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    Spirochaetes Spirochaetes Presentation Transcript

    • SPIROCHAETES Dr Sabrina Moyo Department of Microbiology and Immunology
    • Definition:
      • Long helical, corkscrew-shaped motile bacteria with tight or loose spiral depending on the spp
      • Gram negative measure 0.1 to 3 µm by 5 to 120 µm.
      • Replication is slow (doubling time, 30 hours )
    • Taxonomy
      • The order spirochaetales has two families
      • The family spirochaetaceae- which consist of free living large spiral organisms and
      • The family Treponemataceae has three main genera: causing disease in man namely
      • i) Treponema
      • Ii) Leptospira and
      • Iii) Borrelia 
      • The genus Treponema has two spp that cause human disease
      • i) Treponema pallidum with 3 subspp
      • _ T.P Pallidum -> Syphilis
        • T. p. endemicum -> Endemic syphilis (Bejel)
        • T.p. pertenue -> Yaws
        • ii) Treponema carateum -> Pinta
    • Taxonomy
      • The genus Borrelia has the following medical important spp
      • i) Borrelia burgdorferi -> Lyme disease
      • (borreliosis)
      • Ii) Borrelia recurrentis -> Epidemic relapsing fever
      • (louse borne relapsing fever)
      • Iii) Other Borrelia species -> Endemic relapsing fever
      • (Tick-borne relapsing fever)
      • Iv) B,vincenti -> Together with fusifrom bacteria causes ulcerative gingivo stomatitis (vincent angina)
      • The genus Leptospira interrogans
      • Two spp
      • i) L. interogans - pathogenic
      • Ii) L. biflexa - free living non pathogenic  
      • -> Leptospirosis (Weil’s Disease)
    • Structure
      • The organisms are covered by the outer sheath (glycosaminoglycan)
      • The outer membrane contains peptodiglycan and maintain the structural integrity of the organism
      • The endoflagela (axial filaments) are flagella like organelles in the periplasmic space encased by outer membrane.
      • The endoflagella begins at each end winds around it extending to and overlap at the midpoint
      • Inside the endofloagella is the inner membrane (cytoplasmic membrane) that provide osmotic stability and covers the protoplasmic cylinder
      • They reproduce by tranverse fission .
    • Structure
    • Treponema
      • Morphology
      • Slender spirals measuring 0.2 µm in width and 5-15 µm in length
      • They have tight spirals i.e are regularly spaced at a distance of 1 µm
      • Have pointed and tapering ends,
      • Three to four endoflagella, actively motile, show rotatory corkscrew like movement
      • The spiral are thin require immunofluoresence or dark field illumination, Stained by silver impregnation
      • Microaerophilic surviving at 1-4% Oxygen
      • In proper suspending fluid and presence of reducing agents survive for 3-6 days at 25 o C or in blood at 4 o C for 24 hrs
      • They stain with difficulty except with Giemsa's stain or silver impregnation. 
    • Treponema: Regular spiral coils 1 µm apart
    • Treponema
      • Culture and growth characterisitcs
      • T pallidum has never been cultured continuously in artificial culture media
          • T. pallidum is usually cultured in the testes of rabbits,
          • Non pathogenic /saprophytic Treponema can be cultured anaerobically invitro doubling time is 30 hrs. The saprophytic Reiter strain grows on a defined medium of 11 amino acids, vitamins, salts, minerals, and serum albumin.
          • T pallidum is a microaerophilic organism; it survives best in 1–4% oxygen. In proper suspending fluids and in the presence of reducing substances, T pallidum may remain motile for 3–6 days at 25 °C.
          • In whole blood or plasma stored at 4 °C, organisms remain viable for at least 24 hours, which is of potential importance in blood transfusions.
    • Treponema
      • Reactions to Physical and Chemical Agents
      • Drying kills the spirochete rapidly, as does elevation of the temperature to 42 °C.
      • Treponemes are rapidly immobilized and killed by trivalent arsenical, mercury, and bismuth (contained in drugs of historical interest in the treatment of syphilis.
