Detection Of Infectious Bronchitis Virus Research Articles

Infectious Bronchitis Virus Detection and Isolation from Broiler Chicken

Infectious Bronchitis Virus Detection and Isolation from Broiler Chicken

Development of a sensitive and specific xMAP assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses

BackgroundA serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination.MethodThe microspheres coated with purified recombinant glycoprotein D (gD) of ILTV or nucleocapsid (N) protein of IBV were incubated with serum samples. The simultaneous quantification of ILTV and IBV antibodies were achieved through the interrogation of microspheres by Luminex 200 detection system. .ResultsThis xMAP detection demonstrated no nonspecific reactions with avian influenza virus (AIV), avian leukosis virus (ALV), newcastle disease virus (NDV), and Marek’s disease virus (MDV). The results also demonstrated that the xMAP assay was four times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for ILTV detection and two times more sensitive for IBV detection. A total of 90 chicken serum samples from a chicken farm were tested by xMAP and ELISA assays. The results showed that the coincidence rates were 84.44 and 100% for ILTV and IBV detection, respectively.ConclusionThis study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination.

Evaluation of full S1 gene sequencing of classical and variant infectious bronchitis viruses extracted from allantoic fluid and FTA cards

ABSTRACTSequence variability in the S1 gene determines the genotype of infectious bronchitis virus (IBV) strains. A single RT-PCR assay was developed to amplify and sequence the full S1 gene for six classical and variant IBVs (M41, D274, 793B, IS/885/00, IS/1494/06 and Q1) enriched in allantoic fluid (AF) or the same AF inoculated onto Flinders Technology Association (FTA) cards. Representative strains from each genotype were grown in specific-pathogen-free eggs and RNA was extracted from AF. Full S1 gene amplification was achieved using primer A and primer 22.51. Products were sequenced using primers A, 1050+, 1380+ and SX3+ to obtain short sequences covering the full gene. Following serial dilutions of AF, detection limits of the partial assay were higher than those of the full S1 gene. Partial S1 sequences exhibited higher-than-average nucleotide similarity percentages (79%; 352 bp) compared to full S1 sequences (77%; 1756 bp), suggesting that full S1 analysis allows greater strain differentiation. For IBV detection from AF-inoculated FTA cards, four serotypes were incubated for up to 21 days at three temperatures, 4°C, room temperature (approximately 24°C) and 40°C. RNA was extracted and tested with partial and full S1 protocols. Through partial sequencing, all IBVs were successfully detected at all sampling points and storage temperatures. In contrast, using full S1 sequencing it was not possible to amplify the gene beyond 14 days or when stored at 40°C. Data presented show that for full S1 sequencing, a substantial amount of RNA is needed. Field samples collected onto FTA cards are unlikely to yield such quantity or quality.Abbreviations: AF: allantoic fluid; CD50: ciliostatic dose 50; FTA: Flinders Technology Association; IB: infectious bronchitis; IBV: infectious bronchitis virus

Immunosensor Based on Antibody-Functionalized MoS2 for Rapid Detection of Avian Coronavirus on Cotton Thread.

Infectious bronchitis virus (IBV), an avian coronavirus, significantly affects the performance of both the egg-laying and meat-type birds causing the foremost of economic loss in poultry industry. This paper aims to develop a rapid, low-cost, and sensitive biosensor for IBV detection by using molybdenum disulfide (MoS2). MoS2 is a 2-D nanosheet which has strong high fluorescence-quenching ability when applied to a dye-labeled antibody (Ab). In this paper, we developed an Ab-functionalized MoS2-based fluorescent immunosensor, which utilized the fluorescence resonance energy transfer (FRET) between the MoS2 and fluorescence dye during the Ab–antigen interaction. The assay was performed on a low-cost cotton thread-based microfluidic platform due to the good wicking property and flexibility. Upon the optimization of assay conditions, the immunosensor demonstrated remarkable sensitivity of n}{}4.6times 10^{2}n EID50 per mL and specificity with a dynamic linear response range of 102–106 EID50 per mL for IBV standard solutions. The developed immunoassay successfully detected the IBV spiked chicken serum with satisfactory results. The foregoing presents its potential application for on-farm detection.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I.

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.

