Serovar Copenhageni Research Articles

The Presence of a Virulent Clone of Leptospira interrogans Serovar Canicola in Confirmed Cases of Asymptomatic Dog Carriers in Mexico.

Leptospirosis is a neglected zoonotic disease that commonly affects cattle, pigs, horses, and dogs in many countries. Infection in dogs is usually subclinical, but acute cases of leptospirosis may occur along with systemic failure, which may become fatal. After recovery from an acute infection, dogs may become asymptomatic carriers and shed pathogenic leptospires through urine for long periods of time. Here, a study of ten different cases of leptospirosis is presented, showing the relevance of dogs as asymptomatic carriers of pathogenic Leptospira. The diagnosis was confirmed via isolation and further serological and genetic identification. Four Leptospira isolates (LOCaS28, 31, 34, and 46) were obtained from the kidneys and urine samples of 58 dogs destined for destruction (6.89%) at a Canine Control Center in Mexico City. No spirochetes were observed in the urine samples of those Leptospira-positive dogs examined under dark-field microscopy, and no clinical signs of disease were observed either. Six additional isolates were obtained: two came from asymptomatic carrier dogs (CEL60 and UADY22); another isolate came from an asymptomatic dog that was a pack companion of a clinically ill dog with fatal leptospirosis (AGFA24); and finally, three isolates were taken from dogs that died of leptospirosis (LOCaS59, Citlalli, and Nayar1). Nine out of the ten isolates were identified as being from the serogroup Canicola via cross-absorption MAT using reference strains and specific antisera, and their identity was genetically confirmed as Canicola ST34 via multi-locus sequencing typing (MLST). In contrast, the isolate Nayar1 was identified as serovar Copenhageni ST2. Interestingly, the asymptomatic dogs from which Leptospira isolates were recovered consistently showed high antibody titers in the microscopic agglutination test (MAT), revealing values of at least 1:3200 against serogroup Canicola and lower titer values against other serogroups. Isolates showed different virulence levels in the hamster model. Taken as a whole, all these findings confirmed that dogs may act as asymptomatic carriers of pathogenic leptospires and possibly spread them out to the environment, thus representing an active public health risk. The results also showed that the Canicola ST34 clone is the most prevalent Leptospira serovar in dogs in Mexico, and finally that the old-fashioned MAT is a good alternative for the detection of presumptive Leptospira asymptomatic carrier dogs.

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.

Serological evidence of exposure of healthy dogs to Leptospira in Sydney, New South Wales, Australia.

In 2017, highly fatal canine leptospirosis emerged in Sydney, Australia. Based on results of microscopic agglutination testing (MAT), serovar Copenhageni appeared to be the most common causative serovar. Prior to this, no clinical cases had been reported since 1976. In a serosurvey of healthy dogs in Australian shelters in 2004, 2.4% of 431 New South Wales dogs had serological evidence of exposure to Copenhageni, the most prevalent serovar. The aim of this study was to estimate the current prevalence of Leptospira exposure and associated serovars in healthy Sydney dogs, previously unvaccinated against Leptospira. Serum samples from 411 healthy dogs in leptospirosis hotspots and neighbouring suburbs were collected before vaccination. MAT for 23 serovars was performed at the WHO Leptospirosis Reference Laboratory in Queensland, Australia. The overall seroprevalence was 4.1% (17/411) with low titres (1/50-1/200) detected. Eleven dogs were from known leptospirosis hotspots. Eight dogs were known to hunt rodents. One dog had been in contact with a leptospirosis positive dog 1 year prior. Serovar Topaz was the most prevalent serovar (n = 5) followed by serovars Australis (n = 4), Copenhageni (n = 4), Djasiman (n = 2), Cynopteri (n = 1), Javanica (n = 1), Medanensis (n = 1), and Pomona (n = 1). In conclusion, serological evidence of exposure of dogs in Sydney to Leptospira is low, but apparently has increased since 2004. Positive titres to serovars not previously reported to cause disease in dogs could be due to low virulence of those serovars or cross-reactivity with other serovars.

