Proliferative Enteritis in Leopard Geckos (Eublepharis macularius) Associated with Cryptosporidium sp. Infection

Scott P. Terrell, Elizabeth W. Uhl, and Richard S. Funk

Department of Pathobiology, University of Florida College of Veterinary Medicine, Gainesville, FL (SPT, EWU); Walt Disney World Animal Programs, Bay Lake, FL (SPT); Care Animal Hospital, Brandon, FL (RSF)

Running title: Cryptosporidium in leopard geckos

Abstract. Twenty-three leopard geckos, Eublepharis macularius, with a variable clinical history of weight loss, anorexia, lethargy and diarrhea were submitted either intact or as biopsy specimens to the University of Florida Anatomic Pathology Service. Gross necropsy findings in the intact geckos included marked reduction of subcutaneous adipose tissue stores at the tail base and mild thickening and reddening of the small intestine. Histologic examination revealed Cryptosporidium infection associated with hyperplasia and mononuclear inflammation of the small intestine in all of the affected geckos. Parasites and lesions were only rarely observed in the stomach and large intestine of affected geckos. The histologic and ultrastructural lesions in the small intestine of leopard geckos infected with Cryptosporidium have not been well characterized. This report implicates Cryptosporidium as the cause of disease in the submitted geckos and describes the range of histologic lesions observed.

Introduction

Leopard geckos, Eublepharis macularius, have become popular pets and are a common reptile species in zoological collections throughout the United States. In recent years owners, breeders, and veterinarians working with leopard geckos have described a condition known as "going light", which is characterized by dramatic weight loss over a variable period of time. Affected geckos are lethargic and many have diarrhea characterized by watery white urates. Some of the geckos recover spontaneously; however, a percentage of the affected animals die. Little information on the pathogenesis of this syndrome is available, however it has been associated with intestinal Cryptosporidium infection.^1,14

Cryptosporidium is a genus of protozoan parasites that infects numerous mammalian, avian and reptilian species. It is predominantly a gastrointestinal pathogen, however, respiratory infections also occur in birds. In reptiles Cryptosporidium infections are most commonly described in snakes associated with a chronic clinical course and hypertrophic gastritis. The purpose of the current study was to confirm that leopard geckos affected with the "going light" syndrome are infected with Cryptosporidium and to describe the lesions associated with infection.

Material and Methods

Animals

During the period between 1995-1999, 5 whole leopard geckos and tissues from an additional 17 were submitted to the anatomic pathology service at the University of Florida College of Veterinary Medicine for diagnostic evaluation. Thirteen of 17 geckos submitted to surgical pathology service, and all 5 of the geckos submitted for necropsy were adults. The geckos came from multiple sources including 2 large breeding colonies and a local zoological facility. Typical histories described were severe emaciation, weight loss, lethargy and diarrhea, with approximately 5% of geckos affected and an unknown number of fatalities. Both breeding facilities were "open" facilities, commonly acquiring geckos from outside sources to augment their collection. Geckos at the two breeding facilities were fed a diet of meal worms and crickets, while those at the zoological facility were occasionally fed pinky mice in addition to meal worms and crickets. All of the tissues submitted for histologic examination were obtained from clinically ill animals that had been euthanized by decapitation. The 5 geckos that were submitted for necropsy were euthanized via intra-cardiac injection of sodium pentobarbital.

Light microscopy

The entire stomach, small intestine, and large intestine from 17 geckos were submitted to the surgical pathology service in 10% neutral buffered formalin. Representative sections from each organ were embedded in paraffin, sectioned at 5um, and stained with hematoxylin and eosin (H&E). Representative sections of liver, kidney, heart, lung, brain, stomach, small intestine and large intestine from the 5 necropsied geckos were fixed in 10% neutral buffered formalin and were similarly embedded, sectioned and stained with H&E.

Electron microscopy

For ultrastructural examination, sections of small intestine from 2 of the geckos submitted for necropsy were minced into 1mm squares and fixed in 2% glutaraldehyde. Ultrathin sections were stained with 2% uranyl acetate and Reynold’s lead citrate and were examined on a transmission electron microscope.

