This document provides information about the bacteria Spirochaetes. It discusses their morphology, motility, reproduction, pathogenic species, diseases caused, and laboratory diagnosis. Some key points: - Spirochaetes are elongated, helically coiled, and motile bacteria with endoflagella. They can cause diseases like syphilis, Lyme disease, and leptospirosis. - Pathogenic species include Treponema pallidum (syphilis), Borrelia burgdorferi (Lyme disease), and Leptospira interrogans (leptospirosis). - Laboratory diagnosis involves darkfield microscopy, culture, serology, and

1. Arun Kumar P
Dept of Microbiology
SPIROCHAETES

2.  Elongated, motile, flexible bacteria twisted spirally along the long
axis
 Speira- coiled, chaite- hair
 Endoflagella- polar flagella wound along the helical protoplasmic
cylinder; situated between outer membrane and cell wall
 Aerobic, anaerobic or facultative, some are non-cultivable
 Reproduction by transverse fission
 Pathogenic Spirochaetes- Treponema, Borrelia, Leptospira

4. Periplasmic Flagella Diagram

5. Tightly Coiled Spirochete
AF
OS = outer sheath
AF = axial fibrils
Leptospira interrogans

6. Genus Species Disease
Treponema pallidum ssp. pallidum
pallidum ssp. endemicum
pallidum ssp. pertenue
carateum
Syphilis
Bejel
Yaws
Pinta
Borrelia burgdorferi
recurrentis
Many species
Lyme disease (borreliosis)
Epidemic relapsing fever
Endemic relapsing fever
Leptospira interrogans Leptospirosis
(Weil’s Disease)
Spirochaetales Associated Human Diseases

7. Morphology:-
10µm long and 0.1 to 0.2 µm wide
Ten regular spirals with sharp and angular at regular intervals
of about 1 µm
Actively motile, exhibiting rotation around the long axis,
backward and forward movements and flexion of whole body
During motion, secondary curves appear and disappear in
succession but the primary spirals are unchanged
Cannot seen under light microscope in wet films
Morphology and motility seen under dark ground or Phase
contrast microscope
It can be stained by SLIVER IMPREGNATION or
FONTANA staining methods
Levaditi’s method for tissue section
Treponema pallidum

8. Cultivation of Treponema
 Do not grow in artificial culture media
 Nichol’s strain- virulent strain of T.pallidum maintained by
serial passage in rabbits; isolated from a case of General
Paralysis of Insane in 1912.
 Reiter’s strain- T.phagedenis ( non-pathogenic) used for
group specific tests; grows well in thioglycollate medium with
serum

9. Resistance of Treponema
 Very delicate
 Readily inactivated by drying and heat
 Killed in 1-3 days at 0-4oC
 Remains viable for 10-15 years frozen at –70oC in 10% glycerol,
or in liquid nitrogen (– 130oC)
 Inactivated by contact with distilled water, oxygen, soap,
bismuth, mercurials, arsenicals, common antiseptic agents and
antibiotics

10. Antigenic Structure
 Complex antigenic structure
 3 types of antigens:
 Reagin antigen- hapten extracted from beef heart is used as antigen
to detect these antibodies; antigen is cardiolipin- chemically
diphosphatidyl glycerol
 Group antigen- found in T.pallidum and non-pathogenic strains e.g.
Reiter’s strain
 Species specific antigen- polysaccharide in nature; antibodies seen
only in sera of patients with pathogenic T.pallidum infection

11.  Diseases caused:
Venereal syphilis - T.pallidum
Non-venereal treponematoses:
T.pallidum- endemic syphilis
T.pertenue - yaws
T.carateum - pinta
Transmission- sexual, trans-placental
Primary: chancre- hard, painless, avascular covered by exudate
rich in spirochaetes; regional lymph nodes are discrete, rubbery
and non-tender
Seconadary: 3 months after primary lesion heals- roseolar/
papular skin rashes, condylomata at mucocutaneous junctions,
mucous patches in oropharynx; patient is most infectious in this
stage; ophthalmic, osseous, meningeal involvement
Venereal syphilis
• primary lesion - chancre
– 10 to 60 days
– area of ulceration/
inflammation
– many organisms

