Infectious bronchitis virus (IBV) causes an upper respiratory tract disease in chickens and is highly contagious. Many different types of the virus exist, but only a few types are used as attenuated live vaccines in the commercial poultry industry. Of the vaccine types used, the Arkansas (Ark)–type virus is most frequently reisolated from vaccinated broilers. Previous research has suggested that incomplete clearance of Ark-type vaccine virus plays a role in the inadequate protection observed when vaccinated broilers are challenged with pathogenic Ark virus. In this study, we examine routes of vaccine administration using multiple IBV types including Ark in an effort to understand why Ark vaccines do not provide good protection and persist in commercial broilers. We found that interference between different types of IBV vaccines was not occurring when combined and administered using a commercial hatchery spray cabinet. Also, Ark vaccine virus was not efficacious in 1-day-old broilers when sprayed using a hatchery spray cabinet, but it gave good protection when administrated by eyedrop inoculation. We also found that the amount of Ark vaccine virus was low or undetectable in choanal swabs out to 35 days postvaccination when vaccine was administered by eyedrop or drinking water. Alternatively, a subpopulation of the Ark vaccine isolated from a vaccinated bird, Ark-RI-EP1, showed a peak titer at 7–10 days of age when given by the same routes, suggesting that the Ark-RI-EP1 was more fit with regard to infection, replication in the birds, or both. Moreover, we found that detection of IBV vaccine virus early after administration, regardless of strain or route, correlated with protection against homologous challenge and may thus be a good indicator of vaccine efficacy in the field because humoral antibody titers are typically low or undetectable after vaccination. These experiments provided key findings that can be used to direct efforts for improving the efficacy of IBV Ark-type vaccines given in the hatchery. They also elucidated factors contributing to the persistence of Ark vaccine in the field.
Evaluación de las fallas vacunales con la cepa Arkansas del virus de la bronquitis infecciosa en pollos de engorde.
El virus de la bronquitis infecciosa causa una enfermedad del tracto respiratorio superior de los pollos y es altamente contagiosa. Existen muchos tipos de virus diferentes, pero sólo algunos tipos son utilizados como vacunas vivas atenuadas en la industria avícola. De los tipos de vacunas utilizadas, el tipo Arkansas es el que más frecuentemente se aísla de pollos de engorde vacunados. Las investigaciones anteriores han sugerido que la remoción incompleta del virus de la vacuna Arkansas desempeña un papel en la protección inadecuada observada cuando los pollos vacunados se desafían con una cepa Arkansas patógena. En este estudio, se examinaron las vías de administración de la vacuna utilizando varios tipos del virus de la bronquitis incluyendo Arkansas, en un esfuerzo por entender por qué las vacunas con el tipo Arkansas no proporcionan una buena protección y persisten en los pollos de engorde. Se encontró que no se presentaba interferencia entre diferentes tipos de vacunas de bronquitis infecciosa cuando se combinaban y se administraban usando un gabinete de aspersión en la incubadora. También, la vacuna Arkansas no fue eficaz en pollos de engorde de un día de edad, cuando se aplica por aerosol mediante un gabinete de aspersión en la incubadora, pero dio una buena protección cuando se administran por inoculación ocular. También se encontró que la cantidad del virus de la vacuna Arkansas era baja o no detectable en hisopos recolectados de las coanas a los 35 días después de la vacunación cuando la vacuna se administró por vía ocular o en el agua de bebida. Alternativamente, una subpoblación de la vacuna Arkansas aislada de una ave vacunada, el virus Ark-RI-EP1, mo
Avian infectious bronchitis virus (IBV) is a highly infectious pathogen of chickens that primarily infects epithelial cells of the upper respiratory tract. Infection causes various clinical signs, including nasal discharge, sneezing, watery eyes, weight loss, and lethargy. Depending on virus cell tropism, epithelial cells in the kidney or oviduct can be infected, causing nephritis and decreased egg production, respectively 5. Mortality caused solely by IBV is generally low compared with that of other avian viral pathogens, but young chicks predisposed to IBV infection are more susceptible to secondary bacterial infections that can be lethal 6. IBV is distributed worldwide and costs the poultry industry millions of dollars annually through production decreases; condemnations at the processing plant; and the high cost of prevention measures, including vaccination.
