郑州大学：寄生虫学（一）：en

A LABORATORY GUIDE TO PARASITOLOGY (THE FIFTH EDITION) ZHENG XIAOYING WU ZHONGDAO Department of Parasitology Zhongshan Medical collage , Sun Yat-Sen University July, 2002 CONTENTS Care of the microscope…………………………………….…………………1 Clonorchis sinensis…………………………………….……………….…….2 Paragonimus flukes…………………………………….……………….……5 Fasciolopsis buski…………………………………….……………….….…..7 Schistosoma japonicum…………………………………….………………...8 Tapeworm…………………………………….……………….………….…14 Ascaris lumbricoides & Trichuris trichiura ………………………………..17 Ancylostoma duodenal & Necator americanus……………………………..20 Enterobius vermicularis……………………………………………….…….22 Wuchereria bancrofti & Brugia malayi……………………………………..23 Trichinella spiralis…………………………………….……………….……25 Examination of alimentary helminths…………………………………….…26 Entamoeba histolytica, E.coli & other Amoebae…………………………...27 Giardia lamblia…………………………………….……………….……….30 Trichomonas vaginalis…………………………………….………………...31 Leishmania donovani…………………………………….……………….…32 Plasmodia…………………………………….…………………….…….…35 Opportunistic pathogenic protozoan………………………………….……..40 Mosquitoes…………………………………….……………….…….……...43 Fly…………………………………….…………………….…….…….…...45 Sandfly, fleas, lice & other blood sucking insects…………………………..46 Care of the microscope Do's Do take special care to protect the microscope from dust in hot dry periods. Do take special care to protect the microscope lenses and prisms from fungal growth in hot humid periods. Do clean the immersion oil from the immersion objective each time; use lens tissue dampened with ethanol. Do clean the oculars with lens tissue. Do use the microscope retaining screw fitted at the base of the microscope box to prevent damage to the instrument while in transit. Don't Don't use the tissue used for the oil immersion objective to clean the oculars. Don't dismantle or try to clean parts of the microscope that are difficult to reach. Don't leave the lens parts empty; use the appropriate cover to cover the empty port. Don't exchange lenses from microscopes of different manufacture. CLONORCHIS SINENSIS Objectives and Requirements To study the morphology and the life history of C. sinensis as an example of general feature of trematodes. To study the infection route, infection mode, parasitized site and the pathogenesis of C. sinensis infection. To study the characteristics of ovum of C. sinensis and the pathogenic diagnosis of C. sinensis infection. 4. To learn the basic skill of drawing eggs. Observation and Experiment 1. Adult worm Observe the preserved specimen (Demonstration). Note the size, color, shape of the organism. Spindle-shaped, transparent. Observe the general feature of C. sinensis in stained specimen (Manipulation). First with naked eye, then study under microscope, note and identify following structures. Adherent organs: oral and ventral suckers, compare their size. Digestive system: mouth, pharynx, esophagus and ceca. Excretory system: excretory bladder and pore. Reproductive system: hermaphroditism. Male: observe the number, size, shape and position of testes. Female: observe ovary, seminal receptacle, vitellarium, Mehlis’ gland and uterus. What is meant by hermaphroditism? 2. Larva and intermediate hosts (Demonstration) (1) See live sporocyst, redia and cercaria (or stained sporocyst to replace live one if necessary). (2) See the stained cercaria. Note the eye-spots and characteristics of tail. (3) Live metacercaria. Note its ellipsoidal shape, 138μm×115μm, with two suckers (oral sucker and ventral sucker) and excretory bladder containing black granules. (4) See the first intermediate hosts (Parafossarulus, Alocinma, Bithynia snails), and second intermediate hosts, fresh water fish and crayfish. What are their names? 3. Ovum (Manipulation) Study the ova. Note the shape, color, shell, operculum resting on a rim which takes the shape of distinct shoulders, a small protuberance at abopercular end, an asymmetrical miracidium inside the egg. One of the smallest helminth ova, yellow-brown, thicker eggshell. Material examined: feces and drawn liquid from duodenum. Examining method: see examination of alimentary helminths. (2) See the scanning electron-microscopic (SEM) photograph of ovum (Demonstration). 4. Pathology (1) See specimen, note the parasites in billiary passages (Demonstration). (2) See adult worms inhabiting in billiary passages of a reservoir host (Demonstration). Exercise Draw egg of C. sinensis in detail. Label in full an adult worm of C. sinensis. Draw a life cycle draft of C. sinensis, including final host, intermediate host; adult parasitized site, main injured organs; excretory route of ovum, pathogenic diagnostic methods; the first and second intermediate hosts and in which how the larva development; the infective stage, infection mode and infection route. Thinking 1. Why one can obtain parasitic disease after eaten uncooked fish? 2. Record the results of pressing fish flesh and the dissection of the experimental animal. Reference The fertilization process and egg formation In male, sperm produced in a pair of testes are collected in the seminal vesicle through the vas efferens and vas deferens and are ejected through the ejaculatory duct, around which there is a prostate gland covered by a cirrus sac. In female, a short oviduct comes out of the ovary and joins with the seminal receptacle and Laurer’s canal opening on the dorsal side, and further proceeds to the ootype with surrounding Mehlis’ gland. Then, it becomes the uterus, which extends with many contortions to the common genital pore. Small ducts from the vitelline glands, gradually gather to become the right and left vitelline ducts and then one common vitelline duct, which opens at front of the ootype. An egg cell from ovary and a sperm from testis are united, additional yolk cells provided by the vitelline gland join, and eggshell formation is started in the ootype by the effect of Mehlis’ gland and completed during passage through the uterus. In the egg one miracidium has already been formed. Examine for encysted metacercaria in fish flesh Press a small piece of freshwater fish flesh between two slides, examine for encysted metacercaria of C. sinensis under lower-lens microscope. Isolation of encysted metacercaria Take the 2nd intermediate host-freshwater fish and smash, digest with 250 ml digestive solution per 10 g flesh for 4 h～12 h (Digestive solution: pepsin 9.8 g, 1 N HCl 164 ml, NaCl 17 g, add water to 2 000 ml). Discard rough matter by sieve filtration. After several times sedimentation of the filtrated solution, take the sediment and examine under microscope. 4. Examination adult from infected animal Dissect the guinea pig (or white rat) which were infected with the aid of stomach tube approximately 30 C. sinensis cysts 40 days ago. Expose the common bile duct, gall bladder and search for the adult worms in the billiary tree. PARAGONIMUS FLUKES Objectives and Requirements To study the life cycles of P. westermani, to learn more about the life history of trematodes. To study the main parasitized sites and major pathogenisis. 3. To study the laboratory diagnosis of these flukes. Observation and Experiment 1. P. westermani (1) Adult worm A. See the preserved specimen (Demonstration). B. Study the internal structures of stained specimen (Manipulation). Note the position of the suckers. Are they equal in size? The branched testes are situated in posterior of the body, side by side, and posterior to uterus and ovary. The lobulated ovary is in the opposite side of uterus (fully filled with ova). Are these two female reproductive organs situated anterior or posterior to the worms? C. See the SEM photographs of Paragonimus spines. (2) Larva and intermediate hosts (Demonstration) A. See the first and second intermediate hosts-Melania snail and crayfish. B. See the stained cercaria. C. See the live cysts. D. Isolation of the encysted metacercaria from infected crabs (Students work in groups). Crushed a crab in a mortar. Add some 0.45% NaCl solution or tap water into motar and filter. Allow the filtrate stand and settle, examine sediments for encysted metacercaria under microscope. (3) Egg Morphology of egg (Manipulation). Measures 80～118μm×48～60μm. Note its ellipsoidal shape, uneven thickness of eggshell, slightly oblique operculum and yellowish brown in color. A germinal cell with more than ten yolk cells inside the egg. Materials examined: sputum, feces. Examining methods: direct sputum smear, sputum concentration and direct fecal smear. B. See the SEM photograph of egg. (4) Pathology A. See the preserved infected lungs (Demonstration). Note the capsules and their surrounding damaged tissues. B. See the infected liver (Demonstration). 2. Pagumogonimus skrjabini (1) Adult worm (Demonstration). A. See the preserved specimen. B. See the stained specimen. (2) Intermediate host (Demonstration) See the first intermediate host-snail (Tricula humida). Exercise Draw P. westermani egg in detail and label. Label the adult worm of P. westermani. Write the result report of cyst isolation, to have an analysis of epidemiology of P. westermani. 4. Draw a life cycle draft of P. westermani. Thinking How to explain paragonimiasis is a natural epidemic source disease in mountainous region and forest? And what are the epidemic characteristics of paragonimiasis? Reference Detection of sputum for egg of P. westermani Sputum smear Take a clean slide, place a drop of saline in the center of it, pick up a small amount of the sputum and mix it with the drop of saline, examine under microscope. Egg concentration Collect the sputum of 24 h, pour into a measuring glass and add equal quantity of 10% NaOH, mix, sediment naturally for 6 h～8 h, remove supernatant, take the sediment and examine under microscope. Survey biological hosts of P. westerman in epidemiology Choose one or more brooks, capture Melania snail and stream crab or crayfish . Break up Melania snail and place the internal organs on a slide, open them and put a drop of 0.45% saline, find sporocyst, redia and cercaria under microscope. Dissect stream crab or crayfish and find encysted metacercaria. 