Comparative analysis of a novel N-butanol-prepared antigen vs thermo-resistant and sonicated antigens for human leptospirosis detection.

The diagnosis of human leptospirosis is mainly based on serological assays. Since the extraction by N-butanol has only been studied as an antigen for the diagnosis of cattle leptospirosis, this study aimed to investigate the feasibility of the N-butanol preparation for the diagnosis of human leptospirosis and compare it with sonicated and thermo-resistant antigens in IgM dot-blot test. Paired serum samples from 147 laboratory-confirmed leptospirosis cases were tested. The control group consisted of 148 serum samples from healthy individuals and nonleptospirosis cases. N-butanol antigens from serovar Copenhageni (ButC3) and serovar Patoc (ButP3) showed reactivity with antileptospiral antibodies from patients with confirmed leptospirosis. In the acute phase, sensitivities of IgM dot-blot assay with ButC3 and ButP3 antigens were 47.6% and 51.0%, respectively. In the convalescent phase, sensitivities were 95.9% (ButC3) and 93.2% (ButP3), and no significant differences were observed among the IgM dot-blot tests with other antigens. The specificity of the IgM dot-blot test with ButC3 antigen was good (92.6%), but with ButP3 (83.1%), it was significantly lower than with the other tests. The IgM dot-blot test described in this study is simple to perform and presents reliable visual results. Antigens prepared by N-butanol proved to be valuable diagnostic markers of leptospirosis.

Genetic diversity of Leptospira strains circulating in humans and dogs in France in 2019-2021.

Leptospirosis is a bacterial zoonotic disease. Humans and dogs are susceptible hosts, with similar clinical manifestations ranging from a febrile phase to multiple organ dysfunction. The incidence of leptospirosis in mainland France is relatively high, at about 1 case per 100,000 inhabitants, but our knowledge of the strains circulating in humans and dogs remains limited. We studied the polymorphism of the lfb1 gene sequences in an exhaustive database, to facilitate the identification of Leptospira strains. We identified 46 species-groups (SG) encompassing the eight pathogenic species of Leptospira. We sequenced the lfb1 gene amplification products from 170 biological samples collected from 2019 to 2021: 110 from humans and 60 from dogs. Epidemiological data, including vaccination status in dogs, were also collected. Three Leptospira species displaying considerable diversity were identified: L. interrogans, with eight lfb1 species-groups (including five new lfb1 species-groups) in humans and dogs; L. kirschneri, with two lfb1 species-groups in humans and dogs; and L. borgpetersenii, with one lfb1 species-group in humans only. The lfb1 species-group L. interrogans SG1, corresponding to serovar Icterohaemorrhagiae or Copenhageni, was frequently retrieved from both humans and dogs (n=67/110; 60.9% and n=59/60; 98.3% respectively). A high proportion of the affected dogs developed the disease despite vaccination (n=30/60; 50%). Genotyping with the polymorphic lfb1 gene is both robust and simple. This approach provided the first global picture of the Leptospira strains responsible for acute infections in mainland France, based on biological samples but without the need for culture. Identification of the Leptospira strains circulating and their changes over time will facilitate more precise epidemiological monitoring of susceptible and reservoir species. It should also facilitate the monitoring of environmental contamination, making it possible to implement preventive measures and to reduce the burden of this disease.

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.

Core genome sequencing and genotyping of Leptospira interrogans in clinical samples by target capture sequencing

