Infectious Coryza: Characterization of Bacterium

1. Infectious Coryza: Characterization of Bacterium Prof. Rob Bragg Dept of Microbial, Biochemical and Food Biotechnology University of the Free State Bloemfontein South Africa

2. Infectious Coryza • Caused by Avibacterium paragallinarum(previouslyHaemophilus 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)

3. 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.

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

5. Good selection Bad selection Selection of Chickens

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 1 2 3 lane 1:1742 lane 2 :46-C3 lane 3: Marker 500bp Fig.1: 1% Agarose gel of the PCR amplification of A. paragallinarum reference strains:1742, 46-C3. Amplification of the DNA resulted in a single band of 500bp.

12. 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.

13. 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.

14. Identification

15. 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.

16. 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)

17. 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).

18. Serogroups

19. 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).

20. 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.

21. 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.

22. 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

23. bp A I II bp A III bp A IV V bp A VI 400 400 400 450 300 300 200 250 250 200 50 50 50 50 (1a) (1b) (1c) (1d) Restriction Digests Patterns

24. 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.

25. 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.

26. 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.

27. New Challenge Model

28. 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.

29. Moderate Mild Severe Clinical scores

30. Challenge Model: Disease Profile

31. 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.

32. 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.

33. Virulence of SA Isolates

34. 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.

35. 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.

36. Thank you