Identification
of Metazoan and Protozoan
Parasites
in Tissue Sections
Chris H.
Gardiner, PhD
Captain,
Medical Service Corps, U.S. Navy
Fleet
Hospital Operations & Training Command
Building
63240 Box 555223
Marine
Corps Base Camp Pendleton
Camp
Pendleton, California 92055
46th
Annual AFIP/ARP
Pathology
of Laboratory Animals
Washington,
D.C.
10
August 1999
ANIMAL
KINGDOM
PROTOZOA
MESOZOA
METAZOA
I.
Acoelomate
II. Pseudocoelomate
Phylum Acanthocephala
Phylum Nematoda
III.
Coelomate
Phylum Pentastomida
Phylum
Arthropoda
Class Arachnida - ticks and mites
Class lnsecta
1. Digestive tract absent
...............................................................................................
(2)
digestive tract
present ...............................................................................................
(3)
2. No body cavity
............................................................................................................ cestodes
body cavity
present ......................................
..acanthocephala
3. No body cavity
..........................................
..trematodes
body cavity
present ......................................
(4)
4. Sclerotized openings in cuticle;
digestive
glands lateral to intestine
.pentastomes
no sclerotized
openings; no digestive glands .........................................................
..(5)
5. Striated muscle in bands; much chitin in cuticle; often
with tracheal
tubes
............................................................................................................................ arthropoda
no chitin; no
tracheal tubes ........................................................................................ nematodes
Below is a
"working" key that is to be used in identification of metazoan
parasites in tissue sections. The key consists of characteristics of adult
parasites; however, some characteristics can also be used with identification
of larval forms. All groups of parasites are not included (e.g., leeches) since
few of those omitted are seen by parasitologists.
The
major groups of parasites have many more characteristics that assist in
identification. These will be discussed with each major group studied.
1. No
body cavity; solid with "holes" for structures; body usually
flattened
..5
Body
cavity; coelom or pseudocoelom; body usually cylindrical
...2
2.
Anterior end armed with proboscis; no digestive tract
.ACANTHOCEPHALA
No
proboscis; digestive tract present
3
3.
Cuticle with sclerotized openings; cephalic hooks present;
head and acidophilic glands
present
.PENTASTOMIDA
None of
the above
.4
4. Muscles striated; jointed appendages;
exoskeleton of chitin
...ARTHROPODA
None of
the above ........
.NEMATODA
5. No
digestive tract; presence of calcareous corpuscles; segmented body
CESTODA
Digestive tract; no segmentation or calcareous
corpuscles
..TREMATODA
CHARACTERISTICS
OF TREMATODES IN SECTIONS
Solid
but spongy body
Usually there are no large cavities, not
divided into cortical and medullary regions.
Intestine
present
Usually bifurcated, ceca blind
No
calcareous corpuscles
Integument
- syncytial epithelium, often with spines.
Muscles
- Just below integument there is usually an outer circular layer, a middle
longitudinal layer and an inner diagonal layer. Diagonal layer may be between
circular and longitudinal layers or may be absent. There are also muscle fibers
extending from dorsal to ventral surface of body.
Sex
organs in adults - bisexual except in schistosome group. Vitelline glands supply the yolk for
zygotes.
Egg, -
type, structure, etc.
Suckers
- usually oral and ventral. Have muscle around it.
Excretory
system - often difficult to see but opens posteriorly.
Characters used to identify which trematode
Size
Arrangement
of sex organs, suckers, excretory system, etc.
Eggs -
size, shape, type of operculum, if any; presence or absence of miracidium.
Often eggs may be the only recognizable structure left in the tissues. Only schistosome eggs are not operculated.
Cuticular
spines - number, size, location (on head, body)
Comments
- most trematodes do not use vertebrates as paratenic hosts so there are not
too many zoonoses due to larval trematodes. In the vertebrate they usually
develop to adults or not at all. In Paragonimus the worms (bisexual) are
usually paired in cysts in the lungs and if only a single worm is present in
the body it usually will not encyst but may continue to wander, even into the
subcutaneous tissues or into the brain. We should also remember that liver and lung
flukes normally have a visceral migration phase in their development and
immature worms may occasionally be seen in these locations in autopsy or
surgical specimens.
