Clonal Cultures Research Articles

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells, melanocyte stem cells and neural crest like stem cells. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury. Furthermore, peripheral nerves have been repaired with stem cell grafts, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models. Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long-term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models. Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

Putative silicon transport vesicles in the cytoplasm of the diatom Synedra acus during surge uptake of silicon

We studied the growth of the araphid pennate diatom Synedra acus subsp. radians (Kützing) Skabichevskii using a fluorescent dye N(1),N(3)-dimethyl-N(1)-(7-nitro-2,1,3-benzoxadiazol-4-yl)propane-1,3-diamine (NBD-N2), which stains growing siliceous frustules but does not stain other subcellular organelles. We used a clonal culture of S. acus that was synchronized by silicon starvation. Epifluorescence microscopy was performed in two different ways with cells stained by the addition of silicic acid and the dye. Individual cells immobilized on glass were observed during the first 15-20 min following the replenishment of silicic acid after silicon starvation. Alternatively, we examined cells of a batch culture at time intervals during 36 h after the replenishment of silicic acid using fluorescence and confocal microscopy. The addition of silicic acid and NBD-N2 resulted in the rapid (1-2 min) formation of several dozen green fluorescent submicrometer particles (GFSPs) in the cytoplasm, which was accompanied by the accumulation of fluorescent silica inside silica deposition vesicles (SDVs) along their full length. In 5-15 min, GFSPs disappeared from the cytoplasm. Mature siliceous valves were formed within the SDVs during the subsequent 14-16 h. In the next 8-10 h, GFSPs appeared again in the cytoplasm of daughter cells. The data obtained confirm observations about the two-stage mechanism of silicon assimilation, which includes rapid silicon uptake (surge uptake) followed by slow silica deposition. It is likely that the observed GFSPs are silicon transport vesicles, which were first proposed by Schmid and Schulz in (Protoplasma 100:267-288, 1979).

Single-cell analysis of paralytic shellfish toxins in Alexandrium tamarense by HPLC with post-column fluorescent derivatization

We developed a methodology for analyzing the C-toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic shellfish toxin (PST) detected in a clonal culture of A. tamarense, which is a common causative organism in cases of paralytic shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N)=2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from <1fmol to 700fmolcell−1. This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic shellfish poisoning.

Clonal In Vitro Analysis of Neurotrophin Receptor p75-Immunofluorescent Cells Reveals Phenotypic Plasticity of Primary Rat Olfactory Ensheathing Cells

Clonal in vitro analysis represents a powerful tool for studying cellular differentiation. In the present study, microscope-assisted single cell transfer was combined with immunofluorescence to establish clonal cultures of identified primary rat olfactory ensheathing cells (OECs). During development, OECs originate from the neural crest, a transient population of multipotent cells. Since only neural crest cells have been analyzed at clonal density, it remained unclear whether OECs may retain multipotent features. Neurotrophin receptor p75 (p75(NTR))-immunolabelled rat OECs were seeded at clonal density under visual control using a semiautomated cell selection and transfer device (Quixell™) and emerging clones were analyzed with regard to proliferation and antigenic expression. We demonstrate that OECs from neonatal (P1) and 7 day-old (P7) but not from adult rats formed clones in the presence of OEC- and astrocyte-conditioned media (OEC-CM, A-CM). Cloning efficiency but not in vitro growth of OECs was independent of age but increased upon treatment with OEC-CM. Interestingly, about 75 % of P1 compared to 27 % of P7 OEC clones lost p75(NTR) expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75(NTR) at clonal density and initiated expression of the fibroblast marker Thy-1 may suggest that their developmental potential is greater than previously anticipated. Since microscope-assisted selection of immunofluorescent cells combined with semiautomated transfer guarantees monoclonality in a single step and affords selection of cells according to fluorescent label and/or morphological criteria it may be relevant for a variety of other cell types.

