Leptospiral Vaccine Research Articles

Isolation of Virulent Leptospira Serogroup Australis Field Strains from Symptomatic Dogs for Canine Leptospiral Vaccine Development.

Leptospirosis is a widespread zoonosis caused by spirochaetes belonging to the pathogenic species of Leptospira, which are classified into more than 25 serogroups and 250 serovars. Vaccination can prevent the disease in dogs but offers incomplete efficacy because of a lack of cross-protection between serogroups. The aim of this study was to validate a robust recruitment and sampling process, with the objectives of isolating and typing circulating Leptospira pathogenic strains and then selecting those of proven virulence and pathogenicity for vaccine development. Blood and urine samples from dogs with clinical syndromes compatible with acute leptospirosis were sterilely collected and transported to a reference laboratory for a micro-agglutination test (MAT), PCR, and bacterial isolation. Isolated strains underwent molecular typing using RNA16S, variable-number tandem repeat (VNTR), and pulsed-field gel electrophoresis (PFGE). Subtyping was performed using core genome multilocus sequence typing (CgMLST). Among 64 included dogs, 41 had MAT and/or PCR results compatible with Leptospira infection, and 14 Leptospira strains were isolated. Based on molecular typing, 11 isolates were classified as L. interrogans serogroup Australis, serovar Bratislava, and 3 as serogroup Icterohaemorrhagiae, serovar Icterohaemorrhagiae. CgMLST subtyping revealed a diversity of clonal groups (CGs) distributed in several regional clusters. Besides validating a robust recruitment and sampling process, this study outlines the value of combining PCR and serological testing when suspecting leptospirosis and the usefulness of implementing molecular typing methods to identify circulating field strains. It also confirms the epidemiological importance of the Australis serogroup and allows for the collection of different highly pathogenic strains for vaccine development.

Study on the interference of vaccine antibodies with the serological diagnosis of leptospirosis in cattle

A simple IgG-specific ELISA for Leptospira spp. was compared with the microscopic agglutination test (MAT) to detect IgG antibody responses to a commercial vaccine in cattle. We used an enzyme-linked immunosorbent assay (ELISA) with sonicated Leptospira interrogans serovar copenhageni M 20. After initial vaccination, specific antibodies against Leptospira spp. were detected in 90 % of the animals by IgG-ELISA and 60 % by MAT, while after booster, antibodies were detected in 100 % and 80 % of the animals by IgG-ELISA and MAT, respectively. Both serological MAT and ELISA tests revealed interferences of vaccine antibodies. Disease diagnosis with ELISA and MAT methods should be made two and a half months and four months, respectively, after vaccination to avoid interference of vaccine antibodies. On the other hand, our results suggest that IgG-ELISA may be a useful method to assess the development of IgG antibodies induced by Leptospira vaccine.

Review of leptospirosis in dogs from Mexico: Epidemiology, diagnosis, prevention, and treatment.

Leptospirosis, classified by the World Health Organization as an emerging and neglected disease, is caused by the zoonotic pathogen Leptospira interrogans. This review aims to outline the Mexican epidemic of L. interrogans in dogs, including diagnosis and prevention methods. This review article searched articles from the publishers Wiley, Springer, PubMed, Redalyc, SciElo, and Elsevier. Among the 200 Mexican articles concerning Leptospira epidemiology, diagnosis, treatment, and vaccination, those that failed to meet the set inclusion criteria were excluded. The worldwide study of L. interrogans has focused on this bacterium. In Mexico, up-to-date information on canine prevalence, diagnosis, and vaccine use is scarce. Flow cytometrically detected Salmonella serovars differ from those in current vaccines, emphasizing the importance of broadening vaccine serovar coverage.

