Amilton Clair Pinto Seixas Neto

Protection against lethal leptospirosis after vaccination with LipL32 coupled or coadministered with the B subunit of Escherichia coli heat-labile enterotoxin.

ABSTRACT Leptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species of Leptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) of Leptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.

Subunit Approach to Evaluation of the Immune Protective Potential of Leptospiral Antigens

ABSTRACT Leptospirosis is the most widespread zoonosis in the world. Current vaccines are based on whole-cell preparations that cause severe side effects and do not induce satisfactory immunity. In light of the leptospiral genome sequences recently made available, several studies aimed at identification of protective recombinant immunogens have been performed; however, few such immunogens have been identified. The aim of this study was to evaluate 27 recombinant antigens to determine their potential to induce an immune response protective against leptospirosis in the hamster model. Experiments were conducted with groups of female hamsters immunized with individual antigen preparations. Hamsters were then challenged with a lethal dose of Leptospira interrogans. Thirteen antigens induced protective immune responses; however, only recombinant proteins LIC10325 and LIC13059 induced significant protection against mortality. These results have important implications for the development of an efficacious recombinant subunit vaccine against leptospirosis.

Preliminary Characterization of Mus musculus–Derived Pathogenic Strains of Leptospira borgpetersenii Serogroup Ballum in a Hamster Model

Human and animal leptospirosis caused by Leptospira spp. belonging to serogroup Ballum has increased worldwide in the past decade. We report the isolation and serologic and molecular characterization of four L. borgpetersenii serogroup Ballum isolates obtained from Mus musculus, and preliminary virulence studies. These isolates are useful for diagnosis of leptospirosis and for epidemiologic studies of its virulence and pathogenic mechanisms.

Infection with Leptospira kirschneri Serovar Mozdok: First Report from the Southern Hemisphere.

Leptospirosis is a global zoonosis caused by pathogenic Leptospira spp. In this study, we characterized two Leptospira kirschneri serogroup Pomona serovar Mozdok isolates, one obtained from a dog and the other from a patient with severe leptospirosis, 4 years later. Histopathological analysis showed that both isolates caused severe tissue damage when used to infect hamsters. While L. kirschneri serogroup Pomona serovar Mozdok is endemic in animals in Europe, there is only one report of human leptospirosis in the literature. Although strains belonging to L. kirschneri serogroup Pomona have been identified in cases of human leptospirosis in Europe, serovar Mozdok has not yet been implicated. The 4-year interval between isolations and the fact that this is the first report of serovar Mozdok as the causative agent of human leptospirosis in the southern hemisphere, demonstrates its epidemiological importance to public health. Moreover, the presence of serovar Mozdok in Brazil has the potential to affect vaccine and diagnostic test development.

Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37’s ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.

Highly Virulent Leptospira borgpetersenii Strain Characterized in the Hamster Model

Abstract. A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.

LemA and Erp Y-like recombinant proteins from Leptospira interrogans protect hamsters from challenge using AddaVax™ as adjuvant

BACKGROUND Recombinant subunit vaccines have been extensively evaluated as promising alternatives against leptospirosis. Here, we evaluated two proteins in formulations containing the adjuvant AddaVax™ as vaccine candidates for prevention and control of leptospirosis. METHODS Recombinant proteins rErp Y-like and rLemA were characterized by ELISA to assess their ability to bind extracellular matrix (ECM) components and fibrinogen. Groups of eight hamsters were immunized intramuscularly with rErp Y-like or rLemA mixed with a squalene-based adjuvant (AddaVax), and then vaccine efficacy was determined in terms of protection against a lethal challenge. The humoral immune response was determined by ELISA, and the evidence of sub-lethal infection was evaluated by histopathology and kidney culture. RESULTS rLemA protein binds laminin, fibrinogen, and collagen type IV, while rErp Y-like interacts with fibrinogen. Significant protection was achieved for rLemA and rErp Y-like vaccines, which showed 87.5% and 62.5% survivals, respectively. On day 28, the humoral immune response was significantly greater in the vaccine groups as compared to that in the control group, and the response was predominantly based on IgG2/3. The surviving animals showed negative results in culture isolation but presented with tissue lesions in the lungs and kidneys. CONCLUSION Cumulatively, our findings suggest that LemA and Erp Y-like proteins act as adhesins and are able to protect against mortality, but not against tissue lesions. Moreover, AddaVax is a novel adjuvant with potential for improving the immunogenicity of leptospiral vaccines.

