Laboratory diagnosis of leptospirosis

Percentage of serological positivity examined in 4205 blood sera by serological method microscopic agglutination test (MAT) on the hinterland territory of our laboratory (East Bohemia; 1999-2003) was 0.38-4.7 %. By the PCR method for detection of DNA of pathogenic leptospires (L. interrogans, L. borgpetersenii and L. kirschneri) from 57 samples of different biological materials from patients with fever of unknown etiology positive results were obtained in 4 specimens (7 %; 3 samples of urine and 1 sample of blood). This method was shown to distinguish between pathogenic and nonpathogenic strains and can detect 2.5-10 cells per mL of biological material. As an important presumption of successful detection of pathogenic leptospires a correct collecting of blood, urine samples or liquor is required before starting antibody therapy. The PCR method possesses a clear advantage over other methods, such as MAT, which relies on the detection of antibodies the presence of which cannot be detected until days after infection.     

Etiological structure and clinical epidemiological characteristics of Leptospira infection in Rostov Province based on data from therapeutic institutions from 1960 to 1983

Changes in communal conditions, in economy, as well as in ecology and fauna, which took place in Rostov Province during the last decade (1973-1983) determined shifts in the etiological structure of Leptospira infection and in its course. The study revealed an increase in morbidity caused by L. icterohaemorrhagiae (up to 61%) and L. hebdomadis (up to 22%) with a simultaneous decrease in the isolation rate of L. grippotyphosa and L. pomona (up to 2-3%). In most cases (77%) the diseases caused by leptospires of different serogroups were found to take an icteric course accompanied by the development of hepatorenal insufficiency (46%). The similarity of clinical manifestations in different etiological forms of Leptospira infection was determined by common pathogenetic and pathophysiological features characteristic of the development of the leptospiral infectious process.     

Leptospira population ecology. II. An attempt to assess the number in the soil and the epizootic potential of infected spots

Leptospires released with a carrier's urine were found to inhabit the upper layer of the soil 1 cm deep at a radius of 1-2 cm from the place of their penetration into the soil. A sharp drop in the number of leptospires in the "infected spot" was shown to occur; in 6-12 hours their concentration became no different from that in the intact soil. Most of such spots retained their infective capacity for 6 hours (up to 2 days), though the presence of leptospires at low concentration was registered for 10 days (the term of observation). The infective capacity was probably maintained by the regular addition of leptospires with carriers' urine.     

Morphology of a natural focus of Pomona leptospirosis in a steppe area

The natural focus of Leptospira pomona infection has been found to include areas of the forest-meadow flood plain where the circulation of leptospires is constantly maintained among small mammals (the nucleus of the focus). A high level of Leptospira carriership (17.7%) among the animals has been registered in the central part of the flood plain and near the terraces, these areas having the most favorable conditions for the development of epizootic leptospirosis.     

Biological Effects of Leptospiral Lipids

Lipids were extracted from virulent Leptospira pomona and were purified. These lipids fixed complement in the presence of antiserum to L. pomona but did not stimulate the production of homologous agglutinins in rabbits, mice, or hamsters. When subsequently challenged, all of the mice and hamsters were fully susceptible to L. pomona. The lipid material was neither dermonecrotic nor lethal for mice or hamsters, but 382 mug of lipid from virulent or avirulent leptospires inhibited the growth of normal mice. Leptospiral lipids were toxic for peritoneal macrophages maintained in vitro, and when administered simultaneously with a million lethal doses of L. pomona, the lipids hastened death of the hamsters, presumably by inhibiting phagocytosis early in the course of the infection.     

Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics

Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 10⁶ to 10⁷ bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×10⁸ bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to challenge mice treated with drugs or vaccines, and test the survival, dissemination, and transmission of leptospires between environment and hosts.     

