Development of High-Throughput Methods to Quantify

Cysts of Toxoplasma gondii

D. Aldebert,1 * M. Hypolite,1 P. Cavailles,1 B. Touquet,1 P. Flori,1,2 C. Loeuillet,1

M. F. Cesbron-Delauw1

� AbstractToxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronicallyinfected animals and humans. Cysts can reactivate leading to severe pathologies.They also contribute to the transmission of Toxoplasma infection in humans byingestion of undercooked meat. Classically, the quantification of cyst burden in tis-sues uses microscopy methods, which are laborious and time consuming. Here, wehave developed automated protocols to quantify cysts, based on flow cytometry orhigh-throughput microscopy. Brains of rodents infected with cysts of Prugniaudstrain were incubated with the FITC-Dolichos biflorus lectin and analyzed by flowcytometry and high-throughput epifluorescence microscopy. The comparison ofcyst counts by manual epifluorescence microscopy to flow cytometry or to high-throughput epifluorescence microscopy revealed a good correlation (r 5 0.934, r 50.993, P \ 0.001 respectively). High-throughput epifluorescence microscopy wasfound to be more specific and sensitive than flow cytometry and easier to use forlarge series of samples. This reliable and easy protocol allow the specific detection ofToxoplasma cysts in brain, even at low concentrations; it could be a new way todetect them in water and in contaminate food. ' 2011 International Society for Advance-

ment of Cytometry

� Key termsToxoplasma; cyst; quantification; high-throughput; microscopy; flow cytometry

THE protozoan parasite Toxoplasma gondii is a worldwide parasite. Although its

definitive host is the cat, it can virtually infect all warm-blooded animals including

birds. It is the causative agent of toxoplasmosis, an usually minor and self-limiting

disease, which can lead to dramatic effects on fetuses or on immunocompromised

individuals. Toxoplasmosis is considered as a major cause of abortion in sheep and

goats (1). This anthropozoonosis is transmitted by ingestion of vegetables or water

contaminated with oocysts from infected-cat feces or by the consumption of raw or

uncooked meat containing cysts from infected animals (2). In intermediate hosts

such as sheep, goat, pig, rodent, and human, Toxoplasma cysts persist for life-time

in tissues establishing a chronic infection. In immuno-suppressed host, cyst reacti-

vation may be responsible for severe or fatal encephalitis and/or disseminated toxo-

plasmosis (3).

The detection of cysts in meat is important to develop prevention strategies (4).

Cysts quantification is also performed in experimental animal model of toxoplasmo-

sis to determine the existence of chronic infection, to compare the ability of the

Toxoplasma strains to produce cysts, or to evaluate drugs or vaccine efficacy on the

outcome of toxoplasmosis (5). Several serological tests are available to identify

Toxoplasma infection and to do dating (6). PCR is a sensitive method to detect

infection (7). These assays detect or quantify parasite burden but not specifically

cyst burden. The aim of this study was to develop reproducible high-throughput

1Laboratoire Adaptation et Pathogeniedes Micro-organismes, UMR 5163 CNRS-UJF Grenoble I, France2Pole de Biologie-Pathologie,Parasitology and Mycology Laboratory,University Hospital, Saint Etienne,France

Received 22 December 2010; RevisionReceived 11 July 2011; Accepted 9August 2011

Grant sponsors: ANR French agency(grant IGECONTOX MIME 2007) andCNRS.

*Correspondence to: Delphine Aldebert,Laboratoire Adaptation et Pathogeniedes Microorganismes, UMR 5163 CNRS-UJF, Institut J. Roget, BP 170, 38042Grenoble cedex 9, France

Email: delphine.aldebert@ujf-grenoble.fr

Published online 8 September 2011 inWiley Online Library(wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.21138

© 2011 International Society forAdvancement of Cytometry

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Cytometry Part A � 79A: 952�958, 2011

assays to detect and quantify cysts within tissues. These were

evaluated in comparison with standard laborious micro-

scopic observations.

