Leptospiral DNA Research Articles

Unconventional Sites for Diagnosis of Leptospirosis in Bovine Anicteric Fetuses.

Bovine leptospirosis is an important reproductive disease and abortion is a major sign, leading to economic impacts. Due to its multifactorial etiology, the proper diagnosis of the cause of the abortion is crucial. Necropsy of the fetuses followed by molecular analysis is recommended for diagnosis, and the investigation mainly occurs in the kidneys and liver. This study aimed to analyze unconventional sites for the presence of leptospiral DNA in bovine anicteric aborted fetuses. Five fetuses of the same herd were received for necropsy and diagnosis. Conventional lipL32-PCR was performed in the fetuses' kidneys, livers, lungs, hearts, spleens, subcapsular kidney content, abomasal fluid, and in the cavity's hemorrhagic contents. To complete the investigation, the sera of 30 cows of the herd were collected to perform the serologic screening by Microscopic Agglutination Test. In addition, six subfertile non-pregnant cows from the same herd were selected due to their low reproductive performance, and genital samples (uterine fragment and cervicovaginal mucus) and urine were collected for lipL32-PCR. PCR-positive samples were submitted to a nested PCR of the secY gene and intended for sequencing. The herd presented seroreactive animals (11/30, 36.6%), all against the Sejroe serogroup, with titers between 200 and 1600. In necropsy, four fetuses showed hemorrhagic and anicteric lesions, while one fetus had no macroscopic lesions. Regarding molecular analysis, all the fetuses were positive in lipL32-PCR and the positive sites were the heart, lungs, subcapsular kidney content, thymus, kidneys, liver, and abomasal fluid. Only one fetus presented positive results in the kidney and liver, while three fetuses were positive in the abomasal fluid. Five of six cows were positive for lipL32-PCR, all being positive only in genital samples. Of the fetuses and the cows, seven sequences were obtained and all were identified as Leptospira interrogans serogroup Sejroe serovar Hardjoprajitno. In order to improve the diagnosis of leptospirosis in cows, it is recommended to perform a comprehensive analysis of the samples, beyond the kidneys and liver. Thus, we highly encourage testing multiple organs by PCR to investigate abortions suspected of bovine leptospirosis, particularly in anicteric fetuses.

Leptospira spp. detection in bats from the Peninsula of Yucatán, México

The records of Leptospira spp. in bats from México are scarce. It is known that several species of bats are hosts of pathogenic Leptospira spp.; therefore, they participate in the epidemiological cycle and can generate transmission scenarios to people and animals. The aim was to detect the DNA of Leptospira spp. in bats captured in four sites from the Yucatán Peninsula, México. A kidney fragment was collected from the bats and used to extract genomic DNA. Using a PCR, the DNA of Leptospira spp. was detected. Fifty–four bats of the species Desmodus rotundus, Mimon cozumelae, Pteronotus mesoamericanus, Pteronotus fulvus, Nyctinomops laticaudatus, Peropteryx macrotis, Molossus nigricans, Molossus aztecus, Noctilio leporinus, Saccopteryx bilineata and Mormoops megalophylla were studied. The PCR yielded a total frequency of Leptospira spp. of 44.4% (24/54, 95% CI 36.2–71.7%). Species with at least one positive individual were D. rotundus, M. cozumelae, P. mesoamericanus, N. laticaudatus, P. macrotis, M. nigricans, N. leporinus, M. aztecus, and S. bilineata. A high frequency of Leptospira spp. was found in different species of bats belonging to several trophic guilds. The presence of Leptospira spp. in bats is relevant for Public and Animal Health because the evidence indicates that they can generate zoonotic transmission cycles.

Optimization and comparative analysis of LAMP and PCR techniques for the detection of leptospiral DNA in Golden Syrian hamsters.

Leptospirosis is a zoonotic disease with significant public health and economic impact worldwide. Rapid and accurate diagnosis is essential for effective prevention and treatment. This study optimized a loop-mediated isothermal amplification (LAMP) assay using BFo isothermal DNA polymerase with different colorimetric indicators. LAMP was able to detect DNA from pathogenic and intermediate leptospires, while non-pathogenic leptospires and other non-leptospiral microorganisms were negative. LAMP assay combined with calcein showed a tenfold higher limit of detection (1ng of leptospiral DNA per reaction) than LAMP combined with hydroxynaphthol blue or end-point PCR lipL32 (10ng of DNA per reaction). Animal samples were collected from infected and non-infected Golden Syrian hamsters (Mesocricetus auratus) to evaluate and compare the performance of LAMP and PCR. These techniques showed a substantial agreement according to Cohen's kappa statistic, being both useful techniques for detecting leptospiral DNA in clinical samples. Overall, this study demonstrates that the LAMP assay is a sensitive, specific, rapid, and simple tool for the detection of leptospiral DNA. It has the potential to facilitate the diagnosis of leptospirosis, particularly in low-income regions with limited diagnosis resources.

