Fumagillin

* Objective.-Microsporidia isolated from clinical specimens so far have been identified to level of species by electron microscopy, indirect immunofluorescence (IIF), western blot (WB), and genetic analysis. Recent studies, however, indicate extensive serologic cross-reactions among microsporidian species involved in human disease.

Design and Setting.-In this study, we used IIF and WB techniques to evaluate the reactivity of six different immunoglobulin G monoclonal antibodies (MAbs) raised against Encephalitozoon hellem with six isolates of E hellem that originated from patients with acquired immunodeficiency syndrome. A rabbit isolate of Encephalitozoon cuniculi, and an isolate of Encephalitozoon intestinalis, which was established in cultures from the urine of a patient with acquired immunodeficiency syndrome were also used for comparison.

Results.-Five of the six antibodies, when analyzed by both IIF and WB assays, specifically identified six isolates of E hellem originating from three patients with acquired immunodeficiency syndrome. The sixth MAb, however, reacted with all of the E hellem isolates in the WB assay, but

failed to react with them in the IIF assay. Using the IIF test, five of the six MAbs failed to react with E cuniculi and E intestinalis, even at a dilution of 1:50. The MAbs also did not react in the IIF test with Enterocytozoon bieneusi, Giardia, and Cryptosporidium. These MAbs did react with E cuniculi and E intestinalis in the WB assay, but the banding patterns were very different from those of E hellem, thus facilitating the identification of E hellem from the other microsporidia. The MAbs also reacted, in the IIF test, with E hellem spores in formalin-fixed tissue sections that were heated in a microwave oven.

Conclusions.-Identification of microsporidian agents to the species level is important. Since certain therapeutic agents (eg, fumagillin, albendazole) are efficacious in treating E hellem infections of the cornea, as well as urogenital and respiratory infections caused by E hellem, a quick and definitive identification of the organism is important so that successful therapy may be instituted. An IIF test using the MAbs described here would therefore be invaluable in the quick identification of this parasite.

(Arch Pathol Lab Med. 1998;122:182-186)

The order Microsporida of the phylum Microspora consists of a large group of primitive, obligately parasitic, eukaryotic protozoans that lack mitochondria.1 Microsporidia parasitize members of almost every major phylum of the animal kingdom.2 A salient feature of microsporidia is the presence of a coiled polar tubule in an environmentally resistant spore, which on extrusion injects infective sporoplasm into a suitable host cell. Previously known mainly for their ability to infect insects and rodents, microsporidia are now recognized as important and widely prevalent emerging opportunistic agents causing infection in persons with acquired immunodeficiency syndrome (AIDS).3 During the past 2 years, microsporidiosis has also been recognized in human immunodeficiency virus-seronegative persons4 receiving liver,5 heartlung,6 and kidney-pancreas transplants (D.A.S., Harris Goodman, MD, and Ralph T. Bryan, MD, unpublished data), and as a cause of traveller's diarrhea illness in immunocompetent adults7-9 and children.10 To date, eight microsporidian species have been identified as agents of human disease, producing enteric (Enterocytozoon bieneusi, Encephalitozoon [formerly Septata] intestinalis), ocular (Encephalitozoon hellem, Encephalitozoon cuniculi, Nosema ocularum, and Vittaforma corneae), muscular (Pleistophora species and Trachipleistophora hominis), and disseminated (Encephalitozoon species) infections.11-15 Enterocytozoon bieneusi is the most prevalent microsporidian infecting humans,13 but disseminated infections due to E cuniculi, E hellem, and Encephalitozoon intestinalis are being recognized with increased frequency.16-21

Diagnosis of microsporidiosis to the species level is important because several new drug therapies are efficacious in treating infections caused by some, but not all, microsporidian species.21-25 However, species identification can be difficult and time consuming, requiring specialized techniques (eg, electron microscopy and polymerase chain reaction) currently available in only a few laboratories.26 It would be advantageous to have species-specific monoclonal antisera available for diagnosis of microsporidiosis from both fresh and fixed patient specimens. This report describes the use of immunoglobulin (Ig) G monoclonal antibodies (MAbs) to E hellem that can specifically detect this agent either by indirect immunofluorescence (IIF) or western blot methods.

