Navigating cross-reactivity and host species effects in a serological assay: A case study of the microscopic agglutination test for Leptospira serology.

BACKGROUND: Serology (the detection of antibodies formed by the host against an infecting pathogen) is frequently used to assess current infections and past exposure to specific pathogens. However, the presence of cross-reactivity among host antibodies in serological data makes it challenging to interpret the patterns and draw reliable conclusions about the infecting pathogen or strain. METHODOLOGY/PRINCIPAL FINDINGS: In our study, we use microscopic agglutination test (MAT) serological data from three host species [California sea lion (Zalophus californianus), island fox (Urocyon littoralis), and island spotted skunk (Spilogale gracilis)] with confirmed infections to assess differences in cross-reactivity by host species and diagnostic laboratory. All host species are known to be infected with the same serovar of Leptospira interrogans. We find that absolute and relative antibody titer magnitudes vary systematically across host species and diagnostic laboratories. Despite being infected by the same Leptospira serovar, three host species exhibit different cross-reactivity profiles to a 5-serovar diagnostic panel. We also observe that the cross-reactive antibody titer against a non-infecting serovar can remain detectable after the antibody titer against the infecting serovar declines below detectable levels. CONCLUSIONS/SIGNIFICANCE: Cross-reactivity in serological data makes interpretation difficult and can lead to common pitfalls. Our results show that the highest antibody titer is not a reliable indicator of infecting serovar and highlight an intriguing role of host species in shaping reactivity patterns. On the other side, seronegativity against a given serovar does not rule out that serovar as the cause of infection. We show that titer magnitudes can be influenced by both host species and diagnostic laboratory, indicating that efforts to interpret absolute titers (e.g., as indicators of recent infection) must be calibrated to the system under study. Thus, we implore scientists and health officials using serological data for surveillance to interpret the data with caution.

Study on Seroprevalence and Leptospiral Antibody Distribution Among Livestock Breeders and Farmers in Golestan Province.

BACKGROUND: The high prevalence of leptospirosis in humans is of great public health concern, particularly in tropical and subtropical regions. OBJECTIVE: This study aimed to determine the seroprevalence of leptospiral antibodies and the distribution of serovars in livestock breeders and farmers in Golestan province. METHODS: Seventy samples of serum collected from farmers and ranchers suspected of leptospirosis were examined using an ELISA method for surveying Immunoglobulin M (IgM) anti-leptospiria. Also from samples, DNA was extracted and PCR was performed using by primers for 16s rRNA. Demographic properties of positive patients were analysed. RESULTS: Chi-square statistical test shows a statistically significant difference between the gender and prevalence leptospirosis (p-value = 0.004). Also, by examining the age, it was shown that 68.57% of patients are in the middleaged rang. According to the results obtained from the study and investigation of blood serum IgM-and Immunoglobulin G (IgG) in people suspected of leptospirosis, 3 cases of the patients had IgM higher than 11 and were known to be positive for leptospirosis. Also, by examining the IgG level of patients, 5 cases had intermediate results, and 2 cases were found to be positive for IgG. The PCR results showed that 41.42% of patients tested positive for the 16s rRNA. CONCLUSION: Leptospirosis is a common disease among farmers, and in Golestan province, considering traditional farming methods, it is considered an important infectious disease. Therefore, health and safety measures should be expanded to control and prevent this disease. Also, by employing mechanised agricultural methods, the prevalence of leptospirosis in this region can be significantly reduced.

The role of asymptomatic cattle for leptospirosis dynamics in a herd with imperfect vaccination.

Leptospirosis is an emerging zoonotic disease with high health and economic damage. In this study, we developed a deterministic mathematical model that describes the dynamics of leptospirosis transmission within a cattle herd, incorporating asymptomatic infected and vaccinated compartments. The study examined the transmission role of asymptomatic cattle that contaminate herds without farmers' knowledge. We proved the well-posedness of the proposed model and found the basic reproduction number using the next-generation matrix. Analytically, we demonstrated that the disease-free equilibrium point is locally and globally asymptotically stable when R 0 is less than unity and is otherwise unstable. Graphically, we further established the local asymptotic stability of disease-free and endemic equilibria. Sensitivity analysis showed that the contact rate with asymptomatic infected cattle, ß A , is the most sensitive parameter in the stated model, followed by the recovery rate of asymptomatic infected cattle, σ , and the vaccination rate of susceptible cattle, τ . Numerical simulations revealed that a reduction in contact rate with asymptomatic infected cattle significantly reduced pathogen Leptospira transmission in the herd. In addition, fostering the recovery rate of asymptomatic infected cattle can significantly reduce new infections in the herd. Furthermore, augmenting the vaccination rate among susceptible cattle resulted in a notable decrease in disease prevalence within the herd. Findings of this study underscore the remarkable importance of targeted interventions, such as reducing contact rates with asymptomatic infected cattle, increasing recovery rates using proper treatments, and enhancing vaccination efforts to manage leptospirosis transmission in cattle herds.