BackgroundThe life-threatening pathogen Leptospira interrogans is the most common agent of leptospirosis, an emerging zoonotic disease. However, little is known about the strains that are currently circulating worldwide due to the fastidious nature of the bacteria and the difficulty to isolate cultures. In addition, the paucity of bacteria in blood and other clinical samples has proven to be a considerable challenge for directly genotyping the agent of leptospirosis directly from patient material. Our understanding of the genetic diversity of strains during human infection is therefore limited.MethodsHere, we carried out hybridization capture followed by Illumina sequencing of the core genome directly from 20 clinical samples that were PCR positive for pathogenic Leptospira to elucidate the genetic diversity of currently circulating Leptospira strains in mainland France.ResultsCapture with RNA probes covering the L. interrogans core genome resulted in a 72 to 13,000-fold increase in pathogen reads relative to standard sequencing without capture. Variant analysis of the genomes sequenced from the biological samples using 273 Leptospira reference genomes was then carried out to determine the genotype of the infecting strain. For samples with sufficient coverage (19/20 samples with coverage > 8×), we could unambiguously identify L. interrogans serovars Icterohaemorrhagiae and Copenhageni (14 samples), L. kirschneri serovar Grippotyphosa (4 samples), and L. interrogans serovar Pyrogenes (1 sample) as the infecting strains.ConclusionsWe obtained high-quality genomic data with suitable coverage for confident core genome genotyping of the agent of leptospirosis for most of our clinical samples. The recovery of the genome of the serovars Icterohaemorrhagiae and Copenhageni directly from multiple clinical samples revealed low adaptive diversification of the core genes during human infection. The ability to generate culture-free genomic data opens new opportunities for better understanding of the epidemiology of this fastidious pathogen and pathogenesis of this neglected disease.

Serological and Molecular Evidence of Pathogenic Leptospira spp. in Stray Dogs and Cats of Sicily (South Italy), 2017–2021

Leptospirosis is a zoonosis of public health concern. Its prevalence in stray animals in the South of Italy is unknown. This study aimed to investigate Leptospira spp. prevalence in 1009 stray animals. Out of them, 749 were alive animals, including 358 dogs (316 from Palermo and 42 from Ragusa) and 391 cats (359 from Palermo and 32 from Ragusa), and 260 were corpses (216 dogs and 44 cats) randomly collected in Sicily. Dogs and cats underwent a serological screening by Microscopic Agglutination Test and a molecular investigation by Real-Time PCR targeting lipL32. Corpses were subjected to Real-Time PCR. Serological analyses showed a prevalence of 1.12% (4/358) for dogs and 0.26% (1/391) for cats, with the only positive cat coming from Palermo. Serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni, followed by Canicola and Bratislava, were the most spread among dogs, while the serological positive cat reacted with Hardjo serogroup. Two urine (2/32, 6.25%) and one blood (1/391, 0.26%) samples of cats, all from Ragusa, were positive at Real-Time PCR for pathogenic Leptospira spp. Sequencing analyses showed the presence of L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni in one of the positive urine samples and in the positive blood sample. Analyses on corpses showed a prevalence of 1.85% (4/216) in Sicilian dog kidney samples, while all corpses of cats resulted in negative. Genotyping analysis showed a genetic relatedness between cat and human isolates. Results show Leptospira spp. circulation among Sicilian stray animals. The genetic relatedness between cat and human isolates suggests a possible common infection source.

Feline Susceptibility to Leptospirosis and Presence of Immunosuppressive Co-Morbidities: First European Report of L. interrogans Serogroup Australis Sequence Type 24 in a Cat and Survey of Leptospira Exposure in Outdoor Cats.

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. The role of the cat as a susceptible host and potential environmental reservoir of Leptospira is still not well understood, due to the lack of obvious clinical signs associated with Leptospira spp. infection in this species. This study aims to describe the first European detection of Leptospira interrogans serogroup Australis ST 24 in a young outdoor cat with a severe comorbidity (feline panleukopenia virus). In addition, the results of a preliminary study conducted in 2014-2016 are presented (RC IZSVE 16/12), which reports an investigation of Leptospira exposure of outdoor cats in Northeast Italy by means of serological investigation and molecular evaluation of urine. The animals included in the survey are part of samples collected during active and passive surveillance (diagnostic samples). The study reported a seroprevalence of 10.5% among outdoor cats and the serogroups identified were Grippotyphosa, Icterohaemorrhagiae, Bratislava, Canicola and Ballum. Symptomatic cats reported high MAT titres (ranging from 1:800 to 1:1600) towards antigens belonging to the serovars Grippotyphosa (1:800), Bratislava (1:1600), Icterohaemorrhagiae (1:200) and Copenhageni (1:200-1:800). In one subject, urine tested positive for Leptospira PCR. Cats with high antibody titres for Leptospira and/or positivity on molecular test suffered from immunosuppressive comorbidities (feline immunodeficiency virus and feline leukaemia virus; feline herpesvirus and lymphoma; hyperthyroidism). The overall prevalence of serum antibodies against Leptospira found in free-ranging cats (10.53%, 95% CI: 4.35-16.70%) and the identification of L. interrogans ST 24 in a young cat with immunosuppressive disease (feline panleukopenia virus) suggest the possibility of natural resistance to clinical leptospirosis in healthy cats. In a One Health perspective, further studies are needed to better define the pathogenesis of leptospirosis in cats and their epidemiological role as environmental sentinels or possible carriers of pathogenic Leptospira.

