Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Abstract Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona , were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona . In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Immunochemical studies of opsonic epitopes of the lipopolysaccharide of Leptospira interrogans serovar hardjo

Abstract Leptospiral lipopolysaccharides (LPS) are the main antigens responsible for immunity in leptospirosis. In this investigation we studied the nature of the antigenic determinants of LPS extracted from Leptospira interrogans serovar hardjo (reference strain Hardjoprajitno). The reactions of anti-LPS monoclonal antibodies (mAbs) MUM/F1-4/ hardjo (IgM) and MUM/F1-6/ hardjo (IgG) with whole cell lysates in Western immunoblotting analysis were unaffected by proteinase K treatment. Periodate treatment of the LPS destroyed the binding of MUM/F1-6/ hardjo but preserved that of MUM/F1-4/ hardjo . Alkaline phosphatase decreased significantly the binding of MUM/F1-4/ hardjo to the LPS but only slightly that of MUM/F1-6/ hardjo . On the other hand, phosphodiesterase totally destroyed the binding capacity of both monoclonal antibodies in enzyme immunoassays (EIA). A number of mono- and oligosaccharides was used in EIA inhibition studies. Mannose-6-phosphate and galactose-6-phosphate inhibited the binding of MUM/F1-4/ hardjo (50% inhibition at a concentration of 5 mM) to the antigen, but glucose-6-phosphate did not. Galactosamine and mannosamine inhibited the binding of MUM/F1-6/ hardjo (50% inhibition at a concentration of 3–4 mM), whereas only a weak inhibition was observed with glucosamine. In contrast, N -acetylated amino sugars did not show any inhibition. An O -acetyl group also appears to be involved in the antigen-antibody binding process.     

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.     

Ultrastructure and chemical composition of lipopolysaccharide extracted from Leptospira interrogans serovar copenhageni

Lipopolysaccharide (LPS) from Leptospira interrogans serovar copenhageni was prepared from the aqueous phase of a phenol/water extract. Electron microscopic examination of negatively stained LPS showed a mixture of ribbon-like, round and ring structures. Carbohydrate analysis of the preparations revealed pentoses, hexoses, heptoses, hexosamines, and a 2-keto-3-deoxyonic acid which was chromatographically different from authentic 2-keto-3-deoxyoctonic acid (KDO). The major fatty acids of the LPS were hydroxylauric, palmitic and oleic acids. Although the leptospiral LPS preparations did not contain KDO or hydroxymyristic acid, they were otherwise morphologically and chemically similar to the LPS of other Gram-negative bacteria.     

The immunofluorescent detection of Leptospira interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae infections in human tissues

The identification of Leptospira interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae infections in post-mortem tissues by direct fluorescence microscopy is demonstrated by the use of a serogroup-specific fluorescein isothiocyanate label.     

Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor.

Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.     

Experimental infection of calves and sheep with Leptospira interrogans serovar balcanica

Two of four calves inoculated with Leptospira interrogans serovar balcanica developed low microscopic agglutinating (MA) titres to serovar hardjo. A third calf had an MA titre of 1:1024 by day 19 post-inoculation (PI). Transient leptospiruria was recorded in one calf on days 12 and 13 PI. An in-contact calf did not seroconvert. None of the calves had fever or other clinical signs of disease. Four ewes inoculated with balcanica developed MA titres to hardjo by day 13 PI, and a transient leptospiruria between days 14 and 25 PI. None of the ewes showed any evidence of clinical disease and three of them delivered healthy lambs 22 to 64 days PI. One ewe had mild lesions of focal interstitial nephritis.     

Surface proteomics and label-free quantification of Leptospira interrogans serovar Pomona

