Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis.

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.

Visualization of the cbbL gene in healthy and starved chemoautotrophic nitrifying bacteria.

Three in situ methods of visualizing the cbbL gene in intact cells of nitrifying bacteria at different physiological states (dormant and metabolically active) were compared after epifluorescence microscopy and image analysis. FISH alone showed the weakest signal intensity. Direct in situ PCR, incorporating labeled nucleotides, showed the greatest sensitivity but also the greatest background. The combination of unlabeled in situ PCR followed by FISH showed relatively high sensitivity, along with the lowest background and highest specificity. Although functional gene expression was not examined in this study, visualization of the potential for carbon fixation in heterogeneous cultures of nitrifying bacteria was demonstrated.

Leishmaniasis Tegumentaria Americana: encuesta epidemiologica en una comunidad agricola / Tegumental American Leishmaniasis: epidemiological survey in an agricultural community

Se realizo un estudio epidemiologico sobre Leishmaniasis tegumentaria americana (LTA) en 809 personas del resguardo Indigena de San Andres de Sotavento, departamento de Cordoba, foco endemico tanto de Leishmaniasis cutanea como de Leishmaniasis visceral. La distribucion por sexo de la poblacion encuestada fue de 355 (43.9%) hombres y 454 (56.1%) mujeres; sus indices alergicos fueron de 36.9% y 40.5% respectivamente. La positividad de la intradermorreaccion de Montenegro ascendio en personas de ambos sexos, a medida que aumento la edad