LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.

In vivo cell aggregations of a recent swine biofilm-forming isolate of Leptospira interrogans strain from Argentina / Agregaciones celulares in vivo de Leptospira interrogans producidas por un aislamiento porcino capaz de formar biofilm

Leptospirosis is a zoonosis of ubiquitous distribution caused by spirochetes. Leptospires exist either as saprophytic water-associated organisms or as animal pathogens that can survive in water. Previous works have demonstrated that both saprophytic and pathogenic leptospires are able to produce functional biofilms, which consist of a community of bacteria embedded in an extracellular matrix attached to a surface. This structure is believed to provide protection from environmental aggressiveness. In the present study, we analyzed the capacity of biofilm formation both of a a recent field isolate of Leptospira interrogans serovar Pomona obtained from an aborted swine fetus and of the saprophytic Leptospira biflexa serovar Patoc. We used light microscopy, immunofluorescence, and scanning electron microscopic examinations on glass and polystyrene plate models to evaluate the process in vitro. The ability to form bacterial aggregations in vivo was tested using pregnant guinea pigs infected with both strains. We obtained biofilms both on glass and plastic surfaces. Scanning electron microscopic analysis showed differences in the biofilm structure formed by both strains. L. interrogans serovar Pomona cell aggregations were observed in placental tissues by light microscopy. Biofilms and cell aggregations are consistent with the life of saprophytic strains in water and could help pathogenic strains to colonize the host and lead to abortion in pregnant animals.

La leptospirosis es una zoonosis de amplia distribución causada por el género Leptospira. Las leptospiras existen de manera saprófita asociadas a ambientes acuáticos o como patógenos animales que también pueden sobrevivir en el agua. Trabajos previos demostraron que tanto las leptospiras saprófitas como las patógenas tienen la capacidad de formar biofilms, que consisten en una comunidad de bacterias embebidas en una matriz extracelular adherida a una superficie. Esta estructura tendría la función de proveer protección contra el medioambiente. En este estudio, analizamos la capacidad de formar biofilm en un aislamiento obtenido recientemente de un feto porcino abortado, caracterizado como Leptospira interrogans serovar Pomona, y en la bacteria saprófita Leptospira biflexa serovar Patoc. Se estudió la formación de biofilm en distintas superficies (vidrio y poliestireno), las que se evaluaron por microscopía óptica, inmunofluorescencia y microscopía electrónica de barrido. La capacidad de formar agregaciones bacterianas in vivo se evaluó utilizando un modelo de cobayas preñadas infectadas con ambas cepas. Se obtuvieron biofilms tanto en las superficies plásticas como de vidrio. La microscopía de barrido mostró diferencias en la estructura del biofilm formado entre ambas cepas. Se observaron agregaciones celulares en vasos placentarios de los animales infectados con L. interrogans serovar Pomona. Los biofilms y las agregaciones celulares son compatibles con la vida saprofítica en el agua y podrían favorecer a los microorganismos patógenos en la colonización del hospedador, lo que podría llevar al aborto en los animales preñados.

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.