Molecular diagnosis of infectious bronchitis: recent developments. Richard Currie

Transcription

1 Molecular diagnosis of infectious bronchitis: recent developments Richard Currie

2 The infectious bronchitis virus RNA (nucleic acid) on the inside the genetic finger print Proteins (S1 and S2 spike proteins) on the outside blood tests detect antibodies made against these

3 By looking at the S1 gene and protein, we can fingerprint the virus

4 4/91 Field virus

5 4/91 Field virus nucleic acid sequence XXXX SAMPLE SEQUENCE XXXX SAMPLE SEQUENCE

6 Key point Genetic fingerprints allow us to differentiate viruses from one another We can CLEARLY diagnose different 793B vaccine strains They are not the same They behave differently We can differentiate vaccines from field viruses

7 The use of amino acid sequence to predict epitopes in the hypervariable region of the S1 protein An epitope is the major antigenic determinant of the virus: it s what antibodies bind to

8 The infectious bronchitis virus By understanding the genetic finger print of the live IB vaccines we have, we can differentiate field viruses from vaccine viruses We can determine if the genetic finger print of the vaccine virus changes Is it stable? Is it persistent? Does it colonise the farm and displace field viruses? Which vaccine strain given becomes dominant on-farm? Diversity of genetic finger prints of field viruses can be used to justify the use of combined Ma5 plus 4/91 protectotype vaccination strategies to generate the broadest protection possible

9 The x-ovo diagnostic process Real time PCR Is IB present YES or NO How much is present? Sequencing PCR and amino acid alignments Differentiation of vaccines from field viruses Interpretation, reporting and recommendations

10 European overview

11 Origin of Samples EMEA Broiler Parent 12% Layer 34% Broiler 48% Other 6% No of Submissions 2315 Other Category Includes - Partridge,Layer Parent, Pheasant and Not Classified

12 Most field viruses are QX and 4/91 genotypes % Homology All Countries 01/01/11-21/10/ /91 Ark CR88 D274 H120 Italy-02 Ma5 Other QX Negative SU 100% Sequencing Homology <100% Sequencing Homology Epitope Mismatches <100% Sequencing Homology No Epitope Mismatches Negative Results % Strain Untypeable Results% No of submissions 2555 Mismatches are differences in epitope location and composition between vaccines and field viruses

13 Most field viruses are found in commercial layers - EMEA 4/91 field viruses Layer 59% Broiler Parent 20% Broiler 13% Other 8% No of Submissions 253 Other Category Includes - Layer Parent and Not Classified,

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15 Successful vaccination populates the broiler farm with 4/91 vaccine virus Poland 2012 % Well applied vaccination of 4/91 together with Ma5 leads to the establishment of 4/91 as the dominant strain on-farm, with no field viruses found /91 D274 H120 QX Tabic IB Var V % <100%

16 Field data from France

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18 France 01/11/ /10/2013 France 01/11/ /10/2012 Negative 40% Positive 60% Negative 53% Positive 47% No of Submissions 95 No of Submissions 62

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21 Most field viruses are 4/91 genotypes - France

22 Most field viruses are QX and 4/91 genotypes - Belgium

23 Most field viruses are QX and 4/91 genotypes - Germany % Homology 01/01/ /10/ /91 CR88 D274 H120 M41 Ma5 QX Neg SU 100% <100% Homology Mismatches <100% Homology No Mismatches SU% Neg No of Submissions Mismatches are differences in epitope location and composition between vaccines and field viruses

24 Summary There is a major difference between QX field virus incidence between France and some other EU countries In common with other countries, 4/91 genotype field virus remains the major threat Note that these field viruses are more closely related to the 4/91 genotype than the CR88 or FR94 genotypes An explanation of the difference in QX diagnoses will be given in detail towards the end of the day s presentation

25 What happens when you vaccinate a flock with infectious bronchitis vaccines? It depends on the strains you use

26 H120 only vaccination Origin of Samples Age at Sampling Type of Birds Tracheal & Cloacal 34d Broiler Vaccination History Vaccine Age Dose Route of Application H120 1d 1 Coarse Spray Molecular Analysis Molecular analysis identified the following quantities of IB viral RNA : Tracheal swab: Ct = Cloacal swab Ct = Sequencing Analysis Sequencing analysis characterised this virus as an H120 type infectious bronchitis virus with 100% nucleic acid homology and 100% amino acid homology to the known H120 vaccine vial sequence In general mass type vaccines appear to be recovered in larger quantities from the trachea than cloaca

