Fingerprint patterns were generated for 175 clinical isolates of Leptospira
from eight different countries. Isolates were obtained from humans, rodents/marsupials, and domestic animals (). The PFGE reference library identified 78% (136/175) of the isolates to the serovar level. An additional 15% (27/175) are being investigated further and were tentatively classified as new serovars. The remaining isolates (7%, 12/175) each may not be represented in the PFGE database, or may also represent new serovars and require further analysis. They have yet to undergo further studies as there is currently only one isolate found for each of these. The entire data set of PFGE results is represented in a dendrogram in Figure S1
Serovar identification results and CAAT results of isolates sent to CDC.1
Although some serovars, such as Icterohaemorrhagiae/Copenhageni, were found to occur in most regions included in this study, there were some unique differences in geographic distribution of serovars. Among both rat and human isolates from Thailand, 87% (n
27) were identified as L. interrogans
serovar Bulgarica (). In Brazil, 39% (n
16) of isolates from dogs, swine and cattle were serovar Canicola. Six isolates (14%) from Brazil are being investigated as a new serovar and all were isolated from capybaras. However, serovar Icterohaemorrhagiae/Copenhageni was the most prevalent serovar isolated from human patients in Brazil ().
Selected PFGE patterns of isolates collected from humans and rats in Thailand along with two reference strains of serovar Bulgarica showing two different species and reference serovar Autumnalis.
The most common serovar identified from rats in Peru was Icterohaemorrhagiae/Copenhageni (45%, n
10), but a recently described species (L. licerasiae
isolated from both humans and rats made up 41% of the Peruvian isolates. Human isolates from Egypt were more diverse; serovars Bataviae, Grippotyphosa (L. interrogans
), Icterohaemorrhagiae/Copenhageni, Pyrogenes and Pomona were identified (). The majority of isolates from the United States were submitted from Hawaii, and among these, there are four novel fingerprint patterns by PFGE. Forty-one percent (n
17) of the Hawaiian isolates make up one unknown pattern that is awaiting confirmation of new serovar status within L. interrogans
(species confirmed by 16S rRNA gene sequencing). An additional 10% (n
4) may represent three new serovars of L. noguchii
(species confirmed by DNA hybridization) (, ). Four isolates from Hawaii were identified as closely related to most of the serovars in the Ballum serogroup; reference isolates for serovars Ballum, Castellonis, Guangdong, Arborea, and Soccoestomes are all within three band differences or less from one another in PFGE patterns. Serovar Kenya, the only remaining member of serogroup Ballum, had a distinct pattern that showed greater than 10 band differences from the other reference serovars in serogroup Ballum. Therefore, these four clinical isolates from Hawaii could not be definitely identified to the serovar level without using an additional enzyme, such as SgrAI.
Representative PFGE patterns and MLST types from isolates identified from humans in Egypt and the proportions of each serovar identified among all Egyptian isolates.
PFGE patterns of unidentified, potentially new serovars.
CAAT was performed to validate the use of PFGE as a serovar identification tool. Representative isolates from each country were selected for CAAT analysis. CAAT was performed on 36 isolates identified by PFGE as serovars Canicola, Icterohaemorrhagiae/Copenhageni, Ballum (or related serovar from serogroup Ballum), Bulgarica (L. interrogans), Pomona, Bataviae, Pyrogenes, and Grippotyphosa (L. interrogans). The correlation between PFGE and CAAT was 100% (35/35) (). There was one isolate which could not be fully resolved to the serovar level by either method. This was an isolate from Hawaii that resembled multiple serovars in serogroup Ballum by PFGE. Serologically, the isolate was related to serovar Ballum by CAAT, but could not be definitively identified as serovar Ballum (12.5–25% titer remaining after absorption, greater than the 10% cut off for serovars considered to be the same). Additional reference sera were unavailable at this time and will need to be produced and tested by CAAT, and the PFGE method needs to be optimized with a second enzyme in order to differentiate between serovars of serogroup Ballum. CAAT was unable to distinguish between isolates of serovars Icterohaemorrhagiae and Copenhageni using our reference antisera.
Isolates designated as potential new serovars based on PFGE profiles could not be identified by CAAT and are currently being evaluated at another reference institution for final confirmation of new serovar status (, ).
MLST was also performed on 42 isolates as an additional molecular characterization tool and to evaluate strain phylogeny. Results are displayed in . Three isolates from Brazil were ST37, the same ST type as reference serovars Pomona and Canicola. One isolate from Thailand was of ST34, which is the same as researchers found in Thailand.
Another isolate from Thailand represented both a new ST type as well as a new PFGE pattern. Twelve isolates from Hawaii were of ST51, the same as reference serovar Australis. Eight additional isolates from Hawaii were ST17, which matched the ST type of reference serovars Copenhageni and Icterohaemorrhagiae. Isolates from Egypt were ST17 (n
3); ST37 (n
1); ST50 (n
5), which matches reference serovar Bataviae; ST88 (n
3), which matches our reference strain of serovar Pyrogenes but differs from the three Pyrogenes serovars in the public database (ST types 13, 37, and 49); and ST111 (n
1), which also matches our reference strain of serovar Grippotyphosa but differs from three Grippotyphosa serovars in the public database (ST types 18, 62 and 68). Lastly, the isolate from Guyana and two isolates from Peru were of ST17, which matches serovars Copenhageni and Icterohaemorrhagiae.