Leptospirosis is a re-emerging zoonosis with a global distribution. Surface-exposed outer membrane proteins (SE-OMPs) are crucial for bacterial–host interactions. SE-OMPs locate and expose their epitope on cell surface where is easily accessed by host molecules. This study aimed to screen for surface-exposed proteins and their abundance profile of pathogenic Leptospira interrogans serovar Pomona. Two complementary approaches, surface biotinylation and surface proteolytic shaving, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) were employed to identify SE-OMPs of intact leptospires. For quantitative comparison, in-depth label-free analysis of SE-OMPs obtained from each method was performed using MaxQuant. The total number of proteins identified was 1,001 and 238 for surface biotinylation and proteinase K shaving, respectively. Among these, 39 were previously known SE-OMPs and 68 were predicted to be localized on the leptospiral surface. Based on MaxQuant analysis for relative quantification, six known SE-OMPs including EF- Tu, LipL21, LipL41, LipL46, Loa22, and OmpL36, and one predicted SE-OMPs, LipL71 were found in the 20 most abundant proteins, in which LipL41 was the highest abundant SE-OMP. Moreover, uncharacterized LIC14011 protein (LIP3228 ortholog in serovar Pomona) was identified as a novel predicted surface βb-OMP. High-abundance leptospiral SE-OMPs identified in this study may play roles in virulence and infection and are potential targets for development of vaccine or diagnostic tests for leptospirosis.     

Effects of intrauterine challenge with Leptospira interrogans serovar hardjo on fertility in cattle

The purpose of this study was to determine the effects of Leptospira interrogans serovar hardio on fertility in cattle. Twenty seronegative mature dairy cows were assigned to two groups. Group I (challenged cows) was bred by a seronegative bull followed by intrauterine infusion (within 30 minutes) of Leptospira interrogans serovar hardjo . Group II was bred by the same bull followed by intrauterine infusion of 5 ml of sterile culture medium. Blood samples were collected at two-day intervals to monitor serum antibody titers. Daily blood cultures for 10 days and weekly urine cultures for five weeks were performed to monitor the animals for leptospiremia and leptospiuria. Cows were slaughtered 35 days post-breeding, and their reproductive tracts were examined. All animals remained clinically normal following intrauterine challenge. There was no difference in pregnancy rates (Group I, 7 10 ; Group II, 6 10 ). All embryos, reproductive tracts, and kidneys appeared normal. A microscopic agglutination test (MA) showed that 4 of 10 challenged cows developed serum antibody titers between 8 and 20 days after challenge. However, on the basis of the hamster passive protection test, all challenged cows had serum antibodies present. All blood and urine cultures were negative through the experimental period, as were the final kidney and uterine cultures. In a second experiment, six seronegative cows were infused with killed microorganisms immediately after insemination. Results of a microscopic agglutination test and a hamster passive protection test indicated that these cows did not develop humoral antibodies against serovar hardjo . These results indicated that intrauterine inoculation of Leptospira interrogans serovar hardjo (hamster-adapted strain) following breeding did not affect pregnancy rates despite an intrauterine challenge which caused the development of humoral antibodies.     

Serological and chemical interrelationship of antigens from Leptospira interrogans serovar canicola

Antigens of the outer envelope from Leptospira interrogans serovar canicola (Hond Utrecht IV) were extracted by 50% (v/v) ethanol or by sodium dodecyl sulphate and serological analysis suggested that they were identical. The "fraction 4" extracted by alkali was found to contain glycoproteins of high (retentate) and low (filtrate) molecular weight; the latter behaved like a hapten in serology and in animal immunization experiments. Antibodies were raised in rabbits against this hapten by conjugating it to bovine albumin fraction V. The antiserum was found to react with both the low molecular weight and high molecular weight glycoproteins. This anti-hapten serum contained little or no whole-cell-agglutinating antibodies. The fraction 4 retentate behaved like a complete antigen in serological and immunization studies. Fraction 4 retentate and the outer envelope preparations were serologically related but they were not identical. Chemical studies revealed similarities between the carbohydrate component of the outer envelope obtained by ethanol extraction and fraction 4. The outer envelope extracted by ethanol, fraction 4 and its low and high molecular weight glycoproteins contained arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose, glucosamine and glucuronic acid. Three unidentified peaks were observed in gas-liquid chromatographic analysis of the O-trimethylsilyl derivatives of methyl glycosides of all these samples and one of these peaks co-eluted with the O-trimethylsilyl derivative of 3-O-methylmannose.     

