Evaluating Viral Interference Between Infectious Bronchitis Virus and Newcastle ... ·...

Evaluating Viral Interference Between Infectious Bronchitis Virus and NewcastleDisease Virus Vaccine Strains Using Quantitative Reverse Transcription–Polymerase

Chain Reaction

J. Gelb Jr.,A B. S. Ladman, M. J. Licata, M. H. Shapiro, and L. R. Campion

Department of Animal and Food Sciences, Avian Biosciences Center, College of Agriculture and Natural Resources, University of Delaware,Newark, DE 19716-2150

Received 5 February 2007; Accepted and published ahead of print 5 July 2007

SUMMARY. The potential for infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) replication interference wasevaluated using quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Fourteen-day-old broiler chickens wereinoculated via eyedrop with live commercial vaccine strains of IBV and NDV alone or in combination to directly evaluate IBV andNDV replication in the trachea at 1, 3, and 5 days after vaccination. Commercial NDV vaccine strains used were B1, VG/GA, andC2. The vaccine strains of IBV tested were Massachusetts (Mass) and Arkansas (Ark). The NDV + Mass vaccines used werecommercially manufactured combined products. The NDV + Ark vaccines used were commercial vaccines manufactured as singleentity products that were administered by eyedrop to opposite eyes of each chicken.

As measured by qRT-PCR, the replication of NDV strains B1, VG/GA, and C2 did not interfere with the growth of IBV Massand Ark strain vaccines in the combined vaccine treatment groups. Combination vaccinations using B1 and VG/GA did notinterfere with IBV immunity based on challenge or serum antibody production. In the C2 + Mass vaccination trial, IBV immunityafter challenge was reduced, but it did not seem to be a result of reduced Mass vaccine growth or the ability of the Mass vaccine toinduce serum IBV antibody. In contrast, the replication of IBV strains Mass and Ark interfered with the growth of NDV strains B1,VG/GA, and C2 as measured by qRT-PCR. However, interference with NDV replication was not reflected in a reduction inNewcastle disease challenge of immunity findings when combination Mass + NDV products manufactured by vaccine companieswere tested. Moreover, NDV immunity was not compromised in two of three trials using single entity vaccines of NDV and ArkIBV vaccines manufactured separately but administered simultaneously. However, in one trial, NDV immunity was decreasedwhere a NDV single entity product (C2) was given with an IBV single entity Ark vaccine. This finding emphasizes the importanceof using manufactured combination vaccines whenever possible to avoid potential interference.

RESUMEN. Evaluacion de la interferencia entre cepas vacunales del virus de bronquitis infecciosa y del virus de la enfermedadde Newcastle mediante la prueba cuantitativa de reaccion en cadena por la polimerasa-transcriptasa reversa.

