SpirochetesBy:Dr. Kareema Amine Al-Khafajii ,Assistant Professor, Babylon University ,College Of Medicine, Department Of Microbiology.

Spirochetes are bacteria with a spiral morphology ranging from loose coils to a rigid corkscrew shape. thin (0.1-0.5M) ,long(5-20M), Gram negative bacteria, motile by periplasmic flagella.Morphology and structure:

The spiral morphology of spirochetes is produced by a flexible, peptidoglycan cell wall around which several axial fibrils are wound. The cell wall and axial fibrils are completely covered by an outer bilayered membrane similar to the outer membrane of other Gram-negative bacteria. In some species, a hyaluronic slime layer forms around the exterior of the organism and may contribute to its virulence. Spirochetes are motile, exhibiting rotation and flextion; this motility is believed to result from movement of the axial filaments, although the mechanism is not clear.Many spirochetes are difficult to see by routine microscopy. Although they are Gram-negative, many either take stains poorly or are too thin (0.15Mm or less) to fall within the resolving power of the light microscope. only darkfield microscopy,immunofluorescence, or special staining techniques can demonstrate these spirochetes. Other spirochetes such as Borrelia are wider and readily visible in stained prepration,even routine blood smears.Three genera of Spirochetes are pathogenic to man:

1-Treponema, 2-Borrelia ,3- Leptospira1-The Treponema:

*Classification: Treponema pallidum causing syphilis (venereal disease)Other Treponema:

1-T.palidum subspecies endemicum causing Bejel (endemic syphilis).2 -T.palidum subspecies pertenue causing Yaws

3-T.carateum causing Pinta.Bejel, Yaws and Pinta are non-venereal diseases.Morphology , Structure, and Physiology:

*The microorganism is so thin , tightly coiled with straight end, and not seen by stained smears.

1

*Can be seen only by dark-field microscop,or after binding to specific antibodies-labeled with fluorescent dye.

*Not grow in-vitro, except in selected cultured cells(rabbit epithelial cells), with 30 hours replication time.

*Facultative anaerobes, they can use glucose oxidatively.*Contains 3 periplasmic flagella, inserted at each end of the cell, under the outer

membrane envelope.

Pathogenesis and Clinical Diseasesa) Virulence factors:

1 -outer membrane proteins promote adherence to host cells.2 -hyaluronidase may facilitate perivascular infiltration.

3-coating with host cell fibronectin protect against phagocytosis.4-tissue destruction primarily results from host's immune response to infection.

1- Primary syphilis: painless skin ulcer named ((primary chancre)) occurs at site of skin pentration.a- A chancre contains numerous spirochetes ,and infectious.b-histologicaly endarteritis and periartritis, and infiltrated by PMLs, and macrophages.c-slow growth, and the bacteria disseminated to all tissues mainly skin, mucus membrane and lymph nodes.

2-Secondary syphilis: fever, flue-like illnesses, generalized lymphadenopathy, and mucocutaneous lesions, it is highly infectious .lasted for few weeks ended with latency.

3-Tertiary syphilis: some cases progress to third stage, where granulomata or (( Gumma)) are formed in any tissue lead to sever destruction of skin, bon ,

cartilage…Other will progress to either neorosyphlis or cardiovascular syphilis.

4-Congenital syphilis: in utero infection either lead to latent infection, malformation, or death of fetus, survival with manifestation of rhinitis, then maculopapular rash, and organ tissue destructions will follow if infants are not dead.Epidemiology

*human is the only natural host. No reservoir host.*venereal syphilis is transmitted by sexual contact, injection, blood transfusion, tattoo

or congenital. It's Word wide. *other treponemal infections are transmitted by contact of mucus membranes with

infectious lesions.*Bejel (non-venereal, endemic syphilis) occurs in desert temperate regions of North

Africa, Middle East, and Northern Australia. *Yaws occurs in tropical and desert regions of Africa, South America, and Indonesia.

