Babesia duncani in Culture and in Mouse (ICIM) Model for the Advancement of Babesia Biology, Pathogenesis and Therapy

Materials and Reagents

1. For in vitro culture

   1. 6-well plate (Corning, catalog number: 353046)

   2. 1.7 mL microcentrifuge tubes (Thomas Scientific, catalog number: 1149K01)

   3. Centrifuge tubes (Corning, Falcon tubes, catalog numbers: 430829 [50 mL], 352096 [15 mL])

   4. Pipette tips [USA Scientific, catalog numbers: 1121-3810 (10 μL), 1120-8810 (200 μL), 1111-2721 (1,000 μL)]

   5. Plugged serological sterile pipettes [Corning, Falcon pipettes, catalog numbers: 357543 (5 mL), 357551 (10 mL), 357525 (25 mL)]

   6. Millex filter units [Millipore, catalog numbers: SLGSR33SS (Syringe driven), S2GPU10RE (vacuum driven)]

   7. Pipettes [Eppendorf, catalog numbers: K24694J (1,000 μL), J46084J (200 μL), H16607J (20 μL), I54818J (10 μL)]

   8. Pipette AID (Drummond Scientific)

   9. Aspiration pipettes (Santa Cruz, catalog number: 357781)

   10. DMEM/F-12 (Lonza, catalog number: BE04-687/U1, Basel, Switzerland; or Thermo Fisher Scientific, catalog number: 21331020)

   11. DMEM (Thermo Fisher Scientific, Gibco, catalog number: 11-966-025)

   12. RPMI (Thermo Fisher Scientific, Gibco, catalog number: 11-875-093)

   13. FBS (Thermo Fisher Scientific, Gibco, catalog number: 10438-026, Waltham, MA, USA)

   14. 50× HT media supplement Hybrid-Max^TM (Sigma, catalog number: H0137)

   15. L-glutamine (Thermo Fisher Scientific, Gibco, catalog number: 25030-081)

   16. Antimycotic (antibiotic) (Thermo Fisher Scientific, Gibco, catalog number: 15240-062)

   17. Gentamicin reagent solution (Thermo Fisher Scientific, Gibco, catalog number: 15710-072)

   18. A⁺ human RBCs (American Red Cross or Interstate Blood Bank, Inc.)

   19. B. duncani WA1 strain (BEI Resources, NR-12311)

   20. DMEM/F-12 complete media (250 mL) (see Recipes)

       
       

2. For cryopreservation

   1. Glycerolyte 57 solution (Baxter Healthcare corporation, Deerfield, IL, USA, catalog number: 4A7831)

   2. Cryotube vials (Thermo Scientific, catalog number: 363401)

      
      

3. For slide preparation

   1. Premium microscope slides (Fisherfinest, catalog number: 22038-103)

   2. Hemacolor solution I, fixative (Sigma-Aldrich, catalog number: 65044A-85)

   3. Hemacolor solution II (Sigma-Aldrich, catalog number: 65044B-85)

   4. Hemacolor solution III (Sigma-Aldrich, catalog number: 65044C)

   5. Immersion oil (Cargille, catalog number: 16482)

      
      

4. For mouse infection

   1. BD PrecisionGlide^TM 27G needle (BD, catalog number: 305109)

   2. Lithium heparin–coated blood collection tube (McKesson 574507, Greiner Bio-One, MiniCollect, catalog number: 450477)

   3. Heparinized capillary tubes (Fisher Scientific, catalog number: 22-260-950)

   4. Isoflurane (Covetrus, catalog number: 11695-6777-2)

   5. PEG 400 (Thermo Fisher Scientific, Avantor J.T. Baker U216-07, catalog number: 02-003-646)

   6. 1× PBS diluted from 10× PBS pH 7.4 (Gibco, catalog number: 70011-044)

