Sporulation and Survival of Toxoplasma gondii Oocysts in Seawater

ABSTRACT. We have been collaborating since 1992 in studies on southern sea otters ( Enhdyra lutris nereis ) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otiers. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 1.5 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post-inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22–24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.     

Physical Inactivation of Toxoplasma gondii Oocysts in Water

Inactivation of Toxoplasma gondii oocysts occurred with exposure to pulsed and continuous UV radiation, as evidenced by mouse bioassay. Even at doses of ≥500 mJ/cm², some oocysts retained their viability.     

Detection of Toxoplasma gondii Oocysts in Drinking Water

The world’s largest outbreak of waterborne toxoplasmosis occurred in a municipality in the western Canadian province of British Columbia. When drinking water emerged as a possible source of infection during the outbreak investigation, a laboratory method was needed to attempt detection of the parasite, Toxoplasma gondii. The method developed was based on the current U.S. Environmental Protection Agency method for detection of Cryptosporidium oocysts. Collection of large-volume drinking water samples and cartridge filter processing were unchanged, although identification of Toxoplasma oocysts in the filter retentate was carried out by using a previously described rodent model. Validation of the method developed was tested by using oocysts from a well-characterized Toxoplasma strain.     

Hydrologic and Vegetative Removal of Cryptosporidium parvum,Giardia lamblia,and Toxoplasma gondii Surrogate Microspheres in Coastal Wetlands

Constructed wetland systems are used to reduce pollutants and pathogens in wastewater effluent, but comparatively little is known about pathogen transport through natural wetland habitats. Fecal protozoans, including Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii, are waterborne pathogens of humans and animals, which are carried by surface waters from land-based sources into coastal waters. This study evaluated key factors of coastal wetlands for the reduction of protozoal parasites in surface waters using settling column and recirculating mesocosm tank experiments. Settling column experiments evaluated the effects of salinity, temperature, and water type (“pure” versus “environmental”) on the vertical settling velocities of C. parvum, G. lamblia, and T. gondii surrogates, with salinity and water type found to significantly affect settling of the parasites. The mesocosm tank experiments evaluated the effects of salinity, flow rate, and vegetation parameters on parasite and surrogate counts, with increased salinity and the presence of vegetation found to be significant factors for removal of parasites in a unidirectional transport wetland system. Overall, this study highlights the importance of water type, salinity, and vegetation parameters for pathogen transport within wetland systems, with implications for wetland management, restoration efforts, and coastal water quality.     

Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.Toxoplasma gondii is a ubiquitous parasite found in all classes of warm-blooded vertebrates. Nearly one-third of humans have been exposed to this parasite (15). In immunocompetent adults, acute infection normally results in transient influenza-like symptoms, but in immunocompromised persons retinochoroiditis and encephalitis are more common. Infected individuals can retain the parasite as quiescent tissue cysts for long periods, but invasive infection can occur if the immune status of the infected person deteriorates (42). If women become infected during pregnancy, the parasite can cause abortion or seriously damage the fetus. The potential morbidity from the ingestion of oocysts of T. gondii and the organism''s low infectious dose are a great concern for public health. There are at least four reported waterborne outbreaks of toxoplasmosis (2, 3, 14, 44), and endemic toxoplasmosis in Brazil is associated with the consumption of water or ice contaminated with T. gondii oocysts (1, 23), demonstrating the potential for the waterborne transmission of this disease (15).There is no rapid detection method for T. gondii oocysts recovered from water or other environmental samples. Traditionally, the detection of protozoa in water required their concentration from large volumes of water by filtration or centrifugation, isolation from concentrated particulates by immunomagnetic separation (IMS) or other methods, and detection by immunofluorescence microscopy, the infection of cultured cells, biochemistry, animal infection tests, molecular techniques, or combinations of these (17, 58). For T. gondii oocysts there are no commercially available IMS techniques, no widely available immunofluorescent staining reagents, and no standardized cultivation protocols. The identification of oocysts from environmental samples has included differential floatation and mouse inoculation (27). Recently, IMS techniques have been developed for the isolation of T. gondii oocysts and sporocysts in water (16, 18). Both the oocyst and sporocyst IMS assays, however, had poor specificity, because antibodies cross-reacted with water debris and the sporocyst wall of Hammondia hammondi, Hammondia heydorni, and Neospora caninum (16).PCR is becoming a favored technique for the detection of T. gondii oocysts in water (32, 35, 36, 46, 49, 55) over the conventional mouse bioassay (27, 55), as it reduces the detection time from weeks to 1 to 2 days. Although they have been developed for the detection of T. gondii in clinical specimens (50), no real-time PCR assays have been adapted for the detection of oocysts in water samples, possibly because of expected high concentrations of PCR inhibitors and low numbers of T. gondii oocysts in environmental samples (55).There are several unresolved issues regarding the effectiveness of the PCR detection of T. gondii oocysts in water. The most readily available method for the isolation of T. gondii oocysts from water samples is flocculation or sucrose floatation prior to DNA extraction (35, 36, 49, 55). Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor. For DNA extraction, the phenol-chloroform method or QIAamp mini kit frequently is used (16, 35, 36, 46, 55). When oocysts are recovered from water either by the Environmental Protection Agency (EPA) information collection rule method (53) or EPA Method 1623 (54) without purification by IMS, neither the conventional phenol-chloroform DNA extraction nor the QIAamp mini kit is effective at removing PCR inhibitors (30, 55, 57).Recently, a method was used effectively in the analysis of Cryptosporidium oocysts in surface water, storm water, and wastewater samples (30). This method extracted DNA directly from water concentrates without pathogen IMS, differential flotation, or enrichment cultures, and it utilized a commercial DNA extraction kit, the FastDNA spin kit for soil, and a high concentration of nonacetylated bovine serum albumin in PCR. The FastDNA soil kit has a higher capacity for PCR inhibitor removal than several other commercial extraction kits designed for environmental samples. The use of nonacetylated bovine serum in the PCR neutralizes residual PCR inhibitors that are coextracted with the DNA (30).In the present study, the performance of two published LightCycler real-time PCR assays based on the multicopy B1 gene and 529-bp repetitive element (13, 45) and a newly developed LightCycler real-time PCR assay using a common primer set were analyzed for the detection of T. gondii, using pure DNA and DNA extracted by the aforementioned extraction method (30) from water sample concentrates seeded with known number of oocysts.     

