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Product Name:
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Babesia hegotelforum sp. nov. (in vitro) (Deposited as Babesia sp., Strain MO1)
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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 hegotelforum sp. nov.
Note: Previously referred to as Babesia sp., strain MO1, NR-50441 has been reclassified as Babesia hegotelforum sp. nov.1,2,3
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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.4,5,6 In humans, babesiosis 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.4,5 Five species of Babesia confirmed to infect humans have been identified: B. divergens, B. duncani, B. hegotelforum sp. nov., B. microti and B. venatorum.1,6 B. microti has a worldwide distribution and is the most commonly identified etiologic agent of human babesiosis in the United States.6 Comparative analysis of clinical presentation, host associations, genome-wide sequence divergence and phylogenetic placement identified B. hegotelforum sp. nov. as another cause of babesiosis in the United States.1 In Europe, B. divergens is the primary cause of babesiosis in humans and cattle.4
The small-subunit ribosomal RNA (SSU rRNA) sequence of B. hegotelforum sp. nov. is identical to that of three cases of human babesiosis in the United States, suggesting the cottontail rabbit as the primary reservoir host.1,3,4,7 Developmental forms of B. hegotelforum sp. nov. include ring-like trophozoites, paired pyriforms, tetrads and flower-like multi-merozoite forms containing five to eight merozoites.1 B. hegotelforum sp. nov. parasites reach maximal levels of approximately 8% to 10% parasitemia in vitro and are strictly intraerythrocytic.1
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Material Provided:
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Each vial contains approximately 0.5 mL of infected human erythrocytes in Glycerolyte 57 solution. Please see the Appendix document for cryopreservation instructions.
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Packing/Storage:
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NR-50441 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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Media:
Babesia DMEM/F12 Growth Medium (Appendix)
Human erythrocytes (Appendix)
Incubation:
Temperature: 37°C
Atmosphere: Initially, in a humidified atmosphere of 93% N2, 5% CO2, 2% O2. Once cultures are established, a humidified, ambient atmosphere with 5% CO2.
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 400 × 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 400 × 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 vented cap 25-cm2 cell culture flask (T-25).
8. For continuous culture, add uninfected donor red blood cells (RBCs) to a 10% hematocrit solution.
9. Gently aerate the culture with a 95% air, 5% CO2 mixture through a sterile, 2 mL Pasteur pipet.
10. Incubate the flask at 37°C. Monitor the infection daily by microscopic examination of blood films stained with a 5% Giemsa solution. Subculture when the culture reaches ≥ 6% parasitemia.
Assessment of infection:
1. To determine parasitemia of the culture, prepare thin smears of 1 µL to 3 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. When the culture is at or near peak density, carefully aspirate the supernatant with a sterile, unplugged Pasteur pipet under vacuum, removing as much of the supernatant as possible without aspirating the red blood cells and determine parasitemia.
2. Carefully remove a small cell sample for microscopic examination by Giemsa staining.
3. To the flask, gently add 5 to 10 mL of sterile growth medium warmed to 37°C in a water bath. Mix the media and red blood cells by gently swirling. Aerate the flask, tighten the cap and incubate at 37°C.
4. Monitor the culture every 2 to 3 days. Subculture when the culture reaches ≥ 6% parasitemia. Alternatively, add uninfected RBCs every 3 days to increase the parasitemia within the same culture flask.
Note: During continuous incubation, the growth medium of B. hegotelforum sp. nov. may change color and range from bright red to almost black as a result of oxygen utilization by the parasites and deoxygenation of hemoglobin.9
Please refer to the Appendix document for cryopreservation, medium preparation instructions and 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. Singh, P., et al. “Babesia hegotelforum sp. nov., a Zoonotic Babesia Species Previously Referred to as Babesia sp. MO1.” Emerg. Microbes Infect. 15 (2026): 2637280. PubMed: 41790956.
2. Holman, P. J., Personal Communication.
3. Holman, P. J., et al. “In Vitro Cultivation of a Zoonotic Babesia sp. Isolated from Eastern Cottontail Rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts.” J. Clin. Microbiol. 43 (2005): 3995-4001. PubMed: 16081941.
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. Leiby, D. A. “Transfusion-Transmitted Babesia spp.: Bull's-Eye on Babesia microti.” Clin. Microbiol. Rev. 24 (2011): 14-28. PubMed: 21233506.
7. Herwaldt, B. L., et al. “Babesia divergens-Like Infection, Washington State.” Emerg. Infect. Dis. 10 (2004): 622-629. PubMed: 15200851.
8. Goethert, H. K. and S. R. Telford III. “Enzootic Transmission of Babesia divergens Among Cottontail Rabbits on Nantucket Island, Massachusetts.” Am. J. Trop. Med. Hyg. 69 (2003): 455-460. PubMed: 14695079.
9. Schuster, F. L. “Cultivation of Babesia and Babesia-Like Blood Parasites: Agents of an Emerging Zoonotic Disease.” Clin. Microbiol. Rev. 15 (2002): 365-373. PubMed: 12097245.
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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 hegotelforum sp. nov. (in vitro) (Deposited as Babesia sp., Strain MO1), NR-50441."
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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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