   7. Mouse strains: C3H/HeJ (from Jackson Laboratories, Bar Harbor, ME)

      Note: Our studies have shown that C3H/HeJ mice are susceptible to B. duncani infection following IV inoculation with doses of infected RBCs (iRBCs) ranging between 10² and 10⁷. We found that 10⁷ and 10⁶ iRBC doses elicit an acute increase in parasitemia (P) (up to 35%) within a short span of time [3–5 days post infection (DPI)], whereas mice inoculated with doses of 10²–10⁵ iRBCs show a delayed onset of infection with a lower peak P (Pal et al., 2022). Parasitemia levels in C3H/HeJ mice were always higher in females compared to males at the same infection dose (Aguilar-Delfin et al., 2001, Pal et al., 2022). In contrast, C57BL/6J mice showed 100% survival with no detectable P following inoculation with 10⁴ B. duncani–iRBC. Infection of Balb/cJ with 10⁴ B. duncani–iRBC resulted in increased P over time in all mice; however, approximately 50% of the mice cleared the infection and survived, whereas the other half continued to show detectable P and succumbed to infection (Pal et al., 2022).

   8. 30% isoflurane (50 mL) (inhalation anesthetic; see Recipes)

Equipment

1. Sorvall legend XTR centrifuge (Thermo Scientific, catalog number: 75004521)

2. Microscope (Nikon Eclipse 5Oi)

3. SterilGARD III advance biological safety cabinet (The Baker Company, catalog number: SG603)

4. Water bath (Fisher brand, model: FSGPD15D)

5. Tri gas incubator (Thermo Scientific, model: HERACELL VIOS 160i)

6. Eppendorf MiniSpin (Millipore-Sigma, catalog number: EP022620100)

Software

1. GraphPad Prism version 9.4.1

Procedure

1. In vitro culture of B. duncani WA1 isolate in hRBCs

   1. Prepare media by adding all the components (see Figure 1) in sterile conditions inside a biosafety cabinet (Figure 2).

      Note: In specific circumstances when DMEM/F-12 is not available from the different mentioned sources, DMEM base medium supplemented with missing components (those present in DMEM/F-12) could be successfully used for parasite culture (see Table 1 provided below).

      
      

      
      Figure 1. Representative image showing media components and materials required for B. duncani in vitro culture

      
      

      
      Figure 2. Representative image showing biosafety cabinet BSL-2 grade required for handling and culturing of B. duncani parasite

      
      

      Table 1. Supplement required to complete DMEM base medium to be used as a replacement of DMEM/F-12

      		Catalog number	DMEMb Catalog number: 11965092 (Thermo Fisher Scientific) Conc. (mg/L)	DMEM/F-12 Catalog number: BE04-687F/U1 (LONZA) Conc. (mg/L)
      Amino Acids	L-Alanine	A7219, Sigma	-	4.45
      	L-Asparagine-H2O	A7094, Sigma	-	7.5
      	L-Aspartic acid	A7219, Sigma	-	6.65
      	L-Cysteine hydrochloride-H2O	C6852, Sigma	-	17.56
      	L-Glutamic Acid	G8415, Sigma	-	7.35
      	L-Proline	P5067, Sigma	-	17.27
      Vitamins	Biotin	B4639, Sigma	-	0.004
      	Vitamin B12	V6629, Sigma	-	0.68
      Inorganic Salts	Cupric sulfate (CuSO4·5H2O)	C8027, Sigma	-	0.0012
      	Ferric sulfate (FeSO4·7H2O)	F8633, Sigma	-	0.42
      	Magnesium Chloride (MgCl2) anhydrous	M8266, Sigma	-	28.57
      	Zinc sulfate (ZnSO4·7H2O)	Z0251, Sigma	-	0.43
      Lipids	Linoleic Acid	L1012, Sigma	-	0.044
      	Lipoic Acid	T1395, Sigma	-	0.013
      Other components	Putrescine 2HCl	P5780, Sigma	-	0.081
      	Hypoxanthine and Thymidine mix	H0137-10VL, Sigma	-	0.0112
      			-	0.001468

      
      

   2. Wash hRBCs as follows:

      1. After receiving blood (bag containing approximately 500 mL of total blood) from a donation center, mix gently by inverting the bag 2–3 times. Aliquot blood samples into 50 mL centrifuge tubes and store at 4 °C until washing. Unwashed blood can be stored at 4 °C for up to one month.