Characterization and taxonomic significance of lipopolysaccharides of Leptospira interrogans serovar hardjo

Lipopolysaccharides (LPSs) from Leptospira interrogans serovar hardjo (reference strain hardjoprajitno and strain hardjobovis) were prepared by the hot phenol-water procedure. High yields of LPSs were found in the phenol phase. Gel electrophoresis of the phenol phase LPSs showed similar patterns for all strains in contrast to the different patterns found in the water phase LPSs. Sugar composition was also similar among all strains with rhamnose as the predominant sugar. Mannosamine was detected by high performance thin layer and gas-liquid chromatography. 2-Keto-3-deoxyoctonic acid (KDO) was comparable with authentic KDO by paper chromatography. Periodate oxidation at near neutral pH with or without prior hydrolysis showed that most of the KDO was substituted. The fatty acid composition of strain hardjobovis LPS was slightly different from that of the reference strain hardjoprajitno. Myristic and 3-hydroxymyristic acid were not detected in any of the LPS preparations. In conjunction with genetic and other data, the two strains are sufficiently different to be regarded as members of two separate species sharing common antigens. There is sufficient evidence to rename the hardjoprajitno strain type L. interrogans hardjo-p, and the hardjobovis strain type L. borgpeterseni hardjo-b.     

Monoclonal antibodies detecting different epitopes on the Forssman glycolipid hapten

Two hybridomas, derived by fusing mouse myeloma cells with spleen cells from a rat immunized with mouse mammary tumors, have been shown to produce antibodies that recognize cell surface antigens on mesenchymal cells in a variety of tissues. Evidence presented in this report suggests that these antibodies detect overlapping epitopes on the Forssman glycolipid hapten (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). One antibody (33B12) reacts with the terminal sugar sequence GalNAc alpha 1-3GalNAc and is specific for Forssman. The other antibody (117C9) recognizes the internal sugar sequence GalNAc beta 1-3Gal. The terminal sugar sequence GalNAc beta 1-3Gal in globoside, as well as the internal sugar sequence GalNAc beta 1-4Gal in asialo-GM1, is not recognized as an antigenic determinant by 117C9. Nevertheless, the 117C9 antibody does not react exclusively with the Forssman antigen. In a lipid extract fractionated by Folch partition of mouse mammary tumors, the antibody also detects other glycolipids.     

Monoclonal antibodies to distinctive epitopes on the alpha and beta subunits of the fibronectin receptor

Monoclonal antibodies (MAbs) have been developed that can recognize epitopes that are unique to either the alpha or beta subunit of the fibronectin receptor (FnR). MAbs 11B4 and 7A8 immunoblot the alpha subunit of FnR either in purified form from Chinese hamster ovary (CHO) cells or in nonionic detergent extracts of cells of human and rodent origin electrophoresed under reducing or nonreducing conditions. The MAbs seem to be more reactive to the subunit when it has been electrophoresed under reducing conditions, suggesting that the epitope may be partially masked by the conformation conferred by disulfide bonding. A second set of MAbs, 7E2 and 7F9, is directed to an epitope on the beta subunit that is conformationally dependent upon disulfide bonding, as reduction of the subunit leads to loss of reactivity with both MAbs. Further, 7E2/7F9 immunoblots of nonionic detergent extracts of CHO cells, run under nonreducing conditions, reveal the presence of a third band (90-kDa), immunologically related to the beta subunit, which is not surface-labeled with 125I in intact cells and which does not copurify with the alpha and beta subunits isolated by immunoaffinity purification of FnR using the MAb PB1. The 90-kDa component is not found associated with a plasma membrane fraction prepared by crude cell fractionation, but is abundant in a low-speed pellet containing nuclei and intracellular membranes. This finding suggests that the 90-kDa component is a precursor to the beta subunit. Finally, the epitope of 7E2/7F9 is unique to CHO cells, as cross-reactivity to other cell types cannot be demonstrated by either immunoblotting or immunoprecipitation.     

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura.

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura were analyzed by thin-layer chromatography, gas-liquid chromatography, gas-mass spectrometry and infrared absorption spectrometry. The virulent cells possessed a characteristic lipid pattern consisting of free fatty acid (FFA) (41.8%), one major unidentified phospholipid (14.8%), phosphatidylethanolamine (PE) (12.9%), cholesteryl ester (CE) (9.3%), lysophosphatidylethanolamine (LPE) (4.9%) and diphosphatidyl-glycerol (DPG) (1.1%). Various fatty acids such as hexadecanoic (26.9%), hexadecenoic (15.4%), octadecenoic (26.5%) and octadecadienoic (27.4%) acids were detected in the FFA. The fatty acid composition of the major unidentified phospholipid distinctly differed from those of other lipids including PE, LPE, DPG and CE, and comprised mainly tetradecadienoic (53.6%), tetradecatrienoic (14.0%) and octadecanoic (13.8%) acids. This phospholipid with a large amount of polyunsaturated fatty acids with chain lengths of 14 carbon atoms was detected only in the lipids of the virulent cells.     

