CHYLOUS ASCITES IN A HEDGEHOG (ATELERIX ALBIVENTRIS) Author(s): Yoon-Seok Roh, D.V.M., Ph.D., Eun-Ju Kim, D.V.M., M.S., Ara Cho, D.V.M., Ph.D., Min-Su Kim, D.V.M., Ph.D., Ho-Seong Cho, D.V.M., Ph.D., Chae Woong Lim, D.V.M., Ph.D., and Bumseok Kim, D.V.M., Ph.D. Source: Journal of Zoo and Wildlife Medicine, 45(4):951-954. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2013-0164.1 URL: http://www.bioone.org/doi/full/10.1638/2013-0164.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 45(4): 951–954, 2014 Copyright 2014 by American Association of Zoo Veterinarians
CHYLOUS ASCITES IN A HEDGEHOG (ATELERIX ALBIVENTRIS) Yoon-Seok Roh, D.V.M., Ph.D., Eun-Ju Kim, D.V.M., M.S., Ara Cho, D.V.M., Ph.D., Min-Su Kim, D.V.M., Ph.D., Ho-Seong Cho, D.V.M., Ph.D., Chae Woong Lim, D.V.M., Ph.D., and Bumseok Kim, D.V.M., Ph.D.
Abstract: An African pygmy hedgehog (Atelerix albiventris) was diagnosed as chylous ascites with biliary cirrhosis. Abdomenocentesis revealed a milky fluid with a 324 mg/dl triglyceride level. On serum biochemical examination, the hedgehog had hypoalbuminemia, hypoglycemia, and high blood urea nitrogen. There was no cytologic or genomic evidence of infection, and a blood culture was negative. Histopathologic examination revealed a liver with proliferative bile ducts that were often surrounded by prominent septa of fibrous connective tissue. In the area of ductular reaction, proliferative cells positive for CD66, an embryogenic antigen of epithelial cells, were revealed. The potential association between chylous ascites and liver cirrhosis is undetermined but could be an aspect of future study. This is the first description of chylous ascites in a hedgehog. Key words: Ascites, Atelerix albiventris, chylous, cirrhosis, hedgehog.
BRIEF COMMUNICATION Chylous ascites is an abnormal accumulation of a milk-like peritoneal fluid with triglyceride content above 110 mg/dl in the abdominal cavity.5,11,12 Chylous ascites is uncommon in humans and animals, characterized by the leakage of lymphatic fluid into the abdominal cavity, and has been suggested to be associated with obstruction or abnormalities of lymphatic flow or destruction of lymphatic channels.12,27,30 The most common etiologies of chylous ascites include rupture or obstruction of serosal lymphatic channels by liver cirrhosis or neoplastic cells, congenital defects of the lymphatic flow system, nephrotic syndrome, and traumatic rupture of the lymphatic flow system.12,19,30 Chylous ascites cases have been reported in domestic or zoo animals such as dogs, cats, cattle, horses, and cheetahs (Acinonyx jubatus).6,10,12,13,28 A retrospective study reported hepatic steatosis in 50% of hedgehog necropsies, and most (89%) of hepatic steatoses were submissions from zoos, suggesting that etiology may be related with diet.21 Of note, 57% of hedgehogs with hepatic steatosis had concurrent infectious or neoplastic diseases.21 Moreover, other hepatic diseases such as herpes virus-induced hepatic necrosis and hepatocellular carcinoma have been reported in hedgehog.20,25 From the Veterinary Diagnostic Center (Roh, A. Cho, H.-S. Cho, Lim, B. Kim) and Animal Medical Center (E.-J. Kim, M.-S. Kim), Bio-safety Research Institute and College of Veterinary Medicine (BK21 Plus Program), Chonbuk National University, Jeonju City, 561-756, Republic of Korea. Correspondence should be directed to Dr. B. Kim ([email protected]).
