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17 November 2016 Minimum Infectious Dose Determination of the Arkansas Delmarva Poultry Industry Infectious Bronchitis Virus Vaccine Delivered by Hatchery Spray Cabinet
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The Arkansas Delmarva Poultry Industry (ArkDPI) infectious bronchitis virus (IBV) vaccine is effective when administered by eye drop, where the vaccine virus is able to infect and replicate well in birds and is able to induce protection against homologous challenge. However, accumulating evidence indicates that the ArkDPI vaccine is ineffective when applied by hatchery spray cabinet using the same manufacturer-recommended dose per bird. For this study, we aimed to determine the minimum infectious dose for the spray-administered ArkDPI vaccine, which we designate as the dose that achieves the same level of infection and replication as the eye drop–administered ArkDPI vaccine. To this end, we used increasing doses of commercial ArkDPI vaccine to vaccinate 100 commercial broiler chicks at day of hatch, using a commercial hatchery spray cabinet. The choanal cleft of each bird was swabbed at 7 and 10 days postvaccination, and real-time reverse-transcriptase PCR was performed. We observed that the level of infection and replication with spray vaccination matches with that of eye drop vaccination when chicks received 100 times the standard dose for the commercial ArkDPI vaccine. We further examined the S1 spike gene sequence from a subset of reisolated ArkDPI vaccine virus samples and observed that certain nucleotide changes arise in vaccine viruses reisolated from chicks, as previously reported. This suggests that the ArkDPI vaccine has a certain virus subpopulation that, while successful at infecting and replicating in chicks, represents only a minor virus subpopulation in the original vaccine. Thus, the minimum infectious dose for the ArkDPI vaccine using a hatchery spray cabinet appears to be dependent on the amount of this minor subpopulation reaching the chicks.

In the United States, the Arkansas-type (Ark) infectious bronchitis virus (IBV) is one of the most common IBV serotypes isolated from chickens in the field (5,13). The Arkansas Delmarva Poultry Industry (ArkDPI) strain is the only commercially available attenuated live vaccine against the Ark IBV serotype. It has been shown that ArkDPI vaccine can persist within a flock (6) and that poor vaccine coverage results in rolling replication, which is characterized by lateral transmission of the vaccine virus among birds (9). The ArkDPI vaccine has also been associated with delayed replication within a flock, relative to other serotypes of IBV vaccine (1,6). Indeed, many field isolations of Ark-type IBV appear to be closely related genetically to ArkDPI vaccine virus (13).

Eye drop administration of ArkDPI has been shown to provide good vaccine coverage, as well as good protection against homologous challenge (14). However, when ArkDPI vaccine is delivered by mass applications, such as a hatchery spray cabinet, poor vaccine coverage within the flock and little protection against homologous challenge has been seen (6,14,16). One hypothesis to explain poor vaccine coverage is that its virions may be sensitive to mechanical damage, as the virus particles pass through the lines and nozzle in a hatchery spray cabinet. Though some reduction in titer was observed in the ArkDPI vaccine, mechanical damage is likely not an issue because the reduction in titer was not different from a fully efficacious Massachusetts-type (Mass) IBV vaccine (15). An alternative hypothesis is that the chicks are simply not getting enough ArkDPI vaccine by spray vaccination. To this end, we sought to determine the minimum infectious dose or the dose at which the spray-administered ArkDPI vaccine achieves the same level of infection and replication as the eye drop–administered ArkDPI vaccine. We vaccinated commercial broiler chicks at day of hatch using a commercial hatchery spray cabinet with increasing doses of ArkDPI vaccine and measured virus levels in the chicks at 7 and 10 days postvaccination (dpv) by real-time RRT-PCR.

Reisolation of the ArkDPI vaccine virus from vaccinated chicks has demonstrated that certain nucleotide changes occur in the S1 spike gene, suggesting the existence of a virus subpopulation that is able to more efficiently infect and replicate in chicks (10,11,12,19). To identify what virus subpopulation(s) were replicating in the chicks, we performed S1 spike gene sequencing from swabs taken from vaccinated chicks to examine the genotypes of reisolated ArkDPI vaccine viruses.


