Past Questions Microbiology 4

Front 1

Present pathogenous strains of E. coli. Briefly discuss their virulence factors

Back 1

- Enterotoxigenic E. coli (ETEC)

VF: Fimbriae K99, K88 and F41 and enterotoxins like; LT, STa and STb. Enterocyte adherence via fimbriae, enterotoxins causes secretory diarrhea.

- Enteropathogenic E. coli (EPEC)

VF: Intimin, toxins uncertain. Adherence due to intimin, attachment and lesion results from bacterial attacment to enterocytes and bruch border microvilli. Diarrhea because malabsorption.

- Enterohaemorrhagic E. coli (EHEC)

VF: Stx1 and Stx2. Causes haemorrhages.

- Enteroinvasive E. coli (EIEC)

VF:

- Necrotoxigenic E. coli (NTEC)

VF: Invasins, fimbrial and afimbrial adhesins, toxins like CNF1- and 2. Intestinal colonization to enterocytes and to blood vessels --> edema.

- Enteroaggregative E. coli (EAggEC)

VF:

Front 2

Selective medias to enterobacteriaceae

Back 2

McC - not very permissive

Xylose Lysine Deoxychocolate agar - good for shigella and salmonella

Hektoen agar - differential media between salmonella (black) and shigella (green)

Brilliant green agar - highly selective for salmonella other than S. typhi.

Salmonella Shigella agar - inhibits proteus spp. Used for isolation of salmonella and shigella.

Selenite F broth - enrichment medium for salmonella or shigella.

Front 3

Shipping fever - describe causative agent and the disease.

Back 3

Bovine pneumonic pasteurellosis

Caused by M. haemolytica serotype A1.

- Gram neg

- Coccobacilli to rods

- Non-motile

- Bipolar staining

- Microaerophilic with fermentative metabolism (ferment mannitol, but not D-mannose which diffenrentiate them from Pasturella)

- Fastidious

- Grow on McC

- Phenotypic resemblance to bacteria that belong to Pasturella

- Catalase +

- Oxidase +

Multifactoral, depends on environmental factors, concurrent infections and viruses and other bacterial agents. Also depends on the dose of M. haemolytica and host immune system status.

Causes severe bronchopneumonia.

Depression, fever, anorexia, nasal discharge soft moist cough. Apparent after 6-10 days after stressful episode. When extensive, dyspnea and open-mouth breathing.

Front 4

Isolation and testing of salmonella

Back 4

Specimen collection:

blood, urine, feces, vomit, bile, milk

med mera.

Inoculate in selenite F broth for enrichment and multiplied growth. Then inoculate on BA, McC and SS.

Growth on all 3 = salmonella.

- Biochemical tests:

Lactose neg

Indole neg

Nitrate +

Urease neg

Oxidase neg

Catalase +

H2S neg

Lysine decarboxylation +

- Serotyping - most imp

- Antibiotic susceptibility

Front 5

CAMP test

Back 5

Front 6

General features of actinobacillus

Back 6

- Gram neg

- Rods/coccobacilli

- Fastidious bacteria

- Anaerobic, CO2 required for growth.

- Carbohydrates fermented without gas production.

- Grow on media supplemented with blood and serum

- Temperature 20-42*C

- Most strains on McC but poorly

- Cultures die witin a week - Oxidase +

- Indole neg

- Nitrate reducant

Variably haemolytic on blood agar.

Front 7

How to identify virulent strains of staphylococci?

Back 7

By tube test testing the presence of coagulase (present in all pathogenic strains)

Isolated staphylococcal strains introduced to the tube with rabbit plasma. How fast the clot is formed depends on amount and phase of growth. Clotting occurs between 4-24 h. If negative, 4 h incubation 35*C (room temp) and then read again due to some strains capability of lysing the clot (= false negative result)

Front 8

Pathogenesis of stranglers. How is it transmitted?

Back 8

Stranglers in horses. Caused by Streptococcus equi spp. equi, where the bacteria is commensal to the mucosa of the upper respiratory tract. Opportunistic pathogen.

Pathogenesis:

Attaches to tonsillar crypt cells and adjacent lymphoid nodules. Within hours translocated into local lymphatics.

Fever, malaise, nasal dischargem cough, swelling at mandibular lymph node --> pressure on airway and respiratory difficulty.

Metastatic abscessation --> spread via circulation or lymphatic route to lungs, abdomen, brain (fairly common).

Clinical course short, often no complications but myocarditis and purpura haemorrhagica can follow (PH considered to be immune-mediated disease, occur 1-3 weeks after illness.

