presentation on spirochetes for undergraduate students.

1. Spirochetes
Treponema pallidum

2. Spirochaetes
• Elongated Motile Flexible helical bacteria
• twisted spirally along the long axis
•
•
•
•
•
Spira
=
coil
Chaite
=
hair
Structurally more complex
They are free living organisms found in water and sewage.
Presence of endoflagella situated between outer membrane
and cell wall- responsible for motility

3. Human pathogens
• 3 genera
• Treponema
– Trepos = to turn
– Nema = thread
• Borrelia
• Leptospira

4. Treponema
•
•
•
•
Treponema pallidum Syphilis
Treponema pertenue Yaws
Treponema carateum Pinta
Treponema endemicum
Endemic syphilis

5. Treponema pallidum
•
•
•
•
•
Causative agent of Syphilis
STD
Thin delicate spirochete with tapering ends
10 µm long 0.1-0.2 µm wide
10 regular spirals – sharp angular at regular intervals of 1 µm
Actively motile, exhibiting rotation around long axis –
backward and forward movement with flexion of whole body

6. Staining
• not stained by ordinary
stains
• Giemsa stain – stains light
rose red
• Silver impregnation method
• Negative stain – Indian Ink
• Morphology and motility
can be seen under dark
ground microscope or
phase contrast microscope

7. Structure
• Cell wall – peptidoglycan which gives the cell rigidity
and shape
• Trilaminar cytoplasmic membrane
• Outer membrane – lipid
• Endoflagella – 3-4 in number wind around the axis of
the cell in the space between cell wall and outer
membrane

8. Culture
• Do not grow in artificial media
• Limited growth in tissue culture
• Virulent strains have been maintained by serial testicular
passage in rabbit – Nichol’s strain
• Non pathogenic treponemes show morphological and
antigenic similarities with treponema pallidum – REITER
STRAIN
• Reiter treponemes grow well in Thioglycollate medium
containing serum
• Reiter treponemes have been widely used as the antigen
in group specific treponemal tests for the diagnosis of
Syphilis

9. Resistance
• T.pallidum is a very delicate organism
• Inactivated by drying/heat 41-420C/hr
• Susceptibility to heat was the basis of the ‘fever therapy’ for
syphilis
• Killed in 1-3 days at 0-40 C. So that transfusion syphilis can be
prevented by storing blood for at least 4 days
• Inactivated by contact with oxygen, soap, arsenicals,
mercurials, bismuth, common antiseptics and antibiotics

10. Antigenic structure
• Cardiolipin antigen-common antigen for treponemes
• Group specific antigen-Found in T.pallidum
• as well as in non pathogenic cultivable Reiter
treponemes
• Species specific polysaccharide antigen- antigen
specific for Treponema pallidum

11. Cardiolipin antigen
• Chemically a diphosphatidyl glycerol
• This lipid has been detected in T.pallidum
• Used as antigen in the standard tests for syphilis
[STS] or nonspecific tests for syphilis
– Wassermann test
– Kahn test
– VDRL test

12. REAGIN ANTIBODY
• The antibody that reacts with the Cardiolipin antigen
–Reagin antibody
• It is not known whether the Reagin antibody is
induced by cardiolipin that is present in the
spirochete or released from damaged host tissues
• Reagin antibody detectable 7-10 days after primary
chancre

13. Syphilis
• Sexually transmitted disease
• Infective dose - as few as 60 treponemes
• Incubation period 10- 90 days
3 stages
– Primary
– Secondary
– Tertiary

14. Primary Syphilis
• Presence of Chancre at the site of entry of
spirochete
• Chancre is a painless, relatively avascular,
circumscribed, indurate, superficially
ulcerated lesion
• Common sites – genitalia, mouth, nipples,
• Chancre is covered by a thick, glairy exudate,
very rich in spirochetes

15. Chancre

16. Primary Syphilis…..
• Regional lymph nodes are swollen, discrete,
rubbery and non tender
• It heals in 10-40 days even with out treatment
leaving a thin scar
• Even before the chancre appears, the
spirochetes spread from the site of entry into
the lymph and blood steam
• Patient may be infective

17. Secondary Syphilis
• Sets in 3 months after primary lesion heals
• During this interval patient is asymptomatic
• Secondary lesions are due to wide spread
multiplication of the spirochetes and their
dissemination through blood

18. Secondary Syphilis…..
• Spirochetes are abundant in the lesions
• Patient is most infectious
• Roseolar or papular skin rashes , mucus
patches in the oropharynx
• Condylomata at muco-cutaneous junctions
are the characteristic lesions