Utilizando la prueba cuantitativa de reaccion en cadena por la polimerasa-transcriptasa reversa, se evaluo la posible interferencia enla replicacion entre los virus de bronquitis infecciosa y de la enfermedad de Newcastle. Se inocularon pollos de engorde de 14 dıas deedad por la vıa ocular con cepas vacunales comerciales del virus de bronquitis infecciosa y del virus de la enfermedad de Newcastle solaso en combinacion, con la finalidad de evaluar directamente la replicacion de estos virus en la traquea a los dıas 1, 3 y 5 posteriores a lavacunacion. Las vacunas comerciales contra el virus de la enfermedad de Newcastle utilizadas fueron las cepas B1, VG/GA y C2. Lascepas vacunales evaluadas del virus de bronquitis infecciosa fueron Massachusetts y Arkansas. Las vacunas utilizadas de los virus de laenfermedad de Newcastle + Massachusetts fueron productos combinados fabricados comercialmente. Las vacunas utilizadas de losvirus de la enfermedad de Newcastle + Arkansas fueron productos comerciales fabricados separadamente, administrados por vıa ocularen ojos opuestos para cada ave. Segun se determino mediante la prueba cuantitativa de reaccion en cadena por la polimerasa-transcriptasa reversa, en los grupos que recibieron vacunas combinadas, la replicacion de las cepas del virus de la enfermedad deNewcastle B1, VG/GA y C2 no interfirio con el crecimiento de las cepas vacunales del virus de bronquitis infecciosa Massachusetts yArkansas. Basado en los resultados de desafıo o en la produccion de anticuerpos sericos, las vacunaciones combinadas utilizando B1 yVG/GA no interfirieron con la inmunidad contra el virus de bronquitis infecciosa. En el experimento utilizando las cepas C2 +Massachussets, se redujo la inmunidad posterior al desafıo, sin embargo, esta reduccion no parece ser el resultado de una disminucionen el crecimiento de la vacuna Massachusetts o de la capacidad de la vacuna para inducir anticuerpos contra el virus de bronquitisinfecciosa. En contraste, segun se determino mediante la prueba cuantitativa de reaccion en cadena por la polimerasa-transcriptasareversa, la replicacion de las cepas del virus de bronquitis infecciosa Massachusetts y Arkansas interfirio con la replicacion de las cepasdel virus de la enfermedad de Newcastle B1, VG/GA y C2. Sin embargo, cuando se evaluaron vacunas comerciales ya combinadas conlos virus Massachusetts y el virus de la enfermedad de Newcastle, la interferencia con la replicacion del virus de la enfermedad deNewcastle no se reflejo en una reduccion de la inmunidad contra el desafıo. Adicionalmente, en dos de los tres experimentos utilizandovacunas del virus de la enfermedad de Newcastle y la cepa Arkansas del virus de bronquitis infecciosa, fabricadas individualmente peroadministradas de manera simultanea, la inmunidad contra el virus de la enfermedad de Newcastle no se vio afectada. Sin embargo, enun experimento, la inmunidad contra el virus de la enfermedad de Newcastle disminuyo cuando una vacuna sola preparada con la cepaC2 se administro con una vacuna sola preparada con la cepa Arkansas del virus de bronquitis infecciosa. Este hallazgo enfatiza laimportancia de utilizar vacunas combinadas comerciales cuando sea posible para evitar potenciales interferencias.

Key words: infectious bronchitis virus, Newcastle disease virus, vaccine, viral interference, quantitative reverse transcription–polymerase chain reaction, chicken, B1, VG/GA, C2, Massachusetts, Arkansas

Abbreviations: Ark 5 Arkansas; Conn 5 Connecticut; Ct 5 cycle threshold; dpv 5 days postvaccination; ELISA 5 enzyme-linkedimmunosorbent assay; GMT 5 geometric mean titer; HI 5 hemagglutination-inhibition; PCR 5 polymerase chain reaction;IBV 5 infectious bronchitis virus; Mass 5 Massachusetts; MGB 5 minor groove binding; NDV 5 Newcastle disease virus;qRT-PCR 5 quantitative reverse transcription–polymerase chain reaction; SPF 5 specific-pathogen-free; TPB 5 tryptose phosphate broth

ACorresponding author: E-mail: [email protected]

AVIAN DISEASES 51:924–934, 2007

924

The potential for interference between coronavirus infectiousbronchitis virus (IBV) and paramyxovirus Newcastle disease virus(NDV) exists given that both of these RNA viruses initially infectepithelial cells of the respiratory tract of the chicken and replicate inthe cell’s cytoplasm. Infections of IBV and NDV are controlledusing live strains of each virus. Since the 1950s, the efficacy ofcombination vaccines vs. viruses given separately has been studied.The replication of IBV has been shown to interfere with NDVinfections. Research has shown IBV interfered with the replication ofvirulent (9,10) as well as live vaccine strains (3,7,12,15). Interferencehas also been observed in cell culture (1,4) and embryonatingchicken eggs (1,11,13,18). The interference phenomenon was usedas the basis for developing a laboratory diagnostic test for isolatingIBV field strains, because lesions in chick embryos are difficult torecognize on initial passage (1,11,18). More recently, Cook et al. (5)reported that vaccination with live IBV interfered with thereplication of avian pneumovirus, a different member of the avianparamyxovirus family. In contrast, little effect of NDV on IBVreplication and immunity has been noted (17).

Our research revisited the replication interference potential of IBVand NDV using quantitative reverse transcription–polymerase chainreaction (qRT-PCR) for IBV and NDV to specifically detect theviral genomes in mixed infections. The goals of the research were to1) evaluate qRT-PCR as a tool to assess interference in the chickenand 2) evaluate the potential for interference among NDV and IBVvaccine strains used by the poultry industry today. The VG/GA andC2 strains of NDV and the Arkansas (Ark) strain of IBV have beenintroduced since earlier interference studies were performed.

