Establishment of Continuous In Vitro Culture of Babesia gibsoni by Using VP-SFM Medium with Low-Concentration Serum

ABSTRACT The establishment of in vitro culture methods has greatly facilitated the research of Babesia. However, the current Babesia gibsoni in vitro culture medium requires high concentrations of canine serum, which intensively limits the culture and is unable to satisfy the demands of long-term studies. In this study, AlbuMAX I (2 mg/mL) and 2.5% dog serum (vol/vol) were added to VP-SFM medium to develop a low-concentration serum culture medium named VP-SFMAD (2.5%), and the effectiveness of this medium was assessed by the growth of B. gibsoni. The results showed that VP-SFMAD (2.5%) could support the continuous growth of the parasite, and the parasitemia has no difference with the cultivation in RPMI 1640 with 20% dog serum. In contrast, either a low concentration of dog serum or absence of AlbuMAX I will significantly lower the parasite growth or fail to maintain B. gibsoni growth in the long term. The strategy of reducing the hematocrit was also evaluated, and VP-SFMAD (2.5%) improved the parasitemia to over 50% within 5 days. The high parasitemia is helpful for larger numbers of parasite collection, which is valuable for studying the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites. In addition, VP-SFMAD (2.5%) medium was successfully used for monoclonal parasite screening, which obtained monoclonal strains with parasitized erythrocytes about 3%, which is similar to RPMI-1640D (20%) medium that obtains monoclonal strains on the 18th day. Those results showed that VP-SFMAD can be applied to B. gibsoni continuous long-term, expansion culture, and subclone culture. IMPORTANCE The VP-SFM as a base medium supplemented with AlbuMAX I and a low concentration of canine serum (2.5%) allowed the continuous in vitro culture of Babesia gibsoni at both small and large volumes, which was to meet different experimental needs, such as long-term culture and obtaining high parasitemia and subclone culture. The establishment of in vitro culture systems allows researchers to better understand the metabolism and growth patterns of Babesia. Importantly, several technical problems impeding such studies have been overcome.

The manuscript "Establishment of continuous in vitro culture of Babesia gibsoni by using VP-SFM medium with low concentration serum" by Li et al. describes the methodology for a long-term in vitro culture of a dog Babesia species. The article is well written, but some statistical and technical details are missing particularly for the subcloning. Major points: Figure 2B and 2C: The authors must add the statistical analysis. In the result par on the derivation of subclone of Delat Bgef1a-b isolate, the authors must have full genomic data to demonstrate the success of the subclone isolation. Figure 5B: The authors need to add flow cytometry data to show the purity of the fluorescent subclone strains. Materials and Methods: The sections for the generation of Delta Bgef1a-b transgenic parasites by electroporation and the section for the fluorescence microscopy are missing. Minor points: Line 42: Define PPE, it should be for parasitized erythrocytes. Figure 1B: The authors should highlight in the figure the days with 40% and 20% DS and in the legend specify when fresh RBC are added to decrease the parasitemia. Figure 2B: The authors must add the number of biological replicates in the legend. Line 308: Any references to cite showing that penicillin has no effect on Babesia parasite development?
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Reviewer #1 (Comments for the Author):
The study of babesial parasites is greatly enhanced by the ability to cultivate parasite asexual stages in in vitro culture. Prior methodology for the cultivation of B. gibsoni were not adequate to perform some of the manipulations needed to delve into the biology of the parasite, a problem that the authors appear to have solved. The title and abstract reflect accurately the content of the manuscript and the figures are of acceptable quality (with the exceptions noted). Rewriting to improve English usage is required before this manuscript could be published. No new information on the biology of B. gibsoni is provided, but this manuscript could provide useful information on culture methodology for raising B. gibsoni for study. The manuscript would benefit by addressing the issues below. Response: We feel great thanks for your professional review work on our article that we have used to improve the quality of our manuscript. According to your constructive suggestions, we have made extensive corrections to our previous draft, the detailed corrections are listed below.
1. The Abstract should be rewritten to shorten it by about 1/3. Lines 52-56 could be eliminated altogether without loss. Lines 56-58 could be rewitten as, "Importantly, several technical problems impeding such studies have been overcome.". Response: Thank you for the suggestion. We have removed lines 52-56 and re-written this part to shorten it according to the Reviewer' suggestion, and Lines 56-58 has been rewitten as "Importantly, several technical problems impeding such studies have been overcome.".
