Leptospira strains isolated from cattle in the Amazon region,Brazil, evidence of a variety of species and serogroups with a high frequency of the Sejroe serogroup

In Brazil, there have been few leptospires isolated from cattle, especially in the Amazon, implying that the epidemiology of the disease in this region is still largely unclear. In a previous study, 52 Leptospira isolates were obtained from urine of cattle raised in the Brazilian Amazon and, to achieve a greater understanding of Leptospira infection in cattle of this region, the present study aimed to serologically and molecularly characterizes all these isolates. The laboratory assays used were the microscopic agglutination test (MAT) adopting a panel of polyclonal antisera against Leptospira spp. for serogrouping the isolates, DNA sequencing (secY) and multiple locus variable number tandem repeat analysis (MLVA). The isolates belonged to five species: 20/52 were identified as L. borgpetersenii (38.5 %); 18/52 as L. kirschneri (34.6 %); 9/52 as L. santarosai (17.3 %); 3/52 as L. noguchii (5.8 %) and 2/52 as L. interrogans (3.8 %). With serogrouping, nine different serogroups were detected, with a high frequency of the Sejroe serogroup. MLVA showed that all L. borgpetersenii isolates had a profile compatible with serovar Hardjo; moreover, the other isolates demonstrated a diversity of patterns, and some of them may represent strains not yet characterized. In the Brazilian Amazon, the leptospires circulating in cattle revealed the unique aspects of infections in this area which, in addition to a variety of strains, were characterized by a high frequency of the Sejroe serogroup, highlighting the serovar Hardjo, which has not been reported in other regions of Brazil.     

Annual Variations in <Emphasis Type="Italic">Leptospira</Emphasis> Seroprevalence among Sows in Southern Vietnam

A serological survey was conducted among sows in the Mekong delta in southern Vietnam in 1999 to investigate variations in leptospiral Seroprevalence over a one-year period. In this region, leptospirosis is endemic and a high leptospiral Seroprevalence has been shown in the pig population. In this study, the serology of six Leptospira serovars was analysed by the microscopic agglutination test for 429 sows at five large-scale state farms sampled during the dry period, the rainy period and the early dry period. The serovars included were L. interrogans serovar (sv) autumnalis strain Akiyama A, L. interrogans sv bratislava strain Jez, L. interrogans sv icterohaemorrhagiae strain Kantorowicz, L. interrogans sv pomona strain Pomona, L. borgpetersenii sv tarassovi strain Perepelitsin, and L. kirschneri sv grippotyphosa strain Duyster. Variations in Seroprevalence over the year were found for sv bratislava and sv icterohaemorrhagiae: the Seroprevalence was higher during the dry period compared with the rainy period (p = 0.07 and p = 0.005, respectively) and the early dry period (p = 0.00006 and p = 0.0006, respectively). It is concluded that in regions where water is constantly abundant and where animals are exposed to the outdoor environment all year round there are highly significant variations in leptospiral Seroprevalence over the year.     

Experimental Leptospira interrogans Serovar Kennewicki Infection of Horses

Background: Little information is available about experimental induction of leptospirosis in horses. Objectives: Determine serologic, hematologic responses of horses to Leptospira interrogans serovar Kennewicki infection. Animals: Four adult horses seronegative for leptospirosis. Methods: Experimental and observational study. Horses were challenged with an equine isolate of L. interrogans serovar Kennewicki at 2 different doses and different inoculation sites. After challenge, the horses were monitored for 60 days. Blood, urine, and aqueous humor samples were collected at intervals until euthanasia 60 days after infection. Results: Pyrexia (39.3–40°C) occurred as early as 1 day after challenge with 10 × 10⁸Leptospira divided equally between topical ocular and intraperitoneal injection in 2 horses. Leptospires were recovered from the blood and urine but not from the aqueous humor of the 2 febrile horses. The sera of all 4 challenged horses developed microscopic agglutination test antibody after challenge and remained relatively constant for 21 days. Titer to cross‐reacting strains declined earlier than titer to the challenge strain. Conclusions: Clinical disease in experimentally infected horses can be mild or inapparent in Leptospira infected horses. Repeated serologic testing can allow recognition of the infecting serovar. In febrile horses, Leptospira can be isolated from blood while isolation from the urine can occur after fever has subsided.     

