Claudiomar Soares Brod

Characterization of virulence of Leptospira isolates in a hamster model

Effort has been made to identify protective antigens in order to develop a recombinant vaccine against leptospirosis. Several attempts failed to conclusively demonstrate efficacy of vaccine candidates due to the lack of an appropriate model of lethal leptospirosis. The purposes of our study were: (i) to test the virulence of leptospiral isolates from Brazil, which are representative of important serogroups that cause disease in humans and animals; and (ii) to standardize the lethal dose 50% (LD(50)) for each of the virulent strains using a hamster (Mesocricetus auratus) model. Five of seven Brazilian isolates induced lethality in a hamster model, with inocula lower than 200 leptospires. Histopathological examination of infected animals showed typical lesions found in both natural and experimental leptospirosis. Results described here demonstrated the potential use of Brazilian isolates as highly virulent strains in challenge experiments using hamster as an appropriate animal model for leptospirosis. Furthermore these strains may be useful in heterologous challenge studies which aim to evaluate cross-protective responses induced by sub-unit vaccine candidates.

Shiga toxin-producing Escherichia coli (STEC) isolated from healthy dairy cattle in southern Brazil

Over a period of 1 year, the production of verotoxin was investigated in 1127 Escherichia coli isolated from 243 dairy cattle from 60 small farms in southern Brazil. Vero cell assay was used to detect toxins in culture supernatants from E. coli isolated from bovine feces. Shiga toxin-producing E. coli (STEC) detection rates were 95% (57 of 60) for farms and 49% (119 of 243) for cattle. Prevalence of STEC-positive cattle in the farms ranged from 0 to 100%. Ninety-six percent (315 of 327) of the STEC isolates did not react in the panel of sera used for typing. Twelve isolates, all non-motile, belonged to serogroups previously associated with human diseases, and 67% (8 of 12) were of only two serotypes (O91:H- and sorbitol-fermenting O157:H-). These results indicate that dairy cattle from the region surveyed may be a source of STEC potentially pathogenic for humans.

Condições higiênico-sanitárias no comércio ambulante de alimentos em Pelotas-RS

Street-vending of ready-to-eat (RTE) foods can be a risk to the consumers health, since people usually involved in this activity does not have proper knowledge in safe handling of foods. Despite this, only a few studies have been made on the microbiological quality of these foods and places where they are prepared. In this paper we report on the higienic-sanitary quality of hot-dogs sold by street-vendors from Pelotas,RS. Samples of hot-dogs, water and work surfaces, were collected from 60 street-vending places and taken to the laboratory for analysis. Counts of aerobic mesophilic bacteria (TPC), Staphylococcus coagulase positive (SCP), total coliforms (TC) and coliforms at 45oC (FC) were made on samples of hot-dogs. SPC, TC and FC counts were made on the water and surface samples. Among the 60 samples of hot-dogs 53%, 48%, 37% and 25% were found unsatisfactory for TC, TPC, STA and FC, respectively. Only 3 (5%) water samples were found unsatisfactory according to the TPC standard used, and 27% and 23% did not read the standards for TC and FC. Regarding the surfaces, 70% were found unsatisfactory for TPC, 68% for TC and 67% for FC. Salmonella sp was not detected in any of the samples tested. These results suggest that the hygiene practices of many food street-vending places are not adequate, resulting in a high proportion of read-to-eat RTE) foods with microbiological quality unsatisfactory for consumption.

Seroprevalence of Toxocara Infection in Children from Southern Brazil

Abstract: The seroprevalence of Toxocara canis antibodies in children aged from 1 to 12 yr old was evaluated in Pelotas City, Rio Grande do Sul, Brazil. Human toxocariasis or visceral larva migrans (VLM) was diagnosed with the use of an ELISA based on the T. canis excretory–secretory (TES) antigens; Western blotting was used to confirm the ELISA-positive results. From 427 samples, 50.6% were positive for the presence of anti-TES antibodies. A confirmatory test (Western blot) was carried out on a sample of the ELISA-positive sera (n = 70), and all were positive. The Western blots had specific banding pattern characteristics, where the 30-kDa fraction demonstrated the highest reactivity. This fraction could be important for the specific diagnosis of toxocariasis.

