Leptospira Isolates Research Articles

AVALIAÇÃO DE MÉTODOS DE DESCONTAMINAÇÃO PARA OBTENÇÃO DE CULTURAS PURAS DE LEPTOSPIRAS A PARTIR DE AMOSTRAS MISTAS COM Spirillum spp.

It is challenging to obtain pure cultures of Leptospira from environmental samples, mainly due to the presence of accompanying microbiota that can contaminate cultures, highlighting bacteria of the genus Spirillum that have characteristics similar to leptospires. Thus, cannot be eliminated by the most common decontamination techniques during leptospires isolation process. The purpose of this study was to evaluate five decontamination methods to obtain pure cultures of Leptospira from mixed samples with Spirillum spp. We collected ten samples from untreated domestic sewage dumped of to streets in Macapa city – Brazilian Amazon, and submitted to bacteriological culture for Leptospira isolation. The presence of leptospires was confirmed by visualization under dark-field microscopy and by 16S PCR. In the samples in which Spirillum was observed, five methods of decontamination were tested: 1 – Membrane filtration (0.20 μm and 0.22 μm); 2- Culture in selective medium for Leptospira spp.; 3- Centrifugation; 4- Serial dilution and 5- Plating on solid EMJH medium. Out of all 10 samples, five were positive for leptospires and in three there was the simultaneous detection of leptospires and Spirillum . Plating on solid EMJH medium (5) was the most efficient method, decontaminating all three mixed cultures. Only one culture was decontaminated by the technique of culture in selective medium for Leptospira spp. (2), and the other methods were ineffective. In conclusion, plating on solid EMJH medium method can be used to obtain pure cultures of Leptospira spp. from environmental samples containing Spirillum spp. Keywords: Leptospira; decontamination; mixed cultures; Spirillum .

Influence of Selective Agents (EMJH-STAFF), Sample Filtration and pH on Leptospira interrogans Serovar Icterohaemorrhagiae Cultivation and Isolation from Swine Urine.

Leptospira spp. cause the zoonotic disease leptospirosis, which occurs in numerous mammalians worldwide. Isolation is still important for serotyping and genotyping of Leptospira, which in turn is essential for epidemiological surveillance of leptospirosis and the development of diagnostic tests and vaccines. However, isolation of Leptospira from clinical specimens is inherently insensitive. This study was conducted to examine the influence of selective agents, sample filtration, sample pH and the use of phosphate buffered saline (PBS) buffer for sample storage to improve the success of cultivation and isolation of Leptospira interrogans serovar Icterohaemorrhagiae from swine urine. EMJH (Ellinghausen McCullough, Johnson and Harris) medium including the selective agents sulfamethoxazole, trimethoprim, amphotericin, fosfomycin and 5-fluorouracil (STAFF) increased the success of Leptospira isolation from spiked swine urine samples. Sample filtration yielded only negative results. Isolation in EMJH-STAFF was successful from swine urine with a density as low as 104 Leptospira/mL, and urine with pH ≤ 7 impaired the cultivation rate. Cultivation and isolation were not improved by the addition of PBS to spiked urine samples prior to storage for 24 h at 4 °C. The results of the study demonstrate that cultivation and isolation of leptospires from swine urine can be improved by enhanced methods.

Isolation of an atypical Leptospira strain assigned to the Sejroe serogroup from a water buffalo in Brazil