A rare case of severe leptospirosis infection presenting as septic shock in a non-endemic area: a case report and literature review

BackgroundLeptospirosis is a zoonosis caused by spirochete “genus” leptospira. The clinical presentations of leptospirosis range from an influenza-like presentation of fever and myalgia, to severe forms. Leptospirosis can potentially lead to a misdiagnosis or delay in diagnosis when clinical similarities exist.Case presentationA 63-year-old man presented with fever, shock and thrombocytopenia followed by diffuse pulmonary hemorrhage. Peripheral blood Metagenomic Next-generation Sequencing (mNGS) reported Leptospira interrogans. The patient was treated with piperacillin-tazobactam (TZP) plus doxycycline and improved dramatically after 7 days.ConclusionWe conclude that leptospirosis can potentially lead to a misdiagnosis or delay in diagnosis. Correctly evaluation of thrombocytopenia in acute febrile illnesses facilitates the differential diagnosis of leptospirosis. mNGS can accurately detect Leptospira DNA during the early stage of the infection.

Pathogenic Leptospira Species Are Present in Urban Rats in Sydney, Australia.

Leptospirosis is an emerging disease among people and dogs in Sydney, Australia. However, the routes of Leptospira transmission in these cases, and in particular the possible role of rats as reservoirs of infection in Sydney, are unknown. Rats were collected within the City of Sydney Council area and their kidneys were tested for pathogenic Leptospira DNA by real-time (q)PCR. A subset of rats also had qPCR testing performed on whole blood and urine, and Microscopic Agglutination Testing (MAT) that included a panel of 10 Leptospira serovars from nine different Leptospira serogroups was performed on a subset of serum samples. Based on qPCR testing, the proportion of rats with Leptospira DNA in their kidneys was 9/111 (8.1%). qPCR testing of blood samples (n = 9) and urine (n = 4) was negative. None of the 10 serum samples tested MAT positive. A primary cluster of qPCR-positive locations was detected based on six infected rats, which partially overlapped with a previously identified cluster of canine leptospirosis cases in Sydney. These findings suggest that rats in Sydney might play a role in the transmission of leptospirosis to dogs and people. Further testing of rats in Sydney and investigation into other possible wildlife reservoirs of infection and environmental sources of leptospires are needed.

A predictive risk map for human leptospirosis guiding further investigations in brown rats and surface water

ABSTRACT Leptospirosis is a zoonosis caused by the spirochete Leptospira spp. It is often not clear why certain areas appear to be hotspots for human leptospirosis. Therefore, a predictive risk map for the Netherlands was developed and assessed, based on a random forest model for human leptospirosis incidence levels with various environmental factors and rat density as variables. Next, it was tested whether misclassifications of the risk map could be explained by the prevalence of Leptospira spp. in brown rats. Three recreational areas were chosen, and rats (≥25/location) were tested for Leptospira spp. Concurrently, it was investigated whether Leptospira spp. prevalence in brown rats was associated with Leptospira DNA concentration in surface water, to explore the usability of this parameter in future studies. Approximately 1 L of surface water sample was collected from 10 sites and was tested for Leptospira spp. Although the model predicted the locations of patients relatively well, this study showed that the prevalence of Leptospira spp. infection in rats may be an explaining variable that could improve the predictive model performance. Surface water samples were all negative, even if they had been taken at sites with a high Leptospira spp. prevalence in rats.

Leptospirosis in Aotearoa New Zealand: Protocol for a Nationwide Case-Control Study.

