Page 343 - Avian Virology: Current Research and Future Trends

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Commercial ELISAs are available and used for the serological Clinical specimens including respiratory exudates and tracheal
monitoring of ILT despite the current paradigm that only poor swabs may be used as test materials in most PCRs, although
correlation exists between systemic antibody response and pro- formalin-fixed and paraffin-embedded tissues have shown to be
tection against ILTV. Most commercial ELISA kits use antigens applicable at least in one assay (Humberd et al., 2002). Feather
generated by sonication of ILTV infected cell culture (Noor- pulps/shafts have also been used as a convenient specimen for
mohammadi and Devlin, 2014). However, recent studies have sensitive detection of ILTV by PCR and monitoring of vaccine
used ELISAs based on specific recombinant viral glycoproteins, take (Davidson et al., 2009, 2016). Chicken droppings, isolator
including gB, gC, gD, gG and gI (Shil et al., 2012; Godoy et al., dust and bedding materials can also be used in PCR, but these
2013; Kanabagatte Basavarajappa et al., 2014, 2015). At least in specimens are useful for monitoring viral load in a flock as
vectored ILTV vaccines, a high level of systemic neutralizing anti- opposed to a diagnostic tool (Roy et al., 2015).
bodies against viral gD was correlated with protective immunity Some PCR systems have an additional feature of strain identi-
against ILTV (Kanabagatte Basavarajappa et al., 2014, 2015). fication when combined with a post-amplification technique (see
below). A PCR system targeting the TK (UL23) gene of ILTV
Detection of the virus (Kirkpatrick et al., 2006b) is used as a routine diagnostic tool by
The virus may be isolated and identified in embryonated hen eggs the authors of this chapter although another protocol using ICP4
or cell culture, or detected directly in tracheal exudate by electron gene is used in other diagnostic laboratories (Chacon and Fer-
microscopy, immunofluorescence or ELISA, or by PCR (García reira, 2009).
et al., 2013a).
Strain identification
Isolation and identification Strain identification of ILTV is essential for epizootiological
Isolation of ILTV via embryonated hen eggs or cell culture is no investigations. Differentiation between field and vaccine strains
longer used as a means of routine diagnosis in most diagnostic is also important before an appropriate approach is taken in the
laboratories. This is now replaced with more rapid techniques face of an outbreak. For example, if an ILT outbreak is caused
such as immunofluorescence and PCR. Nevertheless, isolation of by a field strain (as opposed to be a vaccine strain reaction or
ILTV is an essential technique in ILTV research. re-emergence), vaccination with an attenuated vaccine may be
The virus is best grown on the CAM of 10- to 12-day-old recommendable in face of the outbreak.
embryonated hen eggs and induces formation of plaques. A Despite differences in virulence, transmissibility, and size/
number of primary cell cultures can also be used for isolation and morphology of the plaques induced on CAM, all ILTVs are
propagation of the virus, with chicken embryo liver cells appear- thought to be antigenically indistinguishable from each other by
ing to be most sensitive (See ‘Virus propagation’). Cytopathic virus neutralization test, immunofluorescence assay and cross
effects of the virus are presented by cell swelling, increased refrac- protection studies (García et al., 2013a). Hence examination
toriness and fusion (Tripathy and García, 2008). of viral DNA using a range of techniques has been commonly
Confirmation of the identity of the virus isolate can be made utilized for differentiation of ILTV strains. Historically, RFLP
by conventional techniques such as neutralization tests in eggs or of whole viral genomic DNA, or a specific gene have been used
cell culture, electron microscopy, immunofluorescence, or PCR. for strain identification purposes, but these are time consuming.
Also, as they examine only a small number of specific sites of the
Immunological detection genome, they have a very low differentiation power.
Both immunofluorescence and ELISA can be used for the immu-
nological detection of viral antigens in clinical specimens (García PCR based strain identification techniques
et al., 2013a). For immunofluorescence tests, tracheal mucosal A combination of long range PCR and RFLP analysis of multiple
scrapings or cryostat sections can be tested. For ELISA, tracheal genes or genomic regions has been described by multiple workers
exudate is usually tested. and the technique used to establish the origin of emerging ILTVs
in a number of countries (Kirkpatrick et al., 2006b; Oldoni and
Molecular detection by PCR García, 2007; Oldoni et al., 2008; Moreno et al., 2010; Blacker
Both conventional and real time PCR have been described with et al., 2011; Chacón et al., 2010; Kim et al., 2013; Yan et al.,
some reported to be more sensitive than virus isolation (Wil- 2016). The specific genes or genomic regions that are examined
liams et al., 1994; Alexander and Nagy, 1997; Creelan et al., 2006; vary depending on the laboratory, although ICP4 and TK are
Mahmoudian et al., 2011; Vagnozzi et al., 2012a; Zhao et al., most commonly included in the analyses. It is notable however
2013). A loop-mediated isothermal amplification (LAMP) assay that PCR-RFLP of multiple genomic regions is time-consuming
has been described as a simple and rapid tool for the detection (takes approximately 2 days to complete), labour intensive,
of ILTV but it was shown to be less sensitive than conventional and relatively expensive. Additionally, the technique relies on
real time PCR (Xie et al., 2010; Ou et al., 2012). Also, PCR-based nucleotide differences that affect the recognition site of a given
Luminex Suspension Microarray, droplet and nanofluid assays restriction enzyme, and thus variations outside these sites will
have been developed for multiplex high throughput detection of remain undetected. Recent investigations of a number of major
a number of respiratory viruses including ILTV (Laamiri et al., ILTV outbreaks in Australia by the authors of this chapter have
2016; Periyannan Rajeswari et al., 2017; Croville et al., 2018). found ILTVs that were indistinguishable from a current vaccine