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Jun 2020

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Parasitemia Evaluation in Mice Infected with Schistosoma mansoni
曼氏血吸虫感染小鼠的寄生虫病评估   

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Abstract

Schistosomiasis is a neglected tropical disease. Its treatment relies on the use of a single drug, praziquantel. Due to treatment limitations, an alternative for schistosomiasis chemotherapy is required; thus, a better understanding of parasite biology and host-parasite interactions is valuable to aid the identification of new anti-Schistosoma drugs. The parasite has a complex life cycle, which results in challenges regarding the evaluation of Schistosoma mansoni development and mammalian infection establishment. Accordingly, this protocol describes methodologies to evaluate: (1) adult worm growth; (2) reproduction; and (3) granuloma formation; and consequently allows more comprehensive knowledge of S. mansoni development in a natural biological system.

Keywords: Schistosoma mansoni (曼氏血吸虫), In vivo (体内), Granuloma (肉芽肿), Confocal (共焦), Parasitemia (寄生虫血症), Oogram (卵形图)

Background

Schistosomiasis is a neglected tropical disease currently endemic in 78 countries (World Health Organization, 2018). Five Schistosoma species can cause parasitosis and are distributed in Asia, Africa, and the Americas. There are two distinct forms of the disease: urogenital, caused by Schistosoma haematobium; and intestinal, caused by Schistosoma intercalatum, Schistosoma mekongi, Schistosoma japonicum, and Schistosoma mansoni (Uberos and Jerez-Calero, 2012; World Health Organization, 2018). Humans are infected following contact with cercariae released from infected aquatic snails.


Here, we focus on the S. mansoni species. After entering a mammalian host, cercariae differentiate into schistosomula (Hitchcock, 1949; de Waard and Vermeulen, 1961; Stirewalt, 1974), which migrate to intrahepatic vessels and develop in adults (Armstrong, 1965; Miller and Wilson, 1980). After mating, the adult worms migrate to the mesenteric veins, where they complete their development and start laying eggs after the 35th day of infection. A female adult worm lays about 300 eggs per day, which reach tissues and the intestinal lumen and are eliminated along with the feces to the external environment (Moore and Sandground, 1956; Gryseels et al., 2006). However, some eggs become trapped in the liver and induce the formation of granulomatous lesions (Espírito Santo et al., 2008; King and Dangerfield-Cha, 2008), which can lead to fibrosis in host tissues and the promotion of injuries and complications that cause anemia, malnutrition, and loss of quality of life, having a high socioeconomic impact (Burke et al., 2009; Olveda et al., 2017).


Schistosomiasis treatment relies on the use of praziquantel, which displays activity against all species; although, immature worms are not affected (Watson, 2009; Thétiot-Laurent et al., 2013). Additionally, there are reports of adult worms with reduced sensitivity to praziquantel, reinforcing the need for alternative schistosomicidal drugs (Fallon and Doenhoff, 1994; Ismail et al., 1994 and 1996, Engels et al., 2002; Caffrey and Secor, 2011). Therefore, parasite biology and host-parasite interaction studies are a feasible strategy to search for new anti-Schistosoma drugs.


As described, S. mansoni presents a complex life cycle that requires two hosts; thus, a better understanding of parasite biology is challenging, demanding, specialized labor, and time consuming (Clegg, 1965; Michaels and Prata, 1968; Basch, 1981; Irie et al., 1987). Inoculation of animal models with parasites is a way to investigate S. mansoni development in the mammalian host, being an approachable procedure and an important system for the evaluation of the complex host-parasite interaction.


Here, we describe a protocol to assess the development of S. mansoni inside a mammalian host. The following procedures detail how to perform a mouse infection with schistosomula and describe methods to evaluate parasite development, such as growth, reproduction, and infection outcome through granuloma analysis in the liver.


Materials and Reagents

  1. 1-ml insulin syringe with a 27 G needle (BD, catalog number:309623)

  2. 21 G needle (BD, catalog number: 305129)

  3. Plastic Pasteur pipettes (Kasvi, catalog number: K30-300S)

  4. Petri dishes (Sarstedt, catalog number: 82.1473.001)

  5. 6-well plates (Sarstedt, catalog number: 83.3920.300)

  6. 50-ml conical centrifuge tubes (Sarstedt, catalog number: 62.547.254)

  7. 15-ml conical centrifuge tubes (Sarstedt, catalog number: 62.554.205)

  8. Scalpel (CP Lab Safety, catalog number: QC-793-140-EA)

  9. 1-200 µl universal tips (Axygen, catalog number: T-200-Y)

  10. 100-1,000 µl universal tips (Axygen, catalog number: T-1000-B)

  11. Maxymum Recovery® 0.6-ml microfuge tubes (Axygen, catalog number: MCT-060-L-C)

  12. 0.6-ml microfuge tubes (Axygen, catalog number: MCT-060-C-S)

  13. 1.5-ml microfuge tubes (Axygen, catalog number: MCT-150-C-S)

  14. Microscope slides, 26 × 76 mm (KASVI, catalog number: K5-7105)

  15. Microscope slide coverslips, 24 × 24 mm (KASVI, catalog number: K5-2424)

  16. Microscope slide coverslips, 24 × 50 mm (KASVI, catalog number: K5-2450)

  17. High-profile disposable blades 818 (Leica Biosystems, catalog number: 14035838383)

  18. Biopsy cassette (CRALPLAST, catalog number: 2921)

  19. Female Swiss Webster mice, 28 days old

  20. Glasgow Minimum Essential Medium (GMEM) (Sigma-Aldrich, catalog number: G6148)

  21. Glucose (Sigma-Aldrich, catalog number: G5767)

  22. Lactalbumin (Sigma-Aldrich, catalog number: L7252)

  23. HEPES (Sigma-Aldrich, catalog number: H3375)

  24. Fetal bovine serum (Thermo Fisher Scientific, Gibco, catalog number: 16000044)

  25. MEM vitamin solution (Thermo Fisher Scientific, Gibco, catalog number: 16000044)

  26. Schneider’s Insect Medium (Sigma-Aldrich, catalog number: S0146)

  27. Triiodothyronine (Sigma-Aldrich, catalog number: 709611)

  28. Hypoxanthine (Sigma-Aldrich, catalog number: H9636)

  29. Hydrocortisone (Sigma-Aldrich, catalog number: H0888)

  30. Penicillin/streptomycin (Thermo Fisher Scientific, Gibco, catalog number: 15070063)

  31. Sodium heparin (Fisher Scientific, Thermo Fisher Scientific, catalog number: BP252410)

  32. NaCl (Merck Millipore, catalog number: 1064040500)

  33. Xylazine hydrochloride (Sigma-Aldrich, catalog number: X1251)

  34. Ketamine hydrochloride (Sigma-Aldrich, catalog number: 343099)

  35. Sodium thiopental (Sigma-Aldrich, catalog number: T6023)

  36. KOH (Sigma-Aldrich, catalog number: 221473)

  37. Absolute ethanol (Merck Millipore, catalog number 64175)

  38. 36% formaldehyde (Sigma-Aldrich, catalog number: 47608)

  39. Glacial acetic acid (Merck Millipore, catalog number: 64197)

  40. HCl (Merk Millipore, catalog number: 109057)

  41. Carmine (Sigma-Aldrich, catalog number: C1022)

  42. Methyl salicylate (Sigma-Aldrich, catalog number: M6752)

  43. Canada balsam (Sigma-Aldrich, catalog number: 60610)

  44. N-butyl acetate (Sigma-Aldrich, catalog number: 442666-U)

  45. Histological paraffin (Easypath, catalog number: EP-21-20068A)

  46. Hematoxylin (Sigma-Aldrich, catalog number: H3136)

  47. Eosin (Sigma-Aldrich, catalog number: E4009)

  48. Xylene (Sigma-Aldrich, catalog number: 534056)

  49. Entellan (Merck Millipore, catalog number: 1079610100)

  50. Autoclaved tap water

  51. Autoclaved balanced feed for mice

  52. 0.85% NaCl (see Recipes)

  53. 10% KOH (see Recipes)

  54. 1× PBS (see Recipes)

  55. 4% buffered formaldehyde (see Recipes)

  56. 10% formaldehyde (see Recipes)

  57. Alcohol-formalin-acetic Acid (AFA) (see Recipes)

  58. 70% ethanol (see Recipes)

  59. 80% ethanol (see Recipes)

  60. 90% ethanol (see Recipes)

  61. 95% ethanol (see Recipes)

  62. Hydrochloric carmine (see Recipes)

  63. Hydrochloric alcohol (see Recipes)

  64. 1:2 methyl salicylate-Canada balsam (see Recipes)

  65. Supplemented GMEM (see Recipes)

Equipment

  1. Tweezers (CP Lab Safety, catalog number: QC-788-100-EA)

  2. Dissecting scissors (CP Lab Safety, catalog number: QC-791-107-EA)

  3. Perfusion system (AutoMate Scientific, catalog number: 11-140)

  4. 200-ml glass beaker

  5. 5-mm sieve

  6. Manual 2-20 µl single-channel pipette (Eppendorf, catalog number: 3120000038)

  7. Manual 20-200 µl single-channel pipette (Eppendorf, catalog number: 3120000054)

  8. Manual 100-10,00 µl single-channel pipette (Eppendorf, catalog number: 3120000062)

  9. Eppendorf® Centrifuge 5804R (Sigma-Aldrich, catalog number: EP022628146)

  10. Tissue processing PT05 TS (Lupetec)

  11. Inclusion Center CI 2014 (Lupetec)

  12. Aluminum inclusion molds (Lupetec)

  13. Microtome MRP2015 (Lupetec)

  14. Light microscope Axiostar Plus (Zeiss)

  15. Zeiss microscope Stemi SV 11 (Zeiss)

  16. Inverted microscope Axio Vert (Zeiss)

  17. Inverted microscope Eclipse Ti-E (Nikon) with Confocal C2 plus (Nikon)

Software

  1. ImageJ (Schneider et al., 2012) (https://imagej.net/ImageJ1)

  2. NIS-Elements software (Nikon)

Procedure

Notes:

  1. All mouse procedures must be performed according to the animal facility and national guidelines for animal use.

  2. Prior to and during the infection period, mice were maintained with autoclaved water and autoclaved balanced feed “ad libidum”.


