Leptospira sp.

Definition
Leptospira are spiral bacteria (spirochaete) in the family of Leptospiraceae.
There are eight pathogenic, seven non-pathogenic Leptospira species, and
five intermediate species with unknown ability for causing disease.

Ammonium salts are a main source of nitrogen

Leptospirae can survive for weeks in water, particularly at alkaline
pH

Morphology & Identification

Taxonomy

TYPICAL ORGANISM
Tightly coiled, thin, flexible spirochetes 515 m long, with very fine
spirals 0.10.2 m wide; one end is often bent, forming a hook
Enncased in a 3-5 layer outer membrane or envelope. Beneath are
the flexible, helical peptidoglycan layer and the cytoplasmic
membrane; these encompass the cytoplasmic contents of the cell.
The structures surrounded by the outer membrane are collectively
called the protoplasmic cylinder
Actively motile, which is best seen using a dark-field microscope
Has 2 Periplasmic flagella, one originating at each end of the cell.
The free ends of the flagella extend toward the center of the cell
The basal bodies of Leptospira periplasmic flagella resemble those of
Gram ve bacteria
Electron micrographs show a thin axial filament and a delicate
membrane
The spirochete is so delicate that in the dark-field view it may appear
only as a chain of minute cocci. It does not stain readily but can be
impregnated with silver

Leptospira interrogans and L. borgpetersenii are the two most common

species causing diseases in human and animals. The traditional
classification system is based on biochemical and serologic specificity to
differentiate between the pathogenic species, Leptospira interrogans, and
the free-living nonpathogenic species, Leptospira biflexa. The species are
further broken down to more than 200 serovars of L interrogans and more
than 60 serovars of L biflexa. The serovars are further organized into
serogroups of L interrogans and serogroups of L biflexa. A second
classification system is based on DNA-DNA hybridization studies, which have
demonstrated a high degree of heterogeneity within the two species of the
traditional classification.

CULTURE
Grow best under aerobic conditions at 2830C in semisolid medium
(EMJH, others) in 10 mL test tubes with 0.1% agar and 5-fluorouracil
After 12 weeks, the leptospires produce a diffuse zone of growth
near the top of the tube and later a ring of growth at a level in the
tube corresponding to the level of the optimal oxygen tension for the
organisms
GROWTH REQUIREMENTS
Derive energy from oxidation of long-chain fatty acids and vitamins
B1 and B12
Cannot use amino acids or carbohydrates as major energy source

The outer envelope contains large amounts of lipopolysaccharide of

antigenic structure that is variable from one strain to another
Leptospira serovar and serogroups can be seen in the next page

Life cycle & transmission

Antigenic structure

The main strains ("serovars") of L interrogans are all serologically

related and exhibit cross-reactivity in serologic tests
Indicates considerable overlapping in antigenic structure, and
quantitative tests and antibody absorption studies are necessary for
a specific serologic diagnosis

The major reservoirs and carriers: mammalian species, including

rodents, cattle, pigs, domestic and wild animals
Humans are mostly accidental and dead end hosts
The infecting organism is sustained naturally by chronic infection of
the renal tubules of maintenance hosts after primary infection
Organisms are usually transferred from animal to animal by direct
contact. The maintenance hosts or carriers can excrete Leptospira in
their urine for long periods of time or for their entire lives
Leptospires survive weeks or months in moist and warm soil,
stagnant water at neutral or slightly alkaline pH.
Humans are infected via direct contact with infected animal urine,
animal abortion products or most commonly through indirect contact
with infected urine-contaminated water. Oral route has been
reported. Breast milk transmission may occur. Sexual and vertical
transmission in humans occurs rarely.
Humans at risk for leptospirosis are those with occupational
exposure. These include farmers, fishermen, miners, animal
slaughterers, veterinarians, sewage and canal workers, sugar cane
workers, soldiers, etc.
Special events and activities clearly related to the diseases are
recreational water sports, including triathlon, canoeing and whitewater rafting, and natural disasters, such as hurricanes, floods, etc.
Laboratory-acquired infection may occur when dealing with high
concentrations of organisms in culture

Diagnosistic Laboratory Test

SPECIMENS
Aseptically collected blood in a heparin tube, CSF, or tissues for
microscopic examination and culture
Urine should be collected using great care to avoid contamination
Serum is collected for agglutination tests

Pathogenesis

Human infection usually results from leptospires, often in bodies of

water, entering the body through breaks in the skin (cuts and
abrasions) and mucus membranes (mouth, nose, conjunctivae)
After an incubation period of 12 weeks, variable febrile onset during
which spirochetes are present in the bloodstream. The host responds
by producing antibodies that, in combination with complement, are
leptospiricidal. The leptospires are rapidly eliminated from all host
tissues except the brain, eyes, liver and kidneys. They producing
hemorrhage and necrosis of tissue and resulting in dysfunction of
those organs (jaundice, hemorrhage, nitrogen retention)
The illness is often biphasic. After initial improvement, the second
phase develops when the IgM antibody titer rises. It manifests itself
often as "aseptic meningitis" with intense headache, stiff neck, and
pleocytosis of the CSF
Nephritis and hepatitis may also recur, and there may be skin,
muscle, and eye lesions. The degree and distribution of organ
involvement vary in the different diseases produced by different
leptospira)
Hepatitis is frequent in patients with leptospirosis. Kidney
involvement in many animal species is chronic and results in the
shedding of large numbers of leptospirae in the urine; this is
probably the main source of environmental contamination resulting
in infection of humans. Human urine also may contain spirochetes in
the second and third weeks of disease.
Agglutinating, complement-fixing, and lytic antibodies develop
during the infection. Serum from convalescent patients protects
experimental animals against an otherwise fatal infection. The
immunity resulting from infection in humans and animals appears to
be serovar-specific

MICROSCOPIC EXAMINATIONS
Dark-field examination or thick smears stained by the Giemsa show
leptospirae in fresh blood from early infections
Dark-field examination of centrifuged urine
Fluorescein-conjugated antibodies or other immunohistochemical
techniques
CULTURE
Whole fresh blood or urine can be cultured in a semisolid medium.
Because of inhibitory substances in blood, only 1 or 2 drops should
be placed in each of five tubes containing 5 or 10 mL of medium
Up to 0.5 mL of CSF can be used
References:
One drop of undiluted urine can be used
followed
by one
drop each
Mansons
Tropical
Disease
23rd Edition
of 10-fold serially
diluted
urinefor
a
total
of
four
tubes
Jawetz, Melnick, & Adelbergs Medical Microbiology 25 th Edition
Tissue approximately 5 mm in diameter
should be crushed and used
http://www.ncbi.nlm.nih.gov/books/NBK8451/
as the inoculum. Growth is slow, and cultures should be kept for at
least 8 weeks

Serology

Agglutinating antibodies first appear 57 days after infection and

develop slowly reaching a peak at 58 weeks
Very high titers may be attained (>1:10,000). The test is highly
sensitive, but it is difficult to standardize; the end point is 50%
agglutination, which is difficult to determine
Agglutination of the live suspensions is most specific for the serovar
of the infecting leptospires

Immunity
Serovar-specific immunity follows infection, but reinfection with different
serovars may occur.