Deployment of a Reservoir-Targeted Vaccine Against Borrelia burgdorferi Reduces the Prevalence of Babesia microti Coinfection in Ixodes scapularis Ticks.

In the Northeast and upper Midwest of the United States, Babesia microti and Borrelia burgdorferi use Ixodes scapularis ticks as vector and Peromyscus leucopus mice as major reservoir host. We previously established, in a 5-year field trial, that a reservoir-targeted outer surface protein A vaccine reduces the prevalence of B. burgdorferi-infected ticks. We accessed ticks and mouse blood samples collected during the trial, extracted total DNA, and amplified the B. microti 18S rRNA gene. Vaccine deployment reduced the prevalence of ticks coinfected with B. microti and that of mice infected with B. microti. Breaking the enzootic cycle of B. burgdorferi may reduce the incidence of babesiosis.

Fatal Multisystem Inflammatory Syndrome in Adult after SARS-CoV-2 Natural Infection and COVID-19 Vaccination.

We describe a fatal case of multisystem inflammatory syndrome in an adult with onset 22 days after a second dose of mRNA coronavirus disease vaccine. Serologic and clinical findings indicated severe acute respiratory syndrome coronavirus 2 infection occurred before vaccination. The immunopathology of this syndrome, regardless of vaccination status, remains poorly understood.

Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection.

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with Lactobacillus plantarum confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train ex vivo human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis.

Protective Immunity and New Vaccines for Lyme Disease.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

A Multiplexed Serologic Test for Diagnosis of Lyme Disease for Point-of-Care Use.

Single multiplexed assays could replace the standard 2-tiered (STT) algorithm recommended for the laboratory diagnosis of Lyme disease if they perform with a specificity and a sensitivity superior or equal to those of the STT algorithm. We used human serum rigorously characterized to be sera from patients with acute- and convalescent-phase early Lyme disease, Lyme arthritis, and posttreatment Lyme disease syndrome, as well as the necessary controls (n = 241 samples), to select the best of 12 Borrelia burgdorferi proteins to improve our microfluidic assay (mChip-Ld). We then evaluated its serodiagnostic performance in comparison to that of a first-tier enzyme immunoassay and the STT algorithm. We observed that more antigens became positive as Lyme disease progressed from early to late stages. We selected three antigens (3Ag) to include in the mChip-Ld: VlsE and a proprietary synthetic 33-mer peptide (PepVF) to capture sensitivity in all disease stages and OspC for early Lyme disease. With the specificity set at 95%, the sensitivity of the mChip-Ld with 3Ag ranged from 80% (95% confidence interval [CI], 56% to 94%) and 85% (95% CI, 74% to 96%) for two panels of serum from patients with early Lyme disease and was 100% (95% CI, 83% to 100%) for serum from patients with Lyme arthritis; the STT algorithm detected early Lyme disease in the same two panels of serum from patients with early Lyme disease with a sensitivity of 48.5% and 75% and Lyme arthritis in serum from patients with Lyme arthritis with a sensitivity of 100%, and the specificity was 97.5% to 100%. The mChip-Ld platform outperformed the STT algorithm according to sensitivity. These results open the door for the development of a single, rapid, multiplexed diagnostic test for point-of-care use that can be designed to identify the Lyme disease stage.

Lactobacillus plantarum prevents and mitigates alcohol-induced disruption of colonic epithelial tight junctions, endotoxemia, and liver damage by an EGF receptor-dependent mechanism.