19. Roseolar skin rashes

20. Secondary Syphilis……
• There may be ophthalmic, osseous, and
meningeal involvement
• Secondary lesions are highly variable in
distribution, intensity and duration. But they
usually undergo spontaneous healing in some
instance 4-5 years

21. Latent Syphilis
• After the secondary lesions disappear, there is
a period of quiescence known as latent
syphilis
• Diagnosis during this period is possible only by
serological tests

22. Tertiary Syphilis
• After the period of latent syphilis
in many
cases
natural cure
• Others manifestations of tertiary syphilis
• CVS
syphilitic aneurysm
• Chronic granulomata
gummata
• Meningo-vascular meningitis
• Neurological
tabes dorsalis and general
paralysis of insane [GPI]

23. Natural evolution of non venereal
syphilis
• Syphilis acquired non venereally as
occupational in doctors and nurses
• Natural evolution as in venereal syphilis
except the primary chancer is extra genital
usually on fingers

24. Transfusion syphilis
• Syphilis acquired by blood transfusion
• Primary chancer does not occur
• Can be prevented by storing of blood 0 to 40C
at least for 4 days before tranfusion

25. Congenital syphilis
• When infection is transmitted from mother to
foetus trans-placentally
• Can occur at any stage of pregnancy
• A woman with early syphilis can infect her
foetus much more commonly -75to 95%
• The lesion of congenital syphilis usually
develops only after the 4 th month of
gestation – at the time of foetal immune
competence begins

26. • Congenital syphilis can be prevented if the
mother is given adequate treatment before 4th
month of pregnancy
• The obstetric history of an un treated
syphilitic woman is typically one of abortions
and still birth followed by live birth of infants
with stigma of syphilis and finally of healthy
infants

27. Hunterian chancer
• Primary lesion that appears in syphilis
• It is painless relatively avascular circumscribed
indurated superficially ulcerated lesion
• Named after John hunter who produced the
lesion on himself experimentally and
described the evolution of disease

28. Laboratory diagnosis of syphilis
1. Microscopy
2. Detection of antibodies in serum
a)
b)
I.
Test for antibodies reacting with cardiolipin antigen
I.
II.
VDRL( Venereal Disease Research Laboratory test )
RPR ( Rapid Plasma Reagin Test)
I.
RPCF (REITER PROTIEN COMPLIMENT FIXATION TEST)
Not in use now
Test for antibodies reacting with group specific antigen
Test for antibodies reacting with species specific antibodies
I.
II.
III.
IV.
TPI( T.pallidum Immobilisation test)
TPIA (T.pallidum Immune adherence test)
TPA ( T.pallidum Agglutination test )
TPHA ( T.pallidum Haemagglutination assay )

29. VDRL
• Developed in New york
• Test is done on VDRL slide –
• To the Inactivated serum [heating serum at
550C]taken in dilutions 1 drop of cardiolipin antigen is
added
• Mix well in VDRL rotator [ 180 rpm/4mts ]
• Visible clumps/floccules appear on the slide if the
patient serum contain antibody
• Seen under low power microscope
• Serial dilution to determine Ab titer in positive cases

30. VDRL
VDRL slide
VDRL rotator

31. RPR(Rapid plasma reagin test)
• Almost similar to VDRL
• Finely divided carbon particles added to
cardiolipin antigen
1.
2.
3.
4.
unheated serum/plasma can be used
A finger prick sample of blood is sufficient
No need for microscope for reading
commercially available kit
It cannot be used with CSF

32. TRUST
•
•
•
•
Toludine red unheated serum test
Modified RPR test
Commercially available kit
Toludine red particles used instead of carbon
particles
• Automated RPR and automated VDRL- Elisa
have been developed
• Used for large scale test

33. Treatment
• Benzathine penicillin G
– Single injection 2.4 million units in early cases
– Repeated wkly x 3 wks in late cases
• In patients allergic to penicillin
– Erythromycin
– Tetracycline
– Ceftriaxone

34. Side effects
• Jarisch –Herxheimer reaction
• Fever, malaise, exacerbation of symptoms
• Due to liberation of toxic products from the
massive destruction of treponemes or due to
hypersensitivity

35. Immunity
• Re-infections do not appear to occur in a
person already having active infection
• Premunition immunity / Infection immunity
• A patient become susceptible to re-infection
only when his original infection is cured

36. Leptospira

37. • Described by Weil & Stimson
• Leptospires are actively mobile, delicate spirochetes
possessing large number of closely wound spirals &
characteristic hooked ends.
• Leptospires
– Saprophytic species
– Pathogenic species
• Pathogenic species are grouped under leptospira interrogans
& saprophytic species grouped under leptospira biflexa
• Leptospira interrogans classified into serogroups based on
surface antigens.

