Download detection of newcastle disease virus in infected chicken embryos

Bull Vet Inst Pulawy 50, 3-7, 2006
DETECTION OF NEWCASTLE DISEASE VIRUS
IN INFECTED CHICKEN EMBRYOS
AND CHICKEN TISSUES BY RT-PCR
KRZYSZTOF ŚMIETANKA, ZENON MINTA AND KATARZYNA DOMAŃSKA-BLICHARZ
Department of Poultry Diseases,
National Veterinary Research Institute, 24-100 Pulawy, Poland
e-mail: [email protected]
Received for publication November 30, 2005.
Abstract
RT-PCR for the detection of Newcastle disease
virus (NDV) in allantoic fluids of SPF embryonated eggs as
well as in tissues of SPF chickens infected experimentally is
described. The method proved to be specific as all tested
NDVs were detected and no cross reaction with other RNA
viruses was observed. Sensitivity of the method was
established at 105ELD50/0.1 ml. To detect NDV in chicken
tissues, SPF chickens were inoculated with 106 EID50 of 3
NDV reference strains: La Sota (lentogenic), Roakin
(mesogenic) and Italy (velogenic pigeon variant) and 5 d p.i.
various tissue samples were aseptically collected followed by
RT-PCR and virus isolation on SPF embryos. The results
showed high concordance: 93% (La Sota and Italy) to 100%
(Roakin) between both methods.
Key words: chicken embryos, chickens,
Newcastle disease virus, RT-PCR.
Newcastle disease (ND) is a highly contagious
infection of poultry caused by avian paramyxovirus
serotype 1 (Newcastle disease virus, NDV). The disease
is spread worldwide affecting various species of poultry
and other birds (2, 3, 14). However, chickens appear to
be the most susceptible to the disease whereas aquatic
birds, including geese and ducks, are relatively resistant.
NDV differs in virulence and has been grouped into 5
pathotypes:
velogenic
viscerotropic,
velogenic
neurotropic, mesogenic, lentogenic and asymptomatic. It
was shown that NDV virulence is dependent on the
amino acid sequence at the cleavage site of F0 gene. The
presence of multiple basic amino acids at the cleavage
site indicates that the virus isolate is pathogenic (2). In
diagnosis of Newcastle disease, methods recommended
by OIE Manual (16) and EU Council Directive (5)
comprise isolation on SPF embryonated eggs and
identification in the haemagglutination inhibition test.
Recently, as an alternative, OIE regulations have
proposed methods based on molecular biology (16).
Reverse transcription and polymerase chain reaction
(RT-PCR) methods are applied in many laboratories of
the world for the detection and identification of NDV
(1).
The aim of the present study was to apply RTPCR for rapid detection of Newcastle disease virus in
experimentally infected chicken embryos and tissues of
chickens.
Material and Methods
Embryonated eggs. Specific pathogen free
(SPF) eggs were imported from Valo-Lohmann
(Germany).
Viruses. In the study 17 NDV strains were
used: 3 reference strains representing different
pathotypes: La Sota (lentogenic), Roakin (mesogenic)
and Italy (velogenic, pigeon variant) and 14 field strains
isolated in Poland from: chickens - Radom strain
isolated at the beginning of the 70-ties and 3 strains
isolated in 1990-2004, turkeys - 1 strain isolated in
2004, racing pigeons - 7 strains isolated at the late 80ties and beginning of the 90-ties, kindly provided by the
Department of Microbiology, Agricultural University in
Lublin, and feral pigeons - 2 strains recovered in 2002.
Prior to testing in RT-PCR, all the strains were
propagated on SPF embryonated eggs and allantoic fluid
was used for further studies.
Virus isolation assay. Virus isolation was
performed on 9-11 SPF embryonated eggs according to
the Annex III of the Council Directive 92/66/EEC (5).
Experimantel design. Three groups of four 4week-old SPF chickens kept in isolation were inoculated
intraocularly and intranasally with 106 EID50 of the
following strains of NDV: La Sota, Roakin and Italy.
Five days post inoculation (p.i.) tracheal and cloacal
swabs as well as samples from the trachea, lungs, liver,
spleen, heart, brain, kidneys, bursa of Fabricius,
duodenum, caecal tonsils, and rectum were aseptically
collected. Supernatants of the organs (used for viral
isolation as well as for RT-PCR) were prepared
according to the Annex III of the Council Directive
4
allantoic fluid containing lentogenic La Sota strain (108
ELD50/0.1 ml). Subsequently, RNA was isolated and
RT-PCR was performed according to the procedure
described above. The highest dilution with positive RTPCR signal was determined.
Specificity of RT-PCR. To evaluate specificity
of the method, cDNA of the following RNA viruses was
used: paramyxovirus serotype 3, avian influenza virus
(H5N2 and H7N1), infectious bursal disease virus
(vaccinal 228E strain and very virulent 99/150 Polish
field strain), and avian infectious bronchitis virus
(strains M-41 and 4/91). PCR was performed according
to the protocol described above.
