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Diagnosis (pathotype) have yet to be discovered. In situ hybridization has
The changes in the diagnosis of avian adenoviruses and the dis- been also described for the detection of viral DNA in infected
eases caused by them reflect the revolutionary developments in tissues (Latimer et al., 1997). Serological methods are also
instrumentation and methodologies over the last quarter century. employed for the diagnosis of FAdV infection, such as ELISA,
agar gel precipitation test (AGPT), indirect haemagglutination,
Diagnosis of aviadenovirus infections virus neutralization. AGPT was widely used for the detection
Diagnosis of FAdV infections can be carried out by a vast array of antibodies (Ab), since it is fast and economic. Comparison
of methods; the main approaches being virus isolation, the of AGTP offered by diagnostic laboratories, that uses FAdV-1
demonstration of virus particles by electron microscopy (EM), as the antigen with a group specific ELISA demonstrated that
detection of viral proteins and nucleic acid of the virus, and ELISA is considerably more sensitive than the AGPT in early
demonstration of viral specific antibodies in serum samples. stages of infection (Philippe et al., 2007). There are numerous
Although the clinical signs are not distinctive for IBH infec- commercially available ELISA kits to choose from, these tests
tions, the gross and histopathological lesions, particularly the are based on whole virus as antigen. For an ELISA based on
intranuclear inclusion bodies in hepatocytes of the liver are char- two non-structural proteins, 100K and 33K of FAdV-1, it was
acteristic and indicative of infection (Grimes et al., 1977). Virus demonstrated that it can distinguish an acute FAdV infection
isolation from suspensions obtained from faecal samples, tissues, from an inactivated virus-based vaccination response (Xie et al.,
such as liver, kidney, caecal tonsils, pharynx could be done in 2013). Monoclonal Abs against FAdVs have been also reported
primary chicken embryo liver and kidney cells, or hepatoma and shown to improve diagnostic assays (Ahmad and Burgess,
cell lines as described in the section ‘Propagation’. Prior to the 2001). For the detection of type specific antibodies, the more
advent of molecular techniques for definitive diagnosis the pres- accurate virus neutralization (VN) tests (plaque reduction assay
ence of viruses or virus-like particles in tissues was often shown and microneutralization assay) are suggested, although ELISA
by electron microscopy. Virus particles or FAdV antigens in can also be used (Hess, 2000; Philippe et al., 2005).
infected cells can be identified by serotype specific antibodies
in an immunofluorescence test (Hess, 2000). Enzyme-linked Diagnosis of egg drop syndrome and
immunosorbent assay (ELISA) is also described for the detec- haemorrhagic enteritis
tion of FAdVs. A group-specific ELISA by Saifuddin and Wilks For the diagnosis of EDS and EDSV similar methods can be
(Saifuddin and Wilks, 1990b) can detect less than 100 TCID applied that are described for FAdVs. For the isolation of EDSV
50
of adenovirus per gram of liver tissue. Over the last decades, the the most sensitive medium is embryonating duck or geese eggs
newly emerged molecular biological tools, such as restriction originating from SPF or EDSV free flocks. Alternatively, chicken
fragment length polymorphism (RFPL), and the different kinds embryo liver cells support the replication of the virus, while
of polymerase chain reaction (PCR) assays have been applied CEFs are insensitive (Smyth, 2013). The presence of virus can
for the detection of viral nucleic acid and grouping and differ- be shown by haemagglutination using 0.8% chicken red blood
entiation of isolates (Erny et al., 1991; Raue and Hess, 1998; cells (Zsák and Kisary, 1981). The hexon-based PCR combined
Hess et al., 1999; Jiang et al., 1999; Hess, 2000; Raue et al., 2002; with restriction enzyme analysis not only identifies the EDSV
Lüschow et al., 2007; Steer et al., 2009). The early grouping of DNA but differentiates it from fowl adenoviruses (Raue and
FAdVs by RFLP served the basis of the establishment of species Hess, 1998; Dhinakar Raj et al., 2003). More recently a real-
in the genus Aviadenovirus and it was proven correct over and time polymerase chain reaction was developed (Schybli et al.,
over again (Zsák and Kisary, 1984). Direct RFLP of the viral 2014). Antibodies to EDSV can be detected by different meth-
DNA or analysis of PCR products by restriction enzymes is a ods, the most frequented ones are ELISA, haemagglutination
powerful tool for the differentiation of field isolates and helpful inhibition (HI) and VN tests (Adair et al., 1986; Raj et al.,
in epizootiological investigations (Meulemans et al., 2001; Ojkic 2004, 2007; Smyth, 2013).
et al., 2008b; Kaján et al., 2013). Most avian adenovirus PCR The diagnosis of haemorrhagic enteritis (HE), marble spleen
utilizes the hexon gene sequences for primer design, the hexon disease (MSD) and avian adenovirus splenomegaly (AAS) is
pedestals and loops permit the design of oligonucleotides that succinctly described by Pierson and Fitzgerald (2013). All three
generate a PCR product from all twelve serotypes of fowl adenovi- viruses can be propagated in young turkeys; however, a more
ruses. Numerous real-time PCR assays with increased sensitivity convenient option is to use MDTC-RP19 cells (Nazerian and
have been described for both research and diagnostic purposes, Fadly, 1982, 1987). Turkey leucocytes also have been described
making the laboratory turnaround time shorter (Romanova et for the propagation of HEV (van den Hurk, 1990). Viral antigens
al., 2009) and the identification of serotypes accurate (Marek can be detected by AGID, and immunofluorescent or immunop-
et al., 2010a). High-resolution melting-curve analysis technique eroxidase staining of infected cells (Fasina and Fabricant, 1982;
is also available for IBH diagnosis and for virus typing (Steer et Fitzgerald et al., 1992). Traditional, nested and real-time PCR
al., 2009, 2011). Sequencing of the PCR products and providing assay are also described (Hess et al., 1999; Beach et al., 2009b;
phylogenetic trees are offered by many diagnostic laboratories. Mahsoub et al., 2017). For antibody detection the AGID test is
The identification of types of FAdV isolates is done with cer- replaced by the more sensitive ELISA, and they are commercially
tainty; methods, unique sequences associated with virulence available (van den Hurk, 1986; Nazerian et al., 1990).