38. Leptospira
Vector
Disease
Icterohemorrhagica
Rat
Weil’s disease
Canicola
Dog
Canicola fever
Grippothyposa
Field mice
Swamp or marsh fever
Pomona
Pig
Swine heard’s fever
Hebdomadis
Field mice
Seven day fever
Fortbragg
Pretibial fever
Pyrogenes
Pig
Febrile spirochetosis
Bataviae
Rat
Indonesian weil’s disease
Hardjo
Cattle
Dairy farm fever

39. Morphology
•
•
•
•
•
Delicate flexible helical rods 6-20μm long & 0.1 μm thick
Actively motile
Aerobic & microaerophillic
Stain poorly with aniline dyes
Numerous coils close to each other with a distinct hooked
ends resemling umbrella handle.
• Can be stained with with giemsa’s stain
• Better visuvalized using silver impregnation method

41. Culture
• Optimum temperature 25-30°C
• pH 7.2 – 7.5
• Generation time
– In culture  12-16hours
– In animal inoculation  4-8hours
• Media used  EMJH (Ellinghausen, Mc Collough, Johnson, Harris)
• Other media used  enriched media with rabbit serum
– Korthof’s
– Stuarts
– Fletcher’s medium

42. • Animal inoculation
• Chorio-allantoic membrane of chick embryo
• Demonstration in blood of allantoic vessels 4-5days
after inoculation
• Intra peritoneal inoculation of guinea pigs
• Culture of heart blood 10 mins later

43. Clinical features
•
•
•
•
Evidence of inapparent infection in 15-40%
Incubation period is 2-20days
90% of symptomatic patients develop anicteric leptospirosis
10% of symptomatic patients develop weil’s disease ( icteric leptospirosis)
•
Anicteric Leptospirosis
– Flu like syndrome
– Classically severe muscle pain in calves, back & abdomen
– Intense headache in frontal or reterobulbar region
– Pulmonary symptoms – cough & chest pain
– Eye – conjunctival congestion
– Others  pharyngeal congestion, rash, hepatomegaly,
spleenomegaly, mild jaundice.

45. Weil’s Disease

46. •
•
•
Jaundice – severe
– Gives an orange cast to the skin
– Not associated with hepatic necrosis
– Hepatomegaly
– Spleenomegaly in 20 %
Renal dysfunction
– Seen during 2nd week of illness.
– Mainly present with acute tubular necrosis with oliguria or anuria.
– Renal function is completely reversible
Hemorrhagic diathesis
– Epistaxis , Petechiae, Purpura, Ecchymosis
– Rarely severe G.I bleed, adrenal or subarachanoid hemorrhage
– Fatal consequences due to Rhabdomyolysis, Hemolysis, Myocarditis,
Pericarditis, Congestive heart failure, Cardiogenic shock, ARDS, Necrotizing
pancreatirtis& Multi organ failure.

47. Lab Diagnosis





Demonstration of spirochetes in blood in early stages
Demonstration of spirochetes in urine in later stages
Isolation in culture
Animal inoculation
Serological tests.

48. •
Examination of blood
– Useful in the first one week of disease & before antibiotic
administration.
– Dark field microscopy & Immunofluroscence is practically difficult.
– Culture 3-4drops of blood inoculated in EMJH media @ 37°C for
2days and left thereafter at room temperature in dark for 2 weeks.
• Samples from culture examined every 3 days
– Animal inoculation
• Intraperitoneal inoculation into young guinea pigs
– Virulent strain – death in 8-12 days.
– Less virulent strain

49. •
Urine examination
– Appear in urine from 2 week and then intermittently upto 4-6 weeks.
– Urine sample should be examined immediately to prevent lysis by
acidic urine
– Centrifuges urine deposit examined by dark ground illumination

51. Treatment & Prophylaxis
•
•
Penicillin or tetracycline
Jarisch herxheimer like reaction
•
•
•
Rodent control
Disinfection of water
Protective clothing
•
Vaccination is serotype specific  success rate is unpredictable

52. Borrelia

53. borrelia
• Borrelias are a species of spirochetes of which many
are Commensals of buccal & genital mucousa.
•
•
•
•
•
•
Relapsing fever – borrelia recurrentis
Lyme’s disease – borrelia burgdorferi
Vincent’s angina- borrelia vincentii
Neuroborreliosis – borellia garinii
Arthritis & chronic skin lesions – borellia afzelii
Normal commensal – borrelia buccalis.

