Download Molecular characterization of avian infectious laryngotracheitis

Molecular characterization of avian infectious
laryngotracheitis-isolates from Switzerland
C. NEFF*, C. SUDLER and R.K. HOOP
Institute of Veterinary Bacteriology, National Reference Centre for Poultry and Rabbit
Diseases (NRGK), Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 270,
8057 Zürich, Switzerland.
*Corresponding author: [email protected]
Five to twenty annual outbreaks of infectious laryngotracheitis (ILT) occur mainly in
small, backyard and fancy breed flocks in Switzerland. In order to determine the
source of ILT outbreaks and to gain data on the possible role of vaccine strains in
clinical disease, 57 strains of ILT viruses (ILTV) were examined, including field
isolates from Switzerland (n=44) and Southern Germany (n=11) as well as two vaccine
strains of different origin. A fragment of the thymidine kinase gene was amplified by
polymerase chain reaction (PCR) and digested by several restriction endonuclease
(RE) enzymes. Restriction fragment length polymorphism (RFLP) analysis indicated
that 39 of 44 Swiss field strains (89%) were identical to all German field strains and
both vaccine strains. PCR/RFLP could therefore differentiate some, but not all Swiss
field strains from the vaccine strains tested. We conclude that two clones of ILTV have
been circulating in Switzerland during the last twenty years, and that PCR/RFLP has a
practical application in rapid identification of ILTV from clinical samples.
Keywords: infectious laryngotracheitis; PCR/RFLP; poultry; Switzerland; thymidine kinase
Introduction
Avian infectious laryngotracheitis (ILT) is a severe clinical respiratory disease of chickens.
The acute form is characterized by breathing difficulty, coughing up of blood or bloody
mucus, swollen orbital sinuses, high morbidity (up to 100%), variable mortality and
decreased egg production in laying hens. The chronic form of ILT may look like any other
respiratory infection (Albicker-Rippinger and Hoop, 1998). The agent is gallid herpesvirus 1
(GaHV-1), which belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae
(Roizman, 1982). The genome of ILTV is a linear double-stranded DNA of 155 kb in size
(Chang et al., 2000). Latently infected chickens are the primary source of ILT outbreaks.
In Switzerland, ILT is a notifiable disease since 1987. Outbreaks of ILT occur mainly in
small, backyard and fancy breed flocks and occasionally in commercial chickens (Hoop et
al., 1993; Hoop et al., 1986). Although live attenuated vaccines against ILT are available in
Europe, their use is not allowed in Switzerland. The main reasons for the ban are economic
considerations, negligible number of outbreaks, possible reversion of vaccine strains to
virulent virus after sequential bird-to-bird passage (Guy et al., 1991; Guy et al., 1990), difficult
diagnostic analysis of infections carried by vaccinated birds and tedious diagnostic
differentiation between field and vaccine strains.
For identifying ILTV molecular methods have proven to be more sensitive than virus
isolation, being able to detect acute as well as latent infections (Alexander and Nagy, 1997;
Keam et al., 1991; Key et al., 1994; Williams et al., 1994). Several studies documented that
polymerase chain reaction (PCR) technique in conjunction with restriction fragment length
polymorphism (RFLP) analysis can be successfully used to distinguish field and vaccine
strains (Chang et al., 1997; Creelan et al., 2006; Graham et al., 2000; Han and Kim, 2001;
Kirkpatrick et al., 2006; Sellers et al., 2004).
The objective of the present study was to characterize 44 Swiss ILT isolates from
chickens, peacocks and pheasants with acute infection by PCR/RFLP in order to investigate
the diversity of the strains collected over the last twenty years.
Materials and methods
A total of 44 field isolates from chickens, peacocks and pheasants with acute ILT in
Switzerland, collected over the last twenty years, were analyzed. 11 strains from neighboring
Southern Germany and two ILT vaccines were included in the study. The vaccine strains
were of chicken embryo origin (CEO) and tissue culture origin (TCO).
