Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Toxoplasma gondii infection induces lipid metabolism alterations in the murine host

Host lipids have been implicated in the pathogenesis of Toxoplasma gondiiinfection. To determine if Toxoplasmainfection influences the lipid status in the normal host, we assessed serum lipids of Swiss-Webster mice during infection with the BGD-1 strain (type-2) at a series of time points. Mice were bled at days zero and 42 post-infection, and subgroups were additionally bled on alternating weeks (model 1), or sacrificed at days zero, 14 and 42 (model 2) for the measurement of total cholesterol (Chl), high density lipoproteins (HDL), low density lipoproteins (LDL) and triglycerides and adiponectin. At day 42, brains were harvested for cyst enumeration. A significant decrease (p = 0.02) in HDL and total Chl was first noted in infected vs. control mice at day 14 and persisted to day 42 (p = 0.013). Conversely, LDL was unaltered until day 42, when it increased (p = 0.043). Serum LDL levels at day 42 correlated only with cyst counts of above 300 (found in 44% mice), while the change in HDL between days zero and 42 correlated with both the overall mean cyst count (p = 0.041) and cyst counts above 300 (p = 0.044). Calculated per cyst, this decrease in HDL in individual animals ranged from 0.1-17 µmol/L, with a mean of 2.43 ± 4.14 µmol/L. Serum adiponectin levels remained similar between infected and control mice throughout the experiment.

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

"If you know the enemy and know yourself, you need not fear the result of a hundred battles. If you know yourself but not the enemy, for every victory gained you will also suffer a defeat" (SunTzu the Art of War, 544-496 BC). Although written for the managing of conflicts and winning clear victories, this basic guideline can be directly transferred to our battle against apicomplexan parasites and how to focus future basic research in order to transfer the gained knowledge to a therapeutic intervention stratey. Over the last two decades the establishment of several key-technologies, by different groups working on Toxoplasma gondii, made this important human pathogen accessible to modern approaches in molecular cell biology. In fact more and more researchers get attracted to this easy accessible model organism to study specific biological questions, unique to apicomplexans. This fascinating, unique biology might provide us with new therapeutic options in our battle against apicomplexan parasites by finding its Achilles' heel. In this article we argue that in the absence of a powerful high throughput technology for the characterisation of essential gene of interests a coordinated effort should be undertaken to convert our knowledge of the genome into one of the phenome.

Population biology of Toxoplasma gondii: what's out and where did they come from

Until recently, Toxoplasma gondii was considered to be clonal with little genetic variability. In this paper we summarize recent genotyping data from chickens in Brazil, and pigs, lambs and white-tailed deer in the USA, to demonstrate the high genetic diversity and geographical distribution of T. gondii. A total of 149 T. gondii isolates from 13 geographical areas of Brazil and 182 T. gondii isolates from pigs, 53 isolates from sheep and 15 isolates from fetal white-tailed deer from USA were genotyped using the 10 RCFP-PCR genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genotyping of 149 T. gondii isolates from free range chickens in Brazil identified 58 genotype groups. No clonal type II lineage was found. Of the 253 isolates from animals from USA, 18 genotypes were identified, predominantly type II. These studies indicate a higher genetic diversity than previously recognized.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3% of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Advances in understanding immunity to Toxoplasma gondii

Toxoplasma gondii is an important cause of clinical disease in fetuses, infants and immunocompromised patients. Since the discovery of T. gondii 100 years ago, this pathogen and the host's immune response to toxoplasmosis have been studied intensely. This has led to the development of a working model of immunity to T. gondii, and has also resulted in fundamental new insights into the role of various cytokines in resistance to infection. By examining this organism, researchers have identified many of the requirements for resistance to intracellular pathogens and characterized numerous regulatory factors, including interleukin-10 (IL-10) and IL-27, which control inflammatory processes. In the next 100 years of T. gondii immunobiology, researchers will have the opportunity to answer some of the long-standing questions in the field using new techniques and reagents. These future studies will be vital in building a more comprehensive model of immunity to this pathogen and in advancing our understanding of immunoregulation, particularly in humans. Ultimately, the challenge will be to use this information to develop new vaccines and therapies to manage disease in affected patients.

