AFIP Wednesday Slide Conference - No. 28
April 28, 1999
- Conference Moderator: Dr. Don Nichols, Diplomate,
Department of Pathology
National Zoological Park
Washington DC 20008
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Case I - 97-214-3 (AFIP 2655235)
- one electron micrograph photo transparency
- Signalment: Adult, male, hairy-footed hamster (Phodopus
- History: This hamster was noted to have lameness or
ataxia of the left rear leg and impacted cheek pouches. It was
presented to the Department of Animal Health in a moribund condition
and was euthanized.
- Gross Pathology: The carcass was in poor nutritional
condition with little to no subcutaneous and cavitary fat. The
right maxillary incisor was absent, and the mandibular incisors
were slightly overgrown and deviated to the right. Other findings
- Laboratory Results: None.
- Contributor's Diagnosis and Comments: Brain, meningoencephalitis,
suppurative to pyogranulomatous, multifocal, severe.
Etiologic Diagnosis: Algal (protothecal) encephalitis
Etiology: Prototheca sp.
- The lesions in the brain were associated with the presence
of round, 5-10 mm endosporulating eukaryotic organisms. The
walls of these organisms stained with the periodic acid-Schiff
reaction, GMS, and Gridley fungal stain, but the organisms did
not stain with acid-fast or Gram stains. Ultrastructural examination
revealed that they had a thick outer cell wall and contained
up to 3 endospores which had numerous, variably-sized, electron-dense
granules and electron-lucent granules in their cytoplasm. The
light and electron microscopic features of the organisms were
compatible with a species of Prototheca.
Prototheca are colorless, unicellular saprophytic microorganisms
that are believed to be achlorophyllous algae closely related
to green algae in the genus Chlorella. Opportunistic infections
have occasionally been reported in humans, cattle, dogs, cats,
and other species. These infections are often localized to the
skin or subcutis, although disseminated infection can occur.
In cattle, protothecosis typically occurs as chronic mastitis.
- In this hamster, protothecal infection was only detected
in the brain. This animal had spent its entire life inside the
Small Mammal House at the National Zoo. The source and route
of infection were not determined.
- AFIP Diagnosis: Cerebrum: Meningoencephalitis, necrotizing,
pyogranulomatous, multifocal, moderate, with algae, hairy-footed
hamster (Phodopus sungorus), rodent.
- Conference Note: Prototheca are generally regarded
as opportunistic infectious organisms of low pathogenicity, and
dysfunction of the host immune system is usually required before
organisms can invade and act as pathogens. In humans, protothecosis
occurs primarily in two clinical forms: cutaneous infection and
olecranon bursitis. Cutaneous infection, accounting for most
cases, is often progressive and tends to occur in individuals
who are immunosuppressed or have underlying debilitating disease.
Olecranon bursitis accounts for one-third of human cases, and
occurs in healthy individuals, usually associated with trauma.
While Prototheca sp. typically cause mastitis in cattle, in
dogs protothecosis is frequently a systemic disease, and the
organisms have a predilection for the brain and eyes. Cutaneous
disease has been described in cats.
- Three species of Prototheca are recognized: P. stagnora,
P. zopfii, and P. wickerhamii. The latter two are pathogenic
and have similar morphologic characteristics in tissue sections,
although they differ somewhat in size. P. zopfii are 10 to 25
mm in diameter, whereas P. wickerhamii are 1.5 to 11 mm. Also,
the endospores of P. zopfii are larger than those of P. wickerhamii.
- The differential diagnosis considered by conference participants
included Prototheca and Chlorella. Chlorella sp. are green,
chlorophyll-containing algae with otherwise similar morphological
features to those of Prototheca. The chlorophyll is contained
within cytoplasmic chloroplasts that also contain abundant starch.
The starch granules stain intensely with the periodic acid-Schiff
reaction and are diastase sensitive. By electron microscopy,
the chloroplasts consist of a highly organized, twisted or lamellar
component associated with amorphous, electron dense, vacuolated
material representing starch. Prototheca may contain similar,
smaller starch granules in the form of storage plastids. Ultrastructurally,
the storage plastids are homogenous, electron dense, and are
not associated with the lamellar component found in true chloroplasts.
Participants did not identify chloroplasts in the electron micrograph
provided by the contributor, and thus favored the diagnosis of
- Contributor: Department of Pathology, National Zoological
Park, Smithsonian Institution, Washington, DC 20008.
