�Spirochetes

• The spirochetes consist of microorganisms with the general characteristic that their bodies are spiral.
• Depending on the family/species, the spirals can be loose or tight (cork – screw like)
• Three families that make up the spirochetes are:

i) Spirochaetaceae (Gens. Borrelia, Treponema)

1. Leptospiraceae (Gen. Leptospira)
2. Spirilliaceae (Gen. Spirillium)
1. Diseases caused by the spirochetes are generally referred to as spirochaetoses

1.Fam. Spirochaetaceae

1.1 Gen. Borrelia

Consists of different species that cause disease to humans and animals, including:

i) Borrelia burgdoferi (causal agent of lyme disease of humans )

ii) B. duttoni (causal agent of tick-borne relapsing fevers in humans)

iii )B. recurrentis (causal agent of louse borne relapsing fever in humans)

iv) B. anserina (causal agent of fowl spirochaetosis

Morphology:

• The borrelia are generally long spiral microorganisms measuring 5-10 x 3μ with pointed ends (terminal filaments )
• Motile by wavy (snake - like) motion
• The larger borrelia (eg. Borrelia burgdoferi are easy to stain by Giemsa, and are Gram – ve
• The more slender borrelia, eg. B. duttoni , are best visualized by dark field microscopy

Cultural properties/requirements

• Grows well aerobically on nutrient agar, at 37^oC with rabbit serum additives
• B. anserina can also be grown in chick embryo or on ordinary media at 42^oC

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Borrelia burgdoferi

Borrelia duttoni

Borrelia sp. Showing terminal filaments

Borrelioses (i.e diseases caused by Borrelia spp)

i) Lyme disease (first described in 1975)

• Caused by B. burgdoferi in humans – especially in temperate countries
• Vectors of B. burgdoferi are ticks of wild mammals (deer, mice, skunks, foxes…). Humans get infected following skin bites by the forest ticks (Ixodidae spp), infected by the borrelia
• Clinical signs include: initial skin rash at the site of tick bite followed by swollen regional lymph nodes, headaches, fevers, malaise and joint pains (arthritis)

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Ixodidae tick feeding on human skin

Post tick bite-bite skin erythema

• The disease is not fatal but is debilitating
• Diagnosis- based on history of tick bites among hunters, foresters, farmers, pick–nickers…nudists
• Treatment by antibiotics effective (amoxillin, doxycycline)

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ii) Human tick borne relapsing fever (TBRF)

• Caused by B.duttoni, following bites of the nocturnal house soft tick Ornithodorus moubata
• TBRF is common in Tanzania (Dodoma, Kondoa, Shinyanga, Mza, Kigoma, Bk….) – homa ya mapapasi (Kigogo = Makutupa)
• Clinically TBRF presents with malaria like symptoms (periodic fevers, headaches, joint pains, weakness)
• In Tz, children and women are particularly affected
• Diagnosis is based on clinical signs, clinical pathological studies (blood smears) and history, … presence of ticks in houses, and tick bites during sleep

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B. duttoni – Blood smear

Borrelia duttoni –Dark Field microscopy

O. moubata feeding on sleeping persons

Soft tick. O. moubata before and after feeding on blood

Makutupa

Treatment:

Use of antibiotics is effective

Prevention:

-Improve hygiene, allow more light into houses, especially bedrooms, use acaricides (powders and insecticidal sprays in houses)

-Use of impregnated mosquito nets has limited effictiveness

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iii) Human - Louse borne relapsing fever –(LBRF)

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iv) Fowl spirochaetosis

• Caused by Borrelia anserina
• Vectors are blood sucking ticks (Argas persicus, Demanyssius galinae)
• Clinically presents: Septicaemia with severe anaemia, emaciation and diarrhoea in fowl (chickens, ducks, turkeys….)

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• Diagnosis based on clinical signs, clinical - pathological (eg blood smears with spirochaetes) and history/presence of ticks

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• Treatment: Possible with antibiotics, Improve hygiene including removal of ticks - deparasitation
• Control: vaccination (optional) with bacterins raised in chick embryo

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Borrelia anserina - transmission EM picture

Hungry ticks

Engorged Argas sp ticks feeding on a chick

Argas sp feeding on a chicken

Fowl spirochetosis cycle

Cultures of B anserina on solid media

Antigenic properties:

• The borrelia possess a cytotoxic/ hemolytic toxin , which is considered to be the virulence factor in borrelioses.
• H & O antigens exist but appear to have little significance in virulence

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iv) Human - Louse borne relapsing fever –(LBRF)

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1.2. Gen Treponema�

Consists of spirochaete species most of which are non pathogenic, while some cause serious disease to humans and animals including:

i) T. pallidum (causal agent of human syphilis)

ii) T. denticola (causal agent of human periodonthosis)

iii)T. hyodysenteriae (causal agent of swine dysentery)

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1. Treponema pallidum

Is the spirochaetal and causal agent of syphilis, an STI, (aka. yaws, pinta), in humans only.

