This document provides information on Treponema pallidum, the spirochete bacterium that causes syphilis. It discusses the morphology, cultivation, antigenic structure, and pathogenesis of T. pallidum. It also describes the stages of syphilis infection including primary, secondary, and tertiary syphilis. The document concludes with an overview of laboratory diagnosis of syphilis including microscopy, staining techniques, and various serological tests.

1. TREPONEMA PALLIDUM
RUKSANA A JABBAR
BACTERIOLOGY 2ND SEMESTER

2. SPIROCHETES
• structurally complex than other bacteria
• gram negative,elogated ,
f
lexible and twisted.
• many are free living saprophyte ,obligate parasites ,may be
aerobic ,anerobic or facultative
• charaterestic feature :presence of varying number of
endo
f
lagella ,situated between outer membrane and cell wall.
• vary widely in size,long as 500 ,other as short as 5.
• reproduction ;by transverse
f
ission.

3. • belongs to the order spirochetales ,comprising two families
1. spirochaetaceae : spirochaeta,cristispira,treponema ,borrelia
2. leptospiraceae :leptospira
• human pathogens are found in genera treponema,borrelia and leptospira
• spirochaeta are saprophyte found in water ans sewage ,
• cristispira are found in molluscs.

4. TREPONEMA
✴TREPONEMES ; trepos meaning to turn ,and nema meaning thread.
✴short slender spirochetes with
f
ine spirals and pointed or rounded ends
✴some of them are pathogenic ,some are commensals in mouth ,intestines, and
genitalia.
✴treponemes cause following disease in humans:
1. venereal syphilis : T. pallidum
2. endemic syphilis : T. pallidum T. endemicum
3. yaws : T. pertenu
4. pinta : T. carateum

5. TREPONEMA PALLIDUM
✴causative agent of syphills
✴discovered by Schaudinn and Hoffmann 1905: in the chancres and
inguinal lymph nodes of syphilitic patients .
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6. MORPHOLOGY
• Thin,delicate spirochete with tapering ends.
• 10 micrometer long and 0.1 -0.2 micrometer wide
• it has about 10 regular spirals,which are sharp and angular at regular intervals
of 1 m
• actively motile, gram negative,
• morphology and motility can be seen under dark ground or phase contrast
microscope.
Caption

7. live treponema can’t be seen in light microscope.
staining methods
• stained with Giemsa stain — light rose red
• it can be stained by silver impregenation method
• fontana’s method for films
• levaditi’s method for tissue secretions.
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9. ULTRA STRACUTRE
• cytoplasm of T. pallidm is surrounded by trilaminar cytoplasmic
membrane.
• enclosed by cell wall containing thin peptidoglycon.—gives cell rigidity
and shape
• external to this — a lipid rich outer membrane
• endo
f
lagella(3 or 4 )—— space between cell wall and outer membrane
layer.
• do not protrude outside, remain within the outer membrane layer

10. Caption

11. CULTIVATION
✴Do not grow in arti
f
icial media
✴Limited growth — tissue culture cells.
✴Maintain T.pallidum motile and virulent form — for 10 to 12 days in complex
media.
✴T. pallidum strains maintained by serial testicular passage in rabbits.
✴Nicholas strain —diagnostic and research purposes.
✴Reiter strain. —non pathogenic treponemes,shows antigenic and
morphological similarities with T. pallidum.
• this strain well grow in thioglycollate medium

12. RESISTANCE
• T. pallidum very delicate ,rapidly inactivated .
T. pallidum ————————————————————————>>kill
inactivated by antiseptic agents ,soap ,distilled water and contact with oxygen .
transfusion syphilis prevented by —1 to 3 days at 0- -4 degree celsius
stored frozen at -70 degree celsius in 10% glycerol
liquid nitrogen -130 degree celsius
remain viable for 10 to 15 years
drying by heat 41-42 in 1hr,

13. ANTIGENIC STRUCTURE
✴Antigenic structure of T. pallidum is complex .
✴infection induces at least 3 type of antibodies
1. reagin antibody— react with standard or non speci
f
ic test for syphilis.
wassamann ,khan, and VDRL
- hapten extracted from beef heart is used as antigen.
- lipds hapten called cardiolipin ,chemically disphopatidyl
glycerol.
- deteted in T. pallidum.

14. 2.Group Antigens — antigens found in T. pallidum &other nonpathogenic
cultivable treponemes. eg : Reiter treponemes.
3.Polysacchride in nature, species speci
f
ic ,
- the antibodies to this antigen is demonstrated by speci
f
ic T. pallidum
tests.
- positive only with sera of patients infected with pathogenic treponemes.