      2. To wash an aliquot of blood, use incomplete DMEM or RPMI media. Use roughly four volumes of medium per one volume of packed RBCs. Mix the suspension gently to resuspend the RBCs and centrifuge at 1,800 rpm (757 × g) for 10 min at room temperature (RT).

         Note: For washing RBCs, DMEM/RPMI from any source can be used.

      3. Following centrifugation, gently aspirate the medium and carefully remove the buffy coat containing the white blood cells from the top RBC layer. Repeat the washing steps twice.

      4. After the final wash, add an equal volume of incomplete DMEM or RPMI media to make 50% hematocrit (HC).

      5. The washed RBCs (50% HC) can be immediately used for in vitro culture or stored at 4 °C for further use. Washed RBCs can be stored at 4 °C and used for up to two weeks in cell culture.

   3. Thawing cryo-preserved B. duncani–infected erythrocytes:

      1. Take a cryovial of frozen parasite from the liquid nitrogen and immediately put in a 37 °C water bath for a few seconds until the contents of the vial turn into liquid.

      2. Transfer the contents of the thawed vial containing the B. duncani–infected hRBCs to a 50 mL centrifuge tube inside the biosafety cabinet.

      3. Add 200 μL of 12% (w/v) NaCl (0.2× original cryovial volume) dropwise while gently shaking the tube. Incubate for 5 min at RT.

      4. Add 9 mL of 1.6% (w/v) NaCl dropwise while gently shaking the tube.

      5. Centrifuge at 1,500 rpm (526 × g) for 5 min at RT. Remove the supernatant without disturbing the pellet.

      6. Add 25 mL of the incomplete DMEM/F-12 medium to wash the parasite pellet. Centrifuge at 1,500 rpm (526 × g) for 5 min at RT.

      7. Repeat the above step one more time and resuspend the pellet in 3.6 mL of complete DMEM/F-12 media (see Recipes). Add 400 μL of washed 50% HC A⁺ RBCs (step 2) to maintain culture at 5% HC.

      8. Seed the culture into a well of 6-well plate and place it in the incubator at 2% O₂, 5% CO₂, and 93% N₂ atmosphere with 95% humidity setting (see Figure 3).

         
         

      
      Figure 3. Representative image of Tri gas incubator used for B. duncani in vitro culture

      
      

   4. Maintain the culture as follows:

      Replace culture medium every day by aspirating old medium with aspirating pipette and replace with complete DMEM/F-12 medium prewarmed to 37 °C.

      Note: Take extra precaution while aspirating the medium to not disturb the bottom of the plate containing the iRBCs.

      1. Monitor the culture and P level by smear preparation as follows:

         1. Aspirate medium as described above and resuspend culture in fresh medium by gently pipetting a few times.

         2. Take 50 μL culture in a microcentrifuge tube and spin at 2,500 rpm (700 × g) for 2 min at RT.

         3. Carefully remove supernatant with a 200 μL pipette tip (set to 40 μL), leaving behind 10 μL of medium. Resuspend RBCs pellet by gently pipetting a few times.

         4. Place a drop of the resuspended RBCs on a slide and make a smear by sliding another slide at an angle of 25°–45° as shown in Figure 4.

         5. Fix the blood smear for 10 s in the fixative solution I followed by 10 s in solution II. Stain with the solution III for 25 s. Rinse the stained slide in water, air dry, and visualize the slide under the light microscope at 100× (representative image of blood smear with 10% P shown in Figure 5).

         6. Estimate P as follows:

            1. Make a smear from in vitro culture and perform Giemsa staining as described above in steps i–v.

            2. Air dry for 3–4 min. Put a drop of immersion oil and visualize under microscope.

            3. Count iRBCs and total RBCs (tRBCs) (tRBCs = iRBCs + uninfected RBCs) in the field.

            4. Repeat step 3 for different fields until the tRBCs count from adding all the fields is 1,000–2,000 RBCs (for more accuracy).