Morphology and molecular characterization of the epiphytic dinoflagellate Prorocentrum cf. rhathymum in temperate waters off Jeju Island, Korea

Prorocentrum spp. are planktonic and/or benthic species. Benthic Prorocentrum species are of primary concern to scientists and the public because some of them are toxic. We established clonal cultures of 3 strains of Prorocentrum species that were collected from the thalli of a macroalga in the coastal waters off Jeju Island, located at the southern end of Korea. The Korean strains of P. cf. rhathymum, which are morphologically almost identical to the Virgin Island strain of P. rhathymum, were different from P. mexicanum because the former dinoflagellate has one simple collar-like spine in the periflagellar area, while the latter dinoflagellate has a 2- or 3-horned spine. In addition, the sequences of the small subunit (SSU) rDNA of the Korean strains were identical to those of the Malaysian and Floridian strains of P. rhathymum, while the sequences of the large subunit (LSU) rDNA of the Korean strains were 0.1–0.9% different from those of the Iranian and Malaysian strains of P. rhathymum. In phylogenetic trees based on the SSU rDNA sequences, the Korean strains of P. rhathymum formed a clade with the Malaysian and Floridian strains of P. rhathymum and the Vietnamese and Polynesian strains of P. mexicanum. However, in phylogenetic trees based on the LSU rDNA sequences, the Korean strains of P. rhathymum formed a clade with the Iranian strain of P. rhathymum and the Spanish and Mexican strains of P. mexicanum. Therefore, the molecular characterization of the Korean strains does not allow us to clearly classify them as P. rhathymum, nor P. mexicanum, although their morphology has so far been reported to be closer to that of P. rhathymum than P. mexicanum and thus we designated them as P. cf. rhathymum.

An automated system for high-throughput single cell-based breeding

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.

Isolation of clonal cultures of endosymbiotic green algae from their ciliate hosts

Using Paramecium bursaria as a model organism improved protocols have been developed to isolate clonal endosymbiotic algae. This involved micromanipulation of individual protists, rupturing to release endosymbionts followed by enrichment on complex media and a series of plating steps, under low light (PAR ~10μmol photons m−2s−1).

SHORT- VERSUS LONG-TERM RESPONSES TO CHANGING CO2IN A COASTAL DINOFLAGELLATE BLOOM: IMPLICATIONS FOR INTERSPECIFIC COMPETITIVE INTERACTIONS AND COMMUNITY STRUCTURE

Increasing pCO2 (partial pressure of CO2 ) in an "acidified" ocean will affect phytoplankton community structure, but manipulation experiments with assemblages briefly acclimated to simulated future conditions may not accurately predict the long-term evolutionary shifts that could affect inter-specific competitive success. We assessed community structure changes in a natural mixed dinoflagellate bloom incubated at three pCO2 levels (230, 433, and 765 ppm) in a short-term experiment (2 weeks). The four dominant species were then isolated from each treatment into clonal cultures, and maintained at all three pCO2 levels for approximately 1 year. Periodically (4, 8, and 12 months), these pCO2 -conditioned clones were recombined into artificial communities, and allowed to compete at their conditioning pCO2 level or at higher and lower levels. The dominant species in these artificial communities of CO2 -conditioned clones differed from those in the original short-term experiment, but individual species relative abundance trends across pCO2 treatments were often similar. Specific growth rates showed no strong evidence for fitness increases attributable to conditioning pCO2 level. Although pCO2 significantly structured our experimental communities, conditioning time and biotic interactions like mixotrophy also had major roles in determining competitive outcomes. New methods of carrying out extended mixed species experiments are needed to accurately predict future long-term phytoplankton community responses to changing pCO2 .

Annamia toxica gen. et sp. nov. (Cyanobacteria), a freshwater cyanobacterium from Vietnam that produces microcystins: ultrastructure, toxicity and molecular phylogenetics

Nguyen L.T.T., Cronberg G., Moestrup Ø. and Daugbjerg N. 2013. Annamia toxica gen. et sp. nov. (Cyanobacteria), a freshwater cyanobacterium from Vietnam that produces microcystins: ultrastructure, toxicity and molecular phylogenetics. Phycologia 52: 25–36. DOI: 10.2216/10-097.1A freshwater cyanobacterium from Hoamy Reservoir, Hue, Vietnam was isolated into clonal culture (April 2004). Based on general morphology (including size and shape) from light microscopy it was identified as Pseudanabaena cf. moniliformis. However, transmission electron microscopy revealed the thylakoids to be radially arranged, in contrast to the Pseudanabaena group sensu Komárek & Čáslavská (1991), which was characterized by a more or less concentric arrangement of the thylakoids. Furthermore, the phylogenetic relationship of the Vietnamese culture was investigated by sequencing the phycocyanin gene and the gene encoding 16S rRNA. Using morphology, toxicity and gene sequences, we showed that the Vietnamese culture and a culture of Pseudanabaena galeata from the UTEX collection were not related at the generic level. To examine this in greater detail, a search was made for a culture of P. catenata, the type species of Pseudanabaena. Two cultures with that name were available from the SAG culture collection in Göttingen, Germany. Thin sections revealed that both strains were characterized by concentric thylakoids. Based on our polyphasic approach, the Vietnamese material was therefore described as Annamia toxica gen. et sp. nov.

