Histological findings in experimentally infected male calves with chronic besnoitiosis.

The histological findings associated to Besnoitia besnoiti infection were exhaustively studied in target tissues from experimentally and chronically infected calves. Calves were inoculated with 106 bradyzoites via intravenous, subcutaneous and intradermal route. Visible pathognomonic sclera cysts were observed in all infected animals. Tissue cysts were more abundant and lesions were more frequent in calves inoculated via intradermal. The most parasitized tissues were skin, including scrotum (40.81% of positive samples), nostril and nasal turbinate. Tissue cysts were already fully developed as the average tissue cyst diameter was 181.20 µm. Microscopic lesions were mainly detected in skin samples, followed by reproductive and upper respiratory tracts. Mild lesions compatible with both acute (thrombus, oedema and inflammation) and chronic besnoitiosis (skin lesions, hyperkeratosis and dilated sweat glands) coexisted. Vascular damage and inflammation were more frequently observed in skin (including scrotum) followed by testicular parenchyma, epididymis and pampiniform plexus. Histological findings evidenced a subclinical chronic besnoitiosis.

The route of Besnoitia besnoiti tachyzoites inoculation does not influence the clinical outcome of the infection in calves.

In a previous attempt, an experimental model of bovine besnoitiosis was established in calves that were intravenously inoculated with different doses of Besnoitia besnoiti tachyzoites. Despite the fact that all infected calves developed the acute stage of disease, only microscopic findings characteristic of chronic besnoitiosis were reported. In the present study, calves were inoculated by subcutaneous and intradermal routes with B. besnoiti tachyzoites with the aim of developing clinical signs and macroscopic lesions characteristic of chronic besnoitiosis. Nine 3-month-old male calves were randomly distributed into three groups of three animals each. Next, 106 tachyzoites were inoculated by either the subcutaneous (G1) or intradermal route (G2). The negative control group (G3) was inoculated with PBS. Daily clinical monitoring and regular blood collection were performed. At 70 days post-infection (pi), animals were euthanized, and tissues were collected to investigate lesions and parasites. Infected animals developed mild-moderate acute besnoitiosis characterized by lymphadenopathy from four days to 47 days pi, and sporadic fever peaks were only observed in one calf from G2. However, other clinical signs and macroscopic lesions characteristic of chronic besnoitiosis were not detected. Only nine tissue samples were B. besnoiti-DNA-positive, eight of which belonged to reproductive and respiratory tracts tissues from G1. Finally, the kinetics of the immune responses were similar in both infected groups. However, delayed and lower cellular and humoral immune responses were observed in G1 followed by G2 and were compared with intravenously inoculated calves. The differences observed among the three inoculation routes could be due to different effector mechanisms of the host early innate immune response against B. besnoiti. Accordingly, the inoculation route of B. besnoiti tachyzoites does not significantly influence the clinical outcome of the infection in calves. Thus, a further refinement of this experimental model of bovine besnoitiosis is needed to reproduce macroscopic lesions characteristic of chronic stage disease.

Molecular cloning and characterisation of the ribC gene from Bacillus subtilis: a point mutation in ribC results in riboflavin overproduction.

A mutation leading to roseoflavin resistance and deregulated riboflavin biosynthesis was mapped in the genome of the riboflavin-overproducing Bacillus subtilis strains RB52 and RB50 at map position 147 degrees. The chromosomal location indicates that the deregulating mutation in RB52 and RB50 is an allele of the previously identified ribC mutation. We cloned the ribC gene and found that it encodes a putative 36-kDa protein. Surprisingly, RibC has significant sequence similarity to flavin kinases and FAD synthases from various other bacterial species. By comparing the deduced amino acid sequence of RibC from the wild-type parent strain of RB50 with the RibC sequence from the riboflavin-overexpressing RB50 mutant we identified a point mutation that resulted in a Gly to Ser exchange in the C-terminal region of the product.

Cloning of the phytases from Emericella nidulans and the thermophilic fungus Talaromyces thermophilus.

Phytases (EC 3.1.3.8) belong to the family of histidine acid phosphatases. We have cloned the phytases of the fungi Emericella nidulans and Talaromyces thermophilus. The putative enzyme encoded by the E. nidulans sequence consists of 463 amino acids and has a Mr of 51785. The protein deduced from the T. thermophilus sequence consists of 466 amino acids corresponding to a Mr of 51450. Both predicted amino acid sequences exhibited high identity (48% to 67%) to known phytases. This high level of identity allowed the modelling of all available fungal phytases based on the three-dimensional structure coordinates of the Aspergillus niger phytase. By this approach we identified 21 amino acids which are conserved in fungal phyA phytases and are part of the residues forming the substrate pocket. Furthermore, potential glycosylation sites were identified and compared between the aforementioned phytases and the A. niger phytase.

