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Chapter 11 : Culture and Propagation of Microsporidia

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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 infection of a cell culture developed from the ovarian tube lining cells of the silkworm (). Cultures of 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, () 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 . 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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Figures

Image of FIGURE 1
FIGURE 1

Growth of various microsporidia in mammalian cell cultures as seen with a microscope equipped with differential interference contrast (DIC) optics. (Top left) in human lung fibroblast (HLF) cell culture. A fibroblast cell is distended with developing stages and spores of , 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 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 . Note the parasitophorous vacuole (PV) bulging with 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.

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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Image of FIGURE 2
FIGURE 2

(Top left) DIC image of growing on an E6 monolayer. Note the centrifugal growth pattern. Original magnification, ×300. (Top right) DIC image of , a mosquito isolate, growing on an E6 monolayer. Note that the cells are distended with spores that are relatively larger than those of spp. N, host cell nucleus. Original magnification, ×300. (Bottom left) DIC image of , 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 stained with the quick hot Gram chromotrope stain. Original magnification, ×1,250.

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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Image of FIGURE 3
FIGURE 3

Culture smears of (top left and right) stained with the quick hot Gram chromotrope technique and that of (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 (top right) and (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.

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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Image of FIGURE 4
FIGURE 4

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

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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Image of FIGURE 5
FIGURE 5

Transmission electron micrographs of microsporidia. (Top left) . Original magnification, ×4,000. Note various stages growing inside a parasitophorous vacuole (PV) which is unseptated. (Top right) . Note the various developmental stages within a PV which is septated. Original magnification, ×6,000. (Bottom left) . Note several degenerating developing stages (arrowhead) and a single spore in the cytoplasm. Original magnification, ×10,000. (Bottom right) Mosquito-derived 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.

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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Image of FIGURE 6
FIGURE 6

(Top) 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.

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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Tables

Generic image for table
TABLE 1

: isolates, origin, and culture conditions

CSF, cerebrospinal fluid.

No CO (air).

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
Generic image for table
TABLE 2

: isolates, origin, and culture conditions

BAL, bronchoalveolar lavage.

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

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

Cultivated in 5% CO.

No CO (air).

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

MRC-5, FBF, MDCK, E6.

Urine, sputum, nasal wash, BAL.

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
Generic image for table
TABLE 3

: isolates, origin and culture conditions

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

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

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

100 μg of erythromycin per ml.

No C02 (air).

With 10% human AB-positive serum.

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

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
Generic image for table
TABLE 4

Isolation and in vitro cultivation of human microsporidia

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

Duodenal aspirate/biopsy.

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

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

With 2 mM l-glutamine.

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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