Experimental design

One-day-old broiler chicks obtained from a commercial source were vaccinated with a commercially available ArkDPI vaccine using a single-nozzle hatchery spray cabinet. Three separate experiments were conducted. For each experiment, 100 chicks were placed in a hatchery chick basket, spray vaccinated with either a 1×, 10×, or 100× dose in 7 ml then immediately transferred into Horsfall isolation units. For each experiment, some chicks were also vaccinated with ArkDPI vaccine by eye drop, using the manufacturer-recommended dose. In addition, we vaccinated chicks by the eye drop route and using the hatchery spray cabinet method with the manufacturer-recommended dose of a Mass vaccine. Eye drop vaccination was performed on 20 chicks by placing 100 μl of vaccine equivalent to one dose recommended by the manufacturer directly into the eye using a pipette. One dose of ArkDPI and Mass vaccine was calculated to be 1 × 103.5 50% embryo infective doses. Choanal swabs were taken from each bird at days 7 and 10 postvaccination. Typically, challenge virus detection is done at 5 days postexposure; however, our data show that the peak of replication for spray cabinet administration of IBV vaccine viruses is between 7 and 10 dpv (14). Swabs were placed in 1 ml of sterile phosphate-buffered saline (pH 7.4) and stored at –80°C until analysis. Protocols used for the bird experiments were approved by the Institutional Animal Care and Use Committee of the University of Georgia.

Real-time reverse-transcription PCR

Measurement of infectivity and virus replication levels in the chicks was performed by RRT-PCR. Viral RNA was extracted from swab samples using the MagMax-96 Total RNA Isolation Kit (Life Technologies, Thermo Scientific, Waltham, MA) and the Kingfisher magnetic particle processor (Thermo Scientific, Waltham, MA) per manufacturer protocols. Five microliters of each RNA sample was added to 20 μl of master mix from the AgPath-ID One-Step RT-PCR kit (Applied Biosystems, Thermo Scientific). We used the IBV-specific primers, S1 5′ GU391 forward primer (5′-GCT TTT GAG CCT AGC GTT-3′) and S1 3′ GL533 reverse primer (5′-GCC ATG TTG TCA CTG TCT ATT G-3′), and TaqManH dual-labeled probe IBV5′G (5′-FAM-CAC CAC CAG AAC CTG TCA CCT C-BHQ-3′), as described previously (2). The data are reported as the average cycle threshold (Ct) value, the number of PCR cycles at which the fluorescence of the reaction exceeded the minimum level of detection for the assay.

S1 spike gene sequencing and analysis

The S1 spike genes were amplified using the 5′ oligo and 3′ degenerate primers (8) together with the Titan One Tube RT-PCR Kit (Roche, Foster City, CA). Samples were sent to the Georgia Genomics Facility (Athens, GA) for standard Sanger sequencing using the S1 oligo 5′ (5′-TGA AAC TGA ACA AAA GAC-3′) and S1 3′ degenerate (5′-CCA TAA GTA ACA TAA GGR CRA-3′), as previously described (8). Additionally, the Ark99/DPI-Int-F primer (5′-TTT CTG TGA CTA AAT ATC CTA AG-3′) was designed and used to sequence the middle portion of the S1 spike gene. The spike gene for each sample was sequenced with three reads and assembled using Geneious software (Version 8.0.5, Biomatters Ltd., Auckland, New Zealand). Each contig was aligned to the full length S1 spike protein gene of ArkDPI with GenBank accession number ADP06471.2. The frequency of nucleotide sequence changes and corresponding amino acid changes were then determined using Geneious and CodonCode Aligner (Version 5.1.5, CodonCode Corp., Centerville, MA).


ArkDPI is the only commercially available vaccine against Ark-type IBV. The ArkDPI vaccine is efficacious when it is administered by eye drop, but it fails to protect against homologous challenge when it is delivered by a hatchery spray cabinet or by drinking water methods (6,14). Spray cabinet vaccination is associated with a low level of replication that is delayed relative to other IBV vaccine strains (1,6). It, thus, appears that the chicks are not getting an adequate dose when they are vaccinated with a hatchery spray cabinet. The objective of our study was to determine the minimum infectious dose of ArkDPI administered by a hatchery spray cabinet. Herein, we define the minimum infectious dose as the dose of ArkDPI vaccine delivered by spray that achieves the same level of infection and replication in chicks that are vaccinated with the manufacturer-recommended dose by eye drop. “Minimum infectious dose” herein is, thus, evaluated by the vaccine taken as a flock, rather than as individual birds.