            5. Calculate percentage of iRBCs as follows:
               
               Field 1=(9 iRBCs)/(80 tRBCs) Field 2=(10 iRBCs)/(110 tRBCs) Field 3=(11 iRBCs)/(120 tRBCs) Field 5=(8 iRBCs)/(70 tRBCs) Field 6=(12 iRBCs)/(130 tRBCs)
               
               (9+10+11+8+12)/(80+110+120+70+130)= 50/510×100=9.8% 

         
         

         
         Figure 4. Representative image demonstrating angular position of slide and RBC pellet (50 μL of in vitro culture after centrifugation) for smear preparation

         
         

         
         Figure 5. Representative image depicting 10% P of B. duncani–infected human erythrocytes

         
         

         Note: Increasing the number of fields for calculating the proportion of iRBCs and tRBCs and percentage of P levels allows a more accurate determination of P.

         
         

      2. Maintain the parasite culture by inoculating 0.5%–1% P of original culture once the P level reaches 10%–15%.

         Example: For a starter culture of 5 mL with 0.5% P and 5% HC, take 250 μL of the stock culture (10% P and 5% HC) and add 475 μL of 50% HC washed hRBCs and 4.275 mL of complete DMEM/F-12 medium.

      3. For specific assays that require high P, culture the parasites continuously with media replacement after every 24 h until it reaches the desired P. B. duncani in vitro cultures can reach up to 20%–25% P.

         
         

2. Cryopreservation of in vitro cultured B. duncani–iRBCs

   1. Estimate P levels by light microscopy following Giemsa staining of blood smears prepared from in vitro cultured parasites as described in step A4a if P is >5%, then proceed with the following steps of cryopreservation.

   2. Pellet the culture (5 mL) by centrifugation at 1,800 rpm for 5 min at RT.

   3. Aspirate the supernatant leaving behind the RBC pellet.

   4. Add 250 μL of filter-sterilized glycerolyte dropwise while tapping the bottom of the tube to mix.

   5. Gently mix the suspension and put in a cryovial. Label the cryovial describing the parasite strain and % P of the culture on the day of cryopreservation.

   6. Store the cryovial at -80 °C.

   7. For long-term storage (more than six months), take out the cryovial from -80 °C and place in liquid nitrogen.

      
      

3. Purification of B. duncani merozoites

   1. Initiate and maintain B. duncani in vitro culture in hRBCs in DMEM/F-12 medium until P reaches 18%–20% and free merozoite can be observed in blood smears (Figure 6).

      
      

      
      Figure 6. Representative image depicting 18%–20% P with free merozoites indicated by arrows

      
      

   2. To isolate the free merozoites, centrifuge the parasite culture at 1,800 rpm (757 × g) and 37 °C for 5 min.

   3. Use the pellet containing the B. duncani iRBCs for initiating fresh starter cultures or for other assays. The supernatant containing the free merozoites is collected using a pipette in a fresh centrifuge tube.

   4. The supernatant is centrifuged at 4,000 rpm (1,932 × g) and 37 °C for 10 min. The resulting pellet contains the free merozoites.

   5. Resuspend the merozoite pellet in warm DMEM/F-12 in 1:5 ratio (v/v). For example, for a packed pellet of 10 μL, add 50 μL of culture medium.

   6. Estimate the purity of the merozoites by Giemsa staining. The purity of a merozoite preparation is determined by the amount of free merozoites with little to no intact uninfected or infected RBCs. Figure 7 shows an example of a merozoite preparation with no RBCs detected. The merozoite preparations that are more than 95% pure are considered suitable for subsequent cell biological and molecular analyses.

      
      

   
   Figure 7. Representative image showing free merozoites (black arrows). Pink structures in the image represent RBC membranes resulting from lysis of infected erythrocytes.

