Alan J. A. McBride

Carriage of Leptospira interrogans among domestic rats from an urban setting highly endemic for leptospirosis in Brazil

A survey was conducted to identify reservoirs for urban leptospirosis in the city of Salvador, Brazil. Sampling protocols were performed in the vicinity of households of severe leptospirosis cases identified during active hospital-based surveillance. Among a total of 142 captured Rattus norvegicus (Norwegian brown rat), 80.3% had a positive culture isolate from urine or kidney specimens and 68.1% had a positive serum sample by microscopic agglutination test (MAT) titre of > or = 1:100. Monoclonal antibody-based typing of isolates identified that the agent carried by rats was Leptospira interrogans serovar Copenhageni, which was the same serovar isolated from patients during hospital-based surveillance. Leptospira spp. were not isolated from 8 captured Didelphis marsupialis (Opossum), while 5/7 had a positive MAT titre against a saprophytic serogroup. R. rattus were not captured during the survey. The study findings indicate that the brown rat is a major rodent reservoir for leptospirosis in this urban setting. Furthermore, the high carriage rates of L. interrogans serovar Copenhageni in captured rats suggest that there is a significant degree of environmental contamination with this agent in the household environment of high risk areas, which in turn is a cause of transmission during urban epidemics.

Genetic diversity of the Leptospiral immunoglobulin-like (Lig) genes in pathogenic Leptospira spp.

Recent serologic, immunoprotection, and pathogenesis studies identified the Lig proteins as key virulence determinants in interactions of leptospiral pathogens with the mammalian host. We examined the sequence variation and recombination patterns of ligA, ligB, and ligC among 10 pathogenic strains from five Leptospira species. All strains were found to have intact ligB genes and genetic drift accounting for most of the ligB genetic diversity observed. The ligA gene was found exclusively in L. interrogans and L. kirschneri strains, and was created from ligB by a two-step partial gene duplication process. The aminoterminal domain of LigB and the LigA paralog were essentially identical (98.5+/-0.8% mean identity) in strains with both genes. Like ligB, ligC gene variation also followed phylogenetic patterns, suggesting an early gene duplication event. However, ligC is a pseudogene in several strains, suggesting that LigC is not essential for virulence. Two ligB genes and one ligC gene had mosaic compositions and evidence for recombination events between related Leptospira species was also found for some ligA genes. In conclusion, the results presented here indicate that Lig diversity has important ramifications for the selection of Lig polypeptides for use in diagnosis and as vaccine candidates. This sequence information will aid the identification of highly conserved regions within the Lig proteins and improve upon the performance characteristics of the Lig proteins in diagnostic assays and in subunit vaccine formulations with the potential to confer heterologous protection.

Recombinant vaccines against Leptospirosis

Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine.

Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates

The family of leptospiral immunoglobulin-like (lig) genes comprises ligA, ligB and ligC. This study used PCR to demonstrate the presence of lig genes among serovars from a collection of leptospiral strains and clinical isolates. Whilst ligA and ligC appeared to be present in a limited number of pathogenic serovars, the ligB gene was distributed ubiquitously among all pathogenic strains. None of the lig genes were detected among intermediate or saprophytic Leptospira species. It was also shown that, similar to the previously characterized secY gene, a short specific PCR fragment of ligB could be used to correctly identify pathogenic Leptospira species. These findings demonstrate that ligB is widely present among pathogenic strains and may be useful for their reliable identification and classification.

Evaluation of Four Whole-Cell Leptospira-Based Serological Tests for Diagnosis of Urban Leptospirosis

ABSTRACT Four serologic assays for leptospirosis had sensitivities of 72 to 88% and specificities of 88 to 100% in the setting of highly endemic urban transmission, indicating that assays using enzyme-linked immunosorbency and rapid formats may be used as alternatives to the microscopic agglutination test for diagnosing urban leptospirosis. Testing a second sample will be required in cases with an initial negative result, since sensitivity was low (46 to 68%) during the first week of illness.

