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PubMed1993

Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole in Toxoplasma gondii.

The monoclonal antibody (mAb) TG17.179 recognizes an excreted-secreted antigen (ESA) of 28.5 kDa named Gra 2, which is stored in the dense granules of Toxoplasma cells and secreted into the parasitophorous vacuole after host cell invasion. Screening of an expression cDNA library with TG17.179 led to the isolation of several clones, the longest one (clone L) being of 1030 bp. Clone L cDNA was found to be homologous to a previously described composite cDNA encoding a P28 protein of Toxoplasma gondii. Characterization of one genomic clone indicates that the complete GRA 2 gene is about 1.3 kb in length, including an intron of 241 bp. Northern blot and primer extension analyses confirmed the size of the mature messenger (1.1 kb). Amino acid partial sequencing of the native antigen purified by HPLC and metabolic radiolabelings of ESAs perfectly matched the primary amino acid structure deduced from the clone L cDNA. This primary translation product consists of an 185 amino acid polypeptide (19.8 kDa) including a 23 amino acid signal sequence. The presence of many serine and threonine residues may indicate an O-glycosylation. The predicted mature polypeptide shows an internal helical domain with 2 amphipathic alpha-helices. These might be involved in the association of Gra 2 with the membranous network within the parasitophorous vacuole.

Amino Acid Sequence↗
PubMed1994

Cloning of a cDNA encoding the dense granule protein GRA3 from Toxoplasma gondii.

GRA3 is a 30-kDa protein located inside the dense granules of Toxoplasma gondii. Following invasion and exocytosis of dense granules within the parasitophorous vacuole, GRA3 becomes associated with the parasitophorous vacuolar membrane (PVM) and extensions of the PVM which protrude into the cytoplasm. A partial cDNA encoding GRA3 was isolated from a Toxoplasma gondii expression library using polyclonal and monoclonal antibodies to the mature GRA3 protein of tachyzoites. Antibodies affinity purified using the cloned fusion protein reacted with a 30-kDa band on immunoblots and recognized dense granules, the PVM, and PVM extensions by immunofluorescence staining of infected cells. Northern blot analysis indicated the major transcript was of a slightly larger size, and the complete cDNA encoding GRA3 was subsequently obtained. Southern blot analysis suggests that GRA3 is present as a single copy. The cDNA encodes two methionines at the N-terminus followed by an open reading frame with a hydrophobic region of 22 amino acids flanked by charged residues consistent with a signal sequence. Four shorter hydrophobic regions occur but are insufficient to span the membrane. No significant homology was detected to other proteins, including other dense granule proteins. In vitro translation of RNA generated from the cDNA containing either one or two of the N-terminal methionines yielded peptides with apparent M(r) of 35,000 and 37,000 respectively. Translation of RNA from the cDNA containing only the second initiation site in the presence of dog pancreas microsomes resulted in reduction of 4 kDa, sufficient to account for removal of the putative signal sequence.

Amino Acid Sequence↗
PubMed1984

DNA circles with cruciforms from Isospora (Toxoplasma) gondii.

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.

DNA, Bacterial↗
PubMed1992

Prevalence of Toxoplasma gondii antibodies in pastoral camels (Camelus dromedarius) in the Butana plains, mid-Eastern Sudan.

A total of 482 serum samples from pastoral camels in the Butana plains, mid-Eastern Sudan, were tested for Toxoplasma antibodies by the latex agglutination test (LAT). Sixty-seven percent of the camels were seroreactive. The prevalence rate of seroreactivity increased significantly with age (P less than 0.01) and was highest among camels aged over 7 years (74.2%). The prevalence rate of seropositivity decreased proportionally with the level of serum dilution. At dilutions of 1:32 and above, the prevalence rate was 25.9%. There were no sex-linked differences in seroreactivity. This study suggests widespread infection with Toxoplasma gondii among pastoral camels, a finding that warrants a closer look into the possible ways infection is acquired by camels in their arid environment, its economic impact, as well as its public health significance, especially among the nomads who consume cameline milk and liver raw.

Age Factors↗
PubMed1993

Unidentified Toxoplasma-like tissue cysts in the brains of three cats.

