title: Validation of an immunohistochemistry assay to detect turkey coronavirus: a rapid and simple screening tool for limited resource settings.
abstract: The objective of the present study was to develop and apply the direct immunohistochemistry (D-IHC) assay to search for turkey coronavirus (TCoV) antigens in formalin-fixed embedded-paraffin tissues by the use of biotin-labeled polyclonal antibody. Twenty-eight-day-old embryonated turkey eggs (n = 50) were inoculated with TCoV-purified virus, and 3 d after inoculation, sections from ileum, ileum-cecal junction, and ceca were harvested, fixed in neutral formalin, and embedded in paraffin blocks and used as positive control. In addition, a total of 100 field samples from ileum, ileum-cecal junction, and ceca, collected from 30 to 45-d-old turkeys poults experiencing an outbreak of acute enteritis, were used to search for TCoV by the same D-IHC. All results were compared with those obtained by conventional RT-PCR and indirect fluorescent antibody assay (IFA) for all tested samples. Turkey coronavirus was detected in experimentally infected embryo tissues and also in field samples in 100% of ileum-cecal junction and ceca by the 3 detection procedures. With IFA as a reference assay, sensitivity and specificity of D-IHC were 98 and 58%, whereas sensitivity and specificity of reverse transcription-PCR were 96 and 66%, calculated from the total of tested samples from experimental infection. Each of the examined procedures was highly specific (D-IHC, 93%; RT-PCR, 90%), sensitive (D-IHC, 85%; RT-PCR, 86%), and agreement of both D-IHC and RT-PCR was 99 and 100%, respectively, compared with IFA results obtained from all the field samples. These findings demonstrated the utility of D-IHC for direct detection of TCoV from field samples and considering the sensitivity and specificity found here, can be used as an alternative technique.
authors.affiliation: São Paulo State University, Laboratório de Virologia, Departamento de Apoio, Produção e Saúde Animal, CEP, Brazil. tcardoso@fmva.unesp.br
authors.forename: T C
authors.initials: TC
authors.lastname: Cardoso
authors.forename: T L L
authors.initials: TL
authors.lastname: Castanheira
authors.forename: M C B
authors.initials: MC
authors.lastname: Teixeira
authors.forename: A C G
authors.initials: AC
authors.lastname: Rosa
authors.forename: K Y
authors.initials: KY
authors.lastname: Hirata
authors.forename: R D
authors.initials: RD
authors.lastname: Astolphi
authors.forename: M C R
authors.initials: MC
authors.lastname: Luvizotto
mesh_terms: D000818:Animals; D000914:Antibodies, Viral; D004752:Coronavirus, Turkey / Q000302:isolation & purification*; D004196:Disease Outbreaks / Q000662:veterinary; D004753:Enteritis, Transmissible, of Turkeys / Q000821:virology*; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D007150:Immunohistochemistry / Q000191:economics / Q000379:methods / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D014422:Turkeys / Q000821:virology*
chemical_list: D000914:Antibodies, Viral