IBV is a gammacoronavirus, in the family Coronaviridae, and within the order Nidovirales. It is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 27–28 kb. The viral genome encodes the viral replicase complex (1a and 1ab encoding nonstructural proteins 2–16); structural proteins spike (S), envelope, membrane, and nucleocapsid; and several nonstructural proteins. The S protein is a large glycoprotein projected from the virus envelope that consists of an amino-terminal S1 subunit, making up the apical surface of the S protein, and a carboxyl-terminal S2 subunit that anchors the S protein to the viral membrane. The S protein is responsible for viral attachment to cell receptors and virus-host membrane fusion. The location and makeup of the receptor-binding domain in S1 varies among other coronaviruses, but it has not been identified for IBV 5.
Since the first identification of IBV in the 1930s in the United States (reviewed in 11), various serotypes and strains with antigenic variation have been found worldwide 5. There is little to no cross-protection between serotypes or circulating variant viruses, making it extremely difficult to control the disease 10. Different virus types are the result of mutations and recombination in the S protein during virus replication. Mutation, recombination, or both in S protein can change the epitopes that induce neutralizing antibodies and thereby allow new virus types to infect and cause disease even in vaccinated birds.
Currently, the best strategy for control of infectious bronchitis is the use of live attenuated vaccines, despite the fact that vaccinated birds do not obtain cross-protective immunity against heterologous viruses. In broilers in the United States, live vaccines are typically given at 1 day of age in the hatchery by using a spray cabinet and at approximately 2 wk of age in the field by an aerosol sprayer or in the drinking water. In addition, multiple serotypes are combined and used for vaccination in an attempt to induce broader protection 7. Live vaccines stimulate both humoral and cellular immune responses 5 that can cause a vaccine reaction if not administered properly. Vaccine strains of IBV also have been reported to revert to pathogenicity 14. Therefore, diagnosis of circulating viruses and choosing and properly administering the right vaccine type are critical for the control of IBV.
It is generally accepted that birds vaccinated in the hatchery develop sufficient immunity to clear the field boost vaccine virus from the upper respiratory track by 5 days postvaccination 19. However, this clearing does not apply to all IBV serotypes because persistence of the Arkansas (Ark)–type vaccine, one of the most widely used vaccine serotypes in the United States, also was reported 19. The persistence of Ark-type vaccines in commercial broilers can provide the virus with opportunities to undergo mutations, and these mutations can result in a pathogenic phenotype capable of causing a disease outbreak. To this end, variant Ark viruses (Ark-like viruses) have been reported previously 1,16,17,23,24 in vaccinated birds, indicating that the virus is changing. It is not clear why Ark-type vaccines are persisting in commercial broilers; however, it may be due to inadequate priming of the immune response by hatchery vaccination. In this study, we examine different routes of vaccine administration as well as vaccination with multiple IBV types including Ark in an effort to understand why Ark vaccine viruses persist in commercial broilers.
MATERIALS AND METHODS
Vaccines and challenge viruses
Commercially available mono and bivalent live attenuated vaccines of the Ark, Massachusetts (Mass), and Georgia 98 (GA98) types were used in this study. Dr. J. Gelb, Jr. (University of Delaware, Newark, DE) kindly provided the pathogenic Ark-Delmarva Poultry Industry (DPI) Ark/Ark-DPI/81 and the Mass 41 Mass/Mass41/41 strains. The pathogenic GA98 virus GA98/CWL0470/98 was isolated in our laboratory in 1998 20.
Commercial nonvaccinated broiler chicks were obtained from a commercial source at 1 day of age and maintained in positive-pressure Horsfal isolation units at the Poultry Diagnostic and Research Center, University of Georgia, Athens, GA. Feed and water were provided ad libitum.
To examine whether interference occurs between Ark vaccine viruses and other IBV vaccine serotypes, we immunized birds with Mass or GA98 vaccines in combination with the Ark vaccine at the manufacturer's recommended dose. In brief, vaccine stock from the manufacturer was rehydrated in phosphate-buffered saline ([PBS], 1000 doses/ml). Working solutions were then prepared so that the proper number of vaccine doses (one dose/bird) was mixed with PBS in a total volume of 7 ml. Working vaccine solution titers were 1 × 104.6 50% egg infective dose (EID50)/ml for Ark vaccine, 1 × 104.5 EID50/ml for Mass vaccine, and 1 × 105 EID50/ml for GA98 vaccine. One-day-old broilers were divided into six groups: Ark vaccine group, Mass vaccine group, GA98 vaccine group, Ark and Mass combined vaccine group, Ark and GA98 combined vaccine group, and a nonvaccinated control group. Birds were vaccinated using a commercial hatchery spray cabinet delivering 7 ml of vaccine suspension in a single application. Tracheal swabs and tears were collected from five birds in each group at 3, 7, 10, 14, 17, 21, and 28 days of age. Samples were collected from different birds at each time point. At 30 days of age, five birds from each group were challenged intraocularly and intranasally with pathogenic Ark-DPI (1 × 105 EID50/bird), Mass 41 (1 × 105.3 EID50/bird), or GA98 (1 × 105 EID50/bird), and five birds were maintained as nonchallenge controls. At 5 days postchallenge, all birds were examined for clinical signs, tracheal swabs and tears were collected for virus detection, and sera was collected and examined for antibodies against IBV by using a commercial ELISA kit (IDEXX, Portland, ME). Euthanatized birds were examined for lesions, and tracheal tissues were collected for histopathology. Real-time reverse transcriptase (RT)-PCR was used to determine the presence of vaccine and challenge virus in tracheal swabs and tears. Tracheal tissue samples were fixed in 10% neutral buffered formalin, routinely embedded in paraffin, sectioned, and stained for histopathologic examination. Tracheal tissues were prepared, and the lesions were scored from 1 to 4 as described previously 15: 1 = normal, 2 = focal, 3 = multifocal, and 4 = diffuse.
