Ambient ammonia does not appear to inhibit the immune response to infectious bronchitis virus vaccination and protection from homologous challenge in broiler chickens.

Commercial broilers are commonly exposed to gaseous ammonia (NH3) originating from degradation of nitrogen-containing excreta in the litter during the grow-out period. Ammonia concentrations in the air are higher in poorly ventilated houses and appear to coincide with the elevated incidence of respiratory disease occurring during the winter months. This study examined the effect of NH3 on the immune response to infectious bronchitis virus (IBV) vaccination and protection against homologous serotype challenge in commercial broiler chickens. One-day-old chicks were administered IBV vaccine and exposed to 30-60 ppm of NH3. At 28 DOA, birds were challenged oculonasally with a pathogenic homologous IBV, and protection was measured by viral detection, clinical signs, ciliostasis, and presence of airsacculitis. IBV-specific serum IgG and lacrimal fluid IgA titers, as well as Harderian gland (HG) immune cell phenotypes, were evaluated. Ammonia exposure was associated with an increased incidence of airsacculitis among non-vaccinated, challenged birds. Vaccinated, NH3-exposed birds were completely protected from IBV challenge. Ammonia had subtle effects on cilia morphology and function but did not affect vaccine or challenge virus replication and clearance, clinical signs, ciliostasis, tracheal histopathology scores, or immune responses. In the HG of vaccinated birds, the percent of leukocytes, MHC I+/MHC IIhi expression, IgM+ expression, and CD8+ expression was increased, while mucosal IgA and serum IgG titers were nominal. Non-vaccinated, IBV-challenged birds exhibited an increased percent of leukocytes, MHC I+/MHC IIhi expression, and IgM+ expression in the HG at 5 dpc, followed by increased mucosal IgA and serum IgG titers and CD8+ expression at 10-14 dpc. In contrast, vaccinated, IBV-challenged birds had a minimal increase in MHC I+/MHC IIhi expression, and serum IgG antibody titers in vaccinated birds increased rapidly. The results indicate that commercial broilers exposed to moderate levels of ambient NH3 are equally protected against IBV challenge if appropriately vaccinated, and the absence of robust immune activation in vaccinated, challenged birds suggests that the challenge virus was efficiently neutralized before establishing infection. In contrast, ambient NH3 exposure was associated with a higher incidence of airsacculitis in non-vaccinated, challenged birds, despite the apparent lack of differences in the immune response between birds in the NH3-exposed and NH3 control groups.

Comparative sequence analysis of full-length genome of FIPV at different tissue passage levels.

Feline infectious peritonitis virus (FIPV), an alpha Coronavirus, is the causative agent of a fatal immune mediated disease in cats. It is currently unclear if this virus circulates in the field or develops in felines that are infected with Feline enteric coronavirus. To better understand the genomic changes associated with viral adaptation, we sequenced the complete genomes of FIPV WSU 79-1146 at different tissue passage levels: passage 1, passage 8, and passage 50 tissue culture. Twenty-one amino acid differences were observed in the polyprotein 1a/ab between the different passages. Only one residue change was observed in the spike glycoprotein, which reverted back on subsequent passages, four changes were observed in the 3c protein, and one change was observed in each 3a, small membrane, nucleocapsid and 7a proteins. The mutation rate was calculated to be 5.08-6.3 × 10(-6) nucleotides/site/passage in tissue culture suggesting a relatively stable virus. Our data show that FIPV has a low mutation rate as it is passed in cell culture but has the capacity for change specifically in nsp 2, 3c, and 7b as it is passed in cell culture.

Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus.

Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75-43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.

Avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo.

Anti-coronaviral activity of a mixture of oleoresins and essential oils from botanicals, designated QR448(a), was examined in vitro and in vivo. Treatment of avian infectious bronchitis virus (IBV) with QR448(a) reduced the virus titer as measured in two laboratory host systems, Vero E6 cells and embryonating eggs. The effect of QR448(a) on IBV in chickens was also investigated. Administering QR448(a) to chickens at a 1:20 dilution by spray, 2h before challenge with IBV was determined to be the most effective treatment. Treatment decreased the severity of clinical signs and lesions in the birds, and lowered the amount of viral RNA in the trachea. Treatment with QR448(a) protected chickens for up to 4 days post-treatment from clinical signs of disease (but not from infection) and decreased transmission of IBV over a 14-day period. Anti-IBV activity of QR448(a) was greater prior to virus attachment and entry indicating that the effect is virucidal. In addition, QR448(a) had activity against both Massachusetts and Arkansas type IB viruses, indicating that it can be expected to be effective against IBV regardless of serotype. To our knowledge, this is the first report on the in vivo use of a virucidal mixture of compounds effective against the coronavirus IBV.

Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction.

A rapid diagnostic assay for differentiating avian infectious bronchitis virus (IBV) isolates was developed. The basis of the assay is the cleavage of target RNA by RNase H mediated by sequence-specific chimeric oligonucleotides followed by sample to residual ratio quantitation (SRRQ) using RRT-PCR. Four serotype-specific chimeric oligonucleotides were designed, one each for the Massachusetts, Connecticut, Arkansas, and Delaware/Georgia 98 serotypes, and tested for their ability to mediate specific cleavage of target RNA from known homologous and heterologous strains of IBV. Specific cleavage of target RNAs by each chimeric oligonucleotide was verified using agarose gel analysis and RRT-PCR. There were no non-specific cleavage products. Eight different IBV strains representing seven serotypes were tested and each chimeric oligonucleotide mediated cleavage of target RNA only from strains within the serotype that the chimeric was designed against. The SRRQ assay was evaluated on 15 samples without prior knowledge of their grouping and correctly identified the serotype of each sample. The assay is rapid; six samples can be tested in approximately 4 h. In addition, the primer set amplifies all IBV RNAs tested to date and provides a built in control for detecting IBV whether it is typeable or not.

Detection of infectious bronchitis virus by real-time reverse transcriptase-polymerase chain reaction and identification of a quasispecies in the Beaudette strain.

In this report, we describe a real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) diagnostic test for infectious bronchitis virus (IBV) with the use of fluorescence resonance energy transfer (FRET) technology. Two primers that amplify a 383-base pair product between nucleotide positions 703 and 1086 relative to the start codon for the S1 gene of the Massachusetts 41 virus were designed and used to amplify the Beaudette, Massachusetts 41, Florida 18288, Connecticut, Iowa 97, Arkansas DPI, CA/NE95/99, DE/072/ 92, and GA/0470/98 strains of IBV. The primers were specific and did not amplify New Castle disease virus, Mycoplasma spp., or infectious laryngotracheitis virus. For RRT-PCR by FRET, an anchor probe conjugated to fluorescein and a detection probe conjugated to a red fluorophore were designed to anneal to a hypervariable region within the 383-base pair product. The level of sensitivity was 1 x 10(4) RNA molecules used as starting template. After amplification, a melting curve analysis was conducted to specifically identify IBV types. Because of sequence differences in the annealing position of the detection probe, the Arkansas, Connecticut, Beaudette, and Massachusetts 41 strains could be differentiated. No fluorescence was observed for the DE/072/ 92 and GA/0470/98 viruses with the anchor and detection probes. When the Beaudette strain was examined, two melting peaks were observed at 44 C and 51 C, indicating a quasispecies in that laboratory strain of IBV. Routine typing of vaccine strains of IBV was possible with this technology, but high standard deviations associated with the melting curve analysis of the FRET probes described herein made it difficult to use this test reliably for routine typing of IBV field isolates.

Lack of interaction between avian leukosis virus subgroup J and fowl adenovirus (FAV) in FAV-antibody-positive chickens.

Unfounded field speculation has suggested that avian leukosis virus subgroup J (ALV-J) predisposes young meat-type chickens to inclusion body hepatitis caused by fowl adenovirus (FAV). To address this hypothesis, we infected 1-day-old grandparent meat-type chickens carrying maternal antibodies against FAV with a field isolate of FAV associated with inclusion body hepatitis in broilers, ALV-J, or both FAV and ALV-J. We examined the effects of FAV alone or in combination with ALV-J on the basis of clinical signs, overall mortality, growth rate, and gross and microscopic lesions. With such criteria for evaluating possible interactions, we found no significant differences in the dually infected birds in comparison with chickens that received a monovalent challenge with either FAV or ALV-J.

Spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus.

The spike glycoprotein of infectious bronchitis virus (IBV), a coronavirus, is translated as a precursor protein (So), then cleaved into two subunits (S1 and S2) by host cell serine proteases. In this study, we compared the cleavage recognition site of 55 IBV isolates to determine if the cleavage recognition site sequence, which consists of five basic amino acid residues, correlates with host cell range, serotype, geographic origin, and pathogenicity as it does in orthomyxoviruses and paramyxoviruses. The most common cleavage recognition site observed (33 of 55 viruses) was Arg-Arg-Ser-Arg-Arg, representing at least 11 different serotypes. Thus, cleavage recognition site does not appear to correlate with serotype. We also determined that cleavage recognition site sequence does not correlate with pathogenicity because attenuated and pathogenic isolates (different passages of the same virus) contain identical cleavage recognition site sequences. In addition, nephropathogenic strains had the same cleavage recognition site sequence as many nonnephropathogenic isolates. Cleavage recognition site sequence does correlate with viruses in different geographic regions, which may be an important characteristic to examine in epidemiologic studies. An IBV monoclonal antibody neutralization-resistant mutant (NR 18) had an unusual substitution of Ile for Arg at the fourth position, giving the sequence Arg-Arg-Ser-Ile-Arg, which likely prevents cleavage and, thus, destroys the conformationally dependent monoclonal antibody binding epitope. Six residues on the amino-terminal side of the cleavage recognition site are conserved in 31% of the isolates and consist of only one or two basic amino acids. Thus, the number of basic residues around the cleavage recognition site does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with envelope glycoproteins in orthomyxoviruses and paramyxoviruses.

Molecular characterization of infectious bronchitis virus isolates foreign to the United States and comparison with United States isolates.

Eleven infectious bronchitis virus (IBV) isolates foreign to the United States were analyzed by using reverse transcriptase (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) and S1 glycoprotein gene sequencing. Two of the isolates generated RFLP patterns that resembled the Mass 41 strain. Seven novel RFLP patterns were detected among the other nine foreign IBV isolates. Five of the foreign isolates were further analyzed by S1 glycoprotein gene sequencing in our laboratory. Phylogenetic analysis of S1 glycoprotein-deduced amino acid sequences for 4/91 pathogenic, 4/91 attenuated, and Variant 1 were greater than 90% similar to viruses belonging to the 793/B serogroup and, therefore, are possibly serologically related. Variant 2 was only 81.0% similar to viruses belonging to the European serogroup B, and, therefore, predicting its serotype is difficult. Isolates 98-07484 and 97-8123 were genotypically unique and therefore might be serologically unique. With the RFLP patterns and the deduced S1 amino acid sequence data as a reference, none of the IBV isolates foreign to the United States have been detected in the United States.

Identification and analysis of the Georgia 98 serotype, a new serotype of infectious bronchitis virus.

Twenty-five field infectious bronchitis viruses (IBVs) similar to, but genetically distinct from, the DE072 serotype were isolated from several states in the United States from 1990 through 1999 and were examined molecularly and antigenically. A 421-bp sequence in the hypervariable region of the S1 gene was examined, and phylogenetic analysis on that region indicated that these viruses are closely related but fall into unique groups. Cross-virus neutralization testing and entire S1 sequence analysis on selected isolates further confirmed that fact, and we divided the viruses into the DE072 serotype and two other unique groups. In a vaccine protection trial, the commercially available DE072 vaccine showed less than 50% protection against viruses in one of the groups. The majority of the recent isolates belong to that group and share very low antigenic relatedness to the DE072 strain as well as other serotypes of IBV. Consequently, we designated this group as a new serotype, Georgia 98. We developed a restriction fragment length polymorphism analysis that can differentiate this new serotype from all other serotypes of IBV.

Redesign of primer and application of the reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism test to the DE072 strain of infectious bronchitis virus.

