In-vitro antileptospiral activity of Canarium odontophyllum Miq. (Dabai) leaves extract

Aims@#To evaluate the anti-leptospiral activity of Canarium odontophyllum leaves against Leptospira interrogans serovar Bataviae and Leptospira borgpetersenii serovar Javanica. @*Methodology and results@#The extracts (hexane, acetone, methanol and aqueous) used in this study were tested at concentration ranging from 0.049 mg/mL to 25 mg/mL using broth microdilution method. Percentage inhibition (%) was obtained through OD reading at 400 nm. Only methanol extract was incubated with Leptospira to observe population changes under dark field microscope prior to subjected for DNA damaging studies through gel electrophoresis of genomic DNA. Methanol extract showed the highest percentage inhibition of 66% against L.interrogans serovar Bataviae and 74% against L. borgpetersenii serovar Javanica. The IC50 value of methanol extract was 4.60 mg/mL and 2.25 mg/mL against serovar Bataviae and serovar Javanica, respectively. Both Leptospira culture which was treated with IC50 value of methanol extract showed drastic decrease in population compared to untreated Leptospira for both serovar. There was no DNA damage towards serovar Bataviae. However, serovar Javanica exhibited DNA damage as observed from the presence of DNA fragmentation on the gel electrophoresis. @*Conclusion, significance and impact of study@#These findings confirmed that methanol leaves extract from of Canarium odontophyllum has a potential to control leptospirosis.

Using a top predator as a sentinel for environmental contamination with pathogenic bacteria: the Iberian wolf and leptospires

The Iberian wolf (Canis lupus) is the top predator in the Iberian environments in which it lives, feeding on a wide range of species, thus encountering a wide range of disease agents. Therefore, the wolf can serve as sentinel of environmental contamination with pathogens. We investigated the exposure of free-living wolves to 14 serovars of Leptospira interrogans sensu lato. Kidney samples from 49 wolves collected from 2010-2013 in northwestern Spain were analysed by culture, direct immunofluorescence and polymerase chain reaction. Tissue fluids were analysed for antibodies by a microscopic agglutination test. Ten wolves (observed prevalence: 20%, 95% confidence interval = 11-33%) showed evidence of contact with leptospires, eight through direct detection and nine through serology (7 wolves were positive according to both techniques). Titres below the cut-off level were also detected in seven cases. Serovars confirmed were Canicola (n = 4), Icterohaemorrhagiae (n = 3) and Sejroë, Ballum and Grippotyphosa (n = 1 each), indicating that wolves were infected with serovars for which dogs, rodents and ungulates, are the natural hosts and supporting the utility of the wolf and other large predators as environmental sentinels for pathogens.

Aislamiento de Leptospira borgpetersenii de fuentes de agua en Argentina / Isolation of Leptospira borgpetersenii in water sources in Argentina

Introducción: las especies del género Leptospira son los agentes causales de la leptospirosis, enfermedad considerada como la zoonosis de mayor distribución en el mundo.En Argentina reviste carácter endémico. La provincia de Santa Fe registra el mayor número de casos humanos. Desde mediados de la década de 1980, las especies patógenas de leptospiras aisladas de animales y humanos fueron diferenciadas sobre la base de estudios de hibridización ADN-ADN, surgiendo nuevas especies: L. interrogans, L. kirschneri, L. weilii, L. noguchii, L. borgpetersenii, L. santarosai, L. meyeri, L. inadai, L. faineri y L. alexanderi. Objetivo: aislar y caracterizar mediante métodos moleculares, leptospiras de fuentes de agua que vierten en un canal que atraviesa la ciudad de Casilda, Santa Fe, en Argentina. Métodos: se sembraron 6 muestras de agua de las vertientes al canal, previa filtración con filtros Millipore de 0,22 µm, en medios EMJH y Fletcher para aislamiento de leptospiras. Se incubaron en estufa a 30 °C durante 15 días, se observaron semanalmente mediante microscopia de campo oscuro. Se implementó la técnica de reacción en cadena de la polimerasa bajo las condiciones específicas (Sugathan, 2005), con dos juegos de cebadores (Gravekamp, 1993), que permiten detectar la presencia de ADN de leptospiras patógenas. La técnica molecular utilizada para genotipificar fue multiple-locus variable-number tandem repeats analysis (MLVA). Resultados: se obtuvieron 5 aislamientos de Leptospira spp., de los cuales 2 resultaron positivos a la reacción en cadena de la polimerasa, determinando que se trataba de leptospiras patógenas. Mediante la genotipificación por MLVA se pudo observar que uno de los aislamientos patógenos mostró un patrón correspondiente a la especie Leptospira borgpetersenii, no siendo identificable la otra cepa. Conclusiones: en la ciudad del estudio, que tiene alrededor de 40 000 habitantes, se logró identificar por primera vez una cepa de Leptospira borgpetersenii de fuentes de aguas urbanas, con el peligro potencial que esto representa para la población humana y animal.

