Colony polymerase chain reaction of stably transfected Trypanosoma cruzi grown on solid medium

Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Visceral leishmaniasis in the Metropolitan Region of Belo Horizonte, State of Minas Gerais, Brazil

In the last few years the number of human cases of American visceral leishmaniasis in the Metropolitan Region of Belo Horizonte (MRBH), Minas Gerais, Brazil has increased, indicating an elevation in the transmission rate of the disease. The total number of notified human cases in the MRBH since 1994, when the first case was identified, up to 1999 was 345 of which 223 (65%) were from the city itself, indicating an urbanization of the disease in this region of Minas Gerais. The age distribution of visceral leishmaniasis cases in the MRBH shows a higher prevalence in children from 0-4 years old, responsible for 28.9% of the notifications. Clinical and immunological findings from dogs infected with Leishmania chagasi are described. The majority of these animals showed no sign of the disease. Sera from all infected dogs showed detectable Leishmania-induced high titles of antibodies based on the results of an indirect fluorescent antibody test. Samples of isolated Leishmania from human and dogs were characterized as L. (L.) chagasi by biochemical and molecular techniques.

Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi

"Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patterns and in the parasitic peptides recognition patterns of experimentally infected Wistar rats. The rats experimentally infected with T. evansi isolates obtained from coatis, dogs and horses were submitted to indirect immunofluorescence test (IgM e IgG) and Western blotting. The serological titers for IgM and IgG ranged between 1:40 and 1:160. The most recognized polypeptide profiles were in a range of 17 and 74 kDa. Our data suggest that the humoral immune response in Wistar rats is not sufficient for granting an effective control of T. evansi infections.

Antibodies anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae in patients with negative serology for hantavirus. Retrospective study

The seroprevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae in hantavirus seronegative patients, who had symptoms and signs compatible with pneumonia was established. For this purpose we used the indirect fluorescent antibody test. Titers ³ 1:16 for C. pneumoniae and M. pneumoniae were found in 8.6% and 17.1% of the serum, respectively, showing evidence of recent or current infection.

Triatomines Involved in Domestic and Wild Trypanosoma cruzi Transmission in Concepción, Corrientes, Argentina

An entomological and serological survey was performed in three localities of the Department of Concepción, Province of Corrientes, Argentina in 1998 and 1999, to identify triatomines species involved in domestic and wild transmission of Chagas disease. Triatomines were collected by man/hour capture in 32 houses randomly selected and 44 nearby outdoor ecotopes. Trypanosoma cruzi infection in triatomines was assessed by direct microscopic observation (400x) of feces and polymerase chain reaction. Serological techniques used for people were Indirect Hemagglutination Test and Indirect Fluorescent Test. Triatomines were collected in 28.1% of the houses and 31.8% of the wild biotopes. Triatoma infestans (Klug 1834) was exclusively found indoors and T. cruzi infected 60% of them. Triatoma sordida (Stål 1859) was mainly found in extradomestic ecotopes where trypanosome infection rate reached 12.7%. Serological study of 98 local people showed that 29.6% were seroreactive; most of their houses were closed to wild biotopes colonized by T. sordida. Results indicate that there is an active T. infestans mediated transmission of Chagas disease in this zone that yields important human prevalence and that the populations of T. sordida in wild biotopes not only sustain the wild T. cruzi cycle but also represent an actual risk for people living in the area.

A Heminested Polymerase Chain Reaction for the Detection of Brazilian Rabies Isolates from Vampire Bats and Herbivores

A heminested-PCR (hn-PCR) using primers to the nucleoprotein-coding gene in a nested set was evaluated in the detection of Brazilian strains of rabies virus (RV). A representative number of RV nucleoprotein sequences belonging to genotype 1 were aligned. Based on such alignment, primers were directed to highly conserved regions. All 42 clinical samples positive by both fluorescent antibody and mouse inoculation tests were also positive by the hn-PCR. Brain tissue that had been left to decompose, obtained from an experimentally inoculated mouse was tested by hn-PCR and yielded positive results. In conclusion, primers designed here were capable of amplifying Brazilian RV isolates obtained from a rural epidemiological cycle.

IgM-Immunofluorescence Test as a Diagnostic Tool for Epidemiologic Studies of Schistosomiasis in Low Endemic Areas

The high sensitivity and the ability to diagnose schistosomiasis in a very early phase after infection have indicated the detection of IgM antibodies to Schistosoma mansoni gut antigens by the immunofluorescence test (IgM-IFT) as a useful serological test for epidemiological studies in low endemic areas. When applied in a follow-up study for two years, higher rates of seroconversion from IFT negative to positive were observed during the summer months, suggesting seasonal transmission of schistosomiasis in the rural area of the municipality of Itariri (São Paulo, Brazil). In each survey, blood samples from about 600 schoolchildren were collected on filter paper and submitted to IgM-IFT. When the blood samples were classified for the IgM antibody levels, according to the intensity of fluorescent reaction observed at fluorescence microscopy, and correlated to the egg counts in the Kato-Katz positive patients, no association was observed. This observation might suggest that the intensity of fluorescence observed in the IgM-IFT, as an indicator of IgM antibody levels, could not be an useful seroepidemiological marker for classifying areas of low endemicity according to degrees of infection.