Diagnosis of the DE072 strain of infectious bronchitis virus (IBV) by the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) serotype identification test was not possible because the primer used in the RT-PCR did not amplify the S1 gene of the DE072 strain. The 3' end of the polymerase gene and the 5' end of the S2 gene of the DE072 strain were sequenced and compared with the forward and reverse RT-PCR primers, respectively. A 2-bp mismatch at the 3' end of the reverse primer was found. On the basis of these data, a degenerate primer that could amplify the S1 gene of the DE072 strain as well as eight other serotypes of the virus was synthesized. In addition, we were able to differentiate the DE072 strain from all of the other IBV strains examined by RFLP analysis of the RT-PCR product.

Infectious bronchitis virus S2 gene sequence variability may affect S1 subunit specific antibody binding.

The S2 gene of several strains of infectious bronchitis virus (IBV) belonging to the Arkansas, Connecticut, and Florida serotypes was sequenced. Phylogenetic analysis of the S2 gene nucleotide and deduced amino acid sequence data resulted in groups of strains that were the same as groupings observed when S1 sequence data was used. Thus, it appears that S2 subunits are conserved within a serotype but not between serotypes. Although the sequence differences were small, we found that only a few amino acid differences were responsible for different secondary structure predictions for the S2 subunit. It is likely that these changes create different interactions between the S1 and S2 subunits, which could affect the conformation of the S1 subunit where serotype specific epitopes are located. Based on this sequence data, we hypothesize that the S2 subunit can affect specific antibody binding to the S1 subunit of the IBV spike glycoprotein.

Identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus.

Localization of neutralizing, serotype specific epitopes of infectious bronchitis virus has been difficult because these epitopes are conformationally dependent. We identified amino acids involved in a serotype specific, conformationally dependent epitope by analysis of the S1 gene of 13 monoclonal antibody-neutralization-resistant mutants. Substitutions in the predicted amino acid sequence of these mutants were located at residues 304 and/or 386. Most of the substitutions at residue 304 were from threonine to isoleucine, whereas the substitutions at residue 386 were from arginine to proline, histidine, cysteine, or tryptophan. Based on this data, it appears that AA residues at 304 and 386 on the S1 glycoprotein are involved in a virus neutralizing serotype specific epitope.

Further development and use of a molecular serotype identification test for infectious bronchitis virus.

Previously, we developed a rapid serotype identification test for infectious bronchitis virus (IBV) that utilizes the reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism analysis. The RT-PCR is used to amplify the S1 gene from RNA extracted from the virus grown in eggs. Restriction enzyme digestion and electrophoresis of that PCR product is used to determine the serotype of the virus. The purpose of this study was threefold. First, using a modified 5' PCR primer, we altered the procedures of our rapid serotype identification test and amplified the S1 gene of IBV in tracheal swabs collected from specific-pathogen-free leghorn chickens experimentally inoculated with the Arkansas or Mass 41 serotypes of IBV. Direct amplification of IBV in tracheal swabs eliminates the need to isolate the virus in eggs. Second, we attempted to amplify inactivated IBV in allantoic fluid, possibly allowing us to obtain and determine the serotype of isolates originating from outside the U.S.A. Virus inactivated by formalin (0.1% final concentration) could not be amplified by the RT-PCR procedure, but heat-inactivated IBV (56 C for 15 min) was successfully amplified. Third, we developed an internal control for the RT-PCR test by synthesizing RNA runoff transcripts of a cloned truncated S1 gene. The truncated S1 RNA transcripts were added to the RT-PCR reaction and a 1031-bp product was amplified, which could be distinguished from the coamplified S1 gene from viral RNA. The internal RNA control reduces the possibility of obtaining false-negative results in the RT-PCR test.

Production and immunogenicity of multiple antigenic peptide (MAP) constructs derived from the S1 glycoprotein of infectious bronchitis virus (IBV).