MATERIALS AND METHODS

Animals

Experimental procedures were carried out in accordance

with European guidelines and approved by our local ethical

committee. Adult female outbred Swiss White mice were pur-

chased from Janvier laboratory (Le Genest-Saint-Isle, France).

Adult male Lewis, Fischer, Brown Norway, Osborne-Mendel,

Dark Agouti rats were purchased from Janvier laboratory (Le

Genest-Saint-Isle, France). They were maintained in our Spe-

cific-Pathogen-Free animal house facility.

Parasites and Infection

T. gondii Prugniaud-strain and T. gondii Prugniaud b-ga-lactosidase strain were used (8). Cyst stocks were maintained

in chronically infected mice which were sacrificed between 2

and 6 months post infection (pi). Following removal, brains

were gently homogenized with 2 ml phosphate-buffered saline

(PBS). Aliquots of this suspension were used for rat and

mouse oral infections. One month later, blood was collected

from retro-orbital sinus for the detection of anti-Toxoplasma

antibody response by immunofluorescence (9). Brains were

then recovered 2 months pi and homogenized in PBS as

described below. Brains of uninfected mice or rats were used

as controls.

Cyst Detection

Enzymatic detection. For the quantification of Prugniaud

b-galactosidase cysts, each individual mice brain was removed

and homogenized in 4 ml PBS. This suspension was fixed by

adding 4 ml of 10% formaldehyde solution and incubating at

room temperature for 20 min. After one wash in PBS, the pel-

let was suspended in 4 ml of PBS and frozen in liquid nitrogen

for 15 min then warmed at 378C to breakdown the cerebral

tissue. Cysts were labelled with b-galactosidase reagents (9).

Following one night of incubation at 378C, the suspension was

distributed into four wells of six-well culture plates and

scanned by light microscopy (magnification 3200) to count

the blue-stained cysts.

Fluorescent Staining

For the quantification of Prugniaud strain cysts, each rat

brain was removed and homogenized in 16 ml of PBS. Mice

brain was homogenized in 4 ml PBS. Brain suspension was

clarified by gentle incubation in proteinase K buffer (protein-

ase K 0.4 lg/ml, Tris pH8 10 mM, EDTA 1 mM, sodium

dodecyl sulphate 0.2%, sodium chloride 40 mM) for 15 min

at 568C. The reaction was stopped with PMSF 2 mM for 5

min at room temperature. Then, the suspension was washed

with PBS and resuspended with FITC-Dolichos biflorus agglu-

tinin (Vector laboratories, CA USA) 20 lg/ml for 30 min, at

room temperature. After one wash in PBS, rat or mice brains

were, respectively, resuspended in 6 ml or 4 ml of PBS and an-

alyzed by epifluorescence microscopy and flow cytometry.

Flow cytometry description. Data acquisition were per-

formed on a 4-colour FACSCalibur (BD Biosciences)

equipped with 488 nm argon laser and CellQuest Software.

The entire sample was analyzed. A threshold on FITC fluores-

cence was used. Staining cysts (FITC-dolichos biflorus) were

Figure 1. Correlation between two microscopic methods and flow

cytometry to quantify Toxoplasma cysts in brains of rodents.

A: Each brain homogenate from fourteen Swiss White mice

infected with the T. gondii Prugniaud b-galactosidase strain weredivided in two and each half was analyzed either by light micros-

copy after enzymatic substrate revelation (b galactosidase sub-strate) or by epifluorescence microscopy (FITC-Dolichos). B: Each

brain homogenate from Swiss White mice (n 5 14) infected with

the T. gondii Prugniaud b-galactosidase strain was divided intotwo halves and analyzed by light microscopy and flow cytometry

after FITC-Dolichos biflorus labelling. C: Brains from Lewis, Fi-

scher, Brown Norway, Osborne-Mendel, Dark Agouti rats were

treated with proteinase K and labelled with FITC-Dolichos. Plate

containing the samples was analyzed by manual and then auto-

matic epifluorescence microscopy.