The isolation of leptospires from buffaloes worldwide is still limited to a few strains. Thus, the aim of this study was to describe the first Leptospira isolate from buffalo urine, assigned to the Sejroe serogroup, which does not belong to the Wolffi subgroup, traditionally isolated in Brazil. A total of 244 urine samples of water buffaloes (Bubalus bubalis) raised in the Brazilian Amazon were subjected to bacteriological culturing and polymerase chain reaction (PCR) for the detection of leptospires. The obtained isolate was characterized by serogrouping using polyclonal antibodies, partial DNA sequencing, Hardjo-Bovis-specific PCR, multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) and experimental infection in hamsters. PCR was performed on the urine samples; 11/244 were positive (4.5 %) for Leptospira, and only one isolate was recovered (0.4 %). Regarding characterization, the isolate was assigned to the Sejroe serogroup with high titers (12,800) for the Saxkoebing and Sejroe serovar antisera. The isolate was negative for Hardjo-Bovis-specific PCR, and the species Leptospira borgpetersenii was identified by DNA sequencing. The MLVA results showed that the VNTR profile of the isolate was 1−2-5, compatible with that of serovars Sejroe/Istrica. In the experimental infection in hamsters, the animals did not develop clinical signs, and no macroscopic lesions were observed on the organs at necropsy; however, the strain was detected in the kidneys, uterus, and testicles of the animals. The isolate described herein highlights infection by Sejroe strains that may be overlooked in buffaloes and that may be different from those normally isolated and used in serological studies.

Leptospira culture and isolation from tertiary care hospital, Surat

: In a highly globalized world, where boundaries are continually crossed due to different international activities, tropical diseases including Leptospirosis have emerged as significant causes of severe illness and death in untreated patients. To optimize the culture isolation of Leptospira by using different culture methods.: All suspected cases of Leptospirosis regardless of their age/sex were recruited following informed and written consent. Five different blood culture methods have been tried to see better isolation of Leptospira from patient.: Total 326 patients are evaluated over a period of three years. Five different method of blood culture were followed out of that, whole blood culture technique shows better result compaire to other followed technique.: The culture methods are gold standard but time consuming. It has an important role in the study of outbreaks. It provides epidemiological data that is helpful in studying pathogenesis of Leptospira.

Carbon utilization phenome of Leptospira interrogans serovar Manilae strain K64

Background: Leptospirosis, an acute febrile disease caused by the pathogenic species of genus Leptospira, is one of the neglected emerging zoonoses that is of global public health concern. The recent genus-wide sequencing of Leptospira isolates led to the need for better understanding of the complex metabolic mechanisms of this organism. However, majority of the published studies on Leptospira metabolism were still the pioneering works of Baseman and Cox in the 60’s and their contemporaries. Knowledge on the carbon sources that supports the growth of a Leptospira species will not only contribute to the limited metabolic studies but will further support the reported genes and metabolic pathways of this organism. Objective: Thus, this study aimed to describe the carbon utilization phenome of Leptospira interrogans serovar Manilae strain K64, one of the dominantly circulating pathogenic Leptospira in the Philippines. Methods: A previously optimized BiologTM Gen III sole carbon utilization phenotype microarray assay protocol for leptospires was adapted. Results: L. interrogans serovar Manilae strain K64 showed utilization of 29 carbon sources belonging to sugars and sugar derivatives, amino acids, methyl ester, carboxylic acid and fatty acids. These were N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N-acetyl neuraminic acid, D-fructose, D-galactose, 3-methyl glucose, D-fucose, L-fucose, L-rhamnose, inosine, D-fructose-6-phosphate, D-gluconic, D-glucuronic acid, glucuronamide, D-saccharic acid, D-aspartic acid, D-serine, L-alanine, L-arginine, L-histidine, L-pyroglutamic acid, L-serine, D-lactic acid methyl ester, citric acid, D-malic acid, L-malic acid, alpha ketoglutaric acid, alpha ketobutyric acid, and acetoacetic acid. Discussion and Conclusion: The carbon sources utilized by L. interrogans serovar Manilae strain K64 agreed well with the identified genes and metabolic pathways among Leptospira species. Moreover, these 29 carbon sources have been previously reported to be associated in the biosynthesis of peptidoglycan, lipopolysaccharide, histidine, sulfur, amino acids, and isoleucine and in other metabolic pathways such as glycolysis, pentose-phosphate, pyruvate and fatty acid in Leptospira spp.