27 Ma5 plus 4/91 1a Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 6d broiler 10 Vaccination History Vaccine Age Dose Route of Application Nobilis IB 4-91 Hatchery Nobilis IB Ma5 Hatchery Molecular Analysis Molecular analysis identified a large quantity of IB viral RNA (Ct = 21.52). Sequencing Analysis Sequencing analysis characterised this virus as an Ma5 type infectious bronchitis virus with 100% nucleic acid sequence homology and 100% amino acid sequence homology to the known Ma5 vaccine vial sequence Early sampling after combined administration can find Ma5.

28 Ma5 plus 4/91 1b Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 7d broiler Vaccination History Vaccine Age Dose Route of Application Nobilis IB 4-91 Hatchery Nobilis IB Ma5 Hatchery Molecular Analysis Molecular analysis identified the following quantities of IB viral RNA Sample 1 Tracheal Swabs RNA (Ct = 20.06) 4/91 type infectious bronchitis Sample 2 Cloacal Swabs RNA (Ct= 19.49) 4/91 type infectious bronchitis Sequencing Analysis Sequencing analysis characterised this virus as a 4/91 variant infectious bronchitis virus with 100% nucleic acid sequence homology and 100% amino acid sequence homology to the known 4/91 vaccine vial sequence.. Or 4/91

29 Ma5 plus 4/91-2 Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 24d broiler Vaccination History Vaccine Age Dose Route of Application Nobilis IB d 1 ds Spray Nobilis IB Ma5 01d 1 ds Spray Molecular Analysis Molecular analysis identified the following quantities of IB viral RNA Sample 1 Tracheal Swabs RNA (Ct =33.46) - Ma5 type infectious bronchitis Sample 2 Cloacal Swabs RNA (Ct= 24.91) 4/91 type infectious bronchitis Sequencing Analysis Sequencing analysis characterised this virus as Ma5 & 4/91 type infectious bronchitis viruses with 100% nucleic acid sequence homology and 100% amino acid sequence homology to the respective vaccine vial sequences. At times you can find both strains..

30 Ma5 plus 4/91-3 Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 31d broiler 10 Vaccination History Vaccine Age Dose Route of Application Nobilis IB Ma5 Day 1 spray Nobilis IB 4-91 Day 1 spray Poulvac IB QX Day 14 spray Molecular Analysis Molecular analysis identified a large quantity of IB viral RNA (Ct = 22.91). Sequencing Analysis Sequencing analysis characterised this virus as a 4/91 variant infectious bronchitis virus with 100% nucleic acid sequence homology and 100% amino acid sequence homology to the known 4/91 vaccine vial sequence. before 4/91 eventually establishes dominance

31 Microarray results support the effective disappearance of Ma5 when 4/91 dominance is established 3000 Results 2500 PROB 793B ITA02 E 624/I ARK NEV 2000 (MFI) B IT02 624/I ARK BS2-like M41 D274 IS-1494/06- like QX Q1 Negative Control

32 All Countries Broiler (<84 days) Vaccinated with 4/91 and Ma5 at Day of Age 08/09/11-11/02/2014 % /91 Ark D274 H120 Ma5 QX Other SU Neg 100% <100% Homology Mismatches <100% Homology No Mismatches SU % Neg % No of Results - 45

33 Comparison of 4/91 and CR88 vaccine behaviour Differences in persistence

34 100% homologous 4/91 vaccine virus

35 100% homologous CR88 vaccine virus

36 Arkansas - 1 Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 23d Broiler 5 Vaccination History Vaccine Age Dose Route of Application IBmm+Ark Day 0 1 dose Spray IB Primer Day 10 1 dose Spray Molecular Analysis Molecular analysis identified a small quantity of IB viral RNA (Ct = 30.39). Sequencing Analysis Sequencing analysis characterised this virus as an Ark variant infectious bronchitis virus with 100% nucleic acid homology and 100% amino acid homology to the know Ark vaccine vials sequence

37 Arkansas - 2 Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 28d Broiler 10 Vaccination History Vaccine Age Dose Route of Application IBmm+Ark 1d 1ds Spray Molecular Analysis Molecular analysis identified a significant quantity of IB viral RNA (Ct = 27.78). Sequencing Analysis Sequencing analysis characterised this virus as an Ark variant infectious bronchitis virus with 100% nucleic acid sequence homology and 100% amino acid sequence homology to the 4 th most prevalent sub population of viruses from the Ark vaccine vial

