Toxoplasma Gondii DNA Research Articles

Recovery of Toxoplasma gondii DNA in experimentally mummified skin and bones: Prospects for paleoparasitological studies to unveil the origin of toxoplasmosis

Paleoparasitology studies parasite infections by finding the parasites’ remains in preserved organic remains such as natural or artificial mummy tissues, skeletons, teeth, and coprolites, among others. However, some currently important infections like toxoplasmosis have not been studied by paleoparasitology. The reasons include this parasite’s complex life cycle, the resulting difficulties in locating this protozoan in the intermediate host tissues, and the limitation of coprolite studies to felines, the protozoan’s definitive host. The current study thus aimed to produce an experimental model for molecular diagnosis of toxoplasmosis, prioritizing its study in bones and skin, the most abundant materials in archeological collections and sites. The study demonstrated the feasibility of recovering Toxoplasma gondii DNA from desiccated material, including bones and skin, in experimental models both with circulating tachyzoites (RH strain), characteristic of acute infection, and with cysts (ME49 cystogenic strain), characteristic of chronic infection. At present, most individuals with T. gondii infection are in the chronic phase, and the same was probably true in the past. The current study thus expands the odds of finding the parasite in archeological material, enhanced by the nature of the material in which the diagnosis was made. Finding the parasite may help answer questions that are widely debated in the literature on this protozoan’s origin (Old World versus New World). In addition, when conditions do not allow ideal storage of samples for molecular tests, the methodology creates the possibility of testing oven-dried samples transported at room temperature.

Elevated Toxoplasma gondii Infection Rates for Retinas from Eye Banks, Southern Brazil.

We found significantly higher incidence of Toxoplasma gondii DNA in eye bank specimens from Joinville in southern Brazil (13/15, 87%) than in São Paulo (3/42, 7%; p = 2.1 × 10E–8). PCR DNA sequence analysis was more sensitive at locus NTS2 than at locus B1; a high frequency of mixed co-infections was detected.

Assessment and Molecular Characterization of Human Intestinal Parasites in Bivalves from Orchard Beach, NY, USA.

Bivalves have been shown to be carriers of the human intestinal parasites Cryptosporidium parvum and Toxoplasma gondii. The goal of this study is to determine the prevalence of protozoan parasites in mollusks of New York City using a polymerase chain reaction (PCR)-based assay. Four species of mollusks, Mya arenaria, Geukensia demissa, Crassostrea virginica, and Mytilis edulis, were collected from Orchard Beach, NY in the fall of 2014, totaling 159 specimens. Each individual mollusk was dissected to harvest the digestive gland, the mantle, the gills, the foot and the siphon. The tissues were assayed for the presence of Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii DNA by using primers that target parasite-specific genes. C. parvum was found at a prevalence of 50%, 11.3%, and 1%, respectively, in Mya arenaria, G. demissa, and Mytilis edulis. C. parvum DNA was detected in all the tissues of these bivalve species, except the gills. Furthermore, G. lamblia was detected in Mya arenaria, G. demissa, Crassostrea virginica and Mytilis edulis at a prevalence of 37.5%, 4.5%, 60%, and 20.6%, respectively, while T. gondii DNA was not detected.

Molecular prevalence of Toxoplasma gondii DNA in goats' milk and seroprevalence in Northwest Tunisia.

Toxoplasmosis is a worldwide zoonosis with high impact on human and animal health. Consumption of unpasteurized milk is a risk factor of human toxoplasmosis. The aim of this study was to estimate the seroprevalence and molecular prevalence of T. gondii in goats’ milk in Northwest of Tunisia (Jendouba Governorate). A total number of 77 blood samples were collected from six herds were screened with a commercial ELISA kit for T. gondii antibodies. For the same goats’ samples, a nested PCR was performed to detect T. gondii DNA in milk. The seroprevalence of T. gondii infection was 31.2% (±0.05) while the molecular prevalence of this parasite in milk was estimated to 7.8% (±0.03). A very low value of kappa showed that there is not agreement between seroprevalence and parasite prevalence in milk. These results suggest that the consumption of raw milk from naturally infected goats is a potential source of human infection. An extension programme should be implemented to decrease related to goats’ raw milk consumption.

