Development and standardization of the Dot Blot test for serological diagnosis of bovine leptospirosis

Leptospirosis a public health problem and an endemic zoonosis in Brazil, is diagnosed by serological methods. Therefore, low-cost and easy to execute methodologies with good/high sensitivity, such as Dot Blot, are an important diagnostic tool. The aim of this study was to standardize and validation the dot-blot technique for the serological diagnosis of bovine leptospirosis. Several concentrations of antigens applied to nitrocellulose membranes, and different dilutions of conjugated bovine serum were evaluated to develop and standardize the test. The best distinction/contrast between positive and negative samples was observed for 1μL antigen (0.11μg/μL outer membrane protein of the Hardjo serovar (OMPH) and 0.08 μg/μL outer membrane protein of the Wolffi serovar (OMPW)), 1:500 and 1:10000 bovine serum dilution and conjugate, respectively. The Dot Blot test efficiency was 71.87% and kappa index, 0.46 (p<0.0001). The other parameters measured were: sensitivity 91.89%; specificity 59.32%; positive predictive value 58.62%; and, negative predictive value 59.32%. In addition to high sensitivity, other advantages of the Dot Blot technique have been identified, such as practicality, low cost since it does not require sophisticated devices and the fact that the Hardjo and Wolffi OMP also reacted with serovars from other pathogenic serogroups. The results provided positive expectations for the use of Dot Blot as support in the diagnosis of bovine leptospirosis, especially if used as a screening test, stimulating further research for the future development of kits for diagnostic purposes.

Spatial epidemiology of Leptospira sp. exposure in bovines from Veracruz, México

Bovine leptospirosis is a bacterial disease that affects cattle herds, causing economic losses due to reproductive problems, which require expensive treatments. The main source of transmission for cattle is still uncertain, but it has been described that rodents and bats can play an important role in the transmission cycle by being maintenance hosts for the pathogenic species of the bacterium and spreading it through urine. In this study, we characterize possible risk areas for bovine leptospirosis exposure in the state of Veracruz, Mexico, based on the geographical distribution of flying (bats) and terrestrial (rodents and opossums) wild hosts of Leptospira sp. reported in Mexico in addition with climatic, geographic, soil characteristics, land use and human activities variables (environmental variables). We used a generalized linear regression model (GLM) to understand the association between the frequency of anti- Leptospira sp. antibodies (a proxy of exposure to) in cattle herds exposed to Leptospira, the favorability of wild hosts of Leptospira as well as the environmental variables. The parameterized model explains 12.3% of the variance. The frequency of anti- Leptospira sp. antibodies exposoure in cattle herds was associated with elevation, geographic longitude, pH of the soil surface and environmental favorability for the presence of rodents, opossums, and bats. The variation in exposure is mainly explained by a longitudinal gradient (6.4% of the variance) and the favorability-based indices for wild hosts (9.6 % of the variance). Describing the possible risks for exposure to Leptospira in an important and neglected livestock geographical region, we provide valuable information to the selection of areas for diagnosis and prevention of this relevant disease.

Prototype Pentavalent Bovine Leptospira Vaccines Blended with Oil Adjuvant Provides Protection and Prevents Renal Colonization in Guinea Pig Model

Background: Leptospirosis significantly impacts the economy of livestock farmers by causing reproductive failure and production losses in bovines. The control of bovine leptospirosis requires a combination of vaccination programme, biosecurity measures and chemotherapeutic regimens. Vaccination protect the clinical diseases and reduces/prevents renal colonisation of Leptospires and in turn reduces excretion of leptospires in urine which is a major source of environmental contamination. Some serovars of leptospires require cell mediated immunity apart from humoral immunity. This study was undertaken to develop prototype vaccine with prevalent serogroups to induce humoral and cell mediated immunity. Immunogenicity of the prototype vaccines and their safety were assessed experimentally in Guinea pigs.Methods: An inactivated oil adjuvant prototype pentavalent bovine leptospira vaccines with serogroups Australis, Hebdomadis, Hardjo, Javanica and Pomona which were prevalent in Tamil Nadu were developed and compared with the aluminium hydroxide gel adjuvant vaccine. The humoral immune response was assessed measuring antibodies by Microscopic Agglutination Test and Cell Mediated Immune response was assessed by lymphocyte proliferation assay. The ability of these vaccines to protect Guinea pigs against virulent Leptospires challenge was also demonstrated.Result: The prototype vaccine blended with oil adjuvants resulted in higher protective antibody titres of more than 6.64 log2 (≥1:100) than the aluminium hydroxide adjuvant blended vaccine. The protective antibody titres lasted upto 180 days, except for serogroup Javanica (150 days). The Montanide adjuvants were able to produce cell mediated immune response for protection against serovar Hardjo. Challenge study in guinea pigs showed complete protection following vaccination using pentavalent vaccine blended with Montanide adjuvants, while the aluminium hydroxide adjuvant was able to confer only partial protection. The Montanide blended prototype vaccines were also able to prevent renal colonisation of all five serogroups. This study shows the potential of the oil adjuvant blended multivalent vaccine for use in vaccination programmes against bovine leptospirosis.

Laboratory Diagnosis of Bovine Abortions Caused by Non-Maintenance Pathogenic Leptospira spp.: Necropsy, Serology and Molecular Study Out of a Belgian Experience

Bovine leptospirosis is a bacterial zoonotic disease caused by pathogenic Leptospira spp. The pathology and epidemiology of this infection are influenced by the numerous existing serovars and their adaptation to specific hosts. Infections by host-maintained serovars such as Hardjo are well documented, unlike those from the incidental ones. In July 2014, an emerging phenomenon of an increased incidence of icteric abortions associated with leptospiral infection occurred in southern Belgium. First-line serological analyses targeting cattle-adapted serovars failed at initial diagnosis. This study provides a comprehensive description of laboratory findings—at the level of necropsy, serology and molecular diagnosis—regarding icteric and non-icteric abortions (n = 116) recorded during this time (years 2014–2015) and associated with incidental infection by serovars such as Grippotyphosa, Australis and Icterohaemorrhagiae. Based on these tests, a diagnostic pathway is proposed for these types of infection in cattle to establish an affordable but accurate diagnosis in the future. These investigations add insights into the understanding of the pathogenesis of bovine leptospirosis associated with serovars classically described as non-maintenance.