38 Arkansas 3 Ark979 Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 27d Broiler 10 Vaccination History Vaccine Age Dose Route of Application IBmm+Ark 0 days 1 spray Molecular Analysis Molecular analysis identified a significant quantity of IB viral RNA (Ct = 25.38). Sequencing Analysis Sequencing analysis characterised this virus as an Ark variant infectious bronchitis virus with the following substitutions from the known Ark vaccine vial sequence. Nucleic Acid Sequence Position Vaccine Vial Sequence Sample Sequence Comments 979 Thymine Adenine Amino Acid Sequence Position Vaccine Vial Hydrophobicity Index Sample Hydrophobicity Index Hydrophobic/ Hydrophilic Switch Sequence Sequence 327 Tyrosine -1.3 Asparagine -3.5 NO

39 Arkansas - 4 Origin of Samples Age at Sampling Type of Birds Cloacal 28d Broiler Vaccination History Vaccine Age Dose Route of Application H120+D274 Hatchery 1ds Spray Molecular Analysis Molecular analysis identified a large quantity of IB viral RNA (Ct = 23.03). Sequencing Analysis Sequencing analysis characterised this virus as an Ark variant infectious bronchitis virus with the following substitutions from the known Ark vaccine vial sequence. Nucleic Acid Sequence Position Vaccine Vial Sequence Sample Sequence Comments 979 Thymine Adenine 1037 Adenine Guanine Amino Acid Sequence Position Vaccine Vial Hydrophobicity Index Sample Hydrophobicity Index Hydrophobic/ Hydrophilic Switch Sequence Sequence 327 Tyrosine -1.3 Asparagine -3.5 NO 346 Asparagine -3.5 Serine -0.8 NO

40 Arkansas 5a multiple viruses, same flock Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 33d Broiler Vaccination History Vaccine Age Dose Route of Application IB mm Ark Day of age Spray CR88 Day 14 Drinking water Molecular Analysis Molecular analysis identified the following quantities of IB viral RNA Sample 1 Tracheal Swabs RNA (Ct = 30.14).- Ark type infectious bronchitis Sample 2 Cloacal Swabs RNA (Ct= 25.60) Ark type infectious bronchitis Nucleic Acid Sequence TRACHEAL SWAB Position Vaccine Vial Sequence Sample Sequence Comments 903 Cytosine Thymine 979 Thymine Adenine 1036 Adenine Guanine 1037 Adenine Cytosine Amino Acid Sequence TRACHEAL SWAB Position Vaccine Vial Hydrophobicity Index Sample Hydrophobicity Index Hydrophobic/ Hydrophilic Switch Sequence Sequence 327 Tyrosine -1.3 Asparagine -3.5 NO 346 Asparagine -3.5 Alanine 1.8 YES

41 Arkansas 5b multiple viruses, same flock Origin of Samples Age at Sampling Type of Birds Tracheal Cloacal Other 33d Broiler Vaccination History Vaccine Age Dose Route of Application IB mm Ark Day of age Spray CR88 Day 14 Drinking water Molecular Analysis Molecular analysis identified the following quantities of IB viral RNA Sample 1 Tracheal Swabs RNA (Ct = 30.14).- Ark type infectious bronchitis Sample 2 Cloacal Swabs RNA (Ct= 25.60) Ark type infectious bronchitis Nucleic Acid Sequence CLOACAL SWAB Position Vaccine Vial Sequence Sample Sequence Comments 979 Thymine Adenine Amino Acid Sequence CLOACAL SWAB Position Vaccine Vial Hydrophobicity Index Sample Hydrophobicity Index Hydrophobic/ Hydrophilic Switch Sequence Sequence 327 Tyrosine -1.3 Asparagine -3.5 NO

42 European Society for Veterinary Virology 2012

43 What happens when an incomplete dose of vaccine received by the chicken? With 4/91 there is a measurable effect regarding tissue distribution

