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. 2019 Jul 22;30(3):453–461. doi: 10.1007/s13337-019-00537-2

Genotypes II and VIId-based inactivated Newcastle disease vaccine reduces virus shedding

Nehal K Mahmoud 1, Ayman H El-Deeb 2, Mohammed M Emara 2, M A Abd El-Khaleck 1, Hussein A Hussein 2,✉
PMCID: PMC6864008  PMID: 31803813

Abstract

In Egypt, recent outbreaks were reported in NDV-vaccinated flocks. The isolated strain was characterized as class II velogenic genotype VIId of Newcastle disease virus (NDV). In this study, three inactivated NDV vaccine formulations were prepared, the first one is LaSota (genotype II), the second one is genotype VIId and the third one is combined Lasota and genotype VIId at a proportion of 1:1. The challenge trials were conducted in SPF chicks to evaluate the efficacy of the prepared vaccines using 106 EID50/0.5 ml of the Egyptian genotype VIId strain of Newcastle disease virus (NDV-B7-RLQP-CH-EG-12). Our results revealed that all three prepared vaccine formulations conferred 100% protection in the vaccinated chicks. However, the combined vaccine induced the highest haemagglutination inhibition (HI) titers and neutralization indices with significant reduction in virus shedding compared to other vaccine formulations. Histopathology examination of different organs collected from vaccinated chicks post challenge indicated the protective efficacy in vaccinated groups compared to the positive control group where a score of severe lesions was shown. This study reports the efficacy of combined inactivated Lasota and genotype VIId vaccine in reducing virus shedding which is very important in controlling NDV infection in chicken.

Keywords: Neutralization index, NDV genotype VIId, Cross protection, Real time PCR, NDV shedding

Introduction

Despite the intensive vaccination programs that have been developed and used for controlling Newcastle disease (ND), it is still a serious problem facing poultry production sectors worldwide [26]. Newcastle disease is caused by virulent strains of Newcastle disease virus (NDV), an avian paramyxovirus serotype 1 (APMV-1) that belongs to the genus Avulavirus, subfamily Paramyxovirinae, family Paramyxoviridae [11]. Based on the severity of the disease, NDV strains are divided into 5 pathotypes: velogenic viscerotropic, velogenic neurotropic, mesogenic, lentogenic and asymptomatic enteric [2].

The velogenic NDV strains have two basic amino acids, either lysine (K) or arginine (R), at the fusion (F) cleavage site at residues 112–113 and 115–116 as well as a phenylalanine at residue 117 that is cleaved by ubiquitous intracellular proteases. On the other hand, lentogenic strains such as lasota strain have a monobasic cleavage site that is cleaved by extracellular proteases; thus, replication of these strains is restricted to specific tissues [18].

Although all strains of NDV belong to one serotype, antigenic and genetic diversities among them have been reported [15]. To date, genotype V and VII of ND viruses are the most currently circulating worldwide [14]. In Egypt, several outbreaks still occur in vaccinated poultry flocks [8, 20]. Isolation and characterization of such ND strains from recent outbreaks in different vaccinated farms revealed the prevalence of NDV genotype VIId, which is closely related to the Chinese isolates [20]. Mixed infection of this genotype with other respiratory viruses have been reported in the last 8 years [8]. Previous studies reported that genotype VIId is currently endemic in many countries and induces more severe damage in lymphoid tissues when compared to virulent viruses of other genotypes; Genotype VIId damages lymphoid tissue through severe lymphocytic depletion and necrosis of the spleen [27, 31, 34]. Although available commercial vaccines are supposed to induce full protection against challenge with virulent NDV strains, several studies have shown that they do not prevent infection and/or virus shedding [9, 12, 15]. This is considered very critical as it leads to continuous spread of the virus to other vaccinated birds [13].

In this study we prepared three different formulations of inactivated Newcastle disease virus vaccine based on the use of one or mixing of two seed strains of different genotypes. So that we can evaluate their efficacy and compare the level of protection induced by the classical genotype II lasota vaccine with that induced by genotype VIId strain vaccine alone, and also with that induced by mixing both strains in the vaccine following challenge with the Egyptian NDV genotype VIId, which is the causative agent of ND recent outbreaks. Also to identify if the genotype- matched NDV vaccine production is essential and required or the available commercial genotype II lasota vaccine is sufficient to confer protection against genotype VIId infection.

Materials and methods

Viruses

Genotype II lasota strain of NDV was obtained from VSVRI poultry viral vaccine department, Abbassia, Egypt, it was supplied by the Central Veterinary Lab.,Weighybridge, England, and used in vaccine preparation and virus neutralization assays. Genotype II lasota strain is classified as a lentogenic strain of NDV with mean death time (MDT) >  90 h. Genotype VII of NDV (NDV-B7-RLQP- CH-EG-12) is classified as a velogenic NDV with mean death time (MDT) of 52 h and is considered a virulent NDV strain based on the sequence analysis of the F gene cleavage site. It is registered in GenBank with the accession number. (112RKQKR*F117KM288609). Genotype VII was kindly provided by the National Laboratory for Veterinary Control of Poultry Production, Animal Health Research Institute, Egypt, and used in vaccine preparation, virus neutralization assays and as a challenge virus. The viruses were propagated in specific pathogen free (SPF) embryonated chicken eggs (ECEs) and titrated by Reed and Muench method [21]. The harvested viruses were inactivated with binary ethylenimine (BEI) as previously described in a previous study [3].

