Antileptospiral Activity of Serum II. Leptospiral Virulence Factor

A definite relationship exists between the resistance of leptospires to the antibody-complement system and virulence. Leptospires capable of producing either lethal or renal infections in hamsters or guinea pigs were resistant to the leptospiricidal action of antibody and complement. Avirulent leptospires, in contrast to the virulent organisms, were rapidly immobilized and killed by these serum substances. The change of a virulent culture to the avirulent state as a result of growth in culture media was accompanied by the loss of resistance to antibody and complement. Virulent leptospires were phenotypically altered when grown in the presence of the purine analogue, 8-azaguanine. The cells became sensitive to antibody and complement without a corresponding decrease in virulence. The basis for a leptospiral virulence factor, the ability to multiply in vivo, appears to reside in their capacity to resist the leptospiricidal activity of the host antibody-complement system. The immune leptospiricidal assay provides a simple and rapid method of determining the virulence of a culture.     

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.     

Differentiation of Pathogenic and Saprophytic Leptospires I. Growth at Low Temperatures

The minimal growth temperature of the pathogenic leptospires is between 13 and 15 C. The saprophytic leptospires have a minimal growth temperature between 5 and 10 C, or approximately 5 C below that of the pathogens. The capability of the saprophytic leptospires to grow at temperatures below those which allow the growth of the pathogenic leptospires provides a simple method of discrimination. With an inoculum yielding approximately 8 x 10(7) cells per ml in the test medium and an incubation temperature of 13 C, the saprophytic leptospires were easily differentiated from the pathogenic leptospires. All 13 saprophytic leptospires tested grew in the 10% rabbit serum medium at 13 C, whereas none of the 20 pathogens grew during the 30-day incubation period.     

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

Control of immunologically crossreactive leptospiral infection by administration of lipopolysaccharides from a nonpathogenic strain of Leptospira biflexa

In our previous paper (Matsuo, K., Isogai, E., and Araki, Y., Carbohydr. Res., 328: 517-524, 2000), antigenic polysaccharides obtained from the lipopolysaccharide (LPS) fraction of a nonpathogenic leptospira, Leptospira biflexa patoc Patoc I, are shown to be broadly crossreactable with most rabbit antisera elicited by immunization with various pathogenic leptospires. The result led us to test a protective effect of the same LPS in a hamster model system by heterologously challenging with a pathogenic leptospira, L. interrogans manilae UP-MMG. Firstly, a similarity in the antigenic epitopes of L. biflexa and L. interrogans was confirmed by the following assays. In the microscopic agglutination test (MAT), a hamster antiserum elicited by immunization with the L. biflexa-LPS preparation was shown to agglutinate cells of L. interrogans. Contrarily, in the enzyme-linked immunosorbent assay (ELISA), the L. biflexa-LPS preparation was shown to crossreact with a hamster antiserum elicited by immunization with whole cells of L. interrogans. These results suggest that the same or closely related antigens may be present on the cell surfaces of both L. biflexa patoc Patoc I and L. interrogans manilae UP-MMG. Furthermore, in a protective assay, the prior administration of a L. biflexa-LPS preparation resulted in raising a protective response in hamsters against challenge by L. interrogans without any side effect. The protective effect was strongly dependent on the dose amounts and/or administration times of L. biflexa-LPS. Thus, L. biflexa-LPS preparations can use as a potent vaccine against leptospirosis caused by various leptospires.     

Role of serum factors in the phagocytosis of weakly or heavily encapsulated Cryptococcus neoformans strains by guinea pig peripheral blood leukocytes

We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.     

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.     

Electron Microscopy of Immune Disruption of Leptospires: Action of Complement and Lysozyme

Sequential disruption of the sheath of avirulent leptospires of the serotype canicola with antibody and complement was monitored by electron microscopy. Loosening and separation of the sheath from the protoplasmic cylinder was observed as early as 2 min after exposure to complement. Virulent leptospires of this serotype were morphologically intact after 1 hr of exposure to antibody and complement. Similarly, treatment of leptospires of the serotype patoc with normal serum and complement severely damaged the sheath structure. Removal of the sheath of both serotypes permitted lysozyme to act on the wall of the protoplasmic cylinder. Thus, morphological evidence for the location of the mucopeptide-containing structure of these leptospires was obtained. Viable leptospires with intact sheaths were resistant to lysozyme alone. Sections and negatively stained preparations of sheaths of serotypes canicola and patoc revealed three dense layers with two intermediate light zones and an overall thickness of about 110 A. A periodicity of 40 A was observed in sheath fragments produced by complement. The 70 A wallmembrane complex of leptospires of both serotypes consisted of two dense layers with an intermediate light zone. Structures apparent after removal of the outer sheath included membranous bodies or mesosomes, axial filaments attached to terminal knobs at opposite ends of the cell, and electron-dense intracellular bodies.     

