The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 ¼g of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Immune Response to Trypanosoma cruzi Shed Acute Phase Antigen in Children from an Endemic Area for Chagas' Disease in Bolivia

A field study of the immune response to the shed acute phase antigen (SAPA) of Trypanosoma cruzi was carried out in the locality of Mizque, Cochabamba department, Bolivia. Schoolchildren (266), with an average of 8.6 ± 3.6 years, were surveyed for parasitological and serological diagnosis, as well as antibodies directed against SAPA using the corresponding recombinant protein in ELISA. The antibodies against SAPA were shown in 82% of patients presenting positive serological diagnosis (IgG specific antibodies). The positive and negative predictive values were 0.88. Antibodies anti-SAPA were shown in 80.8% of the chagasic patients in the initial stage of the infection (positive IgM serology and/or positive buffy coat (BC) test) and in 81.4% of the patients in the indeterminate stage of the infection (positive IgG serology with negative BC and IgM tests). These results show that the anti-SAPA response is not only present during the initial stage of the infection (few months) but extends some years after infection

Phase 1 Study of an Inactivated Vaccine against American Tegumentary Leishmaniasis in Normal Volunteers in Brazil

A Phase 1 double-blind placebo-controlled study was performed to evaluate a vaccine against American tegumentary leishmaniasis in 61 healthy male volunteers. Side effects and the immune response to the vaccine were evaluated, with 1- and 2- dose schemes, with intervals of 7 or 21 days, each dose containing 1440 mg of protein N antigen of a single strain of Leishmania amazonensis (PH 8) diluted in merthiolated saline (1:10,000). Merthiolated saline and an inert substance were used as placebos. No significant clinical alterations were found following the respective injections in the vaccinated individuals as compared to the placebos, except for local pain, which was associated significantly with injection of the vaccine. The laboratory alterations we observed bore no association with the clinical findings and were unimportant. We observed no differences between the groups with regard to seroconversion or the Montenegro skin test. However, the group that received a single dose of the vaccine and the one that received two doses with a 21-day interval displayed cutaneous induration significantly larger than in the control group, with 100%, 100%, and 66% conversion in the skin test, respectively. We concluded that the vaccine does not present any major side effect that would contraindicate its use in healthy individuals.

Cellulose acetate as solid phase in ELISA for plague

Antigen from Yersinia pestis was adsorbed on cellulose acetate discs (0.5 cm of diameter) which were obtained from dialysis membrane by using a paper punch. ELISA for human plague diagnosis was carried out employing this matrix and was capable to detect amount of 1.3 µg of antigen, 3,200 times diluted positive serum using human anti-IgG conjugate diluted 1:4,000. No relevant antigen lixiviation from the cellulose acetate was observed even after washing the discs 15 times. The discs were impregnated by the coloured products from the ELISA development allowing its use in dot-ELISA. Furthermore, cellulose acetate showed a better performance than the conventional PVC plates.

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2% and 91.1%, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis.

Seasonality and relative abundance of Tabanidae (Diptera) captured on horses in the Pantanal, Brazil

Once a month, from June 1992 to May 1993, collections of tabanids on horse were conducted in the Nhecolândia, Pantanal State of Mato Grosso do Sul, Brazil. Tabanid catches using hand nets were conducted from sunrise to sunset at grassland and cerradão (dense savanna) habitats. A total of 3,442 tabanids from 21 species,12 genera, and 3 subfamilies were collected. Although species abundance varied seasonally depending on habitat, no habitat specificity was observed for the most abundant species. In the grassland, 1,625 (47.2%) tabanids belonging to 19 species were collected, while 1,817 (52.8%) tabanids from 17 species were caught in the cerradão. The number of tabanid species varied from 7 during winter (July/August) to 15 in the spring (October). Tabanus importunus (56%) was the most abundant species, followed by T. occidentalis (8.2%), and T. claripennis (8.1%). The tabanid peak, in October, coincided with the beginning of the rainy season. The population peak of most species, including those with higher vector potential, suggests that the rainy season can be considered as the period of potentially higher risk of mechanical transmission of pathogens by tabanids to horses in the region.

The ue of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.

Differential expression of adhesion moleculesshaping the T-cell subset prevalence during the early phase of autoimmune and Trypanosoma cruzi-elicited myocarditis

The participation of cell adhesion molecules (CAMs) in the establishment of autoimmune and infectious myocarditis is an important matter of investigation and may have therapeutic implication. Trypanosoma cruzi infection induces a CD8-mediated myocarditis in patients with severe cardiomyopathy and experimental animals. Previously, we have proposed that this predominance of CD8+ T-cells is, at least in part, consequence of the differential expression of CAMs on circulating CD8+ lymphocytes. In the present study we investigated the participation of CAMs in shaping the phenotypic nature of the autoimmune CD4-mediated myosin-induced and the CD8-mediated T. cruzi-elicited myocarditis. We provide evidence that the prevalence of a certain T-cell subset inside the inflamed heart reflects the differential profile of the adhesion molecules VLA-4, LFA-1, and ICAM-1 displayed on a large proportion of this particular T-cell population in peripheral blood during the early phase of inflammation. Further, the expression of VCAM-1, ligand for VLA-4, and ICAM-1, counter-receptor for LFA-1, was up-regulated on vascular endothelium and paralleled the entrance of inflammatory cells into the cardiac tissue. Thus, this up-regulated expression of receptors-counter-receptors that regulate T-cell transmigration through the vascular endothelium may have an important role in the pathogenesis of the early phase of both autoimmune and infectious myocarditis.