Transmission through direct contact with nasal discharge or via drainage of abscesses. Often when horses are assembled in one place, like markets, races or competitions. May continue the shed of bacteria 4 weeks after development of clinical signs.

Front 9

Diagnostic methods for identification of mastitis

Back 9

Clinical signs: inflammation of mammary tissue,and clots in the milk

Mastitic milk: clotted, watery, may contain mucus, pus, even blood

Milk samples should be collected carefullyto avoid contamination

Specimens (milk) should be cultured on blood agar, Edwards medium and MacConkey agar and incubated aerobically at 37*C for 24-48h.

Id by haemolysis, biochemical tests and Lancefield grouping.

Front 10

Explain phenomenon of clumping factor

Back 10

It's when coagulase is bound to the bacterial wall, which differentiate from the free coagulase released from pathogenic staphylococcus strains. The clumping factor converts fibrinogen in plasma to fibrin which causes the bacterial cells to clump together. A slide test alike the agglutination test is done (but no antibodies are involved) but it's an auto-reaction.

Front 11

Describe at least 2 selective medias for Salmonella and discuss the outcome of inoculation

Back 11

Hektoen Enteric agar:

Bile salts inhibit gram +

Substrates:

- Lactose

- Salicin

- Thiosulfate/ferric ammonium citrate

- Bromothymol blue

Differential medium. Blue/green = lactose non-fermented. Yellowish = lactose fermented.

Salmonella black colonies (FAC).

Shigella transluscent green colonies.

Brilliant green agar:

Brilliant green dye - inhibit gram + and most members of the enterobacteriaceae except Salmonella.

Substrates:

- Lactose

- Sucrose

- Phenol red

if sugars not fermented = pink colonies. (= salmonella).

If sugars are fermented = yellow/green colonies.

Front 12

Name causative agent in environmental mastitis

Back 12

E. coli, Klebsiella ssp., S. dysgalactiae, M. bovis, C. bovis

Front 13

Shipping fever laboratory diagnosis

Back 13

Specimen:

- Nasal swabs

- Purulent nasal discharge

Post-mortem: pneumonic lung

Direct examination: rods or coccobacilli.

Culture technique:

McC. If heavily contaminated BA with neomycin, bacitrin and acitidone.

Incubate for: 24-48 h aerobically at 37*C.

Id:

- Colonial morphology

--> pinpoint red colonies on McC. No agar, indole negative and have beta-haemolytic colonies.

- growth on McC

- + oxidase test

- + catalase test

Front 14

Characterize selective media for Enterobacteriaceae family

Back 14

Hektoen Agar, Brilliant Green agar, Xyline Lysine deoxycholate agar, Salmonella Shigella agar, Selenite F broth.

Front 15

Species of genus Actinobacillus

Back 15

Species:

- A. lignieresii

- A. pleuropneumoniae

- A. equuli (ssp. equuli, in Eq and Su, and ssp. haemolyticus onlu in horses)

- A. suis

- A. seminisA. actinomycetemcomitans (aggregatibacter actinomycetemcomitans) (do not grow on McC)

Front 16

Characterize genus Actionbacillus, name species of veterinary importance

Back 16

Gram negative

Rods to cocci

Anaerobic, require CO2 for growth

Carbohydrates fermented without gas production

Temp 20-42

Fastidious, need blood and serum

Most strains grow on McC but poorly

Oxidase +

Indole -

Nitrate reductant

Variably haemolytic on BA

A. ligniersii

A. pleuropneumoniae

A equuli (spp. equuli and haemolytica)

A. suis

A. seminis

A. actinomycetemcomitans

Front 17

Five examples of discriminatory features of Pasturella spp. and Mannheimiia haemolytica

Back 17

Pasturella is:

Non-haemolytic

Indole positive

Do not grow on McC

Sweetish odor

Shiny, grayish, moderate size, round colonies

M. haemolytica:

Haemolytic (beta-haemolytic)

Do grow on McC

Indole negative

Pinpoint red colonies on McC

No odor

Front 18

General characteristics of Enterobacteriaceae family

Back 18

• Gram-negative

* facultively anaerobic rods

• Ferment glucose and other sugars

• Reduce nitrate to nitrite

• Oxidase negative

• Catalase positive

• Non-sporeforming

• Tolerate bile salts in McC agar

• Non-fastidious, grow on non-enriched media *Most motile by peritrichous flagella (= flagella projecting in all directions)

Front 19

Haemophilius species of vet. Importance and caused diseases

Back 19

Main pathogens:

- H. parasuis(pigs) - Glässer's disease, meningitis, myositis, pneumonia, septicaemia

- H. paragallinarium(chickens) - Infectious coryza

- H. influenzaemurium(rodents) - respiratory infection, ocular disease

Front 20

What cause cholera and what are the virulence factors

Back 20

Vibrio cholerae.