Biliary cirrhosis is histologically characterized by diffuse periportal to bridging fibrosis associated with marked hepatic architectural remodeling and biliary hyperplasia subsequent to chronic cholangiohepatitis.1,26 The etiology of biliary cirrhosis is thought to be due to either congenital factors or environmental triggers.1,24 Clinical features of biliary cirrhosis include inappetence, cachexia, jaundice, variable liver size, and ascites.9,15 An 11-mo-old male African pygmy hedgehog (Atelerix albiventris) was presented to the Chonbuk Animal Medical Center at Chonbuk National University, Republic of Korea, with abdominal distention, inappetence, and weight loss. On physical examination, the abdomen was soft and distended with a positive fluid-wave sign. Radiography revealed free fluid in the abdomen. Moreover, ultrasound showed no hydronephrosis, and the urinary bladder was intact. Fifty milliliters of milky fluid was obtained using ultrasoundguided paracentesis. Biochemical analysis of the fluid showed a 324-mg/dl triglyceride level, confirming the diagnosis of chylous ascites. Abdominal paracentesis was performed for removal of accumulated fluid every 2 wk (approximately 50 ml each time). Marked serum biochemical abnormalities included an albumin concentration of 1.2 g/dl (reference range 1.8–4.2), glucose of 37 mg/dl (reference range 89–165), blood urea nitrogen (BUN) of 93 mg/dl (reference range 13–54), serum calcium of 9.5 mmol/L (reference range 3.2–7.2), and serum triglyceride of 90 mg/dl (reference range 35.6–40.1).18,29 There was no cytologic evidence of infection, and a blood culture was negative. Therefore, chylous ascites
951
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Figure 1. Representative microscopic lesions from an African pygmy hedgehog (A. albiventris) affected by biliary cirrhosis and renal failure. (A) Marginal bile ductular proliferation was observed (arrow). H&E. Bar ¼ 50 lm. (B) These structures dispersed throughout fibrosis connective tissue (arrow). H&E. Bar ¼ 100 lm. (C) Proliferative cells formed tubular or gland-like structures. H&E. Bar ¼ 20 lm. (D) Cells scored positive for expression of embryogenic marker, fluorescein isothiocyanate–conjugated CD66. Bar ¼ 20 lm. (E) Hepatic necrosis and infiltration of mononuclear cells (arrow) were observed in non-neoplastic area. H&E. Bar ¼ 100 mm. (F) Severe renal tubular necrosis and accumulation of protein-like materials in glomeruli were observed. H&E. Bar ¼ 20 lm.
with liver and renal dysfunction was suspected. The hedgehog’s condition deteriorated rapidly despite fluid therapy (lactated Ringer’s solution, Daihan Pharm. Co., Ltd., Seoul, 156-172, Korea; 10 ml/kg subcutaneously), antibiotics (enrofloxacin, Baytril 50, Bayer Animal Health, Seoul, 156172, Korea; 10 mg/kg subcutaneously), and low lipid diet management. The hedgehog was euthanized due to a poor prognosis and debilitated condition. The hedgehog was submitted to the Veterinary Diagnostic Center at Chonbuk National University for pathologic examination. Tissue samples from various organs were collected, fixed in 10% neutral phosphate-buffered formalin (HT50-1-1, Sigma, St. Louis, Missouri 63103, USA), and processed routinely for histopathology. Tissues sectioned at 5 lm were stained with hematoxylin
and eosin (H&E) and viewed by light microscopy. In addition, several paraffin-embedded sections were subjected to immunofluorescence staining using fluorescein isothiocyanate–conjugated antiCD66 (551479, BD Biosciences, San Jose, California 95131, USA). For identification of infectious etiology, 16S rRNA gene sequencing was performed on formalin-fixed paraffin-embedded or fresh tissue. DNA was extracted from the liver with a commercial kit (TaKaRa DEXPATTM or MiniBESTTM, Takara Bio Inc., Ohtsu-shi, Shiga, 520-2193, Japan) according to the manufacturer’s instructions. Primers 0008F (59-AGAGTTTGATCCTGGCTCAG-39) and 0532R (59TACCGCGGCTGCTGGCAC-39)14 were used to amplify the 500 bp of the 16S rRNA gene. Samples were heated at 948C for 5 min, followed by 35 amplification cycles of denaturation at 948C for 60 sec, primer annealing at 598C for 60 sec, and extension step at 728C for 60 sec, with an additional