Is there a miracidium inside the egg? Do you find operculum or process of eggshell? Materials examined: feces (2) Study the preserved specimen of S. japonicum ova(Manipulation) Ovoidal, 54-63 umX40-58 u m, thin eggshell and lacking the operculum, on the side near one end there is depression from which there extends a small spinose process. (3)See the SEM photograph of egg(Demonstration) (4)See the ova of S. mansoni and S. Hematobium(Demonstration) Compare the ova of these three blood flukes 4. Pathology (1)See the cirrhotic liver and enlarged spleen(Demonstration) Note the size and nodular appearance of the liver and spleen. (2)See the sections showing pathological foci in liver(Demonstration) A. Acute stage, note the infiltration of eosinophils, leucocytes and radiating acidophilic streaks around the eggs B. Chronic stage, note the dead or calcified eggs surrounded by epithelioid cells, giant cells and fibroblasts (3) Dissection of infected experimental animal(Students work in groups) Kill the rabbits infected with S. japonicum for more than 40 days. Note the following features after dissection A. Is there ascites or not? B. Where adult worms live in? C. Observe the lesions of intestine D. Observe the lesions of liver E. Biopsy: Remove a small piece of rectal mucosa from infected rabbit Press it between two slides and examine under microscope. The ova of various developmental stages of embryogenesis are deposited singly or in clumps sometimes in chains following the vessel 5. Laboratory diagnosis (1)Pathogenic examination: fecal examination (2)Hatching of miracidium. A. Principal Under suitable condition, within 24 h-48 h, the miracidium in egg can be hatched According to the tendency to light and to upside of miracidium, which move on the water surface by straight line movement B. Method Use a sieve to filter 30 g of fresh feces. Add water to the filtrate in conical cylinder to 1 000 ml. Let it stand for 15-20 minutes, decant supernatant fluid and add clear
10 Is there a miracidium inside the egg? Do you find operculum or process of eggshell? Materials examined: feces. (2) Study the preserved specimen of S. japonicum ova (Manipulation). Ovoidal, 54～63μm×40～58μm, thin eggshell and lacking the operculum, on the side near one end there is depression from which there extends a small spinose process. (3) See the SEM photograph of egg (Demonstration). (4) See the ova of S. mansoni and S. Hematobium (Demonstration). Compare the ova of these three blood flukes. 4. Pathology (1) See the cirrhotic liver and enlarged spleen (Demonstration). Note the size and nodular appearance of the liver and spleen. (2) See the sections showing pathological foci in liver (Demonstration). A. Acute stage, note the infiltration of eosinophils, leucocytes and radiating acidophilic streaks around the eggs. B. Chronic stage, note the dead or calcified eggs surrounded by epithelioid cells, giant cells and fibroblasts. (3) Dissection of infected experimental animal (Students work in groups). Kill the rabbits infected with S. japonicum for more than 40 days. Note the following features after dissection: A. Is there ascites or not? B. Where adult worms live in? C. Observe the lesions of intestine. D. Observe the lesions of liver. E. Biopsy: Remove a small piece of rectal mucosa from infected rabbit. Press it between two slides and examine under microscope. The ova of various developmental stages of embryogenesis are deposited singly or in clumps sometimes in chains following the vessel. 5. Laboratory diagnosis (1) Pathogenic examination: fecal examination. (2) Hatching of miracidium. A. Principal Under suitable condition, within 24 h～48 h, the miracidium in egg can be hatched. According to the tendency to light and to upside of miracidium, which move on the water surface by straight line movement. B. Method Use a sieve to filter 30 g of fresh feces. Add water to the filtrate in conical cylinder to 1 000 ml. Let it stand for 15～20 minutes, decant supernatant fluid and add clear