Culture-Independent Detection and Identification of Leptospira Serovars.

Pathogenic Leptospira, the causative agents of leptospirosis, comprise >200 serotypes (called serovars). Most have a restricted reservoir-host range, and some, e.g., serovar Copenhageni, are cosmopolitan and of public health importance owing to their propensity to produce severe, fatal disease in humans. Available serotyping approaches-such as multilocus sequence typing, core genome sequence typing, pulsed-field gel electrophoresis, and the cross-agglutination absorption test-are tedious and expensive, and require isolation of the organisms in culture media-a protracted and incredibly inefficient process-precluding their use in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. Here, we have developed a simple yet specific real-time qPCR assay-targeting a Leptospira-unique gene encoding a putative polysaccharide flippase-that provides intraspecies, serotype-defining (i.e., epidemiologically useful) information, and improves upon the sensitivity of preferred lipL32-based qPCR-based diagnostic tests. The assay, dubbed RAgI ("rage one"), is rapid and affordable, and reliably and specifically detects group I pathogenic Leptospira in culture, serum, and urine, with no detectable off-target amplification-even of the genetically related but low virulence group II pathogenic (formerly "intermediate") or nonpathogenic Leptospira. It retained 100% diagnostic specificity when tested against difficult sample types, including field-collected dog urine samples and environmental samples containing varied and complex microbial species-consortia. This assay holds considerable promise in the clinical setting, and for routine epidemiological and environmental surveillance studies. IMPORTANCE Leptospirosis is caused by a diverse group of pathogenic spirochetes comprising over 200 different serotypes. Some are widely reported and of public health importance owing to their propensity to produce severe, fatal disease in humans. Apart from their tedium and expense, current serotyping approaches require isolation of the organisms in culture media-a protracted and incredibly inefficient process-rendering them useless clinically and limiting their utilization in prospective studies or outbreak investigations. The unavailability of culture-independent assays capable of distinguishing Leptospira serotypes remains a crucial gap in the field. The 11108 qPCR-assay overcomes this barrier to progress via direct taxonomic and serotype classification of Leptospira from urine and serum samples, and hence, is the first qPCR-based prognostic test for human leptospirosis.

Pathogenesis and Genomic Analysis of a Virulent Leptospira Interrogans Serovar Copenhageni Isolated from a Dog with Lethal Infection.

Dogs are highly susceptible to leptospirosis and are a public health concern due to their important role as a source of spreading disease, particularly in urban settings. In this study, we present the pathogenesis, serological characterization, and complete genome sequencing of a virulent Brazilian strain (NEG7) of L. interrogans serovar Copenhageni isolated from the urine of a dog that died due to acute leptospirosis. Clinical investigation showed that the dog was presented with icteric mucous membranes, weakness, dehydration, anorexia, and kidney and liver failures. Necropsy followed by histopathological evaluation revealed lesions compatible with liver and kidney leptospirosis. The leptospires recovered from the urine were further characterized by genome analysis, which confirmed that the isolate belonged to L. interrogans serogroup icterohaemorrhagiae serovar Copenhageni. Multiple bioinformatics tools were used to characterize the genomic features, and comparisons with other available Copenhageni strains were performed. Characterization based on absence of an INDEL in the gene lic12008, associated with phylogenetic and ANI (99.99% identity) analyses, confirmed the genetic relatedness of the isolate with L. interrogans serovar Copenhageni. A better understanding of the diversity of the pathogenic Leptospira isolates could help in identifying genotypes responsible for severe infections. Moreover, it can be used to develop control and prevention strategies for Leptospira serovars associated with particular animal reservoirs.

