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Mouse Models of Uncomplicated and Fatal Malaria
无并发症和致命疟疾的小鼠模型

Brian W. HuangBrian W. Huang*Emily PearmanEmily Pearman*Charles C. KimCharles C. Kim  (*contributed equally to this work)
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Abstract

Mouse models have demonstrated utility in delineating the mechanisms underlying many aspects of malaria immunology and physiology. The most common mouse models of malaria employ the rodent-specific parasite species Plasmodium berghei, P. yoelii, and P. chabaudi, which elicit distinct pathologies and immune responses and are used to model different manifestations of human disease. In vitro culture methods are not well developed for rodent Plasmodium parasites, which thus require in vivo maintenance. Moreover, physiologically relevant immunological processes are best studied in vivo. Here, we detail the processes of infecting mice with Plasmodium, maintaining the parasite in vivo, and monitoring parasite levels and health parameters throughout infection.

Keywords: Mouse(鼠标), Malaria(疟疾), Plasmodium(疟原虫), Infection(感染), Immunology(免疫学)

Materials and Reagents

  1. Mice. We perform most of our work in C57BL/6 mice due to the wide availability of transgenic and knockout strains on this genetic background. Slight differences in vendor-specific sub-strains exist, so it is preferable to obtain the mice from the same source throughout a given study. The majority of our studies use 9-12 week old females, since males have higher parasite loads and mortality with P. chabaudi infection in many mouse strains (Laroque et al., 2012).
  2. Plasmodium parasites. Plasmodium parasites can be obtained from the Malaria Research and Reference Reagent Resource (MR4). The most commonly used rodent malaria strains include P. chabaudi AS (MRA-429; used to model uncomplicated malaria), P. berghei ANKA (MRA-311; fatal cerebral malaria), P. yoelii 17XNL (MRA-593; uncomplicated malaria; resolves late relative to P. chabaudi), and P. yoelii YM (MRA-755; lethal malaria without cerebral pathology).
  3. Immersion oil Type A (Cargille, catalog number: 16482 )
  4. Methanol (Thermo Fisher Scientific, catalog number: A412P4 )
  5. Giemsa stain (Acros Organics, catalog number: 295591000 )
  6. 1x PBS (Mediatech Inc., catalog number: 21-040 CV )
  7. Alsever’s solution (MP Biomedicals, catalog number: 092801154 ) (see Recipes) (Note 1)

Equipment

  1. 1 ml syringes (BD, catalog number: 309628 )
  2. 25 g ⅝” needles (BD, catalog number: 305122 )
  3. 25 mm x 75 mm x 1 mm unfrosted glass slides (Premiere, catalog number: 9101-E )
  4. 25 mm x 75 mm x 1 mm frosted glass slides (Globe Scientific Inc., catalog number: 1324W )
  5. Slide staining rack and chamber (Electron Microscopy Sciences, catalog number: 62543-06 and 62541-01 )
  6. Glass cutter (optional) (General, catalog number: 06637383 )
  7. 1.5 ml microfuge tubes (Thermo Fisher Scientific, catalog number: 50809238 )
  8. Sharp surgical scissors
  9. Brightfield microscope equipped with 100x objective
  10. Lens paper (Thermo Fisher Scientific, catalog number: 11-995 )
  11. Lamellar flow hood (optional; if working in a pathogen-free animal facility)
  12. Scale with cup for weighing mice (OHAUS, catalog number: SB36853M )

Software

  1. VersaCount (Kim and DeRisi, 2010)

Procedure

  1. Starting an infection from cryopreserved parasites
    1. Order cryopreserved parasite stocks, provided as infected mouse erythrocytes, from the Malaria Research and Reference Reagent Resource (MR4). MR4 is now administered by the NIAID’s BEI Resources program, and you will need to submit paperwork to obtain security clearance before being able to order parasites. Rodent-restricted Plasmodium parasites are classified as BEI level 1; registration instructions are available at http://www.beiresources.org/RegisterLevel1.aspx.
    2. Cryopreserved parasite stocks will arrive on dry ice. They can be stored at -80 °C in the short term, but will quickly lose viability under these conditions. If they are not used within two days of receipt, store in liquid nitrogen to maximize parasite viability. Cryopreserved parasite stocks can be stored in liquid nitrogen for several years with minimal loss of viability.
    3. On infection day, retrieve the cryovial from storage and place it in a small cooler of dry ice until just before injection.
      Important safety note: Use personal protective equipment to prevent injury from frostbite or cryovial explosion.
    4. Identify one or two mice to be infected. Typically, we use wild type 9-12 week old C57BL/6 female mice.
    5. Prepare a 1 ml syringe with a 25 g ⅝” needle.
    6. Thaw the vial containing cryopreserved infected erythrocytes rapidly in your gloved hand. Reposition the vial frequently to minimize the risk of frostbite.
    7. Load the syringe with the thawed parasites. The parasites are sensitive to lysis and should be loaded slowly, at a rate of approximately 25 µl per second, to minimize shear stress. Do not pass the infected erythrocytes through the needle more than necessary.
    8. Inject 200-250 µl of the cryopreserved parasites per mouse intraperitoneally. We recommend measuring the parasitemia every day starting on day 3 post-infection (with the day following injection being day 1). If increased susceptibility to infection is expected as a possible outcome, begin monitoring earlier. Procedures for measuring parasitemia are described below (Note 2).

