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Journal of Clinical Microbiology, June 2001, p. 2065-2071, Vol. 39, No. 6
Immunology and Disease Resistance
Laboratory,1 Animal Waste Pathogens
Laboratory,2 and Parasite Biology,
Epidemiology and Systematics Laboratory,3 ANRI,
ARS, U.S. Department of Agriculture, Beltsville, Maryland 20705
Received 21 November 2000/Returned for modification 23 January
2001/Accepted 9 March 2001
A highly sensitive and specific method has been developed to
reproducibly detect and quantitate Toxoplasma gondii burden
in animal tissue samples using T. gondii ITS1-derived
primers and a fluorogenic probe via real-time PCR. Assay specificity
was confirmed against a panel of DNA samples from T. gondii
and other common protozoa as well as host animal tissue. This Toxo
TaqMan assay was able to detect as little as 0.1 pg of T. gondii genomic DNA, which is equivalent to 1 T. gondii bradyzoite, and has a dynamic range of detection of from
100 ng to 100 fg of T. gondii DNA. Tissues from
experimentally infected mice and pigs as well as bradyzoite-spiked pig
muscle samples were used to test and standardize this technique.
Positive signals were obtained with T. gondii parasite
concentrations ranging from 4 to 3.7 × 105 parasites
per g of spiked pig tissue, with excellent linearity (R2 = 0.9776). All T. gondii-infected animals were correctly identified by this
technique. Results indicate that this assay is applicable to swine
carcasses and commercial pig products, is compatible with automation
technology for potential slaughterhouse use, and will enable scientists
to diagnose and quantitate T. gondii in animal tissues.
Toxoplasmosis caused by
Toxoplasma gondii is one of the most prevalent parasitic
diseases in human beings and agricultural animals. Mead et al.
(27) have estimated that approximately 225,000 new cases
are reported each year in the United States and assume that 50% of the
cases are due to food-borne transmission of T. gondii. The
National Hospital Discharge survey indicated that toxoplasmosis was the
first diagnosis for approximately 5,000 discharges each year between
1992 and 1996, including 750 deceased patients. Furthermore, 4,000 persons with AIDS will develop toxoplasmic encephalitis in the United
States each year (27). From an economic point of view,
T. gondii infection has a negative impact on society, as
measured through the increase in the costs of chemotherapy for AIDS
patients (13), serological screening for pregnant women, patient care, loss of productivity, and treatment of infected mothers
and children (24). T. gondii can be transmitted
to humans by ingestion of T. gondii oocysts in food or water
or by consumption of tissue cysts in raw or undercooked meat. Infected
pork is considered the most important meat source of T. gondii in the United States (4), and infected lamb is
a major source worldwide (19). Considering the potentially
serious consequences of congenital T. gondii infection in
humans, such as birth defects, retinitis, brain damage, and even death,
it is essential that efforts be directed at preventing food-borne
transmission of T. gondii.
The detection of T. gondii tissue cysts in naturally and
experimentally infected pigs and sheep has been reported for years (8, 19), though the prevalence of T. gondii
infection in U.S. pig populations has been dramatically reduced as
producers modify their management practices (31). However,
the true T. gondii burden in any food product has been
difficult to measure. To date, the most reliable method of inspecting
food for T. gondii has been to demonstrate the presence of
T. gondii tissue cysts by in vivo biological assays
(12, 15). Because these methods are costly and
time-consuming, they are not suitable for slaughterhouse testing or
monitoring of commercial meat products (15).
There have been many reports of molecular detection assays for
toxoplasmosis, but most have been developed for human diagnostics (2, 9, 21) or phylogenetic studies (1, 10,
22). In veterinary medicine, tests for detection of T. gondii by PCR were reported mostly for companion animals
(25, 29) and sheep (11, 28), though
Warnekulasuriya et al. (30) reported using PCR to identify
T. gondii in cured meat products. Several studies have been
directed at quantitating the actual T. gondii burden in
biological fluids or tissues, but these involved time-consuming PCR
protocols (competitive PCR) followed by agarose gel image analysis
(17, 23, 26). Costa et al. (2) addressed the quantitation issue with a real-time PCR analysis of T. gondii in human serum samples in stem cell-transplanted patients;
however, this assay was based on the T. gondii B1 gene and
not the more abundant rRNA gene.