Schistosoma mansoni
- lateral spine - only one with a lateral spine.
Schisotosomes
- common in aquatic turtles.
Heterobilharzia americana
- in mammals in the US.
CESTODES
Tegument
with microvilli; calcareous corpuscles in parenchyma.
No
intestine.
cystic or solid
A. solid
1. sparganum - larval stage (plerocercoid of Spirometra
spp. (pseudophyllidea).
Found most commonly in subcutaneous tissue; no suckers or bothria.
B. cystic
1. cysticercoid - usually occurs
in invertebrate host but in rare instances may be found in mammals. Small bladder and everted scolex. Commonly found in life cycle of Hymenolepis spp.
2. cysticercus - larval stage of
cyclophyllidea - most common is Cysticercus cellulosa
(larva of Taenia solium).
Scolex with four suckers and
armed proboscis.
Calcareous corpuscles most numerous in neck; rare in cyst wall. Look
for the bladder on histo. Other cysticerci may or may not have
hooks. Most common in muscle. Strobilicercus - type of cysticercus.
3. coenurus - larval stage of Multiceps spp.
Identical to cysticercus except this type has numerous scolices. Common in brain. If it has more than one
scolex, it can't be a cysticercus!!
4. hydatid -
a. unilocular - larval stage
of Echinococcus granulosus.
Has thick laminated cyst wall, germinal membrane, and numerous
scolices ( may be collections of scolices in brood capsules). Commonly in lung and liver.
A hydatid cyst contains numerous daughter cysts which, in turn, contain numerous protoscolices. (Not called scolices in hydatid cysts)
Often, all you have left in the lesion is the thick wall. Remember, echinococcal hooklets are acid fast.
b. multilocular - larval
stage of other echinococcal species.
There is no
laminated cyst wall and cyst is lobulated. Commonly in lung and liver.
c. E. vogeli - sort of in between. In NHP - has a multilaminated cyst wall
with multiple rooms.
5. Tetrathyridia - pearly nodules in NHP
Solid body.
NEMATODES
Morphological
features that are helpful in the identification of nematodes
in
tissue
I. Body Wall
Cuticle- 2-3u = thin; 4-6 = thick. (his is the last part of a dead worm to
degenerate in tissue.)
Thickness, number of layers
Striations - transverse, longitudinal
Annulations - presence?
Alae - number, position, shape
Spines etc. - presence?, number, position
Hypodermis -
mesenchyme between cuticle and muscle.
Lateral chords - presence? structure, number
and size of nuclei - May be next to the
lateral alae.
Protrusion of the hypodermis.
Dorsal and ventral chords - structure
Bacillary bands - presence?, position
Meromyarian vs. polymyarian
Platymyarian vs. coelomyarian
Muscle cell structure, nuclei - number
II. Excretory
system
Excretory gland - number, size and position
of nucleus
Excretory pore - position
Lateral canals - presence?, position, number
III. Digestive system
Cephalic
structures and stoma
Labia - number, shape
Papillae - number, position
Teeth, plates, etc. - number,
position
Type -
rhabditoid (pinworm), strongyloid, trichuroid, spiruroid, etc.
Esophageal
glands- type
Diverticulum
- presence, size - think this if you see esophagus and intestine at same time.
Intestine - patent, non-patent (larvae)
Generally brown or black due to heme.
Cells -
number, type, uni- or multi-nucleated, size of nuclei
Cecae -
presence?, size
Anus - terminal or subterminal
IV. Reproductive systems
Female - (generally two tubes)
Uteri - monodelphic; didelphic -
amphidelphic, opisthodelphic, prodelphic; polydelphic
Ovejector - presence?, size, etc.
Ovary - telegonic vs. hologonic
Eggs - shape, size, shell, etc. Ova do not
have shells. Eggs have shells.
(If you see eggs - always a female. If you see sperm, you could be looking at
the male or a cut through the
seminal receptacle and see sperm waiting for eggs to come by after copulation) Also look for eosinophilic globules.
Larvae or prelarvae - presence?,
structure (Eggs are embryonated,
not larvated.)