water. After decantation, transfer sediment to a flask with 500 ml water. Incubation 6 h12 h at 37C and examine the free swimming miracidia If negative, repeat the examination after 24 h 6. Immunodiagnosis (1)Circumoval precipitin(COP)test, cercarien Hullen reaction(CHR) When live eggs, miracidia, or cercariae are mixed into patients'sera, precipitate formation on their surfaces and immobilization of miracidia or cercariae will take place. These reactions are called the circumoval precipitin(COP) test, miracidial immobilization test, and cercarie Hullen reaction(CHR) (2)Fast-ELISA(fast enzyme-linked immunosorbent assay) A Introduction Fast-ELISA is an in-vitro immunodiagnostic test for detection of pathogen infection which combines rapid, reliable and simple characteristics B. Names of reagent No(: Enzyme-coupled reagent; No@2: washing reagent; No@: bottom reagent; No@): developing dye reagent No⑤: diluted serum;No⑥: stopped reagent C. Methods In this application, SEA antigen solution of s japonicum was used to coat microplates overnight at 4C. Plates were incubated for 3-5 minutes at room temperature with one drop of diluted serum or S. japonicum-infected individual serum,or negative serum. The plates were washed three times with washing reagent and incubated for 3-5 minutes with one drop of enzyme (alkaline hosphatase)-coupled reagent at room temperature. Incubate for 3-5 minutes at room temperature and washed five times by tap water. Add one drop of bottom reagent, one drop of developing reagent respectively. After 30 seconds to 3 minutes at room temperature, add one drop of stopped reagent and observe the result D. Criteria of diagnosis Grade the result according to the developing dye degree on a white color +++ the color is much deeper than the positive control ++ the color is similar to the positive control +: the color is darker than the negative control and lighter than the positive control lar to the negative control All sample graded higher than"+ are considered positive 7. Epidemiology(Demonstration) (1)See photographs of patients (2)Environment of endemic area
11 water. After decantation, transfer sediment to a flask with 500 ml water. Incubation 6 h～12 h at 37℃ and examine the free swimming miracidia. If negative, repeat the examination after 24 h. 6. Immunodiagnosis (1) Circumoval precipitin (COP) test, cercarien Hüllen reaction (CHR). When live eggs, miracidia, or cercariae are mixed into patients’ sera, precipitate formation on their surfaces and immobillization of miracidia or cercariae will take place. These reactions are called the circumoval precipitin (COP) test, miracidial immobilization test, and cercarien Hüllen reaction (CHR). (2) Fast-ELISA (fast enzyme-linked immunosorbent assay). A. Introduction. Fast-ELISA is an in-vitro immunodiagnostic test for detection of pathogen infection, which combines rapid, reliable and simple characteristics. B. Names of reagents. No①:Enzyme-coupled reagent; No②: washing reagent; No③: bottom reagent; No④: developing dye reagent; No⑤: diluted serum; No⑥: stopped reagent C. Methods. In this application, SEA antigen solution of S. japonicum was used to coat microplates overnight at 4℃. Plates were incubated for 3～5 minutes at room temperature with one drop of diluted serum or S. japonicum-infected individual serum, or negative serum. The plates were washed three times with washing reagent and incubated for 3 ～ 5 minutes with one drop of enzyme (alkaline phosphatase)-coupled reagent at room temperature. Incubate for 3～5 minutes at room temperature and washed five times by tap water. Add one drop of bottom reagent, one drop of developing reagent respectively. After 30 seconds to 3 minutes at room temperature, add one drop of stopped reagent and observe the result. D. Criteria of diagnosis. Grade the result according to the developing dye degree on a white color background. +++ — ++++: the color is much deeper than the positive control. ++: the color is similar to the positive control. +: the color is darker than the negative control and lighter than the positive control. -: the color is similar to the negative control. All sample graded higher than “+” are considered positive. 7. Epidemiology (Demonstration) (1) See photographs of patients. (2) Environment of endemic area