Structural and functional characterization of Cas2 of CRISPR-Cas subtype I-C lacking the CRISPR component.

The genome of pathogenic Leptospira interrogans serovars (Copenhageni and Lai) are predicted to have CRISPR-Cas of subtypes I-B and I-C. Cas2, one of the core Cas proteins, has a crucial role in adaptive defense against foreign nucleic acids. However, subtype I-C lacks the CRISPR element at its loci essential for RNA-mediated adaptive immunity against foreign nucleic acids. The reason for sustaining the expense of cas genes are unknown in the absence of a CRISPR array. Thus, Cas2C was chosen as a representative Cas protein from two well-studied serovars of Leptospira to address whether it is functional. In this study, the recombinant Cas2C of Leptospira serovars Copenhageni (rLinCas2C, 12 kDa) and Lai (rLinCas2C_Lai, 8.6 kDa) were overexpressed and purified. Due to natural frameshift mutation in the cas2c gene of serovar Lai, rLinCas2C_Lai was overexpressed and purified as a partially translated protein. Nevertheless, the recombinant Cas2C from each serovar exhibited metal-dependent DNase and metal-independent RNase activities. The crystal structure of rLinCas2C obtained at the resolution of 2.60 Å revealed the protein is in apostate conformation and contains N- (1–71 amino acids) and C-terminal (72–90 amino acids) regions, with the former possessing a ferredoxin fold. Substitution of the conserved residues (Tyr7, Asp8, Arg33, and Phe39) with alanine and deletion of Loop L2 resulted in compromised DNase activity. On the other hand, a moderate reduction in RNase activity was evident only in selective rLinCas2C mutants. Overall, in the absence of an array, the observed catalytic activity of Cas2C may be required for biological processes distinct from the CRISPR-Cas-associated function.

Transcriptional analysis of CRISPR I-B arrays of Leptospira interrogans serovar Lai and its processing by Cas6.

In the genome of various Leptospira interrogans serovars, the subtype I-B locus of CRISPR-Cas possesses either one or multiple CRISPR arrays. In silico database (CRISPRCasdb) for predicting CRISPR-Cas reveals seven CRISPR arrays in L. interrogans serovar Lai positioned between the two independent cas-operons. Here, we present the redefined repeat-spacer boundaries of the CRISPR subtype I-B locus of serovar Lai. Such refinement of boundaries of arrays in serovar Lai was done after comparison with the characterized array of another serovar Copenhageni and the manual analysis of CRISPR flanking sequences. Using the reverse transcription-PCR (RT-PCR), we account that the seven CRISPR are transcriptionally active in serovar Lai. Our RT-PCR and quantitative real-time PCR analysis of transcripts in serovar Lai indicated that seven CRISPR of subtype I-B transcribe together as a single precursor unit. Moreover, the cleavage of the two miniature pre-crRNA of the subtype I-B by Cas6 demonstrates the biogenesis of the expected size of mature crRNA essential for the guided interference of foreign DNA. This study features insight into transcription direction and the crRNA biogenesis in serovar Lai essential for RNA-mediated interference of invading nucleic acids.

Transient Presence of Live Leptospira interrogans in Murine Testes.

