Journal of Parasitology MASSIVE MUSCULAR INFECTION BY A SARCOCYSTIS SPECIES IN A SOUTH AMERICAN RATTLESNAKE (CROTALUS DURISSUS TERRIFICUS) --Manuscript Draft-Manuscript Number:
14-642R1
Full Title:
MASSIVE MUSCULAR INFECTION BY A SARCOCYSTIS SPECIES IN A SOUTH AMERICAN RATTLESNAKE (CROTALUS DURISSUS TERRIFICUS)
Short Title:
Sarcocystis sp. in rattlesnake muscle
Article Type:
Short Communications
Corresponding Author:
David S. Lindsay, PhD Virginia Polytechnical Institute and State University Blacksburg, Virginia UNITED STATES
Corresponding Author Secondary Information: Corresponding Author's Institution:
Virginia Polytechnical Institute and State University
Corresponding Author's Secondary Institution: First Author:
John F. Roberts, DVM
First Author Secondary Information: Order of Authors:
John F. Roberts, DVM J Wellehan, DVM J L Weisman, DVM M Rush, DVM A L Childress, DVM David S. Lindsay, PhD
Order of Authors Secondary Information: Abstract:
Massive numbers of sarcocysts of a previously undescribed species of Sarcocystis were observed in the skeletal muscles throughout the body of an adult, female South American rattlesnake (Crotalus durissus terrificus). Examination of tissue sections by light microscopy demonstrated that sarcocysts were present in 20 to 40% of muscle fibers from five sampled locations. Sarcocysts were not present in cardiac muscle, smooth muscle or other organs. Sarcocysts were 0.05-0.15 mm wide, had variable length depending on the viewed orientation and size of the muscle fiber, and had a sarcocyst wall less than 1-µm thick. Sarcocysts were subdivided by septa and had central degeneration in older sarcocysts. Host induced secondary encapsulation or an inflammatory response was not present. By transmission electron microscopy (TEM), the sarcocyst wall was Type I, with a parasitophorous membrane of approximately 100 nanometers in width arranged in an undulating pattern and intermittently folded inward in a branching pattern. The sarcocysts contained metrocytes in different stages of development and mature bradyzoites. The nucleic acid sequence from a section of the 18S small subunit rRNA gene was most closely related to S. mucosa that uses marsupials as intermediate hosts and has an unknown definitive host. This is apparently the third report of muscular Sarcocystis infection in snakes and is the first to describe the ultrastructure of the sarcocysts and use sequencing methods to aid in identification.
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SHORT COMMUNICATIONS
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Massive Muscular Infection by a Sarcocystis Species in a South American
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Rattlesnake (Crotalus durissus terrificus)
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J. F. Roberts, J. F.X. Wellehan Jr.*, J. L. Weisman†, M. Rush‡, A. L. Childress*,
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and David S. Lindsay§, Thompson Bishop Sparks State Diagnostic Laboratory 890
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Simms Road, Auburn University, Auburn, Alabama 36831-2209; *Department of Small
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Animal Clinical Sciences, College of Veterinary Medicine, University of Florida,
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Gainesville, Florida 320608; †Department of Pathobiology, College of Veterinary
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Medicine, Auburn University, Auburn, Alabama 36849; ‡Pathobiology Academic
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Program, School of Veterinary Medicine, St. George's University, St. George's,
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Grenada, West Indies; §Department of Biomedical Sciences and Pathobiology, Virginia-
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Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
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24061-0342. Correspondence should be sent to: [email protected]
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Abstract: Massive numbers of sarcocysts of a previously undescribed species of
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Sarcocystis were observed in the skeletal muscles throughout the body of an adult,
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female South American rattlesnake (Crotalus durissus terrificus). Examination of tissue
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sections by light microscopy demonstrated that sarcocysts were present in 20 to 40% of
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muscle fibers from 5 sampled locations. Sarcocysts were not present in cardiac muscle,
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smooth muscle or other organs. Sarcocysts were 0.05-0.15 mm wide, had variable
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length depending on the viewed orientation and size of the muscle fiber, and had a
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sarcocyst wall less than 1-µm thick. Sarcocysts were subdivided by septa and had
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central degeneration in older sarcocysts. Host induced secondary encapsulation or an
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inflammatory response was not present. By transmission electron microscopy (TEM),
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the sarcocyst wall was Type I, with a parasitophorous membrane of approximately 100
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nanometers in width arranged in an undulating pattern and intermittently folded inward
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in a branching pattern. The sarcocysts contained metrocytes in different stages of
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development and mature bradyzoites. The nucleic acid sequence from a section of the
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18S small subunit rRNA gene was most closely related to S. mucosa that uses
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marsupials as intermediate hosts and has an unknown definitive host. This is
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apparently the third report of muscular Sarcocystis infection in snakes and is the first to
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describe the ultrastructure of the sarcocysts and use sequencing methods to aid in
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identification.
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Members of the genus Sarcocystis are Apicomplexan protozoal parasites that
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are distributed world-wide in vertebrates. They have an obligatory 2-host life cycle with
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a carnivore or omnivore as the definitive host and an omnivore or herbivore as the
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intermediate host (Dubey et al., 1989). Definitive hosts become infected after Ingesting
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sarcocysts containing infective bradyzoites in the muscle tissue of prey. Bradyzoites
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liberated from sarcocysts penetrate in to the lamina propria and transform into male
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(micro) and female (macro) gamonts that develop with in cells in the mucosa.