LEPTOSPIROSE HUMANA: UMA REVISÃO SOBRE A DOENÇA E OS FATORES DE RISCO ASSOCIADOS À ZONA RURAL

eptospirosis is a zoonosis of worldwide distribution and the transmission occurs through direct or indirect contact with carriers. The infection is caused by bacteria of the genus Leptospira and it is a disease of both humans and animals. Rural populations tend to be closer with animals, both wild and domestic, which increases the chance of contact with the agent. Thus, this study reviews literature regarding leptospirosis in the rural areas of the Rio Grande do Sul state (RS), Brazil, in the last 25 years and infers on the risks associated with farming activities. We demonstrate that rural populations, mainly in the southern and central regions of the state, have more risk of acquiring the disease, when compared to urban dwellers. In RS, activities associated with long work hours and lack of use of individual protection equipment, facilitate the contact with the bacteria. Among domestic animals, dogs and cattle are important for the maintenance and transmission of leptospirosis in these regions. Furthermore, sanitary shortcomings such as lack of proper sanitation and waste disposal favor the presence of rodents near the houses, another possible risk factor. This way, education regarding the disease, and proper use of individual protection equipment, seem to be the most needed measures to reduce human infection in the rural areas of the Rio Grande do Sul state, Brazil.

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Campylobacter coli in Swine Slaughtering Flowchart and Research of cdt Genes

Background: Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Campylobacter spp. isolation from pigs during the slaughter and final products have been reported in several countries, including Brazil. However, very little is known about the sources of contamination in the slaughtering flowchart and how these microorganisms are spread in processing plants. Considering the possibility of the pigs carry Campylobacter spp. since the farm or its products are contaminated in the slaughterhouse, this study had as aim to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line. Materials, Methods & Results: Forty animals of 10 lots, four from each lot, were followed during slaughter. Stool samples were collected from the floor of each enclosure where the pigs were housed on the farm and immediately after stunning on slaughterhouse. Samples from carcass surface were collected after removal of the animals from scrap machine, after evisceration and before the refrigeration chamber. It was also collected surface samples from jowls and samples from the scalding tank water before and after the passage of animals. The swabs containing samples were plated onto Columbia agar supplemented with activated charcoal, oxygen reduction solution and antibiotics supplement, and incubated at 42°C for 48 h under microaerobic conditions. The colonies which presented with a shiny and moist appearance were analyzed by Gram staining for identification of Campylobacter by morphology, and then tested for catalase and oxidase. The Campylobacter isolates were identified for species C. jejuni or C. coli by PCR. Bands profiles were determined by rep-PCR and used to compare the strains. Campylobacter was isolated from 19 (9.5%) of the 200 pig samples analyzed, seven (36.8%) of the rectum, seven (36.8%) after evisceration and five (26.3%) before the refrigeration chamber. Campylobacter was not isolated from jowls and from scalding tank water. All isolates were C. coliand cdtnegative.Persistence of strains originating from the farm and cross contaminations during the slaughtering flowchart was identified by the analysis of the bands profiles obtained by rep-PCR. Discussion: C. coli was the species of Campylobacter present in the swine intestinal tract and in the swine slaughterhouse. The animals, once contaminated, can carry the microorganism during the stages of the slaughtering flowchart. The farm where the animals came from is an important source of contamination during processing, however cross contamination also plays a relevant role. The evisceration was considered the most critical stage, due to the greater number of isolates obtained after this procedure, what emphasize the importance of the hygienic-sanitary management in this stage. Campylobacter spp. can survive, despite not being able to multiply, in foods at refrigeration temperatures (-1 to 5°C) for one to three weeks. Therefore, the high percentage of isolates obtained from the carcass before the refrigeration chamber may represent a problem, since the contamination of the carcasses that enter in this sector can be maintained until the food reaches the consumer. There was no similarity between strains isolated from different lots, indicating that there were no persistence of strains both in the farm and in the slaughterhouse.