Leptospira icterohaemorrhagiae in New Zealand.
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The features of leptospiral infection should be sought in all cases of acute renal failure since management depends on the recognition of the clinical syndrome and serological confirmation is usually delayed. Hepatic and renal involvement is usual but renal failure without significant derangement of liver function is described.
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Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hydridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.
Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.
A serologic survey was made of the prevalence of common leptospiral infections in horses in California. A total of 465 serums were tested, using the microscopic agglutination method, against 5 leptospiral serotypes: Leptospira pomona, Leptospira icterohaemorrhagiae, Leptospira canicola, Leptospira grippotyphosa, and Leptospira hardjo. Of the serums tested, 127 (27.30%) were positive against 1 or more of the leptospires, with percentage distribution among the reactors as follows: L pomona, 12.47%; L icterohaemorrhagiae, 10.32%; L canicola, 3.22%; L grippotyphosa, 0.86%; and L hardjo, 0.43%. The prevalence of reactors to leptospiral antigens did not differ with the age or sex of the animals.
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A multiplex polymerase chain reaction (PCR) method using primer sets of G1/G2 and B64-I/B64-II was validated to detect pathogenic leptospira serovars from canine urine samples. The PCR method was found to be specific and sensitive with a detection limit of 100 cells of Leptospira icterohaemorrhagiae per milliliter of urine. The primer set previously designated and erroneously transcribed B64-I/B64-II amplified a DNA fragment of 352 base pairs from Leptospira grippotyphosa and Leptospira sejroe but not from Leptospira autumnalis, Leptospira bratislava, Leptospira canicola, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. From 100 diagnostic canine urine samples, 5 were found positive for Leptospira grippotyphosalsejroe with a PCR product of 352 base pairs and 6 were positive for other pathogenic leptospira serovars with a PCR product of 285 base pairs. One 285-base pair product was sequenced and found to be 99.3% homologous to the G1/G2 PCR fragment sequence reported previously. All 352-base pair PCR products of clinical samples and pure cultures of L. grippotyphosa and L. sejroe were sequenced. The 352-base pair fragment sequences of L. grippotyphosa and L sejroe were identical. Only 2 base pairs were found different between the sequences from pure cultures and those from clinical samples. Serum samples from 3 positive cases that generated a PCR product of 352 base pairs were tested by the microscopic agglutination test, and 2 were found to be positive for L. grippotyphosa (1:10,240 and 1:5,120), 1 was positive for L. grippotyphosa (1:320) or L. icterohaemorrhagiae (1:320). The results of this study suggest that the multiplex PCR with the primer set G1/G2 and the erroneously transcribed B64-I/B64-II may be able to differentiate L. grippotyphosa or L. sejroe from other pathogenic leptospira serovars commonly tested for in Canadian diagnostic laboratories.
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.
The challenge test for leptospira vaccines required by most licensing authorities is difficult to standardise and unreliable. One of the main contributory factors to this, is the difficulty in maintaining the virulence of the challenge strain. This paper describes work carried out to assess the practicality of storing challenge strains of Leptospira canicola and Leptospira icterohaemorrhagiae in liquid nitrogen. The effects of different concentrations of glycerol and dimethyl sulphoxide on the recovery of virulent and avirulent strains of Leptospira canicola and Leptospira icterohaemorrhagiae were investigated. Concentrations of cryopreservant above 5% increased the time taken for the leptospires to grow after recovery from vials stored in liquid nitrogen. In addition, the virulence of five challenge strains were shown to be little affected after 18 passages in vitro.
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Ciprofloxacin activity against Leptospira interrogans serogroup icterohaemorrhagiae was studied in vitro and in an animal model. The MBC of ciprofloxacin was 0.6 microgram/ml. Three of three Syrian hamsters died 8 to 9 days after intraperitoneal challenge with 10(6) leptospires. In contrast, five of six animals given ciprofloxacin 3 or 5 days after challenge survived.
The epidemiology and clinical features of 46 cases of human leptospirosis diagnosed in Israel between 1986 and 1999 were analysed. The median patient age was 37.5 years (range, 16-85 years), and the male/female ratio was 43/3. The most common serogroup found was Leptospira icterohaemorrhagiae. The disease was associated with jaundice in 71% of cases, acute renal failure in 62%, rhabdomyolysis in 52%, pancytopenia in 28%, respiratory failure in 14% and disseminated intravascular coagulation in 5%. Leptospirosis occurs sporadically throughout the year, peaking during the summer months. A shift occurred from predominantly agriculture-related serogroups in the 1970s to urban-related serogroups during the study period reported, with Leptospira icterohaemorrhagiae being the dominant serogroup.
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The capacity of leptospiras to get acclimated in the organism of unusual hosts has been studied. Cultures of Icterohaemorrhagiae from musquashes, house mice and field voles have been isolated of leptospirosis sources under intensive epizootics among rats. A possibility to reproduce Leptospira carrying by the cultures of leptospirae of serogroup icterohaemorrhagiae in musquashes and water voles has been shown. One can conclude that under icterohemorrhagic leptospirosis musquashes and water voles can take the part of additional source of infection but they cannot independently preserve leptospirae in nature as a biological species grey rats being their main hosts.
Leptospirosis is a severe zoonosis in the world. The methods for detecting leptospira are not sensitive and specific so far. The problems in early diagnosis and epidemiological identification of Leptospirosis remain unsolved. Two recombinant DNA fragments of serogroup Icterohaemorrhagiae, Leptospira were selected by repeated molecular cloning and screening in this study firstly. One of them can hybridize with the DNA of various serogroups of Leptospira interrogans; the other can only hybridize with the DNA of serogroup Icterohaemorrhagiae. After the nucleotides sequence analysis, from these 2 recombinant DNA fragments, 2 pairs of polymerase chain reaction (PCR) oligonucleotide primers were synthesized, named primer B 1, 2 and primer B 3, 4 PCRs were carried out with these 2 primers for detecting the microquantity (0.1 ng) of various serovars, serogroup of leptospires. All the DNA of Leptospira interrogans can be amplified by primer B 1, 2, and only the DNA of serogroup icterohaemorrhagiae, Leptospira reacted specially with primer B 3, 4. The DNA of non-pathogenetic Leptospira and some other microbes, however, had no amplification at all. This study is first reported at home and abroad. The results demonstrate that PCR is a very sensitive and specific technique of DNA amplification, which can be used as a powerful tool in the early diagnosis and epidemiological identification of leptospirosis.
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.