Experimental congenital toxoplasmosis in Wistar and Holtzman rats

Congenital toxoplasmosis was evaluated in Wistar and Holtzman rats using two strains of Toxoplasma gondii isolated in Brazil. Pregnant rats were inoculated by subcutaneous or intraperitoneal routes with 10(6) or 8 x 10(6) tachyzoites of N strain (virulent for mice) and by subcutaneous or oral routes with 10(2) or 1.2 x 10(3) cysts of P strain (avirulent for mice). The tissues of rat pups born from these rats were bioassayed for T. gondii infection. T. gondii was not observed in the pups born from rats inoculated with N strain. In the animals inoculated with P strain, congenital toxoplasmosis occurred in 22.8% (Wistar rats inoculated with 10(2) cysts by the subcutaneous route), 11.4% (Wistar rats inoculated with 10(2) cysts by the oral route), 21.2% (Wistar rats inoculated with 1.2 x 10(3) cysts by the oral route) and 2.9% of fetal infection (Holtzman rats inoculated with 10(2) cysts by the oral route). None of the pups born from chronically infected mother were infected with T. gondii.     

Detection of Toxoplasma gondii by polymerase chain reaction in sera of acutely infected mice

The presence of Toxoplasma gondii DNA was detected in sera of acutely infected mice by polymerase chain reaction. Adult mice were inoculated intraperitoneally with 5 x 10(3) T. gondii RH strain tachyzoites. Five mice were killed every 3 hr from 3 to 21 hr post infection (PI) and every day from 1 to 7 days PI. Toxoplasma gondii DNA was first detected in 60% of the infected mice 18 hr PI and in 100% of the animals 21 hr PI and from 1 to 7 days PI. No mice survived longer than 7 days.     

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.     

Experimental infection with the Toxoplasma gondii ME-49 strain in the Brazilian BR-1 mini pig is a suitable animal model for human toxoplasmosis

Toxoplasma gondii causes toxoplasmosis, a worldwide disease. Experimentation with pigs is necessary for the development of new therapeutic approaches to human diseases. BR-1 mini pigs were intramuscularly infected with T. gondii with tachyzoites (RH strain) or orally infected with cysts (ME-49 strain). Haematology and serum biochemistry were analysed and buffy coat cells were inoculated in mice to determine tachyzoite circulation. No alterations were observed in erythrocyte and platelet values; however, band neutrophils increased seven days after infection with ME-49. Serology of the mice inoculated with pig blood leucocytes revealed circulating ME-49 or RH strain tachyzoites in the pigs'' peripheral blood at two and seven or nine days post-infection. The tachyzoites were also directly observed in blood smears from the infected pigs outside and inside leucocytes for longer periods. Alanine-aminotransferase was high at days 21 and 32 in the RH infected pigs. After 90 days, the pigs were euthanised and their tissue samples were processed and inoculated into mice. The mice serology revealed the presence of parasites in the hearts, ileums and mesenteric lymph nodes of the pigs. Additionally, cysts in the mice were only observed after pig heart tissue inoculation. The infected pigs presented similar human outcomes with relatively low pathogenicity and the BR-1 mini pig model infected with ME-49 is suitable to monitor experimental toxoplasmosis.     

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

Horizontal transmission of Toxoplasma gondii in squirrel monkeys (Saimiri sciureus).

The possibility of horizontal transmission of T. gondii was examined in squirrel monkeys. After three monkeys were inoculated perorally with 1.1-2.1 x 10(3) cysts of the T. gondii ME49, the animals were divided into two cages and maintained with one normal monkey for each cage as a cagemate. Two out of the three T. gondii-inoculated monkeys died, and the remaining one monkey was sacrificed in a moribund state one week after infection because of acute toxoplasmosis. Many T. gondii tachyzoites were recovered from broncho-alveolar lavages and were also found histopathologically in the lung, liver, spleen, kidney and lymph nodes and impression smears of tissues from the three T. gondii-inoculated monkeys by Giemsa staining. Anti-T. gondii antibody was examined by immunoblot assay in these animals, and the antibody to T. gondii major surface membrane protein (p30) could be detected after the start of experiment. Furthermore, a specific band of T. gondii NTPase gene was observed by PCR in the liver and lung of infected and cagemate monkeys, and the sequence of the second PCR products obtained from the cagemates, which were clinically normal but gave a positive result in immunoblotting assay, was exactly the same as the sequence of the NTPase gene of T. gondii ME49. These findings suggested that transmission of T. gondii from the infected monkeys to cagemates occurred easily, and since many T. gondii tachyzoites were recovered from the bronchoalveolar lavages of the three T. gondii-inoculated monkeys, we suggest that aerosol infection plays an important role for the enzootic toxoplasmosis in colonies of squirrel monkeys.     

