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Product Name:
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WA1, Clone BdWA1-301 (in vitro)
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Manufacturer:
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BEI Resources
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Taxonomy:
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Protozoa Classification: Babesiidae, Babesia
Species: Babesia duncani
Strain: WA1, Clone BdWA1-301 (in vitro)
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Additional Information:
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Babesia species are intraerythrocytic protozoan parasites of the phylum Apicomplexa that are the causal agents of babesiosis, which is transmitted to both humans and mammals by infected ixodid ticks.3,4 Infection with Babesia species is usually asymptomatic or can result in mild flu-like symptoms that subside within a few days. Severe cases featuring acute anemia, thrombocytopenia, organ failure, or even death may occur among the elderly, splenectomized and immunocompromised individuals.3,4 The majority of human cases of babesiosis in the United States are caused by B. microti, while B. divergens is the primary cause of babesiosis in Europe, though human infections caused by B. divergens-like parasites in the United States have been reported.4,5,6,7 B. duncani infections in the United States have occurred through both tickborne and blood transfusion routes.8
B. duncani, strain WA1, clone BdWA1-301 has demonstrated in vitro and in vivo parasitemia comparable to that of the parental WA1 strain (BEI Resources NR-12311).1
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Material Provided:
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Each vial of NR-59103 contains approximately 0.5 mL of B. duncani-infected human blood in Glycerolyte 57 (1:5). Please refer to the Appendix for cryopreservation instructions.
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Packing/Storage:
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NR-59103 was packaged aseptically in 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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DMEM/F12 Medium containing 20% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 µM hypoxanthine and 16 µM thymidine1
Note: To prevent culture contamination, Penicillin-Streptomycin-Amphotericin B (Antibiotic/Antimycotic) Solution (ATCC® PCS 999 002™) may be added to a final concentration of 100 IU/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B. Gentamicin may also be added to a final concentration of 100 µg/mL.
Human whole blood, Type O+ (Appendix)
Incubation:
Temperature: 37°C
Atmosphere: Humidified atmosphere of 93% N2, 5% CO2, 2% 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 625 × 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 625 × 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 5% hematocrit (HT) suspension every 2 to 3 days.
9. Gently aerate the culture with a 93% N2, 5% CO2, 2% O2 gas mixture through a sterile, cotton-plugged Pasteur pipet and then quickly tighten the cap. Incubate the flask at 37°C.
10. Monitor the parasitemia daily by microscopic examination of blood films stained with a 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. Carefully remove the flask with infected culture from the 37°C incubator without disturbing the RBCs and place it inside a biosafety cabinet.
2. Carefully remove the supernatant with a sterile, unplugged Pasteur pipet under vacuum, or using a sterile pipette. Remove as much of the supernatant as possible without removing the cells.
3. Remove a small cell sample for microscopic examination by Giemsa staining.
4. To the culture flask, gently add prewarmed (37°C) sterile growth medium and uninfected donor RBCs, as needed, for a total of 5% hematocrit. Mix the medium and the cells inside the flask by gentle swirling.
5. Aerate the culture flask with a 93% N2, 5% CO2, 2% O2 gas mixture through a sterile pipette, tighten the cap and incubate the flask in a 37°C incubator.
Note: For rapid increase of parasitemia, changing of the culture medium daily is required for Babesia-infected erythrocyte cultures. Subculture should be performed when the culture is stable and parasitemia reaches 6%.
Please refer to the Appendix for cryopreservation and preparation of human donor 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. Singh, P., A. C. Pal and C. Ben Mamoun. “An Alternative Culture Medium for Continuous In Vitro Propagation of the Human Pathogen Babesia duncani in Human Erythrocytes.” Pathogens 22 (2022): 599. PubMed: 35631120.
2. Conrad, P. A., et al. “Description of Babesia duncani n. sp. (Apicomplexa: Babesiidae) from Humans and Its Differentiation from Other Piroplasms.” Int. J. Parasitol. 36 (2006): 779-789. PubMed: 16725142.
3. Leiby, D. A. “Transfusion-Transmitted Babesia spp.: Bull's-Eye on Babesia microti.” Clin. Microbiol. Rev. 24 (2011): 14-28. PubMed: 21233506.
4. Vannier, E. and P. J. Krause. “Human Babesiosis.” N. Engl. J. Med. 366 (2012): 2397-2407. PubMed: 22716978.
5. Beattie, J. F., M. L. Michelson and P. J. Holman. “Acute Babesiosis Caused by Babesia divergens in a Resident of Kentucky.” N. Engl. J. Med. 347 (2002): 697-698. PubMed: 12200568.
6. Herwaldt, B. L., et al. “A Fatal Case of Babesiosis in Missouri: Identification of Another Piroplasm that Infects Humans.“ Ann. Intern. Med. 124 (1996): 643-650. PubMed: 8607592.
7. Herwaldt, B. L., et al. “Babesia divergens-Like Infection, Washington State.” Emerg. Infect. Dis. 10 (2004): 622-629. PubMed: 15200851.
8. Herwaldt, B. L., et al. “Transfusion-Associated Babesiosis in the United States: A Description of Cases.” Ann. Intern. Med. 155 (2011): 509-519. PubMed: 21893613.
9. Abraham, A., et al. “Establishment of a Continuous in vitro Culture of Babesia duncani in Human Erythrocytes Reveals Unusually High Tolerance to Recommended Therapies.” J. Biol. Chem. 293 (2018): 19974-19981. PubMed: 30463941.
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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 duncani, Strain WA1, Clone BdWA1-301 (in vitro), NR-59103.”
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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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