参见作者原研究论文

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Jun 2018
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Eimeria vermiformis Infection Model of Murine Small Intestine
蠕形艾美耳球虫对小鼠小肠的感染模型   

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Abstract

Eimeria vermiformis is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an E. vermiformis stock solution, preparation of sporulated E. vermiformis to infect mice and determination of oocysts burden. This protocol should help to establish a highly reproducible natural infection challenge model to study immunity in the small intestine. The information obtained from using this mouse model can reveal fundamental mechanisms of interaction between the pathogen and the immune response, e.g., provided by intraepithelial lymphocytes (IEL) at the basolateral site of epithelial cells but also a variety of other immune cell populations present in the gut.

Keywords: Eimeria vermiformis (蠕形艾美耳球虫), Small intestine (小肠), Infection model (感染模型), Coccidiosis (球虫病), Apicomplexan (顶复虫类), Mucosal immunology (黏膜免疫)

Background

Infections of the small intestine are widespread and can cause significant morbidity and mortality worldwide (Munot and Kotler, 2016). Children and the elderly, due to their weaker immune system, suffer the worst consequences. Gaining a better understanding of how the small intestinal immune system functions is therefore of prime importance. However, many of the pathogens used in mouse models are not natural to the host, can cause systemic inflammation or can persist in the tissues for long periods. This can result in uncharacteristic induction of the immune response and inadequate development of protective immunity.

Eimeria vermiformis is a tissue specific, intracellular single cell protozoan, which infects the small intestinal epithelia. Unlike the related pathogen used in infection models, Toxoplasma gondii, this is a natural, self-limiting, murine parasite, which induces a strong immune response and establishes long-lasting protection against subsequent infections. Furthermore, E. vermiformis is monoxenous, its life cycle is completed without the need of a second host species (see Figure 1). It is therefore an ideal pathogen to study the mechanisms of host protection against primary and secondary infection at the small intestinal barrier.

Mice infected with E. vermiformis excrete a non-infectious, unsporulated form of oocysts into the environment. Once in contact with oxygen and moisture, in a process which takes between 2 to 7 days, these oocysts mature into an infectious, sporulated form, which, upon ingestion, disseminates the infection to other animals (Fayer, 1980).

The E. vermiformis infection model has been used in the past and has yielded valuable information in areas such as the role of αβ and γδ T cells that line the small intestinal epithelia (Roberts et al., 1996) and the influence of age of the host in the development of protective immune responses in the intestine (Ramsburg et al., 2003). More recently we have used the E. vermiformis model to highlight the role of mitochondria in controlling the activation state of epithelial-resident T lymphocytes (Konjar et al., 2018).

Here, we present an adapted version of the original protocol used by Long et al. (1976) to purify and sporulate E. vermiformis. We describe in detail the method used to propagate the parasite, infect mice and accurately assess parasite load.


Figure 1. E. vermiformis life cycle. A single sporulated oocyst can initiate an infection when swallowed by its respective host, the mouse. Each mature oocyst holds four sporocysts, each sporocyst gives rise to two sporozoites, which can infect the enterocytes. Inside the host cell, the parasite develops via trophozoite and schizont stages into merozoites. Merozoites can re-infect the epithelial cells and start a new cycle. Eimeria typically undergoes three rounds of asexual multiplication whereupon merozoites give rise to gametes. Male and female gametes can fuse to form an oocyst, protected by a multi-layered cell wall making them highly resistant to environmental pressures. These oocysts shed with the feces. Unsporulated oocysts undergo sporulation upon contact with oxygen and moisture, undergoing meiosis, which takes between 2 to 7 days.