Can caprine arthritis encephalitis virus (CAEV) be transmitted by in vitro fertilization with experimentally infected sperm?

For each of the five fertilization trials of the experiment, frozen semen was prepared for in vitro capacitation at a concentration of 1 × 10⁷ spz/ml and divided into three groups. One group was used as a control, while the two others were inoculated with 100 μl/ml of either culture medium from non-infected cells (placebo group) or cell culture medium containing virus at a concentration of 10⁵ TCID₅₀/ml (infected group). A total of 789 oocytes were used for IVF. For each of the five trials a group of oocytes were used as a non-infected control and were found to be caprine arthritis-encephalitis virus (CAEV) free. The other oocytes were divided in two equal batches. Oocytes in the first batch were in vitro fertilized with CAEV infected sperm (infected group) and the second batch were fertilized with CAEV non-infected sperm (placebo and control groups). After IVF, the zygotes of each group were washed 12 times. The CAEV genome was not detected (using RT-PCR) in the washing media of either the control or placebo groups from each trial. In contrast, the first three washing media from the infected group were consistently found to be positive for the CAEV genome (5/5), whereas subsequent washing media were CAEV-free (P < 0.05). Zygotes obtained using all semen groups tested negative for both the provirus and genome of CAEV. These results clearly show that the first four washes were sufficient to remove viral particles from CAEV infected fertilization media and that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV.     

Bull semen variables after experimental exposure with Bovine Herpesvirus type 5

The influence of Bovine Herpesvirus type 5 (BoHV-5) infection on semen variables and sperm morphology collected from healthy bulls with no reproductive disorder was evaluated in ten ejaculates distributed into two experimental groups: group I, bull semen exposed to 10(2.3) (tissue culture infectious dose) TCID(50)/50 μl of a Brazilian strain of BoHV-5 (US9/BR/2007; GU9457818) and group II, unexposed bull control semen. After experimental infection, the semen was frozen-thawed prior to computerized analysis (CASA) of sperm motility and movement. Also analyzed were sperm phosphatidylserine transposition, acrosomal integrity, mitochondrial function, plasma membrane integrity and Annexin V expression. Viable BoHV-5 particles and their DNA were detected in infected semen after virus isolation and in situ hybridization (ISH) assay. The ISH revealed the BoHV-5 US9 gene in the acrosome and tail of infected spermatozoa. The only remarkable differences between groups I and II were the sperm kinetic variables, whereby infected sperm had a lesser mean velocity (VAP) and curvilinear velocity (VCL) values as compared to controls (P≤0.05). However, the straightness coefficient (STR) and beat cross frequency (BCF) values were higher in infected sperm. These results indicate that BoHV-5 can be found in infected sperm but induces no functional and morphological damage even after freeze-thawing, and, importantly, BoHV-5 can be spread via in vitro and in vivo reproductive biotechnology procedures.     

Laparoscopical intrauterine insemination with different doses of fresh,conserved, and frozen semen for the production of ovine zygotes

