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Schistosoma haematobium Egg Isolation
埃及血吸虫卵细胞的分离   

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Abstract

Schistosoma haematobiumis the etiologic agent for urogenital schistosomiasis, a major source of morbidity and mortality for more than 112 million people worldwide. Infection with S. haematobium results in a variety of immunopathologic sequelae caused by parasite oviposition within the urinary tract, which drives inflammation, hematuria, fibrosis, bladder dysfunction, and increased susceptibility to urothelial carcinoma. Since most of the pathology in schistosomasis is directly attributable to the host reaction to eggs and egg-associated antigens, their isolation and study are important experimental techniques. S. haematobium eggs can be collected from infected tissues for injection into other animals or preparation of crude egg extracts. This protocol describes a simple way to isolate eggs.
Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal materials or any tissues from infected animals.

Materials and Reagents

  1. Schistosoma haematobium-infected hamster tissues (intestines and livers)
  2. NaCl
  3. 1.2% (w/v) NaCl (autoclaved) (see Recipes)
  4. 0.85% (w/v) NaCl (autoclaved) (see Recipes)

Equipment

  1. Waring blender, with variable speed control (Eberbach, model: 8580 semi-micro container and 2-speed Waring power base unit)
  2. Stainless steel sieves (Newark Wire Cloth sieve No. 40, 80, 140, 325; mesh openings of 420 μm, 180 μm, 105 μm, and 45 μm)
  3. Plastic spray bottle (e.g. Curtin-Matheson)
  4. Glass Petri dishes with flat bottoms (Corning Incorporated, 100 x 20 mm)
  5. Pasteur pipets
  6. Hematocytometer
  7. Microscope

Procedure

  1. Sacrifice hamsters at the point of maximal liver and intestinal Schistosoma egg levels (18 weeks post-infection).
  2. Mince the infected tissues (intestines and livers) with scissors, suspend in 1.2% NaCl, and then place in a Waring blender. Blend tissues for 30 sec at low speed. Sieves are assembled from top to bottom (420 μm, 180 μm, 105 μm, and 45 μm) (see Figure 1) with progressively decreasing pore sizes. Pass the blended material through the crudest sieve (420 μm pores).
  3. Collect the tissue remaining on top of the sieve and blend again for 30 sec, this time at medium speed.
  4. Pass the resulting material through the sieveas before, collect the remaining tissue from the upper surface of the sieve, and blend for 30 sec at the highest speed setting. Pass the tissue through the crude sieve an additional time.
  5. Pass the filtrate through the series of stainless steel sieves with decreasing pore sizes, using a plastic spray bottle filled with 1.2% NaCl to rinse the filtrate through the successive screens.
  6. Following repeated washes through the upper sieves, the eggs will be retained on the final sieve (pore size of 45 μm).
  7. In order to reduce contamination with residual hamster tissues, prepare a second set of sieves and pass the egg-containing solution through the crude sieve (420 μm opening) and repeat steps 4 and 5.
  8. Wash the eggs off the final sieve into a glass Petri dish. With gentle swirling, concentrate the eggs in the middle of the dish (eggs look like grains of brown sand by the naked eye), aspirate with a Pasteur pipet, and place them in a tube on ice. Add enough 1.2% NaCl to the Petri dish each time when eggs are removed to keep the volume constant, and repeat swirling and concentrating the eggs until no more can be concentrated in the middle of the dish.
    Note: For best results eggs should be collected using a Petri dish with a perfectly flat bottom.
  9. When no more eggs can be concentrated in the middle of the Petri dish upon swirling, discard the contents of the dish, place the collected eggs back into the dish with fresh 1.2% NaCl and repeat the swirling technique, and collect the concentrated eggs as before.
  10. Finally, count eggs using a hematocytometer.
    After several series of these purification steps, most of the tissue debris and immature eggs will be eliminated. The final egg population will be enriched for mature eggs. Although itis best to use fresh eggs, eggs can be kept at 4 °C for several days in 0.85% NaCl without appreciable loss of viability.


    Figure 1.

Recipes

  1. 1.2% (w/v) NaCl (autoclaved)
  2. 0.85% (w/v) NaCl (autoclaved)
    Note: For downstreamin in vivo studies, Antibiotic-Antimycotic solution (Life Technologies, Gibco®) can be added to the saline solution used in the final step.

Acknowledgments

We acknowledge the following citations from which this protocol was adapted: Fu et al. (2011); Fu et al. (2012). We also acknowledge NIH DK087895 for their funding support.

References

  1. Fu, C. L., Apelo, C. A., Torres, B., Thai, K. H. and Hsieh, M. H. (2011). Mouse bladder wall injection. J Vis Exp (53): e2523.   
  2. Fu, C. L., Odegaard, J. I., Herbert, D. R. and Hsieh, M. H. (2012). A novel mouse model of Schistosoma haematobium egg-induced immunopathology. PLoS Pathog 8(3): e1002605.
  3. Lewis, F., Schistosomiasis, in Current Protocols in Immunology. 2001, John Wiley & Sons, Inc.