Morphology :

• Extremely slender motile spirochete with tightly wound coils, measuring 10-20 x 0.1-0.2μ
• Difficult to stain due to its slender morphology.
• It can not be cultivated in vitro, but growth is achieved well in rabbit testicles.
• Can be maintained in growth medium but auto-lyses within a short period, without multiplying
• Visualization can be achieved using dark field microscopy, as spiral shiny microorganisms, motile in rotational flexuous motion.

Treponema pallidum DF Microscopy

Pathogenesis

1.Syphillis is exclusively a human disease transmitted through skin/mucoasa by coitus and can be localized (P, V, O, A-R) or generalized. It is therefore an STI “par excellence”

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2. Upon contact with microlesions of the skin/mucoasa the spirochete enters the tissue and multiplies locally causing primary syphilis (pustular wound and later a deep scooped, crateriform painless wound ), aka chancre, 3-10d post contact

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Primary syphillis

Pustular -🡪 Chancroid lesion of glans penis & chancre of preputium

Chancre skin (L) and tongue (R)

Chancre of the labia, anorectal and breast (rare)

3. The wound can heal on its own but the spirochaete penetrates lymph nodes --> blood and continues to multiply leading to generalized (secondary) syphillis characterized by skin rashes at different parts of the body after 2-3yrs

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Skin rashes can be severe

4. After an apparent latent period of even up to 10yrs, tertiary syphilis develops, which manifests by cardiovascular disease, sight impairment and neurological disease (general paresis, stroke, blindness, insanity) and death

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NB. Pregnant mothers can infect foetuses (during secondary syphilis) upon crossing of the spirochete through the placenta or during birth in primary syphilis.

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Neonatal syphilis (conjunctivitis &

Dermatitis)

Diagnosis

1. Clinical, clinical pathological & historical is possible during primary and secondary syphilis
2. Microbiological is possible by demonstrating spiral organisms in the chancres
3. Serological - (most common) Venereal Disease Research Laboratory (VDRL) Hemagglutination (VDRL-HA) test or Treponema pallidum hemagglutination (TPHA) Test - used for secondary and tertiary syphilis or Syphilis IFA test

Treatment Treatment of primary and secondary syphilis is effective using penicillin

• No immunotherapy or immunoprophylaxis available

Control/prevention

• (ABC) No sex (Abstinence), Fidelity, or Safe sex (Condom) are the ways to prevent syphilis from spreading

Shiida

Condoms: Come in different colors, sizes and even perfumed ones are available. The choice is yours.

2. Treponema hyodysenteriae

Treponema hyodysenteriae causes swine dysentery, a fatal bloody diarrhoea of swines

Morphology

• It is a loosely spiral mcroorganism, measuring 5-10 x 0.3μ
• Motile in a flexuous (snaky) motion
• Has no fimbria or flagella
• In fresh mocoasal scrapping and fecal smears will show spiral motile bacteria under DF microscope
• Stains by Giemsa and its Gram-ve

T.hyodysenteriae (EM) image

Swine dysentery

Haemorrhagic colitis

Growth requirements/characteristics

• Anaerobic/microaerophilic and difficult/slow growth (37^oC,48h) on ordinary medium
• Additives of serum, glucose & antibiotics (eg. Streptomycin) are essential
• A special medium (called BJ medium) enriched with pig faeces is used for successful growth
• Colonies ≈ 1mm diameter, transluscent & whitish
• Isolation can be achieved from fresh filtered pig faeces or ground pig colon mucoasa

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Biochemical properties: Not reported

Antigenic properties:

• A hemolytic toxin, considered important virulence factor
• Somatic antigens may have a role in pathogenesis

Resistance: Extremely sensitive outside the host body & dies readily when exposed to natural environment

Pathogenesis:

-T. hyodysenteriae invades and proliferates in tissues, invades colon epithelium and damages mucoasa causing diarrhoea with bloody mucus

- T. hyodysenteriae may associate with Fusobacterium sp and Bacterioides sp in causing disease

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Diagnosis:

1. Clinical & pathological: Dysentery, Emaciation, high morbidity (70%) vs low mortility (1%)
2. Direct DF microscopy can reveal presence of spirochates in fecal mucus
3. Serological: ELISA, IFAT

Treatment:

Antibiotics (Erythromycin) in pig feed & sulphonamides

Control/Prevention: Improve hygiene ALSO vaccination with bacterins can be attempted

• NEXTO >

Gen. Leptospira

• Gen. Leptospira has representative species which are agents of a zoonotic condition known as leptospirosis in animals (aka Weils disease in humans).
• The spirochete is maintained for a long time in rodent kidneys and is disseminated through rodent urine into the wet environments.
• Some fishes and reptiles often transmit the leptospires mechanically
• Infection is through the skin and mucoasa (mouth, nose , conjunctiva….) when they come into contact with water contaminated with urine of infected rats
• Leptospirosis is a water-borne occupational zoonosis of fishermen, wetland farmers/foresters, swimmers, butchers and even clinicians…… adventurists