final extension step at 728C for 7 min. The 500-bp DNA amplicon fragment was sequenced directly on both strands. This sequence was compared to sequences in the National Center for Biotechnology Information GenBank database (http://www.ncbi.nlm.nih.gov/blast). A match of 99% with GenBank sequence was considered identification to the species level; 97% matching was considered identification to the genus level.7 Microscopically, ductular structures were characteristically arranged along the margins of the portal tract in portal and periportal regions (marginal bile ductular proliferation, Fig. 1A, C) and often surrounded by prominent septa of fibrous connective tissue (Fig. 1B). Areas of the ductular reaction examined with the fluorescence microscope revealed that proliferative cells are positive for CD66, an embryogenic antigen of epithelial cells (Fig. 1D). However, these cells have no histologic features of malignancy, such as mitotic figure, high N : C ratio, and anisokaryosis. In the surrounding area, focal necrosis and fibrosis, as well as infiltration of mononuclear cells, were observed (Fig. 1E). Renal disease was also noted during histopathologic examination. This finding is consistent with the elevated BUN noted on serum biochemical analysis. Severe damage and calcification were observed in vascular endothelial cells. Renal tubular necrosis and accumulation of protein-like material in glomeruli were found (Fig. 1F). Chylous ascites is either a congenital or acquired disorder. Congenital chylous ascites develops from a defect in the lymphatic channel, such
ROH ET AL.—CHYLOUS ASCITES IN A HEDGEHOG
as lymphatic fistulas communicating with the peritoneal cavity,2 and has not been reported in animals. Acquired chylous ascites is caused by rupture or obstruction of the lymphatic flow system30 and can be of spontaneous or traumatic etiology.27 The most common causes are abdominal cirrhosis and malignancy, accounting for more than two-thirds of all cases in humans.22,23 Other etiologies include infectious diseases, such as tuberculosis and filariasis.3 The pathogenesis of chylous ascites associated with liver disease is complicated. In most cases of chylous ascites due to liver cirrhosis, portal hypertension may cause dilation of, and leakage from, intestinal lymphatic flow.4,12,22 In patients with cirrhosis, small chylomicrons may leak continuously from lymphatic vessels, it seems because of increased lymphatic pressure.17 Portal hypertension increases intestinal lymphatic flow,31 and this increase may overwhelm the drainage capacity of the cisterna chyli, leading to leakage or rupture of small intestinal lymphatic vessels.12,22 Chronic liver diseases, such as liver cirrhosis, frequently have been proposed as possible causes of chylous ascites in humans and animals.4,28,30 Therefore, chylous ascites in this hedgehog may have resulted from liver cirrhosis causing increased pressure of lymphatic flow, leading to intestinal lymphatic dilatation and leakage. Although serum biochemical values of alanine aminotranferease (ALT) and aspartate aminotransferase were within normal limits (data not shown), histopathologic examination revealed obvious biliary cirrhosis accompanied by classical changes affecting portal tracts, including inflammatory infiltrates and proliferation of bile ductular structures. This discrepancy between biochemical and histologic observation can be explained by the fact that 37% of human patients with significant fibrosis and inflammation were diagnosed with persistently normal ALT.16 In addition, liver enzymes of congenital biliary cirrhosis patient are frequently normal in small animal.15 Generally, hypoalbuminemia, hypoglycemia, and elevated BUN levels are features of liver cirrhosis and nephrotic syndrome. In line with these data, liver cirrhosis, renal tubular necrosis, and accumulation of protein-like materials in glomeruli were identified by histopathologic observation. Furthermore, 16S rRNA-based analysis was conducted to rule out other etiology of chylous ascites, especially including tuberculosis and filariasis.3 The gene sequence of this hedgehog’s liver tissue has not shown any major pathogen (data not shown).