Detection ofToxoplasma gondiiDNA by qPCR in the feces of a cat that recently ingested infected prey does not necessarily imply oocyst shedding

Detection of Toxoplasma gondii DNA in cat feces is considered indicative of the presence of T. gondii oocysts. This study aims to demonstrate that the high sensitivity of qPCR can lead to T. gondii DNA detection in cat feces in the absence of oocysts. A cat immune to toxoplasmosis was fed with a mouse experimentally infected with T. gondii. Detection of DNA of this parasite was performed by qPCR on feces passed: (i) on the day the cat ingested the infected prey; (ii) during the three previous days; and (iii) during the three following days. The kinetics of qPCR results are clearly not linked to oocyst shedding and this result demonstrates that qPCR can detect T. gondii DNA related to bradyzoites from an infected prey, in the absence of oocysts. Caution is thus recommended when interpreting T. gondii qPCR results for samples of cat feces.

Endoparasite survey of free-swimming baleen whales (Balaenoptera musculus, B. physalus, B. borealis) and sperm whales (Physeter macrocephalus) using non/minimally invasive methods.

A number of parasitic diseases have gained importance as neozoan opportunistic infections in the marine environment. Here, we report on the gastrointestinal endoparasite fauna of three baleen whale species and one toothed whale: blue (Balaenoptera musculus), fin (Balaenoptera physalus), and sei whales (Balaenoptera borealis) and sperm whales (Physeter macrocephalus) from the Azores Islands, Portugal. In total, 17 individual whale fecal samples [n = 10 (B. physalus); n = 4 (P. macrocephalus); n = 2 (B. musculus); n = 1 (B. borealis)] were collected from free-swimming animals as part of ongoing studies on behavioral ecology. Furthermore, skin biopsies were collected from sperm whales (n = 5) using minimally invasive biopsy darting and tested for the presence of Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti DNA via PCR. Overall, more than ten taxa were detected in whale fecal samples. Within protozoan parasites, Entamoeba spp. occurred most frequently (64.7%), followed by Giardia spp. (17.6%) and Balantidium spp. (5.9%). The most prevalent metazoan parasites were Ascaridida indet. spp. (41.2%), followed by trematodes (17.7%), acanthocephalan spp., strongyles (11.8%), Diphyllobotrium spp. (5.9%), and spirurids (5.9%). Helminths were mainly found in sperm whales, while enteric protozoan parasites were exclusively detected in baleen whales, which might be related to dietary differences. No T. gondii, N. caninum, or B. besnoiti DNA was detected in any skin sample. This is the first record on Giardia and Balantidium infections in large baleen whales.

Functional Analyses of the Toxoplasma gondii DNA Gyrase Holoenzyme: A Janus Topoisomerase with Supercoiling and Decatenation Abilities.

A number of important protozoan parasites including those responsible for toxoplasmosis and malaria belong to the phylum Apicomplexa and are characterised by their possession of a relict plastid, the apicoplast. Being required for survival, apicoplasts are potentially useful drug targets and their attractiveness is increased by the fact that they contain “bacterial” gyrase, a well-established antibacterial drug target. We have cloned and purified the gyrase proteins from the apicoplast of Toxoplasma gondii (the cause of toxoplasmosis), reconstituted the functional enzyme and succeeded in characterising it. We discovered that the enzyme is inhibited by known gyrase inhibitors and that, as well as the expected supercoiling activity, it is also able to decatenate DNA with high efficiency. This unusual dual functionality may be related to the apparent lack of topoisomerase IV in the apicoplast.