(3)Eradication of Oncomelnia snails Exercise al morphology of adult Schistosa 2. Draw an egg of S. japonicum in detail and label 3. Record the result of dissection of infected experimental rabbit 4. Record the result of Fast-ELISA detection Thinking which way and route a person can be infected by S japonicum? What are the differences from other flukes? 2. Can we find the eggs of S. japonicum from the feces of a patient while the adult worms inhabit in the mesenteric vessels? Why? 3. To compare the prevention and cure measures of schistosomasis japonica with other flukes Reference Infection of experimental rabbits( Students work in groups Cover a shaved area of 10 sq. cm with a cover glass up-side-down, which contains 10 live cercariae, add a drop of water to the side of cover glass. After about 10 minutes remove the cover glass and count the remaining cercariae. Observe the affected skin is there any papule or petechiae? 5 weeks later sacrifice the rabbit for observation on adult worms inhabiting the mesenteric veins, and the gross pathological changes in liver and intestines 2. Methods for identification of dead and living eggs Acridine orange method. Place intestinal mucosa scraps (or other tissue)into Kahn-test tube 0.5 ml-I ml of 1: 10 000 acridine orange solution Keep shaking for a while and then incubate at 37C for 2 h for staining. Wash twice with PBS. Take out the tissue, lay flat on glass slide and examine under fluorescent microscope. Live ova orange-red or reddish-green; dead ova yellow 3. Indirect fluorescent antibody method (IFA) for diagnosis of schistosomiasis Japonica (1)Principl IFA is a method for diagnosis of schistosomiasis japonica with high sensitivity and pecificity. It is also of advantages of easy manipulation and saving reaction materials (such as antibody, antigen etc. ) The simplified principle is as follows. The given antigen in tissue section is fixed on the slide by conventional method and allowed to react to the examined serum(or suspension with first antibody). When the antibody is
12 (3) Eradication of Oncomelnia snails. Exercise 1. Label the general morphology of adult Schistosoma. 2. Draw an egg of S. japonicum in detail and label. 3. Record the result of dissection of infected experimental rabbit. 4. Record the result of Fast-ELISA detection. Thinking 1. By which way and route a person can be infected by S. japonicum? What are the differences from other flukes? 2. Can we find the eggs of S. japonicum from the feces of a patient while the adult worms inhabit in the mesenteric vessels? Why? 3. To compare the prevention and cure measures of schistosomasis japonica with other flukes. Reference 1. Infection of experimental rabbits (Students work in groups) Cover a shaved area of 10 sq. cm with a cover glass up-side-down, which contains 10 live cercariae, add a drop of water to the side of cover glass. After about 10 minutes remove the cover glass and count the remaining cercariae. Observe the affected skin, is there any papule or petechiae? 5 weeks later sacrifice the rabbit for observation on adult worms inhabiting the mesenteric veins, and the gross pathological changes in liver and intestines. 2. Methods for identification of dead and living eggs Acridine orange method. Place intestinal mucosa scraps (or other tissue) into Kahn-test tube 0.5 ml～l ml of 1:10 000 acridine orange solution. Keep shaking for a while and then incubate at 37℃ for 2 h for staining. Wash twice with PBS. Take out the tissue, lay flat on glass slide and examine under fluorescent microscope. Live ova orange-red or reddish-green; dead ova yellow. 3. Indirect fluorescent antibody method (IFA) for diagnosis of schistosomiasis japonica (1) Principle IFA is a method for diagnosis of schistosomiasis japonica with high sensitivity and specificity. It is also of advantages of easy manipulation and saving reaction materials (such as antibody, antigen etc.). The simplified principle is as follows. The given antigen in tissue section is fixed on the slide by conventional method and allowed to react to the examined serum (or suspension with first antibody). When the antibody is
