Detection Of Infectious Bronchitis Virus Research Articles

Universal oligonucleotides for the detection of infectious bronchitis virus by the polymerase chain reaction

The universality of seven pairs of oligonucleotides for detection of the coronavirus infectious bronchitis virus (IBV) by reverse-transcription polymerase chain reaction (RT-PCR) was examined using 41 isolates of IBV collected over five decades from Europe, Japan and the USA. Oligonucleotides specific for sequences within the S2 region of the spike (S) gene (Lin et al., 1991a) and nucleocapsid (N) gene (Zwaagstra et al., 1992) gave the appropriate products with all 41 isolates. Oligonu-cleotide pair UTR1 - UTR2 +, corresponding to sequences within the 3 untranslated region (UTR) of the genome, also gave the predicted product with all the isolates. Oligonucleotide pair UTR3 - /UTR4 + was internal to oligonucleotides UTR1 - /UTR2 + and was used in a nested-set arrangement for greater specificity and sensitivity, giving the correct product with the 39 isolates examined. Oligonucleotide pair S1Unil - /S1Uni2 + was used to produce a 1.6 kb cDNA, corresponding to most of the S1 region of the S gene, with 24/24 isolates tested. This oligonucleotide pair was less suited than the others for routine detection of IBV but is recommended for the amplification of the S1 region of the S gene of new isolates for subsequent analysis. Other oligonucleotide pairs, yielding cDNA corresponding to the variable region of the IBV genome where genes 3 and 4 (M) overlap, were selected to be largely specific for Massachusetts serotype isolates, in the context of European strains. RT-PCR analysis using these oligonucleotide pairs showed that a number of field isolate preparations also contained a small amount of Massachusetts serotype virus, probably of vaccine origin and indicative of low level persistent infection. These results suggest that any strain of IBV is likely to be detectable by RT-PCR with at least one of our primer pairs.

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.

A Monoclonal Antibody-Based Immunoperoxidase Procedure for Rapid Detection of Infectious Bronchitis Virus in Infected Tissues

A monoclonal antibody (MAb)-based indirect immunoperoxidase (IP) procedure was applied to detect infectious bronchitis virus (IBV) antigen in frozen tissue sections. With this procedure, IBV antigen could be detected in chorioallantoic membranes (CAMs) within 15 hr postinoculation and in the respiratory tissues of chickens within 96 hr postinoculation. Endogenous peroxidases in chicken tissues were removed by treatment with periodic acid. The IP technique was highly sensitive, convenient, and economical. It allowed simultaneous evaluation of antigen-bearing cells and the general tissue morphology. The IP-stained slides also provided a permanent record.

Detection of Infectious Bronchitis Virus Antigen from Experimentally Infected Chickens by Indirect Immunofluorescent Assay with Monoclonal Antibody

Infectious bronchitis virus (IBV) was detected by indirect immunofluorescent assay with a monoclonal antibody (MAb-IFA). The monoclonal antibody was specific for the nucleocapsid protein of IBV strain M41. The MAb-IFA clearly detected IBV with high specificity in infected chicken kidney cells. The assay furthermore detected IBV in tracheal smears and sliced tracheas from experimentally infected chickens. The positive reaction was found to be longer than that in the virus recovery test. These results indicate that MAb-IFA is a useful method for the detection of IBV from chickens suspected to have infectious bronchitis.

Enzyme-linked immunosorbent assay for the detection of infectious bronchitis virus (IBV) antigen with monoclonal antibody.

Enzyme-linked immunosorbent assay for the detection of infectious bronchitis virus (IBV) antigen with monoclonal antibody.

A fluorescence test in allantoic cells for the detection of infectious bronchitis virus

A fluorescence test in allantoic cells for the detection of infectious bronchitis virus

Enzyme-linked immunosorbent assay for the detection of infectious bronchitis virus antiges.

Enzyme-linked immunosorbent assay (ELISA) to detection of infectious bronchitis virus (IBV) antigen was applied. IBV M41, A-5968 and L2 strains showing strain specificity in virus neutralizing test showed marked cross-reactivity. IBV M41 antigen was detectable from both SPF eggs and chickens inoculated with the virus. Between the positive (purified IBV and infectious allantoic fluid) and negative controls (Newcastle disease virus B1 strain and allantoic fluid from SPF eggs), there were marked differences in ELISA titers. From these results, ELISA was considered to be a speedy and good technic for detecting IBV antigens from test samples infected with the virus experimentally.

Vaccination of Day-Old Broilers against Infectious Bronchitis: Effect of Vaccine Strain and Route of Administration

Nine groups of 10 one-day-old broiler chicks were vaccinated by eye-drop, coarse spray, or intratracheal instillation with combined Newcastle disease-infectious bronchitis live vaccines containing the Massachusetts (Mass) 41, Holland 120, or Connecticut 46 strain of infectious bronchitis virus (IBV). Chickens vaccinated with Mass-41 or Holland 120 were protected against challenge with a Mass strain administered at 4 weeks of age. When immunity to the disease was evaluated by virus recovery, detection of IBV by fluorescent antibody, evaluation of ciliary activity, histopathology, and virus-neutralization titers, the group vaccinated with Connecticut 46 did not show protection. Vaccination at 1 day of age resulted in an antibody response and increased resistance to challenge with Mass-41, regardless of vaccination route.