MATERIALS AND METHODS

Parasites

Six isolates (CDC:0291:V213, CDC:V242, CDC:V257, CDC: V258, CDC:V274, and CDC:V281) of E hellem were grown on monolayers of either monkey kidney cells (E6) or human lung fibroblasts using previously published methods.27,28 Three of these isolates (CDC:V257, CDC:V258, and CDC:V281) originated, respectively, from urine, sputum, and nasal swab samples of a 30-year-old woman with AIDS and disseminated microsporidiosis, and the other two isolates (CDC:V242 and CDC:V274) were from the urine and bronchoalveolar lavage samples, respectively, of a 40-year-old man with AIDS and pulmonary and renal microsporidian infection. The reference isolate (CDC:0291:V213) was cultured from the urine of a male AIDS patient.16,27 A rabbit strain of E cuniculi (designated as ED) and E (Septata) intestinalis (CDC:V297) were also grown in E6 cell cultures. The growth medium consisted of Eagle's minimum essential medium containing 5% heat-inactivated fetal bovine serum, 50 (mu)g/mL gentamicin and 5 (mu)g/mL fungizone (Gibco BRL Life Technologies Inc, Grand Island, NY). The cultures were incubated at 37 deg C in an incubator without carbon dioxide. Parasite Harvest

Spores that were periodically released into the culture medium were collected from several flasks, pooled, and centrifuged at 2500g for 20 minutes at 4 deg C. The pellets were resuspended and washed once in phosphate-buffered saline (PBS) (pH 7.4) containing 0.3% Tween 20. The sedimented culture materials were layered on Percoll29 (Sigma Chemical Company, St Louis, Mo) and centrifuged as described elsewhere to purify spores from the contaminating cell debris. Monoclonal Antibodies

Hybridomas secreting MAbs were produced by immunizing BALB/c mice with E hellem as previously described.28 Briefly, spleen cells from immunized mice that were positive for hightiter antibodies to E hellem by IIF were collected, fused with SP2/0 myeloma cells, established in culture, repeatedly cloned, and screened by IIF for antibodies. Six clones (ED4H6B3/A4, ED4H6B1/G4, ED4H10B11/B12, ED4HlOH5/C12, DA4E9D8/ B3, and DA4BB6E3/G6) were found to be secreting IgG1 antibodies by enzyme-linked immunosorbent assay. These clones were designated MAb 1 through 6 (see Table) for convenience. These clones were injected via the intraperitoneal route into pristane-primed BALB / c mice to produce ascitic fluid that was filtered through membrane filters (0.2 (mu)m pore size), aliquoted, and stored in liquid nitrogen.

Indirect Immunofluorescence Assay

Antibody activity of ascitic fluids from the six hybridoma clones and serum from two immunized rabbits was analyzed by IIF using microsporidian spores obtained from cultures as described elsewhere.28 In brief, spores liberated from host cells were suspended in PBS (0.1 mol/L, pH 7.4) containing 1% formalin to obtain 5 x10^sup 6^ spores/mL. Fifteen microliters of the suspension was then added to 12-well Teflon-coated slides and allowed to dry at room temperature. The slides were wrapped in tissue paper and aluminum foil, dated, and stored at 20 deg C until use. Ascites fluids were serially diluted twofold starting from 1:2 in microtitration plates. Phosphate-buffered saline (0.1 mol/L, pH 7.4) was the diluent. The slides were retrieved from the freezer and allowed to reach room temperature, washed once in PBS before they were covered with 15 (mu)L per well of the appropriately diluted asc tic fluid. The slides were incubated at 37 deg C for 20 minutes, washed three times with PBS, and allowed to dry. The wells were then covered with 15 (mu)L fluorescein isothiocyanate-conjugated goat anti-mouse IgG or goat anti-rabbit IgG before incubating the slides for 20 minutes at 37 deg C . The slides were allowed to dry after a final wash in PBS, mounted with buffered glycerin (pH 9.0), coverslipped, and examined with a microscope equipped with epifluorescence optics. Phosphate-buffered saline and conjugate controls were included with each slide.

