Evaluating Protection Against Infectious Bronchitis Virus by Clinical Signs, Ciliostasis, Challenge Virus Detection, and Histopathology.

In this study, we examined the association among clinical signs, ciliostasis, virus detection, and histopathology for evaluating protection of vaccinated chickens against homologous and heterologous infectious bronchitis virus (IBV) challenge. At 5 days following challenge with IBV, we found a good correlation among clinical signs, ciliostasis in the trachea, challenge virus detection, and microscopic lesions in the trachea, with all four criteria being negative in fully protected birds and positive in fully susceptible birds. In partially protected birds we observed clinical signs and detected challenge virus; however, the ciliated epithelium was intact. In a second experiment, we challenged fully protected, partially protected, and fully susceptible birds with IBV, and then at 5 days postchallenge we gave the birds an opportunistic bacterium intranasally. Twenty Bordetella avium colonies were recovered from one of five fully protected birds, and only five colonies were isolated from two of five partially protected birds without ciliostasis, whereas in birds with ciliostasis, numerous colonies were isolated. Obviously, decreasing IBV infection and replication in the upper respiratory tract will decrease transmission and mutations, leading to variant viruses, and herein we demonstrate that protection of the cilia will decrease secondary bacterial infections, which have been shown to lead to condemnations and increased mortality. Thus, it appears that examining both criteria would be important when evaluating IBV vaccine efficacy.

Hatchery Spray Cabinet Administration Does Not Damage Avian Coronavirus Infectious Bronchitis Virus Vaccine Based on Analysis by Electron Microscopy and Virus Titration.

studies in our laboratory showed that the Arkansas-Delmarva Poultry Industry (Ark-DPI) vaccine given to 1-day-old chickens by hatchery spray cabinet replicated poorly and failed to adequately protect broilers against homologous virus challenge, whereas the same vaccine given by eye-drop did replicate and the birds were protected following homologous virus challenge. To determine if mechanical damage following spray application plays a role in failure of the Ark-DPI vaccine, we examined the morphology of three Ark-DPI vaccines from different manufacturers using an electron microscope and included a Massachusetts (Mass) vaccine as control. One of the Ark-DPI vaccines (vaccine A) and the Mass vaccine had significantly (P < 0.005) fewer spikes than the other two Ark-DPI vaccines. We also found that the Ark-DPI and Mass vaccines had significantly (P < 0.005) fewer spike proteins per virus particle when compared to their respective challenge viruses. This observation is interesting and may provide some insight into the mechanism behind infectious bronchitis virus attenuation. No obvious differences were observed in virus morphology and no consistent trend in the number of spikes per virion was found in before- and after-spray samples. We also determined the vaccine titer before and after spray in embryonated eggs and found that both Ark-DPI and Mass vaccines had a similar drop in titer, 0.40 logi and 0.310 logi, respec10ively. Based on these data, it appears that mechanical damage to the Ark-DPI vaccine is not occurring when delivered by a hatchery spray cabinet, suggesting that some other factor is contributing to the failure of that vaccine when given by that method.

Detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-PCR and correlation with virus detection in embryonated eggs.

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We found that a number of variables, including the virus type examined, primers and probe efficiency and stability, and assay conditions, including thermocycler platform, can affect the data obtained from qRT-PCR assays. These results indicate that qRT-PCR assays can be used to detect IBV challenge virus, but each assay, including the assay conditions and thermocycler, should be individually evaluated if those data are expected to be comparable to virus detection in embryonated eggs.

Evaluation of infectious bronchitis virus Arkansas-type vaccine failure in commercial broilers.

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.

Detection of avian influenza viruses and differentiation of H5, H7, N1, and N2 subtypes using a multiplex microsphere assay.

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase-PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 x 10(5) to 1 x 10(9) copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 x 10(2.5) to 1 x 10(3.2) 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.

Rapid heat-treatment attenuation of infectious bronchitis virus.

