International Journal for ParaSitology VoL 22, No. 3,pp. 385-389, 1992 Printed in Great Britain

0020-7519/92 $5.00 + 0.00 Pergamon Press Ltd 9 1992 Australian Society for Parasitology

RESEARCH NOTE CR Y P T O S P O R I D I U M PAR V U M SPOROZOITE STAINING BY PROPIDIUM IODIDE GREGOIRE COZON,* DOMINIQUE CANNELLA,* FRAN(~OIS BIRON,~" MARIE-ANTOINETtE PIENS,~ MICHI~LE JEANNIN* a n d JEAN- PIERRE REVlLLARD* * INSERM ua0 CNRS URA 1177 UCBL, H6pital E. Herriot, Pavillon P, place d'Arsonval, 69437 Lyon Cedex 03, France i" Service des maladies infectieuses, H6pital de la Croix-Rousse, 93 grande rue de la Croix-Rousse, 69317 Lyon Cedex 04, France ~:Department de Parasitologie, Facult6 de Medecine, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France

(Received 18 November 1991; accepted 14 February 1992) Abstract---Cozos G., CANNELLAD., BIRON F., PIENS M.'A., JEANNINM. and REVILLARDJ.-P. 1992. Cryptosporidium parvum sporozoite staining by propidium iodide. International Journal for Parasitology 22: 385-389. Modified Ziehl-Neelsen (ZN) acid-fast stain is the usual method for detection of Cryptosporidium oocysts in feces. Propidium iodide permitted us to stain free or intra-oocyst sporozoites. With the ZN method only 3-5% of the oocysts purified from three human and one experimentally infected lamb dichromate-preserved feces were stained by carbol fuchsin. These fuchsin-stained oocysts were free of intact sporozoites as identified by propidium iodide staining. Treatment with 10% formalin or 0.5% sodium hypochlofite increased the percentage of acid-fast stained oocysts and thus the sensitivity of acid-fast staining. Treatment with sodium hypochlorite induced intra-oocyst sporozoite alterations as demonstrated by flow cytometric analysis of the oocysts" DNA content. Propidium iodide staining of fixed oocysts is a simple and rapid method to visualize sporozoites and to assess oocyst preservation after different treatments. INDEX KEY WORDS: Cr)'ptosporidium; acid-fast staining; propidium iodide.

Cr)Ttosporiditml parvton is an opportunistic coccidia n parasite of mammals causing gastrointestinal disease (Current, Reese, Ernst, Bailey, H e y m a n & Weinstein, 1983). Oocysts, the environmentally resistant form of the parasite, are excreted in host feces where they are identified by the modified ZiehlNeelsen acid-fast stain, the most commonly used staining procedure (Tzipofi, 1988). In this study, we compared acid-fast and immunofluorescence staining by fluorescein isothiocyanate-conjugated anti-Cr)7~tosporidium monoclonal antibodies (FITC-Mab) with propidium iodide staining of sporozoite D N A in order to identify different populations of oocysts. The data demonstrate that most oocysts of dichromate-treated stools were not acid-fast stained and that stained oocysts did not contain typical and viable sporozoites. Furthermore sodium hypochlorite treatment induced sporozoite alterations demonstrated by flow cytometric analysis while increasing the percentage of acid-fast stained oocysts. Cryptosporidium parvum oocysts were obtained from diarrheal samples of three patients with AIDS known to have cryptosporidiosis and from one

experimentally infected lamb. Feces were stored in 2.5% potassium dichromate (K2Cr2OT) at 4~ from 0.5 to 3--4 months. Oocysts were purified from feces immediately before use by the dichromate sucrose flotation procedure (Current et al., 1983). Briefly, feces were extracted with ether and centrifuged over. a sucrose gradient. The upper layer containing oocysts was washed twice in distilled water. An aliquot of the purified oocyst suspension was airdried on a slide and stained by the modified ZiehlNeelsen acid-fast stain (Henriksen & Pohlenz, 1981). Then the specific anti-Co'ptosporidimn FITC-Mab (Pasteur Diagnostic-Paris) was added. The slide w'as incubated in a humidified chamber for 30 rain at 4~ then rinsed with tap-water and dried. Propidium iodide (Sigma, La Verpilli~re, France) was applied for 30 s (100 p g m l - ' in PBS). Slides were rinsed with tap-water, dried and glycerol-PBS cover-slip mounted and examined by light microscopy and by fluorescent microscopy. Microphotographs were obtained after each step of this staining procedure. For flow cytometric analysis purified oocysts were fixed in 70% ethanol overnight at 4"C. The pellet of centrifugation

385

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386 TABLE l--D IS TRI BUTI ON OF I 0 0 0

