Monoclonal antibodies reacting with pellicular antigens of Toxoplasma gondii tachyzoites have been selected among hybridomas produced against this organism by immunofluorescence assay. These antigens have been further characterized by immunofluorescence on living zoites, Western immunoblotting and immunoprecipitation of lactoperoxidase surface radio-iodinated tachyzoite lysates. The simultaneous characterization of 5 different surface antigens (P43, P35, P30, P23, P22) some of which have already been studied individually allowed a better definition of these antigens and the characterization of a yet undescribed surface molecule (P23).

The development of simple, sensitive and rapid methods for the detection and identification of Toxoplasma gondii is important for the diagnosis and epidemiological studies of the zoonotic disease toxoplasmosis. In the past 2 decades, molecular methods based on a variety of genetic markers have been developed, each with its advantages and limitations. The application of these methods has generated invaluable information to enhance our understanding of the epidemiology, population genetics and phylogeny of T. gondii. However, since most studies focused solely on the detection but not genetic characterization of T. gondii, the information obtained was limited. In this review, we discuss some widely used molecular methods and propose an integrated approach for the detection and identification of T. gondii, in order to generate maximum information for epidemiological, population and phylogenetic studies of this key pathogen.

Humans and other hosts acquire Toxoplasma gondii infection by ingesting tissue cysts in undercooked meat, or by food or drink contaminated with oocysts. Currently, there is no vaccine to prevent clinical disease due this parasite in humans, although, various T. gondii vaccine candidates are being developed. Mice are generally used to test the protective efficacy of vaccines because they are susceptible, reagents are available to measure immune parameters in mice, and they are easily managed in the laboratory. In the present study, pathogenesis of toxoplasmosis was studied in mice of different strains, including Human Leukocyte Antigen (HLA) transgenic mice infected with different doses of T. gondii strains of different genotypes derived from several countries. Based on many experiments, the decreasing order of infectivity and pathogenicity of oocysts was: C57BL/6 background interferon gamma gene knock out (KO), HLA-A*1101, HLA-A*0201, HLA-B*0702, Swiss Webster, C57/black, and BALB/c. Mice fed as few as 1 oocyst of Type I and several atypical strains died of acute toxoplasmosis within 21 days p.i. Some Type II, and III strains were less virulent. The model developed herein should prove to be extremely useful for testing vaccines because it is possible to accurately quantitate a challenge inoculum, test the response to different strains of T. gondii using the same preparations of oocysts which are stable for up to a year, and to have highly reproducible responses to the infection.

Transmission electron microscopy was used to study Toxoplasma gondii infections in the small intestines of Swiss-Webster mice at 2–48 h post-feeding of oocysts (p.f.). Sporozoites passed through intestinal epithelial cells (enterocytes and goblet cells) and infected all cells except red blood cells in the lamina propria. Parasites in intestinal epithelial cells or in cells in the lamina propria were located within a single type of parasitophorous vacuole, which contained exocytosed electron-dense material and well-developed tubulovesicular membranous networks. Sporozoites did not infect intraepithelial lymphocytes (IELs), but at 48 h p.f. IELs had become infected with tachyzoites arising from those that had developed in the lamina propria. At 48 h p.f., the lamina propria contained numerous tachyzoites, much cellular debris, and few intact cells. The intestinal epithelium exhibited limited cytopathological changes except for villar fusion, slight vacuolation, and cell separation at the bases of enterocytes.

The effect of Toxoplasma gondii on neophobic behaviour (the avoidance of novel stimuli) was assessed in four groups of wild rats with naturally occurring Toxoplasma infection. Two groups were placed in individual cages and tested in a series of experiments which examined the effect of Toxoplasma on the rat's reaction to 3 food-related novel stimuli (odour, food- container, food). A trappability study was performed on the other two groups to test whether Toxoplasma had an effect on probability of capture. The results show that low neophobia was significantly associated with positive Toxoplasma titres in 3 out of 4 groups. We suggest that differences between infected and uninfected wild rats arise from pathological changes caused by Toxoplasma cysts in the brains of infected rats. Such behavioural changes may be selectively advantageous for the parasite as they may render Toxoplasma-infected rats more susceptible to predation by domestic cats (the definitive host of Toxoplasma) and, as a side-effect, more susceptible to trapping and poisoning during pest control programmes.

