Toxoplasma gondii: genetic recombination between drug resistant mutants

Mutants resistant to adenine arabinoside (ara-A) or to 5-fluorodeoxyuridine (FUDR) were isolated from a newly isolated oocyst producing strain of Toxoplasma gondii. The selection and characterization of these mutants were carried out in human fibroblast cultures. The ara-A-resistant mutant lacked the enzyme adenosine kinase. The biochemical basis of FUDR resistance remains unknown. Both mutants were used to infect mice to produce brain cysts that contained bradyzoites. Mouse brains that contained cysts were fed to kittens to complete the sexual cycle of T. gondii. Those kittens fed cysts of only one drug-resistant mutant excreted oocysts that yielded no detectable recombinant doubly resistant parasites that could make plaques in the presence of both ara-A and FUDR. Kittens fed a mixture of cysts that contained both mutants excreted oocysts that contained approximately 12% doubly resistant parasites. The reciprocal recombinant, sensitive to both drugs, was also isolated. The doubly resistant recombinant was totally deficient in adenosine kinase activity. This pattern of inheritance is consistant only with a haploid genome for all stages of T. gondii except the zygote formed by fusion of gametes and the unsporulated oocyst. Two FUDR-resistant mutants were also defective in the production of oocysts. These mutants failed to recombine with an ara-A-resistant mutant of proven fertility and thus their inability to make oocysts must result from a defect in the production of both microgametes and macrogametes.     

An Experimental Toxoplasma gondii Dose Response Challenge Model to Study Therapeutic or Vaccine Efficacy in Cats

High numbers of Toxoplasma gondii oocysts in the environment are a risk factor to humans. The environmental contamination might be reduced by vaccinating the definitive host, cats. An experimental challenge model is necessary to quantitatively assess the efficacy of a vaccine or drug treatment. Previous studies have indicated that bradyzoites are highly infectious for cats. To infect cats, tissue cysts were isolated from the brains of mice infected with oocysts of T. gondii M4 strain, and bradyzoites were released by pepsin digestion. Free bradyzoites were counted and graded doses (1000, 100, 50, 10), and 250 intact tissue cysts were inoculated orally into three cats each. Oocysts shed by these five groups of cats were collected from faeces by flotation techniques, counted microscopically and estimated by real time PCR. Additionally, the number of T. gondii in heart, tongue and brains were estimated, and serology for anti T. gondii antibodies was performed. A Beta-Poisson dose-response model was used to estimate the infectivity of single bradyzoites and linear regression was used to determine the relation between inoculated dose and numbers of oocyst shed. We found that real time PCR was more sensitive than microscopic detection of oocysts, and oocysts were detected by PCR in faeces of cats fed 10 bradyzoites but by microscopic examination. Real time PCR may only detect fragments of T. gondii DNA without the presence of oocysts in low doses. Prevalence of tissue cysts of T. gondii in tongue, heart and brains, and anti T. gondii antibody concentrations were all found to depend on the inoculated bradyzoite dose. The combination of the experimental challenge model and the dose response analysis provides a suitable reference for quantifying the potential reduction in human health risk due to a treatment of domestic cats by vaccination or by therapeutic drug application.     

Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat^{1, 2}. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk^3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment ^{5, 6}. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation⁷.     

Development of Toxoplasma gondii Chinese I genotype Wh6 Strain in Cat Intestinal Epithelial Cells

Felids are the unique definitive host of Toxoplasma gondii. The intestine of felid is the only site for initiating Toxoplasma gondii sexual reproduction. T. gondii excretes millions of infectious oocysts from the intestine, which are the primary source of infection. There are many difficulties in developing vaccines and drugs to control oocyst excretion due to the lack of an appropriate experimental model. Here, we established an in vitro feline intestinal epithelial cell (IEC) infection system and an efficient animal model of T. gondii Chinese 1 genotype, Wh6 strain (TgCtwh6). The Kunming mice brain tissues containing TgCtwh6 cysts were harvested 42-day post-infection. The bradyzoites were co-cultured with cat IECs in vitro at a ratio of 1:10. Five 3-month-old domestic cats were orally inoculated with 600 cysts each. The oocysts were detected by daily observation of cat feces by microscopy and polymerase chain reaction. We found that the parasite adhered and invaded cat IECs in vitro, transformed into tachyzoites, and then divided to form rose-like structures. These parasites eventually destroyed host cells, escaped, and finished the asexual reproduction process. Schizonts associated with sexual reproduction have not been observed during development in vitro cultured cells. However, schizonts were detected in all infected cat intestinal epithelial cells, and oocysts were presented in all cat feces. Our study provides a feasible cell model and an efficient infection system for the following studies of T. gondii sexual reproduction, and also lays a foundation to develop drugs and vaccines for blocking excretion and transmission of oocysts.     

