Haematoloechus sp. Infection in Wild-Caught Northern
Leopard Frogs (Rana pipiens)
CHARLIE C. HSU, VMD,1* D. BART CARTER, DVM, MS, DIPLOMATE, ACLAM,2 DONNA A. WILLIAMS, PHD,3
AND CYNTHIA L. BESCH-WILLIFORD, DVM, PHD, DIPLOMATE, ACLAM1
Three male, wild-caught northern leopard frogs (Rana pipiens) died over a 1-week period with no previous history of clinical illness or
disease. Noteworthy necropsy findings in one of the three frogs included depleted fat bodies in the coelomic cavity, indicating a poor
nutritional condition, and a heavy parasite burden in the lungs. The location of infection and morphologic characteristics of the parasite
were consistent with infection by the common lung fluke, Haematoloechus sp. In contrast to the heavy fluke load, only minor microscopic
changes were observed in the lungs. Lesions included mild hypertrophy of the bronchiolar epithelium, with few submucosal inflammatory cells consisting predominately of lymphocytes. Subsequent review of the literature revealed little about the pathologic effects of these
parasites except that small numbers are thought to cause the host little harm. Our findings suggest that even with a large number of
parasites, there is minimal pathologic impact in the lungs. We conclude that heavy lung-fluke infection should not be diagnosed as the
sole or major etiology of death or illness in leopard frogs.
History
From November of 2002 to January of 2003, groups of male, wildcaught northern leopard frogs (Rana pipiens), 6.35 to 7.62 cm in length,
were purchased from a commercial supplier (J. M. Hazen, Alburg, Vt.),
who captured the frogs from Lake Champlain in Vermont. Frogs were
housed in a facility accredited by the Association for Assessment and
Accreditation of Laboratory Animal Care International for use in an
experimental protocol approved by the University of Missouri’s Institutional Animal Care and Use Committee. Primary enclosures were
large polycarbonate plastic cages (48.26 cm × 26.67 cm × 20.32 cm)
with 8 to 10 frogs per box and were enriched with a terra-cotta brick
that extended above the water line to provide a dry resting area. The
water level was maintained approximately 2.54 to 5.08 cm high. Water
changes were performed once a week with tap water that was conditioned (exposed to air for 24 h) prior to placement with the frogs.
The animal room temperature was maintained at 20 to 22°C with
humidity between 30% and 60% and on a 12:12-h light:dark cycle,
but the primary enclosures were kept in an Isotemp Laboratory Refrigerator (Fisher Scientific, Pittsburgh, Pa.) at a temperature of
approximately 15°C. Frogs were orally gavaged once every 2 weeks
with at least 1 ml of pureed meat-based baby food (Vegetable Beef
Dinner, Gerber Products Co., Fremont, Mich.).
Four months after receipt of the last shipment, three frogs from
the colony of approximately 50 frogs were found dead over a 1-week
period. All three frogs came from different primary enclosures and
were not currently on study. Remaining frogs in the colony did not
show signs of clinical disease or illness, and no previous history of
disease or death in the colony had been reported.
Pathologic Findings
Examination of the first two frogs did not reveal any noteworthy
external lesions, and further necropsy or testing was not performed.
Necropsy of the third frog revealed a poor body condition includDepartment of Veterinary Pathobiology, Research Animal Diagnostic Laboratory, University of Missouri–Columbia, 1600 E. Rollins Road, Columbia, Missouri 652111; Office
of Animal Resources, University of Missouri–Columbia, M144 Medical Sciences Building, Columbia, Missouri 652122; Capillary Physiology & Microcirculation Research
Laboratory, MU Sinclair School of Nursing, University of Missouri–Columbia, Columbia, Missouri 65211 3
*Corresponding author
14
CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science
Figure 1. Lung tissue transected to show multiple Haematoloechus sp. (arrows) emerging from the airways.
Figure 2. Haematoloechus sp. (preserved in 70% ethanol) collected from the
lungs of a northern leopard frog (Rana pipiens).
ing prominent pelvic bones and depleted fat bodies within the coelomic cavity. In addition, there was mild reddening of the skin on
the ventromedial aspects of the upper hind legs. The lungs had
multifocal raised brown nodules (diameter, ca. 1 to 2 mm) on the
surface. When the lungs were transected, parasites were found within
the airways (Fig. 1). The parasites were approximately 3 to 6 mm in
length, had dorsoventrally flattened bodies, and a mosaic color pattern of white, brown, and tan (Fig. 2). No other noteworthy lesions
were identified.
Histopathologic review of the intestines, kidneys, liver, spleen, and
testes did not reveal any noteworthy lesions. In the lungs, multiple
cross-sections of large parasites were observed within the airways and
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Figure 3. Mild hypertrophy of the respiratory epithelium (arrowhead) with
few submucosal lymphocytes is adjacent to Haematoloechus sp. parasites (arrows) in the bronchioles of the lung (H&E stain).
in alveolar spaces (Fig. 3). Parasites were characterized by a thin cuticle with short spines and internal organs surrounded by a
parenchymatous matrix. Multiple brown oval operculated eggs (ca.
25 µm × 15 µm) with an eosinophilic center that comprised 70% of
the egg were observed in the parasite uterus. The lung tissue adjacent
to parasites was characterized by very mild hypertrophy of the bronchiolar epithelium with few submucosal inflammatory cells consisting
predominately of lymphocytes. In light of their location, size, histologic characteristics, and egg morphology, the parasites were consistent
with lung flukes of the genus Haematoloechus.
