Isolation of pathogenic Leptospira strains from naturally infected cattle in Uruguay reveals high serovar diversity, and uncovers a relevant risk for human leptospirosis

Abstract

Leptospirosis is a neglected zoonosis with worldwide distribution. The causative agents are spirochete bacteria of the Leptospira genus, displaying huge diversity of serovars, the identity of which is critical for effective diagnosis and vaccination purposes. Among many other mammalian species, Leptospira infects cattle, eliciting acute signs in calves, and chronic disease in adult animals often leading to abortions. In South America, and including in Uruguay, beef and dairy export are leading sources of national income. Despite the importance of bovine health, food safety, and bovine-related dissemination of leptospirosis to humans, extremely limited information is available as to the identity of Leptospira species and serovars infecting cattle in Uruguay and the South American subcontinent. Here we report a multicentric 3-year study resulting in the isolation and detailed characterization of 40 strains of Leptospira spp. obtained from infected cattle. Combined serologic and molecular typing identified these isolates as L. interrogans serogroup Pomona serovar Kennewicki (20 strains), L. interrogans serogroup Canicola serovar Canicola (1 strain), L. borgpetersenii serogroup Sejroe serovar Hardjo (10 strains) and L. noguchii (9 strains). The latter showed remarkable phenotypic and genetic variability, belonging to 6 distinct serogroups, including 3 that did not react with a large panel of reference serogrouping antisera. Approximately 20% of cattle sampled in the field were found to be shedding pathogenic Leptospira in their urine, uncovering a threat for public health that is being largely neglected. The two L. interrogans serovars that we isolated from cattle displayed identical genetic signatures to those of human isolates that had previously been obtained from leptospirosis patients. This report of local Leptospira strains shall improve diagnostic tools and the understanding of leptospirosis epidemiology in South America. These strains could also be used as new components within bacterin vaccines to protect against the pathogenic Leptospira strains that are actually circulating, a direct measure to reduce the risk of human leptospirosis.

Fig 1. Screening of pathogenic Leptospira spp. in urine samples by PCR amplification of the lipL32 gene.

(A) PCR amplification of the lipL32 gene, showing on the left side products obtained from 10 mL of urine without previous washing of the pellet, and on the right side the inhibition controls using pure DNA spiking. (B) Same as (A), except that the urine pellets on the left side were previously washed with PBS pH 7.4. (C) Same as (A) and (B), except that on the left side of the ladder urine pellets were previously washed with PBS pH 7.4 and BSA was included in the PCR mix. Asterisks show PCR reactions with total inhibition. (D) Typical lipL32 amplification results, using optimized conditions as in (C), from randomly chosen urine samples collected in the field. (E) Corresponding inhibition controls for panel (D).

Fig 2. Phylogeny of Leptospira spp. isolates based on secY gene sequence analysis.

Evolutionary history inferred by using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The analysis involved 76 partial sequences of the secY gene including the 40 bovine isolates from Uruguay that we are now reporting. Uruguayan strains from bovine hosts (in blue) and human patients (green) are compared to 32 additional sequences (in red) corresponding to isolates obtained elsewhere and from a variety of hosts, as indicated within brackets. Asterisks indicate the known serovar for isolates where such information is known, following the code: *serovar Pomona **serovar Panama ***serovar Autumnalis ****serovar Hardjo. Isolates obtained in Uruguay are named according to their strain denomination as "IP" (Institut Pasteur Montevideo) or "IH" (Instituto de Higiene) followed respectively by a 7- or a 4-digit number. GenBank accession numbers are indicated in parentheses. Well separated phylogenetic clades have a correspondence to different Leptospira species as indicated toward the right of the figure. The Patoc strain at the bottom of the panel belongs to the saprophytic species L. biflexa.