Contribution of Complement System pathways to the killing of Leptospira spp.

The Complement System (CS) plays an important role in the immune response against leptospirosis and can be activated by the Alternative and Lectin Pathways (Innate Immunity) and by the Classical Pathway (Acquired Immunity). Here we analyzed a broad range of nonpathogenic and pathogenic Leptospira strains considering their interaction with each CS pathway. We determined bacterial survival rate and CS protein deposition in the presence of purified proteins, specific component depleted sera and NHS treated with the chelating agents EDTA (inhibits all three activation pathways) or EGTA (inhibits the Classical and Lectin Pathways). We suggest that the Lectin and the Alternative Pathways have an important role to eliminate saprophytic leptospires since i) approximately 50% survival of both saprophytic strains was observed in the presence of MBL-deficient serum; ii) approximately 50% survival of Leptospira biflexa Patoc I was observed in the presence of NHS – EGTA and iii) C1q-depleted serum caused significant bacterial lysis. In all serovars investigated the deposition of C5–C9 proteins on saprophytic Leptospira strains was more pronounced when compared to pathogenic species confirming previous studies in the literature. No difference on C3 deposition was observed between nonpathogenic and pathogenic strains. In conclusion, Leptospira strains interact to different degrees with CS proteins, especially those necessary to form MAC, indicating that some strains and specific ligands could favor the binding of certain CS proteins.     

An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action

The small cationic outer membrane-disorganizing peptide PMBN sensitized four smooth, encapsulated strains of Escherichia coli (serotypes 02:K1, 04:K12, 018:K1, and 018:K5) to the lethal action of serum. The concentrations of PMBN required were low (0.3 to 1.0 microgram/ml). One E. coli strain (IH 11030; 075:K5) remained virtually resistant to serum and also to anti-075 hyperimmune serum plus complement (C) even in the presence of PMBN. This strain was nevertheless sensitive to the outer membrane permeability-increasing action of PMBN. In the bactericidal system, PMBN could be replaced by high concentrations of lysine20 or protamine but not lysine4. The PMBN-dependent bactericidal activity of GPS was abolished by heating or zymosan treatment that inactivate its C but not by lack of the action of the classical pathway of the C in C4-deficient GPS. PMBN formed a bactericidal system also with normal rabbit, rat, and human serum but not with mouse serum. The bactericidal system against E. coli 018:K1 and its derivative EH 817 (018:K1-) was found to require a factor that can be removed from normal sera by absorption with a rough E. coli strain. This factor could be replaced by specific anti-018 antibodies. The bactericidal activity of fetal calf serum plus PMBN against E. coli 018:K1 was enhanced by normal rabbit or anti-E. coli 018 hyperimmune serum. We suggest that PMBN unshields the deep structures and the hydrophobic membrane milieu of the outer membrane and facilitates the insertion of the membrane attack complex of the C into this milieu.     

Cytotoxins - cause of serum activity as complement in the lymphocytotoxic test

The Ferrone's hypothesis elucidating the effect of rabbit complement in HLA typing using the cytotoxic test, due to the presence of xenocytotoxins to human lymphocytes in the rabbit serum was corroborated by the following experiments: 1. In human serum active as complement in HLA typing non-HLA lymphocytotoxins with optimum activity at 20 degree C were shown. In the guinea pig complement ineffective in HLA typing no cytotoxin to human lymphocytes were found. 2. In the rabbit complement cytotoxins to peripheral human lymphocytes were found when the incubation period of the cytotoxic test was prolonged to 3-4 h. 3. During the cytotoxic test on a model of rabbit immune sera - rabbit lymphocytes human complement showed a substantially higher activity than the rabbit complement. In the human complement xenocytotoxins to rabbit lymphocytes were demonstrated.     

Antigenic specificity of opsonophagocytic antibodies in rabbit anti-sera to group B streptococci.

