Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Serological studies on shistosomiasis mansoni in the Northeast Brasil

Sera from the patients (N = 10) with schistosomiasis mansoni of the hospital of Federal University of Pernambuco, the Schistosoma mansoni egg-positive (N = 51) and -negative (N = 452) inhabitants in Cabo City area, out-patients (N = 37) of the IMIP hospital and Japanese immigrants (N = 127) in Petrolina City area of northeast Brazil as well as Japanese healthy subjects (N = 30) were examined by serological tests including an enzyme-linked immunosorbent assay with antigens prepared from eggs (ELISA-egg) and adult worms (ELISA-adult). The ELISA with egg or adult antigen correctly identified 100% of the uninfected individuals lived in non-endemic area of schistosomiasis. Moreover, when examined cross-reactivity of our ELISA with sera isolated from 78 subjects infected with various intestinal parasitic infections, only one of these sera reacted with the egg and adult antigens. On the examination of 51 sera from the egg-positive subjects, the ELISA-egg revealed the highest sensitivity (98.0%), whereas a large number of false negative reactions of ELISA-adult, Ouchterlony method using adult antigen, circumoval precipitation and immediate intradermal skin test were observed. A low sensitivity of these serologic tests except for ELISA-egg appears to be primarily due to their inability to detect antibody in the sera from egg-positive infantiles. There was no positive correlation between the absorbance values of these two types of ELISA among the sera isolated from ELISA-positive subjects. Rather, by the reactivity of these sera to egg or adult antigen, they could be divided into two subgroups; one reacted more positively with egg antigen and the other with adult antigen. Moreover, it was confirmed that the sera from young subjects (under 20 years old) appear to be highly reactive to the egg antigen than did aged ones. These data suggest that the ELISA with egg antigen, but not with the adult antigen, appears to be useful for the serological survey of schistosomiasis mansoni in the endemic area of northeast Brazil.

Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in the rural sector around Recife, Northeast Brazil

Parasitological examinations were carried out during July to December, 1989, on 485 inhabitants of four villages in São Lourenço da Mata, 25 km northwest of Recife, Pernambuco, Brazil. Approximately 99.6% of the inhabitants were infected with at least one species of intestinal parasites. A high prevalence of Schistosoma mansoni (82.1%), hookworm (80.2%) Trichuris trichiura (69.9%), Ascaris lumbricoides (61.9%) and Entamoeba coli (36.7%) infections were demonstrated. Test tube cultivation revealed that the most common species of hookworm in this region was Necator americanus (88.4%), and also that the prevalence of Strongyloides stercoralis was 5.8%. Three hundred and thirty-four sera were serologically examined for amoebiasis by the gel diffusion precipitation test (GDP) and enzyme-linked immunosorbent assay (ELISA). No positive reaction was observed in all sera as examined by GDP, while 24 sera were positive by ELISA.

Schistosoma mansoni: preclinical studies with 9-acridanone-hydrazones in Cebus monkeys experimentally infected

Derivatives of acridine (9-Acridanone-hydrazones) were tested in Cebus monkeys experimentally infected with Schistosoma mansoni, at the dosages of 50, 25, and 12.5 mg/kg (p.o., single dose). At least, four compounds seemed to be very promising, promoting alterations in the oogram and reducing the worm burden drastically, even at the lowest dose (12.5 mg/kg). No side effects could be detected after drug administration.

Host cell adhesion to Schistosoma mansoni larvae in the peritoneal cavity of naive mice: histological and scanning electron microscopic studies

Cercariae of Schistosoma mansoni inoculated into the peritoneal cavity of naive mice induced host cell adhesion to their surface, but after 90 minutes the number of adherent cells sharply decreased. The cell detachment is progressive and simultaneous to the cercaria-schistosomule transformation. The histological study showed mainly neutrophils in close contact with the larvae. Mononuclear cells and some eosinophils were occasionally seen surrounding the adherent neutrophils. The scanning electron microscopy showed cells displaying twisted microvilli and several microplicae contacting or spreading over the larval surface, and larvae completely surrounded by clusters of cells. These results suggest that the neutrophils recognize molecules on the cercarial surface which induce their spreading

