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

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).

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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).