An opsonophagocytic assay has been developed which requires human polymorphonuclear leukocytes, immune serum, and complement for optimal killing of Group B streptococci. Only with all three of these components was killing of greater than 1.0 log10 of the initial inoculum achieved, using rabbit antisera directed to homologous strains of each of the five known serotypes of Group B streptococci. Titers of specific antisera which opsonized the strains and resulted in greater than 1 log 10 reduction of colony-forming units, ranged from 1:100 (serotype Ib) to 1:3200 (serotype Ia). Cross-reactions between serotype-specific sera and heterologous strains were seen in certain instances. Type Ic strain and serotype Ic antiserum demonstrated cross-reactions with types Ia and Ib which were explainable by known shared antigens among these types. The only other cross-reaction which resulted in greater than 1 log 10 reduction in colony-forming units was when unabsorbed antiserum to strain Ia was used to opsonize a strain of serotype III. Opsonization of 10 serotype III strains was demonstrated with a single type III antiserum. Killing of nine of these strains required polymorphonuclear leukocytes, complement, and antiserum, but one strain, D136C, the reference strain, could be killed (greater than 1 log 10 reduction in colony-forming units) without either complement or specific antiserum. Inhibition studies were performed utilizing large m.w. polysaccharide antigens extracted from each serotype. These antigens inhibited opsonization of homologous strains by homologous antisera with 50% inhibition points ranging between 0.5 and 4 mug.     

Protective effect of rabbit immune serum administered orally to rats infected by a human pathogenic strain of E. coli.

1-week-old rats were inoculated orally with a strain of E. coli (serotype 078) isolated from the blood of a newborn baby who had died of septicemia. During the 3 weeks following inoculation, approximately 50% of the animals died of septicemia and 60% of the surviving rats had pathogenic bacteria in their rectum. Some of the surviving rats were severely impaired in their development. Autopsy showed evidence of active intestinal infection localized mainly in the ileum and cecum. A rabbit anti-E. coli (strain 23) serum (agglutinating titer: 1/2,500) afforded 100% protection when as little as 0.03 mg of serum protein per gram of rat body weight was orally administered in a single dose. The immune serum had an effect both on the mortality rate and on the growth of the rats. However, it never affected the survival of pathogenic bacteria in the rectum, even when administered at a daily dose of 1.5 mg of serum protein per gram of rat body weight on 4 consecutive days. The immune rabbit serum had only a weak bactericidal effect in vitro. The hemagglutination test showed the presence in the immune serum of antibodies against the fimbriae of the pathogenic E. coli strain (titer: 1/1,000). The role of antibody in inhibiting the adherence of bacteria to epithelial cells and/or their progression across the mucous layer are discussed as possible immune mechanisms in the intestinal lumen.     

The activation of the alternative complement pathway by fetal calf serum and mouse thymocytes.

Mouse thymocytes activated the alternative complement pathway of mouse serum in the presence of heated fetal calf serum. The activation required C3 from the fetal calf serum but was independent of antibody either in the murine or bovine serum. No other murine cells tested, including erythrocytes, peripheral blood lymphocytes, lymph node cells, spleen cells, and various cultured cell lines, activated the alternative complement pathway as effectively as thymocytes. In addition, sera from species other than cows could not substitute for fetal calf serum. The C3 deposited on thymocytes was in the form of both C3b (immune adherence positive) and C3bi (conglutinable). We propose that the basis of activation in this system is the specific protection of bovine C3b on mouse thymocyte surface.     

Isolation and identification of the third component of complement of Japanese quails

The identification of the third component of complement (C3) of Japanese quails was attempted by using rabbit antiserum prepared against quail serum-treated zymosan (ZX) as an initial reagent. This antiserum (anti-ZX) had agglutinating activity on rabbit erythrocytes reacted with quail antibody and quail complement (EACq) but not on EAq, and developed two precipitin lines against quail serum at beta- and gamma-regions in crossed immunoelectrophoresis. Subsequently, monospecific antisera to each of these precipitin lines were prepared in rabbits, and quail serum proteins reactive with these antisera were purified by salt precipitation followed by Sephadex gel filtration and DEAE cellulose column chromatography. One protein with a m.w. of 184,000 (184K) resembled mammalian C3 in that: 1) monospecific antiserum (anti-184K protein serum) agglutinated EACq but not EAq; 2) treatment of fresh quail serum with either inulin or zymosan resulted in the conversion of the precipitin line developed against 184K protein from gamma to beta in crossed immunoelectrophoresis; 3) the 184K protein was shown to consist of two polypeptide chains of 110K and 73K linked by disulfide bonds. Furthermore, the 184K protein in serum was cleaved through the incubation with inulin to 174K and 140K proteins that might correspond to C3b and C3c of human complement; 4) the 184K protein bound to zymosan was eluted with hydrazine or methylamine but not with Nonidet P-40, indicating that 184K protein binds to zymosan by a covalent bond but not by a hydrophobic one; and 5) by treatment of fresh quail serum with methylamine, complement reactivity was reduced, although its activity was restored by the addition of purified 184K protein. These results suggest the 184K protein is the quail's equivalent to mammalian C3. When quail serum was reacted with cells that had complement-activating capacity, quail C3 deposited on their membrane as in mammalians; however, no conversion of quail C3 was noted by the reaction with CVF. Antibody to quail C3 failed to cross-react with that in mammals.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Role of complement in immune inactivation of Mycoplasma gallisepticum

