Toxoplasma gondii: from animals to humans

Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Its causative agent, Toxoplasma gondii, is a facultatively heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species. If first contracted during pregnancy, T. gondii may be transmitted vertically by tachyzoites that are passed to the foetus via the placenta. Horizontal transmission of T. gondii may involve three life-cycle stages, i.e. ingesting infectious oocysts from the environment or ingesting tissue cysts or tachyzoites which are contained in meat or primary offal (viscera) of many different animals. Transmission may also occur via tachyzoites contained in blood products, tissue transplants, or unpasteurised milk. However, it is not known which of these routes is more important epidemiologically. In the past, the consumption of raw or undercooked meat, in particular of pigs and sheep, has been regarded as a major route of transmission to humans. However, recent studies showed that the prevalence of T. gondii in meat-producing animals decreased considerably over the past 20 years in areas with intensive farm management. For example, in several countries of the European Union prevalences of T. gondii in fattening pigs are now <1%. Considering these data it is unlikely that pork is still a major source of infection for humans in these countries. However, it is likely that the major routes of transmission are different in human populations with differences in culture and eating habits. In the Americas, recent outbreaks of acute toxoplasmosis in humans have been associated with oocyst contamination of the environment. Therefore, future epidemiological studies on T. gondii infections should consider the role of oocysts as potential sources of infection for humans, and methods to monitor these are currently being developed. This review presents recent epidemiological data on T. gondii, hypotheses on the major routes of transmission to humans in different populations, and preventive measures that may reduce the risk of contracting a primary infection during pregnancy.     

Differences in iNOS and arginase expression and activity in the macrophages of rats are responsible for the resistance against T. gondii infection

Toxoplasma gondii infects humans and warm blooded animals causing devastating disease worldwide. It has long been a mystery as to why the peritoneal macrophages of rats are naturally resistant to T. gondii infection while those of mice are not. Here, we report that high expression levels and activity of inducible nitric oxide synthase (iNOS) and low levels of arginase-1 (Arg 1) activity in the peritoneal macrophages of rats are responsible for their resistance against T. gondii infection, due to high nitric oxide and low polyamines within these cells. The opposite situation was observed in the peritoneal macrophages of mice. This discovery of the opposing functions of iNOS and Arg 1 in rodent peritoneal macrophages may lead to a better understanding of the resistance mechanisms of mammals, particularly humans and livestock, against T. gondii and other intracellular pathogens.     

Determinants of GBP recruitment to Toxoplasma gondii vacuoles and the parasitic factors that control it

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.     

Seroprevalence of Toxoplasma gondii from free-ranging black bears ( Ursus americanus ) from Florida

Toxoplasma gondii is a significant worldwide parasitic protozoan. In the present study, prevalence of antibodies of T. gondii was examined from 29 free-ranging black bears ( Ursus americanus ) from south-central Florida where the host species was listed as state threatened during this project. Overall T. gondii prevalence was found to be 44.8%, specifically 46.2% in male and 43.8% in female U. americanus , using a modified agglutination test (1:25 titer). Seroprevalence differences between sexes were not significant (P > 0.05). Results of the present study add supportive data to the growing body of evidence suggesting that U. americanus has one of the highest T. gondii seroprevalences among all known intermediate hosts. In addition, our data emphasize the importance of understanding parasitic disease dynamics from a conservation perspective.     

Frequency of anti-Toxoplasma gondii antibodies in cats from Valdivia city, Chile

Toxoplasmosis is a parasitic zoonosis. The importance of the cat as related to this parasitosis lies in the fact that it is not only the definite host of the parasite, but responsible for its dissemination through the release of oocysts, which subsequently infect both humans and other animals. The objective of this study was to determine the serological prevalence of Toxoplasma gondii in the feline population of the city of Valdivia (Chile) and to establish the possible epidemiological implications of this prevalence. With these goals in mind the technique of indirect immunofluorescence was implemented to detect anti- T. gondii species--specific IgG. Blood samples from 97 cats (selected using a directed sampling process) from different sectors of Valdivia were collected. The sample included 46 males and 51 females of different ages, Positive and negative control sera obtained from de United States were used to verify the results, observed by UV microscopy. The anti-feline IgG antibody was used as directed by the manufacturer (Sigma). Of the 97 selected cats, 32 were found to be positive with a titre higher than 1:4, a prevalence of 33.0%. The number of infected males as compared to the number of infected females was found to be statistically insignificant, using the chi-square analysis with p less than 0.1. In contrast, a definite correlation between age and seropositivity was found; infection levels were higher in older animals. These results are consistent with those obtained in different studies on this topic that have been performed in different parts of the world. However, they would seem to be in conflict with other, similar studies that have been done recently in Chile; this may be due to the fact that the cats selected for this study were exposed to different climatic conditions than those examined in previous works. In conclusion, it has been determined that in Valdivia exist cats infected by T. gondii, which indicate the presence of the necessary epidemiological conditions for the persistence of this parasitic cycle and the source of infection for humans and other animals.     

