Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

The Toxoplasma gondii (TGR) genes constitute a family of non-coding sequences, three of which have been previously described as possible tools for typing of Toxoplasma gondii isolates. We obtained new isolates of T. gondii from domestic and wild animals, and used these to evaluate the possibility of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources.Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T. gondii isolates could be separated into seven groups. Sequencing the amplified products showed that at least 20 TGR sequences not hitherto described had been found, demonstrating that the TGR gene family comprises a large number of different yet highly homologous sequences. Each isolate had its own unique TGR sequence. The TGR gene family therefore seems a promising target for typing individual T. gondii isolates and for studying the genetic distance between two isolates, which can be used for tracing routes of infection.     

A new set of primers for the detection of Toxoplasma gondii in amniotic fluid using polymerase chain reaction

Abstract A new PCR system including a pair of primers, a probe and an internal control were designed from the B1 gene of Toxoplasma gondii . The system described allowed the detection of less than 10 tachyzoites of the RH strain of T. gondii . Among 21 amniotic fluid samples, this system diagnosed the cases of congenital toxoplasmosis which were simultaneously diagnosed using mice inoculation, in vitro culture, and serology from both amniotic fluid and fetal blood. These results show that these new primers allow for a highly sensitive detection of T. gondii DNA.     

Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control

We have developed a quantitative PCR assay (LightCycler* using the pair of primers JW58 and JW59 for the detection of the 35-fold repeated B gene of oxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of . gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the same primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.106( )parasites in one ml of amniotic fluid (1 to 105( ) . gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.     

Toxoplasma gondii: detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 x 10(4) VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P<0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs.     

K24 T. gondii isolate is a hybrid and has the virulence of lineage I isolates

A permanently high virulence was found in tachyzoites of T. gondii K24 after serial passage in mice (90 passages during 324 days). Virulence tests revealed that a single tachyzoite of the 50th passage represented LD100 for mice. Analysis of genotype of K24 isolate was done by PCR/RFLP with ROP1/Ddel, SAG1/Ddel, 850/Rsal and IGS/Rsal and by RFLP/DNA with TGR1E sequence and Pstl enzyme. K24 isolate had an atypical genotype, with an association of type II (for ROP1, SAG1 genes and TGR1E sequence) and type I (for 850 gene) alleles, and a new pattern observed for IGS. All tested PCR/RFLP did not change through 2, 10, 20, 28, 40, 50, 60, 70, 81 and 90 tested passages. In RFLP/DNA with Pstl enzyme and TGR1E probe, K24 isolate produced a pattern with seven fragments of the size ranging from one to 23 kb and did not change through 7, 56, 70 and 83 tested passages. K24 T. gondii isolate is a hybrid and has the virulence of lineage I isolates.     

Evaluation of PCR methods for 5S-rDNA and p30 genes to detect Toxoplasma gondii in blood and other clinical samples

During the last few years the direct diagnosis of Toxoplasma gondii infection has taken advantage of PCR. The present work tested the sensitivity and specificity of PCR for rDNA and p30 genes. Using ascitic fluid from infected mice rDNA PCR detected 0.5 tachyzoite/ml, while nested p30 PCR 1 tachyzoite/ml. The rDNA amplification was positive in all clinical samples from a single immuno compromised patient (blood, urine and bronchoalveolar fluid). In the same patient nested p30 PCR was positive only in urine and bronchoalveolar lavage (BAL) fluid. The rDNA and p30 amplicons were never found in any amniotic fluids tested. These results could prove the usefulness of rDNA amplification to detect T. gondii in blood.     

Triplex PCR using new primers for the detection of Toxoplasma gondii

Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.     

弓形虫特异DNA片段顺序分析及体外基因扩增

从弓形虫(ZS_2株)基因组文库中筛选出了一个弓形虫特异DNA片段的克隆,对克隆的片段进行了部分顺序分析。根据所得DNA顺序,自行设计并合成特异的寡核苷酸引物对,建立了体外扩增弓形虫特异DNA顺序的聚合酶链反应(PCR)方法。4种不同来源的弓形虫株DNA、人工感染弓形虫的三头幼猪白细胞和胸腺DNA经过PCR扩增,均出现特异的扩增条带;而正常人、正常幼猪外围血白细胞、正常小鼠脾脏、恶性疟原虫、卡氏肺孢子虫、溶组织内阿米巴和人巨细胞病毒DNA均不出现特异的扩增条带。对扩增产物进行了Southern印迹和限制性内切酶图谱分析,证明该PCR产物是弓形虫DNA特异的顺序。该方法可测出少至1pg的弓形虫DNA或1个弓形虫体的裂解液。本文分析的DNA顺序和设计合成的引物顺序数据,经电脑DNA数据库检索,证明无相同的顺序。本方法并具有简便、快速等优点,便于推广应用。     

弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

Detection of Ocular Toxoplasma gondii Infection in Chronic Irregular Recurrent Uveitis by PCR

Toxoplasma gondii is a zoonotic parasite resulting in human infections and one of the infectious pathogens leading to uveitis and retinochoroiditis. The present study was performed to assess T. gondii infection in 20 ocular patients with chronic irregular recurrent uveitis (20 aqueous humor and 20 peripheral blood samples) using PCR. All samples were analyzed by nested PCR targeting a specific B1 gene of T. gondii. The PCR-positive rate was 25% (5/20), including 5% (1) in blood samples, 25% (5) in aqueous humor samples, and 5% (1) in both sample types. A molecular screening test for T. gondii infection in ocular patients with common clinical findings of an unclear retinal margin and an inflammatory membrane over the retina, as seen by fundus examination, may be helpful for early diagnosis and treatment.     

Prevalence to Toxoplasma gondii and Sarcocystis spp. in a reintroduced fisher (Martes pennanti) population in Pennsylvania

Understanding the role of disease in population regulation is important to the conservation of wildlife. We evaluated the prevalence of Toxoplasma gondii exposure and Sarcocystis spp. infection in 46 road-killed and accidentally trapper-killed fisher (Martes pennanti) carcasses collected and stored at -20 C by the Pennsylvania Game Commission from February 2002 to October 2008. Blood samples were assayed for T. gondii antibodies using the modified agglutination test (MAT, 1 : 25) and an indirect immunofluorescent antibody test (IFAT, 1 : 128). For genetic analysis, DNA samples were extracted from thoracic and pelvic limb skeletal muscle from each carcass to test for Sarcocystis spp. using 18s-rRNA PCR primers. Antibodies to T. gondii were found in 100% (38 of 38) of the fishers tested by MAT and in 71% (32 of 45) of the fishers tested by IFAT. PCR analysis revealed that 83% (38 of 46) of the fishers were positive for Sarcocystis spp. Sequence analysis of 7 randomly chosen amplicons revealed the fisher sarcocysts had a 98.3% to 99.1% identity to several avian Sarcocystis spp. sequences in GenBank. Data from our study suggest that a high percentage of fishers in Pennsylvania have been exposed to T. gondii and are infected with Sarcocystis spp.     

Polymerase chain reaction in diagnosis of toxoplasmosis of the central nervous system: effect of methods for isolating DNA from samples of cerebrospinal fluid on results of the reaction

We examined influence of the method of isolation of DNA from cerebrospinal fluid samples on results of PCR in the diagnosis of toxoplasmosis of the central nervous system. Three different protocols of DNA isolation were used for DNA extraction from 360 samples made of cerebrospinal fluid spiked with tachyzoites of Toxoplasma gondii: thermic, enzymatic and enzymatic-filtering. Purified DNA samples were tested by PCR with primers T15 and T16 designed for the B1 gene of the parasite. Enzymatic method of DNA isolation appeared most effective allowing detection of T. gondii DNA in 50% of samples containing single parasite cell.     

A family of repeated DNA sequences in Toxoplasma gondii: cloning, sequence analysis, and use in strain characterization.

A Toxoplasma gondii genomic library was constructed in lambda EMBL3. Repeated fragments were detected by hybridization with radiolabeled total DNA from the parasite and one recombinant was chosen due to its strong hybridization signal. By using electrophoretic and hybridization analysis, four cross-hybridizating restriction fragments were selected and sequenced. The determined nucleotide sequence of these fragments (TGR1A, TGR1E, TGR2, and TGR4) has shown a complex system of conserved and degenerated repeats in which TGR1E corresponds to the most conserved element. This last sequence was used to investigate restriction fragment length polymorphisms among several T. gondii strains by Southern blotting.     