To determine whether dose affects the efficacy of Ark vaccine when delivered using a hatchery spray cabinet, we tested two different doses of Ark vaccine with different numbers of 1-day-old broilers. To ensure that the vaccines were evenly sprayed on all birds, we used only one nozzle in the spray cabinet, and we made a circular-shaped cardboard enclosure approximately 34 cm in diameter that confined a maximum of 40 one-day-old chicks to the spray range of the nozzle. The birds were divided into five groups: 15 birds in the no-vaccine control, 20 birds given 20 doses of vaccine, 20 birds given 40 doses of vaccine, 40 birds given 40 doses of vaccine, and 40 birds given 80 doses of vaccine. Birds in each of the vaccinated groups were sprayed with 7 ml of Ark vaccine suspension. Each group of chicks was housed in separate isolators for 1 hr after vaccination, and 15 birds per group were maintained. The remaining chicks were sacrificed. Choanal swabs and tears were collected at days 3, 7, 10, 14, 17, 21, and 28 days of age from the same birds in each group. At 30 days of age, 10 birds from each group were challenged via the intraocular/intranasal route with pathogenic Ark-DPI (1 × 105 EID50/bird), and five birds per group were kept as nonchallenge controls. At 5 days postchallenge, choanal swabs and tears were collected from all of the birds for virus detection by real-time RT-PCR analysis.
This experiment was designed to determine whether Ark vaccine reisolated from vaccinated broilers at 21 days postvaccination has an advantage over the original commercial vaccine with regard to infection and replication in broiler chicks. The Ark vaccine reisolated at 21 days postvaccination was passed in embryonated eggs one time to increase the virus titer, and the resulting virus isolate was designated Ark-reisolated-egg pass 1 (Ark-RI-EP1). The commercial Ark vaccine and Ark-RI-EP1 virus were titered in embryonated eggs. Replication of the viruses was examined by giving the Ark vaccine or Ark-RI-EP1 (1 × 104.5 EID50/bird) to 10 one-day-old broilers by eyedrop inoculation. A nonvaccinated negative control group also was maintained. Choanal swabs and tears were collected from the same birds (10 birds/each group) at 1, 3, 7, 10, 14, 17, 21, 28, and 35 days of age and analyzed for virus replication using real-time RT-PCR. The nonstructural protein 3 (nsp 3) gene and S1 gene sequences of the Ark vaccine and Ark-RI-EP1 were determined, and consensus sequences were compared with the previously published Ark vaccine viruses to identify changes.
To determine whether vaccine application methods affect vaccine efficacy, we inoculated 1-day-old broilers with either the Ark vaccine or Ark-RI-EP1 by spray, drinking water, or eyedrop. Drinking water vaccine was prepared in cold distilled water with 0.1% powdered skim milk as a stabilizer and was consumed by the birds within 1 hr. The Ark vaccine and Ark-RI-EP1 virus were titered, and a dose of 1 × 103.4 EID50/bird was given by spray, eyedrop, or drinking water. Choanal swabs were collected from the same birds (10 birds/each group) at 3, 7, 10, 14, 17, 21, and 28 days of age. At 30 days of age, 10 birds in each group were challenged via the intraocular/intranasal route with pathogenic Ark-DPI (1 × 105 EID50/bird), and five birds were kept as negative challenge controls. At 5 days postchallenge, choanal swabs and tears were collected for virus detection by real-time RT-PCR, serum was collected and tested for antibodies to IBV by ELISA (IDEXX), and tracheas were collected and fixed in 10% neutral buffered formalin for histopathologic analysis as described above.