Molecular and biologic characteristics of Toxoplasma gondii isolates from wildlife in the United States

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered more virulent in outbred mice and have been isolated predominantly from clinical cases of human toxoplasmosis, whereas types II and III isolates are considered less virulent for mice and are found in humans and food animals. Little is known of genotypes of T. gondii isolates from wild animals. In the present report, genotypes of isolates of T. gondii from wildlife in the United States are described. Sera from wildlife were tested for antibodies to T. gondii with the modified agglutination test, and tissues from animals with titers of 1:25 (seropositive) were bioassayed in mice. Toxoplasma gondii was isolated from the hearts of 21 of 34 seropositive white-tailed deer (Odocoileus virginianus) from Mississippi and from 7 of 29 raccoons (Procyon lotor); 5 of 6 bobcats (Lynx rufus); and the gray fox (Urocyon cinereoargenteus), red fox (Vulpes vulpes), and coyote (Canis latrans) from Georgia. Toxoplasma gondii was also isolated from 7 of 10 seropositive black bears (Ursus americanus) from Pennsylvania by bioassay in cats. All 3 genotypes of T. gondii based on the SAG2 locus were circulating among wildlife.     

Earthworms as paratenic hosts of toxoplasmosis in eastern barred bandicoots in Tasmania

An experimental feeding study was designed to assess the role of earthworms in the transmission of Toxoplasma gondii infection to eastern barred bandicoots (Perameles gunnii). Six animals with no agglutinating antibodies to T. gondii were fed artificially cultured earthworms that had been maintained in autoclaved nutrient-enriched soil. Two animals were given earthworms that had been maintained in soil contaminated with T. gondii oocysts (P89/VEG strain); two animals were fed on earthworms, which initially had been exposed to soil containing T. gondii oocysts then transferred through three changes of sterile soil; two control bandicoots were fed earthworms maintained in sterile soil. Both bandicoots fed earthworms maintained in T. gondii contaminated soil died 11 and 14 days after feeding. The necropsy findings were consistent with acute toxoplasmosis. Bandicoots fed earthworms exposed to oocysts but then transferred through changes of sterilized soil remained healthy as did control animals. All surviving animals remained seronegative over the 6 wk observation period after feeding. These findings confirm that earthworms, a major component of the natural diet of P. gunnii, can transmit T. gondii infection. It appears that oocysts present in the alimentary tracts of the worms, rather than infective stages of T. gondii in worm somatic tissues, are responsible for these infections.     

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.     

Amylopectin biogenesis and characterization in the protozoan parasite Toxoplasma gondii, the intracellular development of which is restricted in the HepG2 cell line

The obligate intracellular protozoan Toxoplasma gondii belongs to the phylum Apicomplexa, which is composed of numerous parasites causing major diseases such as malaria, toxoplasmosis and coccidiosis. The life cycle of T. gondii involves developmental processes from one stage to another with both asexual and sexual parasitic forms. Throughout their life cycle, some apicomplexan parasites accumulate a crystalline storage polysaccharide analogous to amylopectin within the cytoplasm. In T. gondii, both the slowly dividing encysted bradyzoites and the sporozoites of the sexual stage contain a high number of amylopectin granules (AG), while the rapidly replicating tachyzoites are devoid of amylopectin. It is thought that this storage polysaccharide may represent an energy reserve that could fuel the transition from one developmental stage to another one. At present, by comparison to glycogen and plant starch, little is known about the biosynthesis, structure and biological functions of amylopectin in T. gondii. Here, we describe an in vitro system allowing the production and purification of a large amount of amylopectin, which has been subjected to detailed biochemical and structural analyses. Our data indicate that T. gondii synthesizes a genuine amylopectin following changes in the environmental conditions and that this storage polysaccharide differs from glycogen and starch in terms of glucan chain length.     