2. Line 64. "..different numbles.." is meaningless. Please rephrase for meaning. Also, briefly mention what happens once merozoites have invaded the red cell. Response: According to the Reviewer' suggestion We have re-written this part into "The vector tick (Ixodes ricinus) inoculates the infective sporozoites into the blood-stream of the host, where they invade erythrocytes, undergo repeated replication that differentiate into variety of morphology, then escape and invasion new erythrocytes, resulting in destruction of host red blood cells." in lines 68-70.
3. Line 66. What is meant by, "..value-added phase of apical compound parasites.."? It is not clear what the authors were saying. Response: We have corrected the "Excellent in vitro culture systems can be used to study the value-added phase of apical compound parasites," into "Continuous in vitro culture systems can be used to study the proliferation stage of apicomplexan parasites," in lines 70-71.
4. Lines 68-70 could be deleted without loss. Response: Line 68-70 has been deleted. 5. Line 73. Reference (2), referring to the seminal paper by Levy and Ristic, is not even included among the references. Response: We have added this reference by Levy and Ristic in Reference (2).
8. Line 110. Title should read, "..continuous growth of B. gibsoni." Response: Title has been re-written to "Culture initiation and continuous growth of B. gibsoni." In line 120. 9. Why was the experiment shown in Figure 1 not performed as a direct comparison of the two media compositions? It is not possible to directly compare the growth rates of parasites at different times in different media, perhaps with different dog sera. Given how this was done, it could be possible to perform a Z-test analysis on normalized rates of proliferation in the two media, and plotted as cumulative proportions. This would at least provide statistical support for the growth rates being equivalent (or not), although from an experimental point of view it is an inferior approach. Response: Babesia gibsoni WH strain was initially isolated from the blood of infected dogs in Wuhan of China. The initial establishment of culture used different concentrations of serum, and only concentrations up to 40% can maintain the growth of the parasite. After a long period of adaption, the infection rate began to stabilize, then attempts to reduce the serum concentration to 20%, after a period of culture, the parasite began to adapt to this serum concentration. However, attempts to continue to reduce the serum concentration failed, we began to change the medium formula to achieve the purpose of reducing the serum concentration. The Figure 1B was a record of the parasitemia of this parasite in vitro culture for 380 days.
10. Figure 1. Could the authors explain why so many parasites have a highly vacuolated appearance? Response: During in vitro culture of B. gibsoni in our study, the parasite with a highly vacuolated appearance always could be observed. In addition, morphological changes were also observed with a variety of cell morphologies appeared when parasitized erythrocytes were high. This behaviour is probably due to the absence of the normal immune system refer to the paper of Walter et al (2002). B. bovis and B. bigemina with a highly vacuolated appearance were also observed at high density using a perfusion bioreactor (Rojas-Martinez C, 2017,2018Alvarez, 2020). It also could be observed in the continuous in vitro B. bovis culture with a medium free of animal origin components (Álvarez, 2021). And Photographs are often performed from cultures from high parasitemia to obtain sufficient data, which may be the main reason that so many parasites have a highly vacuolated appearance. 11. Lines 131, 207. It would be helpful to state the cell cycling time for this parasite as measured in vitro. Response: Thank you for the suggestion. We tried different ways to measure the cell cycle period in vitro. However, we are not confident about the data. Because we could not synchronize B. gibsoni in vitro as Plasmodium, B. divergens and B. bovis. Using cryo-soft X-ray tomography (cryo-SXT), the visualization of a synchronized B. divergens asexual cycle for the first 24 h showed seven intraerythrocytic (IE) stages, including a single round trophozoite, paired pyriforms (two attached pear-shaped sister cells), double trophozoites (two round unattached cells), double paired pyriforms (two sets of paired sister cells), tetrads or Maltese Crosses (four attached sister cells), quadruple trophozoites (four round unattached cells), and multiple parasites (RBCs containing more than four parasites). However, 24 h later, the life cycle progresses and lose its synchronicity, transforming into asynchronous populations in highly parasitized RBCs (Cursino, 2016). Rossouw et al (Rossouw, 2015) hypothesize B. divergens propagation diagram based on the morphological observations, DNA measurements, temporal distribution and transcriptome expression dynamics. The significant increase in newly infected erythrocytes (ring formations) observed during the first 4-6 hours in culture is markedly quicker than previous reports where the in vitro life cycle of B. divergens parasites was claimed to last around 8 hours under the culture conditions. The average doubling time of B. bovis was roughly 10 h for the purified merozoites that invaded RBCs, while 12.4 h was also observed, which could be due to the difference in culture conditions and the number of parasites that were visualized. Purified B. bovis merozoites started growing upon invasion and lost their synchronicity within one cycle (Hakimi, 2021).  13. Figure 2. Labeling on this figure is much too small to read. Also, it is incorrect to refer to the five subcultures as "..five consecutive generations..". Over 15 days these parasites likely went through 30-50 generations. Response: We have modified figure labeling in Figure 2, and we have changed "five consecutive generations" to "five subcultures". 14. Figure 3B. Figure labeling is much too small to read. Response: We have modified the figure labeling in Figure 3B.