54. Morphology
• Gram negative spirochete, they are large,
motile with 10 axial filaments attached at
both ends of the organism.
• They are refractile spirochetes with irregular
wide open coils.
• They are usually 10-30μm in length and 0.30.7μm wide.

55. Name
East African relapsing
fever
Asian relapsing fever
Causative organism
B.duttonii
Vector
Ornithodoros moubata
b.persica
Ornithodoros tholozanii
Relapsing fever in USA
b.turicatae
o.turicatae
Lyme’s disease
b.parkeri
b.hermsii
b.burgdorferi
o.parkeri
o.hermsii
Ixodes dammcnii – USA
Ixodes pacificus – USA
Ixodes ricinus – Europe &
switz
Vincent’s angina
b.vincentii

56. How to grow?
• Barbour Stoenner Kellymedium ( BSK II)
• Growth best at 34°C
• Spectra of microaerophillic spirochetes( B.Burgdorferi) to
anerobic spirochetes (B.vincentii)
• Borrelia duttoni can be maintained in ticks for a long period in
a sand box kept at room temperature.
• Ticks feed on new born mice once a year. ticks remain
infective and borrelias retain their virulence for many years.
• Borrelias have been preserved in ticks & lice deep frozen at
-76°C

57. Relapsing fever
•
•
•
•
•
•
•
•
•
•
•
Tick borne infection
Soft ticks can survive as long as 10 years with occasional blood meal.
Incubation period 7-10days
Clinical features – patients present with headache, myalgia, chills, nausea, arthralgia,
vomiting, abdominal pain, confusion, dry cough, eye pain, diarrhoea, dizziness,
photophobia, neck pain.
Arthralgia is severe involving small & large joints, but no evidence of evidence of
arthritis.
Fever is generally >104°C and irregular pattern. Tachycardia +, some patients may have
symptoms of meningeal irritation.
Eye – congestion, photophobia, icterus
Skin & mucous membrane – signs of dehydration, petechiae over trunk, extremities &
mucous membrane.
R/S - non productive cough
CVS – high output state with summation gallop
Hepatomegaly, spleenomegaly seen in acute phase, epistaxis, blood tinged sputum
may be seen.

58. •
Fever –
Chill phase – rigors, rising
temperature, hypermetabolism
Flush phase – falling
temperature, diaphoresis &
decreased circulating blood
volume.
Patients may have bouts of fever
with or without spirochetemia in
the first 1 week of convalescence.
One to two relapses can be seen
in louse borne RF
up to 10 relapses in tick borne RF.

59. • Differential diagnosis –
• Typhus fever, typhoid fever, non typhoid
salmonellosis, malaria, dengue,TB, leptospirosis, viral
haemorrhagic fevers.
Doxycycline or tetracycline
• Treatment –
Erythromycin or chloramphenicol
Supportive care

60. Lab diagnosis
• Relapsing Fever
• Sensitivity of staining method is 70%
– Thin & thick smear examination
• Thin smear  Giemsa staining
• Thick smear Leishman staining
 Dark field microscopy

62. Clinical features
• Lyme’s disease
•
•
•
Stage 1 – tick bite elicits classical expanding erythematous skin lesion.
– Spirochete cultivation at this stage is relatively easy
Stage 2 – disseminated infection.
– May present with acute arthritis, purulent meningitis.
– secondary skin lesions , infection of the eye, Hepatitis, Myocardial
damage may occur
Stage 3 (Chronic phase)
– Chronic skin lesions
– Chronic neurologic symptoms
– Chronic arthritis
– Severity of arthritis may come down with every episode

64. • Lyme’s disease
• Examination of clinical specimens  isolates from skin lesions,
blood, CSF & synovial fluid
• Chances of cultivation of Borrelia burgdorferi minimal using BSK
medium
• INDIRECT IMMUNOFLUORESCENT ANTIBODY TEST
– Increase in IgM from 3-6th week of illness confirms diagnosis
– Increase in IgG is slow & sustained which can be used as a
measure of chronicity
– Persistant rise in IgM indicates persistent infection
• Rarely animal inoculation methods are used.

65. • Vincent’s angina
• Microscopic examination of stained smears if exudates from
the ulcers.
• Fusiform bacilli with gram negative bacilli confirms diagnosis
• Fusospirochetosis.
• Culture – swabs of exudates from mouth lesion inoculated
into digest broth (Hartley’s broth enriched with ascetic fluid).
Culture is incubated anerobically at 28-30°C. Culture is
examined daily for growth.