Detection of Neospora caninum-DNA in brain tissues from pigeons in Changchun, Jilin (China)

Neospora caninum is an intracellular protozoan infecting many domestic and wild animals. The domestic chicken (Gallus domesticus) and the sparrow (Passer domesticus) are known as natural intermediate hosts of N. caninum, whereas the role of other birds such as pigeons is still unclear. In the present study, pigeon brain tissues collected in Jilin of China were screened by N. caninum specific-nested PCR to determine whether pigeons functioned as the natural intermediate hosts of N. caninum. The prevalences of N. caninum DNA and Toxoplasma gondii DNA among the brain samples were 30% (63/210) and 13.33% (28/210), respectively. One brain sample was co-infected with N. caninum and T. gondii in naturally infected pigeon. Of the 63 positive samples 42 could be assigned to the NC-PR genotype, 10 to the NC-1 genotypes and 5, 3 and 3 respectively to the each of the three new genotypes identified, indicating genetic polymorphism of N. caninum in pigeons in Jilin of China. The present study expanded the list of intermediate hosts of N. caninum to include pigeons which suggests that pigeons are involved in the transmission of the N. caninum.

Post challenge variability in time gap pertaining to appearance of Toxoplasma gondii DNA in blood and appearance of visible clinical signs in murine model

A marked variation, in terms of time lag gap of the depiction of visible clinical signs and the actual appearance of parasitic stages in body tissues, exists in experimental toxoplasmosis. Keeping this in mind, a study was designed to find out the time gap associated variability in appearance of tachyzoites in blood stream of infected mice. For this purpose, surface antigen 3 (SAG 3) was selected as a candidate to detect toxoplasmosis from blood samples taken at regular intervals from murine models. PCR was standardized to diagnose toxoplasmosis in ten inbred Swiss albino mice after experimental inoculation of 100 tachyzoites of laboratory maintained human RH strain of the parasite. The blood samples were subjected to PCR, in duplicates, using primers directed to the multicopy of SAG 3 gene at regular 12 -h interval post infection. The tachyzoites begin to appear between 36 h post infection and by 48 h blood of all the mice were found to be infected by tachyzoites. Alongside, other visible symptoms begin to appear after 72 h post infection. In conclusion, ample time gap was found regarding the initiation of clinical infection and actual appearance of visible clinical signs that could prove detrimental in final outcome of condition.

Direct Amplification of B1 gene of Toxoplasma gondii DNA using Nested Polymerase Chain Reaction Following Microwave Treatment for Whole Blood Samples

Infection with Toxoplasma gondii is a cause of fetal death since T. gondii can be transmitted to the fetus through the placenta (transplacental) from an infected mother or at vaginal delivery. Blood obtained from women and sheep to confirm their infection with toxoplasmosis by using Enzyme Linked Immunosorbant Assay test (ELISA) to ditective positive specific anti-Toxoplasma (IgM, IgG and IgM, or IgG) antibodies. This study used two methodes to extract DNA (the first one was a standard extraction commercial method (CM-PCR) of genomic DNA using a commercial kit (Promega, USA), and the second one was the direct heat DNA extraction using microwave oven (MW-PCR) for whole blood samples obtained from infected women and sheep. Then nested Polymerase Chain Reaction (n PCR) were used to amplify Toxoplasma B1 gene to detect T. gondii DNA in whole blood samples. The results indecated using of microwave treatment instead of commercial kit to extract DNA is low cost and short time,and complement serology for clinical studies and diagnostic purposes of toxoplasmosis.

Assessment of Toxoplasma gondii levels in zebra mussel (Dreissena polymorpha) by real-time PCR: an organotropism study.