Trypanosomiasis of domestic animals in China

Trypanosomiasis caused by Trypanosoma evansi (surra) is a problem of great economic importance in livestock in China because it affects important working animals: buffaloes in the south and east, and horses and cattle in the north and west. In addition, buffaloes are an important source of meat and leather for the Chinese population. In the north and west, T. evansi is found mainly in camels, whereas in the east, the south and the southwest, it is primarily buffaloes, cattle and horses that are affected by this parasitic flagellate. Although trypanosomiasis is one of the most important parasitic diseases of domestic animals in China, little information on this disease has been published. In this article Zhao-Rong Lun, Yuang Fang, Chang-Ji Wang and Reto Brun evaluate trypanosomiasis of domestic animals in China and review epidemiological studies of the past 40 years.     

Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma's, but only 3% of the host's, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host's immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.     

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.     

Examination of gastrointestinal helminth in livestock grazing in grassland of Bangladesh

To determine association of grassland with parasitic diseases of livestock in Bangladesh, the 'Tracer' animals (two cow calves and two goats) were released for a month in a grassland used for communal grazing of livestock near school premise in Kanthal, Trishal, Mymensingh, Bangladesh. After slaughtering of the tracer animals, their gastrointestinal tract examination revealed six species of nematode and one cestode. The nematode species were Haemonchus contortus, Trichostrongylus axei, Mecistocirrus digitatus, Oesophagostomum spp., Trichuris spp. and Bunostomum sp. The cestode was one of the genus Moniezia. With this preliminary study, grasslands are thought to be one of the main sources of gastrointestinal parasitic diseases of livestock in Bangladesh.     

Seroprevalence of Toxoplasma gondii infection in female sterility patients in China

Toxoplasmosis is an important parasitic disease worldwide and is related to certain psychiatric disorders and sterility. In the present study, serum samples from 882 female sterility patients and 107 pregnant-puerperant women were assayed for anti- T. gondii IgG antibodies using ELISA. The overall T. gondii seroprevalence was 14.8%. In the female sterility patients, 15.9% (140/882) were seropositive and, in the pregnant-puerperant women, 5.6% (6/107) were positive for anti- T. gondii IgG antibodies. There was a significant difference between the 2 groups ( P < 0.05). The samples were further divided into 5 groups based on age, but no significant difference was found among the 5 groups (P > 0.05). Results of the present study argue for more attention to prevention of T. gondii infection in the female population and, in particular, women of childbearing age.     

Epidemiology and control prospects of foodborne parasitic zoonoses in the European Union

In the 27 Member States of the European Union, zoonotic parasites transmitted by food are circulating with different prevalence according to the country, the environmental conditions, the human behaviour, and the socio-economic level. Foodborne parasites can be divided in two main groups according to the way of transmission to humans. These foodborne parasites reach the human beings through the consumption of raw infected food such as muscle tissues of different animal species (Toxoplasma gondii, Sarcocystis hominis, Sarcocystis suishominis, Diphyllobotrium latum, Taenia solium, Taenia saginata, Opisthorchis felineus, Anisakis spp., Pseudoterranova spp., Trichinella spp.), or vegetables (Fasciola hepatica), and contaminated food and water resources (Giardia duodenalis, Cryptosporidium spp., T. gondii, Echinococcus granulosus sensu latu, Echinococcus multilocularis, T. solium, Taenia multiceps). As a general role, the control strategies should be based on the education of the consumers, farmers and shepherds, the improvement of farming conditions, the improvement or the development of more sensitive methods to detect these parasites in slaughtered animals and in foodstuff, a control of sewage sludge on pastures and of drinking water resources, and the reduction of contacts between livestock and wild animals which frequently represent the most important reservoir of these pathogens.     

Serotyping of Toxoplasma gondii in chronically infected pregnant women: predominance of type II in Europe and types I and III in Colombia (South America)

Isolates of Toxoplasma gondii, which is responsible for a wide range of clinical manifestations are grouped into three clonal lineages of different virulence in mice. However, it is not clear whether this genotypic pattern is associated with the clinical profile of the disease in humans nor is the geographical distribution of the genotypes known. This is mainly due to difficulties in obtaining parasitic DNA from patients. The available data are therefore limited and originate from acute or congenital infections or from animals. A non-invasive assay is needed to address issues of strain type, geographical distribution and severity of clinical toxoplasmosis. To serotype T. gondii strains, we have developed an enzyme-linked immunosorbent assay (ELISA) that uses polymorphic polypeptides specific to the three clonal lineages and derived from two dense granule antigens, GRA5 and GRA6. Two hundred and fifty-two sera from chronically infected pregnant women from three different European countries and Colombia were investigated. The analysis of genotype-specific antibody response showed a homogeneous type II distribution in the European samples compared with types I and III but no type II in the Colombian population. Our data concord with those obtained from the genotyping of other isolates from Europe and South America. We demonstrated that, despite some limitation due to antigen and/or antibody specificity, serotyping is a promising assay to investigate the relationship between type of strain and severity of the disease.     