  1. Inoculation of mice with schistosomula

    Note: The following procedure is performed using the schistosomula stage (Figure 1). However, mouse inoculation and further procedures can also be performed using the cercariae stage.

    1. Calculate the volume of medium containing 350 schistosomula. To calculate the number of parasites/volume, multiply 350 by the total volume of the culture medium and divide this result by the total number of parasites cultivated in that culture medium.

      Note: The number of schistosomula to be inoculated per mouse can be up to 450 parasites per animal. Include a control group.

    2. Pipette the previously calculated volume into a Petri dish or onto microscope slides.



      Figure 1. Examples of schistosomula cultivated in culture medium. Representative image of a schistosomula culture obtained at 10× magnification.


    3. Withdraw a sample to check, under the microscope, whether the pipetted volume contains the expected number of schistosomula.

    4. Place the parasites from each group into ten Maxymum Recovery® 0.6-ml microfuge tubes (one tube per mouse). Each microfuge tube should contain 350 schistosomula.

      Note: In the absence of Maxymum Recovery® microfuge tubes, it is possible to use a sterile microfuge tube instead; however, the use of Maxymum Recovery® microfuge tubes ensures that parasite inoculation will be more homogeneous, since it reduces the number of parasites remaining on the walls of the tube.

    5. Make up the volume to 200-300 µl with supplemented GMEM, if necessary.

      Note: The maximum volume to be inoculated into each mouse is 300 µl.

    6. Separate ten female Swiss Webster mice per group.

      Note: The mouse strain can be changed according to the experimental question.

    7. Use a 1-ml insulin syringe with a 21 G needle for each mouse.

    8. Fill the syringe with the schistosomula previously added to the 0.6-ml microfuge tube.

      Note: Use one syringe and needle per animal to avoid inoculation of a disproportionate number of parasites.

    9. Hold the mouse by grasping the skin along its neck.

    10. Inoculate the parasites through the subcutaneous route by injection of the respective syringe contents into the loose skin over the neck.


  2. Euthanasia and perfusion

    Notes:

    1. Since oviposition starts after 35 days of infection, euthanasia should be performed from 40 days after parasite inoculation.

    2. The anesthetics and euthanasia procedures described in this protocol are approved by Brazilian national guidelines following Law 11794/08. The amount and type of sedatives and euthanasia methods should follow the animal use guidelines of each country.

    1. Prepare 1.5 L 0.85% NaCl solution per ten animals.

    2. Add 2,500 U/L sodium heparin to the 0.85% NaCl solution.

    3. Prepare 200 ml 4% buffered formaldehyde solution per ten animals.

    4. Prepare a 2.5% sodium thiopental solution.

    5. Weigh each mouse.

    6. Hold the mouse by grasping the skin along its neck.

    7. Using a 1-ml insulin syringe with a 27 G needle, administer 10 mg/kg xylazine hydrochloride and 150 mg/kg ketamine hydrochloride through the intraperitoneal route by injection into the thigh muscles directed away from the femur.

    8. Wait for the mouse to become anesthetized.

    9. Position the mouse with the abdomen facing upward.

    10. Using a 1-ml insulin syringe with a 27 G needle, administer 150 mg/kg 2.5% sodium thiopental through the intraperitoneal route by injection into the abdomen toward the head.

    11. Press the mouse's feet with tweezers to confirm euthanasia by noting the absence of a movement response.

    12. Clean the abdomen with 70-75% ethanol.

      Note: Perfusion was conducted according to Pellegrino and Siqueira (1956) with some modifications.

    13. Using dissecting scissors make an incision along the midline of the abdomen.

    14. Using dissecting scissors, cut the mouse's diaphragm and rib to expose the heart.

    15. Using dissecting scissors, cut the portal vein.

    16. Place the mouse intestine and portal vein in the 200-ml glass beaker.

    17. Insert the needle of the perfusion equipment into the left ventricle.

    18. Start the perfusion equipment for adult worm recovery by NaCl injection.

      Note: All the liquid that comes out of the portal vein must fall into the beaker since it carries adult worms.

    19. Stop the perfusion equipment when the liquid from the portal vein becomes clear.

    20. Pass the obtained liquid through a 5-mm sieve, which will retain the recovered adult worms.

    21. Transfer the recovered adult worms from the sieve to a 6-well plate. To perform this procedure, turn the sieve upside down and place it on the well. Using a plastic Pasteur pipette and 0.85% NaCl, wash the sieve to release all the worms into the well.

    22. Cover the adult worms with 0.85% NaCl solution.

    23. Remove the mouse intestine and cut it 3-cm downstream from the end of the ileum.

    24. Place the ileum on a microscope slide, cover with a 24 × 24 mm coverslip, and press.

    25. Place the remaining intestine portion in a a 6-well plate.

    26. Remove the mouse liver.

    27. Remove the median lobe and place into a 50-ml conical centrifuge tube previously filled with 200 ml 4% buffered formaldehyde.

    28. Place the remainder of the liver in a 6-well plate.

    29. Repeat this procedure for all animals.

      Note: Label the containers according to the animal: liver, intestine, and worms, and the microscope slide of the ileum.


  3. Assessment of the number of recovered adult worms

    1. Place the recovered adult worms from each mouse in a Petri dish.

    2. Separate the males from the females using a needle or tweezers.

      Note: To differentiate males from females, the size and color of the parasites should be observed. Male adult worms are clear and female adult worms are longer and darker. To recognize the male gynecophoric canal and tubercles, which are characteristics absent in the female, it is necessary to use a magnifier light or light microscope.

    3. Count the males and females recovered from each mouse.

    4. Repeat the procedure for the adult worms recovered from the remaining mice.


  4. Analysis of adult worm length

    1. After counting, transfer the female and male adult worms from each mouse to a 1.5-ml microfuge tube filled with 1 ml AFA solution.

      Note: Adult worms can be maintained in AFA solution for more than a year.

    2. Randomly transfer one female and one male recovered from the same mouse to a microscope slide.

    3. Stretch the adult worms carefully.

    4. Under a Zeiss Microscope Stemi SV 11, take an image of the adult worms.

      Notes:

      1. All images should be taken using the same parameters.

      2. Indicative adult worm images for analysis of length are represented in the original research paper: Tavares, N. C., Gava, S. G., Torres, G. P., de Paiva, C. E. S., Moreira, B. P., Lunkes, F. M. N., Montresor, L. C., Caldeira, R. L. and Mourao, M. M. (2020). Schistosoma mansoni FES Tyrosine Kinase Involvement in the Mammalian Schistosomiasis Outcome and Miracidia Infection Capability in Biomphalaria glabrata.Front Microbiol 11: 963 (37). Link to access:https://www.frontiersin.org/articles/10.3389/fmicb.2020.00963/full.

    5. Transfer the worms back to the 1.5-ml microfuge tube filled with 1 ml AFA solution.

      Note: If the microscope does not have a scale, take an image of a ruler under the microscope using the same parameters as those for the worms.

    6. Repeat the procedure for the adult worms recovered from the remaining mice.

      Note: The number of adult worms to be analyzed will depend on the total number of adult worms recovered. Perform this procedure using the maximum number of adult worms as possible.


  5. Staining adult worms for confocal analysis

    Note: Adult worms are stained and analyzed according to Neves et al. (1998 and 2002).

    1. Use a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette to remove all the AFA solution (Figure 2A).

    2. Fill the 1.5-ml microfuge tube containing the worms with hydrochloric carmine using a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette (Figure 2B).

      Optional: Adult worms can be transferred to a small Petri dish or another container that enables parasite soaking in the hydrochloric carmine.

      Note: The volume of hydrochloric carmine added should completely cover the worms.

    3. Incubate the worms in hydrochloric carmine for 30 min at room temperature (Figure 2B).

    4. Use a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette to remove the excess hydrochloric carmine (Figure 2B).

    5. Add 70% ethanol and quickly remove the ethanol using a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette (Figure 2C).

      Note: This step is important to remove the excess hydrochloric carmine and should be performed quickly to avoid complete removal of hydrochloric carmine.