ABSTRACTAnalysis of Leptospira dissemination and colonization of sex organs in rodents is of significant value as it queries the possibility of mammal-to-mammal venereal transmission. The aim of our study was to evaluate the presence and viability of Leptospira interrogans in testes of mice using models of infection that we previously developed. Using sublethal and lethal doses of bioluminescent strains of L. interrogans serovars Manilae and Copenhageni, we visualized the presence of leptospires in testes of C57BL/6 mice as early as 30 min and up to days 3–4 postinfection. This was confirmed by qPCR for the Copenhageni serovar after lethal infection of C3H/HeJ mice. In this model, no histopathological changes were noticed in testis. We further studied persistence of serovar Copenhageni in C3H/HeJ testes after lethal and sublethal infection, with different doses of leptospires. No viable leptospires were recovered from testes of lethally infected mice. However, we found live culturable Leptospira in testes of 19/19 (100%) sublethally infected mice at the acute phase but not at 15 days postinfection, which corresponds to the chronic phase of renal colonization. The data suggest that colonization of testes with live and potentially infectious leptospires is transient and limited to the spirochetemic phase of infection. Further studies are necessary to evaluate if presence of Leptospira in testes of mice leads to excretion in semen and to venereal transmission to female mice.IMPORTANCE Analysis of venereal transmission of Leptospira is important to determine if direct animal to animal transmission occurs, which could impact measures to prevent and treat leptospirosis. The goal of this study was to determine if live Leptospira colonize mouse testes. We found that colonization of mouse testes with live Leptospira was transient and limited to the acute spirochetemic phase of infection and that transient colonization of the testes was insufficient to cause histopathological changes.

Serological prevalence ofLeptospira serovars among pigs inUkraine during the period of2001-2019.

Leptospirosis isawidespread infection among pigs throughout the world. Inmost cases inUkraine, onlythe microscopic agglutination test (MAT) isused for the diagnosis ofleptospirosis inanimals. Ingeneral, during the period of2001-2019, 2381163 samples ofblood sera from swine were tested inour country and 85338 positive reactions were received, which is3.58% [binomial confidence intervals (BCI), 3.56-3.61%]. Itwas established that the serovars copenhageni- 33.91% (BCI, 33.59-34.23%), bratislava - 14.14% (BCI, 13.90-14.37%), pomona - 8.58% (BCI, 8.39-8.77%), and tarassovi - 7.12% (BCI, 6.95-7.30%) play aleading role inthe aetiological structure ofswine leptospirosis. Alarge number ofpositive reactions toseveral serovars was observed - 29.78% (BCI, 29.47-30.09%) ofthe total number ofpositive cases. Inaddition, the article presents data according toaretrospective analysis ofthe eight serovars circulating among pigs inUkraine. Thus, during the nineteen year period, there was adecrease inthenumber ofpositive reactions tobratislava, pomona, and tarassovi and anincrease inthenumber ofpositive reactions tocopenhageni, polonica, and kabura. Mapping Ukraine's territory for leptospirosis among pigs was carried out. This allows one toidentify zones with arisk ofleptospirosis infections among swine. The maps show that thehighest incidence rates were identified inthe eastern and central parts ofUkraine.

Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization.

Leptospira interrogans is a pathogenic spirochete responsible for leptospirosis, a neglected, zoonotic reemerging disease. Humans are sensitive hosts and may develop severe disease. Some animal species, such as rats and mice can become asymptomatic renal carriers. More than 350 leptospiral serovars have been identified, classified on the basis of the antibody response directed against the lipopolysaccharide (LPS). Similarly to whole inactivated bacteria used as human vaccines, this response is believed to confer only short-term, serogroup-specific protection. The immune response of hosts against leptospires has not been thoroughly studied, which complicates the testing of vaccine candidates. In this work, we studied the immunoglobulin (Ig) profiles in mice infected with L. interrogans over time to determine whether this humoral response confers long-term protection after homologous challenge six months post-infection. Groups of mice were injected intraperitoneally with 2×107 leptospires of one of three pathogenic serovars (Manilae, Copenhageni or Icterohaemorrhagiae), attenuated mutants or heat-killed bacteria. Leptospira-specific immunoglobulin (IgA, IgM, IgG and 4 subclasses) produced in the first weeks up to 6 months post-infection were measured by ELISA. Strikingly, we found sustained high levels of IgM in mice infected with the pathogenic Manilae and Copenhageni strains, both colonizing the kidney. In contrast, the Icterohaemorrhagiae strain did not lead to kidney colonization, even at high dose, and triggered a classical IgM response that peaked at day 8 post-infection and disappeared. The virulent Manilae and Copenhageni serovars elicited high levels and similar profiles of IgG subclasses in contrast to Icterohaemorrhagiae strains that stimulated weaker antibody responses. Inactivated heat-killed Manilae strains elicited very low responses. However, all mice pre-injected with leptospires challenged with high doses of homologous bacteria did not develop acute leptospirosis, and all antibody responses were boosted after challenge. Furthermore, we showed that 2 months post-challenge, mice pre-infected with the attenuated M895 Manilae LPS mutant or heat-killed bacterin were completely protected against renal colonization. In conclusion, we observed a sustained IgM response potentially associated with chronic leptospiral renal infection. We also demonstrated in mice different profiles of protective and cross-reactive antibodies after L. interrogans infection, depending on the serovar and virulence of strains.

