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Gastric Cryptosporidiosis in Freshwater Angelfish (Pterophyllum Scalare)
Brian G. Murphy, Daniel Bradway, Timothy Walsh, George E. Sanders and Kevin Snekvik
J VET Diagn Invest 2009 21: 722
DOI: 10.1177/104063870902100523
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722 Case Reports
J Vet Diagn Invest 21:722–727 (2009)
Gastric cryptosporidiosis in freshwater angelfish
(Pterophyllum scalare)
Brian G. Murphy,1 Daniel Bradway, Timothy Walsh, George E. Sanders, Kevin Snekvik
Abstract. A freshwater angelfish (Pterophyllum scalare) hatchery experienced variable levels of
emaciation, poor growth rates, swollen coelomic cavities, anorexia, listlessness, and increased mortality
within their fish. Multiple chemotherapeutic trials had been attempted without success. In affected fish, large
numbers of protozoa were identified both histologically and ultrastructurally associated with the gastric
mucosa. The youngest cohort of parasitized fish was the most severely affected and demonstrated the greatest
morbidity and mortality. The protozoa were morphologically most consistent with Cryptosporidium. All of the
protozoan life stages were identified ultrastructurally and protozoal genomic DNA was isolated from
parasitized tissue viscera and sequenced. Histological, ultrastructural, genetic, and phylogenetic analyses
confirmed this protozoal organism to be a novel species of Cryptosporidium.
Key words: Angelfish; Cryptosporidium; cryptosporidiosis; fish; protozoa; Pterophyllum scalare.
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The apicomplexan protozoal parasite Cryptosporidium production of sporulated oocysts containing 4 sporozoites
has been identified within numerous vertebrate hosts, not enclosed within a sporocyst.
including mammals, birds, reptiles, and fish.5,11 Crypto- Cryptosporidiosis is recognized as a serious waterborne
sporidia have a life cycle that involves both asexual and disease in humans and other animals for which few effective
sexual reproductive cycles, which is completed within an chemotherapy protocols are currently available.6,14,16
individual host.6 Transmission from one host to another Cryptosporidium typically infects neonatal or young ani-
involves direct fecal-oral transmission often involving mals, often resulting in dysfunction of the alimentary tract.7
ingestion of oocyst-contaminated water. However, recent Relatively little is known about the prevalence and
research indicates that transmission dynamics of crypto- geographic distribution of Cryptosporidium isolates that
sporidia is often more complex than previously thought.14 infect fish.13 Gastric and intestinal cryptosporidiosis have
Oocysts are excreted in the feces. Once ingested, sporulated been previously identified in 14 species of marine and
oocysts excyst within the gastrointestinal tract, releasing freshwater fish.1,2,11,18 Recently, a parasite defined as
infectious sporozoites. The sporozoites attach to and Cryptosporidium molnari was isolated from 2 marine
invade epithelial cells lining the alimentary tract, where teleosts, the gilthead sea bream (Sparus aurata L.) and
they become enclosed within a parasitophorous vacuole, the European sea bass (Dicentrarchus labrax L.). The
which results in the trophozoite stage of the parasite. histologic and ultrastructural features of this organism
Trophozoites undergo asexual proliferation by merogony. have been described in detail.2 Although no molecular
Sexual reproduction occurs by gametogony to generate characterization of C. molnari has been published thus far
both macrogamonts (female) and microgamonts (male).11 by the original research group, a C. molnari–like protozoal
The resultant zygotes undergo sporogony, leading to the organism isolated from a guppy has been histologically and
genetically characterized.13 A recent review of the taxon-
omy of cryptosporidial parasites has been published.5
A freshwater angelfish (Pterophyllum scalare) and discus
From the Department of Veterinary Microbiology and Pathol- (Symphysodon sp.) hatchery in Washington state (USA)
ogy, College of Veterinary Medicine (Murphy, Snekvik) and experienced variable levels of emaciation, poor growth
Washington Animal Disease Diagnostic Laboratory (Bradway, rates, coelomic distention, anorexia, spiraling, listlessness,
Snekvik), Washington State University, Pullman, WA; Smithso- and increased mortality in its fish population. The clinical
nian’s National Zoological Park, Department of Pathology, signs were primarily confined to the larval angelfish, and
Washington, DC (Walsh); the Department of Comparative the adult angelfish were relatively unaffected. Several feed-
Medicine, University of Washington, Seattle, WA (Sanders); and
based and water bath–based pharmaceutical trials (levam-
the U.S. Geological Survey, Western Fisheries Research Center,
Seattle, WA (Sanders). isole, erythromycin, oxytetracycline, and metronidazole)
1
Corresponding Author: Brian Murphy, Department of Pa- were attempted without demonstrable success. During the
thology, Microbiology and Immunology, School of Veterinary peak outbreak, approximately 400 angelfish died in a
Medicine, University of California Davis, 4206 Vet Med 3A, One cohort of 500 larval fish. The aquarium system was
Shields Avenue, Davis, CA 95616-5270. bmurphy@ucdavis.edu organized in a recirculation configuration, utilized chlori-
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Case Reports 723
nated city water, and had a total system volume of 26,500 sequencing of both strands. Nucleotide sequences were
liters, with a flow rate of 490 liters per min and a daily aligned using the AlignX function of Vector NTIi. Align X
water exchange rate of 755 liters. Water quality was calculations for generating the guide tree dendrogram are
maintained with a fluidized sand filter, bubble bead based on the Clustal W algorithm.17 The nucleotide
biofilter, protein skimmer (foam fractionater), ozone sequence of the 18s rRNA polymerase chain reaction
generator, and ultraviolet sterilizers. Angelfish eggs were product of the angelfish Cryptosporidium isolate has been
spawned on slate strips in the breeding tanks and deposited in GenBank under the accession number
transferred to the hatchery, where they were treated with FJ769050.1.
