Culture and Propagation of Microsporidia

Govinda S. Visvesvara; Hercules Moura; Gordon J. Leitch; David A. Schwartz

doi:10.1128/9781555818227.ch11

Chapter 11 : Culture and Propagation of Microsporidia

Authors: Govinda S. Visvesvara¹, Hercules Moura², Gordon J. Leitch⁴, David A. Schwartz⁵

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Affiliations: 1: Division of Parasitic Diseases, M.S.-F/13, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Atlanta, GA 30341-3724.; 2: Division of Parasitic Diseases, M.S.-F/13, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Atlanta, GA 30341-3724; 3: Faculdade de Ciencias Medicas, Universidade do Estado do Rio de Janeiro and Hospital Evandro Chagas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil; 4: Department of Physiology, Morehouse School of Medicine, Atlanta, GA; 5: Department of Pathology, Emory University and Grady Memorial Hospital, Adanta, GA

Content Type: Monograph

Publication Year: 1999

Category: Fungi and Fungal Pathogenesis

Book DOI: 10.1128/9781555818227

Chapter DOI: 10.1128/9781555818227.ch11

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Abstract:

Isolation in culture should always be attempted even when a presumptive diagnosis has been made. This is particularly important for the purpose of establishing a bank of isolates to be used for antigenic, molecular, and biochemical analyses. The first attempt to culture microsporidia was made in 1937 by Trager, who was partially successful in establishing Nosema bombycis infection of a cell culture developed from the ovarian tube lining cells of the silkworm (Bombyx mori). Cultures of Encephalitozoon cuniculi were initiated in several different ways: by adding infected tissue explants to cultured cells, by allowing infected cells in explanted cells to grow, by allowing germination of spores in the presence of cells and thereafter infecting cells, and by scraping infected cells from infected cultures and adding them to fresh cell cultures. The first microsporidian isolated from a human, Vittaforma corneae (Nosema corneum) was cultured from a corneal biopsy that was shipped to the laboratory overnight in Hanks' balanced salt solution (HBSS). Only a few attempts have been made to isolate microsporidia into culture from feces because enteric bacteria and yeast usually overgrow the rich culture medium that is used, which impedes isolation of the fastidious microsporidia, especially Enterocytozoon bieneusi. Inoculated cell cultures should be examined frequently with an inverted microscope preferably equipped with phase-contrast or differential interference-contrast optics. One great advantage of obtaining spores from in vitro cultures is the absence of bacterial and fungal contaminants.

Citation: Visvesvara G, Moura H, Leitch G, Schwartz D. 1999. Culture and Propagation of Microsporidia, p 363-392. In Wittner M, Weiss L (ed), The Microsporidia and Microsporidiosis. ASM Press, Washington, DC. doi: 10.1128/9781555818227.ch11

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Culture and Propagation of Microsporidia

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FIGURE 1

Growth of various microsporidia in mammalian cell cultures as seen with a microscope equipped with differential interference contrast (DIC) optics. (Top left) Encephalitozoon hellem in human lung fibroblast (HLF) cell culture. A fibroblast cell is distended with developing stages and spores of E. hellem, giving the cell a corn-on-the cob appearance. Original magnification, ×600. (Top middle) A low-power (×150) view of the same cell culture showing extensive infection of the monolayer. (Top right) A high-power (×600) view of the same cell culture several months later, exhibiting total destruction of the monolayer. Note the birefringent spores (arrowhead), a number of which have discharged their polar tubules (arrow). (Bottom left) An HLF monolayer inoculated with a Enterocytozoon bieneusi-positive duodenal aspirate from an AIDS patient. Note the corn-on-the cob appearance of the cell, but this time it is due to an adenovirus. Original magnification, ×600. (Bottom right) A monkey kidney cell in an E6 monolayer infected with E. cuniculi. Note the parasitophorous vacuole (PV) bulging with E. cuniculi spores. Some of the spores exhibit a distinct vacuole at one end (long arrow). A spore (arrowhead) has discharged its polar tubule (short arrows). N, host cell nucleus. Original magnification, ×1,250.

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FIGURE 1

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FIGURE 2

(Top left) DIC image of Vittaforma corneae growing on an E6 monolayer. Note the centrifugal growth pattern. Original magnification, ×300. (Top right) DIC image of Nosema algerae, a mosquito isolate, growing on an E6 monolayer. Note that the cells are distended with spores that are relatively larger than those of Encephalitozoon spp. N, host cell nucleus. Original magnification, ×300. (Bottom left) DIC image of N. algerae, isolated from the corneal biopsy of an immunocompetent person, growing within an E6 cell. Note spores and developing stages arranged around the host cell nucleus (N). Original magnification, ×1,250. (Bottom right) An E6 monolayer infected with mosquito-derived N. algerae stained with the quick hot Gram chromotrope stain. Original magnification, ×1,250.

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FIGURE 2

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FIGURE 3

Culture smears of Encephalitozoon hellem (top left and right) stained with the quick hot Gram chromotrope technique and that of E. cuniculi (bottom left and right) after reaction with polyclonal rabbit antiserum in the indirect immunofluorescence test. Note the chains of spores representing polysporoblastic sporogony in both E, hellem (top right) and E. cuniculi (bottom left and right). Note that a spore still in the chain configuration has already extruded its polar tubule (bottom left). Original magnification, ×1,250.

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FIGURE 3

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FIGURE 4

Scanning electron micrographs of microsporidia growing in cell cultures. (Top left) E. cuniculi in E6 cells. (Top right) E. hellem in E6 cells. (Bottom left) Encephalitozoon intestinalis in E6 cells. Note that some spores have already discharged their polar tubules. (Bottom right) an isolate of N. algerae, from a human cornea, growing in HLF cells. Original magnifications, ×5,000.

