Buprenorphine does not affect acute murine toxoplasmosis and is recommended as an analgesic in Toxoplasma gondii studies in mice

Groups of mice were infected with tachyzoites of the RH strain of Toxoplasma gondii, treated with the opioid analgesic buprenorphine, sodium sulfadiazine, a combination of buprenorphine and sodium sulfadiazine, or nothing in the drinking water, on days -1 to 12 postinfection. Mice in the T. gondii-infected buprenorphine-treated group did not live significantly longer (P > 0.05) than mice given T. gondii and not treated with buprenorphine. Clinical observations of mice indicated that buprenorphine treatment reduced distress and pain in mice with acute toxoplasmosis. Mice treated with sodium sulfadiazine alone or sodium sulfadiazine combined with buprenorphine survived the 28-day study. Mice treated with buprenorphine and not infected with T. gondii also survived the 28 days. This study demonstrates that buprenorphine does not adversely interfere with acute T. gondii infection and indicates that buprenorphine can be given to mice to alleviate pain and distress associated with a T. gondii infection, and not adversely influence the results of toxoplasmosis studies. Analgesic (buprenorphine) treatment should now be the standard of care for mice in acute toxoplasmosis studies.     

Hypothalamic-adenohypophyseal origin of reproductive failure in mice following chronic infection with Toxoplasma gondii

Mice chronically infected with Toxoplasma gondii exhibited reproductive failure characterized by a constant diestrous vaginal cytology and ovarian and uterine atrophy. Chronically infected mice were treated with 20 ng of D-Leu6-des-Gly-NH2-Pro-ethylamide (D-Leu6), a structural analog of luteinizing hormone-releasing hormone (LHRH), every 4 hr over a 12-hr period daily, for 3 days. Infected animals treated with D-Leu6 had greater pituitary weight (P less than 0.01), ovarian weight (P less than 0.01), and uterine weight (P less than 0.025), than did infected control mice treated with saline. In addition, a change in vaginal cytology to estrus, metestrus, and proestrus of the D-Leu6-treated animals was observed, although a contiguity of normal estrous cycles and reproductive function was not determined. Comparable basal levels of serum luteinizing hormone (LH) were seen in infected mice and uninfected normal mice. However, the infected animals demonstrated a decreased pituitary responsiveness to D-Leu6 when monitored at 60 (P less than 0.025) and 120 min (P less than 0.010) following intraperitoneal administration of a bolus of 200 ng of the analog. Thus, the observed reproductive failure involves the readily releasable pool of pituitary LH, since basal LH is similar in both groups, and appears to be due to a dysfunction of the hypothalamic-adenohypophyseal axis.     

Efficacy of ponazuril in vitro and in preventing and treating Toxoplasma gondii infections in mice

Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We determined that ponazuril significantly inhibited T. gondii tachyzoite production (P < 0.05) at 5.0, 1.0, or 0.1 microg/ml in African green monkey kidney cells. We used outbred female CD-1 mice to determine the efficacy of ponazuril in preventing and treating acute toxoplasmosis. Each mouse was subcutaneously infected with 1,000 tachyzoites of the RH strain of T. gondii. Mice were weighed daily, and ponazuril was administered orally in a suspension. Mice given 10 or 20 mg/kg body weight ponazuril 1 day before infection and then daily for 10 days were completely protected against acute toxoplasmosis. Relapse did not occur after prophylactic treatments were stopped. Toxoplasma gondii DNA could not be detected in the brains of these mice using polymerase chain reaction (PCR). One hundred percent of mice treated with 10 or 20 mg/kg ponazuril at 3 days after infection and then daily for 10 days were protected from fatal toxoplasmosis. Sixty percent of mice treated with 10 mg/kg ponazuril at 6 days after infection and 100% of mice treated with 20 mg/kg or 50 mg ponazuril 6 days after infection and then daily for 10 days were protected from fatal toxoplasmosis. Relapse did not occur after treatments were stopped. Toxoplasma gondii DNA was detected in the brains of some, but not all, of these mice using PCR. The results demonstrate that ponazuril is effective in preventing and treating toxoplasmosis in mice. It should be further investigated as a safe and effective treatment for this disease in animals.     

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.     

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.     