In Aotearoa New Zealand, 90% of patients with notified leptospirosis (a zoonotic bacterial disease) have been men working in agricultural industries. However, since 2008, the epidemiology of notified cases has been gradually changing, that is, more women are affected; there are more cases associated with occupations traditionally not considered high risk in New Zealand; infecting serovars have changed; and many patients experience symptoms long after infection. We hypothesized that there is a shift in leptospirosis transmission patterns with substantial burden on affected patients and their families. In this paper, we aimed to describe the protocols used to conduct a nationwide case-control study to update leptospirosis risk factors and follow-up studies to assess the burden and sources of leptospirosis in New Zealand. This study used a mixed methods approach, comprising a case-control study and 4 substudies that involved cases only. Cases were recruited nationwide, and controls were frequency matched by sex and rurality. All participants were administered a case-control questionnaire (study 1), with cases being interviewed again at least 6 months after the initial survey (study 2). A subset of cases from two high-risk populations, that is, farmers and abattoir workers, were further engaged in a semistructured interview (study 3). Some cases with regular animal exposure had their in-contact animals (livestock for blood and urine and wildlife for kidney) and environment (soil, mud, and water) sampled (study 4). Patients from selected health clinics suspected of leptospirosis also had blood and urine samples collected (study 5). In studies 4 and 5, blood samples were tested using the microscopic agglutination test to test for antibody titers against Leptospira serovars Hardjo type bovis, Ballum, Tarassovi, Pomona, and Copenhageni. Blood, urine, and environmental samples were also tested for pathogenic Leptospira DNA using polymerase chain reaction. Participants were recruited between July 22, 2019, and January 31, 2022, and data collection for the study has concluded. In total, 95 cases (July 25, 2019, to April 13, 2022) and 300 controls (October 19, 2019, to January 26, 2022) were interviewed for the case-control study; 91 cases participated in the follow-up interviews (July 9, 2020, to October 25, 2022); 13 cases participated in the semistructured interviews (January 26, 2021, to January 19, 2022); and 4 cases had their in-contact animals and environments sampled (October 28, 2020, and July 29, 2021). Data analysis for study 3 has concluded and 2 manuscripts have been drafted for review. Results of the other studies are being analyzed and the specific results of each study will be published as individual manuscripts.. The methods used in this study may provide a basis for future epidemiological studies of infectious diseases. DERR1-10.2196/47900.

Co-Infection by Leptospira montravelensis and Leptospira interrogans Serovar Pomona in Urine Samples of Donkeys and Pigs in Sardinia, Italy.

Saprophytic leptospires are spirochetes enclosed within the non-pathogenic clade of the genus Leptospira, which in turn is subdivided into two subclades S1 and S2. To date, the microorganisms included in these subclades have been isolated from the environment in various parts of the world, and are believed to have no known animal reservoirs. After a case of Leptospira interrogans serovar Pomona was notified to the owner of a farm in Sardinia, all of the farm animals (11 pigs and 3 donkeys) were examined for the presence of Leptospira. Sera of all tested animals resulted positive for antibodies to Leptospira using a microscopic agglutination test (MAT). Moreover, nine (82%) kidney samples from pigs and three urine samples collected from donkeys (100%) tested positive for Leptospira DNA after qPCR. Results obtained after MLST analysis and sequencing of rrs, rpoB, and secY genes, performed on six Leptospira strains isolated in culture, revealed the presence of the genomospecies L. interrogans serovar Pomona in the kidney samples. Conversely, whole-genome sequencing combined with mean nucleotide identity revealed the presence of the saprophytic L. montravelensis in the urine samples. Our results report, for the first time, the isolation of a saprophytic species from mammalian urine, suggesting a new ecological specialization for these bacteria, with a possible transition from free-living to a symbiotic lifestyle. Further studies will have to be conducted to understand the evolution of virulence of these bacteria, potential infectivity, and possible public health implications.

DNA Capture and Enrichment: A Culture-Independent Approach for Characterizing the Genomic Diversity of Pathogenic Leptospira Species.

Because they are difficult to culture, obtaining genomic information from Leptospira spp. is challenging, hindering the overall understanding of leptospirosis. We designed and validated a culture-independent DNA capture and enrichment system for obtaining Leptospira genomic information from complex human and animal samples. It can be utilized with a variety of complex sample types and diverse species as it was designed using the pan-genome of all known pathogenic Leptospira spp. This system significantly increases the proportion of Leptospira DNA contained within DNA extracts obtained from complex samples, oftentimes reaching >95% even when some estimated starting proportions were <1%. Sequencing enriched extracts results in genomic coverage similar to sequenced isolates, thereby enabling enriched complex extracts to be analyzed together with whole genome sequences from isolates, which facilitates robust species identification and high-resolution genotyping. The system is flexible and can be readily updated when new genomic information becomes available. Implementation of this DNA capture and enrichment system will improve efforts to obtain genomic data from unculturable Leptospira-positive human and animal samples. This, in turn, will lead to a better understanding of the overall genomic diversity and gene content of Leptospira spp. that cause leptospirosis, aiding epidemiology and the development of improved diagnostics and vaccines.

