Download Very virulent infectious bursal disease virus (vvIBDV) in vaccinated

Arch. Geflügelk. 2003, 67 (1), 2 – 5, ISSN 0003-9098. Verlag Eugen Ulmer GmbH & Co., Stuttgart
Very virulent infectious bursal disease virus (vvIBDV)
in vaccinated broiler flock: Course of the disease,
identification and characterisation of isolated strain*
Hochpathogenes Infektiöses Bursitis Virus (vvIBDV) in geimpften Broilerherden:
Verlauf der Erkrankung, Identifizierung und Charakterisierung des isolierten Stammes
H. M. Hafez 1, Christine Prusas 1 and R. Raue 2
* Dedicated to Prof. Dr. Gerhard Monreal, on behalf of his seventy-fifth birthday
Introduction
Material and Methods
Infectious bursal disease (IBD) is an acute, highly contagious viral disease of young chickens. It is most often
found in highly concentrated poultry producing areas
throughout the world. IBD is caused by a virus that is
classified as a member of the genus Avibirnavirus of the
family Birnaviridae (Leong et al., 2000), which is characterised by a bisegmented dsRNA genome (Müller et al.,
1979, Kibenge et al., 1988). There are many different
strains of the IBD virus (IBDV), which vary considerably
in their pathogenicity and global distribution. Since 1988,
outbreaks of IBD caused by very virulent (vv) IBDV
strains occurred in broilers in several European countries,
Asia, Africa and recently in Central and South America
(Chettle et al., 1989; van den Berg et al., 1991; Eterradossi et al., 1992; Nunoya et al., 1992; Di Fabio et al.,
1999; Zierenberg et al., 2000). These strains are antigenically similar to the classic virulent strains but can be distinguished by RT-PCR combined with restriction enzyme
analysis (REA) or by genome sequencing (Eterradossi
et al., 1997; 1999, Zierenberg et al., 2000, 2001). In contrast, “variant strains” with a different antigenic profile
were described in the USA. Vaccination failure was incriminated to be responsible for the emergence of antigenic
variation (Rosenberger and Cloud, 1986).
In general, IBDV is resistant to many disinfectants and
environmental factors, and remains infectious for at least
four months in the poultry house environment. Because of
the resistant nature of IBDV, once a poultry house becomes contaminated, the disease tends to recur in subsequent flocks (Lukert and Saif, 1997). Hygienic measures
alone are ineffective and vaccination is essential. Several
vaccines are available. When they are given correctly,
good immunity and protection can be achieved (van den
Berg and Meulemans, 1991). Beside the proper application the major problem with the live vaccination of young
chickens with maternally derived antibodies (MDA) is determining the proper time of vaccination, through monitoring of the antibody level in a breeder flock or its progeny.
The present paper describes an outbreak of IBD in vaccinated broiler flocks.
Detection of viral antigens and antibodies
1
Institute of Poultry Diseases, Faculty of Veterinary Medicine,
Free University Berlin, Germany
Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, Germany
2
Detection of IBD antigen in the bursa of Fabricius (BF)
was performed in an agar gel precipitation test. A portion
of the bursa was homogenised, used as antigen and tested
against a positive polyclonal antiserum. For serological investigations agar gel precipitation tests were used as well.
Virus isolation
The virus isolation trials were carried out in embryonated
SPF-eggs and in chicken embryo fibroblasts using bursa
homogenates from affected birds (Rosenberger et al.,
1998).
Reverse transcription-polymerase chain reaction (RT-PCR)
For further typing of the isolated virus strains, RT-PCR
combined with REA was performed and the variable region of the VP2 gene was sequenced as described previously (Zierenberg et al., 2000, 2001). These techniques
allow to distinguish between field and vaccine strains.
Results
Clinical observations
In a broiler farm with about 180.000 birds kept in 6
houses birds were vaccinated against IBD via drinking
water at day 17 of age, according to the advice of the
hatchery, using TAD1 Gumboro Vac Forte (Lohmann Animal Health). Each dose contained at least 103.0 EID50 embryo-adapted (bursa derived) IBDV.