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospiraborgpetersenii, and Leptospirakirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5′ variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK.     

Monoclonal antibodies against the leptospiral immunoglobulin-like proteins A and B conserved regions

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira that affects humans and a wide variety of animals. Recently the genomes of Leptospira interrogans, Leptospira borgpetersenii and Leptospira biflexa species were sequenced allowing the identification of new virulence factors involved in survival and pathogenesis of bacteria. LigA and LigB are surface-exposed bacterial adhesins whose expression is correlated with the virulence of Leptospira strains. In this study, we produced and characterized five monoclonal antibodies (MAbs) against a recombinant fragment of LigB (rLigBrep) with approximately 54 kDa that comprise the portions of LigA and LigB (domains 2–7). The 5 MAbs obtained were of the IgG1 (2) and IgG2b (3) isotypes and their affinity constants for rLigBrep ranged from 7 × 10⁷ M⁻¹ to 4 × 10⁸ M⁻¹. The MAbs were able to react with the native antigen on the L. interrogans, L. borgpetersenii and Leptospira noguchii surfaces by indirect immunofluorescence, immunoblotting and immunoelectron microscopy. These results demonstrate that the MAbs anti-rLigBrep can be useful to complement genetic studies and to aid studies aiming understanding the role of Lig proteins in Leptospira pathogenesis and the development of Lig-based vaccines and improved diagnostic tests for leptospirosis.     

Multidisciplinary approach in the diagnosis of acute leptospirosis in dogs naturally infected by Leptospira interrogans serogroup Icterohaemorrhagiae: A prospective study

Leptospirosis, a zoonotic disease with worldwide distribution, is caused by spirochetes of the genus Leptospira. In dogs, this disease is frequently misdiagnosed. Few studies have attempted to associate the detection of Leptospira spp. infection with clinicopathological and renal histopathological findings using a multidisciplinary approach. The present study isolated and characterized Leptospira spp. obtained from naturally infected dogs and described relevant clinical and histopathological findings. Blood and urine were collected from 57 dogs with clinical symptomatology suggestive of leptospirosis; 38 cases were confirmed by PCR in urine or by culture or microscopic agglutination testing (titers ≥800). A total of 12 strains of pathogenic Leptospira were isolated from the studied dogs (seven in blood, four in urine and one in both blood and urine samples). All isolates were characterized as Leptospira interrogans serogroup Icterohaemorrhagiae. Of the confirmed cases, almost one-third of the animals had been vaccinated. Our analysis of laboratory testing revealed that azotemia and proteinuria were statistically significant predictors of infection. The main histopathological findings seen in kidney tissues were necrosis, degeneration, tubular regeneration, mononuclear inflammatory infiltrate and congestion. A multidisciplinary approach involving clinicopathological and histopathological characterization of renal involvement can aid in the identification of acute leptospirosis infection.     

Detection and characterization of Leptospira spp. in dogs diagnosed with kidney and/or liver disease in Selangor,Malaysia

Leptospirosis is a serious bacterial disease that affects both humans and animals. A wide range of symptoms have been described in humans; the disease in dogs is commonly associated with kidney and/or liver disease. In Malaysia, information about the common serovars infecting dogs is limited. Therefore, we investigated the occurrences of leptospirosis in 124 pet dogs diagnosed with kidney and/or liver disease. Blood, urine, abdominal effusion, and/or kidney and liver were collected from the dogs. Based on microscopic agglutination testing, 53 of 124 (42.7%) dogs were seropositive for leptospiral exposure. Sera were frequently positive to serovars Bataviae (n = 12), Javanica (n = 10), and Icterohaemorrhagiae (n = 10). Direct detection using PCR showed that 42 of 124 (33.9%) of the whole blood and 36 of 113 (31.9%) urine samples were positive for pathogenic Leptospira spp. By PCR, 2 of 23 (9.1%) kidney and 2 of 23 (9.1%) liver were positive for pathogenic Leptospira spp. Abdominal effusion from 4 dogs were PCR-positive for pathogenic Leptospira spp. The species detected were L. interrogans, L. borgpetersenii, L. kirschneri, and L. kmetyi by partial 16S rRNA sequencing. We further identified and characterized 11 Leptospira spp. isolates from 8 dogs as serovars Bataviae, Javanica, and Australis. The mortality rate of the Leptospira-infected dogs was high (18 of 53; 34%).     