MATERIALS AND METHODS

Specific-pathogen-free (SPF) chicken eggs. Fertile eggs from WhiteLeghorn chickens were purchased from Charles River Laboratories(Wilmington, MA). The eggs were incubated for 9–11 days, and then theywere used for virus isolation attempts after challenge with IBV and NDV.

Broiler chickens. Commercial type broiler chickens were used for theIBV and NDV vaccine interference trials. Broilers were obtained froma commercial hatchery at 1 day of age before vaccinations for IBV andNDV. Chickens were placed in glove-port isolation cabinets (MontairAndersen, Sevenum, the Netherlands) and provided feed and water adlibitum.

Vaccines and challenge viruses. Commercial IBV and NDVcombination and single entity live vaccines were obtained frommanufacturers, and they were diluted as recommended (Table 1). TheNDV vaccine strains evaluated were B1, VG/GA, and C2. IBV vaccinestrains evaluated were Massachusetts (Mass) or Mass + Connecticut(Conn) and Ark. The NDV + Mass vaccines and NDV Mass + Connvaccine used were commercially manufactured combined products. TheNDV + Ark vaccines used were commercial vaccines manufactured assingle entity products that were administered by eyedrop to oppositeeyes of each chicken. Single entity IBV and NDV vaccines wereevaluated because poultry producers use them when commercialcombination products are not available. A nonattenuated La Sotastrain, obtained originally from the NDV repository (University ofWisconsin, Madison, WI) was used for challenging broilers. The La Sotastrain challenge was used because it represents a method of assessingimmunity of broilers in the United States and other countries whererespiratory forms of the disease are common in commercial production.The Mass 41 strain, obtained originally from USDA, NationalVeterinary Services Laboratory (Ames, IA), and the Ark DPI strain(6,8) were used for challenge. The challenge viruses are maintained aslow embryo passage viruses in our laboratory.

qRT-PCR. qRT-PCR was performed on tracheal swabbings frombroilers vaccinated with IBV and NDV vaccines on 1, 3, and 5 dayspostvaccination (dpv). The swabbings were placed in 1 ml of steriletryptose phosphate broth (TPB) (Difco, Detroit, MI) and vortexed.Vaccine-derived IBV and NDV genomic RNA was extracted (RNeasyHMini kit; QIAGEN, Valencia, CA) using 750 ml of swab broth, andRNA was stored at 280 C. Vaccine viral RNA was assayed using qRT-PCR (QuantiTect Probe RT-PCR kit; QIAGEN). Reactions were runin 384-well microtiter plates using ABI Prism 7900 Sequence DetectionSystem (Applied Biosystems, Foster City, CA) and SDS, version 2.3,analysis software (Applied Biosystems). Three primer sets andcorresponding sequence-specific, fluorescent, minor groove binding(MGB) probes were used targeting the NDV phosphoprotein (P) gene,the nucleocapsid (N) gene of the IBV Mass strain, and the subunit of thespike glycoprotein gene (S1) of the IBV Ark strain. The primer andprobe sets were designed using ABI Primer Express 2.0 (AppliedBiosystems). The specificities of the primer and probe sets have beendemonstrated in our laboratory (data not shown). The oligonucleotidesequences for the primers and probes were as follows: NDV P TaqManHForward, 59-GCCAACTTGGGAATGATGAAG-39; NDV P Taq-ManH Reverse, 59-GCCCGTAGATCACTCAGAGATGA-39; NDV PMGB Probe, 59-TTCTGGATCCCGGTTGT-39; Mass N TaqManHForward, 59-GATGGAGGACCTGATGGTAATTT-39; Mass N Taq-ManH Reverse, 59-TCTCCCACTACTGCCACGATT-39; Mass NMGB Probe, 59-TGGGACTTCATTCCTC-39; Ark S1 TaqManH

Table 1. Combination and single entity vaccines used in NDV and IBV interference trials.

