Factors Influencing Tissue Cyst Yield in a Murine Model of Chronic Toxoplasmosis.

Recent advances into the unique biology of Toxoplasma tissue cysts and the bradyzoites they house necessitate optimization of tissue cyst recovery from infected mouse brains. Here, we present data from 83 tissue cyst purifications of Type II ME49 tissue cysts in CBA/J mice performed over a period of 3 years. The effects of infection with both tissue culture tachyzoites as well as ex vivo tissue cysts were assessed. Significant mortality was restricted to tachyzoite infections with female mice being more susceptible. Infection with tissue cysts was associated with both lower overall symptomology and mortality, exhibiting no sex bias. Cumulatively, host sex did not impact overall tissue cyst yields, although tachyzoite-initiated infections generated significantly higher yields compared to tissue cyst-initiated infections. Notably, serial passage of tissue cysts was accompanied with a decreasing trend for subsequent cyst recovery. The time of tissue cyst harvest, a potential reflection of bradyzoite physiological state, had no significant impact on subsequent cyst yield at the selected time points. In aggregate, these data reveal the considerable heterogeneity associated with tissue cyst yield, making the design of adequately powered experiments critical. This is particularly the case for drug studies where overall tissue cyst burden is currently the primary and often sole metric of efficacy, as the data presented here demonstrate that cyst recovery between preparations of untreated animals can mirror and even exceed the reported effects of drug treatment.

Cystoisospora belli, liver disease and hypothesis on the life cycle.

Cystoisospora belli causes chronic diarrhoea, acalculous cholecystitis, cholangiopathy and disseminated cystoisosporosis in patients with AIDS. Clinical manifestations and histological stages during C. belli infection in a patient with AIDS and liver disease were described. It was possible to identify sporozoite-like structures in the villus epithelium of the duodenum, close to the vascularization that underlies the basal membrane and unizoite tissue cysts near to the vascularization in the lamina propria. Unizoite tissue cysts were found inside of sinusoids in the liver communicating with the central vein and with a bile canaliculus and portal spaces. Based on these findings a hypothesis on C. belli life cycle could consider that the route of migration of unizoite tissue cysts up the liver is via the portal blood. The unizoite tissue cysts located in hepatic portal vein could migrated via sinusoid to central vein and general circulation through the venous system. The unizoite tissue cysts could also return via bile canaliculus to bile duct to portal triad. This hypothesis allows to understand the presence of unizoite stages in blood, the pathway by which the bile ducts become infected and unizoites in the liver being able to behave like hypnozoites that favour relapses and treatment failures.

Chronic Toxoplasma gondii Infection Induces Anti-N-Methyl-d-Aspartate Receptor Autoantibodies and Associated Behavioral Changes and Neuropathology.

Anti-NMDA receptor (NMDAR) autoantibodies have been postulated to play a role in the pathogenesis of NMDAR hypofunction, which contributes to the etiology of psychotic symptoms. Toxoplasma gondii is a pathogen implicated in psychiatric disorders and associated with elevation of NMDAR autoantibodies. However, it remains unclear whether parasite infection is the cause of NMDAR autoantibodies. By using mouse models, we found that NMDAR autoantibody generation had a strong temporal association with tissue cyst formation, as determined by MAG1 antibody seroreactivity (r = 0.96; P < 0.0001), which is a serologic marker for the cyst burden. The presence of MAG1 antibody response, but not T. gondii IgG response, was required for NMDAR autoantibody production. The pathogenic relevance of NMDAR autoantibodies to behavioral abnormalities (blunted response to amphetamine-triggered activity and decreased locomotor activity and exploration) and reduced expression of synaptic proteins (the GLUN2B subtype of NMDAR and PSD-95) has been demonstrated in infected mice. Our study suggests that NMDAR autoantibodies are specifically induced by persistent T. gondii infection and are most likely triggered by tissue cysts. NMDAR autoantibody seroreactivity may be a novel pathological hallmark of chronic toxoplasmosis, which raises questions about NMDAR hypofunction and neurodegeneration in the infected brain.