The objective of the present study was to increase the efficiency in the production of ovine zygotes suitable for microinjection via laparoscopical intrauterine insemination. In the first part of the study, 71 ewes of three different breeds were inseminated with one of two different insemination doses (50 x 10(6) or 300 x 10(6) sperm per inseminate) and semen was either freshly diluted, liquid conserved, or frozen/thawed, or females were mated by a fertile ram (controls). In the second part, a total of 46 ewes was inseminated with 300 x 10(6) freshly diluted sperm to verify the findings from part 1 and to unravel effects of breed and age of donor ewe. The oviducts were flushed 24-26 h after insemination and the success of insemination was assessed by microscopical examination. Recovery rates were 78.0+/-26.4 and 72.1+/-24.6% in parts 1 and 2 of the study, respectively. Of these oocytes 62.3 and 62.8% (parts 1 and 2, respectively) were fertilized. In part 1, the highest proportion (64.7%) of pronuclear stages was observed in the group inseminated with 300 x 10(6) freshly diluted semen and was significantly higher compared to the groups inseminated with 50 x 10(6) freshly diluted semen (25.5%, P<0.001), 300 x 10(6) liquid conserved semen (49.0%, P<0.001), or 50 x 10(6) frozen/thawed semen (39.6%, P<0.05). In the control group, the proportion of pronuclear stages amounted to 60.2%. Irrespective of the type of sperm conservation, the overall fertilization rate (zygotes plus 2-cell stages) was higher (P<0.05) following insemination with 300 x 10(6) sperm (68.2%) compared to 50 x 10(6) sperm (56.8%). In part 2, the proportion of pronuclear stages reached 54.2% with an overall fertilization rate of 62.9%. These results were affected by breed and age of the donor as crossbred and younger (<3 years) animals yielded the highest proportion of pronuclear stages. The present study shows that freshly diluted semen at a dosage of 300 x 10(6) sperm yields the highest fertilization rates, the greatest proportion of pronuclear stages and the lowest proportion of mature unfertilized oocytes. Further increases in yields of pronuclear stages can possibly be achieved by selection of sheep from the best suited breed and younger than 3 years of age.     

Toxoplasma gondii does not persist in goldfish (Carassius auratus)

Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are a potential source of Toxoplasma gondii infection. To examine the transmissibility of T. gondii to fish, we observed the development of T. gondii tachyzoites inoculated into oviduct epithelial cells of goldfish (Carassius auratus) microscopically in vitro. Further, the survival period of tachyzoites inoculated into goldfish muscle was bioassayed in mice and through PCR analysis. In cell cultures at 37 C, both RH and Beverley strains of T. gondii tachyzoites had penetrated into cells at 6 hr post inoculation, and were multiplying. In cell cultures at 33 C, many tachyzoites of both strains attached to the host cells, but no intracellular tachyzoites were observed at 24 hr post inoculation. In the T. gondii inoculated goldfish kept at 33 C, tachyzoite DNA was detected in the inoculated region on day 3, but not on day 7. When inoculated goldfish were kept at 37 C, live tachyzoites were seen at the inoculation site on day 3, but not on day 7. These results suggest that T. gondii does not persist in fish.     

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO(2) and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R(2)=63%); percentage of oocytes that interacted with spermatozoa and BCF (R(2)=73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R(2)=64%) and velocity straight line (VSL; R(2)=64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions.     

Tachyzoite-induced life cycle of Toxoplasma gondii in cats

The tachyzoite-induced cycle of Toxoplasma gondii was studied in 46 cats. Tachyzoites of the M-7741 or Me-49 strain of T. gondii were administered orally to cats by pouring into the mouth or by stomach tube, or by intraintestinal inoculation. Ten weaned cats that had been inoculated with tachyzoites directly in the intestine were killed 1, 3, 6, 9, 12, 15, 18, or 25 days later, and their tissues were studied histologically and bioassayed in mice. Toxoplasma gondii was demonstrable in the blood of 8 cats and in other tissues of all these 10. Four out of five 1- to 8-day-old cats fed tachyzoites by stomach tube became infected with T. gondii, and 1 became ill because of toxoplasmosis. All 19 weaned cats fed tachyzoites (poured into the mouth) became infected, and 6 died of acute toxoplasmosis 9-15 days after being fed T. gondii. Six out of 12 weaned cats fed tachyzoites by stomach tube became infected but were asymptomatic. Overall, 12 out of 26 cats observed for 19 days or more shed oocysts with a prepatent period (pp) of 19 days or more, with the sole exception of 1 cat that shed oocysts with a pp of 5 days. Enteroepithelial stages of T. gondii were not found in any cat before oocysts were shed. Cats shed up to 360 million oocysts in a day, and oocysts were shed for 4-6 days.     