VF:

Polysaccharide capsule

- Enterotoxins - LT (activates adenylate cyclase which activates cAMP which lead to hypersecretion of fluids and electrolytes which results in watery diarrhea.

- LPS (core nec to survive bile)

- Siderophores

- Pili - adherence

Front 21

Describe growth of Proteus spp on selective media

Back 21

Swarming bacteria. A multicellular behaviour, a cyclical differentiation of short vegetative cells called swimmer cells and filamentous highly flagallated cells called swarmer cells. Result in a coordinated behaviour.

Front 22

Compare and differentiate between Y. enterocolitica and Y. pseudotuberculosis

Back 22

Y. pseudotuberculosis:

- cause severe systemic disease

- have 10 serotypes (I, II, III most pathogenic)

cause; enteric yearsinosis in farmed deers

- pseudotuberculosis in guinea pgs

- septicaemic yearsiniosis in caged birds

Y. enterocolitica:

- less likely to become systemic disease

- cause disease in humans mostly

- 5 biotypes and 50 serotypes

Both found in intestinal tract of wild mammals. Mechanically spread by birds.

Both cause sporadic abortion in ewes.

Same VF (adhesins, invasins, resistance to phagolysis, induce macrophage apoptosis, enterotoxins like YST (mostly in enterocolitica) and iron acquisation)

Front 23

Vibrio species of veterinary importance and disease they cause

Back 23

V.metschinikovii - cholera like disease in poultry and young birds housed in zoological collections

V. ordalii - Vibriosis in fish. Haemorrhagic septicaemia in eels, salmons and trouts.

V. salmonicida- Cold-water vibriosis. Skin haemorrhages in cultured Atlantic salmon.

Front 24

Media for haemophilus. Explain phenomenon of “satellitism"

Back 24

Chocolate agar contain the necessary X factors (hemin) and V factors (NAD). Satellitism is when you inoculate blood agar with Haemophilus and then streak S. aureus (feeder streak) across. The S. aureus provide the necessary V factors for haemophilus growth - the growth occur only near the feeder streak.

Common in sputum cultures (where both bacterias can be present).

Front 25

Purpose and method of salmonella serotyping

Back 25

The purpose of salmonella serotyping is to differentiate between the 2 400 diff. salmonella serotypes.

Serotyping refers to the differences in microbial surface antigens between different Salmonella serovars. Members of a serogroup is called serovars.

Method:

Serovars id'd by 2 sets of antigens

O - somatic antigens discovered by agglutination test. Gives the serogroup.

H - flagellar antigens. Often biphasic, discovered by 2 rounds of agglutination tests.

Vi - (in some serovars) superficial capsular K-antigens.

Front 26

What are the functions of E.coli in normal flora and how is it transmitted?

Back 26

Fermentation of ingesta. Transmitted fecal-oral route.

Front 27

Lab diagnosis of EHEC

Back 27

Specimen:

feces, urine, blood, milk, tissue samples

Culture:

SMAC (McC with sorbitol)

Incubation 24 h 37*C

Results:

Growth on McC (lac +)

colourless colonies in SMAC, difference between other E. coli that would result in pink colonies - which mean EHEC does not ferment sorbitol (like the other strains do).

Esp. for E. coli O157 (EHEC)

Further on biochemical tests, PCS (genes encoding pathogenicity), serotyping and antibiotic susceptibility

Front 28

Characterize Vibrioaneceae family

Back 28

Gram neg rods (some curved)

Halophilic (thrive in salty conditions)

Non-fastidious but need NaCl complementation

Habitat in salty to breckish waters.

Facultative anaerobes

catalase +

oxidase +

motile

Opportunistic pathogen in fish and in chicken.

V. metschnikovii

V. ordalii

V. salmonicida

Front 29

Isolation of Salmonella from fodder

Back 29

Specimen: Suspected contaminated fodder or animal feed.

Preincubation in buffered peptone water (enrichment broth which supplies bacteria with N compounds and maintain pH)

Incubation for 16-24 h in 37*C

Transfer to SF broth 18-24 h 37*C

Inoculate on SS, BGA, Hektoen agar or XLD agar

Incubate 18-24 h 37*C

Biochemical differentiation and serotyping of result.

