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Enterocytozoon bieneusi as a Cause of Proliferative Serositis in Simian Immunodeficiency Virus-Infected Immunodeficient Macaques (Macaca mulatta).

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 (SIV)-infected macaques (Macaca mulatta, Macaca cyclopis, and Macaca 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.


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.


In situ hybridization was performed on formalin-fixed, paraffin-embedded tissues to demonstrate E bieneusi as previously described.[7,14,15] To determine the cell type that harbored the organisms, IHC was then done on the same sections using an avidinbiotin complex technique.[16,17] Epithelial and mesothelial cells were identified using polyclonal wide-spectrum rabbit anticytokeratin and monoclonal mouse anti-mesothelial cell antibodies listed in Table 1. Macrophages were labeled using mouse monoclonal antibody against human macrophage marker CD68. For doublelabeling studies, a brown chromogen (diaminobenzidine, Sigma, St Louis, Mo) was used to identify the parasites by ISH followed by a gray chromogen (Vector SG, Vector Laboratories, Burlingame, Calif) to label the cells by IHC. Additional antibodies, listed in Table 1, were used to further characterize the lesions.

Table 1. Antibodies Used for Immunohistochemical Analysis
Antibody/Clone Epitope Tissue Stained

Polyclonal rabbit
 anticytokeratin(*) Cytokeratin Epithelial cells
KP-1(*) CD68 Macrophages
HBME-1(*) Mesothelial cells Mesothelial cells
MIB-1([dagger]) Ki-67 Proliferating cells
V9(*) Vimentin Mesenchymal cells

(*) Dako, Carpinteria, Calif.

([dagger]) Immunotech, Westbrook, Me.

PCR and Sequencing

DNA was isolated (Qiagen, Valencia, Calif) from snap frozen abdominal fluid, pleura, and peritoneum obtained at necropsy from 5 of the 7 animals with proliferative serositis. Polymerase chain reaction was performed on DNA, using the outer primers EBIEF1 and EBIER1 as previously described.[7,15] Amplification of DNA was performed in a 50-[micro]L reaction volume containing 1.5 mM Mg[Cl.sub.2], 50 mM KCl, 10 mM Tris/HCl (pH 9.0), 0.2 mM deoxynucleoside triphosphate, 400 pM of each primer, and 2.5 U of Amplitaq DNA Polymerase in a 9600 thermal cycler (PerkinElmer Cetus, Foster City, Calif). Amplification profile consisted of 1 minute at 57 [degrees] C, 2 minutes at 72 [degrees] C, and 30 seconds at 94 [degrees] C for 35 cycles followed by a 10-minute extension at 72 [degrees] C. A total of 1 [micro]L of this reaction was used as template for nested PCR, using the inner primers EBIE636AS (CTG CCT TCG TCC TTG ATC C) and EBIE437S (TGG ATG CTG CAG TTA AAG GG) and identical amplification conditions. Precautions against contamination were followed as previously described.[18] The 607-base pair (bp) outer amplification product from 2 animals was cloned (Invitrogen, San Diego, Calif) and sequenced with forward and reverse primers using an ABI prism automated sequencer (PE Applied Biosystems, Foster City, Calif).


Retrospective analysis by ISH revealed microsporidian organisms in the serosa of the intestines and surrounding reactive tissue in 7 of 16 animals with peritonitis of unknown cause. Lesions in the hepatobiliary tree have previously been described in 4 of these animals (Mm 139-86, 358-88, 163-88, and 506-92).[7] Animals had been experimentally inoculated with SIV and had died with simian AIDS. Viral inoculum, signalment, and survival are summarized in Table 2. All died with AIDS-related lesions, 4 with opportunistic infections in addition to E bieneusi and the others with SIV-associated lesions, including encephalitis, pulmonary arteriopathy, or SIV lymphadenitis.[19]

Table 2. Macaca mulatta With Enterocytozoon bieneusi-Associated Serositis(*)
Animal Viral Inoculum Survival, mo Pleuritis

139-86 SIVmac251 12 Yes
358-88 SIVmac239 38 No
163-88 SIVmac251 39 No
262-89 SIVmac239 25 Yes
506-92 SIVmac239 20 Yes
320-94 SIVmac239 8 Yes
327-96 SIVmac251 30 Yes

Animal Concurrent Lesions

139-86 PCP, SIV lymphadenitis, SIV arteriopathy
358-88 Pneumonia, Cryptosporidium parvum, LPD
163-88 Arteriopathy, pulmonary infarct, Mycobacterium avium
262-89 Enteritis
506-92 CMV, PCP, M avium, SIV arteriopathy, bronchitis,
 SIV encephalitis, LPD
320-94 SIV encephalitis, gastritis, glomerulonephritis
327-96 SIV encephalitis, SIV arteriopathy,
 SIV lymphadenitis, M avium

(*) SIV indicates simian immunodeficiency virus; PCP, Pneumocystis carinii pneumonia; LPD, lymphoproliferative disease; and CMV, cytomegalovirus.