Materials and Reagents

  1. Millex®-GP Filter Unit: Millipore Express® PES membrane 0.22 μm (Merk Millipore Ltd., catalog number: SLGP033RS)
  2. Fine mesh metal sieve (no specific brand) (2x)
  3. Aluminum foil (no specific brand)
  4. 50 ml centrifuge tube, CentriStarTM cap, polypropylene, RNase-/DNase-free, Nonpyrogenic (Corning Science Mexico, catalog number: 430921) 
  5. 15 ml centrifuge tube, CentriStarTM cap, polypropylene, RNase-/DNase-free, Nonpyrogenic (Corning Science Mexico, catalog number: 430791)
  6. 2 ml microcentrifuge tube graduated natural RNase-/DNase-free (Fisher Scientific, catalog number: 05-408-138)
  7. 1,000 μl Pipette tips (no specific brand)
  8. 200 μl Pipette tips (no specific brand)
  9. Fine sand, e.g., for playground and sand pits (Sand can be autoclaved for use in biological containment facilities)
  10. C57BL6/J mouse strain and/or strain and lines of interest 
  11. Eimeria vermiformis (not commercially available)
  12. Sterile tap water
  13. Sodium hypochlorite 14% C12 in aqueous solution (VWR CHEMICALS, catalog number: 27900.365) (Room temperature)
  14. Sodium chloride (NaCl) (Merck KGaA, catalog number: 1. 06404.1000) (Room temperature)
  15. Potassium dichromate (Cr2K2O2) (VWR, catalog number: 26784.297)
  16. Fridge (no specific brand)

Equipment

  1. Fuchs-Rosenthal Counting Chamber (MARIENFELD, catalog number: 0640410)
  2. Two-chamber McMaster Counting Chamber (CHALEX, LLC: www.vetslides.com)
  3. Funnel (no specific brand)
  4. 1 L Beaker (no specific brand)
  5. P1000 Pipette (no specific brand)
  6. P200 Pipette (no specific brand)
  7. Magnetic stirrer (VELP Scientifica, model: F20500162)
  8. Fume hood (Burdinola, model: OR-ST-1500)
  9. Aquarium pump (no specific brand, e.g., 1 W silent pump)
  10. Centrifuge (Eppendorf AG, 22331 Hamburg, model: 5810 R)
  11. Grant-Bio Vortex, PV-1 (Grant Instruments Ltd)
  12. Microscope (Nikon, Japan, model: SE)
  13. Inverted microscope (EU importer-Carl Zeiss Microscopy GmbH, model: Primovert, catalog number: 415519-1100-000)

Procedure

  1. Preparing E. vermiformis stock
    1. Infect mice with E. vermiformis (1,000 oocysts) in 100 μl of tap-filtered water via oral gavage. 
    2. At Day 8 post-infection, place the mice in a cage lined with sand (refer to Procedure B and Figure 3).
      Note: Susceptibility to and patency of infection is mouse strain dependent and should be empirically tested. The protocol described here is typical for the C57BL6/J mouse strain. 
    3. Collect mouse feces from a cage containing five infected animals, on Day 9 post-infection. 
    4. Place feces into a 50 ml Falcon tube, add 40 ml of sterile water and vortex until feces disaggregate. Store in a fridge (2-6 °C).
      Note: Use of harsher methods such as glass beads may damage the parasites.
    5. Repeat procedure, Steps A3-A4, on Days 10 and 11 post-infection.
    6. When ready to prepare the stock, pour the contents of the three Falcon tubes through a fine mesh sieve, into a beaker (to remove larger pieces).
    7. Working in a fume hood, add 180 ml of a 4% potassium dichromate solution (w/v), making up a final concentration of 2.4% (3 x 40 ml + 180 ml = 300 ml end volume).
    8. Add a stir bar to the beaker and place on a magnetic stirrer. Aerate the contents using an aquarium pump on a low setting, enough to create a few air bubbles continuously.
    9. Cover beaker with aluminum foil to reduce evaporation.
    10. Leave the pump on and keep stirring at room temperature in the fume hood for three days. Regularly check stirrer and aeration.
    11. After three days, take a sample and count oocysts in a Fuchs Rosenthal Chamber to check the percentage of sporulation (refer to Procedure C and Figure 2). If sporulation is under 70%, leave the solution for another day and repeat the counting and sporulation assessment the next day or the day after that if required (generally takes 4-5 days).
    12. Once the percentage of sporulation is higher than 70%, place the stock solution in the fridge. Note down the approximate concentration of oocysts for future reference.


      Figure 2. E. vermiformis oocysts. This figure illustrates how “to distinguish sporulated oocyst”. A. Unsporolated or early oocyst. B. Sporulated or late oocyst, which is tetrasporic in which four sporocysts are visible. Recorded at Primovert microscope, taken at 400x magnification (Ocular 10x + objective 40x). Scale bar = 20 μm.