*Pinta occurs in tropical areas of Central and South America.*no seasonal incidence for treponemal disease.

Diagnosis tests of T.palidum infections.1-microscopy (dark field examination or florescent microscopy).

2

2-serology: either non-specific tests (1- VDRL(venereal disease research laboratory test) 2-RPR(rapid plasma regain test)) which measure anti-lipid IgM and IgG formed in the patient in response to lipoidal material released from cells damaged by the infection, as well as to lipids in the surface of T.pallidum. non specific tests show up as positive within 4-6 weeks of infection and decline in positivity in tertiary syphilis or after effective antibiotic treatment of primary or secondary disease. Therefore, these tests are useful for screening. However, they are non-specific and may give positive results in conditions other than syphilis(biological false positive as in; viral infections, collagen vascular disease, acute febrile disease, post immunization, pregnancy, leprosy, malaria and drug misuse.).Specific tests

1-FTA-Abs:Fluorescent treponemal antibody-absorption test.2-TP-PA: T.pallidum particle agglutination test.

3-TPHA: T.pallidum hemogglutination test.4-EIAs:detecting specific IgM, IgG.

These tests should be used to confirm that positive result with a non-specific test is truly to syphilis. Also, because they become positive earlier in the course of the disease, they can be used for confirmation when the clinical picture is strongly indicative of syphilis. They tend to remain positive for many years and may be the only positive test in patients with late syphilis. However they remain positive after appropriate antibiotic treatment and cannot therefore be used as indicators of therapeutic response. They can also give false positive reactions such as autoimmune disease diabetes mellitus, pregnancy….etc

Treatment of Treponema pallidum infections: penicillin is the drug of choice if patient is sensitive to penicillin give erythromycin, tetracycline, or doxycyclin as alternative.

3

Weak Gram-ve, spiral rods.Larger than Treponemes: 0.2-0.5 x 8-30 um

Can be seen by light microscopy, stained by Giemsa or Wright stain.

7-20 periplasmic flagella twisting motility.Microaerophilic, need complex nutrients; some

species could be cultured, but have slow growth rate (18h division time).

2) Borrelia

EM and drawing of a cross-section through Borrelia burgdorferi, the agent that causes Lyme borreliosis. The protoplasmic core of the bacterium is enclosed in a cytoplasmic membrane and conventional cell wall. This in turn is surrounded by an outer envelope, or sheath. Between the protoplasmic core and outer sheath are periplasmic flagella (also called axial fibrils), which are anchored at either end ofthe bacterium and wrap around the protoplasmic core.

Borrelia by Giemsa stain in blood film

Borrelia by dark-field microscopy

4

Clinical diseases caused by Borrelia:.1Relapsing fever:

•Epidemic R.F.: by B. recurrentis; •Endemic R.F.: by 15 other species

.2Lyme disease: by 10 Borrelia species (B. burgdorferi in US and EU, B. garinii in EU and Japan).

.3In relapsing fever, Borrelia can undergo antigenic shift and escape immune clearance periodic febrile and

afebrile periods..4No exotoxins. Endotoxins are responsible for clinical

manifestations..5Immune reactivity against the Lyme disease agents may

be responsible for the clinical disease.

Pathogenesis and clinical diseasePathogenesis and clinical disease

The virulence gene loa22 encodes a protein in the bacteria's external membrane (Pasteur Institute, 2007)

Disease range from asymptomatic and flu-like to severe systemic disease, skin peticheal rash, conjunctival suffusion,

with jaundice and hepatic involvement (Weil’s disease), and renal failure, meningitis, extensive vasculitis, and

myocarditis.All clinical presentations are due to damage to endothelium

of small blood vessels in those organs.Humoral immunity clears infection. Immune reactions

symptoms ?Congenital leptospirosis can also occur.

