Laboratory tools for monitoring and understanding IBDV infection and vaccination

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1 Laboratory tools for monitoring and understanding IBDV infection and vaccination J.J. (Sjaak) de Wit, DVM, PhD, dipl ECPVS GD Deventer, The Netherlands

2 Gumboro-virus (IBDV) Avibirna-virus: segments of dsrna No envelope Very resistant t - 5 days survival in water, feed and manure - 1 days after removal infected flock still present in the chicken house - Sensitive to formaldehyde, chloramine, quats

3 Main proteins: VP: outside capsid, neutralizing epitopes, part pathogenicity VP3: inside capsid VP1: polymerase, part of pathogenicity Letzel et al, 007, Journal of Virology

4 Laboratory tools for monitoring i and understanding IBDV infection and vaccination Vaccination: check the take of the vaccine Challenge: was there a challenge, when, which strain Clinical: despite vaccination, vvibdv, variant strain? Subclinical: vaccinated flock, vvibdv, variant strain? i t t i bi l i l l t i t (d id d b th variant strain : a biological relevant variant (decided by the (vaccinated) chicken)

5 IBDV Serotype 1: Classical-like l lik stains (Faragher, vvibdv, vaccines) Clinical signs, swelling, atrophy of bursa Illness and immunosuppression Variant-like strains (Del E, GLS, etc) No clinical signs, atrophy of bursa Immunosuppression Serotype : turkeys, no clinical symptoms in the chicken, only histopathological lesions

6 Diagnostic tools Needed for: Making the diagnosis Typing strains Understanding breakthroughs Adjusting vaccination programs (vaccine, timing) Monitoring vaccination programs (early warning)

7 Detection of infections and vaccinations Detecting the IBDV strain: Infectious particles (virus isolation) Antigen (staining) Genome (RT-PCR) Detection of the antibody response

8 Vaccination Check the take of the vaccine 3 main kinds of Gumboro vaccines Conventional vaccines (water application) Antibody/antigen complex vaccines Vector-based vaccines

9 Conventional vaccines Application by the drinking water (eye-drop, spray) Too many maternally derived antibodies (MDA) neutralise the vaccine virus Vaccinating too early: vaccine will be neutralised in many (to all) birds by the MDA Vaccinating late: birds with low MDA will be unprotected for a longer time Vaccination date estimation by serology Spreading

10 Conventional vaccines Checking the take: PCR (+ sequencing): positive after a few days for a period of about 1- weeks (bird level) Staining technique: positive after a few days for a period of about 1 week (bird level) Serology: gets positive between 1 and weeks after take

11 Gumboro vaccination

12 Deventer formula, Block et al, Avian Pathology 007 Field experiment 1 16 broiler flocks (Ross, Cobb) Blood 3-1 days (Deventer formula, IDEXX) -5 days prior to vaccination: second blood to check and confirm Vaccination at day of estimation (intermediate vaccine) 0, 7, 14, 1 days post vaccination: PME, 30 blood, 5 bursa (RT- PCR, sequencing, histology)

13 Experiment 1. Serology at 7, 14 and 1 d.p.v. Experiment 1, RT-PCR and histology post vaccination 100 percent positive to % RT-PCR positive histology days post vaccination

14 Deventer formula, Block et al, Avian Pathology 007 Field experiment 0 broiler flocks (Ross, Cobb) Vaccination at general recommendation, intermediate vaccine Blood 3-7 days (retrospective, Deventer formula, IDEXX) retrospective 3 groups: Group 1: vaccinated before the estimated day (-8 to 1 day) Group : vaccinated at the estimated day Group 3: vaccinated after the estimated day (1 to 6 days) 0, 7, 14, 1 days post vaccination: PME, 30 blood samples, technical performance)

15 Experiment. Serology at 7, 14 and 1 d.p.v.

16 Antibody/antigen complex vaccines In ovo or by injection at day 0 Vaccine strain: intermediate plus Replication starts when the MDA level has become low enough (each bird) Replication under field conditions starts t usually after.5 to 4 weeks of age Spreading

17 Antibody/antigen complex vaccines Checking the take: PCR (+ sequencing) : positive after a few days of replication for a period of about 1- weeks (bird level) Staining technique: positive after a few days of replication for a period of about 1 week (bird level) Serology: gets positive between 1 and weeks after start of replication

18 Vector-based vaccines In ovo or by injection at day 0 Replication starts after application (Mareks strain), independent from MDA against IBDV IBDV vaccine: only part of VP Does not spread (missed is unprotected) Can not be combined with other HVT vaccines Protection after about -3 weeks

19 Vector-based vaccines Checking the take: IBDV PCR (+ sequencing): weakly positive in the bursa Vector PCR: feather follicles (Mareks vector) Serology: gets positive between and 3 weeks after the inoculation in Synbiotics ELISA. More slowly positive in classical ELISA s (positive at 4-5 weeks)

20 Laboratory tools for monitoring i and understanding IBDV infection and vaccination Challenge: was there a challenge, when, which strain? Clinical: i l despite vaccination, vvibdv, other classical l strain, variant strain? Subclinical: vaccinated flock, vvibdv, variant strain?