  2. Preparing thin-film blood smears
    One of the most classic assays of malaria research is the thin-film blood smear. In this assay, parasitized red blood cells can be visibly identified by the presence of stained nucleic acid, which is present at comparatively low levels in the anucleate mouse red blood cells. This allows simple assessment of parasitemia-the proportion of red blood cells that are infected-using basic light microscopy. Although parasitemia reflects the combined influence of both parasite load and red blood cell density, its simplicity, sensitivity, and precision make it the most useful measure of parasite load for routine work. We recommend collecting blood smears before 12:00 PM due to the circadian rhythm of some species of Plasmodium parasites and their tendency to cytoadhere to the vasculature in later life cycle stages (Note 3).
    As per our institutional requirements, we record the weight of the mice throughout the study using a scale with 0.1 g resolution. We also score and document the body condition of the mice, as well as other health parameters (e.g., anemia, activity level, hematuria, neurological complications). These measurements may be useful for assessing the progress of the infection and may be required by your institutional guidelines.
    1. Using a pair of sharp surgical scissors, cut off a 0.5 to 1 mm section from the tip of the mouse’s tail. The snip should only remove enough of the tail to obtain a drop of blood.
    2. In one fluid motion towards the distal end of the tail, gently milk the tail to collect a drop of blood at the tip. In some cases, the drop of liquid may lack obvious red blood cells. This can occur as a result of severe anemia, but can also occur if the tail snip is too small. If other infection parameters (e.g. day post-infection, animal activity) are inconsistent with severe anemia, re-snip the tail.
    3. Transfer a 1 to 2 µl drop of blood, directly from the tip of the tail, close to the edge of a microscope slide (Figure 1A).
    4. As the blood will begin to coagulate within several seconds, quickly touch the edge of a second slide to the blood sample at a 45° angle, allowing the blood to spread across the edge (Figures 1B and 1C). Materials can be saved by scoring and snapping unfrosted slides into quarters using a glass scoring tool, and using these to spread the blood (Figure 1D).
      Important safety note: Use caution when snapping glass, as it can result in sharp edges if performed improperly. Dispose of all glass waste according to your institutional guidelines.
    5. Apply gentle pressure and evenly spread the blood across the width of the sample slide to obtain a thin-film blood smear (Note 4) (Figure 1E). Allow the blood to dry for a few seconds.
    6. Place the slides into a rack and submerge the slides in methanol for approximately 5 seconds to fix the smears.
    7. Remove the slides from methanol and allow them to air dry, preferably in front of a fan. While the slides are drying, pipette 1 ml of Giemsa into 250 ml of 1x PBS in a slide staining chamber (Note 5).


      Figure 1. Preparation of thin-film blood smears. A. Transfer a drop of blood onto a microscope slide. B. Side-view: Place the edge of a second slide at a 45° angle, allowing the blood to spread across the edge of the second slide. C. Top-view: Place the edge of a second slide at a 45° angle, allowing the blood to spread across the edge. D. A glass scoring tool and snapped slide fragments. Properly snapped slides should not have sharp edges. E. Place three smears on a single microscope slide for convenience when staining and counting large numbers of smears.

    8. Incubate the slides in the chamber for 10 min to 1 h, depending on the strength of your staining solution. The slide is sufficiently stained when leukocytes present in the smear have dark blue-purple nuclei.
    9. Thoroughly rinse the slides with water and place them in front of a fan to dry. If counting will be performed immediately, the rinsed slides can also be gently blotted dry and analyzed using the procedures described below. The stained slides are stable; if counting will not be performed immediately, the dried slides can be stored in a slide box at room temperature indefinitely.

  3. Counting thin-film blood smears
    For routine monitoring, parasite burden is most conveniently calculated as parasitemia. In order to obtain this number, infected erythrocytes must be distinguished from uninfected erythrocytes, leukocytes, and platelets, and care must be taken to differentiate between reticulocytes (immature red blood cells that still harbor some nucleic acid) and parasitized normocytes (fully mature erythrocytes). Debris and platelets can also pose a challenge for inexperienced researchers, as these particles, when associated with erythrocytes, can have the appearance of parasites. For these reasons, it is important to become very familiar with parasite morphology.
    Notably, different species of malaria parasites have different predilections for different types of red blood cells. P. chabaudi AS parasites display a preference for normocytes, whereas P. berghei ANKA parasites display a preference towards reticulocytes (Sinden et al., 2002). This is most apparent during early infection when parasite numbers are low. At higher loads, parasites tend to be found in all erythrocyte stages. Parasites also differ in other properties with which the researcher should become familiar. For example, P. chabaudi AS parasites cytoadhere, and as a result, ring-stage trophozoites are the main lifecycle stage observed on smears. P. berghei ANKA parasites do not cytoadhere; therefore, all life cycle stages are observed on smears. Our standard practice is to count all stages toward an aggregate parasitemia value; in some cases, it may be preferable to count lifecycle stages separately. See Figure 2 for images of the primary blood stages of Plasmodium parasites.
    1. Place a small drop of immersion oil directly onto the smear and find the focal plane on a brightfield microscope with a 100x objective. The smear can also be coverslipped in a mounting medium prior to microscopic analysis, if long-term preservation of the smear is desired. For routine smear analysis, a coverslip is not required.
    2. Count approximately 500 red blood cells per smear with a tally counter, separately keeping track of infected and uninfected cells. If the parasite life cycle stage is important for your experiment, count the various stages separately (Note 6).
    3. Calculate parasitemia, the percentage of erythrocytes infected with parasites.
      Note: For parasitemia below 0.1% or analyses where high precision is required, more cells should be counted for more precise results. In such cases, estimate the parasitemia by examining up to 10,000 total cells. To do so efficiently, the total number of cells in a given field can be estimated with experience, and successive fields can be summed to reach approximately 10,000 cells, while simultaneously tallying infected cells. At high parasitemia, adequate precision can be obtained from fewer than 500 cells.


      Figure 2. The major Plasmodium life cycle stages observed in blood. Example images of ring trophozoites (commonly referred to as “rings”), late stage trophozoites, and very late stage schizonts are shown. Note that the schizonts in these images have segmented into individual merozoites, which immediately precedes parasite egress. All images are P. berghei ANKA; P. chabaudi AS rings have a similar appearance to P. berghei ANKA rings.

  4. Passaging and initiating experimental infections
    Because it is not practical to maintain mouse malaria parasites in vitro and cryopreserved parasites require one to two passages to return to optimal growth, maintaining an infected passage mouse at all times provides a convenient source of parasites for initiating experiments. In our experience, infections are the most reproducible when parasites are passaged during the ascending phase of parasitemia. The parasitemia levels should be high enough to count accurately and to provide enough inoculum to initiate experiments, but not so high that the parasites have neared or reached the peak of their growth. For non-lethal parasites such as P. chabaudi AS, a range of 5-20% parasitemia gives satisfactory results. For P. chabaudi infection of C57BL/6 mice, this level of parasitemia is typically reached between days 5 and 7 (Note 7). For P. yoelii 17XNL, this level of parasitemia is reached later, typically around day 10 to day 13. For lethal strains such as P. berghei ANKA, passage should be performed at lower parasitemias to avoid loss of the passage parasites due to death of the passage mouse (see below); we typically passage P. berghei ANKA no later than 5 days post infection, and around 5% parasitemia. See Figure 3 for representative parasitemia courses.