Microfilariae - see only in filarids. (Metastrongyle larvae, although they look
like a bag of cells like
microfilariae, have an esophagus).
Also, metastrongyles lay a
uninucleate zygote in tissues which divides and becomes multinucleate
(looks like an egg packet) and then
a larva, all in tissue.
Metastrongyle larvae also always have a
crooked tail.
Male
- (one tube)
Testis - telegonic vs. hologonic
Spicules.-- number, shape, etc.
Gubernaculum - presence?, shape,
etc.
Bursa or other copulatory organ -
presence?, structure
Can see sperm is the caudal
intestine - like a cloaca.
OXYURIDS
-cuticle:
thin; one pair of lateral alae usually present in adults
-muscles:
platymyarian, meromyarian. Pinworm - few muscle cells.
-intestine:
uninucleate cuboidal or columnar cells
-esophagus:
corpus, isthmus and bulb eggs: thick-shelled, usually unembryonated
-examples:
Enterobius, Syphacia, Aspiculurus, Oxyuris
Notes:
If platy and lateral alae = oxyurid (pinworm)
ASCARIDS
-cuticle:
thick; larvae have 1 pair of lateral alae.
Often have small lateral cords.
-muscles:
coelomyarian, polymyarian
-esophagus:
cylindrical; may have ventriculus; mouth has three large lips
-intestine:
many uninucleate columnar cells; low brush border; may have caeca lateral
chords
-large
eggs: thick-shelled, unembryonated
examples: Toxocara, Ascaris,
Parascaris, Neoascaris, Contracaecum Anisakis, Porrocaecum
NOTE: Ascarids never have embryonated eggs (this is how you can always
tell them from spirurids).
Anasakis - big
butterfly bilateral cords.
-cuticle:
may have elaborations, i.e., lateral alae, bosses
-muscles:
coelomyarian, polymyarian
-esophagus:
consists of a anterior muscular and posterior glandular portion
-intestine:
uninucleate cells, long or medium microvillar border
-lateral
chords: often prominent
-eggs:
embryonated, thick-shelled
examples: Gnathostoma, Physaloptera,
Habronema, Thelazia, Gongylonema,
Spirocerca, Tetrameres
NOTES: The eggs are very distinctive - if you can see an egg, you
can make the diagnosis by the egg alone.
Tetrameres - eggs
have small polar filaments.
Gnathostoma -
spines and bulb at anterior end.
Physaloptera - giant
lateral cords - the biggest!!
Draschia - no
thick shells on eggs.
FILARIDS
-cuticle:
may have ridges, bosses, annulations.
May have lateral internal ridges - common in filarids - lateral bulge of cuticle.
-muscles:
coelomyarian, polymyarian
-esophagus:
anterior muscular, posterior glandular
-intestine:
very small. These are the only
nematodes with small intestines.
examples: Dirofilaria, Dipetalonema,
Onchocerca, Stephanofilaria, Brugia
look for microfilariae in utero and in
tissues (e.g., blood, skin)
-cuticle:
external longitudinal ridges in Trichostrongyles; bursa in male; third-stage
larvae have double lateral alae
-muscles:
platymyarian, meromyarian (except Metastrongyles)
-esophagus:
club-shaped; buccal cavity may be cuticularized or not
-intestine:
few multinucleated cells; microvillar border lateral chords: sometimes
look vacuolated
-eggs:
thin-shelled and in early stages of cleavage (except Metastrongyles)
subgroups:
l. Trichostrongyles
- reduced buccal cavity; worms usually red in color; often external longitudinal ridges. examples: Dictyocaulus,
Molineus, Nochtia, Trichostrongylus, Obeliscoides, Nippostrongylus
2. Strongyles - cuticularized buccal
cavity; dense microvilli. examples: Strongylus, Oesophogostomum, Ancylostoma,
Syngamus, Stephanurus
3. Metastrongyles - muscles are
coelomyarian, polymyarian; typical strongyle intestine with microvillar border
shorter than above. examples: Metastrongylus, Filaroides, Parelaphostrongylus,
Angiostrongylus, Parafilaroides, Crenosoma
*look
for eggs vs larvae in uterus and in tissue. look for accessory chords
NOTES: Nippostrongylus - evenly spaced longitudinal ridges which
are unevenly sized and shaped.
