lipL32 Gene Research Articles

Leptospira Survey in Wild Boar (Sus scrofa) Hunted in Tuscany, Central Italy.

Leptospirosis is a re-emerging, worldwide zoonosis, and wild boar (Sus scrofa) are involved in its epidemiology as the reservoir. The aim of this study was to investigate the prevalence of Leptospira with serological, bacteriological, and molecular assays in wild boar hunted in Tuscany (Italy) during two hunting seasons. In total, 287 specimens of sera, kidneys, and liver were collected to perform microscopic agglutination tests (MATs), isolation, and RealTime PCR to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene), and saprophytic (23S rRNA gene) Leptospira. Within sera, 39 (13.59%) were positive to the MAT, and Australis was the most represented serogroup (4.88%), followed by Pomona (4.18%), and Tarassovi (3.14%). Moreover, four Leptospira cultures were positive, and once isolates were identified, one was identified as L. borgpetersenii serovar Tarassovi, and three as L. interrogans serovar Bratislava. Pathogenic Leptospira DNA were detected in 32 wild boar kidneys (11.15%). The characterization through the amplification of the rrs2 gene highlighted their belonging to L. interrogans (23 kidneys), L. borgpetersenii (four), and L. kirschneri (one), while nine kidneys (3.14%) were positive for intermediate Leptospira, all belonging to L. fainei. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis among wildlife in Central Italy.

Leptospira infection and shedding in dogs in Thailand

BackgroundLeptospirosis 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.ResultsTwelve 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).ConclusionsLeptospira 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.

Evaluation of real-time PCR targeting the lipL32 gene for diagnosis of Leptospira infection

BackgroundDifferent diagnostic methods have been used for the laboratory confirmation of leptospirosis. Molecular diagnostic techniques are not only faster and more sensitive than culture analysis, but can also detect a Leptospira infection before the appearance of antibodies. The aim of the present study was to analyze and compare two different PCR approaches applied to blood and urine specimens obtained from patients with clinical manifestations that were suggestive of leptospirosis. Furthermore, the results of these different PCR approaches were compared with the results of culture and serology analyses.ResultsA total of 400 samples (234 blood or 58.5% and 166 urine of 41.5%) from 310 Slovenian patients with clinical manifestations suggestive of leptospirosis were tested using conventional PCR assays targeting the rrs gene and RT-PCR targeting the lipL32 gene. Additionally, culture, serology and sequence analysis were performed for the majority of these samples. The PCR and RT-PCR results were concordant in 376 out of 400 of these samples (94.0%). Conventional PCR was positive for 27 out of 400 samples (6.8%) and RT-PCR was positive for 47 out of 400 samples (11.8%). Culture and microscopic agglutination tests supported these diagnoses.ConclusionsA comparison of the two PCR methods indicated that the RT-PCR targeting of the lipL32 gene was faster, more sensitive and more specific for the determination of Leptospira DNA in these clinical samples.

Diagnostic accuracy of genetic markers and nucleic acid techniques for the detection of Leptospira in clinical samples: A meta-analysis.

BackgroundLeptospirosis is often difficult to diagnose because of its nonspecific symptoms. The drawbacks of direct isolation and serological tests have led to the increased development of nucleic acid-based assays, which are more rapid and accurate. A meta-analysis was performed to evaluate the diagnostic accuracy of genetic markers for the detection of Leptospira in clinical samples.Methodology and principle findingsA literature search was performed in Scopus, PubMed, MEDLINE and non-indexed citations (via Ovid) by using suitable keyword combinations. Studies evaluating the performance of nucleic acid assays targeting leptospire genes in human or animal clinical samples against a reference test were included. Of the 1645 articles identified, 42 eligible studies involving 7414 samples were included in the analysis. The diagnostic performance of nucleic acid assays targeting the rrs, lipL32, secY and flaB genes was pooled and analyzed. Among the genetic markers analyzed, the secY gene showed the highest diagnostic accuracy measures, with a pooled sensitivity of 0.56 (95% CI: 0.50–0.63), a specificity of 0.98 (95% CI: 0.97–0.98), a diagnostic odds ratio of 46.16 (95% CI: 6.20–343.49), and an area under the curve of summary receiver operating characteristics curves of 0.94. Nevertheless, a high degree of heterogeneity was observed in this meta-analysis. Therefore, the present findings here should be interpreted with caution.ConclusionThe diagnostic accuracies of the studies examined for each genetic marker showed a significant heterogeneity. The secY gene exhibited higher diagnostic accuracy measures compared with other genetic markers, such as lipL32, flaB, and rrs, but the difference was not significant. Thus, these genetic markers had no significant difference in diagnostic accuracy for leptospirosis. Further research into these genetic markers is warranted.

