Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon

Abstract

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species "Leptospira licerasiae" serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010(T), which has been deposited into internationally accessible culture collections. By microscopic agglutination test, "Leptospira licerasiae" serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti-L. fainei serovar Hurstbridge at a titer of 1:100. LipL32, although not detectable by PCR, was detectable in "Leptospira licerasiae" serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against "Leptospira licerasiae" serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.

Figure 1. Pulsed field gel electrophoresis analysis of leptospiral isolates obtained from rats and humans in the region of Iquitos, Peru.

Indicated in parentheses is animal source of leptospiral isolate followed by location of trapping (see Methods). Rn, Rattus norvegicus; Rr, Rattus rattus. B, Belen; SJ, San Juan; M, Moralillo.

Figure 2. Phylogram of leptospiral 16S rRNA gene sequences generated by Bayesian phylogenetic analysis with simultaneous runs of 3,000,000 generations.

Bootstrap confidence in assigning branch points is indicated at each node. For clarity, the intermediate clade of leptospires is placed on top, with the L. licerasiae strains first; the intermediates, pathogens and saprophytes groups of Leptospira are indicated at the right. Leptonema is used as the outgroup for comparison. The scale bar (upper left) shows the fractional difference in 16S rRNA gene nucleotide sequences. GenBank accession numbers are indicated to the right of each strain analyzed.

Figure 3. Southern blot to determine the presence of LigA in “L. licerasiae” serovar Varillal.

Lane 1, DIG-labeled marker; lane 2, L. interrogans serovar Icterohaemorrhagiae strain HAI188 (positive control); lane 3, “L. licerasiae” serovar Varillal; lane 4, L. biflexa serovar Patoc I^T.

Figure 4. Western immunoblot of Leptospira interrogans serovar Copenhageni strain L1-130, “L. licerasiae” serovar Varillal, and L. fainei serovar Hurstbridge using rabbit polyclonal antisera to recombinant LipL32 of L. kirschneri serovar Grippotyphosa.

rLipL32, recombinant LipL32 of L. kirschneri serovar Grippotyphosa produced in E. coli.

Figure 5. Real time quantitative PCR analysis of experimental leptospiral infections of hamsters.

HAI188 and HAI156, strains L. interrogans serogroups Icterohaemorrhagiae and Canicola isolated from patients in Iquitos, Peru. VAR10, “L. licerasiae” serovar Varillal strain VAR 010^T. HAI188 and HAI156 caused a severe moribund state at days 4–5; none of the animals with VAR 010^T exhibited any signs of illness. Three 25 mg samples of each tissue were analyzed and error bars indicate the standard deviations of these three samples per tissue.

Figure 6. Seroprevalence of “Leptospira licerasiae” serovar Varillal in acute febrile patients in Iquitos, Peruvian Amazon (n = 881).

Var 10 = “Leptospira licerasiae” serovar Varillal strain VAR 010^T. Criteria for serological diagnosis of acute Leptospira infection: 1) IgM positive by ELISA in either acute or convalescent sera; 2) Conversion in the microscopic agglutination test (MAT) from negative to positive (1∶100 or greater); 3) Single MAT titer of 1:800 or greater; or 4) Four-fold rise in MAT titer.