Leptospirosis as a cause of infertility in Uruguayan beef cattle

Leptospirosis, caused by pathogenic spirochetes of the genus Leptospira spp., is a globally significant zoonotic disease that affects humans and animals. In cattle, leptospirosis is associated not only with overt clinical manifestations but also with reproductive diseases, including infertility. This study assesses the potential correlation between leptospirosis and infertility in Uruguayan beef cattle. A case-control study involved 31 beef herds with no prior history of Leptospira vaccination. In each herd, veterinarians identified 10 non-pregnant (cases) and 25 pregnant cows (controls) using ultrasound, and blood and urine samples were collected from each cow. Serological diagnosis was performed using the Microscopic Agglutination Test (MAT), and quantitative PCR (qPCR) was used to assess Leptospira excretion. Additionally, antibodies against bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis (IBR) were tested. The results demonstrated an association between seropositivity to the Sejroe serogroup (cut-off 1:200) and infertility in cattle (OR=1.31; p-value=0.06). Furthermore, the level of Leptospira excretion (qPCR) in urine was associated with increased infertility risk, with cows excreting over 100 copies per mL of urine having the highest odds of infertility (OR=2.34; p-value<0.01). This study suggests a potential association between leptospirosis and infertility in Uruguayan beef cattle, emphasizing the importance of both serological and molecular diagnostics for assessing reproductive health in cattle herds. Future research should explore the impact of Leptospira serogroups on other reproductive disorders in cattle.

Outer membrane vesicles as nanovaccine candidates against pathogenic Leptospira in experimental Guinea pig model

Leptospira interrogans serovar Hardjo is a long slender bacterium of size 0.1–0.3 μm × 5–50 μm. It is one of the major causes of bovine leptospirosis and is of economical importance because of the reproductive failure, still birth, abortion, and reduced productivity in cattle. It is also a zoonotic disease-causing infection in humans characterized by headaches, fever, chills, sweats and myalgia, lethargy, aching joints, pulmonary haemorrhages, and death in severe cases. Control of the disease involves antibiotic therapy, management and vaccination, of which immunization is the cheapest and effective means of disease prevention. The present study was developed to isolate and characterize the outer membrane vesicles of Leptospira interrogans serovar Hardjo and to evaluate their vaccine potential in guinea pig model. The OMVs were isolated from the culture by sonication and ultracentrifugation. In transmission electron microscopy, the isolated OMVs appeared as small spherical structures of 50–200 nm size. In Western blot and indirect ELISA, antibodies specific to OMVs were observed as indicative of a good humoral immune response elicited by L. interrogans serovar Hardjo OMV. The OMV-based Leptospira vaccine was able to prevent kidney lesions and renal colonization compared to the control and bacterin vaccinated group as proven by histopathology and PCR.

Evaluation of protective efficacy, serological responses, and cytokine modulation induced by polyvalent Leptospira vaccines in hamsters

Whole-cell inactivated vaccines (bacterins) are the only licensed vaccines available for leptospirosis prevention and control, especially in domestic and farm animals. However, despite their widespread use, inconsistencies in their efficacy have been reported. Because immunity induced by bacterins is mainly mediated by antibodies against leptospiral lipopolysaccharides, the involvement of cellular responses is not well-known. The aim of this study was to investigate the efficacy and characterize the humoral and cellular immune responses induced by whole-cell inactivated leptospirosis bacterin formulations containing serovars Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjoprajitno, and Pomona. For the potency test, hamsters were immunized with one dose of polyvalent bacterins (either commercial or experimental) and then challenged with a virulent Pomona strain. Serological (MAT and IgM and IgG-ELISA) and cellular (cytokine transcription in blood evaluated by RT-qPCR) analyses were performed. The results revealed that vaccination with either bacterin formulation was able to protect 90–100% of the hamsters infected with the Pomona serovar, although most of the surviving animals remained as renal carriers. Specific agglutinating antibodies and significant levels of IgM, IgG, and IgG2 (P < 0.05) that were able to react with the six serovars present in the vaccine formulations were produced, indicating that the vaccines can potentially provide immunity against all strains. The protective immunity of these vaccines was mainly mediated by balanced a Th1/Th2 response, characterized by increased IFN-γ, IL-10 and IL-α transcription. These data support the importance of characterizing immunological responses involved in bacterin efficacy and investing in the improvement of these vaccine formulations.