Discussion
Most reports of clinical parasitic diseases in frogs describe nematode infections, and infections with Haematoloechus sp. flukes have
not been reported in the comparative medicine literature. Our report
is intended to provide information about Haematoloechus morphology and life cycle and to minimize the potential for misdiagnosis of
this parasite as the cause of clinical disease when the fluke infection is
found in ill frogs.
Haematoloechus sp. belong to the phylum Platyhelminthes, class
Trematoda, and order Digenea. Species of Haematoloechus reported
to occur in Rana pipiens in North America include H. medioplexus,
H. longiplexus, H. varioplexus, and H. complexus (1-4). Generally,
these lung flukes measure 8.0 mm long by 1.2 mm wide, are digenetic and hermaphroditic, and have dark brown eggs measuring 22
to 29 µm long by 13 to 17 µm wide (1, 5, 6). Flynn (1) states that more
than 40 Haematoloechus species have been found to occur in the lungs
of frogs and toads throughout the world, and this number may be even
greater today. Infection is limited to frogs and toads of the genera
Rana and Bufo and has not been reported to occur in the African
clawed frog (Xenopus laevis), a commonly used research amphibian.
Geographic distribution of trematodes of the order Digenea is dependent on that of snail species, as these trematodes are very
discriminating in their choice of snails as their intermediate hosts
(6). To complete their life cycle, Haematoloechus sp. require two intermediate hosts, a snail and an insect (such as dragonflies or
damselflies) (1, 7, 8). Briefly, adult flukes in the lungs of amphibians
release embryonated eggs, which are passed up the bronchioles and
trachea to the mouth and swallowed. Eggs passed in the feces then
are ingested by snails, in which the eggs hatch and develop to cercariae. The cercariae are released in the feces of the snail and develop
to metacercariae after they are taken up by insect larvae. Infection of
Volume 43, No. 6 / November 2004
an amphibian host occurs when the parasitized adult insect is ingested by a frog, and the metacercariae are released in the stomach,
migrate up the esophagus, and enter the lungs via the trachea, where
they mature to adults.
There is very little published on the pathologic effects of
Haematoloechus sp. lung flukes in frogs. One report states that small
numbers of flukes cause little harm, but mass invasion may cause
serious disease (9), and even as many as 75 flukes have been found in
a single frog (1). Haematoloechus sp. are not zoonotic.
There are no reports of successful elimination of Haematoloechus
sp. with any particular parasiticide. However, empirical treatment
can be attempted in light of efficacy against trematodes in other species. Reported doses of anthelmintics used in amphibians that may be
effective against Haematoloechus sp. include fenbendazole (10 mg/kg
orally once to 100 mg/kg every 10 to 21 days) or praziquantel (8 to
24 mg/kg orally, subcutaneously, or intracoelomically every 14 to 21
days or 10 mg/L water bath for ≤ 3 h every 14 to 21 days) (10-13).
Ivermectin and levamisole, two anthelmintics commonly used in
amphibians, are ineffective against trematode infections (14). Because
the pathologic effects of the flukes are unknown but appear to be
minimal and because two intermediate hosts are required for transmission to other leopard frogs, treatment decisions will vary from
institution to institution in light of the needs of research and husbandry programs.
No other frogs in our colony subsequently died or became ill, suggesting the cluster of deaths represented coincidental, isolated
incidents. To consider the possibility that environmental factors contributed to the deaths, husbandry practices for the frog colony were
evaluated. Water quality may have been a factor, but water quality
evaluation data at the time of death were not recorded; water quality
information including pH and levels of chlorine, ammonia, and nitrite now are recorded at each water change. Housing at a temperature
of 15°C may have altered the frog’s metabolic and immune status.
Although recommended housing temperatures range from 15 to 25°C
(10), temperatures of 0 to 20°C have been reported to be immunosuppressive for amphibians (11). To minimize the potential for
immunosuppression, we suggested limiting the time frogs are housed
at decreased temperatures to 6 months because in the wild, the frogs
experience and can tolerate much lower temperatures for this amount
of time during the winter months. The diet and feeding schedule
also were reviewed, as 1 frog was in poor body condition; the composition of food, amount fed, frequency, and feeding practices were
considered adequate because no other frogs in the colony displayed
clinical signs of malnutrition or disease secondary to gavaging. The
body condition of the frogs was closely monitored by a skilled animal care technician, who adjusted the amount fed accordingly. Gavage
accidents are rare because of the experience of the technician, and
death due to aspiration of food material during feeding was unlikely
in this case, given the lack of food material in the respiratory tree. In
addition, frogs may have harbored undetected bacterial, fungal, viral
or parasitic diseases as they did not come from a closed or well-defined colony. Minor revisions of husbandry practices should provide
environmental conditions for our frog colonies that will minimize
the incidence of stress-related, opportunistic diseases and death.
Finally, the mild reddening of the skin on the ventromedial aspects of the upper hindlegs noted at necropsy was not considered to
be clinically significant because the discoloration was not present when
the dead frog was removed from its primary enclosure. Therefore,
the reddening developed during the few hours of storage in the refrigerator prior to the necropsy and was most likely a post-mortem
change rather than an ante-mortem disease process, such as infection
with Aeromonas hydrophila, the causative agent of redleg disease. Other
frogs in the colony were examined specifically for evidence of redleg,
CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science
15
and no clinical signs of the disease were found.
The primary objective of this report was to familiarize the reader
with the morphologic and histopathologic identification of
Haematoloechus sp., a lung fluke commonly found in wild-caught
Rana pipiens. A secondary objective was to emphasize that heavy
Haematoloechus infection induced minor pathologic changes. Therefore, parasitism by this fluke should not be considered the sole or
major etiology in cases of clinical disease or death in leopard frogs.
Acknowledgment
The author thanks Howard Wilson for his photographic assistance.
References
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