Encephalitozoon cuniculi Levaditi et al. (ATCC® 50602)

Strain Designations: CDC:V282  /  Depositor: GS Visvesvara  /  Biosafety Level: 2

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Strain Designations CDC:V282
Application
Enteric Research
Food and waterborne pathogen research
Opportunistic pathogen research
Biosafety Level 2
Isolation Urine from AIDS patient, Colorado, 1993
Product Format frozen
Storage Conditions Frozen Cultures:
-70°C for 1 week; liquid N2 vapor for long term storage

Freeze-dried Cultures:
2-8°C

Live Cultures:
See Protocols section for handling information
Type Strain no
Genotype III,III,IIIa
Comments
Sporogony
Environmental resistance of spores
flow cytometric analysis
detection of drug-induced effects in infected green monkey kidney cells
Western blot and immunofluorescence analysis
case report
ITS RFLP analysis
Control strain for enterocytozoan identification
Growth Conditions
Temperature: 35°C to 37°C
Atmosphere: 5% CO2
Cell Line: ATCC® CCL-75™ (lung, human), ATCC® CCL-34™ (kidney, canine), or ATCC® CCL-26™ (kidney, African green monkey).
Cryopreservation Harvest and Preservation
  1. To harvest the Encephalitozoon culture, detach any remaining tissue culture cells (infected and uninfected) by scraping the surface of the flask with a cell scraper.
  2. Transfer the cell suspension (including parasites) to 15 mL plastic centrifuge tubes. Centrifuge at 1300 x g for 10 min.
  3. Remove all but 0.5 mL of the supernatant from each tube, resuspend the cell pellets, and pool them to a single tube.
  4. Pass the resulting cell suspension through a syringe equipped with a 27 gauge 1/2 in needle to break up any remaining cells. Adjust the parasite concentration to 2.0 - 4.0 x 107 cells/mL with fresh medium or PBS.
    NOTE: If the concentration of parasites is too low, centrifuge at 1300 x g for 10 min and resuspend in the volume of fresh medium or PBS required to yield the desired concentration.
  5. Prepare a cryoprotective solution containing 20% (v/v) DMSO and 6% (v/v) HIFBS in fresh medium or PBS.
  6. Mix the cell preparation and cryoprotective solution in equal portions. The final concentration will be 1.0 - 2.0 x 107 cells/mL, 10% DMSO, and 3% HIFBS. The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.
    NOTE: To prevent culture contamination, penicillin-streptomycin solution (ATCC® 30-2300) may be added to a final concentration of 50 to 100 I.U./mL penicillin and 50 to 100 µg/mL streptomycin.
  7. Dispense in 0.5 mL aliquots to 1.0-2.0 mL sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
  8. Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1 C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.)
  9. Store frozen ampules in either the vapor or liquid phase of a nitrogen refrigerator.
  10. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.  Do not leave ampule in water bath after thawed.
  11. Immediately after thawing, aseptically transfer contents to a T-25 tissue culture flask containing a fresh monolayer of cells (ATCC® CCL-75™, CCL-34™, or CCL-26™) and 10 mL ATCC® 30-2003 with 3% (v/v) HIFBS.
  12. Outgas the flask for 10 seconds with a 95% air, 5% CO2 gas mixture.
  13. Incubate in a 35-37°C CO2 incubator with the cap screwed on tightly.
Name of Depositor GS Visvesvara
Special Collection NCRR Contract
Year of Origin 1993
References

Didier ES, et al. A microsporidian isolated from an AIDS patient corresponds to Encephalitozoon cuniculi III, originally isolated from domestic dogs. J. Clin. Microbiol. 34: 2835-2837, 1996. PubMed: 8897194

De Groote MA, et al. Polymerase chain reaction and culture confirmation of disseminated Encephalitozoon cuniculi in a patient with AIDS: Successful therapy with albendazole. J. Infect. Dis. 171: 1375-1378, 1995. PubMed: 7751721

Croppo GP, et al. Western blot and immunofluorescence analysis of a human isolate of Encephalitozoon cuniculi established in culture from the urine of a patient with AIDS. J. Parasitol. 83: 66-69, 1997. PubMed: 9057698

Moss DM, et al. Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon. J. Clin. Microbiol. 37: 371-375, 1999. PubMed: 9889221

Moura H, et al. Detection by an immunofluorescence test of Encephalitozoon intestinalis spores in routinely formalin-fixed stool samples stored at room temperature. J. Clin. Microbiol. 37: 2317-2322, 1999. PubMed: 10364604

del Aguila C, et al. Ultrastructure, immunofluorescence, western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS. J. Clin. Microbiol. 36: 1201-1208, 1998. PubMed: 9574677

Bornay-Llinares FJ, et al. Immunologic, microscopic, and molecular evidence of Encephalitozoon intestinalis (Septata intestinalis) infection in mammals other than humans. J. Infect. Dis. 178: 820-826, 1998. PubMed: 9728552

Visvesvara GS, et al. Encephalitozoon cuniculi: light and electron microscopic evidence for di-, tetra, and octosporous sporogony and a note on the molecular phylogeny of Encephalitozoonidae. J. Eukaryot. Microbiol. 46: 110-115, 1999. PubMed: 10361732

Kucerova-Pospisilova Z, et al. Environmental resistance of Encephalitozoon spores. J. Eukaryot. Microbiol. 46: 11S-13S, 1999. PubMed: 10519227

Leitch GJ, et al. Use of fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia. Antimicrob. Agents Chemother. 41: 337-344, 1997. PubMed: 9021189

del Aguila C, et al. In vitro culture, ultrastructure, antigenic, and molecular characterization of Encephalitozoon cuniculi isolated from urine and sputum samples from a Spanish patient with AIDS. J. Clin. Microbiol. 39: 1105-1108, 2001. PubMed: 11230434

Sobottka I, et al. Acute and long-term humoral immunity following active immunization of rabbits with inactivated spores of various Encephalitozoon species. Parasitol. Res. 87: 1-6, 2001. PubMed: 11199842

Xiao L, et al. Genotyping Encephalitozoon cuniculi by multilocus analyses of genes with repetitive sequences. J. Clin. Microbiol. 39: 2248-2253, 2001. PubMed: 11376065

Molestina R, Becnel JJ, Weiss LM. Culture and Propagation of Microsporidia. In Microsporidia: Pathogens of Opportunity, First Edition, Chapter 18: pp. 457-467, 2014. Hoboken, NJ: John Wiley & Sons, Inc.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
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