Basic Swine Bacteriology

 

Enteric Pathogens

Escherichia coli

E

E

E coli tergitol 2

Blood agar

Most pathogenic strains will be β haemolytic or smooth and mucoid

MacConkey’s

Pink colonies – lactose fermenter

Tergitol

Yellow- gold colonies lactose fermenter

A   B    C

 
E

Tests –

A – Urease –ve yellow

B – Simm’s + ve red colour

C – Kligler’s +ve with gas

Oxidase -ve

E coli 1

Gram negative rod

Clostridium perfringens

Clostridia perfringen blood

Requires anaerobic culture.  Note flat spreading colony shape.  Double zone of haemolysis.

Clostridia perfringes 1

Gram positive rods – vegetative and solid rods some spore formation

Blood agar

Brachyspira hyodysenteriae and Brachyspira pilosicoli

Brachyspira sample

Requires anaerobic culture and 42C. A filmy surface growth is typical, rather than distinct individual surface colonies.  β haemolytic Bacteria lie under the surface.

Brachyspira 1

Weakly Gram positive spirochaetes.


 

Salmonella spp.  There are over 2300 types of salmonella

Salmonella blood

Salmonella mac

Salmonella tergitol

Blood agar Salmonella strains are non-haemolytic on blood agar

MacConkey’s

A non-lactose fermenter

Tergitol – a non-lactose fermenter

A   B

 
Salmonella test

Tests –

 

A – Kligler’s Iron agar –H2S + Lactose Blue colour

B  - Lysine Iron Agar + ve Blue colour

Salmonella 1

Gram negative rod

Lawsonia intracellularis

Note L. intracellularis requires cell culture.  It will not grow on agar media

 


 

Respiratory pathogens

Actinobacillus pleuropneumoniae

APP Blood

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Blood agar

Requires staphylococcus streak - Haemolytic - satellitism

MacConkey’s –

No growth

Tergitol - No growth

APP HPS tests

APP left, HPS right plates

 

Differentiation between APP and HPS by cultural characteristics and urease positive – blue coloration and is CAMP positive.

APP colpnies

Haemolytic colonies

APP2

Gram negative rods and coccobacillary forms

 

Organism generally requires NAD (Nicotinamide adenine dinucleotide to grow – V factor) – supplied by a Staphylococcus streak.

Bordetella bronchiseptica

Bordetella bronch blood

Bordetella bronchiseptica mac

Bordetella bronch tergit

Blood agar   - Incubate for at least 48 hours

MacConkey’s -  non lactose fermenter

Tergitol – non lactose fermenter

A     B    C     D  

 
Bordetella poss

A – Kligler’s –ve red

B – Simm’s – Poor reaction little growth

C- urease – Strong positive – pink slant

D - Simmon’s citrate agar – positive – blue color

Oxidase +ve

Bordetella 1

Small gram negative coccobacilli

Pasteurella multocida

Pasateurella multocida A blood

Pasteurella multocida D blood

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Blood agar assisted with Staphylococcus streak to distinguish A and D by producing mucoid colonies

MacConkey’s – no growth

Tergitol – no growth

P. multocida A mucoid colony -  may run together

P. multocida D

non mucoid – dry colony

A   B       C

 
Pasteurella tests

Tests – note distinctive odor – musky

A – Dextrose Broth - +ve red no gas.

B – Lactose –ve yellow

C - Indole positive red

In addition - urease negative

Pasteurella multocida 1

Small Gram negative rod or coccobacillus

Salmonella choleraesuis See enteric pathogens for growth characteristics

Streptococcus suis

Streptococcus suis blood 2

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Blood agar

MacConkey’s – no growth

Tergitol – no growth

Strep suis blood a haem

Note type of haemolysis

α haemolysis - green,

β  haemolysis – clearing

 

Catalase –ve

Strep suis 1

Gram positive small cocci –in chains or pairs


 

Actinobacillus suis

A suis blood

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Blood agar – wide zone of  β haemolysis

MacConkey’s – no growth

Sometimes very small

Tergitol – no growth

A         B    C

 
Actinobacillus suis tests

Tests –

 

No growth on MacConkey’s or Tergitol important differential from E. coli

 

A - Kligler’s – Lactose +ve Dextrose +ve red

B – Simm’s – difficult little reaction

C – Urease +ve red

Actinobacillus suis 1Gram negative rod

Haemophilus parasuis

Haemophilus parasuis blood

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Blood agar – very small colonies around the Staphylococcus streak

MacConkey’s – no growth

Tergitol – no growth

Figure 32

   Can be difficult to grow – requires NAD.  

Reputedly Grows best under CO2 on chocolate agar.

It is urease negative - yellow – useful to distinguish from APP positive - blue.

Haemophilus parasuis 1Gram negative coccobacillus


 

Arcanobacterium pyogenes

Arcanobacterium pyogenes blood

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Blood agar

Catalase -ve

MacConkey’s – no growth

Tergitol – no growth

A pyogenese 1

Gram positive pleomorphic rods – “Chinese letters”

Mycoplasma hyopneumoniae

Mycoplasma’s require special media.  M. hyopneumoniae requires a very long incubation period and a specialized laboratory to grow the organism, which even then is frequently overgrown with other mycoplasma spp.