Vaccine Type Manufacturer/trade name

B1 NDV + Mass IBV CombinationA Schering-Plough/Broilerbron B1B1 NDV + Ark IBV Single entity vaccinesB Newcastle B1 + Broilerbron 99B1 NDV Single Newcastle B1Mass IBV Single BroilerbronArk IBV Single Broilerbron 99VG/GA NDV + Mass + Conn IBV Combination Merial Select/VG-Vac + Mass + ConnVG/GA NDV + Ark IBV Single entity vaccines VG-Vac + ArkVG/GA NDV Single VG-VacMass IBV Single MassArk IBV Single ArkC2 NDV + Mass IBV Combination Intervet/Newhatch C2-MC2 NDV + Ark IBV Single entity vaccines Newhatch C2 + Mildvac-ArkC2 NDV Single Newhatch C2Mass IBV Single Mildvac-MArk IBV Single Mildvac-Ark

ANDV + IBV vaccine manufactured as a combination product given by eyedrop.BNDV + IBV vaccines manufactured as single entity products and administered by eyedrop to opposite eyes of each chicken.

Evaluating NDV–IBV interference by qRT-PCR 925

Forward, 59-GGTACTGCCCCAAGTTGCA-39; Ark S1 TaqManHReverse, 59-CTACTGAGGCCGCACTGAGAT-39; and Ark S1 MGBProbe, 59-TGCTATTGGCTACAGTAAG-39.

Each reaction contained 10 ml of QuantiTect Probe RT-PCR MasterMix (QIAGEN), 1 ml of the appropriate forward primer (0.8 mM stock),1 ml of the appropriate reverse primer (0.8 mM stock), 2 ml of theappropriate MGB Probe (0.2 mM stock), 4.55 ml of RNAse-free water,0.20 ml of QuantiTect RT Enzyme Mix (QIAGEN), 0.25 ml of RNAseinhibitor, and 1 ml of template RNA for a total volume of 20 ml. Testsamples were compared with a standard curve for the appropriateprimer–probe pairings. Standard curves consisted of 10-fold serialdilutions of positive control RNA. Appropriate controls were used toestablish baseline fluorescence. All test and internal controls wereanalyzed in duplicate. Cycle parameters used for all qRT-PCR reactionswere as follows: 1 cycle of reverse transcription at 50 C for 30 min,95 C for 15 min, and 95 C for 15 sec followed by 60 C for 40 sec for40 cycles of PCR. The cycle threshold (Ct) value obtained for eachreaction represented the number of cycles required for the fluorescenceto exceed baseline fluorescence. The Ct value is inversely proportional tothe log10 titer of template RNA in the starting sample.

Infectivity titrations (16) of single entity IBV and NDV vaccines wereperformed in chicken embryos for the purpose of relating infectivitytiters to qRT-PCR findings. RNA extracted from serial 10-fold dilutions(10021024) was analyzed by qRT-PCR. Relative genome titers weredetermined using the formula

log10 titer ~Ct { Ctmax

standard curve

� �;

where Ctmax represents the maximum Ct value of 40 cycles. Data wereanalyzed by analysis of variance.

Vaccine interference trials in broiler chickens. Each NDV vaccinestrain, B1, VG/GA, and C2, was evaluated in a separate experiment. Ineach experiment, approximately 190 broiler chicks (1 day old) wereassigned to 10 treatment groups. At 14 days of age, maternal antibodytiters were determined for IBV using the enzyme-linked immunosorbentassay (ELISA) (Idexx Laboratories, Inc., Westbrook, ME), and for NDVusing the hemagglutination-inhibition (HI) test (14). Broilers were thenvaccinated via eyedrop with a NDV + Mass combination vaccine product,single entity NDV and Ark vaccine products applied simultaneously inopposite eyes, NDV vaccine alone, or IBV vaccine alone using the dosesrecommended by the manufacturer. NDV and IBV challenge controlswere not vaccinated. On 1, 3, and 5 dpv, tracheal swabbings werecollected from three broilers per treatment group and placed in TPB toassess IBV and NDV viral RNA by qRT-PCR. On dpv 36, the chickenswere bled, and sera were tested for NDV HI and IBV ELISA antibodies.Ten to 14 broilers per treatment group were then challenged via eyedropwith either 106 50% embryo lethal dose per bird of the La Sota strainNDV or with 104 50% embryo infectious dose per bird of the Mass 41 orArk DPI strains of IBV. Five days postchallenge, tracheal swabbings wereobtained for virus isolation attempts. Swabbings were placed in 3 ml ofTPB with antibiotics (10,000 IU penicillin G and 10,000 mg ofstreptomycin per ml), and swabbings were stored at 270 C. Swabbingswere thawed, vortexed, incubated at room temperature for 30–45 minbefore inoculating three SPF embryos (10–11 days old) via thechorioallantoic sac with 0.2 ml of the swab tube broth. Mortalities andviable embryos were evaluated 7 days postinoculation for hemagglutinat-ing activity of the allantoic fluids for NDV and typical IBV lesions;stunting, curling, and kidney urates. Chickens from which the challengeviruses were not recovered were considered protected.