Serological Survey and Molecular Detection of Infectious Bronchitis Virus in Broiler Chickens in Diyala Province, Iraq

Serological Survey and Molecular Detection of Infectious Bronchitis Virus in Broiler Chickens in Diyala Province, Iraq

Infectious bronchitis vaccine virus detection and part-S1 genetic variation following single or dual inoculation in broiler chicks

ABSTRACTAn investigation was undertaken of the extent of genetic variation occurring within infectious bronchitis virus (IBV) vaccine strains following vaccination of day-old broiler chicks. Chicks were divided into seven groups, with two groups receiving single Massachusetts (Mass) vaccinations while the other four were inoculated with combinations of different IBV serotypes; Mass, 793B, D274 and Arkansas (Ark). The remaining group was maintained as an unvaccinated control. Following vaccination, swabs and tissues collected at intervals were pooled and RNA was extracted for detection of IBV by reverse transcription polymerase chain reaction. Positive amplicons were sequenced for the part-S1 gene and compared to the original vaccine strain sequences. Single nucleotide polymorphisms, amino acid variations and hydrophobicity changes were identified and recorded for each sampling point. A total of 106 single nucleotide polymorphisms were detected within 28 isolates. The average single nucleotide polymorphism counts of swab isolates were greater than those found in tissue samples. This translated into 64 amino acid changes; however only six resulted in a change to the hydrophobicity properties. All hydrophobic alterations occurred within swab isolates and the majority were recovered at 3 days post vaccination suggesting such changes to be detrimental to early virus survival. Nucleotide deletions were seen only in the group given the combination of Mass and Ark. Of the 16 sequenced samples in this group, 13 contained the same AAT deletion at position 1033 1035 in the Ark strains. Findings presented in this study demonstrate alteration in the S1 nucleotide sequence following co-administration of live IBV vaccines.

Investigation of Infectious Bronchitis Virus Strains by Real Time RT-PCR and S1 Gene Sequencing in Broilers

In this study, detection of infectious bronchitis virus (IBV) from chicken tracheal swab samples by real time RT-PCR, which was reported as a rapid and sensitive virus detection method, was aimed. Phylogenetic relations in different field and variant strains of IBV by sequencing of hyper variable regions (HVRs) of S1 gene of IBV was determined. Tracheal swabs were taken from 120 broiler chickens with respiratory signs, randomly. Samples were inoculated into the allantoic fluids (AF) of SPF embryonated chicken eggs. RNAs were extracted by AF. RNAs were translated to ss cDNAs. Real time RT-PCR was performed with double labelled Taqman probe, IBV 5 GU391 and IBV5 GL533 primer pairs. Positive results were obtained in 33 samples of 120 (27.5 %) by real time RT-PCR. cDNAs from positive samples were used for amplification HVRs (Hyper Variable Regions) in S1 gene region with C2U and Ag0723 primers. Samples were amplified by S1 5’mod and CK2 primers, too. RT-PCR products were sequenced directly. Sequencing results have been compared with NCBI’s nucleotide database. Sequencing results were matched to some variant IBV strains in gen banks database at the rate of 72-86 %. These results support that we are faced with variant IBV strains. Also, we observed a lot of SNP mutations in alignment. This study shows that birds can be infected by IBV variant strains at rate of 27.5 %, although they were vaccinated by classic type vaccine. These results demonstrate that vaccination by classical vaccine strains of IBV cannot provide enough cross protection against variant strains. Therefore, outbreaks of the disease still occur in vaccinated flocks. Also, we found these primers are not enough for all IBV field strains to be sequenced and classified. Thus, different primers should be developed and used primer pairs more than two. In this experiment, we found virus load is more in AF than swab samples because of IBV replication in SPF egg AF. We observed dwarfism and hemorrhages at 14 days old chick embryos

Detection of infectious bronchitis virus 793B, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae in poultry in Ethiopia

A survey was conducted into respiratory infectious diseases of poultry on a chicken breeder farm run by the Ethiopian Institute of Agricultural Research (EIAR), located in Debre Zeit, Ethiopia. Oropharyngeal swabs were collected from 117 randomly selected birds, and blood was taken from a subset of 73 of these birds. A combination of serological and molecular methods was used for detection of pathogens. For the first time in Ethiopia, we report the detection of variant infectious bronchitis virus (793B genotype), avian metapneumovirus subtype B and Mycoplasma synoviae in poultry. Mycoplasma gallisepticum was also found to be present; however, infectious laryngotracheitis virus was not detected by PCR. Newcastle disease virus (NDV) was not detected by PCR, but variable levels of anti-NDV HI antibody titres shows possible exposure to virulent strains or poor vaccine take, or both. For the burgeoning-intensive industry in Ethiopia, this study highlights several circulating infectious respiratory pathogens that can impact on poultry welfare and productivity.

