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.
Photographs of the liver from a horse with disseminated parasitic granulomas due to Hetereobilharzia americana infection. A—Large numbers of small, disseminated granulomas cover the capsular surface of the liver, giving it a diffuse pattern of round, white to pale yellow lesions on a dark background (ie, starry-sky appearance). B—Multiple discrete, white to pale yellow nodules bulge from the cut surface of the liver.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photographs of the liver from a horse with disseminated parasitic granulomas due to Hetereobilharzia americana infection. A—Large numbers of small, disseminated granulomas cover the capsular surface of the liver, giving it a diffuse pattern of round, white to pale yellow lesions on a dark background (ie, starry-sky appearance). B—Multiple discrete, white to pale yellow nodules bulge from the cut surface of the liver.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photographs of the liver from a horse with disseminated parasitic granulomas due to Hetereobilharzia americana infection. A—Large numbers of small, disseminated granulomas cover the capsular surface of the liver, giving it a diffuse pattern of round, white to pale yellow lesions on a dark background (ie, starry-sky appearance). B—Multiple discrete, white to pale yellow nodules bulge from the cut surface of the liver.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photographs of parasitic granulomas due to H americana infection of the same horse as in Figure 1. A—Multiple raised, white nodules form small clusters in the wall of the small intestine that bulge from the serosal surface. B—Multiple discrete, white nodules are visible throughout the cut section of the right caudal lung lobe.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photographs of parasitic granulomas due to H americana infection of the same horse as in Figure 1. A—Multiple raised, white nodules form small clusters in the wall of the small intestine that bulge from the serosal surface. B—Multiple discrete, white nodules are visible throughout the cut section of the right caudal lung lobe.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photographs of parasitic granulomas due to H americana infection of the same horse as in Figure 1. A—Multiple raised, white nodules form small clusters in the wall of the small intestine that bulge from the serosal surface. B—Multiple discrete, white nodules are visible throughout the cut section of the right caudal lung lobe.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
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.
Photomicrograph of a section of a typical eosinophilic granuloma in the liver of the same horse as in Figure 1. The granuloma contains a central coagulum of degenerating eosinophils surrounded by a palisading layer of epithelioid macrophages and multinucleated giant cells. H&E stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photomicrograph of a section of a typical eosinophilic granuloma in the liver of the same horse as in Figure 1. The granuloma contains a central coagulum of degenerating eosinophils surrounded by a palisading layer of epithelioid macrophages and multinucleated giant cells. H&E stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photomicrograph of a section of a typical eosinophilic granuloma in the liver of the same horse as in Figure 1. The granuloma contains a central coagulum of degenerating eosinophils surrounded by a palisading layer of epithelioid macrophages and multinucleated giant cells. H&E stain; bar = 50 μm.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
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.
Photomicrographs of sections of H americana adults (A and B) and eggs (C and D) in the liver of the same horse as in Figure 1. A—Two coiled adult male and female schistosomes within a vessel surrounded by an area of coagulation necrosis. B—Different section taken from the same area showing the larger of the 2 adult schistosomes with numerous spine-studded elevations lining the concave surface of the tegument. This area represents the gynecophoric canal of the male. C—The center of a chronic fibrosing granuloma containing degenerating, yellow-brown schistosome eggshell fragments. D—A chronic fibrosing granuloma containing a single degenerating egg with an outer eggshell (arrowhead) and a multicellular, internal miracidium (arrow). H&E stain; bar = 100 μm (A), 50 μm (B), or 10 μm (C and D).
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photomicrographs of sections of H americana adults (A and B) and eggs (C and D) in the liver of the same horse as in Figure 1. A—Two coiled adult male and female schistosomes within a vessel surrounded by an area of coagulation necrosis. B—Different section taken from the same area showing the larger of the 2 adult schistosomes with numerous spine-studded elevations lining the concave surface of the tegument. This area represents the gynecophoric canal of the male. C—The center of a chronic fibrosing granuloma containing degenerating, yellow-brown schistosome eggshell fragments. D—A chronic fibrosing granuloma containing a single degenerating egg with an outer eggshell (arrowhead) and a multicellular, internal miracidium (arrow). H&E stain; bar = 100 μm (A), 50 μm (B), or 10 μm (C and D).
