Massive Visceral Pentastomiasis Caused by Porocephalus crotali in a Dog

Vet Pathol 46:460–463 (2009) DOI: 10.1354/vp.07-VP-0246-R-BC

Massive Visceral Pentastomiasis Caused by Porocephalus crotali in a Dog M. D. BROOKINS, J. F. X. WELLEHAN, JR., J. F. ROBERTS, K. ALLISON, S. S. CURRAN, A. L. CHILDRESS, AND E. C. GREINER Department of Infectious Diseases and Pathology (MDB, JFR, ECG), and Small Animal Clinical Sciences (JFXW, ALC), College of Veterinary Medicine, University of Florida, Gainesville, FL; Department of Coastal Sciences, The University of Southern Mississippi, Ocean Springs, MS (SSC); and Society for Prevention of Cruelty to Animals of Central Florida, Orlando, FL (KA) Abstract. The testes of a 5-year-old, male, crossbred Schnauzer dog were the indicator organs for detection of massive pentastomiasis. Necropsy revealed numerous additional encysted parasites within the mesenteric lymph nodes, omentum, liver, sub-serosa of the small and large intestines, mesentery, and lungs. The nymphs had a pseudosegmented body, containing large eosinophilic glands and a chitinous cuticle with characteristic pores. Their hook configuration was consistent with that of Porocephalus. A pentastomid-specific 18S rRNA polymerase chain reaction (PCR) was designed and used to amplify template for sequencing. The sequence of the PCR product was 99.7% homologous with the reference sequence for P. crotali. This pentastomid parasite has been reported in North American snakes of genera Crotalus and Agkistrodon. Mammals are intermediate hosts, and snakes are the definitive hosts. Porocephalus crotali has been reported in dogs only once, and molecular methods have not been used previously to identify the species in clinical pentastomiasis. Key words: crotali.

Intermediate host; pentastome; pentastomiasis; polymerase chain reaction; Porocephalus

Pentastomida are a parasitic group of arthropods within the Pancrustacea.1,8,10 Their closest known relatives are branchiuran fish lice, such as Argulus.1,10 Genera Linguatula and Armillifer are most commonly implicated in zoonosis.3,11 Although the term ‘‘porocephalosis’’ has often been used for any visceral pentastomiasis, reported cases of porocephalosis, for which the organism was identified, have been Armillifer sp.13 The most common genera of reptilian pentastomes are Raillietiella, Porocephalus, Kiricephalus, Armillifer, and Sebekia, with the majority found as adults in lungs of snakes, lizards, and crocodilians.15 Adults of Linguatula serrata have been reported to infect nasal passages in dogs.14 There is a single case report of nymphal Porocephalus crotali infection in a dog.18 Porocephalus crotali was first described by Humboldt in 1808. This pentastomid parasite has been reported in the Southeastern United States in snakes of genera Crotalus and Agkistrodon.5,7,16 Adult pentastomes are often an incidental finding in reptiles at necropsy.9 Occult infections can be diagnosed antemortem by observation of eggs in fecal flotation.9 In a typical pentastomid life cycle, eggs are ingested by an intermediate host and develop into nymphs in multiple tissues.15 The definitive reptile host ingests the intermediate host, and the parasite usually matures in the lungs.15,16,19 Adults with mature eggs may be expelled from the trachea and eliminated from the definitive host through oral expulsion. These adults may also be swallowed, resulting in shed eggs in the feces.3,9,19 We describe a case of pentastomiasis in a stray dog as an uncommon host with uncommon tissue distribution for P. crotali infection.

A 5-year-old, intact male, crossbred Schnauzer dog without overt clinical disease was presented by an Alabama rescue group for neutering to the Society for the Prevention of Cruelty to Animals (SPCA) of Central Florida. At neutering, numerous encysted parasites were observed grossly in both testes; euthanasia was elected the following day. At necropsy, the liver, mesentery, mesenteric lymph nodes, lungs, spleen, small intestine, large intestine, and omentum had hundreds of 4 mm 3 3 mm 3 3 mm, C-shaped, densely packed, encysted parasites expanding the subserosa (Fig. 1). There were fewer encysted parasites in the kidneys. The mesenteric lymph nodes were firm and severely enlarged by encysted parasites. The abdominal fat and visceral surface of the abdominal wall contained scattered encysted parasites. No parasites were observed in the heart. Representative samples of each organ were preserved in 10% neutral buffered formalin. Sections of multiple organs were routinely processed for paraffin embedding, sectioned 5 mm thick, and stained with hematoxylin and eosin. Microscopically, multiple 2 mm 3 4 mm to 2 mm 3 2 mm cross-sections of pentastome nymphs were observed within the peripheral parenchyma of the testes (Fig. 2), kidney, liver, spleen, and subserosa of the intestine. In the testes, the seminiferous tubules were centrally compressed. The nymphs had a pseudosegmented body, a chitinous cuticle with pit-like openings to skin glands, chitinous hooked mouth parts, a prominent body cavity, numerous acidophilic glands, metamerically arranged striated muscle, and a digestive tract with villi lined by low columnar epithelium

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Fig. 1. Thoracic and peritoneal cavity; dog. Numerous pentastome nymphs are encysted in subpleural or subperitoneal tissue of the lung, diaphragm, mesentery, and liver. Fig. 2. Testis, subgross photograph; dog. Multiple encysted pentastome nymphs expand the interstitium between seminiferous tubules and tunica albuginea. HE. Bar 5 0.5 cm.