Forebrain-enriched RNAs of the canary: a population analysis using hybridization kinetics.

Canaries and other songbirds offer unique advantages for analyzing the relationship between specific gene regulation and neural plasticity. To establish a quantitative profile of the population of RNAs potentially involved in this regulation, we analyzed the solution hybridization kinetics of canary forebrain cytoplasmic polyadenylated RNA. Hybridization of forebrain cDNA to forebrain RNA provides evidence for RNA species at individual concentrations ranging from less than 10(-6) to about 10(-3) (by fractional mass). Cross-hybridization to RNA from the rest of the brain, together with other studies, defines a subpopulation of about 5000 rare RNAs that are enriched in the forebrain compared to the rest of the brain. Some of these forebrain-enriched RNAs are likely to play a role in regulating the neural plasticity characteristic of the canary forebrain.

Probes for rare mRNAs reveal distributed cell subsets in canary brain.

cDNA clones of 7 low-abundance canary brain RNAs hybridize in situ to different subsets of brain cells. Although these cell sets are distinct, they are dispersed in a variety of brain regions with overlapping anatomical distributions. These cDNA clones were initially selected by their relative hybridization to forebrain and rest-of-brain RNAs and represent a sampling of a much larger population of differentially expressed RNAs present at individual concentrations of 10(-7) to 10(-4) as a fraction of polyadenylated RNA mass. Our results suggest the existence of several thousand low-abundance brain mRNAs likely to be distributed in diverse and overlapping brain cell subsets. Furthermore, our experiments define a simple and general strategy for producing and analyzing molecular probes for subsets of brain cells and provide an initial set of useful reagents for further study of brain organization and development.

Apparent generation of a segmented mRNA from two separate tandem gene families in Trypanosoma cruzi.

Using a cDNA for an abundant Trypanosoma cruzi mRNA as probe, we have cloned and sequenced a gene which is organized in at least 20 nearly perfect tandem repeats of 940 base pairs. The 5' end of the mRNA has been sequenced by primer extension and found to contain a 35 nucleotide mini-exon (or spliced-leader) sequence that is ubiquitous in trypanosome mRNAs. This sequence, however, is not present in the tandem genomic repeats which encode the exon containing the major portion of the mRNA. Previous studies have shown that the 35-nucleotide sequence is encoded by a separate tandem gene family. One model to explain the formation of a segmented mRNA invokes priming of transcription by a small RNA which contains the leader sequence at its 5' end. However, northern blot analysis of total trypanosome RNA reveals a ladder of molecules larger than the mature mRNA, which appear to be faithful multimeric copies of the tandem gene. The discrete sizes of these RNAs correspond to those expected for partially processed precursors. These observations lend credence to the possibility of an alternative model where segmented mRNAs are generated by inter-molecular splicing.

Minichromosomal repetitive DNA in Trypanosoma cruzi: its use in a high-sensitivity parasite detection assay.

We have isolated genomic clones containing members of a tandemly repeated DNA family from Trypanosoma cruzi. This family, which contains a 195-base pair (bp) repeating unit, is the most abundant repetitive DNA in this organism. DNA sequencing analysis of three adjacent tandem repeats as well as two independent nonadjacent repeats showed relatively little sequence heterogeneity. Surprisingly, the three tandem elements contained a 585-bp open reading frame. However, blot hybridization of RNA from epimastigotes as well as blood-form trypomastigotes failed to show evidence for transcription of these sequences. Fractionation of whole T. cruzi DNA in sucrose gradients or in agarose gels followed by hybridization with appropriate radioactive probes showed that the size distribution of DNA bearing the 195-bp repetitive element is distinct from that of kinetoplast DNA as well as from that of DNA bearing tubulin genes. Hybridization of the 195-bp element probe with DNA from six different T. cruzi strains was positive; hybridization with DNA of other protozoa was negative with the single exception of Leptomonas collosoma , which displayed a weak cross-hybridization signal. Clones bearing this repetitive element are shown to be useful as probes for identification and counting of T. cruzi cells by dot-blot hybridization. The sensitivity of this assay permits detection of the DNA of 30 parasites in blood samples.