In the present study we describe the rapid development of an attenuated live vaccine for GA08, a new serotype of infectious bronchitis virus, using a heat-treatment method. Incubation of the GA08 strain of IBV at 56 degrees C and passage in embryonated eggs was used as a method to fast track the attenuation process. The virus was incubated in a 56 degrees C water bath and aliquots were removed every 5 min for up to 1 h, and then each aliquot was inoculated into 10-day-old embryonated eggs. Virus with the longest incubation time that produced lesions in the embryos was harvested, again incubated at 56 degrees C as described and passaged in embryonated eggs. Attenuation of the virus, designated GA08/GA08HSp16/08, was verified in 1-day-old specific pathogen free chicks. A 10x dose of the vaccine was found to be safe for 2-week-old broiler chicks of commercial origin. The efficacy of the heat-treated attenuated virus was determined by vaccinating broiler chicks of commercial origin at 1 and 14 days of age intraocularly/intranasally. Vaccinated birds that were challenged with 10(4.5) median embryo infectious doses of pathogenic GA08 virus/bird at 28 days of age were protected from the disease, and challenge virus was only detected in the trachea of one of 21 birds by real-time reverse transcriptase-polymerase chain reaction at 5 days post challenge. The attenuation process took 10 weeks to complete, which is a substantially shorter time than required to attenuate infectious bronchitis virus by serial passage in embryonated eggs without heat treatment (38 weeks or more).

Infectious bronchitis virus field vaccination coverage and persistence of Arkansas-type viruses in commercial broilers.

To determine the coverage of infectious bronchitis virus (IBV) vaccine field boost in commercial broilers, estimate the relative amount of vaccine virus in the trachea, and follow the clearance of the vaccine, we collected approximately 100 tracheal swabs at various times postvaccination from 10 different flocks and used real-time reverse transcriptase-PCR (RT-PCR) to detect the virus. This allowed us to detect vaccine virus in as few as 3% of the birds in a flock of 20,000 birds with a 95% confidence level. We found that the number of birds positive for IBV vaccine following vaccination in the field resembled a parabolic-shaped curve that peaked around 14 days postvaccination, or it resembled a sinusoidal-type wave with a frequency of about 2 wk. The patterns did not appear to correlate with water or spray vaccination methods, nor did they correlate with the type of backpack sprayer used. The highest number of positive birds in a flock ranged from 66% to 100%. The viral genome copies in the tracheal swabs, as determined by real-time RT-PCR, ranged from 1 x 10(2.6)/ml to 1 x 10(5.2)/ml and, in most studies, had a positive correlation with the number of birds positive for vaccine virus in the flock. On the last sample day of each study, 21, 28, or 35 days postvaccination, from 12% to 66% of the birds were still positive for vaccine virus, and although different IBV vaccine types were used in each study, only Arkansas vaccine virus was identified in selected samples on those days. Arkansas vaccine virus was also the only virus identified in selected samples at 1, 3, and 5 days postvaccination, clearly indicating that Arkansas vaccine virus is persisting in the birds. Protection studies conducted on birds vaccinated with Arkansas- and Delaware-type vaccines and removed from the field at 21 days postvaccination showed complete protection against challenge with Delaware (except for one bird), whereas protection against Arkansas challenge was between 37.5% and 62.5%. Our findings show that introduction of IBV vaccines into a commercial broiler flock do not necessarily follow a seemingly logical pattern of a high number of birds infected followed by clearance from the trachea, but resembled either a parabolic curve or a sinusoidal-type wave. In addition, Arkansas vaccine viruses are clearly persisting in commercial broilers, which may be because of incomplete protection observed for that IBV type.

Biologic characterization of H4, H6, and H9 type low pathogenicity avian influenza viruses from wild birds in chickens and turkeys.

The pathogenesis, virus shedding, and serologic response in specific-pathogen-free (SPF) chickens and commercial turkeys against H4, H6, and H9 type low pathogenic avian influenza viruses (LPAI) from wild birds was examined. Four-week-old chickens and three-week-old turkeys were given 1 x 10(6) EID50 of LPAI per bird, intrachoanally, and examined for clinical signs for 3 wk. Oropharyngeal and cloacal swabs, and fecal samples, were collected at 2, 4, and 7 days postinoculation (PI) for virus detection by real-time RT-PCR. Serum was collected at 7, 14, and 21 days PI and examined for antibodies against avian influenza virus (AIV) by the enzyme-linked immunosorbant assay (ELISA) and hemagglutination inhibition tests. Tissue samples for histopathology were collected from three birds per group at 3 days PI. The hemagglutinin genes of the viruses were sequenced, and phylogenetic analysis was conducted. Clinical signs ranged from no clinical signs to moderate depression, decreased activity, and decreased food and water consumption. Based on virus detection results, SPF chickens were generally found to be shedding more virus from both the oropharynx and cloaca than were commercial turkeys. Microscopic lesion results in both species showed the predominance of lesions in the respiratory and gastrointestinal tract, which is consistent with the fact that these viruses are of low pathogenicity. In chickens and turkeys, oropharyngeal shedding strongly correlated with the lesions found in the upper respiratory tract. Turkeys had fewer lesions in the respiratory tract and more lesions in the gastrointestinal tract compared to chickens. Thirteen LPAI viruses caused seroconversion in commercial turkeys, whereas only 6 LPAI viruses caused seroconversion in SPF chickens. Phylogenetic analysis of the HA genes showed that the H4, H6, and H9 viruses evaluated here represented the full genetic diversity of North American AIVs of their respective subtypes. This data is important for surveillance and control because some of the LPAI viruses (of wild bird origin and examined in this study) that can infect and be shed by chickens and turkeys would be difficult to detect in commercial poultry. Specifically, detection is difficult because these viruses did not cause overt clinical disease or mortality, but only induced mild microscopic lesions and exhibited poor seroconversion.