Experimental Toxoplasma gondii infection in grey seals (Halichoerus grypus)

Laboratory-reared animals were used to assess the susceptibility of seals (Halichoerus grypus) to Toxoplasma gondii infection. Four seals were each orally inoculated with 100 or 10,000 oocysts of T. gondii (VEG strain), and another 4 seals served as negative controls. Occasionally, mild behavioral changes were observed in all inoculated seals but not in control animals. A modified agglutination test revealed the presence of antibodies to T. gondii in sera collected from inoculated seals and mice inoculated as controls. No evidence of the parasite was found on an extensive histological examination of seal tissues, and immunohistochemical staining of tissue sections from inoculated seals revealed a single tissue cyst in only 1 seal. Control mice inoculated with 10 oocysts from the same inoculum given to seals became serologically and histologically positive for T. gondii. Cats that were fed brain or muscle tissue collected from inoculated seals passed T. gondii oocysts in feces. This study demonstrates that T. gondii oocysts can establish viable infection in seals and supports the hypothesis that toxoplasmosis in marine mammals can be acquired from oocysts in surface water runoff and sewer discharge.     

First report of genotyping of Toxoplasma gondii isolates from wild birds in China

Toxoplasma gondii is an important cosmopolitan opportunistic protozoan parasite, which threatens the health of human beings and animals. Genetic characterization of isolates from South America has revealed high genetic diversity. In contrast, isolates from North America and Europe were highly clonal, with 3 major lineages known as the Types I, II, and III. However, limited information on T. gondii genotypes has been reported in The People's Republic of China. Here we conducted a survey to determine genetic diversity of this parasite in wild birds of China. In total, tissues from breast muscle of 178 wild birds, including 98 common pheasants ( Phasianus colchicus ), 35 tree sparrows ( Passer montanus ), 22 house sparrows ( Passer domesticus ), 20 saxaul sparrows ( Passer ammodendri ), and 1 cinnamon sparrow ( Passer rutilans ), were tested for T. gondii infection, 4 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 3 isolates were genotyped with complete data for all loci, and 2 genotypes (Type I and Type II variant) were identified. This is the first report of genetic typing of T. gondii isolates from wild birds from different regions in China. The results suggest that the Type I and II variant strains are circulating in wild birds in China, and these birds are potential reservoirs for T. gondii transmission.     

Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters

Sea otters in California are commonly infected with Toxoplasma gondii. A unique Type X strain is responsible for 72% of otter infections, but its prevalence in terrestrial animals and marine invertebrates inhabiting the same area was unknown. Between 2000 and 2005, 45 terrestrial carnivores (lions, bobcats, domestic cats and foxes) and 1396 invertebrates (mussels, clams and worms) were screened for T. gondii using PCR and DNA sequencing to determine the phylogeographic distribution of T. gondii archetypal I, II, III and Type X genotypes. Marine bivalves have been shown to concentrate T. gondii oocysts in the laboratory, but a comprehensive survey of wild invertebrates has not been reported. A California mussel from an estuary draining into Monterey Bay was confirmed positive for Type X T. gondii by multilocus PCR and DNA sequencing at the B1 and SAG1 loci. This mussel was collected from nearshore marine waters just after the first significant rainfall event in the fall of 2002. Of 45 carnivores tested at the B1, SAG1, and GRA6 typing loci, 15 had PCR-confirmed T. gondii infection; 11 possessed alleles consistent with infection by archetypal Type I, II or III strains and 4 possessed alleles consistent with Type X T. gondii infection. No non-canonical alleles were identified. The four T. gondii strains with Type X alleles were identified from two mountain lions, a bobcat and a fox residing in coastal watersheds adjacent to sea otter habitat near Monterey Bay and Estero Bay. Confirmation of Type X T. gondii in coastal-dwelling felids, canids, a marine bivalve and nearshore-dwelling sea otters supports the hypotheses that feline faecal contamination is flowing from land to sea through surface runoff, and that otters can be infected with T. gondii via consumption of filter-feeding marine invertebrates.