Herein, we describe a highly sensitive and specific method, the Toxo
TaqMan assay, to reproducibly detect and/or quantitate T. gondii burden in animal tissue samples by using real-time PCR. Tissues from experimentally T. gondii-infected mice and pigs
as well as bradyzoite-spiked pig muscle samples were used to test and
standardize this technique.
Sequence analysis.
Alignment of the ITS1 region of the 18S
rRNA gene sequences from T. gondii (GenBank accession no.
X75429.1, RH strain) and other coccidian parasites such as
Neospora caninum (GenBank accession no. AF038861, NC-1
isolate), Hammondia hammondi (GenBank accession no.
AF096499.1), and Hammondia heydorni (GenBank accession no.
AF076858.1) was performed using the Genetics Computer Group (GCG)
sequence analysis software package (Wisconsin Package, version 10.0).
This region was selected because it has been reported to have a high
rate of nucleotide substitutions (1). Primer and probe
sequences for detection of T. gondii and for normalization
with housekeeping genes are shown in Table 1.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2065-2071.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Development of a Real-Time PCR Assay for Detection
of Toxoplasma gondii in Pig and Mouse Tissues
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Sequences of primers and probes used in this
studya
TaqMan assay. The TaqMan assay for T. gondii (Toxo TaqMan) uses a specially designed probe and primers (Table 1) and directly relates the amount of initial target gene to the reporter's fluorescence emission (16). The Toxo TaqMan probe sequence was designed using the software Primer Express (version 1; Applied Biosystems, Foster City, Calif.) and synthesized with the reporter dye FAM (6-carboxifluorescein) at the 5' end and a quencher molecule (TAMRA; 6-carboxytetramethylrhodamine) covalently coupled to the 3' end (Table 1).
For the Toxo TaqMan assay, a 50-µl mixture containing 1 × TaqMan buffer A (Applied Biosystems), 1.5 mM MgCl2, 1 µM each T. gondii ITS1 primer, 200 nM fluorogenic probe, 200 µM each deoxynucleotide triphosphate, and 1 U of AmpliTaq Gold DNA polymerase (Applied Biosystems) was prepared, and up to 1 µg of the template DNA was tested. After activation of the AmpliTaq Gold DNA polymerase for 10 min at 95°C, PCR was performed for 50 cycles of 15 s at 94°C followed by 1 min at 60°C, and the products were analyzed on an ABI Prism 7700 sequence detector (Applied Biosystems). All the data acquisition and data analyses were performed with Sequence Detector Software (SDS version 1.7; Applied Biosystems) and CT values were recorded for statistical analysis on Excel spreadsheets. CT is defined as the cycle number (C) at which the reporter's fluorescence exceeds the threshold value (T), a parameter defined as 10 standard deviations (SD) above the baseline fluorescence from cycles 3 to 15. The mouse
-actin housekeeping gene was detected with a commercially
available TaqMan kit developed for human -actin (Applied Biosystems), using 35 cycles. Conditions for mouse -actin were similar to those described for the Toxo TaqMan assay except the annealing temperature was 52°C, the MgCl2 concentration
was 1.5 mM, and the primer and probe concentrations were 180 and 80 nM, respectively.
Sources of T. gondii. Three strains of T. gondii representing each genotype (RH, genotype I; Me49, genotype II; and VEG, genotype III) were used to infect animals in this study (18). All three infective stages of T. gondii, tachyzoites, bradyzoites, and oocysts, were used to extract genomic DNA or to infect animals (6). To determine minimum infective dose (1 parasite), 10-fold dilutions of tachyzoites, bradyzoites, and oocysts were bioassayed in mice.
Tachyzoites were obtained from peritoneal lavage of mice injected intraperitoneally 3 to 5 days earlier with RH strain tachyzoites. The peritoneal fluid was passed through a 3-µm filter (Nucleopore) to remove host cells. Tachyzoites were pelleted by centrifugation at 1,180 × g for 10 min, and the number of parasites was counted with a hemacytometer. T. gondii genomic DNA was extracted from approximately 108 tachyzoites after overnight incubation with DNA digestion buffer (0.5% sodium dodecyl sulfate [SDS], 25 mM EDTA, 100 mM NaCl, 20 mM Tris-HCl [pH 8.0], and proteinase K [0.1 mg/ml final concentration]). Digested parasites were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and the DNA was precipitated in 0.3 M sodium acetate (final concentration) with 2.5 volumes of 100% ethanol. DNA pellets were solubilized in TE buffer (10 mM Tris-HCl, 1 mM EDTA) and stored at
20°C. The DNA
concentration was estimated by spectrophotometric absorbance at 257 nm.
