SpirochaetalesSpirochaetales Thin, Helical Gram-Negative

Order: Spirochaetales Family: Leptospiraceae –Leptospira interrogans – Leptospirosis Family: Spirochaetaceae –Borrelia ssp. – Relapsing fever –Borrelia burgdorferi – Lyme disease –Treponema pallidum – Venereal Syphilis

Spirochetes “coiled hair” G(-) thin, helical ( x 5-20um) Protoplasmic cylinder –Cell wall peptidoglycan –Cytoplasmic membrane Endoflagellum –Not typical flagella –Attached to each end of protoplasmic cylinder, extend toward opposite end –Also called axial filament Outer sheath - multilayered outer membrane surrounds cylinder

Leptospira: Stain and Culture “thin” “coiled” Bacilli (0.1 x 5-15um), ends hooked shaped Visualize by dark-field microscopy, not reliable as MO very thin Growth media - peptone, beef extract, bovine serum albumin, Tween 80 Incubation up to 28 days

Leptospira: Lab ID Isolate and culture on plating media from patient blood, CSF, urine Serology –ELISA for IgM, IgG –Rapid Slide Agglutination test –Complement Fixation (CF) test – look for 4x rise in titer of paired patient’s serum

Leptospira : Virulence Factors Highly motile – direct invasion, replication in tissues, induce host inflammatory response Endotoxin – LPS Gram(-) cell wall Immune complex – kidney damage (glomerulonephritis) Pathogenicity not well understood

Leptospira interrogans : Leptospirosis Zoonosis - AS infection rodents, wild animals, dogs, farm animals MO shed in urine; contaminate streams, rivers, moist soil Human infection - contact urine, infected animal; ingest contaminated food, water <100 infections/year in USA, mainly Hawaii Often misdiagnosed as “viral syndrome” Route of Transmission: –Recreational exposure contaminated water –Occupational exposure to infected animals (farmers, vets, slaughterhouse workers)

Leptospirosis / Weil’s Disease MO penetrate intact mucosa or skin through small cuts or abrasions; may spread to blood, organs, CNS Infection vary –Asymptomatic, subclinical –Flu-like febrile illness –Weil’s disease - severe systemic disease to blood, liver, kidney, heart, CNS, meningitis Incubation days; followed by fever, chills, severe headache, myalgia, malaise, nausea, vomiting MO multiply rapidly, damage endothelium of small blood vessels, usually not fatal Death ~10%, due to renal failure

L. interrogans : Treatment and Prevention Oral antibiotics - doxycycline, penicillin; given intraveneous (IV) for severe disease Vaccination of livestock and pets to reduce incidence of disease Control of rodent population to reduce MO transmission to humans

Borrelia: Stain and Culture Larger than other spirochetes ( x um) Observe with dark-field microscopy or in blood smear with Giemsa or Wright stain Grown on complex Kelly’s media, but not usually done in diagnostic labs

Borrelia: Lab ID Relapsing Fever (Borrelia ssp) –Microscopy test of choice –Blood smear from patient Lyme disease (B. burgdorferi) –Serology test of choice, MO rarely seen in blood or tissue –Immunofluorescence Assay (IFA) –ELISA -specific IgM, or 4x rise in IgG titer of paired serum

Borrelia : Virulence Factors Antigenic variation –Change surface antigens during infection –Evade host immune recognition –Causes “relapse” of fever episodes Host immune reaction responsible for clinical disease

Borrelia: Relapsing Fever One week incubation; abrupt onset of chills, fever, headache, myalgia Symptoms resolve after 3-7 days; antibodies formed, MOs decrease, leads to afebrile period Fever relapses as MO undergoes antigenic variation; antibodies no longer effective, MOs increase Several relapses may occur, each one less severe than previous one

Endemic Relapsing Fever Many Borrelia species Worldwide; USA (West, rural areas) Transmitted by tick Rodent, mouse, deer, soft tick reservoir Repeated fever relapses Spread in blood to multiple organs Mortality ~5%

Epidemic Relapsing Fever B. recurrentis Africa, South America Transmitted by human lice Human reservoir, crowded, unsanitary conditions Single fever relapse More severe form of disease; heart, brain, liver Mortality as high as 40%

Borrelia burgdorferi : Lyme Disease Worldwide USA - Lyme, CT epidemic of arthritis 1975 Animal reservoir - mouse, deer, tick Transmitted tick to humans Leading vector-borne disease in USA, ~12,000 cases/year Dermatological, rheumatological, neurological, cardiac abnormalities due to immune complex

Lyme Disease Systemic illness, begins with red skin lesion Erythema chronicum migrans (ECM) - lesion expands circular manner Flu-like symptoms - fever, headache, nausea, vomiting, myalgia, fatigue If untreated, may develop: –Days/months – neurologic symptoms, cardiac dysfunction –Months/years – arthritis (most common symptom) –Due to immune complex

Lyme Disease

Borrelia: Treatment and Prevention Relapsing fever –Tetracycline, erythromycin –Eradicate lice – improve hygiene, sanitation, overcrowding living conditions –Avoid ticks, wear protective clothing, use insect repellants (DEET), insecticide Lyme disease – amoxicillin, tetracycline –Avoid ticks as above

Treponema: Stain and Culture Thin, tightly coiled ( x 6-20um) Too thin, not seen Gram stain Visualized - dark-field microscopy, fluorescent antibody or iron staining. T. pallidum does not survive long outside host Pathogenic MOs not culture successfully on lab media Experimental infection in rabbits, grow in tissue culture short time