Introduction: the Leptospira genus species are causative agents of leptospirosis, a disease that is considered the most widely spread zoonotic disease worldwide. In Argentina, leptospirosis is endemic and Santa Fe province has the highest number of human cases. Since mid-1980's, the pathogenic leptospira species isolated from animals and humans have been differenciated through DNA-DNA hybridization tests, resulting in new species: L. interrogans, L. kirschneri, L. weilii, L. noguchii, L. borgpetersenii, L. santarosai, L. meyeri, L. inadai, L. faineri y L. alexanderi. Objectives: to isolate and to characterize by molecular test leptospiras from water poured into a channel that runs through Casilda City in Santa Fe Province, Argentina. Methods: six samples of water from the channel were cultured after having been filtered through 0.22 µm, Millpore filtres in EMJH and Fletcher media to isolate leptospires. They were incubated at 30 °C for 15 days, and weekly observed through dark field microscopy. Polymerase chain reaction assay was used under specific conditions (Sugathan, 2005), with two sets of primers (Gravekamp, 1993), to determine whether the isolates were pathogenic. The molecular technique for genotyping was Multiple-Locus Variable-number tandem repeats Analysis (MLVA). Results: five Leptospira spp. isolates were obtained of which 2 were positive to PCR, all of which determined that they were pathogenic leptospiras. MLVA genotyping allowed the observation of a pattern similar to that of L. borgpetersenii species in one pathogenic isolates, but the other isolate was not identified. Conclusions: in the City where the study was carried out, with a population of about 40,000 inhabitants, a L. borgpetersenii species was identified for the first time in urban water sources, with the potential risk that it may pose for human and animal populations.

Multiple-locus variable-number tandem repeat analysis of reference strains used for the diagnosis of leptospirosis in Argentina / Multiple-locus variable-number tandem repeat analysis de cepas de referencia usadas para el diagnóstico de leptospirosis en Argentina

Leptospirosis is a worldwide zoonosis caused by a spirochete that belongs to the genus Leptospira. In the last years, new methods, such as the PCR-based multiple-locus variable-number tandem repeat analysis (MLVA), have been developed for the genotyping of leptospires. In the present work, the MLVA patterns for all reference strains used in Argentina for bovine, ovine, porcine, equine, caprine and canine leptospirosis diagnosis, as well as in human and wild animal diagnosis, were obtained. MLVA results are presented in such a way that they can be readily used for the identifcation of these strains by the simple and direct comparison of agarose gels. Making the use and interpretation of the MLVA for leptospires typing easier will help increase the use of this method as a routine procedure for human and animal diagnosis, for epidemiological studies, vaccine control and other applications.

La leptospirosis es una zoonosis de distribución global causada por una espiroqueta perteneciente al género Leptospira. En los últimos años se han desarrollado nuevos métodos para la genotipifcación de las leptospiras, entre ellos el denominado multiple-locus variable-number tandem repeat analysis (MLVA). En este trabajo se obtuvieron los patrones de MLVA de todas las cepas de referencia utilizadas en la Argentina para el diagnóstico de leptospirosis en bovinos, ovinos, porcinos, equinos, caprinos y perros, y que también son utilizadas en el diagnóstico de leptospirosis en humanos y en animales salvajes. Los resultados del MLVA se muestran de manera tal que pueden ser fácilmente utilizados para la identifcación de estas cepas por simple comparación visual de geles de agarosa. Al facilitar el uso y la interpretación del MLVA para la tipifcación de leptospiras, se ayudará a difundir la utilización rutinaria de este método en el diagnóstico humano y animal, en estudios epidemiológicos y para el control de vacunas, entre otras aplicaciones.

Characterization of Leptospira borgpetersenii isolates from field rats (Rattus norvegicus) by 16S rRNA and lipL32 gene sequencing

The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 percent, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.