      • Penicillin is treponemicidal in minute concentrations, but the rate of killing is slow, presumably because of the metabolic inactivity and slow multiplication rate of T pallidum (estimated division time is 30 hours).
      • Resistance to penicillin has not been demonstrated in syphilis.
    • Treponema
      • Virulence Factors
      • Several gene products associated with virulent strains,
        • Their roles in pathogenesis are unknown.
      • The outer membrane proteins are associated with adherence to the surface of host cells,
      • Virulent spirochetes produce hyaluronidase, which may facilitate perivascular infiltration.
      • Virulent spirochetes are also coated with host cell fibronectin, which can protect against phagocytosis.
      •  
    • Treponema
      • 1. T.p. pallidum – Syphilis
      • Pathogenesis and pathology
      • Syphilis is an acute and chronic infectious disease caused by Treponema pallidum. 
      • Routes of infection :
      • Mucosal membranes of the vagina or urethra, sometimes scar wound or scratches.
      • Across the placenta to the developing fetus
      • T. pallidum tends to invade the interstitial spaces of tissue at the site of infection and to move rapidly to other locations (active "cork-screw" motility)
    • Treponema
      • Clinical presentation
      • Primary Syphilis:
      • - following penetration of the skin or mucous membranes, a
      • characteristic, painless hard chancre develops at the site of entry
      • within 3 weeks (10-60 days after exposure)at the primary site of
      • initial replication: penis, labia, cervix, anorectal region, the mouth.
            • - The chancre is hard, slightly elevated, round, ulcerous and highly
            • contagious filled with Treponemes.
            • - Simultaneously the organism enters the lymphatics and becomes disseminated.
            • - The chancre heals without treatment in a few weeks(4-6 weeks)
            • due to local immunity, even without treatment, may leave a scar but
            • by that time the organism has already disseminated.
    • Treponema
      • Primary Syphilis:
      • The lymph nodes also become inflamed.
      • Histology endarteritis (inflammation of the inner wall of an artery) and periarteritis
      • (inflammation of membranous sac surrounding the heart with polymorphonuclear
      • leukocytes and macrophages.
      • 1:3 of people exposed becomes infected.
      • Unless syphilis is treated early in this stage, it will progress into secondary syphilis.
    • Primary syphilis
    • Treponema
      • Secondary Syphilis :
      • 4-8 weeks after the chancre heals,
      • Typically there are lesions (filled with treponemes) throughout the body including the skin, mucous membranes, organs, and eyes.
      • Lessions appear as pale red rash appears usually on the palms or soles of feet, or over the entire body.
      • Accompanied by fever, sore throat, headaches, joint pains, poor appetite, weight loss, and hair loss.
      • Sores form around the genitals or anus that secretes extremely infectious fluids.
      • These symptoms usually last 3-6 months, but may not last and then re-appear at any time.
      • Without treatment, syphilis will continue to progress throughout the body.
      • This also heals without treatment and the patient may either spontaneously get well or develop a latent infection
    • Secondary syphilis
    • Treponema
      • The Latency Stage:
      • No obvious symptoms but specific anti-treponemal antibodies are found.
      • T. pallidium is lodging itself into the host's tissue.
      • This stage may last ffrom few months , 3-10 years.
      • Generally individual is not infectious
      • Always passes the disease to the fetus.
      • Approximately 1/3 to ½ may eventually progress to the next stage.
      • 50-70% will not progress to tertiary syphilis.
    • Treponema
      • Tertiary Syphilis:
      • This stage is characterized by granulomatous lesions, called Gummas of which can occur on the skin, internal organs, CNS, bones, eyes, and cardiovascular system. the heart, brain and liver, can be fatal.
            • They are cause by the body’s hyperimmune reaction to remaining spirochetes.
            • When lesions develop in the CNS it is called neurosyphilis and it can lead to paralysis.
            • In the eyes it can lead to blindness, and in the heart to aortic damage or aneurisms.