44 Kidney swabs: Fractional dose versus full dose 4/91: Germany and the UK Drinking water vaccination

45 Comments The slide shows results from 4/91 vaccinated flocks All flocks are fractional dose unless stated D7, D21 and D35 4/91 positive results are spray vaccinated flocks from the UK One full dose 4/91 vaccinated flock in Germany (red circle) tested negative this was drinking water vaccinated and hence this route of administration, and hence administration failure may explain the negative finding In general, fraction dose vaccination does not lead to colonisation of the kidney following vaccination, a finding that would be expected with full dose spray vaccination Hence when clinical problems are found, careful auditing of vaccination to ensure the flock is exposed to a full dose of vaccine, is advised

46 Summary The multiplication of genetic take populations of vaccine viruses can be measured Dominance of the 4/91 strain is expected in the majority of instances Failure to detect the 4/91 strain is a trigger for vaccination application audits and specific sampling of the kidney 7-10 post vaccination to check for 4/91 vaccine replication Genetic and amino acid variation introduction to flocks vaccinated with Arkansas strains has been demonstrated in the UK similar to that seen in the USA Recombinant Ark 491 field viruses have been identified in flocks with clinical egg drop in the UK Correct identification of vaccine and vaccine origin strains is critical to determine correct vaccination strategies

47 Use of amino acid profiles to investigate epidemiology of IB variants that are not homologous with vaccine virus take populations

48 Findings The following graphs show the location and number of amino acid changes that occur on the S1 protein for 4/91, Mass and D274 genotypes Sample sets are subdivided into those obtained from young birds (<18 weeks of age) and laying birds (>18 weeks of age) In most amino acid positions, amino acid changes are very variable. Occasionally they are not.

49 4/91 Non homologous viruses Position of Amino Acid Changes in 4/91 Field Virus <18w of Age Position of Amino Acid Changes in 4/91 Field Virus >18w of Age 25 No of changes 64 No of Submissions No of changes 526 No of Submissions

50 Mass Non homologous viruses Position of Amino Acid Changes in Mass Field Virus <18w of Age Position of Amino Acid Changes in Mass Field Virus >18w of Age

51 D274 Non homologous viruses Position of Amino Acid Changes in D274 Field Virus <18w of Age Position of Amino Acid Changes in D274 Field Virus >18w of Age Position 304 Histidine - Tyrosine Position 304 Histidine - Tyrosine No of Changes No of Changes <18wks No of changes - 53 No of Submissions - 32 >18wks No of changes 53 No of submissions 47

52 Summary 4/91 and Mass field viruses observed in EMEA from show extensive changes relative to the take population of the respective vaccines The major incidence is seen in flocks over 18 weeks of age Introductions in broiler flocks show viruses with much fewer amino acid substitutions This observation is consistent with the introduction of amino acid variation during the course of field infections. The longer the field virus is present in a flock, the greater the amino acid variation that is seen These genotypes act as true field viruses

53 Summary D274 positives are present in relatively minor quantities The amino acid variation in D274 positives is very small 50% of all amino acid substitutions featured a single Histidine Tyrosine mutation at position 304. The mutation is not part of any defined epitope hence of minor immunological relevance Of this specific mutation, 70% of observations occurred in flocks known to be vaccinated with D274 plus H120 live vaccine

54 Summary The most probable diagnosis of the Histidine Tyrosine virus is therefore D274 vaccine origin The finding does not suggest in any way that the D274 strain is reverting to virulence This finding gives further support for the lack of significant D274 field challenge in the EMEA The importance of this finding relates to the prevention of the vaccinating against vaccine strategy

55 Vaccinate against true field viruses Advanced next generation sequencing techniques (NGS) Most field viruses are QX and 4/91 genotypes - Germany Allow clear differentiation of vaccines from field viruses When applied to field outbreaks, can clarify the apparent large number of field viruses found and allow their re-classification In the case of QX, demonstrates the over diagnosis of QX field viruses by around 50% - GERMAN EXAMPLE OPPOSITE % Homology 01/01/ /10/2013 4/91 CR88 D274 H120 M41 Ma5 QX Neg SU 100% <100% Homology Mismatches <100% Homology No Mismatches SU% <100% Homology Vaccine Sub Population No of Submissions - 211

56 QX vaccine in its minor forms is less prevalent in flocks where 4/91 is also given Vaccine origin

57 QX vaccine in its minor forms is more prevalent in flocks where 4/91 is not given Vaccine origin

58 It is normal for live IB vaccines to spread to flocks not vaccinated directly QX vaccine origin positives occur in nonvaccinated flocks as well as QX vaccinated flocks

59 Prevalence of IB vaccines in non vaccinated flocks

60 Summary The presence in the field of minority point mutations present in live QX vaccine vials has been demonstrated by NGS sequencing The mutations are present in QX vaccinated flocks at times where a registered duration of immunity is present. If the viruses were field viruses, the vaccine would be predicted to be protective The mutations are present in the field in countries that use the largest quantities of QX vaccine and absent from countries where usage is minor. The viruses are found in the presence and the absence of live QX vaccine.

