Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Experimental toxoplasmosis in Balb/c mice. Prevention of vertical disease transmission by treatment and reproductive failure in chronic infection

In a study of congenital transmission during acute infection of Toxoplasma gondii, 23 pregnant Balb/c mice were inoculated orally with two cysts each of the P strain. Eight mice were inoculated 6-11 days after becoming pregnant (Group 1). Eight mice inoculated on the 10th-15th day of pregnancy (Group 2) were treated with 100 mg/kg/day of minocycline 48 h after inoculation. Seven mice inoculated on the 10th-15th day of pregnancy were not treated and served as a control (Group 3). Congenital transmission was evaluated through direct examination of the brains of the pups or by bioassay and serologic tests. Congenital transmission was observed in 20 (60.6%) of the 33 pups of Group 1, in one (3.6%) of the 28 pups of Group 2, and in 13 (54.2%) of the 24 pups of Group 3. Forty-nine Balb/c mice were examined in the study of congenital transmission of T. gondii during chronic infection. The females showed reproductive problems during this phase of infection. It was observed accentuated hypertrophy of the endometrium and myometrium. Only two of the females gave birth. Our results demonstrate that Balb/c mice with acute toxoplasmosis can be used as a model for studies of congenital T. gondii infection. Our observations indicate the potential of this model for testing new chemotherapeutic agents against congenital toxoplasmosis.

Inactivated Parapoxvirus ovis induces a transient increase in the expression of proinflammatory, Th1-related, and autoregulatory cytokines in mice

The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/β activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines.

On the tissular parasitism of Trypanosoma cruzi y strain in swiss mice

A review of the tissular parasitism of Trypanosoma cruzi Y strain in Swiss mice was carried out. This strain parasitized preferentially smooth, skeletal and cardiac muscle fibers, with low transitory spleen and liver parasitism, as previously found by some Authors, although differing from other reports. These results can be related to the host genetical constitution and/or the degree of the strain virulence at the time of this study. Furthermore, we discuss that the high macrophagotropism reported for this strain in some instances could be an artificially induced condition resulting from its serial maintenance in mice, either for a longer time and/or by using young animals. The heavy parasitism and inflammation observed in the bladder, pancreas and spermatic duct of some inoculated mice, as well as the testis parasitization, were also noteworthy findings.

Resistance of mice immunized with killed culture trypomastigotes against infection by insect-derived trypomastigotes of Trypanosoma cruzi

Mice immunized with heat or merthiolate-killed culture trypomastigotes of the non-virulent G strain were resistant to the challenge by insect-derived trypomastigotes of the CL strain of Trypanosoma cruzi. No parasitemia was detected, by direct microscopic examination of blood samples, in 90% of immunized mice while all control animals developed a high parasitemia. Trypsinization before heat-inactivation, or fixation with paraformaldehyde, apparently reduced the immunogenicity of the G strain trypomastigotes. Mice immunized with trypomastigotes treated by either of these procedures were not protected against infection by virulent T. cruzi. Analysis of the 13I-labeled surface proteins of G strain trypomastigotes inactivated by the various methods suggests that these components are involved in eliciting protective immunity against T. cruzi infection.

Activity of praziquantel against Hymenolepis nana, at different development stages, in experimentally infected mice

Single doses of praziquantel were administered by oral route, at various time intervals, following the experimental infection of mice with Hymenolepis nana eggs (2000 per animal), to investigate the drug action against different development stages of the parasite. It was shown that either 25 or 50 mg/kg given on the 4th day after inoculation had just a partial effect against the cysticercoids. Moreover, 25 mg/kg given on the 7th day was not able to kill all juvenile forms as well. However, this dose administered on the 10th day, when the parasites had reached maturity taut oviposition was not yet initiated was 100% efficacious. The same degree of efficacy was achieved with the administration of 25 mg/kg on the 14th day when the fully mature worms already lay eggs. These animal findings indicate that in the treatment of human hymenolepiasis praziquantel, 25 mg/kg, should be taken twice, 10 days apart, so that the second dose kills the larval and juvenile forms which have survived the first one. This should be particularly recommended for treating H. nana infection in close communities.