RESULTS

NDV genome levels. Based on qRT-PCR assays, replicationinterference by IBV on NDV vaccine strains B1, VG/GA, and C2was most pronounced on dpv 3 (Fig. 1a–c). By dpv 5, IBV-inducedinterference of NDV was still evident in broilers given VG/GA andC2 strains (Fig. 1b,c), but not in chickens given B1 (Fig. 1a).

Although levels of B1 genome on 3 dpv were numerically reduced byboth the Mass and Ark IBV strains, B1 genome levels weresignificantly (P , 0.05) reduced in broilers given the Ark strain(Fig. 1a). On dpv 1, the VG/GA genome level was numerically higherin broilers vaccinated with VG/GA + Mass and Conn combinationcompared with chickens given VG/GA alone. On dpv 3, VG/GAgenome decreased in broilers given combination IBV vaccine, whereaslevels increased in VG/GA alone vaccinates (Fig. 1b). On dpv 1, C2genome levels in C2 alone vaccinates and C2 + IBV vaccinates werevery low. On dpv 3 and dpv 5, C2 alone genome levels increasedcompared with levels in C2 + IBV vaccinates. On dpv 5, the C2genome level was significantly reduced (P , 0.05) in broilers givenArk as a single entity vaccine with C2 (Fig. 1c).

IBV genome levels. Vaccination with NDV strains B1, VG/GA,and C2 did not interfere with Mass IBV genome levels in broilersgiven combination vaccines (Fig. 2a–c). Vaccination with B1 andC2 did not interfere with Ark genome levels in broilers given singleentity Ark and NDV vaccines simultaneously (Fig. 3a,c). Genomelevels of Ark were significantly (P , 0.05) reduced on dpv 1 inbroilers also vaccinated with VG/GA NDV, but they were similar ondpv 3 and dpv 5 to broilers given Ark vaccine alone (Fig. 3b).

NDV antibody response. Maternal NDV HI serum antibodieswere not detected in broilers at 14 days of age, the day ofvaccination. On dpv 36, NDV serum HI antibody titers of chickensgiven B1 and VG/GA along with IBV vaccines did not differ frombroilers given the same NDV vaccines alone (Fig. 4a,b). In contrast,serum HI antibody titers of broilers given Mass or Ark IBV with theC2 strain were significantly (P , 0.05) reduced compared withchickens given C2 vaccination alone (Fig. 4c).

IBV antibody response. Maternal IBV ELISA serum antibodieswere not detected in broilers at 14-days of age, the day ofvaccination. On dpv 36, IBV ELISA serum antibody titers ofchickens given IBV and NDV vaccines did not differ from broilersgiven the IBV vaccine strains alone (Fig. 5a–c).

NDV challenge of immunity. NDV vaccine strains B1, VG/GA,and C2 manufactured as combination NDV + IBV products resultedin excellent NDV protection, ranging from 82% to 92% (Tables 2–4). The B1 strain also produced excellent NDV protection (91%)when administered simultaneously with the single entity Ark strain(Table 2). Vaccination with VG/GA + single entity Ark resulted in77% protection vs. NDV challenge (Table 3). Vaccination with C2 +single entity Ark resulted in only 36% protection vs. NDV challenge(Table 4). All NDV strains tested produced excellent protection whengiven to broilers as single entity vaccines.

IBV challenge of immunity. Immunity after vaccination withMass or Mass + Conn was not reduced as a result of combining theIBV vaccine with B1 or VG/GA NDV. Sixty-seven percent of B1 +Mass vaccinated broilers and 55% of Mass alone vaccinated broilerswere protected (Table 2). Eighty-three percent of VG/GA + Mass +Conn vaccinated broilers and 92% Mass alone vaccinated broilerswere protected (Table 3). However, Mass protection (55%) in C2 +Mass vaccinated broilers was reduced compared with chickensreceiving Mass vaccine alone (91% protection) (Table 4). Arkvaccination as a single entity product simultaneously administeredwith the NDV vaccines resulted in excellent immunity.

