small gram negative bacteria. brucella small gram negative, coccobacilli non-motile, aerobic,...
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Small Gram negative bacteria
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Brucella• Small Gram negative, coccobacilli• Non-motile, aerobic, non-fermentative• Obligate intracellular• Oxidase and catalase positive• Visualised by Kosters (modified –ZN) stain• Require growth on complex media
• Growth enhanced by CO2 (some require CO2 for growth)
• Species identified by growth characteristics and serotyping• Predilection for male and female reproductive tracts in
animals and cause reproductive disease in mammals• Notifiable category 3 zoonosis
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Diagnosis
• Specimens:– Liquid specimens can be incubated straight.– Scrapings from cotyledons and tissues are
homogenised.– Specimens include urine (males), milk, blood, vaginal
swabs, etc.– Grow at 37°C for 4-15 days.
• Direct microscpy:– From cotyledons using modified Zeihl-Nelsen (MZN)
stain (also known as Koster stain).
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Diagnosis
• Require enriched media to grow.– E.g. blood agar, liver infusion, tryptose, etc
• Colonies are pinpoint, round, smooth, glistening, translucent and non-haemolytic.
• Biochemical tests:– Oxidase (except B. ovis), Catalase positive.– Urease (except B. ovis) positive.– Indole negative.– Reduce nitrate.
• Serological tests:– E.g. serum agglutination test (SAT).
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B. abortus, foetal stomach contents, Koster’s stain
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Koster’s stain
1. Flood the slide with dilute carbol fuchsin and let it act for 5-7 minutes.
2. Wash with water.3. Decolourise with 0.25% acetic acid for up to 30
seconds. The film should be colourless or very faintly pink.
4. Wash with water.5. Counterstain with Loeffler’s methylene blue and
leave for ½-1minute. Brucellae appear as little red bacilli singly, in pairs and in clusters.
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Brucella
Species Main hosts - disease Virulence for humans
B. abortus Cattle. Contagious bovine abortion., orchitis.
Sheep, goats, pigs. Sporadic abortion.
Humans. Undulant fever.
++
B. melitensis Sheep and goats. Orchitis and abortion. Humans. Malta fever.
++++
B. ovis Sheep. Abortion and epididymitis -
B. canis Dogs. Abortion, epididymitis and arthritis. Humans. Mild infection.
+
B. suis Pigs. Abortion, infertility, orchitis and arthritis.
Humans. Undulant fever.+++
B. neotomae Rodents. -
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Brucella pathogenesis
Brucella are facultative intracellular pathogens
Phagosome H+
Acidification
LysosomePhagolysosomefusion
Prevent fusionBrucella sp.
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Pathogenesis of Brucellosis
•Penetrate mucosa•Regional LN
•Phagocytosis/invasion•Proliferation/ Persistence
•Bacteraemia•Dissemination
Persistence Testis Udderexcretion
UterusPlacentitis& abortion
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Brucella infection
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B.melitensis genome
• 2 chromosomes!• Lacking many expected virulence systems
– Type III secretion systems– Fimbriae– Toxins– Capsule
• Putative virulence genes identified– Type IV secretion system
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Brucella pathogenesis
• What we know about Brucella virulence – very little.
• Intracellular pathogen – many cell types• Survives within macrophages
– Inhibits phagolysosome fusion
• Tissue tropism - erythritol– Erythritol preferred carbon source– Present in high concentration in placenta of cattle,
sheep, goats and pigs.
• Siderophore, iron-uptake
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Brucella: persistent infection
• Survival within macrophages• Inhibits phagolysosome fusion– Type IV secretion system
• Don’t alarm the host• Brucella LPS 1000 times less pro-inflammatory than
E.coli LPS
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Bovine brucellosis: Eradication
• 1934 ~ 40% of cattle herds Brucella positive
• A free calf vaccination scheme, S19 vaccine, 1962.
• Voluntary attested herds scheme, 1967.
• Compulsory area eradication, 1971.
• Disease-free, vaccination programme ended 1979
• Brucellosis-free status gained from EU, 1991.