Carbon utilization phenome of Leptospira interrogans serovar Manilae strain K64

Background: Leptospirosis, an acute febrile disease caused by the pathogenic species of genus Leptospira, is one of the neglected emerging zoonoses that is of global public health concern. The recent genus-wide sequencing of Leptospira isolates led to the need for better understanding of the complex metabolic mechanisms of this organism. However, majority of the published studies on Leptospira metabolism were still the pioneering works of Baseman and Cox in the 60’s and their contemporaries. Knowledge on the carbon sources that supports the growth of a Leptospira species will not only contribute to the limited metabolic studies but will further support the reported genes and metabolic pathways of this organism. Objective: Thus, this study aimed to describe the carbon utilization phenome of Leptospira interrogans serovar Manilae strain K64, one of the dominantly circulating pathogenic Leptospira in the Philippines. Methods: A previously optimized BiologTM Gen III sole carbon utilization phenotype microarray assay protocol for leptospires was adapted. Results: L. interrogans serovar Manilae strain K64 showed utilization of 29 carbon sources belonging to sugars and sugar derivatives, amino acids, methyl ester, carboxylic acid and fatty acids. These were N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N-acetyl neuraminic acid, D-fructose, D-galactose, 3-methyl glucose, D-fucose, L-fucose, L-rhamnose, inosine, D-fructose-6-phosphate, D-gluconic, D-glucuronic acid, glucuronamide, D-saccharic acid, D-aspartic acid, D-serine, L-alanine, L-arginine, L-histidine, L-pyroglutamic acid, L-serine, D-lactic acid methyl ester, citric acid, D-malic acid, L-malic acid, alpha ketoglutaric acid, alpha ketobutyric acid, and acetoacetic acid. Discussion and Conclusion: The carbon sources utilized by L. interrogans serovar Manilae strain K64 agreed well with the identified genes and metabolic pathways among Leptospira species. Moreover, these 29 carbon sources have been previously reported to be associated in the biosynthesis of peptidoglycan, lipopolysaccharide, histidine, sulfur, amino acids, and isoleucine and in other metabolic pathways such as glycolysis, pentose-phosphate, pyruvate and fatty acid in Leptospira spp.

Leptospira interrogans serogroup Pomona strains isolated from river buffaloes.

At present, little is known regarding the prevalence of buffalo leptospirosis worldwide, especially with respect to which Leptospira strains may infect this animal species. Furthermore, most investigations into this disease in buffaloes have only been performed with serological studies. In Brazil, particularly in the Amazon, buffalo production is growing and is just as important as cattle production, although few studies have been performed on buffalo compared to cattle. Thus, the aim of this study was to isolate and characterise Leptospira strains from river buffaloes raised in the Brazilian Amazon region. We collected 109 kidney samples from slaughtered buffaloes raised in the Amazon Delta region of Brazil. The samples were analysed by bacteriological culture for the isolation of leptospires, and the obtained isolates were serologically and molecularly characterised by microscopic agglutination test (MAT), DNA sequencing and multiple locus variable-number tandem repeat analysis (MLVA). Five isolates were obtained, and in serogrouping analyses, these isolates were only reactive for the Pomona serogroup, with an observed titre of 25,600. The DNA sequencing results revealed that all the isolates belonged to the species Leptospira interrogans, and the MLVA results showed that the VNTR loci 4, 7 and 10 profile of all the isolates was 4-1-10. In this study, we observed that Pomona serogroup strains circulate in buffaloes in the Amazon, showing that in Brazil, buffaloes can be affected by Leptospira strains other than the Sejroe group, which are adapted to cattle.

Dynamics of experimental infection with Leptospira serogroup Pomona in different sheep breed