Feline Toxoplasmosis from Acutely Infected Mice and the Development of Toxoplasma Cysts*

SYNOPSIS. The development of Toxoplasma cysts was studied in mice inoculated with tachyzoites by several routes. After 1–30 days of infection, murine tissues were examined microscopically, and portions or whole carcasses were fed to mice and cats. The feces of the cats were examined for oocyst shedding. Cyst-like structures containing distinct PAS-positive granules were first seen after 3 days of infection with tachyzoites, and became numerous by 6 days. Argyrophilic walls were first seen after 6 days, and became numerous by 16 days of infection with tachyzoites. Prepatent periods to oocyst shedding (PPO) were either “short” (3–10 days) or “long” (19–48 days). The “short” PPO was found only in cats that had ingested mice infected for 3 days or longer, and was related to the development of PAS-positive granules in T. gondii, and to high, 60–100%, oral infectivity rates for cats. The “long” PPO followed the ingestion of mice infected for only 1–2 days, and was related to tachyzoites without distinct PAS-positive granules and low, 32% or less, infectivity for cats. The “long” PPO followed also the ingestion of oocysts and the parenteral inoculation of tachyzoites, bradyzoites, or sporozoites. Using the “short” PPO as a criterion for detecting cysts in tissues, it was shown that (a) numerous cysts developed in mice 5 days after inoculation with tachyzoites, 7–9 days after inoculation with cysts, and 9–10 days after inoculation with oocysts, and (b) cysts developed faster and more frequently in the brain and muscle than in lungs, liver, spleen, and kidneys of mice inoculated with tachyzoites.     

Toxoplasma gondii: inconsistent dissemination patterns following oral infection in mice

Since Toxoplasma gondii is transmitted in the wild through the ingestion of infective cysts, oral infection is a preferred model for studying the natural mode of parasite dissemination and pathogenesis. Using luciferase-expressing strains of T. gondii and in vivo imaging, we observed different patterns of disease progression in mice depending of the method of oral infection. Oral gavage of infective cysts (e.g., bradyzoites) resulted in an inconsistent pattern of parasite dissemination; in the majority (20/29) of infected mice, luciferase-derived signal (indicating high numbers of Toxoplasma tachyzoites) was first observed in the right chest area. At later time points this signal spread to other parts of the mouse, including the abdominal area. In the remaining mice (9/29), parasites were first observed replicating in the abdominal area, as might be expected. In contrast, when mice were infected naturally (either via ingestion of whole brains from previously infected mice or brain cyst homogenate-soaked bread), parasites were first observed replicating in the abdominal area in all mice examined (10/10). Based on the inconsistency of infections initiated with oral gavage, it is recommended that natural feeding be used to infect mice when a consistent oral infection is desired.     

Toxoplasma gondii: uptake and survival of oocysts in free-living amoebae

Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions with parasitic protozoa remains elusive. In the present study, we assessed whether the free-living Acanthamoebacastellanii, due to its phagocytic activity, can interact with T. gondii oocysts. We report that amoebae can internalize T. gondii oocysts by active uptake. Intracellular oocysts in amoebae rarely underwent phagocytic lysis, retained viability and established infection in mice. Interaction of T. gondii with amoebae did not reduce the infectivity and pathogenicity of oocysts even after prolonged co-cultivation. Our results show that uptake of oocysts by A. castellanii does not restrain the transmission of T. gondii in a murine infection model.     

Evidence for Finely-Regulated Asynchronous Growth of Toxoplasma gondii Cysts Based on Data-Driven Model Selection

Toxoplasma gondii establishes a chronic infection by forming cysts preferentially in the brain. This chronic infection is one of the most common parasitic infections in humans and can be reactivated to develop life-threatening toxoplasmic encephalitis in immunocompromised patients. Host-pathogen interactions during the chronic infection include growth of the cysts and their removal by both natural rupture and elimination by the immune system. Analyzing these interactions is important for understanding the pathogenesis of this common infection. We developed a differential equation framework of cyst growth and employed Akaike Information Criteria (AIC) to determine the growth and removal functions that best describe the distribution of cyst sizes measured from the brains of chronically infected mice. The AIC strongly support models in which T. gondii cysts grow at a constant rate such that the per capita growth rate of the parasite is inversely proportional to the number of parasites within a cyst, suggesting finely-regulated asynchronous replication of the parasites. Our analyses were also able to reject the models where cyst removal rate increases linearly or quadratically in association with increase in cyst size. The modeling and analysis framework may provide a useful tool for understanding the pathogenesis of infections with other cyst producing parasites.     

Waterborne toxoplasmosis - Recent developments

Humans become infected with Toxoplasma gondii mainly by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts from the feces of infected cats. Circumstantial evidence suggests that oocyst-induced infections in humans are clinically more severe than tissue cyst-acquired infections. Until recently, waterborne transmission of T. gondii was considered uncommon, but a large human outbreak linked to contamination of a municipal water reservoir in Canada by wild felids and the widespread infection of marine mammals in the USA provided reasons to question this view. The present paper examines the possible importance of T. gondii transmission by water.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

First isolation of Neospora caninum from the feces of a naturally infected dog.

