Michael A. Lovett

Leptospirosis: a zoonotic disease of global importance

In the past decade, leptospirosis has emerged as a globally important infectious disease. It occurs in urban environments of industrialised and developing countries, as well as in rural regions worldwide. Mortality remains significant, related both to delays in diagnosis due to lack of infrastructure and adequate clinical suspicion, and to other poorly understood reasons that may include inherent pathogenicity of some leptospiral strains or genetically determined host immunopathological responses. Pulmonary haemorrhage is recognised increasingly as a major, often lethal, manifestation of leptospirosis, the pathogenesis of which remains unclear. The completion of the genome sequence of Leptospira interrogans serovar lai, and other continuing leptospiral genome sequencing projects, promise to guide future work on the disease. Mainstays of treatment are still tetracyclines and beta-lactam/cephalosporins. No vaccine is available. Prevention is largely dependent on sanitation measures that may be difficult to implement, especially in developing countries.

Treatment of first episodes of genital herpes simplex virus infection with oral acyclovir. A randomized double-blind controlled trial in normal subjects.

Abstract We performed a double-blind placebo-controlled trial of oral acyclovir in the treatment of first episodes of genital herpes simplex virus infections in 48 young adults (31 women and 17 men...

Alveolar Septal Deposition of Immunoglobulin and Complement Parallels Pulmonary Hemorrhage in a Guinea Pig Model of Severe Pulmonary Leptospirosis

Human patients suffering from leptospirosis present with a diverse array of clinical manifestations, including the more severe and often fatal pulmonary form of the disease. The etiology of pulmonary hemorrhage is unclear. Isolates of Leptospira acquired from patients suffering from pulmonary hemorrhage were used to develop a guinea pig model of pulmonary hemorrhage. Gross findings post-infection confirmed extensive hemorrhage in the lungs and on peritoneal surfaces as the likely cause of death. Immunohistochemistry confirmed the presence of large numbers of leptospires in kidney, liver, intestinal tissues, and spleen, but few inflammatory cells were seen. In marked contrast, few leptospires were detected in infected hemorrhagic lung tissue. Blood chemistries and hematology did not reveal the etiology of the hemorrhage observed. There was no chemical or microscopic evidence for disseminated intravascular coagulation. To ascertain an immunopathologic role during disease, immunofluorescence was performed on infected lung tissues and confirmed the presence of IgM, IgG, IgA, and C3 along the alveolar basement membrane. This suggests that an autoimmune process may be the etiology of fatal pulmonary hemorrhage in leptospirosis.

Characterization of the Outer Membrane Proteome of Leptospira interrogans Expressed during Acute Lethal Infection

ABSTRACT Pathogenic Leptospira species adapt to a wide range of environmental conditions during disease transmission and infection. While the proteome of in vitro cultivated Leptospira has been characterized in several studies to date, relatively little is known of the proteome as expressed by Leptospira during disease processes. Isolates of Leptospira obtained from patients suffering the severe pulmonary form of leptospirosis cause acute lethal infection in guinea pigs and chronic asymptomatic infection in rats. Recent studies have demonstrated that protein and lipopolysaccharide constituents of Leptospira recovered from acutely infected guinea pig tissue differ from that of Leptospira in chronically infected rat tissue and in vitro cultivated Leptospira (J. E. Nally, E. Chow, M. C. Fishbein, D. R. Blanco, and M. A. Lovett, Infect. Immun. 73:3251-3260, 2005). In the current study, the proteome of Leptospira expressed during disease processes was characterized relative to that of in vitro cultivated Leptospira (IVCL) after enrichment for hydrophobic membrane proteins with Triton X-114. Protein samples were separated by two-dimensional gel electrophoresis, and antigens expressed during infection were identified by immunoblotting with monospecific antiserum and convalescent rat serum in addition to mass spectrometry. Results suggest a significant increase in the expression of the outer membrane protein Loa22 during acute infection of guinea pigs relative to other outer membrane proteins, whose expression is generally diminished relative to expression in IVCL. Significant amounts of LipL32 are also expressed by Leptospira during acute infection of guinea pigs.

