Optimization and comparative analysis of LAMP and PCR techniques for the detection of leptospiral DNA in Golden Syrian hamsters.

Leptospirosis is a zoonotic disease with significant public health and economic impact worldwide. Rapid and accurate diagnosis is essential for effective prevention and treatment. This study optimized a loop-mediated isothermal amplification (LAMP) assay using BFo isothermal DNA polymerase with different colorimetric indicators. LAMP was able to detect DNA from pathogenic and intermediate leptospires, while non-pathogenic leptospires and other non-leptospiral microorganisms were negative. LAMP assay combined with calcein showed a tenfold higher limit of detection (1 ng of leptospiral DNA per reaction) than LAMP combined with hydroxynaphthol blue or end-point PCR lipL32 (10 ng of DNA per reaction). Animal samples were collected from infected and non-infected Golden Syrian hamsters (Mesocricetus auratus) to evaluate and compare the performance of LAMP and PCR. These techniques showed a substantial agreement according to Cohen's kappa statistic, being both useful techniques for detecting leptospiral DNA in clinical samples. Overall, this study demonstrates that the LAMP assay is a sensitive, specific, rapid, and simple tool for the detection of leptospiral DNA. It has the potential to facilitate the diagnosis of leptospirosis, particularly in low-income regions with limited diagnosis resources.

First report of pathogenic Leptospira spp. in Tadaridabrasiliensis bats (family Molossidae) and Eptesicus furinalis (family Vespertilionidae) of Argentina. New host species in this country? / Primer reporte de Leptospira spp. patógena en murciélagos Tadaridabrasiliensis (familia: Molossidae) y Eptesicusfurinalis (familia: Vespertilionidae) en Argentina. ¿Nuevas especies hospedadoras en el país?

ABSTRACT Leptospirosis is an endemic disease caused byLeptospiraspp., a bacterium that affects animals and humans. In recent years, the number of reports of leptospirosis in wild animals has increased, which highlights the need to study the infectious agents in these animals. In this study, a duplex PCR for the detection of leptospiral DNA was performed on 50 kidney samples from bats, and a MAT (Microscopic Agglutination Test) for serological detection of anti-leptospiral antibodies was applied to 47 serum samples from bats from different regions of Buenos Aires Province, Argentina. DNA was extracted using Chelex-100 and duplex PCR was performed by targeting the detection of genessecYandflaB, of pathogenicLeptospiraspp. Of the 50 kidney samples, 3 were positive forEumopssp. andTadaridabrasiliensisby duplex PCR. Of the 47 serum samples, 12 were positive for different serovars:Leptospira interrogansserovars Icterohaemorrhagiae, Cynopteri and Bataviae, andLeptospira borgpeterseniiserovar Ballum. This is the first report of the detection of pathogenic leptospires by serology in bats belonging to theT. brasiliensisandEptesicus furinalisspecies in Argentina. In addition, this is the first report of the detection of pathogenic leptospiral DNA by PCR inT. brasiliensisspecies. The detection ofLeptospiraspp. in these wild animals shows that they may play an important role as wildlife reservoirs of leptospires.

RESUMEN La leptospirosis es una enfermedad endémica causada porLeptospiraspp., una bacteria que afecta a animales y a humanos. En los últimos años, el número de reportes de leptospirosis en animales silvestres ha aumentado, lo que resalta la necesidad de analizar los agentes infecciosos en estos animales. En este estudio, se aplicó una reacción en cadena de la polimerasa (PCR) dúplex para la identificación del ADN leptospiral en 50 muestras de riñones de murciélagos y la prueba de aglutinación microscópica (MAT) para la detección serológica de anticuerpos antileptospira en 47 muestras de suero de murciélagos de diferentes regiones de la provincia de Buenos Aires, Argentina. El ADN fue extraído usando Chelex-100 y la PCR dúplex estuvo dirigida a la detección de los genessecYyflaBdeLeptospiraspp. patógena. De las 50 muestras de riñón, tres resultaron positivas por PCR dúplex paraEumopssp. yTadaridabrasiliensis. De las 47 muestras de suero, 12 fueron positivas a diferentes serovares:LeptospirainterrogansserovaresIcterohaemorrhagiae, Cynopteri y Bataviae, yLeptospiraborgpeterseniiserovarBallum. Este es el primer reporte de detección de leptospiras patógenas por serología en murciélagos pertenecientes a las especiesT. brasiliensisyEptesicusfurinalisen Argentina. Además, también es el primero en la localización de ADN leptospiral por PCR en la especieT. brasiliensis.La identificación deLeptospiraspp. en estos animales silvestres muestra que pueden desempeñar un papel importante como reservorios de leptospiras en la fauna silvestre.

