Patho-epidemiological study on Genotype-XIII Newcastle disease virus infection in commercial vaccinated layer farms

Ethical approval

The present study was approved by Institutional Animal Ethics Committee. The authors have taken permission from each poultry farm owner to publish data.

Epidemiological study

The information regarding epidemiological study was obtained by making personal visits to the commercial layer farms situated around Anand, Gujarat where mortality was reported due to ND with regular vaccination programme. The information like name and location of farm, strength of flock, age of the flock when ND was first detected, mortality pattern, duration of mortality (Period for which mortality continues in the farm during outbreak), effect on body weight, production drop, effect on feed consumption, history of vaccination, type of vaccination were recorded. Total 13 layer farms were visited where mortality due to Newcastle disease virus (NDV) was reported.

Gross pathology

During the outbreak of disease in the layer farms, the ailing birds were examined for clinical signs if any and detailed post-mortem was carried out from the carcass in the Department of Pathology and gross pathological lesions were recorded. The gross pathological lesions were recorded and characterized depending upon the severity of the disease as well as concominent other lesions suggestive of secondary bacterial infections like Escherichia coli or Mycoplasma.

Histopathological study

For histopathological examination, tissues from visceral organs like lung, trachea, liver, spleen, kidney, proventriculus, intestine, caecal tonsils, bursa of Fabricious and brain were collected in 10% neutral buffered formalin and processed by paraffin embedding technique. Sections were cut at 5-6 µ thickness with automatic section cutting machine (Leica, Germany) and stained with haematoxylin and eosin (H and E) [9]. The H and E stained sections were observed under the light microscope, and lesions were recorded.

Collection of samples for virological study (NDV propagation in egg embryo)

At the time of post-mortem examination, the carcasses showing gross lesions suggestive of ND were selected for collection of samples for detection of NDV i.e. Propagation and cultivation of NDV in specific pathogen-free egg embryo. After taking necessary aseptic precautions, the pooled tissue samples of trachea, lung, spleen, proventriculus, caecal tonsils and intestine representative of each layer farms were collected separately in sterile petridish for further processing. A total of 13 samples from 13 farms were collected, and the tissues from each farm were pooled and processed as a single sample.

NDV

Reference virus strains (F and R2B) of NDV obtained from the Vaccine Institute, Gandhinagar were used for control study.

Cultivation of NDV in egg embryo

Inoculation in embryo through allantoic route and collection of allantoic fluid carried out as per the method described in OIE Terrestrial Manual 2012 [10].

HA and HI tests

To isolate virus both the test was carried out as per the method described in OIE Terrestrial Manual 2012.

Pathogenicity index

ICPI is an accepted in vivo test for the assessment of virulence of NDV in 1-day-old chicks because of its established accuracy and sensitivity (OIE Terrestrial Manual 2012). During the study, representative samples of different layer farms were inoculated in day old chicks and were pathotyped performing ICPI to evaluate the virulence of the field isolates as stated by Alexander and Senne [11].

The present field isolates obtained during field outbreaks of ND from layer and broiler farms were submitted to Department of Microbiology for F gene sequence and to Department of Animal Biotechnology for whole genome sequencing. The Genome length found was 15192nt. The phylogenetic analysis and evolutionary distances placed these isolates in Genotype XIII (XIIIb) with the available latest sequences in the gene bank.

Epidemiological studies

ND was reported in thirteen layer farms around Anand, Gujarat, India in spite of routine vaccination programme during the period March 2013 to April 2014. Anand is located at 22.57°N 72.93°E. The day temperature in winter is around 83°F and at night is 53°F. Summers are extremely hot, with the day temperature being 115°F, and the night temperature being 90°F. In Gujarat, monsoon is generally hot and utterly humid. The temperature at day is 100°F but at night it falls down to 80°F.

Strength and age of the flock when ND first detected

The strength of the affected flocks in different farms ranged between 4320 and 33764 birds (Table-1). The outbreaks of ND in all the flocks studied were in the age group ranging between 5 and 36 weeks. The disease was noticed as early as 33 days to as later on 252 days of age. All the affected flocks were housed in cage system of management.