   
   

4. Mouse infection

   1. Use 5–6 weeks old female C3H/HeJ mice after one week acclimatization post procurement.

   2. To infect mouse, use either B. duncani in vitro cultured purified merozoites (2 × 10⁷) or human iRBCs (8.5 × 10⁵) and inject by retro-orbital intravenous (IV) route.

      Note: Higher doses of parasite infection will expedite the establishment of infection in mouse. However, results may vary depending on the success of infection and handling.

   3. Calculate the amount of ihRBCs containing 8.5 × 10⁵ parasites as follows:

      1. Calculation of iRBCs in the in vitro culture:

         1 mL of human blood = 5 × 10⁹ RBCs (100% HC)

         1 mL of 5% HC human blood = 2.5 × 10⁸ RBCs

         If the P estimated by Giemsa stain is 10%

         1 mL of 5% HC with 10% P = 10/100 × 2.5 × 10⁸ = 2.5 × 10⁷ iRBCs

      2. Take the desired amount of parasite (8.5 × 10⁵) by serial dilution as follows:

         1. Take 100 μL of above culture (step D3a) containing 2.5 × 10⁶ iRBCs and dilute 1:10 by adding 900 μL of 1× PBS to attain a concentration of 2.5 × 10³ parasites per μL.

         2. For 8.5 × 10⁵ parasites from above diluted culture, take 340 μL (8.5 × 10⁵ ÷ 2.5 × 10⁵ = 340 μL) and pellet down by centrifugation at 1,800 rpm (757 × g) for 5 min at RT. Carefully aspirate the supernatant leaving behind 100 μL of supernatant.

         3. Resuspend by gentle tapping and inject as described below in step D5.

      Note: A serial dilution to take the desired amount of parasite is important, rather than taking a very little amount (e.g., 34 μL) directly from original culture (to avoid pipetting error).

   4. Perform IV infection as follows:

      1. Use a jar with cotton soaked in 30% isoflurane (inhalation anesthetic) diluted in PEG 400 placed at the bottom and covered with a mesh. Place a single mouse inside the sealed chamber containing the above-mentioned inhalation anesthetic (see Figure 8).

         
         

         
         Figure 8. Representative image showing reagents and materials required for mouse infection

         
         

      2. Inject 100 μL of parasite (in PBS) diluted as mentioned above in step D3b and inject via IV route.

      3. Monitor P by microscopy of thin blood smears prepared from blood collected from the tail vein on a regular interval of 24 or 48 h. Plot the P and survival curve using GraphPad Prism.

      4. Blood from an infected mouse can be used to infect another uninfected mouse to maintain the parasite stock. Parasitemia in the stock mouse can be monitored by microscopic examination of Giemsa-stained blood smears.

      5. Calculate the amount of blood required for mouse-to-mouse infection:

         1. Calculation of iRBCs

            1 mL of mouse blood = 10 × 10⁹ RBCs

            If P (estimated by Giemsa stain) is 10%

            1 mL of 10% P = 10/100 × 10 × 10⁹ = 10⁹ iRBCs

         2. Dilution of iRBCs

            If the desired dose of infection is 10⁴ parasites, collect 30 μL of blood from the mouse by retroorbital bleeding in a heparinized blood collection tube and dilute as follows:

            1. Take 10 μL of blood (1.0 × 10⁷ iRBCs) and add 990 μL of 1× PBS to make 1 × 10⁷ iRBC/mL.

            2. Dilute above diluted iRBCs to 1:10 for two more times by taking 100 μL each time from previous dilution and adding 900 μL of 1× PBS (serial dilution).

            3. This will result in 1 mL containing 1 ×10⁵ iRBCs (10² iRBCs/μL) at final step. Use 100 μL (1 × 10⁴ iRBC) of this dilution to inject each mouse.

            Notes:

         1. Calculate the dose for N+2 mice to account for any volume loss. Infection from mouse to mouse is established faster than from in vitro culture to mouse (see Pal et al., 2022).

