SPIROCHAETALES

Katalin Kristóf 2014 Spirochaetales

• 2 Familiae • Spirochetaceae • Treponema • Borrelia • Leptospiraceae • Leptospira

Common feature: ••• thin, coiled, spiral shaped bacteria Length: 10-30 µm ••• Too thin (0,1-0,2 µµµm) to be seen with light microscopy stained with Gram => Darkfield illumination; IF; silver-impregnation

• periplasmic flagellae (endoflagellae) Spirochaetales Associated Human Diseases

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

Apathogen: T. minutum, T. reiteri, T. denticola, T. phagedenis

Plaut-Vincent angina Nonvenereal Treponemal diseases (treponematosis): • Treponema pallidum ssp. endemicum ⇒ bejel (endemic syphilis) - spread person to person by contaminated eating utensils - initial oral lesions, secondary skin lesions - Africa, Asia, Australia (endemic) • Treponema pallidum ssp. pertenue ⇒ yaws - granulomatosous disease, skin lesion - South America, Central Africa, Southeast Asia • Treponema carateum ⇒pinta - skin lesions – spread by direct contact with infected lesions - South America Lues tests – positive! Vincent’s angina Ulcerative tonsillitis causing tissue necrosis often due to extension of acute ulcerative gingivitis

Fusobacterium nucleatum in combination with oral spirochates (Treponema vincentii and others) causes the fusospirochaetal infections Treponema pallidum

Morphology: - thin, coiled spirochetes (0,1 to 0,2 X 6-20 µm) - Three periplasmic flagellae are inserted at each ends (endoflagellae) - Outer membrane proteins: - TrompI, TrompII, TrompIII - Inner proteins:15kDa, 17kDa, 45.5kDa, 47kDa (endoflagellum, cytoplasma membrane, cytoplasma)

Multiplication : by binary transverse fission Cultivation: can not be cultivated on cell-free artificial culture media - Kept alive in rabbit testis ! - rabbit epithelial cells (GT 30h, only a few generations) Treponema pallidum – virulance factors, pathogenesis

• Outer membrane proteins promote adherence to host cells • Hyaluronidase may facilitate perivascular infiltration • Coating of fibronectin protects against phagocytosis • Tissue destruction primarily results from host’s immune response to infection

• Destroy cytoplasma membrane, mitochondrial membrane => Cholesterol, Lecithine, Kardiolipin Ag free ( RPR, VDRL) • Endarteriitis, Periarteriitis» inflammation, necrosis • T-cell dependent late hypersensitivity »Granuloma • Gumma I. Acquired syphilis (venereal disease) Spread: sexually (STD) The “great imitator”! 3 phases:

1) primary phase – 1-2 weeks incubaton period - The initial syphylitic chancre develops at the site where the spirochete is inoculated - Generally on the genitalia, rare: oral cavity, perianal region - localised replication of bacteria =>Papule, macule =>erodes => chancre – ulcus durum: hard, painless ulceration - Painless lymphotic nodes „bubo indolens” - mucocutan lesion is very infectious! - spontaneous remission may occur after 2-6 weeks (50%) 2. Stage develops after 4 to 8 weeks from the primer infection (haematogen spreading) disseminated disease - with generalised mucocutaneous rash , - superficial sores (mucous patches) may occur on mucous membranes of the mouth, vagina, or anus, - while wart-like lesions called condylomata lata may form in moist intertriginous areas.

- Hepatitis - Neurological signs - High fever Highly - micropolyadenopathy contagious! - spontaneous remission may occur; after 1-2 years these symptoms can reoccur latent persistence 3. Stage (late phase) after 3-30 years from the primer infection - all tissues are involved - gumma (granulomatous lesions) in bone, vessels, skin - neurosyphilis: tabes dorsalis, paralysis progressiva (encephalopathy), ataxia, dementia, N. opticus degeneration - cardiovascular syphilis: aortitis, aorta aneurysm (rupture) Progression of Untreated Syphilis

Late benign ‰Gummas in skin and soft tissues

Tertiary Stage II. Congenital (connatal) syphilis

• T pallidum subsp pallidum also damages foetuses (from 4 gw) • Approximately 50 percent of foetuses are aborted or stillborn ; • In early congenital syphilis (before the age of two years ~ II): mucocutaneous lesions, osteochondritis, anaemia, and hepatosplenomegaly. • In late congenital syphilis (> 4 years, ~III) : interstitial keratitis and blindness, tooth deformation (notched incisors and moon molars), eighth-nerve deafness, neurosyphilis, rhagades (fissures at mucocutaneous junctions), cardiovascular lesions, Clutton's joints (fluid accumulation on knee), and bone deformation of the legs, nasal septum, and hard palate.