5

ygoloimedipEygoloimedipE.1esuol ydoB dedworc ni desaercni :

.saera yratinasnu ro

.2skciT.saera larur ni desaercni :

.3 ,yrutnec tsal UE ni saw :FR cimedipE naednA dna ,adnawR ,aipoihtE ni won

..sllihtoof

,esaesid citonooZ :FR cimednE .4ASU nretsew dna ,ediwdlrow

deredisnoc ;ediwdlrow :esaesid emyL .5rotcev gnidael eht -.SU ni esaesid nrob

6

esaesid emyLesaesid emyL.13 .P.I -d 03.2 laitinIsnargim amehtyre 5 : - ro ralucam ,der ,mc 05

elcisev mrof yam ,segralne ,retnec etihw ,ralupap.skeew nihtiw yawa sedaf tI .sisorcen lartnec ro

.3 :smotpmys & sngis ylrae rehtO ,sllihc ,revefyhtaponedahpmyl & ,esialam ,eugitaf ,aiglaym .

.4.srucco noitanimessid suonegotameh ,detaertnu fI

.5 .retfa sraey ro shtom spoleved egats etaL sitirhtrA.yltnettimretni stnioj erom ro eno fo

sisoilerroB fo sisongaiDsisoilerroB fo sisongaiD.1:ypocsorciMhtiw deniats sraems doolb morf revef gnispaler esongaid ot

.niats thgirW ro asmeiG.dohtem dipar ,evitisnes %07

(doolb ni nees ylerar era airetcab) esaesid emyL rof toN.2:erutluC.desu ylerar ,ytivitisnes wol ,htworg wols ,aidem xelpmoc.3raluceloM :sisongaid emyL fo esac ni erutluc naht evitisnes ssel

.esaesid.4:ygoloreS

.1:revef gnispaleR .sisongaid rof elbatius ton yhw ?

.2:emyLeciohc fo dohtem eht … . ?yhw .d emyL rof desu ylnommoc tsom era ASILE dna AFI

5 -:tnemtaerT enilcycarteT yb .F.Rrufec ,enilcycartet ,nillicyxomA yb :emyL emixo

7

seripsotpeL ehTseripsotpeL ehT

.sdne htob ro eno ta kooh a htiw etehcorips delioc ,nihTallegalf cimsalpirep 2 yb elitoM

seborea etagilbO yb dehcirne muidem no ylwols worg ,gnol ,(21B ,2B) senimativ - ta ,ainomma dna sdica yttaf niahc

82 - 03 o.Csnagorretni aripsotpeL cinegohtap namuh eht era xelpmoc

.sniarts ~ era erehT sisoripsotpeL namuh fo sesac ereves 000,005

2 :ytilatroM .raey hcae dlrow eht dnuora -.%5

8

.erutluc ni nworg seripsotpel fo gniniats revliS

.sdne dekooh htiwydob delioc ylthgit eht ecitoN

ME yb aripsotpeL

esaesid lacinilc dna sisenegohtaPesaesid lacinilc dna sisenegohtaP

eneg ecneluriv ehT aol s'airetcab eht ni nietorp a sedocne 22(7002 ,etutitsnI ruetsaP) enarbmem lanretxe

ulf dna citamotpmysa morf egnar esaesiD - ereves ot ekil ,noisuffus lavitcnujnoc ,hsar laehcitep niks ,esaesid cimetsys

tnemevlovni citapeh dna ecidnuaj htiw )lieW ’esaesid s ,( dna ,sitilucsav evisnetxe ,sitigninem ,eruliaf laner dna

.sitidracoym era snoitatneserp lacinilc llA muilehtodne ot egamad ot eud

snagro esoht ni slessev doolb llams fo . snoitcaer enummI .noitcefni sraelc ytinummi laromuH