Tissue cysts of an unidentified Toxoplasma-like parasite were found in three of 103 cats that were diagnosed histologically with toxoplasmosis-like illness at the Angell Memorial Animal Hospital, Boston, MA, between 1952 and 1991. Tissue cysts in these three cats were confined to the brain and two cats had concurrent lymphoid disorders. The tissue cysts of the unidentified parasite were about twice the size of Toxoplasma gondii tissue cysts.

Animals↗
PubMed1995

Immune responses in sheep after immunization with Toxoplasma gondii antigens incorporated into iscoms.

An immunization and infection experiment using 12 sheep was conducted to study the immune responses elicited by an experimental vaccine consisting of Toxoplasma gondii antigens incorporated into immunostimulating complexes (iscoms). Five sheep were immunized subcutaneously with Toxoplasma iscoms. Two doses were given, with a 6 week interval, and 22 days after the second immunization, these five sheep and five non-immunized sheep were inoculated orally with T. gondii oocysts. The two remaining animals served as non-immunized, uninfected controls. The antibody response was analysed by an indirect fluorescent antibody test detecting IgM and an enzyme-linked immunosorbent assay detecting IgG. The first immunization induced low levels of both IgM and IgG, and the second resulted in high levels of IgG but no marked IgM response. After infection, a further increase in IgG was observed in the immunized animals. In the non-immunized sheep, substantial IgM and IgG levels were detected following infection. Immunoblotting analysis indicated that the antibody response to immunization was directed against the same T. gondii antigen as the early antibody response after infection in the non-immunized sheep. Antibodies recognizing the P30 antigen appeared first, followed by antibodies to P22 and other antigens which were probably also of membrane origin. Lymphocyte stimulation tests were performed 15 and 21 days after the last immunization and 105 days after infection. Significant antigen-induced proliferative responses were observed after immunization as well as after infection.

Animals↗
PubMed1994

Seroprevalence of Toxoplasma gondii in Danish farmed mink (Mustela vison S.).

One hundred and ninety-five mink sera randomly selected from 17 Danish mink farms were evaluated for the presence of Toxoplasma gondii antibodies in the latex agglutination test. Six (3%) sera contained T. gondii antibodies in titres of 1:64 or more. The estimated 3% prevalence means that 300,000 mink out of a total mink population of ten million might be infected with Toxoplasma gondii. This large number of possible sero-positive mink in Denmark indicates that there exists a potential risk of acquiring toxoplasmosis by pelting mink.

Agricultural Workers' Diseases↗
PubMed1994

Determination of Toxoplasma gondii in several organs of cattle by carbon immunoassay (CIA) testing.

Samples of muscle, heart, liver and lung were tested for the presence of Toxoplasma gondii using mice as a biologic detector and the carbon immunoassay (CIA) for immunological diagnosis. In positive tissues the percentage of samples positive ranged from 10 to 50% which indicates the importance of cattle in the epidemiology of Toxoplasma in Costa Rica. We discuss these findings in relation to the transmission of the parasite in Costa Rica.

Animals↗
PubMed1996

Seroprevalence of Toxoplasma gondii in cattle, sheep and goats in Iran.

Serum samples from 2000 cows, 3311 sheep and 638 goats from Iran were examined for antibodies to Toxoplasma gondii by use of the latex agglutination (LAT) and indirect hemagglutination tests (IHAT). Antibodies to T. gondii were found in 24.50% sheep and 19.25% of goats. Antibodies to T. gondii were not detected in cow sera by LAT and IHAT in 1:8 and 1:64 dilutions of bovine sera, respectively. Toxoplasma gondii was not found in tissues of 300 aborted fetuses from cows by direct microscopy and bioassay in mice.

Animals↗
PubMed2005

Seroprevalence of Toxoplasma gondii in farm-reared ostriches and wild game species from Zimbabwe.

