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. 2014 Nov;80(22):6926-32.
doi: 10.1128/AEM.02568-14. Epub 2014 Aug 29.

PCR and culture identification of pathogenic Leptospira spp. from coastal soil in Leyte, Philippines, after a storm surge during Super Typhoon Haiyan (Yolanda)

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PCR and culture identification of pathogenic Leptospira spp. from coastal soil in Leyte, Philippines, after a storm surge during Super Typhoon Haiyan (Yolanda)

Mitsumasa Saito et al. Appl Environ Microbiol. 2014 Nov.

Abstract

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof's medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.

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Figures

FIG 1
FIG 1
Locations where the soil samples were collected and results of Leptospira culture and flaB PCR. A total of 23 samples were collected in Tacloban and Palo in Leyte Province, Philippines. The locations of the sites where the soil samples were collected are plotted on the map. Open circle, the primary culture of the collected sample contained no leptospires (1 out of 23 samples; 4%); shaded circles, the primary culture of the collected samples contained leptospires but no DNA of pathogenic Leptospira strains (11 out of 23 samples; 48%); closed circles, the primary culture of the collected samples contained leptospires, and the DNA of pathogenic Leptospira strains was detected (11 out of 23 samples; 48%).
FIG 2
FIG 2
PFGE fingerprint patterns of pathogenic Leptospira isolates. Single-colony isolation was done on the 3 flaB PCR-positive Leptospira cultures (LES-5-S, LES-12-L, and LES-12-S) using solid medium. Five single colonies were picked up from each plate and cultured in fresh liquid Korthof's medium. A total of 15 strains were obtained, and all the strains were found to be flaB PCR positive. Chromosomal DNA of the strains was digested with the NotI restriction endonuclease. The five isolates from each of the three pathogenic leptospiral cultures showed identical fingerprint patterns. The pathogenic leptospires from sample LES-12 were isolated from both liquid medium (LES-12-L) and semisolid medium (LES-12-S). The five isolates from LES-12-L and the five isolates from LES-12-S showed identical fingerprint patterns. Lanes M, 100-bp size markers.
FIG 3
FIG 3
Neighbor-joining phylogenetic tree based on 16S rRNA gene sequence analysis showing the phylogenetic positions of two isolates from this study (MS422 and MS432). The tree was rooted with the 16S rRNA gene of Leptonema illini strain 3055 (GenBank accession number AY714984). GenBank accession numbers are shown in parentheses.

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