Determination of the minimum infectious dose

We performed eye drop vaccination of chicks with the manufacturer-recommended dose of ArkDPI vaccine to serve as the benchmark level of infection and replication. For eye drop vaccination, the number of birds positive for vaccine virus was 88%, and those positive had a mean Ct value of 32.24 ± 0.37 at 7 dpv (Fig. 1A). The number of birds positive for vaccine virus drops to 48%, with a mean Ct value of 34.19 ± 0.44 for positive samples at 10 dpv. In contrast, the percent of birds positive for vaccine virus that were vaccinated with Mass by the eye drop route was 100% at days 7 and 10 postvaccination, with mean Ct values of 28.73 ± 0.30 and 29.74 ± 0.52 for positive samples, respectively. Likewise, spray vaccination with Mass resulted in 100% and 99% of the birds positive for the vaccine virus, with mean Ct values of 27.04 ± 0.16 and 29.34 ± 0.23 for positive samples at 7 and 10 dpv, respectively.

Fig. 1

Determination of the minimum infectious dose for the ArkDPI vaccine administered by hatchery spray cabinet. Choanal swabs were taken at 7 and 10 dpv from 100 chicks vaccinated at day of hatch using the vaccine, dose, and route as indicated. RRT-PCR was performed for all samples to measure virus levels. (A) The number of birds positive for vaccine virus was determined, and the percentage of positive birds are shown. A bird was considered to be positive when the choanal swab Ct value was above the cutoff value of 36 (limit of detection). (B) To measure levels of replication, the mean Ct value was calculated from samples that tested positive for the vaccine virus. The dashed lines indicate the limit of detection with RRT-PCR. Note that all samples are represented on the scatter plot. The number of birds positive for vaccine virus was 88% at day 7 postvaccination, with ArkDPI administered by eye drop (1× dose) or spray at 10× dose, demonstrating that, in this study, the minimum infectious dose for ArkDPI administered by hatchery spray is 100× of the standard dose.


The doses of ArkDPI tested in a hatchery spray cabinet was 1×, 10×, and 100× the manufacturer-recommended dose. As expected, the 1× dose of ArkDPI exhibited a poor level of infection and replication, with 15% testing positive for vaccine virus, and with a mean Ct value of 32.36 ± 0.77 for positive samples at 7 dpv. At 10 dpv at 1× dose, only 22% were positive for the vaccine virus, with a mean Ct value of 30.99 ± 0.59 for positive samples. Likewise, chicks vaccinated with a 10× dose by spray had poor levels of infection and replication with only 27% (Ct = 34.64 ± 0.28) and 19% (Ct = 34.72 ± 0.35) of birds positive for the vaccine virus at 7 and 10 dpv, respectively. We observed much higher levels of infection and replication with chicks vaccinated with a 100× dose, which had approximately 88% (Ct = 31.69 ± 0.23) and 90% (Ct = 31.56 ± 0.25) of the chicks positive for the vaccine virus at 7 and 10 dpv, respectively. The level of infection at 7 dpv was 88% when ArkDPI was delivered by eye drop at the standard dose (1×) or by spray at 100× dose. Thus, the minimum infectious dose of ArkDPI vaccine delivered by hatchery spray cabinet for this study is 100× the manufacturer-recommended dose.

Considering only positive samples, the mean Ct values for ArkDPI was not significantly different between eye drop or spray cabinet administration (Fig. 1B). This suggests that if a bird receives the vaccine, ArkDPI can replicate to an appreciable level. Likewise, the data also suggest that the level of infection is dependent on the dose of vaccine delivered to birds and not on the ability of ArkDPI to replicate in the chick. Note that the mean Ct values for birds vaccinated with Mass, regardless of route of administration, are generally higher than with ArkDPI, indicating that Mass appears to have a higher replicative ability in birds than ArkDPI.

