Friday, November 28, 2008

Enterocytozoon bieneusi as a cause of proliferative serositis in simian immunodeficiency virus-infected immunodeficient macaques part-2

serositis pict-3
Enterocytozoon bieneusi is the most common microsporidian parasite in human patients with acquired immunodeficiency syndrome (AIDS), and since its recognition in 1985, it has been associated with a severe enteropathy and biliary cirrhosis in these patients.1-3 Microsporidia are obligate intracellular parasites that lack mitochondria and use a unique polar tube apparatus to infect the host cell.4,5 Despite its common occurrence, basic aspects of parasite biology and host immunity are poorly understood. There is currently no effective treatment or rational approach to prevention. Until recently, advances have been hampered by the lack of suitable animal models and in vitro systems to cultivate the organism.6

We and others recently described E bieneusi as a spontaneous infection of both immunologically normal and simian immunodeficiency virus (SN)-infected macaques (Maraca mulatta, Maraca cyclopis, and Maraca nemestrina).7,8 The organism can be localized to the cytoplasm of epithelial cells of the gallbladder, bile ducts, and small intestine, where it is associated with a proliferative cholecystitis, cholangiohepatitis, and enteropathy, which closely resembles the conditions seen in human immunodeficiency virus (HIV)-infected human patients. Since its initial recognition, we have shown that this Enterocytozoon is nearly identical to the human-derived E bieneusi at the light, ultrastructural, and genetic level.7-9 Human and rhesus-derived E bieneusi sequences share 99.5% nucleic acid identity over a 2.0-kb fragment of the ribosomal gene complex.10 These observations suggest that rhesus macaques may serve as an animal model of human E bieneusi infection.

Herein we describe a unique proliferative serositis associated with E bieneusi infection of mesothelial cells in immunodeficient rhesus macaques. Identification of the organism within these lesions required the sensitive techniques of in situ hybridization (ISH) and polymerase chain reaction (PCR). This study shows that E bieneusi can disseminate in the immunocompromised host and should be considered in cases of proliferative serositis and pleuritis of unknown cause.

MATERIALS AND METHODS

Animal Housing and Inoculations

Macaques were housed at the New England Regional Primate Research Center in a centralized biolevel 3 containment facility in accordance with standards of the Association for Assessment and Accreditation of Laboratory Animal Care and Harvard Medical School's Animal Care and Use Committee.

Animals were inoculated intravenously with 1 of 2 pathogenic strains of SIV (SIVmac251 or SIVmac239), the history, preparation, and in vivo and in vitro properties of which have been described and reviewed extensively.11-13 Animals inoculated with these viruses were included in a variety of infectivity, pathogenesis, and vaccine studies and received no antiretroviral agents or antimicrobial prophylaxis. The monkeys were monitored closely and euthanized when moribund or deemed necessary by the veterinary staff. Complete postmortem examinations were performed on all animals, and representative samples of tissue were taken for formalin fixation, freezing, and electron microscopy.

Retrospective Analysis

The index case (Mm 320-94) was identified by evaluation of sections of duodenum and common bile duct examined by ISH for E bieneusi RNA. This monkey had severe cholangiohepatitis and choledochitis and a unique proliferative serositis of the intestines. Intracellular Enterocytozoon organisms were identified within this proliferative inflammatory tissue by ISH. Following the recognition of the index case, a retrospective analysis of computerized pathology records was conducted of all SIV infected, immunodeficient macaques who died between September 1991 and March 1999 (n = 225). This review revealed a series of animals with a diagnosis of peritonitis of unknown cause (n = 16). These cases were subjected to further analysis by ISH, immunohistochemistry (IHC), and PCR.

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