present, the immunocomplex will form at the specific site where there is corresponding antigen. And the second antibody against first antibody is labeled with fluorescein (usually isothiocynate, FITC) and consequently allowed to react to the specific immunocomplex to from secondary immunocomplex which can be easily A. Antigen The antigen will be presented in tissue section. S. japonicum adults are removed from the liver and portal system of infected rabbit, after washing, fixed with Rossmans solution overnight. Then, pick out the parasites and wash them with 75% alcohol. The parasites can now be made into tissue section by conventional paraffl embedding method or kept in 75% alcohol solution for further use. Tissue sections are 5 H m in thickness B. First antibody a. 34B7 monoclonal antibody against S japonicum eggs b. Serum from S. japonicum infected rabbits c Normal rabbit serum a. Sheep IgG against rat, labeled with isothiocynate b. Sheep IgG against rabbit, labeled with isothiocynate c Contrast staining solution 10 mg Evan's blue is dissolved in 10 ml 0.5 M PBS pH7. 8, and stored in refrigerator D. PBS 0.05 MPBS pH7. 8; 0.1 M PBS pH7. 8 E. Buffered glycerin Add 1 ml 0. 1 M PBS pH7. 8 to 9 ml glycerin, mix well (3)Manipulation A. Circle the tissue section on the slide with a marker pen and drop 10 u l lst antibody or examined serum onto the tissue within the circle. the slide is then transferred to a damp box at 37C and allowed to react for 30 min B. Gently wash slide with PBS to remove the lst antibody and let to air-dry C. Dilute the second antibody with PBS containing Evan,'s blue to work solution, with Evans blue concentration at 0.01%. Then add 10 u l diluted second antibody to immunocomplex on the slide and transfer again to the damp box at 37C for 30 minutes D. Wash the slide with PBS, air-dry E. Add one drop of buffered glycerin onto the slide and cover it with a coverslip. Take care to avoid air bubbles between the slide and coverslip
13 present, the immunocomplex will form at the specific site where there is corresponding antigen. And the second antibody against first antibody is labeled with fluorescein (usually isothiocynate, FITC) and consequently allowed to react to the specific immunocomplex to from secondary immunocomplex which can be easily observed under fluorescent microscope with violet light source. (2) Materials A. Antigen. The antigen will be presented in tissue section. S. japonicum adults are removed from the liver and portal system of infected rabbit, after washing, fixed with Rossman's solution overnight. Then, pick out the parasites and wash them with 75% alcohol. The parasites can now be made into tissue section by conventional paraffin embedding method or kept in 75% alcohol solution for further use. Tissue sections are 5μm in thickness. B. First antibody. a. 34B7 monoclonal antibody against S. japonicum eggs. b. Serum from S. japonicum infected rabbits. c. Normal rabbit serum. C. Second antibody. a. Sheep IgG against rat, labeled with isothiocynate. b. Sheep IgG against rabbit, labeled with isothiocynate. c. Contrast staining solution. 10 mg Evan's blue is dissolved in 10 ml 0.5 M PBS pH7.8, and stored in refrigerator. D. PBS. 0.05 M PBS pH7.8; 0.1 M PBS pH7.8. E. Buffered glycerin. Add 1 ml 0.1 M PBS pH7.8 to 9 ml glycerin, mix well. (3) Manipulation A. Circle the tissue section on the slide with a marker pen and drop 10μl 1st antibody or examined serum onto the tissue within the circle. The slide is then transferred to a damp box at 37℃ and allowed to react for 30 min. B. Gently wash slide with PBS to remove the 1st antibody and let to air-dry. C. Dilute the second antibody with PBS containing Evan's blue to work solution, with Evan's blue concentration at 0.01%. Then add 10μl diluted second antibody to immunocomplex on the slide and transfer again to the damp box at 37℃ for 30 minutes. D. Wash the slide with PBS, air-dry. E. Add one drop of buffered glycerin onto the slide and cover it with a coverslip. Take care to avoid air bubbles between the slide and coverslip
(4)Observation Observe the resultant slide under a fluorescent microscope. The secondary immunocomplex with second antibody labeled with isothiocynate is present in bright yellow-green color. The occurrence of bright yellow-green color implies the 2nd antibody present in combination with Ist antibody or Ist antibody present in the examined serum Dark red color means negative result Try to analyse the specificity of antigen and antibody according to the difference in intensity and specific fluorescence that you observed 4. Circulating antigen( CAg) detection (1)Introduc iagnosis of schistosomiasis in endemic areas depends mainly on microscopic detection of eggs in stool. The high day-to-day fluctuation in egg excretion necessitates examining stool samples on consecutive days. Adult schistosomes cannot be directly counted in man because they live in