Isolation and molecular characterization of Leptospira interrogans and Leptospira borgpetersenii isolates from the urban rat populations of Kuala Lumpur, Malaysia.
ABSTRACT: Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis.
Project description:Leptospirosis is an emerging infectious disease of global significance, and is endemic in tropical countries, including Malaysia. Over the last decade, a dramatic increase of human cases was reported; however, information on the primary vector, the rat, and the Leptospira serovars circulating among the rat population is limited. Therefore, the present study was undertaken to isolate Leptospira and characterise the serovars circulating in the urban rat populations from selected main cities in Peninsular Malaysia.Rat trappings were carried out between October 2011 to February 2014 in five urban cities which were chosen as study sites to represent different geographical locations in Peninsular Malaysia. Microscopic agglutination test (MAT) and PCR were carried out to identify the Leptospiral serogroup and determine the pathogenic status of the isolates, respectively while pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD)-PCR were used to characterize the isolates.Three rat species were identified from the three hundred and fifty seven rats captured with Rattus rattus, being the dominant rat species (285, 80 %) followed by Rattus norgevicus (53, 15 %) and Rattus exulans (19, 5 %). Only 39 samples (11.0 %) were positive by culture and further confirmed as pathogenic Leptospira by PCR. Significant associations were shown between host infection with locality, season, host-age and species. Based on MAT, two serogroups were identified in the population namely; L. borgpetersenii serogroup Javanica (n?=?16) and L. interrogans serogroup Bataviae (n?=?23). Pulsed-field gel electrophoresis (PFGE) distinguished the two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (41 %), and L. interrogans serovar Bataviae (59 %). RAPD-PCR yielded 14 distinct patterns and was found to be more discriminative than PFGE.This study confirms two Leptospira serovars circulating among the urban rats population in Peninsular Malaysia namely; L. borgpetersenii serovar Javanica and L. interrogans serovars Bataviae. Despite the low number of isolates obtained from the rat population, this study suggests that rodent control programs and disease surveillance may help to reduce the possible risk of disease transmission.
Project description:Leptospira interrogans serovar Bataviae is one of the serovars that can infect dogs. We report the draft genome sequence of Leptospira interrogans serovar Bataviae strain D64, which was isolated from the urine of an asymptomatic dog in Pathum Thani, Thailand, in 2017.
Project description:<h4>Background</h4>The role of rodents in Leptospira epidemiology and transmission is well known worldwide. Rats are known to carry different pathogenic serovars of Leptospira spp. capable of causing disease in humans and animals. Wild rats (Rattus spp.), especially the Norway/brown rat (Rattus norvegicus) and the black rat (R. rattus), are the most important sources of Leptospira infection, as they are abundant in urban and peridomestic environments. In this study, we compiled and summarized available data in the literature on global prevalence of Leptospira exposure and infection in rats, as well as compared the global distribution of Leptospira spp. in rats with respect to prevalence, geographic location, method of detection, diversity of serogroups/serovars, and species of rat.<h4>Methods</h4>We conducted a thorough literature search using PubMed without restrictions on publication date as well as Google Scholar to manually search for other relevant articles. Abstracts were included if they described data pertaining to Leptospira spp. in rats (Rattus spp.) from any geographic region around the world, including reviews. The data extracted from the articles selected included the author(s), year of publication, geographic location, method(s) of detection used, species of rat(s), sample size, prevalence of Leptospira spp. (overall and within each rat species), and information on species, serogroups, and/or serovars of Leptospira spp. detected.<h4>Findings</h4>A thorough search on PubMed retrieved 303 titles. After screening the articles for duplicates and inclusion/exclusion criteria, as well as manual inclusion of relevant articles, 145 articles were included in this review. Leptospira prevalence in rats varied considerably based on geographic location, with some reporting zero prevalence in countries such as Madagascar, Tanzania, and the Faroe Islands, and others reporting as high as >80% prevalence in studies done in Brazil, India, and the Philippines. The top five countries that were reported based on number of articles include India (n = 13), Malaysia (n = 9), Brazil (n = 8), Thailand (n = 7), and France (n = 6). Methods of detecting or isolating Leptospira spp. also varied among studies. Studies among different Rattus species reported a higher Leptospira prevalence in R. norvegicus. The serovar Icterohaemorrhagiae was the most prevalent serovar reported in Rattus spp. worldwide. Additionally, this literature review provided evidence for Leptospira infection in laboratory rodent colonies within controlled environments, implicating the zoonotic potential to laboratory animal caretakers.<h4>Conclusions</h4>Reports on global distribution of Leptospira infection in rats varies widely, with considerably high prevalence reported in many countries. This literature review emphasizes the need for enhanced surveillance programs using standardized methods for assessing Leptospira exposure or infection in rats. This review also demonstrated several weaknesses to the current methods of reporting the prevalence of Leptospira spp. in rats worldwide. As such, this necessitates a call for standardized protocols for the testing and reporting of such studies, especially pertaining to the diagnostic methods used. A deeper understanding of the ecology and epidemiology of Leptospira spp. in rats in urban environments is warranted. It is also pertinent for rat control programs to be proposed in conjunction with increased efforts for public awareness and education regarding leptospirosis transmission and prevention.
