Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni

Leptospira interrogans serovar icterohaemorrhagiae strains Ictero No. I and RGA and serovar copenhageni strains M20, Shiromizu and Shibaura were examined by restriction endonuclease DNA analysis. Fifteen endonucleases (AluI, BamHI, BglII, EcoRI, HaeIII, HhaI, HindIII, KpnI, PstI, SacI, SalI, SmaI, StyI, XbaI and XhoI) were used as the digesting enzymes. Strain Ictero No. I showed endonuclease cleavage patterns which differed from those of the other four strains only when it was digested with enzymes KpnI and HindIII. When digested with KpnI, an extra band of about 5.4 kb was clearly produced, and when digested with HindIII, an extra band of about 25 kb was produced. When the other 13 enzymes were used, no differences were found between the endonuclease cleavage patterns among the five strains. Moreover, strains RGA, M20, Shiromizu and Shibaura could not be distinguished by the restriction endonuclease DNA analysis using all 15 endonucleases. In addition, six newly isolated leptospires from patients with leptospirosis and from Rattus norvegicus were compared with the Ictero No. I and M20 strains, by restriction endonuclease DNA analysis using enzymes KpnI and HindIII. Three leptospires belonging to serovar icterohaemorrhagiae showed the same endonuclease cleavage patterns as the M20 strain. The other three strains, which belong to serovar copenhageni, showed almost the same endonuclease cleavage patterns as the M20 strain; only the Kai ima 702 strain produced an extra band which was not identical to the Ictero No. I-specific extra band when digested with HindIII. The leptospiral restriction endonuclease DNA analysis has revealed taxonomic structures that are unrecognized by serology alone.     

ELISA for the detection of specific IgM and IgG in human leptospirosis

ELISA was used to detect specific IgM and IgG in sera from humans with current or past leptospirosis. A serological pattern of a high IgM titre (greater than or equal to 1280), or moderately increased IgM (160-640) in conjunction with a low IgG titre (less than or equal to 20), with serovar copenhageni antigen was characteristic for approximately two-thirds of the sera from serovar icterohaemorrhagiae patients obtained in the first two months of the disease. The antigen was the supernatant of a heated and centrifuged culture of leptospires. Antigens were prepared from serovars copenhageni, grippotyphosa, hardjo and patoc. Sera from patients with icterohaemorrhagiae, grippotyphosa and hardjo infections showed cross-reactivity when different antigens were used. In past infections the IgG titres were clearly higher with the homologous antigen. ELISA for IgM and IgG allows the rapid diagnosis of acute leptospirosis.     

A new methodological approach for leptospira persistence studies in case of mixed leptospirosis

A new methodical approach for Leptospira persistence studies in case of mixed leptospirosis, based on the use of PCR test systems with different taxonomic specificity for the indication and identification of leptospires, was developed. Two PCR test systems (G and B) were used in experiments on BALB/c white mice to study patterns of the development of mixed infection caused by leptospires of serovar poi (genomospecies L. borgpeterseni) and grippotyphosa (genomospecies L. kirschneri). The conclusion was made of good prospects of this method application in studies on symbiotic relationships of leptospires both in vivo and in vitro.     

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.     

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.     

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.     

Characteristics of the interaction of Leptospira with the host organism in the infectious process in golden hamsters

The comparative evaluation of the interaction of L. icterohaemorrhagiae strain P, L. canicola strain CL and L. hebdomadis strain 650 with golden hamster liver and kidney cells is presented. Three variants of the course of Leptospira infection have been distinguished: (1) the hepato-renal (icteric) variant, caused by the adhesion of leptospires to liver cells with the colonization of their surface and the disaggregation of liver-cell complexes and by the accumulation of leptospires in the kidney interstice; as a consequence, parenchymatous hepatitis and nephroso-nephritis develop, which lead to the death of animals; (2) the renal (anicteric) variant, characterized by the absence of the infective agent and lesions in the liver, by adhesion of leptospires to and their colonization of the nephrothelium of the proximal convoluted tubules of the kidneys; in this case some of the animals die because of renal insufficiency and shock, while in the surviving animals prolonged carrier state develops; (3) the intermediate variant, characterized by the initial process of leptospiral adhesion and colonization in the liver and its subsequent progress in the kidneys.     