Molecular Characterization of Leptospira Species Detected in the Kidneys of Slaughtered Livestock in Abattoirs in Gauteng Province, South Africa.

Leptospira was investigated in kidneys (n = 305) from slaughtered livestock in the Gauteng Province abattoirs, South Africa, using a culture medium to isolate Leptospira, followed by the LipL32 qPCR to detect Leptospira DNA. The SecY gene region was amplified, sequenced, and analyzed for LipL32 qPCR-positive samples or Leptospira isolates. The overall frequency of isolation of Leptospira spp. was 3.9% (12/305), comprising 4.8% (9/186), 4.1% (3/74), and 0% (0/45) from cattle, pigs, and sheep, respectively (p > 0.05). However, with LipL32 qPCR, the overall frequency of Leptospira DNA was 27.5%, consisting of 26.9%, 20.3%, and 42.2% for cattle, pigs, and sheep, respectively (p = 0.03). Based on 22 SecY sequences, the phylogenetic tree identified the L. interrogans cluster with serovar Icterohaemorrhagiae and the L. borgpetersenii cluster with serovar Hardjo bovis strain Lely 607. This study is the first molecular characterization of Leptospira spp. from livestock in South Africa. The reference laboratory uses an eight-serovar microscopic agglutination test panel for leptospirosis diagnosis, of which L. borgpetersenii serovar Hardjo bovis is not part. Our data show that pathogenic L. interrogans and L. borgpetersenii are circulating in the livestock population. Diagnostic use of molecular methods will eliminate or reduce the under-reporting of leptospirosis in livestock, particularly sheep, in South Africa.

Development and Evaluation of Recombinase Polymerase Amplification Assay for Diagnosis of Canine Leptospirosis

Background: Canine Leptospirosis is a life-threatening disease and zoonosis. Usually, PCR assay is carried out for early diagnosis but requires a thermal cycler and post-PCR procedures. This limits its use in resource-limited areas. Hence, the isothermal amplification of nucleic acid by recombinase polymerase amplification assay was developed as a versatile alternative for the diagnosis of canine leptospirosis in this study. Methods: The RPA assay to detect Leptospira DNA was optimized with Leptospira reference strains and its performance characteristics such as analytical, diagnostics and reproducibility were assessed. Result: The limit of detection of RPA assay was estimated as 102 copies of genomic DNA and specific to amplify the pathogenic Leptospira. Out of 150 dog samples screened, Leptospira DNA was detected in 64 (42.6%) by RPA assay and 67 (44.6%) by PCR. The diagnostic sensitivity and specificity of the RPA assay were 92.5% and 97.59% respectively. The RPA assay has a good diagnostic agreement with a kappa value of 0.905. The reproducibility assessment with the third-party testing laboratory revealed a better agreement with a kappa value of 0.81. The simplicity, rapid and less expensive enable this assay to perform at resource-limited laboratories or point-of-care testing.

Evaluation of Leptospira infection and exposure in free-roaming cat populations in northern California and southern Texas.

Leptospirosis is a re-emergent zoonotic bacterial disease associated with renal and hepatic injury. In free-roaming cats in some regions, a high prevalence of Leptospira antibodies has been identified, and pathogenic leptospires have been detected in renal tissue, indicating that they may play a role in Leptospira epidemiology. The objective of this study was to determine the prevalence of Leptospira seroreactivity and urinary shedding of Leptospira DNA in free-roaming cats from northern California and southern Texas. A secondary objective was to compare the results of a point-of-care (POC) assay, designed to detect Leptospira antibodies, with the results of the microscopic agglutination test (MAT) when applied to serum samples from feral cats. Specimens were obtained from free-roaming cats from northern California (n = 52; 2020) and southern Texas (n = 75; 2017). Leptospira quantitative PCR was performed on blood and urine specimens from Californian cats. Serum samples from Californian and Texan cats were subjected to MAT to categorize them as Leptospira antibody-positive or antibody-negative. The performance of the POC assay was assessed using the MAT as the gold standard. Leptospira DNA was not detected in the blood or urine of any cats tested. The results of the MAT were positive in 17.3% (n = 9) of Californian cats and 10.7% (n = 8) of Texan cats (P = 0.3). The median MAT titer was 1:100 (range 1:100-1:200) in Californian cats and 1:200 (range 1:100-1:800) in Texan cats. The POC assay was negative in all specimens. Free-roaming cats in California and Texas are exposed to Leptospira species and may have the potential to act as sentinel hosts. No cats had evidence of current infection, as determined using PCR on blood and urine specimens. The POC test did not reliably detect anti-Leptospira antibodies in these cats. The role of cats in the maintenance or shedding of pathogenic leptospires requires further investigation.

