The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18

More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3 weeks apart, and challenged with T. gondii 3 weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (P < 0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii.     

Distribution of cysts and tachyczoites in calves and pregnant cows inoculated with toxoplasma gondii oocysts

Sixteen calves and 6 cows were each inoculated with 100 000 infective oocytes of the GT-1 strain of Toxoplasma gondii. Cattle were necropsied between 3 and 287 days post-inoculation (DPI) and their tissues were inoculated into mice or fed to Toxoplasma-free cats for the detection of Toxoplasma in bovine tissues. Ten to 10 000-fold more T. gondii were recovered from small intestine and mesenteric lymph nodes of calves at 3 and 6 DPI than from lungs and liver, and the number of T. gondii in bovine tissues was reduced 1000-fold between 6 and 8 DPI. By using the pepsin digestion technique, or feeding tissues to Toxoplasma-free cats, it was demonstrated that T. gondii encysted in bovine tissues as early as 11 DPI and persisted as late as 287 DPI. More Toxoplasma gondii cysts occurred in livers than in any other bovine tissue. Of the 6 cows inoculated at 95–155 days after breeding, 5 delivered normal calves and T. gondii was isolated from only one of these calves. One cow was barren. Toxoplasma gondii was not isolated either by mouse inoculation or by feeding cats tissues from 2 cows killed 132 and 190 DPI. Toxoplasma gondii was not isolated in mice inoculated with tissues of cows killed 98 and 109 DPI, but cats fed on bovine tissues shed T. gondii oocysts. The organism, however, was isolated in mice inoculated with the mesenteric lymph nodes of 1 of the 2 cows killed 162 and 168 DPI, and from the small intestine of the other. Cats fed tissues of these cows later shed T. gondii oocysts.     

A sero-epidemiological survey of Toxoplasma gondii infection in free-range and caged chickens in northeast China

Acquisition of Toxoplasma gondii infections is mainly through ingestion of parasite-contaminated food. T. gondii oocyst distribution in the living environment of human and livestock is directly linked to the prevalence of the parasite infection in humans and domestic animals. In this study, we investigated the sero-prevalence of T. gondii infection in free-range as well as caged chicken in northeast China. The purpose of this study was to investigate the potential prevalence of T. gondii oocysts in the environments. Sera of 308 free-range chickens and 210 caged chickens collected in three areas in northeast China were tested for anti-T. gondii antibodies with ELISA assays. The infection rates of free-range and caged chickens were 34.7% and 2.8% respectively, indicating that the parasite is widely distributed in the environment and poses threatens to the health of people living in those areas.     

Evidence for genetic diversity of Toxoplasma gondii in selected intermediate hosts in Serbia

To contribute to the insight into the worldwide population structure of Toxoplasma gondii, we genetically characterized a total of eight strains isolated from intermediate hosts including humans, sheep and pigeons in Serbia. Although parasite DNA was detected in 28.2% (60/213) of the human samples from 162 patients serologically suspected of active toxoplasmosis, as well as in 5/7 seropositive pigeons and in 2/12 seropositive sheep examined, multilocus PCR-RFLP genotyping, using SAG1, 5′SAG2, 3′SAG2, GRA6, 5′GRA7 and 3′GRA7 as markers, was successful in only four human isolates (of which one was isolated from both the bronchoalveolar lavage fluid and blood samples of a single patient), one sheep and three pigeons. Of the eight isolates, five were type II (62.5%), one was type III, one was atypical, and one had a type I allele at GRA6 as the single locus genotyped. Although type II, as elsewhere in Europe, predominated, these results may suggest a higher genetic diversity of T. gondii in Serbia, reflecting local environmental contamination and also the geographical position of the country in South-East Europe.     

Latitudinal variability in the seroprevalence of antibodies against Toxoplasma gondii in non-migrant and Arctic migratory geese