Enhancement of clinical signs in C3H/HeJ mice vaccinated with a highly immunogenic Leptospira methyl-accepting chemotaxis protein following challenge.

Leptospirosis is the most widespread zoonosis and a life-threatening disease in humans and animals. Licensed killed whole-cell vaccines are available for animals; however, they do not offer heterologous protection, do not induce long-term protection, or prevent renal colonization. In this study, we characterized an immunogenic Leptospira methyl-accepting chemotaxis protein (MCP) identified through a reverse vaccinology approach, predicted its structure, and tested the protective efficacy of a recombinant MCP fragment in the C3H/HeJ mice model. The predicted structure of the full-length MCP revealed an architecture typical for topology class I MCPs. A single dose of MCP vaccine elicited a significant IgG antibody response in immunized mice compared to controls (P < 0.0001), especially the IgG1 and IgG2a subclasses. The vaccination with MCP, despite eliciting a robust immune response, did not protect mice from disease and renal colonization. However, survival curves significantly differed between groups, and the MCP-vaccinated group developed clinical signs faster than the control group. There were differences in gross and histopathological changes between the MCP-vaccinated and control groups. The factors leading to enhanced disease process in vaccinated animals need further investigation. We speculate that anti-MCP antibodies may block the MCP signaling cascade and may limit chemotaxis, preventing Leptospira from reaching its destination, but facilitating its maintenance and replication in the blood stream. Such a phenomenon may exist in endemic areas where humans are highly exposed to Leptospira antigens, and the presence of antibodies might lead to disease enhancement. The role of this protein in Leptospira pathogenesis should be further evaluated to comprehend the lack of protection and potential exacerbation of the disease process. The absence of immune correlates of protection from Leptospira infection is still a major limitation of this field and efforts to gather this knowledge are needed.

Assessing leptospirosis and toxoplasmosis seropositivity in wild boars (Sus scrofa): Implications for public and animal health in Southern Brazil.

Leptospirosis and toxoplasmosis are re-emerging zoonosis caused by infection with pathogenic spirochaetes of Leptospira and the protozoa Toxoplasma gondii, respectively. Wild boars (Sus scrofa), an exotic invasive species in Brazil, could play a role in the diseases' epidemiological cycles, but this issue is still unexplored. This study aimed to evaluate the Leptospira spp. and T. gondii seropositivity in wild boars in Rio Grande do Sul state, south Brazil. Of evaluated animals, 16% (13/80) and 85% (52/61) had antibodies to T. gondii and Leptospira spp., respectively. Sex, weight, age, hunt location and season of hunt were evaluated by their association with seropositivity for both pathogens, but none of them had statistical significance. This study revealed that wild boars should be considered as a potential source of Leptospira spp. and T. gondii dissemination for humans and animal species in shared environments in Rio Grande do Sul state.

Epidemiology of human and animal leptospirosis in Kenya: A systematic review and meta-analysis of disease occurrence, serogroup diversity and risk factors.

BACKGROUND: Leptospirosis is a priority zoonotic disease in Kenya, but an in-depth review of its presence in humans, animals and the environment is lacking. Therefore, we conducted this systematic review and meta-analysis to understand the epidemiological situation to date. METHODOLOGY: We searched for literature in African journals online, AGRIS, Embase, the Leptospira WOAH reference laboratory library, ProMED-mail, PubMed, Scopus, Web of Science, and the institutional repositories of 33 academic institutions and included 66 publications on leptospirosis in Kenya which spanned from 1951 to 2022. The review was registered on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY). FINDINGS: Most investigations were done in rural and urban areas in western, southern, central, and coastal areas in Kenya and the largely pastoral eastern and northern areas were under-represented. A wide host range of domestic animals and wildlife was revealed, and occupational exposure was an important risk factor for humans. The microscopic agglutination test (MAT) was the most frequent test, particularly common in studies conducted during the 1980s and 1990s. However, varying MAT panels and cut-off titres were observed. The overall seroprevalence in cattle was 28.2% (95% confidence intervals [CI]: 12.0-53.0; heterogeneity: I2 = 96.7%, τ2 = 1.4), and 11.0% in goats (95% CI: 5.4-21.2; heterogeneity: I2 = 78.8%, τ2 = 0.4). Molecular tests were seldom used to determine species and illustrate strain diversity. There was a lack of awareness of leptospirosis among farmers and health practitioners. CONCLUSION: The widespread presence of leptospires and inadequate diagnostic capacity demonstrate that leptospirosis is a common but underreported disease in Kenya. Raising awareness and boosting the country's diagnostic capacity is crucial to timely detection and disease control.