DISCUSSION

Quantitative RT-PCR was successfully used to evaluate thereplication interference potential of IBV and NDV after vaccinationof chickens with live vaccines administered together either in

926 J. Gelb Jr. et al.

Fig. 1. Viral genome levels as detected by NDV-specific qRT-PCR in postvaccination tracheal swabs collected from 14-day-old chickens givenNDV strains (a) B1, (b) VG/GA, or (c) C2 in manufactured combination vaccines with Mass, or Mass and Conn strains; or NDV single entityvaccines administered simultaneously with the Ark single entity vaccine. Each data point represents the relative log10 geometric mean titer (GMT)for the sample group (n 5 3). Error bars represent SEM. Data points labeled with the same letter (a–c) are not significantly different (P , 0.05).


Fig. 2. Viral genome levels as detected by IBV Mass-specific qRT-PCR in postvaccination tracheal swabs collected from 14-day-old chickensgiven NDV strains (a) B1, (b) VG/GA, or (c) C2 in manufactured combination vaccines with the Mass, or Mass and Conn strains. Each data pointrepresents the relative log10 GMT for the sample group. Error bars represent SEM. Data points labeled with the same letter (a–c) are not significantlydifferent (P , 0.05).


Fig. 3. Viral genome levels as detected by IBV Ark-specific qRT-PCR in postvaccination tracheal swabs collected from 14-day old chickensgiven single entity NDV strain vaccines (a) B1, (b) VG/GA, or (c) C2, simultaneously administered with the single entity Ark vaccine. Each datapoint represents the relative log10 GMT for the sample group. Error bars represent SEM. Data points labeled with the same letter (a–c) are notsignificantly different (P , 0.05).


Fig. 4. NDV HI geometric mean serum antibody titers of 36-day-old broiler chickens vaccinated at 14 days of age with NDV strains (a) B1, (b)VG/GA, or (c) C2 in manufactured combination vaccines with Mass, or Mass and Conn strains; or NDV single entity vaccines simultaneouslyadministered with the Ark strain. Error bars represent SEM.


Fig. 5. IBV ELISA geometric mean serum antibody titers of 36-day-old broiler chickens vaccinated at 14 days of age with NDV strains (a) B1,(b) VG/GA, or (c) C2 in manufactured combination vaccines with Mass, or Mass and Conn strains; or NDV single entity vaccines simultaneouslyadministered with the Ark strain. Error bars represent SEM. Antibody titers greater than 396 (log10 5 2.59) are considered positive.


manufactured combinations or single entity preparations. Before thedevelopment of PCR technology, the majority of reports relied onassessing interference indirectly on the basis of resulting IBV andNDV immune responses (3,7,10,15,17). Some studies (1,11,18)used NDV HA activity in chick embryos to directly assess IBVinterference on NDV. However, directly evaluating the potentialeffect of NDV on IBV replication has not been reported.

Our findings using qRT-PCR support previous work(1,3,4,9,10,11,12,13,15,18) showing IBV interferes with NDVreplication. Both the Mass and Ark strains of IBV interfered with thereplication of the three NDV live vaccine strains tested; B1, VG/GA,and C2. Interference with the replication of the B1 strain was limitedto dpv 3, whereas the growth of VG/GA and C2 was also reduced ondpv 5. Interestingly, genome levels of the VG/GA strain, whencombined with the Mass + Conn, were higher on dpv 1. It is notknown whether this observation is a result of the VG/GA havinga replication advantage in the presence of IBV or whether themanufacturer recognized the potential for NDV interference andincluded a higher initial concentration of VG/GA in the product.Vaccine manufacturers have used higher release titers of vaccinestrains in combination products where interference is likely to occur(17).