15. PATHOGENISITY
• Natural infection with T. pallidum only I’m humans.
• causative agent of syphilis.

16. SYPHILIS
1. VENERAL SYPHILIS (acquired by sexual contact)
➡primary syphilis
➡secondary syphilis
➡tertiary syphilis
➡late tertiary or quaternary syphilis.
2. NON VENERAL SYPHILIS. (non sexually)
➡acquired syphilis
➡transmitted by blood transfusion
➡congenital syphilis

17. VENERAL SYPHILIS
• aquired by sexual contact.
• site of infection : genital area of both female and male.
• enters the body through minute abrasions on the mucosa or skin.
• During the
f
irst 2 years, the infectivity of a patient to the sexual partner -
primary, secondary, early latent stages.
• generation time — 30-33 hrs.
• symptoms can bee seen after incubation time - about 1 month (10-90
days.)

18. Veneral syphilis
primary syphilis secondary syphilis tertiary syphilis late tertiary
HARD CHANCRE FORMATION
➡at the site .few in genital, and
others include mouth nipple
➡painless avascular ,indurated
circumscribed ,super
f
icially
ulcered lesions.
➡chancre covered by a thick
glairy exudate with very rich
spirochetes
COMPLICATIONS
REGIONAL LYMPH NODES
swollen ,discrete ,rubbery and
nontender
SPREAD FROM SITE OF ENTRY
INTO THE LYMPH AND BLOOD
STREAM
• before chancre appear.
➡heals 10 to 40 dys. without
treatment
➡SETS IN 3 MONTHS AFTER THE
PRIMARY INFECTION HEALS.
➡patient is asymtomatic
➡secondary lesions are due to
multiplication and wide spread of
organisms through disamaination
of blood
PAPULAR OR ROSEOLAR SKIN
RUSHES.
MUCOS PATCHES —
OROPHARNYX
CONDYLOMATA —
MUCOCUTANEOUS JUNCTION
➡ secondary lesions are highly
infectious.Highly veriable in
distribution.
COMPLICATIONS
➡OPTHALMIC ,OSSEOUS,MENIN
GEL INVOLVMENT
➡heals spontaneously
➡some cases take time —4 or 5 yrs.
➡after secondary lesions heals
a period of quiescence
known as latent syphilis.
➡diagnosis possible by
serological tests.
➡many case this is a natural
cure
➡some instance tertiary
syphilis appear after several
years.
➡delayed hypersensitivity due
to the tertiary lesions
contains few spirochetes
➡COMPLICATIONS
CARDIOVASCULAR
LESIONS —ANEURYSMS
CHRONIC GRANULOMATA
(GUMMATA)
MENINGOVASCULAR
MANIFESTATIONS.
➡include NEUROLOGICAL
MANIFESTATIONS—
➡TABES DORSALIS
➡PARALYSIS
➡It develops after a long period of
initial infections.

19. non veneral syphilis
acquired syphilis transmited
by blood
transfusion
congenital syphilis
➡acquired by non venerably
➡occupationally in doctors
and nurse
➡primary chancre develops
extragenital—on the
f
ingers.
➡In rare instances —it may
transmitted through blood
transfusion
➡primary chancre does not
occur.
➡transmitted from mother to foetus
transplacentally
➡transmission take place at any stage OF
pregnancy
➡lesion develops only after 4 months of
gestation period.
PREVENTION
➡mother is given adequate treatment
before 4 month of pregnancy
➡untreated syphilitic women-abortions
and still births.

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22. LAB DIAGNOSIS
• staining and microscope
• culture -do not grow in arti
f
icial media.
• serological tests.
✓non speci
f
ic test
✓group speci
f
ic test
✓speci
f
ic test

23. MAJOR SEROLOGICAL TEST FOR SYPHILIS
A. non speci
f
ic test using cardiolipin antigen (reagin antibody test)
1. wassermann CF reaction
2. khan
f
loculation
3. VDRL
4. RPR
5. automated RPR
6. VDRL -ELISA
B. group speci
f
ic test using cutivable treponemal antigen (reiter strain)test
1. reiter protein cf test RPCF
C. speci
f
ic tests using pathogenic treponemes (T.pallidum)
1. T.pallidum immobilieration assay TPI
2.
f
luorecent treponema antibody absorption test FTA-ABS
3. T.pallidum heamagglutination assay TPHA
4. T.pallidum enzyme immunoassays TP EIA

24. STAINING and MICROSCOPY
• applicable in primary and secondary stages..and congenital syphilis
• Staining done by
➡geimsa staining
➡silver impergnation method
➡fontana method -staining
f
ilms
➡levaditi’s method -tissue sections

25. • specimen : serum and csf ,lesions (congenital syphilis),exudates (pus)
• specimens should be collected with care as the lesions are highly
infectious .
• lesions cleaned with gauze soaked in warm saline
• and the margins gently scraped
• super
f
icial epithelium is abraded.
• gentle pressure is applied to the base of the lesion
• serum that exudes us collected preventing admixture with blood.