3. Infection of P. Westermani for dog Isolation of cysts from crayfish and mixing with meat, feed dogs, each one with 100～200 cysts according to the sizes of dogs. Kill the infected dogs 2～3 months later, open chest and expose the lungs, on which the nodular cysts can be observed, dissect them to find adult worms. FASCIOLOPSIS BUSKI Objectives and Requirements 1. To study the life history and feature in morphology of F. buski. 2. To study the laboratory diagnosis of this fluke. 3. To gain some knowledge of plant vectors and the importance in prophylaxis. Observation and Experiment Adult worm (1) See the preserved specimens (Demonstration). Note the shape, size, color and the positions of oral and ventral suckers and their sizes, large and thick. Study the stained specimen (Manipulation). First with naked eye, then under low power microscope, note the adhesive organ, ceca and reproductive system of both sexes. The intestine is composed of two blind tracts without side branching, and the ventral sucker is very large. 2. Larva, intermediate host and vector (Demonstration) See the snail intermediate host (Planorbis). (2) See the cercaria. See the stained specimen of cyst. See the vector: aquatic plants (caltrop, water chestnut etc.). Ovum See the preserved specimens (Manipulation). Note its large size, 130～140μm×80～85μm, oval shape, pale yellow in color, thin shell, small operculum, an germinal cell surrounded by yolk granules. Materials examined: feces. (2) See the SEM photograph of ovum. Exercise Draw and label F. buski egg in detail. Thinking 1.Draw a life cycle draft of F. buski. SCHISTOSOMA JAPONICUM Objectives and Requirements To understand characteristics of life cycle and morphology of its developmental stages. To observe the pathological changes of schistosomiasis in infected animal. To study morphology of ova, laboratory diagnostic methods and their principle. To gain fundamental knowledge of infective stage, infection mode, methods and significance of immunologic tests commonly used. Observation and Experiment l. Adult worm See the live specimen (Demonstration). See the preserved specimen (Demonstration). Adults are dioecious, a male and a female live together. (3) Study stained specimen (Manipulation). Note the shape, size and color of both sexes, oral and ventral suckers and gynecophoral canal of male, the number, shape, size and arrangement of testes, ovary and uterus of female. Females are long and slender, the posterior half of the body is a little thick and dark brown in color owing to the vitelline gland and intestine. Near the anterior end of the body, there are two suckers. The part of the male body behind the ventral sucker is flattened, and rolled ventrally along the length to form a groove, canalis gynecophorus, into which the female worm fits. What is the meaning of dioecious? See the SEM photograph of adult worm (Demonstration). Larva and intermediate host (Demonstration) See the live miracidia. Note the feature of movement in water. Study the structure of stained specimen of miracidia. See the SEM photograph of miracidia. Identify the intermediate host Oncomelania snail. See the live cercariae. Note the posture of the body resting beneath water surface and its movement. Study stained specimen, note the internal structures and the forked tail. (6) See the SEM photograph of cercariae. Ovum (1) Place a small drop of suspension containing S. japonicum ova on a slide. 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. See the SEM photograph of egg (Demonstration). See the ova of S. mansoni and S. Hematobium (Demonstration). Compare the ova of these three blood flukes. 4. Pathology 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). Acute stage, note the infiltration of eosinophils, leucocytes and radiating acidophilic streaks around the eggs. 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? Observe the lesions of intestine. Observe the lesions of liver. 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. Hatching of miracidium. 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. 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 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). Introduction. Fast-ELISA is an in-vitro immunodiagnostic test for detection of pathogen infection, which combines rapid, reliable and simple characteristics. Names of reagents. No①:Enzyme-coupled reagent; No②: washing reagent; No③: bottom reagent; No④: developing dye reagent; No⑤: diluted serum; No⑥: stopped reagent 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) See photographs of patients. Environment of endemic area. 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 By which way and route a person can be infected by S. japonicum? What are the differences from other flukes? Can we find the eggs of S. japonicum from the feces of a patient while the adult worms inhabit in the mesenteric vessels? Why? 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～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 observed under fluorescent microscope with violet light source. (2) Materials 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 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. Gently wash slide with PBS to remove the 1st antibody and let to air-dry. 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. Wash the slide with PBS, air-dry. 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 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. 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 1.2μg/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 penetration of schistosome cercariae through the skin. 6 Schistosomal Hatching Test If feces containing viable schistosome eggs are diluted with approximately volumes of water, the eggs hatch within a few hours, releasing miracidia. The miracidia are positively phototrophic. The following procedure takes advantage of this characteristic. 1). A stool specimen is homogenized by shaking in normal saline and then strained through two layers of gauze. 2). The material is allowed to sediment, the supernatant is decanted and the sediment resuspended in saline. This process is repeated least twice. I 3). The saline is decanted and replaced with distilled water, and is suspension is placed in a flask. The flask is covered to 1 cm below the level of fluid in the neck of the flask. Additional water is added if necessary. 4) The flask is allowed to stand at room temperature for several hours in subdued light. 5) detect the presence of free-swimming miracidia. The typical miracidium is a tiny, ciliated organism. It is long round form. Miracidium swims ceaselessly during its short life. Free-swimming miracidia are very active, although the miracidia do not have eyespots, they apparently have photoreceptors, and they are positively phototrophic. TAPEWORM: Taenia solium, Taenia saginata, Spirometra mansoni & Echinococus granulosus Objectives and Requirements 1. To study the general morphology and characteristics of life cycle of tapeworm. 2. To study the diagnostic methods and morphological comparison between these tapeworms. 3. To understand the dangerous of taeniasis, cysticercosis, hydatid disease and sparganosis to human being. 4. To study the morphology of hydatid cyst of E. granulosus. Observation and Experiment 1. Taenia Solium and Taenia Saginata. (1) Adult worm A. See the preserved specimen of T. solium (Demonstration). B. See the preserved specimen of T. Saginata (Demonstration). Note the size, ribbon shape, scolex and segmentation. Observe the stained specimen of scolex of T. solium (Manipulation). Spherical, four suckers, with rostellum armed with two rows of hooklets, numbering 25～30. Observe the stained specimen of scolex of T. saginata (Manipulation). Quadrate, four suckers, no rostellum or hooklets. What are the differences between the scoleces of these two tapeworms? E. See the stained specimen of segment of T. solium (Demonstration). See the stained specimen of segment T. saginata (Demonstration). Note each segment has 2 full sets of reproductive organs, one of male and another female. The testes are follicular, about 100 in number. Ovary with two lobes (T. saginata) or three lobes (T. solium). Uterus club shaped. No digestive tract. Observe the ink-stained specimen of gravid segment of T. solium (Demonstration). Count the number of lateral branches of uterus on one side. 7～13 lateral branches on each side of uterus unequal in length. Observe the ink-stained specimen of gravid segment of T. saginata (Demonstration). Count and compare the number of lateral branches with that of T. solium. 15～30 lateral branches, equal in length. Observe the preserved specimen of tapeworm (Demonstration) Press a gravid segment between two slides, examine and count the number of lateral branches under lower-lens microscope. J. See the SEM of T. solium scolex (Demonstration). (2) Larva Study the stained specimen of cysticercus cellulose. Note the rostellum, hooklets, sucker and bladder. See the stained specimen of cysticercus bovis and compare with the scolex of cysticercus cellulose (Demonstration). See the preserved specimen of cysticerci (Demonstration). See cysticercus bovis in cardiac muscle of ox (Demonstration). See cysticercus cellulose in cardiac muscle of pig (Demonstration). (3) Ovum (Manipulation) Observe the live specimen of taenia ova. Note the size, color, characteristics of embryophore and contents of the ova. Nearly spherical, eggshell is easily ruptured, only embryophore is seen. Materials examined: feces or swab of anus. 2. Spirometra mansoni (1) Adult worm (Demonstration) A. See the preserved specimen of adult worm. Study the stained specimen of adult worm. Note globular testes (over 300～500 in number) in the lateral margins of dorsal side, and two lobulated ovary and overlapping uterus in the mid-portion of the segment. See the stained specimen of scolex. See the SEM photograph of S. mansoni scolex. (2) Larva and intermediate hosts (Demonstration) See the procercoid. Observe the live spargana in frog flesh. Note the spargana lodging in muscles, they are long whitish worms. The scolex is similar to that of adult, body unsegmented, strong motility. See the first intermediate host cyclop and second intermediate host frog. (3) Ovum Study ova of S. mansoni. Note its spindle shape, operculum, thin shell, and a germinal cell with numerous yolk granules. (4) Dissection of frog for sparganum (Students work in groups). Sacrifice the frog by destroying the spinal cord with a small drill. Fix the frog on a board ventral side up. Make a midline incision on the abdominal wall. Skin it and search the flesh for spargana. 