    6. Repeat Step E5 (Figure 2C).

    7. Add hydrochloric alcohol and, after a few seconds, remove it using a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette (Figure 2C).

      Note: This step provides contrast of different organs and must be performed quickly to avoid complete hydrochloric carmine removal. The removal of hydrochloric alcohol should occur when the worms turn from an intense red to a pale red.

    8. Add 70% ethanol and incubate for 5 min at room temperature (Figure 2C).

    9. Use a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette to remove the 70% ethanol (Figure 2C).

    10. Add 80% ethanol and incubate for 5 min at room temperature (Figure 2C).

    11. Use a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette to remove the 80% ethanol (Figure 2C).

    12. Add 95% ethanol and incubate for 5 min at room temperature (Figure 2C).

    13. Use a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette to remove the 95% ethanol (Figure 2C).

    14. Add absolute ethanol and incubate for 5 min at room temperature (Figure 2C).

    15. Use a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette to remove the absolute ethanol (Figure 2C).

      Notes:

      1. When pipetting, care should be taken to avoid worm disruption.

      2. The added volume of hydrochloric alcohol and the subsequent ethanol washes should always cover the worms.

      3. Steps E5-E14 can be performed using a small Petri dish or another container. The stained worms can be soaked in the solutions using a 5-mm sieve.

    16. Use a 20-200 µl manual single-channel or a plastic Pasteur pipette to add 1:2 methyl salicylate-Canada balsam solution for at least 24 h. Cover all the worms with the solution and incubate for at least 24 h (Figure 2D and Figure 3A).

      Note: Adult worms can be maintained in this solution for years.

    17. Place the male and female worms separately onto microscope slides (Figure 2E).

    18. Cover the adult worms with 1:2 methyl salicylate-Canada balsam solution using a 20 µl-200 µl manual single-channel pipette or a plastic Pasteur pipette (Figure 2E).

    19. Carefully cover the microscope slide with a 24 × 24 mm coverslip (Figure 2E).

    20. Fill the space between the microscope slide and the coverslip with 1:2 methyl salicylate-Canada balsam solution using a 20-200 µl manual single-channel pipette or a plastic Pasteur pipette (Figure 2E).

    21. Place the slides on a flat surface to dry (Figure 3B).

      Note: The microscope slides should rest on a flat surface to dry for 1-2 months. Observe every day whether more 1:2 methyl salicylate-Canada balsam solution should be added to avoid drying out.



      Figure 2. Schema of staining adult worms for confocal analysis. A. Removal of AFA solution using a manual single-channel pipette. B. Hydrochloric carmine addition, incubation, and removal using a manual single-channel pipette. C. Addition, incubation, and removal of hydrochloric alcohol, 70%, 80%, 95%, and absolute ethanol using a manual single-channel pipette. D. Addition of 1:2 methyl salicylate-Canada balsam solution using a manual single-channel pipette. E. Assembly of slides of stained adult worms.



      Figure 3. Example of stained adult worms. A. Male and female adult worms stained with hydrochloric carmine and covered with 1:2 methyl salicylate-Canada balsam solution. B Microscope slides containing male and female adult worms stained with hydrochloric carmine.


  6. Liver and intestine digestion and egg recovery

    1. Place the remaining liver and intestine of one animal into two different Petri dishes and slice the organs using a scalpel (Figure 4A).

    2. Transfer the sliced liver to a 15-ml Falcon tube (Figure 4A).

    3. Perform this procedure for all mice livers and intestines, separately.

    4. Add 10 ml 10% KOH to dissolve the liver and intestinal tissue; this will not damage the eggs (Figure 4B).

    5. Incubate the livers and intestines in 10% KOH at 4 °C overnight (Figure 4B).

    6. Vortex the tubes (Figure 4C).

    7. Incubate the tubes at 37 °C for at least 1 hour (Figure 4C).

      Note: If the livers and intestines are not completely dissolved, vortex again and incubate the tubes until the organs are completely dissolved.

    8. Centrifuge the tubes for 5 min at 380 × g (Figure 4D).

    9. Discard the supernatant (Figure 4D).

    10. Add 10 ml 0.85% NaCl, resuspend the pellet using a plastic Pasteur pipette, and centrifuge the tubes for 5 min at 380 × g (Figure 4D).

    11. Perform Steps F8-F10 three times (Figure 4D).

    12. After the final centrifugation, discard the supernatant and resuspend the pellet in 1 ml 0.85% NaCl (Figure 4E).

    13. Transfer the contents to a clean 1.5-ml microfuge tube (Figure 4E).

    14. For counting, homogenize the tubes and pipette 10 µl contents to a microscope slide (Figure 4F).

    15. Under a light microscope, count the eggs observed at 5× magnification and record the number (Figure 4F).

    16. Perform Steps F14 and F15 three times.



      Figure 4. Schema of liver and intestine digestion for egg recovery. A. Liver and intestine are sliced using a scalpel, and the chopped organs are transferred to a 15-ml Falcon tube (Steps F1-F2). B. 10% KOH is added to the tissue and incubated overnight (Steps F3-F5). C. The tissue is vortexed and incubated at 37 °C (Steps F6-F7). D. The 15-ml Falcon tube is centrifuged, the supernatant is removed, and 1 ml 0.85% NaCl is added (Steps F8-F11). E. The resuspended eggs are transferred to a 1.5-ml microfuge tube (Steps F12-F13). F. The recovered eggs are counted (Steps F14-F16). G. Representative image of recovered eggs obtained at 10× magnification.


  7. Oogram

    1. Place the microscope slides of the ileum on a light microscope.

    2. For the first 100 eggs observed, count the mature, immature, and dead eggs.

    3. Consider as immature, the eggs from the first to the fourth maturation stage as described by Mati and Melo (2013).

      Note: You can also classify the eggs by stage (1-5, with 5 being an egg containing the developed miracidium; known as mature).


  8. Histology for granuloma analysis

    1. Place the median lobe, previously fixed in 4% buffered formaldehyde, in a Petri dish.

    2. Slice the median lobe into six fragments using a scalpel.

    3. Transfer the fragments to a biopsy cassette.

    4. Keep the remaining portion of liver in 4% buffered formaldehyde.

    5. Close the biopsy cassette.

    6. Label the biopsy cassettes according to the respective animals.

    7. Place the biopsy cassettes into the tissue processing PT05 TS (Lupetec).

    8. Fill the tissue processing PT05 TS (Lupetec) buckets with the respective solution/reagents: 10% formaldehyde, 70% ethanol, 95% ethanol, absolute ethanol, N-butyl acetate, xylene, and histological paraffin.

    9. Set the following steps on the tissue processing PT05 TS (Lupetec).

      1. 10% formaldehyde for 1 h.

      2. 10% formaldehyde for 1 h.

      3. 70% ethanol for 1 h.

      4. 95% ethanol for 1 h.

      5. 95% ethanol for 1 h.

      6. Absolute ethanol for 1 h.

      7. Xylene I for 1 h.

      8. Xylene II for 1 h.

      9. Xylene III for 1 h.

      10. Paraffin I for 1 h.

      11. Paraffin II for 1 h.

      Note: Histological processing can be performed manually following the steps described in Step H9.

    10. Dispense a few drops of histological paraffin onto an aluminum inclusion mold.

    11. Transfer the liver slices from the biopsy cassettes to the mold.

    12. Using the Inclusion Center CI 2014 (Lupetec), dispense histological paraffin onto the liver fragment.

    13. Fit the respective biopsy cassette (the bottom part of the cassette, which contains the identification) on the mold.

    14. Transfer to the chilled plate of the Inclusion Center CI 2014 (Lupetec).

    15. Perform Steps H1-H14 for the other liver samples.

    16. Remove the blocks from the aluminum inclusion molds.

    17. Incubate the blocks at room temperature overnight.

      Note: Paraffin blocks containing tissue can be maintained at room temperature until microscope slide assembly.

    18. Incubate the blocks at -20 °C for 1 h.

    19. Place the blocks on ice.

    20. Fit each block on the Microtome MRP2015 (Lupetec)。

    21. Section the blocks to obtain thin slices (approximately 4-6 µM).

    22. Place the slices in a 50 °C water bath.

      Note: This step is important to stretch the slices; the temperature cannot be higher since the paraffin melts.

    23. Capture the slices with a microscope slide.

    24. Label the microscope slides.

    25. Incubate the microscope slides at 65 °C for 4 h.

    26. Fill four containers with xylene and label each container with: xylene I, xylene II, xylene III, and xylene IV.

    27. Fill four containers with absolute ethanol and label each with: absolute ethanol I, absolute ethanol II, absolute ethanol III, and absolute ethanol IV.