Epidemiological analysis of leptospirosis, dengue, and Co-infection rates among febrile illness cases in Dakshina Kannada, Karnataka.

INTRODUCTION: Leptospirosis and dengue are two significant public health concerns in tropical and subtropical regions, often resulting in severe forms of disease and fatality. This study addresses the pressing public health issues of leptospirosis and dengue in the Dakshina Kannada district of Karnataka, India. Both diseases pose significant health risks and are relatively understudied in this region, making it essential to investigate their prevalence and clinical presentations for targeted healthcare planning. AIM: The primary aim is to determine the frequency of leptospirosis and dengue among febrile illness cases to understand the epidemiological patterns and assess co-infection rates in Dakshina Kannada. METHOD: Between 2020 and 2021, serum samples suspected of leptospirosis were tested using IgM ELISA (n = 1629) and the Microscopic Agglutination Test (MAT) (n = 92) for leptospirosis, while dengue was tested using NS1Ag and IgM antibodies ELISA (n = 1415). Data were collected through medical records and patient interviews. Seasonal trends, gender, and age distributions were analyzed. RESULT: The study found a significant prevalence of leptospirosis (21 %) and dengue (10 %) among febrile illness cases in the study area, with a 1.3 % co-infection rate. Clinically, fever was common to both diseases, but leptospirosis also frequently exhibited symptoms such as abdominal pain, myalgia, and jaundice. MAT screening revealed a predominance of anti-leptospiral antibodies against the Djasiman, Pyrogenes, Hurstbridge, Hebdomadis, and Grippotyphosa serogroups in Dakshina Kannada. CONCLUSION: The study highlights the urgent need for focused public health interventions, improved diagnostic tools, and targeted epidemiological studies to manage these diseases. The findings underscore the necessity of enhancing diagnostic capabilities and public health awareness, particularly considering the significant health risks posed by leptospirosis and dengue in the region.

Spatiotemporal analysis and clinico-epidemiological study for seroprevalence of canine leptospirosis.

Leptospirosis is a worldwide re-emerging zoonotic disease. The study was conducted to estimate the Seroprevalence of canine leptospirosis in a total of 450 dogs, from a total of 97 puppies and 353 adult dogs selected for examination Sampling, started from January to December 2023 in District Kasur in the province Punjab of the country Pakistan. Leptospira IgG ELISA kit manufactured by DRG Instruments GmbH, Germany was used for the screening of canine Leptospira antibodies. Out of 450 tested dogs, 183 dogs (40.67%) were tested positive for Leptospira antibody for the screening of Leptospira antibodies. The estimated Seroprevalence of leptospirosis in various age groups of dogs, were 23.7% (23/97) and 45.3% (160/353), in puppies and adults, respectively (P < 0.05). It was found that out of the sampled dogs, a total of 35/127 (27.6%), 29/100 (29%), 73/130 (56.2%), and 46/93 (49.5%) dogs were tested seropositive for Leptospira antibodies in winter, spring, summer and fall, respectively (P < 0.05).

Accuracy of the Dual Path Platform (DPP) rapid test for the diagnosis of leptospirosis: A multi-center study in six Brazilian states.

