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Diagnosis of Leptospirosis
and
Austrian
epidemiology
MTA Maria Müller Institute for
Veterinary
Disease
Control, Mödling
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Leptospires:
HISTORY
MORPHOLOGY
TAXONOMY
OCCURENCE
TRANSMISSION
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Leptospires
: History
Adolf Weil (1886) his account:„An infectious disease, accompanied by splenomegaly, jaundice and nephritis“
1914 - 1918 war in Europe „Weil‘s disease“ assumed increasing importance:
Hübner & Reiter (1915) successful transmission of „Weil‘s disease“
to guinea pigs and the „flagella like“ bodies in blood films stained with Giemsa
Uhlenhuth & Fromme „spirochaeta interrogenes“
a demonstration of the spirochaete and immunisation with it
Inada & colleages 1914 - 1915 in Japan
coal-miners fell sick
succeeded in transmitting the infection to guinea pigs (blood transfusion)
to demonstrate passive protection with specific immune serum
in studies „mode of infection“ and …..
„Spirochaeta icterohaemorrhagiae“
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Morphology
Order: Spirochaetales
helically coiled (spiralförmig)
thin
motile
propelled by flagellar mechanism
contained wholly within an outer envelope
diverse in chemical and nucleic acid compositions
nutrition and natural habitats generally visualised by darkfield microscopy in wet praparations
not good visible in usual bacterial staining
leptospira is greek origin„lepto“ = fine„spira“ = spiralförmig
Source: www.wikipedia.org
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Morphology
Leptospires range in length
10 - 20µm
coil amplitude of 0,1 - 0,2 µm
two flagella, one arising at each end
At cell division a new flagella is developed at the newly formed end after division
In fluid media they spin rapidly on their long axis in rotational movements and move to and from in both directions
Source: www.wikipedia.org
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Taxonomie
Two methods to classify leptospires:- serological classification:
Leptospira biflexa sensu lato: non pathogenic free living leptospires (about 60 serovars)Leptospira interrogans sensu lato:pathogenic (about 200 serovars in 23 serogroups)
- genomic classification:16 different genospeciesidentification and classification of leptospires by comparingDNA fragments
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Occurence:
Leptospirosis occurs worldwide
acid (pH < 7,0) and dry conditions kill leptospira
Transmission only in wet environments
In cold environments (+4°c) leptospires servive ~21days
Sources are:
Urine, kidneys of infected animals
Surface waters, mud and soil
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Transmission:
Direct transmission:
Blood or body fluids containing leptospires pass from an infected animal to other animals or humans:
transplacental transmission
sexual contacts
suckling milk
Indirect transmission:
Infection of humans and animals from environmental leptospires:
Ponds,lakes,drain water..contaminated by the urine from excretor animals
(rodents,swine,cattle,dogs,…)
Occupational infection:
Exposed are drainers, slaughtermen, farmers, butchers, veterinarians, hunters,…
Non occupational infection:
Infection with contaminated water by animal‘s urine at leisure activities,travel,…(e.g.camping, boating, swimming,….)
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Transmission of leptospires
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Culture
methods
Culturing:
EMJH-Medium: (Ellinghausen, McCollough, Johnson u. Harris)
Serum-free oleic-acid albumin medium
(quality control: clear medium)
Subculturing from and to liquid medium
(0,1->0,2ml)
Growth in liquid medium is proved by gentle shaking
Aseptically transferring (safety cabinet)
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Culture
methods
Temperature:
Cultures are kept in dark at 29°C (5->7 days) to avoid toxic changes
Screw-copped tubes to prevent contamination
Duplicate tubes for several serovars (antigen)
10ml volumes for growth
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Culture
methods
Culture control and preservation
Purification of contaminated cultures(filtration: membrane filter – 0,22µmcentrifugation and resuspension in fresh medium)
Proving of strains (cultures) for identity (serologically)
Preservation of stock cultures:Cryopreservation in liquid nitrogen in a liquid phase
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Culture
of leptospires
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Contaminated
culture
of leptospires
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Microscopic
agglutination
test
Equipment:Materials:
Antisera (reference antisera)
Cultures of leptospires (density and growth control)
Phosphate buffered saline
Safety cabinet
Tubes
Microtiter plates (flat bottom)
Pasteur pipettes (steril)
Tips
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Microscopic
agglutination
test
Testperformance (Screening):
1/25 dilution of serum in phoshate buffered saline (tubes)
Microtiterplate:
A special plate for every animal species (determined by the number of antigens we use for this species)
Human sera are screened in extra plates
In one plate it is possible to check 10 samples
Platelabelling:
Left side: Serovars of leptospires (antigen)
Upper side: Identity number of sample
Right side - column 11: pos.controlserum (connected to serovar)
- column 12: only antigen = neg.control (safety cabinet)
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Microscopic
agglutination
test
Testperformance (Screening)
:
50µl serumdilution in column 1 (sample 1)
50µl serumdilution in column 2 (sample 2) and so on