Vaccines formulation

Vaccines were prepared as water in oil emulsion (W/O) using MONTANIDE™ ISA 70 (incomplete SEPPIC Adjuvant) VG (Vegetative origin) at a ratio of 30/70 (w/w) aqueous/oil ratio. Stable preparations were obtained by mixing the aqueous medium into the MONTANIDE™ ISA 70, at room temperature or less, under vigorous stirring (for 15–30 min) according to the manufacturer’s recommendations. The vaccine formulations prepared were: inactivated Lasota vaccine, inactivated genotype VIId NDV vaccine and a combined inactivated vaccine containing lasota and genotype VIId strains at a proportion of 1:1, the antigen content in all the prepared vaccines was not less than 108.4 EID50/0.5 ml.

Experimental design

Three weeks old SPF chicks (n = 105; n = 35 for each vaccinated and challenged group; n = 15 for positive control and n = 10 for negative control) were purchased from the National specific pathogen free (SPF) poultry project (Kom Oshim, EL-Fayoum, Egypt). All chicks were reared in isolators with feed and water ad libitum. Birds were divided as follows: Group1 was vaccinated with inactivated genotype II Lasota, group2 was vaccinated with inactivated combined Lasota-VII, group3 was vaccinated with inactivated genotype VII NDV, group 4 (n = 15) was left unvaccinated and challenged as a positive control and group5 was neither vaccinated nor challenged as a negative control. Vaccinated chicks received 0.5 ml of the prepared vaccines, subcutaneously.

The use of animals and protocols were approved by the Animal Care and Use Committee of Veterinary Serum and Vaccine Research Institute, Egypt.

Serology

Serum samples were weekly collected from birds for 4 weeks post vaccination and 2 weeks post challenge. The collected sera from all groups were inactivated at 56 °C for 30 min, and stored at − 20 °C.

Serum antibody levels of NDV were determined by Haemagglutination inhibition (HI) and cross HI tests in U-shape microtiter plate using 4 HA units of Lasota and genotype VII strains of NDV, the tests were performed using standard protocols of OIE [33]. Serum neutralization indices were determined by Virus neutralization (VN) and cross VN assays in 9–11 day old SPF-ECE using diluted viruses (Lasota and genotype VII strains of NDV) and constant serum, the procedures were followed as described by Thayer and Beard [28].

Challenge trial

The challenge virus (genotype VII NDV) was propagated in 9–11 day old SPF embryonated chicken eggs and then the harvested fluid was titrated as described by Reed and Muench [21] to prepare the required dose for the challenge test as mentioned in OIE [33]. Birds in groups 1, 2, 3 and 4 were challenged at 28th day post vaccination with 106 EID50/0.5 ml genotype VII NDV by intramuscular route and monitored daily for clinical signs. Three chicks from group 1, 2, 3 and 4 were sacrificed, tracheal swabs were taken for quantitation of virus shedding and the internal organs were collected for histopathological examination.

Quantitation of virus shedding

Tracheal swabs (n = 3) from each group (1, 2, 3 and 4) were collected at the 2nd, 4th, 7th and 10th day post challenge for quantitation of virus shedding. Swabs were dispersed in 800 µl phosphate buffer saline then stored at − 80 °C. Virus shedding was evaluated by Real time RT-PCR [32] and virus infectivity titration in SPF-ECEs [21].

Total RNA was extracted from the collected tracheal swab fluid using QIAamp Viral RNA extraction kit according to the manufacturer’s instructions. The extracted RNAs were tested for presence of NDV–RNA using specific primers and probes targeting M gene amplification as described by wise et al. [32].

The samples which were positive by Real time RT-PCR were then tested for virus isolation and titration in 9–11 day old SPF-ECEs. The virus shedding titers were calculated by Reed and Muench [21].

Histopathological examination

Brain, cecal tonsils, spleen, kidneys, proventriculus and lungs were collected from each group on the 2nd, 4th, 7th and 10th day post challenge and were fixed in 10% neutral buffered formalin and processed for paraffin embedding as previously described [4]. Histopathological sections were stained with haematoxylin and eosin and examined with light microscope.

Statistical analysis

Results represent the mean with standard error of at least triplicate determinations (n = 3). Statistical significance was determined by two-way analysis of variance (ANOVA) with LSD post hoc test using the statistical software program SPSS (version 21.0). Graphs were made using prism 7 software (Graphpad software Inc.,). Significance was reported at the level of P ≤ 0.05.