Biological and molecular characterization of ArkGA: A novel Arkansas serotype vaccine that is highly attenuated, efficacious, and protective against homologous challenge.

Almost all commercial poultry are vaccinated against avian coronavirus infectious bronchitis virus (IBV) using live attenuated vaccines mass administered by spray at day of hatch. Although many different types of IBV vaccines are used successfully, the ArkDPI serotype vaccine, when applied by spray, does not infect and replicate sufficiently to provide protection against homologous challenge. In this study, we examined a different Ark vaccine strain (Ark99), which is no longer used commercially due to its reactivity in one day old chicks, to determine if it could be further attenuated by passage in embryonated eggs but still provide adequate protection. Further attenuation of the Ark99 vaccine was achieved by passage in embryonated eggs but ArkGA P1, P20, and P40 (designated ArkGA after P1) were still too reactive to be suitable vaccine candidates. However, ArkGA P60 when given by spray had little or no vaccine reaction in one day old broiler chicks, and it induced protection from clinical signs and ciliostasis following homologous challenge. In addition, vaccinated and challenged birds had significantly less challenge virus, an important measure of protection, compared to non-vaccinated and challenged controls. The full-length genomes of viruses from egg passages 1, 20, 40, and 60 were sequenced using the Illumina platform and the data showed single nucleotide polymorphisms (SNPs) had accumulated in regions of the genome associated with viral replication, pathogenicity, and cell tropism. ArkGA P60 accumulated the most SNPs in key genes associated with pathogenicity (polyprotein gene 1ab) and cell tropism (spike gene), compared to previous passages, which likely resulted in its more attenuated phenotype. These results indicate that the ArkGA P60 vaccine is safe for spray vaccination of broiler chicks and induces suitable protection against challenge with pathogenic Ark-type virus.

Evaluation of a coccidia vaccine using spray and gel applications.

Coccidiosis is an economically significant disease of poultry caused by species of Eimeria, a parasitic protozoan. Disease can result in poor feed conversion, reduced weight gain, and can lead to the development of necrotic enteritis. For prevention of coccidiosis, poultry are commonly vaccinated with a live, sporulated oocysts mass applied with a vaccination cabinet in the hatchery. Traditionally, coccidia vaccines have been applied by coarse spray in a water based diluent, however, new technology using gel diluents has entered the US market. Gel diluents can have variable viscosities and are "dropped" onto chicks with an applicator bar. It is thought that gel droplets remain intact on the birds for longer than water based droplets, allowing more time for preening and ingestion of oocysts. In this experiment, the efficacy of a commercial coccidia vaccine applied with a water based diluent, a more viscous gel diluent, and a less viscous gel diluent was compared. Fecal samples were collected at multiple time points post-vaccination to quantify vaccine oocyst shedding. Shedding in the first cycle (days 5 to 8 post-vaccination) was related to the number of oocysts received from each application method, where the groups receiving higher doses shed more oocysts. However, a decrease in shedding was seen for the more viscous gel group in the second cycle (days 12 to 15 post-vaccination). Chickens were challenged with Eimeria maxima oocysts and 7 days post-challenge body weight gains and gross and microscopic lesions were recorded to evaluate protection levels for the different vaccine applications. All vaccinated groups appeared to be protected based on body weight gain and lesion scoring. The results of this project indicate that all vaccine applications are effective at protecting against Eimeria maxima challenge when using a proper dose of vaccine that allows for repeated oocyst cycling in the litter post-vaccination.

Molecular and serologic characterization, pathogenicity, and protection studies with infectious bronchitis virus field isolates from California.

