Feb 18, 2015 - AY288998/cockatoo/Indonesia/14698/1990 Ancestral. JN986837/chicken/NL/152608/1993 Ancestral. AY444497/moluccan/Indonesia/904/87 ...
JCM Accepted Manuscript Posted Online 18 February 2015 J. Clin. Microbiol. doi:10.1128/JCM.02818-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Short-Form Paper
Title
Presence of Virulent Newcastle Disease Virus in Vaccinated Chickens Farms In Pakistan
Abstract
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One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens in multiple farms despite of the existence of high average NDV specific antibody titers (>4.75 log2). The data suggest a possible role of vaccinated birds as reservoirs of vNDV.
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Authors: Shafqat Fatima Rehmania, Abdul Wajida,b, Tasra Bibia, Bushra Nazira, Nadia Mukhtara, Abid
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Hussainc, Nazir Ahmad Loned, Tahir Yaquba,b, Claudio L. Afonsod
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a
Quality Operations Laboratory (QOL), University of Veterinary and Animal Sciences, Lahore 54000, Pakistan. b Institute of Biochemistry and Biotechnology (IBBt), University of Veterinary and Animal Sciences Lahore 54000, Pakistan. c Poultry Disease Diagnostic lab, Gakkhar, Gujranwala, Punjab, Pakistan. d Exotic and Emerging Avian Disease Research Unit, Southeast Poultry Research Laboratory, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA.USA. Key Words: Newcastle disease (ND), reservoir, poultry, vaccination Running title: Virulent NDV in vaccinated chickens
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Background. Newcastle disease (ND) is one of the most devastating diseases affecting the
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poultry industry worldwide (1). It is caused by avian paramyxoviruses of serotype 1 (APMV-
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1), also known as Newcastle disease virus (NDV) (1). NDV may be classified into classes
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and subdivided into up to 18 genotypes based on genetic diversity (2). Currently pigeons and
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in cormorants are two well recognized reservoirs of virulent NDV (vNDV) (3, 4), however
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those species normally carry viruses of genotypes Va and VI (1) and the reservoir for other
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highly virulent viruses that commonly circulate in poultry (genotypes Vb, VII, and XII to
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XVIII) is unknown.
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The failure to control ND with vaccination alone in endemic countries underscores the
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importance of understanding mechanisms of vNDV maintenance. It has been shown that
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under experimental conditions vaccines do not prevent replication of highly virulent viruses,
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thus circumstantial evidence suggests that the capacity of vaccinated birds to shed viruses
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may allow vaccinated poultry to act as reservoir (1). As culling is the preferred control
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strategy in the developed world and the epidemiology of vNDV in endemic countries is not
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well understood, evidence supporting a role of vaccinated chickens as reservoir is missing.
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Experimental Details. Three hundred tracheal samples from sick or dead birds and 207
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blood samples from live vaccinated chickens in production were analyzed for the presence of
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vNDV during a 3 months period (March to May, 2013) after a serious outbreak in Pakistan
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(2011-2012) was contained (10). Serum samples were collected mostly at three weeks of age
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from day one vaccinated chickens that were not showing clinical symptoms of NDV.
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Tracheal tissues and blood samples were collected from twenty different districts of Punjab
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for virus isolation and for immune status evaluation, respectively (Table 1 and Fig 1B). One
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hundred and one flocks were tested for presence of vNDV by passaging tracheal samples into
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9-10 day old chicken eggs free from maternal antibodies against NDV. Allantoic fluids were
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tested using a spot haemagglutination (HAI) test and viruses from positives samples were
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isolated and sequenced as described (5). Viral RNA was extracted, cDNA was synthesized
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and sequenced as previously described (6) (10).
