Spirochaetes are elongated, motile bacteria with endoflagella that allow flexing and twisting movements. Treponema pallidum causes syphilis in humans. It has a distinctive morphology and causes primary, secondary, latent, and tertiary stages of infection. Diagnosis involves direct visualization of the spirochete or using serological tests like VDRL, RPR, and treponemal tests that detect antibodies to T. pallidum antigens. Treatment is with penicillin which can cause a Jarisch-Herxheimer reaction.

1. Spirochaetes
Anuradha Bhagwat
Isha Joshi
Sanika Deodhar
Shivani Deshpande

2. CONTENTS
• General Introduction-Spirochaetes
• Treponema
• Treponema Palladium:
• Morphology
• Cultivation
• Antigenic Structure
• Pathogenesis
• Laboratory Diagnosis

3. General Introduction
• Spirochaetes are elongated ,motile,flexible,bacteria twisted along the
long axis.
• Various Species of spirochaetes are:Borrelia,Treponema,Leptospira

4. CELL WALL
Spirochaetes have gram
negative type of cell wall.
The constituents if the cell wall
are:
1. Inner most layer –
Peptidoglycan
2. Lipoprotein layer
3. Outer Membrane-Outer
membrane protein,porins
and phospholipid
4. Outermost layer –
Lipopolysaccharide(not
present here)

5. ENDOFLAGELLA
• Motility of spirochaetes can be explained on the basis of presence of
endoflagella .

6. • Endoflagella are fine fibrils attached subterminally at each pole of the cell and
extend towards the opposite pole between outer membrane and peptidoglycan
layer of the cell wall.

7. • NUMBER OF ENDOFLAGELLA :
• Treponema-3 to 4
• Borrelia-15 to 20
• Leptospira-1
The spiral shape and the serpentine motility depend upon integrity of
the endoflagella .The motility is of three types:
1. Flexion and Extension
2. Corkscrew like rotatory movement
3. Translatory motion

8. TREPONEMA
• Treponema-(trepos meaning to turn and nema meaning thread)
• Thus these are slender spirochaetes with fine sprials and pointed
ends
• Some of them are pathogenic in humans while others occur a
commensals in mouth and genetalia.
• Cannot be cultivated in culture media and are maintained in
susceptible animals.

9. PATHOGENIC
TREPONEMA SPECIES
Vereneal
treponematoses :
Vereneal Syphilis-
Treponema Palladium
Non-Vereneal
treponematoses:
Yaws
Bejel
Pinta

10. YAWS-Caused by
T.pertenue
Causes skin sores and
diffusing growth on legs
around nose and mouth

11. ENDEMIC
SYPHILIS(BEJEL)-Caused
by T.endemicum
Causes mouth sores and
destrucctive lumps in
bone

12. PINTA-T.Carateum
Causes itchy
,thickened,disolored
patches on the skin

13. TREPONEMA PALLADIUM-MORPHOLOGY
• It is thin ,delicate ,elongated,flexible,twisted along the long axis and
motile.
• It is 10um long and 0.1-0.2um wide .
• They possesses 3 to 4 endoflagella attached sub-terminally ,they are
more than half length of the organism so might interdigitate in the
center .
• Transverse Section may show 3 -4 at the terminal and 4-6 at the
center.

14. • MOTILITY IS SEEN BY:
Flexion and extension, Corkscrew-like rotatory movement, Translatory
motion
• Best seen in wet living preparation with dark ground microscope

15. DRIED PREPARATION
• Here it needs to be thickened by silver impregnanation
method:Levaditi for tissue section and Fonatana’s method for staining
films

16. CULTIVATION
• Pathogenic treponemes cannot be cultivated in artificial media.
• Nichol’s strain is maintained in rabbit testis obtained from CSF of a
patient with neuro-syphilis.
• Non pathogenic strain is called Reiter strain shown by T.refringens and
T.phagedenis.
• They can be grown under anaerobic condition in Smith –Noguchi
medium or digest broth enriched with serum.

17. ANTIGENIC STRUCTURE
• The antigenic structure is poorly understood.Infection due to these
treponemes leads to production of 3 types of antibodies.
• On this basis they may be divided as speific and non-specific antigen.

18. SPECIFIC ANTIGEN
• Group specific antigen:Protein in nature Present in both pathogenic
as well as non-pathogenic(Reiter treponeme).Antigen used here to
detect group specificc antibodies is Reiter treponeme.
• Species –specific treponemal antigen:Polysacccharide in
nature,Antgen used here is T.palladium.