Tumor necrosis factor-independent protective effect of recombinant IFN-gamma against acute toxoplasmosis in T cell-deficient mice

rIFN-gamma conferred remarkable resistance against acute infection with Toxoplasma gondii in T cell-deficient (athymic nude) mice. Mice that received an i.p. injection of rIFN-gamma every other day beginning 24 h before infection for a total of eight doses survived significantly longer than untreated control mice although all of the treated mice died after the lymphokine was discontinued. Mice that received 14 doses of rIFN-gamma survived significantly longer than those that received eight doses of the lymphokine although mice started dying soon after the final (14th) injection of rIFN-gamma and eventually all of the treated mice died. Histologic study revealed that the IFN-gamma treatment prevented proliferation of the organisms in all organs examined, including brain, lung, heart, liver, and spleen. The treatment was effective even when started 1 day after infection. Peritoneal macrophages obtained from mice injected with rIFN-gamma were activated and effectively killed tachyzoites of T. gondii in vitro. TNF activity could not be detected in sera of the infected mice during treatment with rIFN-gamma. Administration of anti-TNF antibody did not affect the protective effect of rIFN-gamma against T. gondii infection. These facts indicate that rIFN-gamma can confer resistance to acute infection with T. gondii without collaboration of lymphokines derived from T cells and TNF. This suggests that rIFN-gamma may be effective for therapy of toxoplasmosis in immunosuppressed patients who have impaired activity of T cell function, especially those with AIDS.     

Toxoplasma gondii infection in the brain inhibits neuronal degeneration and learning and memory impairments in a murine model of Alzheimer's disease

Immunosuppression is a characteristic feature of Toxoplasma gondii-infected murine hosts. The present study aimed to determine the effect of the immunosuppression induced by T. gondii infection on the pathogenesis and progression of Alzheimer's disease (AD) in Tg2576 AD mice. Mice were infected with a cyst-forming strain (ME49) of T. gondii, and levels of inflammatory mediators (IFN-γ and nitric oxide), anti-inflammatory cytokines (IL-10 and TGF-β), neuronal damage, and β-amyloid plaque deposition were examined in brain tissues and/or in BV-2 microglial cells. In addition, behavioral tests, including the water maze and Y-maze tests, were performed on T. gondii-infected and uninfected Tg2576 mice. Results revealed that whereas the level of IFN-γ was unchanged, the levels of anti-inflammatory cytokines were significantly higher in T. gondii-infected mice than in uninfected mice, and in BV-2 cells treated with T. gondii lysate antigen. Furthermore, nitrite production from primary cultured brain microglial cells and BV-2 cells was reduced by the addition of T. gondii lysate antigen (TLA), and β-amyloid plaque deposition in the cortex and hippocampus of Tg2576 mouse brains was remarkably lower in T. gondii-infected AD mice than in uninfected controls. In addition, water maze and Y-maze test results revealed retarded cognitive capacities in uninfected mice as compared with infected mice. These findings demonstrate the favorable effects of the immunosuppression induced by T. gondii infection on the pathogenesis and progression of AD in Tg2576 mice.     

Antibodies to immune response gene products inhibit the IgM and IgG antibody response but not the development of resistance to Toxoplasma gondii

We have examined the effects of a monoclonal antibody directed against immune response gene products on the appearance of antibodies and development of resistance to Toxoplasma gondii. In vivo administration of a single dose of anti-I-Ak antibody to C3H/He (H-2k) and not BALB/c (H-2d) mice suppressed both the IgM and IgG response to two different strains of Toxoplasma. Administration of anti-I-Ak antibody to mice 5 days before and 10 days after infection resulted in complete inhibition of IgM and a more pronounced inhibition of IgG response to Toxoplasma. Under these experimental conditions, development of resistance against a subsequent challenge with a virulent strain of Toxoplasma was not affected. The microbicidal and tumoricidal activities of macrophages obtained from anti-I-Ak-treated, Toxoplasma-infected mice and mice infected with Toxoplasma alone were equivalent. Mice treated with anti-I-Ak antibody demonstrated a decreased proliferative response to lipopolysaccharide, a B-cell mitogen. Enumeration of B-cell numbers in anti-I-Ak-treated mice revealed a pronounced decrease in B-cell counts.     

Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone alone or combined with sulfadiazine

The compound 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.     