Genomic characterization and comparative analysis of Leptospira kirschneri serogroup Grippotyphosa UC5/2011, a strain isolated after mare abortion: Implications for genital animal leptospirosis

The genome of a Brazilian strain of Leptospira kirschneri serogroup Grippotyphosa isolated from a mare post-abortion was sequenced and analyzed. High symmetrical identity and few structural differences were found when compared with a European strain of the same serogroup, L. kirschneri serovar Valbuzzi strain 200702274. Genes associated with virulence and antimicrobial resistance were found. Knowledge of the virulence evolution of Leptospira remains limited, especially in diseases of the reproductive sphere. We highlight the importance of virulence studies in the sphere of genital leptospirosis.     

Prevalence of antibodies against Leptospira sp. in snakes,lizards and turtles in Slovenia

Background

Leptospiral infections in poikilothermic (cold blooded) animals have received very little attention and the literature concerning natural infections of these animals is limited. The aim of this study was to determine the prevalence of leptospiral antibodies in reptiles, imported into Slovenia and intended to be pets in close contact with humans. A total of 297 reptiles (22 snakes, 210 lizards and 65 turtles) were tested for specific antibodies against serovars of Leptospira interrogans sensu stricto using the microscopic agglutination test (MAT). Live cultures of different serovars were used as antigens. MAT was performed according to standard procedures and the degree of reaction was interpreted by estimating the percentage of agglutinated leptospires. Samples showing titres of ≥ 50 against one or more serovars were considered as positive.

Results

Antibodies against seven pathogenic serovars of L. interrogans sensu stricto were detected in 46 of 297 reptiles. Among 22 snakes, specific antibodies against pathogenic serovars of three Leptospira species (L. interrogans, L. kirschneri and L. borgpetersenii) at titre levels from 1:50 to 1:400 were detected in 6 snakes. In 31 of 210 lizards, specific antibodies were found in titres from 1:50 to 1:1000 and, finally, among 65 turtles (terrapins and tortoises), 9 had specific antibodies at titre levels between 1:50 and 1:1600. Animals imported from non-EU countries showed significantly higher prevalence (25.0%; 95 confidence interval: 16.7–33.3%) than animals from EU member states (10.4%; confidence interval: 6.1–14.7%).

Conclusions

Reptiles may be considered as potential reservoirs of L. interrogans sensu stricto. Origin of the animals is a risk factor for presence of leptospiral antibodies, especially in lizards. Special attention should be focused on animals from non-EU member states.     

Detection of Leptospira in urine using anti-Leptospira-coated gold nanoparticles

Serological assays for antibody detection have been widely used for Leptospirosis diagnosis. However, antibody is usually undetectable during the first week after infection. Detection of Leptospira DNA can be done by PCR but this technique requires special equipments and the cost is still relatively high. Here we demonstrate that gold nanoparticles can be used to facilitate Leptospira detection. Gold nanoparticles were coated with rabbit antibody specific to Leptospira interrogans serovar Bratislava and these coated particles were used to detect Leptospira in urine. Agglutination of gold particles indicated the presence of Leptospira in samples tested. The sensitivity of detection was 10 leptospires/ml. No agglutination was observed when anti-Leptospira-coated particles were tested with urine containing the organisms commonly found in urine such as Klebsiella pneumoniae, Escherichia coli and Enterococcus faecalis. This assay is very easy to perform and results could be observed with the naked eyes. Fresh or frozen urine samples could be used. The stability of antibody-coated particles was at least 2 months when kept at 4 °C. In conclusion, we have demonstrated that the technique using antibody-coated gold nanoparticles is a promising tool for further validation as a rapid assay for Leptospirosis diagnosis.     

