CLONORCHIS SINENSIS

Transcription

CLONORCHIS SINENSIS

EXPERIMENTAL
GUIDEBOOK OF
MEDICAL
PARASITOLOGY
Fujian medical university
Experimental guidebook of medical parasitology
CONTENTS
Care of the microscope…………………………………….…………………2
Ascaris lumbricoides & Trichuris trichiura …………………………………3
Ancylostoma duodenal & Necator americanus………………………………6
Enterobius vermicularis……………………………………………….……...9
Wuchereria bancrofti & Brugia malayi……………………………………..10
Trichinella spiralis…………………………………….……………….……12
Tapeworm…………………………………….……………….………….…13
Clonorchis sinensis…………………………………….……………….…...17
Paragonimus flukes…………………………………….……………….…..20
Fasciolopsis buski…………………………………….……………….….…22
Schistosoma japonicum…………………………………….……………….24
Examination of alimentary helminths…………………………………….…28
Entamoeba histolytica, E.coli & other Amoebae…………………………...30
Giardia lamblia…………………………………….……………….……….32
Trichomonas vaginalis…………………………………….………………...33
Leishmania donovani…………………………………….……………….…34
Plasmodia…………………………………….…………………….…….…35
Opportunistic pathogenic protozoan………………………………….……..37
Mosquitoes…………………………………….……………….…….……...38
Fly…………………………………….…………………….…….…….…...40
Sandfly, fleas, lice & other blood sucking insects…………………………..41
1
Care of the microscope
Do's
1. Do take special care to protect the microscope from dust in hot dry periods.
2. Do take special care to protect the microscope lenses and prisms from fungal growth
in hot humid periods.
3. Do clean the immersion oil from the immersion objective each time; use lens tissue
dampened with ethanol.
4. Do clean the oculars with lens tissue.
5. 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
1. Don't use the tissue used for the oil immersion objective to clean the oculars.
2. Don't dismantle or try to clean parts of the microscope that are difficult to reach.
3. Don't leave the lens parts empty; use the appropriate cover to cover the empty port.
4. Don't exchange lenses from microscopes of different manufacture.
2
ASCARIS LUMBRICOIDES &
TRICHURIS TRICHIURA
Objectives and Requirements
1. Know the life cycle and common morphology of nematodes.
2. Master how to identify ova of A. lumbricoides and T. trichiura.
3. Study the pathogenesis of A. lumbricoides infection.
Observation and Experiment
l. Ascaris lumbricoides
(1) Adult (Demonstration)
A. 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.
B. See the dissected male and female adult worm of A. lumbricoides.
Fig.1 Adult of Ascaris 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
(3) Study the cross section of A. lumbricoides.
Note the internal and external structures of body wall and body cavity (pseudocele).
A. 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).
B. Intestine: a flat or cylindrical tube, straight, and is lined by a single layer of cells.
C. 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
A. Study the morphology of fertilized ova of A. lumbricoides(Fig.2) (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 (Fig.2) (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).
D. 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)
A. See the gross specimen of intestinal obstruction due to ascariasis
B. See the gross specimen of biliary ascariasis
2. Trichuris trichiura
(1) See the preserved adult worms (Fig.3) (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.
(2) Study the preserved ova (Fig.4) (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
4
egg cell is uncleaved.
Fig.2 Egg of Ascaris lumbricoides: (a) fertilized egg. (b) unfertilized egg.
(1) Pathologic specimens.
Adults parasitic in human cecal mucosa, posterior part of the body being free.
Fig.3
Fig.4
Adult of T.trichiura
Egg of T.trichiura
Exercise
1. Draw the fertilized and unfertilized ova of A. lumbricoides.
2. Draw the ovum of T. trichiura.
5
ANCYLOSTOMA DUODENALE &
NECATOR AMERICANUS
1. Master the differential characteristics of two species of hookworm.
2. Master the morphological characteristics of ovum.
3. Understand the pathogenesis of hookworm diseases, based on the structural
characteristics.
4. Understand the relationship of hookworm epidemiology with natural factors and crops
cultivating.
1. Adult
(1) 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
(Fig.5).
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.
(1) Study the characteristic structure of buccal capsule of Necator americanus (Fig.5).
Note one pair of ventral cutting plates and a pair of dorsal cutting plates.
