Chemical inactivation of Toxoplasma gondii oocysts in water

The protozoan parasite Toxoplasma gondii is increasingly recognized as a waterborne pathogen. Infection can be acquired by drinking contaminated water and conventional water treatments may not effectively inactivate tough, environmentally resistant oocysts. The present study was performed to assess the efficacy of 2 commonly used chemicals, sodium hypochlorite and ozone, to inactivate T. gondii oocysts in water. Oocysts were exposed to 100 mg/L of chlorine for 30 min, or for 2, 4, 8, 16, and 24 hr, or to 6 mg/L of ozone for 1, 2, 4, 8, or 12 min. Oocyst viability was determined by mouse bioassay. Serology, immunohistochemistry, and in vitro parasite isolation were used to evaluate mice for infection. Initially, mouse bioassay experiments were conducted to compare the analytical sensitivity of these 3 detection methods prior to completing the chemical inactivation experiments. Toxoplasma gondii infection was confirmed by at least 1 of the 3 detection methods in mice inoculated with all doses (10(5)-10(0)) of oocysts. Results of the chemical exposure experiments indicate that neither sodium hypochlorite nor ozone effectively inactivate T. gondii oocysts, even when used at high concentrations.     

Antibody levels in goats fed Toxoplasma gondii oocysts

Outbred goats were fed 10(5) Toxoplasma gondii oocysts and were monitored twice weekly for 8 wk for rectal temperature, clinical signs, parasitemia, and antibody levels by indirect fluorescence antibody test (IFAT), latex agglutination test (LAT), and indirect enzyme-linked immunosorbent assay (ELISA). After 8 wk, all goats were killed, and samples of heart, skeletal muscle, brain, lymph nodes, kidneys, and liver were bioassayed in mice. Anorexia, fever, and lethargy were observed from day 3 to day 7 postinfection (PI). Parasitemia was detected by bioassay in 50% of infected goats from day 7 to day 14 PI. Viable T. gondii organisms were isolated from all infected goats. Antibodies to T. gondii were detected in some animals on day 10 PI by IFAT and LAT and on day 14 PI by ELISA. The infected goats were seropositive on day 17 PI.     

Autofluorescence of Toxoplasma gondii and related coccidian oocysts

This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.     

Purification of Toxoplasma gondii oocysts by cesium chloride gradient

We describe the use of a cesium chloride (CsCl) gradient as an improvement for the purification of Toxoplasma gondii oocysts from concentrated suspensions. After concentration by sucrose flotation, this technique gives a > 96% recovery of very pure unsporulated or sporulated oocysts, but requires "fresh" oocysts (< or = 10 weeks of age). This material is suitable for biochemical and immunological analyses of environmentally resistant T. gondii oocysts.     

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

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

Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil

Toxoplasma gondii oocysts spread in the environment are an important source of toxoplasmosis for humans and animal species. Although the life expectancy of oocysts has been studied through the infectivity of inoculated soil samples, the survival dynamics of oocysts in the environment are poorly documented. The aim of this study was to quantify oocyst viability in soil over time under two rain conditions. Oocysts were placed in 54 sentinel chambers containing soil and 18 sealed water tubes, all settled in two containers filled with soil. Containers were watered to simulate rain levels of arid and wet climates and kept at stable temperature for 21.5 months. At nine sampling dates during this period, we sampled six chambers and two water tubes. Three methods were used to measure oocyst viability: microscopic counting, quantitative PCR (qPCR), and mouse inoculation. In parallel, oocysts were kept refrigerated during the same period to analyze their detectability over time. Microscopic counting, qPCR, and mouse inoculation all showed decreasing values over time and highly significant differences between the decreases under dry and damp conditions. The proportion of oocysts surviving after 100 days was estimated to be 7.4% (95% confidence interval [95% CI] = 5.1, 10.8) under dry conditions and 43.7% (5% CI = 35.6, 53.5) under damp conditions. The detectability of oocysts by qPCR over time decreased by 0.5 cycle threshold per 100 days. Finally, a strong correlation between qPCR results and the dose infecting 50% of mice was found; thus, qPCR results may be used as an estimate of the infectivity of soil samples.     

