Leptospira azadi

Taxonomy of leptospira
Domain:Bacteria
Phylum:Spirochaetes
Class:Spirochaetes
Order:Spirochaetales
Family:Leptospiraceae
Genus:Leptospira

THE GENUS LEPTOSPIRA
• The genus Leptospira comprises morphologically similar
thin helical bacteria (spirochetes)
• Trivial name used for all members of the genus is
leptospira or leptospire
• Similar morphologically and culturally, but can be grouped
serologically by agglutinating antigens into characteristic
serovars. 24 serogroups and 250 serovars

THE GENUS LEPTOSPIRA
• 'Leptospira interrogans ‘ major serological complex
suspected to be pathogenic
• 'biflexa complex'other major group, containing
nonpathogenic leptospire
• The genera Leptonema and Turneria were first recognized
because the type strains were morphologically different
from other leptospires

Definitions
o genera Leptospira, Leptonema, and Turneria
o motile, flexible, helical aerobic bacteria, 6-12 pm long and 0.1
pm in diameter
o gram-negative but difficult to visualize, and oxidase-positive and
chemoorganotrophicThe diamino acid in the peptidoglycan α -
diaminopimelic acid and (G + C) ratio is 35-53 mol%.
o one or both ends are hooked, a pair of periplasmic flagella
arising from it subterminally,
o Long chain fatty acids or fatty acid alcohols are used as carbon
and energy source

• Phylogenetic analyses of 16S rRNA genes suggest that Leptospira
species cluster into three groups designated pathogenic,
saprophytic and intermediate
• Pathogenic group in include: alexanderi, borgpetersenii, fainei,
inadai, interuogans, kirschneri, meyeri, noguchii, santarosai, and
weilii
• Saprophytic('nonpathogenic): biflexa, hollandia, and wolbachla

Genetic classification and species
• A species comprises those leptospires whose DNA is 70%
related and whose related DNA sequences contain 5 % or fewer
unpaired bases (divergence)
• There are 12 named species with further groups awaiting
classification
• Analyzed by sequensing and other genetic methods

HISTORICAL PERSPECTIVE
• first described by Adolf Weil in 1886, it has been around for 100 of
years with icteric form of the disease being reported in ancient
China, India, and Europe.
• the first credited account of a leptospire being isolated was in 1916
by Inada et al
• Rats as a reservoir host were identified in Japan in early 1900s, and
basic pathology as well as epidemiology was derived by 1940
• Outbreak being reported with, kayaking, windsurfing, swimming,,
wading through puddles, white-water rafting, and other outdoor
sports played in contaminated water

• Leptospirosis is a potentially fatal disease of humans or
animals caused by one of the pathogenic leptospires. It is
prevalent globally as an acute febrile,
• Sometimes congenital or chronic infection of wild or
domesticated animal
• Leptospires persist in the renal tubules of kidneys in carrier
animals, whence they are excreted in urine into the
environment to contaminate water and soil

• Humans are infected incidentally, affected by an acute
febrile, Sometimes the disease is subclinical. And do not
usually become chronic carriers or transmit infection
• Adult males are at more risk of leptospirosis due to higher
occupational and recreational exposure

HABITAT
• Natural habitat is surface waters, soil, and mud.
• They feed by attachment to surfaces where long-chain fatty
acids are available
• Halophilic leptospire has been isolated from estuarine
waters
• lyophilization can preserve Leptospires

Way of spread
• In the host they circulate and grow in an
aqueous milieu and, passing in urine into
surface waters
• leptospires adapt to environmental conditions
including the salinity and temperatures
• Leptospires infect humans only by accident of
contact with a contaminated environment
• spread from humans to other humans is
almost unknown

MORPHOLOGAY AN D VISUALIZATION
• helical gram-negative bacteria hard to see when
stained by conventional bacteriological stains
• Strong stains (carbol fuchsin) and stains that build up
on the surface (flagellar stains, Giemsa, silver stains,
immunostains) make them appear thicker and
improve visualization
• dark field microscopy The routine method of
observation.
• Leptospires pass through bacteriological filters of
average pore diameter 0.2 pm.