15. Lines 187-190. The statement about octoploids arising from daughter merozoites not leaving the cell may be true, but some form of actual experimental evidence is required to support that statement. The alternative, that multiple merozoites can invade the same erythrocyte, was not ruled out. Response: Thanks for your suggestion. We agree with your idea, and the description of "The statement about octoploids arising from daughter merozoites not leaving the cell" have deleted.
16. Figure 5. What is the source of the mCherry fluorescence? Clearly, these are genetically modified parasites, which is incompatible with the description of parasites in the Methods section. The nature of the modifications and the cultivation history of these parasites must be identified, particularly as these parasites have previously undergone significant in vitro culture adaptation. Also, there is a significant pixel-shift problem with Figure 5B where light and fluorescence images do not align properly. This must be corrected. Response: Considering the Reviewer's suggestion, the sections for the generation of Delta Bgef1a-b transgenic parasites by electroporation and the section have been provided in the "Materials and Methods" (lines 404-420) and "Result" (lines 246-255). During the acquisition of fluorescence images, the parasites are alive and able to move in red blood cells, so there may be a significant pixel-shift problem using living parasites' fluorescence. We have done our best to correct this problem.
Lines 246-255: The plasmid (pBS-ef1a-B) expressing mCherry and PAC was constructed, using 750-bp 5′UTR and 3′UTR of ef-1α B as the promoter and terminator, respectively ( Figure 5A). At 12 days post-selection by 4 nM Puromycin, mCherry-expressing parasites appeared in cultures transfected with plasmids ( Figure  5B). PCR1 (F1R1), PCR2 (F1R2), and PCR3 (F2R1) primer pairs could successfully amplify 3346, 2329, and 2322 bp fragments in the ∆Bgef1a-B strain, respectively, but not in the wild-type (WT) of B. gibsoni ( Figure 5C). Western blot analyses indicated the expression of protein mCherry-PAC in Bgef1a-b knockout strains ( Figure 5D). Lines 404-420: Plasmid constructs and Transfection of parasites. The schematic diagram of the plasmid (pBS-ef1a-B) used in this study is shown in Figure 5B. The reporter gene (mCherry) and drug selection gene (Puromycin) cassettes were driven with Bgef1α 5' UTR (IG-B). Bgef1α 5' UTR (IG-B) and Bgef1α 3' UTR were used as recombination sites cloned into the upstream and downstream of the mCherry-PAC genes. All the PCR primer pairs used for plasmid construction are listed in Table 2. The constructed plasmid was purified using Qiagen® Plasmid Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and was confirmed by sequencing before transfection. Transfection of parasites as previously described (26). In brief, B. gibsoni-infected red blood cells (iRBCs) were centrifuged at low speed and then the culture supernatant is removed. linearized pBS-ef1a-B plasmid (20μg) and iRBCs were mixed and transfected using Lonza buffer SF and program FA113 of Amaxa 4D Nucleofector™ device (Lonza, Cologne, Germany). The transfected mixtures were immediately transferred into a preheated culture containing 10% fresh RBCs. 4 nM Puromycin (4nM) was used to select mCherry-expressing transgenic parasites. After 3 weeks of drug selection, the parasite population was cloned in a 96-well culture plate using limiting dilution as previously described. 18. Line 233. "microaerobic.." should be "microaerophilous..", and the correct reference is again Levy and Ristic (1980). Response: We have changed this word and reference according to the reviewer's suggestion.