92/66/EEC (5). Tracheal and cloacal swabs were
suspended in PBS with antibiotics (1 ml/swab) and after
1 h incubation at room temperature and centrifugation,
supernatants were harvested. All the supernatants were
pooled in batches of four. Additionally, pooled
supernatants of the trachea, lungs, liver, spleen, kidneys,
heart, and brain (pooled sample No. 1) and duodenum,
caecal tonsils, and rectum (pooled sample No. 2) were
also used as separate samples.
RNA isolation. RNA was isolated from
allantoic fluids using commercial RNeasy Mini Kit
(Qiagen, USA) as recommended by the supplier.
Reverse transcription (RT). cDNA was
synthesized using 5 µl of the total RNA, 4 µl of 5x first
strand buffer, 2 µl of 0.1 M DTT, 1 µl of ribonuclease
inhibitor (20 U/µl), 1 µl of 10mM dNTP, 1 µl (200 U)
of Super-Script II reverse transcriptase (Invitrogen,
USA), 0.5 µl of random hexamers (Promega, USA) in a
total volume of 20 µl for 50 min at 42°C.
Primers. A set of primers according to Creelan
et al. (6): NDV/F (5’ - GGT GAG TCT ATC CGG ARG
ATA CAA G – 3’) and NDV/R (5’ - TCA TTG GTT
GCR GCA ATG CTC T– 3’) that flanks the region
encompassing the cleavage site of the fusion protein
gene (F) was used in the study. The expected size of
PCR product was 202 bp. Oligonucleotides were
prepared in the Institute of Biochemistry and Biophysics
in Warsaw.
Polymerase chain reaction (PCR). PCR was
carried out in a total volume of 20 µl containing 2 µl of
cDNA, 2 µl of 10x PCR buffer, 0.5 µl of dNTP, 1.4 µl
of MgCl2 (25 mM), 0.5 µl of Taq polymerase
(Fermentas, Lithuania) and 1 µl of each primer. The
thermocycler conditions were as follows: 2 min at 94ºC
(initial denaturation), followed by 40 cycles of 15 s at
94ºC (denaturation), 30 s at 48ºC (annealing), 30 s at
72ºC (elongation). The PCR ended with a final
elongation for 7 min at 72ºC.
Detection of PCR products. PCR products
were separated in 1.5% agarose gel in 1 x TAE buffer
stained with ethidium bromide, compared with
molecular mass marker and visualized by ultraviolet
(UV) transillumination.
Sensitivity of RT-PCR. The sensitivity of the
RT-PCR was established by the ten-fold diluting of the
M
1
2
3
4
5
6
7
8
Results
All strains previously identified serologically as
NDV also tested positive in RT-PCR test with NDV
specific primers (Fig 1). No cross reaction was found
with other RNA viruses used in the study (data not
shown). Sensitivity of the RT-PCR has been established
at 105ELD50/0.1 ml (Fig. 2).
Table 1 shows the results of RT-PCR and virus
isolation performed on tissue samples collected from
SPF chickens 5 days p.i. By RT-PCR method the
positive results were obtained in all 13 tested samples
(13/13) p.i. with mesogenic Roakin strain, 11 samples
p.i. with velogenic Italy strain and 8 samples p.i. with
lentogenic LaSota strain. Positive result of virological
examination was noted in all samples (Roakin strain), 12
samples (Italy strain) and 10 samples (La Sota strain).
Both pooled samples (number 1 and 2) were positive in
RT-PCR and virus isolation (La Sota, Roakin and Italy
strains). Italy strain was isolated from all samples after
the first embryo passage. Isolation of Roakin strain
required one passage from all samples except for cloacal
and tracheal swabs. La Sota strain was isolated during
the first embryo passage only from the samples collected
from the respiratory tract, brain, bursa of Fabricius and
pooled sample 1. Concordance between RT-PCR and
virus isolation was: 93% (La Sota and Italy strains) and
100% (Roakin strain).
9
10
11 12 13 14
15 16 17 18
202 bp
Fig. 1. Electrophoresis of RT-PCR products: M: marker Gene Ruler 100 bp DNA Ladder (Fermentas, Lithuania), Lane 1:
NDV/Radom/chicken/70; Lane 2: NDV/chicken/89/90; Lane 3: NDV/chicken/18/91; Lane 4: NDV/chicken/548/04; Lane 5:
NDV/pigeon/AR1; Lane 6: NDV/pigeon/AR2; Lane 7: NDV/pigeon/AR3; Lane 8: NDV/pigeon/AR4; Lane 9: NDV/pigeon/AR5;
Lane 10: NDV/pigeon/AR6; Lane 11: NDV/pigeon/AR7; Lane 12: NDV/pigeon/PW/46/02; Lane 13:NDV/pigeon/PW/166/02; Lane
14: NDV/turkey/549/04; Lane 15: NDV/LaSota; Lane 16: NDV/Roakin; Lane 17: NDV/Italy; Lane 18: negative control.