• Can be preventing with penicillin treatment of the Treponema infected pregnant woman! early congenital syphilis late congenital syphilis • Plexus brachialis • Hutchinson's triad: paralysis • interstitial keratitis, • notched incisors • and eighth-nerve deafness

• Tibia deformation

Hutchinson’s teeth – the incisors are smaller than normal, with sloping sides and central semilunar notches. Diagnosis

• Sample: exudates, punctuates from the mucocutan lesion, (1-2 phase) • Microscopy: • Too slender (0,1-0,2 µm) to be seen with light microscopy stained with Gram • Live treponemes can be visualized by using dark-field microscopy • (rapid rotation about its longitudinal axis and bending, flexing, and snapping about its full length) • Fluorescent labelled antitreponemal antibodies • Silver-impregnation

• PCR • Serology Serology

I. Non specific treponemal II. Specific treponemal tests tests reaginic antibodies detection of specific - developed against lipids released from Immobilizin Ab ( treponemal) damaged cells during the early stage of disease and present on the cell surface of the treponema • TPIT

• KKR • FTA-Abs • VDRL • TPHA, TPPA • RPR • TP-ELISA • Western Blot, Immunoblot Ag: Cardiolipin (from extraction of Treponemal Ag: TP proteins, beef heart), lecitin, cholesterol lipoproteins I. Non –treponemal tests • VDRL-test (Venereal Disease Research Laboratory) - Ag = freshly prepared cardiolipin suspension - patient's serum is inactivated at 56 oC, for 30 min - a drop of the cardiolipin suspension is placed on a glass slide - mixed with a drop of the inactivated serum Negative: Cardiolipin suspension remain dispersed. Positive: Cardiolipin forms visible clumps when combining with reagin.

• RPR –test (Rapid Plasma Reagin) Ag = cardiolipin suspension attached to latex particles patient's serum should not be inactivated Negative: Cardiolipin-latex suspension remains intact. Positive: Cardiolipin-latex is agglutinated and sediments as rough granula - Flocculation Flocculation: granules => Ab equally Ag Dilution of patient serum! II. (Specific) treponemal tests 1. FTA-ABS = F luorescent Treponemal Antibody-absorption

• Ag = killed, fixed T.pallidum on glass slide • Overlayed with the patient’s serum, which has been mixed with an extract of nonpathogenic treponemes (T. reiteri ) • fluorescein labelled antihuman immunglobulin (IgG, IgM) • fluorescein microscopy

• The most sensitive and specific

/2.TPI = Treponema pallidum immobilisation test living T.pallidum = Antigen inactivated patient’s serum, complement of guinea pigs The reaction is based on that T. pallidum cells are inhibited in their movement if they are exposed to specific IgG antibodies in the presence of complement. Negative: T. pallidum cells exhibit locomotion. Positive: T. pallidum cells do not show movement. / II. Treponemal tests 3.TPHA, TPPA

T. pallidum ha emagglutination test • T. pallidum particle agglutination • Bird red blood cells sensitized • Gelatin particles sensitized with T. with T. pallidum antigens pallidum antigens (E.coli Tp15, Tp17, Tp 47 - (Bacillus subtilis Tp15, Tp17,Tp47 - recombinant) recombinant)

• Neg/Pos (1:80) • Neg/Pos (1:80) • Titer • Titer • IgG, IgM and IgA • IgG, IgM and IgA II. Treponemal tests: 4. TP-ELISA

• Rekombinant Ag-s Tp15, Tp17, Tp47 II. Treponemal tests: 5. Western blot

• Immunoblot strip • 15, 17, 45.5 and 47 kDa recombinant proteins • IgG, IgM BAP (biologically aspecific positivity) : Sensitivity of serological tests in untreated syphilis

Test Primary Secondary Latent Tertiary

VDRL 78 (74-87) 100 95 (88-100) 71 (37-94) RPR 86 (77-99) 100 98 (95-100) 73 FTA-ABS 84 (70-100) 100 100 96 Treponemal 76 (69-90) 100 97 (97-100) 94 Agglutination EIA 93 100 100

The use of only one type of serologic test is insufficient for diagnosis. Summary of serological tests:

For screening • TPPA/TPHA • FTA-Abs • Tp-ELISA For verification • Western blot

RPR/ VDRL • Determination of the stage • To monitor the effectiveness of therapy • To detect reinfection Treatment, control

• penicillin treatment eradicates all stages, including congenital infection in pregnancy • /Doxycyclin, Azithromycin/

• Jarisch-Herxheimer • lysis of the treponemes causes the release of huge amount antigenes => high fever, anaphylaxia, abortion • (steroid)

Prevention: safe sex • For sex partners of patients with syphilis in any stage: • Draw syphilis serology • Perform physical exam

• Congenital - Can be preventing with penicillin treatment of the Treponema infected pregnant woman – screening! Epidemiology of Borrelia Infections

Borrelia Pediculus humanus recurrentis

Ornithodoros spp. Borrelia spp.