Prevalence of Toxoplasma gondii antibodies and DNA in dogs in Shanghai, China

The aim of this study was to estimate the prevalence of Toxoplasma gondii infection in dogs in Shanghai, China. A total of 1,736 sera (1,178 from the city center and 558 from the outskirts) was collected from healthy dogs and tested for T. gondii infection by indirect hemagglutination; 56 sera (3.2%) were considered positive, with titers > 1∶64. The seroprevalence in dogs from the outskirts of the city (town dogs, 6.0%; countryside dogs, 9.8%) was significantly higher (P > 0.05) than that in city center dogs (1.3%). The age of the dog has an apparent association with T. gondii infection; that is, the seroprevalence ranged from 1.9% (in dogs ≤ 1 year old) to 3.6% (in dogs > 5 years old). There was no significant difference in gender (P ≥ 0.05), that is, 1.4% versus 1.1% for male and female dogs in the city center, 6.2% versus 5.9% in town dogs, and 8.4% versus 11.5% in country dogs, respectively. These results suggest that T. gondii infections are common in dogs from the city center and outskirts of Shanghai, but the T. gondii seroprevalence in dogs is considerably lower than in other regions in PR China. The presence of T. gondii DNA was investigated by nested PCR on 110 blood samples from city center dogs, but no positive samples were found, which may suggest that there were no acute infections of T. gondii in the city center samples. Our results indicate that the control and treatment of toxoplasmosis in Shanghai has been effective. However, it is still essential to further implement integrated strategies to prevent and control T. gondii infection in dogs in both the city center and the outskirts.     

Problems and limitations of conventional and innovative methods for the diagnosis of Toxoplasmosis in humans and animals

Diagnosis of toxoplasmosis is useful for human and animal health. Several techniques are employed for the diagnosis in feline and canine population. Coprological tests for the detection of oocysts in cat faeces are of little significance owing to short patency (15 days). Histological examinations of biological samples show a lack of reliability when the animals are infected with few parasites; the mouse inoculation is the most reliable method even if the detection of cysts in mice brain require 40 days. However tachyzoites of virulent strains can be isolated from peritoneal exudate 3-4 days after inoculation. Samples inoculation in cell cultures (VERO, human fibroblasts) requires specialized laboratories and fails if non viable parasites are present due to tissutal autolysis. Serological tests are the most used diagnostic methods; Dye test and IFAT that require intact tachyzoites are more sensitive and specific compared to IHA, LA, ELISA because, during the infection, the first significant increase of IgM and IgG antibodies was observed against cuticolar antigens. A PCR to identify T. gondii DNA in canine and feline biological samples was developed. The B1 PCR performed on blood samples was less sensitive than when it was performed on other biological fluids requiring 100 tachyzoites, instead of 10. Aqueous humor PCR results could be negative if the infection is low grade or is restricted to the posterior segment or the animal was previously treated with anti-Toxoplasma drugs. SNC disease may be also difficult to diagnose because an high serum IgG titer may be associated with locally production or leakage from serum through a compromised blood-CSF barrier. AB1 PCR was successfully applied for the diagnosis of Toxoplasma abortion in ewes requiring only 10 parasites in placental cotyledon samples; the test compared with mouse inoculation showed similar sensitivity. Discrepancies may have been due to a low and focal distribution of parasites in the tissues or to the presence of non viable parasites if the tissues are autolysed. In regard to diagnostic methods adaptable to slaughter testing, several serological tests have been studied (IFAT, ELISA, IHA) for detection of IgG in sheep, pigs, cattle using also recombinant antigens (gene fragments H4 and H11) to lack the cross reactivity. The problem is the antibodies fall to near background levels as the infection became chronic (6-10 months p.-i.). A highly sensitive and specific method (Toxo Taq Man) has been developed to detect and quantitate T. gondii burden in animal tissue samples (0.1 pg of T. gondii genomic DNA, which is equivalent to 1 bradyzoite) using T. gondii ITS1-derived primers and a fluorogenic probe via Real-Time PCR. This assay is compatible with automation technology for potential slaughterhouse use. The diagnosis of acute infection in human pregnancy is difficult since IgM antibodies can be detected for a very long time after the acute phase; an IgA increase is of more diagnostic value because can be detected only for 6-7 months while the short kinetics of IgE can be useful only to date the infection precisely. In addition an IgG seroconversion is essential for the diagnosis. Among the most reliable tests, IgG avidity test is useful when a single serum sample, in the first months of gestation, is available, but low avidity results may persist for as long as 1 year. For this purpose a panel of serologic tests must be performed (ELISA, EIA, ISAGA, IgG avidity, IFAT, Dye test) for IgM, IgA, IgG and IgE. The serological diagnosis of prenatal infection is difficult since maternal IgG are passively transferred in utero to the foetus and caution must be exercised in interpretation of IgM or IgA results. A technique of Western blots of paired maternal and baby sera for evidencing different bands in the blots of two sera was developed for this purpose (specificity 97-100%, sensitivity 96-98%). The most reliable methods for prenatal diagnosis are PCR, mouse inoculation and cultural techniques performed on amniotic fluid, foetal blood and peripheral maternal blood in pregnants serologically positive. PCR (targets B1, SAG-1, rDNA) with amniotic fluid performed from 18 weeks of gestation is more sensitive and more rapid than conventional diagnostic procedures. PCR has been successfully used to diagnose Toxoplasma encephalitis in immunocompromised patients (cerebral biopsy is the only diagnostic method) and in ocular toxoplasmosis. In this evenience it is useful the study of IgG, IgM, IgA profile of paired serum and aqueous humor (Western blots).     