In this study, we characterized three variant infectious bronchitis virus (IBV) strains isolated in 2003 and 2004 from broiler chickens in California and compared them to previously isolated California variant viruses and to common vaccine serotypes used in the United States. We conducted genetic, serologic, and pathogenicity studies on all three isolates, then tested different vaccines against one of the viruses. Genetically the three variant IBV strains, designated CA557/03, CA706/03, and CA1737/04, were not related to each other. GenBank BLAST database search and phylogenetic analysis of the hypervariable region of the S1 subunit of the spike gene to determine the most closely related viruses to the three variants showed the CA557/03 variant to be 81.8% similar to the CAV/CA56b/91 whereas the CA706/03 and CA1737/04 variant viruses were only distantly related to Dutch/D1466/81 (72.2%), a vaccine strain used in Europe, and Korea/K142/02 (72.7%), a Korean field isolate, respectively. Cross virus-neutralization testing showed that none of the 2003-04 California IBV variant viruses were serologically related to each other or to Ark, Conn, or Mass vaccine strains. In addition the CA1737/04 isolate was also tested against DE072 and found not to be serologically related. All three variant viruses were pathogenic in 1-wk-old broilers and vaccination with Mass/Conn followed by Holland/Conn provided 80% protection against the CA1737/04 virus. The 2003-04 California variant viruses were not compared with variants isolated in California during 1970s and 1980s because, to our knowledge, no genetic information is available and those viruses are no longer obtainable. This study shows that the CA557/03 virus was distantly related to the CAV-type viruses isolated in California in the early 1990s, but that none of the 2003-04 viruses were similar genetically or serologically to the CAL99-type viruses, indicating that new IBV variants continue to emerge and cause disease in commercial chickens in California.

Minimum Infectious Dose Determination of the Arkansas Delmarva Poultry Industry Infectious Bronchitis Virus Vaccine Delivered by Hatchery Spray Cabinet.

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.

Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens.

It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5'-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.

In vitro analysis of a hammerhead ribozyme targeted to infectious bronchitis virus nucleocapsid mRNA.

Hammerhead ribozymes are catalytic RNA molecules that specifically cleave a target RNA molecule. Herein, we report the design, synthesis, and in vitro analysis of a hammerhead ribozyme targeted to the infectious bronchitis virus (IBV) nucleocapsid mRNA. At a concentration of 0.5 or 10 microM, the ribozyme, designated IBV-N-Rz, effectively cleaved target RNAs in trans (37 C, 10 mM MgCl2, 50 mM Tris). Cleavage products were visualized by agarose gel analysis. The time course of the ribozyme reaction was monitored by agarose gel analysis and relative quantitative reverse transcription-polymerase chain reaction. The amount of target RNA continually declined over a 5-hr period, indicating that the ribozyme was truly catalytic. Although stability and delivery problems must be overcome, a hammerhead ribozyme targeted to the IBV nucleocapsid mRNA most likely has antiviral activity and may be an effective therapeutic/prophylactic reagent in the future.

Data from 11 years of molecular typing infectious bronchitis virus field isolates.

In 1993, a new molecular typing method for infectious bronchitis virus (IBV) was introduced. This method uses reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis of the spike gene to obtain RFLP patterns that correlate with serotype. Using that test at the Poultry Diagnostic and Research Center (PDRC, University of Georgia, Athens, GA), we have identified a total of 1523 IBV isolates in the past 11 yr. The data were obtained from clinical samples submitted to our laboratory from birds with clinical signs characteristic of IBV infection. The samples are primarily from the southeastern United States but are also from many other states as well as from outside the United States. Most of the isolations occurred during July, followed by May, April, November, October, and January. The fewest number of isolates identified on an annual basis was 20 in 2003. An unusually high number of isolations occurred in 1997 (318 isolations) and 1999 (246 isolations), which coincided with the GAV variant virus and GA98 variant virus outbreaks respectively. By far, the Ark-DPI strain was the most frequently identified type of IBV and ranged from 23% to 65% of total isolations per year. Ark-like isolates, defined as having a similar but unique RFLP pattern from the Ark-DPI vaccine strain were identified every year of the study except in 1996. In addition, new Ark-like isolates continued to emerge each year (except in the year 2000) beginning in 1997, reflecting the ability of that IBV type to undergo genetic drift. Eighty-two different variant viruses were identified although only two (GAV and GA98) became persistent and caused widespread disease. Some viruses tended to be geographically restricted to a given area (CAV in California and MX97-8147 in Mexico), whereas others were widespread (Ark-DPI, Conn, DE072, and Mass). The Florida, Gray, Holte, Iowa, and JMK types were not detected during the 11-yr period, and no foreign virus types were detected in the United States. These data show that IBV variant viruses are consistently circulating in commercial poultry and are capable of causing disease outbreaks. Our observations highlight the importance of constantly monitoring IBV as well as other coronaviruses like severe acute respiratory syndrome-coronavirus that have the ability to change and emerge to cause disease in a susceptible host.