61 Summary Statistical analysis of the sequences obtained indicates the present of section pressure (consistent with the viruses being of vaccine origin) for positions 429 and 485 (partial sequence numbering)/ codons 117 and 131 (full sequence numbering) The consequences of this finding are the over diagnosis of QX field viruses that should be reclassified as vaccine origin viruses. In Belgium and Germany this is occurring in around 50% of cases of a QX diagnosis The findings do not relate in any way to the presence or absence of a reversion to virulence

62 Conclusion Molecular analysis of clinical samples for the presence of IB can Diagnose the presence of field strains relative to live vaccine strains. Demonstrate differences in behaviour between the major viral genotypes. Identify amino acid changes that are associated with clinical disease. Suggest the application of Protectotype vaccination programmes when field viruses with amino acid characteristics the diverge greatly from vaccine strains are present, thus leading to epitope changes relative to vaccine strains.

63 Conclusion The use of 4/91 based vaccination programmes results in the population of the flock with 4/91 vaccine strain as the dominant strain on farm that is persistent and consistent from a genetic perspective The use of repeat spray in lay vaccination programmes will ensure the maintenance of significant quantities of vaccine virus RNA throughout the laying period and thus provide significant antigen quantities against which a protective immune response will be generated Flocks in lay are confirmed as the major source of infectious bronchitis field viruses. Thus layer flocks in the vicinity of broiler flocks remain a major risk for the introduction of field virus infections

64 Evaluation of the field efficacy of vaccination strategies 1. Broilers

65 What vaccination programmes are associated with field viruses in broilers (>42 days)? EU countries only Negative results in young birds with any correctly administered bronchitis vaccination programme and proper sampling technique should not occur key investigation point SU 8% Neg 18% Short lived broilers 01/02/ /02/2013 Field Virus 76 25% Vaccine Virus %

66 What vaccination programmes are associated with field viruses in broilers (>42 days)?- EU countries only Short lived broilers - all results 01/02/ /02/2013 Other Vaccines 48% 4/91 + anything else 29% 4/91 & Ma5 15% Ma5 +anything else 8%

67 What vaccination programmes are associated with field viruses in broilers (>42 days)? EU countries only Broilers - Field Virus 01/02/ /02/2013 Field Virus with 4/91 + anything else 23% Field Virus with 4/91 & Ma5 4% Other Vaccines 73%

68 Evaluation of the field efficacy of vaccination strategies 2. Birds in lay

69 EMEA field results flocks over 18 weeks of age Results for 18w & Over Submissions 27/04/ /04/2013 Key conclusion: The most likely field virus finding in lay is 4/91 followed by QX

70 Incidence of field viruses Total number of inlay submissions (>18 weeks of age) where 4/91 field virus was confirmed 107 Number of inlay submissions (>18 weeks of age) where 4/91 field virus was confirmed no live IB vaccination in lay 63 Number of inlay submissions (>18 weeks of age) where 4/91 field virus was confirmed and live IB vaccination was used in lay 44 In lay vaccination is associated with the presence of fewer field viruses

71 Administration pattern of Ma5 and 4/91 in lay Distance between 4/91 and Ma5 13/06/ /04/ days; 18; 30% >14 days; 42; 70% Tendency to give Ma5 and 4/91 further apart than optimum for cross protection response

72 Field virus examples 2013

73 Key question How do we know a field virus is causing disease? This is determined by the appearance of clinical signs. The results give supporting indications of the potential of the virus to be a disease causing agent