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Fertilization then occurs and the macrogamont becomes an oocyst. The oocysts
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undergo sporulation to form 2 sporocysts each containing 4 sporozoites. The oocysts
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pass from the lamina propria, often breaking the oocyst wall, and free sporocysts and
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intact oocysts are excreted in the feces. Little to no clinical signs are observed in
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definitive hosts (Dubey et al., 1989). The intermediate host, in turn, becomes infected by
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ingestion of sporocysts in contaminated food or water. Sporozoites migrate from the gut
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and develop asexually by endopolyogeny in endothelial cells of capillaries and small
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vessels. Merozoites usually undergo a second generation of endopolyogeny in vessels
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before entering striated muscle cells to produce sarcocysts. The light and electron
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microscopic structure of sarcocysts are important in determining the species of
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Sarcocystis present in muscle (Dubey et al., 1989; Dubey and Odening, 2001).
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Sarcocysts may lie dormant in the intermediate host for many years.
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Snakes serve as the definitive host for many species of Sarcocystis and excrete
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oocysts/sporocysts in their feces (Matuschka, 1987; Duszynski and Upton, 2009). The
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present short communication is unusual because it reports the occurrence of muscular
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sarcocysts in a snake suggesting that snakes can also serve as intermediate hosts for
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Sarcocystis species. There are apparently only 2 other reports of snakes having
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sarcocysts in their skeletal muscles. Sarcocysts of 2 species, S. pythonis (Tiegs, 1931)
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from an Australian python (Morelia argus) and S. atractaspidis (Parenzan, 1947) from a
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burrowing viper (Atractaspis sp.) and have been reported. The definitive hosts for these
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2 species are unknown, their sarcocyst walls have not been described by transmission
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electron microscopy (TEM), and genetic sequencing has not been reported for either
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species (Matuschka, 1987). This report describes intramuscular sarcocysts in a
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previous unrecognized intermediate host for the genus Sarcocystis.
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A wild caught adult female South American rattlesnake, Crotalus durissus
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terrificus, was confiscated in Tennessee, by federal law enforcement officials and died
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in custody. The snake was imported from its native country as an adult approximately 2
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yr prior to the seizure. This subspecies of Crotalus durissus inhabits the dryer savanna
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of Brazil south of the Amazonian forest and eastern sections of Peru, Bolivia, Paraguay,
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Uruguay and Northern Argentina. Predators and scavengers that consume C. d.
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terrificus may include raptors, marsupials, monitor lizards and ophiophagous snakes
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(Wüster et al., 2005).
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The non-gravid female C. d. terrificus, 84 cm in length and weighing 2.2 kg, was
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presented to the Alabama Department of Agriculture and Industries, Thompson Bishop
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Sparks Diagnostic Laboratory, Auburn, Alabama, in a freshly thawed, moderately
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autolyzed condition. At necropsy, the small intestine and colon were severely distended
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with impacted undigested rodent hair. The lumen of the distal colon was constricted by
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a restrictive “napkin ring” lesion. No gross lesions were observed in skeletal muscle at
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time of necropsy or retrospectively in formalin fixed “wet” tissues. Clostridium sordellii
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and Fusarium sp. were cultured from the colon. The head, 3 transverse body segments
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and the entire viscera were preserved in 10% neutral buffered formalin. Sections of
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multiple organs were routinely processed for paraffin embedding, sectioned at 5 μm
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thick, and stained with hematoxylin and eosin (H&E). Selected tissues were stained with
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routine histochemical stains (Gomori methenamine silver staining [GMS], Giemsa
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staining, Warthin Starry silver staining, periodic acid-Schiff reaction staining [PAS],
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Fite’s acid fast staining).
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Formalin-fixed axial muscle adjacent to a vertebra was minced into 2-3 mm
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cubes and fixed in 3% (v/v) glutaraldehyde in phosphate buffered saline (pH 7.4).
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Muscle cubes were post-fixed in 1% (w/v) osmium tetroxide in 0.1 M phosphate buffer,
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dehydrated in a series of ethanol, passed through 2 changes of propylene oxide, and
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embedded in Poly/Bed 812 resin (Polysciences Inc., Warrington, Pennsylvania). Thin
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sections were stained with uranyl acetate and lead citrate and examined with a 10CA
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TEM operating at 60 kV (Carl Zeiss Micro Imaging GmbH, Jena, Germany). Digital
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images were captured using an ATM camera system (Advanced Microscopy
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Techniques Corp., Danvers, Massachusetts).
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Infected skeletal muscle tissue was processed using methods described by
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Garner et al. (2006). Consensus PCR targeting a region of the 18S SSU rRNA gene
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with previously described primers (Wünschmann et al., 2010) was conducted and
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protocols were used to obtain a sequencing template. The PCR products were resolved
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in 1% agarose gels. The bands were excised and purified using the QIAquick gel
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extraction kit (Qiagen, Valencia, California). Direct sequencing was performed using the
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Big-Dye Terminator Kit (Applied Biosystems, Foster City, California) and analyzed on
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ABI 3130 automated DNA sequencers at the University of Florida Interdisciplinary
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Center for Biotechnology Research Sequencing Facilities, Gainesville, Florida. All
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products were sequenced in both directions. Primer sequences were edited out prior to
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further analyses.
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The skin had diffuse necrotizing epidermitis with a myriad of intralesional fungal
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hyphae and bacteria. By GMS staining, areas of necrotizing epidermitis contained
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sickle-shaped multiseptate macroconidia consistent with Fusarium species. There was
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advanced autolysis of the distal colon. The restrictive colonic lesion contained a
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uniform population of large, polygonal cells consistent with colonic adenocarcinoma and
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special staining of this segment by GMS, Giemsa, Warthin Starry silver, PAS and Fite’s
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acid fast stains were negative for infectious organisms. The cause of death was
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diagnosed as bacterial septicemia and mycotic epidermitis secondary to intestinal
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impaction and dehabilitation.
