Significant differences in the analytic concordance between anti-dsDNA IgG antibody assays for the diagnosis of systemic lupus erythematosus--implications for inter-laboratory testing.

BACKGROUND: Anti-double stranded DNA (anti-dsDNA) autoantibodies are considered hallmark of systemic lupus erythematosus (SLE). METHODS: To determine concordance between assays for the detection of this marker, we analyzed 100 antinuclear antibody (ANA) positive sera with a homogeneous pattern and titers≥1:160 by indirect immunofluorescence assay (IFA) on HEp-2 cells, 100 consecutive anti-dsDNA IgG ELISA-negative as well as 100 healthy control samples using six commercial ELISAs and the Crithidia luciliae immunofluorescence test (CLIFT). RESULTS: The positivity rates for the ELISAs in the ANA positive group ranged from 55.0 to 88.0% with specificities from 84.0 to 98.0%. The CLIFT had a positivity rate of 68.0% and specificity of 84%. In the previously screened anti-dsDNA IgG-negative group, the positivity rates ranged from 1 to 19%. The overall correlations between the ELISAs ranged from 73.0 to 89.5% and varied from 70.0 to 80.0% among specific ELISAs and CLIFT. CONCLUSIONS: Our data show variable degree of concordance between anti-dsDNA IgG ELISAs which may significantly impact inter-laboratory testing as well as the diagnosis and management of SLE patients. Although some of the ELISAs show comparable performance to the CLIFT, the degree of concordance between these assays at high antibody levels suggests that CLIFT is still a relevant confirmatory tool.

A quantitative in vitro cultivation technique to determine cell number and growth rates in strains of Crithidia bombi (Trypanosomatidae), a parasite of bumblebees.

The protozoan parasite Crithidia bombi and its host, the bumblebee Bombus terrestris, are used as a model system for the study of the evolutionary ecology of host-parasite interactions. In order to study these interactions we established a method for in vitro cultivation of single parasite strains. Additionally, a high-throughput method is developed for the determination of cell numbers in cultures by means of optical density (OD) measurements. The protocol for in vitro cultivation allowed for growing different strains on agar plates as well as in culture medium. A calibration curve for the relationship between cell number and OD has been developed. Subsequently, growth rates for different genotypes of C. bombi have been recorded. Significant differences in the growth rates and generation times between these genotypes were demonstrated. As this might be related to the virulence of the parasite, this relationship may be confirmed by in vivo growth rate determination. In comparison with conventional cell counting, the application of OD measurements allows for high-throughput experiments as the time taken to record each sample is reduced by a factor of 30. The in vitro cultivation method allows for controlled infection experiments in order to study host-parasite interactions.

The bacterium endosymbiont of Crithidia deanei undergoes coordinated division with the host cell nucleus.

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.

Molecular divergence defines two distinct lineages of Crithidia bombi (Trypanosomatidae), parasites of bumblebees.

This study provides, for the first time, sequence data for the protozoan flagellates Crithidia bombi and Crithidia mellificae (Kinetoplastea: Trypanosomatidae). We amplified the partial sequences of the small subunit ribosomal RNA (SSU rRNA), glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH), cytochrome b (Cyt b), and the complete internal transcribed spacer region 1 (ITS1) of the ribosomal RNA gene region for 66 clones of C. bombi from Switzerland and Alaska. Furthermore, we sequenced the same stretch of SSU rRNA and gGAPDH for one isolate of C. mellificae from Switzerland. All four molecular markers classified the C. bombi samples into two distinct lineages A and B. Both lineages were found in the two sampling locations. Variation within lineages was small or non-existing. Sequence differences between lineages were 1.64% for SSU rRNA, 4.36% for gGAPDH, and 12.02% for Cyt b. The ITS1-sequences of lineages A and B have diverged so much that no alignment was possible. With regard to ITS1, we additionally found fragment length polymorphism (variation in microsatellite repeat numbers) as well as nucleotide diversity within each lineage. Furthermore, the sequences of SSU rRNA and gGAPDH of C. mellificae were different from both lineages of C. bombi. The separation of lineages A and B, based on sequence differences and phylogenetic reconstruction, is so pronounced as to characterize two species of "C. bombi." We propose to retain C. bombi for the more common lineage A and suggest the name Crithidia expoeki n. sp. for lineage B.

Flagellar and ciliary beating in trypanosome motility.