953
In humans, chronic liver disease has been frequently associated with chylous ascites.8,22,30,31 Similar findings have been reported in a cheetah and domestic cats.12,28 The possibility of a liver disease to the etiology of chylous ascites in hedgehogs, as there is in humans, is unknown but could be an aspect of future study. This report is the first description of a chylous ascites in a hedgehog and forms a base for further studies of the condition. Acknowledgment: This research was supported by Technology Development Program for Bioindustry (Project No. 1121314 and 313005-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
LITERATURE CITED 1. Brown DL, Van Winkle T, Cecere T, Rushton S, Brachelente C, Cullen JM. Congenital hepatic fibrosis in 5 dogs. Vet Pathol. 2010;47:102–107. 2. Browse NL, Wilson NM, Russo F, al-Hassan H, Allen DR. Aetiology and treatment of chylous ascites. Br J Surg. 1992;79:1145–1150. 3. Cardenas A, Chopra S. Chylous ascites. Am J Gastroenterol. 2002;97:1896–1900. 4. Castellote J, Porta F. Chylous ascites secondary to liver cirrhosis. Rev Esp Enferm Dig. 1994;86:912–914. 5. Connally HE. Cytology and fluid analysis of the acute abdomen. Clin Tech Small Anim Pract. 2003;18: 39–44. 6. Cruz AM, Riley CB, MacDonald DG, Ferguson JG. Use of mesenteric lymphangiography in a calf with chylothorax and chyloperitoneum. J Am Vet Med Assoc. 1995;206:1567–1571. 7. Drancourt M, Raoult D. Sequence-based identification of new bacteria: a proposition for creation of an orphan bacterium repository. J Clin Microbiol. 2005; 43:4311–4315. 8. Dumont AE, Mulholland JH. Alterations in thoracic duct lymph flow in hepatic cirrhosis: significance in portal hypertension. Ann Surg. 1962;156:668– 675. 9. Ettinger SJ, Feldman EC. Liver and pancreatic diseases. In: Textbook of veterinary internal medicine: diseases of the dog and the cat. 7th ed. St. Louis (MO): Elsevier Saunders; 2010; p. 273–283. 10. Fossum TW, Hay WH, Boothe HW, Zack PM, Sherding RG, Miller MW. Chylous ascites in three dogs. J Am Vet Med Assoc. 1992;200:70–76. 11. Fossum TW, Jacobs RM, Birchard SJ. Evaluation of cholesterol and triglyceride concentrations in differentiating chylous and nonchylous pleural effusions in dogs and cats. J Am Vet Med Assoc. 1986;188: 49–51.
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12. Gores BR, Berg J, Carpenter JL, Ullman SL. Chylous ascites in cats: nine cases (1978–1993). J Am Vet Med Assoc. 1994;205:1161–1164. 13. Hanselaer JR, Nyland TG. Chyloabdomen and ultrasonographic detection of an intra-abdominal abscess in a foal. J Am Vet Med Assoc. 1983;183:1465– 1467. 14. Imrit K, Goldfischer M, Wang J, Green J, Levine J, Lombardo J, Hong T. Identification of bacteria in formalin-fixed, paraffin-embedded heart valve tissue via 16S rRNA gene nucleotide sequencing. J Clin Microbiol. 2006;44:2609–2611. 15. Kahn CM, Line S. Exotic and laboratory animals. In: The Merck veterinary manual. 10th ed. Whitehouse Station (NJ): Merck & Co., Inc.; 2010. p. 1581–1822. 16. Lai M, Hyatt BJ, Nasser I, Curry M, Afdhal NH. The clinical significance of persistently normal ALT in chronic hepatitis B infection. J Hepatol. 2007;47:760– 767. 17. Malagelada JR, Iber FL, Linscheer WG. Origin of fat in chylous ascites of patients with liver cirrhosis. Gastroenterology 1974;67:878–886. 18. Okorie-Kanu C, Onoja R, Achegbulu E, OkorieKanu O. Normal haematological and serum biochemistry values of African hedgehog (Atelerix albiventris). Comp Clin Pathol [Internet]. 2014 [cited 2014 Jan 14] Available from: doi:10.1007/s00580-013-1870-x 19. Press OW, Press NO, Kaufman SD. Evaluation and management of chylous ascites. Ann Intern Med. 1982;96:358–364. 20. Raymond JT, Garner MM. Spontaneous tumours in captive African hedgehogs (Atelerix albiventris): a retrospective study. J Comp Pathol. 2001;124:128–133. 21. Raymond JT, White MR. Necropsy and histopathologic findings in 14 African hedgehogs (Atelerix albiventris): a retrospective study. J Zoo Wildl Med. 1999;30:273–277. 22. Rector WG Jr. Spontaneous chylous ascites of cirrhosis. J Clin Gastroenterol. 1984;6:369–372.