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.     

Removal of Toxoplasma gondii Oocysts from Sea Water by Eastern Oysters (Crassostrea virginica)

SUMMARY. Toxoplasma gondii infections have been reported in a number of marine mammals. Presently it is not known how these animals acquire T. gondii from their aquatic environment. The eastern oyster, Crassostrea virginica , has been shown to remove Cryptosporidium oocysts from seawater and a similar phenomenon may be occurring with T. gondii oocysts and marine invertebrates. The present study was done to determine if eastern oysters could remove and retain T. gondii oocysts from seawater. Oocysts of the VEG strain of T. gondii (1 × 10⁶ oocysts) were placed in seawater (32 ppt NaCl) containing live eastern oysters. The infected seawater was removed one day postinoculation (PI) and replaced with fresh seawater. Selected oysters were removed at 1, 3 and 6 days PL Hemolymph, gill washes, and oyster tissue were collected separately at each observation time. The oyster tissue was homogenized and all 3 samples fed separately to mice. Toxoplasma gondii positive mice were observed at each time period. The results indicate that T. gondii oocysts can be removed from seawater by eastern oysters and retain their infectivity. Contaminated raw oysters may serve as a source of T. gondii infection for marine mammals and humans.     

Development of a PCR-Enzyme Immunoassay Oligoprobe Detection Method for Toxoplasma gondii Oocysts, Incorporating PCR Controls

Infections caused by Toxoplasma gondii are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of T. gondii infection. T. gondii has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to T. gondii oocysts. Because of the lack of information regarding the prevalence of T. gondii in surface waters, there is a clear need for a rapid, sensitive method to detect T. gondii from water. Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained. Detection of T. gondii nucleic acid by PCR has become the preferred method. We have developed a PCR amplification and detection method for T. gondii oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracil-N-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than 1 day. Fewer than 50 oocysts can be detected following recovery of oocyst DNA. Development of a T. gondii oocyst PCR detection method will provide a useful technique to estimate the levels of T. gondii oocysts present in surface waters.     

Surface antigens of Toxoplasma gondii: variations on a theme

Toxoplasma gondii is an obligate intracellular protozoan parasite with an exceptionally broad host range. Recently, it has become apparent that the number of surface antigens (SAGs) it expresses may rival the number of genera it can infect. Most of these antigens belong to the developmentally regulated and distantly related SAG1 or SAG2 families. The genes encoding the surface antigens are distributed throughout the T. gondii genome, with remarkably little polymorphism observed at each locus. Results from a number of studies have suggested that the surface antigens play an important role in the biology of the parasite. For example, SAG3 null mutants generated by targeted disruption provide convincing evidence that this surface antigen, at least, functions during parasite attachment. Analyses of a SAG1 knockout in rodents, however, indicate that this surface antigen may play a crucial role in immune modulation or virulence attenuation. The current understanding of the SAG1 and SAG2 families will be discussed here.     

