Oocyst shedding by cats fed isolated bradyzoites and comparison of infectivity of bradyzoites of the VEG strain Toxoplasma gondii to cats and mice

Infectivity of bradyzoites of the VEG strain of Toxoplasma gondii was compared in cats and mice. For this, tissue cysts were separated from brains of infected mice using a Percoll gradient, and bradyzoites were released by incubation in acidic pepsin solution. After filtration through a 3-microm filter, bradyzoites were counted and diluted 10-fold in RPMI tissue culture medium. Dilutions estimated to have 1, 10, 100, and 1,000 bradyzoites were fed to cats and inoculated into mice, orally or subcutaneously (s.c.). Three experiments were performed. In experiment 1, 2 of 2 cats fed 1,000 bradyzoites, 1 of 2 cats fed 100 bradyzoites, 1 of 4 cats fed 10 bradyzoites, and 1 of 4 cats fed 1 bradyzoite shed millions of oocysts; 1,000 bradyzoites were infective to all 4 inoculated mice s.c. but not to 4 mice inoculated orally, and 100 bradyzoites were infective to 2 of 4 mice injected s.c. but not to 4 mice inoculated orally. All 16 mice (8 oral, 8 s.c.) injected with 1 or 10 bradyzoites were negative for T. gondii. In experiment 2, 1 of 4 cats fed 10 counted bradyzoites shed oocysts; the same inocula were not infective to 4 mice injected s.c. In experiment 3, 3 of 4 cats fed 1,000 bradyzoites shed oocysts and the inocula were infective to 10 of 10 mice s.c. and 4 of 10 mice orally; 4 of 4 cats fed 100 bradyzoites shed oocysts and the inocula were infective to 6 of 10 mice s.c. and 0 of 10 mice orally; 10 bradyzoites were not infective to cats and mice. Results indicate that bradyzoites are more infective to cats than to mice, and cats can shed millions of oocysts after ingesting just a few bradyzoites.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

Effect of Age and Sex on the Acquisition of Immunity to Toxoplasmosis in Cats*

SYNOPSIS. The effects of age and sex of the cat on oocyst shedding, multiplication of Toxoplasma gondii in tissues of cats, and acquisition of immunity were investigated after oral inoculation of cats with Toxoplasma cysts. Twenty-five cats varying in age from 1 week to 39 months were killed 7-97 days after inoculation with T. gondii. Homogenates of brain, heart, mesenteric lymph nodes, retina, and blood from these cats were inoculated into mice to test for Toxoplasma infectivity. Toxoplasma was isolated more frequently and in higher titers in mice receiving inocula from cats of the youngest age group (1 week old). Toxoplasma gondii was isolated from tissues of only 2 of 21 cats older than 2 months (at the time of inoculation), although all of the animals shed oocysts within 1 week after ingesting the parasites. The number of oocysts shed varied among littermates of the same sex and between sexes. Generally, cats younger than 12 months shed more oocysts than older cats. The number of oocysts shed by older cats varied considerably; males generally shed more oocysts than the females. However, the numbers of cats examined were too small for statistical comparison. Nevertheless, the observations suggest that cats older than 12 months should not be used in experiments where numbers of oocysts shed is critical.     

Toxoplasma gondii: genetic recombination between drug resistant mutants

Mutants resistant to adenine arabinoside (ara-A) or to 5-fluorodeoxyuridine (FUDR) were isolated from a newly isolated oocyst producing strain of Toxoplasma gondii. The selection and characterization of these mutants were carried out in human fibroblast cultures. The ara-A-resistant mutant lacked the enzyme adenosine kinase. The biochemical basis of FUDR resistance remains unknown. Both mutants were used to infect mice to produce brain cysts that contained bradyzoites. Mouse brains that contained cysts were fed to kittens to complete the sexual cycle of T. gondii. Those kittens fed cysts of only one drug-resistant mutant excreted oocysts that yielded no detectable recombinant doubly resistant parasites that could make plaques in the presence of both ara-A and FUDR. Kittens fed a mixture of cysts that contained both mutants excreted oocysts that contained approximately 12% doubly resistant parasites. The reciprocal recombinant, sensitive to both drugs, was also isolated. The doubly resistant recombinant was totally deficient in adenosine kinase activity. This pattern of inheritance is consistant only with a haploid genome for all stages of T. gondii except the zygote formed by fusion of gametes and the unsporulated oocyst. Two FUDR-resistant mutants were also defective in the production of oocysts. These mutants failed to recombine with an ara-A-resistant mutant of proven fertility and thus their inability to make oocysts must result from a defect in the production of both microgametes and macrogametes.     

Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Besnoitia darlingi (Apicomplexa,Sarcocystidae, Toxoplasmatinae): Transmission between Opossums and Cats1

ABSTRACT. Opossums (Didelphis marsupialis), act as intermediate hosts for Besnoitia darlingi and could be infected orally with sporozoites (oocysts) and bradyzoites (tissue cysts), or intraperitoneally (i.p.) with tachyzoites. Infections could presumably be transmitted through cannibalism. Cats (Felis catus), the definitive host, could be infected only with bradyzoites but not sporozoites. Oocysts shed by cats measure about 12 × 12 μm, resemble similarly sized oocysts of Toxoplasma gondii and Hammondia hammondi, and must be differentiated by the appearance of tissue cysts after experimental infection of intermediate hosts. Cats did not form tissue cysts of B. darlingi. Tachyzoites from the related B. jellisoni could be used in the Sabin-Feldman dye test to determine the development of antibody to B. darlingi in opossums after infection.     

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.     

Toxoplasma gondii B1 Gene Detection in Feces of Stray Cats around Seoul,Korea and Genotype Analysis of Two Laboratory-Passaged Isolates

The increasing prevalence of Toxoplasma gondii infection in the human population in the Republic of Korea (= Korea) is due to various reasons such as an increase in meat consumption. However, the importance of cats in transmitting T. gondii infection through oocysts to humans has seldom been assessed. A total of 300 fecal samples of stray cats captured around Seoul from June to August 2013 were examined for T. gondii B1 gene (indicating the presence of oocysts) using nested-PCR. Fourteen (4.7%) of 300 cats examined were positive for B1 gene. Female cats (7.5%) showed a higher prevalence than male cats (1.4%). Cats younger than 3 months (5.5%) showed a higher prevalence than cats (1.5%) older than 3 months. For laboratory passage of the positive samples, the fecal suspension (0.2 ml) of B1 gene positive cats was orally inoculated into experimental mice. Brain tissues of the mice were obtained after 40 days and examined for the presence of tissue cysts. Two isolates were successfully passaged (designated KNIH-1 and KNIH-2) and were molecularly analyzed using the SAG5D and SAG5E gene sequences. The SAG5D and SAG5E gene sequences showed high homologies with the ME49 strain (less virulent strain). The results indicated the importance of stray cats in transmitting T. gondii to humans in Korea, as revealed by detection of B1 gene in fecal samples. T. gondii isolates from cats were successfully passaged in the laboratory for the first time in Korea.     

Toxoplasma gondii: Evidence for the transmission by semen in dogs

Ten male dogs were distributed into three experimental groups for infection with Toxoplasma gondii: GI - three dogs inoculated with 2.0 × 10⁵ P strais oocysts, GII - three dogs infected with 1.0 × 10⁶ RH strain tachyzoites, and GIII - four controls dogs. Several clinical parameters were evaluated. IFAT was performed to detect anti-T. gondii antibodies. Presence of the parasite in semen was evaluated by PCR and bioassay techniques. Tissue parasitism was examined using bioassays and immunohistochemistry in testicle and epididymis fragments collected after orchiectomy. In semen samples collected from these two groups, the presence of T. gondii was verified by bioassays and PCR. T. gondii was detected by immunohistochemistry in tissues (testicle and epididymis fragments) of all six experimentally infected dogs. The T. gondii-positive seminal samples were used in the artificial insemination (AI) of four female dogs free of toxoplasmic infection. Seven days after AI, all of the female dogs presented serologic conversion (IFAT). Fetal reabsorption occurred in two of the dogs, while the others sustained full-term gestation. Several T. gondii cysts were detected in the brains of four offspring. These results suggest that T. gondii can be sexually transmitted in domestic dogs.     