    Figure 3. Parasitemia and survival courses for commonly used Plasmodium strains in mice. A. The course of P. chabaudi AS parasitemia in female C57BL/6 mice. Data were pooled from two independent experiments (n=11). B. The course of P. yoelii 17XNL parasitemia in C57BL/6 mice. Data were from one experiment (n=5). C. The course of P. berghei ANKA parasitemia (n=14; pooled from two independent experiments) and survival (n=28; pooled from three independent experiments) in C57BL/6 mice. Shaded boxes represent our preferred window for passaging the infections. Parasitemias are presented as geometric means with SEM.

    The infection should be passaged as soon after collecting and counting the thin-film blood smear of the passage mouse as possible to minimize the impact of replication and cytoadherence during the interim (Notes 8-9). In addition, an important consideration is that mosquito infectivity can be lost upon serial parasite passage; although we have not tested this ourselves, previous reports have suggested limiting the number of passages to eight (Sinden et al., 2002). Serial passage in mice also results in enhanced virulence compared to mosquito-transmitted parasites (Spence et al., 2013). Thus, new parasite stocks should initially be expanded by infecting several mice, and aliquots of parasites should be cryopreserved. We prefer cryopreservation in Glycerolyte solutions using published protocols (Moll et al., 2013).
    1. Monitor the passage infection daily until the desired parasitemia is reached.
    2. Many parasites are sensitive to thermal fluctuations. Prewarm the sterile Alsever’s solution to 37 °C to minimize shock to the parasites.
    3. Calculate the volume of blood required per 1 ml of Alsever’s solution. Infections are typically initiated by injecting an inoculum of 104 to 106 infected red blood cells in 100 µl of Alsever’s solution per mouse (Sanni et al., 2002). Calculate the volume of blood needed for 107 infected red blood cells per 1 ml of Alsever’s solution using the following equation, where parasitemia is expressed as a fraction:

      Or, more simply, if targeting 106 cells per 100 µl dose:

      For example, a dose of 106 parasites delivered in 100 µl from a passage infection at 10% parasitemia requires 10 µl of whole blood in 1 ml Alsever’s solution.
    4. Euthanize the passage mouse according to your institutional guidelines and harvest blood via cardiac puncture using a 25 g ⅝” needle and a 1 ml syringe (Note 10). If permitted, avoid cervical dislocation before performing cardiac puncture, as this will cause internal bleeding that reduces blood recovery.
      1. Insert the needle approximately 0.5 cm deep into the thoracic cavity (Figure 4A). Pull the plunger to 50 µl to create slight negative pressure.


        Figure 4. Cardiac Puncture. A. Insert the needle approximately 0.5 cm into the thoracic cavity. At this point, pull approximately 50 µl of volume to create a very light vacuum. B. Slowly advance the needle until puncturing the heart. Note the visible flash of blood entering the needle base. C. Slowly pull on the plunger, filling the syringe with blood.

      2. Slowly advance the needle, up to an additional 0.5 to 1 cm, until the needle punctures the heart. This can be observed by a visible flash of blood entering the needle (Figure 4B).
      3. Once the heart has been punctured, slowly pull on the plunger until blood ceases to fill the syringe barrel (Figure 4C). A good rate of draw is approximately 50 µl per second, which avoids stoppage of the blood flow due to a collapsed heart. Once no more blood is readily entering the syringe, more blood can often be obtained by gently twisting and/or moving the needle while pulling gentle vacuum. This should be done quickly, as the blood clots quickly – typically within a minute or two. An effective cardiac puncture on a 9-12 week old female mouse typically yields 300 to 700 µl of whole blood.
      4. Expel the blood from the syringe into a clean 1.5 ml microfuge tube. We typically perform this quickly in the absence of anticoagulant, but tubes containing anticoagulant can be used to reduce the urgency of preparing the inoculum.
      5. Quickly dilute the calculated amount of blood into pre-aliquotted and pre-warmed sterile Alsever’s solution using a pipetteman.
    5. Administer 100 µl of the inoculum to each mouse by intraperitoneal injection using a 25 g ⅝” needle and 1 ml syringe (Note 11). Perform this step rapidly, as parasite viability decreases quickly. We inoculate all mice within 15 min of the original blood draw to maximize reproducibility. Large experiments require multiple experienced handlers performing inoculations. Gently agitate the inoculum regularly, as the erythrocytes settle rapidly.
    6. Monitor the parasitemia as described above.

  5. P. berghei ANKA experimental cerebral malaria
    Infection of C57BL/6 mice with P. berghei ANKA is the most widely used mouse model of cerebral malaria, one of the most severe complications of Plasmodium infection in humans. In this cerebral malaria model, infected mice exhibit neurological symptoms such as ataxia, convulsions, and/or paralysis, and typically die between days 6 to 8 post-infection (Baccarella et al., 2014; Coban et al., 2007). P. berghei has also been used as a model for malaria-associated organ pathology in the liver, lung, and spleen, in addition to the extensively studied cerebral pathology (reviewed by Oca et al., 2013).
    We also note that lethal P. yoelii YM infection follows a similar course as that described here and can largely be performed using the same procedures, although the pathology of this infection is different from that observed in P. berghei ANKA infection of C57BL/6 mice.
    1. P. berghei ANKA replicates quickly, and must be passaged earlier than P. chabaudi AS due to the risk of passage mouse mortality. Begin monitoring the parasitemia of the passage mouse on day 3.
    2. Perform passage infections and/or initiate new experiments during ascending parasitemia levels of approximately 5%, which are typically reached between days 4 and 6. Follow the protocol under section D.
    3. Monitor and record the number of surviving mice daily. Record health parameters as outlined by your institutional guidelines, and record the identifier number of dead mice if needed for association studies (Note 12).
    4. Take thin-film blood smears daily to track parasitemia, if desired. It is recommended to begin monitoring parasitemia no later than day 3.