One hundred and seventy one serum samples from 10 game species from Zimbabwe were tested for IgG antibodies to Toxoplasma gondii infection using the modified agglutination test (MAT). Significantly higher seroprevalences were found in the felidae (Panthera leo) (92% of 26), bovidae (Tragelaphus species) (55.9% of 34) and farm-reared struthionidae (Struthio camelus) (48% of 50) compared to the other groups tested. Among the bovidae, the nyala (Tragelaphus angasii) had the highest seroprevalence of 90% (9/10). Anti-Toxoplasma antibody prevalences in browsers [greater kudu (Tragelaphus strepsiceros) (20% of 10), giraffe (Giraffa camelopardalis) (10% of 10) and elephant (Loxodonta africana) (10% of 20)] were generally in the lower range. No antibodies were detected in the wild African suidae [warthog (Phacochoerus africanus) and bushpig (Potamochoerus larvatus)]. Attempts to isolate T. gondii from the heart muscles of seropositve ostriches by subinoculation in BALB/c mice were unsuccessful.

Agglutination Tests↗
PubMed2005

6-Benzylthioinosine analogues as subversive substrate of Toxoplasma gondii adenosine kinase: activities and selective toxicities.

Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-substituted purine nucleosides act as subversive substrates of T. gondii, but not the human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Herein, we report the testing of newly synthesized 6-benzylthioinosine analogues with various substituents on the phenyl ring of their benzyl group as subversive substrates of T. gondii adenosine kinases. The binding affinity of these compounds to T. gondii adenosine kinase and their efficacy as antitoxoplasmic agents varied depending on the nature and position of the various substituents on the phenyl ring of their benzyl group. p-Cyano-6-benzylthioinosine and 2,4-dichloro-6-benzylthioinosine were the best ligands. In general, analogues with substitution at the para position of the phenyl ring were better ligands than those with the same substitutions at the meta or ortho position. The better binding of the para-substituted analogues is attributed to the combined effect of hydrophobic as well as van der Waals interactions. The 6-benzylthioinosine analogues were devoid of host-toxicity but all showed selective anti-toxoplasmic effect in cell culture and animal models. These results further confirm that toxoplasma adenosine kinase is an excellent target for chemotherapy and that 6-substituted purine nucleosides are potential selective antitoxoplasmic agents.

Adenosine Kinase↗
PubMed2005

Toxoplasma gondii: microneme protein MIC2.

The phylum Apicomplexa contains parasites responsible for a variety of diseases including malaria, cryptosporidiosis, and toxoplasmosis. One of the common features of these parasites is that they contain a set of apical organelles whose sequential secretion is required for the invasion of host cells. Microneme proteins are the main adhesins involved in the attachment to the host cell surface by apicomplexans. The microneme protein MIC2, produced by Toxoplasma gondii, is conserved in apicomplexans and serves as a model to understand the first steps of invasion by the phylum. New data about the structure-function relationship of MIC2 reinforce the critical role of this protein in the successful invasion of cells by Toxoplasma and reveal potential therapeutic targets that may be used to control toxoplasmosis.

Animals↗
PubMed2006

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.

Animals↗
PubMed2006

Toxoplasma gondii: immunogenicity and protection by P30 peptides in a murine model.

Vaccines are promising for the control of toxoplasmosis. Here, we evaluated the immunogenicity of 17 peptides derived from SAG1 surface protein of Toxoplasma gondii in CH3 mice. Only 8 of 16 peptides induced specific antibodies. After a lethal challenge, only the vaccination with 4 of 17 peptides that were from the carboxy terminal end of the protein conferred significant survival. Our work shows that vaccination with peptides from the carboxy-terminal positions of SAG1 major surface protein of Toxoplasma protects mice against a lethal challenge.

Amino Acid Sequence↗
PubMed2006

Identification of Toxoplasma gondii proteins binding human lactoferrin: a new aspect of rhoptry proteins function.

In this paper, we report on the isolation, purification and identification of two Toxoplasma gondii membrane proteins binding human lactoferrin. Parasite membrane proteins were isolated using the commercial Mem-PER Eukaryotic Membrane Protein Extraction System. After purification by lactoferrin affinity chromatography, three protein bands were detected with the molecular mass of 74, 63 and 58 kDa, two of which (63 and 58 kDa) specifically bound biotin labeled human lactoferrin as examined by competitive inhibition. Further identification of latter proteins by ESI/MS/MS amino acid sequencing technique revealed those proteins as Toxoplasma ROP4 (band 63 kDa) and ROP2 (band 58 kDa) antigens known to be involved in many mechanisms essential for the parasite pathogenicity, including host lactoferrin acquisition as determined in this study.