Ixodes spp. Borrelia burgdorferi Borrelia genus

• Morphology: 4- 18 µm, spirochete, have fewer coils. Seven to twenty periplasmic flagella originate at each end and overlap at the center of the cell - twisting motility. Borrelia recurrentis

Reservoir: humans Vector: human body louse Disease: 1. Epidemic (louse-borne) relapsing fever = periodic febrile and afebrile cycles ⇓ bacteria undergo antigenic variation ⇓ specific IgM ⇒ complement-mediated lysis – bacteria disappear from the blood – hidden borrelia can change their outer proteins by gene rearrangement – emerge as novel organism risk: exposed to lice – crowded, unsanitary conditions (war, natural disasters)

2. Endemic (tick-borne) relapsing fever: many Borrelia species ( Borrelia hermsii ) Reservoir: rodents (zoonotic infection) Vector: soft-shelled tick • Symptoms: fever = bacteremia 1 week afebrile period ⇒ fever returns • Diagnosis: Sample: blood (during fever) – Giemsa staining • Treatment: tetracycline, erythromycin Diagnosis: Sample: blood (during fever) – Giemsa staining Antibody detection by indirect immunofluorescence assay

Cultivation: slow growth on artificial culture media microaerophilic, complex nutrition requirement Cultivation does not used to be successful

Treatment: tetracycline , erythromycin

Control - by avoiding the vectors (should wear clothing that covers as much of the skin as possible and use tick repellents) Borrelia burgdorferi

• Reservoir: deers, rodents • Vector: hard-shelled tick (Ixodes ricinus – Europe) • Lyme disease: 1977 group of children develop arthritis in Lyme (Connecticut) 1982 W. Burgdorfer discovered spirochete responsible for the disease Clinical symptoms

1.Localised: erythema chronicum migrans /ECM/ - skin lesion due to tick bite - flat, red border macule with central clearing as it develops - ranging from 5 cm to 50 cm - fades and disappeares within weeks 2. Early disseminated stage - within days and weeks, untreated patient - hematogenous dissemination - headache, fever, arthalgia, myositis - heart failure (heart block, myopericarditis) - meningitis, encephalitis 3. Late manifestation - within months and years - acrodermatitis chronica atrophicans (ACA) /in elderly/ - arthritis - lymphadenomatosis benigna cutis /in children Lyme Borreliosis

Erythema chronicum migrans

Acrodermatitis chronica atrophicans

Lymphadenosis benigna cutis

Lyme arthritis Diagnosis • - clinical symptoms (ECM) • - low number of B. burgdorferi in mucocutan lesions • - from mucocutan lesion nucleic acid amplification ( PCR)

SEROLOGY is important! • Immunofluorescence assay • ELISA • Western blot – confirmation of ELISA

Treatment: • Early Lyme disease: tetracyclines, penicillins. • Arthritic and neurologic disorders: high-dose intravenous penicillin G or ceftriaxone. Prevention: avoid ticks – protective clothing, insect repellents • The spirochetes are present in the midgut of the tick, and 12 to 24 hours is required before the spirochetes are transmitted. Leptospira genus Morphology - thin, coiled spirochete, 20 µµµm, both ends hook shaped Pathogenic species: Leptospira interrogans ⇓ many serovariants (serologically distinct groups) • serovariants ⇒ different animal reservoirs • L. interrogans pomona swine • L. interrogans grippotyphosa mouse • L. interrogans icterohaemorrhagiae rat • L. interrogans canicola dog

• Asymptomatic infection in reservoir hosts – colonise the renal tubules – - spread by the urine of the infected animal – streams, standing waters can be contaminated

• Humans are accidental hosts: leptospira can penetrate the skin

• Occupational infections: farmers, veterinarians, agricultural workers Disease: leptospirosis (zoonosis) Asymptomatic infection Symptomatic infection: Typical biphasic disease 1-2 weeks incubation

1) Acut :flue like symptoms (fever, muscle pain) leptospira in the blood

2) Immune leptospiruric phase: sudden onset of myalgia, headache, abdominal pain, conjunctival suffusion, vascular collapse, thrombocytopenia, hepatic, renal failure aseptic meningitis (no pus in the cerebrospinal fluid)

Weil’s disease: severe form of leptospirosis (mortality: 15-20%) (progressive impairment of hepatic and renal function ; death)

Hepatic involvement with jaundice (icteric disease) (The first human case of leptospirosis was described in 1886) Clinical Progression of Icteric (Weil’s Disease) and Anicteric Leptospirosis

(pigmented part of eye)

Diagnosis Sample: blood, cerebrospinal fluid, urine • silver impregnation • Fluorescein labeled antibody • cultivation - Korthof culture media ⇒ ,,smoke like” growth Containing 10% rabbit serum (long chain fatty acids and vitamins B1 and B12; pH7,4) Slow growing (2 weeks) Low temperature (30 oC) • Serology: Microscopic Agglutination Test detection of specific antibody in the patient’s serum after 2 weeks - serial dilution from the patient’s serum - add equal amount of living leptospira - the bacteria will agglutinate - microscope to detect agglutination • Treatment: doxycycline • Prevention: - Doxycycline can be used as chemoprofilaxis. -Vaccination for high risk individuals (laboratory workers) is availbale. - Materials contaminated with animal urine (e.g. natural water sources) should be avoided. - Human leptospirosis can be controlled by reducing its prevalence in wild and domestic animals. Köszönöm a figyelmet!