Chapter 23 : Intestinal Protozoa (Coccidia), Microsporidia, and Algae

Life cycle of Cryptosporidium spp. illustrating various stages. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch23.f1

Life cycle of Cryptosporidium spp. (Adapted from Current WL, Garcia LS. Clin Microbiol Rev 4:325–358, 1991.) doi:10.1128/9781555819002.ch23.f2

Cryptosporidium spp. (Top) Organisms on the brush border of the mucosal surface (courtesy of Armed Forces Institute of Pathology). (Middle) Organisms on the brush border of the mucosal surface. (Bottom) Organisms on the mucosal surface, human tissue (scanning electron micrograph, ×4,000). (Courtesy of Marietta Voge.) doi:10.1128/9781555819002.ch23.f3

Cryptosporidium. (Top, middle) Oocysts from stool, stained with modified acid-fast stain. Note that in the middle image some of the sporozoites are visible within the oocyst wall. Although the sporozoites are not always seen, the oocysts are immediately infectious when passed. (Bottom) Cryptosporidium and Giardia with combination FA reagent (large organisms are Giardia, small organisms are Cryptosporidium). doi:10.1128/9781555819002.ch23.f4

Diagnostic work flow diagram leading from submission of stool specimen to final permanent-stained smear for Cryptosporidium spp. and Cyclospora cayetanensis. *, Identification of coccidia may be difficult in a direct wet mount unless there are numerous oocysts present. In many laboratories, the use of fecal immunoassays has replaced acid-fast staining techniques for Cryptosporidium spp. The immunoassays can be performed on unconcentrated material (EIA, lateral-flow rapid cartridge) or concentrated material (FA assay). doi:10.1128/9781555819002.ch23.f5

Flow diagram demonstrating sources of waterborne transmission of Cryptosporidium oocysts. doi:10.1128/9781555819002.ch23.f6

Flow diagram demonstrating sources of foodborne transmission of Cryptosporidium oocysts. doi:10.1128/9781555819002.ch23.f7

Flow diagram demonstrating sources of close-contact transmission of Cryptosporidium oocysts. doi:10.1128/9781555819002.ch23.f8

Cyclospora cayetanensis life cycle. doi:10.1128/9781555819002.ch23.f9

Foods associated with Cyclospora cayetanensis contamination and transmission. (Top) Raspberries, raspberry cake; (row 2) pesto, pesto salad; (row 3) snow peas, basil; (bottom) mesclun (baby lettuce leaves for salad). doi:10.1128/9781555819002.ch23.f10

Cyclospora cayetanensis. (Top) Histopathological changes of the intestinal epithelia in an individual with cyclosporiasis (note some blunting of villi). (Middle) Multiple parasitic vacuoles containing meronts type 1 and 2 (arrows). (Bottom) Transmission EM, parasitic vacuole containing fully differentiated meronts. (Courtesy of Ynes Ortega.) doi:10.1128/9781555819002.ch23.f11

Cyclospora cayetanensis oocysts. (Upper) (far left) Unsporulated oocyst with undifferentiated cytoplasm; (A) sporulating oocyst that contains two immature sporocysts; (B) oocyst that was mechanically ruptured with one of its two sporocysts released; (C) one free sporocyst is seen, as well as two free sporozoites, the infective stage of the organism. (Lower) Sporulation of Cyclospora oocysts; Day 0, oocyst passed in fresh stool; Days 5 and 10, sporulated oocysts containing two sporocysts; Rupture, ruptured oocyst, one sporocyst outside, one remaining inside the oocyst wall. (Courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch23.f12

(Upper) Cyclospora cayetanensis autofluorescence using the same filter used for calcofluor white. (Lower) Modified acid-fast stain of C. cayetanensis (note that some oocysts stain quite dark, while some have not retained the stain; this is typical, and the organisms are described as “modified acid-fast variable”). The oocysts measure 8 to 10 μm. A 1% acid decolorizer is recommended—all coccidia will stain well using this approach. A stronger acid may decolorize Cyclospora too much. Note These images were photographed from concentrated, stained fecal material (500 × g for 10 min). doi:10.1128/9781555819002.ch23.f13

Modified acid-fast stain. Oocysts of Cryptosporidium (4–6 μm) (circle) and Cyclospora cayetanensis (8–10 μm) (arrows); one of the Cyclospora oocysts has not retained the stain. The smallest structure (square) is an artifact and measures approximately 1–2 μm. doi:10.1128/9781555819002.ch23.f14