Leptospiral shedding and seropositivity in shelter dogs in the Cumberland Gap Region of Southeastern Appalachia.

BackgroundLeptospirosis, caused by pathogenic Leptospira spp., is a zoonotic infection that affects humans, dogs and many other mammalian species. Virtually any mammalian species can act as asymptomatic reservoir, characterized by chronic renal carriage and shedding of a host-adapted leptospiral serovar. Environmental contamination by chronic shedders results in acquisition of infection by humans and susceptible animals.MethodsIn this study, we investigated if clinically normal shelter dogs and cats harbor leptospires in their kidneys by screening urine samples for the presence of leptospiral DNA by a TaqMan based-quantitative PCR (qPCR) that targets pathogen-associated lipl32 gene. To identify the infecting leptospiral species, a fragment of leptospiral rpoB gene was PCR amplified and sequenced. Additionally, we measured Leptospira-specific serum antibodies using the microscopic agglutination test (MAT), a gold standard in leptospiral serology.ResultsA total of 269 shelter animals (219 dogs and 50 cats) from seven shelters located in the tri-state area of western Virginia, eastern Tennessee, and southeastern Kentucky were included in this study. All cats tested negative by both qPCR and MAT. Of the 219 dogs tested in the study, 26/198 (13.1%, 95% CI: 8.4–17.8%) were positive for leptospiral DNA in urine by qPCR and 38/211 (18.0%, 95% CI: 12.8–23.2%) were seropositive by MAT. Twelve dogs were positive for both qPCR and MAT. Fourteen dogs were positive by qPCR but not by MAT. Additionally, leptospiral rpoB gene sequencing from a sub-set of qPCR-positive urine samples (n = 21) revealed L. interrogans to be the leptospiral species shed by dogs.ConclusionsThese findings have significant implications regarding animal and public health in the Cumberland Gap Region and possibly outside where these animals may be adopted.

Leptospirosis in Northern Sea Otters (Enhydra lutris kenyoni) from Washington, USA.

We diagnosed leptospirosis in six northern sea otters (Enhydra lutris kenyoni) that stranded on beaches in Washington State, US in 2002. Significant gross findings included cyanotic oral mucous membranes, renal swelling, congestion or pale streaks on the cut surface of renal lobules, hematuria, dehydration, lymphadenopathy, pulmonary congestion, and rarely adrenal hemorrhage and congestion. Histopathology showed lymphoplasmacytic tubulointerstitial nephritis with intraluminal spirochetes and immunoreactivity to leptospiral antigens in the renal tubules and interstitium. A quantitative polymerase chain reaction (qPCR) using kidney or urine for the leptospiral lipL32 gene was positive with cycle threshold values indicative of abundant or moderate amounts of nucleic acid. A microscopic agglutination test showed the highest serum antibody titer to serovar Pomona and positive titers to serovars Autumnalis, Bratislava, Hebdomadis, Grippotyphosa, Icterohaemorrhagiae, Pyrogenes, Ballum, Canicola, and Hardjo. Although antibodies to Leptospira interrogans have been previously detected in sea otters, this report describes the pathology of leptospirosis in diseased free-ranging sea otters.

Evidence of Leptospiral Presence in the Cumberland Gap Region.

BackgroundLeptospirosis is a widespread zoonotic disease that causes reproductive losses and/or hepatorenal failure in a number of animal species. Wild reservoirs of the disease, such as rodents, harbor the causative bacterium, Leptospira spp., in their kidneys and contaminate the environment by excreting infected urine. In this study, we tested small wild mammals, environmental water, and livestock in the Cumberland Gap region of southeastern Appalachia for the presence of pathogenic Leptospira or leptospiral antibodies.Methods/ResultsSmall wild mammals (n = 101) and environmental water samples (n = 89) were screened by a real time quantitative PCR that targets the pathogenic Leptospira-specific lipl32 gene. Kidneys from 63 small wild mammals (62.37%) and two water sources (2.25%) tested positive for leptospiral DNA. To identify the infecting leptospiral species in qPCR-positive water and kidney samples, a fragment of leptospiral rpoB gene was PCR amplified and sequenced. L. kirschneri and L. interrogans were the leptospiral species carried by small wild mammals. Furthermore, sera from livestock (n = 52; cattle and horses) were screened for leptospiral antibodies using microscopic agglutination test (MAT). Twenty sera (38.46%) from livestock had antibodies to one or more serovars of pathogenic Leptospira spp.ConclusionsIn conclusion, results from our study show exposure to leptospiral infection in farm animals and the presence of this zoonotic pathogen in the environmental water and kidneys of a significant number of small wild mammals. The public health implications of these findings remain to be assessed.

