DG.

DIRECT
1. Recoltare

prima sptmn - snge, LCR

S2-L3 - urin, dup administrare bic. de sodiu pentru alcalinizare

(pH<7 nefavorabil leptospirelor)

2. Examen Microscopic
nesemnificativ/ examen imunofluorescen
3. Izolarea pe mediu
medii lichide speciale cu ser de iepure sau inoculare subcutan/intraperitoneal
cobai
4. Identificare: caractere morfolo-tinctoriale, de cultur & Antigenice

morfotinctorial: MO, fond ntunecat- filamente subiri, helicoidale,

strlucitoare, n dini de fierestru, mobile, cu crlig la capete

pr. de cultur: nu tulbur mediul, dezvoltare lent (1-6 spt)

pr. antigenice: reacii de aglutinare-liz cu ser specific.

5. Proprieti de patogenitate: septicemie la cobai

5. Antibiograma
DG. INDIRECT
Dg. serologic: Ac sunt detectabili n Z6-10 i ating un maxim n S3-4
Se pot efectua:

RFC folosing Ag din L. nepatogene (biflexa)

reacii de aglutinare liz, pentru determinarea speciei

Diagnostic
eptospira is a flexible, spiral-shaped, Gram-negative spirochete with
internal flagella. Leptospira interrogans has many serovars based on cell
surface antigens.

Leptospira has the general structural characteristics that distinguish

spirochetes from other bacteria (Fig. 35-2). The cell is encased in a threeto five-layer outer membrane or envelope. Beneath this outer
membrane 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 An unusual feature of the
spirochetes is the location of the flagella, which lie between the
outer membrane and the peptidoglycan layer. They are referred to as
periplasmic flagella. The periplasmic flagella are attached to the protoplasmic
cylinder subterminally at each end and extend toward the center of the cell.
The number of periplasmic flagella per cell varies among the spirochetes. The
motility of bacteria with external flagella is impeded in viscous environments,
but that of spirochetes is enhanced. The slender (0. 1 m by 8 to 20 m)
leptospires are tightly coiled, flexible cells (Fig. 35-3). In liquid media, one or
both ends are usually hooked. Leptospires are too slender to be
visualized with the bright-field microscope but are clearly seen by
dark-field or phase microscopy. They do not stain well with aniline
dyes.
The leptospires have two periplasmic flagella, one originating at each
end of the cell. The free ends of the periplasmic flagella extend toward the
center of the cell, but do not overlap as they do in other spirochetes. The
basal bodies of Leptospira periplasmic flagella resemble those of Gramnegative bacteria, whereas those of other spirochetes are similar to the basal
bodies of Gram-positive bacteria. Leptospira differs from other spirochetes in
lacking glycolipids and having diaminopimelic acid rather than ornithine in its
peptidoglycan.
The leptospires have two periplasmic flagella, one originating at each end of
the cell. The free ends of the periplasmic flagella extend toward the center of
the cell, but do not overlap as they do in other spirochetes. The basal bodies
of Leptospira periplasmic flagella resemble those of Gram-negative bacteria,
whereas those of other spirochetes are similar to the basal bodies of Grampositive bacteria. Leptospira differs from other spirochetes in lacking
glycolipids and having diaminopimelic acid rather than ornithine in
its peptidoglycan.
The leptospires are the most readily cultivated of the pathogenic spirochetes.
They have relatively simple nutritional requirements; long-chain fatty
acids and vitamins B1 and B12 are the only organic compounds
known to be necessary for growth. When cultivated in media of pH
7.4 at 30C, their average generation time is about 12 hours.
Aeration is required for maximal growth. They can be cultivated in

plates containing soft (1 percent) agar medium, in which they form

primarily subsurface colonies.
The two species, L interrogans and L biflexa, are further divided into
serotypes based on their antigenic composition. More than 200
serotypes have been identified in L interrogans. The most prevalent
serotypes in the United States are canicola, grippotyphosa, hardjo,
icterohaemorrhagiae, and pomona. Genetic studies have demonstrated
that serologically diverse serotypes may be present in the same genetic
group. At least seven species of pathogenic leptospires have been identified
by nucleotide analysis.
Specific Microbial Characteristics
Leptospira interrogans is a spirochete (coiled, helical-shaped), gram-negative,
aerobic bacteria with a thin body, hooked ends and paired axial flagella [5].
The hooks are used for attachment to the infected tissue. The antigenic
differences in the lipopolysaccharide envelopes that surround their cell walls
are used to classify the different serotypes of the bacteria [5]. The optimal
growth of Leptospira occurs at 28C-30C [5].
Specific tests for identification
Laboratory testing is required to confirm the disease. The laboratory tests
consist of the identification of Leptospira interrogans serotypes in blood,
tissue, urine and/or cerebrospinal fluid. Among these, the testing of
urine is the most reliable approach due to the presence of the
bacteria in this fluid from the onset of symptoms until the third
week of infection [5]. Two FDA-approved commercially available tests are
an indirect hemmaglutination (MRL) and a dipstick test (PanBio) [4].
However, the most reliable test is the microscopic agglutination test
performed by the CDC [5]. Silver staining and immunofluorescence can
also be used to identify leptospira in tissue samples such as kidney, liver,
spleen and muscles [5].