Sensitivity of polymerase chain reaction for detection of known aliquots of Trypanosoma cruzi in the blood of mice: an in vitro study

To evaluate the sensitivity of polymerase chain reaction (PCR) to reveal known number of trypomastigote in the blood of mice, three separate experiments were done. First: To eight samples of 500mul of normal mice blood, one aliquot of 1, 2, 3, 4, 5, 10, and 50 trypomastigotes respectively, were added. Second and third: 10 aliquots with 1 and 10 with 2 trypomastigotes were added to samples of 500mul of normal mice blood. Positive control: 500mul of blood containing 100,000 trypomastigotes. For kDNA minicircles amplification by PCR the primers:S35 and S36 were used. PCR revealed products of 330 b.p in the positive controls. When only one sample with the aliquots of 1 or 2 trypomastigotes was examined, results were negative; results were positive with aliquots of 3 to 50 trypomastigotes. In the 2nd and 3rd experiments, 9/10 aliquots with one parasite and 9/10 with 2 trypomastigotes were positive revealing a high sensitivity of this reaction. In conclusion, the presence of one single parasite in 500mul of blood, is enough for a positive PCR. This method could be used as a complement to the various parasitological cure tests in treated mice, when low volumes of blood are individually examined.

Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice

Post-training intracerebroventricular administration of procaine (20 µg/µl) and dimethocaine (10 or 20 µg/µl), local anesthetics of the ester class, prolonged the latency (s) in the retention test of male and female 3-month-old Swiss albino mice (25-35 g body weight; N = 140) in the elevated plus-maze (mean ± SEM for 10 male mice: control = 41.2 ± 8.1; procaine = 78.5 ± 10.3; 10 µg/µl dimethocaine = 58.7 ± 12.3; 20 µg/µl dimethocaine = 109.6 ± 5.73; for 10 female mice: control = 34.8 ± 5.8; procaine = 55.3 ± 13.4; 10 µg/µl dimethocaine = 59.9 ± 12.3 and 20 µg/µl dimethocaine = 61.3 ± 11.1). However, lidocaine (10 or 20 µg/µl), an amide class type of local anesthetic, failed to influence this parameter. Local anesthetics at the dose range used did not affect the motor coordination of mice exposed to the rota-rod test. These results suggest that procaine and dimethocaine impair some memory process(es) in the plus-maze test. These findings are interpreted in terms of non-anesthetic mechanisms of action of these drugs on memory impairment and also confirm the validity of the elevated plus-maze for the evaluation of drugs affecting learning and memory in mice

Antidepressant-like effect of Hoodia gordonii in a forced swimming test in mice: evidence for involvement of the monoaminergic system

Hoodia gordonii is a plant species used traditionally in southern Africa to suppress appetite. Recently, it has been associated with a significant increase in blood pressure and pulse rate in women, suggesting sympathomimetic activity. The present study investigated the possible antidepressant-like effects of acute and repeated (15 days) administration of H. gordonii extract (25 and 50 mg/kg, po) to mice exposed to a forced swimming test (FST). Neurochemical analysis of brain monoamines was also carried out to determine the involvement of the monoaminergic system on these effects. Acute administration of H. gordonii decreased the immobility of mice in the FST without accompanying changes in general activity in the open-field test during acute treatment, suggesting an antidepressant-like effect. The anti-immobility effect of H. gordonii was prevented by pretreatment of mice with PCPA [an inhibitor of serotonin (5-HT) synthesis], NAN-190 (a 5-HT1A antagonist), ritanserin (a 5-HT2A/2C antagonist), ondansetron (a 5-HT3A antagonist), prazosin (an α1-adrenoceptor antagonist), SCH23390 (a D1 receptor antagonist), yohimbine (an α2-adrenoceptor antagonist), and sulpiride (a D2 receptor antagonist). A significant increase in 5-HT levels in the striatum was detected after acute administration, while 5-HT, norepinephrine and dopamine were significantly elevated after chronic treatment. Results indicated that H. gordonii possesses antidepressant-like activity in the FST by altering the dopaminergic, serotonergic, and noradrenergic systems.

Trypanosoma cruzi: chemotherapy with benznidazole in mice inoculated with strains from Paraná State and from different endemic areas of Brazil

Strains of Trypanosoma cruzi from different geographical areas have shown different levels of susceptibility to trypanocidal drugs. The susceptibility in vivo to benznidazole was investigated in eighteen strains of T. cruzi. Twelve were isolated from chronic chagasic patients from different Chagas’ disease endemic areas. The other six strains were isolated from the northwestern region of Paraná state; two of them from patients three from triatomines (Triatoma sordida) and one from wild reservoir (Didelphis sp.). To test drug the infected mice were divided into two groups of twenty. One group was treated with benznidazole for twenty consecutive days and the other group was used as untreated control. The treatment began after detection of the infection by direct blood examination or haemoculture. The control of cure was done through haemoculture and indirect immunofluorescence test. The drug eliminated the inflammatory lesions of the skeletal muscle of mice considered cured and from the heart of most of them. Moreover, the inflammatory lesions were reduced in treated but not cured animals. The T. cruzi strains studied showed a gradient of drug susceptibility that varied from 0% to 100%. Ten strains were considered sensitive to the treatment (61 to 100% of cure), one strain was partially sensitive (50% of cure) and seven strains were considered resistant to the treatment (0 to 40% of cure). This variation was observed both in strains of T. cruzi isolated from domestic and sylvatic cycles

Pathogenicity of different rabies virus isolates and protection test in vaccinated mice

This study was aimed to evaluate and compare the pathogenicity of rabies virus isolated from bats and dogs, and to verify the efficacy of a commercial rabies vaccine against these isolates. For evaluation of pathogenicity, mice were inoculated by the intramuscular route (IM) with 500MICLD50/0.03mL of the viruses. The cross-protection test was performed by vaccinating groups of mice by the subcutaneous route and challenged through the intracerebral (IC) route. Isolates were fully pathogenic when inoculated by the IC route. When inoculated intramuscularly, the pathogenicity observed showed different death rates: 60.0% for the Desmodus rotundus isolate; 50.0% for dog and Nyctinomops laticaudatus isolates; 40.0% for Artibeus lituratus isolate; 9.5% Molossus molossus isolate; and 5.2% for the Eptesicus furinalis isolate. Mice receiving two doses of the vaccine and challenged by the IC route with the isolates were fully protected. Mice receiving only one dose of vaccine were partially protected against the dog isolate. The isolates from bats were pathogenic by the IC route in mice. However, when inoculated through the intramuscular route, the same isolates were found with different degrees of pathogenicity. The results of this work suggest that a commercial vaccine protects mice from infection with bat rabies virus isolates, in addition to a canine rabies virus isolate.

Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

Reversibility of cardiac fibrosis in mice chronically infected with Trypanosoma cruzi, under specific chemotherapy

This investigation was performed to verify the effect of specific chemotherapy (Benznidazole or MK-346) on the inflammatory and fibrotic cardiac alterations in mice chronically infected with the strains 21 SF (Type II) and Colombian (Type III) of Trypanosoma cruzi. To obtain chronically infected mice, two groups of 100 Swiss mice each, were infected with either the 21 SF or the Colombian strain (2x 10 [raised to the power of] 4 and 5x 10 [raised to the power of] 4 blood forms respectively). The rate of morality in the acute phase was of 80% for both groups. Twenty surviving mice chronically infected with the 21 SF strain and 20 with the Colombian strain were then divided in treated and untreated groups. Excluding those that died during the course of treatment, 14 mice chronically infected with the 21 SF strain and 15 with the Colombian strain were evaluated in the present study. Chemotherapy was performed with Benznidazole (N-benzil-2-nitro-1-imidazolacetamide) in the dose of 100mg/k.b.w/day, for 60 days, or with the MK-436(3(1-methyl-5 nitroimidazol-2-yl) in two daily doses of 250 mg/k.b.w, for 20 days. Parasitological cure tests were performed (xenodiagnosis, haemoculture, subinovulation of the blood into newborn mice), and serological indirect immunofluorescence test. The treated and untreated mice as well as intact controls were killed at different periods after treatment and the heart were submitted to histopathological study with hematoxilineosin and picrosirius staining; ultrastructural study; collagen immunotyping, fibronectin and laminin identification by immunofluorescence tests. Results: the untreated controls either infected with 21 SF or Colombian strain, showed inflammatory and fibrotic alterations that were mild to moderate with the 21 SF strain and intense with the Colombian strain. Redpicrosirius staining showed bundles of collagen in the interstitial space and around cardiac fibers. Increased deposits of mitritial components and collagen fibers, macrophages and fibroblasts appeared at the ultra structural examination. Deposits of fibronectin, laminin, pro-III and IV collagens were seen, most intense in those infected with the Colombian strain. Treated nice, parasitologically cured, presented clear-cut regression of the inflammatory lesions and of the interstitial matrix thickening. Mice infected with the Colombian strain and treated with MK-436, was parasitologically cured in 5/6 cases and showed mild inflammatory infiltration and fibrosis. The mice treated with Benznidazole (Colombian strain) did not cure and showed moderate fibrosis and inflammation. Treatment of the nice infected with the 21 SF with Benznidazole determined parasitological cure of all animals, that showed mild inflammation and fibrosis of the myocardium. The cured mice of all groups and treated but uncured showed collagen degradation at electronmicroscopy and decrease of immunofluorescence pattern of the matrix.

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.

Genotoxic activity of the insecticide Nuvacron (Monocrotophos) detected by the micronucleus test in bone marrow erythrocytes of mice and in CHO cells

The organophosphorus insecticide Nuvacron (Monocrotophos) is a very toxic agent widely utilized in Brazilian agriculture. To evaluate the clastogenic potential of this insecticide, in vivo and in vitro micronucleus (MN) assay experiments were carried out on Swiss mice and on Chinese hamster ovary (CHO) cells, respectively. Nuvacron administered at doses of 2.5 and 5.0 mg/kg induced a statistically significant increase in the frequencies of MN detected in polychromatic bone marrow erythrocytes from animals (six/group) treated ip 24 h before. Exponentially growing CHAO cells were treated continuously (16h) with Nuvacron diluted in water to final concentrations of 1, 10, 100, 200, and 400 mug/ml. Three experiments were carried out using the cytokinesis-block method and a total of 6000 binucleated cells were scored to determine MN frequencies. A statistically significant increase in the frequencies of MN was observed for the cells treated with 1 and 10 mug/ ml Nuvacron. A marked decrease in cell proliferation rates was observed for CHO cultures treated with higher concentrations. These data demonstrate that Nuvacron has a genotoxic effect on both in vivo and in vitro mammalian test systems.

Schistosoma mansoni: protective immunity in mice cured by chemotherapy at the chronic phase of the disease

Aiming at demonstrating a decrease of acquired immunity after chemotherapeutic cure, a group of mice was infected with 25 Schistosoma mansoni cercariae (LE strain). A part of these animals was treated with 400 mg/kg oxamniquine, at 120 days after infection. Challenge infections were carried out at 45, 90 and 170-day-intervals after treatment (185, 210 and 290 days after primoinfection, respectively). Recovery of worms at 20 days after reinfections showed that a residual immunity remains up to 90 days after treatment, and disappears at 170 days after cure. Using the ELISA method, it was possible to detect a decrease of antibody levels (total IgG) in the treated group, when antigens from different evolutive stages of S. mansoni were used. The epidemiological implications of the present results, and the possible mechanisms involved in the decrease of acquired immunity after treatment are discussed.

EFFECT OF BACILLUS OF CALMETTE-GUÉRIN, AVRIDINE AND Propionibacterium acnes AS IMMUNOMODULATORS ON RABIES IN MICE

The cellular and humoral immune responses of mice inoculated with rabies virus and treated with the Bacillus of Calmette-Guérin, Avridine and Propionibacterium acnes were evaluated in this paper. There was a higher percentage of surviving mice in groups submitted to P. acnes treatment. Lower levels of interferon-g (IFN-g) were found in infected mice. The intra-pad inoculation test (IPI) was not effective to detect cellular immune response, contrary to the results found in MIF reaction. The survival of mice did not present correlation with the levels of antirabies serum neutralizing (SN) antibodies titers, IFN-g concentration and MIF response.

Serotyping and evaluation of the virulence in mice of Streptococcus suis strains isolated from diseased pigs

A total of 110 strains of Streptococcus suis, isolated from diseased pigs in Brazil were serotyped and analyzed for virulence. Serotyping of the strains resulted in the following classification: 42 strains of serotype 2 (38.2%), 10 strains of serotype 14 (9.1%), seven strains of serotype 9 (6.4%), three strains each of serotype 7 and 11 (2.7%), two strains each of serotype 1 and 8 (1.8%) and one strain each of serotypes ½, 3, 5, 6 and 10 (0.9%). Cross reactions among serotypes 1, 14 and 7 were observed in 21 strains (19.1%). Only 41.9% of the strains were lethal for mice using the pathogenicity test.

Influence of Trypanosoma cruzi strain on the pathogenesis of chronic myocardiopathy in mice

The murine model of chronic Chaga's myocardiopathy was developed in 201 inbred and outbred mice. The experimental groups consisted of 1st: 73 inbred AKR and A/J mice inoculated with one of the following. Trypanosoma cruzi strains: Peruvian (Type I), 12 SF (Type II) or Colombian (Type III); 2nd: 128 outbred Swiss mice, chronically infected either with Type II or Type III strains isolated from human patients from different geographical areas. All T. cruzi strains were previoulsly characterized by their morphobiological behaviour in mice and by isoenzymatic patterns. For the 1st group the inoculum was 5 x 10**4 for the Peruvian strain and 1 x 10**5 for the 12 SF and Colombian strains. In the 2nd group-Swiss mice the inoculum size varied from 2 x 10**4 to 2 x 10**5. The inbred animals were killed at a 3 time-point scale (90, 180 and 240 days) post-infection. The Swiss mice were killed from 180 to 660 days after infection. The evaluation of parasitemia and serology (xeodiagnosis and indirect immunofluorescent test) was performed. The incidence of macroscopic alterations of the heart and cardiac index were evaluated. Histopathological lesions of the myocardium were graded. The influence of T. cruzi strain on the intensity of cardiac lesions was evaluated by the Chi-square test; the incidence of inflammatory lesions and its relationship to the parasite strain was evaluated by the Fisher test. The influence of the duration of infection was evaluated by using the Gamma Coefficient of Kruskal and Goodman and its measure of significance. Slight to severe microscopic alterations occurred in 85% of the chronically infected nice. There were a clear predominance on the incidence and intensity of inflammatory and fibrotic alterations for the mice infected with Type III strains. Statistical analysis has shown significant differences among the infected groups, in the inflammatory and fibrotic lesions. Macroscopic alterations (right cavities dilatation and apex aneurism of left ventricle), differed in incidence according to mice strains; in Swiss and AKR mice, significant differences were seen in mice infected with different T. cruzi strains, but the A/J mice failed to show significant differences correlated with different parasite strains. The duration of infection, from 90 to 240 days, could not be correlated with the degree of lesions in the several groups.

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.

Effect of treatment with cyclophosphamide in low doses upon the onset of delayed type hypersensitivity in mice chronically infected with Trypanosoma cruzi: involvement of heart interstitial dendritic cells

Acute infection with Trypanosoma cruzi results in intense myocarditis, which progresses to a chronic, asymptomatic indeterminate form. The evolution toward this chronic cardiac form occurs in approximately 30% of all cases of T. cruzi infection. Suppression of delayed type hypersensitivity (DTH) has been proposed as a potential explanation of the indeterminate form. We investigated the effect of cyclophosphamide (CYCL) treatment on the regulatory mechanism of DTH and the participation of heart interstitial dendritic cells (IDCs) in this process using BALB/c mice chronically infected with T. cruzi. One group was treated with CYCL (20 mg/kg body weight) for one month. A DTH skin test was performed by intradermal injection of T. cruzi antigen (3 mg/mL) in the hind-footpad and measured the skin thickness after 24 h, 48 h and 72 h. The skin test revealed increased thickness in antigen-injected footpads, which was more evident in the mice treated with CYCL than in those mice that did not receive treatment. The thickened regions were characterised by perivascular infiltrates and areas of necrosis. Intense lesions of the myocardium were present in three/16 cases and included large areas of necrosis. Morphometric evaluation of lymphocytes showed a predominance of TCD8 cells. Heart IDCs were immunolabelled with specific antibodies (CD11b and CD11c) and T. cruzi antigens were detected using a specific anti-T. cruzi antibody. Identification of T. cruzi antigens, sequestered in these cells using specific anti-T. cruzi antibodies was done, showing a significant increase in the number of these cells in treated mice. These results indicate that IDCs participate in the regulatory mechanisms of DTH response to T. cruzi infection.