Infectious Coryza: Characterization of Bacterium

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Infectious Coryza Characterization of Bacterium

• Prof. Rob Bragg
• Dept of Microbial, Biochemical and Food
  Biotechnology
• University of the Free State
• Bloemfontein
• South Africa

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Infectious Coryza

• Caused by Avibacterium paragallinarum (previously
  Haemophilus paragallinarum).
• Causes drop in egg production in layers which can
  be up to 40
• Normally bacterium requires NAD for growth. (NAD
  independent isolates have been found in South
  Africa since 1990 and recently in Mexico)
• Different serogroups of the bacterium (A, B and C)

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NAD Independent Isolates

• First found in South Africa in 1990.
• NAD independence is plasmid mediated.
• NAD independent isolates appear to be less
  virulent that wild-type strains. (demonstrated
  with wild-type strains and lab produced strains).
• Evidence of immune evasion by NAD independent
  isolates.

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Isolation and Identification of the Bacterium

• Selection of chickens for isolation.
• Growth media and requirements.
• Isolation procedures
• Conventional identification
• PCR
• Other strains

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Selection of Chickens
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Selection of Chickens

• This is critical for isolation.
• Select birds with only very mild clinical signs.
• If birds with severe clinical signs are selected
  A. paragallinarum cultures are often overgrown
  by opportunistic pathogens.

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Growth Media and Requirements

• Blood Tryptose Agar plates (BTA) give best
  results.
• Bacteria require NAD for growth this can be
  added to the medium or can be supplied through
  the use of a feeder culture of Staphylococcus
  aureus.
• Look for typical satellitism.
• Bacteria in micro-aerophilic use candle jar for
  isolation at 37C.

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Isolation Procedures

• Disinfect head.
• Make incision into the sinus cavity.
• Collect sample with sterile swab.
• Streak sample onto BTA plates.
• Inoculate with S. aureus across the inoculum.
• Incubate in a candle jar at 37C overnight.
• Bacteria only survive for 2 to 3 days.

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NAD Independent Isolates

• Same isolation procedures.
• Do not see typical satellitism.
• Colonies will grow on plates without the feed
  culture on passage of the bacteria.
• Colonies very similar to Ornithobacterium
  rhinotracheale.

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Identification

• For diagnostic purposes isolation of bacterium
  showing satellitism from birds with clinical
  signs is sufficient.
• Identification options include biochemical tests
  or PCR.
• PCR tests are preferable and works well. Can be
  done directly on colonies no need to first
  isolate the DNA.

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1
2
3
lane 11742 lane 2 46-C3 lane 3 Marker
??500bp
Fig.1 1 Agarose gel of the PCR amplification of
A. paragallinarum reference strains1742,
46-C3. Amplification of the DNA resulted in a
single band of ?500bp.
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Identification

• There are other NAD requiring organisms which can
  be isolated from chickens the so called
  Haemophilus avium strains.
• Reclassified into Pasteurella avium, P. volantium
  and Pasteurella type A species.
• Regarded as non-pathogenic.
• Can be found quite frequently.

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Identification

• Biochemical tests are possible.
• Must add sterile 0.5 NaCl, 1 sterile chicken
  serum and 0.025 NAD to basal media for
  carbohydrate fermentation tests.

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Identification
Sugar A. para. P. avium P. vol Past
Galactose -
Mannitol -
Sorbitol - -
Xylose - (v) -
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Identification

• NAD independent strain the same a NAD dependent
  A. paragallinarum.
• PCR test detects both variations.
• Biochemical tests are the same.
• Serological tests are the same.
• Only NAD dependence differs.

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Serogroups and Serotyping

• Plate agglutination test three serotypes (A, B
  and C).
• Serotyping scheme based on ability to agglutinate
  Gluteraldehyde fixed red blood cells. Three
  groups found to be the same as serotypes found
  by plate agglutination test.
• Three serogroups (A, B and C)

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Serogroups

• Serogroups currently sub-divided into 9 different
  serovars (A-1 to A-4 B-1 C-1 to C-4).
• Serotyping to serogroup level is relatively
  straight forward HA and HI tests.
• Serotyping to serovar level is difficult (also
  with HA and HI but can be subjective).

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Serogroups
Serogroup Serovar Isolate
A A-1 0083
A A-2 HP-90
A A-3 E3-C
A A-3 HP-14
B B-1 0222
C C-1 H-18
C C-2 Modesto
C C-3 SA-3
C C-4 HP-18
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Serogroups

• Evidence that different serovars are
  geographically isolated (C-3 in Southern Africa,
  C-4 in Australia).
• Evidence of new serovars in other countries
  (South America). (Possibly two new serovar B
  strains, 1 new serovar C strain and one or two
  new serovar A strains).

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Cross Protection

• No cross protection across serogroups.
• Good cross protection between serogroup A
  strains.
• Good to poor cross protection with serogroup C
  strains.
• Cross protection in serogroup C is highly strain
  dependent.

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Cross Protection

• Important to know serogroup and serovar of
  strains in a country for the selection of
  vaccine.
• Serotyping to serovar level with HA and HI test
  is difficult.
• Need alternative methods for serotyping
  molecular based test would be preferable.

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Molecular Serotyping

• Develop a serotyping scheme based on
  haemagglutinin gene of A. paragallinarum (Current
  research project).
• Haemagglutinin gene has been sequenced.
• Ideal situation would be to have serovar specific
  PCR tests specific primer sets for each
  serovar.
• Alternative PCR full gene and do RFLP analysis
  get serovar specific fingerprint

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Restriction Digests Patterns
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Enterobacterial Repetitive Intergenic
Consensus-based PCR

• ERIC-PCR. (Current research project).
• Use long sequence primers and low annealing
  temperature.
• Amplification of regions in bacterial genome.
• Result in genetic fingerprint different
  patterns found for different reference strains.
• Need to investigate significance to cross
  protection.

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Challenge Models

• Most challenge models involve the intra-sinus
  injection of challenge bacteria into all birds in
  the group.
• Record the number of birds showing clinical
  signs after 3 to 5 days.
• Do bacterial re-isolation.
• Calculate protection levels.

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Problems with Challenge Model

• Not a natural route of exposure.
• Always see clinical signs in birds the first few
  days after injection of challenge bacteria,
  irrespective of the level of protection in the
  birds.
• What do you do with these clinical signs?
• Need for a new challenge model.

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New Challenge Model
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Challenge Model

• A total of 10 chickens in adjoining cages per
  test.
• Challenge one bird in the middle and allow for
  natural spread.
• Recorded clinical signs daily.
• Score clinical signs.
• Construct a disease profile using mean daily
  disease scores.

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Clinical scores
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Challenge Model Disease Profile
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New Challenge Model

• Allows for easy comparison between different
  vaccines, virulence of strains, efficacy of
  treatments etc.
• Allows for statistical analysis of the difference
  between the disease levels in vaccinated and
  unvaccinated chickens.

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Virulence of Different Isolates

• Virulence of the four different South African
  serovars was tested using the challenge model.
• Unvaccinated commercial layers were used.
• Groups of chickens were challenged with each of
  the different serovars.

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Virulence of SA Isolates
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Virulence of SA Isolates

• It was found that serovar C-3 is highly virulent.
• Serovar C-2 is less virulent than serovar C-3,
  but is substantially more virulent than serovars
  A-1 or B-1.

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Conclusions

• Knowing the serogroup and serovars of the
  isolates which occur in a country is important.
• Developed a new challenge model which allows for
  statistical comparisons.
• Serovar C-3 has been shown to be highly virulent.
  

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Thank you