Synthetic peptides were prepared as multiple antigenic peptide (MAP) constructs to the S1 glycoprotein of infectious bronchitis virus (IBV). The MAP system has been used in the production of anti-peptide and anti-protein antibodies. It has an advantage over linking peptides to a highly immunogenic carrier molecule because antibodies are not produced to the MAP core matrix of lysine residues. Two 25-residue peptides were synthesized to the Arkansas serotype and two were synthesized to the Massachusetts serotype of IBV. The peptide sequences correspond to amino acid residues 64 to 88 and to residues 117 to 141 for each of the IBV serotypes. A MAP construct for each peptide was prepared by linking 4 copies of a peptide to the immunogenetically inert core matrix of lysine residues. The MAP constructs were used to immunize specific pathogen free chickens. Anti-peptide ELISA titers and the dot immunobinding assay against the homologous peptide were positive for all of the sera tested whereas the anti-whole virus ELISA titers and virus neutralization titers were negative for all of the sera tested. Hyperimmune sera against whole virus did not cross react with synthetic peptides made to the heterologous virus suggesting a possible role for the MAP constructs in a serotype specific dot blot or ELISA test for IBV.

Polymerase chain reaction and a biotin-labeled DNA probe for detection of infectious bronchitis virus in chickens.

Polymerase chain reaction (PCR) and a biotin-labeled DNA probe were used to amplify and detect the genome of infectious bronchitis virus (IBV) from tracheal swabs taken from chickens that were experimentally inoculated with the IBV Beaudette, Arkansas, and Gray strains. The viral genome was successfully detected by PCR and confirmed by dot-hybridization assay using a biotin-labeled DNA probe on days 1, 3, 9, and 14 after exposure. Direct electron microscopy (EM) analysis was used to compare the ability of the two tests to detect IBV from the same tracheal swab samples. The EM analysis did not detect IBV in four of eight necropsy groups that were positive using PCR and the biotin-labeled DNA probe. Although histopathological lesions were observed in the tracheas, no clinical signs or specific antibody response were observed in the birds. The virus was also detected in the allantoic fluid of embryonating chicken eggs that had been inoculated with field samples suspected to be IBV. The field samples were passed four to six times in embryonating eggs, and 10 of 17 samples were positive using PCR and the biotin-labeled probe.

Infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a DNA probe.

A rapid extraction procedure was developed to purify infectious bronchitis virus (IBV) RNA from the allantoic fluid of inoculated embryonating eggs. Reverse transcription of viral RNA and the polymerase chain reaction (PCR) were used to amplify the viral genome from eight different strains of IBV comprising five different serotypes. A biotinylated DNA probe, prepared to a sequence within the PCR amplification product of the Beaudette strain of IBV, was used in a dot-hybridization assay; it detected the amplification products of all of the IBV strains examined. Reverse transcription and PCR amplification were judged to be specific for IBV. This was because amplification products were not detected by agarose gel electrophoresis or by dot-hybridization when template used in the PCR was extracted from allantoic fluid and the chorioallantoic membrane of uninoculated embryonating eggs or from allantoic fluid of embryonating eggs inoculated with other chicken upper respiratory viruses.

Observations on colonial phenotypic variation in Bordetella avium.

Two forms of Bordetella avium colonial morphology on artificial media were observed. The smooth colonial morphology is convex, with an entire edge, and has a glistening mucoid appearance when cultured on 5% bovine blood agar or tryptic soy agar. On the same media, the rough morphology has a crenated edge, a flat surface, and sometimes a wrinkled or ground-glass appearance to the surface. Colonial morphology remained stable when cultures were passaged at 37 C every 48 hours. When different salts or crystal violet were added to the media, the colonial morphology of seven different isolates did not change. Following storage at 4 C on agar, B. avium isolates switched between the smooth and rough colonial morphology, indicating that this is a reversible process. Generally, bacteria with the smooth colonial morphology were motile on sulfide indole motility (SIM) media, whereas bacteria with the rough colonial morphology were non-motile. Only the isolates with the smooth colony type colonized the upper respiratory tract of turkeys, caused clinical signs of the disease, and elicited an agglutinating antibody titer by 21 days postexposure.

Polymerase chain reaction amplification of the genome of infectious bronchitis virus.

The polymerase chain reaction (PCR) was used to amplify a portion of the genome of infectious bronchitis virus (IBV). Two synthetic primers that annealed to segments of the IBV genome in the matrix and nucleocapsid genes facilitated PCR amplification of a 1020-base sequence. Amplification was successful using template DNA from an IBV sequence-containing plasmid and using copy DNA created by reverse transcription of IBV genomic RNA. The PCR product was the expected size and had the expected nucleotide sequence.