Effect of food on immature development, consumption rate, and relative growth rate of Toxorhynchites splendens (Diptera: Culicidae), a predator of container breeding mosquitoes

Food utilization by the larvae of Toxorhynchites splendens (Wiedemann) was studied in the laboratory by offering larvae of Aedes aegypti Linnaeus, Anopheles stephensi (Liston), and Culex quinquefasciatus (Say). Quantitative analyses of data indicated that immature development was significantly faster with increase in food availability. The regression analysis showed that the degrees of the relationship between immature duration (Id) and food availability were higher when offered early instars of prey (first and second instars) than late instars. Consumption rate (Cr) of the predator increased with increase in food availability and this relationship was highly significant when larvae of An. stephensi were offered as food. Consumption rate to food level decreased with increase in the age class of the prey. There was a significant negative correlation between Id and Cr. This aspect helps to increase population turnover of T. splendens in a shorter period when the prey is abundant. Conversely, the predator compensated the loss in daily food intake at low food level by extending Id thereby attains the minimum threshold pupal weight for adult emergence. There was an increase in the relative growth rate (RGR) of the predator when An. stephensi was offered as prey and this was related to the high protein content of the prey per body weight. There was a positive correlation between Cr and RGR. This adaptive life characteristic strategy of this predator is useful for mass-rearing for large scale field release programmes in the control of container breeding mosquitoes is discussed.

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment lenght polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Clinical forms of Trypanosoma cruzi infected individuals in the chronic phase of Chagas disease in Puebla, Mexico

In Mexico, despite the relatively high seroprevalence of Trypanosoma cruzi infection in humans in some areas, reported morbidity of Chagas disease is not clear. We determined clinical stage in 71 individuals seropositive to T. cruzi in the state of Puebla, Mexico, an area endemic for Chagas disease with a reported seroprevalence of 7.7%. Diagnosis of Chagas disease was made by two standardized serological tests (ELISA, IHA). Individuals were stratified according to clinical studies. All patients were submitted to EKG, barium swallow, and barium enema. Groups were identified as indeterminate form (IF) asymptomatic individuals without evidence of abnormalities (n = 34 cases); those with gastrointestinal alterations (12 patients) including symptoms of abnormal relaxation of the lower esophageal sphincter and absent peristalsis in the esophageal body, grade I megaesophagus, and/or megacolon; patients with clinical manifestations and documented changes of chronic Chagas heart disease who were subdivided as follows: mild (8 patients) - mild electrocardiographic changes of ventricular repolarization, sinus bradychardia); moderate (6 patients) - left bundle branch block, right bundle branch block associated with left anterior fascicular block); severe (8 patients) - signs of cardiomegaly, dilated cardiomyopathy); and the associated form (3 cases) that included presence of both cardiomyopathy and megaesophagus. These data highlight the importance of accurate evaluation of the prevalence and clinical course of Chagas disease in endemic and non-endemic areas of Mexico.

15d-PGJ2 modulates acute immune responses to Trypanosoma cruzi infection

The acute phase of Trypanosoma cruzi infection is associated with a strong inflammatory reaction in the heart characterised by a massive infiltration of immune cells that is dependent on the T. cruzi strain and the host response. 15d-PGJ2 belongs to a new class of anti-inflammatory compounds with possible clinical applications. We evaluated the effects of 15d-PGJ2 administered during the acute phase of T. cruzi infection in mice. Mice were infected with the Colombian strain of T. cruzi and subsequently treated with 15d-PGJ2 repeatedly for seven days. The inflammatory infiltrate was examined by histologic analysis. Slides were immunohistochemically stained to count the number and the relative size of parasite nests. Infection-induced changes in serum cytokine levels were measured by ELISA. The results demonstrated that treatment with 15d-PGJ2 reduced the inflammatory infiltrate in the skeletal muscle at the site of infection and decreased the number of lymphocytes and neutrophils in the blood. In addition, we found that 15d-PGJ2 led to a decrease in the relative volume density of amastigote nests in cardiac muscle. T. cruzi-infected animals treated with 15d-PGJ2 displayed a statistically significant increase in IL-10 levels with no change in IFN-γ levels. Taken together, we demonstrate that treatment with 15d-PGJ2 in the acute phase of Chagas disease led to a controlled immune response with decreased numbers of amastigote nests, as measured by the volume density.