Molecular detection of Leptospira spp. in wild boar (Sus scrofa) hunted in Liguria region (Italy)

Leptospirosis is a re-emerging and widespread zoonosis, worldwide distributed, due to a wide variety of wild and domestic animal species able to act as natural or accidental hosts. During last years, in Europe, as in Italy, wild boar (Sus scrofa) population is increased. This animal represents a reservoir for different etiological agents, such as Leptospira. The aim of this investigation was to evaluate the prevalence of Leptospira spp. in wild boar hunted in Liguria region (Italy) during two-year hunting seasons. From 611 hunted wild boar, kidneys were collected. DNA was extracted from each organ and different targets were used to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene) and saprophytic (23S rRNA gene) Leptospira by Taqman-based RealTime-PCR assays. Overall, kidneys were sampled from 282 adults, 155 sub-adults and 174 young wild boar (in total 314 males and 298 females). By RealTime PCR 77 kidneys were positive and, among these, 74 resulted positive for pathogenic (96.10%) and 3 (3.90%) for intermediate Leptospira. No significant differences in pathogenic Leptospira infection ratio were detected between male (11.50%) and female (12.75%). Only 13 sub-adult animals (8.39%) resulted infected by pathogenic Leptospira; 23 young animals (13.22%) and 38 adult animals (13.47%) were positive. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis, which is able to infect other animal species (domestic and wild) including humans. Rarely, intermediate Leptospira could be able to infect wild boar with a renal localization that can contribute to their shedding and circulation.

Update on molecular diagnosis of human leptospirosis

Abstract Background Leptospirosis, caused by pathogenic Leptospira spp., is a widespread zoonotic disease worldwide. Early diagnosis is required for proper patient management and reducing leptospirosis morbidity and mortality. Objective To summarize current literature regarding commonly used and new promising molecular approaches to Leptospira detection and diagnostic tests of human leptospirosis. Method The relevant articles in Leptospira and leptospirosis were retrieved from MEDLINE (PubMed) and Scopus. Results Several molecular techniques have been developed for diagnosis of human leptospirosis. Polymerase chain reaction-based techniques targeting on either lipL32 or 16S rRNA (rrs) gene are most commonly used to detect leptospiral DNA in various clinical specimens. Whole blood and urine are recommended specimens for suspected cases in the first (acute) and the second (immune) phases, respectively. Isothermal amplification with less expensive instrument is an alternative DNA detection technique that may be suitable for resource-limited laboratories. Conclusion Detection of leptospiral DNA in clinical specimens using molecular techniques enhances sensitivity for diagnosis of leptospirosis. The efficient and robust molecular detection especially in the early leptospiremic phase may prompt early and appropriate treatment leading to reduced morbidity and mortality of patients with leptospirosis.

Nanofabricated versatile electrochemical sensor for Leptospira interrogans detection

In this report, a LipL32 gene based nanofabricated electrochemical sensor for the detection of Leptospira interrogans has been developed using carboxylated multiwalled carbon nanotubes with gold nanoparticles (c-MWCNTs/nanoAu) electrode and graphene quantum dots (GQDs). The c-MWCNTs/nanoAu electrode was linked to GQDs using 4-aminothiophenol (ATP). The surface modifications on the electrode surface were delineated using Raman spectroscopy and field emission scanning electron microscopy (FE-SEM). 5'-Amino (NH2) labeled single stranded DNA (ssDNA) probe was immobilized on the surface of c-MWCNTs/nanoAu/ATP/GQD composite electrode. The electrochemical changes of the developed sensor after hybridization with single stranded complementary DNA of L.interrogans were analyzed by differential pulse voltammetry (DPV) using 1mM methylene blue. The sensor showed good linearity with complementary ssDNA concentration ranging from 0.37 to 12ng/μl. The sensor exhibited high specificity to L.interrogans and showed good reproducibility when stored at 4°C.

