Association between Opisthorchis viverrini and Leptospira spp. infection in endemic Northeast Thailand

Abstract

Opisthorchiasis caused by Opisthorchis viverrini is an important foodborne trematodiasis in Thailand, Laos and Cambodia. Interestingly, the opisthorchiasis endemic region overlaps with an area of leptospirosis emergence. Here we report an association between opisthorchiasis and leptospirosis in Thailand. Of 280 sera collected from villagers living around the Lawa wetland complex in Khon Kaen province, 199 (71%) were seropositive for leptospirosis by immunochromatography. Individuals with O. viverrini infection had a significantly higher rate of leptospirosis than those without (P=0.001). Significant higher leptospirosis prevalence was found in males than females (P=0.002). However, females but not males with O. viverrini infection showed a significantly higher seroprevalence of leptospirosis. Twenty-one of 35 environmental samples from the lake (water, mud and fish skin mucus) were positive for Leptospira spp. DNA sequencing, sequence alignment, and phylogenetic analysis of some positive nested PCR products revealed both pathogenic and intermediate pathogenic strains of Leptospira in the samples. Strikingly, O. viverrini metacercariae from the fish were positive for L. interrogans. These results suggest a close association between opisthorchiasis and leptospirosis. Contact with water, mud or eating raw fish harboring liver fluke metacercariae may be risk factors for Leptospira infection.

Keywords: Lawa wetland; Leptospira spp.; Leptospirosis, metacercariae; Opisthorchis viverrini; Thailand.

Fig. 1

A satellite image of Lawa wetland and 6 sites around the big lake from where samples were collected (Yellow spots indicate mud and water collection sites). CKK = Chi Kok Kor, KSR = Kok Sam Ran, DPD = Donpordang, BT = Ban Tad, PAO = Ban Pao, NNK = Nongnakwan, Red circles = fish reservation areas. Scale bar = 1 km

Fig. 2

Grading of immunochromatography. Lane 1 = negative, Lane 2 = +, Lane 3 = ++, Lane 4 = +++, C = internal control, T = test sample

Fig. 3

Immunoblotting of L. interrogans strips with rabbit anti – L. interrogans (2), anti – O. viverrini somatic antigens (3). Lane 4 is O. viverrini somatic antigens probed with anti-O. viverrini somatic antigens. Lane 1 is negative control (PBS).

Fig. 4

Representative nested PCR amplified products of different samples. 1 = 100 bp DNA ladder, lane 2 = mud from control site, lane 3 = water from control site, lane 1 = L. interrogans positive control, Lai strain 56601, lane 2 = water sample CKK, lane 3 = water sample KSR-1, lane 4 = O. viverrini metacercariae, lane 5 = water sample KSR-2, lane 6 = mud from Ban-tad

Fig. 5

Representative pathogenic Leptospira–specific for lipL32 PCR amplified products of different samples. M = 100 bp DNA ladder, lane 1 = L. interrogans positive control, Lai strain 56601, lanes 2–11 = samples from different sources including negative control (DW). OV = O. viverrini, MC = metacercariae, Muscus = fish mucus, w = water sample

Fig. 6

Phylogenetic tree of known Leptospira sequences retrieved from GenBank (accession number indicated after each sequence) and Leptospira sequences identified from the current study. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the Leptospira. Branches corresponding to partitions that were reproduced in less than 50% of bootstrap replicates are collapsed. The bootstrap values are indicated at the branch points. Pathogenic, intermediate and non-pathogenic Leptospira spp. are indicated as vertical lines. Leptopira spp. identified from this study are boxed.