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High Prevalence of Pathogenic Leptospira in Wild Asian Pacific Journal of Tropical Medicine (2011)841-845 841 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm Document heading doi: High prevalence of pathogenic Leptospira in wild and domesticated animals in an endemic area of China Wang Yalin1, Zeng Lingbing1, Yang Hongliang1,4, Xu Jianmin2, Zhang Xiangyan1, Guo Xiaokui1, Pal Utpal3, Qin Jinhong1* 1Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China 2Jiangxi Provincial Center for Disease Control and Prevention, Nanchang 330029, China 3Department of Veterinary Medicine, University of Maryland, College Park, Maryland, 20742, USA 4Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA 80526 ARTICLE INFO ABSTRACT Article history: Objective: Leptospira To assess the prevalence of detected in wildlife and domesticated Received 1 September 2011 Methods: animals in Jiangxi Province, China, in 2009. Urine samples from 28 buffaloes and Received in revised form 15 September 2011 50 50 38 Accepted 12 October 2011 kidney samples from pigs, dogs and rats were collected from Fuliang and Shangrao 2009 (PCR) Available online 20 November 2011 County, Jiangxi Province, China,Leptospira in October . Polymerase chain reaction and culture analyses were used to detectResults: . The cultured isolates were typed using the microscopic (MAT) Keywords: agglutination test . The results showed that rats potentially serve as the main reservoir of leptospiral infection, followed by dogs. Although 16% of rats (6/38) were positive , PCR G1/G2 B64I/B64II Leptospirosis lipLusing culture analysis analysis using the diagnostic primers and or Prevalence 32 50% 24% showed identification asConclusions: and , respectively, of the rat samples as positive for the China presence of leptospiral DNA. PCR-based detection of leptospiral DNA in infected lipL PCR kidney tissues of reservoirs is more efficient when using G1/G2 primers than 32 primers. Culture However, the latter primers have a potential application for detection in urine samples. The Reservoir DNA alarmingly high prevalence of leptospiralLeptospira in the wild rat population near human habitation underscores the utility of routine surveillance, preferably using PCR methods, which are more sensitive than traditional culture-based methods. 1. Introduction animals, whereas direct human-to-human transmission is rarely reported[1]. Many wild or domestic animals, mainly rodents, small marsupials, cattle, pigs and dogs, serve as Leptospirosis, one of the most common and widespread reservoir hosts or carriers of leptospiral pathogens and, zoonoses in the world, is caused by the pathogenic owing to their presence in close proximity to humans, serve leptospiral species. The clinical manifestations of as the most important sources for human infection[2-4]. leptospirosis in humans and animals broadly range from Infected animals may shed leptospiral pathogens via urine flu-like episodes to dysfunction of multiple organs and or other excreta intermittently or regularly for months, years sometimes death. Leptospirosis in mammals is transmitted or even a lifetime[1]. More importantly, even vaccinated by physical contact with infected animals or by exposure animals may still shed infectious organisms in their to water or soil contaminated with the urine of infected urine[1]. Therefore, regular surveillance of carrier hosts for leptospiral infection and persistence is highly warranted for routine evaluations of the risk of human exposure and *Corresponding author: Jinhong Qin, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of prevention of the disease. Medicine, Shanghai 200025, China. China is one of the most endemic regions of global Tel: 86-21-64453285 leptospirosis[5]. Surveillance of leptospirosis in domestic Fax: 86-21-64453285 E-mail: [email protected] and wild animals such as buffaloes, pigs, dogs and rats has Foundation project: supported by grants from, the National Natural Science been routinely performed in many Chinese provinces[6-9]. Foundation of China (30970125,81101264 and 81171587) and the Program of Shanghai 2005 Health Bureau (2008045). The annual infection rate in of trapped rats was Wang Yalin et al./Asian Pacific Journal of Tropical Medicine (2011)841-845 842 5% according to a report from the Chinese Center for 2.4. Culture and isolation of Leptospira Disease Control and Prevention (China CDC)[10]. Thirty- two percent of randomly sampled residents not vaccinated in investigated sites of China were seropositive,Leptospira defined Freshly isolated tissues (two samples from each animal) as having the antibody titer against over 50, were inoculated into 5 