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influenza were used as previously described (7). For sequence analysis of vNDV primers for
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amplification
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CCCATTAGAGGCATACAACAG
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evolutionary relationship among these isolates and other GenBank sequences was determined
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using MEGA6 software (8). All the F gene sequences obtained in this study were deposited in
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GenBank under the Accession Number KF761533 to KF761552 (Table 1). For
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seroprevalence of NDV antibodies, a total of 320 random blood samples from commercial
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farms of each different district were collected, and the Geometric Mean Titres (GMT) and
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standard deviations were calculated (7).
of
a
374
bp
region and
of
the
Universal primers specific for Avian
Fusion
gene
were
designed
R- CAATATAGGGTAGCCGGTGAT).
(FThe
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Status of poultry flocks. All the farms studied practiced routine vaccination at the hatchery.
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In 80% of the farms, birds were vaccinated with a live LaSota vaccine at day 1 via spray or
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eye drop at the hatchery and at days 7 and 14 via water. Twenty per cent of the farms used an
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additional inactivated vaccine dose on day 24. Eighty eight out of 101 evaluated farms were
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found to be positive for the presence of NDV, irrespective of age (broiler and layers) based
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on spot HAI and the matrix PCR test, and of those 20 farms had confirmed presence of
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virulent viruses. The flocks were believed to be free from vNDV, however because NDV is
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endemic in Pakistan, the origin of the outbreaks are unknown. Mortality ranged from 6 to
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50% and morbidity rate was recorded to be up to 60% in some farms, despite vaccination. In
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most severe cases the disease erupted at 21-25 days of age and birds succumbed to death
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within 10-15 days (Table 1). No avian influenza was detected. Mortality due to vNDV
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occurred in both environmentally controlled houses and in open sheds (data not shown).
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Immune status assessed by measuring mean HI titers from serum samples (log2) showed high
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average values ranging from 4.7 to 6.9, and HI of log2 greater than 3 was detected in 99% of
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the 320 birds tested (Table 1).
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Molecular epidemiology. Twenty NDV isolates representative of the different districts were
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selected for genotyping (Table 1, Figures 1A, B and C). Partial and complete (F) gene
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sequences (374 and 1662 base pairs) of viruses from different districts (n=20) were obtained
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and compared with F gene sequences of representative NDV isolates belonging to genotypes
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of class II of NDV, and to previously characterized Pakistani viruses. Maximum likelihood
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phylogenetic analysis based on nucleotide sequences demonstrated that all 2013 NDV
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isolates grouped within genotype VII of class II as subgenotype VIIi (Figure 1A and B).
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Nucleotide sequence of this highly variable region of the F protein revealed 99.6 %
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nucleotide identity among 2013 isolates, and 96.4% identity to 2012 isolates of sub-genotype
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VIIi. All other available NDV GenBank sequences revealed lower nucleotide identity to the
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new isolates, suggesting that all of the 2013 viruses are related to each other and that they
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originated from viruses of the 2012 outbreak. We have recently shown that subgenotype VIIi
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has replaced the existing viruses of genotype XIII and VIId in Pakistan, and was the
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predominant subgenotype in poultry during 2011 and 2012 (9, 10). The virulence of the new
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isolates was confirmed by sequence of the predicted cleavage site of the F protein, which
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indicated that all the isolates carried the112-RRQKRF-117 motif corresponding to the cleavage
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site of virulent NDV.
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Significance. Sequence analysis confirms that one year after a highly virulent outbreak
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occurred in Pakistan, 20 vNDV positive farms in the vast geographic region of Punjab were
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infected with viruses of the same lineage that caused the problem in 2012. This infection
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occurred during a three months period (and continued during 2014) despite of the overall
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relatively high levels of anti NDV antibodies detected in 99% of the samples. Current
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knowledge on NDV vaccines based on experimental studies in laboratory settings that
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mimicked field conditions suggested that herd immunity of >85% with antibody titers of log2
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HI >3 should protect against viral spread (11). However the authors cautioned about the
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limitations of the study due to the reduced amount of data available and to the possible
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difficulties in achieving effective distribution of antibodies titers in large populations of
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poultry. Our results, although limited, present field data that suggest that despite high levels
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of anti NDV antibodies titers of > 3 log2 HI in 99% of the tested birds in different farms and
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localities there was very high incidence of the disease (Table 1). Although it is possible that
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correctly vaccinated chickens may not sustain vNDV under optimal laboratory conditions,
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other factors that may affect the immune status of the flock in the field, such as
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immunosuppression or non-uniform vaccination leading to a rolling infection, may allow the
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persistence of virulent viruses in vaccinated birds. Thus, there is need to re-evaluate the
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robustness of current vaccination methods under field conditions and the role of vaccinated
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birds in virus maintenance.
