Isolation and biological activities of endotoxin from Leptospira interrogans.

Endotoxins extracted with ethylenediaminetetraacetate (EDTA) from Leptospira interrogans serovars icterohaemorrhagiae and canicola and Leptospira biflexa serovar patoc were tested for various biological activities characteristic of endotoxins. The presence of lipopolysaccharide biological activity was demonstrated by the Limulus amoebocyte lysate test, pyrogenicity in rabbits, complement interaction inhibiting the erythrocyte lysis, and chicken-embryo lethality. The lipopolysaccharides did not induce the local Shwartzman reaction. The lipopolysaccharides of serovars icterohaemorrhagiae and canicola were immunogenic in rabbits and were cytotoxic to chicken-embryo fibroblasts.     

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.     

Storage of pathogenic leptospires in liquid nitrogen

The virulence and viability of various serovars of Leptospira interrogans were successfully preserved by storage in liquid nitrogen. Dimethyl sulphoxide at a final concentration of 2.5% (v/v) was added as cryoprotectant to a culture of leptospires grown in Ellinghausen-McCullough-Johnson-Harris medium. Ampoules were cooled at a controlled rate of 1 degree-3 degrees C/min to -70 degrees C, then transferred to the liquid phase of a liquid nitrogen storage unit. Glycerol was discounted as a cryoprotectant as it was found to be approximately 10 times more toxic than dimethyl sulphoxide to four of five serovars used in this study. The viability of nine strains has so far been observed over a period of 8-22 months storage in liquid nitrogen and full viability of all strains has been preserved over this period. Virulence of strains of serovars pomona and hardjo was well preserved, as demonstrated by challenge tests in guinea pigs and domestic pigs.     

Storage of pathogenic leptospires in liquid nitrogen

The virulence and viability of various serovars of Leptospira interrogans were successfully preserved by storage in liquid nitrogen. Dimethyl sulphoxide at a final concentration of 2.5% (v/v) was added as cryoprotectant to a culture of leptospires grown in Ellinghausen-McCullough-Johnson-Harris medium. Ampoules were cooled at a controlled rate of 1°–3°C/min to −70°C, then transferred to the liquid phase of a liquid nitrogen storage unit. Glycerol was discounted as a cryoprotectant as it was found to be approximately 10 times more toxic than dimethyl sulphoxide to four of five serovars used in this study. The viability of nine strains has so far been observed over a period of 8–22 months storage in liquid nitrogen and full viability of all strains has been preserved over this period. Virulence of strains of serovars pomona and hardjo was well preserved, as demonstrated by challenge tests in guinea pigs and domestic pigs.     

Serologic survey for selected microbial pathogens in bison from Kansas.

A serologic survey was conducted on an American bison (Bison bison) herd in Kansas for antibodies against Brucella spp., Leptospira interrogans serovar canicola, pomona, grippotyphosa, icterohaemorrhagiae, and hardjo, Anaplasma spp., bluetongue virus, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. There was an increase in prevalence of bluetongue antibodies from 38% in 1987 to 100% in 1989 in animals greater than or equal to 24-mo-old. Prevalences of antibodies against the other livestock pathogens were either negative or at levels associated with previous vaccination.     

Enzyme patterns in the study of leptospira

An analysis of intracellular and extracellular leptospiral enzymes was made by use of starch-gel electrophoresis with natural and synthetic substrates. Of 37 serotypes examined for extracellular exterase, all had activity of varying mobility and degree. All extracellular preparations were negative for catalase, phosphatase, and naphthylamidase. Intracellularly, five serotypes were examined, including Leptospira biflexa Patoc I, L. biflexa Waz, L. canicola Moulton, L. icterohaemorrhagiae RGA, and L. pomona S91. Among the enzymes detected by this electrophoretic technique were transaminase and catalase, confirming the results of previous investigators. Further, other enzymes heretofore unreported have been detected. These include esterases, phosphatases, lactic, malic, glutamic, succinic, alpha-glycerophosphate, and 6-phosphogluconic dehydrogenases, and a naphthylamidase. The presence of these enzymes suggests the existence of tricarboxylic acid, glycolytic, and pentose-related pathways in Leptospira. In addition, enzyme patterns show promise in leptospiral classification.     

Genome conservation in isolates of Leptospira interrogans.

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.     