Mycophenolic acid induces differentiation of Toxoplasma gondii RH strain tachyzoites into bradyzoites and formation of cyst-like structure in vitro.

The biochemical and structural changes that occur during the conversion of Toxoplasma gondii tachyzoites to bradyzoites and the formation of tissue cyst are not well understood. Maintaining cells infected with T. gondii type II and III strains under stress conditions induces the tachyzoite-bradyzoite in vitro differentiation, along with the formation of cyst-like structures. However, due to the long exposure to such conditions required to induce the differentiation, the severe damages in the host cell and the low encystation frequency, it has been difficult to dissect in more detail these processes. Here, we successfully induced the in vitro formation of Toxoplasma cysts-like structures from tachyzoites of the type I RH strain by treating with mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase. Mycophenolic acid is a drug widely used for HXGPRT positive selection of Toxoplasma mutant strains along with xanthine incubation in the culture medium; under such conditions, formation of tissue cysts has not been reported. We show that the exposure of extracellular tachyzoites to mycophenolic acid in absence of xanthine, followed by host cell invasion, triggered their differentiation into cyst-like structures. The differential expression of CST1, BAG1, and SAG1 molecules, as well as the structural modifications of infected cells, was characterized during the formation of cyst-like structures in vitro. These findings will allow the characterization of signaling pathways involved in tachyzoite to bradyzoite conversion and formation of tissue cysts.

Investigation of tissue cysts in the retina in a mouse model of ocular toxoplasmosis: distribution and interaction with glial cells.

The conversion of tachyzoites into bradyzoites is a way for Toxoplasma gondii to establish a chronic and asymptomatic infection and achieve lifelong persistence in the host. The bradyzoites form tissue cysts in the retina, but not much is known about the horizontal distribution of the cysts or their interactions with glial cells in the retina. A chronic ocular toxoplasmosis model was induced by per oral administration of T. gondii Me49 strain cysts to BALB/c mice. Two months after the infection, retinas were flat-mounted and immunostained to detect cysts, ganglion cells, Müller cells, astrocytes, and microglial cells, followed by observation under fluorescence and confocal microscope. The horizontal distribution showed a rather clustered pattern, but the clusters were not restricted to certain location of the retina. Axial distribution was confined to the inner retina, mostly in ganglion cell layer or the inner plexiform layer. Both ganglion cells, a type of retinal neurons, and Müller cells, predominant retinal glial cells, could harbor cysts. The cysts were spatially separated from astrocytes, the most abundant glial cells in the ganglion cell layer, while close spatial distribution of microglial cells was observed in two thirds of retinal cysts. In this study, we demonstrated that the retinal cysts were not evenly distributed horizontally and were confined to the inner retina axially. Both neurons and one type of glial cells could harbor cysts, and topographic analysis of other glial cells suggests role of microglial cells in chronic ocular toxoplasmosis.

Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil.

The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in free-range chickens from Uberlândia, Minas Gerais state, Brazil, and characterize the genotypic and phenotypic features of two isolates of this parasite, considering the importance of these hosts in the epidemiology of toxoplasmosis. Serum samples from 108 free-range chickens were obtained from ten different districts, and submitted to the modified agglutination test (MAT) for the presence of anti-T. gondii antibodies, and brain and heart tissue samples from infected chickens were processed for mouse bioassay. An overall seroprevalence of 71·3% was found and antibody titres ranged from 16 to 4096. After confirmation of seropositivity by mouse bioassay, the determination of the T. gondii genotypes of two isolates was performed by PCR-RFLP, using primers for the following markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, new SAG2, Apico and CS3. These T. gondii isolates, designated TgChBrUD1and TgChBrUD2, were obtained from heart samples of free-range chickens. The TgChBrUD1 isolate belonged to ToxoDB PCR-RFLP genotype 11 and the TgChBrUD2 isolate belonged to ToxoDB PCR-RFLP genotype 6. Both isolates demonstrated high virulence in a rodent model, with the TgChBrUD1 isolate able to induce brain cysts, in accord with its pattern of multiplication rates in human fibroblast culture. Taken together, these results reveal high prevalence of T. gondii infection in free-range chickens throughout Uberlândia, indicating an important degree of oocyst environmental contamination and the existence of considerable risk for T. gondii transmission to humans by consumption of free-range chicken as a food source.

Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii.

Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and Besnoitia besnoiti were not recognized by mAb K8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.

Presence of Toxoplasma gondii in Pork Intended for Human Consumption in Tropical Southern Mexico.

BACKGROUND: Toxoplasmosis is caused by the protozoon Toxoplasma gondii, which is one of the most widespread parasites that infect animals and humans worldwide. One of the main routes of infection for humans is through the consumption of infected meat containing bradyzoites in tissue cysts. Pork is one of the foremost meat types associated with outbreaks of acute toxoplasmosis in humans. MATERIALS AND METHODS: Sixty blood samples were collected from finished pigs at slaughter and their sera was evaluated by an indirect-IgG ELISA. Matched muscle samples were obtained from the tongue and loin. Whole blood and tissue samples were evaluated to search for T. gondii DNA using a nested-polymerase chain reaction. RESULTS: Seroprevalence of T. gondii was 96.6% (58/60) of sampled pigs. Meanwhile, T. gondii DNA was present in 23.21% of tongue tissue samples (13/56), 7% of loin tissues (4/57), and 0% in blood samples (0/44), respectively. Two pigs were serologically indeterminate. CONCLUSION: This is the first report of the presence of T. gondii DNA in tissue samples obtained from finalized pigs. Results from the present study suggest a high exposure to T. gondii in pigs intended for human consumption from the tropical region of Mexico. Thus, the consumption of some undercooked pork meat meals typical from the southern region of Mexico could represent a significant risk for acquiring infection for the human population.

Naturally acquired bovine besnoitiosis: histological and immunohistochemical findings in acute, subacute, and chronic disease.

The pathogenesis of bovine besnoitiosis, a disease of increasing concern within Europe, is still incompletely understood. In this study, disease progression after natural infection with the causative apicomplexan Besnoitia besnoiti was monitored in histological skin sections of 5 individual female cattle over time. High-frequency skin sampling of 2 cattle with mild and 2 with severe acute, subacute, and chronic besnoitiosis, as well as from 1 animal during subclinical disease, enabled documentation from the beginning of the disease. Skin sections were stained with hematoxylin and eosin and Giemsa, periodic acid-Schiff reaction, and anti-Besnoitia immunohistochemistry. In all 4 clinically affected animals, tachyzoite-like endozoites could be detected for the first time by immunohistochemistry, and tissue cyst evolution was monitored. Besnoitiosis-associated lesions were not detected in the animal showing the subclinical course. Because of the inconsistency of the nomenclature of Besnoitia tissue cyst layers in the literature, a new nomenclature for B. besnoiti cyst wall layers is proposed: tissue cysts consist of a hypertrophied host cell with enlarged nuclei, an intracytoplasmic parasitophorous vacuole with bradyzoites, a sometimes vacuolated inner cyst wall, and an outer cyst wall in more developed cysts. Inner and outer cyst walls can be readily distinguished by using special stains. In 1 animal, extracystic B. besnoiti zoites were immunohistochemically detected during the chronic stage. At necropsy, the 2 severely affected cows displayed large numbers of B. besnoiti cysts in a variety of tissues, including the corium of the claws, contributing mainly to the development of chronic laminitis in these 2 cases.

Life cycle of Cystoisospora felis (Coccidia: Apicomplexa) in cats and mice.