Didelphis albiventris as a carrier of Leptospira sp. in the central nervous tissue in the semiarid region of Northeast,Brazil

Leptospirosis has been investigated in several species of wild animals. The white-eared opossum (Didelphis albiventris) is a mammal common in the brazilian semi-arid, so, this study aimed to investigate its role in the occurrence of the leptospirosis in the region Northeast of Brazil. 12 animals were used, from which samples were collected for the attempt of isolation, molecular detection and serological examination. There was no microbial growth, nor were any anti-Leptospira sp. antibodies found in the serological samples. The PCR detected leptospiric DNA in the central nervous system (CNS) of five animals (41.7 %). The gene in one of the samples was sequenced and showed identity with Leptospira interrogans. The presence of Leptospira sp. in the CNS of Didelphis albiventris does not allow the characterization of the studied animals as reservoirs with potential for transmission of the pathogen in the region, however it represents a site that needs to be further investigated.     

Leptospira interrogans at the Human–Wildlife Interface in Northern Botswana: A Newly Identified Public Health Threat

Leptospirosis is the most widespread zoonosis in the world. In northern Botswana, humans live in close proximity to a diversity of wildlife and peridomestic rodents and may be exposed to a variety of zoonotic pathogens. Little is known regarding the occurrence and epidemiology of L. interrogans in Africa despite the recognized global importance of this zoonotic disease and the threat it poses to public health. In Botswana, banded mongooses (Mungos mungo) live in close proximity to humans across protected and unprotected landscapes and may be a useful sentinel species for assessing the occurrence of zoonotic organisms, such as L. interrogans. We utilized PCR to screen banded mongoose kidneys for leptospiral DNA and identified 41.5% prevalence of renal carriage of L. interrogans (exact binomial 95% CI 27.7–56.7%, n = 41). Renal carriage was also detected in one Selous' mongoose (Paracynictis selousi). This is the first published confirmation of carriage of L. interrogans in either species. This is also the first report of L. interrogans occurrence in northern Botswana and the only report of this organism in a wildlife host in the country. Pathogenic Leptospira are usually transmitted indirectly to humans through soil or water contaminated with infected urine. Other avenues, such as direct contact between humans and wildlife, as well as consumption of mongooses and other wildlife as bushmeat, may pose additional exposure risk and must be considered in public health management of this newly identified zoonotic disease threat. There is a critical need to characterize host species involvement and pathogen transmission dynamics, including human–wildlife interactions that may increase human exposure potential and infection risk. We recommend that public health strategy be modified to include sensitization of medical practitioners to the presence of L. interrogans in the region, the potential for human infection, and implementation of clinical screening. This study illustrates the need for increased focus on neglected zoonotic diseases as they present an important threat to public health.     

Pathogenic Leptospira spp. in bats: Molecular investigation in Southern Brazil

The present study aimed to investigate the frequency of pathogenic Leptospira spp. in Brazilian bats and to determine possible risk factors associated to it. Ninety two bats of 12 species were evaluated. Whole genomic DNA from kidneys was extracted and real-time PCR specific to pathogenic Leptospira spp. was applied. Association between the frequency of specimens positive for Leptospira spp. and sex, age, bat species or family, season of collection, geographic localization and feeding habits was evaluated. The results showed that 39.13% of analyzed bats were found positive for Leptospira spp. Nine bat species had at least one positive result. There was no association among the evaluated variables and frequency of pathogenic Leptospira spp. Although the limitations due to lack of Leptospira spp. isolation, leptospiral carriage was demonstrated in bats of different species from southern Brazil, which reinforces the need for surveillance of infectious agents in wild animals.     