APHASMIDS
-Examples: Trichinella, Trichuris, Capillaria,
Anatrichosoma, Trichosomoides.
-Muscle: polymyarian, coelomyarian
-Bacillary
band - Trichuris only - dark edge comprising up to 30% of the
circumference - this is where they suck up nutrients from.
-No
lateral chords.
-Hypodermal
cords - bilateral in Capillaria - look like bacillary bands. Also in Anatrichosoma.
-Stichosome
- on cross-section, see a large basophilic cell, occasionally with a nucleus
(looks like an egg) surrounding a small round esophagus and filling up most of
the pseudocoelom.
EUSTRONGYLIDS
-Examples:
Dioctophyma, Eustrongyloides
-No
lateral cords
-Little
mesenteries" connecting enteron to musculature.
ACANTHOCEPHALANS
-Look
for hooks around heads.
-Muscles
look different than nematodes; there are small round muscles within the outer
circular musculature.
-Intestine: NONE.
-Eggs
are loose in the body cavity. No
uterus.
ARTHROPODS
Bots and
maggots no difference only in genus of adult dipertan.
-Look for spines.
-Spiracular plate on end of worm
chitinized. Arrangement of spiracles is key to speciation.
-Striated muscle (is an arthropod)
-Virtually impossible to speciate on
histo.
PENTASTOMES
-Often
are C-shaped look in peritoneum of NHPs.
-Have
mouth, anus, digestive system.
-Alimentary
canal is surrounded by eosinophilic glands (only animal with this arrangement)
-Mouth
4 hooks to attach to lung
-Adults
parasitize respiratory system of amphibians, reptiles and some birds.
-Linguatula
nares and lung of dogs.
-Nymphs
found in intermediate host
-Cuticle
sclerotized openings. Stain black on movat. Part of cuticular cells
(which secrete the cuticle). Last
thing to go in autolysis.
-Also
have skeletal muscle.
CLASSIFICATION OF
PR0TOZOANS
Subkingdom
Protozoa
Phylum Sarcomastigophora - flagella or
pseudopodia
Subphylum Mastigophora - flagella in
trophozoites
Class Zoomastigophorea - no
chromatophores
Order
Kinetoplastida - kinetoplast
Family Trypanosomatidae
- body typically leaflike but may be rounded
Genus Leishmania
Genus Trypanosoma
Order
Diplomonadida - bilaterally symmetrical, two similar nuclei
Family
Hexamitidae - body bilaterally symmetrical, six to eight flagella
Genus Spironucleus
(syn., Hexamita)
Genus Giardia
Order
Trichomonadida - four to six flagella, one trailing, no cysts
Family
Trichomonadidae - undulating membrane, trailing flagellum, costa present
Genus Trichomonas
Family
Monocercomonadidae - costa absent, recurrent flagellum free
Genus Histomonas
Subphylum Sarcodina - locomotion by
pseudopodia
Superclass Rhizopodea - pseudopodia,
not axopods
Order Amoebida - naked, no shell
Family
Vahlkampfiidae - nuclear division by promitosis, flagella may be present
Genus Naegleria
Family
Hartmannellidae - nuclear division not premitotic, flagellum absent
Genus Acanthamoeba
Family
Endamoebidae - no flagella, typically in digestive tract
Genus Entamoeba
Phylum Microspora - spores of unicellular origin, with or without polar filaments
Class Microsporididea -
elongated, oval to tubular spores
Order Pleistophoridida -
uninucleate spores
Family Pleistophoridae - in sporogony,
variable number of sporoblasts form
Genus Encephalitozoon
Phylum Myxozoa - spores of multicellular
origin, with two or more polar filaments parasitize poikilotherm vertebrates or
annelids
Class Myxosporea - spore with one
or two sporoplasms and one to six polar capsules, mainly in fish
Order Bivalvulida - spores open in
two valves
Family Myxosomatidae - spore circular or
pyriform
Genus Myxosoma
Phylum Ciliophora - cilia or ciliary organelles in at least one stage
Subphylum
Rhabdophora - ciliary crown around cytostome
Class Litostomatea - monokinetids with
tangential transverse ribbon cilia present in only two orders