    28. Fill a container with hematoxylin.

    29. Fill a container with eosin.

    30. Dip the slides in xylene I for 10 min.

    31. Dip the slides in xylene II for 10 min.

    32. Dip the slides in absolute ethanol I for 10 s.

    33. Dip the slides in absolute ethanol II for 10 s.

    34. Wash the slides with running water for 3 min.

    35. Dip the slides in hematoxylin for 5 min.

    36. Dip the slides in eosin for 30 s.

    37. Wash the slides with water for 2 s.

    38. Wash the slides with absolute ethanol III for 10 s.

    39. Wash the slides with absolute ethanol IV for 10 s.

    40. Incubate the slides at 37 °C for at least 15 min.

    41. Dip the slides in xylene III for 10 s.

    42. Dip the slides in xylene IV for 10 s.

    43. Using a plastic Pasteur pipette, add two drops of entellan to the middle of the slide, making two separate circles.

    44. Dip the extremity of the slide in xylene.

    45. Fit the 24 × 50 mm microscope slide coverslips.

    46. Slightly press the coverslip against the slide.

    47. Incubate the slide at room temperature until completely dry.

      Note: Slides can be stored for years at room temperature.

    48. Place the dry slide on the inverted microscope Axio Vert (Zeiss).

    49. Adjust the 10× objective.

    50. Locate the granuloma.

    51. Take an image of the granuloma.

      Note: For a better reproduction, take images from exudative-productive granulomas.

    52. Take images of 100 granulomas per group.

      Notes:

      1. This number can be modified according to the number of granulomas found.

      2. All images should be taken using the same parameters.

Data analysis

  1. Length analysis of adult worms

    1. Open the image (with a known distance according to the parameters used to take images of adult worms in ImageJ.

    2. Select the “line selection tool” and draw a line to a known distance.

    3. Select “Analyze” and then “Set scale”.

    4. Type the known distance and its respective unit, select “Global”, and then select “ok”.

    5. Open the worm image in ImageJ.

    6. Select “Analyze” and then “Set Measurements”.

    7. Select “length”.

    8. Select the “line selection tool” and draw a line to the worm.

    9. Select “Analyze” and then “Measure”.

      Note: If the worms are not straight, carefully draw a line to each part of the worm and then sum the values to obtain the entire worm length.

    10. Perform the analysis for all images.


  2. Number of eggs recovered from the liver and intestine

    1. Calculate the mean number of eggs from triplicate samples; perform the assessment as described above (Procedure F).

    2. The mean value multiplied by 100 gives the number of eggs per ml (the total number of eggs per animal, recovered from the liver or intestine, will depend on the volume).

    3. Add together the number of eggs recovered from the liver and intestine to yield the total number of eggs per animal.


  3. Fecundity of female adult worms

    To obtain fecundity data, divide the total number of eggs by the number of females recovered from the respective animal.


  4. Oogram analysis

    Calculate the percentage of mature, immature, and dead eggs found in each mouse ileum.


  5. Analysis of the granuloma area

    1. Open ImageJ.

    2. Open an image with a known distance.

    3. Set the scale as described in steps 2-4 of Data analysis B.

    4. Open the granuloma image in ImageJ.

    5. Select “Analyze” and then “Set Measurements”.

    6. Select “area”.

    7. Select the “freehand selection tool” and carefully circle the granuloma.

    8. Select “Analyze” and then “Measure”.

    9. Perform this analysis for all granulomas.


    Note: Indicative results of data analysis A, B, C, D, and E, and the procedures C and E are represented in the original research paper Tavares, N. C., Gava, S. G., Torres, G. P., de Paiva, C. E. S., Moreira, B. P., Lunkes, F. M. N., Montresor, L. C., Caldeira, R. L. and Mourão, M. M. (2020).Schistosoma mansoni FES Tyrosine Kinase Involvement in the Mammalian Schistosomiasis Outcome and Miracidia Infection Capability in Biomphalaria glabrata.Front Microbiol 11: 963 (37). Link to access: https://www.frontiersin.org/articles/10.3389/fmicb.2020.00963/full.

Recipes

Note: All the following recipes can be stored at room temperature.

  1. 0.85% NaCl

    8.5 g NaCl

    Make up to 1 L with distilled water

    Note: Prepare at least 1.5 L per 10 mice.

  2. 10% KOH

    100g KOH

    Make up to 1 L with distilled water

  3. 1× PBS

    137 mM NaCl

    2.7 mM KCl

    10 mM Na2HPO4

    1.8 mM KH2PO4

    pH 7.4

  4. 4% buffered formaldehyde

    111.11 ml formaldehyde

    888.89 ml 1× PBS

  5. 10% formaldehyde

    277.77 ml formaldehyde

    722.23 ml distilled water

  6. Alcohol-formalin-acetic Acid (AFA)

    95 ml absolute ethanol

    3 ml formaldehyde

    2 ml acetic acid

  7. 70% ethanol

    700 ml absolute ethanol

    300 ml distilled water

  8. 80% ethanol

    800 ml absolute ethanol

    200 ml distilled water

  9. 90% ethanol

    900 ml absolute ethanol

    100 ml distilled water

  10. 95% ethanol

    950 ml absolute ethanol

    50 ml distilled water

  11. Hydrochloric carmine

    5 g carmine

    5 ml HCl

    5 ml distilled water

    Make up to 200 ml with 90% ethanol

    Note: Crush the carmine and add to the HCl and water. Place in a water bath for 1 h. Allow to cool. Make up the volume to 200 ml with 90% ethanol.

  12. Hydrochloric alcohol

    100 ml ethanol

    0.5 ml HCl

  13. 1:2 methyl salicylate-Canada balsam

    50 ml methyl salicylate

    100 ml Canada balsam

  14. Supplemented GMEM

    50 ml Glasgow Minimum Essential Medium (GMEM) (prepared and filtered according to the manufacturer’s instructions)

    0.1% glucose

    0.1% lactalbumin

    20 mM HEPES

    2% inactivated fetal bovine serum

    0.5% MEM vitamin solution

    5% Schneider’s Insect Medium

    0.5 μM hypoxanthine

    1 μM hydrocortisone

    1% penicillin/streptomycin

    Note: Store at 4 °C.

Acknowledgments

The authors are grateful for funding from the European Commission’s Seventh Framework Programme for Research, under Grant Agreement no. 602080 (A-ParaDDisE), FAPEMIG (CBB-APQ-0520-13), CAPES Programa PCDD-Programa CAPES/Nottingham University (003/2014), the Productivity Fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) granted to MMM (302518/2018-5), and the fellowship to NCT from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES), Finance Code 001. The authors would also like to thank the Snail and Animal Facility of the René Rachou Institute-Fiocruz for supplying parasites and mice, and the Program for Technological Development in Tools for Health-PDTIS/FIOCRUZ for the use of its facilities.

Competing interests

The authors have no conflicts of interest.

Ethics

The presented animal study was approved by Brazilian national guidelines following Law 11794/08 and by the Ethics Commission for Animal Use (CEUA) of the Oswaldo Cruz Foundation under the number LM-05/18.