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Skeletal muscle examined from the head, tongue, and 3 areas adjacent to
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vertebra had sarcocysts in 20 to 40% of muscle fibers (Fig. 1). Sarcocysts were not
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observed in the cardiac muscle, smooth muscle or other organs nor was an
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inflammatory reaction observed in direct association with sarcocysts. Individual
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sarcocysts were 0.05-0.15 mm in width and up to 2 mm in length. The sarcocyst wall
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was thin, generally measuring less than 1 μm thick and continuous with septa up to 2
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µm in thickness. By H&E staining compartments formed by septa contained faintly
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visible, 1-2 μm in diameter, bradyzoites. By PAS staining, amylopectin granules in
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bradyzoites were visible. No evidence of active merogony, or meronts was observed
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within vascular walls at any location in the body. Some vessel walls did appear sclerotic
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due to increased collagen.
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The sarcocyst wall was Type I (Dubey et al., 1989) (Figs. 2, 3). The
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parasitophorous membrane of the sarcocyst made up the primary sarcocyst wall.
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Portions of the primary sarcocyst wall intermittently folded inward into the sarcocyst in
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an undulating pattern (Fig. 2). Ground substance was immediately beneath the primary
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sarcocyst wall and formed septa of variable thickness that divided the sarcocyst into
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compartments (Figs. 2, 3). The sarcocyst compartments contained metrocytes in
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various stages of development and mature bradyzoites. A secondary cyst wall
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(encapsulation by host cell) was not observed enclosing any sarcocyst (Figs. 1-3). Our
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taxonomic description of the present Sarcocystis species (Figs. 1-3) is as follows:
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Type intermediate host: Crotalus durissus terrificus.
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Type definitive host: unknown.
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Type Locality: Unknown location from South American Continent.
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Type material deposited: Hapantotypes 1 slide H&E stained and a paraffin block of
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embedded muscle are deposited in the United States National Parasite Collection
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(USNPC), Beltsville, Maryland, as USNPC 105448. The DNA sequence from a section
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of the 18S small subunit rRNA is deposited in GenBank.
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Prevalence: 1 of 1, infected when collected in 2007.
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Sarcocyst wall: Type 1 (Dubey et al., 1989) with inward folding of portions of primary
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sarcocyst wall.
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Secondary cyst wall: None present.
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Etymology: Present molecular and transmission data are not sufficient to warrant the
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naming of a new species.
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The final sequence of the 18S SSU rRNA gene product was 681 nucleotides and
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differed from Sarcocystis mucosa (GenBank accession number AF109679) by a single
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nucleotide.
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The snake reported in this study had a massive muscular infection with
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sarcocysts. We estimate the parasite could have made up as much as 3% of the
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snake’s body weight. Even more astounding is the complete absence of an
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inflammatory response. The date and location of capture for this snake are unknown,
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and there is also the question of whether the snake was infected in captivity. It is also
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worthy to speculate if the snake could have been an accidental host for this species of
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Sarcocystis. No evidence of active merogony was observed in capillaries and small
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vessels throughout the body supporting the observation that this was not an acute or
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recent infection.
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Tiegs (1931) described the light microscopic structure of S. pythonis sarcocysts
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in an Australian carpet snake Python spilotes. The sarcocysts were up to 1.1mm in
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length, had a thin wall, contained metrocytes (sporoblasts), and bradyzoites (spores).
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No secondary cyst wall was present. Some sarcocyst were divided in to compartments
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but most were not (Tiegs, 1931). A nucleus was depicted in the drawing for Figure 1
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that was inside the sarcocyst and within the septae (Tiegs, 1931). The sarcocysts we
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observed in the present study in the muscles of C. d. terrificus were divided into
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compartments by septae, had no secondary cyst wall and we did not observe nuceli in
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the septa of sarcocysts.
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The ultrastructure of the Type 1 cyst wall found in the present study is unique in
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that is has intermittent inward folding of the cyst wall (Fig. 2). This may be a defining
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characteristic of the parasite in a natural intermediate host or it may reflect abnormal
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development in an inappropriate intermediate host. The sarcocysts we examined had
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been frozen and fixed in 10% neutral buffered formalin before being fixed in 3% (v/v)
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glutaraldehyde in PBS and processed for TEM. It is highly unlikely these events
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induced significant distortion of the TEM structure of the sarcocyst. Odening (1998) has
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argued that freezing and thawing of sarcocysts and initial fixation in formalin does not
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cause essential loss of structural integrity of sarcocysts when viewed using TEM.
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It is possible that the present species has a reptile as a definitive host, as 6
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dihomoxenous squamate to squamate life cycles have been identified (Odening, 1998;
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Ślapeta et al., 2001). An intraspecific life cycle as observed in some squamate species
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is unlikely in C. d. durissi because they are not ophiophagous and do not cannibalize
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tails or other parts of their species, as do some lizards. A snake to snake life cycle has
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not been reported for any species of Sarcocystis, but this lifecycle is also worthy of
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investigation. There are several species of snakes including the mussurana (Clelia spp.)