Leptospirose genital bovina: achados em touros mantidos em condições do bioma Caatinga

ABSTRACT: Leptospirosis is a disease that causes economic and social impact, as it affects wild and domestic animals and humans. There may be peculiarities in the epidemiology of this disease in the Caatinga biome, Brazil, where the environment is adverse and the etiologic agent, Leptospira spp., requires alternative transmission routes. Considering that in bovine leptospirosis the genital carrier is constantly neglected and the lack of reports on the role of bulls in the epidemiology of the bovine genital leptospirosis (BGL) syndrome, mainly in semiarid conditions such as Caatinga biome, this study aimed to investigate bulls maintained in Caatinga biome conditions as genital carriers of leptospires. Urinary tract (urine, bladder, and kidney) and reproductive tract (vas deferens, cauda epididymis, and vesicular gland) samples were collected from 42 slaughtered bulls. Microscopic agglutination test (MAT), polymerase chain reaction (PCR), and microbiological isolation were included as diagnostic methods. Anti-Leptospira spp. antibodies were found in 17 (40.48%) animals, while 26 animals (61.90%) had at least one organ or urine with leptospiral DNA, and 10 animals (23.81%) were positive at bacteriological culture. Sequenced samples targeting the LipL32 gene showed 99% similarity with Leptospira borgpetersenii. Molecular analysis of samples from the vas deferens and cauda epididymis is recommended for the diagnosis of genital leptospirosis in bulls and, if it is impossible to collect these tissues, semen can be used. In conclusion, this study provides important information relating to bulls from the Caatinga biome, Brazil, as carriers of genital leptospirosis. The results indicate that, even under adverse environmental conditions, leptospires may survive and propagate, mainly due to the characteristic of genital carriers for the sexually spreading of adapted Leptospira species without influence by external variables. Thus, prevention and control strategies for bovine leptospirosis need to include actions aimed at the genital carrier.

Tapered optical fiber DNA biosensor for detecting Leptospira DNA

Objective: To establish a DNA detection platform based on a tapered optical fiber to detect Leptospira DNA by targeting the leptospiral secY gene.Methods: The biosensor works on the principle of light propagating in the special geometry of the optical fiber tapered from a waist diameter of 125 to 12 µm. The fiber surface was functionalized through a cascade of chemical treatments and the immobilization of a DNA capture probe targeting the secY gene. The presence of the target DNA was determined from the wavelength shift in the optical transmission spectrum.Results: The biosensor demonstrated good sensitivity, detecting Leptospira DNA at 0.001 ng/µL, and was selective for Leptospira DNA without cross-reactivity with non-leptospiral microorganisms. The biosensor specifically detected DNA that was specifically amplified through the loop-mediated isothermal amplification approach.Conclusions: These findings warrant the potential of this platform to be developed as a novel alternative approach to diagnose leptospirosis.

Status of Urinary shedders of Leptospires among cattle in Tamilnadu: Implications on zoonosis.

Bovine leptospirosis causes jaundice, mastitis, infertility, abortion, and death of the animal. This research aimed to study the status of urinary shedders of pathogenic Leptospira among the cattle population and identify the infecting serogroup circulating in this region. A total of 305 blood and 305 urine samples were collected from organized farms (n = 44), individually housed animals (n = 81) and animals from the slaughterhouse (n = 180). Microscopic agglutination test was carried out to detect anti‑leptospiral antibodies. Dark‑field microscopic examination and culture of urine were done to detect and isolate the Leptospira. The isolated Leptospira were identified by cross‑agglutination test and gene sequencing. PCR and real‑time PCR were carried out to detect leptospiral genomic DNA in urine samples to detect the shedders. The anti‑leptospiral antibodies were detected in 6.2% of animals. The Leptospira genomic DNA was detected in 9.2% (28 of 305) of urine samples. Of the 28 Leptospira positive urine samples, 39.2% were from animals with clinical signs suggestive of leptospirosis and 60.8% Leptospira positive samples were from slaughterhouse animals. The Leptospira isolated were identified as Leptospira interrogans serogroup Sejroe and Hebdomadis. The present study demonstrates the need to include leptospirosis in cattle health surveillance programmes to prevent leptospirosis by vaccination, preventing renal carriage.