Serological study of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Tarassovi and Ballum in beef cattle, sheep and deer in New Zealand

ABSTRACT Aims To estimate animal-level seroprevalence of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Ballum and Tarassovi, in beef cattle, sheep and deer on New Zealand farms, and herd/flock-level seroprevalence of any serovar when existing same-sera data for serovars Hardjobovis and Pomona were included, and to determine associations between risk factors and animal-level seroprevalence. Methods Banked sera from sheep (n = 82), beef (n = 54) and deer (n = 62) herds/flocks (n = 3,878 animals) from seven regions were analysed using the microscopic agglutination test. Titres of ≥48 were designated positive. Herds/flocks were considered positive if either ≥1, ≥2 or ≥3 animals were positive. Existing same-sera data for serovars Hardjobovis and Pomona were included to establish farm-level any-serovar seropositivity. Factors associated with serological status were analysed using generalised estimating equations. Results Animal-level seroprevalence for serovars Ballum, Copenhageni, and Tarassovi, respectively, was 13.7 (95% CI = 11.7–16.0)%, 12.6 (95% CI = 10.6–14.7)% and 18.0 (95% CI = 15.7–20.5)% for beef cattle, 10.5 (95% CI = 9.0–12.1)%, 16.7 (95% CI = 14.9–18.6)% and 14.0 (95% CI = 12.4–15.8)% for sheep and 6.6 (95% CI = 5.3–8.2)%, 15.5 (95% CI = 13.5–17.7)% and 3.6 (95% CI = 2.7–4.8)% for deer, respectively. Herd/flock-level seroprevalence for Ballum was 86.6, 52.4 and 39.0% for sheep, 85.2, 52.7 and 33.3% for beef cattle and 50.8, 27.9 and 21.3% for deer at definitions ≥1, ≥2 and ≥3 seropositive animals per species, respectively. For Copenhageni, corresponding data were 95.1, 73.2 and 56.1% for sheep, 68.5, 48.2 and 29.6% for beef cattle and 73.8, 57.4 and 41.0% for deer, and for Tarassovi, 80.5, 59.7 and 45.1% for sheep, 83.3, 68.5 and 61.1% for beef cattle, and 42.6, 16.4 and 4.9% for deer. Seropositivity to all serovars was observed from all regions, with some differences in seroprevalence observed between species and regions, but not between islands. Combining with Hardjobovis and Pomona data, herd/flock-level seropositivity for all animal species and all five Leptospira serovars was 100% at definition ≥1 animal positive, and 97.5 and 96.3% for sheep flocks, 87.8 and 97.8% for beef cattle herds, and 89.3 and 75% for deer herds at ≥2 and ≥3 animals positive, respectively. Conclusions Seropositivity to serovars Ballum, Copenhageni and Tarassovi is common in sheep, beef cattle and deer New Zealand and most, or all farms have ≥1 livestock species seropositive to ≥1 serovar. Clinical relevance Serovars Ballum, Tarassovi and Copenhageni should be considered when clinical or subclinical signs of leptospirosis are observed in sheep, beef cattle or deer. Livestock sector workers are potentially at risk of exposure.

Seroprevalence of Leptospira in Racehorses and Broodmares in New Zealand.