Virus detection using real-time RT-PCR
Viral RNA was extracted from swabs and tears by using the MagMax96 total RNA isolation kit (Ambion, Austin, TX) and the KingFisher Automated Nucleic Acid Purification machine (Thermo Fisher Scientific, Waltham, MA) according to the manufacturers' protocols. Real-time RT-PCR analysis was performed using SmartCycler II (Cepheid, Sunnyvale, CA) and the AgPath-ID™ One-Step RT-PCR kit (Ambion) according to the manufacturers' recommendations. The IBV-specific primers and probe for the real-time RT-PCR were published by Callison et al. 4: forward primer IBV5′GU391 (5′-GCT TTT GAG CCT AGC GTT-3′), reverse primer IBV5′GL533 (5′-GCC ATG TTG TCA CTG TCT ATT G-3′), and TaqMan® dual-labeled probe IBV5′G (5′-FAM-CAC CAC CAG AAC CTG TCA CCT C-BHQ-3′). Ark-specific probe and primers also were designed and used in this study: Ark-F′ (5′-GTG AAG TCA CTG TTT CTA-3′), Ark-R′ (5′-AGC ACT CTG GTA GTA ATA C-3′), and a labeled minor groove binding (MGB) probe Ark-P (5′-TET-TRT ATG ACA ACG AAT C-MGBNFQ-3′). The specificity of the Ark primers and probe were verified against Mass, GA98, Connecticut, and Delaware 072 IBV types (data not shown), and the assay standard curve for Ark-specific probe and primers was generated by plotting the cycle threshold (CT) values and log10 of virus copy numbers (y = −0.2709x + 11.9463; y = log10 of virus copy number, x = CT value), with R2 = 0.98. The primers were obtained from Integrated DNA Technologies (Coralville, IA), and TaqMan probe was synthesized by BioSearch Technologies (Novato, CA). The MGB probe was obtained from Applied Biosystems (Foster City, CA). Real-time RT-PCR components and thermocycler parameters were conducted as described previously, and a standard curve for the assay that was previously published was used to calculate the approximate genome copy number for each sample 4.
Sequence analysis of the S1 and the nsp 3 genes
The Ark vaccine and Ark-RI-EP1 S1 genes were amplified by RT-PCR using previously published primers: NEWS1OLIGO5′18 and Degenerate3′ 19. For amplification of the nsp 3 gene, two sets of primers were designed and designated: NSP3-1-F′ (5′-ACT ATA TGT TCT TCC GCT TCA-3′), NSP3-1-R′ (5′-CTT CAC AAT TCT TAA CCC CAC AGT-3′), NSP3-2-F′ (5′-GAT GCT AAT TGG CTT CTT G-3′), and NSP3-2-R′ (5′-AGG GTT TTC TTT CTG TTT GTG TC-3′). Sequencing reactions for S1 genes were performed using the BigDye® Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems) and purified using the Performa DTR Ultra Dye Terminator removal system (Edge Biosystems, Gaithersburg, MD) according to manufacturers' protocols. Nucleotide sequencing was conducted by the Georgia Genomics Facility, University of Georgia, Athens, GA. For nsp 3 genes, gel-purified RT-PCR products were sent to GENEWIZ® (GENEWIZ Inc., South Plainfield, NJ) for nucleotide sequencing. The S1 and the nsp 3 sequences for each virus were assembled using SeqMan and MegAlign programs (DNASTAR, Inc., Madison, WI).
The data were analyzed using JMP Statistical Discovery Software Version 9 (SAS Institute, Inc., Cary, NC). Genome copy number and CT values are presented as mean ± SEM. Means were compared by Student t-test (α = 0.05). Histopathologic scores were analyzed by Kruskal-Wallis test for multiple comparisons test followed by a Dunn post test. Significance is reported at the level of P < 0.05.
Birds in Expt. 1 were spray vaccinated at 1 day of age, challenged at 30 days of age, and the average viral genome copy number per group, analyzed by real-time RT-PCR using IBV-5′G probe, was calculated based on the previously published standard curve for this real-time RT-PCR assay 4. The resulting data are shown in Fig. 1. Tracheal swabs and tears collected from vaccinated and nonchallenged birds at 35 days of age (5 days postchallenge) from each vaccine group also were analyzed to evaluate vaccine virus replication. Replication of Ark vaccine was not detected until 21 days of age, after which replication quickly declined in both tracheal swabs and tears (Fig. 1A,C). The peak titer of Mass and GA98 vaccines given alone (Fig. 1A,C) or in combination with Ark vaccine (Fig. 1B,D) occurred between 10 and 14 days of age. To detect the Ark vaccine virus in combination with other vaccines, an Ark-specific probe (Ark-P) was used, and the average viral genome copy number was calculated based on the linear standard curve. The relative amount of Ark vaccine virus is presented in Fig. 2. Birds given Ark vaccine alone showed a peak titer between 21 and 28 days of age, but birds given a combined vaccine of either Ark with Mass or Ark with GA98 had small amounts of the Ark vaccine in both tracheal swabs and tears, although usually below the limit of detection for this assay. Virus in tracheal swabs from birds given Ark and GA98 vaccines combined with the Ark vaccine was detected at 7 days of age, but no virus was detected at any other time points.