Oocysts were obtained from feces of cats fed tissue cysts of the VEG
strain, as described (7). Bradyzoites were collected from
the brains of female Swiss-Webster mice chronically infected with the
VEG strain (6). All brains were homogenized, and tissue cysts were isolated by centrifugation through an isotonic Percoll gradient. Bradyzoites were released from tissue cysts by short incubation with pepsin-HCl solution at room temperature, neutralized with sodium bicarbonate, and counted with a hemacytometer
(5). Tenfold dilutions were prepared in phosphate-buffered
saline (pH 7.2) and added to 1 g of muscle (biceps femoral)
samples from T. gondii-free pigs. The spiked tissue samples
were pepsin-HCl digested and treated for DNA extraction at a ratio of
1.2 ml/0.1 g of tissue. DNA (500 ng, total input) from each muscle
sample was analyzed in duplicate on 4 different days.
Verification of Toxo TaqMan specificity. Amplification of the 18S rRNA gene for each sample was performed with universal primers (Table 1) to normalize the quality and quantity of DNA between samples. A panel of DNA samples from the related protozoa N. caninum, H. hammondi, Eimeria acervulina, Eimeria tenella, Cryptosporidium parvum, Sarcocystis muris, Sarcocystis tenella (sheep-dog cycle), Sarcocystis cruzi (cattle-dog cycle), as well as T. gondii were tested for primer specificity. DNA samples from muscle and brain of host animals (pig and mouse) were similarly tested. Approximately 150 ng of each parasite DNA was evaluated with 18S rRNA universal primers and with our T. gondii ITS1 primers.
Analyses of T. gondii-infected animals.
T.
gondii DNA was extracted from tissue cysts obtained from
chronically infected mice. Five Swiss-Webster mice were injected subcutaneously with 200 counted bradyzoites of the VEG strain. Six
weeks later, animals were bled, and serology was performed by a
modified agglutination test (MAT) at a 1:50 screening dilution of the
serum (3). Animals were subsequently killed, and whole brains were collected, smears were made to verify tissue cysts, and the
remaining brain was directly processed with DNA digestion buffer
without treatment with pepsin-HCl. DNA was extracted as described above
and stored at 20°C for future use. Brain tissues from four mice
infected with the Me49 T. gondii strain were included in
this experiment and processed in a manner similar to those from
VEG-infected mice.
20°C. In parallel, a
second aliquot was used to infect mice for bioassay confirmation of
T. gondii infection.
Statistical analyses. In order to determine the variability of the assay, intra-assay and interassay (repeatability) precision was measured. Ten replicates of three different T. gondii DNA concentrations (10 pg, 100 pg, and 1 ng) and a T. gondii-spiked muscle sample, randomly distributed through the 96 wells of the thermal cycler, were tested simultaneously. The repeatability of the test (precision between runs) was assessed using the previous T. gondii DNA concentrations, testing four replicates of each on five different days. Variability is shown as the mean ± SD in the graphs and reported as coefficient of variation (CV). Statistical analyses were carried out using Microsoft Excel (Redmond, Wash.).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Assay specificity.
DNA (150 ng) from all parasite and host
samples included in the panel was amplified with the universal 18S rRNA
primers, generating bands of similar intensity (Fig.
1A and data not shown) and confirming the
quality and equal amounts of DNA loaded per reaction. The T. gondii ITS1 primers reacted only with T. gondii DNA,
generating a DNA fragment of the expected size (~333 bp) (Fig. 1B and
data not shown). No PCR products were observed with any other DNA
samples tested. All of these DNA samples were analyzed using the Toxo TaqMan assay, and only the T. gondii DNA gave a strong
positive response, with a CT value of ~16
(Fig. 1C and data not shown).
|
Rn)
and cycle number on the x axis. The amplification plot shows
test results from T. gondii DNA through 50 cycles; other
samples are below the threshold line, indicated in bold.
Assay sensitivity and range of detection.