At day 36 only in two houses (1 and 2) with 56.500
birds a sudden onset of depression, vent pecking, whitish
diarrhoea accompanied with increase in mortality were observed. On necropsy, haemorrhages were found in the
thigh and pectoral muscles. The BF appeared oedematous,
hyperaemic, and had a gelatinous yellowish transudate
covering the serosal surface. The kidneys appeared swollen. Microscopically, lymphocytes, necrosis oedema, hyperaemia, and inflammatory cell infiltration were found in
the BF.
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HAFEZ et al., Very virulent infectious bursal disease virus in vaccinated broiler flock
Virological and serological examination
1
Trials of direct IBDV antigen detection using polyclonal
antibodies in agar gel precipitation test revealed positive
results.
In chicken embryo fibroblasts IBDV was isolated in the
first passage. The isolation in embryonated SPF-eggs was
successful after the second passage (Table 1). Testing of
10 serum samples from each house using Agar gel precipitation test at the time of slaughter revealed positive results of all samples.
Table 1. Results of virological examination
Ergebnisse der virologischen Untersuchung
2
1
M
723
484
406
239
317
2
1
M
GB887-01 E
GB887-01 B
2
3
M
GB887-01 TC
Fig. 1. Results of RT-PCR and restriction enzyme analysis with
BspMI (1) and SacI (2)
Ergebnisse der RT-PCR und der Restriktionsenzymanalyse mit
BspMI (1) and SacI (2)
Sample
Designation of
the isolate
No. of passages
required
Table 2. Summary of the results of RT-PCR combined with REA
Zusammenfassung der Ergebnisse der RT-PCR in Kombination
mit der REA
Embryonated eggs
Chicken embryo
fibroblasts
GB 887/01-E
GB 887/01-TC
2
1
Sample
Designation of
the isolate
Judgment
Bursa homogenate
GB 887/01-B
Molecular biological identification and typing
Embryonated eggs
GB 887/01-E
As the used conventional isolation methods do not allow
to distinguish between field and vaccinal strains, further
investigations using RT-PCR in combination with REA or
sequencing of the variable region in the VP2 gene were
carried out.
The results after RT-PCR combined with REA revealed
that the strain isolated in tissue culture showed a classic
virulent IBDV genotype, whereas the analysis of the bursal material and the allantoic fluids of infected SPF-eggs
indicated the presence of two IBDV strains, a classical
virulent and a very virulent IBDV strain (Fig. 1). The obtained results are summarized in Table 2.
To get further information of the isolated strains, the
variable regions of the VP2 gene were sequenced and
the amino acids were compared (Table 3). In the case
of GB887/01-B and GB887/01-E a faint additional sequence was visible indicating a second virus strain.
However, the amino acid pattern of the main sequence
of GB887/01-B and GB887/01-E revealed the vvIBDV
Chicken embryo
fibroblasts
GB 887/01-TC
classical virulent þ
very virulent
classical virulent þ
very virulent
classical virulent
genotype. As expected, the IBDV strain isolated from
tissue culture (GB887/01-TC) showed the same amino
acid pattern as the vaccine strain LC-75 (TAD1 Gumboro Vac Forte).
Economic parameters
The total mortality at the time of slaughter (day 39) was
6.1% and 5.1% in house 1 and 2, respectively. The total
mortality rate in the non-infected flock (house 3) was 1.9%.
The IBD related mortality rates were 4.5% (house 1) and
2.9% (house 2). In house 1, where the highest mortality rate
was observed, a slight reduction in daily weight gain as
well as in production number were observed (Table 4).
Table 3. Comparison of the amino acids of selected positions of classical virulent (cv) IBDV, very virulent (vv) IBDV, the vaccine strain
LC-75 and the investigated isolates
Vergleich der Aminosäuren an ausgewählten Positionen des klassischen virulenten (cv) IBDV und des hoch virulenten (vv) IBDV, des
Impfstammes LC-75 und der untersuchten Isolate
Amino acid position*
cvIBDV
vvIBDV
LC-75
GB887/01-Bþ
GB887/01-Eþ
GB887/01-TC
222
242
253
256
270
279
284
294
297
299
330
P
V
H
V
T
N
T
L
S
N
R
A
I
Q
I
A
D
A
I
P
S
S
P
V
H
V
A
N
T
L
P
N
R
A
I
Q
I
A
D
A
I
P
S
S
A
I
Q
I
A
D
A
I
P
S
S
P
V
H
V
A
N
T
I
P
N
R
* between amino acid position 206 and 350
þ
main sequence
GB887/01-B : Bursal material
GB887/01-E : strain isolated in embryonated SPF-eggs.
