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Research Article Diversity of Leptospira Spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia Hindawi Publishing Corporation Journal of Tropical Medicine Volume 2017, Article ID 3760674, 8 pages http://dx.doi.org/10.1155/2017/3760674 Research Article Diversity of Leptospira spp. in Rats and Environment from Urban Areas of Sarawak, Malaysia Chai Fung Pui,1 Lesley Maurice Bilung,1 Kasing Apun,1 and Lela Su’ut2 1 Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia 2Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia Correspondence should be addressed to Lesley Maurice Bilung; [email protected] Received 11 November 2016; Revised 9 January 2017; Accepted 6 February 2017; Published 28 February 2017 Academic Editor: Jean-Paul J. Gonzalez Copyright © 2017 Chai Fung Pui et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Various prevalence studies on Leptospira in animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophytic Leptospira in selected animals and environments. This study was therefore conducted to detect Leptospira spp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. Pathogenic Leptospira was present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. Intermediate Leptospira was present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. Saprophytic Leptospira was present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76 Leptospira spp. were isolated. Based on DNA sequencing, the dominant Leptospira spp. circulating in urban areas of Sarawak are pathogenic Leptospira noguchii, intermediate Leptospira wolffii serovar Khorat, and saprophytic Leptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence of Leptospira spp. from rats and the environment, with dominant local serovars in urban areas of Sarawak. 1. Introduction a wide range of clinical manifestation such as renal failure, hepatic failure, severe pulmonary haemorrhage, and even Leptospirosis is a zoonotic disease with worldwide distribu- death [8]. The overall case mortality rate in humans ranges tion and caused by pathogenic Leptospira,whichresultsin from 1 to 5% and the elderly are claimed to have higher infec- significant public health problem worldwide [1, 2]. The genus tion risk of leptospirosis [9]. Incidence rates are often under- Leptospira consists of 20 species, with more than 300 serovars, estimated because of the relative inaccessibility, lack of rapid grouped into 20 serogroups [3]. Based on the pathogenic- diagnostics, and insufficient awareness of leptospirosis [10]. ity, they can be divided into three major clades, namely, Similar prevalence studies on Leptospira spp. had been pathogenic, saprophytic (nonpathogenic), and intermediate conducted in Malaysia in recent years. Leptospira had been (unclear pathogenicity) [2]. The pathogenicity status of inter- isolated from urban rats of Kuala Lumpur [11] as well as soil mediate Leptospira remains a debate matter. For instance, andwaterfromurbansitesinPeninsularMalaysia[12].The hamsters inoculated with intermediate L. inadai and L. licera- presence of Leptospira spp. was also reported in National Ser- siae do not cause any clinical manifestation of leptospirosis vice Training Centres of Peninsular Malaysia [13]. Moreover, although both recovered from patients [4]. Thayaparan et al. [14] reported the presence of Leptospira spp. Humans usually get infected through direct contact with in wildlife around tourism areas in Sarawak. Our previous theinfectedanimalurineviamucousmembraneandexposed study highlighted the presence of Leptospira spp. in national skin or indirect contact by exposure to the contaminated parks [15]. The presence of Leptospira spp. in this area posed soil,water,andfood[5–7].Normally,maintenancehostsare the risk of transmission and infection to humans. Since this asymptomatic while accidental hosts like humans may suffer study on the detection of Leptospira spp. in rats, soil and 2 Journal of Tropical Medicine water from urban areas is first reported in Sarawak, the data UA1 obtained is definitely important to provide an insight on the UA2 status of Leptospira spp. in Sarawak. UA3 Current diagnostic methods for Leptospira spp. usually rely on the demonstration of serum antibodies by micro- UA4 scopic agglutination test (MAT). However, this method is UA5 time-consuming, requires live strain of Leptospira in expo- UA8 UA9 nential culture phase, and gives subjective interpretation of result [16]. The use of PCR is