Small intestinal inflammation following oral infection with Toxoplasma gondii does not occur exclusively in C57BL/6 mice: review of 70 reports from the literature

Small intestinal immunopathology following oral infection with tissue cysts of Toxoplasma gondii has been described in C57BL/6 mice. Seven days after infection, mice develop severe small intestinal necrosis and succumb to infection. The immunopathology is mediated by local overproduction of Th1-type cytokines, a so-called "cytokine storm". The immunopathogenesis of this pathology resembles that of inflammatory bowel disease in humans, i.e., Crohn's disease. In this review, we show that the development of intestinal pathology following oral ingestion of T. gondii is not limited to C57BL/6 mice, but frequently occurs in nature. Using a Pubmed search, we identified 70 publications that report the development of gastrointestinal inflammation following infection with T. gondii in 63 animal species. Of these publications, 53 reports are on accidental ingestion of T. gondii in 49 different animal species and 17 reports are on experimental infections in 19 different animal species. Thus, oral infection with T. gondii appears to cause immunopathology in a large number of animal species in addition to mice. This manuscript reviews the common features of small intestinal immunopathology in the animal kingdom and speculates on consequences of this immunopathology for humankind.

Toxoplasma gondii and the Immunity-Related GTPase (IRG) resistance system in mice: a review

The Immunity Related GTPases (IRG proteins) constitute a large family of interferon-inducible proteins that mediate early resistance to Toxoplasma gondii infection in mice. At least six members of this family are required for resistance of mice to virulent T. gondii strains. Recent results have shown that the complexity of the resistance arises from complex regulatory interactions between different family members. The mode of action against T. gondii depends on the ability of IRG proteins to accumulate on the parasitophorous vacuole of invading tachyzoites and to induce local damage to the vacuole resulting in disruption of the vacuolar membrane. Virulent strains of T. gondiiovercome the IRG resistance system, probably by interfering with the loading of IRG proteins onto the parasitophorous vacuole membrane. It may be assumed that T. gondii strains highly virulent for mice will be disadvantaged in the wild due to the rapid extinction of the infected host, while it is self-evident that susceptibility to virulent strains is disadvantageous to the mouse host. We consider the possibility that this double disadvantage is compensated in wild populations by segregating alleles with different resistance and susceptibility properties in the IRG system.

Veterinary vaccines against Toxoplasma gondii

Toxoplasma gondii has a very wide intermediate host range and is thought to be able to infect all warm blooded animals. The parasite causes a spectrum of different diseases and clinical symptoms within the intermediate hosts and following infection most animals develop adaptive humoral and cell-mediated immune responses. The development of protective immunity to T. gondii following natural infection in many host species has led researchers to look at vaccination as a strategy to control disease, parasite multiplication and establishment in animal hosts. A range of different veterinary vaccines are required to help control T. gondii infection which include vaccines to prevent congenital toxoplasmosis, reduce or eliminate tissue cysts in meat producing animals and to prevent oocyst shedding in cats. In this paper we will discuss some of the history, challenges and progress in the development of veterinary vaccines against T. gondii.

Vaccines against Toxoplasma gondii: challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge.

CD40, autophagy and Toxoplasma gondii

Toxoplasmagondii represents a pathogen that survives within host cells by preventing the endosomal-lysosomal compartments from fusing with the parasitophorous vacuoles. The dogma had been that the non-fusogenic nature of these vacuoles is irreversible. Recent studies revealed that this dogma is not correct. Cell-mediated immunity through CD40 re-routes the parasitophorous vacuoles to the lysosomal compartment by a process called autophagy. Autophagosome formation around the parasitophorous vacuole results in killing of the T. gondii. CD40-induced autophagy likely contributes to resistance against T. gondii particularly in neural tissue.

