Presence of Virulent Newcastle Disease Virus in Vaccinated Chickens in Farms in Pakistan

Abstract

One year after a virulent Newcastle disease virus (vNDV) outbreak in Pakistan, the causative strain was present in vaccinated chickens of multiple farms despite 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.

TEXT

Newcastle disease (ND) is one of the most devastating diseases affecting the poultry industry worldwide (1). It is caused by avian paramyxoviruses of serotype 1 (APMV-1), also known as Newcastle disease virus (NDV) (1). NDV may be classified into two classes and subdivided into up to 18 genotypes based on genetic diversity (2, 3). Currently, pigeons and cormorants are two well-recognized reservoirs of virulent NDV (vNDV) (4, 5); however, those species normally carry viruses of genotypes Va and VI (1), and the reservoir for other highly virulent viruses that commonly circulate in poultry (genotypes Vb, VII, and XII to XVIII) is unknown.

The failure to control ND with vaccination alone in countries where the virus is endemic underscores the importance of understanding the mechanisms of vNDV maintenance. It has been shown under experimental conditions that vaccines do not prevent the replication of highly virulent viruses; thus, circumstantial evidence suggests that the capacity of vaccinated birds to shed the viruses may allow vaccinated poultry to act as a reservoir (1). As culling is the preferred control strategy in the developed world, and as the epidemiology of vNDV in countries where the virus is endemic is not well understood, evidence supporting a role of vaccinated chickens as a reservoir is missing.

Experimental details.

Three hundred tracheal samples from sick or dead birds and 207 blood samples from live vaccinated chickens in production were analyzed for the presence of vNDV during a 3-month period (March to May 2013) after a serious outbreak in Pakistan (2011 to 2012) was contained (6). Serum samples were collected mostly at 3 weeks of age from day-one-vaccinated chickens that were not showing clinical symptoms of NDV. Tracheal tissues and blood samples were collected from chickens across 20 different districts of Punjab for viral isolation in tracheal tissues and for immune status evaluation in blood samples (Table 1 and Fig. 1A). We tested 101 flocks for the presence of vNDV by passaging tracheal samples into 9- to 10-day-old chicken eggs free of maternal antibodies against NDV. Allantoic fluids were tested using a spot hemagglutination (HAI) test, and viruses from the positive samples were isolated and sequenced, as described previously (4). Viral RNA was extracted, and cDNA was synthesized and sequenced, as previously described (6, 7). Universal primers specific for avian influenza were used, as described previously (8). For the sequence analysis of vNDV, primers for amplification of a 374-bp region of the Fusion (F) gene were designed (F-CCCATTAGAGGCATACAACAG and R-CAATATAGGGTAGCCGGTGAT). The evolutionary relationship among these isolates and other GenBank sequences was determined using MEGA6 software (9). To examine the seroprevalence of NDV antibodies, a total of 320 random blood samples from chickens in commercial farms of the different districts were collected, and the geometric mean titers and standard deviations were calculated (8).

TABLE 1.

Selected strain locations and descriptions, accession numbers corresponding to sequenced regions, farm locations and flock sizes, age and number of samples per bird, observed NDV status and mortality, and HI titer data

Strain	Accession no.	Latitude	Longitude	Flock size	Age of bird (days), host	No. of samples collected	% positive for NDV	% mortality	HI titer
									Mean	SD
Chicken/Pak/Bahwalpur/300/2013	KF761551	29.3956	71.6836	36,000	22, broiler	16	94	6	5.7	1.35
Chicken/Pak/Faisal abad/03/2013	KF761538	31.418	73.079	30,000	28, broiler	7	100	16	6.0	0.97
Chicken/Pak/Gujranwala/589/2013	KF761536	32.15	74.1833	36,000	31, layer	29	82	15	6.9	1.06
Chicken/Pak/Gujrat/234/2013	KF761548	32.5738	74.0789	35,000	35, broiler	55	56	15	6.0	0.85
Chicken/Pak/Hafiz abad/611/2013	KF761535	32.07	73.68	6,000	21, broiler	2	100	31	5.4	1.30
Chicken/Pak/Kasur/08/2013	KF761540	31.117	74.4502	77,000	8, broiler	17	53	40	5.7	2.12
Chicken/Pak/Khanewal/55/2013	KF761552	30.303	71.9333	15,000	12, broiler	23	91	7	6.0	0.89
Chicken/Pak/Lahore/02/2013	KF761537	31.545	74.3406	1,00,000	37, broiler	16	100	50	5.8	2.44
Chicken/Pak/Lodhran/62/2013	KF761543	29.5333	71.6333	2,000	28, layer	6	100	5	5.7	0.98
Chicken/Pak/Multan/43/2013	KF761541	30.1978	71.4697	8,000	42, broiler	48	81	45	6.8	1.65
Chicken/Pak/Muzafargarh/160/2013	KF761549	30.0703	71.1933	22,000	21, broiler	8	100	23	5.4	0.81
Chicken/Pak/Nankana Sahib/155/2013	KF761546	31.45	73.7	9,000	25, broiler	6	100	50	6.8	1.69
Chicken/Pak/Norwal/40/2013	KF761550	32.2167	74.95	17,000	26, broiler	17	81	33	5.3	1.13
Chicken/Pak/Okara/118/2013	KF761547	30.8091	73.4477	62,000	29, broiler	9	100	27	6.5	0.63
Chicken/Pak/Rawalpindi/188/2013	KF761534	33.6	73.0333	16,000	22, layer	4	67	40	4.8	1.13
Chicken/Pak/Sahiwal/06/2013	KF761539	30.5833	73.3333	23,000	35, broiler	7	100	43	6.4	1.75
Chicken/Pak/Sargodha/212/2013	KF761533	32.0836	72.6711	3,000	22, broiler	3	100	20	5.1	0.81
Chicken/Pak/Sheikhpura/22/2013	KF761545	31.715	73.985	29,000	24, broiler	14	57	10	5.0	0.73
Chicken/Pak/Sialkut/88/2013	KF761544	32.4972	74.5361	6,000	49, broiler	7	100	7	5.6	1.09
Chicken/Pak/Vehari/46/2013	KF761542	30.0419	72.3528	7,000	22, broiler	6	100	7	5.9	0.85

