Diagnosis of Human Leptospirosis in a Clinical Setting: Real-Time PCR High Resolution Melting Analysis for Detection of Leptospira at the Onset of Disease:

Currently, direct detection of Leptospira can be done in clinical laboratories by conventional and by real-time PCR (qRT-PCR). We tested a biobank of paired samples of serum and urine from the same patient (202 patients) presenting at the hospital in an area endemic for leptospirosis using qRT-PCR followed by high resolution melting (HRM) analysis. The results were compared with those obtained by conventional nested PCR and with the serologic gold standard microscopic agglutination test (MAT). Differences were resolved by sequencing. qRT-PCR-HRM was positive for 46 of the 202 patients (22.7%, accuracy 100%) which is consistent with known prevalence of leptospirosis in the Azores. MAT results were positive for 3 of the 46 patients (6.5%). Analysis of paired samples allowed us to identify the illness point at which patients presented at the hospital: onset, dissemination or excretion. The melting curve analysis of Leptospira species revealed that 60.9% (28/46) of patients were infected with L. interrogans and 39.1% (18/46) were infected with L. borgpetersenii, both endemic to the Azores. We validated the use of qRT-PCR-HRM for diagnosis of leptospirosis and for identification of the Leptospira species at the earliest onset of infection in a clinical setting, in less than 2 hours.publishersversionpublishe

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of \u3c 3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi

Immune Response to Lactobacillus plantarum Expressing Borrelia burgdorferi OspA Is Modulated by the Lipid Modification of the Antigen

Over the past decade there has been increasing interest in the use of lactic acid bacteria as mucosal delivery vehicles for vaccine antigens, microbicides and therapeutics. We investigated the mechanism by which a mucosal vaccine based in recombinant lactic acid bacteria breaks the immunological tolerance of the gut in order to elicit a protective immune response.We analyzed how the lipid modification of OspA affects the localization of the antigen in our delivery vehicle using a number of biochemistry techniques. Furthermore, we examined how OspA-expressing L. plantarum breaks the oral tolerance of the gut by stimulating human intestinal epithelial cells, peripheral blood mononuclear cells and monocyte derived dendritic cells and measuring cytokine production. We show that the leader peptide of OspA targets the protein to the cell envelope of L. plantarum, and it is responsible for protein export across the membrane. Mutation of the lipidation site in OspA redirects protein localization within the cell envelope. Further, we show that lipidated-OspA-expressing L. plantarum does not induce secretion of the pro-inflammatory cytokine IL-8 by intestinal epithelial cells. In addition, it breaks oral tolerance of the gut via Th1/Th2 cell mediated immunity, as shown by the production of pro- and anti-inflammatory cytokines by human dendritic cells, and by the production of IgG2a and IgG1 antibodies, respectively.Lipid modification of OspA expressed in L. plantarum modulates the immune response to this antigen through a Th1/Th2 immune response

Immunization with a Borrelia burgdorferi BB0172-Derived Peptide Protects Mice against Lyme Disease

Lyme disease is the most prevalent arthropod borne disease in the US and it is caused by the bacterial spirochete Borrelia burgdorferi (Bb), which is acquired through the bite of an infected Ixodes tick. Vaccine development efforts focused on the von Willebrand factor A domain of the borrelial protein BB0172 from which four peptides (A, B, C and D) were synthesized and conjugated to Keyhole Limpet Hemocyanin, formulated in Titer Max® adjuvant and used to immunize C3H/HeN mice subcutaneously at days 0, 14 and 21. Sera were collected to evaluate antibody responses and some mice were sacrificed for histopathology to evaluate vaccine safety. Twenty-eight days post-priming, protection was evaluated by needle inoculation of half the mice in each group with 103 Bb/mouse, whereas the rest were challenged with 105Bb/mouse. Eight weeks post-priming, another four groups of similarly immunized mice were challenged using infected ticks. In both experiments, twenty-one days post-challenge, the mice were sacrificed to determine antibody responses, bacterial burdens and conduct histopathology. Results showed that only mice immunized with peptide B were protected against challenge with Bb. In addition, compared to the other the treatment groups, peptide B-immunized mice showed very limited inflammation in the heart and joint tissues. Peptide B-specific antibody titers peaked at 8 weeks post-priming and surprisingly, the anti-peptide B antibodies did not cross-react with Bb lysates. These findings strongly suggest that peptide B is a promising candidate for the development of a new DIVA vaccine (Differentiate between Infected and Vaccinated Animals) for protection against Lyme disease.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