Oogram studies in mice infected with Schistosoma mansoni and treated with dexamethasone

Mice infected with about 90 cercariae of Schistosoma mansoni (LE strain) were treated during five consecutive days with dexamethasone (50 mg/Kg, subcutaneously), starting on the 42th day of infection. Groups of five mice were then daily sacrificed from the first day after onset of treatment until the first day after. The perfusion of the portal system was performed and a piece of the intestine was processed for qualitative and quantitative oograms. This treatment carries to larger numbers of eggs in the tissues of treated mice, when compared with untreated groups. No changes were observed in the kinetics of oviposition, as all stages of viable eggs were observed in the tissues of treated and control mice. These data reinforce the hypothesis of a partial blockade of the egg excretion in immunossupressed mice.

Studies on human anti-rabies immunization in Brazil: I - Evaluation of the 3 + 1 pre-exposure vaccination schedule under field conditions

The currently used pre-exposure anti-rabies immunization schedule in Brazil is the one called 3+1, employing suckling mouse brain vaccine (3 doses on alternate days and the last one on day 30). Although satisfactory results were obtained in well controlled experimental groups using this immunization schedule, in our routine practice, VNA levels lower than 0.5 IU/ml are frequently found. We studied the pre-exposure 3+1 schedule under field conditions in different cities on the State of São Paulo, Brazil, under variable and sometimes adverse circumstances, such as the use of different batches of vaccine with different titers, delivered, stored and administered under local conditions. Fifty out of 256 serum samples (19.5%) showed VNA titers lower than 0.5 IU/ml, but they were not distributed homogeneously among the localities studied. While in some cities the results were completely satisfactory, in others almost 40% did not attain the minimum VNA titer required. The results presented here, considered separately, question our currently used procedures for human pre-exposure anti-rabies immunization. The reasons determining this situation are discussed.

Studies on human anti-rabies immunization in Brazil: II - Preliminary evaluation of the 2-1-1 schedule for human pre-exposure anti-rabies immunization, employing suckling mouse brain vaccine

This study reports preliminary results of virus neutralizing antibody (VNA) titers obtained on different days in the course of human anti-rabies immunization with the 2-1-1 schedule (one dose is given in the right arm and one dose in the left arm at day 0, and one dose is apllied on days 7 and 21), recommended by WHO for post-exposure treatment with cell culture vaccines. A variant schedule (double dose on day zero and another on day 14) was also tested, both employing suckling mouse brain vaccine. A complete seroconversion rate was obtained after only 3 vaccine doses, and almost all patients (11 of 12) presented titers higher than 1.0 IU/ml. Both neutralizing response and seroconversion rates were lower in the group receiving only 3 doses, regardless of the sample collecting day. Although our results are lower than those found with cell culture vaccines, the geometry mean of VNA is fully satisfactory, overcoming the lower limit recommended by WHO of 0.5 IU/ml. The 2-1-1 schedule could be an alternative one for pre exposure immunization, shorter than the classical 3+1 regimen (one dose on days 0, 2, 4 and 30) with only three visits to the doctor, instead of four.

Schistosoma mansoni Sambon, 1907: comparative morphologica studies of some Brazilian strains

The morphology of Schistosoma mansoni adult male worms from three strains which have been maintained in albino mice for several generations, was compared to a strain that has been isolated from the natural host Nectomys squamipes (Rodentia: Muridae) captured in Sumidouro (Rio de Janeiro State) and have been maintained in the same sylvatic rodent under laboratory conditions. Total length of specimens, distance between suckers, the number of testes and extention of testes grouping were the taxonomic characters analysed. The worms recovered from N. squamipes showed expressive differences (p< 0.01) compared to the other strains regarding the considered morphological characters. The strains that were maintained in mice presented statistical differences (p< 0.01) in several characters. Some adult worms besides the normal position of the testes also showed an atypical arrangement of these glands. It can be concluded that the morphology of adult worms may be used to distinguish S. mansoni strains and that morphological changes in adult worms are not induced by successive inoculations of a strain in mice.