therefore gaining more UA6 popularity over MAT now. PCR, as direct genome detection UA7 method, offers various key advantages such as reduced con- UA10 tamination, increased detection accuracy, faster detection, and elimination of the need to use many antigens [6]. Besides, theuseofPCRiscriticalparticularlyatearlystageofinfection and acute stage of illness before antibodies are detectable. The early diagnosis of leptospirosis can reduce the complications due to leptospirosis and prevent fatality [17]. Many different primer sets had been applied in the detec- tion of Leptospira.Inthisstudy,LipL32, rRNA,andrrs genes were targeted as reported in our previous study [15]. LipL32 gene is a major outer membrane lipoprotein of Leptospira. The lipoprotein is highly conserved among pathogenic Lep- tospira and considered as virulence factor where higher levels Figure 1: Location of all the urban areas (UA) examined in this of LipL32 are expressed in Leptospira during acute lethal study (MapCustomizer, 2016). UA1, UA2, UA3, and UA7 are densely infections than Leptospira cultured in vitro [8, 18]. rRNA gene populated villages; UA4 is a famous market near villages; UA5 is a densely populated residential area with prominent hawker centres; is very conserved throughout the bacterial kingdom. The UA6 and UA9 are small villages; UA8 is a public university; and primer set targeting 16S rRNA genes is genus-specific and UA10 is a residential area. useful in epidemiological study [19]. The primer set designed within the rrs gene encoding 16S rRNA is specific to sapro- phytic Leptospira [20]. The objective of this study was to determine the diversity store room. Soil samples were collected from the surrounding of Leptospira spp. in rats, soil, and water samples from urban of housing areas, landfills, open field, and surrounding of areas of Sarawak using PCR. This study also aimed to dissem- lake. Water samples were collected from drain effluent, river, inate information on the dominant Leptospira serovars circu- lake, and puddle. lating in Sarawak so that control and preventive measures for this disease can be taken effectively. 2.2. Rat Trapping and Species Identification. The rats were caught alive using rectangular wire cage traps measuring 15 × × 2. Materials and Methods 12.5 29 cm. Each trapping session took at least three consec- utive days where the cage traps with baits were set up on the 2.1. Study Sites. This study focused on urban areas in Sarawak firstdaybeforecheckingforthecapturingyieldonthecon- which are usually characterized by high density of humans secutive days. All the trapped rats under the family Muridae population. According to Population and Housing Census were euthanized humanely by placing them into a cloth bag 2010 by Department of Statistics Malaysia (2015), urban area containing cotton wool soaked with chloroform. They were is defined as gazetted areas with their adjoining built-up further identified based on phenotypic characteristics such as areas, which had a combined population of 10,000 or more fur colour (ventral and dorsal) and morphological measure- at the time of the Census 2010 or the special development ments such as ear length, hind foot, tail length, and head body area that can be identified, which at least had a population length [21]. of 10,000 with at least 60 % of population (aged 15 years and above) being involved in nonagricultural activities. 2.3. Rat Samples Collection. Selected organs such as kidney The study sites (Figure 1) included public university, vil- and liver were removed from euthanized rats using sterile lages, residential areas, commercial centres, hawker centres, blade. A small piece of tissue was inoculated into modified sem- and markets. The selection of study sites was based on the isolid Ellinghausen-McCullough-Johnson-Harris (EMJH) recent reports of leptospirosis outbreak, environmental expo- media with 100 g/mL 5-fluorouracil. All the enriched sure due to humans activities, the suitability of the habitat cultures were incubated aerobically at room temperature for for the rats breeding, and its likelihood of transmitting the 3 months. They were examined for the presence of Leptospira disease. A total of 107 rats, 292 soil samples and 324 water using polymerase chain reaction (PCR) every month [7, 11]. samples were collected from April 2014 to February 2015. In brief, the cage traps were set randomly near the garbage 2.4. Soil and Water Samples
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