Water quality is a public health concern that calls for relevant biomonitoring programs. Molecular tools such as polymerase chain reaction (PCR) are progressively becoming more sensitive and more specific than conventional techniques to detect pathogens in environmental samples such as water and organisms. The zebra mussel (Dreissena polymorpha) has already been demonstrated to accumulate and concentrate various human waterborne pathogens. In this study, first, a spiking experiment to evaluate detection levels of Toxoplasma gondii DNA in zebra mussel organs using real-time PCR was conducted. Overall, lower DNA levels in the hemolymph, digestive gland, and remaining tissues (gonad and foot) were detected compared to mantle, muscle, and gills. Second, an in vivo experiment with 1000 T. gondii oocysts per mussel and per day for 21 consecutive days, followed by 14days of depuration time in protozoa-free water was performed. T. gondii DNA was detected in all organs, but greatest concentrations were observed in hemolymph and mantle tissues compared to the others organs at the end of the depuration period. These results suggest that (i) the zebra mussel is a potential new tool for measuring T. gondii concentrations and (ii) real-time PCR is a suitable method for pathogen detection in complex matrices such as tissues.

Seropositivity and serointensity of Toxoplasma gondii antibodies and DNA among patients with schizophrenia.

The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.

Toxoplasmosis-associated abortion and stillbirth in Tehran, Iran

Objectives: This study was aimed to evaluate the role of toxoplasmosis in etiology of abortion and stillbirth based on molecular and serological techniques.Material and methods: A total of 110 pregnant women with abortion and stillbirth were enrolled as the case group, and 110 pregnant women with normal delivery were enrolled as the control group. Serological and molecular detections of Toxoplasma gondii were assessed by ELISA and PCR methods.Results: The seroprevalence of IgG was 25.5% in the case group (26.8% in abortion and 21.4% in stillbirth) and 26.4% in the control group. IgM seropositivity was detected in 2.7% of the case group (3.6% in abortion and 0% in stillbirth) and 0.9% of the control group (p = 0.37). Toxoplasma gondii DNA was detected in 6.4% of the case group (7.3% in abortion and 3.6% in stillbirth) and 1.8% of the control group by PCR (p = 0.17). The major risk factor of congenital toxoplasmosis was the history of eating undercooked meat (p = 0.06).Conclusion: Results of this study revealed that the rate of PCR positive in women with abortion and stillbirth was 3.7 times higher than that in normal delivery, but the difference was not statistically significant. These findings suggest that toxoplasmosis can be involved in etiology of abortion and stillbirth.

Detection of Toxoplasma gondii DNA in naturally infected sheep's milk.

The objective of this study was to verify whether Toxoplasma gondii is excreted in the milk of naturally infected sheep. In order to accomplish this, 275 lactating ewes were used; these were bred extensively in 17 estates distributed across nine cities. Polymerase chain reaction amplification was used to detect T. gondii DNA in milk samples, and the indirect immunofluorescence test was employed for the detection of anti-T. gondii IgG antibodies in the sera, with a cut-off value of 1:64. It was possible to verify the presence of the parasite DNA in 6.5% (18/275) of the studied animals. Anti-T. gondii antibodies were present in 41.5% of the animals studied (114/275). There was no correlation between parasite excretion in milk and the presence of IgG in 38.9% of the studied animals (7/18). The high seropositivity and the presence of parasite DNA in the milk led to the conclusion that T. gondii infection is present in the sheep population in southern and southwestern Bahia, and that there is a risk of the human population becoming infected due to the consumption of raw, in natura milk.

Occurrence of anti-Toxoplasma gondii antibodies and parasite DNA in raw milk of sheep and goats of local breeds reared in Northeastern Brazil

The aim of the present study was to detect Toxoplasma gondii DNA in raw milk samples of goats and sheep of local breeds from the semi-arid region of the states of Pernambuco and Paraíba, Northeastern Brazil. Serum and milk samples were collected from 243 animals (186 goats and 57 sheep). The Indirect fluorescent antibody test (IFAT) was used to search for anti-T. gondii IgG antibodies with a cutoff of 64. Subsequently, the raw milk samples were subjected to DNA extraction and PCR to detect DNA of T. gondii. The IFAT results showed a 6.58% (16/243) positivity when all the samples were considered and a positivity of 15.78% (9/57) and 3.76% (7/186) for goats and sheep samples, respectively. The PCR assay detected T. gondii DNA in 2.06% (5/243) of all the samples tested. All the PCR positive samples were from goats. This result shows the importance of adopting measures of flock's sanitary management and avoiding the consumption of raw milk may constitute a potential risk to the health of milk consumers in this region.