19. NON-SPECIFIC ANTIGEN
• A non-specific antibody appears in sera of patients that reacts with
lipid hapten extracted from beef heart .
• This hapten is called cardiolipin.

20. Pathogenesis
Treponemal species pathogenic to man include causative
agents of
• Venereal Syphilis - T. pallidum
• Non- Venereal treponematoses – Yaws T. pertenue
• Endemic Syphilis – T. endemicum
• Pinta – T. carateum

21. • T. pallidum is a strict parasite with a limited life span outside
the body
• Syphilitic infections can be transmitted via
1) Sexual intercourse
2) Materno-foetal transmission – congenital syphilis
3) Intimate person-to-person contact
4) Transfusion of infected blood
5) Accidental finger prick with an infected needle in medical
personnel

22. • Treponemes are present in superficial genital lesions and
pass from one partner to the other either via intact mucous
membrane or minor skin abrasions to multiply at the sight of
entry
• After an incubation period of 10 – 90 days clinical disease
sets in

23. Acquired Syphilis is divided into 4 stages based on period
after which lesions appear and the type of lesions
1. Primary Syphilis
2. Secondary Syphilis
3. Latent Syphilis
4. Tertiary Syphilis

24. Primary Syphilis
The characteristic lesion of primary syphilis is a Chancre.
It is also know as Hunterian Chancre after John Hunter
• It is 1-2cm in diameter and appears 2-4 weeks after exposure
to treponeme
• Painless papule
• Central ulceration
• Avascular
• Circumscribed
• Indurated when fully developed
• Accompanied by Lymphadenitis

25. • The bacteria invade regional lymph nodes ( inguinal in males
and pelvic in females) and through these enter blood
circulation
• The lymph nodes appear swollen, discrete, rubbery and non
tender
• Chancre heals in 3-6 weeks leaving a very thin scar.
• Antibody tests are +ve 1-3 weeks after appearance of
chancre

26. Hard Chancre caused by
T. pallidum is different
from Soft chancre
caused by Haemophilus
ducreyi
(H. ducreyi causes a
non-indurated, purulent,
soft lesion “chanceroid”)

27.  Histology
It is oedematous with dense infiltration of plasma cells ,
lymphocytes and macrophages
Perivascular aggregation of mononuclear cells
Proliferation of vascular endothelium
 Common sites
External genitalia – prepuce, penis, labia, etc
Cervix, perianal areas, anal canal, tongue, lips, cheeks,
nipples,etc
Chancre usually occurs singly, but immunocompromised
individuals may be develop multiple

29. Secondary Syphilis
• Inadequately treated patients develop secondary syphilis
• It sets in 2-6 months after the Chancre heals during which
the patient is asymptomatic
• marked constitutional symptoms include
Mucocutaneous lesions – oropharynx , vagina
Diffuse erythematous lesions - particularly on trunk and
extremities
Condyloma lata – anogenital regions, mucocutaneous
junctions
It is a highly infective stage and lesions heal in 4-5 years.

30. Erythematous lesions
Oropharyngeal
condyloma lata

31. Macules and papules
seen in Secondary
Syphilis

32. Latent Syphilis
• Latent syphilis is asymptomatic
• It is detected after secondary lesions disappear and ONLY by
serological tests.
• It is further classified as
a) Early - < 1 year after secondary syphilis
b) Late - > 1 year after secondary syphilis

33. Tertiary Syphilis
• It develops between 3 years to decades after primary
infection.
• It is a progressive stage prevalent in 3 forms
1) Gummatous Syphilis
2) Cardiovascular Syphilis
3) Neurosyphilis

34. A. Gummatous Syphilis

35. • Syphilitic gumma are solitary, rubbery , pink, small fleshy
masses containing bacteria
• They may be nodular, tumorous or ulcerative
• Lesions may show characteristic central necrosis
• 1mm-1cm in size
• Present predominantly in Brain, testes, bone, liver, etc.