Pathogenesis of alcohol-related diseases such as alcoholic hepatitis involves gut barrier dysfunction, endotoxemia, and toxin-mediated cellular injury. Here we show that Lactobacillus plantarum not only blocks but also mitigates ethanol (EtOH)-induced gut and liver damage in mice. L. plantarum blocks EtOH-induced protein thiol oxidation, and down-regulation of antioxidant gene expression in colon L. plantarum also blocks EtOH-induced expression of TNF-α, IL-1ß, IL-6, monocyte chemotactic protein 1 ( MCP1), C-X-C motif chemokine ligand ( CXCL)1, and CXCL2 genes in colon. Epidermal growth factor receptor (EGFR) signaling mediates the L. plantarum-mediated protection of tight junctions (TJs) and barrier function from acetaldehyde, the EtOH metabolite, in Caco-2 cell monolayers. In mice, doxycycline-mediated expression of dominant negative EGFR blocks L. plantarum-mediated prevention of EtOH-induced TJ disruption, mucosal barrier dysfunction, oxidative stress, and inflammatory response in colon. L. plantarum blocks EtOH-induced endotoxemia as well as EtOH-induced pathologic lesions, triglyceride deposition, oxidative stress, and inflammatory responses in the liver by an EGFR-dependent mechanism. L. plantarum treatment after injury accelerated recovery from EtOH-induced TJ, barrier dysfunction, oxidative stress, and inflammatory response in colon, endotoxemia, and liver damage. Results demonstrate that L. plantarum has both preventive and therapeutic values in treatment of alcohol-induced tissue injury, particularly in alcoholic hepatitis.-Shukla, P. K., Meena, A. S., Manda, B., Gomes-Solecki, M., Dietrich, P., Dragatsis, I., Rao, R. Lactobacillus plantarum prevents and mitigates alcohol-induced disruption of colonic epithelial tight junctions, endotoxemia, and liver damage by an EGF receptor-dependent mechanism.

Sex Matters: Male Hamsters Are More Susceptible to Lethal Infection with Lower Doses of Pathogenic Leptospira than Female Hamsters.

A somewhat contradictory published body of evidence suggests that sex impacts severity outcomes of human leptospirosis. In this study, we used an acute animal model of disease to analyze leptospirosis in male and female hamsters infected side by side with low but increasing doses of Leptospira interrogans serovar Copenhageni. We found that male hamsters were considerably more susceptible to leptospirosis, given that only 6.3% survived infection, whereas 68.7% of the females survived the same infection doses. In contrast to the females, male hamsters had high burdens of L. interrogans in kidney and high histopathological scores after exposure to low infection doses (∼103 bacteria). In hamsters infected with higher doses of L. interrogans (∼104 bacteria), differences in pathogen burdens as well as cytokine and fibrosis transcript levels in kidney were not distinct between sexes. Our results indicate that male hamsters infected with L. interrogans are more susceptible to severe leptospirosis after exposure to lower infectious doses than females.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Borrelia burgdorferi BBI39 Paralogs, Targets of Protective Immunity, Reduce Pathogen Persistence Either in Hosts or in the Vector.

Borrelia burgdorferi genome harbors several paralogous gene families (pgf) that can encode immunogenic proteins of unknown function. Protein-protein interaction assays using a transmission-blocking vaccine candidate, BBA52, as bait identified an interacting partner in spirochetes-a member of pgf 54, annotated as BBI39. We show that BBI39 is a surface-exposed membrane antigen that is immunogenic during spirochete infection, despite the gene being primarily transcribed in the vector with a transient expression in the host only at tick-bite sites. Immunization of rodents with BBI39, or a diverse paralog, BBI36, or their combination impaired pathogen acquisition by the vector, transmission from ticks to hosts, or induction of disease. High-titer BBI39 immunoglobulin G antibodies, which have borreliacidal properties, could be generated through routine subcutaneous or oral immunization, further highlighting use of BBI39 proteins as novel Lyme disease vaccines that can target pathogens in the host or in ticks.

Eyedrop Inoculation Causes Sublethal Leptospirosis in Mice.

