Neospora hughesi Marsh et al. (ATCC® 209622)

Organism: Neospora hughesi Marsh et al.  /  Depositor: University of California, Davis

Permits and Restrictions

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Deposited As Neospora sp.
Strain Designations NE1
Application
Detection of Neospora infection in horses
Biosafety Level 2
Isolation Tachyzoites and bradyzoites from brain stem and spinal cord of naturally infected quarter horse, Equus caballus, California
Product Format frozen
Storage Conditions Frozen: -70°C or colder
Freeze-Dried: 2°C to 8°C
Live Culture: See Protocols Section
Type Strain yes
Comments This strain is contaminated with Mycoplasma orale.
Growth Conditions
Temperature: 35°C to 37°C
Cell Line: ATCC CCL-209 (bovine pulmonary artery endothelium) (Contact ATCC Sales to order)
Alternate Cell Line: Depositor has indicated that the following cell line may alternatively be used: ATCC CCL-81 (kidney, African green monkey)
Cryopreservation Harvest and Preservation
  1. To harvest the Neospora 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 10% (v/v) HIHS 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 5% 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 ATCC® CCL-209™ cells and 10 mL ATCC® 30-2003™ with 5% (v/v) HIHS.
  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 University of California, Davis
Chain of Custody
ATCC <-- University of California, Davis <-- A.E. Marsh and P.A. Conrad
References

Marsh AE, et al. Equine Neospora isolate and its uses. US Patent 6,071,737 dated Jun 6 2000

Marsh AE, et al. Neosporosis as a cause of equine protozoal myeloencephalitis. J. Am. Vet. Med. Assoc. 209: 1907-1913, 1996. PubMed: 8944807

Marsh AE, et al. Description of a new Neospora species (Protozoa: Apicomplexa: Sarcocystidae). J. Parasitol. 84: 983-991, 1998. PubMed: 9794642

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.
  • USDA APHIS VS 16-6 or 16-6A permit must be obtained and a copy of the permit must be sent to ATCC in advance of shipment. The Application Form VS 16-3 (Import controlled material import or transport organisms or vectors) must be submitted to USDA APHIS Veterinary Services to obtain the VS 16-6 or 16-6A permit.
Basic Documentation