Perspective of a new diagnostic for human trichomonosis

Several diagnostic techniques have been employed for the detection of Trichomonas vaginalis. Microtubules constitute the cytoskeleton in eukaryotic cells and are sensitive to antimitotic drugs, such as Taxol (paclitaxel). We used FLUTAX a fluorescent taxoid - to analyze the microtubule distribution in living trophozoites of T. vaginalis in urine and in vaginal discharge. A high intensity of fluorescence was observed in living T. vaginalis, epithelial cells and leukocytes present in urine and vaginal discharge. Our preliminary results show the perspective of a new diagnostic technique for trichomonosis and will contribute to the understanding of the cytoskeleton of T. vaginalis.

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

A contribution to the diagnosis of Capillaria hepatica infection by indirect immunofluorescence test

A highly specific pattern of immunofluorescence was noted when sera from Capillaria hepatica-infected rats were tested against the homologous worms and eggs present either in paraffin or cryostat sections from mouse liver. The pattern was represented by a combined apple green fluorescence of the internal contents of worms and eggs, which persisted in serum-dilutions of 1:400 up to 1:1600. Unequivocal fluorescent pattern was observed from 15 days up to 3 months following inoculation of rats with embryonated C. hepatica eggs and such result was confirmed by the ELISA. After the 4th month of infection, the indirect immunofluorescence test turned negative, probably revealing the extinction of parasitism, however the ELISA was contradictory, disclosing high levels of antibodies in this period . The IIF was also negative when control normal rat sera and sera from rats administered by gavage with immature C. hepatica eggs (spurious infection), or for reactions made against Schistosoma mansoni eggs, although a weakly positive pattern occurred with Fasciola hepatica eggs. The indirect immunofluorescence test may be recommended for use with human sera to detect early C. hepatica infection in special clinical instances and in epidemiological surveys, since it is a simple, inexpensive, and reliable test, presenting excellent sensitivity and specificity. Although the diagnosis is positive only during early infection, this is the period when the symptoms are usually more severe and the need for differential diagnosis is greater.

Dispersal pattern of the sand fly Lutzomyia neivai (Diptera: Psychodidae) in a cutaneous leishmaniasis endemic rural area in Southeastern Brazil

The dispersal pattern of the sand fly Lutzomyia neivai was studied through mark-release-recapture experiments in an American cutaneous leishmaniasis endemic rural area in Southeastern Brazil. Over 6500 specimens were marked with fluorescent powder and released in forest edge and peridomicile habitats from August to November 1999, February and April 2000. Recapture attempts were made using Shannon and CDC traps up to eight successive nights after releases. A total of 493 (7.58%) specimens were recaptured. The number of recaptured males and females of L. neivai in CDC traps was not affected by the distance between the trap and the release points. Approximately 90% of males and females recaptured in CDC traps were caught up to 70 m from the release points. The maximum female flight range recorded was 128 m. The average flight range per day was less than 60 m for males and females. Of the flies released in forest edge, approximately 16% of the recaptured females were caught in Shannon traps in the peridomicile habitat. The results indicate that the movements of L. neivai are spatially focal and the possibility of dispersion from forest to peridomicile habitat may be an important way of contracting leishmaniasis in dwellings.

Frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of Mexico City

A locality in the district of Tlalpan, Mexico City, was selected in order to identify the viral agents in children younger than 5 years of age with acute respiratory infection (ARI). A total of 300 children were randomly selected and were included in this study for a period of 13 months. During this period nasopharyngeal exudates were collected for the isolation of viral agents. Monoclonal fluorescent antibodies were used for viral identification after cell culture. Viral infection was detected in 65% of the specimens. The respiratory syncytial virus (RSV) was the most common virus agent detected. Children required an average of two consultations during the study period. Two high incidence peaks were observed, one during the summer and the other during winter; the most frequent viruses during these seasons were influenza A and RSV, respectively. The largest number of viruses was isolated in the group of children between 1 and 2 years of age and in the group between 4 and 5 years of age. This study demonstrated the presence of ARI and of different viruses in a period of 13 months, as well as the most frequent viruses in children younger than 5 years of age from a community of Mexico City.

Comparison between morphological and staining characteristics of live and dead eggs of Schistosoma mansoni

Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85%. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4% and Neutral Red 1%). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.

The effects of drugs, ions, and poly-l-lysine on the excretory system of Schistosoma mansoni

We have been able to label the excretory system of cercariae and all forms of schistosomula, immature and adult worms with the highly fluorescent dye resorufin. We have shown that the accumulation of the resorufin into the excretory tubules and collecting ducts of the male adult worm depends on the presence of extracellular calcium and phosphate ions. In the adult male worms, praziquantel (PZQ) prevents this accumulation in RPMI medium and disperses resorufin from tubules which have been prelabelled. Female worms and all other developmental stages are much less affected either by the presence of calcium and phosphate ions, or the disruption caused by PZQ. The male can inhibit the excretory system in paired female. Fluorescent PZQ localises in the posterior gut (intestine) region of the male adult worm, but not in the excretory system, except for the anionic carboxy fluorescein derivative of PZQ, which may be excreted by this route. All stages of the parasite can recover from damage by PZQ treatment in vitro. The excretory system is highly sensitive to damage to the surface membrane and may be involved in vesicle movement and damage repair processes. In vivo the adult parasite does not recover from PZQ treatment, but what is inhibiting recovery is unknown, but likely to be related to immune effector molecules.