23. Runyon BA, Akriviadis EA, Keyser AJ. The opacity of portal hypertension-related ascites correlates with the fluid’s triglyceride concentration. Am J Clin Pathol. 1991;96:142–143. 24. Selmi C, Mayo MJ, Bach N, Ishibashi H, Invernizzi P, Gish RG, Gordon SC, Wright HI, Zweiban B, Podda M, Gershwin ME. Primary biliary cirrhosis in monozygotic and dizygotic twins: genetics, epigenetics, and environment. Gastroenterology 2004; 127:485–492. 25. Stack MJ, Higgins RJ, Challoner DJ, Gregory MW. Herpesvirus in the liver of a hedgehog (Erinaceus europaeus). Vet Rec. 1990;127:620–621. 26. Stockhaus C, Van Den Ingh T, Rothuizen J, Teske E. A multistep approach in the cytologic evaluation of liver biopsy samples of dogs with hepatic diseases. Vet Pathol. 2004;41:461–470. 27. Stormo AC, Anastasi GW, Wertheimer HM, Rogers CE. Traumatic chylous ascites. Arch Surg. 1966;92:115–117. 28. Terrell SP, Fontenot DK, Miller MA, Weber MA. Chylous ascites in a cheetah (Acinonyx jubatus) with venoocclusive liver disease. J Zoo Wildl Med. 2003;34:380–384. 29. Thrall MA. Clinical chemistry of mammals: laboratory animals and miscellaneous species. In: Veterinary hematology and clinical chemistry. 2nd ed. Ames (IA): Wiley-Blackwell; 2012. p. 571–580. 30. Tsuchiya M, Okazaki I, Maruyama K, Asakura H, Morita A. Chylous ascites formation and a review of 84 cases. Angiology 1973;24:576–584. 31. Watanabe M, Taketa K, Yonei J, Kubota M, Nagashima H, Akamutsu K, Awai M. A case of liver cirrhosis with chylous ascites and multiple cystic dilatation of the abdominal lymphatic system. Hepatogastroenterology 1980;27:394–400.
Received for publication 23 July 2013
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 45(4): 951–954, 2014 Copyright 2014 by American Association of Zoo Veterinarians
CHYLOUS ASCITES IN A HEDGEHOG (ATELERIX ALBIVENTRIS) Yoon-Seok Roh, D.V.M., Ph.D., Eun-Ju Kim, D.V.M., M.S., Ara Cho, D.V.M., Ph.D., Min-Su Kim, D.V.M., Ph.D., Ho-Seong Cho, D.V.M., Ph.D., Chae Woong Lim, D.V.M., Ph.D., and Bumseok Kim, D.V.M., Ph.D.
Abstract: An African pygmy hedgehog (Atelerix albiventris) was diagnosed as chylous ascites with biliary cirrhosis. Abdomenocentesis revealed a milky fluid with a 324 mg/dl triglyceride level. On serum biochemical examination, the hedgehog had hypoalbuminemia, hypoglycemia, and high blood urea nitrogen. There was no cytologic or genomic evidence of infection, and a blood culture was negative. Histopathologic examination revealed a liver with proliferative bile ducts that were often surrounded by prominent septa of fibrous connective tissue. In the area of ductular reaction, proliferative cells positive for CD66, an embryogenic antigen of epithelial cells, were revealed. The potential association between chylous ascites and liver cirrhosis is undetermined but could be an aspect of future study. This is the first description of chylous ascites in a hedgehog. Key words: Ascites, Atelerix albiventris, chylous, cirrhosis, hedgehog.
BRIEF COMMUNICATION Chylous ascites is an abnormal accumulation of a milk-like peritoneal fluid with triglyceride content above 110 mg/dl in the abdominal cavity.5,11,12 Chylous ascites is uncommon in humans and animals, characterized by the leakage of lymphatic fluid into the abdominal cavity, and has been suggested to be associated with obstruction or abnormalities of lymphatic flow or destruction of lymphatic channels.12,27,30 The most common etiologies of chylous ascites include rupture or obstruction of serosal lymphatic channels by liver cirrhosis or neoplastic cells, congenital defects of the lymphatic flow system, nephrotic syndrome, and traumatic rupture of the lymphatic flow system.12,19,30 Chylous ascites cases have been reported in domestic or zoo animals such as dogs, cats, cattle, horses, and cheetahs (Acinonyx jubatus).6,10,12,13,28 A retrospective study reported hepatic steatosis in 50% of hedgehog necropsies, and most (89%) of hepatic steatoses were submissions from zoos, suggesting that etiology may be related with diet.21 Of note, 57% of hedgehogs with hepatic steatosis had concurrent infectious or neoplastic diseases.21 Moreover, other hepatic diseases such as herpes virus-induced hepatic necrosis and hepatocellular carcinoma have been reported in hedgehog.20,25 From the Veterinary Diagnostic Center (Roh, A. Cho, H.-S. Cho, Lim, B. Kim) and Animal Medical Center (E.-J. Kim, M.-S. Kim), Bio-safety Research Institute and College of Veterinary Medicine (BK21 Plus Program), Chonbuk National University, Jeonju City, 561-756, Republic of Korea. Correspondence should be directed to Dr. B. Kim ([email protected]).