Cystoisospora felis is a ubiquitous apicomplexan protozoon of cats. The endogenous development of C. felis was studied in cats after feeding them infected mice. For this, five newborn cats were killed at 24, 48, 72, 96, and 120 h after having been fed mesenteric lymph nodes and spleens of mice that were inoculated with C. felis sporulated sporocysts. Asexual and sexual development occurred in enterocytes throughout the villi of the small intestine. The number of asexual generations was not determined with certainty, but there were different sized merozoites. At 24 h, merogony was seen only in the duodenum and the jejunum. Beginning at 48 h, the entire small intestine was parasitized. At 24 h, meronts contained 1-4 zoites, and at 48 h up to 12 zoites. Beginning with 72 h, the ileum was more heavily parasitized than the jejunum. At 96 and 120 h, meronts contained many zoites in various stages of development; some divided by endodyogeny. The multiplication was asynchronous, thus both immature multinucleated meronts and mature merozoites were seen in the same parasitophorous vacuole. Gametogony occurred between 96 and 120 h, and oocysts were present at 120 h. For the study of the development of C. felis in murine tissues, mice were killed from day 1 to 720 d after having been fed 10(5) sporocysts, and their tissues were examined for the parasites microscopically, and by bioassay in cats. The following conclusions were drawn. (1) Cystoisospora felis most frequently invaded the mesenteric lymph nodes of mice and remained there for at least 23 mo. (2) It also invaded the spleen, liver, brain, lung, and skeletal muscle of mice, but division was not seen based on microscopical examination. (3) This species could not be passed from mouse to mouse.

Survival of experimentally induced Toxoplasma gondii tissue cysts in vacuum packed goat meat and dry fermented goat meat sausages.

Ingestion of raw or undercooked meat is a potential source of human toxoplasmosis. The aim of this study was to determine the viability of Toxoplasma gondii cysts in vacuum packed (VP) goat meat and in dry fermented sausages (DFS), and evaluate certain physical and chemical parameters, like water activity (aw), pH value, content of salt, dry matter and fat. A portion of muscle tissue from experimentally infected animals was used for production of VP meat with or without addition of 2.5% curing salt, and stored at 4 °C or at -20 °C. Results of bioassay showed that, samples of vacuum packed Toxoplasma positive meat without salt addition were alive after six weeks at 4 °C. Incubation at -20 °C supported the viability after 3 h, but not after 4 h. After 7 days in 2.5% of curing salt, samples of T. gondii VP goat meat were still viable, but not after 14 days at 4 °C. All the DFS samples were not positive for infective cysts which mean that, they do not pose a risk of T. gondii transmission. These data suggest that vacuum packaging increases the survival of T. gondii cysts.

Infection dynamics following experimental challenge of pigs orally dosed with different stages of two archetypal genotypes of Toxoplasma gondii.

Toxoplasma gondii is a food-borne zoonotic parasite widespread in a variety of hosts, including humans. With a majority of infections in Europe estimated to be meat-borne, pork, as one of the most consumed meats worldwide, represents a potential risk for consumers. Therefore, we aimed to investigate the progress of T. gondii infection and tissue tropism in experimentally infected pigs, using different T. gondii isolates and infectious stages, i.e. tissue cysts or oocysts. Twenty-four pigs were allocated to treatment in four groups of six, with each group inoculated orally with an estimated low dose of either 400 oocysts or 10 tissue cysts of two European T. gondii isolates, a type II and a type III isolate. The majority of pigs seroconverted two weeks post-inoculation. Pigs infected with the type III isolate had significantly higher levels of anti-T. gondii antibodies compared to those infected with the type II isolate. Histopathological exams revealed reactive hyperplasia of the lymphatic tissue of all pigs. Additionally, a selected set of nine tissues was collected during necropsy at 50 dpi from each of the remaining 22 pigs for T. gondii DNA detection by quantitative real-time PCR. A positive result was obtained in 29.8 % (59/139) of tested tissues. The brain was identified as the most frequently positive tissue in 63.6 % (14/22) of the animals. In contrast, liver samples tested negative in all animals. The highest mean parasite load, calculated by interpolating the average Cq values on the standard curve made of ten-fold serial dilutions of the genomic DNA, corresponding to 100 to 104 tachyzoites/µL, was observed in shoulder musculature with an estimated concentration of 84.4 [0.0-442.5] parasites per gram of tissue. The study highlights the variability in clinical signs and tissue distribution of T. gondii in pigs based on the combination of parasite stages and strains, with type III isolates, particularly oocysts, causing a stronger antibody response and higher tissue parasite burden. These findings suggest the need for further investigation of type III isolates to better understand their potential risks to humans.