Notes

  1. KSGH is another anticoagulant buffer that can be used for dilution of parasites (Spence et al., 2011). We have not ascertained any significant impact on our infections between using Alsever’s solution and KSGH, but we prefer Alsever’s solution as it is simpler to prepare and is readily commercially available.
  2. Cryopreserved parasites have poor viability, and even injection of a seemingly large cryopreserved inoculum can exhibit slow growth. It is not unusual for infections started from cryopreserved stocks to peak as late as day 12, and at relatively low parasitemias; for this reason, if parasites fail to appear in the blood during the expected window, smears should still be monitored for parasites even on days that are typically considered post-acute for P. chabaudi AS or post-lethal for P. berghei ANKA.
  3. Parasitemia will progressively decline as the day progresses due to more parasites in later life cycle stages cytoadhering to the vasculature. For the most accurate results when comparing parasitemia for multiple days, it is best to collect smears at roughly the same time every morning.
  4. Although traditional protocols push the spreader away from the drop of blood, we pull the spreader across the drop of blood to create a thin-film blood smear. We find this approach to be easier for most individuals to master for preparation of uniform smears.
  5. Other buffers, such as Tris-EDTA, can be used for Giemsa staining. However, the pH impacts the quality of the staining. Higher pH, in particular, will prevent the red color from appearing, although this is not vital for parasite identification. Giemsa preparations also vary across vendors, so it is recommended that the Giemsa concentration and duration of staining be titrated upon receipt of a new bottle.
  6. A convenient replacement for manual tally counters is VersaCount, a free customizable program (Kim and DeRisi, 2010).
  7. Because the course of P. chabaudi approaches the peak approximately one day earlier in males as compared with females, it is possible to “shift” the course of passage forward by a day for convenience in scheduling. We have not observed any significant impact of switching the parasites between male and female mice.
  8. Because of cytoadherence of late stage parasites, if a significant amount of time elapses between gathering the thin blood smear and passaging the infection, the parasitemia used to calculate the inoculum concentration will not accurately reflect the parasitemia of the mouse at the time of passage. This will lead to an overestimation of the number of parasites administered to each mouse, which will increase inter-experiment variability.
  9. Passage of parasites during the descending, post-peak phase of acute parasitemia is possible, but many of these parasites are dying due to the activation of anti-malarial mechanisms, making it difficult to know the precise number of live parasites being delivered. For this reason, we recommend initiating experiments only from parasites harvested during the ascending parasitemia.
  10. Although submandibular blood collection can be used to harvest blood from a passage mouse, cardiac puncture is the preferred technique for obtaining a large, sterile blood sample. Cardiac punctures in 8 week old mice typically yield 300-700 µl of blood, whereas our institutional guidelines only permit submandibular collection up to 50 µl of blood. Submandibular blood collection must also be performed under isoflurane anesthesia, which requires specialized equipment for controlled delivery. The disadvantage to cardiac puncture is that it requires more skill; if it is not performed quickly, the blood can coagulate before sufficient quantities are obtained to prepare the inoculum.
  11. The referenced article published in Journal of Visualized Experiments provides an instructive video demonstrating how to perform an intraperitoneal injection (Machholz et al., 2012).
  12. We find that marking mouse tails with colored permanent markers is an effective, convenient, and minimally traumatic method for tracking mouse identities. This approach is well suited for short-term experiments, since tails will require routine re-marking every 4-5 days. For long-term experiments, tattooing or other forms of identification are preferred.

Recipes

  1. Alsever’s solution
    Sodium chloride
    4.2 g/L
    Sodium citrate dihydrate
    8 g/L
    Citric acid monohydrate
    0.55 g/L
    D-glucose
    20.5 g/L

Acknowledgements

This work was supported by NIH K99 AI085035, NIH R00 AI085035, NCATS UCSF-CTSI UL1 TR000004, and the UCSF Sandler Neglected Tropical Diseases Program. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. We thank Alyssa Baccarella, Joshua Craft, Mary Fontana, Aqieda Bayat, Kim D’Costa, and Nicole Lee for technical assistance.

References

  1. Baccarella, A., Huang, B. W., Fontana, M. F. and Kim, C. C. (2014). Loss of Toll-like receptor 7 alters cytokine production and protects against experimental cerebral malaria. Malar J 13: 354.
  2. Coban, C., Ishii, K. J., Uematsu, S., Arisue, N., Sato, S., Yamamoto, M., Kawai, T., Takeuchi, O., Hisaeda, H., Horii, T. and Akira, S. (2007). Pathological role of Toll-like receptor signaling in cerebral malaria. Int Immunol 19(1): 67-79.
  3. Kim, C. C. and Derisi, J. L. (2010). VersaCount: customizable manual tally software for cell counting. Source Code Biol Med 5(1): 1.
  4. Laroque, A., Min-Oo, G., Tam, M., Radovanovic, I., Stevenson, M. M. and Gros, P. (2012). Genetic control of susceptibility to infection with Plasmodium chabaudi chabaudi AS in inbred mouse strains. Genes Immun 13(2): 155-163.
  5. Machholz, E., Mulder, G., Ruiz, C., Corning, B. F. and Pritchett-Corning, K. R. (2012). Manual restraint and common compound administration routes in mice and rats. J Vis Exp(67): e2771.
  6. Moll, K., Kaneko, A., Scherf, A. and Wahlgren, M. (2013). Methods in malaria research (6th edition). In: Glasgow, E. (ed). UK MR4/ATCC. Manassas.
  7. Oca, M. M. de, Engwerda, C. and Haque, A. (2013). Plasmodium berghei ANKA (PbA) infection of C57BL/6J mice: A model of severe malaria. In: Allen, I. C. (ed). Mouse Models of Innate Immunity. Humana Press, pp 203-213.
  8. Sanni, L. A., Fonseca, L. F. and Langhorne, J. (2002). Mouse models for erythrocytic-stage malaria. Methods Mol Med 72: 57-76.
  9. Sinden, R. E., Butcher, G. A. and Beetsma, A. L. (2002). Maintenance of the Plasmodium berghei life cycle. Methods Mol Med 72: 25-40.
  10. Spence, P. J., Cunningham, D., Jarra, W., Lawton, J., Langhorne, J. and Thompson, J. (2011). Transformation of the rodent malaria parasite Plasmodium chabaudi. Nat Protoc 6(4): 553-561.
  11. Spence, P. J., Jarra, W., Levy, P., Reid, A. J., Chappell, L., Brugat, T., Sanders, M., Berriman, M. and Langhorne, J. (2013). Vector transmission regulates immune control of Plasmodium virulence. Nature 498(7453): 228-231.