Results

Gross Findings

The average weight of the 5 necropsied geckos was 26.5 g with a range from 18.6g to 34.7g. Grossly, there was marked reduction in the width of the base of the tail compared to a normal gecko. Cut section through the tail base revealed a complete absence of subcutaneous fat stores. Coelomic fat stores were present but reduced in 4 of 5 geckos. One gecko had complete absence of coelomic fat stores. In 3 of the 5 geckos the serosal surface of the small intestine was reddened. The wall of small intestine was thickened and the mucosa had a red corrugated appearance. These changes extended throughout the small intestine but were not present in the large intestine, which was distended by liquid white to tan fecal material in all 5 geckos.

Histopathology

Histopathologic findings for each of the 23 geckos examined are summarized in table 1. All of the geckos examined had variable numbers of Cryptosporidium sp. organisms present on the apical aspect of enterocytes lining intestinal villi (Figures 1 and 2). In most cases the organisms were present in small numbers with a patchy distribution among the intestinal villi (14/23). Less commonly, massive numbers of organisms were present diffusely coating villi (5/23). The presence of organisms in the small intestine was associated with hyperplasia of enterocytes lining villi (Figure 1). The hyperplastic lesions were graded as mild, moderate and severe. Normal villus architecture of leopard geckos is characterized by a single layer of enterocytes lining the central core of villus lamina propria. A mild hyperplastic lesion was characterized by duplication of enterocytes and an increase from the normal enterocyte layer thickness to 2 cells in thickness in the infected animals (15/23). Severe hyperplasia was characterized by an increase in the enterocyte layer thickness to 5-10 cells or greater (2/23). Hyperplastic enterocytes were enlarged with vesicular nuclei and prominent acidophilic nucleoli. Rare mitotic figures were observed among hyperplastic enterocytes. In some cases, intestinal villi appeared to be lengthened while in other animals there was evidence of villus thickening and blunting. A mild diffuse mucosal infiltrate of mononuclear inflammatory cells, predominantly lymphocytes, was associated with the presence of Cryptosporidium. Rarely, scattered heterophils were identified in the mucosal lamina propria and submucosa of affected geckos.

Figure 1. Leopard gecko small intestine with Cryptosporidium sp. infection. Note the apical location of the Cryptosporidium sp. organisms (arrows); the marked hyperplasia of the villous enterocytes up to 7 cell layers in thickness (large bar); and the presence of scattered lymphocytes among the villous epithelium. H&E stain, 400x

Figure 2. Leopard gecko small intestine with Cryptosporidium sp. infection. Higher magnification view of Cryptosporidium sp. organisms colonizing the apical aspect of villous enterocytes (arrows), hyperplastic enterocytes and infiltrating lymphocytes. H&E stain, 1000x

Cryptosporidium infection was generally confined to the small intestine, with organisms and mild hyperplastic and inflammatory lesions being observed in the large intestine of only 3 of 23 geckos. In these animals, mucosal hyperplasia similar to that seen in the small intestine was present and associated with Cryptosporidium on the apical aspects of enterocytes. There also were mild infiltrates of lymphocytes within the mucosa. Gastric infection was identified in 1 of the 23 geckos examined. Lesions in the stomach also consisted of epithelial cell hyperplasia and mucosal infiltration by small numbers of lymphocytes.

Ultrastructural findings

Ultrastructural examination was performed on samples of small intestine from a heavily infected gecko. Coccidian parasites with characteristics consistent with Cryptosporidium were present on the apical aspects of multiple enterocytes (Figure 3). The parasites were enclosed in a parasitophoruous vacuole of host (enterocyte) origin. Macrogamete trophozoites and type I meronts containing merozoites were identified. The parasites were attached to the surface of the cell by an undulating feeder organelle, and there was loss of microvilli on the enterocyte surface at the attachment site. Enterocytes were piled up upon one another and had irregular cytoplasmic foldings, increased cytoplasmic volume, and increased numbers of cytoplasmic organelles. Nuclei of hyperplastic enterocytes were markedly enlarged and pale with prominent electron dense nucleoli.