Front 30

Staphylococcus morphological features and characteristics of colonies on blood agar

Back 30

Front 31

Toxins and enzymes produced by Staphylococci

Back 31

Enzymes:

Coagulase

Lipase

Nucleases

Hyaluronidase

Front 32

Characteristic of Staphylococci and Streptococci growth on liquid and solid surfaces

Back 32

Morphology Staphylococci:

Medium sized

White, creamy, yellowish, yellow, orange

Haemolysis type beta:

Complete - S.aureus

Incomplete - S. pseudointermedius and S. intermedius

No haemolysis - S. epidermidis

OF test - aerobic growth

Morphology Streptococci:

Small transluscent colonies

Type of haemolysis

OF test - aerobic growth

Front 33

Virulence factors of Staphylococcus species

Back 33

Coagulase (act thrombin - fibrinogen to fibrin)

Protein A - binds to immunoglobulins (Fab and Fc - inhibit fagocytosis)

Capsular polysaccharide (release of cytokines, interfere with host defense mechanism)

Peptidoglycan and lipotheichoic acid (cytokine release)

Adhesins - MSCRAMMs

Hyaluronidase - spreading factor (so that pathogen can invade deeper tissues)

Toxins

TSST-1 = superantigen, stimulate T cells without antigen recognition.

Staphylococcal enterotoxins (SEs) - vomiting activity

Exfoliative toxins (ETs) - widespread blistering and loss of epidermis (S.pseudointermedius - canine pyoderma)

Haemolysin - Alpha and beta

Leukocidin - Panton-Valentine leukocidin (PVL) necrotizing factor. Poreforming, act on neutrophils. (S. aureus)

Front 34

Differentiation of Staphylococcus species

Back 34

The complete, incomplete or absent haemolysis.

Coagulase slide test - clearest + result in S.aureus. Variable result in S. pseudointermedius.

S. aureus is golden yellow on blood agar. Others white

PCR

Front 35

Test tube detection of coagulase

Back 35

Check for + or neg result for the coagulase enzyme. The clumping factor, dvs. coagulase bound to the bacterial cell wall (not free coagulase). Used to identify pathogenic species.

When bacteria is isolated, introduce into tube with rabbit plasma. Positive result gives clotting which occurs within 4-24 h (incubated 35*C).

After should be held at room temperature and read again after 18-24 h, because some strains lysis the clot and will then give a false negative result at the 1st reading.

Front 36

Differentiation of G+ pyogenic cocci

Back 36

Differences in colony morphology

Catalase test (strepto neg, staphylo pos)

OF test (micrococcus F-)

Resistance to lysostaphin (enzyme that cleaves cross-bridges in cell wall). Staphylo susceptible, Micrococcus resistant

Front 37

What medium is used for isolation of Haemophilus and why

Back 37

Chocolate agar

Because it provides the necessary X factors (hemin) and V factors (NAD) necessary for haemophilus growth.

Front 38

Pyogenic Streptococcus species

Back 38

S. Agalactiae

S. Dysgalactiae

S. Uberis

S. Equi ssp. Zooepidemicus

Front 39

Pathogenic Streptococcus species

Back 39

S. pyogenes - man, scarlet fever

S. agalactiae - cattle sheep goat, chronic mastitis

S.dysgalactiae - cattle, acute mastitis

S. equi ssp. equi - horses, Strangles

S. equi ssp. zooepidemicus - horses, mastitis

S. suis - pigs, septicaemia

S. canis - carnivores, neonatal septicaemia

S. uberis - cattle, mastitis

S. pneumoniae - man primates ect, septicaemia

Front 40

Streptococcal species and pathogenicity

Back 40

S. pyogenes - man, scarlet fever

S. agalactiae - cattle sheep goat, chronic mastitisS.dysgalactiae - cattle, acute mastitis

S. equi ssp. equi - horses, Strangles

S. equi ssp. zooepidemicus - horses, mastitisS. suis - pigs, septicaemia

S. canis - carnivores, neonatal septicaemia

S. uberis - cattle, mastitis

S. pneumoniae - man primates ect, septicaemia

Pathogenicity:

Beta-haemolytic more pathogenic than alpha-haemolytic.

Virulence factors:

• Exotoxins: - Streptolysins (haemolysins) Beta-haemolysin cytotoxic for eukaryotic cells by pore forming in target cell membrane.

CAMP factor (ceramide-binding protein) of S. agalactiae --> haemolysis of staphylococcal sphingomyelinase (cytotoxic for mammary glands)

Pyrogenic exotoxin A - superantigen --> stimulate cytokine production by T cells (streptococcal toxic chock syndrome)

Streptolysin O (SLO) - produce streptococci of A, C and G. Cholesterol-binding toxin. Potent membrane-damaging effects.