Changes in blastogenic responses and antibody titers of mice infected with Toxoplasma gondii]

This study was performed to observe the cell-mediated and humoral immune responses in mice which were infected with Beverley, Fukaya and ME49 strain of Toxoplasma gondii, respectively. The blastogenic responses of splenocytes using [3H]-thymidine and serum antibody titers were measured weekly up to 10 weeks after infection. The blastogenic responses of splenocytes treated with concanavalin A and Toxoplasma lysate were significantly declined in the 3 strain groups as compared with the non-infected group (p less than 0.05), however lipopolysaccharide-treated blastogenic responses were not significantly different between infected and non-infected groups. The serum IgG antibody titers in the three infected groups increased from 2 weeks after infection, and the serum IgM antibody titers increased until 4 weeks after infection. No significant differences were revealed in blastogenic responses and serum antibody titers among the 3 groups. The present study suggested that cell-mediated immune responses were involved in T. gondii infected mice and blastogenic responses of T lymphocytes were inhibited in acute T. gondii infection.     

Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice

The importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis was studied in mice chronically infected with Toxoplasma gondii by using a mAb to this lymphokine. Control mice chronically infected with the ME49 strain that received saline or normal IgG had slight inflammation in their brains whereas those that received the mAb developed severe encephalitis. In contrast to control mice, the mAb-treated mice had many areas of acute focal inflammation and infiltration of large numbers of inflammatory cells in the meninges and parenchyma of their brains. In the areas of acute focal inflammation, tachyzoites and Toxoplasma Ag were demonstrated by immunoperoxidase staining with the use of rabbit anti-Toxoplasma antibody, suggesting that the focal inflammation was induced by Toxoplasma organisms. Acute inflammation was also observed around cysts of Toxoplasma. Immunohistologic staining revealed tachyzoites and Toxoplasma Ag surrounding the periphery of these cysts suggesting cyst disruption had occurred. Mice treated with mAb against IFN-gamma had five times the numbers of cysts in their brains as did control mice. These results clearly indicate that endogenous IFN-gamma plays a significant and important role in prevention of encephalitis in mice chronically infected with Toxoplasma. The mAb-treated mice had the same Toxoplasma antibody titers and the same degree of macrophage killing of Toxoplasma as did untreated controls. These results suggest that IFN-gamma may have a direct role in preventing cyst rupture and toxoplasmic encephalitis.     

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.     

Subcutaneous and intestinal vaccination with tachyzoites of Toxoplasma gondii and acquisition of immunity to peroral and congenital toxoplasma challenge

Mice were immunized s.c. or intraintestinally with two injections of a temperature-sensitive mutant of Toxoplasma gondii (ts4). Nonpersistence of the vaccine strain was documented by subinoculation of tissues of a subgroup of mice 3 mo or more after the second immunization. Mice were immune to other-wise lethal parenteral challenges with tachyzoites of the M7741 strain or to peroral challenge with bradyzoites of the Me49 strain of T. gondii. Although two s.c. or intraintestinal immunizations did not completely protect against development of T. gondii in the brains of mice, fewer cysts developed in the s.c. immunized mice than in control mice (2 +/- 3 cysts/0.01 ml in immunized mice compared with 75 +/- 48 cysts/0.01 ml in controls (p less than 0.002)). Reduction in cyst number after intraintestinal immunization was more variable, but also statistically significant (p less than 0.02). Female mice were first immunized, then mated, and then challenged perorally. Neonates of the s.c. immunized mice were not protected. Neonates of intraintestinally immunized mice were protected in part (36% of 115) against congenital infection compared with controls (7% of 107).     

Effects of specific monoclonal antibodies to dense granular proteins on the invasion of Toxoplasma gondii in vitro and in vivo

Although some reports have been published on the protective effect of antibodies to Toxoplasma gondii surface membrane proteins, few address the inhibitory activity of antibodies to dense granular proteins (GRA proteins). Therefore, we performed a series of experiments to evaluate the inhibitory effects of monoclonal antibodies (mAbs) to GRA proteins (GRA2, 28 kDa; GRA6, 32 kDa) and surface membrane protein (SAG1, 30 kDa) on the invasion of T. gondii tachyzoites. Passive immunization of mice with one of three mAbs following challenge with a lethal dose of tachyzoites significantly increased survival compared with results for mice treated with control ascites. The survival times of mice challenged with tachyzoites pretreated with anti-GRA6 or anti-SAG1 mAb were significantly increased. Mice that received tachyzoites pretreated with both mAb and complement had longer survival times than those that received tachyzoites pretreated with mAb alone. Invasion of tachyzoites into fibroblasts and macrophages was significantly inhibited in the anti-GRA2, anti-GRA6 or anti-SAG1 mAb pretreated group. Pretreatment with mAb and complement inhibited invasion of tachyzoites in both fibroblasts and macrophages. These results suggest that specific antibodies to dense-granule molecules may be useful for controlling infection with T. gondii.     