PCR based genetic marker for the detection of Leptospira interrogans causing leptospirosis

Leptospirosis caused by Leptospira interrogans is a major health concern across the globe. It presents a wide range of clinical manifestations viz. fever, headache, nausea, vomiting and abdominal pain. If left untreated, it can lead to fatal complications. The current methods used for leptospirosis diagnosis are based on the detection of L. interrogans. The methods used for routine leptospirosis diagnosis and the agglutination test which is the gold standard method are time consuming and have shortcomings. Molecular methods such as polymerase chain reaction (PCR) are considered quick, and better in terms of sensitivity and specificity. Keeping in view the importance of PCR in disease diagnosis and pathogen detection the present study is aimed at PCR based detection of Leptospira interrogans using LipL 32 gene specific primers. The primers used in this study amplified partial region of the LipL32 gene under specific conditions. The DNA amplicon of 392 bp confirmed partial amplification of LipL32 gene and showed no homology with any other organism when checked by BLAST. This method seems to be quick, specific and more sensitive as compared to other conventional methods.

Detection of Leptospira spp. using polymerase chain reaction technique from kidney of Rattus norvegicus from Grenada, West Indies

Aim: This study aimed to find out the prevalence of active infection of Leptospira spp. in Rattus norvegicus from Grenada, West Indies, through polymerase chain reaction (PCR). Materials and Methods: One hundred and forty-nine rats were trapped, anesthetized and their kidneys collected aseptically. DNA was extracted from the kidney tissue of each rat. PCR was performed targeting LipL32 gene. Eighteen PCR-positive amplicons for LipL32 gene segment were purified and sent for direct sequencing to the sequencing facility of MCLAB (South San Francisco, USA). Results of sequencing were read and interpreted. The prevalence of Leptospira spp. in relation to sex and age was also recorded. Results: All amplified sequences were compared to the sequences present in GenBank using basic local alignment search tool (BLAST) from the online website National Center for Biotechnology Information, the results revealed that six samples had similarity to Leptospira interrogans strain 1399/2016 and eight samples had similarity with Leptospira borgpetersenii serovar Hardjo-bovis strain L49. Of 149 kidney samples, only 14 were positive for Leptospira spp. by PCR giving an incidence of 9.3%. There was no significant difference found in relation to sex and age. Conclusion: This is the first report confirming active infection of Leptospira spp. in Rattus norvegicus in Grenada using PCR. The presence of active infection in rats can be considered as high risk for humans. Further research to understand the epidemiology of leptospirosis in Grenada is suggested.

First record of Leptospira and Blastocystis infections in captive flying squirrels (Trogopterus xanthipes) from Enshi County, China

In traditional Chinese medicine, the feces of flying squirrels have long been used to promote blood circulation and relieve bodily stasis. However, the excrement of flying squirrels may harbor zoonotic agents that could be hazardous to public health. To understand the occurrence of bacterial and parasitic infections in this species, we investigated selected zoonotic pathogens including Leptospira and Blastocystis in the urine and feces of flying squirrels in China. Urine and fecal samples from flying squirrels were collected from a family-owned flying squirrel farm located in Enshi County, Hubei Province in China. Leptospira and Blastocystis DNA was extracted from the urine and feces of flying squirrels, and used as targets for PCR amplification, using different specific primers. PCR amplification and DNA sequencing showed that 4.4% (3/69) of flying squirrels were positive for Leptospira, while 30.4% (21/69) of the animals were positive for Blastocystis. Notably, 1.4% (1/69) of flying squirrels were found to be co-infected with Leptospira and Blastocystis. Sequence analyses allowed for the detection of 3 Blastocystis subtypes (ST1, ST3 and ST13), and mixed infections of Blastocystis subtype 1 and subtype 3 were found in 4.4% (3/69) of flying squirrels. Phylogenetic analysis of the 16S ribosomal RNA gene (rrs2), the flagellin B gene (flaB), and outer membrane lipoprotein lipL32 gene (LipL32) sequences indicated that the Leptospira species detected in the study was L. interrogans. We concluded that flying squirrels from central China were infected with Leptospira and Blastocystis, suggesting that these animals can be a source of infection for their owners, and using fresh excrement from this animal as traditional medicine could be risky to human health. To the best of our knowledge, this is the first report of Leptospira and Blastocystis infection in flying squirrels from Enshi County, China. Our findings provide new data on the epidemiology of these pathogens in this region.