  2. Preparing E. vermiformis to infect mice
    1. Transfer an appropriate volume containing oocysts from the E. vermiformis stock solution (refer to Procedure A) into a 15 ml Falcon tube.
      Notes:
      1. Usually, stock solution contains approximately 1 x 105 oocysts/ml.
      2. Appropriate volume depends on how many mice you wish to infect and the stock solution obtained. 
    2. Fill up to 15 ml with sterile water and centrifuge for 8 min at 1,800 x g at room temperature. 
    3. Discard the supernatant and vortex to detach the pellet from the bottom of the Falcon tube. 
    4. Fill up again to 15 ml with sterile water and repeat the washing step twice more.
      Note: Typically, the supernatant changes from light yellow to clear. 
    5. Discard the supernatant, vortex the pellet and fill up with Sodium Hypochlorite to 15 ml.
      Note: It is important to thoroughly re-suspend the pellet in the Sodium Hypochlorite since the oocysts are at the bottom and need to float up, to the top of the Sodium Hypochlorite. Use a vortex to mix. 
    6. Centrifuge for 10 min at 1,100 x g with low break (setting 1), room temperature. Take the tube very carefully from the centrifuge and cautiously remove 1-2 ml from the surface ring and transfer to a clean 15 ml Falcon tube.
      Note: For the top 1-2 ml, you may spot a slight cloudiness by eye.
    7. Optional: After removing the top 1-2 ml (Step B6), fill up again with Sodium Hypochlorite, vortex to re-suspend the pellet and repeat the centrifugation for 10 min, at 1,100 x g, low break, at room temperature. Add 1 ml from the surface ring to the previously collected 1-2 ml in the 15 ml Falcon tube.
    8. Fill up the tube containing 2-3 ml of oocysts to 15 ml with sterile water and centrifuge for 8 min at 1,800 x g, at room temperature. 
    9. Discard the supernatant and fill up again to 15 ml with sterile water. Repeat the washing step twice more. 
    10. In the last washing step, discard the supernatant and re-suspend the pellet in 1 ml of sterile filtered tap water. 

  3. Counting sporulated oocysts to infect mice
    1. Dilute the previously prepared E. vermiformis to count in a Fuchs Rosenthal Chamber 0.200 mm depth, using the 10x objective (Figure 3).


      Figure 3. Fuchs Rosenthal Counting Chamber. A. Two-chamber counting slides (0.200 mm depth; 0.0625 mm2). B. In total the grid contains 16 large squares of 1 mm side, enclosed by triple lines. All 16 large squares are subdivided into 16 small squares of 0.25 mm side. Due to the large counting grid and a depth of 0.2 mm the total volume amount is 3.2 μl, so the chamber factor is 312.5.

    2. Count sporulated oocysts in the 16 big squares and multiply by the chamber factor 312.5 (0.2 mm height; volume of 3.2 μl) and the dilution to obtain number of sporulated oocysts/ml. e.g., 60 sporulated oocysts x 312.5 x 10 (dilution 1:10) = 187,500 oocysts/ml = 1.875 x 105 oocysts/ml
      Note: See Figure 2 for distinguishing sporulated and non-sporulated oocysts. 
    3. Adjust to the desired number of oocysts/ml. e.g., 1,000 oocysts/mouse in 100 μl. From the previous example, to prepare 1 ml with the concentration of 1 x 104 oocysts/ml:
      1,000 μl/1.875 x 105 oocysts x 10,000 oocysts = 53.3 μl from the prepared E. vermiformis solution + 946.7 μl of sterile filtered tap water
      Note: Prepare extra volume to account for the loss of volume in the gavage needle. The prepared E. vermiformis solution can be kept in the fridge overnight.
    4. Give 100 μl to each mouse via oral gavage (standard mouse size).

  4. Determining oocyst burden in mouse feces
    1. On Day 6 post infection, place each mouse individually in a cage lined with fine sand (e.g., playground sand).
      Note: Use a thin layer of sand, enough to cover the bottom of the cage (Figure 4).


      Figure 4. Mouse housed in Individually Ventilated Cage (IVC) lined with fine sand

    2. On Day 7, collect the sand with feces from each mouse in each cage. Separate the sand from the feces using a sieve and transfer the feces into a 50 ml Falcon tube using a funnel. Line the cage with clean sand and return the mouse to the cage.
    3. Add 30 ml of sterile water to the Falcon tube and vortex for 1 min. Samples can be stored in the fridge for up to 6 months, prior to oocyst counting.
    4. Repeat Steps C2 and C3 daily until no more oocysts are detected (15-16 days in C57BL6/J mice) for two consecutive days to determine patency (days of oocyst shedding) and accumulative number of oocysts. 
    5. Alternatively, if patency periods are similar between mouse lines or conditions tested and since the majority of oocysts are shed between Days 8-10, a shorter defined period may be chosen and reported.