Indirect immunofluorescence was also performed on tissue sections from the kidney of an AIDS patient obtained at autopsy, as described elsewhere.16,18,28 Following removal of the paraffin coat, the sections were either pretreated for 30 minutes at 37 deg C in a solution containing 2 mg/mL trypsin and 2 mg/mL calcium chloride and washed for 10 minutes in PBS (pH 7.2) or heated twice, 5 minutes at a time, in a microwave oven.30-32 Monoclonal antibodies developed to two unrelated protozoa (Acanthamoeba castellanii and Giardia lamblia) were used as negative controls.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on vertical slab gels, 1 mm thick, containing a gradient of 2.5% to 27% acrylamide (Isolab, Akron, Ohio) and using a continuous denaturing buffer system. Proteins were extracted from spores by dissolving them in a solution containing 2.5% Sodium dodecyl sulfate and 2.25 mol/L urea, and were heat-treated at 65C for 15 minutes in a water bath. Proteins from about 5x106 spores were loaded onto each lane and electrophoresed for about 6 hours. The proteins were either silver-stained by the method of Tsang and Boyer33 or electrophoretically transferred at a constant voltage of 30 V overnight to polyvinylidene difluoride membranes for western blot immunodetection. The membranes were washed four times for 5 minutes each in PBST (PBS, pH 7.4, containing 0.3% Tween 20) and incubated 1 hour at room temperature while being gently rocked in the appropriate ascitic fluid diluted 1:100 in PBS-T. The membranes were then washed three times, 10 minutes per wash, in PBS-T and incubated at room temperature in a 1:1000 dilution of affinity-purified, peroxidase-conjugated, goat anti-mouse IgG. After three changes in PBS-T and one wash in PBS without Tween 20, the membranes were developed for 20 minutes in a solution containing 5 mg 3,3'-diaminobenzidine tetrahydrochloride and 100 mL 3% hydrogen peroxide in 100 mL of PBS (pH 7.4). They were then washed with distilled water and dried at room temperature. Six MAbs were tested against all six isolates of E hellem, E intestinalis (CDC:V297), and E cuniculi (ECLD). Six MAbs were also tested by IIF on fecal smears containing En bieneusi (obtained from heavily infected AIDS patients who were parasitologically confirmed to have En bieneusi), Cryptosporidium, and Giardia. The Meri-flour kit (Meridian Diagnostics Inc, Cincinnati, Ohio) was used as a positive control for Cryptosporidium and Giardia.

RESULTS

Indirect Immunofluorescence

Spores of all six isolates of E hellem fluoresced brightly (4+, in a scale of 0 to 4, where 0 = no fluorescence, 1 = very faint, 2 = faint, 3 = moderate, and 4 = bright) with five (1, 2, 3, 4, and 6) of the six MAbs producing a titer greater than 4096, whereas the heterologous antigens (E cuniculi and E intestinalis) showed no significant reactivity, even at a dilution of 1:50 (Table). MAb 5 (DA4E9D8/B3) showed no reactivity with E cuniculi, E intestinalis, or any of the six isolates of E hellem. MAb 3 (ED4H10B11/B12), although negative for E cuniculi spores, reacted faintly (2+), with the polar tubule of the parasite. Additionally, none of the MAbs reacted with the spores of En bieneusi, Giardia species, or Cryptosporidium species when tested at a dilution of 1:30.

Spores in the tissue sections that were pretreated with trypsin-calcium chloride solution either failed to react or reacted very minimally with the MAbs. However, the spores in the sections that were heated in the microwave oven reacted with all five of the MAbs and produced moderate (3+) fluorescence (Fig 1). Consistency of the reactivity was variable, especially when the tissue specimens were kept in the fixative (formalin) for more than 1 year.