Biliary cirrhosis is histologically characterized by diffuse periportal to bridging fibrosis associated with marked hepatic architectural remodeling and biliary hyperplasia subsequent to chronic cholangiohepatitis.1,26 The etiology of biliary cirrhosis is thought to be due to either congenital factors or environmental triggers.1,24 Clinical features of biliary cirrhosis include inappetence, cachexia, jaundice, variable liver size, and ascites.9,15 An 11-mo-old male African pygmy hedgehog (Atelerix albiventris) was presented to the Chonbuk Animal Medical Center at Chonbuk National University, Republic of Korea, with abdominal distention, inappetence, and weight loss. On physical examination, the abdomen was soft and distended with a positive fluid-wave sign. Radiography revealed free fluid in the abdomen. Moreover, ultrasound showed no hydronephrosis, and the urinary bladder was intact. Fifty milliliters of milky fluid was obtained using ultrasoundguided paracentesis. Biochemical analysis of the fluid showed a 324-mg/dl triglyceride level, confirming the diagnosis of chylous ascites. Abdominal paracentesis was performed for removal of accumulated fluid every 2 wk (approximately 50 ml each time). Marked serum biochemical abnormalities included an albumin concentration of 1.2 g/dl (reference range 1.8–4.2), glucose of 37 mg/dl (reference range 89–165), blood urea nitrogen (BUN) of 93 mg/dl (reference range 13–54), serum calcium of 9.5 mmol/L (reference range 3.2–7.2), and serum triglyceride of 90 mg/dl (reference range 35.6–40.1).18,29 There was no cytologic evidence of infection, and a blood culture was negative. Therefore, chylous ascites
951
952
JOURNAL OF ZOO AND WILDLIFE MEDICINE
Figure 1. Representative microscopic lesions from an African pygmy hedgehog (A. albiventris) affected by biliary cirrhosis and renal failure. (A) Marginal bile ductular proliferation was observed (arrow). H&E. Bar ¼ 50 lm. (B) These structures dispersed throughout fibrosis connective tissue (arrow). H&E. Bar ¼ 100 lm. (C) Proliferative cells formed tubular or gland-like structures. H&E. Bar ¼ 20 lm. (D) Cells scored positive for expression of embryogenic marker, fluorescein isothiocyanate–conjugated CD66. Bar ¼ 20 lm. (E) Hepatic necrosis and infiltration of mononuclear cells (arrow) were observed in non-neoplastic area. H&E. Bar ¼ 100 mm. (F) Severe renal tubular necrosis and accumulation of protein-like materials in glomeruli were observed. H&E. Bar ¼ 20 lm.
with liver and renal dysfunction was suspected. The hedgehog’s condition deteriorated rapidly despite fluid therapy (lactated Ringer’s solution, Daihan Pharm. Co., Ltd., Seoul, 156-172, Korea; 10 ml/kg subcutaneously), antibiotics (enrofloxacin, Baytril 50, Bayer Animal Health, Seoul, 156172, Korea; 10 mg/kg subcutaneously), and low lipid diet management. The hedgehog was euthanized due to a poor prognosis and debilitated condition. The hedgehog was submitted to the Veterinary Diagnostic Center at Chonbuk National University for pathologic examination. Tissue samples from various organs were collected, fixed in 10% neutral phosphate-buffered formalin (HT50-1-1, Sigma, St. Louis, Missouri 63103, USA), and processed routinely for histopathology. Tissues sectioned at 5 lm were stained with hematoxylin
and eosin (H&E) and viewed by light microscopy. In addition, several paraffin-embedded sections were subjected to immunofluorescence staining using fluorescein isothiocyanate–conjugated antiCD66 (551479, BD Biosciences, San Jose, California 95131, USA). For identification of infectious etiology, 16S rRNA gene sequencing was performed on formalin-fixed paraffin-embedded or fresh tissue. DNA was extracted from the liver with a commercial kit (TaKaRa DEXPATTM or MiniBESTTM, Takara Bio Inc., Ohtsu-shi, Shiga, 520-2193, Japan) according to the manufacturer’s instructions. Primers 0008F (59-AGAGTTTGATCCTGGCTCAG-39) and 0532R (59TACCGCGGCTGCTGGCAC-39)14 were used to amplify the 500 bp of the 16S rRNA gene. Samples were heated at 948C for 5 min, followed by 35 amplification cycles of denaturation at 948C for 60 sec, primer annealing at 598C for 60 sec, and extension step at 728C for 60 sec, with an additional final extension step at 728C for 7 min. The 500-bp DNA amplicon fragment was sequenced directly on both strands. This sequence was compared to sequences in the National Center for Biotechnology Information GenBank database (http://www.ncbi.nlm.nih.gov/blast). A match of 99% with GenBank sequence was considered identification to the species level; 97% matching was considered identification