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126 | Brown and Eterradossi

been shown to normally occur between 3 and 5 days (McDougall Antibody detection
and Cook, 1986; Wilding et al., 1986; Jones et al., 1988; Buys et The ELISA assay, immunofluorescence (IF) and serum neutrali-
al., 1989a; Cook et al., 1991, 1993c). Transfer of samples to the zation (SN) tests are all applicable for the detection of antibodies
laboratory should be immediate and samples should be ideally raised against AMPV. However, the ELISA test is by far the most
refrigerated with freeze-packs. If a delay more than 24 hours is commonly used. Its main advantage over the other two tests
unavoidable samples should be frozen at –70°C. is that antibodies reacting in ELISA remain long after those
Most primary isolations are best performed on tracheal organ detected in IF and SN. Antibodies reacting in IF and SN usually
cultures (TOC) that are prepared from SPF chicken, turkey, or cannot be detected more than two weeks after the initial infec-
Muscovy duck embryos (depending on the species from which tion yet antibodies reacting in ELISA can be detected 6–7 weeks
the virus was isolated) just before hatch at 19, 24 and 27–30 days after initial. Numerous commercial and in-house ELISAs for the
post incubation, respectively, or in 6- to 8-day-old embryonated detection of antibodies against whole-AMPV antigen have been
turkey or chicken eggs via the yolk sac. For TOCs virus isolation developed (Grant et al., 1987; Chettle and Wyeth, 1988; O’Loan
is confirmed when ciliostasis in the lumen of the tracheal rings is et al., 1989; Eterradossi et al., 1992, 1995; Mekkes and de Wit,
observed by light microscopy. Successful isolation in eggs results 1998; Turpin et al., 2003; Maherchandani et al., 2004, 2005) as
in haemorrhages and mortality of the embryo (Cook et al., 1999; well as those using recombinant protein as antigen (Gulati et al.,
Panigrahy et al., 2000). Isolation in cell cultures (VERO or QT-35; 2000, 2001; Lwamba et al., 2002b; Luo et al., 2005;). The results
see ‘Propagation’, below) can also be effective and is confirmed of these studies stress that for correct diagnosis the right antigen
by consistent cytopathic effect. Of the different options TOC for the right antibodies should be used (i.e. the infecting AMPV
has been most favoured; however, some viruses may be easier to strain and the ELISA antigen should belong to the same AMPV
isolate in eggs or indeed in cell culture thus it is useful to have all subgroup) and that this can even extend to having an antigen that
techniques available. originates from the same country in which the serum has been
collected (Eterradossi et al., 1995). The good correspondence
Viral antigen between the subgroups of the infecting AMPV strain and the
Detecting viral antigens in tissues, cell cultures or tracheal organ AMPV ELISA antigen is especially important when investigating
cultures can be achieved using immunofluorescence or immu- the usually moderate antibody responses possibly induced by live
nohistochemistry (Baxter-Jones et al., 1987, 1989; Jones et al., attenuated vaccines, as in such studies the use of an heterologous
1988; O’Loan and Allan, 1990; Majó et al., 1995; Hartmann et ELISA antigen may lead one to wrongly conclude that vaccine-
al., 2015) staining and, although limited for diagnostic purposes, induced antibody response is lacking (Eterradossi et al., 1995;
are important in studies concerning virus replication kinetics and Toquin et al., 1996).
pathogenesis studies.

Detection of viral RNA Prevention and control
Viral RNA molecules can be detected using RT-PCR techniques. This section focuses on vaccination for controlling AMPV infec-
RT-PCRs are extremely useful for diagnostic purposes as they are tion as this is currently the main approach however; good farm
highly sensitive, specific and rapid to perform. Classic end point management practices should not be overlooked. Poor hygiene,
AMPV RT-PCR assays, where positive results are observed by the a lack of temperature control, high stocking densities, poor litter
presence of an amplified DNA product in an agarose gel have been quality and poor ventilation can all exacerbate the severity of
developed (Bayon-Auboyer et al., 1999; Cavanagh et al., 1999) disease (Lister, 1998; Jones, 2001; Eterradossi et al., 2015); in
as well as real time RT-PCRs where fluorescent oligonucleotide general, one should attempt to reduce factors of stress as much as
probes or intercalating dyes give out a signal at each amplification possible. Good hygiene practices should extend to delivery and
cycle (Velayudhan et al., 2005; Guionie et al., 2007; Kwon et al., catching personnel.
2010; Cecchinato et al., 2013; Franzo et al., 2014; Lemaitre et al., Both live-attenuated and inactivated vaccines have been devel-
2018). In both systems RT-PCRs that are capable of detecting all oped (Giraud et al., 1987a; Buys et al., 1989a; Cook et al., 1989;
the four known AMPV subgroups use oligonucleotides targeting Cook and Ellis, 1990; Williams et al., 1991a; Kapczynski et al.,
conserved regions in the N open reading frame (ORF) (Bayon- 2008) and are commercially available. Under experimental condi-
Auboyer et al., 1999; Lemaitre et al., 2018). Those that are subgroup tions, live attenuated A or B vaccines have been shown to provide
specific target zones of sequence in ORFs less conserved across the protection against subgroup A, B, C (turkey strain) and D chal-
subgroups (Guionie et al., 2007). In diagnostic terms broad range lenge (Cook et al., 1995, 1999; Eterradossi et al., 1995; Toquin et
RT-PCRs are often performed first so as to have the best chance al., 1996, 1999b). The ability of subgroup A and B live attenuated
of detecting the virus which maybe of one of the four known sub- vaccines to offer protection against challenge with subgroup A, B
groups or indeed an unknown. Subgroup-specific RT-PCRs are or D (proposed type I AMPVs) may not be surprising consider-
then often performed on positive samples followed by nucleotide ing their close genetic and antigenic relationships however, it is
sequencing so as to classify the virus phylogenetically; however, surprising that they also provided protection against challenge
one should keep in mind when interpreting the results that phylo- with the more genetically and antigenically distant subgroup C
genetic relationships between viruses can change depending on the virus (turkey strain) (proposed type II AMPV). This situation
region of the genome amplified and the size of the amplicon used. is further complicated by the fact that live attenuated subgroup