- 1. Chandler FW, Watts JC: Protothecosis and infections caused
by green algae. In: Pathologic Diagnosis of Fungal Infections,
Chandler FW, Watts JC, eds., pp. 43-53, ASCP Press, Chicago,
- 2. Dillberger JE, Homer B, Daubert D, Altman NH: Protothecosis
in two cats. J Amer Vet Med Assoc 192:1557-1559, 1988.
- 3. Jones TC, Hunt RD, King NW: Diseases caused by fungi.
In: Veterinary Pathology, Jones TC, Hunt RD, King NW, eds., 6th
ed., pp. 534-535, Williams & Wilkins, Baltimore, MD, 1997.
- 4. Taniyama H, et al: Disseminated protothecosis caused
by Prototheca zopfii in a cow. Vet Pathol 31:123-125,1994.
Case II - Fac. Med. Vet. da USP (AFIP 2600309)
- Signalment: Juvenile male and female Tracaja turtles
- History: An environmental project aimed at raising
Tracaja turtles, an endangered species in South America, is based
in the Goias State of Brazil. Sixty of these animals were sent
to San Paulo for a research study. These animals were sacrificed
during the research protocol. Ninety percent of these contained
protozoans within the kidneys.
- Gross Pathology: The kidneys were markedly swollen
- Laboratory Results: Urine examination revealed developmental
stages of a protozoan. The largest of these organisms contained
- Contributor's Diagnosis and Comments: Chronic interstitial
nephritis due to a myxosporidian of undetermined generic status.
- Note: The hyaline, refractive spore wall was present
rarely in the large pansporoblasts within the lumen of the tubules.
When stained with Giemsa (enclosed), the polar capsules were
a reddish-purple and were distinguishable from the blue of the
developing spores. Also note the cytoplasm and nuclei of the
mature spore pushed to one side of the spore.
- In the majority of the cases, chronic interstitial nephritis
extending into the parenchyma was present. Heterophils were
common. Tubules were found to contain cellular debris and sloughed
cells. In other cases, only a moderate inflammatory infiltrate
was seen. In infected turtles, sporonts and pansporoblasts were
present within epithelial cells. As the pansporoblasts matured,
they enlarged, ruptured the epithelial cell, and were found within
the lumen of the tubules. Immature spores were formed and gave
rise to mature spores.
- For years, it was thought that myxosporean parasites used
only one host in their life cycle. From research in the last
ten years, it is now known that an intermediate host is required
to complete the life cycle (we refer those interested to references
3, 4, 5). In those organisms for which life cycle studies have
been conducted, the intermediate host has been found to be an
Oligochaeta or Polychaete worm. Life cycles have been elucidated
in both fresh water and marine habitats.
- In the turtles of this case, trophozoites were found in the
epithelial cells and lumens of renal tubules. Spores developed
in disporoblastic pseudoplasmodium in lumens of the renal tubules.
These pseudoplasmodia are very large and contain the hyaline
spores. Spores are found in the urine of infected turtles.
Ongoing studies are attempting to elucidate the intermediate
host. Additional studies are underway to determine the genus
of the myxosporean in question. Myxosporean parasites have been
found in the kidneys of fish, such as sockeye salmon from Canada
and red drum from Florida.
- AFIP Diagnoses:
- 1. Kidney: Tubular ectasia and tubular epithelial hyperplasia,
multifocal, moderate, with numerous intracellular and extracellular
myxosporidia, Tracaja turtle (Podocnemis unifilis), chelonian.
2. Kidney: Nephritis, interstitial, lymphoplasmacytic and heterophilic,
- Conference Note: The class Myxosporea are protozoan
parasites belonging to the phylum Myxozoa. Myxozoans primarily
parasitize invertebrates (mostly annelids) and poikilothermic
vertebrates, the vast majority of which are fish. Thus, most
published reports of disease have been in fish. The myxozoans
that infect fish are all members of the class Myxosporea.
- Myxosporea are transmitted by means of spores to susceptible
hosts. The spores, which have a characteristic morphology that
is the basis for taxonomic classification, are multicellular
spheroid structures that in fish measure approximately 10 mm
in diameter. Spores have one binucleate or two uninucleate sporoplasms,
one to six (usually two) polar capsules, and a shell. One of
the key characteristics of myxosporidia is that all stages, except
during sexual reproduction, are multinucleated forms that have
enveloping (primary) cells that contain enveloped (secondary)
- Most myxosporean infections of fish are relatively harmless,
with only mild host reaction. Heavy infections can be pathogenic,
however, causing mechanical damage due to pseudocysts, or tissue
necrosis and inflammation due to trophozoite feeding. Some of
the diseases in fish caused by myxosporidians include whirling
disease of young salmonids caused by Myxobolus cerebralis; renal
and blood sphaerosporosis in cyprinids caused by Sphaerospora
renicola; and proliferative kidney disease of salmonids caused
by Myxidium lieberkuhni.