(2) See the bursa of A. duodenale (Fig.6)(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.
(3) See the bursa of N. americanus (Fig.6)(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.
(4) See the SEM photographs of buccal cavity and bursa of hookworm.
6
Fig.5
Buccal capsule of two human hookworms (a: Ancylostoma duodenale; b:
Necator americanus)
Fig.6
Copulatory bursa of two human hookworms (a: Ancylostoma duodenale; b:
Necator americanus)
2. Ovum and larva
(1) Study the ova (Fig.7)(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.
(2) See the ova. Note 2-, 4-, 8-cells, morula and embryonated ova (Demonstration).
(3) Study the embryogenesis of rhabditiform larvae (Demonstration).
(4) See the filariform larvae (Demonstration).
(5) See the activities of filariform larvae on water film of soil surface, waiting for
suitable hosts (Demonstration).
(6) See the SEM photographs of ova and filariform larvae.
3. Pathological specimens and tissue section
(1) See the gross specimen of intestine showing hookworm grasping mucous membrane
(Demonstration).
Note the hemorrhagic petechiae and ulcers.
(2) See the section of the head end of hookworm grasping mucosa membrane of small
intestine (Demonstration).
7
The worms support themselves by biting at the root with cutting plate or ventral
teeth, and suck blood.
Fig.7 Egg of human hookworm
Exercise
1. Draw the hookworm ovum.
2. Draw and label the buccal capsules of A. duodenale and N. americanus.
8
ENTEROBIUS VERMICULARIS
(1) Study the structure of E. vermicularis ova and methods used in the diagnosis of E.
vermicularis infection.
(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 (Fig.8) (Demonstration).
(3) 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.
Fig.8
Egg of Enterobius vermicularis
Exercise
1. Draw and label the ovum of E. vermicularis.
9
WUCHERERIA BANCROFTI &
BRUGIA MALAYI
1.Master the morphology of microfilariae and laboratory diagnostic methods.
2. Know the life cycle characteristics and epidemiologic features of filariae.
1. Adult
(1) 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.
Fig.9
Microfilariae (Left: W.bancrofti; right: B.malayi; a: cephalic space; b: body
nuclei; c: terminal nuclei)
2. Larvae
(1) Study the structures of W. bancrofti microfilariae stained specimen (Fig.9)
(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 (Fig.9)
(Manipulation).
Compare the structures with those of W. bancrofti microfilariae. The larva is
10
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. The intermediate host (Demonstration)
Culex quanquefasciatus and Anopheles sinensis.
Exercise
1. Label the microfilariae.
11
TRICHINELLA SPIRALIS
1. Master the diagnostic method of T. spiralis.
1. 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.
2. 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.
Fig.10 Encapsulated larvae in muscular tissue
Exercise
1. Draw the cyst of T. spiralis.
12
TAPEWORM: Taenia solium, Taenia saginata,
Spirometra mansoni & Echinococus granulosus
1. Study the general morphology and characteristics of life cycle of tapeworm.
2. Master the diagnostic methods and morphological comparison between these
tapeworms.
3. Understand the dangerous of taeniasis, cysticercosis, hydatid disease and sparganosis
to human being.
4. Know the morphology of hydatid cyst of E. granulosus.
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.
C. Observe the stained specimen of scolex of T. solium (Fig.11)(Manipulation).
Spherical, four suckers, with rostellum armed with two rows of hooklets, numbering
25～30.
D. Observe the stained specimen of scolex of T. saginata (Fig.12)(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 (Fig.13)(Demonstration).
F. See the stained specimen of segment T. saginata(Fig.13) (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.
G.. 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.
H. 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.
I. Observe the preserved specimen of tapeworm (Demonstration)
Press a gravid segment between two slides, examine and count the number of lateral
13
branches under lower-lens microscope.
J. See the SEM of T. solium scolex (Demonstration).
Fig.11
Scolex of Taenia solium
Fig.12 Scolex of Taenia saginata
Fig.13 Mature proglottid of Taenia solium and Taenia saginata
(2) Larva
A. Study the stained specimen of cysticercus cellulose.
Note the rostellum, hooklets, sucker and bladder.
B. See the stained specimen of cysticercus bovis and compare with the scolex of