3. Echinococus granulosus See the stained specimen of adult worm (Demonstration). Note the four segments (usually) in an adult worm and the difference in maturity, and the characteristics of scolex. See the preserved hydatid cyst in liver (Demonstration). Study the scoleces of hydatid cyst (Demonstration). Note the ovoidal scolex armed with suckers and hooklets. (4) Study the section of hydatid cyst (Demonstration). Note the outermost layer, false cystic wall, resulting from reaction of host, the external layer of true cystic wall is noncellular hyaline cuticular and inner layer is the germinal layer, from which the brood capsules, and daughter cysts and detached germinal fragments are formed. Exercise 1. Draw taenia ova and gravid segment. Label the scolices of T. solium and T. saginata. Record the result of dissecting frog for sparganum. Thinking To compare T. solium and T. saginata based on their morphology, life cycle, pathogenesis and epidemiology (epidemiologic factors and epidemiologic distribution). Why the curative effect examination should be taken for treating tapeworm diseases? What is the curative basis? ASCARIS LUMBRICOIDES & TRICHURIS TRICHIURA Objectives and Requirements To gain some knowledge of the life cycle and common morphology of nematodes. To identify ova of A. lumbricoides and T. trichiura. To study the pathogenesis of A. lumbricoides infection. To further understand the life cycle and pathogenesis of A. lumbricoides through animal experimental observation. Observation and Experiment l. Ascaris lumbricoides (1) Adult (Demonstration) See the preserved male and female adult worm of A. lumbricoides. Note their size, shape and color. The female worm is rather large, being 20～35 cm long. The male worm is rather small and 15～30 cm long, with the tail end turning to the ventral side. The front end of the body in both male and females is equipped with three lips. See the dissected male and female adult worm of A. lumbricoides. (2) Study the transverse section of A. lumbricoides. Note intestinal canal and two sexes of reproductive system. The female has two sets of genital organs. There is a moderate construction at about 1/3 of the body from the front end forming the genial girdle, at the ventral side of which the vulva is open. The anus is located far from the vulva, close to the tail end on the ventral side. The male has a set of genital organs. The cloaca joining the peripheries of the digestive and the genital tract is located near the end. (3) Study the cross section of A. lumbricoides. Note the internal and external structures of body wall and body cavity (pseudocele). The cuticle: A body wall consisting of a protoplasmic syncytial layer called the hypodermis surmounted by an apparently non-living, mainly collagenous cuticle in which no separate cells can be distinguished and underlain by groups of longitudinal muscles. Nuclei are present only in four thickened chords or "line," one dorsal, one ventral, and two lateral. In these chords run nerve fibers. Between the cords there is a single layer of longitudinally spindle-shaped muscle cells of very peculiar structure (striated contractile portion of muscle cell, protoplasmic portion of muscle cell). Intestine: a flat or cylindrical tube, straight, and is lined by a single layer of cells. Cavity: Between the muscles and the gut wall is a relatively spacious body cavity in which the reproductive organs lie, unattached except at their external openings. This cavity is not lined by an epithelium as is a true celome, and it is a pseudocoelomate body cavity which plays an important hydrostatic skeletal role in locomotion. It contains a fluid which serve as distributing medium for digested food and for collection of waste products. It is provided with a small amount of "mesenterial" tissue and a few large phagocytic cells called celomocytes. (4) Ova Study the morphology of fertilized ova of A. lumbricoides (Manipulation). Note the characteristic shape, size, color, and the shell with special reference to albuminoid membrane. 45～75μm×35～50μm, yellow-brown color. B. Study the morphology of unfertilized ova of A. lumbricoides (Manipulation). Compare the appearance and structure with those of fertilized ova. Measuring 88～98μm×39～44μm. C. See the infective stages of ova, including a larva (Demonstration). See the SEM photograph of fertilized and unfertilized ova (Demonstration). (5) See the SEM photograph of lips and copulative spicula (Demonstration). (6) Pathologic specimens (Demonstration) See the gross specimen of intestinal obstruction due to ascariasis See the gross specimen of biliary ascariasis 2. Trichuris trichiura See the preserved adult worms (Demonstration). Note whip-like, the thin anterior part (three-fifths) is occupied by the esophagus and the thick posterior part (two fifths) contains intestine and genital organs. Study the preserved ova (Manipulation). Note the shape, size, color and shell. Observe particularly their bipolar plugs and germinal cell. 50～54μm×22～23μm in size, barrel-shaped, eggshell is brown in color, with pores at both ends that are stoppered with colorless mucoid substance, an egg cell is uncleaved. Pathologic specimens. Adults parasitic in human cecal mucosa, posterior part of the body being free. Exercise 1. Draw the fertilized and unfertilized ova of A. lumbricoides. 2. Draw the ovum of T. trichiura. Reference 1. Experimental A. lumbricoides infection of animal (Students work in groups) Place one female worm Ascaris lying on its ventral surface on a wax pan. Fix with pins at both ends, make an incision at the lateral side of vulva and cut caudally so as to open the body lengthwise from anterior to posterior end. Using a needle, lay the cut edges flat by pinning them to the wax, add a few saline and separate internal organs carefully. Remove about 1.5 cm of one uterus near the vagina with needle, put a sprinkling of ova in a drop of saline and prove that are fertilized under microscope. Put all fertilized ova on a penicillin bottle, and add 2 ml of 2% formalin. Incubate at room temperature or 22℃～33℃ for 3 weeks. Observe and record the embryogenetic development every week, and add 2% formalin to maintain optimal moisture, if necessary. (3) Three weeks later when the ova are mature, remove the formalin by a pipette and mix the ova with a few saline to make up suspension, pick up 0.5 ml ovum suspension with a pipette and inoculate it orally into the stomach of mouse. One week later, dissect the infected mouse, remove the liver and lung, transfer them onto a petri dish, wash blood away with saline, observe the surface carefully for bloody extravasation. Lacerate the organs and isolate the larvae under dissecting microscope. ANCYLOSTOMA DUODENALE & NECATOR AMERICANUS Objectives and Requirements To learn the differential characteristics of two species of hookworm. To learn the morphological characteristics of ovum. To gain some knowledge regarding the environmental factors that might affect the development of hookworm larvae. To study the methods commonly used in laboratory diagnosis and epidemiologic survey. Based on the structural characteristics, to understand the pathogenesis of hookworm diseases. To understand the relationship of hookworm epidemiology with natural factors and crops cultivating. Observation and Experiment 1. Adult See the preserved adult worm (Demonstration). Note the shape, size and gross appearance of bursa of male worm. The body is thin and long. (2) See the male and female worms in copulation (Demonstration). (3) Study the characteristic structure of buccal capsule of Ancylostoma duodenale. Note two pairs of ventral teeth and a pair of accessory teeth. On the dorsal side of the mouth, a pair of small dorsal teeth is seen in the center. Study the characteristic structure of buccal capsule of Necator americanus. Note one pair of ventral cutting plates and a pair of dorsal cutting plates. See the bursa of A. duodenale (Demonstration). The female is with a small mucro at the tail end, while the male is with a characteristic copulatory bursa at the tail end, which looks like an opened umbrella, the ribs of which are called rays. See the bursa of N. americanus (Demonstration). In the male, the copulatory bursa is deep, with the two branches of the lateral ray tightly adhered to each other, and there is a hook on the tip of each spicule. In the female, the vulva opens a little anterior to the center of the body, and no spine exists at the tail end. (7) See the SEM photographs of buccal cavity and bursa of hookworm. 2. Ovum and larva Study the ova (Manipulation). Note the shape, size, color, shell and internal structure. 36～40μm×56～76μm in size, thin eggshell, nearly colorless and transparent. It contains egg cells under cleavage. See the ova. Note 2-, 4-, 8-cells, morula and embryonated ova (Demonstration). Study the embryogenesis of rhabditiform larvae (Demonstration). See the filariform larvae (Demonstration). See the activities of filariform larvae on water film of soil surface, waiting for suitable hosts (Demonstration). See the SEM photographs of ova and filariform larvae. 3. Pathological specimens and tissue section See the gross specimen of intestine showing hookworm grasping mucous membrane (Demonstration). Note the hemorrhagic petechiae and ulcers. See the section of the head end of hookworm grasping mucosa membrane of small intestine (Demonstration). The worms support themselves by biting at the root with cutting plate or ventral teeth, and suck blood. Exercise 1. Draw the hookworm ovum. 2. Draw and label the buccal capsules of A. duodenale and N. americanus. Thinking How does a hookworm infect host? And which dangerous can be caused to the host? What relationship between hookworm infection and natural environment is? Reference 1. Methods used in laboratory diagnosis of hookworm larva. Direct fecal smear and NaCl saturated floatation (the methods are described in details at Examination of Alimentary Helminths on page 268 of the Chinese textbook). See demonstration showing cultivation of larvae. 