Leptospira detection in flood-prone environment of Jakarta, Indonesia.

The study about Leptospira, particularly pathogenic strain, was conducted in the flood-prone area in the Special Capital Region of Jakarta. The aim of this study was to discover and identify the serovars of pathogenic Leptospira in the environment, which might infect human during flooding. Seventy-three samples, consisted of 36 samples of environmental water and 37 samples of soil, were collected from 5 districts of Jakarta. Their pH was measured, and the samples were then cultured in a modified Korthof's medium with 5-fluorouracil (5-FU) addition. Polymerase chain reaction, targeted on 23S rDNA (rrl), FlaB and LipL32 genes, was performed to identify Leptospira genus and differentiate the pathogenic. Identification of the pathogenic Leptospira was done utilising DNA sequencing. Seven samples showed amplification of rrl-gene. flaB and lipl32-PCR assay indicated one positive amplification band, each. Confirmation of flaB and lipl32 amplicons by DNA sequencing and BLAST analysis showed flaB amplicon (G1B; GenBank accession number MK006031.1) had 94% similarity with L.licerasiae (LC005426.1), while lipl32 amplicon was not identified as lipl32 of Leptospira. Based on those results, one intermediate pathogenic and six saprophytic Leptospira were obtained from the environment in Jakarta.

The prevalence of Leptospira among invasive small mammals on Puerto Rican cattle farms.

Leptospirosis, an emerging infectious disease caused by bacteria of the genus Leptospira, is thought to be the most widespread zoonotic disease in the world. A first step in preventing the spread of Leptospira is delineating the animal reservoirs that maintain and disperse the bacteria. Quantitative PCR (qPCR) methods targeting the LipL32 gene were used to analyze kidney samples from 124 House mice (Mus musculus), 94 Black rats (Rattus rattus), 5 Norway rats (R. norvegicus), and 89 small Indian mongooses (Herpestes auropunctatus) from five cattle farms in Puerto Rico. Renal carriage of Leptospira was found in 38% of the sampled individuals, with 59% of the sampled mice, 34% of Black rats, 20% of Norway rats, and 13% of the mongooses. A heterogeneous distribution of prevalence was also found among sites, with the highest prevalence of Leptospira-positive samples at 52% and the lowest at 30%. Comparative sequence analysis of the LipL32 gene from positive samples revealed the presence of two species of Leptospira, L. borgpetersenii and L. interrogans in mice, detected in similar percentages in samples from four farms, while samples from the fifth farm almost exclusively harbored L. interrogans. In rats, both Leptospira species were found, while mongooses only harbored L. interrogans. Numbers tested for both animals, however, were too small (n = 7 each) to relate prevalence of Leptospira species to location. Significant associations of Leptospira prevalence with anthropogenic landscape features were observed at farms in Naguabo and Sabana Grande, where infected individuals were closer to human dwellings, milking barns, and ponds than were uninfected individuals. These results show that rural areas of Puerto Rico are in need of management and longitudinal surveillance of Leptospira in order to prevent continued infection of focal susceptible species (i.e. humans and cattle).

Leptospira in Bats from Hubei Province, China, 2018

To understand the potential role of bats in the epidemiology of Leptospira, we investigated the frequency of Leptospira in bats from Central China in 2018. Sixty bats belonging to four species of two families were captured from rural areas of Xianning City, Hubei Province, China. We extracted DNA from the kidneys of bats, and Leptospira spp. were determined by PCR amplification of the rrs2, flaB, lipL32, and secY genes. We found that 57% (34/60) of bats were positive for Leptospira with at least one of the four genes, and the positive rate of Leptospira in bats was 45% (27/60) with rrs2, 50% (30/60) with flaB, 15% (9/60) with lipL32, and 27% (16/60) with secY. Phylogenetic analysis indicated that bats were infected with two species of Leptospira, including Leptospira interrogans and Leptospira borgpetersenii. A high prevalence of Leptospira spp. in bats suggested that bats were important carriers of Leptospira in China.