Toxoplasma gondii, the coccidian parasite, is known to induce changes in the behaviour of its intermediate hosts. The high prevalence of this parasite in the human population (20–80%) offers the opportunity of studying the influence of the parasite on human behaviour by screening of a normal population. A total of 224 men and 170 women were tested for toxoplasmosis and their personality profiles were measured by Cattell's questionnaire. Highly significant differences between Toxoplasma-infected and uninfected subjects were observed (P < 0·01). For men the factors G (low superego strength), L (protension), O (guilt proneness), and Q2 (group dependency) were positively influenced in infected subjects. For women the prevailing factors were A (affectothymia, P < 0·01), L (alaxia), O (untroubled adequacy) and Q2 (self-sufficiency). To reveal whether toxoplasmosis induces personality factor-shifts or whether certain combinations of personality factors influence the probability of acquiring Toxoplasma infection, we examined the personality profiles of 164 male patients diagnosed with acute toxoplasmosis during the past 13 years. The existence of a positive correlation between the duration of latent toxoplasmosis and the intensity of superego strength decrease (P < 0·02) suggested that the decrease of superego strength (the willingness to accept group moral standards) was induced by T. gondii infection.

A simple, quick and reproducible method consisting of density-gradient centrifugation of homogenized infected mouse brain tissue on Percoll is described for the isolation and purification of cysts of Isospora (Toxoplasma) gondii. A 100% recovery of cysts, with 74·2% in a single fraction with a specific gravity of 1·056, was obtained by overlaying homogenates of infected mouse brains on a pre-formed Percoll gradient and centrifugation at low g forces. With this procedure recovery was independent of the age of the cysts. Titration of purified cystozoites showed there to be no loss of infectivity.

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1–6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.

Manuls or Pallas' cats (Felis manul, syn. Otocolobus manul) are endangered wild cats from Central Asia kept and bred in many zoos. Despite good breeding success young cats frequently die from acute toxoplasmosis. From 1998 to 2002, a breeding pair in the Schönbrunn Zoo in Vienna, Austria, gave birth to 24 kittens; 58% of kittens died between the 2nd and the 14th week of life, mostly due to acute toxoplasmosis. The epidemiology of toxoplasmosis in Pallas' cats was examined and a control strategy to protect the kittens from fatal toxoplasmosis was developed. One 12-week-old kitten from a litter of 6 born in 2001 died of generalized toxoplasmosis. This kitten had shed T. gondii oocysts that were bioassayed in mice. Toxoplasma gondii was isolated in tissue culture inoculated with tissues of these mice. The surviving animals were immediately treated with clindamycin for 16 weeks; they acquired a natural infection and seroconverted by the end of this time without clinical signs.

Feral swine are known reservoirs of various pathogens, including Toxoplasma gondii. Here, we report the first national survey of viable T. gondii in feral swine in the USA. We paired serological surveys with parasite isolation and bioassay to evaluate the prevalence and genetic diversity of these parasites. From 2012–2017, sera and tissues from 1517 feral swine across the USA were collected for the isolation of viable T. gondii. Serum samples were initially screened for antibodies to T. gondii, and then the tissues of seropositive feral swine were bioassayed in mice. Antibodies were detected in 27.7% of feral swine tested by the modified agglutination test (1:25 or higher). Antibody positive rates increased significantly with age, with 10.1% of juveniles, 16.0% of sub-adults and 38.4% of adults testing seropositive. Myocardium (50 g) from 232 seropositive feral swine was digested in pepsin and bioassayed in mice. Viable T. gondii was isolated from 78 feral swine from 21 states. Twelve of the 78 isolates were pathogenic to outbred Swiss Webster mice and 76 of the 78 isolates could be propagated further in cell culture and were genotyped. For genotyping, deoxyribonucleic acid extracted from cell culture-derived tachyzoites was characterized by polymerase chain reaction restriction fragment length polymorphism using the genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genotyping revealed 15 ToxoDB genotypes, including 43 isolates for genotype #5 (haplogroup 12), 11 isolates for #24, four isolates for #2 (haplogroup 3), two isolates for each of genotypes #3 (haplogroup 2), #4 (haplogroup 12), #216, #221, #289 and #297 and one isolate for each of genotypes #1 (haplogroup 2), #39, #66, #260, #261 and #299. Genotype #5 was the most frequently isolated, accounted for 57% (43/76) of the isolates, followed by #24, accounted for 14% (11/76). Genotypes #260, #289, #297 and #299 are new types. Genotype #289 was highly virulent to mice and originated from feral swine collected in Louisiana on the same day at the same location. Genotype #216 was previously demonstrated to be highly virulent to mice. Our results indicate moderate genetic diversity of T. gondii in feral swine in the USA, with the genotype #5 (haplogroup 12) dominant in the continental USA, whereas genotype #24 (10/14) was dominant in Hawaii, suggesting different population structures of the parasites among the two distinct geographical locations.

