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 [r]
Trang 1DETECTION OF NEWCASTLE DISEASE VIRUS
IN INFECTED CHICKEN EMBRYOS AND CHICKEN TISSUES BY RT-PCR
Department of Poultry Diseases, National Veterinary Research Institute, 24-100 Pulawy, Poland
e-mail: ksmiet@piwet.pulawy.pl
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 10 5 ELD50/0.1 ml To detect NDV in chicken
tissues, SPF chickens were inoculated with 10 6 EID 50 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 RT-PCR 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 80-ties and beginning of the 90-80-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
4-week-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
Trang 2202 bp
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
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 RT-PCR 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
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)
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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
Trang 3M 1 2 3 4 5 6 7
Fig.2 Sensitivity of RT-PCR: M: marker Gene Ruler 100 bp DNA Ladder (Fermentas, Lithuania); Lane 1: positive control; Lane 2:
10 7 ELD 50 ; Lane 3: 10 6 ELD 50 ; Lane 4: 10 5 ELD 50 ; Lane 5: 10 4 ELD 50 ; Lane 6: 10 3 ELD 50 ; Lane 7: 10 2 ELD 50
Table 1
Comparison of RT-PCR and virus isolation for the detection of NDV in tissues of infected chickens
Virus
Samples
Virus isolation Passage PCRRT- isolationVirus Passage PCR RT- isolation Virus
Passage
RT-PCR Cloacal
swabs
Tracheal
Bursa of
Caecal
Pooled
Pooled
202 bp
Trang 4Discussion
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
time-consuming 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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