Epitopes mapping for identification of potential cross-reactive peptide against leptospirosis

Leptospira, the pathogenic helical spirochetes that cause leptospirosis, is an emerging zoonotic disease with effective dissemination tactics in the host and can infect humans and animals with moderate or severe illnesses. Thus, peptide-based vaccines may be the most effective strategy to manage the immune response against Leptospira to close these gaps. In the current investigation, highly immunogenic proteins from the proteome of Leptospira interorgan serogroup Icterohaemorrhagie serovar Lai strain 56601 were identified using immunoinformatic methods. It was discovered that the conserved and most immunogenic outer membrane Lepin protein was both antigenic and non-allergenic by testing 15 linear B-cells and the ten best T-cell (Helper-lymphocyte (HTL) with the most significant number of HLA-DR binding alleles and the eight cytotoxic T lymphocyte (CTL)) epitopes. Furthermore, a 3D structural model of CTL epitopes was created using the Pep-Fold3 platform. Using the Autodock 4.2 docking server, research was conducted to determine how well the top-ranked CTL peptide models attach to HLA-A*0201 (PDB ID: 4U6Y). With HLA-A*0201, the epitope SSGTGNLHV binds with a binding energy of −1.29 kcal/mol. Utilizing molecular dynamics modeling, the projected epitope-allele docked complex structure was optimized, and the stability of the complex system was assessed. Therefore, this epitope can trigger an immunological response and produce effective Leptospira vaccine candidates. Overall, this study offers a unique vaccination candidate and may encourage additional research into leptospirosis vaccines. Communicated by Ramaswamy H. Sarma

Updated ACVIM consensus statement on leptospirosis in dogs.

Since publication of the last consensus statement on leptospirosis in dogs, there has been revision of leptospiral taxonomy and advancements in typing methods, widespread use of new diagnostic tests and vaccines, and improved understanding of the epidemiology and pathophysiology of the disease. Leptospirosis continues to be prevalent in dogs, including in small breed dogs from urban areas, puppies as young as 11 weeks of age, geriatric dogs, dogs in rural areas, and dogs that have been inadequately vaccinated for leptospirosis (including dogs vaccinated with 2-serovar Leptospira vaccines in some regions). In 2021, the American College of Veterinary Internal Medicine (ACVIM) Board of Regents voted to approve the topic for a revised Consensus Statement. After identification of core panelists, a multidisciplinary group of 6 experts from the fields of veterinary medicine, human medicine, and public health was assembled to vote on the recommendations using the Delphi method. A draft was presented at the 2023 ACVIM Forum, and a written draft posted on the ACVIM website for comment by the membership before submission to the editors of the Journal of Veterinary Internal Medicine. This revised document provides guidance for veterinary practitioners on disease in dogs as well as cats. The level of agreement among the 12 voting members (including core panelists) is provided in association with each recommendation. A denominator lower than 12 reflects abstention of ≥1 panelists either because they considered the recommendation to be outside their scope of expertise or because there was a perceived conflict of interest.

Challenges and Strategies for Developing Recombinant Vaccines against Leptospirosis: Role of Expression Platforms and Adjuvants in Achieving Protective Efficacy.

The first leptospiral recombinant vaccine was developed in the late 1990s. Since then, progress in the fields of reverse vaccinology (RV) and structural vaccinology (SV) has significantly improved the identification of novel surface-exposed and conserved vaccine targets. However, developing recombinant vaccines for leptospirosis faces various challenges, including selecting the ideal expression platform or delivery system, assessing immunogenicity, selecting adjuvants, establishing vaccine formulation, demonstrating protective efficacy against lethal disease in homologous challenge, achieving full renal clearance using experimental models, and reproducibility of protective efficacy against heterologous challenge. In this review, we highlight the role of the expression/delivery system employed in studies based on the well-known LipL32 and leptospiral immunoglobulin-like (Lig) proteins, as well as the choice of adjuvants, as key factors to achieving the best vaccine performance in terms of protective efficacy against lethal infection and induction of sterile immunity.

Cellular and humoral immune responsiveness to inactivated Leptospira interrogans in dogs vaccinated with a tetravalent Leptospira vaccine.