inaccessible positions in the mesenteric vasculature. Detection of two schistosome adult worm circulating antigens, circulating anodic antigen(CAA), and circulating cathodic antigen( CCa), that show better performance regarding quantitation of infection intensity and follow up after chemotherapy. CAA levels have been found to correlate with S. japonicum worm burden in experimental animals. The circulating soluble egg antigen(CSEA)is 15-20 times more concentrated in eggs than in adult worms (2)Method Polyvinylchloride, flat-bottomed, microtitration plates were coated by incubation overnight at 4C with 100 u l/well of solution containing MAb in 0.035 M PBS, pH 7.8. The plates were washed with 2 mM PBS and nonspecific binding sites were blocked by incubation for one h at 37C with 120 u I/well of 0. 1%(w/v) bovine serum albumin(BSa)in PBS. Plates were washed and incubated for one hr at room temperature with 80 u I/well of two-fold serial dilutions of S japonicum infected mouse serum. or untreated urine from infected individuals. and an antigen standard Control wells were incubated with assay buffer. The plates were washed and incubated for I h at room temperature with 80 u I/well of a solution containing 1.2 u g/ml of MAb-FITC. The plates were washed again and incubated for I h at room temperature with 80 H I/well of anti-FITC/AP. Wash plates thoroughly and incubated overnight at 4C with 80 u I/well of PNPP in DEa buffer and the absorption at 405 nm was measured 5. Cercarial derm Take one mice, fix its four legs on a wooden, shave of a piece of area on abdominal skin, place a slide containing 10 live cecariea on the skin, keep wet. Remove the slide 10 minutes later, observe the located rash, edema and pruritu
14 (4) Observation Observe the resultant slide under a fluorescent microscope. The secondary immunocomplex with second antibody labeled with isothiocynate is present in bright yellow-green color. The occurrence of bright yellow-green color implies the 2nd antibody present in combination with 1st antibody or 1st antibody present in the examined serum. Dark red color means negative result. Try to analyse the specificity of antigen and antibody according to the difference in intensity and specific fluorescence that you observed. 4. Circulating antigen (CAg) detection (1) Introduction Diagnosis of schistosomiasis in endemic areas depends mainly on microscopic detection of eggs in stool. The high day-to-day fluctuation in egg excretion necessitates examining stool samples on consecutive days. Adult schistosomes cannot be directly counted in man because they live in inaccessible positions in the mesenteric vasculature. Detection of two schistosome adult worm circulating antigens, circulating anodic antigen (CAA), and circulating cathodic antigen (CCA), that show better performance regarding quantitation of infection intensity and follow up after chemotherapy. CAA levels have been found to correlate with S. japonicum worm burden in experimental animals. The circulating soluble egg antigen (CSEA) is 15～20 times more concentrated in eggs than in adult worms. (2) Method Polyvinylchloride, flat-bottomed, microtitration plates were coated by incubation overnight at 4℃ with 100μl/well of solution containing MAb in 0.035 M PBS, pH 7.8. The plates were washed with 2 mM PBS and nonspecific binding sites were blocked by incubation for one h at 37℃ with 120μl/well of 0.1% (w/v) bovine serum albumin (BSA) in PBS. Plates were washed and incubated for one hr at room temperature with 80μl/well of two-fold serial dilutions of S. japonicum infected mouse serum, or untreated urine from infected individuals, and an antigen standard. Control wells were incubated with assay buffer. The plates were washed and incubated for 1 h at room temperature with 80μl/well of a solution containing 1.2 μg/ml of MAb-FITC. The plates were washed again and incubated for 1 h at room temperature with 80μl/well of anti-FITC/AP. Wash plates thoroughly and incubated overnight at 4℃ with 80μl/well of PNPP in DEA buffer and the absorption at 405 nm was measured. ５. Cercarial dermatitis Take one mice, fix its four legs on a wooden, shave of a piece of area on abdominal skin, place a slide containing 10 live cecariea on the skin, keep wet. Remove the slide 10 minutes later, observe the located rash, edema and pruritus caused by