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The culture is observed microscopically for evidence of ciliostasis under light microscope. Complete impairment of ciliary activity usually is considered as a positive culture [ 60 ]. Successful growth of IBV has been demonstrated in organ cultures derived from kidney, intestine, proventriculus, and oviduct. However, susceptibility of these organs to IBV can be influenced by the strain of the virus and the amount of virus presence in the sample infective dose.

While a study suggested the universality of using kidney, bursa, and proventriculus in growing IBV, a poor result was obtained when IBV was propagated in cultures derived from different intestinal segments [ 61 ]. An advantage of this method includes easy titration and serotyping of IBV, since no virus adaptation is required [ 62 ]. Possible constraints include lack of affinity of some IBV strain for some organ cells and difficulty in differentiating ciliostasis arising from other viruses, such as Newcastle disease virus and avian adenovirus [ 33 ].

Electron microscopy provides a direct means of detecting and identifying IBV in biological samples based on morphological characteristics of coronavirus. Positive cultures are confirmed based on the presence of coronavirus-like pleomorphic structures with spike projections, following negative staining with phosphotungstic acid Figure 9. Apart from the negative staining method, transmission electron microscopy TEM is also a useful tool which enables the visualization of virus-like particles in infected cells [ 59 , 63 ]. However, this method is often applied to understand viral attachment and entry into the cell but is not a specific diagnostic test [ 35 ].

These methods work based on antigen-antibody reactions [ 64 , 65 ]. Immunoperoxidase methods such as the avidin-biotin complex ABC have been used successfully to localize IBV antigen in tissue samples [ 66 ]. Likewise, indirect immunofluorescent assay is the most frequently used fluorescent technique [ 66 , 67 ].

A pan-coronavirus primer, targeting a conserved region of different coronavirus isolates, could also be used in one-step RT-PCR amplification of IBV strains [ 55 ]. However, amplification and sequencing of the S1 gene provide a reliable means for genotypic classification of new IBV strains [ 74 ]. A serotype-specific PCR assay has been designed to enable differentiation of Massachusetts, Connecticut, Arkansas, and Delaware field isolates [ 73 ].

Full-length sequence of IBV S1 glycoprotein could be targeted for amplification and enzymes analysis [ 72 , 76 ]. RFLP allows differentiation of various known IBV strains, based on their unique electrophoresis banding patterns defined by restriction enzyme digestion [ 72 , 77 ]. The assay was found to be comparable with traditional virus neutralization assay, although some strains such as the Gray and JMK strains were reportedly difficult to differentiate using arrays of restriction enzymes, thus limiting the universal application of this method [ 72 ].

Recently, a high resolution melt curve analysis HRM was also developed to allow differentiation of field from vaccine IBV strains as well as for rapid and sensitive detection of recombinant variants [ 83 , 84 ]. Meir et al. The authors, however, reported variations in sensitivity when either N-gene or S1 genes were targeted as well as when different samples are used for viral amplification.

While these methods are more sensitive than standard RT-PCR, they are more expensive as well and might be beyond the financial capacity of many producers. For genotyping, S1 gene usually is amplified using RT-PCR, sequenced, and subjected to bioinformatics analyses [ 88 , 89 ]. Lack of method standardization among laboratories, particularly with respect to the S1 gene segment length that is used in phylogenetic analysis, limits genotyping to some extent.

Currently, molecular methods such as next generation sequencing NGS have been introduced to sequence whole genomes within limited periods of time, though this approach has been used only in the laboratory. Several respiratory diseases, such as Newcastle disease ND , infectious laryngotracheitis, infectious coryza, avian metapneumovirus aMPV , and avian influenza AI , may produce clinical signs similar to avian infectious bronchitis.

However, certain clinical features, including neurological signs and diarrhoea in ND, high mortality in AI, and pronounced head swelling in coryza, are uncommon in IBV infection and thus may be used in ruling out or arriving at narrowed tentative differential list [ 33 , 90 ]. Ever since the first identification of IBV in s, the poultry industry has suffered a growing number of emerging IBV serotypes.