Project description:Rats are known to be the most important reservoirs and transmission sources of leptospirosis. However, the status of leptospirosis in the Philippines regarding reservoirs and transmission remains unknown. A survey was conducted in Metro Manila and Laguna that analyzed samples obtained from 106 rats. Using the microscopic agglutination test, we found that 92% of rat serum samples were positive for anti-Leptospira antibodies; the most common infecting serovars were Manilae, Hebdomadis, and Losbanos. On the basis of pulsed-field gel electrophoresis and gyrase B gene sequence analyses, four groups of rat kidney isolates were found: L. interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa, and L. borgpetersenii serogroup Javanica. Most isolates were lethal after experimental infection of golden Syrian hamsters. Results showed that these four Leptospira serovars and serogroups are circulating among rats, and that these animals may be one of the possible transmission sources of leptospirosis in the Philippines.
Project description:BACKGROUND:Horses infected with Leptospira present several clinical disorders, one of them being recurrent uveitis. A common endpoint of equine recurrent uveitis is blindness. Serovar pomona has often been incriminated, although others have also been reported. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. A leptospiral DNA fragment that encodes cross-reacting epitopes was previously cloned and expressed in Escherichia coli. RESULTS:A region of that DNA fragment was subcloned and sequenced. Samples of leptospiral DNA from several sources were analysed by PCR with two primer pairs designed to amplify that region. Reference strains from serovars canicola, icterohaemorrhagiae, pomona, pyrogenes, wolffi, bataviae, sentot, hebdomadis and hardjo rendered products of the expected sizes with both pairs of primers. The specific DNA region was also amplified from isolates from Argentina belonging to serogroups Canicola and Pomona. Both L. biflexa serovar patoc and L. borgpetersenii serovar tarassovi rendered a negative result. CONCLUSIONS:The DNA sequence related to the antigen mimicry with equine cornea was not exclusively found in serovar pomona as it was also detected in several strains of Leptospira belonging to different serovars. The results obtained with L. biflexa serovar patoc strain Patoc I and L. borgpetersenii serovar tarassovi strain Perepelicin suggest that this sequence is not present in these strains, which belong to different genomospecies than those which gave positive results. This is an interesting finding since L. biflexa comprises nonpathogenic strains and serovar tarassovi has not been associated clinically with equine uveitis.
Project description:As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species "Leptospira licerasiae" serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010(T), which has been deposited into internationally accessible culture collections. By microscopic agglutination test, "Leptospira licerasiae" serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti-L. fainei serovar Hurstbridge at a titer of 1:100. LipL32, although not detectable by PCR, was detectable in "Leptospira licerasiae" serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against "Leptospira licerasiae" serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.