Molecular detection of toxoplasmosis in wild rats using loop-mediated isothermal amplification assay.

Background and Aim: Toxoplasmosis is caused by the parasite Toxoplasma gondii. Cats are the only known hosts that excrete resistant oocysts. Wild rats serve as crucial reservoirs and intermediate hosts for T. gondii's survival and dissemination. Consuming soil and water containing oocysts can lead to illness. This study aimed to estimate the prevalence of toxoplasmosis in wild rats through molecular detection as an indicator of environmental contamination in Surabaya. Materials and Methods: One hundred rats were collected from the three areas (housing, dense settlements, and traditional markets) and distributed into the five zones: West, East, Central, North, and South of Surabaya. Brain tissue samples were extracted using a Geneaid™ (New Taipei City, Taiwan) DNA isolation kit and analyzed through the loop-mediated isothermal amplification (LAMP) method. Results: The study analyzed brain tissue from 100 wild rats, consisting of 77 Rattus tanezumi and 33 Rattus norvegicus, displaying 30% LAMP positivity. The study revealed that 30% (30/100) of wild rats tested were infected with T. gondii. The molecular prevalence rate in male rats was 32.35% (22/68), compared to females with 25% (8/32). 41.9% of the housing population, 33.3% of traditional markets, and 22.6% of dense settlements had the highest molecular prevalence. The high positive molecular rate at the trapping site can be attributed to cats and dense populations. Conclusion: Thirty percentage wild rats were tested positive for toxoplasmosis in Surabaya, East Java, Indonesia using LAMP method. Implementing strict control and monitoring is crucial in preventing the transmission of diseases from wild rats to humans. It is necessary to carry out further research related to genetic analysis of T. gondii to determine the type of T. gondii that infects animals and humans in Surabaya through bioassay and molecular test.

Constitutive upregulation of transcription factors underlies permissive bradyzoite differentiation in a natural isolate of Toxoplasma gondii.

Toxoplasma gondii bradyzoites play a critical role in pathology due to their long-term persistence in intermediate hosts and their potential to reactivate, resulting in severe diseases in immunocompromised individuals. Currently there is no effective treatment for eliminating bradyzoites. Hence, better in vitro models of T. gondii cyst development would facilitate identification of therapeutic targets for bradyzoites. Herein we characterized a natural isolate of T. gondii, called Tg68, which showed slower in vitro replication of tachyzoites, and permissive bradyzoite development under stress conditions in vitro. Transcriptional analysis revealed constitutive expression in Tg68 tachyzoites of the key regulators of bradyzoite development including BFD1, BFD2, and several AP2 factors. Consistent with this finding, Tg68 tachyzoites expressed high levels of bradyzoite-specific genes including BAG1, ENO1, and LDH2. Moreover, after stress induced differentiation, Tg68 bradyzoites exhibited gene expression profiles of mature bradyzoites, even at early time points. These data suggest that Tg68 tachyzoites exist in a pre-bradyzoite stage primed to readily develop into mature bradyzoites under stress conditions in vitro. Tg68 presents a novel model for differentiation in vitro that will serve as a useful tool for investigation of bradyzoite biology and development of therapeutics.

Constitutive upregulation of transcription factors underlies permissive bradyzoite differentiation in a natural isolate of Toxoplasma gondii.