(Figs. 3, 4). The nymphs were contained in a cyst formed by a single layer of shed cuticle. Cysts were surrounded by fibroblastic proliferation with multifocal infiltration of lymphocytes, plasma cells, and macrophages. Degenerated seminiferous tubules had a wavy basement membrane and intraluminal multinucleated spermatids. The heads of the isolated pentastome nymphs were examined and photographed under a dissecting microscope. The mouth parts had hooks that were oriented in a straight line and head conformation that was not expanded (Fig. 5). Due to the lack of an expanded head, they were not considered to be in the genus Kiricephalus.15 Although parasites of the genus Sebekia sometimes infect mammals, the lack of a U-shaped segment of chitin associated with the pharynx, double nymphal hooks, and simple sclerotized ring morphology of the mouth parts, supported the identification of the genus as Porocephalus.2,6,17 Pentastomid/branchiuran-specific primers were designed from conserved regions of available pentastomid species in different genera, targeting an approximately

Fig. 3. Pentastome nymph, spleen; dog. The crosssection of the anterior section of the nymph includes a hook (H), eosinophilic glands (E), and digestive tract (D). The nymph is contained within a cyst formed by the shed cuticle (arrows). HE. Bar 5 0.5 mm. Fig. 4. Pentastome nymph, cuticle; dog. The cuticle and body wall have sclerotized openings (S) and striated muscle (M). HE. Bar 5 25 mm. Fig. 5. Pentastome nymph head, wet mount; dog. Five hooks are arranged in a straight line and the head lacks expansion.

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425-base pair segment corresponding to bases 638 to 1062 of the small subunit ribosomal RNA gene (SSU) of P. crotali (Genbank accession No. M29931). Because pentastomid 18S sequences available in GenBank were limited to P. crotali and an unidentified Raillietiella sp., Argulus nobilis was included as an ingroup during primer design to ensure the primers would amplify from diverse pentastomids and closely related crustaceans. DNA was extracted from samples using the DNEasy kit (Qiagen, Valencia, CA). A 20 mL reaction was run containing 2 mL extracted DNA, 1 mM forward primer Pent629F 59-CGGTTAAAAAGCTCGTAGTTGG-39, 1 mM reverse primer Pent1011R 59- GGCATCGTTTATGGTTAGAACTAGGG-39, 200 mM each of dATP, dCTP, dGTP, and dTTP, 0.5 U of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), 5% DMSO, and 2 mL PCR buffer plus magnesium (Invitrogen). The mixture was amplified in a thermal cycler (PCR Sprint, Thermo Hybaid, Franklin, MA) with an initial denaturation at 94uC for 5 minutes, followed by 40 cycles of denaturation at 94uC for 30 seconds, annealing at 58uC for 60 seconds, DNA extension at 72uC for 60 seconds, and a final extension step at 72uC for 7 minutes. The PCR product was resolved in a 1% agarose gel. Bands were excised and purified using the QIAquick gel extraction kit (Qiagen). The product was sequenced directly in both directions using the Big-Dye Terminator Kit (Perkin-Elmer, Branchburg, NJ) and analyzed on ABI 377 automated DNA sequencers at the University of Florida Center for Mammalian Genetics DNA Sequencing Facilities. The sequence was compared to known sequences in GenBank (National Center for Biotechnology Information, Bethesda, MD), EMBL (Cambridge, UK), and Data Bank of Japan (Mishima, Shiuoka, Japan) databases using BLASTN.4 Polymerase chain reaction amplification resulted in a product that was 383 base pairs after primer sequences were edited out. The highest BLASTN hit was with P. crotali (GenBank accession No. M29931), with 367 of 383 nucleotides identical. All but one of the differences were due to unresolved positions in the reference sequence. A single nucleotide at position 815 of the reference sequence had a guanine in our sequence and a cytosine in the reference sequence. The sequence was submitted to GenBank under accession number EF583871. This stray dog, which was massively infected with numerous pentastome nymphs, had no clinical signs of disease. The testes were the indicator organs, as encysted nymphs were observed at neutering. There is only a single previous case report of P. crotali infection in a dog,18 making this the second report of a dog as an intermediate host for P. crotali. Previous reports indicate that dogs can be definitive hosts for Linguatula serrata.14 Thus, dogs can serve as intermediate or definitive hosts for different pentastomid species. Pentastomes typically need 2 different hosts to complete their life cycle. We speculate that this dog may have eaten

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numerous infective larvae at one time, had a continuous daily source of infected prey, or eaten a snake with numerous pentastome adults. Porocephalus crotali has been studied in albino rats and mice, and it was found that 6 nymphs within the abdominal cavity of the intermediate host are needed on average to infect the reptilian definitive host.5 This case report demonstrates that pentastomes encyst in a variety of tissues. Identification of pentastomid species is clinically important, as it can direct the clinician toward sources of infection, and direct preventative measures. Morphologic identification of pentastomid species requires specialized training and is challenging when not dealing with adult stages. To our knowledge, this is the first report of use of nucleic acid-based techniques to identify a pentastomid in a clinical case. Consensus PCR is a rapid way to obtain a sequencing template from clinical samples for identification.20 One limitation of this technique is the paucity of reference sequences. Nucleic acid sequence is available for only 3 species: Armillifer armillatus, P. crotali, and an unidentified Raillietiella sp.1,10,12 Only the last 2 are from homologous genes. The data obtained from this case have resolved a number of ambiguities in the P. crotali reference sequence. The homologous sequences from additional morphologically identified pentastomids should be obtained to establish a reference database for identification of additional species.

Acknowledgement We would like to thank Dr. Brian Martin from SPCA of Central Florida for submission of this case. This case report was presented at the 2007 annual meeting of the ACVP, Savannah, Georgia and 2007 South Eastern Veterinary Pathology Slide Conference, Tifton, Georgia.

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6 Esslinger JH: Morphology of the egg and larva of Porocephalus crotali (Pentastomida). 48(3):457–462, 1962 7 Forrester DJ, Shealy RM, Best SH: Porocephalus crotali (Pentastomida) in South Carolina. J Parasitol 56:977, 1970 8 Giribet G, Carranza S, Baguna J, Riutort M, Ribera C: First molecular evidence for the existence of a Tardigrada + Arthropoda clade. Mol Biol Evol 13:76–84, 1996 9 Griener EC, Mader DR: Parasitology. In: Reptile Medicine and Surgery, ed. Mader DR, 2nd ed., pp. 343–364. Elsevier Inc., St. Louis, MO, 2006 10 Lavrov DV, Brown WM, Boore JL: Phylogenetic position of the Pentastomida and (pan)crustacean relationships. Proc R Soc Lond B 271:537–544, 2004 11 Machado MA, Makdissi FF, Canedo LF, Martino RB, Crescentini F, Chieffi PP, Bacchella T, Machado MC: Unusual case of pentastomiasis mimicking liver tumor. J Gastroenterol Hepatol 21: 1218–1220, 2006 12 Mallatt J, Giribet G: Further use of nearly complete 28S and 18S rRNA genes to classify Ecdysozoa: 37 more arthropods and a kinorhynch. Mol Phylogenet Evol 40:772–794, 2006 13 Mapp EM, Goldman LH: Roentgen diagnosis of Armillifer armillatus infestation (porocephalosis) in man. J Natl Med Assoc 68:198–200, 1976

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14 Meshgi B, Asgarian O: Prevalence of Linguatula serrata infestation in stray dogs of Shahrekord, Iran. J Vet Med B 50:466–467, 2003 15 Riley J: The biology of pentastomids. Adv Parasitol 25:45–128, 1986 16 Riley J, Henderson RJ: Pentastomids and the tetrapod lung. Parasitology 119:89–105, 1999 17 Riley J, Huchzermeyer FW: Pentastomid parasites of the family Sebekidae Fain, 1961 in West African dwarf crocodiles Osteolaemus tetraspis Cope, 1851 from the Congo, with a description of Alofia parva n. sp. Onderstepoort J Vet Res 62:151–162, 1995 18 Rogers KS, Miller G, Prestwood AK, Bjorling DE, Latimer KS: Aberrant nymphal pentastomiasis in a dog. J Am Anim Hosp Assoc 21:417–420, 1984 19 Host-parasite relations in some pentastomida. J Parasitol 53(1):202–206, 1967 20 Wellehan JF, Johnson AJ, Harrach B, Benko M, Pessier AP, Johnson CM, Garner MM, Childress A, Jacobson ER: Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses. J Virol 78:13366–13369, 2004 Request reprints from Dr. John F. Roberts, Thompson Bishop Sparks State Diagnostic Lab, PO Box 2209, Auburn, AL 36831-2209 (USA). E-mail: John.Roberts@ vetmed.auburn.edu.

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