Studies were undertaken to evaluate the role of complement in the interaction between mycoplasmas and antiserum. A suspension of the A-1 strain of Mycoplasma gallisepticum in PPLO broth was incubated at 37 C with rabbit immune serum which had been heated for 30 min at 56 C. Samples were removed from the mixture at timed intervals for 1 hr for titration of the mycoplasmas in broth. When normal guinea pig serum was included in the mixture at a final dilution of 1:40, the titer fell rapidly from 10(6) to 10(2) organisms per 0.2 ml. When the guinea pig serum was heated for 30 min at 56 C or was omitted from the mixture, the immune serum did not reduce the titer. The rate of inactivation was related to the final concentration of antiserum and to the incubation temperature. The effect of the guinea pig serum was eliminated by the addition of 0.01 m sodium ethylenediaminetetraacetate or by prior absorption with an unrelated antigen-antibody complex. It was concluded that complement-like substances play an important role in immune inactivation of M. gallisepticum.     

STUDIES IN EXPERIMENTAL EOSINOPHILIA : VI. Uptake of Immune Complexes by Eosinophils

A method is described whereby immune complexes may be visualized in a single cell. Bovine serum albumin labeled with a red-fluorescing dye was joined to a rabbit antiserum labeled with a green-fluorescing dye to yield an immune complex which fluoresced yellow when illuminated by ultraviolet light. Such yellow-fluorescing immune complexes were injected into the peritoneal cavity of guinea pigs, and the peritoneal exudates were examined subsequently. Yellow fluorescent particles were seen in eosinophils obtained from guinea pigs sensitized to hemocyanin and from normal animals. Eosinophils of the blood and of the bone marrow could also take up the complexes in vitro. Neither antigen nor antibody alone was taken up by eosinophils, nor was a mixture of labeled antigen and labeled normal globulin. Similar observations were made with human blood eosinophils. These experiments suggest that eosinophils act as part of the defense against the pathogenic effects of certain immune complexes.     

Specific antibody promotes opsonization and PMN-mediated killing of phagocytosis-resistant Enterococcus faecium

Many clinical isolates of Enterococcus faecium are resistant to neutrophil (PMN)-mediated phagocytosis and killing in the presence of normal human serum. We have now examined the ability of specific polyclonal rabbit antibodies to promote opsonization and killing of phagocytosis-resistant E. faecium. Immune rabbit serum generated against formalin-killed E. faecium TX0016, a phagocytosis-resistant strain, markedly promoted binding of TX0016 organisms to PMNs and PMN-mediated killing. These effects were dramatically reduced by (a) adsorption of immune serum with E. faecium TX0016, but not by adsorption with a strain of E. faecium susceptible to phagocytosis, and (b) incubation of immune serum with carbohydrate purified from TX0016, but not by incubation with a surface protein extract from TX0016. IgG purified from immune serum was unable by itself to promote bacterial binding to PMNs. However, specific IgG was able to promote binding to PMNs and PMN-mediated killing in the presence of normal human serum as a complement source, as were F(ab')(2) and Fab fragments produced from it, and the alternative pathway of complement was sufficient to promote IgG- and F(ab')(2)-mediated opsonization. PMN complement receptor type 3, but not complement receptor type 1, was involved in bacterial binding to PMNs induced by the combination of F(ab')(2) fragments and normal human serum. These results suggest that opsonization by antibodies potentially directed against bacterial carbohydrate, in conjunction with complement activation, has an important role in the host defense against phagocytosis-resistant E. faecium.     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.