Leptospirosis is potentially a fatal zoonosis acquired by contact of skin and mucosal surfaces with soil and water contaminated with infected urine. We analyzed the outcome of infection of C3H/HeJ mice with Leptospira interrogans serovar Copenhageni using an enzootic mode of transmission, the conjunctival route. Infection led to weight loss and L. interrogans dissemination from blood to urine, and spirochetes were detected in blood and urine simultaneously. The infectious dose that led to consistent dissemination to kidney after conjunctival infection was ∼108 leptospires. Interestingly, a lower number of spirochetes appeared to colonize the kidney, given that we quantified ∼105 and ∼10 leptospires per µl of urine and per µg of kidney, respectively. Leptospira-specific IgM and IgG were detected at 15 days postinfection, and isotyping of the Ig subclass showed that the total IgG response switched from an IgG1 response to an IgG3 response after infection with L. interrogans Histological periodic acid-Schiff D staining of infected kidney showed interstitial nephritis, mononuclear cell infiltrates, and reduced size of glomeruli. Quantification of proinflammatory immunomediators in kidney showed that keratinocyte-derived chemokine, macrophage inflammatory protein 2, RANTES, tumor necrosis factor alpha, gamma interferon, and interleukin-10 were upregulated in infected mice. We show that the kinetics of disease progression after infection via the ocular conjunctiva is delayed compared with infection via the standard intraperitoneal route. Differences may be related to the number of L. interrogans spirochetes that succeed in overcoming the natural defenses of the ocular conjunctiva and transit through tissue.

Mouse model for sublethal Leptospira interrogans infection.

Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1ß, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4(+) and double-negative T cells (not CD8(+) cells) and that CD4(+) T cells acquired a CD44(high) CD62L(low) effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection).

Reservoir targeted vaccine against Borrelia burgdorferi: a new strategy to prevent Lyme disease transmission.

A high prevalence of infection with Borrelia burgdorferi in ixodid ticks is correlated with a high incidence of Lyme disease. The transmission of B. burgdorferi to humans can be disrupted by targeting 2 key elements in its enzootic cycle: the reservoir host and the tick vector. In a prospective 5-year field trial, we show that oral vaccination of wild white-footed mice resulted in outer surface protein A-specific seropositivity that led to reductions of 23% and 76% in the nymphal infection prevalence in a cumulative, time-dependent manner (2 and 5 years, respectively), whereas the proportion of infected ticks recovered from control plots varied randomly over time. Significant decreases in tick infection prevalence were observed within 3 years of vaccine deployment. Implementation of such a long-term public health measure could substantially reduce the risk of human exposure to Lyme disease.

Oral immunization with Escherichia coli expressing a lipidated form of LigA protects hamsters against challenge with Leptospira interrogans serovar Copenhageni.

Leptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals that harbor leptospires in their renal tubules and shed the bacteria in their urine. Leptospira interrogans serovar Copenhageni transmitted from Rattus norvegicus to humans is the most prevalent cause of urban leptospirosis. We examined L. interrogans LigA, domains 7 to 13 (LigA7-13), as an oral vaccine delivered by Escherichia coli as a lipidated, membrane-associated protein. The efficacy of the vaccine was evaluated in a susceptible hamster model in terms of the humoral immune response and survival from leptospiral challenge. Four weeks of oral administration of live E. coli expressing LigA7-13 improved survival from intraperitoneal (i.p.) and intradermal (i.d.) challenge by L. interrogans serovar Copenhageni strain Fiocruz L1-130 in Golden Syrian hamsters. Immunization with E. coli expressing LigA7-13 resulted in a systemic antibody response, and a significant LigA7-13 IgG level after the first 2 weeks of immunization was completely predictive of survival 28 days after challenge. As in previous LigA vaccine studies, all immunized hamsters that survived infection had renal leptospiral colonization and histopathological changes. In summary, an oral LigA-based vaccine improved survival from leptospiral challenge by either the i.p. or i.d. route.

Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis.

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, L. interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle.

Intranasal vaccine for Lyme disease provides protection against tick transmitted Borrelia burgdorferi beyond one year.