Natural Antibody in Mammalian Serum Reacting with an Antigen in Some Leptospires

Serum from normal mammals agglutinated and immobilized nonpathogenic Leptospira biflexa and agglutinated avirulent lines of pathogenic serotypes L. icterohaemorrhagiae and L. zanoni. Virulent lines of L. icterohaemorrhagiae and L. zanoni were not affected, nor were any of three strains of L. pomona, one of which was avirulent. The active principle in serum was a beta-macroglobulin which was heat-labile and reduced by 2-mercaptoethanol, and acted in conjunction with complement and lysozyme; it was absorbable from serum by Formalin-treated susceptible leptospires. The Formalin-stable receptor antigen, named "Z antigen," is associated with virulence rather than pathogenicity, but may not be a determinant of virulence.     

The enumeration of Leptospira interrogans serovar pomona by a bioluminescence ATP assay

A rapid method for enumerating viable Leptospira interrogans serovar pomona cells was investigated using a bacterial adenosine triphosphate (ATP) assay. The ATP was assayed by the luciferin-luciferase bioluminescence reaction. Samples of serovar pomona grown in liquid polysorbate 80-bovine albumin (P80-BA) medium for 1-3 days were analysed for ATP content, culture density (nephelometry), direct cell count and most probable number of viable cells (MPNVC) as determined by the dilution tube technique. A linear relationship was found between ATP content and the number of viable cells over the range of 4 X 10(8) to 8 X 10(9) leptospires/ml. Over this range the correlation coefficient for ATP content versus viable cells (0.96) was similar to the coefficient for culture density versus the number of viable cells. The coefficient for direct counts versus the number of viable cells was smaller. The bioluminescence assay of bacterial ATP is a promising method for enumerating viable leptospires in pure culture.     

Interaction of leptospires with murine microglial cells

Leptospires, the agents of leptospirosis, exert tropism for the central nervous system, in the course of mammal infection. We evaluated the interaction between murine microglial cells and strains of pathogenic L. interrogans leptospires and non-pathogenic L. biflexa leptospires, mainly by flow cytometric assays. In the absence of opsonic conditions microglia are capable of ingesting--even quite slowly--the spirochetes and killing the non-pathogenic strain. The adhesion to microglia, which is quick and relevant for all the strains, does not involve the CR3 integrin receptor. These findings suggest that the murine microglia--in non opsonic conditions in vitro--do not effectively clear the pathogenic leptospires.     

Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages

Leptospira interrogans is a spirochaete responsible for a zoonotic disease known as leptospirosis. Leptospires are able to penetrate the abraded skin and mucous membranes and rapidly disseminate to target organs such as the liver, lungs and kidneys. How this pathogen escape from innate immune cells and spread to target organs remains poorly understood. In this paper, the intracellular trafficking undertaken by non-pathogenic Leptospira biflexa and pathogenic L. interrogans in mouse bone marrow-derived macrophages was compared. The delayed in the clearance of L. interrogans was observed. Furthermore, the acquisition of lysosomal markers by L. interrogans-containing phagosomes lagged behind that of L. biflexa-containing phagosomes, and although bone marrow-derived macrophages could degrade L. biflexa as well as L. interrogans, a population of L. interrogans was able to survive and replicate. Intact leptospires were found within vacuoles at 24 h post infection, suggesting that bacterial replication occurs within a membrane-bound compartment. In contrast, L. biflexa were completely degraded at 24 h post infection. Furthermore, L. interrogans but not L. biflexa, were released to the extracellular milieu. These results suggest that pathogenic leptospires are able to survive, replicate and exit from mouse macrophages, enabling their eventual spread to target organs.     