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Figure 1 legend: 1A. Phylogenetic analysis of the partial F gene distances between new and
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past virulent NDV Pakistani isolates based on the Maximum likelihood method. 1B.
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Phylogenetic analysis based on the full fusion gene of selected isolates. 1C. Pakistan map
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showing sites of disease incidence.
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Table 1: Virulent samples description, GenBank accession numbers, farm location, size, age
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of birds, observed mortality, mean and standard deviation of HI titers.
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Acknowledgements
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Funding has been provided by the US Department of State (USDA/ARS/BEP/CRDF) NDV
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31063, USDA CRIS 6612-32000-064 and PARB CGS project No.572. The team is thankful
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to the PARB for technical assistance during the surveillance studies, particularly the Director
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Poultry Research Institute, Rawalpindi, for assigning a team to facilitate the collection of
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samples.
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8
S t ra in Chic ke n/ P a k/ Ba h wa lpur / 300/ 2013 Chic ke n/ P a k/ Fa is a l a ba d/ 03/ 2013 Chic ke n/ P a k/ Gujr a nwa la / 589/ 2013 Chic ke n/ P a k/ Gujr a t / 234/ 2013 Chic ke n/ P a k/ Ha f i z a ba d/ 611/ 2013 Chic ke n/ P a k/ Ka s ur / 08/ 2013 Chic ke n/ P a k/ Kha ne wa l/ 55/ 2013 Chic ke n/ P a k/ La h or e / 02/ 2013 Chic ke n/ P a k/ Lod hr a n/ 62/ 2013 Chic ke n/ P a k/ Mult a n/ 43/ 2013 Chic ke n/ P a k/ Muz a f a r ga r h/ 160/ 2013
Ac c e s s io n No .
Ag e o f b i rd s La t i t u d e
Lo n g i t u d e
Fl o c k s i z e
( In D a y s ) / Ho s t
S a mple s c o lle c t e d
HI t i t r e P o sit iv e %
KF761551
29.3956
71.6836
36,000
22/ Br oile r
16
94
6
KF761538
31.418
73.079
30,000
28/ Br oile r
7
100
16
KF761536
32.15
74.1833
36,000
31/ La ye r
29
82
15
KF761548
32.5738
74.0789
35,000
35/ Br oile r
55
56
15
KF761535
32.07
73.68
6,000
21/ Br oile r
2
100
31
KF761540
31.117
74.4502
77,000
08/ Br oile r
17
53
40
KF761552
30.303
71.9333
15,000
12/ Br oile r
23
91
7
KF761537
31.545
74.3406
100,000
37/ Br oile r
16
100
50
KF761543
29.5333
71.6333
2,000
28/ La ye r
6
100
5
KF761541
30.1978
71.4697
8,000
42/ Br oile r
48
81