Vaccination is commonly used to protect dogs against leptospirosis, however, memory immune responses induced by canine Leptospira vaccines have not been studied. In the present study, antibody and T cell mediated responses were assessed in dogs before and 2weeks after annual revaccination with a commercial tetravalent Leptospira vaccine containing serogroups Canicola and Australis. Vaccination significantly increased average log2 IgG titers from 6.50 to 8.41 in year 1, from 5.99 to 7.32 in year 2, from 5.32 to 8.32 in year 3 and from 5.32 to 7.82 in year 4. The CXCL-10 levels, induced by in vitro stimulation of PBMC with Canicola and Australis, respectively, significantly increased from 1039.05pg/ml and 1037.38pg/ml before vaccination to 2547.73pg/ml and 2730.38pg/ml after vaccination. IFN-γ levels increased from 85.60pg/ml and 178.13pg/ml before vaccination to 538.62pg/ml and 210.97pg/ml after vaccination. The percentage of proliferating CD4+ T cells in response to respective Leptospira strains significantly increased from 1.43% and 1.25% before vaccination to 24.11% and 14.64% after vaccination. Similar responses were also found in the CD8+ T cell subset. Vaccination also significantly enhanced the percentages of central memory CD4+ T cells from 12% to 26.97% and 27.65%, central memory CD8+ T cells from 3% to 9.47% and 7.55%, and effector CD8+ T cells from 3% to 7.6% and 6.42%, as defined by the expression of CD45RA and CD62L, following stimulation with Canicola and Australis, respectively. Lastly, enhanced expression of the activation marker CD25 on T cells after vaccination was found. Together, our results show that next to IgG responses, also T cell responses are induced in dogs upon annual revaccination with a tetravalent Leptospira vaccine, potentially contributing to protection.

High expression of LipL21 protein of Iranian Leptospira interrogans in E. coli, applicable for diagnostic ELISA

Leptospirosis is an emerging infectious zoonotic disease caused by pathogenic Leptospira. The disease is more prevalent among farmers in hot and humid areas of Iran. Lack of clear clinical signs have impeded the diagnosis of leptospirosis. In this study, we attempted to produce a recombinant LipL21 protein of Leptospira based on a dominant pattern of Iranian isolates and to evaluate it in ELISA test. One hundred and sixty-two complete sequences of LipL21 available in GenBank until January 1, 2019 were compared. One dominant LipL21 protein pattern was selected. The codon optimised sequence was cloned into the pET32a+ expression vector. Trx-LipL21 fusion protein was induced, purified and confirmed by 10% SDS-PAGE followed Coomassie blue staining and immune blotting. For evaluation of effectiveness of rLip21 in ELISA test, 200 µg rLip21 with Montanide ISA70 adjuvant was injected subcutaneously in rabbits three times. Results showed that rLipL21 protein was highly expressed in 2YT media in presence of 0.1 mM IPTG after 16 hours incubation at 37 oC. Recombinant protein was purified 36 mg per liter using affinity batch formation method by Ni-NTA resin. ELISA with micro plate coated with 250 ng rLipL21 protein demonstrated prominently diffe­rences between test and control groups (P&lt;0.01). The rLipL21 protein produced large amounts of antibodies in the rabbit. The protein was also able to detect high levels of antibody in animals immu­nised with Leptospira vaccine. The rLipL21 might be a good candidate for diagnosis and evaluation of antibody levels against Leptospira.

Leptospira borgpetersenii Leucine-Rich Repeat Proteins Provide Strong Protective Efficacy as Novel Leptospiral Vaccine Candidates.

Leucine-rich repeat (LRR) proteins are advocated for being assessed in vaccine development. Leptospiral LRR proteins were identified recently in silico from the genome of Leptospira borgpetersenii serogroup Sejroe, the seroprevalence of leptospiral infections of cattle in Thailand. Two LRR recombinant proteins, rKU_Sej_LRR_2012M (2012) and rhKU_Sej_LRR_2271 (2271), containing predicted immunogenic epitopes, were investigated for their cross-protective efficacies in an acute leptospirosis model with heterologous Leptospira serovar Pomona, though, strains from serogroup Sejroe are host-adapted to bovine, leading to chronic disease. Since serovar Pomona is frequently reported as seropositive in cattle, buffaloes, pigs, and dogs in Thailand and causes acute and severe leptospirosis in cattle by incidental infection, the serogroup Sejroe LRR proteins were evaluated for their cross-protective immunity. The protective efficacies were 37.5%, 50.0%, and 75.0% based on the survival rate for the control, 2012, and 2271 groups, respectively. Sera from 2012-immunized hamsters showed weak bactericidal action compared to sera from 2271-immunized hamsters (p < 0.05). Therefore, bacterial tissue clearances, inflammatory responses, and humoral and cell-mediated immune (HMI and CMI) responses were evaluated only in 2271-immunized hamsters challenged with virulent L. interrogans serovar Pomona. The 2271 protein induced prompt humoral immune responses (p < 0.05) and leptospiral tissue clearance, reducing tissue inflammation in immunized hamsters. In addition, protein 2271 and its immunogenic peptides stimulated splenocyte lymphoproliferation and stimulated both HMI and CMI responses by activating Th1 and Th2 cytokine gene expression in vaccinated hamsters. Our data suggest that the immunogenic potential renders rhKU_Sej_LRR_2271 protein a promising candidate for the development of a novel cross-protective vaccine against animal leptospirosis.

