Gondii Oocysts Research Articles

Detection of Toxoplasma gondii oocysts in soils in northwestern China using a new semi-nested PCR assay.

BackgroundToxoplasma gondii is a zoonotic pathogen that can infect a range of animals and humans. Ingestion of T. gondii oocysts in soil is a significant transmission route for humans and animals acquiring toxoplasmosis. In the present study, we developed a new semi-nested PCR method to determine T. gondii oocysts distribution in soils in northwestern China.ResultsThe one tube semi-nested PCR assay was developed to detect the oocysts of T. gondii in soil, targeting the repetitive 529 bp fragment of T. gondii genomic DNA. Then a total of 268 soil samples, including 148 samples from Gansu Province and 120 samples from Qinghai Province, northwestern China, were examined by the semi-nested PCR method. One third of the positive samples were sequenced. The sensitivity of the semi-nested PCR assay was 102 T. gondii oocysts in 5 g soil sample. Investigation of soil samples from northwestern China showed that 34 out of 268 soil samples (12.69%) were T. gondii positive. Sequences of the partial 529 bp fragments varied from 0–1.2% among the sequenced samples. The prevalence of T. gondii oocysts in soil from cities (24/163) was slightly higher than that in soils from pasturing areas (10/105) (P = 0.21). Among the different regions in cities, the prevalence of T. gondii oocysts in soils from parks was 14.15%, whereas that in soils from schools was 19.05%.ConclusionsThe present study firstly reported the prevalence of T. gondii oocysts in soils in northwest China using a novel semi-nested PCR assay, which provided baseline data for the effective prevention and control of toxoplasmosis in this region.

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.

Spatial distribution of Toxoplasma gondii oocysts in soil in a rural area: Influence of cats and land use

Toxoplasma gondii is the protozoan parasite responsible for toxoplasmosis, one of the most prevalent zoonoses worldwide. T. gondii infects humans through the ingestion of meat containing bradyzoites or through soil, food or water contaminated with oocysts. Soil contamination with oocysts is increasingly recognized as a major source of infection for humans, but has rarely been quantified directly. In this study, we investigated the spatial pattern of soil contamination with T. gondii over an area of 2.25km2 in a rural area of eastern France. The frequency and spatial distribution of T. gondii in soil was analyzed in relation with the factors that could influence the pattern of contamination: cats’ frequency and spatial distribution and land use. According to a stratified random sampling Scheme 243 soil samples were collected. The detection of T. gondii oocysts was performed using a recent sensitive method based on concentration and quantitative PCR. Sensitivity was improved by analyzing four replicates at each sampling point. T. gondii was detected in 29.2% of samples. Soil contamination decreased with increasing distance from the core areas of cat home ranges (households and farms). However, it remained high at the periphery of the study site, beyond the boundaries of the largest cat home ranges, and was not related to land use. This pattern of contamination strongly supports the role of inhabited areas which concentrate cat populations as sources of risk for oocyst-induced infection for both humans and animals. Moreover, soil contamination was not restricted to areas of high cat density suggesting a large spatial scale of environmental contamination, which could result from T. gondii oocysts dissemination through rain washing or other mechanisms.

Molecular Detection of Toxoplasma gondii Oocytes in the Soil from the Public Parks of the Arak City, Iran

Background and Aim: Soil structure is mainly composed of sand, silt, c lay, and organic materials. Organisms can live in the soil. The large number of stray cats in the cities of Iran is a major environmental and hea lth problem. Toxoplasma oocysts are shed with the feces of cats, so soil is known as a potential source of transmission of toxoplasmosis. The aim of this stud y was to determine the soil contamination with Toxoplasma gondii oocyst in the public parks of the Arak city. Methods: Sixty soil samples were collected from 15 main park s of Arak city. Four soil samples from the children's playground, a potting place, around trash bins, and around toilets were taken. Oocyst was iso lated from soil by floatation in saturated sucrose. Floating debris was tested by tw o methods: staining by the modified Ziehl-Neelsen technique and PCR. The target of PCR was the 122 bp fragment of the B1 gene. Results: From 60 soil samples of public parks of Arak city, 8 samples (13%) were suspected to Toxoplasma oocyst contamination in staining smears. Only 3 samples (5%) of 60 samples were positive in PCR. The result s showed that the staining method is not a good method to detect oocysts in th e soil because the diversity of oocyst in soil is very high and similar in appearan ce. Conclusion: This study showed soils of public parks in the Arak city were contaminated to oocyst of Toxoplasma. Also molecular method for the detection of parasites in the soil was more suitable than sta ining method.