Strategies for disease control are necessary to reduce incidence of Lyme Disease (LD) including development of safe vaccines for human use. Parainfluenza virus 5 (PIV5) vector has an excellent safety record in animals and PIV5-vectored vaccines are currently under clinical development. We constructed PIV5-vectored LD vaccine candidates expressing OspA from B. burgdorferi (OspAB31) and a chimeric protein containing sequences from B. burgdorferi and B. afzelii (OspABPBPk). Immunogenicity and vaccine efficacy were analyzed in C3H-HeN mice after prime-boost intranasal vaccination with live PIV5-OspAB31 or PIV5-OspABPBPk, subcutaneous (s.c.) vaccination with rOspAB31+Alum, and the respective controls. Mice vaccinated intranasally with live PIV5-AB31 or PIV5-ABPBPk had higher endpoint titers of serum antibody against OspAB31 at 6- and 12- months post vaccination, compared to mice vaccinated s.c. with rOspAB31. Neutralization activity of antibody was maintained up to 18-months post-immunization, with the response greater in live PIV5-delivered OspA vaccines, than that induced by s.c. rOspAB31. Challenge with infected ticks carrying 10-19 strains of B. burgdorferi performed at 4-, 9- or 15-months post-immunization showed increased breakthrough infections in mice vaccinated with s.c. rOspAB31 compared to intranasal PIV5-AB31 or PIV5-ABPBPk at 9- and 15-months, as determined by quantification of serologic antibodies to B. burgdorferi proteins as well as flaB DNA in tissues, and by visualization of motile B. burgdorferi in culture of tissues under dark field microscope. These findings indicate that immunization of mice with PIV5 delivered OspA generates immune responses that produce longer-lasting protection ( > 1 year) against tick-transmitted B. burgdorferi than a parenteral recombinant OspA vaccine.

A parainfluenza virus 5 (PIV5)-vectored intranasal vaccine for Lyme disease provides long-lasting protection against tick transmitted Borrelia burgdorferi in mice.

Lyme disease (LD) is the most prevalent vector borne disease in North America and Europe and its geographic range continues to expand. Strategies for disease control are necessary to effectively reduce incidence of LD including development of safe vaccines for human use. Parainfluenza virus 5 (PIV5) vector has an excellent safety record in animals and PIV5-vectored COVID-19 and RSV vaccines are currently under clinical development. We constructed PIV5-vectored LD vaccine candidates expressing OspA from B. burgdorferi sensu stricto (OspAB31) and a chimeric protein containing sequences from B. burgdorferi and B. afzelii (OspABPBPk). Immunogenicity and vaccine efficacy were analyzed in C3H-HeN mice after prime-boost intranasal (IN) vaccination with PIV5-OspAB31 and PIV5-OspABPBPk, subcutaneous (SC) vaccination with rOspAB31+Alum as well as the respective controls. Mice vaccinated with either PIV5-AB31 or PIV5-ABPBPk intranasally had high endpoint titers of serum antibody against OspA antigen beyond 1 year post vaccination, similar to levels detected in mice vaccinated SC with rOspAB31. Flowcytometric analysis of spleen cells at 9-months post-immunization demonstrated that immunization with the intranasal PIV5 vaccine candidates led to an overall increase in the number of memory B cells, cytotoxic T and cytotoxic effector T cells compared to SC groups. Borreliacidal activity measured by neutralization assay was maintained up to 18 months post-immunization, with the response greater in intranasal PIV5-delivered OspA vaccines, than that induced by SC rOspAB31. Challenge with infected ticks (10-19 strains of B. burgdorferi) performed at 4-, 9- or 15-months post-immunization showed increased breakthrough infections in mice vaccinated with SC rOspAB31 compared to IN PIV5-AB31 or IN PIV5-ABPBPk at 9- and 15-months, as determined by qPCR of B. burgdorferi in tissues, culture of B. burgdorferi from tissues, and antibodies against B. burgdorferi protein VIsE. These data demonstrate that intranasal PIV5-based immunization is superior to parenteral immunization with the same recombinant protein and provides long-lasting protection (> 1 year) against Lyme disease.