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.

Different outcomes of experimental leptospiral infection in mouse strains with distinct genotypes.

The mouse disease model has the advantage of a broad array of immunological and genetic tools available for basic research. Some studies on transgenic and/or mutant mouse strains as models for experimental leptospirosis have been reported; however, the wider use of such models is hampered by a poor understanding of the outcome of experimental leptospiral infection among the different mouse strains available. Here, the outcome of infection by a virulent strain of Leptospira interrogans serogroup Icterohaemorrhagiae strain Cop was studied in four commonly used wild-type mouse strains: A, CBA, BALB/c and C57BL/6. The end points evaluated in this study were survival, presence of kidney lesions, leptospiral load in kidney samples, microscopic agglutination test titre and anti-leptospiral IgG antibody levels. As expected, none of the mouse strains were susceptible to lethal leptospirosis. However, these strains developed specific pathologies associated with sublethal leptospirosis. The A and C57BL/6 strains exhibited a high leptospiral load in kidney samples and the CBA and C57BL/6 strains developed severe inflammatory lesions, whilst the BALB/c strain proved to be the most resistant to subclinical leptospirosis.

Monitoring Leptospira strain collections: the need for quality control

The purpose of this study was to perform a 16S sequence-based quality control of two Leptospira strain collections. 16S rRNA gene sequencing was used to verify two Leptospira reference collections provided by the World Health Organization and maintained at a reference laboratory for leptospirosis in Brazil. Among the 89 serovars evaluated, four conflicting strains were identified in one of the collections. Although 16S rRNA gene sequencing cannot identify Leptospira beyond the species level, it is suitable for the identification of contamination and quality control of leptospiral reference collections. This study highlights the importance of the availability of high-quality 16S rRNA sequences in public databases. In addition, it emphasizes the need for periodical verifications and quality control of Leptospira reference collections.

Characterization of the Immunogenic and Antigenic Potential of Putative Lipoproteins from Leptospira interrogans

The search for a vaccine capable of conferring heterologous protection, through the identification of conserved and cross-protective antigens, remains an ongoing priority in leptospirosis research. In the present study, an in silico analysis was used to identify potentially protective lipoproteins from Leptospira interrogans serovar Copenhageni. Eight putative lipoproteins were selected (LIC10009, LIC10054, LIC10091, LIC11058, LIC11567, LIC13059, LIC13305, and LIC20172), cloned and expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins were used to inoculate mice and the subsequent humoral immune response was evaluated by ELISA. Seven of the potential lipoproteins induced a significant IgG response. Furthermore, all of the recombinant proteins were recognized by antibodies present in the sera of severe leptospirosis patients. These putative lipoproteins exhibited potential for further evaluation as prospective vaccine candidates.

An Imprint method for detecting leptospires in the hamster model of vaccine-mediated immunity for leptospirosis

In determining the efficacy of new vaccine candidates for leptospirosis, the primary end point is death and an important secondary end point is sterilizing immunity. However, evaluation of this end point is often hampered by the time-consuming demands and complexity of methods such as culture isolation (CI). In this study, we evaluated the use of an imprint (or touch preparation) method (IM) in detecting the presence of leptospires in tissues of hamsters infected with Leptospira interrogans serovar Copenhageni. In a dissemination study, compared to CI, the IM led to equal or improved detection of leptospires in kidney, liver, lung and blood samples collected post-infection and overall concordance was good (kappa=0.61). Furthermore, in an evaluation of hamsters immunized with a recombinant leptospiral protein-based vaccine candidate and subsequently challenged, the agreement between the CI and IM was very good (kappa=0.84). These findings indicate that the IM is a rapid method for the direct observation of Leptospira spp. that can be readily applied to evaluating infection in experimental animals and determining sterilizing immunity when screening potential vaccine candidates.