Toxoplasma gondii bradyzoites play a critical role in pathology due to their long-term persistence in intermediate hosts and their potential to reactivate, resulting in severe diseases in immunocompromised individuals. Currently, there is no effective treatment for eliminating bradyzoites. Hence, better in vitro models of T. gondii bradyzoite development would facilitate identification of therapeutic targets for bradyzoites. Herein, we characterized a natural isolate of T. gondii, called Tg68, which showed slower in vitro replication of tachyzoites, and permissive bradyzoite development under stress conditions in vitro. Transcriptional analysis revealed constitutive expression in Tg68 tachyzoites of the key regulators of bradyzoite development including BFD1, BFD2, and several AP2 factors. Consistent with this finding, Tg68 tachyzoites expressed high levels of bradyzoite-specific genes including BAG1, ENO1, and LDH2. Moreover, after stress-induced differentiation, Tg68 bradyzoites exhibited gene expression profiles of mature bradyzoites, even at early time points. These data suggest that Tg68 tachyzoites exist in a pre-bradyzoite stage primed to readily develop into mature bradyzoites under stress conditions in vitro. Tg68 presents a novel model for differentiation in vitro that will serve as a useful tool for the investigation of bradyzoite biology and the development of therapeutics. IMPORTANCE: Toxoplasma gondii is a widespread protozoan that chronically infects ~30% of the world's population. T. gondii can differentiate between the fast-growing life stage that causes acute infection and the slow-growing stage that persists in the host for extended periods of time. The slow-growing stage cannot be eliminated by the host immune response or currently known antiparasitic drugs. Studies on the slow-growing stage have been limited due to the limitations of in vivo experiments and the challenges of in vitro manipulation. Here, we characterize a natural isolate of T. gondii, which constitutively expresses factors that drive development and that is permissive to convert to the slow-growing stage under stress conditions in vitro. The strain presents a novel in vitro model for studying the chronic phase of toxoplasmosis and identifying new therapeutic treatments for chronic infections.

An ex vivo model of Toxoplasma recrudescence reveals developmental plasticity of the bradyzoite stage.

IMPORTANCE: The classical depiction of the Toxoplasma lifecycle is bradyzoite excystation conversion to tachyzoites, cell lysis, and immune control, followed by the reestablishment of bradyzoites and cysts. In contrast, we show that tachyzoite growth slows independent of the host immune response at a predictable time point following excystation. Furthermore, we demonstrate a host cell-dependent pathway of continuous amplification of the cyst-forming bradyzoite population. The developmental plasticity of the excysted bradyzoites further underlines the critical role the cyst plays in the flexibility of the lifecycle of this ubiquitous parasite. This revised model of Toxoplasma recrudescence uncovers previously unknown complexity in the clinically important bradyzoite stage of the parasite, which opens the door to further study these novel developmental features of the Toxoplasma intermediate life cycle.

Tissue cysts and serological detection toxoplasmosis among wild rats from Surabaya, East Java, Indonesia.

Background: The protozoan Toxoplasma gondii is the source of zoonosis toxoplasmosis and causes public health problems throughout the world. Environmental contamination by oocysts excreted by cats as definitive hosts affects the spread of this disease. Wild rats as rodents can be used as an indicator of environmental contamination by oocysts, considering that rats have a habit of living in dirty environments and can be infected by oocysts from the environment. Aim: This study aims to detect toxoplasmosis from tissue cysts and serological tests in wild rats as an indicator of environmental contamination in Surabaya. Methods: A total of 100 wild rats collected from Surabaya were collected in five areas (West, East, Central, North, and South of Surabaya) obtained from three trapping locations: housing, dense settlements, and markets. All samples were examined microscopically for parasitological tests through the brain tissue samples, and the serum was examined using the toxoplasma modified agglutination test to detect the presence of IgG and Immunoglobulin M (IgM). Results: This research used 100 wild rat samples, 77 Rattus tanezumi and 33 Rattus norvegicus, with evidence of 31% in serology and active infection with 19% tissue cyst. The results showed that the seroprevalence of T. gondii in wild rats was 31% (30% for IgG and 1% for IgM). Tissue cysts in the rat brain samples tested were 19% (19/100). The IgG prevalence rate in female rats was 25% (8/32), while for males, it was 32.3% (22/68). The highest seropositive IgG from densely populated settlements was 50%, markets were 25.8%, and housing was 12.1%. The highest seropositive IgM from densely populated settlements was 2.8%. Population density and the presence of cats are factors supporting the high seropositive rate at the trapping location. Conclusion: This study revealed that there has been toxoplasmosis contamination in Surabaya with evidence of 31% in serology and active infection with 19% tissue cyst. It is necessary for controlling with surveillance in cats to prevent transmission in humans.