26. • wet
f
ilms (dark ground microscope)
• exudate and after applying thin coverslips
• examined under dark
f
ield microscopy
• identi
f
ied by its slender spiral structure and slow movement
• advantage
• useful, but negative result occur because of low sensitivity and
concentration 104 per ml in the exudates is required for the test is
positive.

27. • direct
f
luorescent antibody test for T.palidum (DFA TP)
• better and safer method for microscopic diagnosis.
➡smear of the exudate are
f
ixed with acetone
➡sent to laboratory
➡
f
luoroscent tagged anti T.pallidum antiserum

28. • CSF EXAMINATION.
• neurologic ophthalmic manifestations.
• evidence of active tertiary syphilis
eg:gummata lesions.

29. SEROLOGICAL TEST
A. non speci
f
ic test
1. reagin antibody test
• standard test for syphilis
• These tests detect reagin antibody, a non specific antibody that is present in syphilitic
serum.
• It appears in a patient’s serum in 10-14 days after exposure.
• The antigen is cardiolipin antigen which contains alcoholic extract of ox heat muscle to
which cholesterol and lecithin are added .
• washerman compliment fixateon test.-1st reagin antibody test.

30. • watery extract of the liver of syphilitic foetus -as antigen.
• later substitute an alcoholic extract of ox heart tissue lecithin and
cholersterol were added
• replaced by puri
f
ied lipds extract of beef heart (cardiolipin) with lectin
and cholesterol .-by pang born
• then it replaced with simpler
f
locculation test.-use cardiolipin antigen
• CFT -reamined the principle of serological test for syphilis
• wassermann test -no longer in use now

31. 2.1st
f
locculation test khans test
3.it replaced by (VDRL VENERAL DISEASE REASEARCH LABORATORY)
➡inactivated serum (serum heated with 56 for 30 min)
➡mixed with cardiolipin antigen on a special slide
➡rotate for 4 min
➡visible clumbs formed ,cardiolipin combined with reagin antibody.
➡read under low power microscope
➡antibody titre can be determined by testing serial dilution.

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36. 4. RPR (rapid plasma reagin test
• The Rapid Plasma Reagin (RPR) test is a macroscopic, non-
treponemal, flocculation card test.
• that detect antibodies produced against antigens released by damaged host
cells in patients suffering from syphilis
.
• In the test, the RPR antigen is mixed with unheated or heated serum or with
unheated plasma on a plastic-coated card
.
• the antigen used for detection contains 0.03% cardiolipin, 0.21% lecithin, and
0.9% cholesterol in addition to choline chloride, EDTA and charcoal particles.
• If antibodies are present, they combine with the lipid particles of the antigen,
causing them to agglutinate.
• The charcoal particles coagglutinate with the antibodies and show up as
black clumps against the white card
.
If antibodies are not present, the test mixture is uniformly gray.

37. Procedur
e
1.Bring the RPR carbon antigen suspension, controls and samples to room temperature
.
2.Pipette one drop (50 µl) of the test specimen, positive and negative controls onto
separate reaction circles of the disposable slide
.
3.Add one drop of well-mixed RPR reagent next to the test specimen, positive control
and negative control
.
4.Using a mixing stick mix the test specimen and the RPR reagent thoroughly spreading
uniformly over the entire reaction circle
.
5.Rotate the slide gently and continuously either manually or on a mechanical rotor at
180 r.p.m
.
6.Observe for flocculation macroscopically at 8 minutes.

38. Caption
Caption

39. biological false reaction.
• cardiolipin antigen present in both T.pallidam and mammals tissues.
• polyclonal antiphopholipodial antibodies produced against lipodal
antigen present in normal serum.
• this condition that destroy cell nuclei .
• 2 type
• acute and chronic

40. • Acute Reactions.
• lasts less than few weeks or 6 months
• due to various associated infections ,injuries or in
f
lammatory conditions.
• narcotic abuse immunization procedures and pregnancy

41. • CHRONIC REACTIONS
• lasts 6 months
• associated with leprosy,malaria relapsing fever ,hepatitis.
• The prozone phenomenon
• a false-negative response arising from cases in which high antibody
titers interfere with the antigen-antibody lattice network formation
• associated with pregnancy and neurosyphilis.