Toxoplasma gondii is an intracellular coccidian parasite found worldwide and is known to infect virtually all warm-blooded animals. It requires a cat (family Felidae) to complete its full life cycle. Despite the absence of wild felids on the Arctic archipelago of Svalbard, T. gondii has been found in resident predators such as the arctic fox and polar bear. It has therefore been suggested that T. gondii may enter this ecosystem via migratory birds. The objective of this study was to identify locations where goose populations may become infected with T. gondii, and to investigate the dynamics of T. gondii specific antibodies. Single blood samples of both adults and juveniles were collected from selected goose species (Anser anser, A. brachyrhynchus, Branta canadensis, B. leucopsis) at Arctic brood-rearing areas in Russia and on Svalbard, and temperate wintering grounds in the Netherlands and Denmark (migratory populations) as well as temperate brood-rearing grounds (the Netherlands, non-migratory populations). A modified agglutination test was used on serum, for detection of antibodies against T. gondii. Occasional repeated annual sampling of individual adults was performed to determine the antibody dynamics. Adults were found seropositive at all locations (Arctic and temperate, brood-rearing and wintering grounds) with low seroprevalence in brood-rearing birds on temperate grounds. As no juvenile geese were found seropositive at any brood-rearing location, but nine month old geese were found seropositive during spring migration we conclude that geese, irrespective of species and migration, encounter T. gondii infection in wintering areas. In re-sampled birds on Svalbard significant seroreversion was observed, with 42% of seropositive adults showing no detectable antibodies after 12 months, while the proportion of seroconversion was only 3%. Modelled variation of seroprevalence with field data on antibody longevity and parasite transmission suggests seroprevalence of a population within a range of 5.2–19.9%, in line with measured values. The high occurrence of seroreversion compared to the low occurrence of seroconversion hampers analysis of species- or site-specific patterns, but explains the absence of an increase in seroprevalence with age and the observed variation in antibody titre. These findings imply that even though infection rate is low, adults introduce T. gondii to the high Arctic ecosystem following infection in temperate regions.     

Seroprevalence and risk factors associated with Toxoplasma gondii infection in pig farms from Catalonia, north-eastern Spain

Seroprevalence and associated risk factors for Toxoplasma gondii infection in pigs were analyzed in 1202 sera samples, including sows and pigs of three, seven, 11, 15 and 20 weeks of age, from 23 farms in Catalonia, north-eastern Spain. Antibodies were tested by the modified agglutination test (MAT) at titers ?1:25. Antibodies to T. gondii were found in 228 samples (19.0%; 95% CI: 16.8-21.2). The individual prevalence in animals higher than 7 weeks of age was 22.8% (174/762; 95% CI: 16.6-29.0) and the within-farm prevalence ranged from 7.1% to 36.4%. Statistically significant differences were found among age classes. The risk factors significantly associated with T. gondii seroprevalence were the presence of cats, percentage of mortality at weaning and the presence of outdoor facilities in the farms. The seroprevalence observed in the present study indicates widespread exposure to T. gondii among domestic pigs in Catalonia, which may have important implications for public health.     

Seroprevalence of Toxoplasma gondii and Neospora caninum in wild and domestic ruminants sharing pastures in Galicia (Northwest Spain)

The prevalence of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and cELISA, respectively, in 160 roe deer (Capreolus capreolus), 177 sheep and 178 cattle sharing pastures in Galicia (Northwest Spain). The seroprevalence for T. gondii was 13.7% in roe deer, 57% in sheep and 7.3% in cattle. The seroprevalence for N. canimum was 6.8%, 10.1% and 24.1% in roe deer, sheep and cattle, respectively. Statistically significant differences were observed between sheep and the other species for T. gondii and between cattle and the other ruminants for N. caninum. Only 19/515 animals were positive for both, T. gondii and N. caninum. Statistically significant differences were observed among different geographical areas for T. gondii but not for Neospora, seroprevalence being higher in the coastal area lower than in other areas. This study reveals a widespread exposure to T. gondii in Galician ruminants, and therefore, those species, particularly sheep, should be regarded as a potential source of infection for humans.     

Factors affecting seroprevalence of Toxoplasma gondii in the endangered Iberian lynx (Lynx pardinus)