cysticercus cellulose (Fig.14)(Demonstration).
C. See the preserved specimen of cysticerci (Demonstration).
14
D. See cysticercus bovis in cardiac muscle of ox (Demonstration).
E. See cysticercus cellulose in cardiac muscle of pig (Demonstration).
Fig.14
Cysticercus cellulosae
(3) Ovum (Manipulation)
Observe the live specimen of taenia ova (Fig.15). 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.
Fig.15
Taenia egg
2. Spirometra mansoni
(1) Adult worm (Demonstration)
A. See the preserved specimen of adult worm.
A. Study the stained specimen of adult worm (Fig.16).
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.
B. See the stained specimen of scolex.
C. See the SEM photograph of S. mansoni scolex.
(2) Larva and intermediate hosts (Demonstration)
A. See the procercoid.
B. 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.
15
C. See the first intermediate host cyclop and second intermediate host frog.
Fig.16 Mature proglottid of Spirometra mansoni
(3) Ovum
Study ova of S. mansoni. Note its spindle shape, operculum, thin shell, and a
germinal cell with numerous yolk granules.
3. Echinococus granulosus
(5) See the stained specimen of adult worm (Fig.17)(Demonstration).
Note the four segments (usually) in an adult worm and the difference in maturity,
and the characteristics of scolex.
(6) See the preserved hydatid cyst in liver (Demonstration).
(7) 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.
1. Label the scolices of T. solium and T.
saginata.
Fig.17 Adult worm of Echinococcus granulosus
16
CLONORCHIS SINENSIS
1. Understand the morphological features of adult parasite.
2. Master the characteristics of eggs.
3. Know the life cycle of Clonorchis sinensis.
4. Distinguish the intermediate host.
1. Adult worm
(1) Observe the preserved specimen (Demonstration).
Note the size, color, shape of the organism. Spindle-shaped, transparent.
(2) Observe the general feature of C. sinensis in stained specimen(Fig.18)
(Manipulation).
First with naked eye, then study under microscope, note and identify following
structures.
A. Adherent organs: oral and ventral suckers, compare their size.
B. Digestive system: mouth, pharynx, esophagus and ceca.
C. Excretory system: excretory bladder and pore.
D. 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?
17
Fig.18 Adult worm of Clonorchis sinensis
3. Ovum (Manipulation)
(1) Study the ova(Fig.19). 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).
18
Fig.19
Egg of Clonorchis sinensis
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
1. Draw egg of C. sinensis in detail.
2. Label in full an adult worm of C. sinensis.
3. 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.
19
PARAGONIMUS FLUKES
1. Understand the main morphological features of the adult parasites.
2. Master the characteristics of the eggs.
3. Know the life cycle of Paragonimus flukes.
4. Distinguish the intermediate host.
1. P. westermani
(1) Adult worm
A. See the preserved specimen (Demonstration).
B. Study the internal structures of stained specimen （Fig.20）(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
D. 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.
20
B. See the infected liver (Demonstration).
Fig.20 Morphology of Paragonimus westermani
Exercise
1. Draw P. westermani egg in detail and label.
2. Label the adult worm of P. westermani.
3. 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.
Fig.21 Egg of Paragonimus westerman
21
FASCIOLOPSIS BUSKI
1. Study the life history and feature in morphology of F. buski.
2. Master the laboratory diagnosis of this fluke.
3. To gain some knowledge of plant vectors and the importance in prophylaxis.
1. 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.
(1) Study the stained specimen (Fig.22)(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)
(2) See the snail intermediate host (Planorbis).
(2) See the cercaria.
(3) See the stained specimen of cyst.
(4) See the vector: aquatic plants (caltrop, water chestnut etc.).
3. Ovum
(1) See the preserved specimens (Fig.23)(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
2. Draw and label F. buski egg in detail.
22
Fig.22 Adult worm of Fasciolopsis buski
Fig.23 Egg of Fasciolopsis buski
23
SCHISTOSOMA JAPONICUM
1.
2.
3.
4.
TUnderstand characteristics of life cycle and morphology of its developmental stages.
Observe the pathological changes of schistosomiasis in infected animal.
Study morphology of ova, laboratory diagnostic methods and their principle.
