1. Infectious bronchitis virus, European vision
J.J. (Sjaak) de Wit, DVM, PhD, dipl ECPVS
GD Deventer
the Netherlands

2. Infectious bronchitis virus

3. Infectious bronchitis virus
Corona Virus, a ssRNA virus
- Relatively high rate of mutations (0,0012 subst per nt per jaar)
- Also recombinations
Many serotypes/genotypes:
Massachusetts (M41, H120), D274, D1466, D3128, Ark, Conn, Delaware, Florida, California, Holte, 793B (4/91, CR88), D388 (QX), B1648/D8880, Gray, T-strain, etc, etc
Many genetic variants in Latin America

5. Summary IBV variants
In many countries, variants are the majority of the detected field strains
Selection by lower cross-protection?
Some variants stay for a longer time, others come and go (and reappear)

6. Disease IBV depends on: pathotype strain (variation within serotypes)
type of chicken
age
climate: ammonia, dust, E. coli, ORT
co-infections (viruses, Mycoplasma)
protection: vaccination, serotype, protectotype

7. Damage IBV in broilers decreased feed intake and growth
sneezing, gasping, rales, nasal discharge
Flip-overs: sudden deaths by choking
swollen head syndrome
airsacculitis by secondary bacterial infections
(E. coli, O. rhinotracheale, etc)
condemnations
nephritis: mortality, very wet litter

8. Damage IBV in layers/breeders
decreased feed intake
cage : sometimes sneezing, wet eyes ground : sometimes SHS
drop egg production, egg quality decreased (shell, protein, dirty), hatchability
peritonitis (coli)
weak bones (indirect)
false layers
kidneys

9. Egg shell and inner quality problems by IBV

10. False layers (IBV D388/QX)
Look and act like laying birds, pelvic bones are wide open, no egg
Irreversible damage of the oviduct, active ovary
Very early infection with a (very) pathogenic IBV strain in chickens without relevant maternally derived antibodies against that strain

11. Implications of presence of relevant variants
More/severe outbreaks despite normal vaccinations
respiratory disease
increase secondary bacterial infections
drops in egg production
nephritis
egg quality: shell, color, hatchability
false layers

12. IBV strain typing protectotype (primary, secondary) serotype (VNT)
epitope type (Mab, IFA, ELISA)
genotype (RT-PCR, RFLP, sequencing)

13. Protectotyping
Most relevant for the chicken: is the vaccinated chicken well protected against challenge?
Yes: vaccine and challenge strain are of the same protectotype
Requires animal experiments: expensive

14. Serotyping
Does a antiserum against a certain strain neutralize the other strain (virus neutralization test):
Yes: same serotype
Correlation with protectotype: more serotypes in 1 protectotype
Some antigenic variation within a serotype
Many serotypes: expensive
No information about pathotypes

15. Serotyping Dr Jane Cook

16. Epitope typing by MoAbs
Typing by panel of Moabs (mainly against S1)
Antigen ELISA, IPT, IFT
Difficult and limited availability of Moabs, laborious
Good for screening
Sero-type
Moabs
48.4
51.2
48.2
43.4
104.10
Mass + -
D274
D1466
D8880
793B
D388

17. Genotyping Characterization of strains based on (part of) the genome
Usually on part of S1 (2 highly variable regions (HVR) of about 300 bp)
2 main approaches:
General S1 RT-PCR, cDNA is used for typing by
RFLP (restriction enzymes)
Sequencing, differentiating field strains from vaccines
Genotype-specific RT-PCR’s

18. Relation IBV genotype –serotype - protectotype
Genotype: primary AA sequence in part of genome, genetic relation between strains
Serotype/protectotype: 3D structure, biological function of whole virus
Translation of primary AA sequence into 3D structure and biological function of IBV is not (yet) possible

20. Each variant a new vaccine?
Different protectotype?
Relevant variant?
Big area?
For longer period? (vaccine development takes years)
Broilers (live vaccine) and layers/breeders (live and/or killed vaccine)
Problem
How many vaccines are you going to produce and use?
Broilers: 1 or 2 IBV vaccinations is maximum