Effect of Immunization of Cats with Isospora felis and BCG on Immunity to Reexcretion of Toxoplasma gondii Oocysts*

SYNOPSIS. The effect of pretreatment with Isospora felis and bacillus Calmette-Guérin (BCG) on the reexcretion of Toxoplasma gondii occysts was studied in 16 coccidia-free cats. The following conclusions were drawn: (A) Chronically T. gondii-infected cats reexcreted T. gondii oocysts after superinfection with I. felis, and this reexcretion was prevented in cats infected with I. felis before T. gondii infection. (B) Administration of BCG before Toxoplasma infection had no apparent effect on the outcome of the infection.     

Cytoskeleton of Toxoplasma gondii

The cytoskeleton of Toxoplasma gondii was studied by electron microscopy using whole mounts of detergent-extracted parasites and thin sections of routine preparations, tannic acid-stained organisms, and detergent-extracted parasites. In whole mounts, the spiral arrangement of the 22 pellicular microtubules closely corresponded to the pattern of surface ridges seen previously by scanning electron microscopy and reflected the torsion of the parasite body during locomotion. The microtubules had free posterior ends and were anchored anteriorly in the polar ring, presumed to be a microtubule organizing center (MTOC). The insertions of the microtubules were supported by blunt projections of the polar ring, forming a cogwheel pattern in transverse view. The internal microtubules had 13 protofilaments and were twice the length of the conoid. They extended through the conoid and ended at the anterior preconoidal ring, presumably a second MTOC. The subunits of the conoid were arranged in a counterclockwise spiral when traced from base to tip, as were the pellicular microtubules. We postulate that as the conoid moves, the polar ring complex moves along the spiral pathway of the conoid subunits. Retraction of the conoid would then rotate the polar ring, producing the torsion of the body we observed by SEM.     

Toxoplasma gondii and mucosal immunity

Toxoplasma gondii, an intracellular parasite infects the host through the oral route. Infection induces a cascade of immunological events that involve both the components of the innate and adaptative immune responses. Alteration of the homeostatic balance of infected intestine results in an acute inflammatory ileitis in certain strains of inbred mice. Both the infected enterocytes as well as the CD4 T cells from the lamina propria produce chemokines and cytokines that are necessary to clear the parasite whereas CD8 intraepithelial lymphocytes secrete transforming growth factor beta that reduces the inflammation. In this review, we describe the salient features of this complex network of interactions among the different components of the gut-associated lymphoid tissue cell population that are induced after oral infection with T. gondii.     

Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondi in faeces collected from dogs in Germany

Faecal samples of 24,089 dogs were examined coproscopically in two veterinary laboratories in Germany between March 2001 and October 2004. In 47 dogs, oocysts of 9–14 μm size were found. Their morphology was similar to those of Hammondia heydorni and Neospora caninum. Samples of 28 of these dogs were further examined by inoculation into gerbils: seven isolates induced a specific antibody response against antigens of N. caninum NC-1 tachyzoites. This response suggests that the isolates contained N. caninum. In addition to H. heydorni (12 times isolated), Toxoplasma gondii occysts (twice) and Hammondia hammondi oocysts (twice) were observed in dog faeces. The latter findings suggest that coprophagia with a subsequent intestinal passage by dogs plays a role in the dissemination of coccidian parasites for which cats are definitive hosts. Five of the seven N. caninum (NC-GER2, NC-GER3, NC-GER4, NC-GER5, NC-GER6) and the two T. gondii isolates (TG-dgGER1, TG-dgGER2) were successfully passaged into cell culture and are now available for detailed characterization. In contrast to oocysts of other parasites, N. caninum oocysts were predominantly found between January and April (Fisher exact; P=0.038). In the sera of dogs shedding N. caninum, no reactions against the immunodominant antigens with apparent molecular weights of 19, 29, 30, 33 and 37 kDa of N. caninum tachyzoites were observed 3–5 weeks after shedding. However, the animals recognized a 152-kDa N. caninum antigen. Compared with those identified as H. heydorni, T. gondii or H. hammondi, N. caninum oocyst isolates were significantly smaller in length with the 75th percentiles ≤10.7 μm when measured in concentrated sucrose solution and smaller length–width ratios with the 75th percentiles ≤1.06. It may thus be possible to develop criteria for a preliminary identification of N. caninum in dog faeces based on the oocyst morphology.     

Toxoplasma gondii tachyzoite-bradyzoite interconversion

During infection in the intermediate host, Toxoplasma gondii undergoes stage conversion between the rapidly dividing tachyzoite that is responsible for acute toxoplasmosis and the slowly replicating, encysted bradyzoite stage. This process of tachyzoite-bradyzoite interconversion is central to the pathogenesis and longevity of infection. Recent research has identified several stage-specific genes and proteins. However, despite recent advances in the understanding of Toxoplasma cell biology, more research is necessary to elucidate the complex events occurring during tachyzoite-bradyzoite interconversion. Here, a brief summary of this process is provided and a new method to characterize gene expression during interconversion is introduced.