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。     

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.     

Role of serodiagnosis in recognizing atypical forms of leptospirosis in Krasnodar Territory

The work deals with the epidemiological situation with respect to Leptospira infections in the Krasnodar territory. The work demonstrates that, in comparison with 1960-ies characterized by the prevalence of diseases caused by L. pomona and L. grippotyphosa, in 1970-ies the increase of the specific proportion of infections caused by L. icterohaemorrhagiae was registered. In recent 5 years (1980-1984) this leptospirosis constituted 73.5-94.8% of the total morbidity rate in the territory. Such situation was caused by active prophylactic measures in cattle breeding, as well as by the increase of the number of leptospirosis foci, appearing as the result of human activities and providing favorable conditions for the life and multiplication of Norway rats, the main source of infection caused by L. icterohaemorrhagiae as indicated by the fact that up to 23.9% of the Norway rats examined in this study proved to be contaminated. The detection of cases of leptospirosis among febrile patients and mistakes in clinical diagnosis confirm the necessity of the serological examination of febrile patients with the acute onset of the disease with temperature reaching 38 degrees C and higher for 3-5 days and the clinical picture of the disease being unclear in order to find out patients presenting with the atypical clinical picture of leptospirosis.     

The impact of Leptospira seropositivity on reproductive performance in sows in southern Viet Nam

A serologic survey was conducted among sows in the Mekong delta in southern Viet Nam to investigate associations between leptospiral seropositivity and reproductive performance. Data were collected from a total of 339 sows in lactation or gestation, from four large-scale state farms on three occasions. The seroprevalence for Leptospira interrogans serovar (sv) autumnalis was 32%, for L. interrogans sv bratislava 29%, for L. kirschneri sv grippotyphosa 13%, for L. interrogans sv icterohaemorrhagiae 27%, for L. interrogans sv pomona 5%, and for L. borgpetersenii sv tarassovi 13%. The reproductive parameters number of days from weaning to service (WSI), number of piglets born, number of piglets born dead, and number of piglets born weak, were evaluated. Seropositivity for sv tarassovi was associated with 0.8 more dead piglets per litter (P = 0.06), and sv grippotyphosa with a 1 day longer WSI (P = 0.06). There were no significant associations between reproductive performance and sv autumnalis, sv bratislava, sv pomona, and sv icterohaemorrhagiae. It is concluded that seropositivity for Leptospira can be associated with impaired reproductive performance even in areas where a high degree of immunity among sows is expected.     

Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae

We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.     

Analysis of differential proteomes in pathogenic and non‐pathogenic Leptospira: Potential pathogenic and virulence factors

Leptospirosis is a bacterial zoonotic disease caused by spirochetes in the genus Leptospira. To date, factors determining the pathogenicity and virulence of leptospires remain unclear. We performed a gel‐based proteomic analysis to evaluate differential leptospiral proteomes in the pathogenic L. interrogans (serovars Australis, Bratislava, Autumnalis, and Icterohaemorrhagiae) and the non‐pathogenic L. biflexa (serovar Patoc). Quantitative proteome analysis and MS protein identification revealed 42 forms of 33 unique proteins whose levels were significantly greater in the pathogenic serovars compared with the non‐pathogenic serovar. Among the four pathogenic serovars, the more virulent serovar Icterohaemorrhagiae (which is most commonly associated with severe leptospirosis in patients) had significantly greater levels of 14 forms of 12 unique proteins, when compared with the other three pathogenic serovars. Some of these identified proteins may serve as the pathogenic and/or virulence factors of leptospirosis.     

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.     

Repetitive sequence of Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1: a sensitive probe for demonstration of Leptospira interrogans strains.