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that are native to the geographic range of C. d. terrificus, have immunity to rattlesnake
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venoms, and have a diet largely composed of snakes in the family Viperidae (Cerdas
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and Lomonte, 1982; Pinto and Lema, 2002)
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The Sarcocystis fauna of South America is likely under reported because of the
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huge diversity of animal species on the continent. Because S. mucosa is a close genetic
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match for this species in this snake, a comparison of these 2 species with possible
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phylogenetic implications is warranted. Sarcocystis mucosa is a species of Sarcocystis
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with unique characteristics in that the intermediate hosts are marsupials (wallabies and
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pademelons), it is macroscopic, it occurs in the esophagus and gastrointestinal tract, it
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may occur in smooth muscle and it has secondary host response encapsulation by the
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intermediate host (O’Donoghue et al., 1987; Jakes, 1998). A secondary host
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encapsulation response is only reported for approximately 15 of the approximately 200
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named species of Sarcocystis (Odening, 1998). The sarcocyst membrane of S. mucosa
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is Type 13 and contains widely spaced and mushroom like with microtubules that reach
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into the ground substance (Jakes, 1998). The sarcocysts observed in the present study
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have a Type 1 cyst wall with minimal ground substance, no microtubules or mushroom-
201
like projections and was found only in skeletal muscle. The sarcocysts of the 2 species
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are similar because they both form relatively large sarcocysts that are divided into
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compartments by septa. It is postulated that the intermediate host of S. mucosa is a
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carnivorous marsupial (Jakes, 1998). It is possible a carnivorous marsupial, possibly an
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opossum, could be the definitive host for the Sarcocystis observed in the present study.
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We would like to thank Kristen Bullard, Smithsonian Institution Libraries,
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Washington D.C. for research assistance and Sara Rawlinson, Melissa Roseman and
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Heather Busby of Alabama Department of Agriculture and Industries, Thompson-
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Bishop-Sparks Diagnostic Lab, Auburn, AL for technical expertise. We are grateful to
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Kathy Lowe, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg,
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Virginia for assistance with transmission electron microscopy and James Harrison,
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Kentucky Reptile Zoo, Slade, Kentucky for identifying the species of snake in this study.
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LITERATURE CITED
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Dubey, J. P., and K. Odening. 2001. Toxoplasmosis and related infections. In Parasitic
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diseases of wild mammals, W. M. Samuel, M. J. Pybus, and A. A. Kocan (eds.). Iowa
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State University Press, Ames, Iowa, p. 478-519.
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_____, C. A. Speer, and R. Fayer. 1989. Sarcocystosis of animals and man. CRC
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Press, Boca Raton, Florida, 215 p.
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Duszynski, D. W., and S. J. Upton. 2009. The biology of the Coccidia (Apicomplexa) of
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snakes of the world. A scholarly handbook for identification and treatment.”
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CreateSpace, a subsidiary of Amazon.com, 470 p.
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Garner, M. M., C. H. Gardiner, J. F. Wellehan, A. J. Johnson, T. McNamara, M. Linn, S.
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P. Terrell, A. Childress, and E. R. Jacobson. 2006. Intranuclear coccidiosis in tortoises:
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Nine cases. Veterinary Pathology 43: 311–320.
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Jakes, K. A., 1998. Sarcocystis mucosa in Bennett’s Wallabies and Pademelons from
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Tasmania. Journal of Wildlife Diseases 34: 594-599.
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Matuschka, F. R. 1987. Reptiles as intermediate and/or final hosts of sarcosporidia.
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Parsitology Research 73: 22-32.
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O’Donoghue, P. J., D. L. Obendorf, M. G. O’Callaghan, E. Moore, and B. R. Dixon.
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1987. Sarcocystis mucosa (Balnchrd 1885) Labbe’ 1889 in an unadorned rock wallabies
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(Petrogale assimilis) and Bennett’s wallabies (Macropus rufogriseus). Parasitology
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Research 73: 113-120.
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Odening, K. 1998. The present state of species-systematics in Sarcocystis Lankester,
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1882 (Protista, Sporozoa, Coccidia). Systematic Parasitology 41: 209-233.
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Parenzan, P. 1947. Sarcosporidiosi (psorospermosi) da nuova specie (Prot.:
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Sarcocystis atractaspidis n. sp.) in reptile (Atractaspis). Bollettino della Societa dei
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naturalisti in Napoli 55: 117-119.
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Pinto, C., and T. De Lema. 2002. Feeding behavior and diet of snakes, genera Boiruna
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and Clelia (Serpentes, Colubridae). Iheringia Serie Zoologia 92: 9-19.
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Ślapeta, J. R., D. Modŕy, J. Votýpka, M. Jirku, B. Koudela, and J. Lukeś. 2001. Multiple
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origin of the dihomoxenous life cycle in sarcosporidia. International Journal for
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Parasitology 31: 413-417.
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Tiegs, O. W., 1931. Note on the occurrence of Sarcocystis in muscle of python.
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Parasitology 23: 412-414.
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Wünschmann, A., F. X. Wellehan Jr., A. Armien, W. J. Bemrick, D. Barnes, G. A.
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Averbeck, R. Roback, M. Schwabenlander, E. D’Almeida, R. Joki, et al. 2010. Renal
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infection by a new coccidian genus in big brown bats (Eptesicus fuscus). Journal of
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Parasitology 96: 178-183.
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Wüster, W., J. E. Ferguson, J. A. Quijada-Mascareñas, C. E. Pook, M. G. Salomão, and
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R. S. Thorpe. 2005. Tracing an invasion: Land bridges, refugia and the phylogeography
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251
of the Neotropical rattlesnake (Serpentes: Viperidae: Crotalus durissus). Molecular
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Ecology 14: 1095-1108.
253 254
Figure 1. Hematoxylin and eosin stained section of skeletal muscle from Crotalus
255
durissus terrificus demonstrating numerous myocytes that contain sarcocysts (S). Bar
256
= 100 µm.