Project description:Leptospirosis is a zoonosis known to be endemic in the Mekong Delta of Vietnam, even though clinical reports are uncommon. We investigated leptospira infection in rats purchased in food markets during the rainy season (October) (n=150), as well as those trapped during the dry season (February-March) (n=125) in the region using RT-PCR for the lipL32 gene, confirmed by 16S rRNA, as well as by the microscopic agglutination test (MAT). Results were compared with the serovar distribution of human cases referred from Ho Chi Minh City hospitals (2004-2012) confirmed by MAT (n=45). The MAT seroprevalence among rats was 18.3%. The highest MAT seroprevalence corresponded, in decreasing order, to: Rattus norvegicus (33.0%), Bandicota indica (26.5%), Rattus tanezumi (24.6%), Rattus exulans (14.3%), and Rattus argentiventer (7.1%). The most prevalent serovars were, in descending order: Javanica (4.6% rats), Lousiana (4.2%), Copenageni (4.2%), Cynopterie (3.7%), Pomona (2.9%), and Icterohaemorrhagiae (2.5%). A total of 16 rats (5.8%) tested positive by RT-PCR. Overall, larger rats tended to have a higher prevalence of detection. There was considerable agreement between MAT and PCR (kappa=0.28 [0.07-0.49]), although significantly more rats were positive by MAT (McNemar 29.9 (p<0.001). MAT prevalence was higher among rats during the rainy season compared with rats in the dry season. There are no current available data on leptospira serovars in humans in the Mekong Delta, although existing studies suggest limited overlapping between human and rat serovars. Further studies should take into account a wider range of potential reservoirs (i.e., dogs, pigs) as well as perform geographically linked co-sampling of humans and animals to establish the main sources of leptospirosis in the region.
Project description:BACKGROUND:Leptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and dogs, but prevalence of Leptospira shedding in dogs in Thailand is unknown. The aim of this study was to determine urinary shedding of Leptospira in dogs in Thailand, to evaluate antibody prevalence by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA), and to assess risk factors for Leptospira infection. In Northern, Northeastern, and Central Thailand, 273 stray (n?=?119) or client-owned (n?=?154) dogs from rural (n?=?139) or urban (n?=?134) areas were randomly included. Dogs that had received antibiotics within 4?weeks prior to sampling were excluded. No dog had received vaccination against Leptospira. Urine was evaluated by real-time polymerase chain reaction (PCR) specific for lipL32 gene of pathogenic Leptospira. Additionally, urine was cultured for 6?months in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Antibodies were measured by ELISA and MAT against 24 serovars belonging to 15 serogroups and 1 undesignated serogroup. Risk factor analysis was performed with backwards stepwise selection based on Wald. RESULTS:Twelve of 273 (4.4%; 95% confidence interval (CI): 2.0-6.8%) urine samples were PCR-positive. In 1/273 dogs (0.4%; 95% CI: 0.01-1.1%) Leptospira could be cultured from urine. MAT detected antibodies in 33/273 dogs (12.1%; 95% CI: 8.2-16.0%) against 19 different serovars (Anhoa, Australis, Ballum, Bataviae, Bratislava, Broomi, Canicola, Copenhageni, Coxi, Grippotyphosa, Haemolytica, Icterohaemorrhagiae, Khorat, Paidjan, Patoc, Pyrogenes, Rachmati, Saxkoebing, Sejroe). In 111/252 dogs (44.0%; 95% CI: 37.9-50.2%) immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies were found by ELISA. Female dogs had a significantly higher risk for Leptospira infection (p?=?0.023). CONCLUSIONS:Leptospira shedding occurs in randomly sampled dogs in Thailand, with infection rates comparable to those of Europe and the USA. Therefore, the potential zoonotic risk should not be underestimated and use of Leptospira vaccines are recommended.
Project description: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.
Project description:BACKGROUND: Leptospirosis is a zoonotic disease caused by the genus, Leptospira. Leptospira interrogans is the most common genomospecies implicated in the disease. Epidemiological investigations are needed to distinguish outbreak situations or to trace reservoirs of the organisms. Current methodologies used for typing Leptospira have significant drawbacks. The development of an easy to perform yet high resolution method is needed for this organism. METHODS: In this study we have searched the available genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 for the presence of tandem repeats. These repeats were evaluated against reference strains for diversity. Six loci were selected to create a Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) to explore the genetic diversity within L. interrogans serovar Australis clinical isolates from Far North Queensland. RESULTS: The 39 reference strains used for the development of the method displayed 39 distinct patterns. Diversity Indexes for the loci varied between 0.80 and 0.93 and the number of repeat units at each locus varied between less than one to 52 repeats. When the MLVA was applied to serovar Australis isolates three large clusters were distinguishable, each comprising various hosts including Rattus species, human and canines. CONCLUSION: The MLVA described in this report, was easy to perform, analyse and was reproducible. The loci selected had high diversity allowing discrimination between serovars and also between strains within a serovar. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made.