2. Methods used in epidemiologic survey of hookworm. Baermann's technique. Put triple-layered gauze on a sieve. To the gauze add the soil sample. Put the sieve on a funnel with clamped rubber tube connecting its lower end. Fill the funnel with 40℃ water until its level is raised to touch the lower portion of the soil. After 20 minutes, open the clamp and let out the lowest portion of water to a beaker or centrifuged tube. Let the collected water stand for 10～20 minutes or centrifuge for 1～2 minutes, then examine the sediment for larvae. (2) Pad method. Soak a pad of six to seven layers of gauze in 40℃ water. Cover the soil with the warm pad to attract the larvae. Cover the pad with a petri dish to keep the former warm. After 20 minutes, rinse the pad in water repeatedly. Examine sediment for larvae. ENTEROBIUS VERMICULARIS Objectives and Requirements Study the structure of E. vermicularis ova and methods used in the diagnosis of E. vermicularis infection. Observation and Experiment (1) See the preserved adult worm of E. vermicularis (Demonstration). Note more or less spindle-shaped, whitish in color. The female worm is 8 mm～13 mm long and the male is only 2 mm～5 mm. The male has its posterior end strongly curved ventral, and the female tail portion is sharply pointed. (2) See the whole mount of adult worms (Demonstration). Study the preserved specimen of ova (Manipulation). Note their shape, size, color, shell and content. 50～60μm×20～30μm in size, elongate-ovoidal, flattened on the ventral side, thick and transparent eggshell, fully embryonated. Why the shape looks different in lateral and dorsal views? (4) See the SEM photograph of the anterior end of adult. (5) See the swab method for diagnosis of E. vermicularis (Demonstration). Wet a cotton swab with normal saline and rub corrugations in perianal area. Then stir the swab in NaCl saturated solution in a test tube (or penicillin bottle), and add NaCl saturated solution up to rim of test tube, cover the test tube with a clean slide in contact with the solution. Let it stand for 5～10 minutes. Examine the slide for ova. Exercise 1. Draw and label the ovum of E. vermicularis. Thinking 1. Why there are more infections of E. vermicularis in rural area than in city? Reference Angiostrongylus cantonensis The rat lungworm, A. cantonensis, is widespread, and the infection seems to be increasing in rats and bandicoots in the tropics and subtropics, very probably through the agency of infected rats traveling as stowaways on ships.It has been found in rats in New Orleans and in Egypt, where human cases are not known to occur. Human infections that cause eosinophilic meningitis have been reported from Hawaii, Tahiti, Japan, mainland China, Taiwan,Thailand, Vietnam, Malaysia, Indonesia, Vanuatu, American Samoa, and the Ivory Coast. Unconfirmed cases have been noted from other Pacific islands, Hong Kong, the Philippines, Papua New Guinea, Australia, New Caledonia, Réunion, Mauritius, Cuba, and Puerto Rico. A. cantonensis is a slender worm, up to 25 mm long. Larval stages develop in slugs and land snails. When eaten by rats, the larvae migrate to the meninges and develop in the brain for about a month. Young adults then migrate to the pulmonary artery, where they attain maturity. The incidence of this infection in rats and snails may be quite high in endemic areas. In human hosts, Angiostrongylus does not complete its developmental cycle. When third-stage larvae are ingested, they penetrate into blood vessels in the intestinal tract and are carried to the meninges but are unable to migrate to the lungs, as they do in rats. Rarely, worms develop to the young adult stage in the meninges, but they soon die, and it is the death of the larvae or young adults and the inflammatory reaction provoked by the dead worms that causes the characteristic signs and symptoms of human infection. A presumptive diagnosis may be made in patients from areas where the disease is endemic on the basis of meningitis with blood and spinal fluid eosinophilia. Lesions may be seen in the meninges by computed tomography (CT), with serologic confirmation of the infection by ELISA. Sources of human infection are slugs, land snails, or fresh-water prawns and other paratenic (transport) hosts (Fig. 9-28), which are often consumed raw in islands of the Pacific, in Thailand, and in Vietnam. In Thailand and Malaysia snails of the genus Pila are eaten raw, either mixed with vegetables or as a form of medication. The giant African land snail, Achatina fulica, has spread throughout the Pacific islands and is apparently a common vector. Raw prawns are a frequent article of diet in Tahiti, and larvae found in them are infective to laboratory rats. The contamination of fresh vegetables by carnivorous land planarians that have fed on infected snails appears to be another important means of infection in New Caledonia, and perhaps elsewhere. Symptoms. The incubation period of the disease varies and apparently can be as long as 47 days. Infection in man is usually benign and se]f-limited, although fatalities have occurred. Symptoms of meningitis or meningoen- cephalitis--headache and stiff neck, often with sensorial changes--are of abrupt onset. A radiculomyeloencephalitis, with pains and paresthesias of the lower trunk and legs, bowel and bladder dysfunction, and one death, were seen in 16 of 21 Korean fishermen who ate giant African land snails in Samoa and in none of five who ate cooked snails. A CSF pleocytosis of more than 500 per ul occurred in 80 per cent of infected persons, and on autopsy worms were found in the subarachnoid space of the lumbar cord, with invasion of the white and gray matter. The spinal fluid usually contains 100 to 2000 white blood ceils per ul generally accompanied by a marked eosinophilia. Other common symptoms include nausea and vomiting, fever, and, early in the infection, abdominal pain, malaise, and constipation. A blood eosinophilia is also common; total leukocyte counts are moderately elevated. Eye invasion is marked by visual impairment, ocular pain, b]epharo- spasm, circumcorneal injection, keratitis, cells and flares in the anterior chamber and vitreous, iritis, and retinal edema. Living worms have been noted, and on occasion removed surgically. Pathogenesis. Little is known about the effects of this parasite on the central nervous system, since most patients recover uneventfully. On autopsy, sections of immature Angiostrongylus have been seen in the cerebrum and cerebellum, as well as the spinal cord, associated with infiltrates of eosino- phi]s,-monocytes, and foreign-body giant cells. Marked tissue necrosis has been seen in some areas in connection with dead worms. Immature worms have been found in spinal fluid obtained by lumbar puncture; adult worms have been found in the eye and the pulmonary artery. Treatment. Trials of specific therapy are still inconclusive. Thiabenda- zole and mebendazole have some effect in animal infections, but thiabenda- zole was found to be ineffective in reported human cases. Symptomatic treatment is all that is needed in the majority of these infections, but where specific anthelminthic treatment seens necessary, mebendazole, 100mg twice daily for t days, is a recommended investigational regimen for adults. WUCHERERIA BANCROFTI & BRUGIA MALAYI Objectives and Requirements l. To study the life cycle characteristics and epidemiologic features of filariae. 2.To study the morphology of microfilariae and laboratory diagnostic methods. Observation and Experiment 1. Adult See the preserved adult worm of Wuchereria bancrofti (Demonstration). Adults look like thin and long threads. (2) See the preserved adult worm of Brugia malayi (Demonstration). The adults resemble that of W. bancrofti in morphology, but is thinner and shorter. (3) See the preserved adult worm of Loa loa (Demonstration). The adults take a thread-like form, and there are small lumps on the body surface. (4) See the preserved adult worm of Dirofilaria immitis (Demonstration). The adults look like thin white noodles, and the tail of the male is coiled. 2. Larvae Study the structures of W. bancrofti microfilariae stained specimen (Manipulation). Note the shape, appearance, sheath, cellular column and caudal nuclei. The larva is sheathed by egg membrane, the excretory pore is adjacent to the excretory cell, one of the genital cells (G-cells) is far apart from the others, and there is no nucleus at the tail end. (2) Study the structures of B. malayi microfilariae stained specimen (Manipulation). Compare the structures with those of W. bancrofti microfilariae. The larva is sheathed and shorter, the excretory cell is located separately from the excretory pore, all of the fore genital cells are situated far ahead of the anus, the first cell (G-1) being especially large, and the tail end has two terminal nuclei. (3) Observe the microfilariae in unstained blood film. (4) See the infective larvae in mosquito thoracic muscles (Demonstration). (5) See the infective larvae in mosquito proboscis (Demonstration). 3. The intermediate host (Demonstration) Culex quanquefasciatus and Anopheles sinensis. 4. Pathogenesis (Demonstration) (1) See the photograph showing the patient with elephantiasis of lower extremity and scrotum. (2) See the photograph showing the patient with hydrocele. Exercise 1. Label the microfilariae. Thinking 1. What are the epidemiologic distribution characteristics of filariasis of W. bancrofti? 