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analyzed on a bivariate dot plot of FSC versus FITC (Fig. 2).

The logarithmic mode was used for each one.

High-throughput epifluorescent microscopy detection. The

imaging station supports the IX2 system from Olympus

microscope. The illumination system MT20 allows fast wave-

length switching and attenuation for a rapid image acquisition

using a sensitive digital camera (ORCA B&W firewire 1.4MP).

The station is equipped with a 43 objective and filter blocks

BP 460-495/DM505/BA510-550. Automated image acquisition

and data analysis were performed with ScanR software.

Statistical analysis. Spearman’s rank correlation test (r) was

done to determine the association between different techni-

ques, the online BIOSTATGV software was used, P\ 0.01 was

considered significant. As both the sensitivity and specificity

of a test vary with the threshold, the XLSTAT software (version

2010.3.05) was used to draw the ROC curve.

RESULTS

Toxoplasma cysts are usually detected by light microscopy.

However, it is difficult to detect small cysts among brain

aggregated components. The in situ enzymatic detection of b-galactosidase in Toxoplasma expressing the b-gal gene has beenused to improve cyst numeration (8,9). This published

method is, however, limited to b-gal transgenic parasites.

Here, we used the Dolichos biflorus lectin properties to bind

the N-acetylgalactosamine of the cyst wall (10) and to develop

two fluorescence methods to stain cysts from any Toxoplasma

strains. Before labelling brain cysts with the fluorescent Doli-

chos lectin agglutinin, a step of mild proteinase K digestion

was added for cerebral tissue clarification without cyst wall

destruction. Under these conditions, the lack of very low non-

specific labelling allowed easy detection of cysts. Cyst quantifi-

cation was performed on 14 brains of infected mice. Each

brain was homogenized, then, one half was used for enzymatic

labelling, the other half for fluorescent labelling. Comparison

of the two methods revealed a significant correlation between

cyst numeration by light and fluorescent microscopy (r 50.806, n 5 14, P\ 0.0017, thus validating our protocol of cyst

labelling with FITC Dolichos lectin (Fig. 1A).

Microscopic methods for cyst quantification are labori-

ous and the reproducibility is user-dependant. We, therefore,

developed an automated method to count cysts by flow cyto-

metry. The preparation of samples were similar to that used

for fluorescent microscopy except that samples diluted a fur-

ther fourfold in PBS before flow cytometry analysis to avoid

plugging the lines. Brains of noninfected mice were used to

determine analysis parameters. Brains from noninfected mice

determined the background (Fig. 2A). Cyst number was

obtained in the gate. (Fig. 2B)

Flow cytometry cyst count was compared with count

obtained by the epifluorescent microscopic method. The analysis

was performed on a total of 14 individual infected mice brains. A

high correlation was found (r 5 0.934, n 5 14, P\ 0.001) indi-

cating that flow cytometry can be used for cyst quantification

(Fig. 1B). We further assessed the repeatability and reproducibil-

ity with the brain of four and five infected mice, respectively. Ten

samples from each brain were analyzed for the repeatability assay.

The coefficients of variation ranged between 10.0 to 24.1% (Fig.

3A). To assess the reproducibility, each individual infected brain

was homogenized with PBS and divided into four samples which

were labelled and analyzed on 4 consecutive days. The coeffi-

cients of variation ranged between 8.0 and 35.7% (Fig. 3B).

Figure 2. Flow cytometry numeration of Toxoplasma cysts. Brain homogenates from Swiss White mice were labelled with FITC-Dolichos

biflorus agglutinin after proteinase K treatment. Samples were diluted fourfold in PBS and analyzed by flow cytometry. Threshold was

done on fluorescence. The region chosen to determine the cyst number was established on FITC fluorescence and forward scatter (FSC)

parameters. Samples from uninfected mice were used to establish the background (A). The cysts were counted in the positive gate (B).