45
KF761549
30.0703
71.1933
22,000
21/ Br oile r
8
100
23
KF761546
31.45
73.7
9,000
25/ Br oile r
6
100
50
KF761550
32.2167
74.95
17,000
26/ Br oile r
17
81
33
KF761547
30.8091
73.4477
62,000
29/ Br oile r
9
100
27
Chic ke n/ P a k/ Na n ka na
S t . De v
Mo r t a l i t y % 5.7
1.35
6.0
0.97
6.9
1.06
6.0
0.85
5.4
1.30
5.7
2.12
6.0
0.89
5.8
2.44
5.7
0.98
6.8
1.65
5.4
0.81
6.8
1.69
S a hib/ 155/ 2013 Chic ke n/ P a k/ Nor wa l/ 40/ 2013 Chic ke n/ P a k/ Oka r a / 118/ 2013 Chic ke n/ P a k/ Ra w a lpindi/ 188/ 2013 Chic ke n/ P a k/ S a hi wa l/ 06/ 2013 Chic ke n/ P a k/ S a r godha / 212/ 2013 Chic ke n/ P a k/ S he i khpur a / 22/ 2013 Chic ke n/ P a k/ S ia l kut / 88/ 2013 Chic ke n/ P a k/ Ve h a r i/ 46/ 2013
KF761534
33.6
73.0333
16,000
22/ La ye r
4
67
40
KF761539
30.5833
73.3333
23,000
35/ Br oile r
7
100
43
KF761533
32.0836
72.6711
3,000
22/ Br oile r
3
100
20
KF761545
31.715
73.985
29,000
24/ Br oile r
14
57
10
KF761544
32.4972
74.5361
6,000
49/ Br oile r
7
100
7
KF761542
30.0419
72.3528
7,000
22/ Br oile r
6
100
7
5.3
1.13
6.5
0.63
4.8
1.13
6.4
1.75
5.1
0.81
5.0
0.73
5.6
1.09
5.9
0.85
Table 1: Selected samples location, description, accession number corresponding to sequenced region, farm location and size, age of birds, observed mortality, mean and standard deviation of HI titers.
Figure 1A
66
KF761539/Chicken/Pak/Sahiwal/06/2013 KF761533/Chicken/Pak/Sargodha/212/2013 KF761538/Chicken/Pak/Rawalpindi/188/2013 KF761549Chicken/Pak/Muzafargarh/160/2013 KF761541/Chicken/Pak/Multan/43/2013 KF761543/Chicken/Pak/Lodhran/62/2013 98 KF761535/Chicken/Pak/Hafiz abad/611/2013 KF761536/Chicken/Pak/Gujranwala/589/2013 KF761538/Chicken/Pak/Faisal abad/03/2013 KF761551/Chicken/Pak/Bahwalpur/300/2013 KF761542/Chicken/Pak/Vehari/46/2013 KF761552/Chicken/Pak/Khanewal/55/2013 KF761546/Chicken/Pak/Nankana Sahib/155/2013 KF761548/Chicken/Pak/Gujrat/234/2013 KF761540/Chicken/Pak/Kasur/08/2013 85 KF761537/Chicken/Pak/Lahore/02/2013 VIIi KF761550/Chicken/Pak/Norwal/40/2013 98 62 KF761547/Chicken/Pak/Okara/118/2013 KF761545/Chicken/Pak/Sheikhpura/22/2013 KF761544/Chicken/Pak/Sialkut/88/2013 66 KF113352/Chicken/Pak/UVAS/212/2012 63 KF113353/Pheasant/Pak/Lahore/136/2012 KF113344/Chicken/Pak/Okara/103/2011 KF113342/Chicken/Pak/Lahore/50/2011 KF113351/Chicken/Pak/UVAS/211/2012 KF113350/Chicken/Pak/Lahore/200/2012 KF113339/Chicken/Pak/Lahore/30/2011 79 KF113340/Chicken/Pak/Lahore/32/2011 KF113341/Chicken/Pak/Lahore/43/2011 90 KF113343/Chicken/Pak/Gujranwala/56/2011 KF113345/Chicken/Pak/Gujranwala117/2011 KF113346/Chicken/Pak/Khyber Puk. Khawa/118/2011 KF113347/Chicken/Pak/Khyber Puk. Khawa/119/2011 KF113348/Chicken/Pak/Khyber Puk. Khawa/162/2012 