Gain fundamental knowledge of infective stage, infection mode, methods and
significance of immunologic tests commonly used.
l. Adult worm
(1) See the live specimen (Demonstration).
(2) See the preserved specimen (Demonstration).
Adults are dioecious, a male and a female live together.
(3) Study stained specimen (Fig.24)(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?
(4) See the SEM photograph of adult worm (Demonstration).
2. Larva and intermediate host (Demonstration)
(1) See the live miracidia.
Note the feature of movement in water.
(2) Study the structure of stained specimen of miracidia(Fig.25).
(3) See the SEM photograph of miracidia.
(4) Identify the intermediate host Oncomelania snail.
(5) 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(Fig.25).
(6) See the SEM photograph of cercariae.
24
Fig.24 Adult worm of Schistosoma japonicum
3. 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.
(1) See the SEM photograph of egg (Demonstration).
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.
25
Fig.25 Egg and larva of Schistosoma japonicum
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
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).
26
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.
Exercise
1. Label the general morphology of adult Schistosoma.
2. Draw an egg of S. japonicum in detail and label.
27
EXAMINATION OF ALIMENTARY
HELMINTHS
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.
2． 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.
3． 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
1. Record the result of self-stool examination.
2. Try to compare the advantages and disadvantages of the three methods used in the
practice.
Reference
1. Quality control for fecal examination.
Reliable and accurate parasite identification depends on:
28
(1) Collecting satisfactory specimens.
(2) Preparing and maintaining reagent correctly.
(3) Careful performance of appropriate techniques and thorough examination of
finished preparation.
29
ENTAMOEBA HISTOLYTICA, E. COLI &
OTHER AMOEBAE
1. Master morphological structures of trophozoite and cyst of E. histolytica.
2. Master laboratory diagnostic methods of E. histolytica.
3. Understand the pathogenesis of E. histolytica.
4. Know how to differentiate E. hisiolytica from E. coli.
1. E. histolytica
(1) Trophozoite
A. Study the iron-haematoxylin stained specimen of E. histolytica (Fig.26)
(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.
B. 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
A. Study the iron-haematoxylin stained specimen of E. histolytica cysts (Fig.26)
(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.
30
B. 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.
Fig.26 Trophozoite and cyst of Entamoeba histolytica
2. E. coli
(1) 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.
(2) 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.
Exercise
1. Draw the trophozoite and cyst of E. histolytica and cyst of E. coli.
31
GIARDIA LAMBLIA
1. Master the general structure of G. lamblia trophozoite and cyst.
2. Know the diagnostic method.
1. 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.
2. 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.
32
TRICHOMONAS VAGINALIS
1. Master the general structure of T. vaginalis trophozoite.
2. Understand the diagnostic method.
1. Study the stained trophozoites of T. vaginalis (Fig.27) (Manipulation)
Broad end of the body, 4 flagella, a posterior flagellum, a short flagellum, a nucleus
near the origin of the anterior position, axoneme.
2. See the live trophozoites of T. vaginalis (Demonstration)
Fig.27
Trophozoite of Giardia lamblia
Exercise
l. Draw the trophozoite of T. vaginalis.
33
LEISHMANIA DONOVANI
1. Master the morphological structures of amastigotes and promastigotes of L. donovani.
2. Understand methods used in the diagnosis.
1. 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.
2. 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.
3. 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)
Fig.28
Amastigote and promastigote of Leishmania donovani
Exercise
1. Draw the macrophage contains amastigotes, and a free promastigote of L. donovani.
34
PLASMODIA
1.Master the life cycle of Plasmodia and understand their pathogenic mechanism.
2. Master laboratory diagnostic methods of malarial parasites.
3. Study morphological structures of Plasmodia, identify morphological structures of