Epidemiological Study of Pathogenic Leptospira in Raccoons (Procyon lotor) in a Suburb of Tokyo, Japan.

Leptospirosis is a zoonosis that affects humans and animals worldwide. Raccoons (Procyon lotor), adopted in urban environments, may act as potential reservoirs of Leptospira. We investigated the prevalence of pathogenic Leptospira in the kidney and urine samples of raccoons living in Tokyo, as well as anti-leptospiral antibodies in their serum, and aimed to examine the factors that expose raccoons to Leptospira. Polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect leptospiral DNA and anti-leptospiral antibodies, respectively. Thirty-six of 156 raccoons (23.1%) were positive by PCR, and 16 of 165 raccoons (9.7%) were positive by ELISA. The prevalence and seroprevalence rates differed depending on the raccoon dispersal period. We used univariable logistic regression to estimate the environmental factors associated with pathogenic Leptospira and anti-leptospiral antibodies in raccoons. Significant differences were observed in the PCR results for the seasons (spring−summer) (p = 0.01), average monthly temperature (p < 0.01), and average monthly rainfall (p < 0.01). No significant difference was seen in the ELISA results, but raccoons in larger urban areas tended to have higher seroprevalence rates (p = 0.06). We identified a pattern of leptospiral spread in raccoon dispersal and environmental factors that expose raccoons to Leptospira.

Leptospira spp. Prevalence in Cats from Southern Italy with Evaluation of Risk Factors for Exposure and Clinical Findings in Infected Cats.

Leptospirosis is a worldwide zoonotic disease, but feline leptospirosis is rarely reported. This study aimed at investigating Leptospira spp. prevalence in cats from southern Italy, evaluating risk factors, clinical findings and laboratory data associated with infection. The serum of 112 cats was investigated by microscopic agglutination test (MAT), detecting anti-Leptospira antibodies against 14 pathogenic serovars. Blood and urine samples were tested by a real-time polymerase chain reaction targeting the lipL32 gene of pathogenic Leptospira. Antibodies against serovars Poi, Bratislava, Arborea, Ballum, Pomona and Lora were detected in 15.3% (17/111) of cats (titers range: 20–320). Leptospira spp. DNA was found in 3% (4/109) of blood and 9% (10/111) of urine samples. The spring season was the only risk factor for urinary Leptospira DNA shedding. Laboratory abnormalities significantly associated and/or correlated with Leptospira spp. positivity were anemia, monocytosis, neutrophilia, eosinopenia, increased alanine aminotransferase activity, hypoalbuminemia and hyperglobulinemia. In the investigated areas, cats are frequently infected by Leptospira spp. and can represent an additional reservoir or sentinel for a risk of infection. Moreover, some laboratory changes could be compatible with a pathogenic effect of Leptospira spp. in the feline host.

Carbon quantum dots functionalized tapered optical fiber for highly sensitive and specific detection of Leptospira DNA

In this paper, we report on the development of DNA biosensor using carbon quantum dots (CQDs) to enhance Leptospira DNA detection. Leptospira is a bacterium that invades human body and cause leptospirosis disease. Leptospirosis has attracted attention mainly due to several cases of human mortality which have distressed health professionals. This biosensor system employs tapered optical fiber as a sensing transducer. The CQDs were functionalized at the tapered region via in situ deposition (drop-casted) before incubation using commercialized chemicals to link the 16 s ribosomal RNA gene (rrs) probe DNA. Detection was achieved with the occurrence of hybridization between the probe DNA and different concentrations of complementary DNA (Leptospira DNA). This biosensor with limit of detection (LOD) of 1.0 fM demonstrated better sensitivity at 1.8295 nm/nM and higher affinity as its dissociation constant (Kd) of 0.552 × 10−09 M is lower compared to previous reported biosensors without CQDs. High specificity biosensor is validated when minimal shift was observed for non-complementary DNA. This novel biosensor was also able to distinguish between genomic DNA of Leptospira serovars and Clostridium difficile as control sample. The proposed biosensor has successfully exhibited the prominent role of nanomaterial for sensor enhancement. Consequently, the biosensor approach in this study has emphasized the potential for the development of more sensitive and reliable leptospirosis diagnostic testing in the future.