12.  Latent: Period of quiescence for several years, can only be
diagnosed by serological tests
 Tertiary: Cardiovascular lesions- aneurysms, chronic
granulomata and meningovascular lesions; may represent
manifestations of delayed hypersensitivity
 Quarternary/ Late Tertiary: Tabes dorsalis or General Paralysis
of Insane

13. NON-VENEREAL SYPHILIS
 Congenital syphilis:
 Woman with early syphilis is more infective to her fetus
 Lesions usually develop after 4th month of gestation- suggests that
pathogenesis requires immune response from the fetus
 Can be treated if mother is treated adequately before 4th month of
pregnancy
 Occupational syphilis: primary chancre is absent or extragenital,
usually on fingers

14. LAB DIAGNOSIS OF SYPHILIS
Microscopy
 Applicable in primary and secondary syphilis,
and in congenital syphilis with superficial
lesions
 Serum exuding from base of lesion should be
collected
 Wet films examined under dark ground or
phase contrast microscopy
 Direct fluorescent antibody test( DFA-TP)-
better and safer than DGM

15. SEROLOGICAL TESTS
Standard tests for Syphilis/ reagin Ab tests (Non-specific)
Cardiolipin is purified extract of beef heart with lecithin and cholesterol
 Wasserman’s Complement Fixation Test- no longer in use
 Kahn Test- Tube flocculation test
 VDRL- Slide flocculation test, requires inactivated serum, seen under low
power lens of microscope
 Rapid Plasma Reagin test- Antigen contains fine carbon particles; so
reaction easily visible to naked eye, not suitable for testing CSF
 Reagin tests- negative 6-18 months after treatment

16. BIOLOGICAL FALSE POSITIVE (BFP) REACTIONS
 Positive reactions with cardiolipin tests, negative with specific
treponemal tests, in absence of present/ past infection, no technical
error
 Seen in 1% normal sera
 Acute BFP- acute infections, injury, inflammation
 Chronic BFP- relapsing fever, hepatitis, tropical eosinophilia

17. Specific T. pallidum Tests
 Strain used- virulent Nichol’s strain
 Treponema pallidum Immobilization (TPI)- positive when 50% organisms
immobilized; most specific but complex test
 Fluorescent Treponemal Antibody (FTA) test- Indirect immunofluorescent
test; Nichol’s strain used as smears on slides
 FTA-ABS - Test serum preabsorbed with sonicate of Reiter’s treponemes;
standard reference test
 Treponema pallidum haemagglutination (TPHA)- Antigen used: tanned
erythrocytes sensitized with sonicate of Reiter’s treponeme; simpler, more
economical than FTA-ABS, standard kits available; so standard confirmatory test

18. Nonvenereal Treponemal Diseases
 Bejel, Yaws & Pinta
 Primitive tropical and subtropical regions
 Primarily in impoverished children
Treponema pallidum ssp. endemicum
 Bejel (endemic syphilis)
• Initial lesions: nondescript oral lesions
• Secondary lesions: oral papules and mucosal patches
• Late: gummas (granulomas) of skin, bones & nasopharynx
 Transmitted person-to-person by contaminated eating utensils
 Primitive tropical/subtropical areas (Africa, Asia & Australia)

19. Treponema pallidum ssp. pertenue
Papillomatous Lesions of Yaws: painless
nodules widely distributed over body with
abundant contagious spirochetes.
 Yaws: granulomatous disease
• Early: skin lesions (see below)
• Late: destructive lesions of skin, lymph nodes & bones
 Transmitted by direct contact with lesions containing abundant spirochetes
 Primitive tropical areas (S. America, Central Africa, SE Asia)

20. Treponema carateum
Pinta: primarily restricted to skin
• 1-3 week incubation period
• Initial lesions: small pruritic
papules
• Secondary: enlarged plaques persist
for months to years
• Late: disseminated, recurrent
hypopigmentation or
depigmentation of skin lesions;
scarring & disfigurement
Transmitted by direct contact with
skin lesions
Primitive tropical areas
(Mexico, Central & South America)
Hypopigmented Skin
Lesions of Pinta:
depigmentation is commonly
seen as a late sequel with all
treponemal diseases

21. 21
Leptospirosis

22. LEPTOSPIRA
 Several are saprophytic, many are parasitic in rodents
 Infection in natural host- asymptomatic
 Genus Leptospira--- L.interrogans(pathogenic)
L.biflexa(saprophytic)
 6-12 m long x 0.1 m thick
 Hooked ends( umbrella handles)
 Numerous closely placed coils
 Seen under Dark ground/ Phase contrast microscopy as
actively motile
 Stained best by silver impregnation

