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

Infectious bronchitis virus

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

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)

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

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

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

Egg shell and inner quality problems by IBV

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

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

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

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

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

Serotyping Dr Jane Cook

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

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

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

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

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

Protection inhibits response

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

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

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)

More attention seems to work: IgM results in time (spray, the Netherlands) Type of birds 1998 2006-2007 2009-2010 Broilers 17% 38% - Pullets 46% 57% Broiler breeders 52% 77% GPS 61%

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

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

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

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%

Summary kidney protection post Q1 challenge virus peak percentage IBV replication at day 5 and 8 post challenge No vaccine Ma5 Ma5 + 4/91 Q1 12.216 20 10 Q1 12.185 Q1 12.124 50 40 Q1 12.078 nt

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)

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

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

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

Box and Ellis, Avian Pathology 1985 Mass challenge (aerosol) of 4x 26-28 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

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%

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

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

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?

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

Level of D388/QX virus neutralizing antibodies in breeders (non-D388 vaccines) (De Wit et al., Avian Pathology 2011, pp463-471) 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

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

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

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

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

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

Thank you for your attention