Analysis of 1840 Equine Intraocular Fluid Samples for the Presence of Anti-Leptospira Antibodies and Leptospiral DNA and the Correlation to Ophthalmologic Findings in Terms of Equine Recurrent Uveitis (ERU)-A Retrospective Study.

Simple SummaryIn horses, the chronic intraocular leptospiral infection has been shown to cause equine recurrent uveitis (ERU). This inflammatory ophthalmic disease recurs for years and usually leads to blindness. Only recently it was found that biofilm formation of the leptospires in the vitreous cavity leads to uveitis recurrences and prevents effective elimination of the infection by antibiotics or by the immune system. The most effective treatment is vitrectomy (lavage of the vitreous cavity), which mechanically removes the biofilm infection. This surgery has been performed in horses for more than 30 years, and thousands of intraocular specimens have been analyzed for antibodies directed against leptospires and by PCR for leptospiral DNA. For the present study, medical records were retrospectively analyzed. Complete medical and laboratory records were available for 1800 intraocular specimens from horses treated from 2002 to 2017 (1387 specimens from ERU-eyes, 237 specimens from eyes affected with another type of uveitis, and 216 specimens from healthy eyes). In 83% of intraocular samples from ERU eyes, antibodies were detectable, and especially the detection of immunoglobulin A (IgA) seems to play an important role. In 72% of the intraocular specimens, leptospiral DNA was detectable by PCR. No antibodies were detectable in the samples from eyes with another type of uveitis or in the samples from healthy eyes. A PCR was positive in only one sample from a healthy eye. These results with a very high number of intraocular specimens demonstrate the great importance of an intraocular leptospiral infection for ERU. It can be concluded that for a reliable diagnosis of intraocular leptospiral infection or to reliably exclude an infection, multiple tests should be applied.In the equine clinic of the LMU in Munich, therapeutic vitrectomies have been routinely performed in horses for three decades. The vitreous samples obtained during vitrectomies were usually tested for anti-Leptospira antibodies and for more than 20 years also by PCR for leptospiral DNA. If the indication for surgery was ophthalmologically inconclusive, an aqueous humor was collected preoperatively and examined for evidence of leptospiral infection. In this study, medical records from 2002 to 2017 were analyzed. Records for 1387 eyes affected by equine recurrent uveitis (ERU) and 237 eyes affected by another type of uveitis met the inclusion criteria. A total of 216 samples from healthy eyes were used as controls. In 83% of intraocular samples from ERU eyes, antibody titers of 1:100 or higher were detectable by microscopic agglutination test (MAT). Similarly, 83% of intraocular samples had anti-Leptospira antibodies detected by ELISA. In 72% of the intraocular specimens, leptospiral DNA was detectable by PCR. No antibodies were detectable in the samples from eyes with another type of uveitis or in the samples from healthy eyes. A PCR was positive in only one sample from a healthy eye. These results with a very high number of intraocular specimens demonstrate the great importance of an intraocular leptospiral infection for ERU. It can be concluded that for a reliable diagnosis of intraocular leptospiral infection or to reliably exclude an infection multiple tests should be applied.

Bovine genital leptospirosis: Evidence of ovarian infection by Leptospira interrogans

Leptospirosis in ruminants causes reproductive failures leading to important economic losses. This study assessed the occurrence and genetically identified Leptospira spp. in the follicular fluid (FF) of naturally infected live cows. A total of 251 asymptomatic cows from different commercial dairy herds were subjected to ovum-pick up technique for follicular fluid sampling. PCR was performed for Leptospira spp. detection and phylogenetic analysis was later implemented for sequencing. From 251 samples analyzed, 67 (26.7 %) were lipL32-PCR positive, confirming the presence of leptospiral DNA on FF. Furthermore, it was possible to amplify and sequence nine strains after secY nested-PCR. All of them were identified as L. interrogans, with 100 % of identity with strains belonging to Sejroe serogroup. Our findings reveal a high occurrence of infection of Leptospira in the ovarium of asymptomatic cows, highlighting the importance of considering the silent leptospirosis syndrome when screening animals for assisted reproductive biotechniques.