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Human brucellosis
Algeria
Argentina
Australia
Azerbaijan
Bosnia-Herzegovina
Colombia
Germany
Greece
Iran
Israel
Italy
Jordan
Mexico
Portugal
Spain
Syria
Tajikistan
Tunisia
Turkey
United Kingdom
United States
0 5000 10000 15000 20000 25000
Human brucellosis cases 2003
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Brucella screening
• Usually serology – many different tests• Detect antibody in serum (milk) against Brucella LPS• Tests
– Milk ring test– Rose Bengal plate test– ELISA– Serum agglutination test (SAT)
• False positives– Antibodies cross react with LPS of other bacteria– Yersinia enterocolitica O9
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Brucella screening
Test Name Sample Sensitivity % Specificity % Use RBT (Rose Bengal test. Also known as Buffered Brucella antigen test)
Serum H M
Formerly used as screening test for animals not contributing to bulk milk supplies; superceded by ELISA.
ELISA*
Serum H H Prescribed. Screening test for for animals not contributing to bulk milk supplies.
SAT (serum agglutination test)
Serum M H Use in EU. Confirmatory test for Elisa reactors.
CFT* (Complement fixation test)
Serum M H Prescribed. Confirmatory test for Elisa reactors. ELISA* Milk H H GB screening test for dairy herds
Milk ring test (MRT)
Milk NA NA Formerly used for screening dairy herds: now superceded by ELISA
Culture
swab/milk/ foetal
tissues
Depends upon sample quality. Should be high 100 % Definitive
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Brucella vaccines
• Live vaccines, not genetically defined
• “Smooth” strains– B.abortus S19 - Cattle– B. melitensis Rev.1 -sheep goats
• “Rough” strains– B.abortus RB51 rifampicin R - Cattle
• Problems– Anti-LPS antibodies – screening– Infection of humans
Lipid A
O side-chain
Core
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Bordetella
• Small gram negative coccobacilli• Strictly aerobic • Oxidase and catalase positive
• Motile (except B.pertussis and B.parapertussis)• Don’t ferment carbohydrates (growth is strongly
inhibited in acidic medium)• Grow on MacConkey (except B.pertussis and
B.parapertussis)• Respiratory tract pathogens mammals and birds
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Diagnosis
• Culture on blood agar for 24-48h.
• Colonies are pinpoint and smooth.
• Growth on the selective Smith-Baskerville (SB) medium is enhanced. Colonies appear green surrounded by bluish discolouration.
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Bordetella infections
Bacteria Host Disease
B.bronchiseptica Dogs
Cats
Pigs
Many sp.
Kennel cough
Respiratory infections Progressive atrophic rhinitis with P.multocida, bronchopneumonia
Respiratory infections
B.parapertussis Sheep
Humans
Respiratory infections
Whooping cough
B.avium Turkeys Turkey coryza
B.pertussis Humans Whooping cough
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Natural case of B. bronchiseptica infection
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Adhesion of B.bronchiseptica to ciliated respiratory cells
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Bordetella virulence factors
Fimbriae
Filamentous
Haemagglutinin (FHA)
Pertactin
AdhesinsAttach to
host tissues
Adenylate cyclase toxin
Tracheal cytotoxin
Dermonecrotic toxin
Type III secretion system
ToxinsTissue damage
Counteract
host defences
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calmodulin
[cAMP]• Phagocytosis
• Chemotaxis
•Nitric oxide
•Adenylate
•cyclase
•RTX
•toxin
•Adenylate cyclase toxin
•TCT •LPS
•Tracheal cytotoxin (TCT)
Exotoxin & adhisin
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Regulation of Bordetella virulence
BvgS
BvgA
Temp
Virulence genes
expressedBvgA P
37OC
BvgA
25OC
Virulence genes
not expressed
Virulence genes
never expressed
Mutation
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Tight adhesion to cilia.
Production of toxins.
Paralyse cilia
Pathogenesis
Initial attachment of B. bronchisepticato ciliated cells.
PMT
Destruction of turbinates
Loss of cilia.
Interference with innate and specific immune response.
Accumulation of mucus.
•Colonisation with toxigenic
•P.multocida types A or D
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Moraxella bovis
• Gram negative short plump rods often in pairs• Strict aerobe, “twitching-motility”• Oxidase and catalase positive• Do not grow on MacConkey. Do not utilise sugars• Urease and nitrate negative• Infectious bovine keratoconjunctivitis, (new forest eye,
pink eye)• Virulence factors
– Fimbriae (pili)– Haemolysin – RTX toxin– Lactoferrin + transferrin binding protein – Fe uptake– Capsule, degradative enzymes
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Infectious bovine keratoconjunctivitis