         2. A dose response infection (ranging from 10²–10⁷ iRBCs) was tested in mouse-to-mouse transmission of B. duncani and successful infection was achieved at all doses (Pal et al., 2022). Compared to higher doses, subsequent lower doses led to delays in the establishment of infection. However, results may vary depending on the handling.

      6. Any animal showing signs of distress should be humanely euthanized per approved IACUC protocol by CO₂ asphyxiation followed by cervical dislocation.

Data analysis

For in vitro studies (Figure 9), percent P on the indicated days (days 0, 3, 6, 9, 12, and 15) is determined by counting the number of iRBCs out of approximately 3,000 tRBCs per blood smear prepared from cultures grown in different media, as described in step A4a. The values are then plotted using GraphPad prism. Statistical significance (P-value) is calculated using two-way ANOVA.

  For in vivo studies (Figure 10), P is estimated as described in step A4.a.vi from thin blood smears prepared from mouse blood at the indicated time points (DPI 1, 3, 4, 6, 7, 8, 9, 10, and 11). The number of iRBCs in a total of approximately 2,000 tRBCs is determined for each mouse. At least three mice are used per group. Data are then plotted using GraphPad prism 9.4.1 software. Kaplan-Meier survival curves are generated using GraphPad prism 9.4.1.

Figure 9. In vitro propagation of B. duncani WA1 in different growth media. (A) Continuous in vitro growth of B. duncani WA1 in different media for a period of 15 days in hRBCs. The cultures were diluted (D) on days 3, 6, and 9 (indicated by arrows). (B) Representative images of Giemsa-stained smears of in vitro cultured B. duncani WA1 parasites showing different stages, including rings (R), double rings (DR), filamentous forms (FF), and tetrads (T). (C) Graph showing percentage of different developmental stages of B. duncani WA1 in DMEM/F-12 and Claycomb media. Data presented are mean ± SD of two independent experiments performed in biological duplicates. No significant differences (p > 0.99, two-way ANOVA) were found between the different stages in the two media [adapted and modified from Singh et al. (2022)].

Figure 10. Lethal B. duncani infection in immunocompetent mice. (A) Parasitemia profile over time in female C3H/HeJ mice (n = 3/group) following infection with B. duncani parasites from in vitro culture in hRBCs (purple; 8.5 × 10⁵ iRBC/mouse), or B. duncani parasitized mouse RBCs (mRBC) collected from infected mice (green; 1 × 10⁴ iRBC/mouse). Uninfected mice profile depicted in red. E: euthanized. (B) Kaplan-Meier plot of percent survival of uninfected mice (red), mice infected with in vitro cultured B. duncani parasites (purple), or mice infected with B. duncani parasitized mouse blood (green) at indicated doses [adapted and modified from Pal et al. (2022)].

Recipes

1. DMEM/F-12 complete media (250 mL)

   DMEM/F-12 188.5 mL

   FBS 50 mL

   HT media supplement (50×) hybrid max 5 mL

   L-Glutamine 2.5 mL

   Antimycotic 2.5 mL

   Gentamycin 2.5 mL

2. 30% isoflurane (50 mL)

   Isoflurane 15 mL

   PEG 400 35 mL

Acknowledgments

Funding: The published protocols by Pal and colleagues (Pal et al., 2022) and Singh and colleagues (Singh et al., 2022) were supported by the National Institutes of Health grants (AI123321, AI138139, AI152220, and AI136118), the Steven and Alexandra Cohen Foundation (Lyme 62 2020), and the Global Lyme Alliance to CBM.

Competing interests

None of the named authors have any conflict of interest, financial or otherwise.

Ethics

All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) at Yale University (Protocol #2020-07689). Animals were acclimatized for one week after arrival before the start of an experiment. Animals that showed signs of distress or appeared moribund were humanly euthanized using approved protocols.

  Institutional Review Board Statement: All animal studies conducted were approved by the Institutional Animal Care and Use Committees (IACUC) at Yale University (Protocol #2020-07689).

  Institutional Biosafety Statement: All studies involving the use of human blood and Babesia parasites in culture were approved by the Institutional BioSafety Committee at Yale University.

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