Treponema: Lab ID Detect antibody made by infected host Two serology antibody test used: –Nonspecific (Nontreponemal) –Specific (Treponemal) In lesions, use specific fluorescent antibody to ID MO

Treponema: Nonspecific Serology Test Nontreponemal test – use to screen Very sensitive, but not specific Inexpensive, easy to do Detect antibody against lipids MO cell surface Detect non-treponemal antibody –Called Reagin or Wasserman antibody –React with cardiolipin-lecithin of normal tissue –Other diseases also give positive test Several test (use beef heart cardiolipin) –VDRL test (Venereal Disease Research Laboratory) –Rapid Plasma Reagin card test (RPR) –Automated Reagin Test (ART)

Treponema: Specific Serology Test Specific Treponemal test – confirm positive screening result Very specific, expensive, more difficult to perform Several test for antibody in patient –Fluorescent Treponemal antibody absorption test (T. pallidum antigen + patient’s serum; use fluorescein-labeled anti-human IgG to detect) –Treponema pallidum particle agglutination test –ELISA

Treponema : Virulence Factors Outer membrane proteins – adherence Hyaluronidase – perivascular spreading Molecular mimicry – outer sheath contains molecules resemble those on human cells; allows MO to evade host defense recognition Tissue destruction and lesions – due to host immune response (immune complexes, hypersensitivity)

Treponema pallidum: Syphillis Worldwide epidemic USA (#3 STD after Chamydia, Neiserria) Strict human pathogen Transmission by direct sexual contact; exposure ~30% risk of infection Congenital infection from infected pregnant mom (bacteremia) to fetus

Syphillis Venereal, sexual contact – primary lesion in genital tract Non-venereal, non-sexual – lesion on oral, mucous membranes Indirectly – infection by common usage of eating and drinking utensils Untreated natural course of disease occurs in several stages

Primary Syphillis (Weeks) Penetration skin or mucous membranes - painless hard chancre develops site of entry within 3 weeks; highly contagious, filled with treponemes MO enters lymphatics, disseminated Chancre heals without treatment in few weeks due to local immunity, but MO has disseminated

Secondary Syphillis (Years) 1-2 months after primary stage Flu-like symptoms, sore throat, headache, fever, myalgia, lymphoadenopathy, rash Lesions (with treponemes) throughout body; skin, mucous membranes, organs, eyes Resolves slowly without treatment; patient gets well or develops latent infection Latent infection – no symptoms, specific treponemal antibodies; may last 3-10 years; biological balance between MO and host No treatment ~30-50% progress to next stage

Tertiary Syphillis (3-20 years) Diffuse, chronic inflammation, destroy any organ tissue (i.e. vascular, brain, eye) Granulomatous lesions, called gummas of the skin, internal organs, CNS, bones, eyes, CV system; by hyperimmune reaction to spirochetes Cardiovascular syphilis - aortic damage, aneurisms Neurosyphilis - paralysis Eyes - blindness

Congenital / Other Syphilis T. pallidum – Congenital infection –Cross placenta to infect fetus –Most born without clinical disease (latent infection) –May result in multi-organ malformation, death –In pregnant woman with primary or secondary stage disease, usually results in stillbirth T. pertenue - Yaws or tropical syphilis –S. America, Africa, S.E. Asia –Transmitted by direct contact infected lesions –Granulomatous disaese T. carateum – Pinta –C. & S. America –By direct contact with infected lesions –Skin disease with hyperpigmentation in patches, lesions

Treponema: Treatment and Prevention Penicillin remains drug of choice Public Health prevention: –Education – social, economic, moral –Surveillance - identify and treat infected patients and partners

Case Study: Spirochete An 18-year-old woman complained of knee pain that started 2 weeks previously. Three months earlier, soon after vacationing in Connecticut, she noticed a circular area of redness on her lower leg; it was approximately 10 cm in diameter. During the next 2 weeks, the area enlarged and the border became more clearly demarcated; however, the rash gradually disappeared. A few days after the rash disappeared, the woman experienced the onset of headaches, an inability to concentrate, and nausea. These symptoms also gradually abated.

Case Study: Spirochete The pain in her knee developed approximately 1 month after these symptoms disappeared. On examination of the knee, mild tenderness and pain were elicited. A small amount of serous fluid was aspirated from the joint, and it had an elevated white blood cell count. Antibodies to Borrelia burgdorferi were present in the patient’s serum (titers of 1:32 and 1:1024 for IgM and IgG, respectively), confirming the clinical diagnosis of Lyme arthritis.

Case Study: Questions 1. What are the initial and late manifestations of Lyme disease? 2. What are the limitations of the following diagnostic tests for Lyme disease: microscopy, culture, and serology? How do these compare with the diagnostic tests for other relapsing fevers? 3. Name two examples each of nontreponemal and treponemal tests for syphilis. What reactions to those tests would you expect in patients with primary, secondary, and late syphilis?

Case Study: Questions 4. What are the reservoir and vectors for syphilis, epidemic and endemic relapsing fever, Lyme disease, and leptospirosis? 5. What diagnostic tests can be used for the diagnosis of leptospirosis?

Class Assignment Textbook Reading: Chapter 23 The Spirochetes Key Terms Learning Assessment Questions

Final Exam Tue., March 20, :30 – 10:30 am Mycobacterium thru Ureaplasma Lecture, Reading, Key Terms, Learning Assessment Questions Case Study 7, 8, 9, 10 (Mycobacterium, Clostridium, Chlamydia, Legionella) Exam Format: –Multiple Choice –Terms –True/False Statements –Short Essay Review, Review, Review! Repetition is the key to retention