23. CULTURE OF LEPTOSPIRA
 Aerobic, microaerophilic
 Cultivated in media with rabbit serum
 Liquid and semi-solid ( Korthof’s, Stuart’s, Fletcher’s)
 Semi-synthetic medium( Ellinghausen McCullough Johnson Harris-
EMJH medium)
 Grow on chorio-allantoic membrane of chick embryo
 Intra-peritoneal inoculation of guinea pigs

24. LEPTOSPIRA- RESISTANCE
 Killed in 10 minutes at 50oC, 10 seconds at 60oC
 Sensitive to acid and bile
 Destroyed by chlorine and most antiseptics and disinfectants
 Survival in water depends on pH, salinity, temperature, nature,
amount of pollution

25. ANTIGENS OF LEPTOSPIRA
 Genus- specific antigen common to all members of the genus
 Classification into serogroups and serovars based on surface
antigens
 L.interrogans  serogp icterohemorrhagiae  22 serovars:
 e.g. icterohemorrhagiae, pomona, canicola, grippotyphosa,
copenhageni

26. LEPTOSPIRA- PATHOGENICITY
 It is a zoonosis; humans infected when water contaminated with urine of
carrier animals enters body through cuts/ abrasions/ intact mucosa
 Incubation period- 10 days
 Ranges from mild undifferentiated pyrexia  Weil’s syndrome( severe
disease with hepatorenal damage which can be fatal)

27. CLINICAL FEATURES
 Fever with rigors, vomiting, headache, conjunctival suffusion, calf pain,
purpuric haemorrhages
 Albuminuria, jaundice seen in 10-20%
 Aseptic meningitis or abdominal symptoms may predominate
 Mild virus-like syndrome
 Anicteric leptospirosis: Systemic with aseptic meningitis
 Icteric leptospirosis: Overwhelming disease (Weil’s disease)
Vascular collapse
Thrombocytopenia
Hemorrhage
Hepatic and renal dysfunction
NOTE: Icteric refers to jaundice (yellowing of skin and mucus
membranes by deposition of bile) and liver involvement

28.  Leptospirosis, also called Weil’s disease in humans
 Direct invasion and replication in tissues
 Characterized by an acute febrile jaundice & immune complex
glomerulonephritis
 Incubation period usually 10-12 days with flu-like illness usually progressing
through two clinical stages:
i. Leptospiremia develops rapidly after infection (usually lasts about 7
days) without local lesion
ii. Infects the kidneys and organisms are shed in the urine (leptospiruria)
with renal failure and death not uncommon
 Hepatic injury & meningeal irritation is common
Pathogenesis of Icteric Leptospirosis

29. LAB DIAGNOSIS- EXAMINATION OF BLOOD
 Only in early stages before antibiotic is given
 Dark ground/ Phase contrast microscopy of blood films
 Culture on EMJH/ other medium  may take weeks to months
 Inoculation into young guinea pigs
 Identification by agglutination with type specific sera
EXAMINATION OF URINE
 From 2nd to 4th/6th week
 Centrifuged deposit- neutralised with alkaline solution
 Inoculation into guinea pigs

30. SEROLOGY
 Antibodies in serum  appear at end of 1st week, increase till 4th week
 Genus specific tests: Antigen used is L.biflexa Patoc 1 strain-
-Sensitized Erythrocyte Lysis
-Complement Fixation
-Indirect Immunofluorescence
-ELISA (IgM, IgG)
-IgM specific dipstick
 Type specific tests: identify serovar
-Macroscopic Agglutination Test
-Microscopic Agglutination Test- more specific, done in reference
laboratories

31.  Diagnosis in animals- Culturing pieces of kidneys
 Examination of water
 Treatment of leptospirosis - Penicillin
- Doxycycline for prophylaxis

32. Comparison of Diagnostic Tests for Leptospirosis

33. Introduction
 Borrelia spp are large, motile, refractile spirochetes
with irregular wide open coils.
 Measuring about 0.2-0.3um in diam. & 3-20um in length.
 3-10 loose coils with 15-29 periplasmic flagella.
 Gram negative & stained well with Giemsa stain.