to the genus level.7 Microscopically, ductular structures were characteristically arranged along the margins of the portal tract in portal and periportal regions (marginal bile ductular proliferation, Fig. 1A, C) and often surrounded by prominent septa of fibrous connective tissue (Fig. 1B). Areas of the ductular reaction examined with the fluorescence microscope revealed that proliferative cells are positive for CD66, an embryogenic antigen of epithelial cells (Fig. 1D). However, these cells have no histologic features of malignancy, such as mitotic figure, high N : C ratio, and anisokaryosis. In the surrounding area, focal necrosis and fibrosis, as well as infiltration of mononuclear cells, were observed (Fig. 1E). Renal disease was also noted during histopathologic examination. This finding is consistent with the elevated BUN noted on serum biochemical analysis. Severe damage and calcification were observed in vascular endothelial cells. Renal tubular necrosis and accumulation of protein-like material in glomeruli were found (Fig. 1F). Chylous ascites is either a congenital or acquired disorder. Congenital chylous ascites develops from a defect in the lymphatic channel, such
ROH ET AL.—CHYLOUS ASCITES IN A HEDGEHOG
as lymphatic fistulas communicating with the peritoneal cavity,2 and has not been reported in animals. Acquired chylous ascites is caused by rupture or obstruction of the lymphatic flow system30 and can be of spontaneous or traumatic etiology.27 The most common causes are abdominal cirrhosis and malignancy, accounting for more than two-thirds of all cases in humans.22,23 Other etiologies include infectious diseases, such as tuberculosis and filariasis.3 The pathogenesis of chylous ascites associated with liver disease is complicated. In most cases of chylous ascites due to liver cirrhosis, portal hypertension may cause dilation of, and leakage from, intestinal lymphatic flow.4,12,22 In patients with cirrhosis, small chylomicrons may leak continuously from lymphatic vessels, it seems because of increased lymphatic pressure.17 Portal hypertension increases intestinal lymphatic flow,31 and this increase may overwhelm the drainage capacity of the cisterna chyli, leading to leakage or rupture of small intestinal lymphatic vessels.12,22 Chronic liver diseases, such as liver cirrhosis, frequently have been proposed as possible causes of chylous ascites in humans and animals.4,28,30 Therefore, chylous ascites in this hedgehog may have resulted from liver cirrhosis causing increased pressure of lymphatic flow, leading to intestinal lymphatic dilatation and leakage. Although serum biochemical values of alanine aminotranferease (ALT) and aspartate aminotransferase were within normal limits (data not shown), histopathologic examination revealed obvious biliary cirrhosis accompanied by classical changes affecting portal tracts, including inflammatory infiltrates and proliferation of bile ductular structures. This discrepancy between biochemical and histologic observation can be explained by the fact that 37% of human patients with significant fibrosis and inflammation were diagnosed with persistently normal ALT.16 In addition, liver enzymes of congenital biliary cirrhosis patient are frequently normal in small animal.15 Generally, hypoalbuminemia, hypoglycemia, and elevated BUN levels are features of liver cirrhosis and nephrotic syndrome. In line with these data, liver cirrhosis, renal tubular necrosis, and accumulation of protein-like materials in glomeruli were identified by histopathologic observation. Furthermore, 16S rRNA-based analysis was conducted to rule out other etiology of chylous ascites, especially including tuberculosis and filariasis.3 The gene sequence of this hedgehog’s liver tissue has not shown any major pathogen (data not shown).
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In humans, chronic liver disease has been frequently associated with chylous ascites.8,22,30,31 Similar findings have been reported in a cheetah and domestic cats.12,28 The possibility of a liver disease to the etiology of chylous ascites in hedgehogs, as there is in humans, is unknown but could be an aspect of future study. This report is the first description of a chylous ascites in a hedgehog and forms a base for further studies of the condition. Acknowledgment: This research was supported by Technology Development Program for Bioindustry (Project No. 1121314 and 313005-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
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Received for publication 23 July 2013