Feline Toxoplasmosis from Acutely Infected Mice and the Development of Toxoplasma Cysts*

SYNOPSIS. The development of Toxoplasma cysts was studied in mice inoculated with tachyzoites by several routes. After 1–30 days of infection, murine tissues were examined microscopically, and portions or whole carcasses were fed to mice and cats. The feces of the cats were examined for oocyst shedding. Cyst-like structures containing distinct PAS-positive granules were first seen after 3 days of infection with tachyzoites, and became numerous by 6 days. Argyrophilic walls were first seen after 6 days, and became numerous by 16 days of infection with tachyzoites. Prepatent periods to oocyst shedding (PPO) were either “short” (3–10 days) or “long” (19–48 days). The “short” PPO was found only in cats that had ingested mice infected for 3 days or longer, and was related to the development of PAS-positive granules in T. gondii, and to high, 60–100%, oral infectivity rates for cats. The “long” PPO followed the ingestion of mice infected for only 1–2 days, and was related to tachyzoites without distinct PAS-positive granules and low, 32% or less, infectivity for cats. The “long” PPO followed also the ingestion of oocysts and the parenteral inoculation of tachyzoites, bradyzoites, or sporozoites. Using the “short” PPO as a criterion for detecting cysts in tissues, it was shown that (a) numerous cysts developed in mice 5 days after inoculation with tachyzoites, 7–9 days after inoculation with cysts, and 9–10 days after inoculation with oocysts, and (b) cysts developed faster and more frequently in the brain and muscle than in lungs, liver, spleen, and kidneys of mice inoculated with tachyzoites.     

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.     

Toxoplasma gondii seroprevalence in goats,cats and humans in Russia

Toxoplasmosis, a most common zoonosis, is caused by the protozoan parasite Toxoplasma gondii. However, there is little epidemiological information on T. gondii infections in humans and livestock animals in Russia. Therefore, in this study, the seroprevalence of T. gondii in goats in Russia was investigated. A total of 216 goats from 32 farms were investigated and 95 of them were seropositive for T. gondii. The difference in seroprevalence between the examined regions was not statistically significant. We next collected serum samples from 99 cats and 181 humans in Kazan city, the state capital of the Republic of Tatarstan, Russia, and examined their T. gondii seroprevalences. Thirty-nine of the 99 cat samples and 56 of the 181 human samples showed seropositivity. Logistical regression analysis revealed that the cat breeding history of the human subjects, but not their sex or age is a significant risk factor for T. gondii seropositivity. These findings suggest that the natural environment in Russia may be widely polluted with T. gondii oocysts shed by cats, and ingestion of these oocysts provides a major route for human infection with this parasite.     

Antibody kinetics and exposure to Toxoplasma gondii in cats: a seroepidemiological study

Domestic cats are the most important definitive hosts for Toxoplasma gondii, the agent of an important global zoonosis. Serial sera from cats orally inoculated either with T. gondii tissue cysts (n = 3) or sporulated oocysts (n = 3) and from 65 client-owned cats, plus sera from 1,757 client-owned cats presented to veterinarians in Switzerland were analysed for an antibody response to T. gondii by ELISA. Risk factors for seropositivity and prevalence were estimated with a generalised linear and beta regression model. The first model examined the association of an OD₄₀₅ value as the dependent variable, with gender, age, and outside access as possible independent variables. In the second model, we first analysed the data assuming a bimodal distribution representing two overlapping distributions of OD₄₀₅ values from positive and negative cats, enabling the assignment of a probability of true infection status to each cat. Mean probabilities of true infection status across groups represent an estimate of true prevalence. These probabilities were then regressed against age, gender and outside access. Antibody kinetics in cats orally inoculated with tissue cysts, shedding oocysts, did not differ significantly from those of cats inoculated with sporulated oocysts without detectable oocyst excretion, suggesting extraintestinal parasite invasion and exposure to tachyzoites in both situations at an early stage of infection. Analysis of serial serum samples suggested a persisting long-term humoral immune response. Of the client-owned cats, 42.4% (95% confidence interval (CI): 40.1–44.6) had a positive true infection status. This was higher (56.3% (95% CI: 53.2–59.6)) in cats with outside access than in those without (22.1% (95% CI: 18.9–25.4)). In the first model, the factors age (P < 0.0001), gender (male: P = 0.046), and outside access (P < 0.0001) were independently associated with significantly higher OD₄₀₅ values. In the second model, the probability of having a positive true infection status increased with age (P < 0.0001), was higher with outside access (P < 0.0001) and in outdoor male cats (P = 0.0006).     

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.     