Clearance of the challenge virus in vaccinated and nonvaccinated birds was determined by real-time RT-PCR on RNA extracted from tracheal swabs and tears collected at 5 days postchallenge (Fig. 3). Birds vaccinated with Ark and challenged with the homologous virus (Fig. 3A,C) were not adequately protected, as indicated by detection of high amounts of viral RNA, and no statistically significant difference was observed between that group and nonvaccinated birds challenged with the same virus. Birds vaccinated with either Mass or GA98 were protected against homologous challenge (Fig. 3A,C). In combined vaccine groups, birds were adequately protected against the homologous Mass or GA98 virus, and they also showed a slightly better protection against Ark-DPI challenge compared with that of birds vaccinated with Ark alone (Fig. 3B,D). In most groups, virus load detected in tears was 10- to 100-fold higher than load in tracheal swabs. The number of virus-positive birds, corresponding CT values, and histopathologic scores for each group are shown in Table 1. Vaccine virus was detected in most of the vaccinated groups on the 2 days before challenge (28 days of age), although at a very low level close to the limit of detection; and birds that were vaccinated and not challenged were also positive for virus at necropsy (35 days of age), indicating that vaccine virus was still present. The CT values in the nonvaccinated/challenged groups were between 22.8 and 26.4, whereas the vaccinated and challenged groups had higher CT values, indicating less virus was present. The histopathologic scores of groups challenged with Ark-DPI were higher than those of any other group and showed no statistical difference with birds in the nonvaccinated-challenge groups.
IBV real-time RT-PCR mean CT value ± SEM and histopathologic score for Expt. 1 at 5 days postchallenge.
All of the sera collected from birds at 1 day of age were positive for maternal antibodies. To determine whether the maternal antibodies were neutralizing, Ark, Mass, and GA98 vaccines were used in virus neutralization (VN) tests in embryonated eggs. Good protection against both Ark (average titer, 91.2) and Mass (average titer, 97.7) was observed and comparatively low maternal antibodies against GA98 were observed (average titer, 4.79). There was little or no specific IBV humoral antibody detected at 5 days postchallenge in any of the groups (data not shown).
In Expt. 2, five groups of either 20 or 40 birds were given a 1× or 2× dose of vaccine by hatchery spray cabinet, and then vaccine virus levels in tracheal swabs and tears were measured by real-time RT-PCR from 3 to 28 days of age. A low level of vaccine virus was detected at 21 days of age in two and six of 15 birds in choanal swabs and tears, respectively, in the group of 20 birds that received 40 doses of vaccine. Vaccine virus was not detected in any other group before challenge.
The birds were challenged at 30 days of age, and the clinical signs and clearance of the homologous Ark-DPI challenge virus are presented in Table 2. Clinical signs at 5 days postchallenge were observed in ≥80% of birds in all of the groups except the group where 40 doses of Ark-DPI vaccine were given to 20 birds; this latter group had only two of 10 birds positive for clinical signs. The Ark-DPI challenge virus was detected in all of the birds in all groups except the nonchallenged negative control group; however, in the group of 20 birds that received 40 doses of vaccine, higher CT values in both choanal swabs and tears were recorded, indicating less challenge virus was present compared with that of the other challenge groups.
Clinical signs, IBV real-time RT-PCR mean CT value ± SEM for Expt. 2 at 5 days postchallenge.
In Expt. 3, the levels of commercial Ark vaccine and the vaccine virus reisolated from broilers at 21 days postvaccination and passed one time in embryonated eggs (Ark-RI-EP1) were examined in eyedrop-vaccinated birds by real-time RT-PCR (Fig. 4). An equal dose of both vaccines was given. The levels of Ark vaccine peaked at 3 days of age followed by a gradual decline until 35 days of age. The highest Ark-RI-EP1 virus levels were at 1 day of age and then declined until 10 days of age. A slight rise in copy number was observed for the Ark-RI-EPI virus at 14 days of age. Both viruses were below the limit of detection by 35 days of age when the experiment was terminated. These data are in contrast to the Ark vaccine replication pattern in Expt. 1 (Fig. 1), where Ark vaccine was administrated using a hatchery spray cabinet and not detected until 21 days of age.