To estimate the
sensitivity of the Toxo TaqMan assay, amplification of 10-fold
dilutions of T. gondii genomic DNA was performed. Dilution
series containing from 100 ng to 10 fg of T. gondii DNA were
tested in duplicate. Figure 2A shows a
typical display of a Toxo TaqMan assay provided by the ABI Prism 7700 instrument. Positive signals (CT values) were
found for all dilutions except 10 fg of T. gondii DNA. Thus,
in our hands, a detection limit of 100 fg of T. gondii DNA,
or DNA equivalent to 1 bradyzoite, was achievable. The PCR products
generated through the Toxo TaqMan assay were electrophoresed in an
agarose gel and stained with ethidium bromide. In agreement with the
CT values, bands of the expected sizes were
detected down to 100 fg of T. gondii DNA, although the bands
for 1 pg and 100 fg were dim (Fig. 2B, lanes F and G). Figure 2C shows
the mean CT values for eight replicates of the
series of T. gondii DNA concentrations. The
CT values, ranging from 15.52 ± 0.21 for 100 ng
of T. gondii genomic DNA to 38.41 ± 0.64 for 100 fg,
showed reproducible linearity over a 10,000,000-fold range
(R2 = 0.9984). A significant coefficient of
correlation was found for the mean CT values and
T. gondii DNA concentrations (r = 0.9793).
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Precision of Toxo TaqMan assay.
The intra-assay precision
(white bars) (Fig. 3) was measured on 10 replicates of three T. gondii DNA concentrations and spiked pig samples tested on the same day and expressed as mean
CT values. Results showed low variability, with
a CV of <1.75% for the lowest T. gondii genomic DNA
concentration (10 pg) and <1% for the T. gondii-spiked
sample. The interassay precision (black bars) (Fig. 3), expressed as
mean CT values for 20 replicates (four
replicates on five different days), presented a CV of 4.5% for
T. gondii genomic DNA and a CV of 1.7% for the T. gondii-spiked sample.
|
Toxo TaqMan assay results with bradyzoite-spiked pig muscle
samples.
To test whether T. gondii could be detected in
pig tissues, DNA (500 ng, total input) isolated from 1-g samples of pig
muscle spiked with known numbers of T. gondii bradyzoites
was analyzed. All samples gave positive signals, with mean
CT values ranging from 27.2 to 46.5 (Fig.
4A). The Toxo TaqMan assay was linear for
a 1,000,000-fold range of bradyzoite concentrations
(R2 = 0.9776) (Fig. 4B). A significant
coefficient of correlation (r = 0.68) between number
of bradyzoites and CT values was observed.
|
Toxo TaqMan assay results from animals experimentally infected with
T. gondii.
The Toxo TaqMan assay results on samples
from experimentally infected pigs and mice corresponded well with their
serological status; all infected and control animals were identified
correctly (Table 2). Good correlation
between CT values and MAT titers was observed
for the pig samples (r = 0.719). All Toxo TaqMan CT values from mouse DNA samples were normalized
against CT values generated with the -actin
TaqMan kit (Table 2). These data confirmed that the Toxo TaqMan assay
was positive when tested against each of the three T. gondii
genotypes.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study presents results for a new molecular Toxo TaqMan assay. Sensitivity at 100 fg of T. gondii DNA is similar to that reported elsewhere despite differences in the target gene (2, 21, 25). The assay exhibits a linear range of detection (R2 = 0.9985) similar to that found by Costa et al. (2). Furthermore, the Toxo TaqMan assay shows a twofold better sensitivity than other tests (20, 28, 29), which detected ~10 parasites in diverse biological samples. In these other studies, T. gondii DNA extractions were performed from a variety of samples, such as serum (2), aqueous humor (25), amniotic fluid (17), and cell culture (20), where the contaminating host-derived DNA level is low, and therefore cannot be equated to the Toxo TaqMan assay, which was performed on DNA recovered from as much as 50 g of original pig tissue.
When applied to bradyzoite-spiked muscle samples, the Toxo TaqMan test successfully detected as few as ~4 bradyzoites per g of pig tissue. This sensitivity level is substantially better than that reported by Warnekulasuriya et al. (30) on cured meats (5 × 103 tachyzoites/g), who attributed this low sensitivity to the high salt content of the cured meat. Considering that the Toxo TaqMan assay was linear (R2 = 0.9776) and detected from as few as 4 to as many as 3.7×105 parasites per g of tissue, the range of detection is not a limitation. Other investigators have reported a range of detection of only 102 to 104 T. gondii organisms using a quantitative competitive PCR with the B1 gene (23) or with a highly repetitive DNA sequence as the target gene (17). Costa et al. (2) detected from 1 to ~400 parasites in 200-µl serum samples of stem cell-transplanted patients using a LightCycler-based PCR test.