2. To describe the pathogenesis of W. Bancrofti and B. Malayi. Reference Laboratory diagnostic methods for filaria (1) Thick blood film Sterilize the ear lob with 75% alcohol and puncture with needle. Place on a clean slide two to three drops blood approximate 60 mm3 and spread it with the corner of another slide to equal the diameter of two-fen coin. Let it dry. Put it in water, the thick film becomes whitish in color and examine it under microscope. (2) Examination of live microfilariae (Demonstration) Place two drops of patient's blood (or dog infected with Dirofilaria) and cover with coverslip. Note their wiggle among the RBC. (3) Examination of venous blood for microfilariae (Demonstration) Mix 1ml of venous blood (taken at night) with o.1 ml 3.8% sodium citrate in a centrifugal tube, then add 9 ml of distilled water to break the RBC. Centrifugalize for 2 minutes at 3 000 rpm. Decant the supernatant and examine the sediment for microfilariae. TRICHINELLA SPIRALIS Objectives and Requirements To learn the diagnostic method of T. spiralis. To understand the life cycle and pathogenesis of T. spiralis through animal experiment. Observation and Experiment See the preserved adult worms (Demonstration) The adults look like pieces of thread, being 2.2 mm～3.0 mm long in the female and 1.2 mm～1.5 mm long in the male. See the stained specimen of cyst (larva) in muscular tissue (Demonstration) Larvae that reach striated muscles invade muscular fibers to be encysted. With the development of the larva, the cyst wall is thickened to form a cyst of characteristic spindle form, its major axis being in parallel with the muscle fibers. Usually, each cyst has one coiled larva. 3. See the live cyst (Demonstration) 4. Experimental infection of mice and examination (Students work in groups) Take buccal muscle or diaphragm of infected mice, cut into pieces and feed the normal mice. Four weeks later, kill the infected mice. Examine (l) buccal muscle, (2) diaphragm and (3) heart muscles by placing a small piece of the referred tissue between two slides and examining under microscope for cysts. (4) dissect and wash the small intestine to find the adults. Exercise 1. Draw the cyst of T. spiralis. 2. Record the result of the infection experiment and give a brief analysis. Thinking Which kinds of domestic animals can be the reservoir host of human parasites? What are the differences existed in the distribution of biohelminth and geohelminth? EXAMINATION OF ALIMENTARY HELMINTHS Objectives and Requirements 1 To learn methods commonly used in stool examination. 2. Self-stool examination for helminth eggs and therapy. Methods 1．Direct fecal smear. Transfer a small amount of fecal sample to 2 drops of saline on a slide. Mix to obtain a fairly dense uniform smear free of large lumps. Some practice is necessary to judge the density, that is: one can see the strokes of words in the newspaper through the transparent smear. Identify parasite ova and distinguish them from food residues such as various kinds of plant cells, yeast, pollen, plant fiber etc. The fecal smear must be kept wet during examination. NaCl saturated solution floatation. Mix small lumps of fecal material (about peanut-sized) with small amount of NaCl saturated solution in a penicillin bottle. Then add up to the rim of bottle, cover with a coverslip and allow to contact with it (avoid any air bubble). After 20 minutes, examine the coverslip on a slide for ova. Sedimentation. Mix 5 g～10 g of fecal material with small amount of water in a beaker (or test tube). Dilute with water and filter away the bulky undigested residues. Let the filtrate stand for 15～20 minutes. Discard the supernatant and add water again, thus repeat several times until the supernatant is clear. Decant the supernatant fluid and examine sediment for ova. Exercise Record the result of self-stool examination. Try to compare the advantages and disadvantages of the three methods used in the practice. Reference Quality control for fecal examination. Reliable and accurate parasite identification depends on: Collecting satisfactory specimens. Preparing and maintaining reagent correctly. Careful performance of appropriate techniques and thorough examination of finished preparation. ENTAMOEBA HISTOLYTICA, E. COLI & OTHER AMOEBAE Objectives and Requirements 1. To study morphological structures of trophozoite and cyst of E. histolytica. 2. To study laboratory diagnostic methods of E. histolytica. 3. To understand the pathogenesis of E. histolytica. 4. To learn differentiating E. hisiolytica from E. coli. Observation and Experiment 1. E. histolytica (1) Trophozoite Study the iron-haematoxylin stained specimen of E. histolytica (Manipulation). Find the parasite under high power, then observe with oil immersion lens. Note ectoplasm and pseudopodia, endoplasm with fine granules. Some of the trophozoites may contain RBCs which appearance varies with process of digestion. Food vacuoles can also be found in the endoplasma. The spherical nucleus has definite nuclear membrane, the inner surface of which is lined with uniform and closely packed chromatin (peripheral chromatin). The central karyosome is deeply stained. Study the living trophozoites in fecal material (Manipulation). With a toothpick, remove some material from mucus or flecks of blood in patient's stool (or from culture medium), mix with saline on a slide, cover and examine. It is advisable to use rather weak illumination. Observe with care the appearance, motility and the cytoplasmic inclusion, e. g. RBC (if specimen obtained from patient) or starch (if specimen obtained from culture). The fecal samples must be examined immediately after collection or be kept at optimal temperature so as to maintain the motility of the trophozoite; it is particularly so during winter. (2) Cyst Study the iron-haematoxylin stained specimen of E. histolytica cysts (Manipulation). Using oil immersion lens, study the spherical cyst with a hyaline cystic wall, the number of nucleus may be single, two or quadrate. The structure of nucleus is the same as trophozoite. The glycogen is readily dissolved in stained specimen and only vacuole is left. Chromatoid bodies are rod-like masses with round ends. In fully mature cysts, the chromatoid bodies are often lacking. Study the iodine stained specimen of cysts (Manipulation). Stain fresh fecal material by adding a drop of iodine solution to the edge of coverslip. The cysts stain yellowish, the glycogen is brownish yellow, the nuclei are hyaline unstained sphere-like. (3) Cultivation of trophozoite (Students work in groups). With sterilized technique, add 0.5 ml of inactivated serum and a small sprinkling of rice starch and a few drops of penicillin solution to the medium, inoculate the fecal sample containing trophozoites (about 0.1 ml). Incubate at 30℃ for one week and examine. (4) Pathologic specimens (Demonstration). See the gross lesions in the intestine. Note the ulcers vary from pin point to the size of rice grain, with central erosion surrounded by elevated edematous tissues forming a niche of a volcano. Superficial ulcers are formed due to detachment of necrotic mucosa, or large ulcerated patches due to coalescence of small ulcers. Mucosa between ulcers is usually normal. See the section of intestinal lesions. Note flask-shaped ulcer, numerous trophozoites and infiltration of leukocytes can be seen. See the amebic hepatic abscess. Abscess of various size can usually be seen in the right lobe, singly and discrete. The abscesses are surrounded by necrotic tissue, thus the edge of the abscess appears irregular and spongy, while the abscess fluid is brown in color. (5) See the photographs of trophozoite and cyst. 2. E. coli See the iron haematoxylin stained E. coli trophozoites (Demonstration). Compare the size of trophozoite, the nuclear chromatin, position of karyosome and endoplasmic inclusions with those of E. histolytica. Study the iron haematoxylin stained specimen of E. coli cysts (Manipulation). Note and compare the size, number of nuclei, nuclear structure and chromatoid bodies with those of E. histolyitca cyst. 3. Other non-pathogenic amoebae in human alimentary tract See the E. gingivalis trophozoites (Demonstration). Note the distinct ectoplasm and central or slightly eccentric large karyosome. See the live Endolimax nana trophozoites (Demonstration). Note the small size, sluggish motility, blunt pseudopodia large central or eccentric karyosome, peripheral chromatin seldom seen; See demonstration of E. nana cyst. Note small size, oval shape, l～4 nuclei, karyosome large and irregular without peripheral chromatin, usually no chromatoid bodies. See the live Iodamoeba butschlii trophozoites (Demonstration). Note the small size, spherical shape, sluggish motility, blunt pseudopodia, large central karyosome surrounded by refractive granules, usually no peripheral chromatin. Food vacuole in endoplasm contains bacteria. See the I. butschlii cysts (Demonstration). Note the irregular or spherical shape, one nucleus, large eccentric karyosome with peripheral chromatin, no chromatoid bodies. The most conspicuous feature is the large glycogen vacuole, stains golden brown with iodine. See the E. polecki cysts This is an incidental parasite of man. Note spherical shape, one nucleus, central or eccentric karyosome, dense uniform peripheral chromatin, rod-like chromatoid bodies. Exercise Draw the trophozoite and cyst of E. histolytica and cyst of E. coli. Thinking How does amebic dysentery be transmitted? How to diagnose the intestinal lesion and the exo-intestinal lesion of E. histolytica? GIARDIA LAMBLIA Objectives and Requirements 1. To study the general structure of G. lamblia trophozoite and cyst. 2. To learn the diagnostic method. Observation and Experiment Study the iron haematoxylin stained trophozoites (Manipulation). Note inverted pear-shape, convex dorsal surface and concave ventral surface (sucking disc). Two nuclei with large karyosome at the anterior, two axostyles and four pairs of flagella. Study the iron haematoxylin stained cysts (Manipulation). Note shape, well defined wall and two to four eccentrically located nuclei. 