mL of Korthoff medium with 250 毺g according to the microscopic agglutination test (MAT)[10]. of 5-fluorouracil. One milliliter of buffalo mid-stream urine This suggests that human exposure to leptospiral infection was inoculated into 5 mL of Korthoff medium with 250 毺g in certain habitats may be extremely high. In addition, of 5-fluorouracil, and 10-fold serial dilutions were made ℃in because most of the earlier diagnostic methods to determine 2 additional tubes. Samples were then incubated at 28 leptospiral prevalence used relatively less sensitive and examined for spirochete growth for up to two months by approaches, such as culture analysis and MAT, the actual examination under a dark microscope. [11,12] infection rate could be underestimated . Therefore,Leptospira the 2.5. MAT use of more sensitive methods for the detection of infection is warranted to replace or complement currently available, less efficient and laborious methods. The isolates were typed by MAT. The procedure In the current study, we evaluated the prevalence of was performed, as detailed, with the following minor [1] leptospiral organisms in domestic and wild animals in an modifications . Briefly, all isolates were examined7 and endemic province of China in 2009Leptospira. We also compared diluted, using saline, to approximately 10 cells/mL. routine culture-based diagnosis of with PCR- The diluted cells were then added to serially diluted based diagnosis using multiple primer sets in isolated standard rabbit serum (National Institute for the Control kidney and urine samples from the reservoir hosts. of Pharmaceutical and Biological Products, China) in 96℃- well flat-bottom microtiter plates and incubated at 37 2. Materials and methods for 2 h. The agglutination result was evaluatedLeptospira by dark- field microscopy at 100伊 magnification. species [14] 2.1. Ethical approval included in the antigen panel are listed in Table 1 . 2.6. PCR detection This study was reviewed and approved by the Laboratory ℃ Leptospira Animal Sciences Center of the Shanghai Jiao Tong Kidney samples were stored at -80 until use. University School of Medicine and Jiangxi CDC (Permit DNA was extracted from the kidney using the DNeasy Tissue Number: SYXK2008-0050). The study was conducted Kit (Qiagen) according to the product manual. The urine adhering to the regulations for the administration of affairs samples were centrifuged for 5 min at 5 000 rpm to discard concerning experimental animals in China. Wild rats were the cell debris, and the supernatant was subjected to further humanely euthanized soon after being trapped, and all centrifugation at 12 000 rpm for 20 min to collect the pellet efforts were made to minimize suffering. Verbal informed within℃ 2 h of the initialLeptospira collection; the pellet was stored at consent was obtained from each livestock owner prior to the -20 until use. DNA was extracted from the sample collection. collected urine pellets by centrifugation at 12 000 rpm using DNA DNA 2.2. Sample collection the Micro Kit (Qiagen). The from 15 reference strains was isolated using the bacterial DNA isolation kit (Watsonbiot, China). The DNA concentration was tested by A total of 164 samples collected from Fuliang and Shangrao spectrophotometer. County, Jiangxi Province, China, in October 2009 were Three primer pairs were used to detect the leptospiral DNA G1/G2 B64I/B64II tested in Apodemusthe current agrarius study. Thirty-eightRattus losea wild rats, which . Two pairs were and lipL ,lipL which are’ widely [15] AAA included and , were trapped used . Another primer pair’ was lipL 32 ( ’ 32F: 5 - by the trap-night method, with rat clips from 9 paddy CTT TCG ATT TTG GCT’ AT -3 and 32R: 5 - TGT TTT TGC fields in Fuliang County. To decrease the possibility of’ AAT TCT TCA GG -3 ), which amplifies a 758 bp product. The contamination, the kidneys were isolated from the animal s primerlipL pair was designedLeptospira based on a sequence alignment dorsal aspect[13]. Morning urine samples from 28 buffaloes of 32 sequences of species, including 4 NCBI were collected from 6 paddy fields in Fuliang County, and sequenced pathogenicLeptospira leptospiral strains deposited in kidney samples from 50 pigs and 50 dogs were collected and 6 draft reference strains sequencedlipL by our from freshly slaughtered animals in 2 slaughterhouses in research groupLeptos (unpublishedpira data). Also, the 32 gene Shangrao County. sequence of kirschneri strain MORU UT130, G1/G2 2.3. Reference strains and culture conditions which lipLcould not amplify, was included in our primer design. The 32 primer pair was targeted to aLeptospira specific, yet conserved
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