Small Gram negative bacteria

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

3

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).

4

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).

5

B. abortus, foetal stomach contents, Koster’s stain

7

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.

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. -

9

Brucella pathogenesis

Brucella are facultative intracellular pathogens

Phagosome H+

Acidification

LysosomePhagolysosomefusion

Prevent fusionBrucella sp.

Pathogenesis of Brucellosis

•Penetrate mucosa•Regional LN

•Phagocytosis/invasion•Proliferation/ Persistence

•Bacteraemia•Dissemination

Persistence Testis Udderexcretion

UterusPlacentitis& abortion

11

Brucella infection

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

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

14

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

15

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.

16

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

17

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

18

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

20

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

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

22

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.

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

Natural case of B. bronchiseptica infection

Adhesion of B.bronchiseptica to ciliated respiratory cells

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

calmodulin

[cAMP]• Phagocytosis

• Chemotaxis

•Nitric oxide

•Adenylate

•cyclase

•RTX

•toxin

•Adenylate cyclase toxin

•TCT •LPS

•Tracheal cytotoxin (TCT)

Exotoxin & adhisin

28

Regulation of Bordetella virulence

BvgS

BvgA

Temp

Virulence genes

expressedBvgA P

37OC

BvgA

25OC

Virulence genes

not expressed

Virulence genes

never expressed

Mutation

29

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

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

Infectious bovine keratoconjunctivitis