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954 Automatic Assays to Count Toxoplasma Cysts

Finally, both the sensitivity and specificity of the flow

cytometric assay were determined in comparison with results

obtained using the b-galactosidase detection. Twenty four

individual brains were analyzed, i.e., 12 from infected and 12

from noninfected mice. The 12 brains from infected mice were

found positive by flow cytometry, and 11 brains from nonin-

fected mice were also detected positive. In the negative sam-

ples, flow cytometry revealed between 1 to 4 cysts. These

results gave a sensitivity of 100% and a specificity of 8%. As

both the sensitivity and specificity of a test vary with the

threshold, the XLSTAT software (version 2010.3.05) was used

to draw the ROC curve. A specificity of 90.9% and a sensitivity

of 100% were obtained if the brains contained more than 70

cysts. Hence, it appeared that flow cytometry is a good auto-

matic method to count cysts in brains with high cyst burden

([70 cysts).

To improve the lack of specificity of flow cytometry for

brains with low cyst burden, we next examined the perform-

ance of high-throughput microscopy. Brain samples from

inbred rat strains known to produce either very few Toxo-

plasma cysts or no cyst burden were analyzed (11). Rat brains

were labelled as previously described with FITC-Dolichos, and

the Olympus ScanR screening station was used for automated

image acquisition and data analysis of samples. To obtain

accurate autofocus for successful automated image

acquisition, Human Foreskin Fibroblasts stained with Hoechst

were plated onto six-well plates and overlaid with labelled

brain samples. The automated image acquisition was per-

formed at 340 magnification. The entire area of the wells was

observed and data analysis was performed using a flow cyto-

metric approach. All FITC events were detected (Figs. 4A and

4D), and cysts were selected on the basis of their area and cir-

cularity (Figs. 4B and 4E). A gate corresponding to cysts was

drawn and quantification was automatically realized for each

well. In contrast to flow cytometry, a gallery view of all images

of the gated data population can be created to allow a visual

control of data point (Figs. 4C and 4F). We could manually

exclude nonspecific events as images are linked reciprocally to

any data point. The performance of high-throughput micros-

copy was evaluated by comparing the cyst number with man-

ual fluorescent microscopy. Eleven rats were infected with the

Prugniaud Toxoplasma strain. The brain of each rat was ana-

lyzed successively with manual and then automatic micros-

copy. We detected between 2 and 413 cysts per brain, and a

high correlation was observed between automated and manual

assays (r 5 0.993, n 5 11, P \ 0.001) (Fig. 1C). We also

observed a good correlation for the five brains containing less

than 70 cysts per brain (r 5 0.903). Thirteen negative brains

were screened with both assays. All were negative using the

manual technique, and the few false positives seen with the

automated method could easily be eliminated through obser-

vation of the control gallery (Figs. 4C and 4F).

We next performed some retention tests to find out

whether the availability of the instruments or the screening of

a large series of samples could be a limitation. One half of

infected brain was immediately stained and analyzed, whereas

the other half was stored in liquid nitrogen with 90% fetal calf

serum and 10% dimethyl sulfoxide. These frozen samples were

labelled and analyzed 1 month later. The important lost of

cysts after freezing led us to try an alternative conservation

process (Fig. 5A). Brains were immediately labelled after

recovery. One half of the brain was analyzed immediately,

whereas the other half was stored at 48C for 1 or 2 weeks

before analysis. We also tested whether labelling could be per-

formed on brain samples after 1 month of storage at 48C (Fig.

5B). The labelling and the analysis were performed on these

samples. A high preservative performance was observed with

storage at 48C prior or after labelling.

DISCUSSION

Here, we have developed and validated a sensitive high-

throughput assay which is, to our knowledge, the first report

of an automated count of Toxoplama cysts from brain tissue.