Molecular Characterization of Infectious Bronchitis Virus in Chicken

INFECTIOUS BRONCHITIS (IB) is a highly contagious ………disease of serious economic importance in the poultry industry worldwide. Diagnosis of Infectious Bronchitis Virus (IBV) infections in Egypt during 2013 - 2016 among poultry flocks. A total of 41 chicken flocks were examined (40 broiler flock and one breeder flock). Results revealed that 21 chicken flocks were found positive IBV by N gene conventional RT-PCR and real time RT-PCR. The samples were isolated and propagated in commercial embryonated chicken eggs (ECE) with result revealed that only one sample that showed curling and dwarfing of the embryo after the second passage while 10 samples were HA positive that suggest mixed infection of IBV with other viruses such as Newcastle Virus or Avian Influenza Virus. Furthermore, the allantoic fluids of the positive samples were subjected to specific S1 PCR to differentiate strains of IBV. The absence of positive reaction of S1gene conventional RT-PCR may confirm the involvements of other respiratory pathogens in chickens as Newcastle disease, avian influenza and mycoplasmosis that masked IBV infections. In conclusion, our results confirmed the detection of IBV in chicken flocks in Egypt which responsible for the respiratory problems in poultry farms and diagnosis with Real-time PCR because it is more rapid, sensitive, and reproducible, and the risk of carryover contamination is minimized compared to conventional PCR.

Molecular Detection of Infectious Bronchitis Virus Isolated from Commercial Breeder Farms in Chittagong District, Bangladesh

Molecular Detection of Infectious Bronchitis Virus Isolated from Commercial Breeder Farms in Chittagong District, Bangladesh

Detection and identification of infectious bronchitis virus by RT-PCR in Iran.

Infectious bronchitis virus (IBV) causes severe diseases in poultry with significant economic consequences to the poultry industry in Iran. The aim of this study was the detection and identification of IBV by reverse transcription(RT)-PCR in Iran. Ten IB virus strains were detected by testing trachea, cecal tonsil, and kidney tissues collected from broiler and layer farms in Iran. In order to detect infectious bronchitis virus, an optimized RT-PCR was used. Primers targeting the conserved region of known IBV serotypes were used in the RT-PCR assay. Primers selectively detecting Massachusetts and 793/B type IB viruses were designed to amplify the S1 gene of the virus and used in the nested PCR test. Our findings indicate the circulation of at least three genotypes of IB viruses (Massachusetts, 793/B, and variant 2) among poultry flocks.

Genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in Egypt.

To characterize the circulating infectious bronchitis virus (IBV) strains in Egypt depending on the sequence of the spike-1 (S1) gene [hypervariable region-3 (HVR-3)] and to study the pathotypic features of these strains. In this work, twenty flocks were sampled for IBV detection using RRT-PCR and isolation of IBV in specific pathogen free (SPF) chicks during the period from 2010 to 2015. Partial sequencing and phylogenetic analysis of 400 bp representing the HVR-3 of the S1 gene was conducted. Pathotypic characterization of one selected virus from each group (Egy/Var-I, Egy/Var-II and classic) was evaluated in one day old SPF chicks. The chicks were divided into 4 groups 10 birds each including the negative control group. Birds were inoculated at one day by intranasal instillation of 10(5)EID50/100 μL of IBV viruses [IBV-EG/1212B-2012 (Egy/Var-II), IBV/EG/IBV1-2011 (Egy/Var-I) and IBV-EG/11539F-2011 (classic)], while the remaining negative control group was kept uninfected. The birds were observed for clinical signs, gross lesions and virus pathogenicity. The real-time rRT-PCR test was performed for virus detection in the tissues. Histopathological examinations were evaluated in both trachea and kidneys. The results revealed that these viruses were separated into two distinct groups; variant (GI-23) and classic (GI-1), where 16 viruses belonged to a variant group, including 2 subdivisions [Egy/Var-I (6 isolates) and Egy/Var-II (10 isolates)] and 4 viruses clustered to the classic group (Mass-like). IBV isolates in the variant group were grouped with other IBV strains from the Middle East. The variant subgroup (Egy/Var-I) was likely resembling the original Egyptian variant strain (Egypt/Beni-Suif/01) and the Israeli strain (IS/1494/2006). The second subgroup (Egy/Var-II) included the viruses circulating in the Middle East (Ck/EG/BSU-2 and Ck/EG/BSU-3/2011) and the Israeli strain (IS/885/00). The two variant subgroups (Egy/Var-I and Egy/Var-II) found to be highly pathogenic to SPF chicks with mortalities up to 50% than those of the classic group which was of low virulence (10% mortality). Pathogenicity indices were 25 (Egy/Var-II), 24 (Egy/Var-I) and 8 (classic); with clinical scores 3, 2 and 1 respectively. These findings indicated that the recent circulating Egyptian IBVs have multiple heterogeneous origins in marked diversifying nature of their spread, with high pathotype in specific pathogen free chicks.