Host immune response to Toxoplasma gondii and Ascaris lumbricoides in a highly endemic area: evidence of parasite co-immunomodulation properties influencing the outcome of both infections

Toxoplasmosis and ascaridiasis evoke polar Th-1 and Th-2 host immune responses, respectively. A study to investigate the specific cytokine profile production by in vitro cultures of peripheral blood mononuclear cells from individuals living under precarious sanitary conditions in a highly endemic area for the parasites Toxoplasma gondii and Ascaris lumbricoides was conducted. High levels of both IFN-³ (Th-1) and IL-13 (Th-2) were observed in groups of co-infected individuals presenting toxoplasmic ocular lesions. Significantly lower IL-10 and TGF-² levels were produced by co-infected individuals in comparison with groups of individuals not infected with A. lumbricoides and either positive or negative for T. gondii living under good sanitary conditions (control groups). The possible influence of co-parasitism on the clinical presentation of ocular toxoplasmosis is discussed.

The sero-prevalence of Toxoplasma gondii in British marine mammals

Serum samples from 101 stranded or bycatch cetaceans from British waters were screened for Toxoplasma gondii-specific antibodies using the Sabin Feldman Dye Test. Relatively high seropositivity was recorded in short-beaked Delphinus delphis and this study presents the first documented case of Toxoplasma in a humpback whale Megaptera novaeangliae.

Toxoplasma gondii in animals used for human consumption

Food-borne toxoplasmosis in humans may result from exposure to different stages of Toxoplasma gondii, in particular from the ingestion of tissue cysts or tachyzoites contained in meat, primary offal (viscera) or meat-derived products of many different animals, or the ingestion of sporulated oocysts that are contained in the environment and may contaminate food and water. Although the potential for transmission of the parasite to humans via food has been known for several decades, it is not known which routes are most important from a public health point of view. It is likely that transmission of the parasite to humans is influenced not only by the potential contamination of various food sources, but also by the individual behaviour of consumers in different ethnic groups and geographical regions. Most current methods for detection of T. gondii in meat-producing animals, in products of animal origin, or in the environment are insufficient because they do not allow quantification of infectious stages. Hence, most studies report only qualitative data from which it is difficult to assess the true risk of infection in individual cases. There is a need for quantitative data so that efficient strategies to reduce food-borne transmission of T. gondii to humans can be developed.

Congenital toxoplasmosis: evaluation of serological methods for the detection of anti-Toxoplasma gondii IgM and IgA antibodies

A study was carried out to evaluate the presence of serological markers for the immunodiagnosis of the vertical transmission of toxoplasmosis. We tested the sensitivity, specificity and predictive values (positive and negative) of different serological methods for the early diagnosis of congenital toxoplasmosis. In a prospective longitudinal study, 50 infants with suspected congenital toxoplasmosis were followed up in the ambulatory care centre of Congenital Infections at University Hospital in Goiânia, Goiás, Brazil, from 1 January 2004-30 September 2005. Microparticle Enzyme Immunoassay (MEIA), Enzyme-Linked Fluorescent Assay (ELFA) and Immune-Fluorescent Antibody Technique (IFAT) were used to detect specific IgM anti-Toxoplasma gondii antibodies and a capture ELISA was used to detect specific IgA antibodies. The results showed that 28/50 infants were infected. During the neonatal period, IgM was detected in 39.3% (11/28) of those infected infants and IgA was detected in 21.4% (6/28). The sensitivity, specificity and predictive values (positive and negative) of each assay were, respectively: MEIA and ELFA: 60.9%, 100%, 100%, 55.0%; IFAT: 59.6%, 91.7%, 93.3%, 53.7%; IgA capture ELISA: 57.1%, 100%, 100%, 51.2%. The presence of specific IgM and IgA antibodies during the neonatal period was not frequent, although it was correlated with the most severe cases of congenital transmission. The results indicate that the absence of congenital disease markers (IgM and IgA) in newborns, even after confirming the absence with several techniques, does not constitute an exclusion criterion for toxoplasmosis.