3. See the SEM photographs of G. Lamblia (Demonstration). Exercise l. Draw the trophozoite and cyst of G. lamblia. Thinking Why people infected G. lamblia may suffer diarrhea? TRICHOMONAS VAGINALIS Objectives and Requirements 1. To study the general structure of T. vaginalis trophozoite and cyst. 2. To learn the diagnostic method. Observation and Experiment Study the stained trophozoites of T. vaginalis (Manipulation) Broad end of the body, 4 flagella, a posterior flagellum, a short flagellum, a nucleus near the origin of the anterior position, axoneme. See the live trophozoites of T. vaginalis (Demonstration) Examination of trophozoites of T. vaginalis Direct wet film, using vagina excretory. Exercise l. Draw the trophozoite of T. vaginalis. Thinking What are the transmission routes of T. vaginalis? Reference Direct examination of vaginal and urethral smears (1) With a sterile cotton swab, collect the vaginal or urethral discharge. Put the swab immediately into a sterile tube containing about 3 ml of sterile saline. (3) Smears on a slide, and smear for staining can be made if desired. Finding of T. vaginalis in vaginal secretions, prostatic secretions and urine. Centrifuged or sediment material If a swab in saline is received, remove the excess fluid from the swab by squeezing it against the side of the tube. Centrifuge the tube for 2 minutes. If a centrifuge is not available, let the tube stand for 10 minutes to allow any sediment to settle on the bottom. With a pipette remove the supernatant fluid. Take a drop of the sediment and put on a microscope slide. Cover with a coverslip and examine with 10×and 40×objectives for motile flagellates. LEISHMANIA DONOVANI Objectives and Requirements 1. To study morphological structures of amastigotes and promastigotes of L. donovani. 2. To understand methods used in the diagnosis. 3. To understand the pathogenesis of L. donovani from parasitized site. Observation and Experiment Study the stained smear of amastigotes (or non-flagellar forms) (Manipulation) Look for reticulo-endothellial cells harboring the amastigotes. Note the number of amastigotes in one host cell. They are very small and have the appearance of granules in the cytoplasm of macrophages. When the host cells are ruptured, amastigotes are discrete. Study carefully under microscope with oil immersion lens, note the discrete amastigotes, its size in respect to the size of host cell. Identify the blue cytoplasm, red nucleus, basal body and kinetoplast. See the stained specimen of promastigote (or flagellar from) (Demonstration) Note the spindle shape, flagellum, nucleus, basal body, rhizoplast and kinetoplast, the last three can hardly be separated from each other and often seen as one purple dot. See the live promastigote (Demonstration) Place a drop of culture medium on a slide. Cover and note the rapid, lashing movement of the flagellum that actively propels the parasite forward. 4. See the SEM photographs of promastigote and amastigote (Demonstration) 5. See adult sandfly, the vector of L. Donovani (Demonstration) Exercise Draw the macrophage contains amastigotes, and a free promastigote of L. donovani. Thinking A person suffered from visceral leishmaniasis, how to diagnose it pathogenically? Reference Culture of L. donovani Inoculation of NNN culture medium with spleen juice, blood, or bits of excised dermis is a reliable procedure. Here we particularly recommends seeding 3 or 4 NNN culture tubes with the sediment obtained by centrifuging 2 ml～5 ml of blood added to four times its volume of citrated saline. The tubes are incubated at 22℃～24℃, and flagellates appear in 7 days or later in 90 per cent of untreated cases. Materials. Difco blood agar base 8 g Distilled water 200 ml Defibrinated rabbit blood 0.6 ml in each 5 ml of medium Preparation of defibrinated rabbit blood. Collect 20 ml of rabbit blood into a sterile flask containing about 100 glass beads of 4 mm diameter. Defibrinate the blood by rotating the flask for 5 minutes. Add 200 u of penicillin, 200 mg of gentamicin, and 2 mg of streptomycin per ml of defibrinated blood. Preparation. Pour 200 ml of water into a flask, add the agar to the water, mix, and warm the flask in boiling water until the agar is completely dissolved. Dispense the medium in 5 ml amounts into screw-cap bottle (20 ml capacity). Sterilize by autoclaving (with caps loosened) at 121℃ for 15 minutes and allow the agar to cool to 45℃～50℃. Add 0.6 ml of sterile defibrinated rabbit blood to each bottle and mix gently. Allow the medium to solidify with the bottles in a sloped position. Leave the bottles in an upright position at room temperature for 24 h to allow fluid of condensation to form. The bottles should be stored at 4℃～6℃ until required. Note: The medium should be prepared in aseptic working conditions. Use. Inoculate about 0.1 ml of specimen aseptically into the fluid of condensation of each of 2 bottles at room temperature. Incubate the cultures at 24℃(±2℃) in the dark. Examine every 4 days. Transfer a drop of the culture using a sterile wire loop to a slide for examination for promastigotes. Note: Negative cultures must be subcultured after 8 days into fresh medium and examined every 4 days for a further 20 days. Detection of Leishmania in lymph node aspirates Parasites may be demonstrated in aspirates of spleen (98% positivity), bone marrow (54%～86%), or enlarged lymph nodes (64%). When the spleen is small and soft or impalpable, bone-marrow or lymph-node aspiration is recommended. For diagnosis of Leishmania it is especially using bone-marrow or lymph-node aspiration. Prepare the syringe; pull the piston as far back as possible. Ask the person to sit down. Disinfect the chosen site on the neck with 70% ethanol. With left hand, take the gland between the thumb and index finger and make it stand out. Holding the needle between thumb and finger, introduce it at right angles into the center of the gland. First pierce the skin, then penetrate the center of the gland. With left hand, gently knead the gland. With right hand, revolve the needle in both directions. The glandular fluid will ooze into the needle. The operation should last about one minute. Withdraw the needle in one rapid movement, holding index finger over the hub. Apply a swab dipped in disinfectant to the point of entry. Attach the needle to the syringe, with the piston pulled back. Push the piston gently half way down the barrel to discharge the glandular fluid in the needle on to the slide. Cover the preparation with a coverslip. Examine at once under the microscope at a magnification of approximately 400×, using the 40× objective. Wait until the convection currents stop. Then examine the preparation. PLASMODIA Objectives and Requirements 1.To study the life cycle of Plasmodia and understand their pathogenic mechanism. 2. To study laboratory diagnostic methods of malarial parasites. 3. To study morphological structures of Plasmodia, to identify morphological structures of developing stages of erythrocytic schizogony and gametocytes of P. vivax, and to differentiate the ring-form and gametocytes of P. vivax from P. falciparum. 4. To identify of insect vector of malaria—Anopheles. 5. To learn simple method of experiment with Plasmodium. Observation and Experiment 1. P. vivax (Erythrocytic stage) (Manipulation) Ring form Compare its size in respect to the infected RBC. Note the delicate blue-stained ring of cytoplasm and a red chromatin dot. Trophozoite Compare the small and large trophozoites, do they have definite form? Note the vacuole, changes of chromatin, pigment, alteration of infected RBC and Schuffner's dots. The cytoplasm with the nucleus begins to enlarge while the ring form becomes irregular in shape and sometimes projects pseudopodia. Schizont Note the number and arrangement of chromatin, distribution of cytoplasm and aggregation of pigment granules. It becomes oval or round body, and its nucleus begins to segment. The number of the nucleus in the mature schizont is from 12～24, usually 16. Gametocytes Note the size, shape, position of chromatin, distribution of pigment granules and alteration of infected RBC. What are the morphological differences between male and female gametocytes? 2. P. falciparum (Erythrocytic stage) (Manipulation) Ring form Note its small size and delicate ring, about 1/5 the diameter of erythrocyte. Sometimes there are two chromatin dots in one ring form or multiple infection in one RBC. Gametocytes Note their particular sausage or crescent shape, position of chromatin, distribution of pigment granules and alteration of infected RBC. 3. P. malariae (Erythrocytic stage) (Demonstration) (l) See the ring form. Note the large chromatin dot, dense cytoplasm and its size in respect to infected RBC. (2) See the trophozoite. Note the band form, its cytoplasm compact, pigment coarse and early appearance. See the schizont. Note mature schizont, the merozoites 6～12 in number, arranged as "rosette" central mass of dark brown pigment granules. See the gametocytes. Resemble those of P. vivax, except smaller in size, coarse dark pigment granules, alteration of infected RBC rare. See the SEM photographs of P. vivax and P. falciparum 5. Exo-erythrocytic stage (Demonstration) 6. Development of Plasmodia in mosquito host (Demonstration) (1) See the oocyst in the intestinal wall of mosquito. (2) See the sporozoites in the salivary gland smear of infected mosquito. 7. Inoculation of P. berghei (Students work in groups) Obtain blood from infected mouse by cardiac puncture (or orbital puncture), dilute in 1 ml saline inject 0.1 ml diluted blood to a mouse intraperitoneally. Rear the mouse and examine next week. 8. Preparation of blood film of malarial parasites and staining (Manipulation) (l) Preparation of both thin and thick blood films at one slide. Collect two drops of blood from the cut tail of infected mouse and place on one end of a clean slide. Spread the drop near end to the size of two-fen coin with the corner of another slide to make up thick film. Holding another slide at an angle of 30°～45°, and in contact with the other drop of blood, the first touch the drop of blood and let it spread along the line of contact between the slides. Then push the slide along, with a smooth, rapid movement, thus drawing the blood out to form a thin film. Then let it dry. Its thickness will depend on the size of the drop, the angle between the slides and rapidity with which the smear is made. (2) Giemsa's staining. Fix the dried thin film with methyl alcohol (avoid fixing the thick film) and let it dry. Cover with 2% Giemsa's solution on the smears. Let stay 30~60 minutes. Wash by pouring neutral distilled water over the slide until color does not run from it to a noticeable extent. Drain and stand on end to dry. 9. Vector of malarial parasites—Anopheles (Demonstration) (1) An. sinensis (2) An. Minimus (3) An. dirus 10. See the SEM photographs of merozoite (Demonstration) Exercise l. Draw ring form, trophozoite, schizont and gametocytes of P. vivax. 