Seroprevalences of <i>Toxoplasma gondii</i> and <i>Neospora caninum</i> in Pet Rabbits in Japan

ABSTRACTThe potential contamination of Toxoplasma gondii and Neospora caninum oocysts in the human environment is a concern from the public health viewpoint. However, estimation of their seroprevalences in humans cannot be performed in a manner that distinguishes between oocysts and tissue cysts as a source of infection. Rabbits are considered popular pet animals in Japan that can acquire natural infections by the aforementioned parasites only through the ingestion of oocysts. Therefore, this study was conducted to estimate the seroprevalences of T. gondii and N. caninum in pet rabbits in Japan as an indicator of the possible oocyst contamination in the environment surrounding human beings. Serum samples of 337 rabbits were examined by different serological methods. Enzyme-linked immunosorbent assays were performed to measure the titer of IgG and IgM antibodies. Samples revealed to be seropositive by ELISA were further analyzed by a latex agglutination test, Western blotting and an indirect immunofluorescence assay. The rates of seropositivity for T. gondii were 0.89% (3/337) and 0.29% (1/337) in IgG and IgM ELISA, respectively. SAG1 and SAG2 were detected as major antigens by the positive rabbit sera in Western blotting associated with strong staining observed by IFA in T. gondii tachyzoites. Regarding N. caninum, none of the serum samples showed a specific reaction in both Western blotting and the IFA. The results of this study indicate low seroprevalences of toxoplasmosis and neosporosis in pet rabbits in Japan, suggesting low oocyst contamination in the human environment.

Attempted Detection of Toxoplasma gondii Oocysts in Environmental Waters Using a Simple Approach to Evaluate the Potential for Waterborne Transmission in the Galápagos Islands, Ecuador

Toxoplasmosis is a health concern for wildlife and humans, particularly in island ecosystems. In the Galápagos Islands, exposure to Toxoplasma gondii has been found in marine avifauna on islands with and without domestic cats. To evaluate potential waterborne transmission of T. gondii, we attempted to use filtration and epifluorescent microscopy to detect autofluorescent T. gondii oocysts in fresh and estuarine surface water samples. T. gondii oocyst-like structures were microscopically visualized but were not confirmed by polymerase chain reaction and sequence analyses. Further research is needed to refine environmental pathogen screening techniques and to evaluate disease risk of waterborne zoonoses such as T. gondii for wildlife and humans, particularly in the Galápagos and other naive island ecosystems.

Bioaccumulation of human waterborne protozoa by zebra mussel (Dreissena polymorpha): Interest for water biomonitoring

Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii are ubiquitous pathogens, which waterborne transmission has been largely demonstrated. Since they can be found in various watercourses, interactions with aquatic organisms are possible. Protozoan detection for watercourses biomonitoring is currently based on large water filtration. The zebra mussel, Dreissena polymorpha, is a choice biological model in ecotoxicological studies which are already in use to detect chemical contaminations in watercourses. In the present study, the zebra mussel was tested as a new tool for detecting water contamination by protozoa. In vivo exposures were conducted in laboratory experiments. Zebra mussel was exposed to various protozoan concentrations for one week. Detection of protozoa was realized by Taqman real time qPCR. Our experiments evidenced C. parvum, G. duodenalis and T. gondii oocyst bioaccumulation by mussels proportionally to ambient contamination, and significant T. gondii prevalence was observed in muscle tissue. To our knowledge, this is the first study that demonstrates T. gondii oocyst accumulation by zebra mussel. The results from this study highlight the capacity of zebra mussels to reveal ambient biological contamination, and thus to be used as a new effective tool in sanitary biomonitoring of water bodies.

