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strain (unpublished data) using this methodology. It is therefore field isolates of ILTV through eggs and/or cell culture, and also
possible that the combination of PCR RFLP currently used in the development of vaccines which utilize other viral vectors to
diagnostic laboratories may not be totally reliable for strain iden- express immunogenic proteins from ILTV. Other approaches to
tification of ILTVs (see ‘Vaccines’). ILTV vaccine development, including generation of ILTV dele-
A TaqMan Real-time PCR assay has been developed and vali- tion mutants, are currently being explored. The history, challenges
dated for the detection and differentiation of field strain from a and advancements in the area of ILTV vaccine development and
gG deficient vaccine strain in Australia (Shil et al., 2015). use has been recently reviewed (Coppo et al., 2013; García, 2017)
and are further described below.
Multilocus sequencing and whole genome
sequencing (WGS) Attenuated vaccines
Nucleotide sequence analysis of PCR amplicons generated from Attenuated, or modified live, ILTV vaccines have been produced
individual ILTV genes has been described by several workers for since the 1960s and are used widely in major poultry produc-
strain identification purposes (García et al., 2013a). However, ing regions worldwide. These vaccines were produced by serial
SNPs are relatively rare in ILTV genomes and genetic diversity passage of ILTV isolates through eggs, and/or cell culture, to
appears to occur most commonly through recombination of large attenuate the virus. Most attenuated vaccine strains of ILTV are
regions of the genome (Lee et al., 2013; Menendez et al., 2014). A propagated in eggs (CEO vaccines) although they can also be
recent study (Choi et al., 2016) has shown that sequence analysis produced in tissue culture (tissue culture origin vaccines, TCO
of multiple genes including UL54, UL52, gB, ICP18.5, ICP4, and vaccines). Different countries typically produce and use different
gJ genes had a better strain differentiation power than PCR-RFLP attenuated vaccine strains of ILTV. An overview of the attenuated
of multiple genomic regions described above. vaccine strains that have been generated and used in different
The first complete genome sequence of ILTV was published countries has recently been published (Menendez et al., 2014).
in 2011 (Lee et al., 2011a). Since then the whole genome These attenuated vaccines have served poultry industries well, but
sequence from a large number of ILTVs has been determined they do have a number of limitations. These limitations include
and compared to those previously available in the GenBank (Lee reversion to virulence following in vivo passage (Guy et al., 1991),
et al., 2011b, 2013; Chandra et al., 2012; Lee, S.W. et al., 2012; incomplete attenuation resulting in vaccine viruses that can cause
Spatz et al., 2012; García et al., 2013b; Kong et al., 2013; Agnew- clinical signs of disease (Guy et al., 1990; Kirkpatrick et al., 2006a;
Crumpton et al., 2016; Piccirillo et al., 2016). The information Oldoni et al., 2009; Coppo et al., 2011), the ability to establish
has been extremely useful in exploring the relationship between latency with reactivation (Hughes et al., 1991a), and the ability
ILTV isolates on a global scale and in determining the mechanism to regain virulence through recombination with other vaccine
behind the emergence of new ILTVs with increased virulence and strains (Lee, S.W. et al., 2012). In addition, interference between
transmissibility in a number of countries. These studies have also vaccines administered at the same time has been observed, with a
been instrumental in defining genetic markers for accurate strain TCO ILT vaccine, but not a CEO ILT vaccine, showing reduced
identification of ILTVs especially vaccine and field strains. vaccine efficacy when administered concurrently with Newcastle
disease virus (NDV) and IB vaccines (Vagnozzi et al., 2010).
Future of the diagnostics There are also challenges associated with delivering attenu-
Multi-locus and WGS are highly useful tools for strain identifica- ated vaccines to poultry flocks. Methods of delivery that are
tion purposes although they are still considered time consuming, suitable for mass vaccination are favoured due to cost and labour
require extensive interpretation of the results, and may be prone constraints. These methods typically include administration via
to misinterpretation. With further advancement of whole genome drinking water, spray, or via eye drop. Delivery by drinking water,
sequencing technology, including significant reductions in cost in particular, is associated with a number of limitations, includ-
and turnaround time, emergence of in-house systems, and new ing non-uniform delivery to chickens (Fulton et al., 2000) with
DNA extraction tools, it is expected that WGS will become a individual chickens who do not drink the water containing the
routine technique for strain identification of ILTV isolates in the vaccine remaining unvaccinated, and chickens who drink less
near future. than desired receiving less than the full dose of vaccine. Further-
more, the distribution of live vaccine through the drinking lines
in poultry sheds may also be non-uniform (Fulton et al., 2000). In
Vaccines addition, when vaccine is delivered to chickens via drinking water,
Vaccination and appropriate biosecurity procedures are the most the vaccine virus does not easily reach the tissues that can best
effective methods of controlling disease caused by infection support viral replication (laryngeal, tracheal and conjunctival
with ILTV. Vaccines to control ILTV have been used since 1934 surfaces), which can impact the efficacy of the vaccines (Robert-
(Brandly and Bushnell, 1934). This was the first time that vacci- son and Egerton, 1981) and can potentially allow vaccine viruses
nation had been used to control a major viral disease in poultry. to circulate and gain virulence (Samberg et al., 1971). Vaccine
Initially, vaccination involved application of virulent virus to the delivery via eye-drop allows more uniform delivery of the vaccine
cloaca of chickens (Brandly and Bushnell, 1934). Since then (Fulton et al., 2000) and delivers the vaccine directly to tissues
ILTV vaccine development has progressed and has included that can support viral replication (Robertson and Egerton, 1981),
the generation of live attenuated vaccines by serial passage of however the labour costs associated with this method of delivery