257
Figure 2. Transmission electron micrographs of Sarcocystis sp. in axial skeletal
258
muscle from Crotalus durissus terrificus. (A) The primary sarcocyst wall is composed of
259
the parasitophorous vacuole membrane ornamented with electron-dense projections.
260
Note inward folding of primary cyst wall into the ground substance (Gs). Note the
261
absence of a secondary cyst wall and absence of microtubules in the inward folds of
262
the primary cyst wall. The outer margin of the primary cyst wall is bordered by
263
sarcoplasm of the skeletal muscle cell (Sk). Septa (S) divide the sarcocyst into
264
compartments containing bradyzoites (Bz). Uranyl acetate and lead citrate. Bar = 10
265
µm. (B) Higher magnification of a sarcocyst in axial skeletal muscle (Sk) from the
266
rattlesnake demonstrating characteristic inward folding of areas in the primary
267
sarcocyst wall, ground substance (Gs), septa (s) and groups of bradyzoites (Bz). Note
268
the absence of a secondary cyst wall and absence of microtubules in the inward folds
269
of the primary cyst wall. Uranyl acetate and lead citrate. Bar = 5 µm.
270
Figure 3. Transmission electron micrographs of Sarcocystis sp. in axial skeletal muscle
271
(Sk) from Crotalus durissus terrificus.demonstratin the features of a Type 1 sarcocyst
272
wall. No inward folding of the primary sarcocyst wall is present in these figures. (A)
273
Groups of bradyzoites (Bz) are below the ground substance (Gs) of the primary
12
13
274
sarcocyst wall and the sarcocyst is divided in to compartments by septa (S). Uranyl
275
acetate and lead citrate. Bar = 2 µm. (B) Interphase of the primary sarcocyst wall and
276
cytoplasm of a skeletal muscle (Sk) cell. Note the host cell mitochondrion (M) next to
277
the primary sarcocyst wall and the ground substance (Gs) that is immediately beneath
278
it. Uranyl acetate and lead citrate. Bar = 500 nm.
13
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14-642R1
Full Title:
MASSIVE MUSCULAR INFECTION BY A SARCOCYSTIS SPECIES IN A SOUTH AMERICAN RATTLESNAKE (CROTALUS DURISSUS TERRIFICUS)
Short Title:
Sarcocystis sp. in rattlesnake muscle
Article Type:
Short Communications
Corresponding Author:
David S. Lindsay, PhD Virginia Polytechnical Institute and State University Blacksburg, Virginia UNITED STATES
Corresponding Author Secondary Information: Corresponding Author's Institution:
Virginia Polytechnical Institute and State University
Corresponding Author's Secondary Institution: First Author:
John F. Roberts, DVM
First Author Secondary Information: Order of Authors:
John F. Roberts, DVM J Wellehan, DVM J L Weisman, DVM M Rush, DVM A L Childress, DVM David S. Lindsay, PhD
Order of Authors Secondary Information: Abstract:
Massive numbers of sarcocysts of a previously undescribed species of Sarcocystis were observed in the skeletal muscles throughout the body of an adult, female South American rattlesnake (Crotalus durissus terrificus). Examination of tissue sections by light microscopy demonstrated that sarcocysts were present in 20 to 40% of muscle fibers from five sampled locations. Sarcocysts were not present in cardiac muscle, smooth muscle or other organs. Sarcocysts were 0.05-0.15 mm wide, had variable length depending on the viewed orientation and size of the muscle fiber, and had a sarcocyst wall less than 1-µm thick. Sarcocysts were subdivided by septa and had central degeneration in older sarcocysts. Host induced secondary encapsulation or an inflammatory response was not present. By transmission electron microscopy (TEM), the sarcocyst wall was Type I, with a parasitophorous membrane of approximately 100 nanometers in width arranged in an undulating pattern and intermittently folded inward in a branching pattern. The sarcocysts contained metrocytes in different stages of development and mature bradyzoites. The nucleic acid sequence from a section of the 18S small subunit rRNA gene was most closely related to S. mucosa that uses marsupials as intermediate hosts and has an unknown definitive host. This is apparently the third report of muscular Sarcocystis infection in snakes and is the first to describe the ultrastructure of the sarcocysts and use sequencing methods to aid in identification.
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SHORT COMMUNICATIONS
2
Massive Muscular Infection by a Sarcocystis Species in a South American
3
Rattlesnake (Crotalus durissus terrificus)
4
J. F. Roberts, J. F.X. Wellehan Jr.*, J. L. Weisman†, M. Rush‡, A. L. Childress*,
5
and David S. Lindsay§, Thompson Bishop Sparks State Diagnostic Laboratory 890
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Simms Road, Auburn University, Auburn, Alabama 36831-2209; *Department of Small
7
Animal Clinical Sciences, College of Veterinary Medicine, University of Florida,
8
Gainesville, Florida 320608; †Department of Pathobiology, College of Veterinary
9
Medicine, Auburn University, Auburn, Alabama 36849; ‡Pathobiology Academic
10
Program, School of Veterinary Medicine, St. George's University, St. George's,
11
Grenada, West Indies; §Department of Biomedical Sciences and Pathobiology, Virginia-
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Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
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24061-0342. Correspondence should be sent to: [email protected]
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Abstract: Massive numbers of sarcocysts of a previously undescribed species of
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Sarcocystis were observed in the skeletal muscles throughout the body of an adult,
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female South American rattlesnake (Crotalus durissus terrificus). Examination of tissue
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sections by light microscopy demonstrated that sarcocysts were present in 20 to 40% of
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muscle fibers from 5 sampled locations. Sarcocysts were not present in cardiac muscle,
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smooth muscle or other organs. Sarcocysts were 0.05-0.15 mm wide, had variable
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length depending on the viewed orientation and size of the muscle fiber, and had a
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sarcocyst wall less than 1-µm thick. Sarcocysts were subdivided by septa and had
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central degeneration in older sarcocysts. Host induced secondary encapsulation or an
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inflammatory response was not present. By transmission electron microscopy (TEM),
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the sarcocyst wall was Type I, with a parasitophorous membrane of approximately 100
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nanometers in width arranged in an undulating pattern and intermittently folded inward
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in a branching pattern. The sarcocysts contained metrocytes in different stages of
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development and mature bradyzoites. The nucleic acid sequence from a section of the
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18S small subunit rRNA gene was most closely related to S. mucosa that uses
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marsupials as intermediate hosts and has an unknown definitive host. This is
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apparently the third report of muscular Sarcocystis infection in snakes and is the first to
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describe the ultrastructure of the sarcocysts and use sequencing methods to aid in
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identification.