Most of the information about leptospirosis was obtained from experimental infections with rodents, which in spite of being relevant, do not provide all the answers about the disease in animals and human beings, given the variability of interactions which exist between the agent and the different hosts. Therefore, the objective of the present study was to verify the pathogenesis of the infection in native breeds of sheep of Brazil challenged with a strain of the serogroup Pomona. A total of 10 sheep were divided into two groups with five animals according to the breed. In each group four sheep were challenged by intraperitoneal route with a strain of the serogroup Pomona and one was used as control. Sheep were monitored for 60 days, with collection of blood for serologic diagnosis, as well as vaginal fluid and urine for microbiological and molecular analyses. After this period the animals were submitted to euthanasia and necropsy, with collection of tissues for microbiological, molecular and histopathologic diagnosis. All the challenged sheep presented anti-Leptospira antibodies. Crossbred sheep presented a lower concentration of titers and the antibodies were detected for a shorter period of time when compared to the Santa Inês sheep, with statistical difference in the concentration of the titers on the days 15 (p<0.05), 45 (p<0.05) and 60 (p<0.05) post-infection. There was no significant difference between the groups when comparing the positivity rates of the microscopic agglutination test (MAT) (p>0.05). Eight positive reactions in the urine and vaginal fluid PCR were detected in both groups, being four (50%) in urine and four (50%) in the vaginal fluid, however without statistical difference (p>0.05). In both groups there was a greater proportion of PCR positive samples in kidneys (71.4%) in relation to uterus (28.6%), however without significant difference (p>0.05). There was no significant difference between the groups when comparing the positivity rates of the PCR (p>0.05). The isolation of leptospires from the urine and kidney of a crossbred sheep was possible. Therefore, it is possible that the native sheep, especially the crossbred, may have a relation of resistance with strains of the serogroup Pomona. However the intensity and duration of this relation need to be elucidated and longer-lasting investigations of natural and experimental infections are necessary in order to determine the epidemiological nature of this relation.

Insight into the Epidemiology of Leptospirosis: A Review of Leptospira Isolations from "Unconventional" Hosts.

Simple SummaryThe isolation of Leptospira is the most important test to assess infection in animal species. Several animals play a role as maintenance-host for specific serovars and in the last 30 years the incidence of leptospirosis has constantly increased in well-known reservoirs as well as in “unconventional” hosts. The emergence and the identification of Leptospira infection in such “unconventional” hosts could be related to several factors including problematic or inaccurate sampling modes during the Leptospira isolation, newly identified Leptospira strains, underestimated leptospirosis cases and climatic changes causing modifications of ecological niches. The aim of this review was to report the Leptospira isolations of the last 60 years from animals that could be considered “unconventional” hosts. Thus, the identification of “unconventional” hosts is crucial because they almost surely play an important role in the epidemiology of Leptospira infection.Leptospirosis is a re-emerging worldwide zoonotic disease. Even though the primary serological test for diagnosis and surveying is the microscopic agglutination test (MAT), isolation remains the gold-standard test to detect Leptospira infections. The leptospirosis transmission is linked to maintenance and accidental hosts. In the epidemiology of Leptospira some serovar are strictly related to specific maintenance hosts; however, in recent years, the bacterium was isolated from an even wider spectrum of species. The aim of this review is to report the isolation of Leptospira strains in animals which could be recognized as “unconventional” hosts, analyzing studies from 1960 to 2020 that highlighted the Leptospira isolation. This scientific literature aimed to provide evidence of infection in several animal species including of the Carnivora, Didelphimorphia, Rodentia, Cetacea, Cingulata, Afrosoricida, Chiroptera and Primate orders, as well as in Reptilia and Amphibia classes. In conclusion, the spreading of Leptospira is attention-worthy because the infection could occur in all the animal species ranging in a specific area. Further screening and isolations are needed to collect all necessary data to gain a complete understanding of leptospirosis epidemiology and its modifications.

Isolation and molecular confirmation of leptospires from dogs in Himachal Pradesh, India

Leptospirosis is a common but largely underreported zoonotic disease in India. It has been recognized as a re-emerging infectious disease causing deaths in dogs as well as humans. Leptospires have been isolated from both clinically ill and apparently healthy animals from different geographical regions but there is no information on the presence of leptospires in various clinical conditions of animals in the northern state of Himachal Pradesh, India. Here, using dark field microscopy, culture isolation and molecular biology-based tests, we confirm leptospirosis in three clinically affected dogs with chronic renal involvement. The study reports the first isolation and molecular confirmation of leptospires from clinically affected dogs in Himachal Pradesh, India.

TruenatTM - micro real-time-polymerase chain reaction for rapid diagnosis of leptospirosis at minimal resource settings.