Effects of an Anatoxin-a(s)-Producing Strain of &amp;lt;i&amp;gt;Anabaena spiroides&amp;lt;/i&amp;gt; (Cyanobacteria) on the Survivorship and Somatic Growth of Two &amp;lt;i&amp;gt;Daphnia similis&amp;lt;/i&amp;gt; Clones

The toxicity of an anatoxin-a(s) producer strain of Anabaena spiroides (ITEP-024) was estimated through sub-chronic bioassays with two clones of Daphnia similis (Labtox and Itajuba), both with intact cells and aqueous extracts of lyophilized material. Animals were grown as clonal cultures in the lab with mineral water plus 20% lake water. The concentrations used in the bioassays were 0.125, 0.25, 0.375, 0.50 and 1.00 mg·L-1 for intact cell cultures and 10, 25, 50 and 100 mg·L-1 for aqueous extracts. Controls with nutritive food were used. The bioassays lasted 72 hours for the aqueous extracts and 96 hours for the intact cell cultures, with measurements of survivorship every day and body length at the beginning and at the end of the bioassays. Both kinds of samples affected survivorship and growth rate of both Daphnia clones, with the intact cell samples being more effective than aqueous extracts. Regarding survivorship, the clone Itajuba was more sensitive to aqueous extracts than the clone Labtox (LC50 = 54.4 and 83.1 mg·L-1, respectively). No significant difference was found between clones in the intact cell bioassays. Regarding growth rates, a significant difference was found between clones in both samples. However, growth rate was significantly decreased in much lower concentrations of intact cells than in aqueous extracts of cyanobacteria. A stimulus of growth was found in the lower concentration (10 mg·L-1) of aqueous extracts, which is consistent with a hormetic response. In spite of its known high neurotoxicity to mice, ITEP-024 strain caused any effect on mobility of both clones. The effects on survivorship and growth of Daphnia caused by ITEP-024 strain in much higher concentrations of aqueous extracts suggests that uptake of toxins from the water are not so effective as the uptake trough the gut when intact cells are ingested.

Psammamonas australis gen. et sp. nov. (Raphidophyceae), a new dimorphic, sand-dwelling alga

Grant B., Waller R.F., Clementson L.A. and Wetherbee R. 2013. Psammamonas australis gen. et sp. nov. (Raphidophyceae), a new dimorphic, sand-dwelling alga. Phycologia 52: 57–64. DOI: 10.2216/12-070.1A marine raphidophyte Psammamonas australis gen. et sp. nov. was described. Clonal cultures were established from field samples collected during the summer of 2009 from Wild Dog Beach, Victoria, Australia. Cells of Psammamonas australis had two distinct morphologies, a motile swimming phase and an adhesive benthic phase. Motile cells were irregularly ellipsoid to fusiform and were 25–35 μm long and 10–15 μm wide. The cells possessed two heterokont flagella arising from a groove and five to six nonoverlapping plastids. Benthic cells were typically irregularly discoid and were strongly adhered to the substrate either as single cells or close-fitting rafts. P. australis employed amoeboid movement during both the formation and the release of the multicelled rafts. Phylogenetic analysis based on the nuclear-encoded SSU ribosomal DNA sequence as well as the morphological evidence indicated that P. australis represented a new genus.

Influence of cell size and DNA content on growth rate and photosystem II function in cryptic species of Ditylum brightwellii.