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Members of the genus Sarcocystis are Apicomplexan protozoal parasites that
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are distributed world-wide in vertebrates. They have an obligatory 2-host life cycle with
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a carnivore or omnivore as the definitive host and an omnivore or herbivore as the
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intermediate host (Dubey et al., 1989). Definitive hosts become infected after Ingesting
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sarcocysts containing infective bradyzoites in the muscle tissue of prey. Bradyzoites
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liberated from sarcocysts penetrate in to the lamina propria and transform into male
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(micro) and female (macro) gamonts that develop with in cells in the mucosa.
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Fertilization then occurs and the macrogamont becomes an oocyst. The oocysts
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undergo sporulation to form 2 sporocysts each containing 4 sporozoites. The oocysts
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pass from the lamina propria, often breaking the oocyst wall, and free sporocysts and
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intact oocysts are excreted in the feces. Little to no clinical signs are observed in
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definitive hosts (Dubey et al., 1989). The intermediate host, in turn, becomes infected by
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ingestion of sporocysts in contaminated food or water. Sporozoites migrate from the gut
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and develop asexually by endopolyogeny in endothelial cells of capillaries and small
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vessels. Merozoites usually undergo a second generation of endopolyogeny in vessels
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before entering striated muscle cells to produce sarcocysts. The light and electron
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microscopic structure of sarcocysts are important in determining the species of
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Sarcocystis present in muscle (Dubey et al., 1989; Dubey and Odening, 2001).
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Sarcocysts may lie dormant in the intermediate host for many years.
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Snakes serve as the definitive host for many species of Sarcocystis and excrete
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oocysts/sporocysts in their feces (Matuschka, 1987; Duszynski and Upton, 2009). The
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present short communication is unusual because it reports the occurrence of muscular
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sarcocysts in a snake suggesting that snakes can also serve as intermediate hosts for
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Sarcocystis species. There are apparently only 2 other reports of snakes having
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sarcocysts in their skeletal muscles. Sarcocysts of 2 species, S. pythonis (Tiegs, 1931)
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from an Australian python (Morelia argus) and S. atractaspidis (Parenzan, 1947) from a
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burrowing viper (Atractaspis sp.) and have been reported. The definitive hosts for these
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2 species are unknown, their sarcocyst walls have not been described by transmission
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electron microscopy (TEM), and genetic sequencing has not been reported for either
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species (Matuschka, 1987). This report describes intramuscular sarcocysts in a
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previous unrecognized intermediate host for the genus Sarcocystis.
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A wild caught adult female South American rattlesnake, Crotalus durissus
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terrificus, was confiscated in Tennessee, by federal law enforcement officials and died
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in custody. The snake was imported from its native country as an adult approximately 2
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yr prior to the seizure. This subspecies of Crotalus durissus inhabits the dryer savanna
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of Brazil south of the Amazonian forest and eastern sections of Peru, Bolivia, Paraguay,
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Uruguay and Northern Argentina. Predators and scavengers that consume C. d.
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terrificus may include raptors, marsupials, monitor lizards and ophiophagous snakes
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(Wüster et al., 2005).
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The non-gravid female C. d. terrificus, 84 cm in length and weighing 2.2 kg, was
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presented to the Alabama Department of Agriculture and Industries, Thompson Bishop
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Sparks Diagnostic Laboratory, Auburn, Alabama, in a freshly thawed, moderately
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autolyzed condition. At necropsy, the small intestine and colon were severely distended
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with impacted undigested rodent hair. The lumen of the distal colon was constricted by
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a restrictive “napkin ring” lesion. No gross lesions were observed in skeletal muscle at
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time of necropsy or retrospectively in formalin fixed “wet” tissues. Clostridium sordellii
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and Fusarium sp. were cultured from the colon. The head, 3 transverse body segments
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and the entire viscera were preserved in 10% neutral buffered formalin. Sections of
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multiple organs were routinely processed for paraffin embedding, sectioned at 5 μm
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thick, and stained with hematoxylin and eosin (H&E). Selected tissues were stained with
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routine histochemical stains (Gomori methenamine silver staining [GMS], Giemsa
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staining, Warthin Starry silver staining, periodic acid-Schiff reaction staining [PAS],
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Fite’s acid fast staining).
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Formalin-fixed axial muscle adjacent to a vertebra was minced into 2-3 mm
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cubes and fixed in 3% (v/v) glutaraldehyde in phosphate buffered saline (pH 7.4).