Grossly, peritonitis with visceral adhesions was described in all cases. In 3 cases, ascites was noted. The serosal surfaces of the intestines were thickened, edematous, and rough, and the splenic and hepatic capsules were sometimes thickened. The omentum and mesentery were also thickened and adherent to the viscera. Pleural adhesions were noted in 3 cases.

Histologically, there was extensive thickening of the serosal surfaces characterized by proliferation of mesothelial cells, lymphangiectasia, edema, fibrin, and a mild nonsuppurative inflammatory cell infiltrate (Figure 1). The mesothelial cell hyperplasia was characterized by basophilia, cytomegaly, and loss of normal simple squamous architecture of the serosa. By means of ISH, E bieneusi could be seen in the proliferative tissue (Figure 2, A and B). In more advanced cases, proliferative serositis was accompanied by vascular proliferation and organization of fibrin. In the later stages, the proliferating mesothelial cells were engulfed and surrounded by dense bands of fibrous connective tissue, producing extensive fibrous adhesions. The pleuritis, seen in 5 of 7 cases of serositis, was morphologically similar to the peritonitis, and E bieneusi organisms were visible within the pleural mesothelial cells. In one case, E bieneusi could be seen in the bronchiolar epithelium and was accompanied by a lymphocytic, neutrophilic, and eosinophilic bronchiolitis.


In all cases, there was some degree of cholecystitis and choledochitis characterized by epithelial proliferation, a lymphoplasmacytic infiltrate, and occasional neutrophils, morphologic features commonly recognized with Enterocytozoon infection of immunodeficient macaques. In the most severe cases, there was also cholangitis and cirrhosis.

The plasmodial stages of E bieneusi with their characteristic cleftlike inclusions were easily seen by ISH within the thickened serosa of the intestine and lung (Figure 2, A). Immunohistochemical staining of the same sections showed that most of the organisms were located in the cytoplasm of serosal cells positive for cytokeratin (Figure 2, B). In one case of peritonitis, a few macrophages in the same sections also contained microsporidla (Figure 2, C). Plasmodia were also found within cells that did not label for either CD68 or cytokeratin, suggesting infection of mesothelial cell precursors (Figure 2, D).

Immunohistochemistry using antibodies to cytokeratin and mesothelial cells demonstrated the thickening, disruption, and disorganization of the serosal mesothelial cells. A monoclonal antibody to the proliferative marker Ki-67, MIB-1, also showed the increased mitotic activity of these cells. Immunohistochemistry revealed that many of the proliferative cells were not positive for either cytokeratin or mesothelial cell antibody. These cells were vimentin positive, suggesting a stage of differentiation between fibroblasts and mature mesothelial cells. Immunohistochemistry using anti-CD68 showed an increase in the number of macrophages in the reactive tissue.

To confirm the identity of the microsporidian identified in tissue sections by ISH, PCR was performed on DNA isolated from pleural and peritoneal tissue and abdominal fluid from 5 of 7 animals with proliferative serositis. These tissues were positive for E bieneusi DNA (Figure 3). Cloning and sequencing of the 607-bp outer PCR product revealed 100% identity to sequences previously obtained from rhesus macaque fecal samples (Genbank accession number AF023245) and to sequences obtained from human patients with AIDS (Genbank accession number AF024657 and L16868).



We describe a unique proliferative serositis associated with E bieneusi infection of mesothelial cells in immunodeficient SIV-infected rhesus macaques. Retrospective analysis revealed E bieneusi proliferative serositis in 7 of 16 cases of peritonitis of unknown origin, suggesting that the organism may represent a common cause of peritonitis in the immunocompromised host. The morphologic findings of nonsuppurative inflammation accompanied by proliferating mesothelium was highly suggestive of Enterocytozoon infection and could be confirmed by ISH and PCR.

This study confirms previous observations of the increased sensitivity of ISH compared with conventional techniques in identifying E bieneusi infections.[7,15,20] Routine cytochemical stains used to demonstrate microsporidia (Weber's modified trichrome, Gram stain, silver stains) revealed only rare organisms within the proliferating serosa. In contrast, ISH demonstrated characteristic plasmodial stages within the proliferating mesothelial cells. We and others have previously noted the increased sensitivity of ISH compared with conventional staining techniques.[7,20] In contrast to histochemical techniques that rely on specific anatomic structures, such as chitin, within the spore wall to stain organisms, ISH detects nucleic acid sequences that are present in all developmental stages.[15,20] Comparison of serial sections of infected small intestine often reveals a far greater number of infected cells by ISH than other staining techniques, an observation confirmed by ultrastructural examination.