Simple SummaryLeptospirosis is a zoonotic disease, caused by bacteria (Leptospira), that is frequently reported to occur in horses worldwide. Leptospirosis has been associated with abortions in mares, can cause a painful eye condition, and it poses a zoonotic risk to equine workers and veterinarians. Epidemiological data on the occurrence of leptospirosis or the frequency of exposure to the bacteria in horses in New Zealand are lacking. A survey was conducted to determine the seroprevalence of Leptospira in Thoroughbred racing and breeding horses in New Zealand. Horse owners were surveyed, and a blood sample was taken from the horses enrolled in the study to determine the frequency of five different types of Leptospira found to cause leptospirosis in humans and livestock in New Zealand. The results showed that a quarter of the horses sampled had previously been exposed to Leptospira. Several management factors, such as grazing horses alternately with cattle or sheep, increasing horse age, and breeding horses, were linked to exposure to Leptospira in this group of horses. Given the level of exposure found, horses may play a role in the epidemiology of leptospirosis in New Zealand.A cross-sectional survey was conducted to determine the seroprevalence of Leptospira in a cohort of horses and to evaluate potential risk factors for Leptospira seropositivity in horses in New Zealand. The convenience sample included 499 Thoroughbred racing and breeding horses from 25 commercial properties in North Island, New Zealand. A questionnaire was used to collect demographic data on horses and property-level information on grazing and management practices, pest (rodent) management, access to natural waterways, other livestock on the property, and possible contact with wildlife. The microscopic agglutination test was used to test sera for serovars Ballum, Copenhageni, Hardjo (bovis), Pomona, and Tarassovi. Logistic regression was used to investigate the risk factors for Leptospira seropositivity to at least one serovar and for each serovar individually. A total of 124 (25%, 95% confidence interval (CI) 21–29%) horses had positive titres to any one of the five serovars. The seroprevalence of Ballum, Copenhageni, Hardjo (bovis), Pomona, and Tarassovi was 5% (95% CI 3–7%), 9% (95% CI 7–12%), 6% (95% CI 4–8%), 6% (95% CI 4–8%), and 6% (95% CI 4–8%), respectively. Broodmares, compared to racehorses and alternately grazing horses with sheep, increased the odds of exposure to any one serovar, whilst grazing the same time as sheep and alternately grazing horses with cattle increased the odds of exposure to Ballum and Hardjo (bovis), respectively. Historical exposure to Leptospira in racing and breeding horses was identified, and risk factors were consistent with pasture-based exposure.

Cross-sectional evaluation of multiple epidemiological cycles of Leptospira species in peri-urban wildlife in California.

To perform a cross-sectional survey to estimate prevalence of and potential risk factors for Leptospira spp infection and exposure in peri-urban wildlife throughout California. 723 animals representing 12 wildlife species. Blood and urine samples were obtained from wildlife in California from 2007 to 2017. Live animals were captured in humane traps, anesthetized, and released. Carcasses were donated by wildlife services and necropsied for urine, blood, and kidney tissue samples. Samples were tested for antibodies against 6 serovars of Leptospira spp with a microscopic agglutination test and for pathogenic Leptospira spp DNA with a real-time PCR assay targeting the LipL32 gene. Potential risk factors for Leptospira spp exposure were assessed by logistic regression. Genetic relatedness of Leptospira spp were assessed with DNA sequencing of the rrs2 gene and multiple locus sequence analysis. Statewide Leptospira spp seroprevalence was 39.1%, and prevalence of positive PCR assay results for Leptospira spp DNA was 23.0%. Risk factors for Leptospira spp exposure included being an adult, being from northern California, and being a western gray squirrel, coyote, striped skunk, raccoon, gray fox, or mountain lion. Antibodies against serovar Pomona predominated in most species, followed by serovar Copenhageni. Complete rrs2 sequences were identified as Leptospira interrogans and multiple locus sequence type analysis revealed sequence type 140. Pathogenic Leptospira spp appeared to be common and widespread among peri-urban wildlife in California. Our data highlight the potential for exposure to infectious disease for both humans and domestic animals at the urban-wildland interface.

New strategies for Leptospira vaccine development based on LPS removal.

Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.