21 Detection of infections Detecting the IBDV virus: Infectious particles (virus isolation) Antigen (staining) Genome (RT-PCR) Detection of the antibody response

conversion Can be very difficult to diagnose especially in - Can be

22 Symptoms of IBDV infections - High variability in: - Clinical signs - Mortality - Immunosuppression - growth depression - Higher feed conversion Can be very difficult to diagnose especially in - Can be very difficult to diagnose, especially in vaccinated birds or with variant strains

virus titre: first 3-6 days in bursa Detectable virus: 1- weeks in

23 Clinical outbreak Acute phase of infection (inflammation of bursa) Maximal amount of virus at time of clinical signs Max. virus titre: first 3-6 days in bursa Detectable virus: 1- weeks in non-protected birds Less and shorter in (partially) protected birds Seroconversion

24 Subclinical case No visible signs, so when to sample? Frequent sampling (e.g. weekly) of 5 bursa s at week 1,, 3, and 4. If suspicion (retrospectively) of infection: testing pools of (small) bursa of every week for the presence of virus (+ typing) Seroconversion

25 IBDV strain typing protectotype serotype (VNT) epitope type (Moab, IFA, ELISA) genotype (RT-PCR, RFLP, sequencing,)

26 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

27 Serotyping - serotypes (virus neutralization test): - serotype 1: Gumboro disease - serotype : non-pathogenic - High correlation with protectotype - Some antigenic variation within serotype 1 - No information about pathotypes (mild, intermdiate, classical, vv)

28 Epitope typing Typing by panel of Moabs (mainly against VP) Antigen ELISA, IPT, IFT Difficult and limited availability of Moabs, laborious Very good for screening

29 Genotyping Characterization of strains based on (part of) the genome Usually on part of VP (highly variable region) of about 345 bp) By sequencing or RFLP (restriction enzymes) Translation of the (little part of the) genomic information into the biological and antigenic function of a strain is only partially possible Screening

30 RFLP RFLP = Restriction Fragment Length Polymorphism RT-PCR followed by restriction with endonucleases: cut DNA at very specific places Product of RT-PCR is treated with enzymes that cut it in pieces: grouping on the number and seize of pieces

31 PCR Reference Chart: Field and Vaccine Viruses Daral J. Jackwood (USA) Molecular Group 1 IBD strains and vaccines Delaware A 1084A (Merial SVS-510) BstN1 enzyme Fragment sizes Mbo1 enzyme Fragment sizes Delaware E Maryland variant 89/03 (Intervet hatchery vaccine) 3 STC (APHIS standard challenge) Variant Vax-BD (Schering) IBD Blen (Merial) 4 Baxendale S706, D-78 (Merial, Intervet) Bursavac, Univax (Schering) ViBursa G&CE (Vineland) 5 Edgar, Lukert Bursine, B-, B+ (Fort Dodge) Indiana variant ViBursa-L, Bioburs, Bursamune 6 RS593 (USA, Intervet) V877 (Australia, Fort Dodge) U.S.A. field isolates Hipragumboro-GM97 New 44 Many wild type viruses Any possible pattern on Mbo1 New other Many wild type viruses Any possible pattern on BstN1 Any possible pattern on Mbo1

32 RFLP Provides only data of a few percent of the RT-PCR product Does not provide information which change has taken place Silent mutations (not relevant for the virus) can result in another genotype

33 Sequencing Product of RT-PCR is analysed for the sequence of all nucleotides. The sequence of nucleotides can be translated into the sequence of amino acids (AAs) Comparison with known sequences of other strains Percentage of similarity Vaccines can be differentiated from field strains Most vaccines can be differentiated from each other Phylogenic tree

34 Main proteins: VP: outside capsid, neutralizing epitopes, part pathogenicity VP3: inside capsid VP1: polymerase, part pathogenicity Letzel et al, 007, Journal of Virology