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photomicrographs of sections of H americana adults (A and B) and eggs (C and D) in the liver of the same horse as in Figure 1. A—Two coiled adult male and female schistosomes within a vessel surrounded by an area of coagulation necrosis. B—Different section taken from the same area showing the larger of the 2 adult schistosomes with numerous spine-studded elevations lining the concave surface of the tegument. This area represents the gynecophoric canal of the male. C—The center of a chronic fibrosing granuloma containing degenerating, yellow-brown schistosome eggshell fragments. D—A chronic fibrosing granuloma containing a single degenerating egg with an outer eggshell (arrowhead) and a multicellular, internal miracidium (arrow). H&E stain; bar = 100 μm (A), 50 μm (B), or 10 μm (C and D).
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
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
Photomicrographs of sections of chronic fibrosing granulomas due to H americana infection in the lung and small intestine of the same horse as in Figure 1. A—Multiple discrete and coalescing granulomas containing concentric layers of collagen and rare central eggshell fragments (not shown) in a caudal lung lobe. B—Multiple chronic, coalescing granulomas containing abundant fibrous tissue in the tunica muscularis and serosa of the small intestine. H&E stain; in both panels, bar = 500 μm.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photomicrographs of sections of chronic fibrosing granulomas due to H americana infection in the lung and small intestine of the same horse as in Figure 1. A—Multiple discrete and coalescing granulomas containing concentric layers of collagen and rare central eggshell fragments (not shown) in a caudal lung lobe. B—Multiple chronic, coalescing granulomas containing abundant fibrous tissue in the tunica muscularis and serosa of the small intestine. H&E stain; in both panels, bar = 500 μm.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
Photomicrographs of sections of chronic fibrosing granulomas due to H americana infection in the lung and small intestine of the same horse as in Figure 1. A—Multiple discrete and coalescing granulomas containing concentric layers of collagen and rare central eggshell fragments (not shown) in a caudal lung lobe. B—Multiple chronic, coalescing granulomas containing abundant fibrous tissue in the tunica muscularis and serosa of the small intestine. H&E stain; in both panels, bar = 500 μm.
Citation: Journal of the American Veterinary Medical Association 239, 8; 10.2460/javma.239.8.1117
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.
Discussion
Heterobilharzia americana, a trematode in the family Schistosomatidae, is an important cause of chronic diarrhea, weight loss, gastrointestinal and liver disease, and hypercalcemia in dogs and is predominantly found in the Gulf Coast and south Atlantic region of the United States,16 where infections have been reported in Texas,17–20,a Louisiana,21 Florida,22,23 and, more recently, North Carolina.24 The geographic range of the trematode and its intermediate hosts, the lymnaeid freshwater snails Fossaria (Lamnaea) cubensis and Pseudosuccinea columella, includes Florida, Georgia, Louisiana, Mississippi, North Carolina, South Carolina, Texas, and, perhaps most surprisingly, Kansas, following importation of infected raccoons from Texas and Florida.25 The natural definitive host is the raccoon, although a number of other wild and captive mammals, including the bobcat, armadillo, Brazilian tapir, beaver, coyote, mountain lion, mink, nutria, opossum, red wolf, swamp rabbit, and white-tailed deer, have been naturally infected.25 The importance of schistosomiasis in dogs as an emerging disease in the United States is underscored by recent studies24,26,b of H americana infection in North Carolina, Kansas, Alabama, Georgia, and Tennessee and an increasing incidence of the disease in Texas.a
The life cycle of H americana and the pathogenesis of schistosomiasis in dogs have been documented,27 and we assume that infections in horses share many of the same features. Mature eggs shed from the feces of an infected raccoon hatch and release free-swimming miracidia once they come in contact with freshwater. The miracidia swim near the water surface, where they attach themselves to an appropriate snail and penetrate the snail's soft mantle. The miracidia then undergo development in the snail, resulting in mother and daughter sporocysts, with the eventual emergence from the snail as free-swimming cercariae. Susceptible mammals standing, wading, or swimming in freshwater are exposed to the cercariae, which are capable of penetrating the host's intact skin. The cercariae migrate first to the lungs and then shortly after to the liver, where they undergo most of their growth and development. Mature worms eventually migrate to the mesenteric veins, although mature males and females in copula may be found in both the mesenteric and intrahepatic veins.27 Eggs released from the female into the mesenteric veins release proteolytic enzymes through pores in the eggshell that allow them to break through the vessel walls and migrate to the intestinal tract.28 Following penetration of the intestinal wall, they enter the intestinal lumen and are shed in the feces. The eggs, which may also circulate as emboli in the vessels, induce a severe granulomatous response when they become lodged in visceral organs, and although the immune response destroys the eggs, it also results in most of the pathological findings associated with this disease.