19. Line 291. It should be stated when the parasites were isolated in Wuhan, and any prior cultivation history. This sounds as though the authors were working with a primary isolate, but this is clearly not the case. Figure 5 dispels that idea. Response: As suggested by the reviewer, we have added more information about this parasite. "B. gibsoni WH strain was originally isolated from a naturally infected dog in Wuhan of China in 2017. The strain underwent a 280-day domestication process before stable in vitro culture, during which the growth of the parasite was unstable with the highest parasitemia changed greatly from 3-8%, and the subculture cycle was 5-8 days." in lines 361-365. 20. Table 1 could be eliminated, as the information is provided in the text. Alternatively, refer to Table 1 and eliminate most of the associated verbiage from the text. Response: Thanks for your excellent suggestion. Table 1 was retained and most of the associated verbiage was eliminated from the text. 21. Line 334. "emersion" should be "immersion". Response: We have changed this word according to the reviewer's suggestion.
The manuscript "Establishment of continuous in vitro culture of Babesia gibsoni by using VP-SFM medium with low concentration serum" by Li et al. describes the methodology for a long-term in vitro culture of a dog Babesia species. The article is well written, but some statistical and technical details are missing particularly for the subcloning. Response: Thanks very much for reviewing this manuscript. I really appreciate all your comments and suggestions. Please find my itemized responses in below and my revisions in the re-submitted files.
Major points: Figure 2B and 2C: The authors must add the statistical analysis. Response: Figure 2 has been revised to better present the statistical analysis. In the result par on the derivation of subclone of Delat Bgef1a-b isolate, the authors must have full genomic data to demonstrate the success of the subclone isolation. Response: Thanks very much for the reviewer's comments, the double-cope gene ef1a including ef1a-A and ef1a-B, which started by a bidirectional promoter (ef1a-IG). It cannot be verified with genomic data in the way that conventional methods, such as the ORF of ef1a-B were amplified by PCR because the sequences of ef1a-A and ef1a-B were identical and reversed. We have added "The plasmid (pBS-ef1a-B) expressing mCherry and PAC was constructed, using 750-bp 5′UTR and 3′UTR of ef-1α B as the promoter and terminator, respectively ( Figure 5A). At 12 days post-selection by 4 nM Puromycin, mCherry-expressing parasites appeared in cultures transfected with plasmids ( Figure 5B). PCR1 (F1R1), PCR2 (F1R2), and PCR3 (F2R1) primer pairs could successfully amplify 3346, 2329, and 2322 bp fragments in the ∆Bgef1a-B strain, respectively, but not in the wild-type (WT) of B. gibsoni ( Figure   5C). Western blot analyses indicated the expression of protein mCherry-PAC in Bgef1a-b knockout strains ( Figure 5D)." in lines 246-255. Figure 5B: The authors need to add flow cytometry data to show the purity of the fluorescent subclone strains. Response: As suggested by the reviewer, we have added flow cytometry data in line and Figure 6B. Red fluorescence was detected in almost all parasites using flow cytometry, which indicated that parasites obtained using VP-SFM AD (2.5%) medium were monoclonal after 18 days of culture without drug screening ( Figure 6B). As shown in lines 261-264. Lines 261-264: In addition, red fluorescence was detected in almost all parasites using flow cytometry, which indicated that parasites obtained using VP-SFM AD (2.5%) medium were monoclonal after 18 days of culture without drug screening ( Figure 6B). Figure 6. Limiting dilution cloning of parasites using VP-SFMAD (2.5%).
