Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites: preliminary characterization of its genome

Garberi, Juan C.; Blanco, Jorge C. G.; Angel, Sérgio O.; Pzsenny, Viviana; Arakelian, M. C.; Pastini, A C

doi:10.1590/s0074-02761990000400007

Cited by 5 publications

(6 citation statements)

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“…Oocysts and eggs were recovered according to 10 with major modifications as follows: The buffered sucrose solution (PBS-Ca buffer, PBS, PH 7.4 plus 1 mM CaCl 2 ) is an ideal density gradient material owing to low cost, inertness, stability, high solubility, and non-toxic nature. Furthermore, removal of fine debris and sucrose from separated oocysts and eggs is best achieved by repeated washings with distilled water or a suitable buffer under vacuum filtration.…”

Section: Methodsmentioning

confidence: 99%

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Electrical cream separator coupled with vacuum filtration for the purification of eimerian oocysts and trichostrongylid eggs

El‐Ashram

Suo

2017

Sci Rep

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Several methods have been proposed for separation of eimerian oocysts and trichostrongylid eggs from extraneous debris; however, these methods have been considered to be still inconvenient in terms of time and wide-ranging applications. We describe herein an alternative way using the combination of electrical cream separator and vacuum filtration for harvesting and purifying eimerian oocysts and haemonchine eggs on large-scale applications with approximately 81% and 92% recovery rates for oocysts and nematode eggs obtained from avian and ovine faeces, correspondingly. The sporulation percentages as a measure of viability in the harvested oocysts and eggs from dry faecal materials are nearly 68% and 74%, respectively, and 12 liters of faecal suspension can be processed in approximately 7.5 min. The mode of separation in terms of costs (i.e. simple laboratory equipments and comparably cheap reagents) and benefits renders the reported procedure an appropriate pursuit to harvest and purify parasite oocysts and eggs on a large scale in the shortest duration from diverse volumes of environmental samples compared to the modified traditional sucrose gradient, which can be employed on a small scale.Many studies have been devoted to coccidiosis owing to significant economic losses to the poultry and many other domestic livestock industries throughout the world. Coccidiosis is a severe infection caused by the apicomplexan parasite of genus Eimeria in a host and predilection site specific manner 1-4 . Furthermore, among the gastrointestinal parasites that cause losses to the farming industry, for example, Ostertagia, Trichostrongylus, Nematodirus and Cooperia, Haemonchus contortus or the barber's pole worm is the predominant nematodes that infect small ruminants 5 .Coprologic examinations have long been conceded as an effective way for parasite identification that animal and human contracted with them. The most commonly employed technique in veterinary medicine, medical and clinical laboratory for separation of oocysts and eggs is the faecal floatation test. The technique depends on the differences in the specific gravity of the oocysts and eggs, the fluid floatation medium and debris. In other words, the differential density exists between them. Centrifugal flotation methods are clearly superior to non-centrifugation flotation procedures in terms of rapidity and efficiency.A considerable amount of literature has been published on eimerian oocyst and nematode egg separation from faecal materials. Techniques for the concentration and purification of oocysts and eggs from faecal samples include saturated salt solution flotation, sucrose density, zinc sulfate, and Percoll discontinuous density gradient centrifugation. Sucrose gradient ultracentrifugation has been exploited to separate subcellular fractions of Eimeria tenella sporozoites 6 . More recent attention has focused on the isolation of the endocytic organelle from macrophages by sucrose gradient 7 . In 1987, Arrowood and Sterling 8 published a paper in which they emplo...

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Section: Methodsmentioning

confidence: 99%

“…In a study which set out to purify Toxplasma gondii tacyzoites, Garberi et al . 10 found that tachyzoites obtained by two consecutive discontinuous sucrose gradient separation maintained its biological activity.…”

mentioning

confidence: 94%

Electrical cream separator coupled with vacuum filtration for the purification of eimerian oocysts and trichostrongylid eggs

El‐Ashram

Suo

2017

Sci Rep

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“…Three days after the inoculation, fluids were collected in cold phosphate-buffered saline, pH 7·2, containing 1 m m CaCl 2 (PBS-Ca). Highly purified tachyzoites, the rapidly multiplying form of the parasite, were obtained by two consecutive discontinuous sucrose gradients as reported previously (Garberi et al 1990). Briefly, sucrose step gradients were prepared by carefully layering equal volumes of 40–50–60% sucrose in PBS-Ca solution in 40 mL centrifuge tubes.…”