Life Cycle of Hammondia hammondi (Apicomplexa: Sarcocystidae) in Cats

Hammondia hammondi and Toxoplasma gondii are feline coccidians that are morphologically, antigenically, and phylogenitically related. Both parasites multiply asexually and sexually in feline intestinal enterocytes, but H. hammondi remains confined to enterocytes whereas T. gondii also parasitizes extra‐intestinal tissues of the cat. Here, we studied multiplication of H. hammondi in feline intestine and compared with T. gondii cycle. Five parasite–free cats were inoculated orally with tissue cysts and free bradyzoites from skeletal muscles of gamma interferon gene knockout mice and killed at 1, 3, 4, 6, and 7 d later. At 1 and 3 d post inoculation (DPI), numerous individual intracellular bradyzoites were detected in histological sections of small intestine. At 4 DPI only schizonts were found and they were located in enterocyte cytoplasm above the host cell nucleus. At 6 and 7 DPI both schizonts and gamonts were seen and they were located in enterocytes. Ultrastucturally, schizogonic and gametogonic development of H. hammondi was similar to T. gondii. However, in H. hammondi merozoites rhoptries were longer, and coiled and contained more micronemes than in T. gondii. Ultrastructural development is illustrated in detail.     

Waterborne toxoplasmosis - Recent developments

Humans become infected with Toxoplasma gondii mainly by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts from the feces of infected cats. Circumstantial evidence suggests that oocyst-induced infections in humans are clinically more severe than tissue cyst-acquired infections. Until recently, waterborne transmission of T. gondii was considered uncommon, but a large human outbreak linked to contamination of a municipal water reservoir in Canada by wild felids and the widespread infection of marine mammals in the USA provided reasons to question this view. The present paper examines the possible importance of T. gondii transmission by water.     

Serotyping of Toxoplasma gondii in Cats (Felis domesticus) Reveals Predominance of Type II Infections in Germany

Background

Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany.

Methodology

To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7).

Findings

Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III.

Conclusions

Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.     

Interferon-Gamma Release Assay: An Effective Tool to Detect Early Toxoplasma gondii Infection in Mice

Early diagnosis of Toxoplasma gondii infection before the formation of tissue cysts is vital for treatment, as drugs available for toxoplasmosis cannot kill bradyzoites contained in the cysts. However, current methods, such as antibody-based ELISA, are ineffective for detection of early infection. Here, we developed an interferon-gamma release assay (IGRA), measuring the IFN-γ released by T lymphocytes stimulated by Toxoplasma antigen peptides in vitro, for the detection of T. gondii infection in mice. Splenocytes isolated from infected mice were stimulated by peptides derived from dense granule proteins GRA4 and GRA6 and rhoptry protein ROP7, and released IFN-γ was measured by ELISA. Results showed that both acute and chronic infection could be detected by IGRA. More importantly, IGRA detected infection as early as the third day post infection; while serum IgM and IgG were detected 9 days and 13 days post infection, respectively. Our findings demonstrated that an IGRA-positive and ELISA-negative sample revealed an early infection, indicating the combination of IGRA and ELISA can be employed for the early diagnosis of T. gondii infection in human beings, cats and livestock.     

Control of human toxoplasmosis

Toxoplasmosis is caused by Toxoplasma gondii, an apicomplexan parasite that is able to infect any nucleated cell in any warm-blooded animal. Toxoplasma gondii infects around 2 billion people and, whilst only a small percentage of infected people will suffer serious disease, the prevalence of the parasite makes it one of the most damaging zoonotic diseases in the world. Toxoplasmosis is a disease with multiple manifestations: it can cause a fatal encephalitis in immunosuppressed people; if first contracted during pregnancy, it can cause miscarriage or congenital defects in the neonate; and it can cause serious ocular disease, even in immunocompetent people. The disease has a complex epidemiology, being transmitted by ingestion of oocysts that are shed in the faeces of definitive feline hosts and contaminate water, soil and crops, or by consumption of intracellular cysts in undercooked meat from intermediate hosts. In this review we examine current and future approaches to control toxoplasmosis, which encompass a variety of measures that target different components of the life cycle of T. gondii. These include: education programs about the parasite and avoidance of contact with infectious stages; biosecurity and sanitation to ensure food and water safety; chemo- and immunotherapeutics to control active infections and disease; prophylactic options to prevent acquisition of infection by livestock and cyst formation in meat; and vaccines to prevent shedding of oocysts by definitive feline hosts.