Neospora caninum is a major cause of abortion in cattle worldwide. Cattle become infected with N. caninum by ingesting oocysts from the environment or transplacentally from dam to fetus. Experimentally, dogs can act as definitive hosts, but dogs excrete few oocysts after ingesting tissue cysts. A natural definitive host was unknown until now. In the present study, N. caninum was isolated from the feces of a dog. Gerbils (Meriones unguiculatus) fed feces from the dog developed antibodies to N. caninum in the Neospora caninum agglutination test, and tissue cysts were found in their brains. Neospora caninum was isolated in cell culture and in gamma-interferon gene knockout mice inoculated with brain homogenates of infected gerbils. The DNA obtained from fecal oocysts of the dog, from the brains of gerbils fed dog feces, and from organisms isolated in cell cultures inoculated with gerbil brains was confirmed as N. caninum. The identification of N. caninum oocyst by bioassay and polymerase chain reaction demonstrates that the dog is a natural definitive host for N. caninum.     

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1^?/?NOS2^?/? than RAG1^?/? mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8⁺ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome–lysosome fusion could be involved in the T. gondii cyst elimination.     

Waterborne toxoplasmosis investigated and analysed under hydrogeological assessment: new data and perspectives for further research

We present a set of data on human and chicken Toxoplasma gondii seroprevalence that was investigated and analysed in light of groundwater vulnerability information in an area endemic for waterborne toxoplasmosis in Brazil. Hydrogeological assessment was undertaken to select sites for water collection from wells for T. gondii oocyst testing and for collecting blood from free-range chickens and humans for anti-T. gondii serologic testing. Serologic testing of human specimens was done using conventional commercial tests and a sporozoite-specific embryogenesis-related protein (TgERP), which is able to differentiate whether infection resulted from tissue cysts or oocysts. Water specimens were negative for the presence of viable T. gondii oocysts. However, seroprevalence in free-range chickens was significantly associated with vulnerability of groundwater to surface contamination (p < 0.0001; odds ratio: 4.73, 95% confidence interval: 2.18-10.2). Surprisingly, a high prevalence of antibodies against TgERP was detected in human specimens, suggesting the possibility of a continuous contamination of drinking water with T. gondii oocysts in this endemic setting. These findings and the new proposed approach to investigate and analyse endemic toxoplasmosis in light of groundwater vulnerability information associated with prevalence in humans estimated by oocyst antigens recognition have implications for the potential role of hydrogeological assessment in researching waterborne toxoplasmosis at a global scale.     

Releasing latent Toxoplasma gondii cysts from host cells to the extracellular environment induces excystation

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects a wide variety of mammals and birds. Although T. gondii infects the brain and muscles in its latent cyst form containing bradyzoite stage parasites during chronic infection, when a chronically infected host becomes immunodeficient or is preyed upon by a predator, the latent cyst undergoes excystation. However, it is not yet known how T. gondii recognises the triggers of excystation in the microenvironment surrounding the cyst. In this study, we incubated T. gondii cysts from host cells in several solutions containing a variety of ionic compositions. Excystation occurred in a solution with an ionic composition which mimicked that of the extracellular environment. However, excystation did not occur in a solution that mimicked the intracellular environment. We also found that the specific Na⁺/K⁺ ratio and the presence of Ca²⁺, mimicking the extracellular environment, are required to trigger excystation. To examine whether the stage conversion of bradyzoite to tachyzoite occurs prior to egress, we constructed a gene-modified T. gondii strain expressing a green fluorescent protein specifically in the tachyzoite stage. During the process of cyst reactivation of this strain, green fluorescence was detected prior to excystation. This suggests that stage conversion from bradyzoite to tachyzoite occurs prior to cyst disruption. These results indicate that T. gondii bradyzoites monitor the ionic composition of their surroundings to recognise their expulsion from host cells, to effectively time their excystation and stage conversion.     

Toxoplasma gondii: Impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF−/− mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF−/− mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.     

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology

Toxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons'' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite.     

Toxoplasma gondii: Flat-mounting of retina as a new tool for the observation of ocular infection in mice

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli β-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.     

Eimeria acervulina: The influence of inoculation dose on transmission between broiler chickens

The course and clinical appearance of an Eimeria species infection in chicken flocks depend on the response of an individual bird to infection and on population-dynamics of the infection in the flock. Differences in ingested numbers of oocysts may affect oocyst load in the flock and the subsequent infectious dose for not yet infected birds. To study the link between numbers of oocysts excreted by infected birds and transmission of Eimeria acervulina, experiments were carried out with 42 pairs of broiler chickens using inoculation doses with 5, 50, 500 or 50,000 sporulated oocysts. In each pair one bird was inoculated and the other bird was contact-exposed. All contact birds became infected, which occurred on average within 34 h after exposure to an inoculated bird. Although a higher inoculation dose resulted in higher oocyst excretion in inoculated and contact-infected birds, only small non-significant differences in transmission rates between groups were found.