The goal of this study was to characterize how natural routes of infection affect the kinetics of pathogenic Leptospira dissemination to blood and kidney. C3H/HeJ mice were sublethally infected with L. interrogans serovar Copenhageni FioCruz L1-130 (Leptospira) through exposure of a dermis wound and through oral and nasal mucosa, in comparison to uninfected mice and to mice infected via standard intraperitoneal inoculation. In striking contrast to oral mucosa inoculation, transdermal and nasal mucosa infections led to weight loss, renal colonization and inflammation, as previously observed for conjunctival and intraperitoneal infections. However, the timing at which Leptospira gained access to blood, as well as Leptospira' colonization of the kidney and shedding in urine, differed from intraperitoneal infection. Furthermore, a comparative analysis of transcription of proinflammatory mediators in kidney and total immunoglobulin isotyping in serum from infected mice, showed increased innate immune response markers (KC, MIP-2, TNF-α) and lower Th1 associated IFN-γ in kidney, as well as lower Th1 associated IgG2a in mice infected through the nasal mucosa as compared to intraperitoneal infection. We conclude that the route of infection affects the timing at which Leptospira gains access to blood for dissemination, as well as the dynamics of colonization and inflammation of the kidney.

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.

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 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 in kidney and high histopathological scores after exposure to low infection doses (∼10 bacteria). In hamsters infected with higher doses of (∼10 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 are more susceptible to severe leptospirosis after exposure to lower infectious doses than females.

The laborious microscopic agglutination test (MAT) is the gold standard serologic test for laboratory diagnosis of leptospirosis. We developed EIA based serologic assays using recombinant proteins (rLigA, rLigB, rLipL32) and whole-cell extracts from eight Leptospira serovars as antigen and assessed the diagnostic performance of the new assay within each class, against MAT positive (MAT+) human sera panels from Portugal/PT (n = 143) and Angola/AO (n = 100). We found that a combination of recombinant proteins rLigA, rLigB and rLipL32 correctly identified antigen-specific IgG from patients with clinical and laboratory confirmed leptospirosis (MAT+) with 92% sensitivity and ~ 97% specificity (AUC 0.974) in serum from the provinces of Luanda (LDA) and Huambo (HBO) in Angola. A combination of whole cell extracts of L. interrogans sv Copenhageni (LiC), L. kirschneri Mozdok (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc (LbP) accurately identified patients with clinical and laboratory confirmed leptospirosis (MAT+) with 100% sensitivity and ~ 98% specificity for all provinces of Angola and Portugal (AUC: 0.997 for AO/LDA/HBO, 1.000 for AO/HLA, 0.999 for PT/AZ and 1.000 for PT/LIS). Interestingly, we found that MAT+ IgG+ serum from Angola had a significantly higher presence of IgD and that IgG3/IgG1 isotypes were significantly increased in the MAT+ IgG+ serum from Portugal. Given that IgM/IgD class and IgG3/IgG1 specific isotypes are produced in the earliest course of infection, immunoglobulin G isotyping may be used to inform diagnosis of acute leptospirosis. The speed, ease of use and accuracy of EIA tests make them excellent alternatives to the laborious and expensive MAT for screening acute infection in areas where circulating serovars of pathogenic Leptospira are well defined.

Toll-Like Receptor (TLR) 4, the LPS receptor, plays a central role in the control of leptospirosis and absence of TLR4 results in lethal infection in mice. Because human TLR4 does not sense the atypical leptospiral-LPS, we hypothesized that TLR4/MD-2 humanized transgenic mice (huTLR4) may be more susceptible to leptospirosis than wild-type mice, and thus may constitute a model of acute human leptospirosis. We infected huTLR4 mice, which express human TLR4 but not murine TLR4, with a high dose of L. interrogans serovar Copenhageni FioCruz (Leptospira) in comparison to C57BL/6J wild-type (WT) and, as a control, a congenic strain in which the tlr4 coding sequences are deleted (muTLR4Lps-del). We show that the huTLR4 gene is fully functional in the murine background. We found that dissemination of Leptospira in blood, shedding in urine, colonization of the kidney and overall kinetics of leptospirosis progression is equivalent between WT and huTLR4 C57BL/6J mice. Furthermore, inflammation of the kidney appeared to be subdued in huTLR4 compared to WT mice in that we observed less infiltrates of mononuclear lymphocytes, less innate immune markers and no relevant differences in fibrosis markers. Thus, huTLR4 mice showed less inflammation and kidney pathology, and are not more susceptible to leptospirosis than WT mice. This study is significant as it indicates that one intact TLR4 gene, be it mouse or human, is necessary to control acute leptospirosis.