      • Gumma lesions whose pathogenesis is uncertain frequently lead to destruction of soft tissue or bone.
      • Occur for an extended period from as few as 2 to over 40 years from onset of infection.
    • Tertiary syphilis
    • Treponema
      • Congenital Syphilis:
      • Occurs when the treponemes cross the placenta during the fifth month to infect the unborn fetus (occurs usually when mom is in the latent stage).
      • This can result in damage to mental development or other neurological symptoms or the child may be born with generalized syphilis.
      • In a pregnant woman who has a primary of secondary stage of the disease, this usually results in stillbirth
      • Multi-organ deformities or latent infections.
      • Most infected infants are born without clinical evidence of disease,
        • May develop rhinitis followed by a widespread papular rash.
        • Late bony destruction and cardiovascular syphilis
    • Treponema
      • Diagnosis
      • Specimen: Secretions (in ulcers), serum, cerebral spinal fluid
      • 1. Direct examination (Microscopy)
      • Dark field illumination: motile spirochetes
      • Direct Immunofluorescence antibody staining (FA)
        • Stained using flourescein-labelled antitreponeme serum : fluorescent spirochetes
      • 2. Culture: Efforts to culture T. pallidium have been generally unsuccessful.
      • 3. Serology
      • Non-specific; non-treponemal tests
      • Specific treponemal tests.
    • Treponema
      • Non-treponemal tests:
      • Detect IgG and IgM antibodies (reagin antibodies) developed against lipids from damaged cells during the early stage of the disease.
      • The antigen used for the nontreponemal tests is cardiolipin, derived from beef heart.
        • Venereal Disease Research Laboratory (VDRL)
        • Rapid plasma reagin (RPR) test.
        • Coagulation of cardiolipin antigen by the patient's serum.
        • Both tests are rapid,
        • VDRL test can be used to test cerebrospinal fluid from patients with suspected neurosyphilis
        • Become negative after successful treatment
      • Treponemal tests
        • Specific antibody tests used to confirm positive reactions with the VDRL or RPR tests.
        • Can be positive before the nontreponemal tests in early syphilis.
        • Remain positive when the nonspecific tests revert to negative in some patients who have late syphilis.
      • -> Fluorescent Treponemal Antibody Absorption (FTA-ABS) test
      • -> Treponemal pallidum Hemagglultination Test (TPPA, TPHA)
    • Treponema
      • Treatment, Prevention and Control
      • Penicillin is the drug of choice since in 1943
        • Benzathine penicillin or penicillin G
      • Tetracycline, erythromycin, and chloramphenicol
      • Penicillin or chloramphenicol in neurosyphilis.  
      • Epidemiology
      • Prevalence in Tanzania 4-7% in pregnant women
      • Associated with HIV transmission  
      • Prevention:
      • Health Education to include abstinence, condoms use
    • Treponema
      • Other diseases related to syphilis
      • Caused by organisms related to T pallidum,
      • Give positive treponemal and non treponemal reactions
      • Spread by direct contact only not (sexually)
      •  
      • 1. Bejel
      • Caused by T pallidum subspecies endemicum
      • Endemic in Africa, Middle east, SE Asia
      • Highly infectious skin lesions; late visceral complications
      • Rx Penicillin
    • Treponema
      • 2. Yaws
      • Caused by T pallidum subspecies pertenue
      • Endemic in children in tropical countries
      • Ulcerative lesion on the arms and legs
      • Scar formation and bone lesions are common
      • A variant of syphilis??
      • Rx Penicillin
      • 3. Pinta
      • Caused by T carrateum
      • Endemic in all age groups in Mexico, Central and S America
      • Non ulcerative papule on the skin followed by hyper-pigmented lesions
      • Rx Penicillin
    • Borrelia
      • Morphology:
      • Irregular/loose spiral 5-8
      • The spiral are 10-30 µm long and 0.3 µm wide.