- One of the first Myxosporean infections described was Hoferellus
(Mitraspora) cyprini, the cause of kidney enlargement disease
in young cyprinids. The descriptions of the microscopic lesions
associated with H. cyprini bear some resemblance to the lesions
observed in the turtles. Trophozoites are associated with the
epithelium of renal tubules (or ureters), and undergo endodyogeny,
becoming large plasmodia. Trophozoites initially congregate
on the apical surface of the epithelium, but become progressively
incorporated into the cell layer as epithelial hyperplasia and
papillary growth occurs. In the terminal stages of infection,
the tubules are markedly enlarged, displacing the swimbladder,
and distending the abdomen.
- Contributor: Departamento de Patologia, Faculdade
de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo,
- 1. Gardiner CH, Fayer R, Dubey JP: Myxozoa. In: An Atlas
of Protozoan Parasites in Animal Tissues, Agriculture Handbook
No. 651, pp. 14-15, U.S. Department of Agriculture, Washington
- 2. Kreier JP: Myxosporidia. In: Parasitic Protozoa, Kreier
JP, ed., vol. IV, pp. 115-154, Academic Press, New York, NY,
- 3. Markiw ME, Wolf K: Myxosoma cerebralis (Myxozoa: Myxosporea):
Etiological agent of salmonid whirling disease requires tubificid
worm (Annelida: Oligochaeta) in its life cycle. J Protozool 30:561-564,
- 4. Wolf K, Markiw ME: Biology contravenes taxonomy in the
Myxozoa: New discoveries show alteration of invertebrate and
vertebrate hosts. Science 225:1449-1452, 1984.
- 5. Ruidisch S, El-Matbouli M, Hoffman RW: The role of tubificid
worms as an intermediate host in the life cycle of Myxobolus
pavlovskii (Akhmerov, 1954). Parasitol Res 77:663-667, 1991.
- 6. Kent ML, Whitaker DJ, Dawe SC: Parvicapcula minibicornis
(Myxozoa, Myxosporea) from the kidney of sockeye salmon (Oncorhynchus
nerka) from British Columbia, Canada. J Parasitol 83:1153-1156,
- 7. Landsberg JH: Kidney myxosporean parasites in red drum
Sciaenops ocellatus (Sciaenidae) from Florida, USA, with a description
of Parvicapsula renalis. Diseases of Aquatic Organisms 17:9-16,
- 8. Garden O: The myxosporea of fish: A review. British Vet
J 148:223-239, 1992.
- 9. Noga EJ: Myxozoan infections: General features. In: Fish
Disease: Diagnosis and Treatment, Noga EJ, ed., pp. 173-178,
Mosby-Year Book, St., Louis, MO, 1996.
Case III - 97-3720 (AFIP 2641212)
- Signalment: Adult slender tree frog (Litoria adelensis).
- History: Slender tree frog tadpoles had been introduced
into a large pond built to reproduce the natural wetland habitat
of an endangered species, the Western Swamp Tortoise in the Perth
Zoological Gardens. Whilst intended as a feed source, some tadpoles
had survived, and a colony had developed within the pond environs.
Mortalities were first observed in slender tree frogs within
the pond, initially with no premonitory clinical signs noted
by the zoo attendants. As the outbreak progressed, several moribund
and depressed frogs that appeared reluctant to move when approached
were seen, and mortalities in tadpoles were noted.
- Gross Pathology: There was an increased volume of
clear coelomic fluid and multiple raised pale foci, 0.5 mm to
1.0 mm in diameter which were randomly distributed over the surface
and extended into the parenchyma of the kidney, liver and spleen.
One frog had a unilateral opacity of the cornea with mild exophthalmia,
and another had an irregular pale area at the apex of the heart
in addition to pale foci within the liver and kidney.
- Laboratory Results: Mucor amphibiorum was cultured
from kidney, liver and skin.
- Contributor's Diagnosis and Comments: Liver: Hepatitis, necrotizing
and granulomatous within intralesional Mucor amphibiorum sphaerules.