In Expt. 4, different groups of birds were vaccinated by a hatchery spray cabinet, drinking water, or eyedrop using Ark vaccine or Ark-RI-EP1 and then were monitored for the level of vaccine virus by real-time RT-PCR (Fig. 5). Low levels of the Ark vaccine virus (1 × 102 to 1 × 102.5 genome copies) were detected in choanal swabs and tears from birds vaccinated by eyedrop (Fig. 5A,C), with a slight rise observed at 17 and 21 days of age. The Ark vaccine virus was not detected in the drinking water– and spray-vaccinated groups (Fig. 5A,C). In birds given the Ark-RI-EP1 virus via drinking water or eyedrop, the replication pattern peaked between 7 and 10 days of age and then declined to undetectable levels by 21 days of age (Fig. 5B,D). However, no Ark-RI-EP1 virus was detected from either choanal swabs or tears in the spray vaccinated birds (Fig. 5B,D).
To assess protection, we challenged the birds at 30 days of age with pathogenic Ark-DPI (Table 3; Fig. 6). None of the vaccinated and nonchallenged birds had clinical signs or significant histopathologic lesions. A small amount of virus was detected in one nonchallenged bird vaccinated via drinking water. In the birds vaccinated with Ark by eyedrop and challenged with Ark-DPI, only one of 10 birds showed clinical signs, whereas the spray- and drinking water–vaccinated and challenged groups had eight of 10 and nine of 10 birds with clinical signs, respectively. A low level of challenge virus was observed in choanal swabs and tears in the group given Ark vaccine virus by eyedrop (Fig. 6A,C), whereas relatively high levels of virus, (statistically different from the eyedrop-vaccinated group) were found in the spray- and drinking water–vaccinated and challenged groups. Histopathologic scores of all the challenged birds vaccinated with Ark were significantly different from those of the negative control group, with the exception of the group vaccinated by eyedrop.
Clinical sign, IBV real-time RT-PCR mean CT values ± SEM, and histopathologic score for Expt. 4 at 5 days postchallenge.
Groups given the Ark-RI-EP1 virus by eyedrop or drinking water and challenged with Ark-DPI had fewer birds with clinical signs (zero and two of 10, respectively) compared with the challenged birds vaccinated with Ark-RI-EP1 by spray (eight of 10 with signs). Low levels of virus were detected in two nonchallenged birds from each of the eyedrop- and drinking water–vaccinated groups. Challenge virus was detected in all of the challenged groups, but birds that received the Ark-RI-EP1 virus by eyedrop or in the drinking water had fewer positive birds (four and six of 10, respectively), and significantly less virus was detected in those groups compared with the other challenge groups (Fig. 6B,D). Histopathologic scores were statistically significant only in the birds receiving no vaccine and challenged or vaccinated by spray and challenged compared with negative controls. Based on histopathology, all other groups were protected.
The S1 and the nsp 3 gene sequences for the commercial Ark vaccine and the Ark-RI-EP1 virus are shown in Table 4. For S1, previously published subpopulations were compared, and the most closely related sequence was included with our viruses in Table 4. The Ark-RI-EP1 S1 gene sequence had nine nonsynonymous point mutations and a three-nucleotide deletion compared with Ark vaccine. For the nsp 3 gene, there was only a single nonsynonymous mutation between Ark-RI-EP1 and the Ark vaccine.
Difference in S1 gene and nsp 3 gene of parental commercial Ark-DPI vaccine and Ark-RI-EP1.
In this study, we examined the lack of protection observed in birds given a commercially available Ark vaccine by hatchery spray cabinet. We conducted four experiments, with the first experiment designed to examine whether other IBV vaccine types are interfering with the Ark vaccine when given simultaneously. To distinguish Ark vaccine virus when it was combined with Mass or GA98 vaccine, two separate probes, a universal IBV probe (IBV-5′G) and Ark-P, were used. The second experiment was designed to determine whether a more focused spray at either a 1× or 2× dose would improve the efficacy of the Ark vaccine given by hatchery spray cabinet. There is also a possibility that a subpopulation of the vaccine, as reported by van Santen and Toro 27, would be more fit for replication in the birds and perhaps produce better immunity. Thus, the third and fourth experiments were designed to examine an isolate of Ark vaccine obtained from broilers at 21 days postvaccination for infection, replication, and efficacy against Ark challenge in chicks.
