Small Gram negative bacteria

1. Small Gram negative bacteria

2. 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 • Notifiablecategory 3zoonosis

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).

6. 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.

8. Brucella

9. Lysosome Phagolysosome fusion Acidification Prevent fusion Brucella sp. Phagosome H+ Brucella pathogenesis Brucella are facultative intracellular pathogens

10. Pathogenesis of Brucellosis • Penetrate mucosa • Regional LN • Phagocytosis/invasion • Proliferation/ Persistence • Bacteraemia • Dissemination Udder excretion Testis Uterus Placentitis & abortion Persistence

11. Brucella infection

12. 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

13. 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 Brucella pathogenesis

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

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

20. O side-chain Core Lipid A 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

21. 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.

23. Bordetella infections

24. Natural case of B. bronchiseptica infection

25. Adhesion of B.bronchiseptica to ciliated respiratory cells

26. Fimbriae Attach to host tissues Adhesins Filamentous Haemagglutinin (FHA) Pertactin Adenylate cyclase toxin Tissue damage Counteract host defences Toxins Tracheal cytotoxin Dermonecrotic toxin Type III secretion system Bordetella virulence factors

27. Adenylate cyclase toxin • Tracheal cytotoxin (TCT) • Adenylate • cyclase • RTX • toxin • TCT • LPS • Nitric oxide calmodulin [cAMP] •  Phagocytosis •  Chemotaxis Exotoxin & adhisin

28. Virulence genes expressed BvgA P 37OC 25OC Mutation Virulence genes not expressed BvgA Virulence genes never expressed Regulation of Bordetella virulence Temp BvgS BvgA

29. PMT Destruction of turbinates Initial attachment of B. bronchiseptica to ciliated cells. Tight adhesion to cilia. Production of toxins. Paralyse cilia • Colonisation with toxigenic • P.multocida types A or D Pathogenesis Loss of cilia. Interference with innate and specific immune response. Accumulation of mucus.

30. 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

31. Infectious bovine keratoconjunctivitis