Populations of pathogenic Leptospira of varying virulence in nature

The study of geographically remote populations of Norway rats (Rattus norvegicus) revealed that in one of these populations a highly virulent population of Leptospira copenhageni, serogroup icterohaemorrhagiae, and in another population of rats a faintly virulent population of these microorganisms circulated simultaneously. At the same time in vitro experiments with Leptospira cultures showed the absence of the constant probability of sharp changes in the level of their virulence in time.     

Growth of Pathogenic Leptospira in Chemically Defined Media

A protein-free chemically defined medium for cultivation of pathogenic Leptospira was developed. The medium permitted continued serial subculturing of 9 serogroups (52 strains) of the 12 serogroups (61 strains) tested. Growth was initiated from small inocula, and the growth rate and maximal cell yields were similar to those on serum-containing media. The nutritional requirements of serogroups L. canicola, L. pomona, and L. grippotyphosa were studied in a basal medium composed of inorganic salts, a fatty acid, vitamin B(12), and thiamine. All strains tested utilized ammonium chloride as the sole nitrogen source. A fatty acid, vitamin B(12), and ferrous ions were essential. Growth was stimulated by thiamine, potassium, and calcium ions.     

Etiological structure of icterohemorrhagic leptospirosis in gray rats (Rattus norvegicus)

In 93 Leptospira strains isolated from Norwegian rats serovar determination was made. As a result, leptospires circulating among Norwegian rats were found to belong mainly to serovar copenhageni, group Icterohaemorrhagiae, while leptospires of serovar icterohaemorrhagiae, even if occurring, were found only in the animals inhabiting pigsties. Leptospirosis epizooty among rats, caused by L. icterohaemorrhagiae, took its course independently of leptospirosis epizooty among mice, caused by L. hebdomadis, and simultaneously with it.     

Serological Investigations of Leptospiral Deoxyribonucleases

Enzymoserological comparison of a selection of leptospira strains tested with sera from rabbits immunized with unpurified DNase of Leptospira interrogans, serotype canicola, indicates the production of DNase of serologically very similar properties by the serotypes canicola, autumnalis, icterohemorrhagiae and pomona. The DNase produced by serotype hyos was serologically different from the others, while the serotypes grippotyphosa and bataviae did not produce DNases at all. The method used made it possible to differentiate between leptospiral DNase and normally occurring DNases in the serum samples. Neither leptospira DNase nor specific leptospira-DNase-antibodies could be detected in dog sera with high agglutinationlysis titres after natural infection.     

The number of large ribosomal RNA genes in Leptospira interrogans and Leptospira biflexa

We determined the number of large ribosomal RNA genes in five strains of Leptospira by hybridization of 15 restriction endonuclease digests of genomic DNA to the [32P]-labeled fragment of 23s rRNA gene. Almost all the restriction gels gave two radioactive bands. The conclusion from these results is that there are at least two rRNA genes in these leptospiral strains. Furthermore, the hybridization patterns of L. icterohaemorrhagiae strains Ictero No. I and RGA are almost identical. The number of rRNA genes and taxonomic relationships of these leptospires were discussed.     

Influence of leptospirosis on reproductive performance of sows in Brazil

The purpose of this study was to investigate the association between seropositivity for the most frequent Leptospira serovars and reproductive losses in sows in Brazil. Serum samples from 351 sows from 18 herds (in the state of Rio de Janeiro, Brazil) with low reproductive efficiency were tested (microscopic agglutination) for antibodies against serovars of Leptospira. Antibodies were detected in serum samples of 66.1% of all sows, most frequently serovar icterohaemorrhagiae (43.1%), followed by pomona (18.1%) and tarassovi (9.9%). Seroreactivity to icterohaemorrhagiae and pomona were associated (P<0.05) with impaired reproductive performance (and substantial economic loss). Seroreactivity for pomona was associated (P<0.05) with stillborn piglets and mummified fetuses, whereas seroreactivity to icterohaemorrhagiae was associated (P<0.05) with the number of piglets born dead.     

Phylogenetic analysis of Leptospira strains of pathogenic serovars using 23S rDNA gene sequences.