High prevalence of Toxoplasma gondii oocyst shedding in stray and pet cats (Felis catus) in Virginia, United States

BackgroundThe protozoan Toxoplasma gondii is the causative agent of toxoplasmosis, with complications varying from mental disease to death. While human infection can occur via ingestion of tissue cysts from infected meat, most human infection comes from oocysts. Cats are the only definitive host, and thus shedding of oocysts by cats provides the ultimate source of toxoplasmosis.MethodsWhile most studies in the area use seroprevalence to monitor Toxoplasma incidence in cat populations, this provides only a history of infection. This study used PCR detection of oocysts from cat feces to more accurately estimate the numbers of cats producing oocysts and thus posing an active health risk. DNA sequencing was use to confirm the identity of the PCR products.ResultsOf the 49 cats tested, 9 yielded PCR products of the expected size. Six of the nine were determined by sequence analysis to be false positives, while three products were true positives. Overall, 6% of cats examined were found to be actively shedding oocysts.ConclusionsThe incidence of oocyst shedding in the cat population studied was significantly higher than expected and higher than found in most cat populations world-wide. Of equal importance, the primers tested were shown to produce PCR products of multiple sizes and non-target products of expected size. We detected false positives at a higher rate than true positives, emphasizing the need for confirmatory analysis. Further research may produce better protocols for Toxoplasma detection from cat fecal samples.

Serial Monoxenous Transmission ofToxoplasma gondiiin Cats

Abstract : Oral administration of Toxoplasma gondii oocysts to cats (i.e., monoxenous transmission) typically induces patent infections in fewer than half of test subjects. In the present study, oral administration of T. gondii oocysts to 5 kittens induced a patent infection in 2 of them, but only 1 kitten shed enough oocysts to enable further study. Those monoxenously-produced oocysts were administered to another kitten, which produced a second generation of monoxenous oocysts, and then those were used to induce a third generation of monoxenous oocysts. These results provide a rationale to develop a strain of T. gondii that has efficient direct transmission. The isolate of T. gondii that was able to be passaged in this manner has been designated the Dubey strain and cultured tachyzoites have been donated to a repository.

Hydrological modelling of Toxoplasma gondii oocysts transport to investigate contaminated snowmelt runoff as a potential source of infection for marine mammals in the Canadian Arctic

Toxoplasma gondii (T. gondii) is a zoonotic protozoan that sometimes causes serious illness in humans and other animals worldwide, including the Canadian Arctic. Wild and domestic felids, the only hosts able to shed T. gondii oocysts, are practically non-existent in the Canadian Arctic. So here the hypothesis that T. gondii oocysts, shed in the southern areas of the boreal watershed, could contaminate the Arctic coastal marine environment via surface runoff, particularly during the spring snowmelt period, was explored. A watershed model was applied to simulate the hydrological transport of T. gondii oocysts during the snowmelt period and test the possible efficiency of river-to-sea transport as a potential source of marine organisms' exposure to this pathogen. Simulations were run for two pilot watersheds with the ultimate aim of extrapolating the results across the Canadian Arctic watersheds. Results suggest that daily stream flow concentrations of T. gondii oocysts at the river outlet are likely to be very low. However, accumulation of oocysts in the estuarine areas may be large enough to contaminate estuarine/marine filter-feeding molluscs and snails on which seals and other marine mammals may feed. Potential maximum concentrations of T. gondii oocysts in runoff are reached at the beginning of the snowmelt period with maxima varying with discharge rates into rivers and how far upstream oocysts are discharged. Meteorological conditions during the snowmelt period can affect simulated concentrations of oocysts. These findings support the hypothesis that T. gondii oocysts carried in snowmelt runoff could be a source of T. gondii infection for marine mammals in the Canadian Arctic, and for Arctic human populations that hunt and consume raw meat from marine mammals.

Evaluation of a commercial ELISA kit for detection of antibodies against Toxoplasma gondii in serum, plasma and meat juice from experimentally and naturally infected sheep