Small but consistent numbers of horses with disseminated hepatic granulomas similar to those of the horse of the present study are seen in the necropsy service at Texas A&M University each year. Of 1,672 horses that were necropsied at our institution from January 2004 through July 2010, 38 (2.3%) had a morphologic diagnosis of hepatic parasitic granulomas included in the necropsy report, which represents between 5 and 6 affected horses/y. Although H americana has been implicated as a possible cause of disseminated hepatic granulomas in horses,29 a definitive cause has never been established.
Our previous attempts to amplify H americana–specific DNA from formalin-fixed liver granulomas of other horses have all been unsuccessful. We believe this was attributable to a combination of the negative effect of formalin fixation on the yield of extracted schistosome DNA and the small amount of DNA remaining in the liver because of the chronic nature of the disease. In the horse of the present study, fresh tissue was used and positive PCR products were obtained from 2 of 6 granulomas that were carefully dissected from the surrounding liver tissue. In a single previous attempt at our institution to amplify H americana DNA, in which fresh liver from another affected horse and a different set of primers were used, a 1,187-bp fragment of the H americana SSU rRNA gene was obtained (GenBank accession No. DQ230918). We conclude, therefore, that the use of fresh tissue may be critical for obtaining positive PCR amplification and that isolation of the granulomas by careful dissection prior to DNA extraction may also be necessary.
There has been only 1 previous study29 on fibrosing granulomas in an equine liver that were postulated to be the result of chronic schistosomiasis of undeterminable origin. The gross and histopathologic changes were nearly identical to those observed in the horse of the present study, and the authors suggested that H americana or another species of schistosome may be involved. Of 11 horses examined in that study,29 only 1 had hepatic granulomas containing broken or empty eggshells consistent with schistosome eggs, supporting our contention that in most instances, by the time a diagnosis of hepatic schistosome infection in horses is made, the changes are long-standing and the trematode eggs that incited the lesion are no longer present. We have also found that in many instances, the hepatic granulomas are an incidental finding unrelated to some primary disease as it was in this horse, which had a metastatic squamous cell carcinoma.
On first observation of an affected liver, via histologic examination of hepatic biopsy specimens, imaging studies, surgical intervention, or postmortem examination of the abdominal cavity, the changes associated with this disease can be quite impressive; however, they can also be misleading because only the most severely affected horses appear to develop clinically relevant hepatic dysfunction. Hepatic dysfunction may in fact be more severe during the time of initial infection when the liver is being seeded with schistosome eggs and a marked inflammatory response to their presence ensues. It may also be important to note that the most severely affected horses that we have observed at necropsy have been those with some other chronic disease, either infectious, inflammatory, or neoplastic, and less frequently horses euthanized because of colic, trauma, or other acute conditions.
We believe that schistosomiasis in horses due to H americana is an underdiagnosed disease that is likely to continue to spread to new areas as the geographic range of infected raccoons and dogs as well as the snail intermediate hosts continues to expand.30,31 Although the formation of small numbers of randomly scattered eosinophilic and fibrosing granulomas in the liver may be a common response to localized injury due to other parasitic insults, such as ascarid32 and strongyle33 migration and perhaps tissue-invading ciliated protozoa,34 the presence of large numbers of disseminated granulomas arranged in the distinctive shotgun pattern of small, round, white to pale yellow nodules on a dark background (ie, starry-sky appearance) observed in this horse, particularly when found in horses raised in the southeastern United States, is something we propose is more likely associated with H americana infection.
ABBREVIATION
| SSU | Small subunit |
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.
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