Molecular detection and characterization of pathogenic leptospira species in environmental samples of selected districts in Perak, Malaysia
Leptospirosis is an endemic zoonotic disease in Malaysia caused by pathogenic species of genus Leptospira. Most cases of human leptospirosis are resulted from environmental exposure to water and soils contaminated with bacteria shed in urine of infected carrier animals. Epidemiological informatio...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/84236/1/FPSK%20%28p%29%202019%2022%20UPM%20ir.pdf |
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| Summary: | Leptospirosis is an endemic zoonotic disease in Malaysia caused by
pathogenic species of genus Leptospira. Most cases of human leptospirosis
are resulted from environmental exposure to water and soils contaminated with
bacteria shed in urine of infected carrier animals. Epidemiological information
about leptospirosis and Leptospira in the Perak state is scarce despite high
disease incidence and mortality rate. To assess public health risk for
leptospirosis, this study aims to determine the cross-sectional prevalence of
pathogenic Leptospira in recreational and residential public places, as well as
to characterize the genetic diversity of pathogenic Leptospira isolated from
environmental samples. A total of 228 environmental water and shore soils
samples were collected from 20 amenity forests and wet markets, filtered, and
subjected to cultivation of leptospires in enriched EMJH medium. Presence of
pathogenic Leptospira was confirmed by specific amplification of lipL32 gene
by polymerase chain reaction (PCR). Results showed a high prevalence of
pathogens (11 %, n = 25) throughout Perak, with highly varied localised
prevalence among 13 positive sampling sites (6.7 - 41.7 %). Further distribution
analysis implies a higher exposure risk in amenity forests than wet markets,
through soil than water, as well as in the districts Kampar and Kinta than
Batang Padang, Kuala Kangsar, Kerian, Larut, Matang & Selama. Unexpectedly the localised prevalence was not significantly associated with
provision of waste management and site cleanliness. In addition to that, the
total absence of pathogen at sites BF, GF, KW, LI, MM, PF, and SS in this
present study has no relation to environmental parameters of samples,
including temperature, pH, and water salinity. On another hand, seeking a
sensitive molecular detection tool for accurate surveillance has driven this
study to compare performance of other pathogen-specific diagnostic PCR
assays on the positive samples. Unexpectedly a notably low and varied
detection sensitivity (12 - 83 %) was determined among environmental isolates
in relation to reference Leptospira strains sourced from human or animal hosts.
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Among genetic markers studied, lipL32 and flaB have been most prevalent,
followed by gyrB and lfb1, whereas PCRs targeting secY and ligB showed high
false-negativity. The absence of amplification was most likely attributed to
mismatch in primer-annealing sites owing to high sequence polymorphisms. Phylogenetic analysis of partial 16S rRNA gene sequences revealed a
subclade formed by all environmental isolates (except intermediate KF4) within
the ‘pathogens’ clade, suggesting a fair distance from described host- associated Leptospira strains despite the low bootstrap values. A noticeable
clustering of isolates sourced from similar sampling site, district, nor ecological
niches was not observed. Through comparative polymorphic nucleotide
analysis, ten pathogenic isolates were found closest to L. kmetyi and L. alstonii
which have been prevailing in Malaysia, while the others probably represent
novel species. In conclusion, genetically diverse pathogenic Leptospira spp. was widely distributed in Perak. Determining the virulence potential and whole- genome sequence for these atypical pathogenic isolates is important to
validate the risk of leptospirosis, evolutionary relationship and reclassification of genus Leptospira. |
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