Leptospirosis is a zoonotic disease with significant global impact and a challenging diagnosis. The utilization of adequately validated rapid tests is relevant for the opportune identification of the disease and for reduction in fatality rates. The present study analyzes the accuracy and reliability of the Dual Path Platform (DPP) assay -produced in Brazil by the Oswaldo Cruz Foundation (Fiocruz)- for diagnosing leptospirosis. Firstly, a serological panel was constructed in the Brazilian Reference Laboratory for Leptospirosis using samples routinely handled by reference laboratories of six Brazilian states. It consisted of 150 positive (according to MAT and IgM-ELISA) and 250 negative samples for leptospirosis. Subsequently, the panel samples were distributed to the reference laboratories for the performance of DPP assays in triplicate. Different measures were used in the assessment of diagnostic quality. Predictive values were estimated for different pre-test probability settings. Sensitivities varied between 67.33 % and 74.00 % and specificities between 93.20 % and 98.40 % in the states, and there were adequate agreements between them. Accuracies were lower for the samples of patients with less than 7 days of symptoms. In contexts of prevalence values up to around 25 %, positive and negative predictive values were around 90 %. However, in situations of high pre-test probabilities, NPVs were low. This study improves understanding of the use of DPP in diagnosing leptospirosis, particularly its application in healthcare settings. As long as the time of symptoms onset and clinical and epidemiological contexts are adequately considered for the interpretation of results, DPP is a valid option to be used in the leptospirosis diagnostic routine.

Development of a new accurate lateral flow immunoassay for diagnosis of human leptospirosis.

PURPOSE: The current diagnostic methods for leptospirosis diagnosis are technically complex and expensive, with limited applicability to specialized laboratories. Furthermore, they lack diagnostic accuracy in the acute stage of the disease, which coincides with a period when antibiotics are highly effective. New simple and accurate tests are mandatory to decentralize and improve diagnosis. Here, we introduced a new lateral flow immunoassay (Lepto-LF) for human leptospirosis. METHODS: We conducted a double-blinded assay using 104 serum samples from patients with confirmed or discarded diagnosis for leptospirosis. The diagnostic performance of Lepto-LF was estimated across different ranges of days from onset of symptoms (dpo), considering the diagnostic algorithm as reference standard. Additionally, it was compared with the screening methods enzyme-linked immunosorbent assay (IgM-ELISA) and the slide agglutination test using temperature-resistant antigen (SATR). RESULTS: Lepto-LF exhibited perfect diagnostic performance with a Youden´s index J = 1 from 6 dpo in the acute phase. IgM-ELISA gave slightly lower accuracy with J = 0.91 and 95.5% of both sensitivity and specificity; while SATR showed a markedly inferior yield (J = 0.41, sensitivity = 95.5%, specificity = 45.5%). The performances remained consistent in the convalescence phase of the disease (> 10 dpo). CONCLUSION: Lepto-LF was found to be a reliable test for simple, rapid and early diagnosis of leptospirosis, resulting a promising tool for decentralizing leptospirosis diagnosis and enabling timely treatment of patients. In addition, Lepto-LF may be employed as confirmatory test, especially in remote areas and vulnerable contexts where the standard MAT is not available.

Systematic review and meta-analysis on seroprevalence of leptospirosis in non-human primates worldwide.

Leptospirosis is a zoonotic disease whose transmission is linked to multiple factors involving the interface between animals, humans, and the environment. This disease is of great importance for public health, as it profoundly affects the health aspects of the population and animals. Considering the importance of non-human primates in this epidemiological chain, the objective of this research was to conduct a systematic literature review with meta-analysis, providing information on leptospirosis in non-human primates (NHPs) and an update on the infection situation in Brazil and other countries. Thus, a search was performed in five databases, initially finding 3332 studies, of which 32 met the eligibility criteria and were used for the systematic review. According to them, the most prevalent serogroup in non-human primates was Icterohaemorrhagiae, which is adapted to rodents as primary hosts. A wide distribution of the infection was found in the regions of both wild and captive animals. Through meta-analysis, the seroprevalence rate of leptospirosis in non-human primates was 27.21% (CI 17.97-38.95%). Cochran's Q test (p < 0.01) identified heterogeneity between studies, classified as high by the Higgins and Thompson test (I2 = 92.4%). Therefore, seroepidemiological and Leptospira isolation studies in non-human primates are important to investigate and monitor the suspected impact of these species as maintainers or transmitters of the pathogen to humans and other wild and domestic animals, in addition to demonstrating the need for standardization related to control and prevention measures.

Spatial and seasonal distribution of Leptospira spp. seropositive horses in the Northeast region of Brazil.