Hot safranin stain of Cyclospora cayetanensis oocysts (photographed at a high magnification). Note the oocysts are unsporulated. (Courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch23.f15

Life cycle of Cystoisospora belli. (Illustration by Gwen Gloege.) doi:10.1128/9781555819002.ch23.f16

( 1 ) Immature oocyst of Cystoisospora belli; ( 2 ) mature oocyst of C. belli. (Illustration by Nobuko Kitamura.) doi:10.1128/9781555819002.ch23.f17

(Left) Immature oocyst of Cystoisospora belli, wet preparation. (Right) Immature oocyst of C. belli, modified acid-fast stain (note that the entire oocyst has stained). doi:10.1128/9781555819002.ch23.f18

(Left) Mature oocyst of Cystoisospora belli, wet preparation. (Right) Mature oocyst of C. belli, modified acid-fast stain. doi:10.1128/9781555819002.ch23.f19

Cystoisospora belli. (Upper) Immature oocyst photographed by fluorescence microscopy; (lower) mature oocyst photographed by fluorescence microscopy (courtesy of the CDC Public Health Image Library). doi:10.1128/9781555819002.ch23.f20

Cystoisospora belli. Sections of the upper jejunum showing various developmental (asexual and sexual) stages of C. belli (×1,000, hematoxylin and eosin stain). (A) Trophozoites (arrows), spherical in shape. (B) An immature schizont that has undergone nuclear division (arrow). Many eosinophils are infiltrated in the lamina propria. (C) A mature schizont with about six merozoites (left arrow) and a merozoite which has entered an enterocyte (right arrow). Eosinophil infiltrations are also seen in this figure. (D) Two merozoites in an enterocyte (long arrow), two macrogamonts (short arrows), and a developing trophozoite (lower left) are seen in the jejunal epithelial layer. (Courtesy of http://creativecommons.org/licenses/by-nc/3.0/.) doi:10.1128/9781555819002.ch23.f21

Cystoisospora belli. Sections of the upper jejunum showing various sexual stages of Cystoisospora belli (×1,000, hematoxylin and eosin stain). (A) A developing microgamont (arrow) with multiple nuclei that have migrated to the periphery. (B) A mature microgamont with multiple nuclei (arrow) in an enterocyte of the crypt layer. (C) A probable early macrogamont (arrow) in an enterocyte. (D) An old macrogamont or macrogamete (arrow) in a jejunal epithelial cell. (Courtesy of http://creativecommons.org/licenses/by-nc/3.0/.) doi:10.1128/9781555819002.ch23.f22

Life cycle of Sarcocystis spp. (Illustration by Gwen Gloege.) doi:10.1128/9781555819002.ch23.f23

Sarcocystis spp. (Upper, left) Sporulated oocyst in unstained wet mount; (right) sporulated oocyst in wet mount viewed under UV microscopy, magnification ×400 (courtesy of the CDC Public Health Image Library). (Lower) Classic sarcocystosis in cut beef (courtesy of the University of Sydney, Centre for Veterinary Education, http://vetbook.org/wiki/cow/index.php/Sarcocystis_spp). doi:10.1128/9781555819002.ch23.f24

(Top) Sarcocystis spp. in human muscle. (Middle) Higher magnification showing individual elongate bradyzoites. (Bottom) Numerous sarcocysts in longitudinal and cross section in muscles of sheep tongue. Note the lack of direct inflammatory response to sarcocysts. (Bottom image courtesy of reference 51 .) doi:10.1128/9781555819002.ch23.f25

Life cycle of microsporidia. (From Gardiner CH et al, An Atlas of Protozoan Parasites in Animal Tissues, U.S. Department of Agriculture handbook no. 651, U.S. Department of Agriculture, Washington, DC, 1988.) doi:10.1128/9781555819002.ch23.f26