36. B. Cardiovascular Syphilis
• More serious lesions of heart and aorta are seen in this case
Inflammatory response to spirochetes in arterial walls
Chronic inflammation of Tunica adventitia of elastic arteries
Resultant endarteritis and periarteritis of vaso vasorum
Ischaemia of t. media and t. adventitia
Weakening of wall of aorta and resultant dilatation
Aortic Aneurysm

37. Hepar Lobatum
Aorta

38. C. Neurosyphilis
• It may manifest as
1. Meningovascular Syphilis
2. Tabes dorsalis affecting the spinal cord - Tabes dorsalis is a
slow degeneration of the nerve cells and nerve fibers that
carry sensory information to the brain
3. General paresis of the insane - a severe neuropsychiatric
disorder. It is a result of cerebral atrophy caused due to
chronic meningoencephalitis

40. Argyll Robertson Pupil

41. Congenital Syphilis
It is seen in a foetus of more than 16 weeks gestation who is
exposed to maternal spirochaetemia.
Features include
• Saddle - shaped nose deformity
• Hutchinson’s teeth
• Interstitial keratitis
• Corneal opacity
• Epiphysitis
• Periostitis

43. Treatment
• T. pallidum is particularly sensitive to Pencillin
• In early cases, 2.4 million units of Benzathine
penicillin is adequate
For late syphilis this maybe repeated weekly for 3
weeks
• Doxycyline is administered in patients allergic to Penicillin
• Ceftriaxone is effective particularly in Neurosyphilis
 Antibiotic therapy particularly with penicillin may induce
Jarisch-Herxheimer Reaction due to release of endotoxins
following lysis of spirochaetes . Its onset is within 1-2 hours and
reaction subsides in 12-24 hours

44. • Fever
• Chills
• Myalgia
• Tachycardia
• Hypervetilation
• Vasodilation
• Hypotension

45. Laboratory diagnosis

46. CLINICAL DIAGNOSIS OF SYPHILLIS
Demonstration of treponemes
in exudate.
demonstration of
treponemes in tissue
Demonstration of treponemal
antigen in the lesion
Serological tests

47. 1)Demonstration of treponema pallidum in
exudate
A) DARK GROUND MICROSCOPY:
The surface of the lesion is cleaned with
gauze dipped in warm normal saline and the
margins are gently scraped.
Gentle pressure is applied to base of lesion
till serum exudates.
The material is collected directly on
coverslip, placed on a glass slide &
examined under dark ground microscope.
T. Pallidum is recognized by slender
structure, regularity of its spirals and
pointed ends.
DISADVANTAGE: This method cannot be
used for oral lesions as the non pathogenic
treponemes can be confused with
T.pallidum.

48. B) DIRECT FLUORESCENT ANTIBODY STAINING FOR T.
PALLIDUM (DFA-TP)
 This method provides a more definite
diagnosis.
 The smear made on glass slide is fixed by
acetone and sent to laboratory.
 This is then stained by fluorescein labeled
pathogen specific antibody and examined
under fluorescence microscope.
 T.Pallidum appears as distinctive, sharply
outlined structure which has apple green
fluorescence.
 ADVANTAGE: This can be used to diagnose
oral lesions which is not possible by dark
ground microscopy.

49. 3)DEMONSTRATION OF TREPONEMAL
ANTIGEN IN THE LESIONS
A) ENZYME IMMUNOASSAY
B)POLYMERASE CHAIN REACTION

50. 4) SEROLOGICAL DIAGNOSIS
Based on serological diagnosis:
 nontreponemal/standard test for syphillis (STS)
 Treponemal tests
Nontreponemal tests:
1) Wasserman & kahn test
2) Veneral disease research laboratory (VDRL)
3) Rapid plasma reagin (RPR)
Antigen used in these tests is an alcoholic extract of beef
heart tissue (cardiolipin) to which lecithin and cholesterol
are added.

51. 2)VDRL TEST
It is performed as a slide test on glass side which consists of
inactivated patient serum+ antigen (cardiolipin,
lecithin,cholesterol)
Mixture is rotated for 4 mins mechanically after which
flocculation can be detected under low power microscope.
This test is used as screening test in syphillis.
Positive in 70% primary and 90% secondary syphillis
Patients with late syphillis are non reactive.
ADVANTAGE: standard test for testing of CSF.

52. VDRL TEST

53. 3)RAPID PLASMA REAGIN (RPR) TEST
VDRL antigen is adsorbed on finely divided carbon particles
& suspended in choline chloride.
Antigen is also stabilized by EDTA.
One drop patient serum+ 1 drop of modified antigen taken on
disposable plastic card by a disposable stick.
Card is rocked gently for 8 mins
+ve test: flocculation of -ve test: absence of
Carbon visible to naked black aggregates
eye( black aggeregates) with uniform grey
background.

54. If the test is positive the quantitative tests should be done
by doubling dilutions of serum (1:2, 1:4, 1:6, 1:8,…….. 1:32).
Most labs use this test as screening for syphillis.
Sensitivity and specificity: same as VDRL test but is is rapid
and easy to perform.
DISADVANTAGE: cannot be used to test CSF.