Survey indicates circulation of 4/91 and QX-type infectious bronchitis viruses in Hungary in 2014 - Short communication.

Understanding the epidemiology and improving vaccinal protection against the highly variable chicken infectious bronchitis virus (IBV) requires the knowledge of circulating IBV serotypes/genotypes in defined geographic areas. Accordingly, the authors initiated a survey among the major poultry producers in Hungary in order to reveal the prevailing IBV serotypes in the country. Tracheal swabs and organ samples (caecal tonsils, kidneys, and trachea) were collected from broiler, layer, and meat-type breeder flocks, and were subjected to IBV detection by virus isolation and polymerase chain reaction (PCR). The IBV-positive samples were further characterised by nucleotide sequencing and phylogenetic analysis of a portion of the S1 IBV gene. Seventeen out of the 26 submitted samples proved to be positive for IBV. Sequence analyses revealed ten 4/91 and six QX serotypes, and a single D274 type IB virus. One sample contained a mixture of QX and Massachusetts serotype viruses. Presumably most of the 4/91 and D274 type viruses were vaccine strains. The proportion of QX type viruses and their observed variation are in good agreement with the situation in a few other European countries. The detected viruses clustered largely according to their geographic origin, with a few exceptions. If updated regularly, the preliminary 'virus map' will be useful for the adjustment of vaccination protocols.

Assessment of the potential relationship between egg quality and infectious bronchitis virus infection in Australian layer flocks

ObjectiveThis investigation aimed to determine if there was a relationship between the production of eggs with poor internal quality, as measured by poor Haugh units, by Australian layer flocks and the detection of infectious bronchitis virus (IBV) in the hens. Other risk factors including flock size, flock type, flock age, chicken breed and vaccination frequency were also assessed.MethodsThe study group comprised 17 flocks from 14 farms. Data relating to the factors investigated were requested on a regular basis. The Haugh unit data were used to grade eggs as good or poor based on the age and flock at the time of data collection. Cloacal swabs were collected from 20 chickens in each flock approximately every 6 weeks.Results IBV was detected from a majority of the flocks and in 68% of cases the IBV strain detected was an A‐vaccine‐related field strain. Three variant strains were detected. Detection of IBV in a flock, the farm type and flock size were identified as potential risk factors for the production of eggs with poor Haugh units.ConclusionIBV is prevalent in Australian layer flocks, but infection was primarily subclinical. The results complement previous reports indicating that there are many potential risk factors for the production of eggs with poor Haugh units.

In this issue - April 2014

In this issue - April 2014

Detection of variant infectious bronchitis viruses in broiler flocks in Libya

A number of broiler flocks with respiratory disease and high mortality in five broiler farms in Libya were sampled for detection of infectious bronchitis virus (IBV). Twelve IBV strains from these farms were detected by reverse transcription polymerase chain reaction (RT-PCR) and differentiated by nucleotide sequencing of the hypervariable region of the S1 gene. A pair-wise comparison of the sequences showed two distinctive patterns. Those from farms 1, 2, 4 and 5, formed a separate cluster with 94–99% relatedness to the Egyptian IBV strains CK/Eg/BSU-2/2011, CK/Eg/BSU-3/2011 and Eg/1212B. Sequences from the farm 3 formed another cluster with 100% relatedness to Eg/CLEVB-2/IBV/012 and IS/1494/06. This appears to be the first report on the co-circulation these variant IBVs in Libya.