Seroprevalence of Leptospira spp. in Working Horses Located in the Central Region of Chile

Urban working horses live in close contact with their owners. They are usually kept in periurban areas of big cities and cohabit with other animals under precarious sanitary conditions, whereas army horses are kept under controlled management and work. These characteristics leave urban working horses in higher risk of exposure to Leptospira spp. and could become a zoonotic risk for their owners. The aim of this study was to determine the frequency of seropositive working horses to diverse serovars of Leptospira spp. and compare them to a group of army horses. The microscopic agglutination test was used to assess the serum of 426 horses (160 working horses and 266 army horses) against two serovars of Leptospira borgpetersenii (Hardjo and Ballum) and four of Leptospira interrogans (Pomona, Canicola, Icterohaemorrhagiae, and Autumnalis). In the urban working horses group, 30.63% of horses were positive to at least one serovar at titers above 1:100, whereas 23.31% of the army horses were positive. The most frequent serovar in the working horse group was Ballum followed by Canicola, whereas in the army group was Autumnalis followed by Ballum. The serovars Hardjo, Pomona, and Icterohaemorrhagiae were not present in the army horses, whereas all serovars studied were detected in urban working horses. Although no horses studied presented clinical signs of leptospirosis, the study confirms exposure to Leptospira spp. and the importance of studying in more detail the livelihood conditions in which working horses are kept and possible risk of transmission to their owners.     

Prevalence of Leptospira interrogans antibodies in free-ranging Tayassu pecari of the Southern Pantanal, Brazil, an ecosystem where wildlife and cattle interact

We surveyed a wild population of white-lipped peccaries (Tayassu pecari) in the Brazilian Pantanal for evidence of Leptospira interrogans. Serum samples from 71 free-ranging T. pecari were obtained between 2003 and 2005 in the southern Pantanal of Mato Grosso do Sul state. We used microscopic microagglutination to test for antibodies against 14 L. interrogans serovars (antibody titers ≥1:100 were considered seropositive). Seventy percent of captured animals tested positive for leptospirosis antibodies. Antibodies against icterohaemorrhagiae and autumnalis serovars were the most prevalent. We used log-linear analyses to test for associations among seropositivity, age class, and sex of captured animals. Seropositivity was strongly associated with animal age class, but independent of sex. Forty-six percent of animals less than 2 years old, 63% of adults during peak reproductive years, and 100% of the oldest age class were seropositive. A nonparametric multivariate procedure (MRPP) showed that the composition of serovar antibody types changed with age, and ANOVA models demonstrated that antibody titers increased with age, suggesting long-term exposure to a greater number and variety (i.e., serovar types) of L. interrogans infections. This study presents the first quantitative survey of antibodies against L. interrogans serovars in a T. pecari population of the Pantanal. The high prevalence of leptospirosis antibodies in free-ranging white-lipped peccaries and the potential impacts on reproduction and population dynamics emphasize the need for further studies investigating the roles of Pantanal wildlife and livestock in the transmission and maintenance of L. interrogans in the environment.     

Molecular genotyping by pulsed-field gel electrophoresis of restricted genomic DNA of strains of taylorella equigenitalis isolated in Ireland and in the United States

The profiles, after digestion with ApaI or NotI and pulsed-field gel electrophoresis (PFGE), of genomic DNA from 18 strains of Taylorella equigenitalis isolated in Ireland were of three different types. The 13 strains in one of these types gave PFGE profiles identical to that of an American prototype strain, Kentucky 188, but different from strain NCTC11184^T and from a strain isolated in Japan.Eight additional strains isolated in the United States gave four distinct types of genomic PFGE profiles. All four types were different from that of T. equigenitalis NCTC11184^T or that of the Japanese strain. The profile of three strains of one type was identical to that of Kentucky 188.     

Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil

The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries.     

Molecular Characterization by LSSP‐PCR and DNA Sequencing of a Pathogenic Isolate of Leptospira interrogans from Brazil

We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31–21%), Hardjo Norma (46–40%), Hardjo hardjobovis (18–10%), Mini (8–4%) and Wolffi (7–4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three‐dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single‐specific primer polymerase chain reaction (LSSP‐PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.