In Expt. 1, we found no interference between Ark vaccine and other vaccine types (Mass or GA98) when bivalent vaccines (Ark and Mass or Ark and GA98) were used, based on protection level. In fact, birds vaccinated with bivalent vaccines acquired better protection against pathogenic Ark-DPI virus than birds vaccinated with Ark alone. In addition, Ark vaccine virus replication slightly peaked at 7 days of age in tracheal swabs when combined with GA98, whereas the peak of virus replication was at 21–28 days of age when Ark vaccine was given alone. In bivalent vaccine groups, the predominant vaccine virus detected was either Mass or GA98. The earlier peak titers for the Ark vaccine when that vaccine was given in combination with GA98 could explain the slightly better protection against Ark-DPI challenge observed in those birds. It is not clear why the Ark vaccine replication peak occurred earlier when given in combination with GA98, but it is possible that a synergistic effect occurred where the GA98 vaccine created a suitable environment for infection, replication, or both of the Ark vaccine. In addition, the better protection against pathogenic Ark-DPI viruses in bivalent vaccine groups supports a possible synergistic effect. Birds with a vaccine virus replication peak between 7 and 10 days of age showed better protection against homologous challenge regardless of vaccine type. It may be possible to apply this observation to assess protection against IBV in the field, because little or no ELISA titers are observed after vaccination.
In our experiments, low levels of vaccine viruses were still present in the tracheas and tears of some birds by 35 days of age, regardless of vaccine type or delivery method. Alvarado et al. 1 showed Ark vaccine virus was detected in the trachea and the cecal tonsils up to 28 days postvaccination in hatchery spray–vaccinated broilers. Naqi et al. 23 also reported the shedding of IBV Mass type vaccine virus up to 63 and 77 days after the initial exposure via the ocular route. In addition, Jackwood et al. 17 reported IBV vaccine was not fully cleared in commercial broilers with field boost vaccination and that only Ark vaccine was consistently identified in the vaccinated birds. These studies reinforce the importance of proper vaccination; however, even under the best condition, hatchery spray cabinet delivery of the Ark vaccine may still result in persistence of the vaccine viruses in the birds.
As expected, maternal antibodies were detected in the commercial broiler chicks at 1 day of age. Although reports indicate that maternal antibodies to IBV do not interfere with IBV vaccination at 1 day of age 8,9, it is possible that high neutralizing maternal antibodies specific for the Ark virus could affect efficacy of that vaccine. Nonetheless, we conducted VN tests and confirmed that neutralizing maternal antibodies were indeed present for the Ark, Mass, and GA98 viruses. Because neutralizing maternal antibodies were detected for all three IBV vaccine types and at approximately the same titer for Ark and Mass, it seems that something else must be contributing to the poor efficacy of the Ark vaccine when given by hatchery spray cabinet. We found little or no circulating antibodies against IBV by ELISA at 5 days postchallenge. It has been reported that low levels of humoral antibodies did not always indicate a lack of protection against IBV and that mucosal immunoglobulin (Ig) A in the upper respiratory tract plays an important role in preventing infection 13,21,26. We did not examine mucosal IgA.
In Expt. 2, we examined either a 1× or 2× dose given to groups of either 20 or 40 birds and found that only the group of 20 birds vaccinated with 40 doses of Ark vaccine showed better protection and detectable levels of vaccine virus at 28 days of age compared with the other groups. It is not clear why only this group had evidence of vaccine virus replication, but it is possible that delivering a 2× dose in 7 ml (0.35 ml/bird) is a critical combination of dose and volume compared with groups of 40 birds sprayed with a 2× dose in 7 ml (0.175 ml/bird) or birds that received a 1× dose. In addition, delivering a 2× dose by spray likely does not equal the same dose delivered by eyedrop.
Selection of subpopulations has been described for the IBV Ark-type vaccines after only one passage in chickens (22). In Expt. 3, we isolated an Ark vaccine virus at the peak titer in broilers (21 days postvaccination), passaged it one time in embryonated eggs to increase the titer, and designated it Ark-RI-EP1. Assuming that Ark-RI-EP1 would be more fit to replicate in birds than the original Ark vaccine, we gave the virus to birds by eyedrop and found that the replication pattern was nearly identical to the original Ark vaccine, with a peak titer of the viruses in the trachea at 3 days of age and a decline to undetectable levels by 21 days postvaccination. These data were in contrast to Ark vaccine administrated using a hatchery spray cabinet where the virus was not detected until 21 days postvaccination, presumably because of the route of inoculation.