Effects of high pressure processing on Toxoplasma gondii oocysts on raspberries

Oocysts are the environmentally resistant life stage of Toxoplasma gondii. Humans can become infected by accidentally ingesting the oocysts in water or from contaminated produce. Severe disease can occur in immunocompromised individuals, and nonimmune pregnant women can infect their offspring. Chronic infection is associated with decreased mental functions, vision and hearing problems, and some mental disorders such as schizophrenia. High pressure processing (HPP) is a commercial method used to treat food to eliminate pathogens. Treatment of produce to eliminate viable T. gondii oocysts would provide a means to protect consumers. The present study was done to better define the effects of HPP on oocysts placed on raspberries. Raspberries were chosen because they are a known source of a related human intestinal parasite, Cyclospora cayetanensis. Raspberries were inoculated with 5 x 10(4) oocysts of the VEG strain of T. gondii for 20 hr prior to HPP. Individual raspberries were exposed to 500 MPa, 400 MPa, 340 MPa, 300 MPa, 270 MPa, 250 MPa, 200 MPA, 100 MPa, or no MPa treatment for 60 sec in a commercial HPP unit (1 MPa = 10 atm = 147 psi). Treatment of raspberries with 340 MPa for 60 sec was needed to render oocysts spot inoculated on the raspberries noninfectious for mice. Treatment of raspberries with 200 MPa or less for 60 sec was not effective in rendering oocysts noninfectious for mice.     

Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica)

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.     

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.     

Sporulation and survival of Toxoplasma gondii oocysts in different types of commercial cat litter

Toxoplasma gondii oocysts are environmentally resistant and can survive outdoors for many months in dry and cold climates. In the present study, sporulation and survival of T. gondii oocysts was studied in different types of cat litters commercially available in the United States. Oocysts sporulated within 2-3 days in all types of cat litters and occasionally remained viable for 14 days. Results indicate that cat litter should be changed daily to prevent sporulation and infectivity to people.     

Examination of attachment and survival of Toxoplasma gondii oocysts on raspberries and blueberries

The consumption of Toxoplasma gondii oocysts on fresh produce may be a means of its transmission to humans. Cats shed T. gondii oocysts, which contaminate produce directly or contaminate water sources for agricultural irrigation and pesticide and fertilizer applications. Cyclospora cayetanensis is a related coccidial parasite, and outbreaks of diarrhea caused by C. cayetanensis have been associated with the ingestion of contaminated raspberries. The oocysts of these coccidians are similar in size and shape, indicating that they may attach to and be retained on produce in a similar manner. In the present study the attachment and survival of T. gondii oocysts on 2 structurally different types of berries were examined. Raspberries and blueberries were inoculated individually with 1.0 x 10(1) to 2.0 x 10(4) oocysts of sporulated T. gondii. Berries inoculated with 2.0 x 10(4) oocysts were stored at 4 C for up to 8 wk. Oocyst viability and recovery were analyzed by feeding processed material to mice. Mice fed T. gondii-inoculated berries stored at 4 C for 8 wk developed acute infections. In other experiments mice fed raspberries inoculated with > or = 1.0 x 10(1) oocysts became infected, whereas only mice fed blueberries inoculated with > or = 1.0 x 10(3) oocysts became infected. This study demonstrates that T. gondii oocysts can adhere to berries and can be recovered by bioassays in mice and that raspberries retain more inoculated oocysts than do blueberries. The results suggest that T. gondii may serve as a model for C. cayetanensis in food safety studies.     

Effects of high pressure processing on infectivity of Toxoplasma gondii oocysts for mice

High pressure processing (HPP) has been shown to be an effective non-thermal method of eliminating non-spore forming bacteria from a variety of food products. The shelf-life of the products is extended and the sensory features of the food are not or only minimally effected by HPP The present study examined the effects of HPP using a commercial scale unit on the viability of Toxoplasma gondii oocysts. Oocysts were exposed from 100 to 550 MPa for 1 min in the HPP unit and then HPP treated oocysts were orally fed to groups of mice. Oocysts treated with 550 MPa or less did not develop structural alterations when viewed with light microscopy. Oocysts treated with 550 MPa, 480 MPa, 400 Mpa, or 340 MPa were rendered noninfectious for mice. Mice fed oocysts treated with no or 100 to 270 MPa became infected and most developed acute toxoplasmosis and were killed or died 7 to 10 days after infection. These results suggest that HPP technology may be useful in the removal of T. gondii oocysts from food products.