References

  1. Armstrong, J. C. (1965). Mating Behavior and Development of Schistosomes in the Mouse. J Parasitol 51: 605-616.
  2. Basch, P. F. (1981). Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. J Parasitol 67(2): 179-185.
  3. Burke, M. L., Jones, M. K., Gobert, G. N., Li, Y. S., Ellis, M. K. and McManus, D. P. (2009). Immunopathogenesis of human schistosomiasis. Parasite Immunol 31(4): 163-176.
  4. Caffrey, C. R. and Secor, W. E. (2011). Schistosomiasis: from drug deployment to drug development. Curr Opin Infect Dis 24(5): 410-417.
  5. Clegg, J. A. (1965). In Vitro Cultivation of Schistosoma mansoni. Exp Parasitol 16: 133-147.
  6. de Waard. and Vermeulen, N. E. (1961). Penetration of the mammalian skin by cercariae of Schistosoma mansoni. Trop Geogr Med 13: 82-88.
  7. Engels, D., Chitsulo, L., Montresor, A. and Savioli, L. (2002). The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Trop 82(2): 139-146.
  8. Espírito Santo, M. C., Azeredo, L. M., Teles, H. M., Gryschek, R. C., Ferreira, C. S. and Amato Neto, V. (2008). Abdominal ultrasound in the evaluation of fibrosis and portal hypertension in an area of schistosomiasis low endemicity. Rev Inst Med Trop Sao Paulo 50(2): 117-119.
  9. Fallon, P. G. and Doenhoff, M. J. (1994). Drug-resistant schistosomiasis: resistance to praziquantel and oxamniquine induced in Schistosoma mansoni in mice is drug specific. Am J Trop Med Hyg 51(1): 83-88.
  10. Gryseels, B., Polman, K., Clerinx, J. and Kestens, L. (2006). Human schistosomiasis. Lancet 368(9541): 1106-1118.
  11. Hitchcock, D. J. (1949). Penetration characteristics of Schistosoma mansoni cercariae. J Parasitol 35(2): 216.
  12. Irie, Y., Tanaka, M. and Yasuraoka, K. (1987). Degenerative changes in the reproductive organs of female schistosomes during maintenance in vitro. J Parasitol 73(4): 829-835.
  13. Ismail, M. M., Taha, S. A., Farghaly, A. M. and el-Azony, A. S. (1994). Laboratory induced resistance to praziquantel in experimental schistosomiasis. J Egypt Soc Parasitol 24(3): 685-695.
  14. Ismail, M., Metwally, A., Farghaly, A., Bruce, J., Tao, L. F. and Bennett, J. L. (1996). Characterization of isolates of Schistosoma mansoni from Egyptian villagers that tolerate high doses of praziquantel. Am J Trop Med Hyg 55(2): 214-218.
  15. King, C. H. and Dangerfield-Cha, M. (2008). The unacknowledged impact of chronic schistosomiasis. Chronic Illn 4(1): 65-79.
  16. Mati, V. L. and Melo, A. L. (2013). Current applications of oogram methodology in experimental schistosomiasis; fecundity of female Schistosoma mansoni and egg release in the intestine of AKR/J mice following immunomodulatory treatment with pentoxifylline. J Helminthol 87(1): 115-124.
  17. Michaels, R. M. and Prata, A. (1968). Evolution and characteristics of Schistosoma mansoni eggs laid in vitro. J Parasitol 54(5): 921-930.
  18. Miller, P. and Wilson, R. A. (1980). Migration of the schistosomula of Schistosoma mansoni from the lungs to the hepatic portal system. Parasitology 80(2): 267-288.
  19. Moore, D. V. and Sandground, J. H. (1956). The relative egg producing capacity of Schistosoma mansoni and Schistosoma japonicum. Am J Trop Med Hyg 5(5): 831-840.
  20. Neves, R. H., Oliveira, S. A., Machado-Silva, J. R., Coutinho, E. and Gomes, D. C. (2002). Phenotypic characterization of Schistosoma mansoni adult worms recovered from undernourished mice: a morphometric study focusing on the reproductive system. Rev Soc Bras Med Trop 35(4): 405-407.
  21. Neves, R. H., Pereira, M. J., de Oliveira, R. M., Gomes, D. C. and Machado-Silva, J. R. (1998). Schistosoma mansoni Sambon, 1907: morphometric differences between adult worms from sympatric rodent and human isolates. Mem Inst Oswaldo Cruz 93 Suppl 1: 309-312.
  22. Olveda, D. U., Inobaya, M., Olveda, R. M., Vinluan, M. L., Ng, S. K., Weerakoon, K., McManus, D. P., Ramm, G. A., Harn, D. A., Li, Y., Lam, A. K., Guevarra, J. R. and Ross, A. G. (2017). Diagnosing schistosomiasis-induced liver morbidity: implications for global control. Int J Infect Dis 54: 138-144.
  23. Pellegrino, J. and Siqueira, A. F. (1956). [A perfusion technic for recovery of Schistosoma mansoni from experimentally infected guinea pigs]. Rev Bras Malariol Doencas Trop 8(4): 589-597.
  24. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  25. Stirewalt, M. A. (1974). Schistosoma mansoni: cercaria to schistosomule. Adv Parasitol 12: 115-182.
  26. Tavares, N. C., Gava, S. G., Torres, G. P., de Paiva, C. E. S., Moreira, B. P., Lunkes, F. M. N., Montresor, L. C., Caldeira, R. L. and Mourao, M. M. (2020). Schistosoma mansoni FES Tyrosine Kinase Involvement in the Mammalian Schistosomiasis Outcome and Miracidia Infection Capability in Biomphalaria glabrata. Front Microbiol 11: 963.
  27. Thétiot-Laurent, S. A., Boissier, J., Robert, A. and Meunier, B. (2013). Schistosomiasis chemotherapy. Angew Chem Int Ed Engl 52(31): 7936-7956.
  28. Uberos, J., and Jerez-Calero, A. (2012). “Imported Schistosomiasis,” in Schistosomiasis. Pediatrics Service, Universitary Hospital "San Cecilio", Granada, Spain. ISBN: 978-953-307-852-6.
  29. Watson, M. (2009). Praziquantel. J Exot Pet Med 3(18): 229-231.
  30. World Health Organization. (2018). Global Health Estimates 2016: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2016. 2019-05-03.


简介

[摘要]血吸虫病是一种被忽视的热带病。其治疗依赖于单一药物吡喹酮的使用。由于治疗的局限性,需要血吸虫病化疗的替代方法;牛逼HUS,更好地理解寄生虫生物学和主机-寄生虫的相互作用是有价值的,以帮助在识别新的抗血吸虫药物小号。Ť他寄生虫有一个复杂的生命周期,这导致challeng ES有关的评价小号chistosoma氏的发展和 哺乳动物感染的建立。因此,该协议描述methodolog IES来评估:(1)成虫生长; (2)繁殖;(3)肉芽肿的形成;因此允许更全面的了解曼氏血吸虫在开发一个天然的生物系统。

[背景]血吸虫病是一种被忽视的热带病,目前在78个国家/地区流行(世界卫生组织,2018年)。五种血吸虫可引起寄生虫病,分布在亚洲,非洲和美洲。该疾病有两种截然不同的形式:泌尿生殖器,由血吸虫血红蛋白引起;肠内血吸虫,湄公血吸虫,日本血吸虫和曼氏血吸虫引起的肠道和肠道感染(Uberos and Jerez-Calero,2012; World Health Organization,2018)。人类感染下从受感染的淡水螺释放出尾蚴接触。

在这里,我们专注于曼氏血吸虫小号pecie小号。进入后一个哺乳动物宿主,尾蚴不同entiate在对童虫(希区柯克,1949;德Waard和Vermeulen的,1961; Stirewalt,1974) ,其迁移到肝内血管和发展成人(阿姆斯特朗,1965;米勒和Wilson,1980)。交配后,成虫蠕虫迁移到肠系膜静脉,在感染的第35天后,它们完成了发育并开始产卵。一位女成虫奠定了约300个鸡蛋每一天,它达到组织和肠腔和消除沿随粪便排出到外部环境(摩尔和Sandground,1956年; Gryseels等,2006)。然而,一些蛋变得截留在肝脏和诱导的形成肉芽肿性损害(圣埃斯皮里托等人,2008;王和菲尔德-CHA,2008) ,这可导致在宿主组织纤维化和promoti上的损伤和并发症会导致贫血,营养不良和生活质量下降,对社会经济产生重大影响(Burke等,2009; Olveda等,2017)。

血吸虫病治疗依赖于使用吡喹酮,其显示针对所有物种中的活性; 人虽然,未成熟的虫不受影响(沃森,2009;Thétiot-劳伦。等人,2013年)。此外,有报道说成虫对吡喹酮的敏感性降低,从而增加了对其他杀血吸虫药的需求(Fallon和Doenhoff,1994; Ismail等,1994和1996; Engels等,2002; Caffrey和Secor,2011)。 。因此,寄生生物和宿主-寄生虫相互作用的研究是一个可行的策略,以搜索为新的抗血吸虫药物。

如前所述,曼氏葡萄球菌存在着一个复杂的生命周期,需要两个宿主。因此,对寄生虫生物学的更好理解是具有挑战性的,艰巨的,需要专门的劳动和耗时的(Clegg,1965; Michaels和Prata,1968; Basch,1981; Irie等,1987)。我的动物模型的noculation与寄生虫是调查的方式曼氏血吸虫发展的哺乳动物宿主,是一个平易近人的程序和重要系统的evaluat离子复杂的主机-寄生虫的相互作用。

在这里,我们描述了评估哺乳动物宿主内曼氏链球菌发展的方案。以下过程详细介绍了如何用血吸虫进行小鼠感染,并描述了通过肝脏肉芽肿分析评估寄生虫发育(例如生长,繁殖和感染结果)的方法。

