Date Published: May 23, 2019
Publisher: Public Library of Science
Author(s): Daniela Santos Almeida, Lucas Nogueira Paz, Daiana Santos de Oliveira, Danielle Nascimento Silva, Paula Ristow, Camila Hamond, Federico Costa, Ricardo Wagner Portela, Alessandra Estrela-Lima, Melissa Hanzen Pinna, Kalimuthusamy Natarajaseenivasan.
The study aimed to evaluate the histopathological characteristics of renal lesions in chronically infected sheep and with low titers of anti-Leptospira antibodies from a slaughterhouse. In the serological analysis, 24.74% (48/194) presented seroreactivity with a titer equal to or greater than 100. Among these seroreactive sheep, titers of 100 were predominant (58.33%, 28/48), with the highest titer being 1,600 (2.08%, 1/48). Serogroup Sejroe (sv. Hardjo) was the most frequent at 35.42% (17/48). Leptospiral DNA was verified in 4.12% (8/194) of the kidney samples tested, and no urine sample was positive. All the samples corresponded to the pathogenic species L. interrogans. The eight amplicons with 202-nucleotides were identical with two mismatches (presented 100% of identity) using the PCR targeting to secY gene. Histological sections of PCR-positive kidneys were submitted to direct detection by the anti-LipL32 immunohistochemistry (IHC) technique. The Leptospira spp. antigen was evident in 62.5% (5/8) of the kidneys. Positive staining was observed in the cytoplasm of tubular cells and in the form of brownish aggregates that adhered to tubular epithelial cells and projected into the lumen. Inflammatory lymphoplasmacytic infiltrate, ranging from mild to moderate, with multifocal distribution, was the predominant finding in seroreactive animals (33.33%, 16/48). The demonstration of the leptospiral antigen lining the renal tubules through IHC of naturally infected sheep confirmed by PCR characterizes renal colonization in a species with the presence of histological changes compatible with leptospirosis.
Leptospires live in the wild in mammalian reservoirs, which are chronically infected in the kidneys and eliminate the bacteria in the urine, polluting the environment . Rodents are considered the main carriers of the agent, among which the sewage rat (Rattus norvegicus) stands out as the most important reservoir in the urban ecosystems of the world [2, 3]. In rural environments, other species described as reservoirs of leptospirosis include cattles, swine, horses and canines, as well as wild animals [1, 4, 5]. Transmission occurs through direct contact with the urine of infected or indirectly from animals with contaminated environments, followed by the penetration of leptospires into skin lesions or mucous membranes .
Of the 194 sera evaluated by MAT, 24.74% (48/194) presented seroreactivity according to the cutoff titer of 100. The titers of 100 were predominant, representing 58.33% (28/48) of the positive reactions, followed by titers of 200 (33.33%, 16/48), 400 (6.25%, 3/48) and 1,600 (2.1%, 1/48). Sejroe (sv Hardjo) was the most prevalent, representing 35.42% (17/48) of the reactive samples, followed by Australis (sv Bratislava) and Pomona, both at 10.42% (5/48) (Table 1).
The present study detected ovine chronic renal carriers of L. interrogans through serological, molecular and histological techniques. In this study, ovine carriers presented renal changes compatible with chronic infection characterized by the presence of lymphoplasmacytic inflammatory infiltrate. Additionally, this study is the first to demonstrate Leptospira immunolabeling by IHC, using anti-Lipl32 antibody in sheep and could be used as a basis for future studies aimed at elucidating the mechanisms of renal colonization and pathogenesis of leptospirosis in these animals.
Sheep detected as carriers of leptospires presented renal damage compatible with chronic infection, characterized by the presence of inflammatory lymphoplasmacytic infiltrate, which varied from mild to moderate. In these animals there was a predominance of low titers of anti-Leptospira antibodies and specific immunostaining of the anti-LipL32 antibody in most sheep kidneys but no biofilm was detected.