(Top) Diagram of the internal structure of a microsporidian spore. The spore coat has an outer electron-dense region called the exospore (Ex) and an inner, thicker, electron-lucent region, the endospore (En). A unit membrane (P) separates the spore coat from the spore contents. The extrusion apparatus—anchoring disk (A), polar tubule (Pt), lamellar polaroplast (Lp), spongiform polaroplast (Sp), and tubular polaroplast (Tp)—dominates the spore contents and is diagnostic for microsporidian identification. The posterior vacuole (Pv) is a membrane-bound vesicle, which sometimes contains a “membrane whirl,” a “glomerular-like” structure, flocculent material, or some combination of these structures. The spore cytoplasm is dense and contains ribosomes (R) in a tightly coiled helical array. The nucleation may consist of a single nucleus or a pair of abutted nuclei, a diplokaryon (Dn). The size of the spore depends on the particular species and can vary from less than 1 μm to more than 10 μm. The number of polar tubule coils also varies from a few to 30 or more, again depending on the species observed. (Middle) EM of Anncaliia (Brachiola, Nosema) connori spore in adrenal gland showing the coiled polar filament (arrows) and two nuclei. Note the polar tubules are in a single row. (Magnification, ×30,000; courtesy of reference 55 ). (Bottom) Transmission electron micrograph of a microsporidian spore with an extruded polar tubule inserted into a eukaryotic cell. The spore injects the infective sporoplasm through its polar tubule. (Courtesy of the CDC Public Health Image Library; Massimo Scaglia, Laboratory of Clinical Parasitology, Institute of Infectious Diseases, University-IRCCS San Matteo, Pavia, Italy.) doi:10.1128/9781555819002.ch23.f27

Spores in enterocytes. Note the position of the spores between the cell nucleus and the lumen of the intestine. Top and middle photographs taken with a 40× objective; bottom image taken with the 100× oil immersion objective. Note the dark-staining dot within the spores in the bottom photograph; this dot stains PAS positive. The tissue was stained with Giemsa stain. doi:10.1128/9781555819002.ch23.f28

Section of appendix showing Anncaliia connori spores in muscularis. The anterior end of the spore has a PAS-positive granule (arrows). (PAS, ×1,260; Armed Forces Institute of Pathology photograph.) doi:10.1128/9781555819002.ch23.f29

(Upper) Anncaliia connori spores in wall of ileum (90 min Grocott methenamine silver stain, ×1,300). (Lower) A. connori in myocardium (Ziehl-Neelsen stain, ×1,440). (Armed Forces Institute of Pathology photographs.) doi:10.1128/9781555819002.ch23.f30

Microsporidian spores seen in an eye infection. (Top) Hematoxylin and eosin stain. (Middle) PAS stain; note the dark staining dot at the end of each spore. (Bottom) Silver stain; note spores are outlined. doi:10.1128/9781555819002.ch23.f31

Spores in cytoplasm of intact jejunal enterocyte of a man with intractable diarrhea and malabsorption; transmission electron micrograph of a jejunal suction biopsy specimen. Note the dark oval developing spores; at higher magnification, some of the elements of the polar tubules would be visible. (Courtesy of Owen RL, in Farthing MJG, Keusch GI [ed], Enteric Infection: Mechanisms, Manifestations and Management, Chapman & Hall, Ltd., London, United Kingdom, 1987.) doi:10.1128/9781555819002.ch23.f32

Microsporidian spores. (Top) Spores stained with modified trichrome stain (Ryan blue); note the diagonal or horizontal “stripes” that are evidence of the polar tubule. (Middle) Calcofluor white staining of urine sediment; note the small oval intracellular and extracellular spores. This is a nonspecific stain but is more likely to represent a true positive than when seen in a stool specimen containing many fluorescing artifacts. (Bottom) Yeast artifacts; note the large size, lack of the polar tubule, and evidence of budding (arrow). doi:10.1128/9781555819002.ch23.f33

Microsporidian spores. This image shows a positive FA test using Encephalitozoon-specific reagent. Note the fluorescing spores, indicating the organisms are within the genus Encephalitozoon. doi:10.1128/9781555819002.ch23.f34

Sporangia containing sporangiospores (endospores) of Prototheca wickerhamii. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch23.f35

Sporangia containing developing sporangiospores (endospores) of Prototheca spp. (Courtesy of the CDC Public Health Image Library.) doi:10.1128/9781555819002.ch23.f36

Prototheca wickermanii. (Upper) Histopathological section of tissue sample obtained from the patient's skin and stained with PAS. Magnification, ×400. (Lower) Isolate on horse blood Columbia agar after incubation at 35°C for 96 h, showing smooth, creamy colonies (courtesy of reference 9 ). doi:10.1128/9781555819002.ch23.f37