  5. Counting total oocysts in infected mouse feces
    1. To count, vortex the 50 ml Falcon tube containing the feces in 30 ml volume of an individual mouse from one day vigorously to disaggregate the feces.
    2. Prepare 3 independent dilutions for each sample in saturated sodium chloride (final volume 1 ml, e.g., dilution 1:10–100 μl of feces + 900 μl of sodium chloride).
      Note: It is important to cut the tip end of the 200 and 1,000 μl tips (1 cm), in order to take up and transfer the fecal pieces from the sample. 
    3. Adjust the dilution to count up to 100 oocysts in each counting chamber.
      Note: On the peak of infection higher oocysts numbers will be encountered. Prepare serial dilutions, not exceeding more than 3 dilution steps (refer to Table 1).

      Table 1. Guide to dilute E. vermiformis in mouse feces to count in a McMaster Chamber. Dilutions are dependent on the day post-infection (d.p.i.). These dilutions are representative for C57BL6/J mice infected with 1,000 oocysts. 


    4. Fill up the McMaster counting chamber (Figure 5) and wait ~2 min for the oocysts to float up in the saturated sodium chloride solution.


      Figure 5. Two-chamber McMaster counting slide. 0.15 cm depth; 1 cm2.

    5. Count total oocysts inside the grid areas using the 10x objective (include oocysts on the left and top grid lines).
    6. Take the average of the triplicate counts and multiply by the dilution factor (chamber volume of 150 μl and original volume of 30 ml; dilution factor is 200x) as well as any additional dilutions used to obtain the total number of oocysts/mouse/day (Figure 6).
      Formula: Average of oocysts counted from three chambers x 200 x additional dilution = number of oocysts/mouse per day


      Figure 6. Characteristic oocyst burden of C57BL6/J mice. Counts of oocysts in the feces of mice inoculated with 1,000 sporulated oocysts of E. vermiformis. Three animals were analyzed per time point. Error bars show standard error of the mean (SEM).

Acknowledgments

This work was supported by the European Union H2020 ERA project (No. 667824–EXCELLtoINNOV) for work in the Veldhoen laboratory and Fundação para a Ciência e a Tecnologia (FCT) Studentship SFRH/BD/131605/2017 to P.F-C. The authors like to thank Ana Figueiredo Duarte for designing Figure 1.
Application of the method described here can be found in reference Konjar et al. (2018).

Competing interests

The authors declare no competing financial interests.

Ethics

All animal experimentations complied with regulations of the Direção-Geral de Alimentação e Veterinária Portugal and local ethical review committee and guidelines at Instituto de Medicina Molecular | João Lobo Antunes.

References

  1. Fayer, R. (1980). Epidemiology of protozoan infections: The coccidia. Vet Parasitol 6(1): 75-103.
  2. Konjar, Š., Frising, U. C., Ferreira, C., Hinterleitner, R., Mayassi, T., Zhang, Q., Blankenhaus, B., Haberman, N., Loo, Y., Guedes, J., Baptista, M., Innocentin, S., Stange, J., Strathdee, D., Jabri, B. and Veldhoen, M. (2018). Mitochondria maintain controlled activation state of epithelial-resident T lymphocytes. Sci Immunol 3(24): eaan2543.
  3. Long, P. L., Millard, B. J., Joyner, L. P. and Norton, C. C. (1976). A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet Lat 6(3): 201-217. 
  4. Munot, K. and Kotler, D. P. (2016). Small Intestinal Infections. Curr Gastroenterol Rep 18(6): 31.
  5. Ramsburg, E., Tigelaar, R., Craft, J. and Hayday, A. (2003). Age-dependent requirement for gammadelta T cells in the primary but not secondary protective immune response against an intestinal parasite. J Exp Med 198(9): 1403-1414.
  6. Roberts, S. J., Smith, A. L., West, A. B., Wen, L., Findly, R. C., Owen, M. J. and Hayday, A. C. (1996). T-cell alpha beta + and gamma delta + deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium. Proc Natl Acad Sci U S A 93(21): 11774-11779.