GB887/01-TC: strain isolated in chicken embryo fibroblasts.
Typical amino acids for the vvIBDV genotype are in bold.
Amino acids responsible for the adaptation to tissue culture are in italic.
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HAFEZ et al., Very virulent infectious bursal disease virus in vaccinated broiler flock
Table 4. Economic parameters
Wirtschaftliche Parameter
Parameters
House 1
House 2
House 3
No. of birds
Age at vaccination (day)
Age at onset of clinical signs
Total mortality rate (%)
IBD related mortality rate
Daily weight gain (g)
Feed conversion rate
Production number
28.594
17
36
6.1
4.5
43.23
1 : 1.83
221.92
28.305
17
36
5.1
2.9
44.79
1 : 1.81
234.94
28.305
17
No signs
1.9
(0.1)*
43.35
1 : 1.85
229.94
* non –– IBD related mortality
Discussion
An effective IBDV control has to involve an effective vaccination program for breeder, broiler and pullet flocks.
When the vaccine is applied correctly, good immunity
and protection can be achieved (van den Berg and
Meulemans, 1991). The vaccination program for each region or company should be established on the basis of
the evaluation of the circulated field virus strain, its antigenicity and pathogenicity. In addition, several other aspects such as the poultry density in an area and management or environmental associated stress should be taken
into account.
Beside the proper application the major problem with
the live vaccination of young chickens with MDA is determining the proper time of vaccination, through monitoring
of the antibody level in a breeder flock or its progeny (Lukert and Saif, 1997). However, if the MDA titres are not
uniform in a flock, multiple costly vaccinations may be
required (Kouwenhoven and van den Bos, 1994). Some
causes of poorly uniform MDA titres are, poor vaccine administration in broiler breeders and mixing of broilers
from different breeder flocks. The best way to reduce
these problems is to avoid as much as possible mixed
hatches from different breeder flocks. When it is not possible, it is advisable to mix offspring from flocks with
identical or very similar MDA titres (Kouwenhoven and
van den Bos, 1994). Furthermore, specially in the USA
and some parts of South America, the reason of the outbreaks in vaccinated flocks may be due to use of vaccine
that does not include all types of “variant” strains.
The more frequent reason for outbreaks in vaccinated
flocks is incorrect application of the vaccine. In the present field investigation birds in all houses are vaccinated
at day 17 according to the advise of the hatchery based on
antibody titre of the breeder flock. It is clearly that the
chickens in the two houses (1 and 2) were infected with a
vvIBDV strain accompanied with clinical signs, lesions
and increased mortality rates in spite of the vaccination at
day 17. The results described here indicate either a failure
in the vaccination procedure in these houses or that the
birds of these houses might originated from other breeder
flocks with different MDA titres.
Summary
In a broiler farm with about 180.000 birds kept in 6
houses, birds were vaccinated against IBD via drinking
water at day 17 of age, according to the advice of the
hatchery, using TAD1 Gumboro Vac Forte (Lohmann Animal Health). At day 36 only in two houses (1 and 2) with
56.900 birds a sudden onset of depression, vent pecking,
whitish diarrhoea accompanied with increase in mortality
were observed. On necropsy, haemorrhages were found in
the thigh and pectoral muscles. The BF appeared oedematous, hyperaemic, and had a gelatinous yellowish transudate covering the serosal surface. Trials of direct IBDV
antigen detection using polyclonal antibodies in agar gel
precipitation test revealed positive results. In chicken embryo fibroblasts IBDV was isolated in the first passage.
The isolation in embryonated SPF-eggs was successful
after the second passage. Further investigations using RTPCR in combination with restriction enzyme analysis and
sequences of variable region VP2 was carried out. The result revealed that isolated virus in tissue culture was classic virulent IBDV (Vaccinal strain). Examination of the
bursa homogenate as well as the isolated virus in chicken
embryo resulted in the detection of vvIBDV as well as the
classic strain (Vaccinal strain).