Figure 3. Leopard gecko small intestine with Cryptosporidium sp. infection. Electron micrograph demonstrating Cryptosporidium sp. organisms (arrows) attached to the apical aspect of villous enterocytes via feeder organelle. Organisms are associated with loss of microvilli and formation of a pedestal. Lymphocytes are present among hyperplastic enterocytes. Transmission electron micrograph, Bar = 2 microns.

Discussion

Cryptosporidium is an apicomplexan parasite found predominantly within the gastrointestinal tract and respiratory tract of many classes of animals including mammals, birds, reptiles and fish. In mammals, Cryptosporidium infections involve the gastrointestinal tract and are associated with clinical signs including profuse watery diarrhea, dehydration, anorexia and weight loss. Young animals and immune compromised animals are more susceptible to infection.³ The organisms are found in the small intestine associated with villous atrophy, crypt hyperplasia, focal epithelial cell necrosis and mononuclear inflammatory cell infiltrates.¹⁰

In birds, Cryptosporidium infections are found in both the respiratory and gastrointestinal tracts with respiratory infections being much more common.^6,10 Similar to mammals, young birds are more susceptible to infection. Clinical signs in infected birds include respiratory distress, coughing, sneezing, and nasal congestion.⁹ The organisms can be found in multiple locations in the upper respiratory tract, lungs and air sacs associated with hypertrophy and hyperplasia of respiratory epithelial cells and loss of ciliated respiratory epithelial cells.^6.10 Gastrointestinal infections in birds are associated with diarrhea, dehydration and weight loss. Organisms are commonly observed in the small intestine but are also identified in areas such as the cecum, colon, cloaca and the bursa of Fabricius.¹⁰ Histologic lesions in the gastrointestinal tract include villous atrophy and fusion, hypertrophy and hyperplasia of mucosal epithelial cells and hyperplasia of crypt epithelial cells.¹⁰

Cryptosporidium infections in lizards, tortoises and snakes have been reported with snakes being most likely to be clinically affected.^5,8,10 Clinical signs of cryptosporidial infection in snakes are characterized by anorexia, weight loss, regurgitation and diarrhea. In contrast to infections in mammals and birds, adult snakes are commonly affected and the disease is usually chronic in nature. Organisms are localized to the stomach and are associated with hyperplasia and hypertrophy of gastric epithelium and gastric glands with mononuclear inflammatory cell infiltration into the submucosa.

Cryptosporidium infections in lizards are usually subclinical, although disease associated with gastric infection has been observed in a chameleon.^2,11 Cryptosporidium organisms have been observed in the cloaca of clinically ill Madagascar giant day geckos (Phelsuma madagascariensis grandis), however there was not a clear association between the presence of organisms and disease.^11,12 Cryptosporidium organisms have also been observed in the ear canal of iguanas (Iguana iguana).⁴

Recently, there have been two reports of Cryptosporidium infection in leopard geckos associated with anorexia, weight loss, emaciation, diarrhea and sometimes death of affected animals.^1,14 Coke and Tristan (1998) described identification of Cryptosporidium oocysts in fecal smears of multiple animals and histologic evidence of Cryptosporidium infection in the stomach (1 animal) and stomach and small intestine (1 animal). Histologic lesions associated with these infections were not described. Welsh et al., (1999) described cryptosporidial organisms on the apical surface of epithelial cells of the stomach and intestine with infiltration by heterophils and mixed mononuclear infiltrates. Oocysts were also detected in fecal smears from affected animals.

The reports of cryptosporidial infections in the small intestine of the leopard geckos (including this report), the cloaca of the Madagascar giant day geckos; and the ear canal of iguanas are the only known reports of extra-gastric cryptosporidial infection in reptiles. Upton (1990) commented that the presence of cryptosporidial organisms in such an unusual location (the cloaca) in the Madagascar giant day geckos suggested these organisms were likely a new species of Cryptosporidium. It seems possible that the Cryptosporidium organisms infecting predominantly the small intestine of leopard geckos may represent yet another new species.