A portable immunosensor provides sensitive and rapid detection of Borrelia burgdorferi antigen in spiked blood.

There are no assays for detecting B. burgdorferi antigen in blood of infected Lyme disease individuals. Here, we provide proof-of-principle evidence that we can quantify B. burgdorferi antigen in spiked blood using a portable smartphone-based fluorescence microscope that measures immunoagglutination on a paper microfluidic chip. We targeted B. burgdorferi OspA to develop a working prototype and added examples of two antigens (OspC and VlsE) that have diagnostic value for discrimination of Lyme disease stage. Using an extensively validated monoclonal antibody to OspA (LA-2), detection of OspA antigen had a broad linear range up to 100 pg/mL in 1% blood and the limit of detection (LOD) was 100 fg/mL (= 10 pg/mL in undiluted blood), which was 1000 times lower than our target of 10 ng/mL. Analysis of the two other targets was done using polyclonal and monoclonal antibodies. OspC antigen was detected at LOD 100 pg/mL (= 10 ng/mL of undiluted blood) and VlsE antigen was detected at LOD 1-10 pg/mL (= 0.1-1 ng/mL of undiluted blood). The method is accurate and was performed in 20 min from sample to answer. When optimized for detecting several B. burgdorferi antigens, this assay may differentiate active from past infections and facilitate diagnosis of Lyme disease in the initial weeks of infection, when antibody presence is typically below the threshold to be detected by serologic methods.

The year that shaped the outcome of the OspA vaccine for human Lyme disease.

The expansion of Lyme borreliosis endemic areas and the corresponding increase of disease incidence have opened the possibility for greater acceptance of a vaccine. In this perspective article, we discuss the discovery of outer surface protein A (OspA) of B. burgdorferi, and the subsequent pre-clinical testing and clinical trials of a recombinant OspA vaccine for human Lyme disease. We also discuss in detail the open public hearings of the FDA Lyme disease vaccine advisory panel held in 1998 where concerns of molecular mimicry induced autoimmunity to native OspA were raised, the limitations of those studies, and the current modifications of recombinant OspA to develop a multivalent subunit vaccine for Lyme disease.

Necroptosis Contributes to Persistent Inflammation During Acute Leptospirosis.

Leptospirosis is an emerging infectious disease. Recently, canine and human leptospirosis outbreaks were reported in California and New York, respectively. In this study we evaluated the role that cell death processes play in the inflammatory response to Leptospira. Groups of male C3H/HeJ mice were infected with pathogenic L. interrogans and non-pathogenic L. biflexa for 24 and 72 hours; inflammatory processes were characterized for apoptosis and necroptosis by flowcytometry of spleen cells and were further assessed for expression of biomarkers of necroptosis by western blot. We found that pathogenic L. interrogans promotes apoptosis in myeloid neutrophils and monocytes at 24h and 72h post-infection, whereas L. biflexa promotes apoptosis of myeloid monocytes only at 24h post-infection. It is interesting that the immune cells undergoing the common programmed cell death pathway (apoptosis) are the cell types which were not increased in frequency in spleen of mice infected with L. interrogans (neutrophils) and L. biflexa (monocytes) in our previous study. The same trend was observed with pathogenic L. interrogans inducing necroptosis of myeloid neutrophils in addition to monocytes and macrophages at 24h and/or 72h post-infection, whereas L. biflexa promoted this pro-inflammatory cell death process in monocytes and macrophages only at 24h post-infection. Thus, early apoptosis and necroptosis of these cell types may explain its absence in frequency in spleen. Furthermore, at 24h and 72h, expression of the necroptosis molecular biomarkers p-MLKL, p-RIP1 and p-RIP3 was increased post infection with pathogenic L. interrogans. These data suggest that the underlying cell death processes involved in immune responses to pathogenic Leptospira contribute directly to persistent inflammation during the early stages of leptospirosis.