简介

小鼠模型已经证明了用于描绘疟疾免疫学和生理学的许多方面的机制的效用。 最常见的疟疾小鼠模型使用啮齿动物特异性寄生虫物种伯氏疟原虫 。 yoelii 和 P。 chabaudi ,其引起不同的病理学和免疫应答,并用于模拟人类疾病的不同表现。 体外培养方法对于啮齿动物疟原虫寄生虫不是很好发展,因此需要在体内维持。 此外,生理相关的免疫过程最好在体内研究。 在这里,我们详细的感染小鼠与疟原虫,维持寄生虫体内,并监测寄生虫水平和健康参数整个感染的过程。

关键字:鼠标, 疟疾, 疟原虫, 感染, 免疫学

材料和试剂

  1. 老鼠。我们在C57BL/6小鼠中执行我们的大部分工作,因为在这种遗传背景上转基因和敲除菌株的广泛可用性。存在供应商特异性亚株的轻微差异,因此优选在整个给定研究中从相同来源获得小鼠。我们大多数研究使用9-12周龄的女性,因为男性有更高的寄生虫负荷和死亡率与P。 charoudi 感染(Laroque et al。,2012)。
  2. 寄生虫。疟原虫寄生虫可以从疟疾研究和参考试剂资源(MR4)获得。最常用的啮齿动物疟疾菌株包括P. chabaudi AS(MRA-429;用于模拟单纯性疟疾),伯氏疟原虫(ANG)(MRA-311;致死性脑疟疾) yoelii 17XNL(MRA-593;单纯性疟疾;相对于 p。chabaudi 晚期),和 yoelii YM(MRA-755;致死性疟疾,无脑病变)。
  3. 浸没油A型(Cargille,目录号:16482)
  4. 甲醇(Thermo Fisher Scientific,目录号:A412P4)
  5. 吉姆萨染料(Acros Organics,目录号:295591000)
  6. 1x PBS(Mediatech Inc.,目录号:21-040CV)
  7. Alsever的解决方案(MP Biomedicals,目录号:092801154)(参见配方)(注1)

设备

  1. 1ml注射器(BD,目录号:309628)
  2. 25g⅝"针(BD,目录号:305122)
  3. 25mm×75mm×1mm未霜玻璃载玻片(Premiere,目录号:9101-E)
  4. 25mm×75mm×1mm磨砂玻璃载玻片(Globe Scientific Inc.,目录号:1324W)
  5. 载玻片染色架和室(Electron Microscopy Sciences,目录号:62543-06和62541-01)
  6. 玻璃切割器(可选)(一般,目录号:06637383)
  7. 1.5ml微量离心管(Thermo Fisher Scientific,目录号:50809238)
  8. 锋利的手术剪刀
  9. 明场显微镜配备100倍物镜
  10. 透镜纸(Thermo Fisher Scientific,目录号:11-995)
  11. 薄层流动罩(可选;如果在无病原体的动物设施中工作)
  12. 用于称量小鼠的杯子(OHAUS,目录号:SB36853M)

软件

  1. VersaCount(Kim和DeRisi,2010)

程序

  1. 开始感冒来自低温保存的寄生虫
    1. 命令低温保存的寄生虫库存,作为感染的小鼠提供 红细胞,来自疟疾研究和参考试剂资源 (MR4)。 MR4现在由NIAID的BEI资源计划管理   您将需要提交文书工作以获得安全许可 能够命令寄生虫。 啮齿类动物限制的疟原虫寄生虫 分类为BEI 1级; 注册说明 在 http://www.beiresources.org/RegisterLevel1.aspx
    2. 冷冻保存   寄生虫库存将在干冰上到达。 它们可以储存在-80℃下   短期内,但将很快失去在这些条件下的生存能力。   如果它们在收到后两天内没有使用,存放在液体中 氮以最大化寄生虫活力。 冷冻保存寄生虫 可以储存在液氮中几年,损失最小 可行性。
    3. 在感染日,从存储中取出冷冻小瓶,并将其放置在干冰的小冷却器中,直到即将注射。
      重要安全提示:使用个人防护装备,防止冻伤或冻存爆炸造成的伤害。
    4. 确定一个或两个小鼠被感染。 通常,我们使用野生型9-12周龄的C57BL/6雌性小鼠
    5. 准备一个1毫升注射器用25克⅝"针
    6. 快速解冻含有冷冻保存的感染的红细胞的小瓶   在你戴手套的手。 经常重新放置小瓶以最小化 冻伤风险。
    7. 加载注射器解冻的寄生虫。 寄生虫对裂解敏感,应缓慢加载,在a 速率约25μl/秒,以使剪切应力最小化。 不要   使感染的红细胞通过针头超过所需的量
    8. 每只小鼠注射200-250微升的冷冻寄生虫 腹膜内。 我们建议每天测定寄生虫血症 感染后第3天开始(注射后1天   第1天)。 如果预期对感染的易感性增加 可能的结果,开始更早监测。 测量程序 寄生虫血症如下所述(注2)

  2. 准备薄膜血涂片
    疟疾研究最经典的测定之一是薄膜血涂片。在该测定中,寄生的红细胞可以通过染色核酸的存在可视地鉴定,所述染色核酸在无核小鼠红细胞中以相对低的水平存在。这允许使用基本光学显微镜简单评估寄生虫血症 - 被感染的红细胞的比例。虽然寄生虫血症反映寄生虫负荷和红细胞密度的组合影响,其简单性,灵敏度和精度使其成为日常工作中寄生虫负荷的最有用的措施。我们建议在下午12:00之前收集血液涂片,因为一些种类的疟原虫寄生虫的昼夜节律,以及它们在后续生命周期阶段中细胞外到脉管系统的倾向(注3)。 根据我们的制度要求,我们使用0.1g分辨率的刻度记录整个研究中小鼠的体重。我们还评分和记录小鼠的身体状况,以及其他健康参数(例如,贫血,活动水平,血尿,神经性并发症)。这些测量可能有助于评估感染的进展,并且可能是您的机构指南所要求的
    1. 使用一对锋利的手术剪刀,切断0.5到1毫米的部分 从鼠标的尾部的尖端。剪刀应该只去除足够的 尾巴获得一滴血。
    2. 在一个流体运动中 尾部的尾端,轻轻地挤奶尾巴收集一滴 血液在尖端。在一些情况下,液滴可能缺乏明显的红色  血细胞。这可能是严重贫血的结果,但也可以 如果尾剪切太小则发生。如果其他感染参数(例如感染后的天数,动物活动)与严重的不一致 贫血,重新剪断尾巴。
    3. 转移1到2微升的血液,直接从尾部,靠近显微镜载玻片的边缘(图1A)。
    4. 因为血液会在几秒钟内开始凝结,很快  以45°角接触第二载玻片的边缘到血液样品, 允许血液扩散穿过边缘(图1B和1C)。 材料可以通过刻痕和捕捉未冻结的幻灯片保存 使用玻璃刻痕工具,并使用这些来传播血液  (图1D)。
      重要安全注意事项:抓住时请小心 玻璃,因为如果执行不当可能会导致尖锐的边缘。处置 的所有玻璃废物。
    5. 应用温和的压力,并均匀地扩散血液的宽度 样品载玻片获得薄膜血涂片(注4)(图1E)。   让血液干燥几秒钟。
    6. 将幻灯片放入架子,并淹没幻灯片在甲醇中大约5秒钟以固定涂片。
    7. 从甲醇中取出载玻片,让它们风干, 优选地在风扇的前面。 当载玻片干燥时,移液管1ml 的吉姆萨放入250ml 1×PBS的玻片染色室中(注5)