This study adds mesothelial cells to the increasing range of host cells infected by E bieneusi, which includes biliary, intestinal, and respiratory epithelial cells and possibly macrophages of the lamina propria. Other microsporidial species (Encephalitozoon sp, Septata sp, and Nosema sp) are much more likely to disseminate than E bieneusi. Enterocytozoon cuniculi has been reported as the cause of peritonitis diagnosed by ultrastructural examination in one patient with AIDS and found in multiple tissues in other HIV-infected patients.[3,21-23] A hematogenous route of dissemination is suspected. Reports of E bieneusi infection in humans have been limited to the epithelium of the intestinal and hepatobiliary tracts, although E bieneusi has been seen rarely in macrophages and nasal and bronchial epithelium.[24-26] Rare organisms in alveolar macrophages are associated with lysosomes and in various stages of degeneration.[4]

The gallbladder is the likely reservoir of E bieneusi infection in both immunocompetent and immunodeficient macaques.[27] During the progressive phases of AIDS, organisms are also commonly found in small intestinal epithelium, where they are associated with varying degrees of villous atrophy and crypt hyperplasia. As in biopsy specimens from human patients,[28] various stages have been recognized in the duodenal lamina propria and rarely within the mesenteric lymph node.[7] This suggests that a possible means of dissemination might be through the lymphatic system, from the villous lacteals to the dilated lymphatics in the serosa. Frequent anastomoses between the lymphatics of the parietal peritoneum and the parietal pleura, on either side of the diaphragm, may provide a route for the spread of infection to the pleura. Macrophages might provide a means of transport for spores that, unless in clusters, are not easily seen. Once delivered to the serosa, productive infection of mesothelial cells may begin.

These findings indicate that E bieneusi may disseminate and represent a common cause of peritonitis of unknown origin in the immunocompromised host. We have previously shown extensive genetic and ultrastructural similarities between E bieneusi derived from rhesus macaques and humans.[7,9] Furthermore, we have successfully transmitted E bieneusi obtained from a human patient with AIDS to SIV-infected macaques and demonstrated similar morphologic alterations in humans and animals.[7,29] The extensive similarities between the human and rhesus-derived E bieneusi suggest that a similar origin should be considered in cases of proliferative serositis of unknown origin in immunodeficient human patients.

Financial support was provided by the Public Health Service grants RR 00168, RR0700, and RO1AI41889.


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[15.] Carville A, Mansfield K, Widmer G, et al. Development and application of genetic probes for the detection of E. bieneusi in formalin fixed stools and intestinal biopsies of infected patients. Clin Diagn Lab Immunol. 1997;4:1-3.

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[19.] Simon MA, Chalifoux LV, Ringler DJ. Pathologic features of SIV-induced disease and the association of macrophage infection with disease evolution. AIDS Res Hum Retroviruses. 1992;8:327-337.

[20.] Velasquez JN, Camevale S, Labbe JH, Chertcoff A, Cabrera MG, Oelemann W. In situ hybridization: a molecular approach for the diagnosis of the microsporidian parasite Enterocytozoon bieneusi. Hum Pathol. 1999;30:54-58.

[21.] Call A. Cytological and taxonomical comparison of two intestinal disseminating microsporidioses. AIDS. 1993;7:S12-S16.

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[23.] Zender HO, Arrigoni E, Eckert J, Kapanci Y. A case of Encephalitozoon cuniculi peritonitis in a patient with AIDS. Am J Clin Pathol. 1989;92:352-356.

[24.] Eeftinck Schattenkerk JKM, Van Gool T, van Ketel RJ, et al. Clinical significance of small-intestinal microsporidiosis in HIV-1-infected individuals. Lancet. 1991;337:895-898.

[25.] Weber R, Kuster H, Keller R, et al. Pulmonary and intestinal microsporidiosis in a patient with the acquired immunodeficiency syndrome. Am Rev Respir Dis. 1992;146:1603-1605.

[26.] Weber R, Bryan RT, Schwartz DA, Owen RL. Human microsporidial infection. Clin Microbial Rev. 1996;7:426-461.

[27.] Mansfield KG, Carville A, Hebert D, et al. Persistent infection of normal rhesus macaques (Macaca mulatta) with Enterocytozoon bieneusi localizes to the hepatobiliary tree. J Clin Adicrobiol. 1998;36:2336-2338.

[28.] Schwartz DA, Abou-Elella A, Wilcox CM, et al. The presence of Enterocytozoon bieneusi spores in the lamina propria of small intestinal biopsies with no evidence of disseminated microsporidiosis. Arch Pathol Lab Med. 1995;119: 424-428.

[29.] Tzipori S, Carville A, Widmer G, Kotler D, Mansfield KG, Lackner A. Transmission and establishment of a persistent infection of E. bieneusi derived from a human with AIDS in SIV-infected rhesus monkeys. J Infect Dis. 1997;175:1016-1020.

Accepted for publication May 1, 2000.

From the New England Regional Primate Research Center, Harvard Medical School, One Pine Hill Dr, Southborough, Mass.

Reprints: Keith Mansfield, DVM, Harvard Medical School, New England Regional Primate Research Center, PO Box 9102, Southborough, MA 01772-9012 (e-mail:
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Author:Chalifoux, Laura V.; Carville, Angela; Pauley, Douglas; Thompson, Brendon; Lackner, Andrew A.; Mansf
Publication:Archives of Pathology & Laboratory Medicine
Date:Oct 1, 2000
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