简介

Eimeria vermiformis 是一种组织特异性细胞内原生动物,感染小肠小肠上皮,已被广泛用作研究粘膜免疫学的球虫模型。 该小鼠感染模型对于研究宿主保护小肠中原发性和继发性感染的机制是有价值的。 在这里,我们描述了 E的生成。 vermiformis 原液,制备孢子 E。 vermiformis 感染小鼠并测定卵囊负担。 该方案应有助于建立高度可重复的自然感染挑战模型,以研究小肠的免疫力。 使用该小鼠模型获得的信息可揭示病原体与免疫应答之间相互作用的基本机制,例如,由上皮细胞基底外侧部位的上皮内淋巴细胞(IEL)提供,但也有多种 肠道中存在的其他免疫细胞群。

【背景】小肠感染很普遍,可能导致全球范围内的严重发病率和死亡率(Munot和Kotler,2016)。由于免疫系统较弱,儿童和老人遭受的后果最严重。因此,更好地了解小肠免疫系统的功能是至关重要的。然而,小鼠模型中使用的许多病原体对宿主来说不是天然的,可引起全身性炎症或可长期持续存在于组织中。这可能导致免疫反应的不典型诱导和保护性免疫的不充分发展。

Eimeria vermiformis 是一种组织特异性细胞内单细胞原生动物,可感染小肠上皮细胞。与感染模型中使用的相关病原体弓形虫不同,这是一种天然的,自限性的小鼠寄生虫,可诱导强烈的免疫反应,并对随后的感染建立长效保护。此外, E. vermiformis 是单一的,它的生命周期完成而不需要第二种宿主(参见图1)。因此,它是研究宿主保护小肠屏障的原发性和继发性感染机制的理想病原体。

感染 E的小鼠。 vermiformis 将一种非传染性,未形成孢子的卵囊分泌到环境中。一旦与氧气和水分接触,在一个需要2至7天的过程中,这些卵囊成熟为一种传染性的孢子形式,一旦摄入,就会将感染传播给其他动物(Fayer,1980)。

E. vermiformis 感染模型过去曾被用过,并且在小肠上皮细胞中αβ和γδT细胞的作用等方面产生了有价值的信息(Roberts et al。,1996 )以及宿主年龄对肠道保护性免疫反应发展的影响(Ramsburg et al。,2003)。最近我们使用了 E. vermiformis 模型突出线粒体在控制上皮细胞T淋巴细胞活化状态中的作用(Konjar et al。,2018)。

在这里,我们提出了Long et al。(1976)使用的原始协议的改编版本来净化和孢子化 E. vermiformis 。我们详细描述了用于繁殖寄生虫,感染小鼠并准确评估寄生虫负荷的方法。


图1. E. vermiformis 生命周期。单个孢子形成的卵囊可以在被各自宿主小鼠吞食时引发感染。每个成熟的卵囊都有四个孢子囊,每个孢子囊都会产生两个子孢子,它们可以感染肠细胞。在宿主细胞内,寄生虫通过滋养体和裂殖体阶段发育成裂殖子。裂殖子可以重新感染上皮细胞并开始新的循环。艾美球虫通常经历三轮无性繁殖,因此裂殖子产生配子。雄性和雌性配子可融合形成卵囊,由多层细胞壁保护,使其具有高度抗环境压力。这些卵囊与粪便一起流下。未经孢子化的卵囊在接触氧气和水分时经历孢子形成,经历减数分裂,需要2到7天。

关键字:蠕形艾美耳球虫, 小肠, 感染模型, 球虫病, 顶复虫类, 黏膜免疫

材料和试剂

  1. Millex ® -GP过滤单元:Millipore Express ® PES膜0.22μm(Merk Millipore Ltd.,目录号:SLGP033RS)
  2. 细网金属筛(无特定品牌)(2x)
  3. 铝箔(没有特定品牌)
  4. 50 ml离心管,CentriStar TM 帽,聚丙烯,RNase- / DNase-free,Nonpprogenic(Corning Science Mexico,目录号:430921) 
  5. 15 ml离心管,CentriStar TM 帽,聚丙烯,RNase- / DNase-free,Nonpprogenic(Corning Science Mexico,目录号:430791)
  6. 2 ml微量离心管分级天然RNase- / DNase-free(Fisher Scientific,目录号:05-408-138)
  7. 1,000μl移液器吸头(无特定品牌)
  8. 200μl移液器吸头(无特定品牌)
  9. 细砂,例如,用于操场和沙坑(沙子可以高压灭菌用于生物防护设施)
  10. C57BL6 / J小鼠品系和/或菌株和感兴趣的品系 
  11. Eimeria vermiformis (不可商购)
  12. 无菌自来水
  13. 次氯酸钠14%C12水溶液(VWR CHEMICALS,目录号:27900.365)(室温)
  14. 氯化钠(NaCl)(Merck KGaA,目录号:1。06404.1000)(室温)
  15. 重铬酸钾(Cr 2 K 2 O 2 )(VWR,目录号:26784.297)
  16. 冰箱(没有特定品牌)