Leptospirosis is an anthropozoonosis of economic and public health importance, caused by bacteria of the genus Leptospira. Horses are deemed important in its transmission chain due to their proximity to humans, and because the species is often asymptomatic, making these animals potential silent reservoirs. In this context, the objectives of this study were to determine the prevalence of seropositive horses for Leptospira spp., and to identify the presence of Leptospira spp. serogroups and antibody titers, the occurrence of areas with higher density of infection cases and demographic characteristics associated with seropositivity in the states of Paraíba (PB), Pernambuco (PE), Rio Grande do Norte (RN) and Ceará (CE), in the Northeast region of Brazil, during rainy (May and June) and dry (October and November) seasons from 2017 to 2019. Using the microscopic agglutination test (MAT), 1152 equine serum samples from 225 municipalities were analyzed. Anti-Leptospira antibodies were detected in 23.9 % (95 % CI= 21.4 - 26.3 %) of the samples in the three-year period, with a frequency of 30.4 % (95 % CI= 26.7 - 34.2 %) during the rainy period (with greater emphasis on the Ballum serogroup) and 17.4 % (95 % CI= 14.3 - 20.5 %) in the dry period (with greater emphasis on the Sejroe serogroup). Age of horses ≥ 6 years (6-10 years, 11-15 years and ≥ 16 years), rainy season, and animal belonging to Pernambuco state were factors with higher seropositivities. Regarding spatial distribution, a higher percentage of seropositive animals was observed in Pernambuco (P < 0.05), in interstate border areas, and large urban centers, with a spatial cluster detected in the dry season of 2018 with relative risk of 2.8 (P = 0.049) times higher in municipalities within the cluster. It is suggested that measures for controlling rodents and contact with wild animals in equine farming, both in rainy and dry periods, combined with care regarding the use of pastures shared with cattle and the adoption of immunoprophylaxis are important in preventing and controlling leptospirosis in horses in the Northeast region of Brazil.

Leptospirosis in central India: A retrospective study to explore burden of tropical illness.

Leptospirosis, an underdiagnosed zoonotic disease in India, was studied retrospectively in Madhya Pradesh, Central India. Between 2018 and 2019, 2617 samples from patients with hepatitis-related symptoms were collected. Of these, 518 tested negative for hepatitis and other tropical viral diseases under the VRDL project were analyzed for leptospira IgM using ELISA. 68 (13.12%) were positive for leptospirosis. Common symptoms included fever (97.45%) and jaundice (42.27%), with renal involvement in 30.88% of cases. Higher incidence was observed in the 31-60 age group, especially during monsoon and post-monsoon seasons. The study highlights the need for increased clinician awareness and inclusion of leptospirosis in screening panels to differentiate tropical illnesses in India.

Leptospirosis: A dual threat - predisposing risk for renal transplant and trigger for renal transplant dysfunction.

Leptospirosis (LTPS) is a bacterial infection that affects humans, often with mild or no symptoms. It is estimated that approximately 10 % of patients with LTPS may experience multi-organ dysfunction, including renal abnormalities. In regions where LTPS is widespread, a considerable number of instances involving acute kidney injury (AKI) and chronic kidney disease (CKD) of unknown etiology (CKDu) have been reported. Additionally, studies have shown a correlation between kidney graft dysfunction in patients with stable kidney transplants after LTPS. These findings indicate that exposure to LTPS may increase the likelihood of kidney transplantation due to the onset of both acute and chronic kidney injuries. Simultaneously, it poses a potential risk to the stability of kidney grafts. Unfortunately, there is limited scientific literature addressing this issue, making it difficult to determine the negative impact that LTPS may have, such as its role as a risk factor for the need of kidney transplantation or as a threat to individuals who have undergone kidney transplants. This study aims to shed light on the immune mechanisms triggered during LTPS infection and their importance in both kidney damage and allograft dysfunction.

Mapping serogroup distribution and seroprevalence of leptospirosis in livestock of Assam, Northeastern State of India: Unveiling uncommon Leptospira serogroups.