Leptospiral Leucine-Rich Repeat Protein-Based Lateral Flow for Assessment of Canine Leptospiral Immunoglobulin G.

The recombinant, modified leucine-rich repeat protein rhKU_Sej_LRR_2271 has been suggested as a candidate for leptospiral vaccine development since it was predicted to be a transmembrane protein containing leucine-rich repeat motifs and immunogenic epitopes. The immunogenic epitopes showed binding affinities with lower IC50 values than peptides of known antigenic proteins, e.g., LipL32. Moreover, this protein was immunoreactive with hyperimmune sera against several serovars. In this study, we aimed to develop a lateral flow strip test using the rhKU_Sej_LRR_2271 protein for the detection of anti-leptospiral IgG in dogs. The lateral flow assay was performed with 184 dog plasma samples and evaluated with a culture method, 16S ribosomal RNA gene (rss) analysis real-time PCR, and LipL32 ELISA. The culture method failed to detect leptospires in the dog blood samples. Six of nine symptomatic dogs gave positive results with the real-time PCR assay. The lateral flow assay and LipL32 ELISA gave positive results with 59 and 50 dogs, respectively. The sensitivity, specificity, and accuracy of the rhKU_Sej_LRR_2271 lateral flow strip test were 70.00, 82.09, and 78.80%, respectively, when compared with LipL32 ELISA. There was a significant association between the LipL32 ELISA and the rhKU_Sej_LRR_2271 lateral flow assay. The rhKU_Sej_LRR_2271 lateral flow strip test has therefore demonstrated a good potential to detect anti-leptospiral IgG in dogs.

Immune response at a vaccine-challenge study using beagle dogs and locally isolated Leptospira spp

Determination of the immune response of dogs by measuring the antibody levels (utilizing MAT) and levels of cytokines (TNF-α, IL-4 and IFN-γ) post-vaccination with locally produced killed whole-celled Leptospiral vaccine and post-challenge with a locally isolated Leptospira icterohaemorrhagiae Copenhageni strain. For assessment of immunity of the vaccine serum antibodies were detected before and after vaccination and challenge in three studies. The effects of the challenge were determined by a variety of parameters including reisolation of the challenge Leptospira spp. via blood, urine, and kidney samples. The challenge strain did not produce generalised infection but elevated circulating antibody levels in both the control and vaccinated dogs in any of the three studies, however leptospires were reisolated from the urine of the control dogs but not the vaccinated dogs. Cytokine levels (TNF-α, IFN-γ and IL-4) were detected post-challenge in the vaccinated dogs to determine the immune profile response. The whole-killed cell vaccine in this study did not prevent leptospireamia but prevented leptospiruria in vaccinated dogs after a challenge with a live Leptospira icterohaemorrhagiea Copenhageni. The vaccine-challenge showed increased antibody (MAT) levels due to vaccination and infection (through challenge). Cytokine production (TNF-α, IFN-γ and IL-4) by the host immune system was observed post-challenge with live leptospires.

An Assessment and Comparison of the Efficacy of Two Licensed Tetravalent Leptospira Vaccines for Dogs Using an Improved Challenge Model.

An objective of the present studies was to compare the protective effects of two European licensed canine tetravalent leptospirosis vaccines, Nobivac L4 and Versican Plus L4. Four similar challenge studies in beagle dogs were performed. In each study the dogs were assigned to one of three treatment groups as follows: group 1, Nobivac L4; group 2, Versican Plus L4; group 3, non-vaccinated control group. Two vaccinations were followed by a Leptospira challenge. Strains of the following serogroups were used for challenge: study 1, Grippotyphosa; study 2, Icterohaemorrhagiae; study 3, Canicola; study 4, Australis. Parameters of efficacy were antibody titres; body temperature; clinical signs; cultures of Leptospira bacteria from the blood, urine, kidney and liver; rapid urinalysis; macroscopic and histopathological examination at necropsy. It was concluded that compared to vaccination with Versican Plus L4, vaccination with Nobivac L4 resulted in generally better control of leptospirosis disease parameters after the challenge including a complete prevention of the clinical signs following a Grippotyphosa and Icterohaemorrhagiae challenge. In contrast, vaccination with Versican Plus L4 only prevented infection by Australis and shedding by Grippotyphosa and Australis but it did not lead to any statistically significant reduction of either infection or shedding following an Icterohaemorrhagiae challenge.

Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1β, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog.

Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Leptospira Vaccine Strains.

Canine Leptospira vaccines contain inactivated strains of pathogenic Leptospira, the causative agents of leptospirosis. For an effective response to vaccination, activation of the innate immune system via pattern recognition receptors such as TLRs is crucial. However, it is not known which TLRs are activated by Leptospira in dogs. To investigate the involvement of canine TLR2, TLR4, and TLR5 in the recognition of Leptospira, we stimulated canine moDC and reporter cells expressing canine TLR2 with either whole-inactivated bacteria or purified LPS of Leptospira strains, representing the serogroups generally used in canine leptospirosis vaccines. Using the endotoxin neutralizing reagent polymyxin B and TLR4 antagonist RS-LPS, we demonstrate that Leptospira LPS and canine TLR4 are involved in IL-1β production as well as in the uptake of inactivated Leptospira in canine moDC. Furthermore, polymyxin B only partially inhibited IL-1β production induced by inactivated Leptospira, suggesting that next to TLR4, also other TLRs may be involved. The observed activation of canine TLR2-expressing reporter cells by inactivated Leptospira strains indicates that TLR2 could be one of these TLRs. Next, we analyzed TLR2 and TLR4 activating capabilities by the same Leptospira strains using human and mouse TLR-expressing reporter cells. Inactivated Leptospira and leptospiral LPS activated not only mouse, but also human TLR4 and this activation was shown to be LPS dependent in both cases. Additionally, inactivated Leptospira activated mouse and human TLR2-expressing reporter cell lines. In our study, we could not identify significant species differences in the recognition of Leptospira by TLR2 and TLR4 between dog, human and mouse. Lastly, we show that these inactivated Leptospira strains are recognized by both mouse and human TLR5 reporter cells only after exposure to additional heat-treatment. Unfortunately, we were not able to confirm this in the canine system. Our data show that TLR2 and TLR4 are involved in the recognition of Leptospira strains used in the production of canine Leptospira vaccines. This study contributes to the understanding of Leptospira-induced innate immune responses in dogs, humans, and mice. Future studies are needed to further explore the role of canine TLR2, TLR4 and TLR5 in the induction of vaccine-mediated immunity against Leptospira.

In silico screening and epitope mapping of leptospiral outer membrane protein—Lsa46

Leptospirosis is one of the neglected diseases caused by the spirochete, Leptospira interrogans. Leptospiral surface adhesion (Lsa) proteins are surface exposed outer membrane proteins present in the pathogen. It acts as laminin and plasminogen binding proteins which enable them to infect host cells. The major target for the development of vaccine in the current era focuses on surface exposed outer membrane proteins, as they can induce strong and fast immune response in hosts. Therefore, the present study mapped the potential epitopes of the Leptospiral outer membrane proteins, mainly the surface adhesion proteins. Protein sequence analysis of Lsa proteins was done by in silico methods. The primary protein sequence analysis revealed Lsa46 as a suitable target which can be a potent Leptospiral vaccine candidate. Its structure was modelled by threading based method in I-TASSER server and validated by Ramachandran plot. The predicted epitope’s interactions with human IgG, IgM(Fab) and T-cell receptor TCR(αβ) were performed by molecular docking studies using Biovia Discovery studio 2018. One of the predicted B-cell epitopes and the IgG showed desirable binding interactions, while four of the predicted B-cell epitopes and T-cell epitopes showed desirable binding interactions with IgM and TCR respectively. The molecular dynamic simulation studies carried out with the molecular docked complexes gave minimized energies indicating stable interactions. The structural analysis of the entire simulated complex showed a stable nature except for one of the Epitope-IgM complex. Further the binding free energy calculation of eight receptor-ligand complex predicted them energetically stable. The results of the study help in elucidating the structural and functional characterization of Lsa46 for epitope-based vaccine design. Communicated by Ramaswamy H. Sarma.