DNA content and cell volume have both been hypothesized as controls on metabolic rate and other physiological traits. We use cultures of two cryptic species of Ditylum brightwellii (West) Grunow with an approximately two-fold difference in genome size and a small and large culture of each clone obtained by isolating small and large cells to compare the physiological consequences of size changes due to differences in DNA content and reduction in cell size following many generations of asexual reproduction. We quantified the growth rate, the functional absorption cross-section of photosystem II (PSII), susceptibility of PSII to photoinactivation, PSII repair capacity, and PSII reaction center proteins D1 (PsbA) and D2 (PsbD) for each culture at a range of irradiances. The species with the smaller genome has a higher growth rate and, when acclimated to growth-limiting irradiance, has higher PSII repair rate capacity, PSII functional optical absorption cross-section, and PsbA per unit protein, relative to the species with the larger genome. By contrast, cell division rates vary little within clonal cultures of the same species despite significant differences in average cell volume. Given the similarity in cell division rates within species, larger cells within species have a higher demand for biosynthetic reductant. As a consequence, larger cells within species have higher numbers of PSII per unit protein (PsbA), since PSII photochemically generates the reductant to support biosynthesis. These results suggest that DNA content, as opposed to cell volume, has a key role in setting the differences in maximum growth rate across diatom species of different size while PSII content and related photophysiological traits are influenced by both growth rate and cell size.

41 RAPID PRODUCTION OF α-1,3-GALACTOSYLTRANSFERASE-DEFICIENT MINIATURE PIGS BY NUCLEAR TRANSFER FOLLOWING NEONATAL SKIN BIOPSY AND LOSS OF HETEROZYGOSITY

Production of α-1,3-galactosyltransferase (αGT)-deficient pigs is a fundamental task to overcome hyperacute rejection in pig-to-human xenotransplantation. However, the procedure to produce such pigs requires a great deal of cost, time, and labor. Heterozygote αGT knockout (KO) pigs should be bred at least for 2 generations to ultimately produce homozygote progenies. Here, we show that αGT-deficient miniature pigs could be produced in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Miniature pig fetal fibroblasts were transfected with αGT-targeting vector and treated with neomycin. Eighty-seven colonies were screened by PCR, and 2 among them were identified as homologous recombinants with monoallelic disruption of αGT (targeting efficiency = 2.3%). One of the two colonies was chosen and used for subsequent NT. In total, 559 nuclear transfer embryos reconstructed with αGT gene-targeted fetal fibroblasts were transferred to 5 surrogate gilts. Three gilts became pregnant and developed to term with 3 live piglets delivered. Being analyzed by PCR, all piglets born were found to be αGT gene targeted. Fibroblasts were isolated from ear skin biopsies of a 10-day-old αGT gene-targeted miniature pig. To increase the number of mitotic recombination that occurs spontaneously during cell proliferation, fibroblasts were cultured for 6 to 8 passages. Then, these cells were treated with IB4-conjugated Dynabeads; IB4 lectin binds to galactose-α-1,3-galactose (αGal), the antigen formed by αGT, and is responsible for hyperacute rejection (HAR). Using a magnetic column, fibroblasts bound to IB4 lectin were removed. Remaining cells lacking αGal were cultured further until colony formation. A total of 22 fibroblast colonies were screened by PCR, and 15 among them were confirmed as loss of heterozygosity (LOH) with biallelic disruption of αGT by mitotic recombination (LOH efficiency = 68%). One of these clonal cultures of αGT-deficient fibroblasts was chosen and used for subsequent NT. In total, 729 nuclear transfer embryos reconstructed with αGT-deficient neonatal ear skin fibroblasts were transferred to 4 surrogate gilts. Two gilts became pregnant, and 3 live piglets were produced. Based on PCR, both αGT alleles were targeted in all piglets born. Only 8 months were required from the production of heterozygote to homozygote KO in contrast to approximately 2 years consumed for conventional breeding of heterozygotes. The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by neonatal skin biopsies and subsequent selection of mitotic recombinants. Such a procedure may be beneficial for the production of other homozygote KO animals, especially in species that require substantial periods of gestation such as pigs.

Low Molecular Weight Dextran Sulfate Binds to Human Myoblasts and Improves their Survival after Transplantation in Mice