Leptospirosis is a significant zoonotic disease affecting livestock, leading to reproductive issues and economic losses. Despite its endemic status in India, research has predominantly focused on coastal regions, leaving the North Eastern Region (NER) underexplored. This study aims to investigate the seroprevalence and serogroup distribution of leptospirosis in livestock across Assam, a major state in the North Eastern Region (NER) of India. Serum samples (n=811) from cattle, buffalo, sheep, goats, and pigs were collected between 2016 and 2019 and screened using the Microscopic Agglutination Test (MAT) for 24 serogroups. The overall seroprevalence was 22.9 % (186/811), with highest prevalence in cattle (26.2 %) and buffalo (25 %), followed by small ruminants (19.8 %) and pigs (18.6 %) . Notably, uncommon serovars such as Mini (28.8 %), Manhao (12.4 %), and Cynopteri (7.5 %) were identified, indicating a unique epidemiological pattern in Assam. High seroprevalence was observed in districts like Bongaigaon (66.7 %), Kamrup Metropolitan (50.0 %), and Nalbari (40.0 %), emphasizing the need for targeted intervention strategies. The presence of these uncommon serogroups, typically found in neighbouring countries and other regions, suggests potential transboundary transmission from these countries. This study provides valuable insights into the seroprevalence and serogroup distribution of leptospirosis in Assam's livestock, highlighting the need for region-specific surveillance and control measures. These findings underscore the importance of understanding the local epidemiological landscape to develop effective disease management and prevention strategies, ultimately reducing the impact of leptospirosis in the NER of India.

Identification of circulating miRNA biomarkers in leptospirosis for early detection: A promising diagnostic approach.

Leptospirosis is a zoonotic disease of global significance, contributing to morbidity and mortality worldwide. It is endemic to tropical regions, with outbreaks during monsoons. The disease manifestations are similar to that of other febrile illness such as dengue, malaria hence often misdiagnosed and underreported. The zoonoses if undetected, progresses to cause severe life-threatening complications also known as Weil's disease. Routine diagnostic tests are based on the detection of antibodies in patient serum and are not accurate during the initial phase of the infection. Therefore, it is necessary to detect novel biomarkers that can be used in early detection of leptospirosis. Circulating miRNAs are known to be promising biomarkers for various diseases including cancer, tuberculosis, influenza; hence in this study the potential of miRNAs as biomarkers for leptospirosis was evaluated. A total of 30 leptospirosis cases were screened for the differential expression of 10 miRNA by RT-qPCR assay. The differential expression was calculated by relative quantification using healthy individuals as controls. Among the 10 miRNA,3 miRNA, miR-28-5p, miR-302c-3p and miR-302a-3p were reported to exhibit a significant trend of upregulation. Further their role in immune pathways and biological processes was investigated by KEGG analysis and Gene Ontology. The 3 miRNAs were observed to target various immune response pathways, thus confirming their role in host immune response. Based on the results obtained in this study, miR-28-5p, miR-302c-3p and miR-302a-3p can be considered as potential biomarkers for the detection of leptospirosis.

Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential.

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine. KEY POINTS: • Several T- and B-cell epitopes were identified in all the three proteins. • Four different adjuvants were used in vaccine formulations. • Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

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.

Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

AIMS: Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. METHODS AND RESULTS: In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35 kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P < 0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P < 0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. CONCLUSIONS: These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

Seroprevalence of Leptospira antibodies in dogs and cats from Santa Fe, a city in East-Central Argentina endemic for leptospirosis.

This study examines household pets as potential epidemiological links between environments contaminated with pathogenic leptospires and humans in Santa Fe, Argentina. The aims of our study were: (a) to characterize the habits and exposure to environmental sources of leptospirosis in the population of dogs and cats attending to municipal spay and neutering campaigns in Santa Fe, Argentina, (b) to assess the seroprevalence of anti-Leptospira antibodies in asymptomatic dogs and cats, (c) to evaluate factors that could increase seropositivity, and (d) to identify spatial clusters of seropositive dogs and cats in the capital city of Santa Fe. From May to November 2022, a cross-sectional serosurvey was conducted during municipal spaying/neutering campaigns. Eligible household dogs and cats were over 6 months old, apparently healthy, and not vaccinated against leptospirosis in the past 6 months. We used microagglutination test (MAT) to assess anti-Leptospira antibodies using a panel of 10 reference strains. We used generalized linear mixed effects models (GLMM) to examine individual and census tract-level risk factors for seropositivity, and local Moran's I statistic for spatial clusters. Results showed higher leptospiral antibody prevalence in dogs (18.2 %) than cats (3.6 %, p = 0.002). Dogs with street access had higher likelihood of being seropositive (OR: 3.8, 95 % CI: 1.2; 11.9), and areas with chronic poverty showed an elevated risk of presenting seropositive animals (RR: 4.0, 95 % CI: 1.1; 14.4). Spatial analysis didn't reveal significant seropositivity clusters among census tracts. These findings shed light on widespread Leptospira seropositivity in pets in this endemic region. Understanding seroprevalence and risk factors can guide public and veterinary health strategies, emphasizing increased leptospirosis vaccination for dogs in vulnerable areas and promoting responsible pet care.