孕牛外周血中胎儿Y-特异性序列的检测

根据公牛Sry特异性序列设计两对寡核苷酸引物。并对20份妊娠晚期母牛外周血DNA样品进行PCR扩增,结果有9份样品检测出有Sry片断(阳性),其余11份为阴性。经分娩牛犊性别验证,在20份被检测的样品中,有16份PCR检测结果与犊牛实际性别相符,其余4例不符。我们的实验结果说明,胎儿细胞可以进入到孕牛的外周血中去。     

Prevalence of Toxoplasma gondii in slaughtered pigs in the state of Pernambuco, Brazil

The object of this study was to investigate Toxoplasma gondii antibodies and parasite DNA in pigs in the state of Pernambuco, Brazil. Blood samples were collected from 305 slaughtered pigs in 11 municipalities, and their sera were tested for T. gondii antibodies using the indirect fluorescent antibody test (IFAT, cutoff 1:64); 38 (12.5%) samples were positive. Attempts were made to detect T. gondii DNA in the heart tissue of seropositive pigs using the B 1 gene and PCR; 21 (55.2%) of the 38 hearts were positive. This is the first detection of T. gondii DNA in tissues of serologically positive swine in the State of Pernambuco, Brazil.     

Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States: risk assessment to consumers

The prevalence of viable Toxoplasma gondii was determined in 6,282 samples (2,094 each of beef, chicken, and pork) obtained from 698 retail meat stores from 28 major geographic areas of the United States. Each sample consisted of a minimum of 1 kg of meat purchased from the retail meat case. To detect viable T. gondii, meat samples were fed to T. gondii-free cats and feces of cats were examined for oocyst shedding. Initially, 100 g of meat from 6 individual samples of a given species were pooled (total, 600 g), fed to a cat over a period of 3 days, and feces were examined for oocysts for 14 days; the remaining meat samples were stored at 4 C for 14 days (until results of the initial cat fecal examination were known). When a cat fed pooled samples had shed oocysts, 6 individual meat samples from each pool were bioassayed for T. gondii in cats and mice. Toxoplasma gondii isolates were then genetically characterized using the SAG2 locus and 5 hypervariable microsatellite loci. In all, 7 cats fed pooled pork samples shed oocysts. Toxoplasma gondii oocysts were detected microscopically in the feces of 2 of the cats; 1 isolate was Type II and the second was Type III. Analyzed individually, T. gondii was detected by bioassay in 3 of the 12 associated samples with genetic data indicating T. gondii isolates present in 2. The remaining 5 pooled pork samples had so few oocysts that they were not initially detected by microscopic examination, but rather by mouse bioassay of cat feces. Two were Type I, 1 was Type II, and 2 were Type III. None of the cats fed chicken or beef samples shed oocysts. Overall, the prevalence of viable T. gondii in retail meat was very low. Nevertheless, consumers, especially pregnant women, should be aware that they can acquire T. gondii infection from ingestion of undercooked meat, and in particular, pork. Cooking meat to an internal temperature of 66 C kills T. gondii.     

Real-time PCR-based quantification of Toxoplasma gondii in tissue samples of serologically positive outdoor chickens

This study aimed to quantify Toxoplasma gondii in tissue samples of serologically positive chickens using real-time polymerase chain reaction (PCR). Of 65 chickens evaluated, 28 were positive for T. gondii antibodies. Brain and heart samples were collected from 26 seropositive chickens and DNA was extracted using Trizol? and amplified using real-time PCR with SYBR? Green. Parasite DNA was detected in 24 of the 26 samples analyzed; the number of positive tissue samples and the parasite quantity did not differ between tissue types. The results confirmed the analytical sensitivity of parasite detection in chicken tissue samples and demonstrated the possibility of using other molecular systems for genotypic analysis.     

Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA

Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR.