Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m(3)G antibodies

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m(3)G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m(3)G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Para State, Amazon, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação da vacinação anti-rábica e da suplementação com probiótico na resposta imune humoral em bovinos

This study evaluated the humoral immune response of a new rabies vaccine developed by the Instituto Butantan (potency of 3.27 UI/ml) in primovaccinated cattle and the effect of probiotic on this response. Thirty-four 15-month old Nelore cattle were randomly divided into 2 groups (17 animals/group). All the animals were vaccinated on day 0 (zero) and then animals in one group received probiotic added to a mineral mixture (GP) while the others were given only the mineral mixture (GC). Blood samples were collected on days 0, 75 and 150 for rabies neutralizing antibodies titers by seroneutralization assay on BHK21 cells (RFFIT). Protective antibody titers (>= 0.5 UI/mL) were found in 82.4% of the animals from GP and in 76.5% of the animals from GC and no statistical difference (p>0.05) between antibody titers in GP and GC was detected on days 75 and 150. It was also observed that in both groups antibody titers was decreased on day 150 (p<0.01). In conclusion, the tested rabies vaccine promotes efficient soroconversion and keeps antibody levels in primovaccinated cattle, but probiotic does not affect the humoral anti-rabies immune response.

Indirect fluorescent test for detection of anti-Paracoccidioides brasiliensis antibodies using coated bentonite particles as antigen

An indirect fluorescent test was developed for detecting antibodies to Paracoccidioides brasiliensis using bentonite particles as antigen (Bent-IF). The bentonite particles were coated with P. brasiliensis polysaccharide antigen and tested with sera from paracoccidioidomycosis patients (36 sera), normal blood donors (32 sera) and patients with non-mycotic diseases (29 sera). The titres given by the positive sera were compared with those of complement fixation (CF), immunodiffusion (ID) and immunofluorescent test using yeast forms of the fungus as antigen (conventional-IF). All normal blood donors' sera gave a negative Bent-IF, conventional-IF, ID and CF tests. All paracoccidioidomycosis sera were reactive in conventional-IF and gave concordant results in Bent-IF. There was no correlation between CF and Bent-IF titres. 27·6% of sera from patients with non-mycotic diseases gave weak titres in both IF-tests. The present data indicate that the Bent-IF is a sensitive and simple serodiagnostic technique comparable with the conventional P. brasiliensis antibody test. © 1983.

Characterization of myeloid or lymphoid acute leukemia by a chemiluminescence assay: Comparison with immunocytochemistry using an antimyeloperoxidase antibody

A simple and sensitive chemiluminescence assay for the demonstration of the activity of intracellular myeloperoxidase (MPO) is described, which is useful for the distinction between myeloid and lymphoid commitment in blasts from acute leukemia patients. When the cut-off point was settled at 13 mV of chemiluminescence all cases of acute myeloid leukemia (AML) were distinguished from those of acute lymphoid leukemia. In addition, this technique was able to demonstrate MPO activity in AML poorly differentiated (FAB-M0) which usually does not stain for MPO in classical cytochemistry preparations and could be negative also by immunocytochemistry with anti-MPO monoclonal antibody. Therefore the method here described presented a higher sensitivity than the immunocytochemistry procedure with anti-MPO.

Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor

Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.

IgG, IgM and IgA antibody response for the diagnosis and follow-up of paracoccidioidomycosis: Comparison of counterimmunoelectrophoresis and complement fixation

IgG, IgM and IgA antibodies to GP43 (glycoprotein fraction of Paracoccidioides brasiliensis) were measured by ELISA in 63 samples from 23 patients with paracoccidioidomycosis before and twice after chemotherapy was started. Antibodies against P. brasiliensis were detected by indirect immunofluorescence (IF) (IgG, IgM and IgA isotypes), counterimmunoelectrophoresis (CIE) and complement fixation. Two control groups composed of 19 healthy individuals and 12 patients with other diseases (six with histoplasmosis, three with tuberculosis and three with other mycoses). The highest efficiency percentages were found with IgG and IgA- ELISA (100%), IgG-IF (96.2%), CIE (94.4%) and the lowest with CF (75.9%). Highest positive and negative predictive values (100%) were observed for IgG and IgA ELISA. IgG and IgM-ELISA antibodies are more often found in patients with acute than chronic disease (P = 0.01). Four to six months after treatment follow-up showed decreased levels of IgG and IgM-ELISA for acute cases and decreased titres of CIE for chronic cases in relation to pretreatment levels. This study suggests that IgG-ELISA anti-GP43 represents a good marker to monitor clinical response to therapy.

Toxocariasis: Serological diagnosis by indirect antibody competition elisa

Toxocariasis is caused by infection of man by Toxocara canis and Toxocara, cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE) employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody. in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%).

Action of anti-bothropic factor isolated from Didelphis marsupialis on renal effects of Bothrops erythromelas venom

Acute renal failure is the most common complication in the lethal cases caused by snakebites in Brazil. Among the Brazilian venom snakes, Bothrops erythromelas is responsible for the majority of accidents in Northeastern Brazil. Didelphis marsupialis serum could inhibit myonecrotic, hemorrhagic, edematogenic hyperalgesic and lethal effects of envenomation determined by ophidian bites. In the present study, we evaluated the action of the anti-bothropic factor isolated from D. marsupialis on the renal effects promoted by B. erythromelas venom without systemic interference. Isolated kidneys from Wistar rats were perfused with Krebs-Henseleit solution containing 6% bovine serum albumin. We analyzed renal perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), and the percentages of sodium and potassium tubular transport (%TNa +, %TK +). The B. erythromelas venom (10 μg mL -1) decreased the PP (ct=108.71±5.09 mmHg; BE=65.21±5.6 mmHg*) and RVR (ct=5.76±0.65 mmHg mL -1 g -1 min -1; BE=3.10±0.45 mmHg mL -1 g -1 min -1*) . On the other hand, the GFR decreased at 60 min (ct 60=0.76±0. 07 mL g -1 min -1; BE 60=0.42±0.12 mL g -1 min -1*) and increased at 120 min (ct 120=0.72±0.01 mL g -1 min -1; BE 120=1.24±0.26 mL g -1 min -1*). The UF increased significantly when compared with the control group (ct=0.14±0.01 mL g -1 min -1; BE=0.47±0.08 mL g -1 min -1*). The venom reduced the %TNa + (ct 90=79.18±0.88%; BE 90=58.35±4.86%*) and %TK + (ct 90=67.20±4.04%; BE 90=57. 32±5.26%*) The anti-bothropic factor from D. marsupialis (10 μg mL -1) incubated with B. erythromelas venom (10 μg mL -1) blocked the effects on PP, RVR, %TNa +, and %TK +, but was not able to reverse the effects in UF and GFR promoted by venom alone. However, the highest concentration of D. marsupialis serum (30 μg mL -1) reversed all the renal effects induced by the venom. In conclusion, B. erythromelas venom altered all the renal functional parameters evaluated and the anti-bothropic factor from D. marsupialis was able to inhibit the effects induced by the venom in isolated kidney. © 2005 Elsevier Ltd. All rights reserved.

Serological profile of anti-Toxoplasma gondii antibodies in apparently healthy dogs of the city of Botucatu, São Paulo state, Brazil

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, a coccidian protozoan of worldwide distribution. The seroprevalence in canine population can be an alternative for measuring T. gondii urban spreading. A total of 780 blood samples from dogs were collected, during the yearly anti-rabies campaign, carried out by the Department of Veterinary Hygiene and Public Health, School of Veterinary Medicine and Animal Husbandry (FMVZ), São Paulo State University, UNESP, together with the county health authorities, in August 1999. Using Indirect Fluorescent Antibody Test (IFAT) for detecting antibodies anti-T. gondii in the sera samples, we observed that 258 dogs (33.1%) were positive. The associations between the serological results and the epidemiological variables were studied. Statistically significant differences were not found regarding sex (32.2% male and 34.3% female reactors). Dogs without a defined breed showed seropositivity statistically higher than the pedigreed group. The occurrence of infection was considered higher with age.

Toxoplasma gondii: Comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7 × 107 viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10 = 2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as gold standard and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISA × IFAT (κ = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 ± 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs. © 2005 Elsevier Inc. All rights reserved.

Presença de anticorpos anti-Neospora caninum e Toxoplasma gondii em cães com leishmaniose visceral da região de Araçatuba, São Paulo, Brasil

Dogs that had positive and negative sera to Leishmania chagasi from the region of Araçatuba, São Paulo, Brazil, were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies as potential co-infecting agents. Blood samples were collected from 204 dogs and out of them 98 were carriers of leishmaniosis. Sera were tested for the presence of anti-L. chagasi antibodies by ELISA, and anti-T. gondii and anti-N. caninum by an indirect fluorescent antibody test (IFAT). Age, gender, and association between the presences of anti-L. chagasi antibodies and seroprevalence to N. caninum and T. gondii were analyzed by chi-square test. Out of the 204 sera investigated, 36 (17.6%) were positive for N. caninum (IFAT=50) and 75 (36.8%) to T. gondii (IFAT=16) with titers that varied from 50 to 6400 for N. caninum, and from 16 to 16384 for T. gondii. The copresence of anti-L. chagasi, N. caninum and T. gondii antibodies was observed in 17 (8.3%) dogs. Antibodies to N. caninum were observed in four (3.8%) out of 106 dogs that were negative for L. chagasi, and in 32 (32.6%) out of the 98 dogs that were positive for L. chagasi. Anti-T. gondii antibodies were found in 40 (41.0%) and in 35 (33.0%) of the 98 positive dogs and in 106 negative dogs for L. chagasi, respectively. An association between the presence of antibodies against L. chagasi and a positive response to N. caninum (p<0.001) was observed. The gender and age of the dogs did not show an association between the presence of antibodies and any of the agents studied (p>0.05), with the exception of age and presence of anti-L. chagasi antibodies, in which only a slight association was observed (p=0.038). Within this interaction, a higher number of dogs, older than four years, were positive for this agent when compared to other age groups.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 × 10 4 VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) × IFA (ah) (r = 0.62, P = 0.05), MAT(s) × MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) × r-ELISA (ah) (r = 0.14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. © 2007 Elsevier Ltd. All rights reserved.