Semen changes in boars after experimental infection with porcine reproductive and respiratory syndrome (PRRS) virus

Eleven boars seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) were trained for semen collection: five boars were inoculated intranasally with 6 x 10(6)TCID(50)/ml of PRRSV (Group A); four boars were inoculated intranasally with 6 x 10(4)TCID(50)/ml (Group B); and two boars were used as uninfected control (Group C). Semen samples were collected at 7-d intervals from 49 d prior to experimental inoculation with PRRSV to 70 d after inoculation, and were examined for sperm volume, sperm concentration, sperm morphology, sperm motility and for the presence of PRRSV. The infection in boars was demonstrated by the reisolation of PRRSV from the serum of all inoculated boars. Rectal temperatures and general health of the boars were clinically normal throughout the trial. Differences were observed in the quality of semen collected from boars after experimental infection with PRRSV. This infection induced a significant decrease in sperm motility and in spermatozoa with normal acrosomes. Of the semen samples tested for virus isolation in swine alveolar macrophages PRRSV was only isolated in 1 boar from Group B. The virus was detected in an additional semen sample in Group A by the production of an antibody titer in a biological assay. All attempts to detect PRRSV by RT-PCR in semen samples were unsuccessful. Nevertheless, from our study it is possible to suggest that the PRRSV can occasionally be transmitted in the semen during the initial phase of the disease.     

Serologic response of the opossum Didelphis virginiana to a temperature-sensitive mutant (ts-4) of Toxoplasma gondii.

A study was designed to measure the Toxoplasma gondii-specific IgM and IgG antibody responses of opossums inoculated with tachyzoites of the temperature-sensitive mutant of T. gondii, ts-4, and to examine its persistence in the tissues. Four young opossums seronegative for anti-Toxoplasma gondii IgM and IgG antibodies immediately after capture and 4 wk later were injected subcutaneously with 1.8 x 10(6) ts-4 tachyzoites; a fifth opossum (also seronegative) received an injection of saline only. Serum was collected weekly and titered by modified direct agglutination for anti-Toxoplasma gondii IgM and IgG. IgM titers were detectable from week 1 to week 6 postinoculation (PI). IgG was measurable by week 3 and remained high for 30 wk PI when the opossums were killed and examined. The control opossum did not develop a specific antibody response. At necropsy major lesions were not found. No anti-Toxoplasma gondii IgG was detected in serum collected from mice injected with tissues prepared from the opossums at necropsy, and no T. gondii was found on impression smears made at necropsy from these mice. Modified direct agglutination performed with or without 0.2 M 2-mercaptoethanol worked well for measuring specific IgM and IgG antibodies in experimentally infected opossums.     

Evaluation of the mood-stabilizing agent valproic acid as a preventative for toxoplasmosis in mice and activity against tissue cysts in mice

Toxoplasma gondii is a common intracellular protozoan infection of humans worldwide. Severe disease can occur in immunocompromised individuals and the in the fetuses of nonimmune pregnant women. Chronic infection is associated with vision and hearing problems, and functional mental alterations, including schizophrenia. The mood-stabilizing agent valproic acid has been shown to inhibit the development of T. gondii in vitro at dosages that are normally achieved in the serum and cerebral spinal fluid of human patients and to have positive effects on the behavior of rats chronically infected with T. gondii. The present study was done to examine the in vivo activity of valproic acid against acute toxoplasmosis in mice. Two studies were done with valproic acid given in the drinking water at concentrations of 1.5 mg/ml (Experiment 1) or 3.0 mg/ml (Experiment 2). In a third experiment (Experiment 3), valproic acid was injected intraperitoneally (i.p.) at doses of 200 or 300 mg/kg every 12 hr. Valproic acid was not effective in preventing acute toxoplasmosis. All mice treated with valproic acid died or were killed and did not (P > 0.05) live significantly longer than the controls. Tachyzoites were demonstrated in the tissues of infected valproic-acid-treated mice. A fourth study was done to determine if valproic acid has activity against T. gondii tissue cysts in chronically infected mice. Mice were chronically infected with the ME-49 strain of T. gondii for 8 wk and then treated orally with valproic acid at approximately 6.6 mg/ml (800 mg/kg/day) in the drinking water for 10 wk (amount was varied due to increasing mouse weights). No significant differences (P > 0.05) were present in tissue cyst numbers in valproic-acid-treated T. gondii chronically infected mice and in mice chronically infected with T. gondii but not given valproic acid. Our results indicate that valproic acid, although effective in vitro against T. gondii tachyzoites, is not effective as a preventative in mice inoculated with T. gondii tachyzoites. Additionally, no activity against tissue cysts was observed in chronically T. gondii-infected valproic-acid-treated mice.     