The method is based on the Dolichos biflorus lectin property to

bind to N-acetylgalactosamine on the Toxoplasma cyst wall of

any Toxoplasma strain (10). A commercial FITC-Dolichos

biflorus lectin is available. The labelling may be done in one

Figure 3. Repeatability and reproducibility of flow cytometric

method. A: Repeatability: Each infected Swiss White mouse brain

(n 5 4) was separated into 10 samples which were labelled with

FITC-Dolichos and analyzed by flow cytometry. B: Reproducibility

: Each mouse brain (n 5 5) was separated into four samples,

labelled with FITC-Dolichos and analyzed by flow cytometry at 4

consecutive days. Each sample is represented by one dot. The

line represents the mean.

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Cytometry Part A � 79A: 952�958, 2011 955

step with low nonspecific labelling if appropriate digestion of

cerebral tissue is performed. We have found that mild protein-

ase K treatment is a critical step to obtain highly sensitive

labelling without loss of cysts.

The performance of flow cytometry was evaluated first,

as this equipment is currently available in many laborato-

ries. We demonstrated that flow cytometry analysis gave a

good correlation with the standard microscopic method.

Repeatability and reproducibility were also comparable to

microscopy. The variability of cyst distribution in the sample

required to analyse 60% of the sample (data not shown). We

also showed that flow cytometry is a powerful tool to count

cysts from brain containing more than 70 cysts. These results

were comparable to those obtained from studies of bacterial

counts in food samples, water, and environment. Those reports

showed that although flow cytometry allows a rapid detection

of microorganisms and avoids most of the problems encoun-

tered with culture, it cannot be applied to the enumeration of

rare events (12).

To quantify low cyst burden in brain, we have developed

an alternative method based on high-throughput microscopy,

which permits acquisition of thousands of images. Automa-

tion of all aspects of microscope control due to advances in

computer hardware and software, made the development of

this method possible. The high-throughput technology has

been already used to measure host cell attachment and inva-

sion by the human pathogen Toxoplasma gondii (13). Here, we

described for the first time an automatic microscopic assay to

enumerate Toxoplasma cyst from brain. An entire rat brain

could be analyzed, and during the scanning of the six-wells

plate, 1680 images could be acquired. The images were then

automatically analyzed with the software (Olympus, ScanR).

The cysts were automatically detected by their fluorescence,

area, and circularity. The possibility to obtain a gallery of

Figure 4. High-throughput method. Brains from Lewis, Fischer, Brown Norway, Osborne-Mendel, Dark Agouti rats were treated with pro-

teinase K and labelled with FITC-Dolichos. Plate containing the samples was scanned by automatic epifluorescence microcope. The fluo-

rescence events were detected (A, D) and cysts were identified with circularity factor and area (B, E). The number of events in the box was

automatically obtained. To verify that all events found in the box were cysts, a gallery corresponding to selected events could be obtained

(C, F). (A, C) a positive brain. (D, F) a negative brain. events corresponding to no cysts.

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956 Automatic Assays to Count Toxoplasma Cysts

selected events permitted to exclude nonspecific events. The

high-throughput microscopy gave a good correlation with

manual epifluorescence microscopy even for brains containing

rare events making it a highly specific and sensitive method.

Moreover, the manual microscopic method is time consuming,

laborious, and user-dependant. It can be replaced by high-

throughput microscopy that gives comparable results and can

easily be used on a large series of samples. In addition, the con-

servation test demonstrated that samples could be stored at 48Cfor several weeks before analysis.

In summary, we have developed two complementary

automated fluorescence methods to count Toxoplasma cysts in

brain samples. Flow cytometry, largely present in laboratories,

appeared to be limited to samples with high cyst number and

adequate size to be aspirated by the flow cytometry needle.

The high-throughput microscopy is also quantitative, and

more specific and sensitive than flow cytometry. These techni-

ques should be preferred to bioassay or PCR when determina-

tion of cyst number is critical. These assays could be a new

way to analyse Toxoplasma cysts from water or meat.

ACKNOWLEDGMENT

The text was re-read by Jean Gagnon and Barhie Bellete.

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