Background & objectives:The biological spectrum of leptospirosis ranges from acute undifferentiated febrile illness to severe fatal syndrome or a combination of syndromes. Diagnosis on clinical grounds alone is difficult and depends on laboratory support. However, no confirmatory tests are available, which is rapid and can be performed with minimum facilities available. The objectives of this study were to evaluate the diagnostic utility, accuracy and reproducibility of a rapid real time-PCR based method (Truenat™) for early diagnosis of leptospirosis, and its usage in low resource settings.Methods:The Truenat™ test was performed using plasma sample collected from confirmed patients and controls. DNA was extracted from plasma samples and the reaction was performed as per the manufacturer’s instructions. Leptospiral isolates were also used to assess the performance using different serovars.Results:Evaluation of the Truenat™ test with RT-PCR as the gold standard showed that Truenat™ had a sensitivity of 97.4 per cent and a specificity of 98.6 per cent. The overall agreement with RT-PCR was 98.2 per cent.Interpretation & conclusions:Our results showed that the test would be a useful tool for early diagnosis of leptospirosis in settings with minimal facilities and the test results could be obtained within an hour. This indicates that a specific therapy can be instituted during the early phase of the disease even at peripheral healthcare facilities as well during the outbreaks.

Potentially Pathogenic Leptospira in the Environment of an Elephant Camp in Thailand.

Leptospira is the causative agent of leptospirosis, a globally emerging zoonotic disease. The infection is commonly acquired through contact with the contaminated environment. To extend the knowledge on environmental source of leptospirosis, we investigated the presence of Leptospira in an elephant camp setting where the interaction between humans, animals, and the shared environment occur particularly when engaging in recreational activities. In this study, a total of 24 environmental samples were collected from an elephant camp area in western Thailand. All samples were processed for Leptospira isolation using the EMJH medium. The identification of Leptospira species was carried out by partial 16S rRNA and secY gene sequencing. Of those 24 samples, 18 samples (75%) were culture-positive for Leptospira. The recovered leptospires were mostly derived from water and soil sampled from a river and a mud pond, the main areas for recreational activities. The majority of the isolates were classified into “Pathogens” clade (89%, 16/18) and more than half of the isolates (61%, 11/18) contained species of the “Saprophytes” clade. Notably, two soil isolates from the river beach sampling area were found to contain leptospiral DNA with high similarity to the pathogenic L. interrogans and L. santarosai. The evidence of diverse Leptospira species, particularly those belonging to the “Pathogens” clade, suggest that the shared environments of an elephant camp can serve as potential infection source and may pose a risk to the elephant camp tourists and workers.

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.

Seasonal variation of Leptospira species in pond and paddy field water samples in Thrissur district

Leptospirosis is an acute anthropozoonotic infection occurring worldwide caused by a pathogenic spirochete belonging to the genus Leptospira. The highest occurrence of leptospirosis in tropical countries is directly related to heavy rainfall and flooding. The changing rainfall pattern in the Western Ghats with rainless days during the monsoon months is reportedly affecting normal climatic condition of Kerala. Leptospires are able to survive in an environment with high humidity and moderate temperature.. The present study was undertaken to study the seasonal variation in occurrence of Leptospira spp. in pond and paddy field water in Thrissur district of Kerala. The samples were collected in two seasons i.e. monsoon (June to September) (Season-1) and summer (February to May) (Season-2) for the identification of Leptospira spp. The water samples were subjected to PCR for identification of the organism. The temperature and pH of water samples were recorded at the time of collection. The different physicochemical parameters of water in these two sources were in the range of pH (6.5- 7.9), conductivity (85- 150 µs/cm), salinity (0.02- 0.12 psu), turbidity (5-50NTU), dissolved oxygen (6-8 mg/L). Out of the 80 samples analysed, 52 (23 from monsoon and 29 from summer) samples detected 16Sr RNA at 430 bp and hence was positive for Leptospira species. The pathogenicity of the positive isolates were analysed by detecting the virulence gene viz., lipl 32, lipl 21 and lipl 41 respectively. The results revealed that out of the total isolates of Leptospira, 23 and 21 isolates were positive for lipl 21 and lipl 41 in monsoon and summer seasons respectively. Out of the total 85 paddy field water samples collected, 17 samples were found to be positive for Leptospira spp. The virulence gene lipl 21 was detected in three samples in summer season. However, lipl 32 and lipl 41 genes were not detected in any of the samples. The results of the present study demonstrated the ubiquitous nature of the organism in environment in the both seasons. The contaminated environment can hence be one of the sources for the increase in the frequency of human and animal leptospirosis in Kerala. Keywords: Leptospira spp., Leptospirosis, Paddy field water quality, lipl 32, lipl 21 and lipl 41