Shape and structure
• about 10-20 μm long
• peptidoglycan complex loosely referred
to as cell wall arranged as a flexible
hollow tubular right-handed helix
• amplitude of the coil is about 0.10-0.15
μm and its wavelength is about 0.5 μm
• Leptonema is generally longer (13-15
pm) and Turrneria is shorter (3.5-7.5 pm
long) and more tightly coiled

SPHEROPLASTS
• In some cultures leptospires appear in a
'granular' form. The granules are small cystic
structures, about 1.5- 2.0 mm in diameter
• Once thought to be a cystic stage of a life
cycle, they are really spheroplasts whose
formation can be induced with ethanol,
detergents, sodium deoxycholate, high salt
conditions, lysozyme or mild heat And also
be observed in tissues and in phagocytes

Flagella
• Leptospiral flagella originate in a disc rotor and
hooked proximal end structure in the cell wall,
similar to other gram-negative bacteria.
• with differential or gradient centrifugation can see
the leptospiral Flagella are seen as tight flat coils
• The isolated flagellum is composed of a helically
wound central core of proteins, surrounded by an
outer sheath

• Seven different flagellar proteins have been
recognized
• Theres a 34 kDa protein associated with
the core, of 11.3 nm, and a 36 kDa protein
associated with the sheath, measuring 27.5
nm
• 32 kDa FlaB protein leptospira similar in
sequence to FlaB proteins described in
Treponema pallidum
• The FlaB protein structure and its gene
sequences are highly conserved
throughout the species;

CULTURAL CHARACTERISATION
• TEMPERATURE:
optimum temperature range of 28-30'C, with extremes of 11-42C.
Growth at 11-13"C has been proposed as a phenotypic test for L.
biflexa
Pathogens grow in mammalian hosts at febrile body temperatures,
and in chick embryos and young chicks around 40-42C but do not
grow well in laboratory media at these temperatures

• pH:
Leptospira will grow in the range pH 6.5-8.4
• Osmolarity
Leptospira will grow in the range 0.05 – 0.8 M, molarity of
salts
• OXIDATION-REDUCTIOPNO TENTIAL:
Leptospires are aerobic bacteria that do not tolerate reducing
conditions or anaerobiosis, when the oxidationreduction
potential is less than about Eh - 0.250 mV at pH7.2.

Growth
• Leptospira strains grow slowly, colonies can take from 3-7
days to 3 weeks to appear.
• Leptospires are grown routinely in liquid media, Some
strains may not grow at all on solid media
• leptospires cultures of liquid media at 30'C with a
doubling time of 6-8 h under optimum conditions
• Leptonema grows rapidly in medium to reach maximum
density on incubation for I8-:72h at 30'C.

Growth requirements
• Leptospires require an oxygen source
• Pathogenic leptospires require long-chain (C12 –C18)
unsaturated fatty acids, as a carrbon source
• Leptospira biflexa strains can grow on long- or short-chain,
saturated or unsaturated fatty acids
• The only essential nitrogen source is ammonia probably
producing ammonia by deamination.

OTHER REQUIREMENTS FOR GROWTH
• all the pathogen group require pyrimidines.
• L. biflexa and other nonpathogens and Leptonema can
synthesize their own purines and pyrimidines. Turneria
parva requires purines.
• Phosphates, sulfates, ferric iron or hemoglobin (or
heme),calciuma nd magnesium, thiamine and
cyanocobalamin are essential

Cultur media
• media contain heat-labile proteins or other ingredients and
are sterilized by filtration
• used of rabbit or other animal serum
• Incubation in 28-30 c Cultures should be checked for
growth or contamination after 3-4 days and subcultured
after 7-21 days

culture media
• Oleic acid-albumin (OA) media and serum media based on 1
percent BSA and Tween 80.
• Special selective and indicator media: containingo one or more
of cycloheximide , bacitracin, 5-fluorouracil nalidixic acid,
polymyxin-B sulphate, polymyxin B ,rifampin (10 pglml) or
vancomycin
Recommended to reduce contaminationon primary isolation or
to purify contaminated culture
• Protein-free and low-protein media developed for vaccine
production