Lack of effective diagnostic methods and vaccines that could easily tackle the menace caused by multiple IBV serotypes is partly blamed for the serious economic losses as results of infectious bronchitis disease. Conventional detection assays such as virus neutralization and virus isolation have been used extensively, but, due to lack of sensitivity and specificity of serological assays and laborious nature of virus isolation methods, these assays have gradually been replaced by the new sensitive and specific assays such as RT-PCR, RFLP, and qRT-PCR that enable rapid genotyping and identification of new IBV strains.

However, there is a need for standardization across laboratories with respect to the type and length of target gene to be considered for genotyping so as to ensure common understanding of genotype distributions in order to guide vaccine selection for prevention and control. Faruku Bande conceived the idea, collected and studied published papers, drafted the review paper, and made all uncited photos from cases handled in his Ph. Siti Suri Arshad provided photos used in Figures 1 , 5 , and 9. All authors have read and agreed with submission of final paper to the journal.

Dennis F. Lawler also provided paper-editing assistance. Advances in Virology. Indexed in Web of Science. Journal Menu. Special Issues Menu. Subscribe to Table of Contents Alerts. Table of Contents Alerts. Abstract Infectious bronchitis IB is one of the major economically important poultry diseases distributed worldwide. Introduction Infectious bronchitis IB causes significant economic losses to the poultry industry worldwide [ 1 , 2 ]. Pathogenesis Infectious bronchitis virus infects primarily the respiratory system. Host Susceptibility Although domestic fowl Gallus gallus and pheasant Phasianus spp.

Age and Breed Predisposition Chickens of all ages and breed types are susceptible to IBV infection, but the extent and severity of the disease is pronounced in young chicks, compared to adults. Infection and Transmission The virus is transmitted via the respiratory secretions, as well as faecal droplets from infected poultry.

Incubation Period Generally the short incubation period for IBV varies with infective dose and route of infection. Clinical Course and Manifestations In the host, initial infection occurs at epithelia of Harderian gland, trachea, lungs, and air sacs. Figure 2: Irregularity in the shape and sizes of eggs from natural IBV infected breeder chickens a. Watery albumen from IBV infected chicken b left compared to normal egg b right. Figure 3: Gross lesions observed on respiratory organs of chicken naturally infected with IBV.

Presence of mucoid secretion, congestion, and hyperaemia in the trachea a ; mild focal areas of lung consolidation b. Figure 4: Histopathological changes in the trachea of naturally IBV infected chicken.

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Note: the marked infiltration of lymphocytes within the epithelia black arrow b and evidence of mucosal secretions of goblet cells yellow arrow a. Figure 5: Gross lesions in kidney of chicken following experimental infection with a nephropathogenic infectious bronchitis virus. Note: swelling and congestion of the kidney arrow courtesy: Siti Suri Arshad.

Figure 6: Chicken showing natural IBV infection.

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Accumulation of egg yolk in abdominal cavity a ; slightly enlarged, pale, friable liver b and multiple petechial haemorrhages on the serosal surfaces of proventriculus c , gizzard d , and small intestine e. Figure 7: Cystic oviduct in week-old chicken experimentally infected with a CR88 infectious bronchitis virus strain. Note the distention of the entire oviduct and fluid accumulation arrow. Note evidence of dwarfism and curling of the toes in IBV infected embryo right compared to a noninfected control embryo left.

Figure 9: Negative staining electron microscope showing spherical shape of virus with typical spike projections arrow surrounding the virion of avian infectious bronchitis virus courtesy: Siti Suri Arshad. Figure Electropherogram showing 1. Figure Neighbour joining phylogenetic analysis based on nucleotide acid sequence of S1-spike gene of classical and variant IBV strains identified in different countries. The tree was drawn with MEGA5 software using bootstrap replicates.

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