Western Blot

Antigens extracted from all six isolates of E hellem reacted strongly with five (MAbs 1, 2, 3, 4, and 6; see Table) of the six MAbs and exhibited remarkably similar profiles, sharing seven major bands at approximately 120, gO, 48, 40, 28, 25, and 16 kd. Encephalitozoon cuniculi antigens also reacted well with these MAbs. They produced bands at approximately 90, 45, 40, 28, and 25 kd, and displayed an overall banding pattern that was different from the patterns obtained for E hellem. Encephalitozoon intestinalis antigens reacted the least with these MAbs and produced two major bands at approximately 45 and 25 kd, as well as several indistinct bands. Figure 2, A illustrates the reactions of MAb 4 (ED4HlOH5/C12), which is representative of the other four MAbs, with the antigens of various isolates. MAb 5 (DA4E9D8/B3) reacted appreciably with all of the E hellem antigens tested; however, it produced darkly staining bands at about 120, 75, 20,18, and 15 kd, a pattern very different from those produced by the other five MAbs (Fig 2, B). Encephalitozoon cuniculi antigens also reacted with MAb 5 (DA4E9D8/B3) and produced two additional bands at about 28 and 17 kd. Encephalitozoon intestinalis antigens reacted poorly with MAb 5 and produced bands at approximately 28,17, and 15 kd. Overall, the banding patterns obtained with MAb 5 were not as strong as for the other five MAbs. E6 cells, used as a negative control in all experiments, reacted faintly with MAb 1 (ED4H6B3/A4) only and produced a band at about 38 kd (not shown).

COMMENT

In an earlier report,28 we described preliminary results of the usefulness of MAb 3 (ED4H10B11/B12). We describe here the successful production of several MAbs that may be used as specific diagnostic reagents to identify E hellem in a variety of samples from patients with microsporidiosis. These MAbs were characterized using both IIF and western blot techniques.

The results clearly show that these MAbs react selectively with all isolates of E hellem at titers of 1:50 or greater, yet show no reactivity with either E cuniculi or E intestinalis in the IIF test. The reasons MAb 3 reacted faintly (2+) with the polar tubule of E cuniculi in the IIF test are not clear, although it can be hypothesized that this particular MAb recognizes an epitope in the polar tubules common to both E hellem and E cuniculi. Also, these MAbs produced specific banding patterns when reacted with all isolates of E hellem in the western blot. Although E cuniculi cross-reacts with these MAbs, the banding patterns produced are very different. Encephalitozoon intestinalis, on the other hand, reacts minimally with these MAbs. A significant observation was that all six isolates of E hellem, obtained from different tissue sites of three patients originating from different geographic areas, are identical in their protein profile and western blot patterns. Also, isolates of E hellem originating from different tissues of the same patient were found to be homogeneous in their banding patterns.

Currently, the identification of human microsporidia to the species level depends on the use of time-consuming electron microscopy and molecular techniques.13,19 Electron microscopy, however, cannot differentiate E hellem from E cuniculi in formalin-fixed tissue sections.19,26 Serologic reagents (polyclonal and monoclonal antibodies) that have been developed so far34-37 do not unequivocally identify E hellem. In this report, we show that five of the six MAbs developed in our laboratories may be used to specifically identify E hellem derived both from cell cultures and from formalin-fixed tissue sections of patients with disseminated microsporidiosis caused by this species. Microwave heating of formalin-fixed, paraffin-embedded tissue sections prior to treatment with fluorescein isothiocyanate-conjugated monoclonal or polyclonal antibody appears to enhance staining of the antigen.30.32 The fluorescent properties of the spores, however, may vary if the tissue sections are fixed in formalin for a prolonged period. Finally, we have shown that these MAbs failed to react with spores of E intestinalis and En bieneusi, as well as with cysts of Giardia and oocysts of Cryptosporidium in fecal smears, thus emphasizing the specificity of these MAbs for definitive clinical diagnosis.

Diagnosis of microsporidiosis to the species level is important because certain therapeutic agents are efficacious in treating infections caused by some, but not all, microsporidian species. For example, corneal and systemic infections caused by E hel/em can be successfully treated with fumagillin and albendazole, respectively.4,13,17 Hence, a quick and definitive identification, preferably by IIF using the MAbs described here, of E hellem in corneal, respiratory, or urogenital samples will be greatly advantageous in the successful treatment of microsporidiosis due to E hellem.

Accepted for publication August 25, 1997.

From the Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Ga (Drs Croppo, Visvesvara, and Wallace); the Department of Physiology, Morehouse School of Medicine, Atlanta, Ga (Dr Leitch); and the Departments of Pathology and Medicine (Infectious Diseases), Emory University School of Medicine, Atlanta, Ga (Dr Schwartz).

Reprint requests to Mail stop F-13, Biology and Diagnostic Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341-3724 (Dr Visvesvara).

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