Subclass Trichostomatia - no oral
toxicysts
Family
Balantidiidae - cytostome and oral cavity; long body; cilia holotrichous
Genus Balantidium
Subphylum Cyrtophora - ciliates with varied kinetids
Class Oligohymenophorea
Subclass Hymenostomatia - usually
holotrichous
Order Hymenostomatida - body
kinetome has preoral structure
Family Ichthyophthiriidae - watchglass
organelle near buccal cavity
Genus Ichthyophthirius
Phylum Apicomplexa - motile stage with apical
complex
Class Sporozoasida - stage with
simple resistant spore, sexual reproduction
Order Eucoccidiorida - asexual and
sexual phases
Family Klossiellidae - monoxenous,
microgametes without flagella
Genus Klossiella
Family Haemogregarinidae - heteroxenous,
in circulatory system of vertebrates, in
digestive tract of invertebrates
Genus Haemogregarina
Genus Hepatozoon
Family Eimeriidae - monoxenous, typically
in intestinal epithelial cells, sporogony
outside
Genus Eimeria
Genus Isospora
Genus Caryospora (atypical)
Family Crytposporidiidae - monoxenous, in
microvillar border, sporulation inside or
outside of host, oocysts with four naked sporozoites
Genus Cryptosporidium
Family Sarcocystidae - heteroxenous, producing
oocysts with two sporocysts in
intestine of definitive host, asexual stages in intermediate host
Genus Toxoplasma
Genus Cystoisospora
Genus Besnoitia
Genus Hammondia
Genus Sarcocystis
Genus Frenkelia
Family Calyptosporidae - heteroxenous,
invertebrate intermediate host, definitive
hosts poikilothermic
Genus Calyptospora
Family Plasmodiidae - heteroxenous, zoite
motile, sporozoites naked
Genus Haemoproteus
Genus Leucocytozoon
Genus Hepatocystis
Genus Plasmodium
Family Babesiidae - probably no sexual
reproduction
Genus Babesia
Genus Entopolypoides
Family Theileriidae - erythrocytic stages
smaller than Babesiidae
Genus Theileria
(syn., Cytauxzoon)
Protozoan
of undetermined taxonomic status
Genus Pneumocystis
SUMMARY OF CHARACTERISTICS
-
amastigotes are spherical to ovoid and measure 2 by 5 um.
- amastigotes contain a
round nucleus and a rod-shaped kinetoplast. In smears the
kinetoplast stains well; in histological section it is often inapparent.
- tough to diagnose if
you only have a few.
Trypanosoma
- amastigotes are 2X larger than Leishmania
- kinetoplasts are larger and stain
more intensely in histological section than Leishmania
- trypomastigotes are pleomorphic
- watch out for macrophages with Leishmania
migrating through cardiac
vessels
- T. cruzi C-shaped
on blood smear.
- trophozoites are binucleate.
- in histological section
trophozoites may be found adhering to intestinal epithelium
- easy to see eyes on smear, tough
on histo.
Pentatrichomonas, Spironucleus, Trichomonas, etc.
- look for undulating membrane,
costa, etc.
- measure size
- know site and host
- Spironucleus one nucleus.
- trophozoites are usually in
clusters in tissue
- stain poorly with H&E; usually
well with PAS
- nuclei are often inapparent
- no flagella in tissue
- lots of necrosis; look at edge of
necrosis
- rarely dont take PAS
- measure these also
- nuclei contain peripheral
chromatin plaques and a small, central endosome
- E. invadens often
has basophilic pseudopodia, never find pseudopodia on E. histolytica
Acanthamoeba, Naegleria
-
free-living amoeba
- nuclei contain a very large endosome with
a clear area at its periphery
- definitely not an Entamoeba
nucleus, but other features the same.
-
spores range from 2-15 um in length
-
mature spores are usually larger than immature
-
annular ring is visible with a variety of stains
-
gram stains are useful in identification; stains poorly with H&E
-
mature spores are acid-fast
-
spores contain a PAS-positive polar
granule in a capsule
-
no other organism has a polar filament.
