Application of Real-Time Fluorescent PCR for Quantitative Assessment of Neospora caninum Infections in Organotypic Slice Cultures of Rat Central Nervous System Tissue

Abstract

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

Neospora caninum is an important cyst-forming coccidian parasite with a high level of veterinary clinical relevance. Infection takes place either through oral uptake of oocysts or bradyzoite-containing tissue cysts or through transplacental passage of rapidly proliferating tachyzoites from the mother to the fetus. N. caninum is well known for causing congenital infections in cows which can lead to abortion and/or severe damage of the fetus. In addition, N. caninum infections cause neurological symptoms in dogs (1, 7).

Dissemination of the pathogen into many different tissues takes place due to the infection of, and proliferation within, cells of the reticuloendothelial system, such as macrophages and lymphocytes. However, the predilection site for primary parasite proliferation and for the establishment of the hypobiotic, bradyzoite-containing tissue cyst stage is the central nervous system (CNS) (3). Tachyzoites can rapidly multiply, and repeated processes of host cell invasion, proliferation, host cell lysis, and subsequent infection of neighboring cells, in combination with immunopathological events, produce significant necrotic lesions within affected tissues. As a consequence, severe neuromuscular disease occurs due to the destruction of neural 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, hypobiotic bradyzoite stage of the parasite, do not cause any host reaction, although formation of granulomas around degenerating tissue cysts or bradyzoites has been observed. Cyst rupture most likely occurs now and then and can cause foci of inflammation (3).

In the last few years, diagnosis of neosporosis was much improved by the development of PCR tests, which allow highly sensitive detection of the parasite through the amplification, and subsequent demonstration, of parasite-specific DNA sequences (reviewed in reference 4). One of the most commonly used diagnostic PCRs includes a set of primers which are targeted to the repetitive genomic sequence Nc5 (10, 11). In the present study, a quantitative assay, based on the Nc-5 PCR test, was developed. This assay relies on a dual fluorescent hybridization probe system and the real-time PCR LightCycler Instrument, which allows online detection of amplified DNA. We applied this quantitative PCR for measurement of N. caninum proliferation in organotypic rat brain slice cultures (13) which were infected with N. caninum tachyzoites, and these measurements were compared to the assessment of parasite infection intensities by immunohistochemistry.

MATERIALS AND METHODS

Parasites and infection of organotypic rat brain slice cultures.

Tachyzoites of the NcSweB1 isolate (12) were maintained by continuous passage in Vero cell cultures. They were separated from their host cells using PD-10 columns (Pharmacia) according to the method of Hemphill (6). Organotypic slice explants of rat brain cortex were prepared essentially as described by Stoppini et al. (13). The tissue samples corresponding to serial slices were allowed to recover from explantation trauma for 1 week before infection was initiated. For infection, slice cultures were overlaid with 10⁶ freshly isolated and purified NcSweB1 tachyzoites in 300 μl 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 identically without parasites. The infected slices were then further maintained at 37°C for 1 to 5 days prior to analysis.

Immunohistochemistry.

For immunohistochemical monitoring of parasite proliferation, tissue slices were fixed overnight in 5 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.2, at 4°C, placed into 18% sucrose in PBS for 24 h, and then cut at 10- to 20-μm intervals on a cryostat (Cryocut 1800; Leica Instruments, Nussloch, Germany) and mounted onto poly-l-lysine-coated slides. 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 were visualized by applying a polyclonal rabbit anti-N. caninum antiserum and a goat anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate (Sigma) as previously described (8). Specimens were subsequently stained with a monoclonal antibody directed against glial fibrillary acidic protein (Chemicon International Inc) and a goat anti-mouse immunoglobulin G conjugated to Texas red (Sigma). They were then embedded in a mixture of glycerol-gelvatol containing 1.4-diazobicyclo[2.2.2]octan (Merck) as an antifading reagent and were inspected on a Nikon Eclipse E800 digital confocal fluorescence microscope. Processing of images was performed using the Openlab 2.07 software (Improvision, Heidelberg, Germany).

Processing of DNA samples and LightCycler-based quantitative PCR.