关键字:曼氏血吸虫, 体内, 肉芽肿, 共焦, 寄生虫血症, 卵形图



材料和试剂


1 -毫升我nsulin注射器与一个27克Ñ eedle (BD,目录号:309623)
2 1个G Ñ eedle (BD,目录号:305129 )
巴斯德塑料移液器(Kasvi,目录号:K30-300S)
培养皿ES (Sarstedt的,目录号:82.1473.001)
6-我们升升板(Sarstedt的,目录号:83.3920.300)
50 -毫升Ç onical Ç entrifuge吨ubes(Sarstedt的,目录号:62.547.254)
15 -毫升Ç onical Ç entrifuge吨ubes(Sarstedt的,目录号:62.554.205)
手术刀(CP大号AB小号afety,目录号:QC-793-140-EA)
1-200微升ù niversal提示(爱思进,目录号:T-200-Y)
100-1 ,000微升ü NI通用开的提示(爱思进,目录号:T-1000-B)
Maxymum恢复® 0.6 -毫升米ICRO夫格管(Axygen公司,目录号:MCT-060-LC)
0.6 -毫升米ICRO夫格管(Axygen公司,目录号:MCT-060-CS)
1.5 -毫升米ICRO夫格管(Axygen公司,目录号:MCT-150-CS)
显微镜载玻片,26 × 76 mm(KASVI,目录号:K5-7105)
显微镜载玻片盖玻片,24 × 24 mm(KASVI,目录号:K5-2424)
显微镜载玻片盖玻片,24 × 50 mm(KASVI,目录号:K5-2450)
高p设定档d isposable b lades 818(徕卡生物系统公司,目录号:14035838383)
活检盒(CRALPLAST,目录号:2921)
28天大的女性Swiss Webster m ice
格拉斯哥最低必需培养基(GMEM)(Sigma-Aldrich,目录号:G6148)
葡萄糖(Sigma-Aldrich,目录号:G5767 )
乳清蛋白(Sigma-Aldrich,目录号:L7252)
HEPES(Sigma-Aldrich,目录号:H3375)
胎牛血清(Thermo Fisher Scientific,Gibco,目录号:16000044)
MEM维生素溶液(Thermo Fisher Scientific,Gibco,目录号:16000044)
施耐德的昆虫培养基(Sigma-Aldrich,目录号:S0146)
Triiodothyronine(Sigma-Aldrich,目录号:709611)
次黄嘌呤(Sigma-Aldrich,产品目录号:H9636)
氢化可的松(Sigma-Aldrich,目录号:H0888)
青霉素/链霉素(Thermo Fisher Scientific,Gibco,目录号:15070063)
肝素钠(Fisher Scientific,Thermo Fisher Scientific,目录号BP252410)
NaCl(默克密理博(Merck Millipore),目录号:1064040500)
盐酸赛拉嗪(Sigma-Aldrich,目录号:X1251)
盐酸氯胺酮(Sigma-Aldrich,目录号:343099)
叔戊二醇钠(西格玛奥德里奇(Sigma-Aldrich),目录号:T6023)
KOH(Sigma-Aldrich,目录号:221473)
无水乙醇(默克密理博(Merck Millipore),目录号64175 )
36%˚F ormaldehyde(Sigma-Aldrich公司,目录号:47608)
冰醋酸酸(默克,目录号:64197)
HCl(Merk Millipor e,目录号:109057)
胭脂红(Sigma-Aldrich ,目录号:C1022)
水杨酸甲酯(Sigma-Aldrich,目录号:M6752 )
加拿大香脂(Sigma-Aldrich,目录号:60610 )
N- b utyl乙酸酯(Sigma-Aldrich公司,目录号:442666-U )
组织学p araffin (的EasyPath,目录号:EP-21-20068A)
苏木(Sigma-Aldrich,目录号:H3136 )
曙红(Sigma-Aldrich,目录号:E4009 )
二甲苯(Sigma-Aldrich,目录号:534056 )
Entellan(默克密理博(Merck Millipore),目录号:1079610100)
高压灭菌自来水
高压灭菌的用于冰的平衡进料
0.85%NaCl(请参阅食谱)
10%KOH(请参阅食谱)
1 × PBS(请参阅食谱)
4%甲醛缓冲液(请参阅食谱)
10%甲醛(请参阅食谱)
酒精˚F ormalin-一个cetic酸(AFA)(见食谱)
70%乙醇(请参阅食谱)
80%乙醇(请参阅食谱)
90%乙醇(请参阅食谱)
95%乙醇(请参阅食谱)
盐酸胭脂红(见食谱)
盐酸酒精(请参阅食谱)
1:2水杨酸甲酯-加拿大香脂(请参阅食谱)
补充的GMEM(请参阅食谱)

设备


Tweeze RS(CP实验室小号afety ,目录号:QC-788-100-EA)
解剖剪(CP实验室小号afety ,目录号:QC-791-107-EA)
灌注系统(AutoMate Scientific ,目录号:11-140)
200 -毫升玻璃烧杯中
5 -毫米筛
手册2-20微升单-道移液器仪(Eppendorf ,目录号:3120000038)
手册20-200微升单-道移液器仪(Eppendorf ,目录号:3120000054)
手册100-10,00微升单-道移液器仪(Eppendorf ,目录号:3120000062)
的Eppendorf ®离心机5804R(Sigma-Aldrich公司,目录号:EP022628146)
组织处理PT05 TS(Lupetec)
包含中心CI 2014(Lupetec)
铝夹杂物模具(Lupetec)
切片机MRP2015(Lupetec)
光学显微镜Axiostar Plus(Zeiss)
蔡司米icroscope STEMI SV 11(Zeiss)对
倒置显微镜Axio Vert(Zeiss)
倒置显微镜Eclipse Ti-E(Nikon),带共焦C2 plus(尼康)

软件


ImageJ (Schneider等人,2012)(https://imagej.net/ImageJ1)
NIS-Elements软件(尼康)

程序


ñ OTES:

必须根据动物设施和国家动物使用指南执行所有鼠标操作。
PR IOR到和在感染期间,小鼠维持蒸压水和高压灭菌平衡饲料“广告libidum ” 。

用血吸虫接种小鼠
注意:下面的过程我s,使用的童虫阶段进行(图1) 。但是,也可以使用尾c阶段进行小鼠接种和其他操作。

计算含有350个血吸虫的培养基的体积。要计算寄生虫的数量/体积,请将350乘以培养基的总体积,然后将结果除以该培养基中培养的寄生虫的总数。
注意:每只小鼠要接种的血吸虫数量最多可达每只动物450个寄生虫。包括一个对照组。

吸管previo usly计算体积中以在P ETRI菜或到显微镜载玻片上。



图1.在培养基中培养的血吸虫的实例。的代表性图像一个获得童虫培养在10 ×放大率。


取出样品以在显微镜下检查移液体积是否包含预期数量的血吸虫。
放置寄生虫从每个组成十个Maxymum恢复® 0.6 -毫升米ICRO夫格管(一个每只小鼠管)。È ACH微夫格管应包含350童虫。
注:在没有Maxymum恢复的®微夫格管小号,有可能使用一个无菌的微量夫格管代替; ħ H但是,使用Maxymum的恢复ÿ ®微夫格管小号确保寄生虫接种将更加均匀,因为它减少了的数量剩余寄生虫ø n中的壁的管第。

弥补了体积200-300微升与补充GMEM,如果需要的话。
注意:每只小鼠的最大接种量为300 µl。

每组分离十只雌性Swiss Webster雌性小鼠。
注意:m个乌斯菌株可根据实验问题而改变。

使用1 -毫升我nsulin注射器用21号针对每只小鼠。
填充以前添加到0.6童注射器-毫升微夫格管。 
注意:每只动物用一根注射器和一根针避免接种不成比例的寄生虫。

抓住鼠标沿脖子的皮肤来握住鼠标。
接种通过皮下途径通过injecti寄生虫上的各自的注射器内容小号到松弛的皮肤在颈部。

安乐死与灌注
注意小号:

由于在感染35天后开始产卵,因此应在寄生虫接种后40天开始实施安乐死。
在此协议中所描述的麻醉和安乐死过程由巴西批准伊恩以下法律11794/08国家指导方针。在一个安装和镇静剂和安乐死方法的类型应遵循的动物使用指南小号每个国家的。
P repare 1.5L的0.85%NaCl溶液中每十只动物。
将2500 U / L肝素钠添加到0.85%NaCl溶液中。
每十只动物准备200毫升4%缓冲甲醛溶液。
制备2 0.5%小号裂果吨hiopental溶液。
称重每只鼠标。
抓住鼠标沿脖子的皮肤来握住鼠标。
使用1 -毫升我nsulin注射器与一个27克Ñ eedle ,辖10mg / kg的X ylazine盐酸盐和150毫克/公斤ķ纱罗盐酸盐通过injecti腹膜内途径上进入的大腿肌肉直接编从股骨远。
等待鼠标被前来麻醉。
与腹部朝上鼠标位置病房。
使用1 -毫升我nsulin注射器与一个27号针,辖150毫克/公斤的2.5%小号裂果吨hiopental通过injecti腹膜内途径上成向头部腹部。
按下鼠标的脚钳小号来确认euthan为IA时指出没有一个运动响应。
用70-75%的乙醇清洁腹部。
注:P erfusion物进行根据吨ö Pellegrino的并与一些Siqueira(1956)的修改。

使用解剖剪刀沿腹部的中线切开。
用解剖剪刀切开鼠标的横diaphragm膜和肋骨,露出心脏。
用解剖剪刀切开门静脉。
P花边在200中的小鼠肠和门静脉-毫升玻璃烧杯中。
将灌注设备的针头插入左心室。
启动用于通过NaCl注射恢复成虫的灌注设备。
注意:所有从门静脉流出的液体都必须落入烧杯中,因为它带有成虫。

当门静脉的液体变澄清时,停止灌注设备。
通过所获得的液体通过5 -毫米筛,其将保留回收成虫。
将回收的成虫从筛子转移到6孔板中。为了执行第是程序,转筛倒置和PLAC Ë它就做好了。使用塑料巴斯德吸管和0.85%NaCl清洗筛子,将所有蠕虫释放到孔中。
用0.85%NaCl溶液覆盖成虫。
取出小鼠肠和剪3 -厘米从回肠末端下游。
将回肠放在显微镜载玻片上,盖上24 × 24 mm盖玻片,然后按。
放置剩余的肠部我N的AA 6孔板中。
去除小鼠肝脏。
除去中叶和地方变成50 -毫升锥形离心管中预先填充用200ml 4%缓冲的甲醛。
放置保持DER肝脏我n中的6孔板中。
对所有动物重复此过程。
注意:根据动物标签的容器:肝,肠,和蠕虫,并且显微镜载玻片的回肠。


评估成虫的数量
将每只老鼠的恢复的成年蠕虫放在培养皿中。
使用针头或镊子将雄性与雌性分开。
注意:要区分男小号从女性小号,寄生虫的大小和颜色应得到遵守。雄成虫是透明的,雌成虫则更长和更暗。要识别女性所不具备的男性妇科阴茎和结节,必须使用放大镜或光学显微镜。