Use of the Leptospira sp. ligB C-terminus coding region as a diagnostic tool of animal leptospirosis.

Leptospirosis is the most widespread zoonosis worldwide, and it can cause reproductive failures in livestock, while in humans may vary from a mild fever to multi-organ failure and death. Due to this, in this study, we evaluated the usefulness of the segment encoding LigB C-terminus region, only present in pathogenic as target for a diagnostic PCR. This new PCR yielded a 100 % positivity for pathogenic Leptospira species and no cross-reactivity was found with intermediate or non-pathogenic species, or with other microorganisms, demostrating its high analytical specificity. The estimated analytical sensitivity was higher in serum samples than in blood or urine samples (6-9 × 102 lept/mL and 6-9 × 105 and 6-9 × 106 lept/mL, respectively). Multiple sequence alignment of the target region from different pathogenic Leptospira species confirmed that this gene region is highly conserved among these species, with few single nucleotide polymorphisms. The ligb-ct PCR here developed appears as a useful tool for the molecular diagnosis of leptospirosis.

First report of pathogenic Leptospira spp. in Tadarida brasiliensis bats (family Molossidae) and Eptesicus furinalis (family Vespertilionidae) of Argentina. New host species in this country?

Leptospirosis is an endemic disease caused by Leptospira spp., a bacterium that affects animals and humans. In recent years, the number of reports of leptospirosis in wild animals has increased, which highlights the need to study the infectious agents in these animals. In this study, a duplex PCR for the detection of leptospiral DNA was performed on 50 kidney samples from bats, and a MAT (Microscopic Agglutination Test) for serological detection of anti-leptospiral antibodies was applied to 47 serum samples from bats from different regions of Buenos Aires Province, Argentina. DNA was extracted using Chelex-100 and duplex PCR was performed by targeting the detection of genes secY and flaB, of pathogenic Leptospira spp. Of the 50 kidney samples, 3 were positive for Eumops sp. and Tadarida brasiliensis by duplex PCR. Of the 47 serum samples, 12 were positive for different serovars: Leptospira interrogans serovars Icterohaemorrhagiae, Cynopteri and Bataviae, and Leptospira borgpetersenii serovar Ballum. This is the first report of the detection of pathogenic leptospires by serology in bats belonging to the T. brasiliensis and Eptesicus furinalis species in Argentina. In addition, this is the first report of the detection of pathogenic leptospiral DNA by PCR in T. brasiliensis species. The detection of Leptospira spp. in these wild animals shows that they may play an important role as wildlife reservoirs of leptospires.

New enzyme-linked immunoassay for the detection of specific antibodies against multiple Leptospira serogroups in bovine sera.

Leptospirosis is a zoonotic disease with worldwide endemicity in Argentina, it is a significant public health problem in low-income populations. Bovine leptospirosis is a serious economic problem for cattle production, causing abortions, reduced milk yield, mortality in calves and decreased daily weight gain. We developed an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni M 20. We evaluated its performance for the detection of specific antibodies against multiple Leptospira serogroups in bovine. The microscopic agglutination test (MAT) was used as the gold standard. The performance of this ELISA was evaluated with a panel of sera (118 MAT confirmed positive and 97 MAT negative). The overall sensitivity was close to 85.6 % and the specificity was 83.5 %, according to the MAT reference method. Analytical specificity of the IgG-ELISA was evaluated using 50 bovine serum samples from animals showing serum antibodies against other pathogens that cause abortion in bovine, such as Brucella sp., Neospora sp. and Bovine Viral Diarrhea (BVD), and no cross-reaction was observed. This IgG-ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in bovine.