Wild felids are considered important in maintaining the sylvatic cycle of Toxoplasma gondii. Although, T. gondii antibodies have been reported in several species of wild felids, little is known of the epidemiology and risk factors associated with T. gondii infection in wild cats. The Iberian lynx (Lynx pardinus) is the most endangered felid species in the world. In the present study, seroprevalence and associated risk factors for T. gondii infection in a large population of Iberian lynx in Spain were determined. Serum samples from 129 Iberian lynx collected from 2005 to 2009 and 85 wild rabbits (Oryctolagus cuniculus), sharing the habitat with the Iberian lynx, were tested for antibodies to T. gondii by the modified agglutination test (MAT) using a cut-off value of 1:25. Antibodies to T. gondii were found in 81 of 129 (62.8%) Iberian lynx. Seroprevalence to T. gondii in Iberian lynx significantly increased with age (P < 0.001). T. gondii seroprevalences were similar in free-ranging (66.7% of 93) and wild-caught captive lynx (69% of 84) but significantly lower in captive-born lynx (22.5% of 40). Seroprevalence was higher in lynx with concurrent Cytauxzoonfelis (88% of 25) but not with concurrent Feline Leukemia Virus (FeLV) infection (53.8% of 13). There were no significant differences in seroprevalence between sexes, geographic region and year of sample collection (2005–2009). Oocysts of T. gondii were not detected microscopically in fecal samples from 58 lynx. Wild rabbits are considered the most important food for the lynx. Antibodies to T. gondii were found in 14 (11.9%) of 85 rabbits tested. The present results indicate that T. gondii infection is widespread in the two areas where Iberian lynx survive in Spain. The fact that four captive-born lynx seroconverted was indication of contact with T. gondii also in the Captive Breeding Centers, hence, control measures to prevent T. gondii infection would be necessary in these centers.     

Effects of synbiotic-based Bifidobacterium animalis in female rats experimentally infected with Toxoplasma gondii

The aim of this study was to assess the effects of a synbiotic composed of Bifidobacterium animalis and fructooligosaccharides on female rats infected with Toxoplasma gondii. Female Wistar rats, treated or not with dexamethasone, were daily supplemented with synbiotics for 21 days. After 15 days of supplementation, the rats were orally infected with 10⁴T. gondii bradyzoites. Blood samples were collected to measure the levels of IFN-γ, IL-10 and T. gondii antibodies. All synbiotic-supplemented rats survived until the end of the experiment; however, non-supplemented dexamethasone-treated rats died between the fifth and the eighth days after T. gondii infection. Dexamethasone-treated rats supplemented with synbiotics (P < 0.05) were capable of synthesizing IFN-γ, and this immunological response was essential to ensure their survival. In addition, brain cysts were found in one rat not supplemented with synbiotics. Results suggest that the synbiotic composed of B. animalis and fructooligosaccharides may be beneficial to toxoplasmosis control.     

Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats

There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA - extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method - using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results.     

Immunization of mice against Streptococcus suis serotype 2 infections using a live avirulent strain.

In this study, the IgG response of mice injected with two virulent strains and one avirulent Streptococcus suis capsular type 2 strain was compared by Western blotting. The serum from mice immunized against the avirulent strain could recognize most proteins of the various strains tested and similar results were obtained with serum from mice injected with virulent strains. The live avirulent strain was injected twice (days 0 and 10) to groups of five mice, and four virulent strains from different geographical origins were used to challenge the animals. All mice, except one in one group, survived the challenge. These results suggest that a live avirulent strain could be used for immunization of swine, the natural host.     

Microscopic analysis of calcium ionophore activated egress of Toxoplasma gondii from the host cell

Toxoplasma gondii invades and destroys nucleated cells of warm blooded hosts in a process which involves several steps: recognition, adhesion, penetration, multiplication inside a parasitophorous vacuole (PV) and egress. The last one is the least understood. Parasite egress from LLC-MK2 cells infected with the RH strain of T. gondii was artificially triggered with 4BrA23187 calcium ionophore. The combination of videomicroscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM) showed that egress does not result from host cell rupture due to overloading with tachyzoites. Videomicroscopy showed that upon calcium ionophore administration parasite rosettes disassemble, the contour of the parasitophorous vacuole disappears and each tachyzoite takes a separate route to the extracellular medium. FESEM and TEM showed the fragmentation of the intravacuolar network, the fragmentation of parasitophorous vacuole membrane and individual tachyzoites with extruded conoids migrating through the cytosol, tightly surrounded by remnants of parasitophorous vacuole membrane or free in the cytosol. Both videomicroscopy and FESEM showed that a single parasite can cross the host cell membrane without disrupting it, while a large number of parasites, egressing simultaneously, rupture the membrane and the cell as a whole. These data suggest that invasion and egress share less similarities than previously believed.     

Seroprevalence of and risk factors for Toxoplasma gondii in sheep raised in the Jaboticabal microregion, São Paulo State, Brazil

Seroprevalence of and risk factors for toxoplasmosis in sheep from different properties in the Jaboticabal microregion, São Paulo State, Brazil were determined. Antibodies to Toxoplasma gondii were found in sera of 52.0% of 488 sheep tested by indirect fluorescent antibody test (IFAT ? 64). T. gondii seropositivity in sheep was significantly associated with gender of the sheep, pasturing system, contact with cats, and the use of mineral supplements and the type of feed.     