3 to 5 ml of methylene blue per gallon of water. Free- Grossly evident visceral lesions were not apparent in any
swimming ‘‘larval’’ fish were transferred to another tank at fish. Small numbers of aerobic bacterial organisms were
2–3 weeks of age. isolated from the kidneys of approximately 2 fish per
Two separate shipments of moribund fish, approximately cohort, including Aeromonas hydrophilia, Pseudomonas
3 weeks apart, were shipped live from the hatchery to the putrefaciens, Sphingomonas paucimobilis, and Micrococcus
diagnostic laboratory. Affected fish included individually spp. Severe intestinal capillariasis was diagnosed in a single
bagged cohorts of younger than 60 days, 150 days old, and large, moribund angelfish (older than 1 year).
older than one year. Upon arrival at the diagnostic Thirty angelfish were examined histologically in multiple,
laboratory, the fish were observed and humanely eutha- stepped sagittal sections. Large numbers of Cryptosporid-
nized with a buffered solution of MS222 anesthetic,a dosed ium organisms were identified via light microscopy within
to affect. Each euthanized cohort was divided into 4 lots: the gastric mucosal epithelium and adjacent gastric lumina
frozen at 280uC, fixed with 10% buffered formalin, fixed of multiple fish (Fig. 1). Myriad cryptosporidial organisms
with 2.5% gluteraldehyde, or submitted fresh for microbial ranging from 1 to 4.1 mm in diameter were identified along
culture of the visceral tissues. A clinically normal adult the apical surface of the gastric epithelium. In severely
plecostomus fish (Hypostomus plecostomus) housed in the affected individuals, more than 100 gastric Cryptosporidium
same tank as the affected angelfish for the purpose of algae organisms could be identified per 1,0003 field (200-mm
control was humanely euthanized, as previously described. length of mucosal epithelium). Both sporulated and
The plecostomus was necropsied, and the tissues were unsporulated oocysts were identified embedded deep within
examined histologically. the cytoplasm of the gastric epithelial cells. The sporulated
The tissues were fixed in 10% buffered formalin and oocysts were nearly spherical and measured 3.4 mm 6
decalcifiedb for 1–4 hr before being embedded in paraffin. 0.4 mm (diameter, n 5 64). Sporulated oocysts contained
Longitudinal sections were routinely processed and stained basophilic sporozoites. The oocysts occurred singly and in
with hematoxylin and eosin. All of the tissue sections were clusters of 4–10 within cytoplasmic vacuoles surrounded by
examined by veterinary pathologists (BGM, KRS, TFW). zones of clearing in gastric epithelial cells.
Gastric protozoa were measured from multiple individual In severely parasitized fish, the gastric lumen was filled
fish and from different cohorts with an ocular micrometer with large numbers of desquamated, degenerate epithelial
at a magnification of 1,0003. For transmission electron cells; macrophages; sporulated oocysts; and granular
microscopy, small pieces of gastric tissue (,1 mm3) were acellular debris. Scattered, parasitized gastric epithelial
fixed in 2.5% (v/v) glutaraldehyde and routinely processed cells had pyknotic nuclei, cytoplasmic hypereosinophilia,
for electron microscopy. Ultrathin sections were cut with a and vacuolation (epithelial cell degeneration). The subja-
microtome and examined with a transmission electron cent lamina propria was often multifocally infiltrated by
microscope.c Black and white photographs were taken with moderate numbers of lymphocytes, macrophages, and
film and scanned into a digital format. eosinophilic granular cells (Fig. 1). Cryptosporidium
Frozen fish viscera were minced, and DNA was organisms were not identified within the intestine,
extracted and purified from both frozen tissue and esophagus, or pharynx in any of the examined fish. A
formalin-fixed, paraffin-embedded sections utilizing a clinically normal plecostomus fish housed in the same
DNA extraction kit.d Polymerase chain reaction was tank as the affected angelfish had no histologic evidence
utilized to amplify a 695-nucleotide region of the of alimentary cryptosporidiosis.