设备

  1. Fuchs-Rosenthal Counting Chamber(MARIENFELD,目录号:0640410)
  2. 双室McMaster计数室(CHALEX,LLC: www.vetslides.com )
  3. 漏斗(没有特定品牌)
  4. 1 L烧杯(没有特定品牌)
  5. P1000移液器(无特定品牌)
  6. P200移液器(无特定品牌)
  7. 磁力搅拌器(VELP Scientifica,型号:F20500162)
  8. 通风橱(Burdinola,型号:OR-ST-1500)
  9. 水族箱泵(没有特定品牌,例如,1 W静音泵)
  10. 离心机(Eppendorf AG,22331 Hamburg,型号:5810 R)
  11. Grant-Bio Vortex,PV-1(Grant Instruments Ltd)
  12. 显微镜(尼康,日本,型号:SE)
  13. 倒置显微镜(欧盟进口商 - Carl Zeiss Microscopy GmbH,型号:Primovert,目录号:415519-1100-)

程序

  1. 准备 E. vermiformis 股票
    1. 用 E感染小鼠。通过口服强饲法在100μl自来水中加入vermiformis (1,000个卵囊)。 
    2. 在感染后第8天,将小鼠置于衬有沙子的笼子中(参见方法B和图3)。
      注意:感染的易感性和通畅性取决于小鼠的菌株,应根据经验进行测试。此处描述的方案是C57BL6 / J小鼠品系的典型方案。 
    3. 在感染后第9天,从含有5只感染动物的笼子中收集小鼠粪便。 
    4. 将粪便放入50毫升Falcon管中,加入40毫升无菌水并涡旋直至粪便解聚。存放在冰箱中(2-6°C)。
      注意:使用更严酷的方法(如玻璃珠)可能会损坏寄生虫。
    5. 在感染后第10天和第11天重复步骤A3-A4。
    6. 准备好准备原料时,将三个Falcon管的内容物通过细网筛倒入烧杯中(以去除较大的碎片)。
    7. 在通风橱中工作,加入180毫升4%重铬酸钾溶液(w / v),最终浓度为2.4%(3 x 40 ml + 180 ml = 300 ml终体积)。
    8. 向烧杯中加入搅拌棒并置于磁力搅拌器上。使用低水位的水族箱泵对内容物进行充气,足以连续产生一些气泡。
    9. 用铝箔盖上烧杯以减少蒸发。
    10. 将泵打开并在室温下在通风橱中搅拌三天。定期检查搅拌器和通风。
    11. 三天后,取样并在Fuchs Rosenthal室中计数卵囊以检查孢子形成的百分比(参见程序C和图2)。如果孢子形成低于70%,则将溶液再保留一天,并在第二天或之后的第二天(如果需要)重复计数和孢子形成评估(通常需要4-5天)。
    12. 一旦孢子形成的百分比高于70%,将储备溶液放入冰箱。记下卵囊的近似浓度,以备将来参考。


      图2. E. vermiformis 卵囊。 这个图说明了如何“区分孢子形成的卵囊”。 A.未呕吐或早期卵囊。 B.孢子形成或晚期卵囊,其为四孢子,其中可见四个孢子囊。在Primovert显微镜下记录,以400x放大倍数(Ocular 10x +物镜40x)拍摄。比例尺=20μm。