Mortality

Mortality due to ND in all the thirteen flocks was ranged between 6.53 and 53.88%. The overall mortality was 21.21%. Among different layer birds mortality in layer chicks ranged from 13.26% to 53.88%, in growers 9.14-19.21%, and in layers 6.53-9.65% (Table-1). Younger flocks had higher mortality due to low vaccinal immunity resulting from less number of vaccinations. The death was reported daily without any control in spite of vaccination and medication. The duration of mortality recorded in all the layer farms was as short duration as 13 days at Yogi poultry Farm, Sarsa and as long duration as 46 days at Bhoomi poultry Farm, Sarsa. The overall duration of mortality was 23 days (Table-1).

Vaccination history

As per available records on each of the affected farms it was noticed that the affected layer flocks were vaccinated with Lasota (D/W) and lentogenic ND Killed 0.25 ml (S/C) at neck region in 1st week (Flock no. 1, 2, 3) further Lasota boostering was done at 5^th week (Flock no. 4, 5, 6, 7 and 8), R₂B 0.5 ml (I/M) at 10th week (Flock No. 9, 10 and 11), Lasota (D/W) and Lentogenic ND killed 0.5 ml (S/C) during prelay stage at 20^th week and Lasota (D/W) at 35^th weeks of age (Flock No. 12 and 13). The vaccination schedule was followed as per standard guidelines using cold chain.

Effect on feed consumption

The entire affected layer flocks revealed reduction in feed intake ranging from 3.46% to 46.91%. In the majority of the farms (10/13) feed intake was reduced above 17%, whereas in three farms it was reduced below 10% in comparison to the standard feed intake (Table-2).

Effect on body weight

The entire affected layer flocks revealed depression in body weight ranging from 13.85% to 42.37%. Majority of farms (11/13) revealed depression in body weight above 19% during the course of the disease as compared to standard body weight as per the guidelines given by hatchery (Table-2).

Seasonal distribution

During the study period, it was observed that majority of the outbreaks (11/13) were reported in the summer months of April to June while only two farms showed the presence of disease in the month of January and February. The seasonal distribution pattern indicated that birds were more prone for the disease in summer months than winter months (Table-2).

Effect on production

Out of total thirteen flocks affected with the disease, only two farms were in laying phase. As per the available data of production performance, both these flocks revealed drop in egg production during the course of the disease. The drop in egg production was from 89.28% to 56.44% in Keval Poultry farm Sarsa, and from 89.4% to 68.0% in Honest Poultry Farm, Flock No-A, Sarsa. Production reached up to 84% after 6 weeks at Keval Poultry farm Sarsa, and up to 86% after 4 weeks at Honest Poultry Farm, Flock No-A, Sarsa. But none of the farms reached up to original production more than 86% during laying phase. Both these laying flocks showed 20-30% drop in hen day egg production. There was a slow recovery after a month but failed to reach original production level. The appearance of lethargy eggs and eggshell breakage which are usually seen with classical ND outbreaks were not severe in the present outbreaks.

Clinical signs

At the beginning, the disease appeared suddenly with high mortality without any clinical signs in the entire affected layer. Subsequently clinical signs appeared gradually. They include reduced feed intake, listlessness, increased respiration, Greenish diarrhoea with soiled feathers of vent, dehydration, loss in body weight, conjunctivitis, prostration and increasing mortality. Some of the flocks also showed oedema around eyes and head. Greenish diarrhoea (Figure-1) was frequently seen in birds that survived early in infection. Mortality continued for 2-3 weeks and reduced with appearance of torticollis. Birds with torticollis (Figure-2) showed lingering mortality. Among the laying flocks there was 20-30% drop in hen day egg production which showed slow recovery after 5-6 weeks but failed to reach original production level.

Figure-1

Layer bird affected with Newcastle disease showing greenish diarrhoea and depression.

Figure-2

Layer bird affected with Newcastle disease showing characteristic symptoms of torticollis (twisted neck and paralysis).

Pathological studies

Gross pathology

Gross pathological lesions observed in all the layer flocks were typically of velogenic viscerotropic ND. The lesions were characterized by emaciation and dehydration of carcass with deep congestion of breast musculature, multifocal to diffuse haemorrhages around proventricular gland, necrotic (diphtheritic) haemorrhagic ulcers throughout the intestine and caecal tonsils (Figure-3). In addition to the disseminated multiple foci of necrosis, pin-point haemorrhages were observed in the spleen parenchyma. Severe congestion of trachea and lungs was a prominent feature in the majority of layer chicks. In addition, the laying flocks showed degenerated, misshapen and haemorrhagic ova with lesion of egg peritonitis.