Effect of rIFN-gamma and IL-2 treatments in mouse and nude rat infections with Toxoplasma gondii.

Mice and nude rats lethally infected with T. gondii and treated with recombinant rat interferon-gamma (rIFN-gamma) or recombinant human interleukin-2 (rIL-2) were protected against death, when compared with untreated infected controls. In mice rIFN-gamma and rIL-2 played an important role in "prophylactic treatment", but not in "curative therapy". The survival rate was 42% in mice treated with 3 doses of 20,000 U of rIFN-gamma at days -2, -1, 0 before challenge and up to 66% in mice treated with 3 doses of 10,000 U of rIFN-gamma at days -2, 0, +2 before and after infection. Whereas the survival rate was 33% in mice that received 3 doses of 500 U rIL-2 at days -2, -1, 0 before infection, or -2, 0, +2 before and after infection respectively, up to 50% of the mice treated with 3 doses of 1,000 U rIL-2 at days -2, -1, 0 survived. In nude rats rIFN-gamma had a slight effect in "prophylactic treatment", whereas rIL-2 was active only in "curative treatment". The survival rate was 25% both in nude rats treated with doses of 400,000 U of rIFN-gamma at days -3, 0 before challenge, or with doses of 5,000 U of rIL-2 at days +2, +6, +9 after infection. These results lead us to hypothesise that the mechanism by which the lymphokine treatment exerts a protective effect on Toxoplasma infected mice is different from that on nu/nu rats. We conclude that these cytokines may play a notable role in modulating the host's immune defence against T. gondii infection.     

Toxoplasma gondii: detection of MIC10 antigen in sera of experimentally infected mice

We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.     

Prevalence of Toxoplasma gondii in rats (Rattus norvegicus) in Grenada, West Indies

Cats are important in the natural epidemiology of Toxoplasma gondii, because they are the only hosts that can excrete environmentally resistant oocysts. Cats are infected with T. gondii via predation on infected birds and rodents. During 2005, 238 rats (Rattus norvegicus) were trapped in Grenada, West Indies, and their sera along with tissue samples from their hearts and brains were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test (MAT, titer 1:40 or higher); only 2 (0.8%) of 238 rats were found to be infected. Brains and hearts of all rats were bioassayed in mice. Toxoplasma gondii was isolated from the brain and the heart of only 1 rat, which had a MAT titer of 1:320. All of 5 mice inoculated with the heart tissue, and the 5 mice inoculated with the brain tissue of the infected rat remained asymptomatic, even though tissue cysts were found in their brains. Genetically, the isolates of T. gondii from the heart and the brain were identical and had genotype III by using the SAG1, SAG2, SAG3, BTUB, and GRA6 gene markers. These data indicate that rats are not important in the natural history of T. gondii in Grenada.     

Effect of Eimeria falciformis infection on the development of toxoplasmosis in mice

White mice previously infected with 10(2), 10(3) or 10(4) Eimeria falciformis oocysts on days 0, 5, 10 or 30 were inoculated per os with 10(1), 10(2), 10(3) or 10(4) Toxoplasma oocysts. While the results obtained for mice with higher Toxoplasma inocula were consistent, animals with 10(1) and 10(2) oocysts previous inoculation with Eimeria showed important differences related with those infected only with Toxoplasma. For example, survival time was higher in animals infected with both parasites, especially if inoculated with Eimeria 30 days before Toxoplasma infection. Furthermore the number of T. gondii cysts found in the animals previously infected with Eimeria was lower compared with mice inoculated with Toxoplasma only. Body weight of mice infected with Toxoplasma previous infection with Eimeria was almost normal in relation to those infected only with Toxoplasma, indicating a probable pathological effect due to the parasite, more evident in "non immunized" mice.     

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.     

Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.