The single flagellum of Leishmania and Trypanosoma parasites is becoming an increasingly attractive model for the analysis of flagellar function-driven largely by the abundance of genomic and proteomic information available for the organelle, the genetic manipulability of the organisms and the importance of motility for the parasite lifecycle. However, as yet, there is a paucity of published data on the beating of any genetically malleable trypanosomatid species. Here we undertook an in-depth analysis using high-speed videomicroscopy of the beating of free-swimming Leishmania major cells in comparison to Crithidia species (for which there is some existing literature). In so doing, we describe a simple and generally-applicable technique to facilitate the quantitative analysis of free-swimming cells. Our analysis thoroughly defines the parameters of the expected tip-to-base symmetrical flagellar beat in these species. It also describes beat initiation from points other than the flagellum tip and a completely different, base-to-tip highly-asymmetric beat that represents a ciliary beat of trypanosomatid flagella. Moreover, detailed analysis of parameter interrelationships revealed an unexpected dependency of wavelength on oscillator length that may be the result of reversible constraint of doublet sliding at the tip or resonance of the flagellar beat.

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa.

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.

Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids.

Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure. However, the ultrastructural localization of nucleic acids within these protozoa remains uncertain. We have applied cytochemical and immunocytochemical approaches to precisely identify DNA and RNA in lower endosymbiont-bearing trypanosomatids. Using the Terminal deoxynucleotidyl Transferase (TdT) immunogold technique, we showed that nuclear DNA is seen associated with the nuclear envelope during the trypanosomatid cell cycle. By combining the TdT technique with the acetylation method, which improves the contrast between structures containing fibrils and granules, we have demonstrated that the nucleolus of endosymbiont-bearing trypanosomatids is composed of two constituents: a granular component and a DNA-positive fibrillar zone. Moreover, we revealed that DNA of endosymbiotic bacteria consisted of electron-dense filaments which are usually in close contact with the prokaryote envelope. Using a Lowicryl post-embedding immunogold labeling procedure with anti-RNA antibodies, we showed the presence of RNA not only over the cytoplasm, the interchromatin spaces and the nucleolus, but also over the kinetoplast and virus-like particles present in Crithidia desouzai.

Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species.

Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Gelatin-SDS-PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms. Our survey indicates that the proteolytic profiles of C. deanei and C. desouzai are identical; that C. oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus. This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C. deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.

Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates.

Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.

[Cloning of the flagellate Crithidia oncopelti on a solid nutrient medium of indeterminant composition not containing hemin]. / Klonirovanie zhgutikonostsa Crithidia oncopelti na plotnoi pitatel'noi srede neopredelnnogo sostava, na soderzhashchei gemin.

On a simple solid medium of indefinite content without hemin, colonies of Crithidia oncopelti were received. The clonal nature of them was proved. The survival in some experiments varied from 10 to 70%. Young colonies, 6 days after inoculation, were 0.3- 2.0 mm in diameter. C. oncopelti differed from Herpetomonas megaseliae and Crithidia harmosa in the morphology of colonies. To further genetical study of C. oncopelti, a possibility of cloning the object on a simple solid medium is an important prerequisite.

Morphologic and biochemical characterization of Crithidia brasiliensis sp. n.

A trypanosomatid with a choanomastigote stage and, therefore, belonging to the genus Crithidia, was isolated in culture from the alimentary tract of the hemipteran genus Zelus. The trypanosomatid was able to grow at 37 C, a characteristic reported to date from only 2 other members of Crithidia, C hutneri and C. luciliae thermophila. Subsequently, the flagellate was cloned for biochemical studies which involved cleaving of kDNA by restriction endonucleases and analyses of the isoenzyme and histone patterns. In all the attributes revealed by the foregoing methods, the organism from Zelus differed from the latter 2 congeneric species. On these and morphologic grounds, this organism appears to belong in a new species for which the name Crithidia brasiliensis sp. n. is proposed.

[Ribosomal RNA synthesis in the kinetoplasts of 2 species of Crithidia]. / O sinteze ribosomnoi RNK v kinetoplastakh dvukh vidov kritidii.

A study was made of the possibility of ribosomal RNA synthesis in kinetoplasts of two crithidian species--C. oncopelty and C. fasciculata. The RNA synthesized by isolated kinetoplasts in sucrose gradients was resolved into the following five fractions; 23S, 16S, 12S, 9S and 6--4S. Since pure kinetoplast ribosomes contain 23S and 16S RNA, a supposition was made that the first two transcripts might be ribosomal RNAs. It is suggested that the kinetoplast DNA contains genes for rRNAs and that the synthesis of these RNAs occurs in the kinetoplast.