- Histopathologically, frogs showed a multisystemic, multifocal
and coalescing granulomatous inflammatory reaction centered about
Mucor amphibiorum sphaerules. The sphaerules ranged from 10
µm in diameter in those with no internal structures, to
larger than 30 µm diameter mother sphaerules which contained
between four and eight daughter sphaerules.2,3 The eosinophilic
refractile capsule of the sphaerules stained strongly by the
periodic acid-Schiff reaction.
- Mucor amphibiorum is a fungus which replicates in soil and
in culture as aerial mycelia and sporangiospores typical of other
members of the Mucor genera. In tissues, it occurs solely as
unique circular structures or sphaerules which contain between
2 and 11 daughter sphaerules. Mucor amphibiorum was first isolated
in 1972 from an Australian green tree frog (Litoria caerulea)
held in a collection in Germany.1 It was believed that L. caerulea
may have been infected prior to importation from Australia.
However, the collection also contained frogs imported from South
America, and the exact source of the infection could not be determined.
- Infection by Mucor amphibiorum has been reported in Australia
in skin lesions in the platypus (Ornithorhynchus anatinus) in
Tasmania, and as a multisystemic infection in cane toads (Bufo
marinus) and a green tree frog (Litoria caerulea) in Queensland.2,3
The detection of Mucor amphibiorum in Western Australia indicates
the fungus is more widely distributed than previously believed
and raises concerns over the susceptibility of other Australian
anuran species to this fungus.
- AFIP Diagnosis: Liver: Hepatitis, granulomatous, necrotizing,
diffuse, severe, with numerous fungal spherules, slender tree
frog (Litoria adelensis), amphibian.
- Conference Note: Mucor amphibiorum is unusual among
the Zygomycetes in that it is a dimorphic fungus, occurring in
tissues in a spherical form, while hyphae and reproductive structures
are found only in the external environment. The fungus was classified
as Mucor sp. by Schipper in 1978 based on morphology. Within
tissues, the organism reproduces by forming numerous daughter
spherules that are released upon degeneration of the wall of
the mother spherule.
- Mucor amphibiorum is a primary pathogen in anurans, and several
routes of infection are suspected or described, depending upon
the species affected. In infected cane toads from Australia,
ingestion of contaminated soil was suspected as the route of
infection, with dissemination to the liver via the hepatic portal
system and subsequent hematogenous spread to multiple organs.
Infected toads can excrete fungus in their feces. In a single
case report of Mucor amphibiorum in a free-ranging green tree
frog from Australia, a respiratory route of infection was suspected;
dissemination to multiple organs was also present. In platypuses,
animals appear to be infected percutaneously and the infection
- The infectivity and dissemination of M. amphibiorum are probably
related to its temperature tolerance. The maximum temperature
at which the fungus grows in vitro is 36°C. In the platypus,
whose body temperature is 32°C, lesions occur primarily in
the dermal tissues of the body and on the extremities. In poikilothermic
anurans, however, organisms become disseminated and are able
to grow in various organs.
- In tissue sections, organisms that occur as spherical forms
similar to M. amphibiorum include Prototheca sp. and Coccidiodes
immitis. The mother spherules of M. amphibiorum are larger than
the cells of Prototheca, although the smaller spherules may be
similar in size to the algae. In culture, Prototheca retain
their spherical form, whereas M. amphibiorum forms a mycelial
phase. Coccidiodes immitis can be distinguished from M. amphibiorum
by the endospores, which are smaller and more numerous than spherules.
- Contributor: Agriculture Western Australia- Division
of Veterinary and Biomedical Sciences, Murdoch University, South
Street, Murdoch, Western Australia 6150.
- 1. Obendorf DL, Peel BF, Munday BL: Mucor amphibiorum infection
in platypus (Ornithorhynchus anatinus). J Wildl Dis 29:485-487,
- 2. Berger L, Speare R, Humphrey J: Mucormycosis in a free-ranging
green tree frog from Australia. J Wildl Dis 33:903-907, 1997.
- 3. Speare R, Berger L, O'Shea P, Ladds PW, Thomas AD: Pathology
of mucormycosis of cane toads in Australia. J Wildl Dis 33:105-111,
- 4. Speare R, Thomas AD, O'Shea P, Shipton WA: Mucor amphibiorum
in the toad, Bufo marinus, in Australia. J Wildl Dis 30:399-407,
Case IV - 97-263 (AFIP 2635662)
- Signalment: Adult, male freshwater turtle (Chrysemas
- History: The turtle was one of several purchased from
a commercial vendor for research purposes, which included in
vitro intracellular recording of the cerebellum using a patch-clamp
technique. Several of the turtles died during the quarantine
period, and the investigator indicated that a small parasitic
worm was found moving within one of the brain preparations.