Although IBV vaccination interfered with NDV replication asmeasured by qRT-PCR, reduced NDV immunity did not result inmost instances. The B1, VG/GA, and C2 strains in products

manufactured as NDV and IBV combinations induced high levels ofNDV protective immunity. However, interference with NDVimmunity was observed in broilers vaccinated with single entityproducts of C2 and Ark IBV. Similarly, Thornton and Muskett (15)reported that experimentally mixed IBV and NDV productsconferred lower immunity than manufactured combined vaccines.It is important to recognize that using single entity vaccines togethermay not be advisable, because such practice could result in reducedefficacy. In contrast, combination vaccines are formulated bymanufacturers so as to take each component’s viral characteristics(immunogenicity and vaccine strain titer) into consideration tominimize or prevent interference. The effects of interference onvaccine performance have been reported to be dose dependent, andthey may be avoided by maintaining a dose of the NDV vaccinefraction that is 100–1000 times higher compared with the IBVfraction (17).

The NDV vaccine strains, as evaluated using qRT-PCR, did notinterfere with the replication of IBV vaccines strains Mass and Ark.Broilers vaccinated with the VG/GA NDV vaccine had significantly(P , 0.05) lower Ark genome levels on dpv 1, but they recovered bydpv 3 and dpv 5 to levels comparable with Ark alone vaccinates. Thevaccines used in this trial were single entity products containing VG/GA and Ark and the reduction of Ark genome on dpv 1 may reflectof a lower concentration of the strain in the vaccine itself. It is clearthat replication of the Ark strain was not impaired beyond dpv 3;

Table 2. Challenge of immunity results of 36-day-old broilers vaccinated by eyedrop with NDV B1 and IBV vaccine strains Mass or Ark at14 days of age.

Treatment group Challenge virusA % protectionB

1. B1 + Mass combination vaccineC La Sota 90 (9/10)2. B1 + Ark single entity vaccinesD La Sota 91 (10/11)3. B1 + Mass combination vaccine Mass 41 67 (8/12)4. B1 + Ark single entity vaccines Ark DPI 83 (10/12)5. Mass vaccine alone Mass 41 55 (6/11)6. Ark vaccine alone Ark DPI 91 (10/11)7. B1 vaccine alone La Sota 100 (12/12)8. NDV challenge control La Sota 0 (0/12)9. Mass challenge control Mass 41 0 (0/11)

10. Ark challenge control Ark DPI 0 (0/10)AChallenge viruses: La Sota, NDV; Mass 41 IBV and Ark DPI, IBV.B% protection 5 percentage of chickens from which the challenge virus was not reisolated/total (no. protected/total).CB1 + Mass vaccine manufactured as a combination product.DB1 + Ark vaccines manufactured as single entity products were administered by eyedrop to opposite eyes of each chicken

Table 3. Challenge of immunity results of 36-day-old broilers vaccinated by eyedrop with NDV VG/GA and IBV vaccine strains Mass + Connor Ark at 14 days of age.


1. VG/GA + Mass + Conn combination vaccineC La Sota 92 (12/13)2. VG/GA + Ark single entity vaccinesD La Sota 77 (10/13)3. VG/GA + Mass + Conn combination vaccine Mass 41 83 (10/12)4. VG/GA + Ark single entity vaccines Ark DPI 100 (14/14)5. Mass + Conn vaccine alone Mass 41 92 (11/12)6. Ark vaccine alone Ark DPI 100 (11/11)7. VG/GA vaccine alone La Sota 100 (12/12)8. NDV challenge control La Sota 0 (0/13)9. Mass challenge control Mass 41 0 (0/12)

10. Ark challenge control Ark DPI 0 (0/13)AChallenge viruses: La Sota, NDV; Mass 41 IBV and Ark DPI, IBV.B% protection 5 percentage of chickens from which the challenge virus was not reisolated/total (no. protected/total).CVG/GA + Mass + Conn vaccine manufactured as a combination product.DVG/GA + Ark vaccines manufactured as single entity products were administered by eyedrop to opposite eyes of each chicken.


thus, if interference occurred, it was transient. Moreover, VG/GA +Ark vaccinated broilers were 100% protected upon Ark DPIchallenge (Table 3).