It is difficult to find T. gondii tissue cysts in large
animal species for several reasons, including sampling bias and
preferred parasite sites. Dubey et al. (7) have estimated
that less than 1 tissue cyst/50 g of tissue is likely to be found in
T. gondii-infected pigs. Thus, it is possible that when
performing any test for tissue cyst detection, false-negatives can
result from insufficient sample size or improper sample acquisition
(12). In our study, it was assumed that the pigs
inoculated with T. gondii might harbor low number of tissue
cysts despite a moderate infective dose (500 oocysts). Therefore, for
the tests in this report, a digestion-concentration method was applied
for some samples, such as the 50 g of brain or tongue, to reduce
the actual amount of host tissue undergoing DNA extraction. Continuing
studies in our laboratory are focusing on how long after infection
T. gondii can be detected in pigs or in stored pig tissue
samples. Because CT values in the Toxo TaqMan
assay correspond to the number of bradyzoites per gram (coefficient of
correlation = 0.68), it should be possible to construct a
standard curve using samples spiked with known numbers of T. gondii tachyzoites or bradyzoites and from this extrapolate the
T. gondii burden in unknown samples once the
CT values have been determined.
The ability of our Toxo TaqMan assay to correctly identify T. gondii-infected animals with a high degree of sensitivity was demonstrated for both parasite-infected mice and pigs, and it was also
shown to work on multiple stages of infection (Table 2). These results
were corroborated by both serological response of all infected animals
and presence of tissue cysts in the brain of infected mice.
Furthermore, a significant association between antibody titers of pigs
and CT values was observed (r = 0.719). Like other quantitative methods (2, 17,
23), the Toxo TaqMan assay is dependent on the initial DNA
input; thus, for standardization, the use of a normalization
housekeeping gene is strongly recommended. In the future, such analyses
could be carried out using multiple TaqMan assays with different
fluorogenic probes.
At present, serological testing is the only practical tool to identify T. gondii infection in pigs for either food animal inspections or epidemiological studies (14). Mouse and cat bioassays accurately confirm T. gondii infection, but these bioassays require use of live animals for 6 to 8 weeks and are costly and time-consuming. With the development of the Toxo TaqMan assay, we hope to supplement or even replace current bioassay or indirect serology testing with a more direct assay. With combined tissue digestion, fast DNA preparation, and the Toxo TaqMan assay, it is expected that T. gondii detection could be completed within 24 h or less. This newly developed assay will thus help to monitor T. gondii contamination in commercial meat products, and ultimately reduce food-borne transmission of this harmful parasite. Moreover, the Toxo TaqMan assay will provide an objective tool for quantitating T. gondii burden for vaccination studies and clinical trials for therapeutic treatments.
The Toxo TaqMan assay is a very sensitive and specific assay that can be used to detect T. gondii in a variety of biological samples using T. gondii-specific ITS1 primers and a fluorogenic probe via real-time PCR. The detection response was linear over a 10,000,000-fold range of T. gondii DNA concentrations (R2 = 0.9985) as well as parasite numbers (R2 = 0.9776), and the assay was able to detect as little as 0.1 pg of T. gondii genomic DNA. The Toxo TaqMan assay presents several important advantages over other methods of detection and quantitation of T. gondii DNA. First, the Toxo TaqMan is performed in a closed tube with no post-PCR manipulations, thereby reducing potential PCR product carryovers and post-PCR processing time. Second, the assay is quick; results can be confirmed within 1 day. Third, the assay response is sensitive and linear over a broad range of DNA concentrations. Finally, sample processing is compatible with current PCR-based automation technology (16).
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ACKNOWLEDGMENTS |
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We thank Diane Hawkins-Cooper, Sam Shen, Oliver Kwok, and Alexander Domingo for excellent technical assistance and Susan Liddell and Mark Jenkins for kindly providing parasite DNA samples and help with DNA sequence analyses.
This work was supported by funds from the ARS, USDA, and grants awarded by the National Pork Producers Council (NPPC 00-132 and 00-024).
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FOOTNOTES |
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* Corresponding author. Mailing address: Immunology and Disease Resistance Lab, ANRI, ARS, USDA, Bldg. 1040, Rm. 105, Beltsville, MD 20705. Phone: (301) 504-8201. Fax: (301) 504-5306. E-mail: jlunney{at}anri.barc.usda.gov.
Present address: Virology Area, Faculty of Veterinary Sciences,
University of Buenos Aires, (1427) Buenos Aires, Argentina.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, June 2001, p. 2065-2071, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2065-2071.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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