BackgroundToxoplasmosis is one of the most common food borne zoonoses worldwide, and can be a serious life-threatening disease in the congenitally infected fetus and in immunosupressed patients. Among food animals, sheep along with goats and pigs possess the highest incidence of T. gondii cysts in meat, and play a major role as a source of human infection.MethodsIn this study, a new commercial ELISA kit (PrioCHECK® Toxoplasma Ab SR, Prionics Schlieren-Zurich, Switzerland) for the detection of anti-T. gondii antibodies in serum, plasma and meat juice of sheep, was evaluated by comparing it with the indirect fluorescent antibody test (IFAT), indirect haemagglutination test (IHA) and real-time PCR, on samples from experimentally inoculated and naturally exposed sheep.ResultsThe commercial ELISA detected the infection status in 50% and 100% of sheep orally inoculated with 10,000 T. gondii oocysts (n = 6), from two or three weeks post infection (wpi), respectively, both on serum and plasma samples. Meat juice from all experimentally inoculated sheep collected at slaughter (12 wpi) showed positive ELISA values. In naturally exposed sheep (n = 396), the ELISA showed a very good agreement with IFAT (kappa = 0.91-1.0) and IHA (kappa = 0.96-1.0) performed on serum; and a positive correlation was observed between ELISA values and IFAT titers. By a Receiver Operating Characteristics (ROC) curve analysis, the commercial ELISA had relative sensitivities between 93.33% and 100%, and relative specificities between 96.87% and 100% respect to IFAT and IHA, depending on the considered cut-off value and animal groups tested. Furthermore, the ELISA correctly recognized all animals reacting positive in real-time PCR. The ELISA results on meat juice agreed with those on serum samples in all experimentally inoculated animals, and in 94 out of 96 (97.9%) naturally exposed sheep, when meat juice was tested at a 1:10 dilution.ConclusionThe commercial ELISA kit evaluated in this study could represent a valuable tool to improve the surveillance and reporting system for T. gondii in sheep populations at the farm level or for diagnosis at the slaughterhouse, contributing to the control of this widespread zoonosis.

Assessment of diagnostic accuracy of a commercial ELISA for the detection of Toxoplasma gondii infection in pigs compared with IFAT, TgSAG1-ELISA and Western blot, using a Bayesian latent class approach

Serological methods are the most commonly used diagnostic tools to detect Toxoplasma gondii infections in pigs. In the absence of a readily available ‘gold standard’, an estimation of diagnostic accuracy is difficult to assess. A commercial ELISA (PrioCHECK® Toxoplasma Ab porcine ELISA, Prionics, Schlieren, Switzerland) for the diagnosis of T. gondii infection in pigs was evaluated in naturally infected animals from two distinct populations; indoor and outdoor living animals. An assessment of diagnostic accuracy, using a Bayesian latent class approach with adjustment for within indoor and outdoor farm clustering using random effects, was performed. Tests used for comparison were: IFAT; ELISA using native affinity-purified P30 (SAG1) T. gondii tachyzoite surface antigen (TgSAG1-ELISA); and Western blot with T. gondii tachyzoites lysate. The data set comprised 297 pig serum samples across outdoor (n=149) and indoor (n=148) farms in Argentina. The estimated sensitivity and specificity for the commercial ELISA were 98.9% (95% credible interval: 96.2; 100) and 92.7% (95% credible interval: 87.7; 96.6), respectively. The analysis of sera and plasma from pigs (n=6) experimentally inoculated with 5,000 T. gondii oocysts revealed a pronounced antibody response beginning 2weeks p.i. until the end of the observation period (11weeks p.i.) in all animals. Meat juice obtained from inoculated animals after euthanasia also tested positive. These results suggest that the PrioCHECK® Toxoplasma Ab porcine ELISA may be a useful tool to perform serological diagnosis of T. gondii infections in pigs to control Toxoplasma infection in pigs and humans.

Molecular and Biological Characterization of First Isolates ofHammondia hammondifrom Cats from Ethiopia

Toxoplasma gondii oocysts are morphologically and antigenically similar to oocysts of another feline coccidian, Hammondia hammondi. The distinction between H. hammondi and T. gondii is important from an epidemiological perspective because all isolates of T. gondii are potentially pathogenic for humans and animals, whereas H. hammondi is not known to cause clinical disease in any naturally infected intermediate or definitive hosts. In the present report, H. hammondi (designated HhCatEt1 and HhCatEt2) oocysts were found microscopically in the feces of 2 of 36 feral domestic cats (Felis catus) from Addis Ababa, Ethiopia. Oocysts were orally infective to Swiss Webster and gamma interferon gene knockout mice; the inoculated mice developed tissue cysts in their muscles. Laboratory-raised cats fed mouse tissues of infected mice shed H. hammondi oocysts with a prepatent period of 5 days. The DNA extracted from sporulated oocysts reacted with H. hammondi-specific primers, and sequences were deposited in GenBank (accession nos. JX477424, and KC223619). This is the first report of isolation of H. hammondi from cats from the African continent.

