Laboratory diagnostics: Actinobacillus pleuropneumoniae (APP)

Assays available

Gross pathology

• Evaluates the presence of tissue lesions which can almost be diagnostic for the disease.
• Location of lesions: lung
• Necro-hemorrhagic areas of consolidation in the lung accompanied by fibrinous pleuritis (see Figure 1).

• Pros:
  • Presumptive diagnosis can be made after gross examination of lungs.
• Cons:
  • Actinobacillus suis can cause some similar lesions although less severe.
  • Cannot identify serotype.

Bacterial culture

• Isolation of live organism
• Sample types: lung
• Pros:
  • Can do in any lab (including in-house)
  • Biotype I requires NAD (usually provided via Staphylococcus aureus nurse colony)
  • Biotype II does not require NAD
  • Relative low cost
• Cons:
  • Not always easy to grow
  • Cannot serotype
  • Pigs previously treated with antibiotics can prevent bacterial growth

Antimicrobial susceptibility

• Tests in vitro ability of live organism to grow under specific concentrations of different antimicrobials
• Sample types: lung
• Pros:
  • Identification of susceptibility or resistance of specific isolate to common antimicrobials
  • Identification of antimicrobial resistance trends
• Cons:
  • Requires a bacterial isolate
  • In vitro testing may be slightly different than in vivo results
  • Some specific antimicrobials may not be tested or require separate, special testing
  • Moderate cost

Polymerase chain reaction (PCR) General or Serotyping

• Detects presence of specific sequence of bacterial nucleic acid (DNA)
• Sample types: lung and tonsil
• Pros:
  • High sensitivity
  • General
    • Target ApxIV toxin genes
    • Present in all APP serotypes
  • Serotyping
    • Target different parts of ApxI, ApxII, and ApxIII toxin genes
    • See Table 1 for serotyping and toxin production
  • Moderate cost
    • Can often do pooling of tissue samples to lower cost while minimizing loss of sensitivity.
• Cons:
  • Require several specific PCR primers to determine serotyping
  • Not all isolates are typable
    • Currently 19 serotypes identified and growing
  • Not all assays validated for testing tonsil tissue

Table 1: Toxin production for each serotype.

	Toxin Production
Serotype	ApxI	ApxII	ApxIII	ApxIV
1	X	X		X
2		X	X	X
3		X	X	X
4		X	X	X
5	X	X		X
6		X	X	X
7		X		X
8		X	X	X
9	X	X		X
10	X			X
11	X	X		X
12		X		X
13		X		X
14	X			X
15		X	X	X
16	X	X		X
17		X		X
18		X		X
19		X		X

Enzyme-linked immunosorbent assay (ELISA) LPS-O-Antigen

• Detects presence of antibodies
• Sample types: serum
• Target: LPS-O-Antigen
• Pros:
  • Can be useful in helping serotyping
  • High sensitivity
• Cons:
  • Large amount of cross reactivity between serotypes due to share capsular and LPS-O antigen epitopes
    • The following serotypes cross react due to shared LPS-O antigens:
      • Cross reaction between serotypes 1, 9, and 11
      • Cross reaction between serotypes 3, 6, 8, 15, 17, and 19
      • Cross reaction between serotypes 4, 7, and 18
  • Unable to determine specific serotype with just one assay
  • Very expensive and impractical to test for all serotypes

Enzyme-linked immunosorbent assay (ELISA) ApxIV antigen

• Detects presence of antibodies
• Sample types: serum
• Target: ApxIV Antigen
• Pros:
  • High sensitivity
  • Detects all APP serotypes
• Cons:
  • Unable to determine specific serotype
  • Unable to determine virulence of isolate

Result interpretation

Gross pathology

• Positive: Often presumptive diagnosis is sufficient
• Negative: No gross lung lesions

Bacterial culture

• Positive:
  • Biotype 1: NAD dependent
  • Biotype 2: NAD independent
• Negative: Negative or animal possibly previously treated with antibiotics

Antimicrobial susceptibility

• Susceptible: possible good choice for treatment if antimicrobial can reach target tissue
• Resistant: select different antimicrobial
• MIC: are done to ensure the antimicrobial selected achieves the listed MIC value in the target organ

PCR

• Positive:
  • Organism present
  • Can identify presence of different genes which can help serotype
• Negative:
  • Negative or too late after infection.
  • Animal possibly previously treated with antibiotics
  • Not all isolates can be serotyped (Non-typable)

ELISA LPS-O-Antigen

• Positive: help serotype or identify group of serotypes
• Negative: negative, too late after infection, or animal possibly previously treated with antibiotics

ELISA ApxIV antigen

• Positive: Organism present
• Negative: negative, too late after infection, or animal possibly previously treated with antibiotics

Scenarios

Growing pigs with sudden death or respiratory disease (acute)

• Necropsy of 1-3 recently dead or euthanize coughing pigs. Grossly evaluating lung for necro-hemorrhagic areas of consolidation accompanied by fibrinous pleuritis.
• Submit affected lungs for bacterial culture and APP PCR general testing and if positive then for PCR serotyping.

Establish negative status of herd

• Sample 30 of largest finishing pigs on farm and test 2-3 times a year using ApxIV ELISA.