Epidemiological Aspects in the Leptospira spp. and Toxoplasma gondii Infection in Horses from Botucatu,São Paulo,Brazil

Leptospirosis and toxoplasmosis are zoonoses with high importance because of the economic and public health impact. This study was aimed to determine the seroprevalence of leptospirosis and toxoplamosis in 714 serum samples of horses from different farms from Botucatu, São Paulo, Brazil. The samples were researched for Toxoplasma gondii antibodies by indirect immunofluorescence antibody test (IFAT) and for Leptospira spp. antibodies by microscopic agglutination test. Of 714 serum samples, 128 (17.9%; 95% CI: 15.3%-20.9%) were positive for one or more serovars of Leptospira spp., with icterohaemorrhagiae, canicola, and castellonis as the most prevalent serovars, whereas 42 (5.9%; 95% CI: 4.4%-7.9%) were positive for T gondii, of which 33 samples (78.57%; 95% CI: 64.0%-88.2%) presented a titer of 16, 7 (16.7%; 95% CI: 8.4%-30.7%) a titer of 64, and 1 (2.38%; 95% CI: 0.6%-12.3%) a titer of 256. No significant difference was found among the results obtained and the associated variables such as age and sex.     

Feline immunodeficiency virus, feline leukemia virus and Toxoplasma gondii in stray and household cats in Kerman-Iran: Seroprevalence and correlation with clinical and laboratory findings

This study was carried out to determine the seroprevalence of feline leukemia virus (FeLV), feline immunodeficiency virus (FIV) and Toxoplasma gondii (T. gondii) infection among stray and owned cats in southeastern Iran and to identify the influence of age, sex, lifestyle, health status, and laboratory findings on seropositivity. The overall infection rate for FIV, FeLV, and T. gondii was 19.2%, 14.2%, and 32.1% respectively. Results of the multivariate logistic regression analysis showed that old adults more likely to be seropositive than juveniles for FIV, FeLV, and T. gondii (adjusted odds ratios [ORs], 1.84, 1.56, and 2.57 respectively). Anemic and diseased cats ([ORs], 6.62 and 0.9) were at a greater risk of testing positive for FeLV. Male cats were 4.91 times as likely to have FIV as were female and hyperglobulinemia was significantly more prevalent in FIV-infected cats ([ORs], 3.4). In conclusion, FIV and FeLV seem to be endemic in Iran and retroviral-associated immunosuppression may be a risk factor for active toxoplasmosis in infected cats.     

The role of rodents and other wildlife in the epidemiology of swine toxoplasmosis

Two swine production units and their contiguous wildlife populations were used for this study. These herds were located 7 miles apart in the major swine producing area of south central Georgia. Herd A had a Toxoplasma gondii antibody prevalence of 27.6% in all ages of swine sampled, with an increased incidence of 13.6% over a 5-month period. This herd was maintained under a semi-range condition. The swine in Herd B were maintained exclusively in concrete floored, enclosed buildings. This herd had a 0.85% T. gondii prevalence. Rodents and a few other wildlife and domestic species were trapped in or around both herds. These animals were examined for Toxoplasma antibodies using the same test procedure utilized for swine sera, the indirect immunofluorescent (IIF) test. Additionally, rodent tissues were homogenized and suspensions prepared for interperitoneal (I/P) inoculation into CF₁ laboratory mice.Rodents and wildlife species examined were: Mus musculus, Peromyscus leucopus, Rattus norvegicus, Sigmodon hispidus, Procyon lotor, and Didelphis marsupalis. Feral and domestic animals other than swine that also were tested for the presence of T. gondii antibodies included two cats, two horses, and a dog. The overall prevalence of T. gondii antibodies in all non-porcine species examined was 67% for those animals in and around the premises of Herd A, and 63% for those in and around Herd B.Toxoplasma infectivity of rodents whose tissues were processed and inoculated into laboratory mice correlated well with the results of the IIF test on the serum of these wild rodents. No tachyzoites of T. gondii were found in the peritoneal exudate of laboratory mice post I/P inoculation with wild rodent tissue, with one exception.While there was no significant difference in Toxoplasma infectivity in non-porcine species on these two premises, management practices appeared to be the determining factor in swine infection with T. gondii. Excluding wildlife precluded infection.     