Introduction: Myoblasts are skeletal muscle progenitors cells responsible for skeletal muscle growth and regeneration. As such and because myoblasts faces the complex challenges of clinical application, myoblast transplantation appears to be a promising approach for treatment of extensive skeletal muscle destruction occurring in severe myopathies such as Duchenne muscular dystrophy. Nevertheless, even in autologous combination, such approaches are impaired by the rapid death, limited migration and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant in vitro and in vivo. These beneficial effects of DXS in modulating innate immunity make this substance a possible candidate to improve the success of myoblast transplantation. Methods: Clonal cultures of human myoblasts were prepared from human muscle biopsies obtained during corrective orthopedic surgery. Myoblasts were 100% positives for the myogenic markers CD56 and desmin as assessed by flow cytometry. For in vivo experiments, human myoblasts were transduced with a lentiviral vector encoding the renilla luciferase bioluminescence marker gene (Rluc). Non-invasive bioluminescence imaging was used to quantify survival of engrafted human myoblasts after transplantation in immunodeficient mice. All research with human samples and mice was performed in compliance with the institutional guidelines, national as well as international guidelines. Results: In vitro, DXS binds dose dependently to human myoblasts and significantly inhibits staurosporine mediated apoptosis and necrosis. DXS pretreatment also protects human myoblasts from natural killer cell-mediated cytotoxicity in a dose dependent manner. At a multiplicity of infection of 1, 80% of the cells expressed the renilla luciferase transgene after lentiviral infection. Rluc transduced myoblasts kept their myogenic capacity to proliferate and to differentiate in vitro as assessed by fusion indexes. In vivo, we observed a two-fold improvement in survival of human myoblasts that were pretreated with DXS (10 mg/ml) as compared to untreated cells, at 1 day and at 3 days after transplantation (77.9 ± 10.1% vs 39.4 ± 4.9%; p = 0.0009 and 38.1 ± 8.5% vs 15.1 ± 3.4%; p = 0.01, respectively). No significant difference was observed at 7 days (8.0 ± 2.1 % vs 3.6 ± 0.7%; p = 0.06) and at 14 days post injection with values that were indistinguishable from background levels. Conclusion: Taken together, we provide evidence that DXS act as a myoblast protectant in vitro and in vivo, able locally to prevent early cell death. This protective effect of DXS was efficient up to 72h after pretreatment. DXS might represent a useful therapeutic reagent to improve the success of myoblast cell transplantation. More experiments are needed to evaluate if combination of DXS pretreatment of human myoblasts with systemic DXS administration of the recipient may be envisaged to enhance myoblast transplantation success.

Antihistomonal effects of artemisinin and Artemisia annua extracts in vitro could not be confirmed by in vivo experiments in turkeys and chickens

Five different Artemisia annua-derived materials (i.e. dry leaves, pure artemisinin, and hexane, dichloromethane or methanol extracts of leaves) were screened for their in vitro activities against six clonal cultures of Histomonas meleagridis. Except for the methanol extract, all tested materials displayed in vitro activity against all tested protozoal clones. Neither the dry plant material, extracts nor artemisinin showed any antibacterial activity against the xenic bacteria accompanying the six H. meleagridis clones at concentration levels identical to the antihistomonal setting. The dichloromethane extract of dry leaves (Ext-DCM) (minimal lethal concentration=1.0 mg/ml) and artemisinin (half-maximal inhibitory concentration=1.295 mg/ml) had the most promising antihistomonal properties and were therefore subsequently tested in a standardized experimental infection model in both turkeys and chickens infected with clonal H. meleagridis. There were no differences between treatment groups, where all infected turkeys showed severe clinical histomonosis and demonstrated severe typhlohepatitis typical for histomonosis. Consistent with the infection model used, the infected chickens did not show any adverse clinical signs but contracted severe lesions in their caeca 7 and 10 days post infection (d.p.i.), liver lesions were absent to mild after 7 d.p.i. and progressed to severe lesions at 10 d.p.i.; thus no differences between treatment groups were observed. In conclusion, neither artemisinin nor Ext-DCM was able to prevent experimental histomonosis in turkeys and chickens at the given concentrations, which is contrary to the antihistomonal effect noticed in vitro even though the same clonal culture was used. The results of this study therefore clearly demonstrate the importance of defined in vivo experimentation in order to assess and verify in vitro results.

Effects of different nitrate and phosphate concentrations on the growth and toxin production of an Alexandrium tamarense strain collected from Drake Passage