cryptosporidial 18s ribosomal RNA (rRNA) gene using All of the cryptosporidial life stages were identified
18S forward primer (59-AGTCATAGTCTTGTCT- ultrastructurally.2 Extracytoplasmic trophozoites were lo-
CAAAGATT-39) and 18S reverse primer (59-TTAA- cated in apical epithelial cell–derived parasitophorous
CAAATCTAAGAATTTCACC-39), as previously de- vacuoles with basilar feeder organelles (Fig. 2). Epithelial
scribed.9 Extracted sterile diethylpyrocarbonate-treated cell surfaces were covered by remnants of microvilli.
water was used as a negative control, and water was used Meronts containing either 4 (type II) or 8 (type I, Fig. 3)
as a no-template control for all polymerase chain merozoites were also detected. Apical to luminal macro-
reactions. The polymerase chain reaction amplicons were gamonts were distinguished from trophozoites by the
visualized on 1.5% agarose gels containing ethidium numerous cytoplasmic amylopectin (polysaccharide) gran-
bromide and purified with the use of a DNA gel ules (Fig. 4). Microgamonts were rarely identified relative
extraction kit.e The amplicon DNA was sequenced by a to the other developmental stages, as has been found
local vendorf using a commercial sequencing kit,g with previously with C. molnari.2 Both sporulated and unsporu-
analysis on a DNA sequencer.h Sequencing reactions were lated oocysts were embedded deep within the epithelial cell
done in duplicate, and sequences were confirmed by the cytoplasm, often occurred in clusters, and also contained
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724 Case Reports
Figure 1. Angelfish gastric mucosa. Mucosal cryptosporidia (black arrowhead) and associated infiltration of the lamina propria by
lymphocytes, macrophages, and eosinophilic granular cells (grey arrowheads). Light microscopy, hematoxylin and eosin–stained
histologic sections, 6003 magnification. Bar 5 50 mm.
Figure 2. Angelfish gastric mucosa. Multiple sections of apical trophozoites within parasitophorous vacuoles. Note the feeder
organelle (arrows) and remnant microvilli (arrowhead). Transmission electron micrograph, 6,0003 magnification. Bar 5 1 mm.
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Case Reports 725
Figure 6. Phylogenetic dendrogram for sequences of 18s rRNA from various cryptosporidial isolates (a) and nucleotide sequence
of polymerase chain reaction–amplified 18s rRNA (angelfish isolate, b).
cytoplasmic amylopectin granules (Fig. 5). Sporulated these species. At present, there is disagreement among
oocysts contained 4 sporozoites and a residual body filled taxonomists as to the precise number of species within the
with amylopectin granules. The sporulated oocysts were genus Cryptosporidium. To resolve these controversies,
naked (lacked a sporocyst), as has been described some taxonomists have argued the need for polyphasic
previously for C. molnari–infected fish.2 typing (i.e., an integrated approach using standardized
The 18s rRNA sequence was compared with published morphologic, biologic, and molecular methods [e.g., genetic
sequences for a C. molnari–like organism (GenBank methods] for describing Cryptosporidium species).3 Cur-
accession no. AY524773) using Vector NTI software.i rently, C. molnari is the only recognized species that
Phylogenetic analysis of the 18s rRNA locus for a variety parasitizes fish accepted as a valid cryptosporidial species.14
of cryptosporidial species (C. molnari–like, C. serpentis, Until more molecular and biologic data become available
C. muris, C. andersoni, C. parvum, C. baileyi, C. bovis, and for cryptosporidia of piscine species, some researchers feel
C. hominis) determined the angelfish isolate to be most that there is insufficient data for establishing valid names
closely related to, although distinct from, the C. molnari– for those cryptosporidia-like parasites in fish.5
like isolate (87% similarity; Fig. 6). Many of the previously described Cryptosporidium
After the first description of C. muris in 1907,8 multiple infections in fish have not been associated with clinical
protozoa of this genus were named after the host in which signs of disease.11 However, evidence of acute cellular
they were found.4 Recent morphologic, genetic, and cross- injury has been associated with infections with C. molnari
transmission studies have subsequently invalidated many of in fingerlings and juvenile marine fish2 and Cryptosporid-
r
Figure 3. Angelfish gastric mucosa. Luminal type I meront containing 8 merozoites (a) adjacent to apical trophozoite (b).