DNA was prepared according to a standard protocol of Sambrook et al. (1989). Extracted
DNA was used in a PCR to amplify a 2.1 kb fragment of the thymidine kinase gene of ILTV.
PCR oligonucleotide sense and antisense primers were designed on the basis of the
published DNA sequence of the thymidine kinase gene of ILTV by Griffin and Boursnell
(1990). The resultant PCR products were digested by the RE enzymes HaeIII and MspI and
separated by electrophoresis in a agarose gel. The size of RE fragments was compared with
molecular weight standards.
Results and discussion
PCR/RFLP analysis indicated that 39 of 44 Swiss field strains (89%) showed the same
RFLP pattern and were identical to all German field strains and both vaccine strains. The
remaining five Swiss field strains showed a different cleavage pattern (Table 1).
Table 1 Summary of results of RFLP analysis of ILTV field and vaccine strains.
Gene
Enzymes
Field strains
Switzerland
Southern Germany
Thymidine kinase
HaeIII
39 / 1 *
11 / 1
5/2
MspI
39 / 3
11 / 3
5/4
* Number of ILT strains tested / different numbers indicate different RFLP patterns.
Vaccine strains
CEO
TCO
1/1
1/1
1/3
1/3
In this paper, we describe the detection of ILTV by PCR/RFLP targeted to the thymidine
kinase gene in samples collected over the last twenty years from outbreaks of acute ILT in
chickens, peacocks and pheasants in Switzerland.
A full comparison of the RE cleavage products showed that two different RFLP patterns
were found in the 44 Swiss field isolates and the 11 isolates from ILT outbreaks of Southern
Germany. One group contained 39 Swiss and all Southern German field isolates, the other
group comprised five Swiss field isolates. Using the same molecular methods as described in
this report, two different RFLP patterns were also observed among field isolates from
outbreaks of acute ILT in chickens in Taiwan (Chang et al., 1997).
The two vaccine strains from different origin produced identical DNA fragments, in
contrast to the findings of Chang et al. (1997). They compared four different vaccine strains,
one TCO vaccine from Japan and three CEO vaccines from Holland, Iowa and Kansas. As
described in their protocol, the amplification and digestion of the thymidine kinase gene with
the enzymes HaeIII and MspI allowed to differentiate the TCO vaccine from the CEO
vaccines with identical RE cleavage products. In our study, vaccine strains, the TCO vaccine
from Spain and the CEO vaccine from the Netherlands, showed the same RFLP pattern.
These results suggest that TCO vaccine strains may vary in virulence.
The discrimination of virulent ILTV strains from less virulent and CEO vaccine strains by
PCR/RFLP of the thymidine kinase gene and the correlation of different RFLP patterns with
varying virulence of ILTV were described in a previous study from Han and Kim (2001).
Other authors also reported that the virulence of herpesviruses is associated with the
thymidine kinase gene (Han et al., 2002; Mettenleiter, 1991). Increased virulence for the
CEO vaccine, but not for the TCO vaccine, after continuous passages in specific-pathogenfree chickens was observed in a survey from Guy et al. (1991). The authors suggested that
vaccine strains could be involved in field outbreaks.
One of the aims of this project was to determine the source of ILT infections in commercial
and backyard chickens, peacocks and pheasants in Switzerland and to gain data on the
possible role of vaccine strains in clinical disease. Neither the classification of our Swiss field
isolates in virulent and less virulent strains nor the differentiation between field and vaccine
strains was possible. For a more specific characterization and reliable identification of ILTV
field and vaccine strains by PCR/RFLP the examination of several gene regions with a
number of RE enzymes seems to be necessary. Nevertheless, the present study showed
that two various clones of ILTV have been circulating in Switzerland during the last twenty
years. In fact, our findings revealed a practical application of PCR/RFLP in rapid identification
of ILTV in clinical samples, but for epidemiological investigations further studies will be
required.
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