We have studied the activity of the polyether ionophorous antibiotic monensin against the cyst form (bradyzoite) of Toxoplasma gondii. The experiments were conducted in vitro using 2 methods; cysts produced either in mice or in cell culture were exposed to monensin in vitro, and the infectivity of the parasites was then assessed in vivo or in vitro. The data obtained from these 2 systems of evaluation showed that monensin inhibits the infectivity and the viability of the bradyzoites. Its activity was time and concentration dependent. The first effects were observed at very low drug concentrations (i.e. 0·0001 μg/ml). Immunofluorescence and electron microscopy analysis showed significant cytological alterations of the monensin-treated bradyzoites: they were swollen, had a large number of vacuoles in their cytoplasm and were found lysed at higher concentrations in ionophore.

In recent years, an extensive collection of Toxoplasma gondii samples have been typed using a set of 10 PCR-RFLP genetic markers. Here we summarize the data reported until the end of 2012. A total of 1457 samples were typed into 189 genotypes. Overall, only a few genotypes dominate in the northern hemisphere, which is in stark contrast to the southern hemisphere where hundreds of genotypes coexist with none being notably dominant. PCR-RFLP genotype #1 (Type II clonal), #2 (Type III), #3 (Type II variant) and #10 (Type I) are identified globally. Genotypes #2 and #3 dominate in Africa, genotypes #9 (Chinese 1) and #10 are prevalent in Asia, genotypes #1, #2 and #3 are prevalent in Europe, genotypes #1, #2, #3, #4 and #5 dominate in North America (#4 and #5 are collectively known as Type 12). In Central and South America, there is no clear dominance of any genotype even though a few have relatively higher frequencies. Statistical analysis indicates significant differences among populations in Africa, Asia, Europe, North America, and Central and South America, with only Europe and North America exhibiting similar diversity. Collectively, the results revealed distinct population structures and geographical patterns of diversity in T. gondii.

To assess the role of white-tailed deer (Odocoileus virginianus, WTD) in the epidemiology of toxoplasmosis, we conducted a national survey of WTD across the USA for Toxoplasma gondii infection. To do this, we combined serology with parasite isolation to evaluate the prevalence and genetic diversity of T. gondii in this game species. From October 2012 to March 2019, serum and tissues were collected from 914 WTD across the USA. Serum samples were screened for antibodies to T. gondii, and then the tissues of seropositive WTD were bioassayed in mice. Antibodies were detected in 329 (36%) of 914 WTD tested by the modified agglutination test (positive reaction at 1:25 or higher). Viable T. gondii was isolated from the heart of 36 WTD from 11 states. Three of the 36 isolates were pathogenic but not highly virulent to outbred Swiss Webster mice and all 36 isolates could be propagated further in cell culture and were genotyped. For genotyping, DNA extracted from cell culture-derived tachyzoites was characterized by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using the genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genotyping revealed seven ToxoDB PCR-RFLP genotypes, including 24 isolates for genotype #5 (haplogroup 12), four isolates for #2 (type III, haplogroup 3), three isolates for genotypes #1 (type II, haplogroup 2), two isolates for genotypes #3 (type II, haplogroup 2) and one isolate each for #39, #221 and #224. Genotype #5 was the most frequently isolated, accounting for 66.6% (24 of 36) of the isolates. Combining the 36 isolates from this study with previously reported 69 isolates from WTD, 15 genotypes have been identified. Among these, 50.4% (53/105) isolates belong to genotype #5. Our results indicate moderate genetic diversity of T. gondii in WTD. The results also indicate that undercooked venison should not be consumed by humans or fed to cats.

To date, little is known about the dynamics of vertical transmission of Toxoplasma gondii in Australian marsupials. Studies in mice demonstrate that vertical transmission of T. gondii is common and that chronically infected mice can transmit T. gondii to successive generations. In this study, PCR and immunohistochemistry were used to detect T. gondii in chronically infected marsupial dams and their offspring. T. gondii was detected in the unfurred pouch young of 2 out of 10 chronically infected western grey kangaroos (Macropus fuliginosus) and in the unfurred pouch young of a brush-tailed bettong (Bettongia penicillata). Results of the study suggest that vertical transmission of T. gondii can occur in chronically infected Australian marsupials.