2. Draw ring form and gametocytes of P. falciparum. 3. Label SEM photographs of merozoite. 4. Record the result of artificial infection of P. berghei in mice. Thinking According to the life cycles of Plasmodium, explain the paroxysm, recrudescence and relapse. What are the epidemic characteristics of malaria? Reference Acridine orange fluorescent stain Fix the thin smear with methyl alcohol, then immerse it in 0.01% acridine orange solution for 4 minutes, rinse with water, then immerse in distilled water for one minute. Add one drop of water and cover it, examine under fluorescent microscope. DNA of chromatin shows yellowish green fluorescence, while RNA of cytoplasm orange-yellow fluorescence. Indirect fluorescent antibody test (l) Antibody: rabbit-anti-human IgG fluorescent antibody to be diluted to 1:8 (or 1:16) with PBS, pH8.0. (2) Antigen sample: dry blood films made with blood collected right after clinical attacks. Hemolyse the slide in 0.1N HCl for 5 minutes, wash in running water, immerse in PBS for 5 minutes, then air dry. (3) Serum for examination: serum sample to be diluted with PBS to 1:20. (4) Staining. Spread one drop of diluted serum sample on the antigen blood film, put in a moistened box and transfer to an incubator at 37℃ for 30 minutes to allow antigen-antibody reaction. Wash the blood film with PBS for 1 minute, immerse in PBS for 5 minutes, repeat once, then air dry. Add one drop of antibody containing Evans blue (in dilution 1:10 000) on the blood film where Ag-Ab reaction took place, put the slide in a moistened box into incubator at 37℃ for 30 minutes. Wash away the excessive fluorescent antibody as step B. Mount the dried blood film with carbonate or phosphate buffered glycerin, or just add a small drop of PBS (pH8.0) onto it and cover. Examine under fluorescent microscope. Criteria of diagnosis. Grade the result according to the fluorescence density on the schizont or trophozoite. +++ — ++++: fluorescence on plasmodial cytoplasm is bright to brilliant, structures clear. ++: fluorescence bright, structures clear. +: cytoplasm clearly visible but structures not very clear. ±: only faint fluorescence visible on the plasmodial cytoplasm and RBC shadow, parasite structures not clear. -: no fluorescence visible on plasmodial cytoplasm. All sample graded higher than “+” are considered positive. ParaSight-F rapid manual diagnostic test of P. falciparum Introduction Current diagnostic methods are based on microscopic examination which takes 5～10 minutes for staining and up to 20 minutes for reading, and requires a well-maintained microscope and an experienced microscopist. New techniques, such as hybridization with DNA probes, are too sophisticated for routine use in the field. (2) Test principal ParaSight-F test is a qualitative diagnostic test of P. falciparum, which is based on the detection by monoclonal antibody of a species-specific soluble antigen histidine-rich protein (HRP-Ⅱ), a special glycoprotein of P. falciparum secreted during the parasite's erythrocytic cycle, with a peak during schizont rupture in whole blood and which can be performed without special equipment. A visual reading is given by a polyclonal antibody coupled with dye-loaded liposomes; when positive, a pink line appears. A rapid dipstick antigen capture assay for the diagnosis of P. falciparum (1) Principal Recent advances in the diagnosis of P. falciparum infection have made it possible to consider supplementing light microscopy with a standardized dipstick antigen capture assay based on the detection of HRP-Ⅱ. The stability, reproducibility, and ease of use of the assay clearly indicate that it has potential for application in the management of malaria, particularly at the peripheral health care level, provided its accuracy can be assured and that it can be made affordable. A specific assay for P. falciparum is now commercially available and a similar assay for P. vivax is under development. Steps Add lysing fluid to tube. Take blood sample from fingerprick. Mix blood with anticoagulant in capillary tube. Transfer blood sample to lysing fluid. Place dispensing tip on sample tube. Place lysed blood sample in well. Stand dipstick in blood sample. Add detection agent to well. Add washing fluid to well. In positive cases, a pink line develops almost simultaneously at the monoclonal antibody deposit site with a pink broken line above it as the reagent control. In negative cases, only the pink broken line occurs. ICT Malaria P.f / P.v test (1) Introduction Malaria diagnosis has been a difficult process requiring the skills of highly trained microscopists to detect Plasmodium in stained blood films. In addition to detection of parasites, the ability to differentiate Plasmodium falciparum from other malarial species is important for the early and effective treatment of infection with this potentially lethal organism. The introduction of the ICT Malaria P.f / P.v test combines rapid and reliable diagnosis of malaria with the ability to differentiate P. falciparum and P. vivax infections. Test principle The ICT Malaria P.f / P.v test is an in-vitro immunodignostic test for the detection and speciation of P. falciparum and P. vivax in whole blood. Based on immunochromatographic technology, this test uses antibodies to identify specific antigens associated with P. falciparum and P. vivax infections. A finger prick blood sample is all that is required for the step procedure which gives results in eight minutes. Malarial antigens are detected using colloidal gold labeled antibodies. In a positive test, pink lines develop in the test window. The location of these lines identifies the Plasmodium species detected, while development of a procedural control line indicates that the test has been performed correctly. Quality control for blood examination Equipment must be clean. Films must be of correct density. Films must be allowed to dry in a horizontal position and for the correct time to ensure good results. The stain dilutions and the buffered water used for staining must be accurately prepared and the stock stain must be of good quality. The staining procedure should be followed very carefully. OPPORTUNISTIC PATHOGENIC PROTOZOAN Pneumocytis carinii, Toxoplasma gondii & Cryptosporidium Objectives and Requirements To obtain a primary understanding of the relation between the immunity of infected host and opportunistic pathogenic protozoan. To observe the structural features of several species of opportunistic pathogenic protozoan. Observation and Experiment 1. Pneumocytis carinii (1) See the stained specimen (Demonstration) Trophozoite, note its size, various shape of nucleus, and the color of both cytoplasm and nucleus. Cyst, note its shape, size and the number of sporozoites within it. (2) Animal experiment (Students work in groups) Introduction The establishment of parasitic infection implies that the parasites come together with their hosts and the interaction begins. As soon as the parasites enter the host, first thing is that they have to struggle against the host immunity before they multiply enough to injure the host and usher in clinical symptoms. In some cases, for example in the opportunistic pathogenic protozoan, the outcome of the interaction is usually establishment of the balance between the parasites and their host. That is the certain numbers of the parasites survive within the host but none of the related clinical symptoms are observed, which is called suppressive infection. However, the balance requires existence of certain conditions of both host and the parasites. Any change of the related conditions would result in the appearance of imbalance. For instance, the decrease of the host immunity might lead to dramatic multiplication of the protozoan in the state of suppressive infection, and consequently the occurrence of acute attack. The protozoan of these characteristics, including P. carinii, T. gondii and species of Cryptosporidium, are called opportunistic pathogenic protozoan. This is why the P. carinii infection is fatal to the AIDS patients. In this experiment, the immunity of observed animals are artificially suppressed by administration of immuno-suppressed drugs before they are infected with P. carinii, and the observations are carried out on the changes in the body weight, physiological state, pulmonary pathological change of the hosts, as well as examination for pathogens in the hosts. B. Materials ① Experimental animal: white rat. ② Homogenate of infected lung tissue with P. carinii. ③ Immuno-suppressed drugs: dexamethasone, cortisone acetate. ④ Nourishment: low protein diet; drinking water with tetracycline 1 mg/ml. C. Methods ① Experimental Group. Six white rats are used in the experimental group. The animals are orally administrated with l mg/L dexamethasone in drinking water or intradermally injected with cortisone 12.5 mg/100g body weight two times a week for six weeks. Two weeks after drug administration, three of them are transtracheally inoculated with 0.15 ml homogenate of infected lung tissue with P. carinii. At sixth week, the animals are stopped feeding of immuno-suppressed drug and one or two out of both transtracheally inoculated infected and the other non-infected subgroups are dissected respectively for observations on the pulmonary pathological changes and examination for P. carinii by stained smear of lung tissue. The remainders of the two subgroups are left till they are dissected at the eighth week for the same observation as conducted at the sixth week. Each of the animals treated with immuno-suppressed drug is muscularly inoculated once a week with PG 40 000 u and streptomycin 20 000 u. Control Group. Three white rats are used in the control group. They are free from the immuno-suppressed drug and two of them are transtracheally inoculated with 0.15 ml homogenate of infected lung tissue with P. carinii. All three animals are then dissected at the sixth week for the same observations as experimental group. Stop immuno-suppressed drug at the 6th week, dissect one or two rats out of both experiment and control