55. BIOLOGICAL FALSE POSITIVE REACTIONS
Antibodies against cardiolipin may be detected in absence
of T. pallidum infection giving rise to false positive rections
(BFP).
These are of 2 types:
A. ACUTE/TRANSIENT BFP REACTION:
After acute febrile infectious diseases.
B. CHRONIC BFP REACTION:
 Autoimmune diseases
 Infectious mononucleosis, eosinophilia
 Leprosy
 Malaria
 Relapsing fever
 Hepatitis

56. Treponemal Tests
• They detect specific antibody to T. pallidum proteins
• Used to confirm the infection and determine whether the disease is
active
• False positive results can occur and may be due to other infections

57. Treponemal
Tests
Using Cultivable
Treponemes
-RPCF test
Using
pathogenic T.
pallidum
antigen
Test using
live T. pallidum
-TPI test
Tests using
killed T. pallidum
-TPA test
-TPIA test
-FTA test
FTA-ABS test
IgM FTA-ABS test
Tests using
T.pallidum extract
-TPHA test
-EIA test

58. (a) Using cultivable treponemes
Reiter protein complement fixation (RPCF) test
• Antigen is derived from endoflagella of T.
phagedenis (Reiter treponeme)
• Less sensitive than cardiolipin test in early syphilis
but more sensitive in late syphilis
(b) Using pathogenic T.pallidum antigen
• All require live, killed or extracts of T. pallidum
grown in rabbit’s serum, testes : Nichol’s strain

59. • Suspension of treponemes, complement + Test serum
-----> Incubate anaerobically
• Examine under Dark-ground microscope
• In presence of antibody : Treponemes found to be
immobilized
i. Treponema pallidum immobilization (TPI) test
I. Tests using live T. pallidum

60. ii. Treponema pallidum immune adherence (TPIA)
test
• Suspension of
inactivated
treponemes + Test
serum,
complement, fresh
heparinised whole
blood ----->
Incubate
• In presence of antibody :
Treponemes adhere to erythrocytes
• In absence : No immune
adherence

61. i. Treponema pallidum agglutination (TPA) test
• Suspension of T.pallidum (inactivated by formalin) +
Test serum -----> Incubate
• Examined under Dark-ground microscope
• In presence of antibody : Treponemes found to be
agglutinated
II. Tests using killed T. pallidum

62. • Indirect immunofluorescence antibody test
• Diluted patient’s serum is absorbed with extract of
Reiter treponemes -----> layered on a slide to which T.
pallidum (pallidum) has been fixed
• In presence of antibody: antibody will coat the
treponeme
• Has high specificity (92%-99%) and sensitivity
• Modification: IgM FTA-ABS test: detects IgM
antibodies in congenital syphilis, distinguishing it from
IgG
iii. Fluorescent treponemal antibody- absorption (FTA-ABS) test

63. FTA-ABS test

64. i. Treponema pallidum haemagglutination (TPHA)
test
• T.pallidum antigen absorbed on RBC surface + patient
serum
• In presence of antibody : haemagglutination is seen
• May be performed in microlitre plates : Microlitre
haemagglutination T.pallidum (MHA-TP) test
• Test sensitivity : 65%
III. Tests using T. pallidum extract

65. TPHA test
MHA-TP test

66. ii. Enzyme immunoassay (EIA) test
• Ultrasonicate of T. pallidum antigen coated on tubes
or ferrous metal beads.
• Antibody in patient serum is detected by EIA.
• Test sensitivity : 90%
• Test specificity : 98%

67. Prophylaxis
• Source of infection : infected individual , for 3-5 yrs during early
syphilis.
• Transmission is by direct contact.
• Avoidance of sexual contact with infected individual.
• Use of antibiotic prophylaxis : penicillin

68. Borrelia vincentii
• Morphology : 7 -18µm long, 0.2 -
0.6µm wide, with 3-8 loose open
coils of variable size, motile
• Cultural characteristics: Gram
negative, stained with dilute carbol
fuchsin, methyl violet, Giemsa &
Leishman stains, obligate anaerobe,
cultured in digest broth enriched
with ascitic fluid.
• Pathogenesis: gives rise to ulcerative
gingivostomatitis or oropharyngitis
(vincent’s angina)
• Diagnosis: smears from ulcerative
lesions stained with dilute carbol
fuchsin