Must have a PAS
-
EM - see lots of burn out. Thick spore
coat does not fix well.
-
Encephalitozoon - generally same size throughout lifespan.
- spores ovoid to elongate
- spores usually contain two capsules
- capsules stain intensely with
Giemsa; spores are acid-fast
Ciliates
- cilia are usually visible but can be
indistinct when organism is surrounded
by tissue
- usually a large macronucleus is present
- in lung in a herbivore - probably terminal
aspiration.
- poorly stained with H&E; GMS is the
stain of choice
- smears from lung should be stained with
Giemsa; cysts contain up to eight intracystic bodies
- protozoan.
Apicomplexa
- numerous genera
- sporulated oocysts used to differentiate
genera
- oocysts generally sporulate after passed.
- a microgametocyte forms many
microgametes; each has
flagellum; a macrogameotcyte forms one macrogamete.
- a macrogamete contains eosinophilic
globules
- oocyst has shell; sometimes
collapsed
- schizonts are either large or
small, can have
megaloschizonts
Eucoccidian
all look alike on EM.
Polar
ring - mouth.
Rhoptries
- secrete digestive juices.
Micronemes
- skeleton.
- many species
- usually in intestinal epithelial cells
- schizonts small or large
- resemble Eimeria
- also in epithelial cells.
- so many rupture at once - get into blood
and liver.
- sexual forms in birds and reptiles
- intermediate hosts are rodents
- forms caryocyst in rodents, skin of dogs.
- intracellular in intestinal epithelial
cells; appear to be on surface
- usually in intestine but also in
respiratory and renal epithelium
- very small in size
- oocysts are acid-fast, sometimes may be so
in histological section
- trophozoites are not acid-fast.
- sexual forms in intestinal lamina propria
- asexual stages first in endothelial cells;
then muscles
- oocyst is sporulated in lamina propria
- massive numbers of sporocysts seen in
immunosuppressed birds
==> lethal.
- Intestine ==> reproduces one time
in macrophage ==> endothelial
cells where it has an asexual reproduction.
- Only type of coccidian in which
oocysts sporulate in tissue
-
Sarcocysts in endothelium have never been reported in man.
- asexual schizonts in CNS of rodents
- schizonts are lobed and contain
zoites
- look like little flowers in the
brain on low mag.
- adults in vultures.
- asexual cysts cause hypertrophy and increase in number
of host cell nuclei
- Looks just like Eimeria in dog gut.
- sexual stages in feline intestinal
epithelium
- asexual stages in many hosts
-
ultrastructurally tachyzoites are within vacuoles within macrophages
-
occasionally PAS-positive.
- asexual cyst with thick wall
- PAS-positive.
- in kidneys of horses and rodents
- zoites bud from a residium -
endopolygeny
-
multinucleate resistant stage which passes out in urine.
- infected erythrocytes in reptiles and
birds
- zoites bud from a residium
- one zoite sits in an RBC.
- in
many mammals
- zoites
bud from a residium
- white
blood cells
- in
many species of animals; not only mammals
-
schizonts in endothelial cells or hepatocytes
-
erythrocytes contain developmental stages; pigment may be apparent
- ring
forms, dots, etc.
- can
look like Toxoplasma, Sarcocystis, Haemoproteus
- NHPs
-
asexual cysts found in the liver
-
trophozoites and gametocytes are in erythrocytes
- cysts
generally have "fingers"
Theileria (Cytauxzoon)
-
schizonts in many cell types
-
trophozoites in erythrocytes - look like piroplasms
-
trophozoites in erythrocytes
- trophozoites in erythrocytes resemble Hepatocystis;
however, are usually appliqued and
erythrocytes bulge at site
- no
gametocytes found in peripheral blood
- schizonts
are sinuous and common in the lung in endothelium
-
erythrocytes contain trophozoites and gametocytes; pigment present
- looks
like Sarcocystis, Plasmodium
-
trophozoites/gametocytes in leukocytes or erythrocytes
- small schizonts or megaloschizonts are present in the
liver and brain
- cause the nucleus of the endothelial cell to
hypertrophy.