Neotropical wild rodents Akodon and Oligoryzomys (Cricetidae: Sigmodontinae) as important carriers of pathogenic renal Leptospira in the Atlantic forest, in Brazil

Leptospirosis is an important worldwide zoonosis, caused by a bacterium of the genus Leptospira. For a better understanding of the disease, it is relevant the application of the one health concept. The Atlantic Forest is considered a biodiversity hotspot, with a great endemism of species and despite its importance and proximity to urban areas, the potential role of its fauna as carriers of infectious agents is still poorly understood. Although it is well-known that rats and mice are key reservoirs of leptospires, particularly Rattus norvegicus, wild rodents have also been revealed as reservoirs of leptospiral strains. Thus, the purpose of this study was to investigate the role of the sigmodontine rodents from genera Akodon and Oligoryzomys as pathogenic Leptospira carriers in the Atlantic Forest. We studied 52 Akodon spp. and 15 Oligoryzomys spp. from three areas. Overall 30% were PCR-positive for pathogenic Leptospira, 27% (14/52) Akodon spp. and 40% (6/15) Oligoryzomys spp. DNA sequencing of LipL32 gene confirmed nine species as pathogenic Leptospira. This remarkable incidence of leptospiral carriage within wild genera emphasizes the role of these rodents as carriers of leptospires throughout in this environment.

Identification of Leptospira spp. from environmental sources in areas with high human leptospirosis incidence in the Philippines

ABSTRACTLeptospira is the causative agent of leptospirosis, which is considered an emerging major threat to public health due to its increasing frequency reported worldwide. In the Philippines, the prevalence of the disease continuously rises, particularly in urban areas. Because leptospirosis is commonly transmitted through contact with contaminated environment, water and soil samples were collected in regions in the Philippines where high incidence of human leptospirosis cases was reported recently. Of the 54 samples screened for the presence of Leptospira, 35% were found positive through 23S rRNA gene PCR-based detection. None were found positive when primers targeting lipL32, lipL41, and ompL1 genes were used. Most of these isolates were collected from rural areas. However, 16S rRNA gene sequencing identified all isolates to be L. yanagawae and L. meyeri, which are nonpathogenic. Despite the lack of evidence of the presence of pathogenic species in the environmental sources, the results still suggest that leptospires persist in these areas. These data are crucial for environmental monitoring and identification of contaminated areas where humans may be at risk of infection.

First Real-Time PCR in Morocco for Human Leptospirosis Using TaqMan Probes Targeting the LipL32 Gene

Background: Currently, qPCR has been used as a rapid diagnostic method for human leptospirosis. Previous studies have indicated that qPCR has high sensitivity in the early days of the illness. Objectives: The aim of this study was to evaluate qPCR as a diagnostic method for human leptospirosis in the National Institute of Hygiene, Rabat, Morocco. Methods: From 2004 to 2016, 67 sera related to 67 patients with clinical signs mimic to leptospirosis were sent to the laboratory of Bacteriology for routine diagnosis and confirmation. SAT, ELISA IgM, ELISA IgG, and qPCR were used for the diagnosis. Results: High positivity was observed by SAT (88.24%), ELISA IgM (58.82%), and real-time PCR (17.64%), in sequence. No negative results by serological tests had positive results by real-time PCR. Forty-six patients were males (68.68%) and 21 were females (31.34%). The high incidence observed was from Sidi Qacem (40%). Conclusions: SAT and ELISA IgM are useful for the diagnosis of human leptospirosis in Morocco and they can provide prompt and low-cost diagnosis, especially when resources are limited.

Pathogenic Leptospira species are widely disseminated among small mammals in Atlantic Forest biome.

Leptospirosis is a common worldwide bacterial zoonosis and has been studied in One Health approaches. Small mammals are described as the most important maintenance reservoirs of several pathogens in nature, including leptospires. The aim of this study was to identify infection by leptospires among small mammals on the Atlantic forest biome and evaluate their potential as carriers of these spirochetes. A total of 153 small mammals belonging to orders Rodentia and Didelphimorphia (distributed on 17 genera and 22 species) were captured. Blood and kidney samples were collected from animals and a conventional PCR targeted on lipL32 gene was conducted on renal tissues. Species identification was performed in eight samples by sequencing of rrs gene. A total of 28% of the animals presented lipL32 PCR-positive, and four pathogenic Leptospira species (L.interrogans, L.borgpetersenii, L.santarosai and L.noguchii) were identified. This study highlights the role of small mammals as carriers of leptospires on the Atlantic Forest representing a potential source of pathogenic Leptospira spp infection for both humans and domestic animals.