In vivo apoptosis of hepatocytes in guinea pigs infected with Leptospira interrogans serovar icterohaemorrhagiae

To investigate the contribution of the previously demonstrated in vitro apoptosis to the pathogenesis of leptospirosis, guinea pigs were infected with Leptospira interrogans serovar icterohaemorrhagiae strain Verdun and sequentially killed to collect target organs involved in the natural history of the disease (liver, kidneys, lungs, spleen and heart). The combination of histopathological procedures and a specific TUNEL assay showed a significant Leptospira-induced programmed cell death of hepatocytes with a peak at 48 h post inoculation. Hepatocyte nuclei showed morphological changes including fragmented and condensed nuclei. This phenomenon occurred early in the course of the disease at a time where infecting leptospires were present at a low density between the liver parenchyma cells.     

Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai

It has been suggested that leptospiral hemolysins are important in the virulence and pathogenesis of leptospirosis. We have isolated an Escherichia coli clone carrying the 7.8kb DNA insert from a genomic library of Leptospira interrogans serovar lai by plaque hybridization using a sequence derived from the sphingomyelinase C gene (sphA) of L. borgpetersenii. The clone showed a clear beta-hemolytic zone on sheep blood agar and high hemolytic activities on both human and sheep erythrocytes in liquid assays. The clone carried at least two genes responsible for the hemolytic activities, encoded by two open reading frames of 1662 and 816 nucleotides, which are named sphH and hap-1 (hemolysis associated protein-1), respectively. The SphH showed 75% homology to the SphA at the amino acid level, and the Hap-1 showed no significant homology in major databases. Interestingly, however, E. coli cells harboring sphH did not show sphingomyelinase or phospholipase activities. Moreover, SphH-mediated hemolysis was osmotically protected by polyethylene glycol 5000, suggesting that the hemolysis is likely to be caused by pore formation on the membrane. The SphH was successfully expressed in E. coli as a histidine (His)-SphH fusion protein. Both sphH and hap-1 were highly conserved among the Leptospira species, except for the absence of sphH in non-pathogenic L. biflexa serovar patoc. We concluded that the SphH is a novel hemolysin of a pathogenic Leptospira species, which may be a putative pore-forming protein.     

Isolation of an antigenic oligosaccharide fraction from Leptospira interrogans serovar canicola with a monoclonal antibody

An oligosaccharide fraction containing the antigenic determinant of lipopolysaccharide antigen (TM antigen) from Leptospira interrogans serovar canicola, recognized by a monoclonal antibody (CT3) which agglutinates serovars canicola and broomi, was isolated by formic acid and successive sulphuric acid hydrolyses. Separation of the antigenic compounds was done by Bio-Gel P-2 and Sephadex G-25 gel filtration, and high-performance liquid chromatography with two different columns. The fraction finally obtained was a mixture of two oligosaccharides, both of which migrated as a single spot having a slightly higher mobility than an authentic tetrasaccharide (stachyose) on thin layer chromatography. The fraction contained rhamnose, arabinose and two major and two minor unknown sugars which were shown to be N- or O-acetylated and/or O-methylated sugars by nuclear magnetic resonance. The fraction inhibited the binding of CT3 antibody with TM antigen in enzyme-linked immunosorbent assay and microscopic agglutination of serovar canicola with the antibody. The inhibitory activity was destroyed by periodate oxidation or mild alkaline treatment, but was resistant to sodium borohydride reduction.     

Serological evidence of the persistence of infection with Leptospira interrogans serovar Hardjo in cattle in Mongolia

Serum samples were collected randomly from Mongol breed cattle in three geographically distinct provinces of Mongolia. Antibodies specific to Leptospira interrogans serovar Hardjo were detected by microscopic agglutination test (MAT) at titres of 100 or more in 80.4% (86/107) of the samples from Dornod Province, but in only 28.9% (13/45) in Arkhangai and 23.5% (12/51) in Khuvsgul Provinces, respectively. Treatment of 9 serum samples from Dornod, positive to serovar Hardjo in MAT and negative in the homologous immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), with 2-mercaptoethanol (2-ME) indicated that those animals were recently infected.