Biological ability of Malpura rams to counter heat stress challenges and its consequences on production performance in a semi-arid tropical environment

A study was conducted to assess the biological ability of Malpura rams to counter heat stress challenges and its impact on the productive performance in a semi-arid tropical environment. The eighteen adult Malpura rams (average body weight 55.0 kg) were divided into two groups, GI (n = 9; Control) and GII (n = 9; heat stress). The study was conducted for a period of 60 days. The results showed that body weight, body condition score, heart girth and feed intake were significantly (p < 0.05) lower in GII as compared to GI rams. The physiological responses i.e. respiration rate, pulse rate, rectal temperature and sweating rate (body and scrotum) were significantly (p < 0.05) higher in rams exposed to heat stress. Among the endocrine parameters, plasma T₃, T₄ and testosterone concentrations were significantly (p < 0.05) lower in GII rams, while cortisol concentration was significantly (p < 0.05) higher in GII rams. In addition, heat stress significantly (p < 0.05) reduced semen volume, total sperm motility, sperm concentration and progressive sperm motility in GII rams. The results indicated that Malpura rams adapted to the heat stress condition, however, while doing so their production performances were compromised.     

Experimental infection of Peromyscus californicus with Toxoplasma gondii

Eight female Peromyscus californicus were infected with 10(2) or 10(4) Toxoplasma gondii culture-derived tachyzoites (Type II or X) isolated from southern sea otters. All but 2 mice survived infection and developed antibodies to T. gondii. The 2 fatally infected mice were inoculated with 10(4) tachyzoites of the Type X strain. Parasite detection by immunohistochemistry (IHC) and DNA amplification with 2 polymerase chain reaction (PCR) methods was compared for brain, heart, lung, liver, spleen, biceps muscle, and tongue, at a mean of 41 days postinfection. Parasites were detected most commonly by IHC in spleen (8/8) and brain (6/8). DNA amplification by PCR was most successful from brain, heart, and spleen.     

Immune responses of mice intraduodenally infected with Toxoplasma gondii KI-1 tachyzoites

Toxoplasma gondii Korean isolate (KI-1) tachyzoites were inoculated intraduodenally to BALB/c mice using a silicon tube, and the course of infection and immune responses of mice were studied. Whereas control mice, that were infected intraperitoneally, died within day 7 post-infection (PI), the intraduodenally infected mice survived until day 9 PI (infection with 1 × 10(5) tachyzoites) or day 11 PI (with 1 × 10(6) tachyzoites). Based on histopathologic (Giemsa stain) and PCR (B1 gene) studies, it was suggested that tachyzoites, after entering the small intestine, invaded into endothelial cells, divided there, and propagated to other organs. PCR appeared to be more sensitive than histopathology to detect infected organs and tissues. The organisms spread over multiple organs by day 6 PI. However, proliferative responses of splenocytes and mesenteric lymph node (MLN) cells in response to con A or Toxoplasma lysate antigen decreased significantly, suggesting immunosuppression. Splenic CD4(+) and CD8(+) T-lymphocytes showed decreases in number until day 9 PI, whereas IFN-γ and IL-10 decreased slightly at day 6 PI and returned to normal levels by day 9 PI. No TNF-α was detected throughout the experimental period. The results showed that intraduodenal infection with KI-1 tachyzoites was successful but did not elicit significant mucosal immunity in mice and allowed dissemination of T. gondii organisms to systemic organs. The immunosuppression of mice included reduced lymphoproliferative responses to splenocytes and MLN cells to mitogen and low production of cytokines, such as IFN-γ, TNF-α, and IL-10, in response to T. gondii infection.