Identification of avian coronavirus in wild aquatic birds of the central and eastern USA.

Coronaviruses (CoVs) are worldwide in distribution, highly infectious, and difficult to control because of their extensive genetic diversity, short generation time, and high mutation rates. Genetically diverse CoVs have been reported from wild aquatic birds that may represent a potential reservoir for avian CoVs as well as hosts for mutations and recombination events leading to new serotypes or genera. We tested 133 pooled samples representing 700 first-passage (in eggs) and 303 direct cloacal swab transport media samples from wild aquatic birds in the US that were avian influenza-negative. We isolated RNA from frozen samples and performed reverse transcriptase-PCR using a published universal CoV primer set. Of the samples tested, one from a Ruddy Turnstone (Arenaria interpres) was positive for CoV, showing nucleotide sequence similarity to a duck coronavirus (DK/CH/HN/ZZ2004). These data indicate a possible low prevalence of CoVs circulating in wild aquatic birds in the eastern half of the US.

Typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the S1 gene.

A universal primer set was developed that amplifies a region covering hypervariable region (HVR) 1 and HVR 2 in the S1 gene of the infectious bronchitis virus (IBV). The universality of this primer set was confirmed by testing the reference strains of different serotypes or variants of the IBV present in the United States. An approximately 450-bp region containing HVR 1 and HVR 2 of 7 untyped field isolates obtained in 1999 and 2000 was amplified. Direct sequencing followed by phylogenetic analysis on that region allowed us to type those field isolates that were not typable by reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). Furthermore, it was found that typing by phylogenetic analysis of that region correlates with virus neutralization results. Together with RT-PCR and RFLP, this method will serve as a fast typing method for IBV diagnosis.

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp.

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.

Attenuation, safety, and efficacy of an infectious bronchitis virus GA98 serotype vaccine.

In 1998, novel strains of infectious bronchitis virus (IBV) were identified in chickens from the southeastern United States and classified as a new serotype designated Georgia 98 (GA98). Because of the widespread nature of the GA98 virus in the southeastern United States and the lack of adequate protection with the DE072 vaccine, we developed a specific vaccine for the GA98 serotype. The GA98/0470/98 isolate of IBV was passaged in embryonating chicken eggs 70 times, and attenuation of the virus was determined in specific-pathogen-free chicks. Pass 70 of the GA98/0470/98 strain of IBV when given at 1 day of age by coarse spray and at 14 days of age in the drinking water at 1 x 10(4.5) 50% embryo infectious dose/bird protected against the homologous GA98 challenge as well as provided good protection against the DE072-type virus. In addition, the vaccine was shown to be adequately attenuated and safe at a 10x dosage.

Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein.

We have constructed a DNA vaccine (pDKArkS1-DPI) expressing the S1 glycoprotein (Arkansas DPI) of infectious bronchitis virus (IBV) to examine protective immunity after in ovo and intramuscular DNA immunization. Birds receiving in ovo DNA followed by live virus vaccination at 2 wk of age were 100% protected from clinical disease. Birds receiving only live virus vaccine or only in ovo DNA vaccination were < or = 80% protected. IBV was detected up to 10 days postchallenge in unvaccinated control groups, whereas birds receiving in ovo DNA and live virus vaccination cleared IBV from tracheal samples before day 5 postchallenge. Transcription of the S1 gene was confirmed in lung tissue after in ovo vaccination by an antisense riboprobe, and the S1 protein was detected by immunohistology in the heart and bursa. In a separate experiment, birds were injected intramuscularly with either 50, 100, or 150 microg of the DNA vaccine at 1 day of age and then again with either 100, 200, or 300 microg of the DNA vaccine, respectively, at 14 days of age. At 10 days postchallenge, no clinical signs were observed and no challenge virus was reisolated from the birds vaccinated with 150 microg and 300 microg of DNA. Between DNA-vaccinated birds and nonvaccinated control birds, no statistical differences were observed for IBV-specific serum antibodies as detected by enzyme-linked immunosorbent assay or the virus neutralization test. These data indicate that DNA vaccination with the S1 gene either in ovo or intramuscularly can provide birds with some protection against clinical disease after homologous IBV challenge.