Whole genome sequencing and de novo assembly of three virulent Indian isolates of Leptospira.

Leptospirosis is a re-emerging bacterial zoonosis caused by pathogenic Leptospira, with a worldwide distribution and becoming a major public health concern. Prophylaxis of this disease is difficult due to several factors such as non-specific variable clinical manifestation, presence of a large number of serovar, species and asymptomatic reservoir hosts, lack of proper diagnostics and vaccines. Despite its global importance and severity of the disease, knowledge about the molecular mechanism of pathogenesis and evolution of pathogenic species of Leptospira remains limited. In this study, we sequenced and analyzed three highly pathogenic species of Indian isolates of Leptospira (interrogans, santarosai, and kirschneri). Additionally, we identified some virulence-related and CRISPR-Cas genes. The virulent analysis showed 232 potential virulence factors encoding proteins in L. interrogans strain Salinem and L. santarosai strain M-4 genome. While the genome of L. kirschneri strain Wumalasena was predicted to encode 198 virulence factor proteins. The variant calling analysis revealed 1151, 19,786, and 22,996 single nucleotide polymorphisms (SNPs) for L. interrogans strain Salinem, L. kirschneri strain Wumalasena and L. santarosai strain M-4, respectively, with a maximum of 5315 missense and 12,221 synonymous mutations for L. santarosai strain M-4. The structural analyses of genomes indicated potential evidence of inversions and structural rearrangment in all three genomes. The availability of these genome sequences and in silico analysis of Leptospira will provide a basis for a deeper understanding of their molecular diversity and pathogenesis mechanism, and further pave a way towards proper management of the disease.

Correction: Genus-wide Leptospira core genome multilocus sequence typing for strain taxonomy and global surveillance.

[This corrects the article DOI: 10.1371/journal.pntd.0007374.].

Leptospira transport medium (LTM): A practical tool for leptospires isolation

The isolation of Leptospira is challenging, since the bacteria of this genus are susceptible to adverse environmental conditions and may not remain viable for extend periods in urine samples. This study attempted to develop and evaluate a simple and practical method to isolate leptospires from bovine urine samples. A culture medium for sample transport, named Leptospira Transport Medium (LTM), was described and validated using reference serovars of Leptospira spp. in addition to autochthonous strains isolated in Brazil. We evaluated LTM in the field, by collecting 215 urine samples from slaughtered cattle and immediately seeding them in LTM and Fletcher's medium, used as control. The cultures were sent to a laboratory within 10 days for further processing. Moreover, 16S PCR was also performed on the urine samples directly to detect Leptospira DNA. Using LTM enabled 52 isolates (24.2%) to be obtained in pure culture, and contamination was only observed in 15/215 samples (7.0%). Regarding the samples in Fletcher's medium, 10 (4.6%) isolates were obtained. With 16S PCR performed in the urine samples, 31 samples (14.4%) were determined to be positive. LTM was developed and used in a simple and practical way and can significantly improve the isolation of leptospires from urine samples, as well as being highly useful in remote areas, not only in Brazil but also in other countries where few easily accessible laboratories are available. Furthermore, LTM can be prepared by laboratories and provided to veterinarians and technicians for urine collection in the field.

Isolation and propagation of leptospires at 37\u2009\xb0C directly from the mammalian host

The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28–30 °C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain IC:20:001 in semi-solid media at 29 °C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29 °C. In addition, HAN supported primary isolation at 37 °C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29 °C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37 °C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37 °C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.

