Dogs are highly susceptible to the leptospiral infection, notably stray and sheltered dogs. Unsanitary conditions often observed in dog shelters may predispose the introduction and spread of leptospires among sheltered populations, potentially increasing the chances for the inadvertent adoption of asymptomatically infected animals. The present work describes a longitudinal study using a multidisciplinary approach for the identification of chronically infected dogs and the characterization of potentially pathogenic strains circulating among stray and sheltered dog populations in São Paulo, Brazil. A total of 123 dogs from three populations were included. The initial evaluation consisted of blood and urine quantitative PCR testing (qPCR), the detection of specific antibodies by microscopic agglutination test (MAT), physical examination and hematological and serum biochemistry analyses. The qPCR-positive dogs were prospectively examined, and reevaluations also included culture from urine samples. Positive qPCR samples were subjected to 16S rRNA and secY gene phylogenetic analysis. The recovered strains were characterized by Multilocus Sequence Typing, polyclonal serogroup identification and virulence determination. Leptospiruria was detected in all populations studied (13/123), and phylogenetic analysis revealed that 10 dogs had L. interrogans infection. Three dogs (3/13) had L. santarosai infection. The secY phylogenetic analysis revealed that the L. santarosai sequences clustered separately from those obtained from other hosts. Ten leptospiruric dogs were reevaluated, and three dogs presented persistent leptospiruria, allowing culturing from two dogs. The strains were characterized as L. interrogans serogroup Canicola (virulent) and L. santarosai serogroup Sejroe (not virulent). Serum samples were retested by MAT using the DU92 and DU114 strains as antigens, and no increased seroreactivity was detected. Asymptomatic L. santarosai infection was observed in all populations studied, suggesting a possible role of dogs in the chain of transmission of this leptospiral species. The results suggest a genetic distinction between lineages of Brazilian L. santarosai maintained by dogs and other animal hosts. Our findings revealed that dogs could act as maintenance hosts for distinct pathogenic Leptospira, highlighting also that asymptomatically infected dogs can be inadvertently admitted and adopted in dog shelters, potentially increasing the risks of zoonotic transmission.

BackgroundDogs presenting with acute leptospirosis may present non-specific clinical and laboratory findings, and the definitive diagnosis may require additional confirmatory tests, including bacterial culture, for the direct or indirect identification of the pathogen. The present study describes the diagnosis of leptospirosis in suspected dogs based on the use of multiple diagnostic tests, including serological, molecular and bacteriological tests, along with the characterization of the recovered leptospiral strains.ResultsUrine, serum and blood samples were collected from 33 dogs with suspected clinical leptospirosis treated at the University of São Paulo Veterinary Hospital Service (Hovet FMVZ-USP) between 2013 and 2016. Only dogs with high blood urea nitrogen and creatinine levels in association with multiple clinical manifestations of the disease were included. Leptospiral culture, PCR and serology (Microscopic agglutination test - MAT) were performed in blood and urine samples taken from all suspected dogs at clinical presentation, and an additional prospective MAT titration was performed in seven dogs. Infection could be identified exclusively by PCR in 10 dogs (30.3%), exclusively by MAT in four dogs (12.1%) and by both tests in four dogs, totaling 18 dogs (54.5–95%CI: 37.6–71.5). Six out of eight MAT-confirmed cases presented with the highest titers against the Icterohaemorrhagiae serogroup. Leptospires were recovered from urine samples from two PCR-positive dogs, and both strains could be characterized by Multilocus Sequence Analysis and serogrouping as L. interrogans serogroup Icterohaemorrhagiae. Both isolates were shown to be pathogenic in the hamster model.ConclusionsThe simultaneous use of MAT and PCR was able to increase the diagnosis of leptospirosis in clinically suspected cases. Despite the increasing incidence of new serovars affecting dogs being reported in different locations, our results suggest that leptospiral strains belonging to the Icterohaemorrhagiae serogroup are still a major causative agent of canine leptospirosis in São Paulo, Brazil.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1547-4) contains supplementary material, which is available to authorized users.