计算从每只小鼠中恢复的雄性和雌性。
重复次Ë程序从剩余的老鼠恢复的成虫。

成虫蠕虫长度分析
计数后,转移的雌性和雄性成虫从每只小鼠的1.5 -毫升微夫格装入1ml AFA溶液管。
注意:成虫可以在AFA解决方案中维护一年以上。

从同一只小鼠中回收的一只雌性和一只雄性随机转移到显微镜载玻片上。
小心地伸展成虫。
在Zeiss显微镜Stemi SV 11下,拍摄成年蠕虫的n幅图像。
注意:

所有图像均应使用相同的参数拍摄。
原始研究论文中提供了用于指示长度的指示性成虫蠕虫图像:Tavares,NC,Gava,SG,Torres,GP,de Paiva,CES,Moreira,BP,Lunkes,FMN,Montresor,LC,Caldeira,RL和Mourao ,MM(2020)。曼氏血吸虫FES酪氨酸激酶参与哺乳动物血吸虫病的结果和褐飞虱的Miracdiadia感染能力。微生物学杂志(Front Microbiol)11:963(37)。链接访问:https : //www.frontiersin.org/articles/10.3389/fmicb.2020.00963/full。
转移蠕虫回到1.5 -毫升微夫格装入1ml AFA溶液管。
不E:如果显微镜没有规模,采取N个图像一个RUL的ER在显微镜下使用相同的参数为那些被虫子。

对从其余小鼠中回收的成年蠕虫重复此过程。
注意:要分析的成虫数量取决于回收的成虫总数。使用最大数量的成虫蠕虫执行此过程。


染色dult蠕虫š共聚焦分析
注意:根据Neves等人对成虫进行了重新染色和分析。(1998年和2002年)。

使用20 - 200微升手动单道移液器或塑料巴斯德吸管除去所有的AFA溶液(图2A)。
填充1.5 -毫升微夫格使用含用盐酸胭脂红蠕虫管20 - 200微升手动单道移液器或塑料巴斯德吸管(图2B)。
可选:甲dult蠕虫可以被转移到一个小的培养皿中或其它容器,使在盐酸胭脂红寄生虫浸泡。

注:添加盐酸应该胭脂红的体积完全覆盖的蠕虫。

在室温下将蠕虫在盐酸胭脂红中孵育30分钟(图2B)。
使用20 - 200微升手动单道移液器或塑料巴斯德吸管以去除过量的盐酸胭脂红(图2B)。
添加70%乙醇,并迅速用除去乙醇20 - 200微升手动单道移液器或塑料巴斯德吸管(图2C)。
注意:这一步是很重要的,以除去多余的盐酸胭脂红,应该立即执行,以避免完全去除的盐酸胭脂红。

重复小号TEP E5(图2C)。
加盐酸和醇,在几秒钟后,使用将其删除20 - 200微升手动单道移液器或塑料巴斯德吸管(图2C)。
注意:此步骤提供了不同器官的对比,必须迅速执行以避免完全去除盐酸胭脂红。当蠕虫从深红色变成浅红色时,应除去盐酸。

加入70%乙醇,并在室温下孵育5分钟(图2C)。
使用20 - 200微升手动单道移液器或塑料巴斯德吸管除去70%的乙醇(图2C)。
加入80%乙醇,并在室温下孵育5分钟(图2C)。
使用20 - 200微升手动单道移液器或塑料巴斯德吸管除去80%的乙醇(图2C)。
加入95%乙醇,并在室温下孵育5分钟(图2C)。
使用20 - 200微升手动单道移液器或塑料巴斯德吸管除去95%的乙醇(图2C)。
加入无水乙醇,在室温下孵育5分钟(图2C)。
使用20 - 200微升手动单道移液器或塑料巴斯德吸管除去无水乙醇(图2C)。
笔记:

移液时,应注意避免蠕虫干扰。
所添加的盐酸酒精量和随后的乙醇洗涤液应始终覆盖蠕虫。
小号TEPS E5- ë 14可以使用一个小的培养皿或另一个容器中进行。将染色的蠕虫可以在使用5溶液中浸泡-毫米筛。
使用20 - 200微升手动单信道或塑料巴斯德吸管添加1:至少24小时2水杨酸甲酯,加拿大香脂溶液。覆盖所有与蠕虫的溶液并孵育至少24小时(图2D和图3A )。
注意:此解决方案中的成虫蠕虫可以维持多年。

放置雄性和雌性蠕虫分开ö n要显微镜载玻片(图2E)。
用20 µl-200 µl手动单道移液器或塑料Pasteur移液器用1:2的水杨酸甲酯-加拿大香脂溶液覆盖成虫。
小心覆盖显微镜载玻片用一个24 × 24毫米盖玻片(图2E)。
填充所述显微镜载玻片之间的空间的盖玻片1:使用2水杨酸甲酯,加拿大香脂溶液20-200微升手动单道移液器或塑料巴斯德吸管(图2E)。
将幻灯片放在平坦的表面上晾干(图3B)。
注意:显微镜载玻片应放在平面表面到干˚F或1 - 2个月。观察每天是否更1:2水杨酸甲酯,加拿大树胶溶液应加入以避免干荷兰国际集团Ò UT 。


卡明

图2.架构的染色成虫共聚焦分析。A.使用手动单通道移液器去除AFA解决方案。B.使用手动单道移液器添加,孵育和去除盐酸胭脂红。C.加法,温育,并使用手动单通道移液器去除盐酸醇,70%,80%,95%,和无水乙醇的。D.使用手动单道移液器添加1:2的水杨酸甲酯-加拿大香脂溶液。幻灯片E.大会的染色成虫。


vermes corados.tif

图3.染成虫的示例。A.中号ALE,并用盐酸胭脂红染色,并用1覆盖雌性成虫:2水杨酸甲酯,加拿大香脂溶液。B含有载有盐酸胭脂红的成年和成年蠕虫的显微镜载玻片。


肝肠消化和卵恢复
放置一个动物的剩余肝和肠中,以2二个fferent培养皿ES和切片器官使用解剖刀(图4A)。
转移切片肝脏15 -毫升猎鹰管(图4A)。
执行此过程的所有小鼠肝脏和小肠小号,分别。
加入10毫升10%KOH以溶解intestin肝脏和人组织; 这不会损坏鸡蛋(图4B)。
将肝和肠在4 °C下于10%KOH中孵育过夜(图4B)。
涡旋管(图4C)。
将试管在37 °C下孵育至少1小时(图4C)。
注意:如果肝脏和肠子没有完全溶解,请再次涡旋并孵育试管,直到器官完全溶解。

将试管以380 × g的速度离心5分钟(图4D)。
丢弃上清液(图4D)。
加入10 ml 0.85%NaCl,用塑料巴斯德吸管重新悬浮沉淀,然后将离心管以380 × g离心5分钟(图4D)。
执行小号TEPS F8- ˚F 10三次(图4D)。
在后最终离心,弃去上清液并重新悬浮沉淀在1毫升0.85%的NaCl(图4E)。
传送内容小号到干净1.5 -毫升微夫格管(图4E)。
用于计数,均化管和移液管将10μl内容小号至显微镜载玻片(图4F)。
在光学显微镜下,计数放大5倍观察到的卵并记录其数目(图4F)。
执行小号TEPS F14和F15三次。

蒂夫

图4.肝脏和肠消化的卵回收示意图。A.肝和肠的切片使用手术刀,和切碎器官转移到15 -米升Falcon管(小号TEPS F1-F2)。B. 10%KOH中加入组织和培养过夜(小号TEPS F3-F5)。C. Ť他组织涡旋孵育d在37 ℃下(小号TEPS F6-F7)。D. Ť他15 -米升Falcon管离心,将上清液除去,1 ml的0.85%的NaCl中加入(小号TEPS F8-F11)。E.将重悬的蛋被转移到一个1.5 -米升微夫格管(小号依照步骤F12-F13)。F. Ť他恢复蛋进行计数(小号TEPS F14-F16)。G.以10 ×放大倍数获得的回收卵的代表性图像。


Oogram
放置显微镜载玻片的回肠上的光学显微镜。
对于观察到的前100个卵,计算成熟,未成熟和死卵。
如Mati和Melo (2013 )所述,从第一到第四成熟阶段的卵被认为是不成熟的。
注意:您还可以按阶段对鸡蛋进行分类(1-5到5 ,其中5是包含发育的miracidium的鸡蛋;称为成熟蛋)。


