Neglected leptospirosis in raccoons (Procyon lotor) in Indiana, USA.

BACKGROUND: Leptospirosis is a globally important zoonotic disease occurring clinically and subclinically in humans and animals. OBJECTIVES: To determine whether raccoons in Indiana carried leptospires in their kidneys. ANIMALS AND METHODS: Thirty-four raccoons were live-trapped from two forest patches in central Indiana. Following euthanasia, a portion of kidney (2 cm(2)) from each raccoon was homogenized and used for leptospiral culture. Leptospiral cultures were subjected to DNA extraction and various polymerase chain reaction (PCR) procedures reported previously. Serum sample from each raccoon was collected and antibody titers to leptospiral serovars were determined by microscopic agglutination test (MAT). RESULTS: All leptospiral cultures were positive for Leptospira by various PCR procedures. The PCR with the primers targeting the conservative region of LipL32 gene was the most sensitive PCR in the detection of pathogenic leptospires. The variable LipL32 PCR amplicons were sequenced and compared to the reference strains available in GenBank. Twelve kidney cultures had Leptospira interrogans, eight had Leptospira kirschneri and two had Leptospira borgpetersenii. They were predominantly Grippotyphosa serogroup. Antileptospire antibodies were detected in 16 out of 34 raccoons (47.1%) by MAT. There were titers ≥ 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%). CONCLUSIONS: Raccoons in Indiana carry leptospiral organisms in kidneys and the leptospires are predominantly L. interrogans species and of the Grippotyphosa serogroup. CLINICAL IMPORTANCE: The raccoons serve as reservoir hosts that exposure sources to wildlife, livestock, pets and humans.

Polymerase chain reaction-based discrimination of viable from non-viable Mycoplasma gallisepticum.

The present study was based on the reverse transcription polymerase chain reaction (RT-PCR) of the 16S ribosomal nucleic acid (rRNA) of Mycoplasma for detection of viable Mycoplasma gallisepticum. To determine the stability of M. gallisepticum 16S rRNA in vitro, three inactivation methods were used and the suspensions were stored at different temperatures. The 16S rRNA of M. gallisepticum was detected up to approximately 20-25 h at 37 °C, 22-25 h at 16 °C, and 23-27 h at 4 °C. The test, therefore, could detect viable or recently dead M. gallisepticum (< 20 h). The RT-PCR method was applied during an in vivo study of drug efficacy under experimental conditions, where commercial broiler-breeder eggs were inoculated with M. gallisepticum into the yolk. Hatched chicks that had been inoculated in ovo were treated with Macrolide 1. The method was then applied in a flock of day 0 chicks with naturally acquired vertical transmission of M. gallisepticum, treated with Macrolide 2. Swabs of the respiratory tract were obtained for PCR and RT-PCR evaluations to determine the viability of M. gallisepticum. This study proved that the combination of both PCR and RT-PCR enables detection and differentiation of viable from non-viable M. gallisepticum.