50µl pos.controlserum (corresponding to antigen) in column 11
Safety cabinet:
50µl of the corresponding antigen in row 1 (well 1->12)
50µl of the corresponding antigen in row 2 and so on
-> final dilution 1:50
Column 12 = only antigen (leptospira) = neg.control
Shaking softly (plateshaker)
Incubation 29°C (2->2,5 hours) in a humid chamber
Examination of each well by dark-field microscopy for agglutination
50% agglutination at one or more antigens is examined by titration
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Microscopic
agglutination
test
Testperformance (Agglutination)
Tubes:1/25 dilution of serum in phosphate buffered saline
Microtiterplate:
An extra plate for each antigen
Platelabelling:
Left side: Seradilutions 1:50 -> 1:6400
Upper side: Identity number of sample (serumdilution)
Right side (column 11): pos.controlserum for corresponding antigen
Right side (column 12): antigen control
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Microscopic
agglutination
test
Testperformance (Agglutination):Row A: 100µl dilution in the corresponding well of row A
50µl phosphate buffered saline in row B ->H
Dilution from row A ->H (volume 50µl)
Dilutionsteps : 1:50 –> 1:6400
Safety cabinet:
50µl of the corresponding antigen in each well
Shaking softly (microplateshaker)
Incubation 29°C (2-2,5 hours)
Examination of each well by dark-field microscopy for agglutination (row A-H)
As endpoint is defined this serum dilution that shows 50% agglutination of leptospires
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Screening
1 2 3 4 5 6 7 8 9 10 11 12
Pat.1 Pat.2 Pat.3 Pat.4 Pat.5 Pat.6 Pat.7 Pat.8 Pat.1 Pat. 10
+Ko -Ko
A=ict. 50µl Pat 1+L.ict. wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
B=can. 50µl Pat1+L.can. wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
C=brat. 50µl Pat1+L.brat.
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
D=gripp. 50µl Pat1+L.gripp.
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
E=tarr. 50µl Pat1+L.tarr. wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
F=pom. 50µl Pat1+L.pom wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
G=wolffi 50µl Pat1+L.wolff
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
H=hard. 50µl Pat1+L.hard.
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
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Platelabeling
for
Screening:
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Agglutination:
1L.bratisl.
2 3 4 5 6 7 8 9 10 11 12
Pat.1 Pat.2 Pat.3 Pat.4 Pat.5 Pat.6 Pat.7 Pat.8 Pat.1 Pat. 10
+Ko -Ko
A=1: 50 100µl wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
B=1: 100 50µl + 50µl PBS
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
C=1: 200 50µl + 50µl PBS
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
D=1: 400 50µl + 50µl PBS
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
E=1: 800 50µl + 50µl PBS
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
F=1:1600 50µl + 50µl PBS
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
G=1:3200 50µl + 50µl PBS
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
H=1:6400 50µl + 50µl PBS
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
wie Pat.1
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Microscopic
agglutination
test:
positive control negative control
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Leptospirosis: Serological
examination
of veterinarians
0
0,5
1
1,5
2
2,5
3
3,5
L.bratis.
L.batav.
L.canicola
L.hardjö
L.saxköb.
L.sejrö
137 blood samples tested
10 veterinarians had antibodies against leptospira – 4 of them with positive titer
1 veterinarian with high antibody titers against L.saxköbing and L.bataviae- he had to stay in hospital
9 veterinarians reported about different clinical signs
2 veterinarians with positive titers were practising with swine and all of them practising in slaughtering
Summary: veterinarians are exposed with infections to zoonosis (example: leptospirosis)
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Leptospirosis-hunting
feral
animals
Blood samples from 147 hunters and 108 wild boars were tested
11 serovars of Leptospira
15 hunters (10%) had antibodies with positive, 26 hunters suspect titers(see first diagramm)
A control group (50 persons – mainly city dwellers) no leptospira antibodies
Samples of 36 wild boars (30%) antibody positive, 16 samples suspect titers(see second diagramm)
Conclusion:High risk of leptospira infection in hunters compared to other occupational groups.
0
5
10
15L.a.ball.
L.bratisl.
L.grippo.
L.hardjö
0
10
20
30
0
L.a.ball.
L.bratisl.
L.grippo.
L.ictero.
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Leptospirosis
2009 (Human samples):
0
5
10
15 L.ictero.
L.brat.
L.canicola
L.autumn.
L.ballum
L.wolffi
45 human blood samples tested
41 samples/styria, 3 samples/upper Austria, 1 sample/Vienna
16 serovars of leptospires
In 26 samples antibodies against leptospira found
Organigram 1: Antibody titer in 22 samples 1:50 (=suspect):
L.icterohäm.,L.bratislava, L.canicola
Organigram 2: Antibody titer in 6 samples >1:100 (=positive):
L.iterohäm., L.bratislava, L.autumnalis
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Plate
for
Microagglutinationtest
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Leptospirosis
2008 (Human samples):
0
5
10
15 L.ictero.
L.brat.
L.austr.ball.
L.ballum
L.grippo.
L.saxköb.
85 human blood samples tested
76 samples/styria, 6 samples/upper Austria, 3 samples/Vienna
16 serovars of leptospires
In 41 samples antibodies against leptospira found
Organigram 1: Antibody titer in 34 samples 1:50 (=suspect):
L.icterohäm.,L.bratislava, L.grippotyphosa, L.ballum
Organigram 2: Antibody titer in 7 samples >1:100 (=positive):
L.bratislava, L.icterohäm., L.grippotyphosa