21. Each variant a new vaccine?
Alternative for new vaccines:
Optimal use of available vaccines
Revaccination (not too soon unless the first vaccination was not well applied)
Well performed vaccination(s) that are well taken by the chicken might also induce more cross-protection (strain dependent)
Eye drop
choose for a (“one”) live variant vaccine that provides broad protection (in combination with Mass vaccine) against many variants
Examples: Ma5-4/91 (Cook), MM-Ark (Gelb)
Booster with inactivated vaccines for layers and breeders

22. Protection inhibits response

23. Better use of available vaccines
In the field: many variables that can give a lot of bias, source of a lot of different opinions about the same subject
Monitoring of vaccinations requires continuous attention: a movie tells more than a picture

24. Field trial
IgM response of 360 flocks (broilers, breeders, grandparents, pullets) that were vaccinated at about 2 weeks of age with an IBV vaccine by coarse spray
Application (water, spray, disc, ……..)
Equipment
Dosage (vaccine, water)
Temperature of the house, of the water
Additives
Other vaccinations close to the IBV vaccination
Etc
Goal: detecting critical factors for the application under field conditions to be able to increase the average efficacy of IBV vaccinations

26. Field trial, practical translation
Better results when:
Ventilation off during spray (15.5%, P=0,037)
(enough) Light on during spray (41%, P=0,009)
Second IBV vaccination not within 2 weeks (2,5% per extra day, P=0,005)
cold water was used (3,2% per °C, P=0,021)
Flock size/housing type: unclear
Cages significantly lower responses than floor housing (31% vs 53%, P=0,01)
Bigger flocks significantly lower results (1% per 1000 extra birds, P=0,04)

27. More attention seems to work: IgM results in time (spray, the Netherlands)
Type of birds
1998
Broilers
17%
38% -
Pullets
46%
57%
Broiler breeders
52%
77%
GPS
61%

28. Broad protection by live vaccines
Most important for young birds
Existing live vaccines can differ in immunogenicity and cross protection against local “variant” strains:
Animal experiments
Broaden protection against many variants by vaccination with Mass and a selected variant vaccine

29. Cook et al, Avian Pathology 1998
At least 85% protection

30. Cook et al, 1998 Avian Pathology
Challenge
Country
Protection against challenge (day 35) in trachea post vaccination
Ma5 (day 1)
4/91 (day 14)
Ma5 + 4/91
difference
M41
100
4/91 UK
D207
Holland 84 80 91 +7
D1466 20 26 59
+39
Arkansas
USA 75 72 85
+10
TM86
Japan 79 71 93
+14
FB 3 77 94
+17
A1121
Taiwan 53 43 90
+37
890/80
S. Africa 47 21 89
+42
50/96
Brazil 37 78 -
57/96 88 76
62/96 82 81 87 +5
64/96 64
22/97
Honduras 55 30
+27

31. 4 Chilean Q1 strains, experiments GD
Genetically very close
For the chicken different
No sign of replication in the proventriculus
Respiratory protection by Mass (Ma5): 76.5, 92.5, 44 and 97.5%
Respiratory protection by Ma5 – 4/91: 100, 99, 100 and 99%

32. Summary kidney protection post Q1 challenge
virus
peak percentage IBV replication at day 5 and 8 post challenge
No vaccine
Ma5
Ma5 + 4/91 Q 20 10 Q Q 50 40 Q nt

33. Demands variant vaccine (to broaden the cross protection)
Mass must not protect against attenuated variant vaccine
Tested in animal experiments with local strains
Attenuated variant must broaden the protection against all/many variants (variants can come and go)

34. Layers and breeders
Need a long lasting protection against the relevant field strains of that area
Main goal: protection against damage of the egg production and egg quality