Figure-3

Proventriculus showing diffuse haemorrhages along with haemorrhagic and necrotic patches on intestine and caecal tonsils.

Histopathology

Proventriculus

The lesions obtained were focal to diffuse haemorrhages within mucosal ridges as well as in the glandular regions of proventriculus and were suggestive of haemorrhagic proventriculitis (Figure-4). Along with haemorrhages, there was diffuse infiltration of lymphocyte in the mucosa with shortening and sloughing of proventricular papillae.

Figure-4

Section of proventriculus from a layer bird showing focal to diffuse haemorrhages in the mucosal and glandular region. H and E stain ×150.

Spleen

The lesions in the splenic parenchyma includes mild to moderate congestion, multifocal areas of lymphoid necrosis (Figure-5) and reticuloendothelial cell hyperplasia. The lesions were consistent and observed in the majority of flocks.

Figure-5

Section of spleen from a layer bird showing mild congestion and multi-focal areas of lymphoid necrosis. H and E stain ×150.

Brain

Lesions in the brain parenchyma though not consistent but were indicative of non-purulent encephalitis with neuronal degeneration. They included perivascular infiltration of lymphocytes, neuronal degeneration and foci of glial cells in the brain parenchyma (Figure-6). The lesions were observed only in few flocks of grower birds.

Figure-6

Section of brain from a layer bird showing neuronal degeneration and focal area of gliosis. H and E stain ×300.

Virus propagation in egg embryo

All the 13 flock samples (n=13) were inoculated in 9-11 days old embryonated SPF eggs, which resulted in death of all embryos following incubation up to 72 h post-inoculation, thereafter allantois fluid was harvested. The allantois fluid was then subjected to centrifugation at 10000 rpm for 10 min for removal of cellular part. The embryos were taken out one by one in separate petri dishes and washed with nephron sparing surgery for observation of gross lesions produced by the virus. There were multiple haemorrhagic lesions produced throughout the surface of the body of the embryos particularly at the head region (Figure-7).

Figure-7

Egg embryos of SPF chicks infected with Newcastle disease virus showing haemorrhagic lesions on the body surface.

HA and HI tests

The test revealed the HA titers in all the 13 field samples and two vaccine sample in between the range of 2⁴-2⁸. HA was confirmed by presence or absence of mat formation of the RBCs (Table-3). The titration was considered as positive at the highest dilution giving complete HA. The positive haemagglutinating allantoic fluids were tested by HI test with known NDV anti-serum and it showed the HI titers of 2³-2⁹ (Table-4). The HI titer was considered as the highest dilution of serum causing complete inhibition of 4 HA units of the antigen. The agglutination was assessed by tilting the plates, and streaming of RBCs was compared with control wells (positive-serum, virus and PBS controls) to record the results. Results of HA and HI confirmed the presence of NDV in allantoic fluids obtained from all the 13 of embryonated eggs inoculated with field samples.

Along with all the 13, NDV F and R₂B (Reference strain) allantoic fluid samples were found positive for HA activity. Among these, two samples (15.38%) had HA titer of 1:16 or below, ten samples (76.92%) had titer between 1:16 and 1:64 and one samples (7.69%) had titer of 1:128 and above. Higher titers indicated more concentration of the virus in the respective samples.

HA positive 13 samples and the reference R2B and F strain allantoic fluid samples were subjected to HI test for the confirmation of NDV by using constant antigen (4HA units) and serially diluted known NDV anti-serum. All the 13 samples and the reference R2B and F strain allantoic fluid sample were found positive for HI activity. Among these eight samples (61.53%) had HI titer of 1:16 and below, three samples (23.07%) had titer between 1:16 and 1:64, and two samples (15.38%) had titer between 1:128 and 1:512. This approach was necessary to confirm the specific nature of HA activity. The finding of present study i.e. HA and HI activity of field samples further confirmed that all the 23 flocks were established cases of virulent NDV (VNDV) infection and the disease occurred on these farms in spite of regular prevention programme.

Pathotyping by ICPI

As per OIE, the ICPI test is scoring systems that evaluate illness or death in chickens. The values in the ICPI test range from 0 to 2.0. The most virulent viruses approach the ICPI value near 2.0, while lentogenic strains are usually close to 0.0. For velogenic strains, the cut-off value of ICPI score is 0.7. The ICPI value found during the present study from all thirteen samples was 2.0 and indicative of velogenic nature of the field NDV strains.