Detection and Characterization of Leptospira Infection and Exposure in Rats on the Caribbean Island of Saint Kitts.

Simple SummaryLeptospirosis is a widespread zoonotic and potentially life-threatening disease in humans and animals. Many animal species maintain Leptospira, the bacterial agent causing this disease, in the kidneys and they shed the bacteria in urine. These animals act as a source of infection and environmental contamination. Leptospira infection has been previously reported in several animals on the Caribbean island of Saint Kitts, yet, no data is currently available on rats, a significant reservoir host of this pathogen. The main goal of this study was to detect Leptospira infection and exposure in two species of rats on Saint Kitts, by using a complementary set of diagnostic tools. Infecting Leptospira strains were subsequently characterized with a combination of serologic, molecular, and genomic techniques. Results show a relatively high prevalence of infection with L. interrogans (serogroup Icterohaemorrhagiae) and L. borgpetersenii (serogroup Ballum), with evidence supporting mixed infection with both species in some rats. Our study suggests the use of multiple diagnostic tests to enhance the results of Leptospira surveillance studies and diagnostic investigations. In this study, we detected and characterized Leptospira infection and exposure in rats on the Caribbean island of Saint Kitts for the first time. We detected Leptospira infection in 17/29 (59%), 14/29 (48)%, and 11/29 (38)% of rats by RT-PCR, culture, and immunofluorescence assay, respectively. Whole genome sequencing (WGS) and analysis and serogrouping of 17 Leptospira strains isolated from rats revealed their close relationship with L. interrogans serogroup Icterohaemorrhagiae (n = 10) and L. borgpetersenii serogroup Ballum (n = 7). WGS, serogrouping, and additional PCR tests on rat kidneys confirmed mixed infections with L. interrogans and L. borgpetersenii in the kidneys of three rats. Microscopic agglutination test (MAT) was positive for 25/29 (87%) of the rats tested, and the response was restricted to serovars Icterohaemorrhagiae {24/29(83%)}, Mankarso {4/29(14%)}, Copenhageni {4/29(14%)}, Grippotyphosa {2/29(7%)}, and Wolffi {1/29(3%)}. Interestingly, there was no agglutinating antibody response to serovar Ballum. We observed a similar pattern in the serologic response using Leptospira isolates obtained from this study with each of the rat sera, with strong response to L. interrogans isolates but minimal reactivity to L. borgpetersenii isolates. Our findings suggest the use of multiple complementary diagnostic tests for Leptospira surveillance and diagnosis to improve the accuracy of the data.

Short communication: Uterine leptospiral infection is strongly associated to strains of serogroup Sejroe on experimentally infected hamsters

Leptospira infection is influenced by the host species and the bacterial strain involved. Important differences on their tissue distribution are referred, also depending on the host and the studied strain. Considering tissue distribution of leptospires the majority of the studies focus on a single strain, or strains from the same serogroup with different inoculation doses. Nevertheless, none had associated different serogroups with their tissue distribution. Thus, the present study aims to investigate the hypothesis that tissue distribution of Leptospira spp. on experimentally infected hamsters may vary according to the strain serogroup. Females of Golden Syrian hamsters were used for the experimental infection with Leptospira isolates (n = 13) belonging to serogroups Icterohaemorrhagiae (n = 8) and Sejroe (n = 5). PCRs were conducted in renal, hepatic and uterine tissue. Seven out of the thirteen studied hamsters presented acute clinical signs and were euthanized before the 21° day p.i. (strains VF237, VF52, U81, UFF-SG001, UFF-G19, UFF-B15, L1-130), while the others presented a subclinical infection. Regarding to the PCR results it was possible to observe that, all but one strain (UFF-SG001), which was detected on all the studied tissues, presented some differences on their tissue distribution. All strains could be detected on at least one tissue and a significant association was observed for the occurrence of the strains of serogroup Sejroe in uterus (p = 0.044). The results of the present study indicate that strains of serogroup Sejroe are strongly associated to uterine infection on experimentally infected hamsters.