35. Live and / or inactivated vaccines
local protection (most important for broilers),
low antibody titres,
protection lasts not very long (field),
Inactivated vaccines:
low local protection,
higher level of antibodies (after live priming)
longer period of protection

36. Induction of a high antibody response (HI, VNT) useful?
Low number of papers about protection against IBV in the production period
E.g. Box et al, several papers

37. Box and Ellis, Avian Pathology 1985
Mass challenge (aerosol) of 4x layers at 38 weeks of age
No vaccine
Inac at 3 and 16 weeks
H120 (wk 3) and inac at 16 weeks
H120 (wk 3) and H52 at 15 weeks

39. Mean HI M41 titre at Mass challenge wk 38
Box and Ellis, Avian Pathology, 1985
Live priming
inac
Mean HI M41 titre at Mass challenge wk 38
Drop in egg production no
2.7
72%
H120, H52
4.1
12%
2x Inac M41
6.8
27%
H120
Inac M41
7.9 2%

40. Conclusions of all experiments by Box et al
Boosting with inactivated vaccines increased the protection against damage of the oviduct/ovary after homologous IBV challenge
A higher level of protection against egg drop after IBV challenge is correlated with a higher HI-titre (more is better)
Best results after live priming and boosting with inactivated vaccine

41. Relevance for protection of a difference in level of virus neutralizing antibodies in laying birds
Vaccination/challenge experiments GD with 6 serotypes in layers
1 log2 higher titre resulted on average in a 9% lower drop in egg production after challenge

42. Protection against variant strains
For many variants a homologous priming + inactivated vaccine with same strain is not available
Broad heterologous priming useful?
Antigens in the inactivated vaccine?

43. Level of virus neutralizing antibodies against Chinese Q1 genotype
Vaccination programmes
Live priming (heterologous)
Antigens in inactivated IBV vaccine
(heterologous)
Ma5
Inac M41
Ma5 and 4-91
Inac M41+D274
Inac M41+ D274

45. Level of D388/QX virus neutralizing antibodies in breeders (non-D388 vaccines) (De Wit et al., Avian Pathology 2011, pp )
Group
Mean log2 VNT titer (SD)
Day 0
4 weeks post first Inact
10 weeks post first Inact A
Live*
<7
7.0 (1.6) B
Live, Inact IB2
8.9 (1.2)
8.8 (1.3) C
Live, Inact IB3
9.0 (1.7)
9.3 (2.2)
* H120/D274 and 4/91
Heterologous live priming and heterologous inactivated vaccines induced a high level of virus neutralizing antibodies against the D388/QX variant
More antibodies correlated with more protection against challenge

46. Conclusion priming/boosting
broad live priming is helpful to induce more virus neutralizing antibodies against more strains
more strains in inactivated vaccine might be helpful to induce more neutralizing antibodies against more strains
also depends on strains, amount and quality of antigen per dose, adjuvant and application

47. General conclusion of challenge trials in layers and breeders at GD
the average level of cross-protection against variants might be increased by
A broad heterologous live priming
A broad heterologous boosting by an inactivated vaccine
However, this is a general rule, vaccine (programme) dependent, variant dependent: no guarantee

48. IBV vaccinations, (very) general rules
Young birds:
In general, vaccines within the same protectotype provide high protection against homologous challenge
Suitable combinations of vaccines might also induce high levels of cross-protection: animal experiment
Not every variant requires a special vaccine

49. IBV vaccinations, (very) general rules
Laying birds
Live priming is important
Broad heterologous priming can be useful too
Boosting with inactivated vaccines helps to get higher (virus neutralizing) antibody titres (related with higher protection), even when the antigen(s) are heterologous to the challenge virus

50. IBV vaccinations, (very) general rules
Laying birds
More strains in inactivated vaccine might be helpful to induce more virus neutralizing antibodies against other strains (in general)
also dependent on strains, amount and quality of antigen per dose, adjuvant and application
for specific strain: you have to test
Not every variant needs its own vaccine

51. Thank you for your attention