Fate and Transport of Toxoplasma gondii Oocysts in Seasonally Snow Covered Watersheds: A Conceptual Framework from a Melting Snowpack to the Canadian Arctic Coasts

Toxoplasma gondii is a zoonotic protozoan that causes serious illness in humans and infects animals worldwide, including the Canadian Arctic. Indeed, high prevalence of infection amongst Inuit has been recorded, possibly due to consumption of raw infected seal meat. Here we explore the hypothesis that T. gondii oocysts contaminate the coastal marine environment via surface runoff from across the boreal watershed, particularly during the snowmelt period. We propose a conceptual framework of the different processes governing the fate and transport of T. gondii oocysts from the melting snowpack to the Canadian arctic coast via the freshwater runoff. This framework identifies the feasibility of a transmission pathway of oocysts from contaminated soil to the marine environment, but also the complexity and multiplicity of mechanisms involved. In addition, the framework identifies knowledge gaps for guiding future studies on T. gondii oocysts. Furthermore, this work could be used as a tool to investigate the possible estuarine contamination by other faeces-borne pathogens transported via the spring freshet in seasonally snow covered watersheds.

Prevalence of faecal-borne parasites in colony stray cats in northern Italy.

Endoparasitic infections are common in stray cats. Many of these parasites are responsible for zoonoses, and stray cats can be a source of environmental contamination. The prevalence of parasites in 139 stray colony cats in the city of Milan, northern Italy, was investigated by faecal examination. The overall prevalence of endoparasites was 50.4%, with 11 different parasites found. Parasites with zoonotic potential were detected in 49.6% of cats. Concurrent infections with two or more zoonotic parasites were recorded in 14.3% of cats. Among the parasites found, the most common was Toxocara cati (33.1%; P <0.0001). The other species found by coproscopic examination were: Ancylostoma tubaeformae (7.2%), Isospora species (4.3%), Trichuris vulpis (2.9%), Dipylidium caninum (2.9%), Aelurostrongylus abstrusus (2.9%), Eucoleus aerophilus (syn Capillaria aerophila) (1.4%), Spirometra species (1.4%), Taenia pisiformis (0.7%) and Hymenolepis nana (0.7%). Coproantigen specific for Giardia duodenalis was detected in 2.9% of the samples. Pseudoparasites (eggs of mites) were found in 4.3% of the samples. No sample contained Toxoplasma gondii oocysts, despite the fact that 70 cats tested positive for T gondii-specific IgG antibodies, and none of the diarrhoetic samples tested positive for Cryptosporidium species oocysts. Variables linked to infection were body condition score (BCS), the presence of diarrhoea and infection with G duodenalis. Cats infected with G duodenalis were more likely to have a low BCS (odds ratio (OR) = 11.5, P = 0.02) and diarrhoea (OR = 30.7, P = 0.0007). The results of the present study confirm that endoparasitic infections, most of which have zoonotic potential, are distributed in stray colony cats of Milan.

Isolation and genetic characterization of Toxoplasma gondii from mute swan (Cygnus olor) from the USA

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study, antibodies to T. gondii were determined in serum samples from 632 mute swans (Cygnus olor) collected from different areas of the USA. Sera were tested by T. gondii modified agglutination test; 54 (8.5%) of 632 samples were seropositive with titers of 25 in 28 sera, 50 in 22 sera, 100 in three samples, and 200 or higher in one swan. Hearts from 14 seropositive swans were bioassayed in mice and viable T. gondii (designated TgSwanUs1–3) were isolated from the hearts of three. These three T. gondii isolates were further propagated in cell culture, and DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers (SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico). Results of genotyping revealed that two strains (TgSwanUs1, TgSwanUs2) were Type III (ToxoDB PCR-RFLP genotype #2), and TgSwanUs3 was a new genotype designated here as ToxoDB PCR-RFLP genotype #216. Pathogenicity of oocysts derived from these three strains was determined in Swiss Webster (SW) outbred mice. All mice infected with oocysts and tachyzoites of the atypical isolate (TgSwanUs3) died of acute toxoplasmosis, irrespective of the dose. Oocysts of the remaining two isolates were less pathogenic but differed from each other; 10 oocysts of the TgSwanUs1 killed all inoculated mice whereas 1 million oocysts of the TgSwanUs2 were needed to kill all infected SW mice. Isolation of T. gondii from mute swan indicates that the local waters were contaminated by T. gondii oocysts, and that mouse T. gondii virulent strains are circulating in wildlife. Mute swan is a new host record for T. gondii.

