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      Characterization of leptospira borgpetersenii isolates from field rats (rattus norvegicus) by 16s rrna and lipl32 gene sequencing

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          Abstract

          The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 %, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.

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          16S ribosomal DNA amplification for phylogenetic study.

          D J R Lane, D A Pelletier, S M Barns … (1991)
          A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
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            The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection.

            D Haake, J Matsunaga, C Bolin … (2000)
            We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.
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              Differentiation of pathogenic and saprophytic letospires. I. Growth at low temperatures.

              Keven R. Johnson, Jamie Harris (1967)
              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.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Braz J Microbiol
                Journal ID (iso-abbrev): Braz. J. Microbiol
                Journal ID (publisher-id): bjm
                Journal ID (pmc): bjm
                Title: Brazilian Journal of Microbiology
                Publisher: Sociedade Brasileira de Microbiologia
                ISSN (Print): 1517-8382
                ISSN (Electronic): 1678-4405
                Publication date (Print): Jan-Mar 2010
                Publication date (Electronic): 1 March 2010
                Volume: 41
                Issue: 1
                Pages: 150-157
                Affiliations
                [1 ]Division of Medical Microbiology, Department of Microbiology, School of Life Sciences, Bharathidasan University ,Tiruchirappalli–620024,Tamilnadu, India
                [2 ]National Centre for Cell Sciences, Maharastra ,Pune–411 007., India
                [3 ]Regional Medical Research Centre, Indian Council of Medical Research, WHO Collaborative Centre for Diagnosis, Research, Reference and Training in Leptospirosis ,Port Blair–744 101, Andaman and Nicobar Islands, India
                [4 ]Tamilnadu Veterinary and Animal Sciences University , Chennai–600 007, India
                Author notes
                * Corresponding Author. Mailing address: Division of Medical Microbiology, Department of Microbiology, School of Life Sciences Bharathidasan University, Tiruchirappalli – 620 024, India.; Tel: +914312407082 Fax: +914312407045.; E-mail: natarajaseenivasan@ 123456rediffmail.com / natarajaseenivasan@ 123456gmail.com
                Article
                Publisher ID: S1517-838220100001000022
                DOI: 10.1590/S1517-838220100001000022
                PMC ID: 3768625
                PubMed ID: 24031475
                SO-VID: 044b7781-b310-4492-8bf6-1ca113470984
                Copyright © © Sociedade Brasileira de Microbiologia
                License:

                All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License

                History
                Date received : 26 December 2008
                Date revision received : 15 July 2009
                Date accepted : 21 August 2009
                Categories
                Subject: Veterinary Microbiology
                Subject: Research Paper

                Keywords: leptospirosis,leptospira borgpetersenii,lipl32,16s rrna
                Data availability:
                Keywords: leptospirosis, leptospira borgpetersenii, lipl32, 16s rrna

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