? smotpmys.rucco osla nac sisoripsotpel latinegnoC

9

sisoripsotpeL fo ygoloimedipE sisoripsotpeL fo ygoloimedipE

a si sisoripsotpeL sisonooz tsrif) .dlrow eht tuohguorht dnuof si taht .(7091 ni derevocsid

era sriovreser niaM stnedor airetcab eht robrah hcihw ,star yllaicepse , eniru rieht ni airetcab eht etercxe ,selubut laner ni retawhserf

lios yddum dna secafrus detcefni teg slamina & nam yllatnedicni.lios ro retaw hcus ot tcatnoc no

sa hcus slamina noinapmoc ro kcotsevil sa hcus ,slaminA & naM aiv detcefni era ,sgod sucum rieht hguorht ro niks rieht ni skaerb

senarbmem .etawhserf ot) erusopxe lanoitaercer yb rehtie si noitcefni namuH ro (r

.(slamina ot) erusopxe lanoitapucconosrep oN -ot -.daerps nosrep

noitinifed yB stsoh latnedicni ni dehsilbatse ton si egairrac cinorhc ,.(slamina dna nam)

snosaes mraw ni eroM …?yhw..

10

Diagnosis of Leptospirosis Diagnosis of Leptospirosis Specimen taken:

Blood and CSF: Leptospires are detected in blood and CSF at first 10 days of infection. Urine: after first week and for 3 months. Serum

.1Microscopy: not used for the very thin nature of leptospires, even by dark-field microscopy.

Fluorescent-labeled antibody staining is used..2Culture:

Special media: Fletcher, or Tween80-albumin mediaAerobic

Special temp.: 28-30 oCLong incubation: up to 4 months

.3Serology: Microscopic agglutination test (MAT) is the reference test. The test detects agglutinating antibodies specific to leptospira antigens, in patient’s serum.

(Using live Leptospires). Patient serum positive titer is ≥1/200.Other serologic tests are less sensitive and specific (for screening only).

Cross-reaction with other spirochetal serologic tests occur.

11

Leptospira

Leptospirosis has Worldwide distribution. Perhaps no other disease has such a long list of synonyms as leptospirosis. It has been known as Weil`s disease, mud-fever, trench-fever,ricefield –fever, cane cutter`s fever, swamp fever, flood fever, autumnal fever, seven days fever of Japan,spirochetal jaundice, canicola fever etc etc….. Reservoirs The wild, domesticated as well as cagd game animals can act as reservoirs of human being. Mode of transmission:Infection is acquired through contact with environment contaminated with urine of an animal who is a carrier or is suffering from disease caused by leptospires. Infection may also arise from bathing or accidental immersion in the fresh water of lakes, rivers or canals contaminate with the urine of the infected livestock that has been using the water for drinking. The leptospira belong to family spirochetaceae. The genus Leptospira comprises of many serogroups which are further subdivided into serovars which possess related serological characteristics. Morphologically and culturally the Leptospires cannot be differentiated. However , they can be classified serologically. The pathogenic members are identified as serovars belonging to species L.interrogans and are described as L.interrogans serovars L.icterohaemorrhagia. some people prefer to assign complete species status to serovars and describe this serovar as L.icterohaemorrhagia.

Pathogenesis

The site of entry into the host is through mucosal surfaces. Important portals of entery are fresh or partially healed abrasions of the skin and intact mucosa of the buccal cavity, nasal passages or conjunctiva. No lesion is caused at the site of entry or in the regional lymph nodes by the leptospires. The organisms quickly enter the blood stream where they multiply and this process is accompanied by the development of transient fever. Simultaneously, the bacteria start acting upon other organs and subsequent symptoms pertain to the affected organ.Leptospires take just a few days to establish in organs like ,liver, spleen, kidney, and the pathological changes are initiated there. By the time the immune system of the body gets activated (as evidenced by the production of serum antibodies) the leptospires get established in the parenchyma of the liver and spleen and in tubular region of the kidneys where these may persist.

- animal pathogenicity.is a test used for the diagnosis of leptospirosis. After inoculation of infected blood or CSF of experimental animal ,it will develop rapidly fatal illness characterized by fever, haemorrhage and loss of weight, and then death may occur within 4-7days. Leptospirae can easily be isolated from kidney and often from the brain and other tissues at the height of infection but prior to the death of the

animal. After death, it is usually difficult to isolate Leptospirae.

12