Enteroviruses (EVs) from the D species are the causative agents of a diverse range of infectious diseases in spite of comprising only five known members. This small clade has a diverse host range and tissue tropism. It contains types infecting non-human primates and/or humans, and for the latter, they preferentially infect the eye, respiratory tract, gastrointestinal tract, and nervous system. Although several Enterovirus D members, in particular EV-D68, have been associated with neurological complications, including acute myelitis, there is currently no effective treatment or vaccine against any of them. This review highlights the peculiarities of this viral species, focusing on genome organization, functional elements, receptor usage, and pathogenesis.

AbstractToll-Like Receptor (TLR) 4 plays a central role in the control of leptospirosis and TLR4 deficiency results in lethal infection in mice. Human TLR4 however does not recognize Leptospira spp which may allow for their escape from immune recognition. We hypothesized that the replacement of murine TLR4 with human TLR4 may produce an immunocompetent mouse model of sublethal leptospirosis. We infected TLR4/MD-2 humanized transgenic mice (huTLR4) developed on a C57BL/6J TLR4/MD-2 double knock-out background with a sublethal dose of L. interrogans serovar Copenhageni (Leptospira). We found that Leptospira disseminated to target organs of huTLR4 mice as shown by the presence of spirochetes in blood, urine and kidney after infection. We did a comparative study between huTLR4 mice and wildtype C57BL/6J (muTLR4) to find out if the differences observed in the humanized TLR4 transgenic model were due to the transgene modification or to other factors. Replacing murine TLR4 with human TLR4 resulted in considerable higher shedding of Leptospira in urine in huTLR4 mice. Regarding inflammation of the kidney, MIP-2 and iNOS were significantly decreased in huTLR4 in comparison to muTLR4, whereas TNF-α and IFN-γ were increased, but there was no difference in RANTES that was increased in both groups of infected mice. In conclusion, our data suggests that human TLR4 may mediate higher susceptibility to leptospirosis that results in kidney pathology associated with downregulation of local MIP-2, antimicrobial iNOS and RANTES which are insuficient to restrict Leptospira and reduce shedding in urine. The TLR4 humanized mouse model of sublethal leptospirosis described is a novel tool to test therapies and vaccines against this neglected re-emerging zoonotic disease.

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira which affect both humans and animals. 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 how male and female hamsters infected side-by-side with low but increasing doses of L. interrogans under the same exposure conditions develop Leptospirosis. We found that female hamsters were considerably more resistant to Leptospirosis given that 87.5% survived infection; male hamsters did not gain weight and 93.7% succumbed to infection with the same infectious doses. Analysis of bacterial burden in kidney of male hamsters showed that infection with the lowest dose (103) resulted in a 4Log increase of L. interrogans, whereas females infected with the same dose had a reduction of ~1Log, after 28 days of infection. Non-surviving hamsters had signs of compromised renal function (higher levels of creatinine in blood), as well as increased levels of anti-inflammatory IL-10 and innate response pro-inflammatory CCL3, CxCL10 and TNF-α in kidney, as well as ColA1 which is a marker of kidney fibrosis. In endemic areas, humans are more often exposed to the lower infection doses which were lethal to males, than the higher doses which were lethal to males and females. It is possible that the frequency of lower infectious doses in nature and increased biological susceptibility both contribute for aggravated outcomes of leptospirosis in males.