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Muscle cubes were post-fixed in 1% (w/v) osmium tetroxide in 0.1 M phosphate buffer,
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dehydrated in a series of ethanol, passed through 2 changes of propylene oxide, and
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embedded in Poly/Bed 812 resin (Polysciences Inc., Warrington, Pennsylvania). Thin
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sections were stained with uranyl acetate and lead citrate and examined with a 10CA
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TEM operating at 60 kV (Carl Zeiss Micro Imaging GmbH, Jena, Germany). Digital
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images were captured using an ATM camera system (Advanced Microscopy
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Techniques Corp., Danvers, Massachusetts).
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Infected skeletal muscle tissue was processed using methods described by
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Garner et al. (2006). Consensus PCR targeting a region of the 18S SSU rRNA gene
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with previously described primers (Wünschmann et al., 2010) was conducted and
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protocols were used to obtain a sequencing template. The PCR products were resolved
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in 1% agarose gels. The bands were excised and purified using the QIAquick gel
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extraction kit (Qiagen, Valencia, California). Direct sequencing was performed using the
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Big-Dye Terminator Kit (Applied Biosystems, Foster City, California) and analyzed on
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ABI 3130 automated DNA sequencers at the University of Florida Interdisciplinary
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Center for Biotechnology Research Sequencing Facilities, Gainesville, Florida. All
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products were sequenced in both directions. Primer sequences were edited out prior to
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further analyses.
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The skin had diffuse necrotizing epidermitis with a myriad of intralesional fungal
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hyphae and bacteria. By GMS staining, areas of necrotizing epidermitis contained
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sickle-shaped multiseptate macroconidia consistent with Fusarium species. There was
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advanced autolysis of the distal colon. The restrictive colonic lesion contained a
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uniform population of large, polygonal cells consistent with colonic adenocarcinoma and
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special staining of this segment by GMS, Giemsa, Warthin Starry silver, PAS and Fite’s
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acid fast stains were negative for infectious organisms. The cause of death was
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diagnosed as bacterial septicemia and mycotic epidermitis secondary to intestinal
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impaction and dehabilitation.
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Skeletal muscle examined from the head, tongue, and 3 areas adjacent to
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vertebra had sarcocysts in 20 to 40% of muscle fibers (Fig. 1). Sarcocysts were not
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observed in the cardiac muscle, smooth muscle or other organs nor was an
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inflammatory reaction observed in direct association with sarcocysts. Individual
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sarcocysts were 0.05-0.15 mm in width and up to 2 mm in length. The sarcocyst wall
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was thin, generally measuring less than 1 μm thick and continuous with septa up to 2
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µm in thickness. By H&E staining compartments formed by septa contained faintly
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visible, 1-2 μm in diameter, bradyzoites. By PAS staining, amylopectin granules in
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bradyzoites were visible. No evidence of active merogony, or meronts was observed
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within vascular walls at any location in the body. Some vessel walls did appear sclerotic
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due to increased collagen.
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The sarcocyst wall was Type I (Dubey et al., 1989) (Figs. 2, 3). The
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parasitophorous membrane of the sarcocyst made up the primary sarcocyst wall.
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Portions of the primary sarcocyst wall intermittently folded inward into the sarcocyst in
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an undulating pattern (Fig. 2). Ground substance was immediately beneath the primary
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sarcocyst wall and formed septa of variable thickness that divided the sarcocyst into
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compartments (Figs. 2, 3). The sarcocyst compartments contained metrocytes in
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various stages of development and mature bradyzoites. A secondary cyst wall
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(encapsulation by host cell) was not observed enclosing any sarcocyst (Figs. 1-3). Our
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taxonomic description of the present Sarcocystis species (Figs. 1-3) is as follows:
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Type intermediate host: Crotalus durissus terrificus.
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Type definitive host: unknown.
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Type Locality: Unknown location from South American Continent.
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Type material deposited: Hapantotypes 1 slide H&E stained and a paraffin block of
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embedded muscle are deposited in the United States National Parasite Collection
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(USNPC), Beltsville, Maryland, as USNPC 105448. The DNA sequence from a section
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of the 18S small subunit rRNA is deposited in GenBank.
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Prevalence: 1 of 1, infected when collected in 2007.
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Sarcocyst wall: Type 1 (Dubey et al., 1989) with inward folding of portions of primary
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sarcocyst wall.
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Secondary cyst wall: None present.
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Etymology: Present molecular and transmission data are not sufficient to warrant the
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naming of a new species.
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The final sequence of the 18S SSU rRNA gene product was 681 nucleotides and
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differed from Sarcocystis mucosa (GenBank accession number AF109679) by a single
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nucleotide.
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The snake reported in this study had a massive muscular infection with
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sarcocysts. We estimate the parasite could have made up as much as 3% of the
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snake’s body weight. Even more astounding is the complete absence of an
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inflammatory response. The date and location of capture for this snake are unknown,
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and there is also the question of whether the snake was infected in captivity. It is also
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worthy to speculate if the snake could have been an accidental host for this species of
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Sarcocystis. No evidence of active merogony was observed in capillaries and small
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vessels throughout the body supporting the observation that this was not an acute or
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recent infection.
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Tiegs (1931) described the light microscopic structure of S. pythonis sarcocysts
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in an Australian carpet snake Python spilotes. The sarcocysts were up to 1.1mm in
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length, had a thin wall, contained metrocytes (sporoblasts), and bradyzoites (spores).
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No secondary cyst wall was present. Some sarcocyst were divided in to compartments
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but most were not (Tiegs, 1931). A nucleus was depicted in the drawing for Figure 1
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that was inside the sarcocyst and within the septae (Tiegs, 1931). The sarcocysts we
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observed in the present study in the muscles of C. d. terrificus were divided into
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compartments by septae, had no secondary cyst wall and we did not observe nuceli in
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the septa of sarcocysts.