- Hyaluronidase - DNAse - Streptokinase

- Protease

- Polysaccharide capsule, antiphygocytic (S. pyogenes, S. pneumoniae and some strains of S. qui)

- Cell wall M proteins --> antiphagocytic also

• Enzymes Dissolve fibrin

• Binding of plasma components - avoid IMS or phagocytic effect.

Front 41

Identification of Streptococcus responsible for mastitis

Back 41

Streptococcus agalactiae.

BA and McC. No growth on McC.

Colonies mucoid transluscent, with beta-heamolysis. Incubation 37*C for 24-48 h

Front 42

Morphology and characteristics of Pasteurella

Back 42

- Small

- Non-motile

- Gram neg

- Rods/coccobacilli

- Oxidase +

- Facultative anaerobes

- Most species catalase +

- Fastidious

- Bipolar staining

- Most commensals of the upper respiratory tract mucosa, some respiratory pathogens.

- Survival in environment relatively short.

Vet importance:

Pastuerella multocida

Bibersteinia trehalosi

Morph:

Round, shiny, grayish, moderate sized colonies. Smell sweetish.

Front 43

Major pathogenic species of Pasteurella, as well as those of minor veterinary importance

Back 43

Vet importance:Pastuerella multocida Bibersteinia trehalosi

Minor:

P. aerogenes

P. anatis

P. avium

P. caballi

P. canis

P. dagmatis

P. gallinarium

P. limphangitis

P. pneumotropica

Front 44

Pasteurella species and pathogenicity

Back 44

Vet importance:Pastuerella multocida Bibersteinia trehalosi

Minor:P. aerogenesP. anatisP. aviumP. caballiP. canisP. dagmatisP. gallinariumP. limphangitisP. pneumotropica

Endogenous infections.Commensal in upper respiratory tract, invade tissues of immunosuppressed animals.Transmission --> exogenous by direct contact or through aerosol.Importance in disease development:

- Adhesions

- Avoidance of phagocytosis

- Fimbriae, enhance mucosal attachment

- Hyaluronic capsule, antiphagocytosis role

DiseaseSepticaemic pasteurellosis --> severe endotoxaemia and disseminated intravascular coagulation which cause serious illness which can prove fatal. Endotoxin is pyrogenic.

Front 45

Vibrio pathogenicity

Back 45

Virulence factors for V. cholerae

- Capsular

- adherence + protection

- Polysaccharide

- Toxins - similar to LT enterotoxin of E. coli. 2 component toxin; B subunit (mediates entry) and A subunit activate cellular adenylate cyclase, causing cAMP accumulation and result in hypersecretion of electrolytes and fluids.

- LPS - core necessary to survive bile.

- Siderophores - acquisation of iron

- Pili - adherence

Front 46

What diseases are caused by Actinobacillus, which species causes each disease?

Back 46

A. ligniersii- wood tongue in bovine

A. pleuropneumoniae- pleuropneumonia in pigs

A equuli (spp. equuli and haemolytica)- septicaemia and sleepy foal disease

A. suis- septicaemia, pneumonia

A. seminis- epididymitis

A. actinomycetemcomitans- epididymitis

Front 47

Characteristics and pathogenicity of Aeromonas and Plesiomonas

Back 47

Aeromonas:

Invade host cell, disseminate to any organ. Death due to massive septicaemia and toxic etracellular products of the organism, which interefere with host blood supply and result in tissue necrosis.Virulence factors

- Toxin - cytotoxin, similar to Shiga toxin

- Surface proteins

- Extracellular degradative enzymes.

- Heamolysins - aerolysins, poreforming toxin that causes ion leakage --> cell lysis

- Cytotoxic enterotoxin, heat-stable cytotoxic toxin and heat-labile enterotoxin --> enteropathogenicity.

- Flagellae - swimming

- Pili - adhesins

- Capsule

- Wide range of enzymes (serine protease, lipases, aminopeptidases, metalloproteases and phospholipases.

- Siderophores

Plesiomonas:

- Cholera-like toxin

- Haemolysin

- role in iron acquisation

- Heat-stable enterotoxin

Front 48

Front 49

Lab diagnosis of A. lignieresii

Back 49

Wash pus or exudate with distilled water on a petri dish. Suspend the white granules in 10% KOH on a slide and cover with a glass slip. Observe under microscope the clubs and the area that surrounds the clubs that the granulomatous create (low magnitude). Can also be stained for better vision.