74 Use of phylogenetic tree to establish the presence of a virus that differs significantly

75 4/91 Field virus broilers epitope mismatch with vaccine Individual tracheal (Ct 27.91) and cloacal (Ct 21.62) swab analysis. H120 at day 7, 4/91 at day 20 Vaccination History Vaccine Age Dose Route of Application HB1+IB 7 days 1ds DW Bursine plus 12 days 1ds DW ND Inac. 18 days 1ds Inj. 4/91 20 days 1ds Eye drop Respiratory clinical signs, mortality, enteritis. Large quantity of 4/91 field virus (9 amino acid substitutions different from the vaccine) shedding prior to onset of immunity of 4/91 vaccine. Recommendation to move 4/91 plus Ma5 application to day of age

76 QX field virus EU cloacal swabs - epitope mismatch with vaccine 67 week old layers vaccinated with 4/91 at 14d & 10w, Ma5 at 1d, 7w & 14w Ct Value Vaccination History Vaccine Age Dose Route of Application Nobilis IB d; 10 w 1 ds aerosol Nobilis IB H120 Nobilis IB Ma5 1d; 7w; 14w 1 ds aerosol Nobilis IB inac (M41) 16w 1 ds inj Ct value not consistent with aggressive infection

77 QX field virus EU cloacal swabs - epitope mismatch with vaccine 53 week old layers vaccinated with 4/91 at 14d & 8w, Ma5 at 35d and Primer at 1d & 10w Ct Value Vaccination History Vaccine Age Dose Route of Application Nobilis IB d,8 wks 1ds Spray, drinking water Nobilis IB H120 Nobilis IB Ma5 35d 1ds Drinking water H120+D274 (IB Primer) 1 d, 10 wks 1ds Spray, drinking water No clear IB related clinical signs sampling from another house on the same farm showed small quantities of untypable virus. Protectotype vaccination programme is not optimised for QX protection

78 4/91 field virus EU cloacal swabs - epitope mismatch with vaccine 74 week old broiler parents vaccinated with 4/91 at 14d & 10w, H120 at 14w & 56w, Ma5 at 35d and IB Primer at 1d and 19w: Ct Value Vaccination History Vaccine Age Dose Route of Application Nobilis IB d;10w 1 ds spray H120 14w;56w 1ds spray Nobilis IB Ma5 35d 1 ds spray H120+D274 (IB Primer) 1d; 19w 1 ds spray IB88 IBmm+Ark Nobilis IB inac (M41) Olvac A+B (Nev14+Nev24) inac 69w 1ds inj Nobilis IB multi inac (M41+D274) 16w 1ds inj No 4/91 vaccine in close proximity to field challenge

79 4/91 field virus EU cloacal swabs - epitope mismatch with vaccine 53 week old broiler parents vaccinated with 4/91 at 14d & 10w, Ma5 at 8w and IB Primer at 1d: Ct Value Vaccination History Vaccine Age Dose Route of Application Nobilis IB days, Coarse spray 1 ds 10 weeks fine spray Nobilis IB H120 Nobilis IB Ma5 8 weeks 1 ds Fine spray H120+D274 (IB Primer) 1 day 1 ds Coarse spray IB88 IBmm+Ark Nobilis Rhino CV 6 weeks 1 ds Coarse spray NobilisTRT Live Other (please specify) Nobilis RT+IB multi+g_nd 18 weeks 1d Inj.i.m. No 4/91 vaccine in close proximity to field challenge

80 MASS Type field virus EU cloacal swabs - epitope mismatch with vaccine 64 week old layers vaccinated with 4/91 at 2w & 9w, Ma5 at 5w & 11w: Ct Value Vaccination History Vaccine Age Dose Route of Application Nobilis IB ,9 wks 1 ds spray Nobilis IB H120 Nobilis IB Ma5 5, 11 wks 1 ds Drinking water H120+D274 (IB Primer) IB88 IBmm+Ark Nobilis Rhino CV NobilisTRT Live ( Nemovac) 14 wks 1 ds spray Other (please specify) Ceva Bron 1 d 1 ds spray

81 QX field virus EU cloacal swabs - epitope mismatch with vaccine 64 week old layers vaccinated with 4/91 at 2w & 9w, Ma5 at 5w & 11w Ct Value Vaccination History Vaccine Age Dose Route of Application Nobilis IB ,9 wks 1 ds spray Nobilis IB H120 Nobilis IB Ma5 5, 11 wks 1 ds Drinking water H120+D274 (IB Primer) IB88 IBmm+Ark Nobilis Rhino CV NobilisTRT Live ( Nemovac) 14 wks 1 ds spray Other (please specify) Ceva Bron 1 d 1 ds spray

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