      图1.薄膜血涂片的制备。 A。 转移一滴 血液到显微镜载玻片上。 B.侧视图:放置一秒的边缘 以45°角滑动,允许血液扩散穿过其边缘 第二张幻灯片。 C.顶视图:将第二张幻灯片的边缘以45°放置 角度,允许血液扩散到边缘。 D.玻璃刻痕   工具和捕捉的幻灯片片段。 正确抓住幻灯片不应该 有锋利的边缘。 E.将三块涂片放在单个显微镜载玻片上   染色和计数大量涂片时方便。

    8. 孵育在室中的幻灯片10分钟到1小时,取决于 您的染色溶液的强度。 幻灯片是充分 当存在于涂片中的白细胞具有深蓝紫色时染色 核。
    9. 用水彻底冲洗载玻片并将其放入 前面的风扇要干燥。 如果计数将立即执行,则 冲洗的载玻片也可以轻轻地吸干并使用分析 程序。 染色的载玻片是稳定的; 如果计数 不会立即执行,干燥的载玻片可以存储在a 滑动盒在室温下无限期。

  3. 计数薄膜血涂片
    对于常规监测,寄生虫负荷最方便地计算为寄生虫血症。为了获得这个数目,必须将感染的红细胞与未感染的红细胞,白细胞和血小板区分开,并且必须注意区分网织红细胞(仍然含有一些核酸的未成熟红细胞)和寄生的正常细胞(完全成熟的红细胞) 。碎片和血小板也可能对缺乏经验的研究人员构成挑战,因为这些颗粒当与红细胞相关时可以具有寄生虫的外观。由于这些原因,重要的是变得非常熟悉寄生虫形态 值得注意的是,不同种类的疟疾寄生虫对不同类型的红细胞具有不同的倾向。 p。 chabaudi AS寄生虫显示对正常细胞的偏爱,而 P。 berghei ANKA寄生虫表现出对网织红细胞的偏爱(Sinden等人,2002)。这在寄生虫数量低的早期感染期间是最明显的。在较高的负荷下,寄生虫倾向于在所有红细胞阶段中发现。寄生虫在研究者应该熟悉的其它性质方面也不同。例如, P。 chabaudi AS寄生虫细胞质,因此,环状期滋养体是在涂片上观察到的主要生命周期阶段。 p。 berghei ANKA寄生虫不是细胞质的;因此,在涂片上观察到所有生命周期阶段。我们的标准做法是统计所有阶段的总体寄生虫血症值;在一些情况下,可以优选单独地对生命周期阶段进行计数。参见图2的疟原虫寄生虫的原始血液阶段的图像。
    1. 将一滴浸油直接放在涂片上,找到  焦平面在具有100倍物镜的明场显微镜上。的 涂片也可以在微观上在封固介质中盖片 分析,如果需要长期保存涂片。对于 常规涂片分析,不需要盖玻片
    2. 计数 使用计数计数器每个涂片约500个红细胞, 分别跟踪感染和未感染的细胞。如果 寄生虫的生命周期阶段对你的实验很重要,算 (注6)。
    3. 计算寄生虫血症,感染寄生虫的红细胞的百分比。
      注意:对于低于0.1%的寄生虫血症或高精度的分析 需要更多的细胞计数以获得更精确的结果。在这样的  病例,通过检查高达10,000个总细胞估计寄生虫血症。 为了有效地这样做,给定字段中的单元格的总数可以是 估计有经验,连续字段可以总计到达 约10,000个细胞,同时计数感染 细胞。在高寄生虫血症时,可以获得足够的精确度 少于500个单元格。


      图2.主要的疟原虫生命周期 在血液中观察到的阶段。环状滋养体的实例图像(通常 称为"环"),晚期滋养体和非常晚期 裂缝。注意这些图像中的裂缝 分割成单独的裂殖子,其紧接在之前 寄生虫出口。所有图像为 P。 berghei ANKA; p。 chabaudi AS环 具有与 P类似的外观。 berghei ANKA环。

  4. 传代和启动实验性感染
    因为在体外维持小鼠疟疾寄生物是不实用的,并且冷冻保存的寄生虫需要一至两次传代才能恢复最佳生长,所以始终保持感染的传代小鼠为启动实验提供了方便的寄生虫来源。在我们的经验中,当寄生虫在寄生虫血症的上升阶段期间传代时,感染是最可重复的。寄生虫血症水平应当足够高以准确计数并提供足够的接种物来启动实验,但不高到使寄生虫接近或达到其生长的峰值。对于非致死寄生虫如P. chabaudi AS,范围为5-20% 寄生虫血症产生令人满意的结果。对于 p。 chabaudi 感染C57BL/6小鼠,这种水平的寄生虫血症通常在第5天和第7天之间达到(注7)。对于 p。约氏疟原虫17XNL后,这种水平的寄生虫血症在晚些时候达到,通常在第10天至第13天。对于致死菌株如P.P。 berghei ANKA,应在较低寄生虫率下进行传代,以避免由于传代小鼠死亡而导致的传代寄生虫损失(见下文);我们通常通过 p。 berghei ANKA不晚于感染后5天,和约5%寄生虫血症。参见图3的代表性寄生虫血症。