肉芽肿分析的组织学
放置中叶,预先固定我ñ 4%缓冲的甲醛,在皮氏培养皿。
用手术刀将中叶切成六个碎片。
转移片段一个b iopsy盒。
保持肝脏的剩余部分我N 4%缓冲的甲醛。
关闭b iopsy盒。
标签的b iopsy盒小号根据各个动物小号。
放置b iopsy盒进入吨问题处理PT05 TS(Lupetec)。
填充吨与相应的溶液/试剂问题处理PT05 TS(Lupetec)铲斗:10%甲醛,70%乙醇,95%乙醇,无水乙醇,乙酸正丁酯,X亚基,和ħ istological石蜡。
设置在以下步骤牛逼问题处理PT05 TS(Lupetec)。
10%甲醛1小时。
10%甲醛1小时。
70%乙醇放置1小时。
95%乙醇放置1小时。
95%乙醇放置1小时。
绝对乙醇1小时。
二甲苯I 1小时。
二甲苯II 1小时。
二甲苯III 1小时。
石蜡I 1小时。
石蜡II 1小时。
注意:^ h可以执行istological处理手动以下中所描述的步骤小号TEP H9。

将几滴组织学石蜡分配到铝夹杂物模具上。
转移从肝脏切片b iopsy盒到所述模具中。
使用包容中心CI 2014(Lupetec),将组织学石蜡分配到肝脏碎片上。
适合RESP ective b iopsy盒在模具上(磁带盒,其中包含所述识别的底部部分)。
转移到CI Inclusion Center CI 2014(Lupetec)的冷却板中。
执行小号TEPS H1 - ^ h 14为其他肝脏样本。
从铝夹杂物模具中取出砖块。
在室温下孵育块过夜。
注意:包含组织的石蜡块可以保持在室温下,直到显微镜载玻片组装为止。

在-20 °C下孵育模块s 1小时。
将块放在冰上。
将每个样品块安装在Microtome MRP2015(Lupetec)上
将块切成薄片(约4-6 µM)。
放置切片我Ñ一个50 ℃的水浴。
注意:此步骤对于拉伸切片很重要;Ť他的温度不能高呃,因为石蜡熔化小号。

用显微镜载玻片捕获切片。
标记显微镜载玻片。
孵育显微镜SLI d ES在65 ℃下4小时。
填充四个容器与X亚基和标记与每个容器:X亚基I,X亚基II,X亚基III,和X亚基IV。
填充四个容器用无水乙醇和标签各自具有:一bsolute乙醇I,一个bsolute乙醇II,一个bsolute乙醇III,和无水乙醇IV。
将苏木精装满容器。
用曙红填充一个容器。
浸在幻灯片X撑我10分钟。
浸在幻灯片中X亚基II 10分钟。
浸在幻灯片中一个bsolute乙醇我10秒。
沾在滑动一个bsolute乙醇II为10秒。
用流水洗涤载玻片3分钟。
浸在幻灯片ħ ematoxyl我n,用于5分钟。
浸在幻灯片ë osin 30秒。
用水洗涤载玻片2秒钟。
用洗涤载玻片一个bsolute乙醇III为10秒。
用洗涤载玻片一个bsolute乙醇IV为10秒。
将玻片在37 °C下孵育至少15分钟。
浸在幻灯片X撑III为10秒。
浸在幻灯片中X亚基IV为10秒。
使用塑料巴斯德吸管中,加入两滴Ë ntellan到中间的滑动,使得两个单独的圆。
浸在幻灯片的肢体X撑。
装上24 × 50 mm的显微镜载玻片盖玻片。
轻轻将盖玻片按在幻灯片上。
在室温下孵育载玻片,直到完全干燥。
注:幻灯片可以STOR多年在室温下编辑。

放置干燥滑动ø n中的我nverted显微镜Axio上韦尔(蔡司)。
调整10倍物镜。
找到肉芽肿。
拍摄肉芽肿的n幅图像。
注意:为获得更好的繁殖效果,请从渗出性肉芽肿中拍摄图像。

拍摄每组100个肉芽肿的图像。
笔记:

该数目可以根据发现的肉芽肿的数目进行修改。
所有图像均应使用相同的参数拍摄。

数据分析


成虫的长度分析小号
根据参数打开图像(具有已知距离š用于拍摄的图像成虫在ImageJ的。
选择“线选择工具”并绘制一条已知距离的线。
选择“分析”,然后选择“设置比例” 。
输入已知距离及其相应的单位,选择“全局” ,然后选择“确定” 。
在ImageJ中打开蠕虫映像。
选择“分析”,然后选择“设置度量” 。
选择“长度”。
选择“线选择工具”并为蠕虫画一条线。
选择“分析”,然后选择“测量”。
注意:如果蠕虫不是直的,请小心地在蠕虫的每个部分画一条线,然后将这些值相加以获得整个蠕虫的长度。

对所有图像进行分析。

从回收的卵数的肝脏和小肠
计算平均数量鸡蛋的小号˚F ROM一式三份样品; 执行的,如上所述(评估P rocedure F)。
平均值乘以100可得出每毫升的卵数(每只动物从肝脏或肠中回收的卵总数取决于体积)。
添加一起数的卵中回收从肝脏和肠以得到每只动物的蛋的总数。

雌成虫繁殖力小号
要获取繁殖力数据,请用鸡蛋总数除以从相应动物中回收的雌性数量。


Oogram分析
计算在每个小鼠回肠中发现的成熟,未成熟和死卵的百分比。


分析的肉芽肿区
打开ImageJ。
打开已知距离的图像。
设置的比例如在步骤2中所描述-数据分析B的第4
在ImageJ中打开肉芽肿图像。
选择“分析”,然后选择“设置度量”。
选择“区域”。
选择“徒手选择工具”,并仔细盘旋肉芽肿。
选择“分析”,然后选择“测量”。
对所有肉芽肿执行此分析。

注:指示结果d ATA分析A,B,C,d,和E,以及程序C和E都代表我n中的原创性研究论文塔瓦雷斯,NC,加瓦,SG,托雷斯,GP,德派瓦,CES,莫雷拉,BP,Lunkes,FMN,蒙特莎,LC,卡尔代拉,RL和MOUR ã O,MM(2020)。曼氏血吸虫FES酪氨酸激酶参与哺乳动物血吸虫病的结果和褐飞虱的Miracdiadia感染能力。微生物学杂志(Front Microbiol)11:963(37)。链接访问:https://www.frontiersin.org/articles/10.3389/fmicb.2020.00963/full。


菜谱


注意:以下所有食谱都可以在室温下保存。

0.85%氯化钠
8.5克氯化钠

用蒸馏水补足1 L

注:P repare至少1.5升每10只小鼠。

10%氢氧化钾
100克KOH

用蒸馏水补足1 L

1 × PBS
137毫米氯化钠

2.7毫米氯化钾

10毫米Na 2 HPO 4

1.8毫米KH 2 PO 4

pH值7.4

4%缓冲甲醛
111.11毫升甲醛

888.89毫升1 × PBS

10%甲醛
277.77毫升甲醛

722.23毫升蒸馏水

酒精˚F ormalin-一个cetic酸(AFA)
95毫升无水乙醇

3毫升甲醛

2毫升一个cetic一个CID
70%乙醇
700毫升无水乙醇

300毫升蒸馏水

80%乙醇
800毫升无水乙醇

200毫升蒸馏水

90%乙醇
900毫升无水乙醇

100毫升蒸馏水

95%乙醇
950毫升无水乙醇

50毫升蒸馏水

盐酸胭脂红
5克Ç armine

5毫升盐酸

5毫升蒸馏水

用90%e醇补充200 ml

注意:粉碎胭脂红并加入HCl和水。P花边水浴1个小时。待其冷却。用90%的乙醇补足200毫升的体积。

盐酸酒精
100毫升乙醇

0.5毫升HCl

1:2水杨酸甲酯-加拿大香脂
50毫升米水杨酸乙酯

100毫升加拿大香脂

补充的GMEM
50毫升格拉斯哥最低必需培养基(GMEM)(根据制备并过滤的厂家专业读者的指令)

0.1%葡萄糖

0.1%乳白蛋白

20毫米HEPES

2%灭活的胎牛血清

0.5%MEM维生素溶液

5%施耐德昆虫培养基

0.5μM次黄嘌呤

1μM氢化可的松

1%青霉素/链霉素

注意:存放在4 °C 。


致谢


作者是感谢来自欧盟委员会第七框架计划资助为[R esearch,在格兰特一个greement没有。602080(A-ParaDDisE),FAPEMIG(CBB-APQ-0520-13) ,披肩Programa PCDD-Programa披肩/诺丁汉大学(003/2014),在生产力˚F从ellowship Conselho国立DesenvolvimentoCientíficoËTecnológico(的CNPq)授予MMM(302518 / 2018-5) ,并牛逼从他奖学金到NCT的Coordenação德Aperfeiçoamento德Pessoal德NIVEL高级,巴西(CAPES),金融码001笔者也想感谢小号钉和一个nimal ˚F acility在勒内·拉彻研究所,还有Fiocruz为供应寄生虫小号和M冰,并在工具健康PDTIS / FIOCRUZ为使用其设施的计划技术发展。


利益争夺


作者有没有冲突小号的兴趣。


伦理


所提出的动物研究是通过巴西伊恩以下11794/08法的国家准则和伦理委员会的动物使用(CEUA)的数量LM下,奥斯瓦尔多克鲁斯基金会- 05/18。


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引用:Tavares, N. C. and Mourão, M. M. (2021). Parasitemia Evaluation in Mice Infected with Schistosoma mansoni. Bio-protocol 11(10): e4017. DOI: 10.21769/BioProtoc.4017.
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