35 AA sequence in a part of the HVR (38 60) of VP of several reference and field IBDV strains (data GD Deventer) Strains /70 Faragher T S L S I G G E L V F Q T S V Q G L V L G A T Delaware E T S L S V G G E L V F K T S V Q S L V L G A T vvdv86 T S L S I G G E L V F Q T S V Q G L I L G A T Field 1 T S L S I G G E L V F H T S V H G L A L D A T Field T S L S V G G E L V F Q T S V Q S L V L S A T Field 3 T S L S V G G E L V F Q T S V Q G L I L G A T Field 4 T S L S V G G E L V F Q T S V H G L V L G A T Field 5 T S L S V G G E L V F Q T S V N G L V L G A T Field 6 T S L S V G G E L V F Q T S V Q G L V L G A T Field 7 T S L S V G G E L V F Q T S V Q G L V L N A T

36 Comparison of HVR of Gumboro strains Vaccine A Vaccine B Vaccnie C Vaccine D Vaccine E IBDV09 9,7 97,6 91,1 89,4 91,9 91,1 9,7 9,7 93,5 89,4 91,1 90, IBDV040 93,5 98,4 91,9 90, 9,7 91,9 93,5 93,5 94,3 90, 91,9 91,1 IBDV04 9,7 91,9 91,1 96,7 91,9 91,1 9,7 93,5 9,7 89,4 90, 87,8 IBDV06 91,9 94,3 91,9 91,1 91,1 91,9 91,9 96,7 95,9 90, 99, 90, IBDV063 91,99 94,3 91,99 91,11 91,11 91,99 91,99 96,7 95,99 90, 99, 90, IBDV063x 91,9 94,3 91,9 91,1 91,1 91,9 91,9 96,7 95,9 90, 99, 90, IBDV066 91,9 94,3 91,9 91,1 91,1 91,9 91,9 96,7 95,9 90, 99, 90, IBDV067 91,9 94,3 91,9 91,1 91,1 91,9 91,9 96,7 95,9 90, 99, 90, IBDV079 9,7 91,9 91, ,9 91,1 9,7 9,7 91,9 88,6 90, 87,0 IBDV080 9,7 94,3 91,99 89,4 91,99 91,99 9,7 9,7 93,5 91,11 89,4 91,11 IBDV086 97,6 9,7 99, 90, 96,7 99, 97,6 94,3 95,1 91,9 90, 91,9 IBDV088 98,4 93, ,1 97, ,4 95,1 95,9 91,1 91,1 91,1 IBDV089 95, ,5 91,9 94,3 93,5 95,1 95,1 95,9 91,9 93,5 9,7 IBDV090 99, 94,3 97,6 91, ,6 99, 94,3 95,1 90, 90, 90, IBDV09 95, ,5 91,99 94,3 93,5 95,1 95,1 95,99 91,99 93,5 9,7 IBDV094 98,4 93, ,1 97, ,4 95,1 95,9 91,1 91,1 91,1 Vaccine F Vaccine G 5/70 FD consensus s Delaware E DV86 1A-00-17

37 IBDV serology estimate optimal age(s) of vaccination check the response to vaccination check for (subclinical) infections in unvaccinated and ( ) vaccinated chickens (monitoring for next flock and diagnosis)

38 IBDV serology VNT: very sensitive, very specific, expensive AGPT: pos/neg, very specific ELISA: single dilution to titre calculation, l fast, commercially available, variability in sensitivity and specificity

39 Check for (subclinical) i l) infections in unvaccinated and vaccinated chickens Vaccinated birds: increased titres at slaughter is an indication (not a proof) for challenge: look for other signs: e.g. feed conversion, wet litter, bursa abnormalities at post mortems, immunosuppression, etc Normal titres is no proof for the absence of a (late) infection No clear cut-off

40 ELISA results at 6 weeks in vaccinated unsuspected vs IBDV-suspected broilers flocks control suspected > 3.5

41 General limitations of IBDV serology Late infections are missed (antibodies are always 1- weeks behind the infection) serology has difficulties in detecting infections in vaccinated birds: indication serology can not differentiate between different challenge viruses

42 Checking the take of vaccinations and the presence of (subclinical) infections Costs time and money Does not make your life more easy in the short term. But in the long term, it helps you to make more rational decisions based on knowledge, instead of belief or trust Serology: do not only use it when there are already outbreaks. Monitoring at slaughter: information about vaccination and indication of field pressure: prevent the problems

43 Thank you very much for your attention

Gumboro Disease: where are we with IBDV epidemiology. J.J. (Sjaak) de Wit, DVM, PhD, dipl ECPVS GD Deventer, The Netherlands

Gumboro Disease: where are we with IBDV epidemiology J.J. (Sjaak) de Wit, DVM, PhD, dipl ECPVS GD Deventer, The Netherlands Gumboro-virus (IBDV) Avibirna-virus: 2 segments of dsrna Non enveloped virus