Nitrate (N) and phosphate (P) are believed to be two of the most important nutrients for the growth and toxin production for Alexandrium species. The study of the growth and toxicity characteristics of the Alexandrium spp. under the change of N and P can help us to understand the dynamics of algal bloom and toxification events in natural environments. A strain of Alexandrium tamarense (designated as Kci) was successfully isolated from the Drake Passage in 2001 and the clonal culture has been kept in our laboratory (Ho et al., 2003, 2012). In order to extend our understanding on the growth physiology and toxicity of this A. tamarense strain, growth and cellular toxin content were examined in unialgal batch cultures under different concentrations of N and P. The effects of variable N, P concentrations on growth, cellular toxicity (fg STXeq. cell−1), and toxin composition (% molar) were determined in both exponential and stationary growth phases. The toxin profile, determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), was found to be remained relatively stable and was consistently dominated by the N-sulfocarbamoyl C-toxins (>90%) under different conditions and growth phases. There were also trace amounts of other carbamate gonyautoxins consistently expressed. The cellular toxicity varied under different N and P concentrations, as well as different growth stages. A positive correlation was observed between cellular toxicity and N concentrations, but the toxicity was enhanced when P was depleted. Both cell densities and growth rate of the cells were severely suppressed under N- or P-depletion. However, the biovolume of the cells tended to be larger at N- or P-depleted cultures. Results from the present study provide valuable insight for the ecophysiology of Alexandrium species in the coastal ecosystem of Drake Passage.

Mendelian Inheritance Pattern and High Mutation Rates of Microsatellite Alleles in the Diatom Pseudo-nitzschia multistriata

The diatom Pseudo-nitzschia multistriata exhibits a diplontic life cycle composed of an extensive phase of vegetative cell division and a brief phase of sexual reproduction. To explore genotypic stability, we genotyped seven polymorphic microsatellite loci in 26 monoclonal strains over 3-16 months in a culture maintenance regime. Moreover, to assess inheritance patterns of the microsatellite alleles, we genotyped 246 F1 strains resulting from four mating experiments between parental strains of know genotype. Results generally conformed expectations according to Mendelian inheritance patterns, but deviations were detected indicating mutations during sexual reproduction. A total of forty-two mutations were detected in the clonal cultures over time. Microsatellites with more core-repeats accumulated mutations faster. The mutation rate varied significantly across loci and strains. A binomial mass function and a computer simulation showed that the mutation rate was significantly higher during the first months of culture (μ≈3×10(-3) per locus per cell division) and decreased to μ≈1×10(-3) in the strains kept for 16 months. Our results suggest that genetic mutations acquired in both the vegetative phase and sexual reproduction add to the allelic diversity of microsatellites, and hence to the genotypic variation present in a natural population.

Escherichia coli strongly supports the growth of Histomonas meleagridis, in a monoxenic culture, without influence on its pathogenicity

Based on clonal cultures of Histomonas meleagridis, monoxenic cultures have, to our knowledge for the first time, been established in a liquid medium. The faecal flora was exchanged for defined bacterial strains by selective destruction of the initial bacteria with a variety of antibiotics, keeping the flagellate alive. The growth of the protozoan parasite was found to depend on the bacteria, especially on their energy metabolism. Escherichia coli was found to strongly support the growth of the parasite, whereas Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa were less efficient. Confocal laser microscopy showed that H. meleagridis could take up green fluorescent protein-tagged E. coli DH5α, suggesting that bacteria serve as a food supply for the protozoa. By exchanging the bacterial flora for E. coli strain DH5α in H. meleagridis cultures that underwent continuous in vitro passages, it was possible to show that the in vivo attenuation process was independent of the bacteria. Furthermore, the gut flora in infected turkeys had no negative effect on the protozoan’s virulence. Consequently, attenuation depends not on the bacteria in the culture but on the in vitro passages. Finally, the experiments provided evidence that the infection of turkeys with H. meleagridis enabled infection of the liver with E. coli.

One Perkinsus species may hide another: characterization of Perkinsus species present in clam production areas of France

Although clam populations in France are known to be infected with protozoans of the genus Perkinsus, no molecular characterization was previously performed on these parasites. Considering that several members of this genus have been associated with mortalities of molluscs worldwide, a study was undertaken in order to characterize these parasites in France. For that purpose, clams, Ruditapes philippinarum and R. decussatus, collected from different production areas and found to be infected with Perkinsus sp. in thioglycolate culture medium, were selected for PCR-RFLP tests and sequencing. Perkinsus olseni was detected in all the investigated areas and results also suggested the presence of P. chesapeaki in Leucate, a lagoon on the Mediterranean coast and in Bonne Anse in Charente Maritime, on the Atlantic coast. Clonal cultures from both detected species were produced in order to describe and compare in vitro stages. Differences in size between both Perkinsus spp. were noticed especially for schizonts and zoosporangia. Lastly, in situ hybridization tests allowed confirmation of the presence of both species in the same R. decussatus population and even in same clams. This is the first detection of P. chesapeaki in Ruditapes species and outside North America, which questions its introduction into Europe.