JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/02/$04.00�0 DOI: 10.1128/JCM.40.1.252–255.2002

Jan. 2002, p. 252–255 Vol. 40, No. 1

Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Application of Real-Time Fluorescent PCR for QuantitativeAssessment of Neospora caninum Infections in Organotypic

Slice Cultures of Rat Central Nervous System TissueNorbert Muller,1* Nathalie Vonlaufen,1 Christian Gianinazzi,2 Stephen L. Leib,2

and Andrew Hemphill1*Institute of Parasitology, University of Berne, Langgass-Strasse 122, 3012 Berne,1 and Institute for Infectious Diseases,

University of Berne, Friedbuhlstrasse 51, 3010 Berne,2 Switzerland

Received 14 May 2001/Accepted 9 September 2001

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used todevelop a quantitative PCR assay which allows the determination of infection intensities within differentexperimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescenthybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assaywas successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures ofrat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasitequantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploringdifferent aspects of cerebral N. caninum infection.

Neospora caninum is an important cyst-forming coccidianparasite with a high level of veterinary clinical relevance. In-fection takes place either through oral uptake of oocysts orbradyzoite-containing tissue cysts or through transplacentalpassage of rapidly proliferating tachyzoites from the mother tothe fetus. N. caninum is well known for causing congenitalinfections in cows which can lead to abortion and/or severedamage of the fetus. In addition, N. caninum infections causeneurological symptoms in dogs (1, 7).

Dissemination of the pathogen into many different tissuestakes place due to the infection of, and proliferation within,cells of the reticuloendothelial system, such as macrophagesand lymphocytes. However, the predilection site for primaryparasite proliferation and for the establishment of the hypobi-otic, bradyzoite-containing tissue cyst stage is the central ner-vous system (CNS) (3). Tachyzoites can rapidly multiply, andrepeated processes of host cell invasion, proliferation, host celllysis, and subsequent infection of neighboring cells, in combi-nation with immunopathological events, produce significantnecrotic lesions within affected tissues. As a consequence, se-vere neuromuscular disease occurs due to the destruction ofneural cells in the brain and within cranial and spinal nerves,affecting the conductivity of the neural tissue (2, 9). In contrast,N. caninum tissue cysts, containing the slowly dividing, hypo-biotic bradyzoite stage of the parasite, do not cause any hostreaction, although formation of granulomas around degener-ating tissue cysts or bradyzoites has been observed. Cyst rup-ture most likely occurs now and then and can cause foci ofinflammation (3).

In the last few years, diagnosis of neosporosis was much

improved by the development of PCR tests, which allow highlysensitive detection of the parasite through the amplification,and subsequent demonstration, of parasite-specific DNA se-quences (reviewed in reference 4). One of the most commonlyused diagnostic PCRs includes a set of primers which aretargeted to the repetitive genomic sequence Nc5 (10, 11). Inthe present study, a quantitative assay, based on the Nc-5 PCRtest, was developed. This assay relies on a dual fluorescenthybridization probe system and the real-time PCR LightCyclerInstrument, which allows online detection of amplified DNA.We applied this quantitative PCR for measurement of N. cani-num proliferation in organotypic rat brain slice cultures (13)which were infected with N. caninum tachyzoites, and thesemeasurements were compared to the assessment of parasiteinfection intensities by immunohistochemistry.

MATERIALS AND METHODS

Parasites and infection of organotypic rat brain slice cultures. Tachyzoites ofthe NcSweB1 isolate (12) were maintained by continuous passage in Vero cellcultures. They were separated from their host cells using PD-10 columns (Phar-macia) according to the method of Hemphill (6). Organotypic slice explants ofrat brain cortex were prepared essentially as described by Stoppini et al. (13).The tissue samples corresponding to serial slices were allowed to recover fromexplantation trauma for 1 week before infection was initiated. For infection, slicecultures were overlaid with 106 freshly isolated and purified NcSweB1 tachyzoitesin 300 �l of RPMI 1640 culture medium without serum for 1 h at 37°C, 5% CO2,followed by two washes in RPMI 1640. Control cultures were treated identicallywithout parasites. The infected slices were then further maintained at 37°C for 1to 5 days prior to analysis.