The leptospiral genome and its
elements
• G + C content of 35-51 mol%
• genome size originally estimated to be 4500- 5000 kb
• Both pathogenic and saprophytic leptospires contain two
ribosomal 23S rRNA genes but only one 16S rRNA, for 5S
rRNA,

The leptospiral genome and its elements
 no compound transposons have been
found in the genus Leptospira,
 Genome sequencing has indicated
IS1533. An IS3-like element, designated
1S1500, was present only in pathogenic
leptospires
 LPS biosynthetic locus is bounded by two
IS7533 elements
 presence of these IS elements may
regulate gene expression leading to
differences in LPS structure

The leptospiral genome and its
elements
• Genetic analysis of Leptospira has been impeded by the
lack of genetic exchange systems
• isolated three bacteriophages, whose replication was limited
to the saprophyte L. biflexa.
• phage LE1, was shown to replicate as a plasmid in L.
biflexa and was used as the basis for the first l. biflexa- E.
coli plasmid shuttle vector, pGKLep4

• two report of gene inactivation by
recombination-mediated allelic replacement in
Leptospira.
1. The kanamycin resistance gene from
pGKLep4 was used to inactivate the flaB
gene, encoding the flagellar subunit
protein, in L. biflexa
Mutants were nonmotile and lacked
flagella and hooked ends, but retained their
helical shape
2. Inactivation of recA in L. biflexa resulted in
reduced growth rate and altered nucleoid
morphology

Lipopolysaccharid
LPS comprises the major surface component of leptospires and is the
target for agglutinating and opsonizing antibodies thus important for
serological classiflcation of leptospires.
The structure of LPS similar to that of typical gram negative LPS (keto-deoxy-
octulonate (KDO) xylose ….)
rfb loci involved in the biosynthesis of the LPS O-antigen contains at
least 31 (ORF). Encode enzymes involved in the biosynthesis of
activated sugars, glycosyl transferases, and sugar processing and
transport proteins
Leptospiral LPS appears to be assembled via the classical Wzy (Rfc)
dependent pathway.

Protein and lipoprotein antigens
• Ompl-l: is a transmembrane protein with porin activity
which exists in a typical trimeric form in the leptospiral
outer membran
• lipoproteins, which are well conserved across the
pathogenic species(major OMP, LipLs, as well as OmpL1,
LipL41(synergystic), lipL31,48,…) but LipL36 is not
produced during infection

Other localized structures and
components
• A glycolipoprotein (GLP) with a high content of toxic lipids
(palmitoleic and oleic acids) is cytotoxic and lethal for laboratory
animals. produce antibodies to GLP
• GLP inhibits Na*, K*ATPase
• Peptidoglycan was cytotoxic, inducing the release of TNFα from
peripheral blood PMN

PATHOGENICITY AN D VIRULENCE FACTORS
• Pathogenic species are dermonecrotic and cytotoxic.
• L. biflexa and avirulent pathogenic leptospires kill with
immunoglobulins ,lysozyme and complement
• Virulent leptospires able to survive in macrophages, in
which they induce apoptosis

virulence
• LipL36 was synthesized at 30'C but not at 37"C
• LipL4l, LrpL32, and Ompl-l, are produced only at 37'C and
during growth in an animal infection model
• Virulent leptospires survive because the antigens reacting with
these opsonic immunoglobulins are not expressed or not
available on the surface
• Opsonizing antibodies (LPS epitopes) (avirulent
leptospira)appear, 3-10 days after inoculation lead to clearance
by reticuloendothelial Phagocytosis