Parasitological, Molecular, and Histopathological Investigation of the Potential Activity of Propolis and Wheat Germ Oil against Acute Toxoplasmosis in Mice.

Toxoplasmosis is one of the most common parasitic zoonoses that affects all vertebrates. The drugs most commonly used against toxoplasmosis have many side effects, making the development of new antiparasitic drugs a big challenge. The present study evaluated the therapeutic effectiveness of novel herbal treatments, including propolis and wheat germ oil (WGO), against acute toxoplasmosis. A total of 50 albino mice were divided into five groups: group 1 (G1) (non-infected and non-treated); group 2 (G2) (infected without treatment); group 3 (G3) (treated with propolis); group 4 (G4) (treated with WGO); group 5 (G5) (treated with a combination of propolis and WGO). The effects of the herbal substances on different organs, mainly liver, spleen, and lungs, were investigated using parasitological, molecular, and histopathological examinations. The results of parasitological examination demonstrated statistically significant (p < 0.05) differences in the parasitic load between treated groups (G3, G4, and G5) compared to the control positive group (G2). These differences were represented by a significant reduction in the parasite load in stained tissue smears from the liver obtained from the animals treated with propolis (G3) compared to the parasite load in the positive control group. Similarly, animals (G4) treated with WGO exhibited a significant reduction in the parasite load versus the positive control group, while the lowest parasite load was found in G5, treated with propolis and WGO. Quantification of the parasite burden through molecular methods (PCR) revealed similar findings represented by reduction in the parasite burden in all treated groups with WGO and propolis as compared to the control group. Importantly, these previous parasitological and molecular findings were accompanied by a marked improvement in the histopathological picture of the liver, spleen, and lungs. In conclusion, propolis and WGO showed a good combination of therapeutic efficacy against acute toxoplasmosis.

Behavioral Changes Induced by Latent Toxoplasmosis Could Arise from CNS Inflammation and Neuropathogenesis.

Chronic infection with Toxoplasma gondii, a neurotropic parasite, has been linked to multiple behavioral changes in rodents and humans. The pathogenic mechanisms underlying these correlations are not known. I discuss here from animal studies the distribution of tissue cysts, the constant immune surveillance, the critical role of cyst burden, and the time-dependent consequences, which I believe are crucial to explaining the behavioral changes. In line with the brain-wide distribution of tissue cysts and chronic neuroinflammation, infected mice displayed a broad range of behavioral phenotypes. Many studies suggest that behavioral changes in mice are directly associated with tissue cyst presence or cyst burden and the host immune response. Cyst burden may not exert direct effects; however, the mechanisms causing behavioral and neuropathological changes are potentially the consequence of cyst burden over time, such as the neuroinflammation required to control the reactivation of tissue cysts. The reduction of neuroinflammation has proven that neuropathogenesis and behavioral abnormalities can be reversed, at least partially, in infected mice. Overall, Toxoplasma-induced behavioral changes are likely to be an indirect consequence of the host immune response in a parasite burden-dependent manner.

Immunomagnetic separation of Toxoplasma gondii and Hammondia spp. tissue cysts generated in cell culture.

Toxoplasma gondii is commonly transmitted among animals and humans by ingestion of infected animal tissues or by consumption of food and water contaminated with environmentally-resistant oocysts excreted by cats. Tissue cysts and oocysts have different walls, whose structures and compositions are poorly known. Herein, we describe an immunomagnetic separation (IMS) method that was successfully used for purification of T. gondii tissue cysts generated in cell culture. We used an IgG monoclonal antibody (mAb) that reacts against antigens in tissue cyst walls. Many in vitro produced cysts were obtained by this IMS; >2,000 T. gondii cysts were isolated from a single culture flask of 25 cm2. Tissue cysts from two Hammondia spp., H. hammondi, and H. heydorni, produced in cell culture were also separated using this method. As a reference, purification of tissue cysts by Percoll gradients was used. Percoll was able to separate T. gondii tissue cysts produced in mice but was not suitable for purifying T. gondii tissue cysts produced in vitro. The IMS described here should favor proteomic studies involving tissue cysts of T. gondii.