RESUMO
Leptospirosis is a neglected zoonotic disease that commonly affects cattle, pigs, horses, and dogs in many countries. Infection in dogs is usually subclinical, but acute cases of leptospirosis may occur along with systemic failure, which may become fatal. After recovery from an acute infection, dogs may become asymptomatic carriers and shed pathogenic leptospires through urine for long periods of time. Here, a study of ten different cases of leptospirosis is presented, showing the relevance of dogs as asymptomatic carriers of pathogenic Leptospira. The diagnosis was confirmed via isolation and further serological and genetic identification. Four Leptospira isolates (LOCaS28, 31, 34, and 46) were obtained from the kidneys and urine samples of 58 dogs destined for destruction (6.89%) at a Canine Control Center in Mexico City. No spirochetes were observed in the urine samples of those Leptospira-positive dogs examined under dark-field microscopy, and no clinical signs of disease were observed either. Six additional isolates were obtained: two came from asymptomatic carrier dogs (CEL60 and UADY22); another isolate came from an asymptomatic dog that was a pack companion of a clinically ill dog with fatal leptospirosis (AGFA24); and finally, three isolates were taken from dogs that died of leptospirosis (LOCaS59, Citlalli, and Nayar1). Nine out of the ten isolates were identified as being from the serogroup Canicola via cross-absorption MAT using reference strains and specific antisera, and their identity was genetically confirmed as Canicola ST34 via multi-locus sequencing typing (MLST). In contrast, the isolate Nayar1 was identified as serovar Copenhageni ST2. Interestingly, the asymptomatic dogs from which Leptospira isolates were recovered consistently showed high antibody titers in the microscopic agglutination test (MAT), revealing values of at least 1:3200 against serogroup Canicola and lower titer values against other serogroups. Isolates showed different virulence levels in the hamster model. Taken as a whole, all these findings confirmed that dogs may act as asymptomatic carriers of pathogenic leptospires and possibly spread them out to the environment, thus representing an active public health risk. The results also showed that the Canicola ST34 clone is the most prevalent Leptospira serovar in dogs in Mexico, and finally that the old-fashioned MAT is a good alternative for the detection of presumptive Leptospira asymptomatic carrier dogs.
RESUMO
The wide variety of pathogenic Leptospira serovars and the weak protection offered by the available vaccines encourage the search for protective immunogens against leptospirosis. We found that the secretin GspD of the type II secretion system (T2S) of Leptospira interrogans serovar Canicola was highly conserved amongst pathogenic serovars and was expressed in vivo during infection, as shown by immunohistochemistry. Convalescent sera of hamsters, dogs, and cows showed the presence of IgG antibodies, recognizing a recombinant version of this protein expressed in Escherichia coli (rGspDLC) in Western blot assays. In a pilot vaccination study, a group of eight hamsters was immunized on days zero and 14 with 50 µg of rGspDLC mixed with Freund's incomplete adjuvant (FIA). On day 28 of the study, 1,000 LD50 (Lethal Dose 50%) of a virulent strain of Leptospira interrogans serovar Canicola (LOCaS46) were inoculated by an intraoral submucosal route (IOSM). Seventy-five percent protection against disease (p = 0.017573, Fisher's exact test) and 50% protection against infection were observed in this group of vaccinated hamsters. In contrast, 85% of non-vaccinated hamsters died six to nine days after the challenge. These results suggest the potential usefulness of the T2S secretin GspD of Leptospira as a protective recombinant vaccine against leptospirosis.