Virulence
• The primary lesions in leptospirosis consist of damage to the
endothelial cells of small blood vessels, leading to leakage of
plasma and hemorrhages.
• The consequences of the damage to blood vessels are ischemia to
the cells and organs dependent on the disturbed
• Renal tubular necrosis is common lead to localization of
leptospires on the luminal surface of the tubular cells, where
they grow and are excreted in urine

virulence
• Leptospira may have one or more toxins, one of which is a
cytotoxic and antigenic GLP

unusualunsaturated fatty acids of leptospiral origin acting as competitive inhibitors
of the incorporation of normally occurring fatty acids in the target cell membrane
• LPS does not appear to play a significant part in
pathogenes
• haemolysins( sphingomyelinases) produce holes in
erythrocyte and probably other cell membranes

LABORATORY ISOLATION AND
IDENTIFICATION
• Leptospires are so hard to see and slow to grow that
conventional bacteriological diagnostic procedures are not
practicable
• Dark field microscopy rapid diagnostioc test . culture taking up
to 3-4 weeks for growth and days to weeks for identification.
• Immunofluorescent staining is rapid and speciflc if the serovar
or serogroup
• Molecular method

Identification of isolates; typing methods
• Colony type : typing by the morphology of colony
• The usual method for testing serological identity is by
microscopic agglutination test (MAT).
• Serological identification should be done at serovar level if
possible
• Molecular typing method: molecular typin based on
sequencing of 16SrRNA, ITS, and RFLP , PFGE,(NotI or
SgrAI)

• At subspecies level, the first widely
used method was restriction
endonuclease analysis (REA) of
whole genomic DNA
• insertion sequences; some of
these have been used as targets for
identification and molecular
typing schemes(diferent in
number like IS1500)

Treatment
• All classes of antibiotics except
chloramphenicol and rifampin kill
leptospires.
• Penicillin and doxycycline are used
widely in therapy
• Resistance to any antibiotic has not
appeared as a clinical problem,
probably because there is no human-to-
human transmission

Refrences
1. TOPLEY TILSON'S MICROBIOLOGY& MICROBIALI NFECTIONS . This tenth edition published
In 2005 by Hodder Arnold, an imprint of Hodder Education and n mcmllcr of the Houcr Headlinc
Group.
2. PAUL N. LEVETT*. Leptospirosis. CLINICAL MICROBIOLOGY REVIEWS, 0893-8512/01/$04.0010
Apr. 2001, p. 296–326
3. Ehsanollah Sakhaee , Gholam Reza Abdollah pour. Detection of leptospiral antibodies by microscopic
agglutination test in north-east of Iran. Asian Pacific Journal of Tropical Biomedicine (2011)227-229
4. Bahari, A.1*; Abdollahpour, G.2; Sadeghi-Nasab, A. et al. A serological survey on leptospirosis in aborted
dairy cattle in industrial farms of Hamedan suburb, Iran. Iranian Journal of Veterinary Research, Shiraz
University, Vol. 12, No. 4, Ser. No. 37, 2011
5. Y. Khousheh, A. Hassanpour, 1 2 3G.R. Abdollahpour and 4S. Mogaddam. Seroprevalence of
Leptospira Infection in Horses in Ardabil-Iran. Global Veterinaria 9 (5): 586-589, 2012 ISSN 1992-6197
© IDOSI Publications, 2012 DOI: 10.5829/idosi.gv.2012.9.5.6657
6. Aghaiypour ∗, K., Safavieh, S. Molecular detection of pathogenic Leptospira in Iran. Archives of Razi
Institute, Vol. 62, No. 4, Autumn (2007) 191-197.
7. A. DOOSTI, R. AHMADI & A. ARSHI. PCR DETECTION OF LEPTOSPIROSIS IN IRANIAN
CAMELS.Bulgarian Journal of Veterinary Medicine (2012), 15, No 3, 178−183.
8. H Honarmand 1, *S Eshraghi 2, MR Khorramizadeh eet al. Distribution of H uman Leptospirosis in
Guilan Province, Northern IranIranian J Publ Health, Vol. 36, No.1, 2007, pp.68-72

Leptospira azadi

In this document