KF113349/Chicken/Pak/Kasur/191/2012 AY288998/cockatoo/Indonesia/14698/1990 Ancestral JN986837/chicken/NL/152608/1993 Ancestral AY444497/moluccan/Indonesia/904/87 Ancestral 61 48 80 DQ858357/China/YG03/2006 VIIf 51 AY028995/China/CH/A7/96/2001 43 90 DQ485256/Chicken/China/Gu/2000 VIIe EF589134/China/H2/2007 87 95 DQ485269/China/Chi/Gu13/2004 VIId EF579731/Broiler/Shandong/YS/2006 87 DQ417112/China/SSX03/2003 VIIg 85 DQ485271/China/Gu14/2005 VIIb 63 DQ227247/China/SDD01/2001 67 99 259879777/Avian/Mauritania/2006 XVIII 30 515794290/Chicken/Ivory Coast/2007 JX393313/Culex/Indonesia/JKT/1979 Ancestral JN942041/Cockatoo/India/7847/1982 Ancestral 20 JQ267583/Chicken/IranEMM/3/2009 51 JQ267580/Chicken/IranEMM/6/2011 84 JQ267581/Chicken/Iran-EMM/5/2011 GU182323/Chicken/SPVC/Karachi/NDV/43/2008 97 KF113338/Chicken/Pak/University Diagnostic Lab/12/2010 JQ517285/Chicken/NDV/UDL8/2011 82 JN682190/Chicken/CP/Islamabad2/2010 66 GU182331/Chicken/SPVC/Karachi/NDV/33/2007 XIII GU182331/Chicken/SPVC/Karachi/NDV/33/2007 65 57 JN682191/Chicken/CP/Islamabad3/2010 95 JN682184/Chicken/BYP/Lahore/2010 JN682185/Chicken/CP/Rawalpindi1/2010 25 JN682186/Chicken/CP/Islamabad1/2010 74 JN682187/Chicken/BYP/Rawalpindi/2010 JN682210/Chicken/BYP//2010 70 JN682189/Chicken/CP/Rawalpindi/2/2010 49 78 JN682211/Chicken/CP/2010 JN682188/Chicken/CP/Attock/2010 64 JN627507/GD1003/373838901/2010 XII JN627508/GD450/373838903/2011 99 GU182329/Chicken/SPVC/Karachi/NDV/26/2005 JX186997/Chicken/Dominican Republic/867-2/2008 JX915242/Chicken/Dominican Republic/28138-4/1986 99 0.02
XVI
Figure 1B
100
99
85 KF113345.1-chicken-Pakistan-2011 KF113348.1-chicken-Pakistan-2012 KF113350.1-chicken-Pakistan-2012 KF113351.1-chicken-Pakistan-2012 KF113339.1-chicken-Pakistan-2011 JX532092.1-peacock-Pakistan-2012 JX854452.1-pheasant-Pakistan-2011 100 HQ697254.1-chicken-Indonesia-2010 KF792018.1-chicken-Israel-2011 KF792019.1-chicken-Israel-2012 KF792020.1-parrot-Israel-2012 93 KF113344.1-chicken-Pakistan-2011 KF113341.1-chicken-Pakistan-2011 KF113342.1-chicken-Pakistan-2011 HQ697257.1-chicken-Indonesia-2010 KF113347.1-chicken-Pakistan-2012 KF113349.1-chicken-Pakistan-2012 KF113343.1-chicken-Pakistan-2011 Chicken/Pak/Gujranwala/649/2013 Chicken/Pak/Gakkhar/609/2013 96KF792021.1-chicken-Israel-2013
KF767105.1-lory-Indonesia-1988 JN986837.1-chicken-Netherlands-1993 AY288998-cockatoo-Indonesia-1990 100
97 100
0.01
JX393313.1-mosquito pool-Indonesia-1979 KF767104.1-cockatoo-Indonesia-1988 KF767106.1-parrot-Indonesia-1976 AB605247.1-chicken-Bali-2007 100 JX193074.1-egret-China-2011 KF026013.1-chicken-Malaysia-2011 HQ697261.1-chicken-Bali-2010 100 HQ697255.1-chicken-Indonesia-2010 HQ697256.1-chicken-Indonesia-2009 88
Figure 1C
