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.     

Toxoplasma gondii infections in chickens – performance of various antibody detection techniques in serum and meat juice relative to bioassay and DNA detection methods

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100?times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature.     

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 survey of Neospora caninum and Toxoplasma gondii infection in urban rodents from Brazil

Toxoplasma gondii is a protozoan parasite that infects humans and other warm-blooded animals; it uses feral and domestic cats as the definitive hosts. Neospora caninum is a protozoan parasite of animals whose life cycle is very similar to T. gondii but uses canids as definitive hosts. Small rodents play an important role in the life cycle of T. gondii , and a few findings indicated that they may be natural intermediate hosts for N. caninum . The present study was aimed at identifying infections by T. gondii and N. caninum in urban rodents. Infections by T. gondii were quantified using isolation of the parasite by bioassay in mice; molecular methods were also used for both parasites. Overall, 217 rodents were captured. Brain and heart tissues of all rodents were bioassayed in mice for the detection of T. gondii infection. Brain and heart tissues of 121 rodents had the DNA extracted for molecular analysis. Toxoplasma gondii was isolated by bioassay from a single rodent. From the 121 rodents tested for the presence of T. gondii DNA, 2 animals were positive. In contrast, DNA of N. caninum was not detected in any of the samples. In conclusion, the surveys of N. caninum and T. gondii infection in Rattus rattus , Rattus norvegicus , and Mus musculus captured in urban areas of S?o Paulo reveal a striking low frequency of occurrence of these infections.     

Prevalence of Toxoplasma gondii infection diagnosed by PCR in farmed red foxes, arctic foxes and raccoon dogs

The aim of this study was to compare Toxoplasma gondii infection in three canid species: red fox Vulpes vulpes, arctic fox Vulpes lagopus and raccoon dog Nyctereutesprocyonoides kept at the same farm. Anal swabs were taken from 24 adult and 10 juvenile red foxes, 12 adult arctic foxes, three adult and seven juvenile raccoon dogs. Additionally, muscle samples were taken from 10 juvenile red foxes. PCR was used to detect T. gondii DNA. T. gondii infection was not detected in any of the arctic foxes; 60% ofraccoon dogs were infected; the prevalence of the parasite in material from red fox swabs was intermediate between the prevalence observed in arctic foxes and raccoon dogs. It is possible that susceptibility and immune response to the parasite differ between the three investigated canid species. T. gondii DNA was detected in muscle tissue of five young foxes. The results of this study suggest that T. gondii infection is not rare in farmed canids.     

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.     

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.     

Detection of Infectious Laryngotracheitis Virus by Real-Time PCR in Naturally and Experimentally Infected Chickens

Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek''s disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens.     

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in S?o Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits.     

Investigating the possible role of benthic macroinvertebrates and zooplankton in the life cycle of the haplosporidian Bonamia ostreae

Bonamia ostreae is a protistan parasite of the European flat oyster, Ostrea edulis. Though direct transmission of the parasite can occur between oysters, it is unclear if this represents the complete life cycle of the parasite, and the role of a secondary or intermediate host or carrier species cannot be ruled out. In this preliminary study, benthic macroinvertebrates and zooplankton from a B. ostreae-endemic area were screened for the presence of parasite DNA, using polymerase chain reaction (PCR). Eight benthic macroinvertebrates and nineteen grouped zooplankton samples gave positive results. Certain species, found positive for the parasite DNA, were then used in laboratory transmission trials, to investigate if they could infect na?ve oysters. Transmission of B. ostreae was effected to two na?ve oysters cohabiting with the brittle star, Ophiothrix fragilis.     

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。     

Genotyping of Toxoplasma gondii associated with abortion in sheep

Genotypes of Toxoplasma gondii in naturally infected tissues from 13 ovine abortions from 10 farms in the United Kingdom, from 2 wild rodents captured on 1 farm, and from 2 isolates in laboratory mouse brains (made from the hearts of healthy, infected lambs) all conformed to the type II lineage determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the SAG2 gene. PCR was applied directly to tissue extracts without parasite isolation or DNA extraction. These results demonstrate the feasibility of direct analysis of T. gondii lineages from samples derived either from clinical cases or asymptomatic infections.     

Parasite load in guinea pig foetus with real time PCR after maternofoetal transmission of Toxoplasma gondii

Parasite loads of different tissues were assessed in guinea pig foetus after maternal infection. Twelve female guinea pigs were infected with 100 cysts of the 76 K strain of Toxoplasma gondii by the oral route. Inoculation was performed 20 +/- 5 days (G20) or 40 +/- 5 days (G40) after the beginning of gestation. Gestational age was determined by progesterone assay. Maternal and foetal organ samples were taken 60 days after the beginning of gestation. Parasite loads (from placenta, amniotic fluid (AF), cord blood (CB), foetal brain, liver, lung and spleen) were assessed by a real-time PCR quantification using fluorescence resonance energy transfer (FRET) hybridization probes on the Light Cycler. Congenital transmission was proven by the presence of parasites in blood or tissue samples of the foetus in 84.6% (11/13) and 100% (16/16) of cases after inoculation on G20 and G40, respectively. The quantitative analysis of our results after inoculation at G20 and G40 has allowed us to determinate the positive parasitic loads as a function of the origin of the sample and the period of inoculation. The parasite loads expressed as log (parasite/g) were low in AF and CB samples: 1.49 +/- 0.50 and 1.05 +/- 0.10 at G20 and 1.21 +/- 0.36 and 1.20 +/- 0.42 at G40 respectively. In contrast the placenta and the different foetal tissues had higher parasite burdens: 2.89 +/- 0.54 to 5.30 +/- 0.51 at G20 and 2.81 +/- 0.71 to 3.65 +/- 0.59 at G40. All the placentae were positive for parasites even in the two cases with no proven transmission. Real time quantitative PCR using the hybridization probe was a very sensitive and reproducible technique to study the kinetics of congenital toxoplasmosis in the guinea pig model wich is close to that of humans.     