Schistosoma mansoni: importance of skin and pulmonary phases to concomitant immunity in albino mice

Fourteen-day-old schistosomula obtained from mice previously infected were surgically transferred to the portal vein of receptor mice. Another group of mice was infected with cercariae by transcutaneous route. After 90 days, those groups were challenged with 100 cercariae, transcutaneously, as well as a control group. Two weeks later the animals were perfused and mature and immature worms counted separately. Statistically significant differences were observed in the recovery of immature worms, when the control group was compared with those twice infected. No statistical difference was detected between the group infected transcutaneously, and that infected by worm inoculation in portal vein. Results demonstrated that suppression of skin and lung migration of the parasite does not interfere with the development of the so called concomitant immunity.

Anaphylaxis with Schistosoma mansoni extracts in normal and infected mice

Methods generally utilized for studies on anaphylaxis to protein antigens such as determination of histamine release to the blood, hemoconcentration, histamine release from peritoneal mast cells and passive cutaneous anaphylaxis (PCA) were used to investigate some aspects of the anaphylaxis to parasite antigens in Schistosoma mansoni infected mice. The release of histamine to the blood and significant rates of hemoconcentration were induced by intravenous injection of schistosomula or cercarial extracts into 10-13 weeks infected mice. Cercarial, schistosomula, worm tegument and soluble egg antigens were able to trigger histamine release from peritoneal mast cells from chronically infected mice. In spite of the PCA reaction beeing detected within 2 hours of sensitization (IgG1antibodies) in 6 of 8 tested sera from chronically infected mice, no detectable reactions were obtained after 48 hours sensitization (IgE antibodies). Although IgE was not detected in the circulation, by the PCA technique, the results indicate that the infected mice contained IgE antibodies bound to their mast cells.

Hexachlorophene as a topically applied chemical for prophylaxis against Schistosoma mansoni infections in mice

Treatment of mouse tail skins with hexachlorophene (1.25% w/v) in absolute methanol or 70% isopropanol suppressed Schistosoma mansoni infections by more than 95% even when the application was performed up to three days prior to exposure to cercarial suspensions by tail immersion. Treatment with concentrations of 0.313% or higher one day prior to exposure provided at least 98% protection when the treated surface was not subjected to water washes of greater duration than 1/2 hour. Tail immersion application of 1.25% hexachlorophene one day prior to exposure still provided 87-92% protection after 3 hours water wash. Wipe application of 1.25% hexachlorophene three days prior to exposure still provided 93% protection following 3 hours water wash. High cercarial recoveries from exposure tubes at the end of exposure periods indicated high antipenetrant activity for hexachlorophene. Sufficient hexachlorophene leached from treated tail skins into the surrounding water to affect subsequently added cercariae so that they were no longer infective to untreated mice.

Protection of athymic (Nu/Nu) BALB/c mice against Plasmodium berghei by splenocytes from normal (Nu/ +) BALB/c mice

Athymic BALB/c (Nu/Nu) mice died at 7-13 days after inoculation (DAI) of Plasmodium berghei NK65, whereas their heterozygous (Nu/+) littermates died at 7-8 DAI. Nude (Nu/Nu) mice, reconstituted with 2 x 10(7) splenocytes from uninfected heterozygous (Nu/+) littermates at 20 days before parasite inoculation (DBI), died about 2 days earlier than control nude mice; nude mice reconstituted at 10 or 2 DBI lived 2 to 4 days longer than control nudes; and nude mice reconstituted 2 DAI lived even longer and some survived. These findings indicate that P. berghei NK65 induces at least two T-cell dependent immune phenomena, one suppressive and the other stimulatory. Reconstitution of nude mice with T-cells from BALB/c (Nu/+) mice appeared to reduce or bypass suppressive T-cell activities which allowed the formation of a protective immune response by some of the nude mice.