- Gross Pathology: There was focal necrosis within the
pancreas. There were white spots within the liver and spleen.
- Laboratory Results: None.
- Contributor's Diagnoses and Comments:
- 1. Granulomatous pancreatitis - trematode ova.
2. Embolized trematode ova - small intestine and pancreas.
- The lesions are compatible with Spirorchid blood fluke infection.
There are 16 genera within the family Spirorchidae, all of which
affect turtles. Although monogenetic and digenetic fluke infections
are common in freshwater turtles and considered relatively nonpathogenic,
the Spirorchid blood flukes are an exception. Spirorchis scriptae
was first described in 1923. Three species of blood flukes have
recently been associated with mortalities in stranded green turtles.
The adult flukes generally live in the heart and larger arteries
where they produce numerous ova that are widely distributed to
body organs. The adult flukes have been associated with focal
intimal villus proliferation and thromboembolic disease. The
pathogenicity of the flukes varies somewhat between the species,
and is also related to widespread deposition of the ova which
form granulomas. Spirorchis parvus are capable of invading various
tissues including the pancreas and the central nervous system.
- AFIP Diagnoses:
- 1. Pancreas: Pancreatitis, granulomatous, with abundant
necrotic debris, freshwater turtle (Chrysemas picta), chelonian.
2. Small intestine and pancreas: Granulomas, multiple, with
- Conference Note: In the pancreas, and within the serosa,
tunica muscularis and submucosa of the small intestine, there
are numerous trematode eggs that lack an operculum, have golden-brown
shells that often appear triangular in histologic section, and
occasionally contain multiple, small, round basophilic structures
(intact miracidia). Some eggs are not associated with any host
response, while others are surrounded by immature fibrous connective
tissue and/or a few multinucleate giant cells. Adult schistosomes
are present in vessels of the small intestine and pancreas in
- In most slides, at least one of the serial sections of the
pancreas is focally replaced by extensive granulomatous inflammation
with abundant eosinophilic, necrotic material; sections of a
degenerate larval nematode may also be present in this area on
some slides. In some sections, bacilli are found at the periphery
of areas of necrosis and granulomatous inflammation, within the
surrounding pancreatic mesentery, and occasionally within mesenteric
vessels. These were found to be Gram-negative with tissue Gram
stains performed at the AFIP. An acid-fast stain and a GMS did
not demonstrate acid-fast bacteria or mycotic organisms.
- The schistosomes are the only trematodes that do not have
operculated eggs. The schistosomes are also the only group of
trematodes that have separate sexes; other trematodes are hermaphroditic.
Important aspects of the pathogenicity of spirorchid infections
in turtles include dissemination of trematode eggs to many organs
and tissues, occlusion of vessels by the eggs, and migration
of eggs through vessel walls inciting an initial acute inflammatory
response followed by granuloma formation. Passage of eggs from
vessels in the intestine through the epithelium and into the
lumen allows invasion of bacteria, and secondary bacterial infections
are likely responsible for the deaths of some turtles infected
by spirorchid flukes. The Gram-negative bacteria present in
this turtle were likely secondary to spirorchid infection, and
may have contributed to the lesions and inflammatory response
in the pancreas. The presence of larval nematodes in some sections
may have also contributed to pancreatic lesions.
- Contributor: Department of Comparative Medicine, M.S.
Hershey Medical Center, Penn State University, 500 University
Drive, H054, Hershey, PA 17033.
- 1. Gordon AN, Kelly WR, Cribb TH: Lesions caused by cardiovascular
flukes (Digenea: Spirorchidae) in stranded green turtles (Chelonia
mydas). Vet Pathol 35:21-30, 1998.
- 2. Holliman RB, Fischer JE, Parker JC: Studies on Spirorchis
parvus (Stunkard, 1923) and its pathological effects on Chrysemas
picta picta. J Parasitol 57:71-77, 1971.
- 3. Johnson CA, et al.: Fatal trematodiasis in research turtles.
Lab Anim Sci 48:340-343, 1998.
- Course Coordinator:
- Ed Stevens, DVM
Captain, United States Army
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615
- * The American Veterinary Medical Association and the American
College of Veterinary Pathologists are co-sponsors of the Registry
of Veterinary Pathology. The C.L. Davis Foundation also provides
substantial support for the Registry.
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