Detection of Antibody to Turkey Coronavirus by Antibody-Capture Enzyme-Linked Immunosorbent Assay Utilizing Infectious Bronchitis Virus Antigen

Abstract

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.

Immunohistochemistry Assay to Detect Turkey Coronavirus (TCoV) from Experimentally Infected Poults

“…In addition, basal lamina was infiltrated with mononuclear cells, presented submucosa oedema compatible with viral enteritis (results not shown). These findings have been described for PEMS, however they are common to other enteric pathogens [6][7][8] . Regarding to the intestines analyzed here, they were flaccid, thin-walled, and filled with loose contents, and the disease was acute, with symptoms appearing and lasting for about 3 weeks In order to determine the efficiency of biotinlabeled antibody produced, the direct ELISA was performed and the IBV antigen reacted specific against the respective antibody, producing an optical density pf 1.0 (DO), whilst the non-labeled antibody produced any OD [9][10][11][12] .…”

“…In fact, Coronaviruses are in the family Coronaviridae, which are enveloped, positive-stranded RNA virus that infects a wide range of mammalian and avian species [6] . So far, Turkey Coronavirus (TCoV) and infectious bronchitis virus (IBV) belong to antigenic group III and share antigenic similarity [7] . This biological characteristic allows using antigen, as well as, antibodies raises against IBV, to develop and apply immunological tools for the TCoV diagnosis [8][9][10] .…”

Coronaviruses in poultry and other birds

“…Some 25 years ago it was suggested that TCoV was closely related to BCoV of Group 2 (Dea et al ., 1990). We now know that this is not the case and that TCoV is unequivocally closely related to IBV, both in terms of gene and protein sequences (Breslin et al ., 1999a,b;Cavanagh et al ., 2001), and also antigenically (Guy et al ., 1997;Breslin et al, 2000;Loa et al ., 2000).…”

A Reverse Transcriptase—Polymerase Chain Reaction Assay for the Diagnosis of Turkey Coronavirus Infection

“…They are pleomorphic positive-sense single-stranded RNA viruses measuring 60-220 nm in diameter. 3,11 The characteristic feature of the coronaviral morphology is the presence of long (12-24 nm), widely spaced petal-or pear-shaped surface projections, which impart to the virus the appearance of a solar corona. 14 Three structural proteins have been identified in the virus.…”

“…5,11 The most widely used test for the serologic diagnosis of coronaviral enteritis is the indirect fluorescent antibody test. These procedures are labor intensive and time consuming.…”