The 23S ribosomal DNAs were amplified from 11 strains of Leptospira interrogans sensu lato by polymerase chain reaction (PCR) and sequenced. The PCR products of about 290-bp DNA fragments indicated more than 97% sequence similarity to each other. The phylogenetic tree based on the 23S ribosomal DNAs obtained in this study revealed that 11 strains of L. interrogans examined composed a cluster distinct to that of L. weilii and L. borgpetersenii, confirming that these strains were similar to strain Moulton of L. interrogans serovar canicola in 23S rDNA sequence.     

不同毒力钩端螺旋体引起THP-1细胞中细胞因子表达的研究

本文旨在通过观察不同毒力钩端螺旋体激活人单核巨噬细胞株THP-1中细胞因子信号途径的差异,探讨天然免疫在钩端螺旋体病发病机制中的作用.将3种不同毒力的钩端螺旋体--赖型有毒株Lai株、赖型减毒株IPAV株及非致病性Patoc型Patoc 1株与诱导后的细胞相互作用,在作用0、2、12、24、48 h 后收集标本,应用实...     

Lipid metabolic changes in experimentally induced leptospiral infection with serovars australis, canicola and icterohaemorrhagiae

Changes in lipid fractions were evaluated in young guinea pigs when infected with 1 ml of 7 day old live cultures of leptospira interrogans serovars australis, canicola and icterohaemorrhagiae. Statistically significant elevation in triglycerides, very low density lipoprotein and phospholipid and a significant reduction in high density lipoprotein (HDL) in all the groups was observed. Cholesterol and low density lipoprotein showed ascending trend in icterohaemorrhagiae group, whereas they were normal in other groups. The results suggest that increase in triglycerides, phospholipid and decrease in HDL in a suspected case of leptospirosis may be considered as markers.     

钩体凋亡相关基因的特征分析

应用生物信息学方法预测钩体与凋亡相关基因 ,并对其编码蛋白的结构特征进行深入分析。结果发现问号钩体黄疸出血型赖株 3对凋亡相关基因 ,其编码蛋白一级结构 ,保守区域和结构域同大肠埃希菌凋亡基因同源性很高。这提示凋亡相关基因存在于钩体中 ,可能在钩体流行和生态方面具有重要意义 ,深入研究可望发现新型抗菌靶基因。     

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 studies on Leptospira strain Ictero No. I using monoclonal antibodies.

In order to elucidate the full serological characteristics of strain Ictero No. I, the first strain of Leptospira isolated by Inada and Ido in 1914, 17 monoclonal antibodies against the strain were produced by cell fusion technology. The antibody-producing hybridomas were designated IMAs 1 to 17. The reactivities of the monoclonal antibodies produced by the hybridomas were determined by the microscopic agglutination test. One of the 17 monoclonal antibodies, IMA 1, reacted with strains Ictero No. I, LT 1131 and Naam, but not with other strains of the serogroup Icterohaemorrhagiae including strain RGA used in the present study. Moreover, the reactivity of the antigenic determinant of IMA 1 was inactivated by treatment at 56 C for 30 min. The 16 other monoclonal antibodies, IMAs 2 to 17, showed different reaction patterns against Leptospira strains of the serogroup Icterohaemorrhagiae. All of the 16 antibodies reacted with both Ictero No. I and RGA strains. The antigens against antibodies IMAs 2 to 17 were thermostable. The present study thus clarified the presence of a thermolabile antigen in strain Ictero No. I, and allowed correct distinction between the serotype of strain Ictero No. I and that of strain RGA using IMA 1. Therefore, we propose that strain Ictero No. I represents serovar icterohaemorrhagiae, as originally designated by Inada and Ido. Moreover, strain Ictero No. I should be designated as the type strain of Leptospira interrogans.     

An application of monoclonal antibodies to identification of leptospires of serogroup icterohaemorrhagiae found in China

For the purpose of improving the procedures of identification of leptospires, a set of 5 monoclonal antibodies with different serological reactivity against serovars of Leptospira interrogans Icterohaemorrhagiae serogroup isolated in China was developed. One hundred and eight strains isolated from epidemic fields in 5 provinces in southern China were distinctly identified into 4 serovars of Icterohaemorrhagiae serogroup by the monoclonal antibody procedure, i.e., 98 isolates were identified as serovar lai, 7 as icterohaemorrhagiae, 2 as copenhageni, and 1 as H2. Factor antiserum procedure was used at the same time as control for typing these strains and an identical result was obtained.