Immunohistochemistry. For immunohistochemical monitoring of parasiteproliferation, tissue slices were fixed overnight in 5 ml of 4% paraformaldehydein phosphate-buffered saline (PBS), pH 7.2, at 4°C, placed into 18% sucrose inPBS for 24 h, and then cut at 10- to 20-�m intervals on a cryostat (Cryocut 1800;Leica Instruments, Nussloch, Germany) and mounted onto poly-L-lysine-coatedslides. Unspecific binding sites were blocked by incubation of slices in PBS–3%bovine serum albumin–50 mM glycine, pH 7.2, for 2 h at 24°C. Tachyzoites werevisualized by applying a polyclonal rabbit anti-N. caninum antiserum and a goatanti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate (Sigma)as previously described (8). Specimens were subsequently stained with a mono-clonal antibody directed against glial fibrillary acidic protein (Chemicon Inter-

* Corresponding author. Mailing address: Institute of Parasitology,Langgass-Str. 122, P.O. Box 8466, CH-3001 Berne, Switzerland.Phone: 41 31 631 2474. Fax: 41 31 631 2622. E-mail for Norbert Muller:nmueller@ipa.unibe.ch. E-mail for Andrew Hemphill: hemphill@ipa.unibe.ch.

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national Inc) and a goat anti-mouse immunoglobulin G conjugated to Texas red(Sigma). They were then embedded in a mixture of glycerol-gelvatol containing1.4-diazobicyclo[2.2.2]octan (Merck) as an antifading reagent and were inspectedon a Nikon Eclipse E800 digital confocal fluorescence microscope. Processing ofimages was performed using the Openlab 2.07 software (Improvision, Heidel-berg, Germany).

Processing of DNA samples and LightCycler-based quantitative PCR. DNAwas extracted from entire brain slices by using the DNAeasy Kit (Qiagen, Basel,Switzerland) according to the standard protocol suitable for tissue samples. DNAwas eluted in 100 �l of AE buffer (elution buffer from the kit) and subsequentlyboiled for 5 min. For quantitative PCR, forward primer Np21plus and reverseprimer Np6plus were used. These primers had been designed to amplify a 337-bpsequence of the Nc5 region of N. caninum (11). Detection of DNA amplificationproducts through fluorescence resonance energy transfer on the LightCyclerInstrument (Roche Diagnostics, Basel, Switzerland) was achieved by hybridiza-tion of Nc5-specific 5�-LC-Red 640-labeled Np 5LC (5�-TCCCTCGGTTCACCCGTTCACACAC-3�) detection probe and 3�-fluorescein-labeled Np 3FL (5�-CACGTATCCCACCTCTCACCGCTACCA-3�) anchor probe (TIB MOLBIOL,Berlin, Germany). The resonance energy transfer was over a 3-base gap betweenthe two probes. PCR amplification was performed with 1 �l of 1:5-diluted sampleDNA (see also below) using the LightCycler DNA Master Hybridization Probeskit (Roche Diagnostics) in a standard reaction supplemented with MgCl2 to afinal concentration of 3 mM and containing a 0.5 �M concentration of eachprimer plus a 0.3 �M concentration of each probe. After denaturation of DNAfor 30 s at 95°C, amplification was done in 50 cycles (5 cycles including dena-turation [95°C, 1 s], annealing [63°C, 5 s], and extension [72°C, 20 s], plus 10cycles including denaturation [95°C, 1 s], “touch-down” annealing [63 to 53°C;temperature reduction, 1°C per cycle], 5 s; extension [72°C, 20 s], plus 35 cycles

including denaturation [95°C, 1 s], annealing [53°C, 5 s], and extension [72°C, 20s]; ramp rates in all cycle steps were 20°C/s) with 1 �l of 1:5-diluted DNA samples(see above). Fluorescence was measured at the end of each annealing phase inthe “single” mode with the channel setting F2/1. Fluorescence signals from theamplification products were quantitatively assessed by applying the standardsoftware (version 3.5.3) of the LightCycler Instrument. Quantitation of PCRproducts was achieved by plotting the fluorescence signals versus the cycle num-bers at which the signals crossed the baseline (see Fig. 2A). Adjustment of thebaseline was performed by using the “minimize error” mode. Positive sampleswere identified by a fluorescence signal which accumulated to values above thebaseline within 50 cycles of reaction. As external standards, samples containingDNA equivalents from 100, 10, and 1 parasite were included. Linearity amongthe standard reactions was reflected by the correlation coefficient, which wascalculated by computer program to be 1. Lack of PCR-inhibitory effects andoverall comparability of the different standard and sample reactions were evi-denced by demonstrating the quasi-identity of the slopes from the amplificationplots (monitoring amplification rates) at the baseline crossing points (see Fig.2A). Furthermore, reproducibility of the test system was demonstrated by prov-ing an overall low variation within three independent runs of the standardreactions representing 100 (interassay coefficient of variation, 7.8%), 10 (13.3%),and 1 (17.2%) parasite, respectively.