CryptosporidiumOOCYSTS (IDENTIFIED BY F I T C - M A B )

FOUR FECAL SAMPLES, ACCORDING TO TttEIR STAINING BY PROPIDIUM IODIDE

Fuchsin-positive oocysts Altered sporozoites

Intact sporozoites

PURIFIED FROM

(PI) AND CARBOL FUCIISIN

Fuchsin-negative oocysts PI-

PI+

a

Human feces 1 Human feces 2 tluman feces 3 Lamb feces b Human feces 1 Formalin 15 min Formalin 2 h Na hypochlorite 15 min Na hypochlorite 2 h*

37 30 47 50

2 2 I 1

79 46 118 147

882 922 834 802

37 28 133 236

2 1 0 0

79 201 106 52

882 770 764 7i2

762

160

76

2

a, Staining procedure on purified oocysts from three different human feces samples and from one experimentally infected lamb feces. b, Staining procedure on purified oocysts from one human feces sample after treatment with 10% buffered formalin or with 0.5% sodium hypochlorite. * All oocysts were more or less intensively acid-fast stained and appeared collapsed. The propidium iodide fluorescence is heterogeneous and only the highly stained oocysts are reported here as propidium iodide-positive oocysts. (10 min at 400 g) was incubated in a PBS RNAse A III solution (250 k.u. ml -I) (Sigma) for 5 min. After washing in PBS, oocysts were incubated for 30 s in a propidium iodide solution (100 Itg ml -t in PBS), rinsed immediately with PBS and then the FITCMab was added. The suspension was analyzed in a FACScan cytometer (Becton Dickinson, Mountain View, CA). All the oocysts were stained by the specific anti-C. p a r v u m FITC-Mab which did not show cross-reaction with 38 other microorganisms tested by the manufacturer. In the four dichromate-preserved feces samples studied, only 3-5% of the specific Mabstained oocysts were carbol fuchsin-stained (Table I). Staining with propidium iodide visualized intraoocyst sporozoite D N A as well as the D N A of the free sporozoites obtained after the excystation procedure (incubation of purified oocysts in a 0.25% trypsin, 0.75% sodium taurocholate in PBS solution, 60 min in a 37~ water bath) (Fig. 1, upper panel). In dichromate-preserved stools carbol fuchsin-negative unexcysted oocysts could be divided into two populations as regards their staining pattern with propidium iodide (Fig. 1, lower panel). One population contained sporozoites (80-92% of carbol fuchsinnegative ooeysts) whereas the second population was free of sporozoites (5-15% o f carbol fuchsin-negative oocysts) (Table 1) corresponding to the

excysted oocysts. The first population was more refringent in light microscopy than the second. In fuchsin-positive oocysts propidium iodide staining was either undetectable or pale and homogeneous (Fig. I, lower panel) and no intact sporozoite could be visualized (Table 1). Incubation of purified oocysts in 10% (v/v) buffered formalin increased the percentage of oocysts stained by fuchsin from 4 to 13% and decreased accordingly the number of oocysts containing typical sporozoites. Incubation in 0.5% sodium hypochlorite for 1 5 m i n or-2 h increased the percentage of fuchsin-sthined oocysts to 24 and 92%, respectively (Table 1). Carbol fuchsin staining was often pale and atypical after the long incubation in sodium hypochlorite. Flow cytometric analysis showed that incubation of oocysts with sodium hypochlorite resulted in a shrinkage as evidenced by a decrease of light scattering at forward angles (Fig. 2bI) along with a decrease in propidium iodide fluorescence intensity (Fig. 2blI), indicating the presence of empty oocysts (propidium iodidenegative) with reduced D N A content and a small number o f intact oocysts. Two hours of 0.5% sodium hypochlorite treatment inhibited excystation ability of a dichromate-preserved oocyst suspension (data not shown). Preservation o f stools with 10% formalin rather than with dichromate was reported to increase the

Research Note

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FZG. 1. Purified Cryptosporidium oocysts after staining with the modified acid-fast technique followed by the propidium iodide and the anti-Cryptosporidi,ml FITC-Mab staining. Upper panel: excysted oocysts. Free sporozoites are stained with propidium iodide and empty oocyst walls are stained only by the FITC-Mab. Lower panel: unexcysted oocysts. (a) Oocysts stained by the modified acid-fast procedure. (b) The same oocysts visualized by immunofluorescence staining with propidium iodide and with the FITC-Mab conjugate. Note the acid-fast stained oocysts with a pale and homogenous fluorescence (arrows) (bar = 10/~m). sensitivity of the Ziehl-Neelson acid-fast stain for Cryptosporidium oocyst detection (Garcia, Bruckner, Brewer & Shimizu, 1983). In the present study with dichromate-preserved stools no more than 3-5% of oocysts, as identified by indirect immunoftuorescence with specific Mab, were stained by carbol fuchsin. Formalin fixation (2 h) or a short exposure to 0.5% sodium hypochlorite, known to be non-toxic against ooeysts (Campbell, Tzipori, Hutchinson & Angus,