5
M
1
2
3
4
5
6
7
202 bp
Fig.2. Sensitivity of RT-PCR: M: marker Gene Ruler 100 bp DNA Ladder (Fermentas, Lithuania); Lane 1: positive control; Lane 2:
107ELD50; Lane 3: 106ELD50; Lane 4: 105ELD50; Lane 5: 104 ELD50; Lane 6: 103 ELD50; Lane 7: 102 ELD50.
Table 1
Comparison of RT-PCR and virus isolation for the detection of NDV in tissues of infected chickens
Virus
Samples
Cloacal
swabs
Tracheal
swabs
Trachea
La Sota strain
Roakin strain
Virus
Passage RT- Virus
Passage
RTisolation
PCR isolation
PCR
Italy strain
Virus
Passage
isolation
RTPCR
-
-
-
+
2
+
+
1
+
+
1
+
+
2
+
+
1
+
+
1
+
+
1
+
+
1
+
Lungs
+
1
+
+
1
+
+
1
+
Liver
+
2
-
+
1
+
+
1
+
Spleen
+
2
+
+
1
+
+
1
+
Heart
-
-
-
+
1
+
+
1
+
Brain
+
1
+
+
1
+
+
1
+
Kidney
+
2
+
+
1
+
+
1
+
Bursa of
Fabricius
Duodenum
+
1
+
+
1
+
+
1
-
+
2
+
+
1
+
-
-
-
Caecal
tonsils
Rectum
-
-
-
+
1
+
+
1
+
-
-
-
+
1
+
+
1
+
Pooled
sample No. 1
Pooled
sample No. 2
+
1
+
+
1
+
+
1
+
+
2
+
+
1
+
+
1
+
6
Discussion
As Newcastle disease is one of the most
important infectious diseases of poultry, rapid detection
and identification of the virus is crucial for the effective
control of the disease. Conventional diagnostic methods
such as virus isolation on SPF embryonated eggs
followed
by
serological
identification
in
haemagglutination-inhibition test is laborious and timeconsuming. The speed of the diagnosis can be
considerably increased by using methods based on
molecular biology e.g. reverse transcription –
polymerase chain reaction (1, 6, 7, 10- 13).
RT-PCR for the detection of NDV was first
described by Jestin & Jestin in 1991 (10) and to date it
has been successfully developed in different
modifications (1) e.g. using universal primers to detect
all NDVs (6, 7), pathotype specific primers that enable
rapid differentiation of the pathotype (12) or nested PCR
(11, 13). In our study we have applied RT-PCR method
at first for the detection and identification of NDV in
allantoic fluids of infected embryonated eggs and then in
tissues of experimentally infected chickens with NDV
strains of different virulence: lento-, meso- and
velogenic.
Our study on the sensitivity of the test indicates
that at least 105ELD50 should be present in 0.1 ml of the
allantoic fluid to obtain a positive result. The sensitivity
can be improved by the development of a modification
of RT-PCR e.g. “nested” RT-PCR (11, 13). On the
other hand, during infection with NDV strains, even
with lentogenic ones, the ELD50 value of the virus in
infected tissues is usually higher that the sensitivity
threshold established in our studies (8, 9, 15). Indeed,
applied RT-PCR method proved to be useful for the
detection of NDV directly in chicken tissues and a high
degree of correlation with virus isolation test was
observed. Collection of samples was carried out 5 d
post infection because the high amount of the virus was
expected to be found in a variety of organs at that very
time. Velogenic and to some extent mesogenic NDV
strains are pantropic and can be found in many organs,
what was confirmed by our studies (Roakin: 13/13
samples by RT-PCR and virus isolation, Italy: 11/13
samples by RT-PCR and 12/13 by virus isolation).
Lentogenic strains are generally less invasive and can be
found usually in the respiratory and digestive tracts (2,
4, 14, 17) but some of them spread to internal organs,
especially during early phase of infection (7-9, 15). It is
presumed
that
their
multiplication
rate
in
parenchymatous organs is lower than for meso- or
velogenic strains (9). In our study La Sota strain was
detected in 8/13 and 9/13 samples by RT-PCR and virus
isolation, respectively. The need to perform 2 passages
to obtain a positive result in case of samples collected
from the liver, spleen, kidneys and duodenum suggests
the LaSota strain multiplies in lower titres in these
organs.
Regardless of the virulence of the strains,
pooled samples of different organs were always positive
in either test. Due to the fact that NDV strains of
different virulence show diversity in tissue predilection
in different time post infection, it is recommended to
pool samples of various organs rather than examine
selected tissues. It should also be taken into account that
the viral titres decline relatively quickly in certain
organs.
A significant reduction of time required to
complete the RT-PCR test (8-12 h) in comparison with
the standard virus isolation (up to 2 weeks) seems to be
the greatest advantage of the RT-PCR method.
Moreover, the use of primers encompassing cleavage
site of F0 gene enables to establish virulence of an
isolate, either by direct sequencing or by restriction
enzyme analysis and can be used alternatively to in vivo
method (1 day-old chicks inoculation) of the virulence
assessment.
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