      • Highly flexible and move both by rotation and twisting
      • Stain readily with bacteriological stains as wel as Giemsa or Wright’s stains because they are larger can be easly stained
      • Larger than treponemes
      • Culture and growth characteristics
      • Organism can be cultured in fluid media containing blood, serum, or tissue In fluid medium with blood, serum or tissues
      • Multiply is rapid in chick embryos (chorioallantoic membrane)
      • At 4 o C survives for several months in infected blood or in culture
      • In some ticks but not lice can pass from generation to generation
    • Borrelia: Irregular spirals
    • Borrelia
      • Antigenic structure
      • Has variation in antigenic structure
      • The antigenic structure of the organisms changes in the course of a single infection.
      • The antibodies produced initially act as a selective factor that permits the survival only of antigenically distinct variants.
      • The relapsing course of the disease appears to be due to the multiplication of such antigenic variants, against which the host must then develop new antibodies.
      • Ultimate recovery (after three to ten relapses) is associated with the presence of antibodies against several antigenic variants.
    • Borrelia
      • 1. Relapsing fever
      • Relapsing fever is caused by arthropo-borne spirochates of the genus Borrelia and clinically charcterised by recurrent episodes of fevre and septicaemia
      • Relapsing fever – two types
      • i) Epidermic relapsing fever - Human body louse transmit B.recurrentis. This is a more severe form of the disease that the endemic form.
      • Ii) Endemic relapsing fever - soft ticks of the genus Ornithodorus hemsii transmit spp of Borrelia ( B.hemsii )
    • Borrelia
      • Pathology and pathogenesis
      • B reccurentis causes epidemic relapsing fever
      • Transmitted by lice
      • Incubation period of 3-10 days
      • Sudden onset of high fever with chills high temp for 3-5 days, severe headache, myagias, arthragias,and lethargy, photophobia and cough
      • Borrellia in blood-Relapsing nature due to antigenic variation
      • A febrile period of 4-10 days followed by send attacks
      • Truncal skin rash of 1-2 days duration are common at the end of primary febrile episode
      • There are 3-10 recurrences of diminishing severity
      • Attacks are probably terminated by agglutination antibodies and lytic effects
      • The duration and intensity of the symptoms progressively decrease with each episode 
    • Borrelia
      • Diagnosis
      • Specimens
      • Blood specimens are obtained during the rise in fever, for
      • smears and animal inoculation
      • 1. Microscopy
      • Blood taken during the febrile episode for smear or animal inoculation
        • Thick and thin smears using Wright’s stain
        • Large, loosely coiled spirochetes
      • 2. Animal inoculation
      • Intra peritoneal inoculation of white mice or young ratsStained film from tai blood after 2-4 days 
      • 3. Culture
      • Grow on fletchers semisolid media. Cultures are rarely perfomed in most clinical lab because the media are not readly available and the organism grow slowly on them
    • Borrelia sp. in blood smear
    • Borrelia
      • Treatment
      • Relapsing fever has been succesfully treated with Tetracycline, Erythromycin, and penicillin.
      • mortality of treated RF is less than 5%
      • mortality of un treated RF is up to 40%
    • Borrelia
      • Epidemiology and control
      • RF Occurs through out the world
      • The distribution of EpRF is largely determined by socio-economic and ecologic factors where as distribution of EnRFis governed by the biology of ticks
      • For endemic Rf small mammals, Rodents and ticks are the main reservoirs
      • For epidermic RF humans are the only host
      • Avoidance to exposure to ticks and lice and delousing (cleanness and insecticides)
      • Elimination of athropod vectors where insectisides can be used in dwelling and surrounding areas
      • Good personal hygine, delousing procedures
      • Vaccins are not available
    • Borrelia
      • 2. Lyme disease.