42. • B group speci
f
ic treponema test
• test using cultivable treponemes as antigen were developed (Employed the Reiter
treponemes.)
• RPCF test (reiter protein compliment
f
ixation)
➡using a lipopolysaccharide -protein complex derived from the treponeme
➡sensitivity speci
f
icity lower than other T palladium tests.
➡RPCF free from BFP reaction..but still gave some false reaction
➡RPCF and reiter treponeme tests are not now in general use

43. C speci
f
ic T. palladium test.
• use the virulent Nichols strain of T.pallidum
• maintained by serial inoculation in rabbit tests.

44. 1.TPI Treponema pallidum immobilisation test
• 1st introduced in 1949
➡These are tests that detect antibody to T. pallidum subsp. pallidum and other species.
➡ The test uses live treponemes which when mixed with the patient’s serum, the
antibody in the serum immobilizes the organisms when examined under the
darkground microscope..
➡
• The disadvantage is that there is always the risk of infection to the worker, and
extreme complexity
• test is as positive —- 50 or more treponemes immobilised
• negative —20 or less.
• false reaction occur -antitreponemal drug may show false positive reaction
• TPI golden standard test in serology
• TPIA ,TPA TEST ARE NOT USED.

45. • 2.FTA ABS f(luorescent trepnemal antibody absorption test)
• (FLOURESCENT TREPONEMAL ANTIBODY TEST) FTA
• this fat abs modi
f
ied from FTA
• The test uses Nicholl’s strain as antigen
f
ixed on a slide.
• Diluted patient’s serum is added on to the antigen, excess washed off and
the smear treated with anti human immunoglobulin conjugate.
• After incubating and washing, the slide is examined under the
f
luorescent
microscope.
Caption

46. • 3.TPHA treponema pallidum heamagglutination assay
• test is similar to the FTA in sensitivity.
• Antigen is Nicholl’s strain coated with tanned turkey or chicken or sheep red blood cells ( sensitised cells).
A non sensitized cell suspension is used as control.
• In the presence of treponemal antibody, the treponemes adhere to the sensitized red cells and settle at
the bottom of the micro titre plate well as orange to red layer.
• It is easy to perform, fast and cheap. simpler and standerd confirmation test.
• MHA TP microheamagglutination test —-this procedure now used in this test
• VDRL CSF -neurosyphilis
• TPHA AND FTA ABS ——-conforming the diagnosis of syphilis

47. • 4 .EIA
• rapid agglutination test.
• using T.pallidum antigens and are available commercially
• using latex particles coated with 3 immunodominet proteins of T.pallidum
• obtained by recombination technology.
• its speci
f
ic test more sensitive.

48. • detection of Ig M
• detateble by the 2nd week of infection.
• neonatal serum contain IgM -confrms congenital syphilis.

49. EPIDEMIOLOGY
• world wide distribution
• transmitted sexually or vertically.
• most contagious to sex partners during primary and secondary stages
• discovery of penicillin eradicated this disease.
• increase has occurred in its incidence due to
❖changing customs
❖habits
❖values in society
• AIDS pandemic has had an impact of syphilis

50. PROPHYLAXIX
• Avoidance of sexual contact with infected partner.
• use of physical barriers
• antiseptics pottasium permanganate
• antibiotics
• no vaccine available;

51. TREATMNET
• PENICILLIN is effective
• single injection of 2.4 million unit of benzathine penicillin G -in early case .
• late syphilis -this amount repeated weekly for 3 weeks.
• in patients allergic to penicillin -erythromycin ,tetracycline
• neurosyphilis -ceftriaxone
• primary and secondary syphilis — bed rest
• congenital syphilis-prevented by adeqative tretment for mother before 4
month of pregnancy

52. • jarisch herxheimer reaction.
• self limited reaction to antitreponemal therapy.
• fever,malaise vomiting chills .
• occur within 24 hrs after therapy
managed with aspirin and bed rest —in primary secondary syphilis .

53. REFERENCE
• ANADANARAYANAN 6TH EDITION
• https://archive.archaeology.org/9701/newsbriefs/syphilis.html
• https://microbenotes.com/rapid-plasma-reagin-rpr-test/
• microbe note.com

54. THANK YOU