DNA was extracted from entire brain slices by using the DNAeasy Kit (Qiagen, Basel, Switzerland) according to the standard protocol suitable for tissue samples. DNA was eluted in 100 μl of AE buffer (elution buffer from the kit) and subsequently boiled for 5 min. For quantitative PCR, forward primer Np21plus and reverse primer Np6plus were used. These primers had been designed to amplify a 337-bp sequence of the Nc5 region of N. caninum (11). Detection of DNA amplification products through fluorescence resonance energy transfer on the LightCycler Instrument (Roche Diagnostics, Basel, Switzerland) was achieved by hybridization 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 between the two probes. PCR amplification was performed with 1 μl of 1:5-diluted sample DNA (see also below) using the LightCycler DNA Master Hybridization Probes kit (Roche Diagnostics) in a standard reaction supplemented with MgCl₂ to a final concentration of 3 mM and containing a 0.5 μM concentration of each primer plus a 0.3 μM concentration of each probe. After denaturation of DNA for 30 s at 95°C, amplification was done in 50 cycles (5 cycles including denaturation [95°C, 1 s], annealing [63°C, 5 s], and extension [72°C, 20 s], plus 10 cycles 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, 20 s]; ramp rates in all cycle steps were 20°C/s) with 1 μl of 1:5-diluted DNA samples (see above). Fluorescence was measured at the end of each annealing phase in the “single” mode with the channel setting F2/1. Fluorescence signals from the amplification products were quantitatively assessed by applying the standard software (version 3.5.3) of the LightCycler Instrument. Quantitation of PCR products was achieved by plotting the fluorescence signals versus the cycle numbers at which the signals crossed the baseline (see Fig. 2A). Adjustment of the baseline was performed by using the “minimize error” mode. Positive samples were identified by a fluorescence signal which accumulated to values above the baseline within 50 cycles of reaction. As external standards, samples containing DNA equivalents from 100, 10, and 1 parasite were included. Linearity among the standard reactions was reflected by the correlation coefficient, which was calculated by computer program to be 1. Lack of PCR-inhibitory effects and overall comparability of the different standard and sample reactions were evidenced by demonstrating the quasi-identity of the slopes from the amplification plots (monitoring amplification rates) at the baseline crossing points (see Fig. 2A). Furthermore, reproducibility of the test system was demonstrated by proving an overall low variation within three independent runs of the standard reactions representing 100 (interassay coefficient of variation, 7.8%), 10 (13.3%), and 1 (17.2%) parasite, respectively.

FIG. 2.

LightCycler-PCR for quantitative assessment of N. caninum in organotypic rat brain tissue samples. (A) Typical example from three independent 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 at which the signals crossed the baseline (indicated as a horizontal line), and standards (dotted curves) were used for calculation of the parasite numbers within the samples. (B) Parasite growth kinetics, expressed as mean values plus standard deviations from three independent determinations. Values are given as parasite numbers detected in the various reactions (left scale). The extrapolated numbers of parasites corresponding to the entire section of brain slice are indicated on the right.

RESULTS AND DISCUSSION

Two sets of 10 serial rat brain slices were incubated in presence of 10⁶ N. caninum tachyzoites per set. Between days 1 and 5, postinoculation samples were investigated for infection intensities by examination of slices either through immunohistochemistry (Fig. 1) or quantitative PCR (Fig. 2). In addition, an uninfected control sample (representing day zero) was analyzed by each technique. Semiquantitative immunohistological evaluation revealed a progressive increase of the intracellular parasite numbers (Fig. 1). These results were largely confirmed by PCR on a quantitative level. For quantitative PCR-based determination of parasite numbers at the different time points postinoculation, corresponding data from the DNA amplification plots were compared with the standard plots representing DNA equivalents from approximately 100, 10, and 1 parasite(s) (Fig. 2A). By assessment of parasite numbers as means (plus standard deviations) from values determined in three independent PCR runs, infection intensities were revealed to continuously increase to a number of approximately 48,000 parasites per slice at day 5 postinoculation (Fig. 2B).

FIG. 1.

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

Taken together, the present results showed that inoculation of organotypic rat brain slice cultures with an appropriate number (approximately 10⁶) of N. caninum tachyzoites resulted in continuous parasite growth over a period of at least 5 days. The investigation revealed that the proliferation rate can be precisely monitored by using the highly sensitive Nc5-PCR (11) for quantitative detection of accumulating parasites. In contrast, immunofluorescence detection of parasites allows only a semiquantitative assessment of parasite proliferation.

The excellent operating characteristics make the quantitative PCR assay a versatile tool for studying, ex vivo and under experimentally controlled conditions, a large variety of biological parameters relevant during the cerebral phase of an N. caninum infection. Accordingly, PCR-based parasite quantitation of organotypic brain tissue samples may become an important experimental model for generation of novel information on those processes that cause neuronal pathogenicity in bovine and canine neosporosis. In addition, PCR-based quantitation of N. caninum can be applied to determine infection intensities in tissues and body fluids originating from both experimentally infected and naturally infected samples and thus is useful for epidemiological and clinical studies, as well as for research applications, such as the assessment of the efficacy of treatment and/or vaccination strategies to be developed in the future.