The underlying cause of the seemingly high susceptibility of leopard geckos to infection and the development of severe disease associated with Cryptosporidium is unknown. Cryptosporidium infections in mammals and birds are common in juvenile animals and are often associated with immune suppression or severe stress.^3,10 In this report the majority of affected animals were adults. Many of the affected animals came from intensive breeding operations where stress associated with crowded conditions is possible. Based upon observations in mammals and birds, underlying viral infection or immune suppression are also possible predisposing factors. Investigation of underlying viral infection or immune suppression was not pursued.

The consistent presence of organisms associated with pathologic lesions in clinically ill leopard geckos suggests Cryptosporidium is the cause of a "going light" syndrome, which is characterized clinically by diarrhea, emaciation, anorexia, weight loss and death in a percentage of affected animals. The histologic lesions in the small intestine of affected geckos are similar to those seen in snakes with gastric Cryptosporidium infections. The presence of organisms in the small intestine of a reptile is unusual and raises the possibility that leopard geckos are uniquely susceptible to intestinal infection or that the Cryptosporidium infecting leopard geckos is a unique species. Cryptosporidium species are determined by oocyst size and morphology. Measurements of oocyst morphology from 11 species of reptiles suggest that at least 5 species of Cryptosporidium may infect reptiles.^7,13 Future studies will attempt to identify which species of Cryptosporidium is infecting leopard geckos and to determine the source of infection.

References

1. Coke RL, Tristan TE: Cryptosporidium infection in a colony of leopard geckos, Eublepharis macularius. In: Proceedings of the Association of Reptilian and Amphibian Veterinarians, pp. 157-165, 1998

2. Dillehay, DL, Boosinger TR, Mackenzie S :Gastric cryptosporidiosis in a chameleon. Journal of the American Veterinary Medical Association 189:1139, 1986

3. Fayer R, Speer C, Dubey JP: The general biology of Cryptosporidium. In: Cryptosporidium and Cryptosporidiosis (edited by Fayer, R. ). CRC Press Inc., New York, NY, 1997

4. Fitzgerald SD, Moisan PG, Bennett R: Aural polyp associated with cryptosporidiosis in an iguana (Iguana iguana). Journal of Veterinary Diagnostic Investigation 10:179-180, 1998

5. Funk RS: Implications of cryptosporidiosis in emerald tree boas, Corallus caninus. In: Proceedings of the Joint Conference of the American Association of Zoo Veterinarians and the American Association of Wildlife Veterinarians (Edited by Junge, R.E.): 360-361. Oakland, CA,1988

6. Goodwin MA: Cryptosporidiosis in birds - a review. Avian pathology 18:365-384, 1989

7. Graczyk TK, Cranfield MR: Diagnosis of subclinical Cryptosporidiosis in captive snakes based on stomach lavage and cloacal sampling. Veterinary Parasitology 67:143-151, 1996

8. Heuschele WP, Oosterhuis J, Janssen D, Anderson MP, Benirschke H: Cryptosporidial infections in captive wild animals. Journal of Wildlife Diseases 22:493-496, 1986

9. Lindsay DS, Blagburn BL: Cryptosporidiosis in birds. In: Cryptosporidiosis of Man and Animals (Edited by Dubey, J.P., Speer, C.A. and Fayer, R) pp. 149-156. CRC Press, Boston, MA, 1990

10. O’Donoghue PJ: Cryptosporidium and Cryptosporidiosis in man and animals. International Journal of Parasitology 25:139-195. 1995.

11. Upton SJ: Cryptosporidium spp. in lower vertebrates. In: Cryptosporidiosis of Man and Animals (Edited by Dubey, J.P., Speer, C.A. and Fayer, R) pp. 149-156. CRC Press, Boston, MA, 1990.

12. Upton SJ, Barnard SM: Two new species of coccidia (Apicomplexa: Eimeriidae) from Madagascar geckonids. Journal of Protozoology 34: pp. 452, 1987

13.Upton SJ, McAllister CT, Freed PS, Barnard SM: Cryptosporidium spp. in wild and captive reptiles. Journal of Wildlife Diseases 25:20-30, 1989

14. Welsh RD, Cooper VL, Talent LG: Fluid urates in geckos. Laboratory Animal 28:17-18, 1999

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