Toxoplasma gondii infections are common in humans and animals worldwide. Domestic free-range chickens (Gallus domesticus) are excellent sentinels of environmental contamination with T. gondii oocysts because they feed on the ground. Chickens can be easily infected with T. gondii; however, clinical toxoplasmosis is rare in these hosts. Chickens are comparatively inexpensive and thus are good sentinel animals for T. gondii infections on the farms. Here, the authors reviewed prevalence, the persistence of infection, clinical disease, epidemiology and genetic diversity of T. gondii strains isolated from chickens worldwide for the past decade. Data on phenotypic and molecular characteristics of 794 viable T. gondii strains from chickens are discussed, including new data on T. gondii isolates from chickens in Brazil. This paper will be of interest to biologists, epidemiologists, veterinarians and parasitologists.

Infections by the protozoan parasite Toxoplasma gondii are widely prevalent in humans and animals in Brazil. The burden of clinical toxoplasmosis in humans is considered to be very high. The high prevalence and encouragement of the Brazilian Government provides a unique opportunity for international groups to study the epidemiology and control of toxoplasmosis in Brazil. Many early papers on toxoplasmosis in Brazil were published in Portuguese and often not available to scientists in English-speaking countries. In the present paper we review prevalence, clinical spectrum, molecular epidemiology, and control of T. gondii in humans and animals in Brazil. This knowledge should be useful to biologists, public health workers, veterinarians, and physicians. Brazil has a very high rate of T. gondii infection in humans. Up to 50% of elementary school children and 50–80% of women of child-bearing age have antibodies to T. gondii. The risks for uninfected women to acquire toxoplasmosis during pregnancy and fetal transmission are high because the environment is highly contaminated with oocysts. The burden of toxoplasmosis in congenitally infected children is also very high. From limited data on screening of infants for T. gondii IgM at birth, 5–23 children are born infected per 10 000 live births in Brazil. Based on an estimate of 1 infected child per 1000 births, 2649 children with congenital toxoplasmosis are likely to be born annually in Brazil. Most of these infected children are likely to develop symptoms or signs of clinical toxoplasmosis. Among the congenitally infected children whose clinical data are described in this review, several died soon after birth, 35% had neurological disease including hydrocephalus, microcephaly and mental retardation, 80% had ocular lesions, and in one report 40% of children had hearing loss. The severity of clinical toxoplasmosis in Brazilian children may be associated with the genetic characteristics of T. gondii isolates prevailing in animals and humans in Brazil.

Toxoplasma gondii is an obligate intracellular parasite of phylum Apicomplexa. To facilitate high-efficiency invasion of host cells, T. gondii secretes various proteins related to the moving junction (MJ) complex from rhoptries and micronemes into the interface between the parasite and host. AMA1/RON2/4/5/8 is an important MJ complex, but its mechanism of assembly remains unclear. In this study, we used the CRISPR-Cas9 system to generate a derivative of T. gondii strain RH with a null mutation in TgRON4, thought to be an essential MJ component. Deficiency of TgRON4 moderately decreased invasion ability relative to that of the wild-type parasite. In addition, expression of the endogenous N-terminal fragment of RON5 decreased in the mutant. Together, the results improve our understanding of the assembly mechanism of the MJ complex of T. gondii and raise the possibility of developing new therapeutic drugs that target this complex.

Small marine snails and abalone have been identified as high- and low-risk prey items, respectively, for exposure of threatened southern sea otters to Toxoplasma gondii, a zoonotic parasite that can cause fatal encephalitis in animals and humans. While recent work has characterized snails as paratenic hosts for T. gondii, the ability of abalone to vector the parasite has not been evaluated. To further elucidate why abalone predation may be protective against T. gondii exposure, this study aimed to determine whether: (1) abalone are physiologically capable of acquiring T. gondii; and (2) abalone and snails differ in their ability to concentrate and retain the parasite. Abalone were exposed to T. gondii surrogate microspheres for 24 h, and fecal samples were examined for 2 weeks following exposure. Concentration of surrogates was 2–3 orders of magnitude greater in abalone feces than in the spiked seawater, and excretion of surrogates continued for 14 days post-exposure. These results indicate that, physiologically, abalone and snails can equally vector T. gondii as paratenic hosts. Reduced risk of T. gondii infection in abalone-specializing otters may therefore result from abalone's high nutritional value, which implies otters must consume fewer animals to meet their caloric needs.