The total mortality rate at the time of slaughter (day 39)
was 6.1% and 5.1% in house 1 and 2, respectively. The
total mortality in the non-infected flock (house 3) was
1.9%. The IBD related mortality rates were 4.5% (house
1) and 2.9% (house 2). In house 1, where the highest mortality rate was observed, a slight reduction in daily weight
gain as well as in production number was detected. The
results described here indicate either a failure in the vaccination procedure in these houses or that the birds of these
houses might originated from other breeder flocks with
different maternal derived antibody titres.
Key words
Infectious bursal Disease, Diagnosis, Vaccination, Vaccination failure
Zusammenfassung
Hochpathogenes Infektiöses Bursitis Virus (vvIBDV) in geimpften Broilerherden: Verlauf der Erkrankung, Identifizierung und Charakterisierung des isolierten Stammes
In einer Broilerfarm mit etwa 180000 Tieren, die in 6 Ställen gehalten wurden, erfolgte die Impfung gegen IBD über das Trinkwasser entsprechend der Hinweise aus der Brüterei am 17. Lebenstag mit TAD1 Gumboro Vac Forte (Lohmann Animal
Health). Am 36. Tag traten in 2 Ställen (1 & 2), mit ca. 56900
Tieren, plötzlich Depressionen, Kloakenpicken und weißlicher
Durchfall, verbunden mit einem Ansteigen der Mortalität, auf.
Bei der Zerlegung wurden Hämorrhagien in der Schenkel- und
Herzmuskulatur festgestellt. Die Bursa Fabricii war ödematös und
hyperämisch. Die Schleimhaut der Bursa Fabricii war mit einer
gallertigen gelblichen Flüssigkeit bedeckt. Versuche des direkten
viralen Antigennachweises mit polyklonalen Antikörpern im
Agar-Gel-Präzipitationstest führten zu positiven Ergebnissen. Das
Virus wurde nach einer Passage auf Hühnerembryofibroblasten ––
Zellen isoliert. Die Isolierung in embryonierten SPF-Eiern gelang
nach 2 Passagen. Weitere Untersuchungen unter Verwendung einer RT-PCR in Verbindung mit einer Restriktionsenzymanalyse
und einer Sequenzierung der variablen VP2 Region wurden
durchgeführt. Die Ergebnisse zeigten, dass es sich bei dem Virus,
das in Zellkulturen isoliert wurde, um den klassischen virulenten
IBDV- Stamm (Vakzine- Stamm) handelte. Bei der Untersuchung
des Bursahomogenates wie auch des im embryonierten Hühnerei
isolierten Virus wurden sowohl Hochpathogenes Infektiöses Bursitis Virus (vvIBDV) wie auch der klassische Stamm (Vakzine
Stamm) nachgewiesen.
Die gesamte Mortalitätsrate zum Zeitpunkt der Schlachtung
(39. Tag) lag bei 6,1% in Stall 1 bzw. 5,1% in Stall 2. Die gesamte Mortalität der nicht infizierten Herde (Stall 3) betrug 1,9%.
Die Mortalitätsraten, die durch IBD verursacht wurden, lagen bei
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HAFEZ et al., Very virulent infectious bursal disease virus in vaccinated broiler flock
4,5% (Stall 1) und 2,9% (Stall 2). In Stall 1 mit der höchsten
Mortalitätsrate wurden eine geringe Reduktion der täglichen Gewichtszunahme sowie eine Reduktion der Produktionszahlen festgestellt. Die hier beschriebenen Ergebnisse sprechen dafür, dass
entweder Fehler bei der Durchführung der Impfung unterlaufen
sind oder die Tiere dieser Ställe aus anderen Elterntierherden
stammten und abweichende maternale Antikörpertiter besaßen.
Stichworte
Infektiöse Bursitis, Diagnose, Vakzination, Impffehler
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Correspondence: Prof. Dr. Hafez Mohamed Hafez, Institute of Poultry Diseases, Free
University Berlin, Koserstr. 21, 14195 Berlin, Germany;
e-mail: [email protected]