Walter Lilenbaum

Biofilm formation by saprophytic and pathogenic leptospires

Leptospires exist as saprophytic organisms that are aquatic or as pathogens that are able to survive in water. Leptospirosis is transmitted to humans through environmental surface waters contaminated by the urine of mammals, usually rodents, which are chronically infected by pathogenic strains. The ecology of Leptospira spp. prompted us to evaluate if these spirochaetes were able to form biofilms. This study investigated the characteristics of biofilm development by both saprophytic and pathogenic Leptospira species using microscopic examinations and a polystyrene plate model. Biofilms were formed preferentially on glass and polystyrene surfaces. Electron microscopic images showed cells embedded in an extracellular matrix. The formation of such a biofilm is consistent with the life of saprophytic strains in water and may help pathogenic strains to survive in environmental habitats and to colonize the host.

Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres > or =400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants.

Factors associated with bovine leptospirosis in Rio de Janeiro, Brazil

Serum samples from 379 adult cows with lowered fertility farmed in the Rio de Janeiro region of Brazil. They were examined for Leptospira antibodies. A questionnaire was completed for each herd. Antibodies were detected in 46.9% of these cows, mainly to serovar hardjo. Main risk factor associated with seropositivity was co-grazing with other species, mainly pigs (OR = 3.17; p<0.04). Absence of or infrequent veterinary assistance (OR = 1.74) has also been suggested to be associated to the overall seroprevalence to leptospirosis. We suggest that a successful control programme for bovine leptospirosis should include a complete investigation of herd management practices, which could influence in the occurrence of the infection.

Potential application of new diagnostic methods for controlling bovine Tuberculosis in Brazil

Bovine tuberculosis, a chronic infection in cattle caused by Mycobacterium bovis, remains an economic and public health problem for several countries. Due to its economic impact on international trade, contagious nature, and implications for human health, global programs to eradicate the disease were implemented worldwide. Those programs are based on slaughtering PPD-reactive animals. Despite the National Programs in Brazil, complete eradication has not been achieved, and the disease remains, albeit at a lower prevalence. The central purpose of this review is to address diagnostic tests for tuberculosis. Considering the course of the infection in cattle, at least two tests, ideally complementary to one another, may be necessary for an adequate diagnosis: the first based on the cellular response, and the second capable of identifying anergic animals by detection of specific anti-M.bovis antibodies.

Identification of Mycobacterium bovis Isolates by a multiplex PCR

Isolates from suggestive bovine tuberculosis lesions were tested by a multiplex polymerase chain reaction (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, specific for M. bovis and Mycobacterium tuberculosis complex respectively. The m-PCR successfully identified as M. bovis 88.24% of the isolates.

Leptospirosis antibodies in mammals from Rio de Janeiro Zoo, Brazil.

Serum samples from 77 animals belonging to 38 species and 19 families in Rio de Janeiro Zoo, Brazil were tested for antibodies against serovars of Leptospira interrogans by microscopic agglutination test. Antibodies were detected in serum samples of 37.7% of all animals belonging to 10 families. Seropositivity was more common in the Carnivora Canidae (7/9), Procyonidae (5/9) and the Edentata Myrmecophagidae (5/9). Icterohaemorrhagiae was the most common serogroup. Seropositivity was less common in primates.

Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro, Brazil.

Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.

The role of horses in the transmission of leptospirosis in an urban tropical area.

The objective of this study was to demonstrate the presence of leptospires in equine urine, as evidence for a potential role of horses in transmission of this organism. Thoroughbred horses (aged 2-5 years, n = 276) from Rio de Janeiro, Brazil, were studied. After a severe storm, the premises of the animals remained flooded for 72 h. Blood samples for serology were collected on days 20 and 35 (day of storm = day 0). On day 20, 132 (47·8%) horses were seroreactive (titre ≥200) and, of these, 23 (31·0%) had increased antibody titres on day 35. Furthermore, 34 urine samples (for PCR and culture) were collected from seroreactive horses on day 35. Copenhageni was the most frequent serovar (88·8% of reactive titres). Although none of the urine samples were culture positive, 12 (35·2%) were PCR positive. This is apparently the first report of evidence of leptospires in urban horses. Furthermore, we suggest that these animals can play a role in the transmission of leptospirosis in urban areas.

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.

Serology and PCR for bovine leptospirosis: herd and individual approaches

LEPTOSPIROSIS is an important zoonotic bacterial infection of livestock that causes abortions, stillbirths, infertility and reduction of milk production, determining economic hazards. Cattle act as the maintenance host of serovar Hardjo (serogroup serjoe), which is the most common strain of Leptospira in many rural regions (Hernandez-Rodriguez and others 2011). The standard serological diagnosis of leptospirosis is the microscopic agglutination test (MAT), which is recommended for use on a herd-screening basis (Lilenbaum and others 2009). Since leptospiral cultivation is laborious and time-consuming, other methods have been applied for the direct identification of leptospires (Marianelli and others 2007). PCR is increasingly being reported as a rapid and definitive diagnostic tool in many species, such as buffaloes (Marianelli and others 2007), small ruminants (Lilenbaum and others 2009) and cattle (Hernandez-Rodriguez and others 2011). The aim of the present study was to compare the use of MAT and PCR for the diagnosis of leptospirosis in cattle. Three dairy herds (A, B and C) from Rio de Janeiro, Brazil, with high levels of reproductive failure, …