A 4.8-kilobase (kb) repetitive sequence element generated with KpnI digestion was cloned from the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No. 1. The sequence, repeated in tandem, was located on the 280-kb fragment between the FseI and AscI sites on the chromosome by hybridization using the 4.8-kb fragment as a probe. We cloned the fragment containing the element for the Ictero No. 1 strain in a lambda EMBL3 bacteriophage DNA, and one out of 5 clones was sequenced. Within the sequenced 9-kb segment that partially repeated, 9 putative open-reading frames and 2 transfer RNA genes, for alanine and isoleucine, were identified. A similarity search for the products deduced from the sequenced data revealed that the repeated sequence includes both beta-oxidation enzymes, acyl-CoA dehydrogenase and enoyl-CoA hydratase, and hydroxythiazole kinase protein homologues. Hybridization experiments against different leptospiral strains using the element as a probe showed a similar sequence in the strains of L. interrogans and L. kirschneri, but not in any strains of L. borgpetersenii, L. weillii, L. meyeri or L. biflexa. Results indicated that the highly repeated element in the Ictero No. 1 strain exists as a well conserved sequence, though at a moderate level of repetition, in certain strains of L. interrogans and L. kirschneri. PCR amplification targeting the repetitive element was successful and indicated that the procedure provides a sensitive and specific probe to detect leptospires.     

Serovar Diversity of Pathogenic Leptospira Circulating in the French West Indies

Background

Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012.

Methods and Findings

Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles.

Conclusions

The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.     

Serological analysis of serogroup icterohaemorrhagiae using monoclonal antibodies

Eighteen serovars (19 strains) of serogroup Icterohaemorrhagiae were serologically analyzed using 18 monoclonal antibodies against serovar copenhageni Shiromizu, M20 and serovar icterohaemorrhagiae RGA strains. The reaction patterns of the serovars against these monoclonal antibodies were different. According to these results, we divided the serovars, except for serovar tonkini, into the following three subgroups: Subgroup 1 reacted to many monoclonal antibodies including serovars icterohaemorrhagiae, copenhageni, hualien, monymusk, mankarso, and budapest. Subgroup 2 fell between subgroups 1 and 3 including serovars dakota, naam, bogvere, birkini, smithi, ndambari, gem, ndahambukuje and mwogolo. Subgroup 3 reacted to only a few monoclonal antibodies: serovars weaveri and sarmin. Serovar tonkini did not react to any of the monoclonal antibodies used. There is a possibility that serovar tonkini does not belong to serogroup Icterohaemorrhagiae. Further studies on the serological reactions of each strain revealed that it was impossible to distinguish the RGA strain from the serovar hualien LT11-31 strain, indicating that they may be identical. It was also observed that serovar copenhageni and monymusk seemed to be closely related. Serovars birkini and smithi, and serovars ndambari and gem were alike in their serological reactivities. Among the 18 monoclonal antibodies, RGAMA-1 was a unique antibody which reacted only to serovar icterohaemorrhagiae and serovar hualien, indicating that it must be the serovar icterohaemorrhagiae specific antibody. On the other hand, SHIRMA-2, 5, 6 reacted to all the serovars except for serovars weaveri, sarmin, and tonkini. These antibodies exhibited a broad reaction spectrum.     

The use of methanol extract of Leptospira interrogans in complement fixation tests for leptospirosis

Methanol extracts were obtained from L. interrogans serovars icterohaemorrhagiae and canicola and L. biflexa serovar patoc. Human sera from 167 normal individuals and 40 patients with different infectious diseases tested by complement fixation tests showed negative reactions. Sera from 100 patients with a suspicion of leptospirosis were tested by complement fixation tests and microscopic agglutination reactions. Agreement of 84% was found for those two reactions. Positive microscopic agglutination tests at a dilution 1:20-1:400 with negative complement fixation tests were observed in 5% of patients and negative microscopic agglutination with complement fixation tests in the range of 1:20-1:1280 were observed in 11% of the cases.     

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.