  2. 准备 E. vermiformis 感染老鼠
    1. 从 E转移含有卵囊的适当体积。将vermiformis 原液(参见程序A)加入15 ml Falcon试管中。
      注意:
      1. 通常,储备溶液含有约1 x 10 5 卵囊/ ml。
      2. 适当的体积取决于您希望感染的小鼠数量和获得的储备溶液。 
    2. 用无菌水填充至15毫升,并在室温下以1,800 x g 离心8分钟。 
    3. 弃去上清液并涡旋,将颗粒从Falcon管的底部分离。 
    4. 用无菌水再次填充至15ml并重复洗涤步骤两次。
      注意:通常情况下,上清液会从淡黄色变为清澈。
    5. 弃去上清液,涡旋沉淀并用次氯酸钠填充至15ml。
      注意:重要的是将颗粒彻底重新悬浮在次氯酸钠中,因为卵囊位于底部,需要漂浮到次氯酸钠的顶部。使用涡旋混合。 
    6. 在室温下以1,100 x g 离心10分钟,低断裂(设定1)。从离心机中小心地取出管子,小心地从表面环上取下1-2毫升,然后转移到干净的15毫升Falcon管中。
      注意:对于前1-2毫升,您可能会发现眼睛有轻微的浑浊。
    7. 可选:取出顶部1-2毫升(步骤B6)后,再次用次氯酸钠填充,涡旋重新悬浮沉淀,重复离心10分钟,在1,100 xg ,低断裂, 在室温下。从表面环中加入1ml至15ml Falcon管中先前收集的1-2ml。
    8. 将含有2-3ml卵囊的试管用无菌水填充至15ml,并在室温下以1,800 x g 离心8分钟。 
    9. 弃去上清液,再用无菌水再充满15毫升。再重复两次洗涤步骤。 
    10. 在最后的洗涤步骤中,弃去上清液并将沉淀重新悬浮在1ml无菌过滤的自来水中。 

  3. 计算孢子形成的卵囊感染小鼠
    1. 稀释先前准备的 E. vermiformis 使用10x物镜计算Fuchs Rosenthal Chamber 0.200 mm深度(图3)。


      图3. Fuchs Rosenthal计数室。 :一种。双室计数载玻片(0.200mm深度; 0.0625mm 2 )。 B.总的来说,网格包含16个1毫米边的大正方形,由三条线包围。所有16个大方块被细分为16个0.25mm侧的小方块。由于计数网格较大,深度为0.2 mm,总体积为3.2μl,因此腔室系数为312.5。

    2. 计算16个大方块中的孢子形成的卵囊,并乘以室因子312.5(0.2mm高度;体积3.2μl)并稀释以获得孢子形成的卵囊数/ ml。
      例如,60个孢子形成的卵囊x 312.5 x 10(稀释1:10)= 187,500个卵囊/ ml = 1.875 x 10 5 卵囊/ ml
      注意:参见图2,区分孢子形成和未形成孢子的卵囊。 
    3. 调整到所需的卵囊数/ ml。
      例如,100μl中的1,000个卵囊/小鼠。从前面的例子中,准备1毫升浓度为1×10 4 卵囊/ ml:
      来自制备的 E的1,000μl/ 1.875×10 5个卵囊x 10,000个卵囊=53.3μl。 vermiformis 溶液+946.7μl无菌过滤自来水
      注意:准备额外的体积以解释灌胃针的体积损失。准备好的 E。 vermiformis 溶液可以在冰箱中保存过夜。
    4. 通过口服管饲法(标准小鼠大小)给每只小鼠100μl。

  4. 确定小鼠粪便中的卵囊负担
    1. 在感染后第6天,将每只小鼠单独放入衬有细沙(例如,操场上的沙子)的笼子中。
      注意:使用一层薄薄的沙子,足以覆盖笼子的底部(图4)。


      图4.鼠标放置在内衬细沙的单独通风笼(IVC)中

    2. 在第7天,从每只笼子中的每只小鼠收集带有粪便的沙子。使用筛子将沙子与粪便分开,并使用漏斗将粪便转移到50ml Falcon管中。将笼子放在干净的沙子上并将鼠标放回笼子中。
    3. 向Falcon管中加入30ml无菌水并涡旋1分钟。在卵囊计数之前,样品可以在冰箱中储存长达6个月。
    4. 每天重复步骤C2和C3,直到不再检测到卵囊(在C57BL6 / J小鼠中15-16天)连续两天以确定通畅(卵囊脱落的天数)和卵囊的累积数量。 
    5. 或者,如果小鼠品系或测试条件之间的通畅期相似,并且由于大多数卵囊在第8-10天之间脱落,可以选择并报告较短的确定时期。