Pathogenic Leptospira spp. Seroprevalence and Herd-Level Risk Factors Associated with Chilean Dairy Cattle.

Simple SummaryLeptospirosis is a ubiquitous distributed infectious disease present in wild and domestic animals that can be transmitted to humans. This study aimed to estimate the burden of the bacteria in dairy cows from southern Chile and identify the factors associated with the herd-level status. We studied 147 herds and 4876 lactating cows from the area, and an infected herd was defined when at least one serologically positive reactor to MAT was detected. An epidemiological survey was applied to the herd’s owner. The estimated overall individual prevalence was 5.3% (95% CI 2.9–7.7), the overall herd-level prevalence was 42.2% (95% CI 34.2–50.2), and there was variation in both between different herd sizes. L. borgpetersenii serovars Hardjo and Tarassovi and L. interrogans serovar Pomona were the more frequent serovars in Leptospira non-vaccinated herds. Attenuated Leptospira vaccine usage was assessed as a factor that decreases the risk of a farm being infected (OR = 0.04; 95% CI = 0.02–0.11), and variables that increase that risk were using bulls for mating (OR = 3.43; (95% CI = 1.1–10.1) and continuous calving distribution (OR = 3.4; 95% CI = 1.3–8.8). The results from this study will contribute to unravelling the infection burden in the main dairy area of the country and designing control strategies.Leptospirosis is a ubiquitous distributed zoonotic infectious disease present in wild and domestic animals. This study aimed to estimate within-herd and herd-level seroprevalence against pathogenic Leptospira spp. in dairy cows from southern Chile and identify risk factors associated with the herd-level status. We used a multi-stage strategy combined with a stratified sample strategy for randomly sampling 147 herds and 4876 lactating cows. We considered as infected a herd with at least one positive reactor to MAT. In addition, an epidemiological survey was applied to the herd’s owners and a logistic regression (LR) model was constructed to analyze it. The overall within-herd prevalence was 5.9% (95% CI 4.9–6.8), the overall herd-level prevalence was 42.2% (95% CI 9.2–47.9), and there was variation in both between different herd sizes. L. borgpetersenii serovars Hardjo and Tarassovi and L. interrogans serovar Pomona were the more frequent serovars in non-vaccinated herds. A factor that decreases the risk of a farm being infected was Leptospira vaccine usage (OR = 0.04; 95% CI = 0.02–0.11), and variables that increase risk were using bulls for mating (OR = 3.43; 95% CI = 1.1–10.1) and continuous calving distribution (OR = 3.4; 95% CI = 1.3–8.8). The study’s results will contribute to unravelling the infection burden in the main dairy area of the country and designing control strategies.

Protective efficacy of whole-cell inactivated Leptospira vaccines made using virulent or avirulent strains in a hamster model

Whole-cell inactivated vaccines remain the only licensed vaccines used to control human and animal leptospirosis worldwide. Although they are protective against lethal infections, the efficacy of these vaccines has been divergent. The manufacturing process often involves the use of standard bacterial strains subjected to serial in vitro passages, with a risk of loss of virulence, and may affect the immunogenicity and consequently decrease protection. Thus, the objective of this study was to perform a comparative analysis of the efficacy of in-house bacterins produced with standard (avirulent) and virulent strains. Hamsters were immunized with killed bacteria produced using avirulent and virulent strains of L. interrogans serovars Copenhageni and Canicola. Vaccine efficacy was determined in terms of protection against lethal homologous or heterologous challenges. The results showed that immunization with both avirulent and virulent Canicola strains resulted in 100% protection against homologous challenge. Conversely, Copenhageni bacterins produced using an avirulent strain conferred only 25–37.5% protection against homologous challenge (P > 0.05), while virulent Copenhageni bacterin conferred 100% protection (P < 0.001). A single vaccine dose was sufficient to induce protection, and administration of a prime boost significantly reduced the bacterial load in the kidneys and improved the humoral immune response to the virulent Copenhageni strain. These findings suggest that the maintenance of virulent strains in bacterin formulations is essential for improving the immunogenicity and efficacy of leptospirosis vaccines.