 

Other pathogens

Erysipelothrix rhusiopathiae

Erysipelothrix rhusiopathiae blood

Mac no growth 3

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Blood agar – small colonies

MacConkey’s – no growth

Tergitol – no growth

Erysipelothrix rhusiopathiae tests

Tests -  Produces hydrogen sulfide (black streak) along stab line in Kligler’s iron agar.

 

Erysipelasis 1Slender Gram positive rod

Actinobaculum suis (Eubacterium suis)

Gram positive pleomorphic rods

Urinary Actinomyces suis colonies

Urinary A

Tests – urease positive

Blood agar – requires anaerobic culture.  Flat colonies fried egg shaped

Pleomorphic rods

Staphylococcus spp.  – Example - Staphylococcus hyicus

Staph blood

Mac no growth 2

Tergitol no growth 3

Blood agar

MacConkey’s No growth

Tergitol No growth

Staphlylococcus 1

Gram positive cocci – in clumps resembling grapes

 

Catalase positive


 

Basic antimicrobial tests

Kirby-Bauer

Kirby Bauer sensitivity

Note each disc has its own zone of inhibition

Minimum Inhibition Concentration - MIC

MIC tests 1

MIC tests 1a

Automated system

View of the plate – demonstrating growth (white areas) and inhibition (clear)

MIC test 2

E test MIC strip.

 

The zone of clearing visibly seen on the plate.

 


Specific Tests:

 

Gram’s Stain:

Always place organisms in centre and always use “frosted side up”

  1. Make a thin smear of the organism with a tiny droplet of water on a slide
  2. Dry – best if air dried
  3. Fix by gently hearing 2-3 times through the flame
  4. Flood smear with Crystal violet for 5 seconds, hold side perfectly level, then dump
  5. Wash with distilled water
  6. Flood smear, with Gram’s iodine for 5 seconds
  7. Wash with distilled water
  8. Decolorise with acetone-alcohol about 3 seconds
  9. Wash with distilled water
  10. Counterstain with safranin for 5 seconds
  11. Wash with distilled water
  12. Blot slide dry, face down on a paper towel.

 

Acid Fast:  Zeil-Neilson

  1. Make a thin smear of the organism with a tiny droplet of water on a slide
  2. Fix by gently hearing 2-3 times through the flame
  3. Flood smear with Carbol fuchsin
  4. Heat flooded slide over flame for 2-3 minutes
  5. Wash with distilled water
  6. Decolorise with acetone-alcohol about 3 seconds
  7. Wash with distilled water
  8. Counterstain with methylene blue for 30 seconds
  9. Wash will distilled water
  10. Blot slide dry, face down on a paper towel.

 

Catalase Test:

1.   Dip a capillary tube into 3% H202

2.  Touch a colony

3.   Observe the tube for bubble indicating a positive reaction

Do not contaminate the bacterial colony with blood agar as red blood cells contain catalase thus resulting in a false result.  Old cultures can lose their catalase activity.

 

Oxidase Test:

1.      Hold a piece of oxidase test paper with forceps and touch onto an area of heavy growth

2.      Rapid (within 10 seconds) purple positive.  If within a minute delayed positive

Note an oxidase organism will also be catalase positive


 

 

Basic Swine Bacteriology – Summary Table

 

 

 

Organism

 

Gram Stain

Growth

Sugars and reactions

Anaerobe only

Hamolytic Blood agar

MacConkey

Tergitol

Catalyse

Oxidase

Dextrose broth

Kliglers

Kliglers iron

Lactose

Lysine

Simms

Simms Citrate

Urease

Actinobaculum suis

+B

+

N

-

-

-

-

 

 

 

 

 

 

 

+

Actinobacillus pleuropnumoniae

-CB

 

Y

-

-

V

V

 

 

 

 

 

 

 

+

Actinobacillus suis

-B

 

β

-

-

+

V

+

 

 

+

 

 

P

+

Arcanobacterium pyogenes

+B

 

N

-

-

-

-

 

 

 

 

 

 

 

 

Bordetella bronchiseptica

-CB

 

N

+NL

+NL

+

+

 

-

 

 

 

P

 

+

Brachyspira

+S

+

β

-

-

-

-

 

 

 

 

 

 

 

 

Clostridium perfringens

+B

+

Y

-

-

-

-

 

 

 

 

 

 

 

 

Erysipelothrix rhusiopathiae

+B

 

N

-

-

-

-

 

 

+

 

 

 

 

 

Escherichia coli

-B

 

N+β

+L

+L

+

-

 

+G

 

 

 

+

 

-

Haemophilus parasuis

-CB

 

N

-

-

+

-

 

 

 

 

 

 

 

-

Pasteurella multocida

-CB

 

N

-

-

+

+

+

 

 

-

 

 

+

-

Salmonella

-B

 

N

+NL

+NL

+

-

 

 

+

 

+

 

 

 

Staphylococcus

+C

 

 

-

-

+

-

 

 

 

 

 

 

 

 

Streptococci

+C

 

α/ β

-

-

-

-

 

 

 

 

 

 

 

 

 

Code:

+ and green = Positive.     and red = Negative. 

Gram stain:  B = Bacillus/coccobacillus.  C = Coccoid. CB = Coccobacillus. S = Spirochaete.

Sugars and reactions: V= variable G = Gas.  P = Poor  L = Lactose NL= Non Lactose

Haemolytic Y = Yes and type α or β.  N= No. 

 

Gram’s staining of the main organisms