Dengue type 2 outbreak in the south of the State of Bahia, Brazil: laboratorial and epidemiological studies

During March 1994 cases of a exanthematic acute disease were reported in the municipalities of Itagemirim, Eunápolis and Belmonte, state of Bahia. Dengue fever was confirmed by serology (MAC-ELISA) and by dengue virus type 2 isolation, genotype Jamaica. Signs and symptoms of classic dengue fever were observed with a high percentual of rash (73.8%) and pruritus (50.5%). Major haemorrhagic manifestations were unfrequent and only bleeding gum was reported. Dengue virus activity spreaded rapidly to important tourism counties like Porto Seguro, Ilhéus, Santa Cruz de Cabrália, Prado, Alcobaça and others, representing a risk for the spreading of dengue virus into the country and abroad.

Studies on prevalence of Strongyloides infection in Holambra and Maceió, Brazil, by the agar plate faecal culture method

Prevalence of Strongyloides stercoralis infection in three areas of Brazil was surveyed by a recently developed faecal culture method (an agar plate culture). The Strongyloides infection was confirmed in 11.3% of 432 subjects examined. The diagnostic efficacy of the agar plate culture was as high as 93.9% compared to only 28.5% and 26.5% by the Harada-Mori filter paper culture and faecal concentration methods, when faecal samples were examined simultaneously by these three methods. Among the 49 positive samples, about 60% were confirmed to be positive only by the agar plate culture. These results indicate that the agar plate culture is a sensitive new tool for the correct diagnosis of chronic Strongyloides infection.

SCHISTOSOMIASIS MANSONI IN THE "BAIXADA OCIDENTAL MARANHENSE", STATE OF MARANHÃO, BRAZIL: CROSS-SECTIONAL STUDIES PERFORMED IN 1987 AND 1993

A cross-sectional study on the prevalence of schistosomiasis mansoni in three sites of the "Baixada Ocidental Maranhense" was carried out in 1993 in: Alegre (in the municipality of São Bento), Aliança (in Cururupu) and Coroatá II (in the municipality of São João Batista). Results were compared to those of another study performed at the same sites and in similar conditions, in 1987. The entire population of the three sites, with few exceptions, was submitted to fecal tests using the Kato-Katz method and immediate intradermal tests for schistosomiasis in both studies. Subjects with positive results in one of these tests were clinically evaluated by a physical examination. In 1993, the total of 827 subjects were submitted to fecal examination and 826 to intradermal test. Schistosoma mansoni eggs were found in the feces of 154 (18.6%) subjects, while 478 (57.9%) subjects presented a positive intradermal test. Stool examination was carried out in 367 subjects in Alegre with a positivity rate of 14.9%; the intradermal test, performed in 366 subjects, was positive in 47.5% of the cases. In Aliança, 277 subjects had their feces examined and were submitted to an intradermal test, with a positivity rate of 34.4% and 70.7%, respectively. Finally in Coroatá II, 183 inhabitants submitted to fecal and intradermal tests had positivity rates of 2.2% and 59.0%, respectively. When the present data were compared to those obtained in the survey performed in 1987, a significant decrease in the prevalence of infection by S. mansoni was observed in Alegre and Coroatá II, and a prevalence increase in Aliança.

PCR with Paracoccidioides brasiliensis specific primers: potential use in ecological studies

The precise microenvironment of Paracoccidioides brasiliensis has not yet been discovered perhaps because the methods used are not sensitive enough. We applied to this purpose the polymerase chain reaction (PCR) using three sets of specific primers corresponding to two P. brasiliensis genes. This fungus as well as several other fungi, were grown and their DNA obtained by mechanical disruption and a phenol chloroform isoamylalcohol-based purification method. The DNA served for a PCR reaction that employed specific primers from two P. brasiliensis genes that codify for antigenic proteins, namely, the 27 kDa and the 43 kDa. The lowest detection range for the 27 kDa gene was 3 pg. The amplification for both genes was positive only with DNA from P. brasiliensis; additionally, the mRNA for the 27 kDa gene was present only in P. brasiliensis, as indicated by the Northern analysis. The standardization of PCR technology permitted the amplification of P. brasiliensis DNA in artificially contaminated soils and in tissues of armadillos naturally infected with the fungus. These results indicate that PCR technology could play an important role in the search for P. brasiliensis’ habitat and could also be used in other ecological studies.