Trypanosoma (Herpetosoma) rangeli Tejera, 1920: intracellular amastigote stages of reproduction in white mice

The method, site, and stage of multiplication of Trypanosoma (Herpetosoma) rangeli Tejera, 1920 has not hitherto been known. "We have now observed many intracellular nests or pseudocysts, containing amastigotes and trypomastigotes of this parasite in the heart, liver, and spleen of suckling (5.0 g) male white mice (NMRI strain) inoculated i.p. with 9 x 10(4) metatrypomastigotes/g body weight from a 12-day-old culture of the "Dog-82" strain of T. rangeli. At the peak of parasitemia (1.9 x 10(6) trypomastigotes/ml blood, 3 days post-inoculation) various tissues were taken for sectioning and staining. The heart was most intensely parasitized. The amastigotes were rounded or ellipsoidal, with a rounded nucleus and the kinetoplast in the form of a straight or curved bar; the average maximum diameter of 50 measured amastigotes was 4.2 p. Binary fission was seen in the nucleus and kinetoplast of some amastigotes; no blood trypomastigotes were seen in division. The above characteristics, as well as the location of the pseudocysts in the tissues, are similar to T. cruzi. Comparison of these results with those reported for other Herpetosoma suggest study of the taxonomic position of T. rangeli.

Schistosomiasis mansoni: evidence for a milder response in germfree mice

Germfree (GF and conventional (CV) mice were infected intraperitoneally with GF cercariae of Schistosoma mansoni and kept for six weeks. Twenty four hours before killing, they were injected with [³H]-thymidine. Schistosoma worms, harvested after perfusion of portal system, were counted as well as eggs from liver and intestines. Liver was also used for DNA, protein, and collagen determinations. [³H] -Thymidine incorporation and collagen determinations were used to establish the indices given by the difference between their contents in infected and control animals and expressed per thousand eggs in liver. The recovery of worms in GF mice was around twice as much as in CV ones, and the total number of eggs was higher in the liver of GF animals. No hypertrophy of liver cells was observed by the ratio protein/DNA, but [³H]-thymidine incorporation into DNA was higher than in controls in both GF and CV infected animals. The [³H]-thymidine and collagen indices were lower in GF animals which indicate a more discrete cellular replication and smaller collagen content in relation to the number of eggs present in livers of these mice. It was concluded that the disease seems to be less severe in GF animals.

American trypanosomiasis (Chagas' disease) in conventional and germfree rats and mice

Germfree (GF) and conventional (CV) CFW (LOB) mice and Wistar and Sprague-Dawley rats were infected with Trypanosoma cruzi. The disease was more severe in the GF than in the CV animals as revealed by: (1) an earlier and more intense parasitemia; (2) a more precocious mortality; (3) a twice enlarged spleen: (4) a more intense cell and tissue parasitism; (5) visceral signs of cardiac failure.

Cutaneous leishmaniasis in germfree, gnotobiotic, and conventional mice

Cutaneous leishmaniasis was much more severe in conventional than in gnotobiotic mice as revealed by macro and microscopic examination. An inoculum of Leishmania mexicana amazonensis was used.

: acquired resistance in mice by implantation of young irradiated worms into the portal system

In two distinct experiments, immature S. mansoni worms (LE strain, Belo Horizonte, Brazil), aged 20 days, obtained from the portal system of white outbred mice, were irradiated with 14 and 4 Krad, respectively. Afterwards, the worms were directly inoculated into the portal vein of normal mice. Inoculation was performed with 20 irradiated worms per animal. Fifty days after inoculation, the mice that received 4 and 14 Krad-irradiated worms and their respective controls were infected with S. mansoni cercariae (LE strain), by transcutaneous route. Twenty days after this challenge infection, the animals were sacrificed and perfused for mature irradiated (90-day-old) and immature (20-day-old) worm counts. Analysis of the results showed that statistically significant protection against cercariae occurred in both groups with irradiated worms.