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

Changes in Lipopolysaccharide O Antigen Distinguish Acute versus Chronic Leptospira interrogans Infections

ABSTRACT Leptospirosis is the most geographically widespread zoonotic disease in the world. A severe pulmonary form of leptospirosis (SPFL) is being recognized with increased frequency. We have reported that human SPFL isolates of Leptospira cause acute lethal infection with prominent pulmonary hemorrhage in guinea pigs. We have found that the same SPFL strains cause asymptomatic infection and chronic renal shedding in rats, where infection is restricted to the renal tubules. To address the antigenic composition of host tissue-derived Leptospira (HTL), motile leptospires were purified from guinea pig liver by centrifugation on Percoll density gradients and compared to Percoll-purified in vitro-cultivated Leptospira (IVCL). The lipopolysaccharide O antigen (Oag) content of guinea pig liver-derived HTL was markedly reduced compared to that of IVCL, as demonstrated both by immunoblotting with a monoclonal antibody that was serovar specific for Oag and by periodate-silver staining. Confocal microscopy of HTL in guinea pig liver and kidney with the Oag-specific monoclonal antibody provided further evidence that diminution of the Oag content occurred in situ during lethal infection. In contrast, the Oag content of HTL in chronically infected rat renal tubules was indistinguishable from that of IVCL. These findings suggest that there may be regulation of Oag synthesis by Leptospira specific to the animal host infected. The hypothesis that the Oag content is related to whether lethal infection or chronic renal tubular colonization occurs remains to be tested.

Temporal Analysis of the Antigenic Composition of Borrelia burgdorferi during Infection in Rabbit Skin

ABSTRACT The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.

Purification of treponema pallidum, nichols strain, by percoll density gradient centrifugation

The purification of motile and virulent Treponema pallidum, Nichols strain, from rabbit testicular tissue is reported. Suspensions of T. pallidum were overlayed onto 20-ml cushions of 43% Percoll and in-situ density gradients were formed by centrifugation at 34,800 g for 30 min. Gradient fractionation indicated that T. pallidum banded at a density of 1.051 g/cc3 and that soluble proteineous testicular components remained in the upper portion of the gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the removal of host testicular and serum components. Purified suspensions of T. pallidum were > 95% actively motile and fully virulent, and >50% motility could be maintained in vitro for up to five days. As determined by electron microscopy, Percollpurified T. pallidum was structurally unaltered and contained much less tissue debris than did crude extracts or T. pallidum prepared by differential centrifugation. The Percoll purification method has been applied successfully to physiology, recombinant DNA, and antigenic structure studies, and to the preparation of antigen for the fluorescent treponemal antibody-absorbed (FTA-Abs) test for syphilis.

Antigenic Composition of Borrelia burgdorferi during Infection of SCID Mice

ABSTRACT The general concept that during infection of mice the Borrelia burgdorferi surface protein composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct compositional analysis. In this report we describe novel means for studying the antigenic composition of host-adapted Borrelia (HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated Borrelia (IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the antigenic composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic B. burgdorferi antigens from tissue provides a powerful tool for determining the antigenic composition of HAB.

Serologic analysis of first-episode nonprimary genital herpes simplex virus infection: Presence of type 2 antibody in acute serum samples

Acute serum samples from patients with nonprimary first-episode genital herpes were evaluated for the presence of type-specific antibody to herpes simplex virus type 1 and type 2. A standard microneutralization assay and western blot analysis, which identified the response to individual polypeptides of type 1 and type 2 herpes, were performed in both unadsorbed serum samples and serum samples adsorbed with either type 1 or type 2 antigens to remove cross-reacting antibodies. Of 24 serum samples examined, seven were found to contain only type 1 antibodies, 11 had type 2 antibodies alone, and six serum samples had both type 1 and type 2 antibodies. It is believed that a majority of the 17 patients whose acute serum samples already contained type 2 antibody were experiencing their first clinically apparent recurrence of a previous asymptomatic type 2 infection. Physicians should, therefore, acknowledge that patients with first-episode nonprimary herpes 2 infection may not have had a recent sexual contact with a herpes simplex virus-infected partner.