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FIGURE 4

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FIGURE 5

Transmission electron micrographs of microsporidia. (Top left) E. cuniculi. Original magnification, ×4,000. Note various stages growing inside a parasitophorous vacuole (PV) which is unseptated. (Top right) E. intestinalis. Note the various developmental stages within a PV which is septated. Original magnification, ×6,000. (Bottom left) E. bieneusi. Note several degenerating developing stages (arrowhead) and a single spore in the cytoplasm. Original magnification, ×10,000. (Bottom right) Mosquito-derived N. algerae within an E6 cell. Note several electron-dense spores and a number of developing stages. Original magnification, ×2,500. M, meront; S, spore; SB, sporoblast; N, host cell nucleus.

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FIGURE 5

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FIGURE 6

(Top) E. bieneusi within a disintegrating E6 cell. Note a spore (asterisk), several sporo-blasts with polar tubules in cross-sectional (arrowhead), and stacked (small arrow) and coiled (large arrow) profiles. Original magnification, ×12.000. (Reprinted with permission from the Society of Protozoologists.) (Bottom) E6 cell containing a mass of adenoviral particles (AV). Magnification, ×6,000.

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FIGURE 6

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Tables

Culture and Propagation of Microsporidia

/content/10.1128/9781555818227.chap11.tab11-1

TABLE 1

Encephalitozoon cuniculi: isolates, origin, and culture conditions

a CSF, cerebrospinal fluid.

b No COs (air).

10.1128/9781555818227/tab11-1_thmb.gif

10.1128/9781555818227/tab11-1.gif

TABLE 1

Encephalitozoon cuniculi: isolates, origin, and culture conditions

a CSF, cerebrospinal fluid.

b No COs (air).

/content/10.1128/9781555818227.chap11.tab11-2

TABLE 2

Encephalitozoon hellem: isolates, origin, and culture conditions

a BAL, bronchoalveolar lavage.

b HLF, human lung fibroblast; FBF, fetal bovine fibroblast.

c FBS, fetal bovine serum; MEM, minimal essential medium; FCS, fetal calf serum; EMEM, Eagles MEM.

d Cultivated in 5% CO2.

e No CO2 (air).

f Fetal calf serum plus 200 IU of penicillin, 200 μg of streptomycin, and 2.5 μg of amphotericin B.

g MRC-5, FBF, MDCK, E6.

h Urine, sputum, nasal wash, BAL.

10.1128/9781555818227/tab11-2_thmb.gif

10.1128/9781555818227/tab11-2.gif

TABLE 2

Encephalitozoon hellem: isolates, origin, and culture conditions

a BAL, bronchoalveolar lavage.

b HLF, human lung fibroblast; FBF, fetal bovine fibroblast.

c FBS, fetal bovine serum; MEM, minimal essential medium; FCS, fetal calf serum; EMEM, Eagles MEM.

d Cultivated in 5% CO2.

e No CO2 (air).

f Fetal calf serum plus 200 IU of penicillin, 200 μg of streptomycin, and 2.5 μg of amphotericin B.

g MRC-5, FBF, MDCK, E6.

h Urine, sputum, nasal wash, BAL.

/content/10.1128/9781555818227.chap11.tab11-3

TABLE 3

Encephalitozoon intestinalis: isolates, origin and culture conditions

a Nasal, nasopharyngeal aspirate; BAL, bronchoalveolar lavage; intest, duodenal aspirate/biopsy.

b HLF, human lung fibroblast; HEL, human embryonic lung; HMDM, human monocyte-derived macrophage.

c MEM, Eagle's minimum essential medium; FBS, fetal bovine serum; FCS, fetal calf serum.

d 100 μg of erythromycin per ml.

e No C02 (air).

f With 10% human AB-positive serum.

g RK13, MDCK, HT-29, Caco-2,Vero, I 047.

10.1128/9781555818227/tab11-3_thmb.gif

10.1128/9781555818227/tab11-3.gif

TABLE 3

Encephalitozoon intestinalis: isolates, origin and culture conditions

a Nasal, nasopharyngeal aspirate; BAL, bronchoalveolar lavage; intest, duodenal aspirate/biopsy.

b HLF, human lung fibroblast; HEL, human embryonic lung; HMDM, human monocyte-derived macrophage.

c MEM, Eagle's minimum essential medium; FBS, fetal bovine serum; FCS, fetal calf serum.

d 100 μg of erythromycin per ml.

e No C02 (air).

f With 10% human AB-positive serum.

g RK13, MDCK, HT-29, Caco-2,Vero, I 047.

/content/10.1128/9781555818227.chap11.tab11-4

TABLE 4

Isolation and in vitro cultivation of human microsporidia

a MEM, minimal essential medium; FBS, fetal bovine serum; FCS, fetal calf serum; DMEM, Dulbecco's modified MEM.

b Duodenal aspirate/biopsy.

c SIRC, MDCK, MRC-5, XEN, L-929, FHM.

d COS-1, RK-13, MDCK, myoblasts G-7 and L6-C10.

e With 2 mM l-glutamine.

10.1128/9781555818227/tab11-4_thmb.gif

10.1128/9781555818227/tab11-4.gif

TABLE 4

Isolation and in vitro cultivation of human microsporidia

a MEM, minimal essential medium; FBS, fetal bovine serum; FCS, fetal calf serum; DMEM, Dulbecco's modified MEM.

b Duodenal aspirate/biopsy.

c SIRC, MDCK, MRC-5, XEN, L-929, FHM.

d COS-1, RK-13, MDCK, myoblasts G-7 and L6-C10.

e With 2 mM l-glutamine.