简介

血吸虫血吸虫病是血管性血吸虫病的病原体,是世界范围内超过1.12亿人的发病和死亡的主要来源。 用 S。 血红蛋白导致由泌尿道内的寄生虫产卵引起的各种免疫病理后遗症,其驱动炎症,血尿,纤维化,膀胱功能障碍和对尿路上皮癌的易感性增加。 由于血吸虫病的大多数病理学直接归因于宿主对卵和卵相关抗原的反应,因此它们的分离和研究是重要的实验技术。 可以从感染的组织收集血细胞的卵以注射到其他动物中或制备粗蛋提取物。 本协议描述了一种简单的分离鸡蛋的方法。
血吸虫是一种生物危害。 在处理血吸虫材料或感染动物的任何组织时,工人应随时戴上乳胶手套。

材料和试剂

  1. 血吸虫感染的仓鼠组织(肠和肝脏)
  2. NaCl
  3. 1.2%(w/v)NaCl(高压灭菌)(见配方)
  4. 0.85%(w/v)NaCl(高压灭菌)(见配方)

设备

  1. Waring搅拌机,可变速度控制(Eberbach,型号:8580半微容器和2速Waring电源基本单元)
  2. 不锈钢筛(Newark Wire Cloth sieve No.40,80,140,325;网孔为420μm,180μm,105μm和45μm)
  3. 塑料喷雾瓶(如 Curtin-Matheson)
  4. 具有平底的玻璃培养皿(Corning Incorporated,100×20mm)
  5. 巴斯德移液器
  6. 血细胞计数器
  7. 显微镜

程序

  1. 在最大肝脏和肠血吸虫卵水平(感染后18周)时杀死仓鼠。
  2. 用剪刀剁碎感染的组织(肠和肝),悬浮在1.2%NaCl中,然后置于Waring混合器中。 低速混合组织30秒。 筛子从顶部到底部(420μm,180μm,105μm和45μm)(参见图1)组装,具有逐渐减小的孔径。 使混合的材料通过粗糙的筛子(420μm孔)
  3. 收集保留在筛子顶部的组织并再次混合30秒,这一次在中等速度。
  4. 使所得材料通过之前的多孔菌,从筛子的上表面收集剩余的组织,并在最高速度设置下混合30秒。让组织通过粗筛子再次。
  5. 将滤液通过一系列孔径不断减小的不锈钢筛,使用装有1.2%NaCl的塑料喷雾瓶通过连续筛选冲洗滤液。
  6. 在通过上筛重复洗涤后,蛋将保留在最终筛(孔径为45μm)上。
  7. 为了减少残留仓鼠组织的污染,准备第二组筛并将含蛋溶液通过粗筛(420μm开口),并重复步骤4和5.
  8. 将蛋从最后的筛子洗涤到玻璃培养皿中。轻轻旋转,将鸡蛋集中在盘子的中间(鸡蛋看起来像棕色沙粒,用肉眼),用巴斯德吸管抽吸,并将它们放在冰管中。在每次取出蛋以保持体积不变时,向培养皿中加入足够的1.2%NaCl,并重复旋转和浓缩蛋,直到不再能够集中在培养皿的中间。
    注意:为了获得最佳效果,应使用底部完全平坦的培养皿收集鸡蛋。
  9. 当在旋转时没有更多的蛋可以集中在培养皿的中间时,丢弃培养皿的内容物,将收集的蛋放回具有新鲜的1.2%NaCl的培养皿中,并重复旋转技术,并如前所述收集浓缩的蛋 。
  10. 最后,使用血细胞计数器计数鸡蛋 在几个系列的这些纯化步骤后,大部分组织碎片和未成熟的卵将被消除。 最终的卵群将富集成熟的卵。 虽然最好使用新鲜的鸡蛋,鸡蛋可以在4℃下保存几天,在0.85%NaCl,没有明显的生存能力的损失。


    图1 。

食谱

  1. 1.2%(w/v)NaCl(高压灭菌)
  2. 0.85%(w/v)NaCl(高压灭菌) 注意:对于体内研究的下游,可以将抗生素 - 抗真菌溶液(Life Technologies,Gibco )加入到最后步骤中使用的盐水溶液中。

致谢

我们承认以下引用本协议的改编:Fu em et al。(2011); Fu等人(2012)。 我们也承认NIH DK087895的资金支持。

参考文献

  1. Fu,C.L.,Apelo,C.A.,Torres,B.,Thai,K.H.and Hsieh,M.H。(2011)。 鼠标膀胱壁注射 J Vis Exp (53 ):e2523。   
  2. Fu,C.L.,Odegaard,J.I.,Herbert,D.R.and Hsieh,M.H。(2012)。 血吸虫血吸虫卵诱导的免疫病理学的一种新型小鼠模型 /em> Pathog 8(3):e1002605。
  3. Lewis,F.,Schistosomiasis,in Current Protocols in Immunology。 2001,John Wiley& Sons,Inc.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Fu, C. and Hsieh, M. H. (2012). Schistosoma haematobium Egg Isolation. Bio-protocol 2(20): e279. DOI: 10.21769/BioProtoc.279.
  2. Fu, C. L., Odegaard, J. I., Herbert, D. R. and Hsieh, M. H. (2012). A novel mouse model of Schistosoma haematobium egg-induced immunopathology. PLoS Pathog 8(3): e1002605.
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ngwewondo adela
university of buea
I WISH TO HAVE THE PROTOCOL FOR THE ISOLATION OF SCHISTOSOMA MANSONI EGGS, MALRIA PARASITE, ONCHOCERCA VOLVULUS
2/27/2014 6:13:06 AM Reply