FIG 1.

(A) Phylogenetic analysis of the partial F gene distances between new and past virulent NDV Pakistani isolates based on the maximum likelihood method. (B) Phylogenetic analysis based on the full fusion gene of selected isolates. (C) Pakistan map showing sites of disease incidence.

Status of poultry flocks.

All of the study farms practiced routine vaccination at the hatchery. In 80% of the farms, birds were vaccinated with a live LaSota vaccine at day 1 via spray or eye drop at the hatchery and at days 7 and 14 via water. Twenty percent of the farms used an additional inactivated vaccine dose on day 24. Eighty-eight of the 101 evaluated farms were positive for the presence of NDV, irrespective of bird age (broiler and layers), based on spot HAI and the matrix PCR test; of those 88, 20 farms had a confirmed presence of virulent viruses. The flocks were believed to be free of vNDV; however, because Pakistan is an area of NDV endemicity, the origin of the outbreaks is unknown. Avian mortality ranged from 6% to 50%, and the morbidity rate was recorded to be up to 60% in some farms despite vaccination. In the most severe cases, the disease erupted at 21 to 25 days of age, and the birds succumbed to death within 10 to 15 days (Table 1). No avian influenza was detected. Mortality due to vNDV occurred both in environmentally controlled houses and in open sheds (data not shown). The immune status, assessed by measuring the mean hemagglutination inhibition (HI) titers from serum samples (log₂) showed high-average values ranging from 4.7 to 6.9, and an HI titer of log₂ >3 was detected in 99% of the 320 birds tested (Table 1).

Molecular epidemiology.

Twenty NDV isolates representative of the different districts were selected for genotyping (Table 1 and Fig. 1A through C). Partial and complete F gene sequences (374 and 1,662 bp, respectively) of viruses from different districts (n = 20) were obtained and compared with F gene sequences of representative NDV isolates belonging to the genotypes of class II of NDV and to previously characterized Pakistani viruses. The maximum likelihood phylogenetic analysis based on nucleotide sequences demonstrated that all NDV isolates from 2013 were grouped within genotype VII of class II as subgenotype VIIi (Fig. 1A and B). The nucleotide sequence of this highly variable region of the F protein revealed a 99.6% nucleotide identity among 2013 isolates of subgenotype VIIi and a 96.4% identity among 2012 isolates from the same subgenotype. All other available NDV GenBank sequences revealed a lower nucleotide identity with the new isolates, suggesting that all of the viruses from 2013 are related to each other and that they originated from viruses of the 2012 outbreak. We have recently shown that the subgenotype VIIi has replaced the existing viruses of genotype XIII and VIId in Pakistan and was the predominant subgenotype in poultry during 2011 and 2012 (6, 10). The virulence of the new isolates was confirmed by sequence of the predicted cleavage site of the F protein, which indicated that all of the isolates carried the ¹¹²RRQKRF¹¹⁷ motif corresponding to the cleavage site of virulent NDV.

Significance.

Sequence analysis confirms that, 1 year after a highly virulent outbreak occurred in Pakistan, 20 vNDV-positive farms in the vast geographic region of Punjab were infected with viruses of the same lineage that caused the problem in 2012. This infection occurred during a 3-month period (and continued during 2014) despite the overall relatively high levels of anti-NDV antibodies detected in 99% of the samples. Current knowledge about NDV vaccines based on experimental studies in laboratory settings that mimicked field conditions suggests that herd immunity of >85% with antibody titers of log₂ HI >3 should protect against viral spread (11). However, the authors cautioned about the limitations of the study due to the reduced amount of data available and to the possible difficulties in achieving effective distribution of antibodies titers in large populations of poultry. Our results, although limited, present field data that suggest that, despite high levels of anti-NDV antibody titers of >3 log₂ HI in 99% of the tested birds in different farms and localities, there was very high incidence of the disease (Table 1). Although it is possible that correctly vaccinated chickens may not sustain vNDV under optimal laboratory conditions, other factors that may affect the immune status of the flock in the field, such as immunosuppression or nonuniform vaccination leading to a rolling infection, may allow the persistence of virulent viruses in vaccinated birds. Thus, there is a need to reevaluate the robustness of current vaccination methods under field conditions and the role of vaccinated birds in viral maintenance.

Nucleotide sequence accession numbers.

All of the F gene sequences obtained in this study were deposited in GenBank under the accession numbers KF761533 to KF761552 (Table 1).