developing stages of erythrocytic schizogony and gametocytes of P. vivax, and
differentiate the ring-form and gametocytes of P. vivax from P. falciparum.
4. Identify insect vector of malaria—Anopheles.
5. Know simple method of experiment with Plasmodium.
1. P. vivax (Erythrocytic stage) (Manipulation)
(1) Ring form
Compare its size in respect to the infected RBC. Note the delicate blue-stained ring
of cytoplasm and a red chromatin dot.
(2) 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.
(3) 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.
(4) 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)
(1) 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.
(3) Gametocytes
Note their particular sausage or crescent shape, position of chromatin, distribution of
pigment granules and alteration of infected RBC.
35
4. 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.
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.
36
OPPORTUNISTIC PATHOGENIC PROTOZOAN
Toxoplasma gondii
& Cryptosporidium
1. Obtain a primary understanding of the relation between the immunity of infected
host and opportunistic pathogenic protozoan.
2. Observe the structural features of several species of opportunistic pathogenic
protozoan.
1. Cryptosporidium
(l) See the stained oocysts (Demonstration).
Note its size, shape and number of sporozoites within it.
2. 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
1. 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.
37
MOSQUITOES
1. Master the morphological characteristics of developing stages of mosquitoes.
2. Understand mosquitoes as vectors of mosquito-borne diseases.
3.Know differential characteristics of Anopheles, Culex and Aedes.
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.
2. 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).
3. Structure of mosquito heads (Fig.29)
(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).
38
Fig.29 Structure of mosquito head
4. 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.
5. See the live larvae of three genera (Demonstration)
Note the siphon or spiracles and resting position of larvae.
6. 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).
A. See the plasmodial oocysts on the intestinal wall of mosquito.
B. 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.
(1) 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.
39
FLY
1. Understand the basic morphology of different stages of common flies and their role as
vector.
Observations and Experiment
1. 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.
5. Study the structure of leg (Demonstration)
Note the hair appearance of leg terminating in pad and claws. Observe carefully
minute hairs on the pad.
6. 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)
Exercise
1. Label the head and leg of fly.
40
SANDFLY, FLEAS, LICE & OTHER
BLOOD-SUCKING INSECTS
1. Understand the basic morphology of different stages of sandfly, the vector of
pathogen of kala azar.
2. Master the morphological characteristics of flea, louse, bedbug, tick and mite.
3. Understand the relation of above arthropods to the diseases.
1. Sandfly (Demonstration)
(1) See the adult.
(2) See the eggs.
(3) See the larva.
(4) See the pupa.
2. Flea (Demonstration)
(1) 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.).
(2) 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.
41
(2) See the eggs.
Note the ellipsoidal, operculated white eggs firmly attached to the hairs or fibers.
4. Cockroach (Demonstration)
(1) See Periplaneta americana.
(2) See Blattella germanica.
5. Tick (Demonstration)
(1) 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.
(2) 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
A. 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).
B. See the whole mount of larvae (Demonstration).
Note its tiny size, oval shape, capitulum, scutum and body hairs.
(3) 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.
(4) Demobex
See the whole mount of adult (Demonstration).
7. Bedbug (Demonstration)
(1) 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?
Exercise
1. Report the result of examining demodex.
42

CLONORCHIS SINENSIS

Transcription

Similar documents

PETA

RESEARCH PROPOSAL SURVEY LAB to - iupui gk-12

promo bebek betutu website

How to Tie a Woggle

lnstallation - ConservationMart.com

Develop a lifelong love of reading in every student

Turkey - EOPOWER

Key - Scioly.org

RÉSoNANSS: a regional contribution to

Real Time Control of Hand Prosthesis Using Surface EMG