Transmission electron micrograph, 6,0003 magnification. Bar 5 1 mm.
Figure 4. Angelfish gastric mucosa. Multiple luminal macrogamonts (arrowheads) and numerous apical trophozoites.
Transmission electron micrograph, 3,0003 magnification. Bar 5 2 mm.
Figure 5. Angelfish gastric mucosa. A cluster of sporulated (white arrowhead) and unsporulated (black arrowhead) oocysts
embedded within the cytoplasm of a gastric epithelial cell. Transmission electron micrograph, 2,0003 magnification. Bar 5 3.5 mm.
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726 Case Reports
ium-infected cichlids from a lake in Israel.12 Subacute to The University of Melbourne, Australia, personal com-
chronic cellular injury has been identified in a C. molnari– munication, 2009). An 87% sequence similarity was
like infection in guppies.13 Cryptosporidium molnari has identified in a comparison of the 18s rRNA sequences
been associated with mortalities in some stocks of between the C. molnari–like parasite and the freshwater
cultured gilthead sea bream. More recently, coinfections angelfish isolate described in the current study. A Basic
with bacteria (Vibrio harveyi) and C. molnari resulted in Local Alignment Search Tool (http://www.ncbi.nlm.nih.
greater histopathologic gastric damage relative to infec- gov/blast/Blast.cgi) search, as well as a guide tree
tions with C. molnari alone.15 In the study described dendrogram generated for 18s rRNA gene sequences
herein, small numbers of bacterial organisms were from a variety of cryptosporidial species, indicate that the
inconsistently isolated from the viscera of multiple fish. C. molnari–like isolate is most closely related to the
This finding, along with the lack of histologic lesions angelfish isolate. An 87% similarity between the 2 isolates
consistent with bacterial infection (e.g., necrosis, granu- suggests that the angelfish isolate described in the present
lomas), suggests that bacterial coinfection did not play study is a new species of Cryptosporidium that shares
an important role in fish morbidity. In the present many common morphologic and niche features with C.
study, histopathologic gastric changes were most pro- molnari. Whether this angelfish isolate warrants designa-
nounced in the larval and juvenile angelfish. Clinically, tion as a new species awaits sequencing results from
this cohort of fish demonstrated the greatest morbidity cryptosporidial organisms recently described in other
and mortality. piscine hosts.
Cryptosporidiosis has previously been described in Acknowledgements. The authors thank Ruth Brown
plecostomus fish.10 For this reason, a plecostomus fish from for her expert assistance with the ultrastructural compo-
a tank with the affected juvenile fish was euthanized and nents of this study. The authors also acknowledge and are
examined both grossly and histologically. No evidence of grateful to Cary Swanson for submitting this interesting
alimentary cryptosporidiosis was identified in this animal. case.
The mechanism of transmission between angelfish tanks was
not determined but is likely to be the result of fecal-
contaminated water passing through multiple tanks. The Sources and manufacturers
recirculation system lacked a system for in-line particulate
a. Tricaine methanesulfonate, Argent Chemical Laboratories,
filtration before the water reached the various ultraviolet
Redmond, WA.
sterilizers. As a result, the particulate size was most likely
b. Decal Stat, Decal Chemical Corporation, Tallman, NY.
greater than the optimal 20–50 mm recommended for c. Hitachi H-600, Hitachi Medical Systems, Twinsburg, OH.
adequate ultraviolet exposure and kill rates for bacterial d. QIAamp, Qiagen, Valencia, CA.
and protozoan pathogens. e. Freeze‘n’Squeeze Kit, Bio-Rad, Hercules, CA.
No previously published report describing cryptosporid- f. Amplicon Express, Pullman, WA.
iosis in angelfish was identified in a search of the veterinary g. Amersham DYEnamic ET Terminator Cycle Sequencing Kit,
peer-reviewed literature. In addition, review articles defin- GE Healthcare, Stockholm, Sweden.
ing piscine cryptosporidiosis do not reference any such h. ABI 373 DNA Sequencer, Applied Biosystems, Foster City,
cases.2,11 Several features of the Cryptosporidium organisms CA.
described in the current study are consistent with the i. Invitrogen, Carlsbad, CA.
previously described C. molnari. As with C. molnari, these
organisms are fish parasites, occur within the gastric
mucosa, are associated with gastric epithelial cell degener- References
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