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Product Name:
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Rouen 87 (in vitro)
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Manufacturer:
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BEI Resources
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Taxonomy:
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Protozoa Classification: Apicomplexa, Babesia
Species: Babesia divergens
Strain: Rouen 87 (also referred to as Rouen 1987)1
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Material Provided:
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Each vial of NR-52008 contains approximately 0.5 mL of B. divergens-infected human blood in Glycerolyte 57 (1:5). Please refer to Appendix I for cryopreservation instructions.
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Packing/Storage:
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NR-52008 was packaged aseptically in screw-capped plastic cryovials and is provided frozen on dry ice. The product should be stored at -130°C or colder, preferably in the vapor phase of a liquid nitrogen freezer. If liquid nitrogen storage facilities are not available, frozen cryovials may be stored at -70°C or colder for approximately one week.
Note: Do not under any circumstances store vials at temperatures warmer than -70°C. Storage under these conditions will result in the death of the culture.
To ensure the highest level of viability, the culture should be initiated immediately upon receipt. Any warming of the product during shipping and transfer must be avoided, as this will adversely affect the viability of the product. For transfer between freezers and for shipping, the product may be placed on dry ice for brief periods, although use of a portable liquid nitrogen carrier is preferred. Please read the following recommendations prior to using this material.
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Safety Precautions:
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All blood cultures should be handled with appropriate safety precautions necessary for the handling of bloodborne pathogens. Personnel must be trained in accordance with their institutional policy regarding bloodborne pathogens.
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Growth Conditions:
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RPMI 1640 medium adjusted to contain 10% (v/v) heat-inactivated human serum (pooled Type A), 25 mM HEPES, 2 mM L-glutamine, 2 g/L D-glucose, 27 µg/mL hypoxanthine, 4.4 g/L sodium bicarbonate and 5 µg/mL gentamicin (optional); (Appendix II)
Human erythrocytes (Appendix III)
Incubation:
Temperature: 37°C
Atmosphere: 90% N2, 5% CO2, 5% O2
Propagation:
1. Place the frozen vial in a 35°C to 37°C water bath until the culture is completely thawed. Transfer the vial to a biological safety hood and wipe the outside surface of the vial with 70% ethanol.
2. Immediately after thawing, aseptically transfer the contents of the vial to a sterile 50 mL conical centrifuge tube using a 1 mL pipette.
3. Add dropwise a 12% sodium chloride (NaCl) solution to reach approximately a 1:5 ratio of NaCl to cell mixture (approximately 0.2× the original culture volume). Allow the vial to incubate for 5 minutes at room temperature.
4. Using a 10 mL pipette, add dropwise while shaking 10 volumes of a 1.6% NaCl solution (10:1 ratio of NaCl to original culture volume).
5. Centrifuge at 1000 × g for 5 minutes. Remove the supernatant, leaving approximately 0.5 mL to 1 mL of supernatant in the tube. Resuspend the cells by gently swirling the tube.
6. Add dropwise while shaking 10 volumes of growth medium. Centrifuge at 1000 × g for 5 minutes and carefully remove the supernatant.
7. Add 5 mL of growth medium (warmed to 37°C) and transfer the culture to a non-vented cap 25-cm2 cell culture flask (T-25).
8. For continuous culture, add uninfected donor red blood cells (RBCs) to a 1% to 2% hematocrit solution.
9. Gently aerate the culture with a 90% N2, 5% CO2, 5% O2 mixture through a sterile, cotton-plugged Pasteur pipet. Incubate the flask at 37°C.
10. Incubate the flask at 37°C. Monitor the infection daily by microscopic examination of blood films stained with a 5% Giemsa solution.
Assessment of infection:
1. To determine parasitemia of the culture, prepare thin smears of 3 µL to 5 µL of cell culture samples on microscopic slides. Fix in methanol, allow to air dry. Stain with a 5% Giemsa solution, allowing the slides to incubate in the stain for 40 minutes. Prepare fresh Giemsa stain on a daily basis.
2. Examine the slides under a microscope at 1000× magnification for the presence of intracellular parasite forms.
3. Count the number of infected red blood cells (RBC) versus the total number of red blood cells under oil immersion and determine the % parasitemia:
% parasitemia = (Infected RBC/Total RBC) × 100
Note: A minimum of 500 red blood cells should be counted.
Maintenance:
1. Remove the flask with infected culture from the 37°C incubator and place inside a biosafety cabinet.
2. Carefully remove the supernatant with a sterile, unplugged Pasteur pipet under vacuum. Remove as much of the supernatant as possible without taking the cells.
3. Add 25 mL of sterile warm (37°C) complete medium to the flask, gently mix and aerate, then quickly tighten the cap and place the flask in the 37°C incubator until the next change of medium.
Note: Changing of the culture medium daily is required for Babesia-infected erythrocyte cultures.
Please refer to Appendix I for cryopreservation, Appendix II for complete medium preparation instructions and Appendix III for preparation of human erythrocytes instructions.
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Disclaimers:
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You are authorized to use this product for research use only. It is not intended for human use. Use of this product is subject to the terms and conditions of the BEI Resources Material Transfer Agreement (MTA). The MTA is available on our Web site at www.beiresources.org. While BEI Resources uses reasonable efforts to include accurate and up-to-date information on this product sheet, neither ATCC® nor the U.S. Government makes any warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. Neither ATCC® nor the U.S. Government warrants that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, use and disposal. ATCC® and the U.S. Government are not liable for any damages or injuries arising from receipt and/ or use of this product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, the U.S. Government, ATCC®, their suppliers and contributors to BEI Resources are not liable for damages arising from the misidentification or misrepresentation of products. |
References:
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1. Precigout, E., et al. “Analysis of Immune Responses of Different Hosts to Babesia divergens Isolates from Different Geographic Areas and Capacity of Culture-Derived Exoantigens to Induce Efficient Cross-Protection.” Infect. Immun. 59 (1991): 2799-2805. PubMed: 1713201. Erratum in: Infect. Immun. 60 (1992): 1728.
2. Cuesta, I., et al. “High-Quality Draft Genome Sequence of Babesia divergens, the Etiological Agent of Cattle and Human Babesiosis.” Genome Announc. 2 (2014): e01194-14. PubMed: 25395649.
3. González, L. M., et al. “Comparative and Functional Genomics of the Protozoan Parasite Babesia divergens Highlighting the Invasion and Egress Processes.” PLoS Negl. Trop. Dis. 13 (2019): e0007680. PubMed: 31425518.
4. Kumar, A., J. O’Bryan and P. J. Krause. “The Global Emergence of Human Babesiosis.” Pathogens 10 (2021): 1447. PubMed: 34832603. Erratum in: Pathogens 11 (2022): 607.
5. Renard, I. and C. B. Mamoun. “Treatment of Human Babesiosis: Then and Now.” Pathogens 10 (2021): 1120. PubMed: 34578153.
6. Yang, Y., et al. “Emerging Human Babesiosis with "Ground Zero" in North America.” Microorganisms 9 (2021): 440. PubMed: 33672522.
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Citation:
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Acknowledgment for publications should read “The following reagent was obtained through BEI Resources, NIAID, NIH: Babesia divergens, Strain Rouen 87 (in vitro), NR-52008.”
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Biosafety Level:
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2
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories (BMBL). Current Edition. Washington, DC: U.S. Government Printing Office.
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