Amino Acid Sequence↗
PubMed2004

Mechanisms underlying the manipulation of host apoptotic pathways by Toxoplasma gondii.

The establishment of a productive infection by an obligate intracellular pathogen is dependent on subversion of cellular defences. Apoptosis, or programmed cell death, is a property of metazoan cells that plays a critical role in inhibiting the proliferation of invasive organisms and viruses thereby protecting uninfected cells and limiting damage to the host organism. Not surprisingly, manipulation of the machinery of apoptosis plays a critical role in the pathogenesis of several intracellular pathogens. Toxoplasma gondii, arguably one of the most successful protozoan pathogens, has evolved several strategies to inhibit both the initiation and propagation of the apoptotic cascade. Recent work from several groups indicates an exquisite level of sophistication in the mechanisms to inhibit apoptosis along its diverse pathways. Much of this ability appears to centre around the manipulation of host transcription, specifically of genes involved in the pro-survival/anti-apoptotic response effectively manipulating the infected cell into a highly anti-apoptotic state. The implications of these observations extend beyond Toxoplasma biology to the broader area of microbial pathogenesis and cell signalling in mammalian cells.

Animals↗
PubMed2004

An unusual genotype of Toxoplasma gondii is common in California sea otters (Enhydra lutris nereis) and is a cause of mortality.

Toxoplasma gondii-associated meningoencephalitis is a significant disease of California sea otters (Enhydra lutris nereis), responsible for 16% of total mortality in fresh, beachcast carcasses. Toxoplasma gondii isolates were obtained from 35 California otters necropsied between 1998 and 2002. Based on multi-locus PCR-restriction fragment length polymorphism and DNA sequencing at conserved genes (18S rDNA, ITS-1) and polymorphic genes (B1, SAG1, SAG3 and GRA6), two distinct genotypes were identified: type II and a novel genotype, here called type x, that possessed distinct alleles at three of the four polymorphic loci sequenced. The majority (60%) of sea otter T. gondii infections were of genotype x, with the remaining 40% being of genotype II. No type I or III genotypes were identified. Epidemiological methods were used to examine the relationship between isolated T. gondii genotype(s) and spatial and demographic risk factors, such as otter stranding location and sex, as well as specific outcomes related to pathogenicity, such as severity of brain inflammation on histopathology and T. gondii-associated mortality. Differences were identified with respect to T. gondii genotype and sea otter sex and stranding location along the California coast. Localised spatial clustering was detected for both type II (centred within Monterey Bay) and x (centred near Morro Bay)-infected otters. The Morro Bay cluster of type x-infected otters overlaps previously reported high-risk areas for sea otter infection and mortality due to T. gondii. Nine of the 12 otters that had T. gondii-associated meningoencephalitis as a primary cause of death were infected with type x parasites.

Animals↗
PubMed2004

Genetic diversity, clonality and sexuality in Toxoplasma gondii.

The majority of Toxoplasma gondii strains from a variety of human and animal sources have been grouped into three highly clonal but closely related lineages. The low occurrence of nucleotide differences among the three predominant lineages and their unusual dimorphic allelic composition suggest that they have arisen from a recent common ancestry. Less than 1% of the previously studied strains contain unique genotypes and high divergence of DNA sequence, and therefore are considered 'exotic' or 'atypical' strains. The seemingly low genetic diversity in T. gondii may have been underestimated because most parasite strains in previous studies were collected from human patients and domestic animals in North America and Europe. To investigate the genetic diversity of T. gondii, we analysed parasite strains isolated from remote geographical regions by multilocus microsatellite sequencing and phylogenetic analysis. The genetic diversity indices, the molecular analysis of microsatellite genotypes and the constructed phylogram considered together suggest that the global T. gondii population is highly diversified and not characteristic of a clonal organism. The most parsimonious hypothesis is that T. gondii presents a complex population structure with a mix of clonal and sexual propagation as a function of the environmental conditions. The comparison between domestic strains data on one hand and wild strains data on the other hand is in favour of more frequent sexual recombinations in wild environment even though Toxoplasma subpopulation in human and domestic animals is largely clonal.

Animal Diseases↗