Cytologic detection of Toxoplasma gondii in the cerebrospinal fluid of a dog and in vitro isolation of a unique mouse-virulent recombinant strain

Parasites resembling Neospora caninum or Toxoplasma gondii were detected by cytologic examination of cerebrospinal fluid (CSF) from a dog with neurologic disease. The dog became severely ill and was euthanized. Canine tissue homogenates were used for direct parasite isolation in cell culture, bioassay in 2 mouse lineages, and PCR. T. gondii was isolated in monkey kidney cells, and species identity was confirmed by PCR. Inoculated parasites were highly virulent for mice, which developed clinical signs and were euthanized immediately. PCR-RFLP for T. gondii using the cultured isolate (TgDgBA22) was conducted with 12 genetic markers, and a unique recombinant strain was identified. Detection of T. gondii by CSF cytology, although described in humans, had not been reported previously in dogs, to our knowledge, and was crucial for the diagnosis of toxoplasmosis in the examined dog.     

Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant

The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund's modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund's complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (±S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P < 0.01) as compared with the group 4 (control: mean 3181 ± 97.5) were 77.25% (724 ± 98) in group 1, 88.02% (381 ± 97.5) in group 2, 38.92% (1943 ± 130.3) in group 3, 44.31% (1771.4 ± 102) in group 5, 59.28% (1295.2 ± 99.1) in group 7 and 55.69% (1409.5 ± 89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P < 0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P < 0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate.     

East coast fever: 2. Cross-immunity trials with a Kenya strain of Theileria lawrencei

Cross-immunity trials were carried out on cattle which were immunized against theileriosis either by chemoprophylaxis or by sub-lethal challenge. In the first of two experiments, animals were immunized against either one of two strains of Theileria parva or one of two strains of Theileria lawrencei. They were then challenged with a Kenya strain of T. lawrencei (T. lawrencei KB 5). The former animals were not protected against the challenge, whereas the latter were either partially protected, when a Tanzanian strain of T. lawrencei had been used for immunization, or completely protected when another Kenyan strain of T. lawrencei had been used.In the second experiment, animals were immunized by chemoprophylaxis against T. lawrencei (KB5) and challenged with T. parva (one strain) or T. lawrencei (five strains). The cattle were protected against challenge with the identical or related T. lawrencei strains, but only partially protected against the T. parva strain and a Tanzanian strain of T. lawrencei.It appears that cattle cannot be immunized for field exposure to theileriosis throughout East Africa by chemoprophylaxis using T. lawrencei (KB5) alone, but may be able to withstand field exposure in a variety of situations if a combination of different theilerial strains is used in the vaccination procedure.     

Toxoplasma gondii in invasive animals on the Island of Fernando de Noronha in Brazil: Molecular characterization and mouse virulence studies of new genotypes

This study aimed to genetically characterize and to determine virulence from Toxoplasma gondii samples from invasive animals in the Island of Fernando de Noronha, Brazil. Blood samples were collected from 21 tegu-lizard (Salvator merianae), 12 rock-cavies (Kerodon rupestris) and 154 black-rats (Rattus rattus) from the Island and MAT (cutoff 1:25) detected anti-T. gondii antibodies in 0% of the tegus (0/21); 58.3% of the rock-cavies (7/12) and 22.7% of rats (35/154). Tissue samples (brain, heart, liver and lung) from positive animals in MAT were collected for molecular analysis and for bioassay in Swiss Webster mice. After observation period, mice were euthanized, and serological detection and tissue cyst search in the brain were performed. The brain of positive animals for serological detection or tissue cyst search was cultured in MARC-145 cells for maintenance of the T. gondii isolate. No isolate was obtained from rock cavies. Nine isolates were obtained by bioassay of 35 seropositive black rats. DNA samples were extracted from rat tissues and from parasite isolates in cell culture, and genotyped using 10 PCR-RFLP markers. ToxoDB genotypes #78 (1) from rat tissue and #146 (4), #163 (2), #260 (2) and #291 (1) from cell culture were detected. Markers of genes ROP18 and ROP5 were analyzed and in vivo virulence test was conducted in mice. Analysis revealed two allele combinations, 3/1 and 3/3, indicating non-lethal T. gondii strains, which is supported by mouse virulence test.