Freezing and storage at −20 °C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis

Nucleic acid-based testing has become crucial for toxoplasmosis diagnosis. For retrospective (forensic or scientific) studies, optimal methods must be employed for DNA long-term storage. We compared Toxoplasma gondii detection before and after DNA storage using real-time PCR. No significant differences were found depending on duration or storage conditions at −20 °C or −80 °C.

Molecular detection of Toxoplasma gondii DNA in milk and risk factors analysis of seroprevalence in pregnant women at Sharkia, Egypt

Molecular detection of Toxoplasma gondii DNA in milk and risk factors analysis of seroprevalence in pregnant women at Sharkia, Egypt

Multisystemic toxoplasmosis associated with a type II-like Toxoplasma gondii strain in a New Zealand fur seal (Arctocephalus forsteri) from New South Wales, Australia

We report the first confirmed case of toxoplasmosis in an Australian pinniped. Presence of Toxoplasma gondii DNA was detected in the brain of a free-ranging subadult New Zealand fur seal (Arctocephalus forsteri) with nonsuppurative meningoencephalitis, hypophysitis, posterior uveitis, retrobulbar cellulitis, and myocarditis associated with protozoan cysts and tachyzoites. The emaciated seal stranded moribund on a beach in northern Sydney in New South Wales. Histopathology coupled with specific immunohistochemistry and PCR assays confirmed the presence of T. gondii. The T. gondii sample (NZfs8825) identified in this study has an identical genotype as the type II (ToxoDB PCR-RFLP genotype #1) based on the direct sequencing and virtual RFLP of multilocus DNA markers including SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Direct sequencing of T. gondii B1 DNA marker from the T. gondii sample (NZfs8825) identified a type II-like strain, based on presence of non-archetypal B1 gene polymorphisms previously reported as unique to Australia. This study suggests that T. gondii oocysts originating from mainland Australia, which has a large population of feral cats, may act as a disease threat to native marine fauna. Therefore, emerging toxoplasmosis in the Arctic has a relevant parallel in the Southern Ocean within Australian waters with yet unknown relevance to Antarctica.

Red foxes (Vulpes vulpes) in Romania are carriers of Toxoplasma gondii but not Neospora caninum.

Brain samples from 182 red foxes (Vulpes vulpes) from Romania were examined using a standard PCR technique. Results provide evidence of Toxoplasma gondii (11 foxes=6.0%) and Neospora caninum (1 fox=0.5%) DNA in red foxes from Romania. No coinfections were found.

Produce immunogenic monocyte derived-dendritic cells by Toxoplasma gondii proteins (TUM4P.904)

Abstract Background: DCs based immunotherapy is considered as a promising approach for activating immune responses against tumor antigens. It has been believed that TLR ligands derived from microorganisms are more potent in activating DCs. In this study, the effects of Toxoplasma gondii (T.gondii) protein and DNA components on human DCs maturation were evaluated. Method: T.gondii Tachyzoites were obtained by intraperitoneal passage in mice. Protein and DNA components were extracted by relevant kits. Subsequently, protein lysate was fractionated by increasing amounts of ammonium sulfate and after that by ion-exchange chromatography. Protein fractions and DNA components were added to human monocyte derived DCs. After 20h, phenotype markers (CD80, CD83, CD86 and HLA-DR) on DCs were determined by flow cytometry. Also, IL-12p70 and IL-10 concentration in DCs supernatants were evaluated. Ultimately, the effects of different fractions on phagocytosis activity of DCs were compared. Results: We found that T.gondii protein components are more potent in activating DCs than DNA components. Moreover, there is a considerable difference in phenotype markers and phagocytic activity between DCs matured by different protein fractions. Also, the ability of some protein fractions is higher in activating DCs to produce IL-12p70 and lower IL-10. Conclusion: The protein fractions with high ability can be used to produce potent DCs for instructing immune responses against tumor antigens.