groups, observe the pathological changes in the lungs and smear on slide for examination of parasites under microscope. ④ The remainders are left for two weeks more before they are dissected and observed as described in step C. Both experimental and control groups of animals are weighed and observed for physiological state once a week. The results of weighing and observation are recorded. The route of the experiment is outlined as the following graph. Experimental group Control group 2 weeks normal diet 2 weeks normal diet +cortisone transtracheal transtracheal inoculation inoculation normal of P.carinii of P.carinii diet 4 weeks 4 weeks 4 weeks 4 weeks dissect stop stop dissect dissect dissect drug drug 2 weeks 2 weeks dissect dissect 2. Cryptosporidium (l) See the stained oocysts (Demonstration). Note its size, shape and number of sporozoites within it. 3. Toxoplasma gondii (l) See the stained tachyzoites and pseudocysts (Demonstration). Note its size, shape, and the color of both cytoplasma and nucleus. Pseudocyst is factually a macrophage containing tachyzoites with the number from several to tens. (2) See the stained cysts (Demonstration). Note its size, shape, cystic wall and the number of bradyzoites within it. Exercise Based on the recorded results, to write the experimental report on the relationship between opportunistic pathogenic protozoan infection and the immunity of the host, with analysis, discussion and conclusion. Reference The pathogenic examination of T. gondii and animal inoculation Liquid material: Blood, using blood film. Cerebrospinal fluid (CSF) or ascites, examining the sediment after centrifugation. The live trophozoites are crescentic in shape, move actively. Solid material: Examined by cutting section pathologically. If negative, using animal inoculation. Method: Grinding tissue to become paste-like, add saline to dilute it, inject into mouse abdomen, tap the abdomen 7 days later and examine ascites for trophozoites. MOSQUITOES Objectives and Requirements 1. To study morphological characteristics of developing stages of mosquitoes. 2. To understand mosquitoes as vectors of mosquito-borne diseases. 3. To learn differential characteristics of Anopheles, Culex and Aedes. Observation and Experiment 1. See three genera of live adult mosquitoes (Demonstration) (1) Anopheles (2) Culex (3) Aedes Note the differences in color, wing spots, position of head at rest, abdominal segments covered with scales of various colors. Study the desiccated specimens of adults of three genera of mosquitoes (Demonstration) Identify organs and appendages of head, thorax and abdomen. A pair of compound eyes, antennae, maxillary palps and a proboscis (mouth parts) of head; the thorax bearing 3 pairs of legs and one pair of wings; 10 segmented abdomen (the last two segments are modified to male genitalia). Structure of mosquito heads (1) Study whole mount of head of Anopheles (Manipulation). Identify compound eyes, 15 jointed antennae with intersegmental hairs. The mouthpart consists of a tubular labium terminating with two tiny labella, one labium and one hypopharynx, one pair of mandibles and one pair of maxillae with all these to form a cannula during blood sucking. Differentiate the male and female Anopheles. Note the head for the plumose antennal hairs of males and pilose hairs of females. Compare the length of antenna in respect to that of proboscis of both sexes. (2) See Culex head, both male and female, and compare (Demonstration). See the egg of Anopheles, Culex and Aedes (Demonstration) Which of them is floating on water in form of raft or scattered, with or without floats? Note the shape and size. See the live larvae of three genera (Demonstration) Note the siphon or spiracles and resting position of larvae. See the live pupae of three genera (Demonstration) Note the large anterior portion-cephalothorax with two respiratory trumpets which extend above the surface film and enable the pupae to obtain its air supply and the curve abdomen consists of visible segments assuring a "comma" shape. 7. See the photographs of breeding and resting places of mosquitoes (Demonstration) 8. Study the relation of mosquito to the diseases (1) Transmitting malaria (Demonstration). See the plasmodial oocysts on the intestinal wall of mosquito. See the plasmodial sporozoites in the salivary gland smear of infected mosquito. (2) Transmitting filariasis (Demonstration). See the infective filarial larvae in labium of infected mosquito. Transmitting Dengue virus (Demonstration). See the fluorescent stained specimen of Dengue virus in the salivary gland smear of the infected mosquito. Exercise l. Label head of Anopheles. Thinking How to identify the arthropod of Arachnida and Insecta? Which kinds of arbo-diseases (entomophilous diseases) can be prevented by eliminating mosquitoes in city? FLY Objectives and Requirements 1. To understand the basic morphology of different stages of common flies and their role as vector. Observations and Experiment Study the desiccated specimen of housefly (Manipulation) Identify head, thorax and abdomen. Note a pair of compound eyes and proboscis in the head; thorax bearing three pairs of legs and a pair of wings, 4 longitudinal black strips on the dorsum of thorax, abdomen segmented. 2. See the dry specimen of Chrysomyia megacephala (Demonstration) 3. See the dry specimen of Sarcophagidae species (Demonstration) 4. Study the external structures of head (Demonstration) Note the sucking mouthpart consisting of three parts. The proximal part, the rostrum, bears a pair of spin maxillary palps and is considered as a part of head proper; the middle region, the haustellum, is supposed to be homologous to labium; the expanded distal part is the oral plates made up of fleshy labella with tracheal structures. Study the structure of leg (Demonstration) Note the hair appearance of leg terminating in pad and claws. Observe carefully minute hairs on the pad. See the fly larvae (Demonstration) Note the shape, size, segmentation, spiracular plate and movement of living larvae. 7. See the preserved specimen of pupae (Demonstration) 8. See the fly eggs (Demonstration) 9. See the helminthic ova carried by housefly footpads (Demonstration) Exercise 1. Label the head and leg of fly. SANDFLY, FLEAS, LICE & OTHER BLOOD-SUCKING INSECTS Objectives and Requirements 1. To understand the basic morphology of different stages of sandfly, the vector of pathogen of kala azar. 2. To learn the morphological characteristics of flea, louse, bedbug, tick and mite. 3. To understand the relation of above arthropods to the diseases. Observation and Experiment 1. Sandfly (Demonstration) (1) See the adult. (2) See the eggs. (3) See the larva. (4) See the pupa. 2. Flea (Demonstration) See the adult. Note the shape, size and color. The whole body is laterally compressed with bristles and spines in posterior direction, genal or/and pronotal comb in some species, suctorial mouth parts, wingless and three pairs of legs (Genal comb-dark brown teeth on the ventral margin of gena. Pronotal comb–dark brown on the posterior margin of the dorsum of the first thoracic segment.). See the powerful leg terminating in two curved claws. The last pair is being greatly elongated for leaping. (3) See the eggs. 3. Louse (Demonstration) (1) See the whole mount of body louse. (2) See the whole mount of head louse. (3) See the whole mount of crab louse. Note the general features of these three species. The head bears a pair of eyes, a pair of 5-jointed antennae and an extensile piercing mouthpart. The thorax is composed of three fused segments. Each of which bears a pair of legs terminating in a single hook-like claw and tibial process for grasping hairs or fibers of clothes. The body and head lice differ only in size, the crab louse is small in size, indistinct segmented abdomen and large heavy claws. See the eggs. Note the ellipsoidal, operculated white eggs firmly attached to the hairs or fibers. 4. Cockroach (Demonstration) See Periplaneta americana. See Blattella germanica. 5. Tick (Demonstration) See the whole mount of adult hard tick. Note its large size, fused cephalothorax, piercing mouthpart and the chitinous (shield-shaped) covering the entire dorsal surface in the male and anterior part in the female; 4 pairs of legs. See the whole mount of adult soft tick. Note its large size, and fused cephalothoracic portion. The mouthpart is situated ventral to anterior end and can not be visible in dorsal view. No scutum. 6. Mite (Demonstration) (1) Sarcoptes scabiei See the whole mount of adult. Note its small size, oval shape, dorsal surface of the body with transverse ridges, spines and bristles. The mouthpart consists of toothed chelicerae. Pedipalps and labial palp fused to the hypostome, 4 pairs of short but stout legs, the first two pairs terminating in long tubular processes each with a bell shaped sucker and claws. (2) Leptotrombidium deliense A. See the eggs (Demonstration). See the whole mount of larvae (Demonstration). Note its tiny size, oval shape, capitulum, scutum and body hairs. Collect and observe live larvae. Anesthetize one mouse and inspect the ears for larval mites. Remove them, if any, with dissecting pen to a petri dish containing water, observe under low power lens. D. See the live nymphs (Demonstration). E. See the whole mount of adult (Demonstration). F. See the live larvae grouping at the tip of a conical object (Demonstration). Dermatophagoides (Demonstration) See the whole mount of adult. Note its small size, compact wrinkles of integument, two anterior pairs and two posterior pairs of legs terminating in long tubular processes each with a bell shaped sucker and claws. Demobex A. See the whole mount of adult (Demonstration). B. Self-survey for Demodex. 7. Bedbug (Demonstration) See the whole mount of adult. Note dorsoventrally flattened shape, color, body covered with short serrated hairs; a pair of prominent compound eyes, a slender and flexible mouthpart of head; three pairs of legs of thorax; ten segmented abdomen. Do you find the stink-glands between the basal parts of the second and third legs? (2) See the eggs. (3) See the larvae. Exercise 1. Report the result of examining demodex.