RESULTS AND DISCUSSION

Two sets of 10 serial rat brain slices were incubated inpresence of 106 N. caninum tachyzoites per set. Between days1 and 5, postinoculation samples were investigated for infec-

FIG. 1. Monitoring of parasite proliferation following infection of organotypic cultures with 106 N. caninum tachyzoites (days 1 to 5). Parasiteswere detected by immunolabeling using a polyclonal anti-N. caninum antiserum followed by detection with a fluorescein isothiocyanate-conjugatedanti-rabbit antibody. The brain tissue was counterstained employing a monoclonal antibody directed against glial fibrillary acidic protein followedby staining with an anti-mouse–Texas red conjugate.

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tion intensities by examination of slices either through immu-nohistochemistry (Fig. 1) or quantitative PCR (Fig. 2). In ad-dition, an uninfected control sample (representing day zero)was analyzed by each technique. Semiquantitative immunohis-tological evaluation revealed a progressive increase of the in-tracellular parasite numbers (Fig. 1). These results were largelyconfirmed by PCR on a quantitative level. For quantitativePCR-based determination of parasite numbers at the differenttime points postinoculation, corresponding data from theDNA amplification plots were compared with the standardplots representing DNA equivalents from approximately 100,10, and 1 parasite(s) (Fig. 2A). By assessment of parasitenumbers as means (plus standard deviations) from values de-termined in three independent PCR runs, infection intensitieswere revealed to continuously increase to a number of approx-

imately 48,000 parasites per slice at day 5 postinoculation (Fig.2B).

Taken together, the present results showed that inoculationof organotypic rat brain slice cultures with an appropriatenumber (approximately 106) of N. caninum tachyzoites re-sulted in continuous parasite growth over a period of at least 5days. The investigation revealed that the proliferation rate canbe precisely monitored by using the highly sensitive Nc5-PCR(11) for quantitative detection of accumulating parasites. Incontrast, immunofluorescence detection of parasites allowsonly a semiquantitative assessment of parasite proliferation.

The excellent operating characteristics make the quantita-tive PCR assay a versatile tool for studying, ex vivo and underexperimentally controlled conditions, a large variety of biolog-ical parameters relevant during the cerebral phase of an N.

FIG. 2. LightCycler-PCR for quantitative assessment of N. caninum in organotypic rat brain tissue samples. (A) Typical example from threeindependent PCR runs, including amplification plots representing standard reactions (dotted lines) for 100 (left), 10 (middle), and 1 parasite (right)or reactions representing samples taken at days 1 (open circles), 2 (closed circles), 3 (open squares), 4 (closed squares), and 5 (crosses)postinoculation with 106 parasites. Quantitation of PCR products was achieved by plotting the fluorescence signals versus the cycle numbers atwhich the signals crossed the baseline (indicated as a horizontal line), and standards (dotted curves) were used for calculation of the parasitenumbers within the samples. (B) Parasite growth kinetics, expressed as mean values plus standard deviations from three independent determi-nations. Values are given as parasite numbers detected in the various reactions (left scale). The extrapolated numbers of parasites correspondingto the entire section of brain slice are indicated on the right.

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caninum infection. Accordingly, PCR-based parasite quantita-tion of organotypic brain tissue samples may become an im-portant experimental model for generation of novel informa-tion on those processes that cause neuronal pathogenicity inbovine and canine neosporosis. In addition, PCR-based quan-titation of N. caninum can be applied to determine infectionintensities in tissues and body fluids originating from bothexperimentally infected and naturally infected samples andthus is useful for epidemiological and clinical studies, as well asfor research applications, such as the assessment of the efficacyof treatment and/or vaccination strategies to be developed inthe future.

ACKNOWLEDGMENTS

We thankfully acknowledge the expert technical assistance ofFranziska Simon (Institute for Infectious Diseases). We also thankBruno Gottstein, Heinz Sager (Institute of Parasitology), and MartinTauber (Institute for Infectous Diseases) for their support.

This study was financed through grants of the Swiss National ScienceFoundation (no. 32-56486.99 and 32-61654), the National Institutes ofHealth (NS-35902), and the Foundation Research 3R.

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