Isolation and identification of leptospires from raccoons (Procyon lotor) in Indiana, USA

Ching Giap Tan, Purdue University

Abstract

The present study was conducted to determine whether raccoons ( Procyon lotor) carry leptospires in their kidneys and thus are capable of infecting livestock and other domesticated animals in landscapes where these species co-occur. Isolation of leptospiral organisms and identification of leptospiral species and serogroups from raccoon kidneys using culture in conjunction with PCR and sequencing was performed. Thirty-four raccoons were live-trapped from a single forest patch in a highly agricultural region of central Indiana, USA. A portion of kidney (2cm2) from each raccoon was homogenized and used for leptospiral culture. Leptospiral cultures were subjected to DNA extraction and various PCR procedures. Serum sample from each raccoon also was collected and antibody titers to leptospiral serovars were determined by microscopic agglutination test (MAT). Twenty-nine out of 34 raccoon kidney cultures (85.3%) were found to have characteristic thin slender and curved spiral-shaped leptospiral organisms with hooked ends by dark field microscopy (DFM). All leptospiral cultures (34/34), 2 including those negative for leptospires by DFM, were positive for Leptospira by various PCR procedures. The PCR with primers targeting the conservative region of LipL32 gene was the most sensitive PCR in detection of pathogenic leptospires. Twenty-two raccoon kidney cultures (64.7%) were positive for leptospirosis by PCR amplification with the primers flanking the variable region of LipL32 gene. The PCR amplicons were directly sequenced and the sequences were compared to those of the reference strains available in GenBank. Twelve kidney cultures had Leptospira interrogans, 8 had L.kirschneri and 2 had L. borgpettersenii. In addition, serogroup-specific primers designed from the O-antigen gene were used to determine leptospiral serogroups. They were predominantly Grippotyphosa serogroup. Anti-leptospire antibodies were detected in 16 out of 34 raccoons (47.1%) by MAT. There were titers of ≥ 1:80 in 16 raccoons (47.1%) and titers ≥ 1:400 in 3 raccoons (8.8%). The highest leptospiral serovar-specific seroreactivity among 34 raccoons was L.interrogans Bratislava (38.2%) and L.interrogans Grippotyphosa (32.4%), respectively. The results indicated that the raccoon kidneys carry leptospiral organisms that can be detected and identified by culture in conjunction with PCR and sequencing which the leptospires were identified predominantly L. interrogans species and of the Grippotyphosa serogroup. Given the high densities of raccoons that exist in urban and agricultural ecosystem, the results of this study suggest that raccoons likely are an important reservoir for the maintenance and transmission of leptospiral organisms between wildlife and livestock, domesticated animals, and humans.

Degree

M.S.

Advisors

Lin, Purdue University.

Subject Area

Veterinary services

COinS