Growth of Leptospira pomona and Its Effect on Various Tissue Culture Systems

Miller, Robert E. (University of Nebraska College of Medicine, Omaha), Norman G. Miller, and Roberta J. White. Growth of Leptospira pomona and its effect on various tissue culture systems. J. Bacteriol. 92:502-509. 1966.-Leptospira pomona strain 3341 was grown in association with primary fetal bovine kidney (PBK) and human embryonic skin-muscle fibroblastic (HE) cells in Eagle's minimal essential medium (MEM) with 5% sheep serum. Growth curves of leptospires in PBK and HE cell cultures showed no substantial increase in growth above that obtained in Eagle's MEM in the absence of tissue culture cells. This suggested that no stimulatory growth factors for leptospires were produced by the tissue cells. Fibroblastic cells of the PBK monolayer showed separation, deterioration, and, finally, complete disintegration. Epithelial-like cells remained unaffected. HE cells showed the same cytopathic effect as PBK fibroblastic cells, indicating that this effect was not limited to PBK fibroblastic cells. Warthin-Starry stains of PBK and HE cell monolayers showed masses of leptospires adhering to fibroblastic cells, whereas only a few were seen on epithelial-like cells. Large numbers of leptospires on the surface of fibroblastic cells are very likely associated with the cytopathic effect. Dislodgment of leptospires from fibroblastic cells did not increase the total number of spirochetes in the culture. This indicated that leptospiral growth did not occur on the surface of these cells.     

Bactericidal activity of normal sera from various animal species against pathogenic and saprophytic leptospires

The leptospirocidal activity of 5 animal species against L. interrogans, L. biflexa and L. kazachstanica I and II, belonging to different serogroups and serovars, was studied. Cattle and sheep sera had no lytic effect on 36.9-40.1% of pathogenic Leptospira strains, but other pathogenic strains, as well as saprophytes, were lyzed by these sera. L. pomona and L. grippotyphosa exhibited high resistance to cattle serum, the latter being also resistant to sheep serum.     

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti‐leptospiral strategies.     

IN VIVO AND IN VITRO OBSERVATIONS OF LEPTOSPIRA POMONA BY ELECTRON MICROSCOPY.

Miller, Norman G. (University of Nebraska College of Medicine, Omaha) and Richard B. Wilson. In vivo and in vitro observations of Leptospira pomona by electron microscopy. J. Bacteriol. 84:569-576. 1962.-Leptospira pomona 3341 was observed by electron microscopy, after the preparation of thin sections from culture material and from infected hamster tissue. The external membrane of low electron density envelops the entire leptospire and appears to be quite flexible, as suggested by its many folds. The spiral protoplasmic body is tubular in structure with a relatively dense wall and a central area of low electron density. Occasionally, very dark circumscribed bodies were seen imbedded in the protoplasmic wall. Detailed morphology is presented of a knoblike structure located at the end of the axial filament. Bifurcation of the axial filament could be demonstrated in leptospires from cultures. Leptospires were observed free or enclosed in vesicles within the cytoplasm of liver parenchymal and renal tubule cells. Erythrocytes located in kidney tissue also contained leptospires within the cytoplasm. The appearance of intracellular leptospires is much the same as those seen extracellularly or from culture.     

Diagnosis and seroprevalence of leptospirosis in California sea lions from coastal California

The sensitivity and specificity of the microscopic agglutination test (MAT) as a method for detection of exposure to Leptospira spp. in California sea lions (Zalophus californianus) were determined. Sera came from individuals that demonstrated clinical signs of renal disease, had lesions suggestive of leptospirosis at necropsy, and had visible leptospires in silver stained kidney sections as positive controls. Sera from unexposed captive individuals were used as negative controls. The test was 100% sensitive at 1:3,200 for confirming renal infection and 100% specific at negative < 1:100 for detection of Leptospira interrogans scrovar pomona antibodies by MAT in California sea lions. Leptospira interrogans serovar pomona was used as a screening serovar because it has been isolated previously from the kidneys and placentas of California sea lions, and there appears to be cross-reactivity between serovar pomona and other serovars. Sera from 225 free-ranging California sea lions presented to one of three participating California (USA) coastal marine mammal rehabilitation centers in 1996 were then evaluated for antibodies to serovar pomona using the MAT. The overall seroprevalence was 38.2% (86/225), although the prevalence varied among locations from 100% (38/38) in animals at the Marine Mammal Care Center (Fort MacArthur, California, USA) to 0% (0/14) at SeaWorld California (San Diego, California). At The Marine Mammal Center (Sausalito, California) [prevalence 27.8% (48/173)], the majority of seropositive animals were subadults and adults, and males were 4.7 times more likely to be seropositive to serovar pomona than females. When combining results from all three centers, subadult and adult animals were more likely to be seropositive than pups and juvenile sea lions, and the highest proportion of seropositive animals presented during the autumn months. Serum elevations of blood urea nitrogen, creatinine, phosphorus, and/or calcium were associated with seropositivity to serovar pomona. We found no association between potassium or sodium levels and seropositivity.     

PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte,Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof''s medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.     

Evidence of Leptospira interrogans infection in California sea lion pups from the Gulf of California

Forty-two urine and 96 blood and serum samples were obtained from California sea lion (Zalophus californianus) pups from the Gulf of California during the 2000 reproductive season. Antibody prevalence to 13 serovars of Leptospira interrogans was determined by microagglutination tests (MAT); presence of pathogenic leptospires was detected by polymerase chain reaction (PCR). Samples with antibody titers > or = 1:25 or 115 bp fragments on ethidium bromidestained 1.5% agarose gels were considered positive. Antibody prevalence was 54% overall with highest prevalence against serovar cynopteri (50% of all positive reactions). Highest antibody titers (1:50) were detected against serovars cynopteri and pomona. Polymerase chain reaction products were observed in two of 42 urine samples, six of 96 blood samples, and one of 96 serum samples. Presence of PCR products in blood and serum was demonstrated in pups that were seronegative. Kruskall-Wallis tests and corresponding post hoc Tukey tests (alpha = 0.05) showed that prevalence of leptospirosis was significantly different among all rookeries. The high seroprevalence (54%), low antibody titers (maximum 1:50), absence of pups showing clinical signs indicative of the disease, and lack of recent reports of increased mortality of sea lions in the Gulf of California are suggestive of the presence of enzootic host-adapted serovars. Crowding in rookeries as well as the presence of bats and rodents on some of the islands may explain infection by L. interrogans (sensu lato) and some of the differences in seroprevalence among reproductive rookeries.     

Lesions and immune responses produced in hamsters and guinea pigs inoculated with some strains of leptospira

Pathogenic lesions and immune responses in hamsters and guinea pigs produced by three leptospiral serovars, viz. autumnalis, grippotyphosa, and pomona, and their pool were experimentally studied. Hepatic lesions precede renal localisation. The infections were documented by the demonstration of leptospires and histopathological study. The 2-Me sensitive IgM was responsible for MAT titres in the early immune response.     

Responses of human endothelial cells to pathogenic and non-pathogenic Leptospira species

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.     

A rapid and quantitative method for the detection of Leptospira species in human leptospirosis

Prompt laboratory diagnosis of leptospirosis infection facilitates patient management and initiation of therapy. A cost effective real-time PCR assay using SYBR Green I was developed for detection of pathogenic leptospires in serum specimens. Specific PCR products were obtained only with DNA of pathogenic Leptospira genomospecies. LightCycler PCR ability to distinguish between species was possible using melting curves, providing an approach for identification with a specific Tm assigned to a single species or set of species. Assay sensitivity was approximately 50 leptospires/ml, corresponding to one to two genome copies in a PCR mixture. Fifty-one patients who had clinical symptoms consistent with leptospirosis were tested both with a previously described rrs amplification and our real-time assay. Our LFB1 real-time assay confirmed the diagnosis for 25 patients (49%, 25/51) and revealed an estimated density of 8.0x10(1)-3.9x10(4) leptospires/ml of blood. The total assay time for 12 clinical samples from sample to data analysis was less than 3 h. These data illustrate the potential of our LFB1 real-time assay for the rapid detection of leptospires in serum samples and their subsequent quantification in a single run.