      • - Caused by Borrelia burgdorferi and transmitted by ticks
      • Lyme disease named after the town of Lyme, Connecticut where the cluster of cases in children were identified
      • Transmitted by the bite of small Ixodes tick
      • Characterized by skin lesion (erythema migrans, flu like symptoms , arthritis and arthralgia
    • Borrelia
      • Morphology:
      • B burgdorferi is spiral organism 20-30 µm x 0.2- 0.3 µm
      • Distance between turns are 2 -4 µm
      • Highly motile with 7-11 endoflagella
      • Stain with aniline dyes and silver impregnation
      • Grows well in complex liquid medium
      •  
    • Borrelia
      • Pathogenesis and Clinical findings
      • Transmission-> injection of organism in tick saliva or regurgitation of tick’s midgut contents
      • This is a systemic illness that may begin with the appearance of a red skin lesion called erythema chronicum migrans (ECM) because the lesion expands in a circular manner.
      • Produce characteristic skin lesion
      • Migration through the blood and lymph to the rest of the body
    • Borrelia
      • Pathogenesis and Clinical findings
      • There are early manifestation
        • Skin lesion (erythema migrans) 3-4 weeks after tick bite
        • Flue like illness
      • Late manifestation (weeks to months)
        • Arthralgia, arthritis, meningitis, facial palsy and cardiac disease
      • Late manifestation (months to years)
        • Chronic skin, nervous system or joint involvement
        • ?deposition of antigen antibody complexes
    • Lyme disease
    • Borrelia
      • Diagnosis
      • Clinically- Unique skin lesion
      • Laboratory
        • Blood, csf, joint fluid
      • Smears are insensitive
      • Culture takes 6-8 weeks
      • Molecular probes using PCR, is rapid, sensitive and specific
      • Serology using immunofluoresence and ELISA
      • Treatment
      • Early disease: Doxycycline or amoxycilin 20-30 days
      • Late disease: Ceftriaxone
    • Borrelia
      • Epidemiology and prevention
      • Transmission: Small ticks
      • Mice and deer are the main reservoirs
      • Prevention is by avoidance of exposure to ticks
      • Insecticides
    • Leptospira
      • Morphology
      • Tightly coiled, thin flexible spirochetes 5-15µm long with very fine spirals 0.1-0.2 µm wide
      • On end is often bent, forming a hook
      • Has very thin axial filament
      • Not readily stained but can be silver impregnated
      •  
    • Leptospira: Tightly coiled, thin and flexible
    • Leptospira
      • Culture
      • Grows aerobically at 28-30 o C in serum containing semisolid medium (Fletcher’s).
      • Pathology and clinical findings
      • This disease is acquired by contact with the urine of an infected animal or ingestion of contaminated food or water.
      • It is more a disease of animals other than man.
      • Infection can vary from asymptomatic to fulminant.
      • The CNS, liver, and kidneys are most commonly infected.
      • Many infections are mild and asymptomatic
      • IP 1-2 weeks
      • Febrile onsets with spirochetes in blood
      • Then establish in several systems (liver , kidneys)
      • Result into hemorrhage and organ dysfunction
      • Aseptic meningitis can occur
    • Leptospira
      • Diagnosis
      • Specimens: blood, csf, tissues, urine
      • Microscopy: Dark filed examination show leptospira
      • Culture in Fletcher’s medium: slow growth (8 weeks)
      • Animal inoculation:
        • Sensitive
        • Intra peritoneal in young hamster, guinea pigs
        • Demonstrate leptospira in peritoneal fluid
        • Death in 8-14 days with hemorrhagic lesions in organs
      • Serology for agglutinating antibodies in EIA
        • Very sensitive but hazardous
      •  
    • Leptospira
      • Treatment
      • Mild form: Oral tetracycline, ampicilin, amoxycilin
      • Moderate and severe form: Intravenous penicillin
      •  
      • Epidemiology and control
      • Mainly animal infection and human are accidentally infected
      • Rats, mice rodents, swine, dogs and cattle are principal source of infection
      • They excrete leptospira in urine feces
      • Leptospira survive in stagnant water for several weeks
      • Drinking, swimming, bathing, food contamination can lead to infection
    • Leptospira
      • Prevention
      • Prevent exposure to potentially contaminated water
      • Rodents control
    • Leptospira
      • THANK YOU!