S1 gene sequence analysis

The S1 spike is a structural protein found on the surface of coronavirus particles. It is primarily responsible for binding to host tissues (3) and is the main target of virus-neutralizing antibodies (4,7). Moreover, the highest sequence diversity in the viral genome is found in hypervariable regions within the S1 spike gene (4,20).

Reisolations of ArkDPI from vaccinated chicks have shown that many nucleotide sequence changes occur at the S1 spike gene, specifically at hypervariable regions (10,12,19). The rapid emergence of these viruses indicates that the ArkDPI vaccine contains virus subpopulations, which appear to have differences in the ability to infect or replicate or both in chickens. For our study, we examined the sequence of the S1 spike genes from chicks vaccinated with ArkDPI via the eye drop route or via hatchery spray cabinet at various doses. Each sequence was assembled and compared with the reference ArkDPI S1 spike gene in GenBank. As expected, we observed specific nucleotide changes in the reisolated ArkDPI vaccine virus (Fig. 2). Interestingly, the frequency of nucleotide changes was similar between groups. For instance, position 127 in the reference ArkDPI strain is a T, whereas in all of the groups tested, most of the samples (≥50%), position 127 contains a C. These predominant sequence differences observed in reisolated ArkDPI vaccine virus (Table 1) are evidence of a virus subpopulation that is competent at infecting and replicating in birds, as previously proposed (10,12,18,19). Our data suggest that poor vaccine coverage with spray-administered ArkDPI is a result of having an insufficient number of virus particles belonging to the competent virus subpopulation that is successful in infecting and replicating in chicks. When chicks are vaccinated with a critical number of vaccine virus particles, the minimum infectious dose of the competent virus subpopulation is achieved, which is, in this study, a 100× dose. Note that we did not observe an increase in clinical signs after vaccination with high doses at 10× or 100×.

Fig. 2

A summary of the polymorphisms found in the S1 gene of the ArkDPI vaccine virus. The height of the letters indicates the proportion of samples with the nucleotide base indicated by the letter. Grey letters indicate nucleotides that are found in the reference ArkDPI sequence (top row), while black letters indicate a nucleotide change from the reference sequence. Single letter International Union of Pure and Applied Chemistry codes are used for the nucleotide bases. Additionally, the Greek letter delta (Δ) indicates a deletion. The minimum number samples considered per group are 12 for eye drop at 7 dpv, 2 for eye drop at 10 dpv, 4 for 10× spray at 7 dpv, 7 for 10× spray at 10 dpv, 10 for 100× spray at 7 dpv, and 13 for 100× spray at 10 dpv.


Table 1

Nucleotide changes and their corresponding amino acid changes (enclosed in parenthesis) in the S1 gene of vaccine virus reisolated from vaccinated chicks.A



Rows with bold entries indicate those positions in which the nucleotide or amino acid in the reisolated ArkDPI is the predominant (≥50%) nucleotide or amino acid.

In summary, we have demonstrated the minimum infectious dose for the ArkDPI vaccine administered by hatchery spray cabinet is 100× the manufacturer-recommended dose. Reisolated ArkDPI vaccine viruses showed similar nucleotide changes in the S1 spike gene, regardless of route of administration or dose. This indicates that a certain virus subpopulation is competent to infect and replicate in chicks, providing further evidence of the existence of these virus subpopulations in the ArkDPI vaccine.

This study highlights the importance of developing well-defined vaccination protocols and implementing them properly. Indeed, it has been demonstrated that vaccination of birds with less than a full dose does not protect birds from homologous challenge with virulent IBV (17). Clearly, it is not economically feasible to vaccinate birds with 100× dose of ArkDPI vaccine. There is, thus, a need to further enhance existing vaccination strategies or to develop an alternative Ark-type IBV vaccine.


Funding for these experiments was provided by the U.S. Poultry and Egg Association, Project No. 680.



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Christina M. Leyson, Deborah A. Hilt, Brian J. Jordan, and Mark W. Jackwood "Minimum Infectious Dose Determination of the Arkansas Delmarva Poultry Industry Infectious Bronchitis Virus Vaccine Delivered by Hatchery Spray Cabinet," Avian Diseases 61(1), 123-127, (17 November 2016).
Received: 28 July 2016; Accepted: 1 October 2016; Published: 17 November 2016

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