurrent evolutionary forces. / text