    图3.小鼠中常用的疟原虫菌株的寄生虫病和存活期。 A. chabaudi 在雌性C57BL/6小鼠中的寄生虫血症。从两个独立实验汇集数据(n = 11)。 B. P的过程。约氏疟原虫在C57BL/6小鼠中的17XNL寄生虫血症。数据来自一个实验(n = 5)。 C. P的过程。 b小鼠的ANKA寄生虫血症(n = 14;来自两个独立实验)和存活(n = 28;来自三个独立实验)。阴影框代表我们首选的传染感染窗口。 parasitemias用SEM显示为几何平均值。

    在收集和计数传代小鼠的薄膜血涂片后,应尽快传染感染,以尽量减少临床期间复制和细胞粘附的影响(注8-9)。此外,一个重要的考虑是蚊子传染性可以在串行寄生虫传代时丧失;虽然我们没有自己测试,以前的报告已经建议限制通道数为八(Sinden等人,2002年)。小鼠中的连续传代也导致与蚊子传播的寄生虫相比增强的毒力(Spence等人,2013)。因此,新的寄生虫群最初应通过感染几只小鼠而扩增,并且寄生虫的等分试样应该冷冻保存。我们优选使用公开的方案在甘油酯溶液中冷冻保存(Moll等人,2013)。
    1. 每天监测传代感染,直到达到所需的寄生虫血症。
    2. 许多寄生虫对热波动敏感。预热 灭菌的Alsever的溶液,37°C,以尽量减少对寄生虫的冲击
    3. 计算每1ml Alsever's所需的血液体积 解。感染通常通过注射接种物引发  10 4 至10 6 感染的红细胞在100μlAlsever溶液中  小鼠(Sanni等人,2002)。计算所需的血量 10 感染的红细胞每1ml Alsever's溶液使用 以下方程式表示,其中寄生虫血症以分数表示:

      或者,更简单地,如果靶向10 6 细胞/100μl剂量:

      例如,来自传代的100μl的剂量的10 6 6个寄生虫  在10%寄生虫感染时,需要10μl全血1ml Alsever的解决方案。
    4. 安乐死根据你的通道鼠标  机构指南和收获血液通过心脏穿刺使用a 25 g⅝"针和1 ml注射器(注10)。 如果允许,请避免 宫颈脱位,然后进行心脏穿刺,因为这将 引起内出血,减少血液恢复
      1. 插入 针大约0.5厘米深进入胸腔(图4A)。 拉柱塞至50微升以产生轻微的负压。


        图4.心脏穿刺。A.将针头插入约0.5厘米 进入胸腔。 此时,拉约50微升 体积创建非常轻的真空。 B.缓慢推进针直到   穿刺心脏。 注意可见闪光的血液进入 针座。 C.慢慢地拉动柱塞,用注射器填充 血液。

      2. 缓慢推进针,最多再增加0.5到   1厘米,直到针刺入心脏。 这可以通过a 可见闪烁的血液进入针头(图4B)
      3. 一旦 心脏已被刺破,慢慢拉上柱塞直到血液停止 以填充注射器筒(图4C)。 好的抽奖率是 大约每秒50μl,这避免了血流的停止 由于萎缩的心脏。 一旦没有更多的血液容易进入 注射器,通常通过轻轻扭曲和/或可以获得更多的血液 移动针头同时拉动轻柔的真空。 这应该做 迅速,因为血液迅速凝结 - 通常在一两分钟内。 通常在9-12周龄的雌性小鼠上进行有效的心脏穿刺 产生300至700μl的全血
      4. 驱逐血从 注射器置入干净的1.5ml微量离心管中。 我们通常执行此操作 迅速在无抗凝血剂,但管内含有 抗凝剂可用于降低制备的紧迫性 接种
      5. 快速稀释计算量的血液进入 预分装和预热无菌Alsever's溶液,使用a pipetteman。
    5. 每个小鼠管理100微升的接种物 腹腔注射使用25克⅝"针和1毫升注射器(注 11)。 快速执行此步骤,因为寄生虫活力快速下降。   我们在15分钟内接种原始血液抽取的所有小鼠 最大限度地提高重复性。 大型实验需要多个经验   处理程序执行接种。 轻轻搅拌接种物 定期,因为红细胞快速沉淀
    6. 监测寄生虫血症,如上所述。

  5. p。 berghei ANKA实验性脑疟疾
    C57BL/6小鼠用EM的感染。 berghei ANKA是最广泛使用的大脑疟疾的小鼠模型,是人类疟原虫感染的最严重的并发症之一。在这种大脑疟疾模型中,感染的小鼠表现出神经症状,例如共济失调,抽搐和/或麻痹,并且通常在感染后第6天至第8天死亡(Baccarella等人, et al。,,2007)。 p。除了广泛研究的脑病理之外,berghei还用作肝,肺和脾中与疟疾相关的器官病理学的模型(Oca等人综述 2013)。
    我们还注意到致死的。 yel感染遵循与本文所述类似的过程,并且可以大部分使用相同的程序进行,尽管该感染的病理学与在p中观察到的不同。 berghei ANKA感染C57BL/6小鼠
    1. p。 berghei ANKA快速复制,且必须早于 P传递。  chabaudi AS,因为传代小鼠死亡的风险。开始 在第3天监测传代小鼠的寄生虫血症
    2. 在进行期间感染和/或开始新的实验 升高的寄生虫血症水平为约5%,这通常是 达到第4天和第6天之间。按照第D节的协议。
    3. 每天监测和记录存活小鼠的数量。 记录健康 参数由您的机构指导方针概述,并记录 标识符死亡小鼠的数量,如果需要进行关联研究(注 12)。
    4. 每天进行薄膜血涂片跟踪寄生虫血症,如果 需要。 建议开始监测寄生虫血症不晚于   第3天。