Molecules to modeling: Toxoplasma gondii oocysts at the human-animal-environment interface.

Environmental transmission of extremely resistant Toxoplasma gondii oocysts has resulted in infection of diverse species around the world, leading to severe disease and deaths in human and animal populations. This review explores T. gondii oocyst shedding, survival, and transmission, emphasizing the importance of linking laboratory and landscape from molecular characterization of oocysts to watershed-level models of oocyst loading and transport in terrestrial and aquatic systems. Building on discipline-specific studies, a One Health approach incorporating tools and perspectives from diverse fields and stakeholders has contributed to an advanced understanding of T. gondii and is addressing transmission at the rapidly changing human-animal-environment interface.

Detection of Toxoplasma gondii oocysts in different water resources by Loop Mediated Isothermal Amplification (LAMP)

Human toxoplasmosis is potentially contracted due to consumption of contaminated drinking water and represents an increasing public health risk worldwide. Toxoplasma gondii oocysts can be resistant to standard disinfection processes, including UV radiation. Increased awareness of the risk of waterborne toxoplasmosis outbreaks has led to an increase in research interest in the detection of oocysts in environmental water systems. Ninety-five environmental water samples from the Lower Rhine area in Germany have been included in the study and examined for the presence of Toxoplasma. Water samples were filtered or flocculated by aluminum sulfate and purified by sucrose density gradient. DNA was then extracted, and the DNA samples were then examined by LAMP analysis. T. gondii DNA was detected in eight out of 83 (9.6%) influent and effluent samples obtained from wastewater treatment plants. All samples (n=12) from the surface, ground, raw and tap waters tested negative. The purpose of this work was to investigate the occurrence and distribution of Toxoplasma oocysts on the Lower Rhine in Germany. Our study provides evidence that the assay is a sensitive, specific, rapid and cost effective method for the detection of T. gondii and is useful for both the investigations of cases of waterborne outbreaks and for identifying the source of contamination.

β-1,3-Glucan, Which Can Be Targeted by Drugs, Forms a Trabecular Scaffold in the Oocyst Walls ofToxoplasmaandEimeria

ABSTRACTThe walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls.

Ante-Mortem Diagnosis, Diarrhea, Oocyst Shedding, Treatment, Isolation, and Genetic Typing ofToxoplasma gondiiAssociated with Clinical Toxoplasmosis in a Naturally Infected Cat

Toxoplasma gondii infections are common in humans and other animals, but clinical disease is relatively rare. It is unknown whether the severity of toxoplasmosis in immunocompetent hosts is due to the parasite strain, host variability, or to other factors. Recently, attention has been focused on the genetic variability among T. gondii isolates from apparently healthy and sick hosts. Whether T. gondii genetic makeup plays a part in the pathogenesis of clinical feline toxoplasmosis is uncertain because little is known of genetic typing of strains associated with clinical feline toxoplasmosis. A 6-mo-old domestic male cat was hospitalized because of lethargy, anorexia, fever, and diarrhea. Numerous (6 million in 1 sample) T. gondii oocysts were found in feces of the cat and antibodies to T. gondii (titer 1:800) were found in its serum by the modified agglutination test. The cat was medicated orally with Clindamycin for 10 days; it became asymptomatic after 10 days and was discharged from the hospital. Viable T. gondii (designated TgCatUs9) was isolated from feces (oocysts) by bioassays in mice. Genetic typing using the DNA extracted from the brains of infected mice and 10 PCR-restriction fragment length polymorphism (RFLP) markers revealed Type II allele at the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, and PK1 loci and Type I at the L358 and Apico loci; therefore, this isolate belongs to the ToxoDB PCR-RFLP genotype no. 4, which is grouped into the Type 12 lineage that is dominant in wildlife from North America. To our knowledge, this is the first T. gondii isolate characterized genetically from a sick cat in the USA.