As expected given the qRT-PCR results, NDV vaccination didnot interfere with or enhance IBV immunity when administered incombination with Mass or as a single entity vaccine with Ark. SerumIBV ELISA antibody titers were not reduced in broilers given NDVvaccines. Challenge of IBV immunity results indicated broilersreceiving combination NDV and IBV vaccines had levels ofprotection against Mass 41 and Ark DPI that were comparablewith those given single entity IBV vaccines. However, in the C2 +Mass vaccine trial, higher IBV immunity was noted in broilers giventhe Mass single entity vaccine (91% protection) than those receivingthe C2 + Mass combination vaccine (55% protection) (Table 4).The reason for the decrease in IBV protection observed in broilersvaccinated with C2 + Mass is not clear, but it does not seem to bea result of reduced Mass vaccine replication (Fig. 2c) or the ability ofthe Mass vaccine to induce IBV ELISA serum antibody (Fig. 5c).This trial could be repeated to clarify this finding. A report indicatedNDV vaccine strains given in combination with IBV have had littleor no impact on IBV immunity (17).

Strains VG/GA and C2 were developed as alternatives to the B1strain to avoid respiratory disease-associated side effects that mayresult from vaccination of young broilers. The qRT-PCR findingsdemonstrate that the replication of these milder NDV vaccines issusceptible to IBV interference. For the C2 vaccine, reduced NDVHI serum antibody production in broilers given the C2 + Masscombination product is likely due to interference. However, in themost important measure of immunity, challenge protection, bothC2 and VG/GA induced excellent NDV protective immunity whenused with IBV in combination-manufactured products.

The limited focus of our study is not meant to be the basis ofrecommending any of the NDV + IBV vaccines tested for use bypoultry producers. Our study examined IBV and NDV simulta-neous infections under highly defined conditions that are notrepresentative of poultry industry standard practices. Vaccinationwas delayed for 14 days to reduce the influence of maternal antibodylevels. Maternal antibodies, especially for NDV, have been shown tointerfere with developing immunity after vaccination by a differentmechanism; neutralization of the vaccine virus (2). Furthermore,broilers in our study were vaccinated only once, whereas hatcheryvaccination followed by field revaccination is commonplace in mostproduction areas of the United States and in other countries. Whendeveloping an NDV and IBV vaccination program for chickens,

strain(s) selection, the number and timing of vaccination(s), and themethod(s) of application all must be considered.

Cook et al. (5) used nonquantitative RT-PCR to show that IBVvaccination interfered with avian pneumovirus vaccine replication.In our study, qRT-PCR was probably more sensitive for examiningvirus–virus interactions. In the future, qRT-PCR could be used toevaluate the potential for interference for NDV and IBV andperhaps other vaccine combinations used in human and veterinarymedicine. For vaccine formulation, it may be possible to assessinterference and perhaps establish appropriate doses of the viralcomponents to develop more effective vaccines.

REFERENCES

1. Beard, C. W. Infectious bronchitis virus interference with Newcastledisease virus in monolayers of chicken kidney cells. Avian Dis. 11:399–406.1967.

2. Box, P. G. The influence of maternal antibody on vaccination againstNewcastle disease. Vet. Rec. 77:246. 1965.

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Table 4. Challenge of immunity results of 36-day-old broilers vaccinated by eyedrop with NDV C2 and IBV vaccine strains Mass or Ark at14 days of age.


1. C2 + Mass combination vaccineC La Sota 82 (9/11)2. C2 + Ark single entity vaccinesD La Sota 36 (4/11)3. C2 + Mass combination vaccine Mass 41 55 (6/11)4. C2 + Ark single entity vaccines Ark DPI 100 (11/11)5. Mass vaccine alone Mass 41 91 (10/11)6. Ark vaccine alone Ark DPI 100 (11/11)7. C2 vaccine alone La Sota 91 (10/11)8. NDV challenge control La Sota 0 (0/11)9. Mass challenge control Mass 41 0 (0/11)

10. Ark challenge control Ark DPI 0 (0/11)AChallenge viruses: La Sota, NDV; Mass 41 IBV and Ark DPI, IBV.B% protection 5 percentage of chickens from which the challenge virus was not reisolated/total (no. protected/total).CC2 + Mass vaccine manufactured as a combination product.DC2 + Ark vaccines manufactured as single entity products and administered by eyedrop to opposite eyes of each chicken.


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ACKNOWLEDGMENTS

This research was funded in part by grant 580 from the U.S. Poultryand Egg Association to J.G. and B.S.L. Support was also provided by theDelaware Agricultural Experiment Station, Newark through RegionalResearch funds as a contribution to project NC-1019.


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