Section: Methodsmentioning

confidence: 99%

Unmethylated CpG motifs inToxoplasma gondiiDNA induce TLR9- and IFN-β-dependent expression ofα-defensin-5 in intestinal epithelial cells

Santamaria¹,

Caballero²,

Corral

2015

Parasitology

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The gut epithelial barrier is a strategic place to prevent, or at least to limit, parasite dissemination upon oral infection with Toxoplasma gondii. Innate immunity to this pathogen results from delicate interactions involving different components of the infecting agent and the host. We herein aimed to examine the molecular mechanism by which protozoan DNA boosts the production of α-defensin-5 (DEFA-5), the main antimicrobial peptide at the target site of infection. The present study shows that DEFA-5 is rapidly upregulated in intestinal epithelial cells following intracellular Toll-like receptor 9 (TLR9) activation by unmethylated CpG motifs in DNA from T. gondii (CpG-DNA). Concomitantly, CpG-DNA purified from the pathogen markedly increased TLR9 mRNA expression levels in the Caco-2 cell line. We further verified that DEFA-5 production was dependent on interferon-β released from these cells upon treatment with CpG-DNA prepared from tachyzoites. Our results suggest that, in protozoan DNA-stimulated intestinal epithelial cells, the TLR9/interferon-β/DEFA-5 pathway may initiate an innate anti-T. gondii response without the need of parasite invasion. These findings highlight the key role of the gut epithelium in Toxoplasma recognition and amplification of local host defence against this microbe, thereby contributing to gain insight into immunoprotective mechanisms and to improve therapeutic strategies.

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“…The extraction of T. gondii DNA and the isolation and cloning of R522 sequence in pCR2.1-TOPO have been described earlier (Kusumawati et al, 2010;Sri Hartati et al, 2006). Briefly, tachyzoites of an Indonesian isolate IS-1 of T. gondii (Iskandar, 1988) were grown in the peritoneal cavity of Balb/c mice and collected from peritoneal exudate 6 days post-inoculation and purified according to Garberi et al (1990). DNA was extracted using PureLink Genomic DNA Kit (Invitrogen).…”

Section: Extraction Of Toxoplasma Dna and Isolation And Cloning Of Thmentioning

confidence: 99%

Development of Dot-blot Hybridization Based on 522 bp Repetitive Sequence (R522) for Detection of Toxoplasma gondii

Kusumawati¹,

Widodo²,

Wanahari³

et al. 2014

Biosci., Biotechnol. Res. Asia

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Toxoplasmosis, arising from infection by Toxoplasma gondii, is one of the most common parasitic diseases in humans and other warm-blooded animals. In humans, infections are usually asymptomatic but severe disease can occur in immunocompromized individuals and newborns. Due to the importance of the disease and in order to take suitable measures, an early diagnosis of the disease is essential, particularly for pregnant women and in industry of domestic animals. The genome of T. gondii contains repeat sequences B1 and R522 which constitute ideal targets for genome-based detection methods. The 522 base pairs repeat sequences R522 are the most promising due to the high copy number, evaluated to be 200 to 300 units within the genome. We developed a simple dot-blot hybridization based on R522 sequences. The method is simple and does not require sophisticated devices. The test of the method, using cloned R522 as target, showed that the parasite detection method was sensitive and proved to be promising for use in routine health controls as well as for the survey of Toxoplasma infections.

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Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites: preliminary characterization of its genome

Cited by 5 publications

References 0 publications

Electrical cream separator coupled with vacuum filtration for the purification of eimerian oocysts and trichostrongylid eggs

Electrical cream separator coupled with vacuum filtration for the purification of eimerian oocysts and trichostrongylid eggs

Unmethylated CpG motifs inToxoplasma gondiiDNA induce TLR9- and IFN-β-dependent expression ofα-defensin-5 in intestinal epithelial cells

Development of Dot-blot Hybridization Based on 522 bp Repetitive Sequence (R522) for Detection of Toxoplasma gondii

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