笔记

  1. KSGH是另一种可用于稀释寄生虫的抗凝血剂缓冲液(Spence等人,2011)。我们没有确定使用Alsever的解决方案和KSGH之间对我们感染的任何重大影响,但我们更喜欢Alsever的解决方案,因为它更容易准备并且易于商业化。
  2. 冷冻保存的寄生虫具有差的生存力,并且甚至注射看起来大的冷冻保存的接种物可以表现出缓慢的生长。从低温保存的股票开始的感染到第12天晚上达到高峰,并且在相对较低的寄生虫感染的情况下并不罕见;因为这个原因,如果寄生虫在期望的窗口期间未出现在血液中,则甚至在通常被认为是急性后的P的天上仍应监测寄生虫。 chabaudi AS或post-lethal for P。 berghei ANKA。
  3. 由于在后期生命周期阶段细胞粘附到脉管系统中的更多寄生虫,寄生虫血症将逐渐减少。为了在比较多天的寄生虫血症时获得最准确的结果,最好在每天早上大致相同的时间收集涂片。
  4. 虽然传统的协议推动撒布器远离血液滴,我们拉伸撒布器跨越血液滴,以创建薄膜血涂片。我们发现这种方法对于大多数个体来说更容易掌握以准备均匀的涂片
  5. 其它缓冲液,例如Tris-EDTA,可用于Giemsa染色。然而,pH影响染色的质量。更高的pH,特别是将防止出现红色,虽然这对于寄生虫鉴定不是至关重要的。 Giemsa的制剂也因供应商而异,因此建议在接收新瓶时滴定Giemsa浓度和染色持续时间。
  6. 手动计数器的方便替换是VersaCount,一个可自由定制的程序(Kim和DeRisi,2010)。
  7. 因为 p的过程。 chabaudi 在男性中比女性大约提前一天达到峰值,为了方便调度,有可能将通过的过程"移动"一天。我们没有观察到在雄性和雌性小鼠之间切换寄生虫的任何显着影响
  8. 由于晚期寄生虫的细胞粘附,如果在收集细血涂片和传染传染之间经过了大量时间,用于计算接种物浓度的寄生虫血症将不能准确地反映在传代时小鼠的寄生虫血症。这将导致过高估计施用给每只小鼠的寄生虫数量,这将增加实验间变异性。
  9. 在急性寄生虫血症的下降,后期阶段期间寄生虫的传代是可能的,但是由于抗疟疾机制的激活,许多这些寄生虫死亡,使得难以知道传递的活寄生虫的精确数量。因此,我们建议只从在上升性寄生虫血症期间收获的寄生虫开始实验
  10. 虽然颌下血液收集可用于从传代小鼠收获血液,但是心脏穿刺是获得大的无菌血液样品的优选技术。 8周龄小鼠的心脏穿刺通常产生300-700μl的血液,而我们的制度指导仅允许颌下收集高达50μl的血液。颌下血液收集也必须在异氟烷麻醉下进行,这需要用于控制递送的专门设备。心脏穿刺的缺点是它需要更多的技能;如果不能快速进行,血液可以在获得足够的量以制备接种物之前凝结
  11. 在"可视化实验杂志"中发表的参考文献提供了说明性视频,其展示了如何进行腹膜内注射(Machholz等人,2012)。
  12. 我们发现标记具有彩色永久标记的鼠标尾是一种有效,方便,最小创伤的跟踪鼠标身份的方法。这种方法非常适合短期实验,因为尾部需要每4-5天进行常规重新标记。对于长期实验,优选纹身或其他形式的识别

食谱

  1. Alsever的解决方案
    氯化钠
    4.2 g/L
    柠檬酸钠二水合物
    8 g/L
    柠檬酸一水合物
    0.55g/L
    D-葡萄糖
    20.5克/升

致谢

这项工作由NIH K99 AI085035,NIH R00 AI085035,NCATS UCSF-CTSI UL1 TR000004和UCSF Sandler被忽视的热带病项目支持。 其内容完全由作者的责任,并不一定代表NIH的官方意见。 我们感谢Alyssa Baccarella,Joshua Craft,Mary Fontana,Aqieda Bayat,Kim D'Costa和Nicole Lee的技术帮助。

参考文献

  1. Baccarella,A.,Huang,B.W.,Fontana,M.F.and Kim,C.C。(2014)。 Toll样受体7的丧失改变了细胞因子的产生,并防止实验性脑疟疾。 Malar J 13:354.
  2. Coban,C.,Ishii,KJ,Uematsu,S.,Arisue,N.,Sato,S.,Yamamoto,M.,Kawai,T.,Takeuchi,O.,Hisaeda,H.,Horii,T.and Akira ,S。(2007)。 Toll样受体信号在大脑疟疾中的病理作用。Int Immunol 19(1):67-79。
  3. Kim,C.C。和Derisi,J.L。(2010)。 VersaCount:可定制的手动计数软件,用于细胞计数。源代码Biol Med 5(1):1.
  4. Laroque,A.,Min-Oo,G.,Tam,M.,Radovanovic,I.,Stevenson,M.M.and Gros,P.(2012)。 在自交系小鼠中对疟原虫感染易感性的基因控制 chabaudi AS菌株。基因免疫 13(2):155-163
  5. Machholz,E.,Mulder,G.,Ruiz,C.,Corning,B.F.and Pritchett-Corning,K.R。(2012)。 在小鼠和大鼠中手动约束和常见的化合物给药途径。 Exp (67):e2771。
  6. Moll,K.,Kaneko,A.,Scherf,A.and Wahlgren,M。(2013)。 疟疾研究方法(第6版)。 In:Glasgow,E.(ed)。 UK MR4/ATCC 。马纳萨斯。
  7. Oca,M. M. de,Engwerda,C。和Haque,A。(2013)。 伯氏疟原虫 ANKA(PbA)感染C57BL/6J小鼠:严重疟疾的模型。 In:Allen,IC(ed)。 先天免疫的小鼠模型。 Humana Press,pp 203-213。
  8. Sanni,L.A.,Fonseca,L.F.and Langhorne,J。(2002)。 红细胞期疟疾的小鼠模型 方法Mol Med 72:57-76。
  9. Sinden,R.E.,Butcher,G.A。和Beetsma,A.L。(2002)。 维护疟原虫生活史 > Methods Mol Med 72:25-40。
  10. Spence,P.J.,Cunningham,D.,Jarra,W.,Lawton,J.,Langhorne,J.and Thompson,J.(2011)。 啮齿动物疟疾寄生虫的转化 。疟原虫 em> Nat Protoc 6(4):553-561。
  11. Spence,P.J.,Jarra,W.,Levy,P.,Reid,A.J.,Chappell,L.,Brugat,T.,Sanders,M.,Berriman,M.and Langhorne,J.(2013)。 载体传导调节疟原虫毒力的免疫控制 < em> Nature 498(7453):228-231。
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引用:Huang, B. W., Pearman, E. and Kim, C. C. (2015). Mouse Models of Uncomplicated and Fatal Malaria. Bio-protocol 5(13): e1514. DOI: 10.21769/BioProtoc.1514.
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