Molecular detection of infectious bronchitis virus and it is relation with avian influenza virus (H9) and Mycoplasma gallisepticum from different geographical regions in Iraq

Infectious bronchitis virus (IBV), Avian influenza virus (AIV) and Mycoplasma gallisepticum (MG) have been recognized as the most important pathogens in poultry cause acute respiratory infection and serous economic problems in Iraq and many other countries all over the world. This study was conducted to investigate the distribution of these diseases in commercial chicken flocks in different geographical region in middle part of Iraq by using qPCR. Tracheal swabs and tissue specimens from trachea, lung and kidney were taken from 38 different cases from commercial broiler chicken flocks in (Najaf, Hilla, Muthana and Theqaar governorates) in the period from November 2010 to June 2011, all these flocks were showed respiratory symptoms and mortality about 20-90%. The results showed that 92.1% of samples collected from these flocks were infected with IBV, 20% of samples were infected with IB alone and 45.71% of samples with IB combined with both GM and AIV subtype H9 and 25.71% of samples were positive to both IBV and AIV(H9). No samples were positive to AIV (H9) or MG alone. Because of importance of respiratory diseases as a most common conditions noted in commercial flocks in Iraq and no previous study detecting this pathogens by molecular techniques, this study come to detect and confirm the diagnosis of this pathogens by qPCR as new technique used in this field in Iraq.

Detection of infectious bronchitis virus serotypes by reverse transcription polymerase chain reaction in broiler chickens.

Infectious bronchitis (IB) is a highly contagious disease of the respiratory and urogenital tract of chickens, caused by infectious bronchitis virus (IBV), a member of the family Coronaviridae. The disease is common throughout the world where chickens are produced commercially. PCR on reverse transcribed RNA is a potent technique for the detection of IBV. In comparison with classical detection methods, PCR-based techniques are both sensitive and fast. Dozens of serotypes and genotypes of IBV have been detected, and many more will surely be reported in future. This research was conducted to identify the infectious bronchitis virus with group specific primers of avian Coronaviruses in Zabol, southeast of Iran. Tracheal swabs were collected from eleven commercial broiler flocks and these swabs were used for RNA extraction. General primers included XCE2+ and XCE2- that amplify all IBV serotypes were used. Primers MCE1+, BCE1+ and DCE1+ was used to amplifying the specific nucleotide sequence of Massachusetts, 4/91 and D274 serotypes, respectively. The results of this study showed that 36.36% of the sampled flocks were positive to IBV by RT-PCR. Moreover, the Massachusetts was the identified serotype of infectious bronchitis virus. The results provide the first molecular evidence for the presence of infectious bronchitis virus and Massachusetts serotype in Zabol.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-36) contains supplementary material, which is available to authorized users.

Development of a Microplate Lectin-Capture RT-PCR (MLC-RT-PCR) for the Detection of Avian Infectious Bronchitis Virus

Rapid, sensitive and specific methods are necessary to confirm the diagnosis of outbreaks of avian infectious bronchitis virus (IBV) infection. The amplification of IBV genome by reverse transcription followed by polymerase chain reaction (RT-PCR) has been one of the most used methods for the detection of this virus in clinical samples. To reduce the time and the number of steps in the molecular diagnosis of IBV, we developed a sensitive and rapid detection method based on viral capture by a lectin (Concanavalin A—Con A) in the microplate wells, followed by RT-PCR to amplify the S1 gene. The detection limit of IBV was 103 EID50/ml for the amplification of 5’part of the S1 gene, and 104 EID50/ml for the amplification of full S1 gene. This technique was specific for IBV detection, and no amplified products were detected for other avian viral pathogens (bursal infectious disease virus, avian metapneumovirus and Newcastle disease virus). The MLC-RT-PCR was as sensitive as conventional RT-PCR, and virus isolation method for the detection of IBV in tissue samples collected from experimentally infected birds. The MLC-RT-PCR technique demonstrated a great potential for the rapid and specific diagnosis of IBV.