• Humans and literally all warm blooded animals and some fishes are susceptible
• Clinical leptospirosis is variably from initial fevers, headaches, general malaise
• Advanced leptospirosis includes: jaundice, pettechial hemorrhage of mucoasa, hemoglobinuria, abortions, liver and kidney failure
• If untreated it causes death due to severe anaemia

Rodents: Reservoirs of Leptospira spp

Transmission of leptospirosis: Rat >water> humans and animals

Wetland farmers

Flood victims

Consumption of products contaminated with rat urine

Most mammalian species are susceptible to leptospirosis

Taxonomy of the leptospires

The genus Leptospira were initially divided into 2 groups, based on their potential to cause disease:

1. Leptospira interrogans group with a number of pathogenic species, which are slow growing ( 7+ days at 13-15^oC),

2. Leptospira biflexa group consisting of saprophytic species containing fast growing (2-3days at 5-10^oC) - saprophytic leptospires

The two groups can further be differentiated by:

i) Growth requirements, (eg. L. interrogans group do not survive below 10^oC, or in > 0.1M NaCl)

Species and of genus Leptospira

Today, within the genus Leptospira the described 20 species based on DNA hybridization of which:

• 13 are pathogenic
• 3 are intermediate/opportunistic
• 4 are saprophytic

Pathogenic species include:

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• The species of the genus Leptospira are further classified into serogroups and about 200 serovariants (serovars)
• In the classification of the leptospira the genus, species and serovariant are mentioned: eg. Leptospira interrogans serovar australis
• However, descriptively and in scientific reporting most leptospirologists skip the species name and commonly refer to the genus and serovariant when describing this bacterium (i.e Leptospira australis)

• Morphology of leptospires
• Long, slender (6-20 x 0.1-0.2μ) spiral microorganisms, with an axial filament around which the spirals are coiled. The spiral body ends in loose curved hooked presentations called terminal filaments.
• The leptospira exhibit an active back and forth flexuous motility
• Their motion are propelled by the terminal filaments

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Leptospira sp. Tightly coiler spirals (EM)

Staining characteristics:

• Leptospira being slender organsms are hard to stain by ordinary dyes, however, if Gram stain is attempted, leptospires stain weak Gram-ve
• Visualization is best achieved by:

i) observing a culture of the leptospires under a “dark-Field”

microscope or EM

ii)histologically by silver impregnation

iii)Immunohistologically by Immunofluorescence antibody technique (IFAT)

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Leptospira sp (DF Microscopy)

Leptospira sp Immunofluorescence (IFAT)staining

Cultural properties/requirements

• Very fastidious
• Aerobic environment, and best achieved in a semisolid media (eg. Fletcher’s, Korthoff, or in liquid medium (eg. Ellinghausen Mac Cullough-Johnson & Harris medium -EMJH) with additions of albumin or rabbit serum
• Growth temperature is wide, achieved from 29-30^oC up to 37^oC and above
• Growth is slow, turbidity in liquid medium achieved after 4days, with constant aeration (light shaking) but may take up to 30d

Antigenic properties

1. The axial filament is a proteinaceous Ag common to all species of the genus
2. Somatic Ags, including surface LPS (endotoxin) are variable with species and the LPS has been used as the key to classify the leptospires into serogroups and serovariants
3. The LPS itself and a hemolysin are important virulence factors

Resistance

-Variably resistant to environmental factors

eg.

• Survival in tissues is possible over a wide range of Temperature ( from 5^oC up to 37^oC),
• Survival is commonly possible in environmental water bodies at alkaline pH, which area not over contamination with faster growing microorganisms or fermenting organic matters

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The disease (leptospirosis) is diagnosed by:

i) Clinical manifestations fever (aka pyrexia of unknown origin –PUO), jaundice, hemoglobinuria mucosal/skin pettechia

ii) Bacteriologically: culture of suspect urine, blood, kidney tissue

iii) Serologically: microagglutination vs species/ serovariant- specific serum or ELISA)

iv) Molecular by PCR

v) Histopathologically by demonstration of spirochetes in kidney tissue, liver sections and in blood during febrile phase of disease

vi) Experimental infection of hamsters with suspect urine or blood or isolated leptospires

Clinical leptospirosis: Jaundice, skin pettechia

Dog jaundice

jaundice

Abortion due to leptospirosis are , usually without retention of the placenta

• Disease Treatment :

i) Use of antibiotics (penicillin, streptomycin, ciproflaxin)

• Control/prevention

i) Remove rodents

ii) Water sanitation & avoid spending time in floods

iii) Avoid rat infested waters and pools

iv)Use protective gear in fishing hunting and sporting

v) Vaccination of herds in disease endemic areas

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