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

Immunochallenge reduces risk sensitivity during foraging in white-footed mice

Foraging behaviour has the potential to impact interactions among species in a community and is influenced, in part, by individual condition and fear of predation. These relationships are exemplified by white-footed mice, Peromyscus leucopus, in mixed hardwood forest communities of the northeastern U.S.A., whose foraging behaviour can influence the population ecology of several prey items and the disease ecology of human Lyme disease and its tick vector. We examined whether dosage of an immunogen influenced foraging behaviour in wild white-footed mice at foraging arenas. Low-dose mice preferentially favoured safe (covered) food patches over risky food patches, whereas high-dose mice showed no preference based on patch safety. Immunochallenge did not alter foraging time in patches of equal risk. The results reveal fitness costs of an immune response, namely that immunochallenged mice favour increased energy consumption over safety from predation, presumably leading to greater mortality. Acceptance of risky patches for foraging by immunochallenged mice suggests that mice mounting this immune response will forage in a greater proportion of their home ranges and encounter a greater number of patchily distributed prey items and ticks carrying pathogens

Antibody response to oral administration of recombinant <i>L. plantarum</i>: serological IgG.

<p>C3H-HeJ mice were inoculated intragastrically with <i>L. plantarum</i> expressing OspA (LpA) or the mutant OspA_D17 (LpA_D17). Control mice were inoculated with <i>L. plantarum</i> (Lp). Serum samples were collected at days 0, 14, 28, 49 and 68, and specific serological anti-OspA total IgG (A), IgG1 (B) and IgG2a (C) antibodies were measured by indirect ELISA. The results are expressed as Optical Density at 450 nm (OD₄₅₀). The average of triplicate samples per mouse was determined and the error bar indicates standard deviation. *<i>p</i><0.05, **<i>p</i><0.01. Results are representative of one of three independent experiments. <i>n</i> = 4 mice per group.</p

Evaluation of protein export and lipidation.

<p>OspA- and OspA_D17-expressing <i>L. plantarum</i> were disrupted with a French® press, the insoluble material (cell envelope) was extracted with Triton X-114 and partitioned into detergent and aqueous phases. Protein fractions were analyzed on a SDS-PAGE and tested by immunoblot with OspA-specific monoclonal antibody LA2.2 (A). Protein was quantified by densitometry. The results were plotted as a percentage of the total OspA content for each recombinant <i>Lactobacillus</i> (B). TE, total extract; D, detergent phase; A, aqueous phase; OspA- and OspA_D17-expressing <i>L. plantarum</i> (LpA and LpA_D17, respectively). *<i>p</i><0.05.</p

Production of cytokines in human MDDC co-cultured with recombinant <i>Lactobacillus</i>.

<p>Monocytes were isolated from human PBMCs by MACS using the Monocyte Isolation Kit II. Monocytes were derived to immature DCs (MDDCs) by cultivating the cells in the presence of 100 nM GM-CSF and 10 nM IL-4 for 5 days. MDDCs (2×10⁵ cells/well) were co-cultured with UV-killed recombinant <i>Lactobacillus</i> expressing OspA (LpA) or the mutant OspA_D17 (LpA_D17) at MOI 10:1 colony-forming units per DC. 100 ng/ml <i>Escherichia coli</i> O111:B4 lipopolysaccharide (LPS) and <i>L. plantarum</i> (Lp) were used as positive and negative control, respectively. After 48 h of stimulation, supernatants were collected and TNF (A), IL-12 (B), IFNγ (C), IL-6 (D) and IL-10 (E) cytokine production was measured by sandwich ELISA (Quantikine). *<i>p</i><0.05,**<i>p</i><0.001. Results are representative of one of three independent experiments.</p