Spirochaetes

MBBS students

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

3. Periplasmic Flagella Diagram

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

5. 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

6. 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

7. 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

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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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)

18. 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)

19. 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

20. 21 Leptospirosis

21. 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

22. 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

23. LEPTOSPIRA - RESISTANCE Killed in 10 minutes at 50 o C, 10 seconds at 60 o C 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

24. 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

25. 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)

26. 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

27. 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: Leptospiremia develops rapidly after infection (usually lasts about 7 days) without local lesion 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

28. 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

29. 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

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

31. Comparison of Diagnostic Tests for Leptospirosis

32. 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 .

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

34. 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

35. 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.

36. 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.

37. 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

38. 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)

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

40. 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

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

42. 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.

43. Vincent Angina 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’.

44. 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 .

45. Thank You