  5. 计算受感染小鼠粪便中的总卵囊数量
    1. 为了计数,在一天内剧烈地将含有粪便的50ml Falcon管在30ml体积的单个小鼠中涡旋以解聚粪便。
    2. 在饱和氯化钠中制备3个独立稀释液(最终体积1 ml,例如,稀释1:10-100μl粪便+900μl氯化钠)。
      注意:切割200和1,000μl吸头(1 cm)的尖端非常重要,以便从样品中吸取并转移粪便。
    3. 调整稀释度,计算每个计数室中最多100个卵囊。
      注意:在感染高峰期会遇到更高的卵囊数。准备连续稀释液,不超过3个稀释步骤(参见表1)。

      表1.稀释指南 E.小鼠粪便中的vermiformis 可以计入McMaster Chamber。 稀释取决于感染后的一天(d.p.i.)。这些稀释液代表感染1,000个卵囊的C57BL6 / J小鼠。 


    4. 填满McMaster计数室(图5),等待~2分钟,让卵囊在饱和氯化钠溶液中漂浮。


      图5.双室麦克马斯特计数载玻片。 0.15厘米深度; 1厘米 2 。

    5. 使用10x物镜计算网格区域内的总卵囊(包括左侧和顶部网格线上的卵囊)。
    6. 取三次计数的平均值并乘以稀释因子(室容积150μl,原始体积30ml;稀释倍数为200x)以及用于获得卵囊总数/小鼠/天的任何其他稀释液(图6)。
      公式:从三个室计算的卵囊平均值×200倍额外稀释度=每天卵囊数/小鼠数


      图6.C57BL6 / J小鼠的特征性卵囊负荷。 小鼠粪便中的卵囊数量接种了1000个孢子形成的卵囊 E. vermiformis 。每个时间点分析三只动物。误差棒显示平均值的标准误差(SEM)。

致谢

这项工作得到了欧盟H2020 ERA项目(编号667824-EXCELLtoINNOV)的支持,该项目用于Veldhoen实验室和FundaçãoparaaCiênciaea Tecnologia(FCT)学生SFRH / BD / 131605/2017到P.F-C的工作。作者要感谢Ana Figueiredo Duarte设计图1.
这里描述的方法的应用可以在参考文献Konjar 等人(2018)中找到。

利益争夺

作者声明没有竞争性的经济利益。

伦理

所有动物实验均符合葡萄牙Diretrão-Geral deAlimentaçãoeVeterinária的规定以及Instituto de Medicina Molecular的当地伦理审查委员会和指南。 JoãoLoboAntunes。

参考

  1. Fayer,R。(1980)。 原生动物感染的流行病学:球虫病。 兽医寄生虫 6(1):75-103。
  2. Konjar,Š。,Frising,UC,Ferreira,C.,Hinterleitner,R.,Mayassi,T.,Zhang,Q.,Blankenhaus,B.,Haberman,N.,Loo,Y。,Guedes,J.,Baptista ,M.,Innocentin,S.,Stange,J.,Strathdee,D.,Jabri,B。和Veldhoen,M。(2018)。 线粒体维持上皮细胞驻留T淋巴细胞的受控活化状态。 Sci Immunol 3(24):eaan2543。
  3. Long,P.L.,Millard,B.J.,Joyner,L.P。和Norton,C.C。(1976)。 用于研究和诊断禽球虫病的实验室技术指南。 Folia Vet Lat 6(3):201-217。 
  4. Munot,K。和Kotler,D。P.(2016)。 小肠感染。 Curr Gastroenterol Rep 18(6 ):31。
  5. Ramsburg,E.,Tigelaar,R.,Craft,J。和Hayday,A。(2003)。 针对肠道寄生虫的初级但非次级保护性免疫应答中gammadelta T细胞的年龄依赖性要求。 J Exp Med 198(9):1403-1414。
  6. Roberts,S.J.,Smith,A.L.,West,A.B.,Wen,L.,Findly,R.C.,Owen,M.J。和Hayday,A.C。(1996)。 T细胞alpha beta +和gamma delta +缺陷小鼠显示异常但不同的自然表型,广泛感染肠上皮细胞。 Proc Natl Acad Sci USA 93(21):11774-11779。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Figueiredo-Campos, P., Ferreira, C., Blankenhaus, B. and Veldhoen, M. (2018). Eimeria vermiformis Infection Model of Murine Small Intestine. Bio-protocol 8(24): e3122. DOI: 10.21769/BioProtoc.3122.
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