34. Some medically
important
borrelia-
B. recurrentis
– Relapsing
fever
B. burgdorferi-
Lyme’s
disease
B. vincenti-
Vincent
Angina.

35. Relapsing Fever
Characteristic Louseborn Tickborn
Epidemology Epidemic Usually endemic
Agent B. Recurrentis B. hermesii, B. turicatae,
B. parkeri
Route of entry Crushing & rubbing on
abraded skin
Through bite
Shedding in saliva &
discharges
No Yes
Transovarial
transmission
No Yes
Clinical features More severe Less severe

36. Borrelia recurrentis-
Morphology
-
Irregular spiral with one or both ends pointed.
Possesses 5-10 loose spiral coils at interval of about 2mm
Cultural characteristics-
Microaerophilic, temp- 28-30°C
Cultivation is difficult but can be cultivated on ‘modified Kelly’s
medium’
Grows well on CAM of chick embryos.
Inoculated in mice & rats intraperitoneally.

37. Antigenic properties-
Readily undergoes antigenic variation in vivo.
Therefore occurrence of relapses in the disease.
Antigenic variation is due to DNA rearrangements in linear
plasmids.
Recovery after no. of relapses is due to development of
immunity to all antigenic variants.

38. Clinical features-
 Onset is typically abrupt (I.P.- 2-10 days)
 High fever (40°C ) ( borrelia are demonstrable)
 Shaking chills, delirium, severe muscle aches, pain in bone & joints
 Hepatosplenomegaly
 Neurologic complications
 Fever subsides in 3-5 days
 Afebrile period (4-10 days)(disappearence)
 Relapse(reappearence)
 3-10 relapses
 Disease subsides

39. Lab diagnosis
Borrelia can be found in blood during fever
Drop of blood- Dark ground OR Phase contrast
microscopy
Blood smears- Giemsa/Leishman/dilute Carbol fuchsin
Inoculation of 1-2 ml blood into white mice & smear is
prepared from blood collected from tail of vein after 2
days, observed daily for 2 weeks.
Fluoroscent procedures
Serology & cultures are unreliable.
False positive reaction for syphilis(VDRL)

40.  Identified in 1975 in Lyme , Connecticut , USA.
 Is a most common vector born disease in USA
 Causitive agent- Borrelia burgdorferi
-B.garinii, B.afzeli

41.  Epidemology-
 Vector- Ixodid tick
 Borrelia grows mainly in midgut of the tick.
 Infection occurs by regurgitation of the gut content during
biting.
 Most commonly found in North eastern states in USA.
 No vertical transmission in ticks.
 Most effective tick stage of transmission is - nymph

42. Clinical disease-
I.P.-3-30 days.
Three stages-
1) Localized infection-
-’Erythema chronicum migrans’.
-macule at the site of bite with redness, induration.
1) Disseminated infection-
-fever, headache, myalgia , arthralgia,
lymphadenopathy.
-Most common lesions are meningitis & arthritis.
3) Persistant infection-
-Chronic skin lesions, chronic neurologic symptoms & chronic
arthritis.

43. Lab diagnosis-
 Culture- modified Kelly’s medium
-Most effective in early Lyme’s disease
 Morphologic detection- silver impregnation method
- Insensitive method.
 Molecular detection- more sensitive method
 Serologic detection- diagnostic method of choice.
-EIA, Immunofluoroscence, Immunoblot tech.
 Cross reactions-
-specific treponemal Ag, HIV, EBV, ricketssial
infections.

45. VincentAngina
Caused by borrelia vincenti .
Is a mouth commensal but may, under predisposing
conditions such as malnutrition, viral infections, give rise
to ulcerative gingivostomatitis or oropharyngitis (Vincent
angina)
In this B. vincenti is always associated with fusiform
bacilli (fusobacterium fusiforme)
Symbiotic infection is called as ‘fusospirochetosis’.

47. This symbiotic infection can be demonstrated in some of the
lung abscess, phagedenous skin ulcers & gangrenous
balanitis.
 Morphology-
 Motile spirochetes, 5-20um × 0.2-0.6um wide with 3-8 coils.
 Easily stained with dilute carbol fuchsin & is Gram negative.
Demonstration of spirochetes & fusiform bacilli in stained
smears
Culturing is difficult.
Molecular methods.

48. Thank You