Evidência do cão como reservatório da leptospirose humana: isolamento de um sorovar, caracterização molecular e utilização em inquérito sorológico

Canine leptospirosis has been known as Stuttgart disease since 1898, and dogs are considered to be the second principal source of infection in man. The isolation of a pathogenic serovar from dog urine that was diagnosed clinically and laboratorial as having leptospirosis and its utilization to test serological samples of human and canine cases of leptospirosis, has demonstrated its importance to the ecosystem of the southern region of Brazil. The results of the serological microscopic agglutination test indicated that 100% of human serum samples from 12 patients from the serum bank of 2001 at the Center for Control of Zoonoses, that had titers between 25 and 3,200 with the canicola serovar, and 72% of 105 canine serum samples from the same serum bank, also reacted with the new isolate. The mean and median titers of the human serum samples tested with the battery of antigens recommended by WHO was 630 and 100 respectively, and when tested with the isolate it was 1,823 and 400. In the dog sera, the values were respectively 347 and 100 with the battery, and 1,088 and 200 with the isolate.

Leptospira noguchii and human and animal leptospirosis, Southern Brazil.

To the Editor: Pathogenic leptospires, the causative agents of leptospirosis, exhibit wide phenotypic and genotypic variations. They are currently classified into 17 species and >200 serovars (1,2). Most reported cases of leptospirosis in Brazil are of urban origin and caused by Leptospira interrogans (3). Brazil underwent a dramatic demographic transformation due to uncontrolled growth of urban centers during the last 60 years. Urban slums are sites of poor sanitation that favors rat-borne transmission of leptospirosis among humans. Thus, this may explain the major involvement of serovar Copenhageni (L. interrogans). The predominance of L. interrogans is likely due to the underestimation of rural cases of leptospirosis.

Aglutininas anti-leptospíricas em cães na área de influência do centro de controle de zoonoses, Pelotas, RS, Brasil, no ano de 1995

During the year of 1995 we examined 425 serum samples from dogs in the area of influence of the Center for Zoonosis Control, performing the microscopic agglutination test for diagnosis of leptospirosis. Of these, 148 (34.8%) gave positive reactions with titers equal or higher than 1:100, for the following serovars: L. canicola (58.1%), L. icterohaemorrhagiae (20.9%), L. copenhageni (11.4%), L. grippotiphosa and L. castellonis (2.7%) L. andamana and L. autumnalis and L. pyrogenes with (1.4%). The prevalence of infection was higher in March, August, September, October and November, which coincided, with the higher temperatures and pluvial precipitation observed the month before the outbreaks. This observation stress the importance of these two factors for the survival of leptospira in the environment.

Leptospira borgpetersenii from free-living white-eared opossum (Didelphis albiventris): first isolation in Brazil.

Leptospirosis is a zoonotic disease that occurs all over the world, caused by bacteria of the genus Leptospira. Marsupial and didelphidae families are considered susceptible to infection caused by a wide range of Leptospira serovars for which they serve as reservoirs. Thirty-three free-living white-eared opossums (Didelphis albiventris) were captured in Southern Brazil and bodily fluids were collected. From the urine samples it was possible to obtain an isolate identified as Leptospira borgpetersenii by rpoB gene sequencing and belonging to serovar Castellonis by Multilocus Variable-Number Tandem-Repeat Analysis. This is the first report of the isolation of Leptospira spp. from the white-eared opossum in Brazil. In addition, the new strain was also virulent in the hamster model of lethal leptospirosis. The microscopic agglutination test (MAT) was used for detecting the presence of antibodies against Leptospira spp. in white-eared opossum, human, cattle and canine sera using a panel of 59 Leptospira strains that included the new isolate. The inclusion of the new strain in the MAT battery increased the MAT sensitivity for canine sera. These findings suggest that the white-eared opossum is an important reservoir of pathogenic Leptospira spp.

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outermembrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/cmice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT mediumand screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM.MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cellantigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and non-pathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted withnon-pathogenic Leptospira. The MAbs described have potential for use in diagnostic tests for leptospirosis.

Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool

Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.