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The ultrastructure of the Type 1 cyst wall found in the present study is unique in
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that is has intermittent inward folding of the cyst wall (Fig. 2). This may be a defining
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characteristic of the parasite in a natural intermediate host or it may reflect abnormal
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development in an inappropriate intermediate host. The sarcocysts we examined had
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been frozen and fixed in 10% neutral buffered formalin before being fixed in 3% (v/v)
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glutaraldehyde in PBS and processed for TEM. It is highly unlikely these events
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induced significant distortion of the TEM structure of the sarcocyst. Odening (1998) has
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argued that freezing and thawing of sarcocysts and initial fixation in formalin does not
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cause essential loss of structural integrity of sarcocysts when viewed using TEM.
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It is possible that the present species has a reptile as a definitive host, as 6
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dihomoxenous squamate to squamate life cycles have been identified (Odening, 1998;
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Ślapeta et al., 2001). An intraspecific life cycle as observed in some squamate species
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is unlikely in C. d. durissi because they are not ophiophagous and do not cannibalize
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tails or other parts of their species, as do some lizards. A snake to snake life cycle has
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not been reported for any species of Sarcocystis, but this lifecycle is also worthy of
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investigation. There are several species of snakes including the mussurana (Clelia spp.)
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that are native to the geographic range of C. d. terrificus, have immunity to rattlesnake
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venoms, and have a diet largely composed of snakes in the family Viperidae (Cerdas
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and Lomonte, 1982; Pinto and Lema, 2002)
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The Sarcocystis fauna of South America is likely under reported because of the
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huge diversity of animal species on the continent. Because S. mucosa is a close genetic
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match for this species in this snake, a comparison of these 2 species with possible
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phylogenetic implications is warranted. Sarcocystis mucosa is a species of Sarcocystis
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with unique characteristics in that the intermediate hosts are marsupials (wallabies and
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pademelons), it is macroscopic, it occurs in the esophagus and gastrointestinal tract, it
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may occur in smooth muscle and it has secondary host response encapsulation by the
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intermediate host (O’Donoghue et al., 1987; Jakes, 1998). A secondary host
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encapsulation response is only reported for approximately 15 of the approximately 200
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named species of Sarcocystis (Odening, 1998). The sarcocyst membrane of S. mucosa
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is Type 13 and contains widely spaced and mushroom like with microtubules that reach
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into the ground substance (Jakes, 1998). The sarcocysts observed in the present study
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have a Type 1 cyst wall with minimal ground substance, no microtubules or mushroom-
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like projections and was found only in skeletal muscle. The sarcocysts of the 2 species
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are similar because they both form relatively large sarcocysts that are divided into
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compartments by septa. It is postulated that the intermediate host of S. mucosa is a
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carnivorous marsupial (Jakes, 1998). It is possible a carnivorous marsupial, possibly an
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opossum, could be the definitive host for the Sarcocystis observed in the present study.
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We would like to thank Kristen Bullard, Smithsonian Institution Libraries,
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Washington D.C. for research assistance and Sara Rawlinson, Melissa Roseman and
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Heather Busby of Alabama Department of Agriculture and Industries, Thompson-
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Bishop-Sparks Diagnostic Lab, Auburn, AL for technical expertise. We are grateful to
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Kathy Lowe, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg,
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Virginia for assistance with transmission electron microscopy and James Harrison,
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Kentucky Reptile Zoo, Slade, Kentucky for identifying the species of snake in this study.
213
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253 254
Figure 1. Hematoxylin and eosin stained section of skeletal muscle from Crotalus
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durissus terrificus demonstrating numerous myocytes that contain sarcocysts (S). Bar
256
= 100 µm.
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Figure 2. Transmission electron micrographs of Sarcocystis sp. in axial skeletal
258
muscle from Crotalus durissus terrificus. (A) The primary sarcocyst wall is composed of
259
the parasitophorous vacuole membrane ornamented with electron-dense projections.
260
Note inward folding of primary cyst wall into the ground substance (Gs). Note the
261
absence of a secondary cyst wall and absence of microtubules in the inward folds of
262
the primary cyst wall. The outer margin of the primary cyst wall is bordered by
263
sarcoplasm of the skeletal muscle cell (Sk). Septa (S) divide the sarcocyst into
264
compartments containing bradyzoites (Bz). Uranyl acetate and lead citrate. Bar = 10
265
µm. (B) Higher magnification of a sarcocyst in axial skeletal muscle (Sk) from the
266
rattlesnake demonstrating characteristic inward folding of areas in the primary
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sarcocyst wall, ground substance (Gs), septa (s) and groups of bradyzoites (Bz). Note
268
the absence of a secondary cyst wall and absence of microtubules in the inward folds
269
of the primary cyst wall. Uranyl acetate and lead citrate. Bar = 5 µm.
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Figure 3. Transmission electron micrographs of Sarcocystis sp. in axial skeletal muscle
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(Sk) from Crotalus durissus terrificus.demonstratin the features of a Type 1 sarcocyst
272
wall. No inward folding of the primary sarcocyst wall is present in these figures. (A)
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Groups of bradyzoites (Bz) are below the ground substance (Gs) of the primary
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13
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sarcocyst wall and the sarcocyst is divided in to compartments by septa (S). Uranyl
275
acetate and lead citrate. Bar = 2 µm. (B) Interphase of the primary sarcocyst wall and
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cytoplasm of a skeletal muscle (Sk) cell. Note the host cell mitochondrion (M) next to
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the primary sarcocyst wall and the ground substance (Gs) that is immediately beneath
278
it. Uranyl acetate and lead citrate. Bar = 500 nm.
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