Polymerase chain reaction for detection ofToxoplasma gondii in human biological samples

Using the polymerase chain reaction (PCR), Toxoplasma gondii from gene TGR1E with primers TGR1E-1, TGR1E-2 (standard PCR), and from B1 gene with primers TM1, TM2, TM3 (hemi-nested PCR) was detected in biological samples from 347 individuals (441 biological materials). Of the total of 441 biological materials, T. gondii DNA was detected in 5.2 %; it was positive in the following samples: blood (n = 6), blood from newborns (2), biopsies (2) and samples of progenitor cells (2) (from candidates for bone marrow transplantation). DNA of T. gondii was also revealed in 11 samples (8.3 %) of 120 cases of pregnant women during prenatal examinations. A positive result in the blood was also found in two cases of newborn babies from mothers who were infected in later pregnancy. The positive PCR examination was confirmed by serological methods (ELISA and complement fixation test). Agreement of PCR results and the detection of antibodies against toxoplasma was found in 83.3 %. Rapid PCR examination for the confirmation of acute parasitemia T. gondii is particularly important for the patients in whom the infection may cause serious consequences (e.g., for fetus in pregnant women or for patients suffering from imunosuppression).     

Kinetics of parasite burdens in blood and tissues during murine toxoplasmosis

A sensitive real-time PCR technique was used to examine the distribution of Toxoplasma gondii in the blood and tissues of mice during acute and chronic infection. Groups of Swiss Albino mice, inoculated i.p. with 10(2) or 10(6) tachyzoites of the RH strain as a typical type-1 strain, or fed 10 cysts of the Me49 strain as a typical type-2 strain, were killed at different time points post-infection (p.i.), and blood and organs including the lungs, brain and liver were harvested for DNA extraction. Toxoplasma DNA was quantified by a real-time PCR targeted at the 529bp gene fragment, with a detection limit of a single parasite per g/ml of tissue. The results showed a strain- and dose-dependent spread of Toxoplasma. In infection with type-1 parasites, in case of a high infective dose, Toxoplasma DNA was detected within 24h p.i. in all analyzed tissues including the brain. Conversely, in case of a low infective dose, parasitaemia was undetectable early p.i., at a time when Toxoplasma DNA was detected in the tissues, but reached very high levels as infection progressed. With both infective doses, pre-death parasite burdens were higher in the blood than in the tissues, whereas the same loads in the lungs suggest that reaching these Toxoplasma burdens may be critical for survival. In infection with Me49 parasites, steady high parasite burdens were noted up to the end of the experiment at d42 only in the brain, parasitaemia was low but detectable throughout, and Toxoplasma DNA was completely cleared only from the liver. These data are important to better understand the pathogenesis of toxoplasmosis, and also as baseline data for the experimental evaluation of novel chemotherapeutics.     

Measurement of the efficacy of vaccines and antimicrobial therapy against infection with Toxoplasma gondii

To facilitate studies of vaccines and antimicrobial agents effective against Toxoplasma gondii infection, an assay system was developed to semi-quantitate parasitaemia using PCR amplification of T. gondii DNA obtained from the blood of mice infected with the parasite. A competitive internal standard DNA fragment of the B1 gene of T. gondii was generated and used in PCR so that the amplified product could be semi-quantitated and false negative results could be avoided. The PCR assay system was used to analyse the levels of parasitaemia in immunised and antimicrobial agent treated mice at various times after infection with T. gondii. The results of these studies indicate that this highly sensitive detection method is a rapid and reliable procedure that can be employed to assess the abilities of vaccines or antimicrobial agents to provide protection early following T. gondii infection.     

Characterization of Toxoplasma gondii from domestic animals from Minas Gerais, Brazil

In an attempt to isolate and characterize Toxoplasma gondii from the State of Minas Gerais, Brazil, musculature samples from 72 pigs, 25 dogs, 28 free-range chickens and 50 chickens produced in industrialized farms were collected. Antibodies to T. gondii have not been detected in pigs, but were found in nine (40.9 %) out of 22 dogs, and in 15 (53.6 %) of 28 free range chickens. T. gondii was not isolated from pigs and industrialized chickens, but from eight dogs and 11 free range chickens. In order to determine T. gondii virulence, female BALB/c mice were inoculated with 10(3), 10(2), 10(1) and 10(0) tachyzoites of the 19 isolates. The strains RH (virulent) and ME49 (non-virulent) were used as references. Isolates were divided into three groups according to the virulence phenotype: five isolates were classified into virulent in mice, one into non-virulent and 13 into intermediate virulent. Nested-PCR of T. gondii SAG2 locus amplified DNA from 21 out of 22 DNA samples directly extracted from heart of free range chickens. These samples were genotyped through a PCR-RFLP assay. Seventeen (80.9 %) were classified into type I; one (4.8 %) into type III and three (14.3 %) into type I or II.     

Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations. Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.     

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.