A 22-year-old American Paint Horse gelding from the Gulf Coast region of Texas was examined at the Texas A&M University Veterinary Medical Teaching Hospital because of regrowth of a large necrotic and ulcerated perirectal mass just ventral to the anus. A mass in the same location had been surgically removed at a private veterinary clinic 11 months earlier. At that time, the mass was assumed to be a squamous cell carcinoma, although a histologic examination was not performed.
On evaluation, a large (15.0 × 12.5-cm) triangular-shaped skin defect had formed over the mass in the region between the rectum and ischial arch, creating a 10-cm-deep hole. The mass and associated soft tissues were contaminated with tarry fecal material and fly larvae. The horse had also developed persistent paraphimosis and was recently having some difficulty with urination but was otherwise bright and alert and had no other clinical abnormalities.
Following cleansing and superficial debridement, smears were made from scrapings of the mass, which on cytologic evaluation contained neoplastic cells that varied from round to oval and spindle shaped, with marked cellular atypia suggestive of a mesenchymal neoplasm. Scrapings of tissue were also processed for histologic examination and were found to contain islands and trabeculae of neoplastic epithelial cells with central areas of acantholysis consistent with an acantholytic squamous cell carcinoma. Passage of a stallion catheter revealed an intact urethra, which could be digitally palpated through the soft tissue hole created by the tumor. Because of the invasive and malignant nature of the tumor, its close proximity to the urethra, and the considerable amount of tumor-related tissue necrosis, the horse was anesthetized with xylazine (1 mg/kg [0.45 mg/lb], IV) followed by diazepam (0.1 mg/kg [0.045 mg/lb], IV) and ketamine hydrochloride (2 mg/kg [0.91 mg/lb], IV); once under anesthesia, the horse was euthanized with potassium chloride (2 mEq/kg, IV). A complete necropsy was performed.
At postmortem examination, the most striking feature noted upon opening the abdominal cavity was the presence of hundreds of small (1- to 2-mm-diameter), round, pale yellow to white, slightly raised, firm, granulomas disseminated uniformly over the capsular surface of all liver lobes (Figure 1). The granulomas extended throughout the parenchyma of the liver on all cut sections. Small clusters of up to 10 to 20 firm, slightly raised, 1- to 2-mm granulomas were also found on the serosal surface of the duodenum and jejunum (Figure 2). Bilaterally, the cranioventral aspect of the caudal lung lobes was firm and contained multiple white, slightly raised, 1- to 1.5-mm-diameter granulomas on cut section. The perirectal squamous cell carcinoma, which was the primary and expected lesion in this horse, surrounded the base of the penis and compressed the bulbospongiosus muscle but did not invade the urethra.
Samples of all tissues were fixed in neutral-buffered 10% formalin at the time of necropsy. Selected tissues were later trimmed, dehydrated, embedded in paraffin, and stained with H&E by use of routine histologic procedures. Histologic examination of the perirectal mass revealed the presence of an acantholytic squamous cell carcinoma. Additional lesions included a gastric leiomyoblastoma and metastatic acantholytic squamous cell carcinoma in the lungs, mediastinum, and pericardial sac that were unrelated to the pulmonary granulomas.
Histologic examination of the liver revealed marked, subacute to chronic inflammatory changes that are typically associated with parasite migration. These included numerous well-circumscribed granulomas containing a central eosinophilic core of necrotic cells surrounded by a peripheral rim of large numbers of palisading epithelioid macrophages and variable numbers of multinucleated giant cells (Figure 3). These areas were further surrounded by scattered eosinophils and moderate numbers of lymphocytes and plasma cells, with circumferential areas of fibrosis. Some of the granulomas that were considered more chronic in nature contained variable amounts of mineralization, while many others consisted primarily of lamellated layers of collagen with only small areas of inflammation.
One small area of the liver, however, was substantially different from all other areas. On gross examination, it contained a single 1-cm-diameter granulomatous-appearing lesion that was up to 10 times as large as all of the other granulomas. Histologically, this area contained a thick fibrous capsule that surrounded a well-circumscribed area of coagulation necrosis. Within the center of the lesion was a cyst-like space that was presumed to be a blood vessel and contained a pair of coiled adult male and female trematodes, whose internal structure was morphologically compatible with classic descriptions of members of the family Schistosomatidae.1–3 Each of the schistosomes lacked a body cavity and had a syncytial tegument (cuticle) that surrounded a parenchyma filled with loosely arranged mesoderm cells (Figure 4). The larger and more flattened of the 2 worms, which was presumed to be a male cut in transverse section, was 800 × 300 μm wide and had incurved lateral edges, which if fully extended would make it up to 1.7 mm wide. The smaller and more rounded worm was likely a female that was held within the male's gynecophoric canal, the central groove formed by the curved lateral margins where the female lives in permanent copula with the male. The positioning of the female adjacent to the male is believed to be the result of the tissue section being taken just proximal or distal to the point where the female enters or exits the canal. Alternatively, tissue processing or sectioning artifact may have artificially separated the 2. Numerous small, spine-studded elevations lined the concave outer surface of the tegument of the larger schistosome, adding further support to the view that this area represents part of the gynecophoric canal, where the pointed spines are used to hold the female in place.
Additional evidence for hepatic schistosome infection in this horse included the presence of fragments of thin yellow-brown membranes resembling trematode eggshells within the center of several of the granulomas (Figure 4). One of the granulomas contained a single schistosome egg with an outer eggshell and internal miracidium. Although it was somewhat collapsed and degenerate, the morphology of the egg, which had a diameter of 35 μm, lacked a spine or hook on its thin eggshell, and contained a single multicellular miracidium, was compatible with eggs of the schistosome Heterobilharzia americana.4,5 In almost all instances, egg and eggshell fragments were found within the center of granulomas and were surrounded by an abundance of fibrous tissue with little remaining inflammation. A similar pattern of multiple chronic granulomas with rare eggshell fragments was found in the affected areas of the lungs and wall of the small intestine (Figure 5). The marked hepatic, pulmonary, and small intestinal granuloma formation in this horse was similar to what has been observed following infections with H americana or Schistosoma spp in other mammals.6,7
To test the theory that the hepatic granulomas were the result of infection with H americana, fresh liver tissue from this horse was used to harvest DNA from 6 granulomas for PCR amplification. The primers used8 were designed to amplify DNA from a portion of the H americana 18S SSU rRNA gene. The DNA from a previously sequenced fragment of H americana recovered from an infected dog was used as a positive control. Polymerase chain reaction amplification of the harvested DNA resulted in amplification of identical 487-base pair fragments from 2 of the 6 granulomas. Amplified DNA was not obtained from the remaining 4 granulomas. The 2 amplified products were sequenced directly (GenBank accession No. HM363369). Each of the products had 100% sequence identity with a previously reported partial sequence for the H americana SSU rRNA gene, which was obtained following experimental infection of a golden hamster (Mesocricetus auratus) with H americana (GenBank accession No. AY157220).9
The SSU rRNA gene is 1 of 3 genes that has been used extensively to phylogenetically classify members of the Schistosomatidae family.9 The Heterobilharzia SSU rRNA sequence from the horse of the present study had close sequence identity with several other schistosome species. Their life cycle, biologic characteristics, host specificity, and geographic restriction, however, make their presence unlikely in this horse. The SSU rRNA sequence (GenBank accession No. AY157221) of Schistosomatium douthitti, the only other species of schistosome besides H americana that is known to infect mammals in the United States, had 98% sequence identity (481/487 bp), but that species is restricted to rodent hosts (muskrats and mice) in the northern United States and southern Canada.10 The SSU rRNA sequence (GenBank accession No. AY157233) of Schistosoma margrebowiei had 97% sequence identity (477/487 bp), but that species is found in antelope in Africa.11 The SSU rRNA sequence (GenBank accession No. AY157231) of Schistosoma indicum had 97% sequence identity (477/487 bp), and that species infects horses and ruminants but is found in South Asia.12 Lastly, the SSU rRNA sequence (GenBank accession No. AY157229) of Schistosoma incognitum had 97% sequence identity (477/487 bp), but that species is found in pigs, dogs, and rodents in South Asia, Indonesia, and Thailand.13–15 The sequence of the Heterobilharzia SSU rRNA gene fragment did not have close sequence identity to the avian schistosomes (genus Trichobilharzia), which are typically associated with schistosomiasis caused by cercarial penetration of human skin.
Snowden K, White S, Lewis B, et al. Distribution and characterization of Heterobilharzia americana infections in the dog in Texas (abstr), in Proceedings. 22nd Annu Meet World Assoc Adv Vet Parasitol 2009;99.
Wilson N, Johnson EM, Brunker J, et al. Heterobilharziasis in a dog (abstr), in Proceedings. 52nd Annu Meet Am Assoc Vet Parasitol 2007;93.
1. Chitwood M, Lichtenfels JR. Identification of parasitic metazoa in tissue sections. Exp Parasitol 1972; 32:407–519.
2. Price EW. A synopsis of the trematode family Schistosomidae, with descriptions of new genera and species. Proc U S Natl Museum 1929; 75:1–39.
3. Soulsby EJL. Trematodes (phylum: Platyhelminthes). In: Helminths, arthropods and protozoa of domesticated animals. 7th ed. Philadelphia: Lea & Febiger, 1982;8–87.
4. Bartsch RC, Ward BC. Visceral lesions in raccoons naturally infected with Heterobilharzia americana. Vet Pathol 1976; 13:241–249.
6. Anderson WI, King JM, Uhl EM, et al. Pathology of experimental Schistosoma mansoni infection in the Eastern woodchuck (Marmota monax). Vet Pathol 1991; 28:245–247.
7. Kagan IG, Meranze DR. The histopathology of the liver in mice experimentally infected with Schistosomatium douthitti. J Infect Dis 1957; 100:32–39.
8. Corapi WV, Ajithdoss DK, Snowden KF, et al. Multi-organ involvement of Heterobilharzia americana infection in a dog presented for systemic mineralization. J Vet Diagn Invest 2011; 23:826–831.
9. Lockyer AE, Olson PD, Ostergaard P, et al. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships of Schistosoma Weinland, 1858. Parasitology 2003; 126:203–224.
10. Malek EA. Geographical distribution, hosts, and biology of Schistosomatium douthitti (Cort, 1914) Price, 1931. Can J Zool 1977; 55:661–671.
11. Fransen J, Vercruysse J, Southgate VR, et al. Histopathologic findings of Schistosoma margrebowiei in experimentally infected laboratory animals. Vet Pathol 1992; 29:559–561.
12. Sharma DN, Dwivedi JN. Pulmonary schistosomiasis in sheep and goats due to Schistosoma indicum in India. J Comp Pathol 1976; 86:449–454.
13. Farhati K, de Pecoulas PE, Rajguru-Kazemi M. Les schistosomes d'animaux d'Asie. Med Mal Infect 1995; 25:107–110.
14. Carney WP, Van Peenen PFD, Ibrahim PB, et al. Schistosoma incognitum from Cikurai, West Java, Indonesia. Int J Parasitol 1977; 7:361–366.
15. Bunnag T, Thirachandra S, Impand P, et al. Schistosoma incognitum and its zoonotic potential role in Phitsanulok and Phichit provinces, northern Thailand. Southeast Asian J Trop Med Public Health 1983; 14:163–170.
16. Fabrick C, Bugbee A, Fosgate G. Clinical features and outcome of Heterobilharzia americana infection in dogs. J Vet Intern Med 2010; 24:140–144.
17. Fradkin JM, Braniecki AM, Craig TM, et al. Elevated parathyroid hormone-related protein and hypercalcemia in two dogs with schistosomiasis. J Am Anim Hosp Assoc 2001; 37:349–355.
19. Ruth J. Heterobilharzia americana infection and glomerulonephritis in a dog. J Am Anim Hosp Assoc 2010; 46:203–208.
20. Troy GC, Forrester D, Cockburn C, et al. Heterobilharzia americana infection and hypercalcemia in a dog: a case report. J Am Anim Hosp Assoc 1987; 23:35–40.
21. Malek EA, Ash LR, Lee HF, et al. Heterobilharzia infection in the dog and other mammals in Louisiana. J Parasitol 1961; 47:619–623.
22. Rohrer CR, Phillips LA, Ford SL, et al. Hypercalcemia in a dog: a challenging case. J Am Anim Hosp Assoc 2000; 36:20–25.
24. Flowers JR, Hammerberg B, Wood SL, et al. Heterobilharzia americana infection in a dog. J Am Vet Med Assoc 2002; 220:193–196.
25. McKown RD, Veatch JK, Fox LB. New locality record for Heterobilharzia americana. J Wildl Dis 1991; 27:156–160.
26. Johnson EM. Canine schistosomiasis in North America: an underdiagnosed disease with an expanding distribution. Compend Contin Educ Pract Vet 2010; 32:E1–E4.
27. Lee HF. Life history of Heterobilharzia americana Price 1929, a schistosome of the raccoon and other mammals in southeastern United States. J Parasitol 1962; 28:728–739.
28. Jones TC, Hunt RD, King NW. Diseases caused by parasitic helminths and arthropods. In: Veterinary pathology. 6th ed. Philadelphia: Williams & Wilkins, 1997;601–680.
29. Buergelt CD, Greiner EC. Fibrosing granulomas in the equine liver and peritoneum: a retrospective morphologic study. J Vet Diagn Invest 1995; 7:102–107.
30. Cheriuyot HK, Jordan HE. Potential for the spread of Fasciola hepatica in cattle in Oklahoma. J Am Vet Med Assoc 1990; 196:1090–1094.
31. Madsen H, Frandsen F. The spread of freshwater snails including those of medical and veterinary importance. Acta Trop 1989; 46:139–146.
32. Brown PJ, Clayton HM. Hepatic pathology of experimental Parascaris equorum infection in worm-free foals. J Comp Pathol 1979; 89:115–123.
33. Klei TR, Torbert BJ, Ochoa R, et al. Morphologic and clinicopathologic changes following Strongylus vulgaris infections of immune and nonimmune ponies. Am J Vet Res 1982; 43:1300–1307.
34. French RA, Meier WA, Zachary JF. Eosinophilic colitis and hepatitis in a horse with colonic intramucosal ciliated protozoa. Vet Pathol 1996; 33:235–238.