To further examine the influence that the route of inoculation has on the dynamics of replication in the birds, we examined three different routes of vaccination by using both the Ark vaccine and Ark-RI-EP1 virus in Expt. 4. Similarly to the previous experiment, Ark vaccine and Ark-RI-EP1 were detected within the first week after vaccination when the viruses were administrated by eyedrop. However, spray vaccination showed a delay (>3 wk) in detecting both the Ark vaccine and Ark-RI-EP1 virus in the birds. Thus, it seems that the Ark-RI-EP1 virus is not more fit than the original Ark vaccine for infection and replication in broilers when administered by hatchery spray cabinet. When the viruses were given by drinking water, the Ark vaccine virus was undetectable out to 35 days of age, whereas the Ark-RI-EP1 virus had a peak titer at 7 days of age. This observation suggests that there are differences between the Ark vaccine and the Ark-RI-EP1 with regard to infection, replication, or both in the birds. This difference was further reinforced by the greater level of protection observed in birds given the Ark-RI-EP1 vaccine in drinking water.
To further examine differences between the Ark vaccine and the Ark-RI-EP1 virus, we examined the S1 gene and the nsp 3 gene sequences. The S1 gene analysis showed nine nonsynonymous differences between the Ark vaccine and the Ark-RI-EP1 consensus sequences. In addition, there was a three-nucleotide deletion resulting in the loss of Asn at residue 345 in Ark-RI-EP1. Compared with all previously published subpopulations of Ark vaccines 12,22,27, Ark-RI-EP1 is very similar to C3 (NCBI accession EU359626) 12, but it has two additional mutations at nucleotide positions 188 (C->T) and 233 (C->T). Ark-RI-EP1 shares three amino acid changes (Tyr43His, Ser213Ala, and Tyr326Asn) and the loss of Asn at amino acid position 345 with previously published vaccine viruses EU283047, EU283051, EU283053, and EU283056 22. In this previous study, reisolated Ark vaccines from different manufacturers were compared, and it was indicated that minor double peaks existed at these three positions in original vaccines and a deletion of Asn345 was observed in all reisolated Ark vaccine viruses. As noted by McKinley et al. 22, it is possible that these changes are the result of selection of a more fit subpopulation and a deletion of three nucleotides coding for Asn345 could be important for in vivo replication of Ark-type vaccine. In the previous studies, the reisolated vaccine viruses were collected up until 14 days postvaccination. The differences between those viruses and our reisolated vaccine virus indicate that a different major subpopulation of the Ark vaccine was selected in the birds by 21 days postvaccination, suggesting that subpopulations may be changing during the course of an infection.
Nonstructural replicase genes have been documented to be associated with the pathogenicity of infectious bronchitis virus 2,3, and many changes, especially in the nsp 3 gene, have been reported 25. The nsp 3 gene sequence of Ark-RI-EP1 showed one nonsynonymous mutation resulting in a glycine-to-aspartic acid change compared with the Ark vaccine virus. Only one amino acid sequence change between Ark-RI-EP1 and the Ark vaccine in the nsp 3 gene suggests that the attenuation of Ark-RI-EP1 was maintained, as we observed in the vaccinated birds.
In conclusion, it seems that hatchery spray cabinet vaccination of broilers with Ark-type vaccine is not sufficient to immunize birds against homologous Ark-DPI challenge virus, although eyedrop vaccination with that same vaccine and the same dose did induce protective immunity. This finding indicates that the vaccine virus is immunogenic. Hatchery spray cabinet vaccinators typically deliver 100 doses of vaccine to 100 birds in a total of 7 ml. We found that a 2× dose of the Ark vaccine in a total of 7 ml given to 20 birds was sufficient to induce some protection, whereas a 2× dose in 7 ml given to 40 birds was not, suggesting that the combination of dose and vaccine volume is important. Finally, an isolate of Ark vaccine, Ark-RI-EP1, from a broiler at 21 days of age may have been more fit to infect and replicate in the birds, although we saw no differences in infection, replication, or protection compared with the original Ark vaccine when delivered by hatchery spray cabinet. Clearly, the efficacy of Ark-type IBV vaccines when delivered by hatchery spray cabinet is not acceptable, and the key to protection against Ark and perhaps reducing vaccine persistence in the field lies in developing a sound method of immunization in the hatchery.
We thank Benjamin Jackwood, Joshua Jackwood, Carey Stewart, and Drs. Brian Jordan, Sharmi Thor, and Jamie Phillips for help with the bird studies.
Arkansas-Delmarva Poultry Industry
50% egg infective dose
infectious bronchitis virus
minor groove binding
National Center for Biotechnology Information
Poultry Diagnostic and Research Center