1982), increased the number of fuchsin-positive oocysts which, however, remained below 24%. Thus for clinical diagnostic applications, acid-fast staining although more simple and less expensive would appear to grossly underestimate the number of Cryptosporidium oocysts depending on the specimens, and the indirect immunofluorescence method remains more accurate and more sensitive (Rusnak, Hadfield, Rhodes & Gaines, 1989).

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F,G. 2. Flow cytometry analysis of Cryptosporidium oocysts stained with propidium iodide. (a) Oocysts incubated in PBS. (b) Oocysts incubated for 2 h in 0.5% sodium 9hypochlorite. (c) Oocysts incubated for 2 h in 10% formalin. Histograms displayed the forward angle light scatter parameter (I) of FITC-Mab positive-gated oocysts and the logarithmic intensity of propidium iodide fluorescence (II) among anti-Cryptosporidium FITC-Mab positive-gated oocysts.

Staining o f nucleic acids with propidium iodide permitted an unequivocal identification o f both free sporozoites after excystation and sporozoites contained within methanol-fixed oocysts. Most of the dichromate-preserved oocysts contained intact sporozoites. Furthermore we report that, besides typical empty oocyst walls stained by the monoclonal antibody, many oocysts were found to contain altered sporozoites after sodium hypochlorite treatment as shown by a pale and homogenized fluorescence under the microscope and a decrease and heterogeneous distribution of D N A content with the flow cytometric analysis. Furthermore, excystation inability after sodium hypochlorite treatment leads us to think that these altered forms are quite unlikely to contain infectious viable sporozoites. The great variability o f altered form incidence in different specimens may account at least in part for the wide variation o f oocyst numbers required to induce experimental infection in the neonatal mouse (Arrowood, Mead, M a h r t & Sterling, 1989; Ungar, Burris, Queen & Finkelman, 1990). In conclusion the propidium iodide staining is a simple and rapid method to visualize free sporozoites or the sporozoites contained in acid-fast stained oocysts. It allows the identification o f empty oocysts

and permits a rapid assessment o f oocyst preservation after long-term storage.

Acknowledgenlents--The authors thank M. Naciri (INRA, Tours, France) for providing the dichromate-preserved feces from an experimentally infected lamb. This work was supported in part by grants from: Hospices civils de Lyon; Universit6 Claude Bernard, Facult6 Lyon Nord. D. Cannella is supported by Agence Nationale de Recherches sur le SIDA. REFERENCES ARROWOODM. J., MEADJ. R., MAHRTJ. L. & STERUNGC. R. 1989. Effects of immune colostrum and orally administered antisporozoite monoclonal antibodies on the outcome of Cryptosporidium parvum infections in neonatal mice. htfection and Immunity 57: 2283-2288. CAMPBELL I., TZ,POR[ S., HUTClUNSONG. & ANGUS K. V. 1982. Effect of disinfectants on survival of Cr)Tto-

sporidium oocysts. The Veterinary Record 111:414-415. CURRENTW. L., REESEN. C., Egr~sr J. V., BARLEYW. S., HEYMAN M. B. & WEINSTE,r~W. M. 1983. ttuman cryptosporidiosis in immunocompetent and immunodeficient persons. The New EnglandJournal of Medicine 308: i 252-1257. GARCIAL. S., BRUCKNERD. A., BREWERT. C. & SHIMIZUR. Y. 1983. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. Journal of ClinicalMicrobiology 18: 185-190.

Research Note HENRIKSEN S. A. & POHLENZ J. F. L. 1981. Staining of cryptosporidia by a modified Ziehl-Neelson technique. Acta Veterinaria Scandinavia 22: 594-596. RUSNAKJ., HADFIELDT. L., RHODESM. M. & GAINESJ. K. 1989. Detection of Cryptosporidium oocysts in human fecal specimens by an indirect immunofluorescence assay with monoclonal antibodies. Journal of Clinical Micro-

389

biology 27:1135-1136. TZIPORI S. 1988. Cryptosporidiosis in perspective. Advances in Parasitology 27:63-129. Ur~GARB. L. P., BURRXSJ. A., QUEENC. A. & FINKELMANF. D. 1990. New mouse models for chronic Cryptosporidium infection in immunodeficient hosts. Infection and Immunity 58: 961-969.