Materials and Methods: The sections for the generation of Delta Bgef1a-b transgenic parasites by electroporation and the section for the fluorescence microscopy are missing. Response: As suggested by the reviewer, we have added the sections for the generation of Delta Bgef1a-b transgenic parasites by electroporation and the section for the fluorescence microscopy in "Materials and Methods". As showed in lines 404-420. Lines 404-420: Plasmid constructs and Transfection of parasites. The schematic diagram of the plasmid (pBS-ef1a-B) used in this study is shown in Figure 5B. The reporter gene (mCherry) and drug selection gene (Puromycin) cassettes were driven with Bgef1α 5' UTR (IG-B). Bgef1α 5' UTR (IG-B) and Bgef1α 3' UTR were used as recombination sites cloned into the upstream and downstream of the mCherry-PAC genes. All the PCR primer pairs used for plasmid construction are listed in Table 2. The constructed plasmid was purified using Qiagen® Plasmid Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and was confirmed by sequencing before transfection. Transfection of parasites as previously described (26). In brief, B. gibsoni-infected red blood cells (iRBCs) were centrifuged at low speed and then the culture supernatant is removed. linearized pBS-ef1a-B plasmid (20μg) and iRBCs were mixed and transfected using Lonza buffer SF and program FA113 of Amaxa 4D Nucleofector™ device (Lonza, Cologne, Germany). The transfected mixtures were immediately transferred into a preheated culture containing 10% fresh RBCs. 4 nM Puromycin (4nM) was used to select mCherry-expressing transgenic parasites. After 3 weeks of drug selection, the parasite population was cloned in a 96-well culture plate using limiting dilution as previously described.
Minor points: Line 42: Define PPE, it should be for parasitized erythrocytes. Response: We have changed "PPE" to "parasitized erythrocytes". Figure 1B: The authors should highlight in the figure the days with 40% and 20% DS and in the legend specify when fresh RBC are added to decrease the parasitemia. Response: As suggested by the reviewer, we have changed Figure 1. They are data from in vitro culture that lasted 380 days, and fresh RBCs are added to decrease the parasitemia when the parasite grows for 3 or 4 days. Figure 2B: The authors must add the number of biological replicates in the legend. Response: We have added "Data represent mean values measured in triplicates." in the legend.
Line 308: Any references to cite showing that penicillin has no effect on Babesia parasite development? Response: We did not know if penicillin has effect on Babesia parasite development. While Penicillin has potent combined antimicrobial effects against gram-positive and gram-negative bacteria and can be used to prevent bacterial contamination of cell cultures. The use of Penicillin (100 U/mL) was a reference to previous culture protocols in Toxoplasma and Babesia. Christiansen, C., Maus, D., Hoppenz, E., Murillo-León, M., Hoffmann, T., Scholz, J., Melerowicz, F., Steinfeldt, T., Seeber, F., & Blume, M. (2022). The revised manuscript has been reviewed and the improvements are noted. However, several of reviewer #1's concerns remain and these include the need for statistical inputs, clarity in statements, more details for plasmid construction and improved image overlays. The authors are requested to further improve their manuscript before a decision on its suitability for publication can be considered.
Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. This revised manuscript is now improved over the original. The authors are thanked for addressing most of the concerns of the reviewers, although some remain. Despite these improvements there remain points in need of improvement before this manuscript could be ready for publication. Most of these points, listed below, could be addressed at the keyboard. A few require more work.
[1] line 63. Unlike mosquitoes and malaria, ticks transmitting Babesia do not "inoculate sporozoites into the bloodstream..". In tick feeding, the mouthparts create a damaged dermal wound into which blood and lymph pool, and into which sporozoites are released. Sporozoites must find their way to the circulation from that pool. Please rewrite this statement for accuracy.
[2] line 120. I initially misunderstood the nature of the results shown in Figure 1. The authors' description is now clearer. Regardless, statistical support should be provided comparing the relative growth rates of the parasites in the two different media, based upon recorded growth records and dilutions involved in subcultures. This would allow a more rigorous statement of comparative growth during establishment and between media.
[3] line 138 (new ms). What is meant by, "..instability in contamination rates.."? This is not clear as written, nor in context. Contamination would refer to the growth of something unintended in the culture, for example bacterial or fungal growth.
[5] lines 205, 358. At least skeletal detail should be given about the construction of plasmid pBS-ef1a-B, and the accession number for EF1a-B from the reference genome.
[6] original comment 11. It would still be helpful to state an approximate cell cycling time for this parasite, even if not completely accurate, to help the reader assess and anticipate growth characteristics.
[7] original comment 13. The labeling on all figures remains too small and needs to be redone.
[8] original comment 16. The authors have not addressed this concern satisfactorily. There remains a significant pixel-shift problem wherein the DIC and various fluorescent images do not align properly. This is not due to parasite movement, which would manifest as blurred images, but rather to non-identical redirection of light by the fluorescence filter cubes relative to the plane of the image.
Reviewer #2 (Comments for the Author): No further comments.
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To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
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