Results

Serology

Serum samples HI titers and virus neutralization indices were determined using NDV homologous and heterologous antigens (Fig. 1). The observed results of HI test revealed that the mean of NDV antibody titer in group3 was significantly lower than group1 and group2 in the 2nd and 3rd weeks post vaccination. During the 4th week post vaccination, a little difference was observed between all vaccinated groups by using Lasota antigen in HI test (Fig. 1a).

Fig. 1.

Fig. 1

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Mean of HI titer log2 and neutralization indices of serum from vaccinated chicks with different prepared inactivated vaccines. a Lasota strain specific HI test. b Genotype VIId strain of NDV specific HI test. c Lasota strain specific VN test. d Genotype VIId strain of NDV specific VN test. Group (1): lasota vaccinated group. Group (2): Combined (lasota and genotype VIId of NDV) vaccinated group. Group (3): Genotype VIId of NDV vaccinated group

On the other hand, during the 3rd and 4th weeks post vaccination, difference observed between group1 and group2 by using genotype VII NDV antigen in HI test; Where, the highest antibody titers were observed in group2 (Fig. 1a, b). The mean of NDV antibody titer increased significantly during 1st and 2nd weeks post challenge.

The results of VN test showed that during the 4th week post vaccination, the mean of neutralization indices was significantly lower in group3 than group1. During the 5th week post vaccination the difference was observed between group1 and group 2 using Lasota strain in virus neutralization test (Fig. 1c).

Using genotype VII NDV strain in virus neutralization test, the mean of neutralization indices was significantly different between group1 and group2 during the 4th week post vaccination. The highest neutralization indices were observed in group2 during the 3rd and 4th weeks post vaccination (Fig. 1d).

Homologous antigens and homologous viruses were found to record higher HI antibody titers and neutralization indices compared to heterologous ones.

Protective efficacy of different inactivated NDV formulations

All chicks in the negative control group remained normal during the course of the experiment while the non-vaccinated-challenged chicks in group4 displayed conjunctivitis, severe depression, paralysis in lower extremities and greenish diarrhea during the 2nd to the 4th day post challenge with 100% mortality within 4–5 days post challenge. Post mortem examination revealed severe congested viscera with hemorrhagic spots on the tips of proventriculus. The protection % was 100% in all vaccinated groups where neither symptoms nor mortalities were observed.

Quantitation of virus shedding

Tracheal swab samples were taken from three chicks (n = 3) per group (1, 2, 3, 4) on the 2nd, 4th, 7th, and 10th day post challenge and were tested for viral shedding. All tracheal swabs taken from group4 (non-vaccinated challenged group) were positive (3/3). All three samples collected on the 2nd day post challenge from group1 were negative (0/3), but the virus shedding was positive in 2/3, 3/3 and 2/3 of the tested chicks on the 4th, 7th and 10th day post challenge; respectively. In case of group3, the virus shedding in the swabs collected on the 2nd, 4th, 7th and 10th day post challenge was positive in 3/3, 2/3, 3/3 and 2/3 of the tested chicks, respectively. In group2, the shedding results revealed that the samples collected on the 7th and 10th day post challenge were positive in 1/3 and 2/3 of the tested chicks; respectively While the samples taken on the 2nd and 4th day post challenge were negative (0/3).

Results of mean virus titers Log10 detected in tracheal swabs on the 2nd day post challenge revealed a significant difference in groups 3 and 4 compared to Group 1 and 2. While there was a difference observed in group 2 compared to group 3 and 4 on the 4th day post challenge. Also, on the 7th day post challenge there was a difference observed between group2 and other vaccinated groups as shown in Table 1.

Table 1.

Quantification of viral shedding

Groups Days post challenge
2nd 4th 7th 10th
Virus titer (Log10)
rRT-PCR Isolation rRT-PCR Isolation rRT-PCR Isolation rRT-PCR Isolation
Group 1 0a 0a 3.66 ± 1.8ab 0b 6.66 ± 0.33b 1.99 ± 1.2a 4.33 ± 2.1a 1.82 ± 0.9a
Group 2 0a 0a 0a 0b 2 ± 2a 0a 3.33 ± 1.76a 0a
Group 3 5.33 ± 0.33b 2.1 ± 1b 5.33 ± 2.66b 2.35 ± 1.19c 6.66 ± 0.33b 1.4 ± 1.4a 3 ± 1.5a 0a
Group 4 5.66 ± 0.66b 5.66 ± 0.6c 8.67 ± 0.33b 8.67 ± 0.33a NS NS
Group 5 0a 0a 0a 0b 0a 0a 0a 0a
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Mean of virus titer log10 EID50%/0.1 ± S.E of NDV from the tracheal swabs post challenge measured by rRT-PCR and infectivity titration. Means with different superscript letters (a, b, c) within the same column are significantly different at P value  < 0.05 between chicken groups. Group 1: lasota vaccinated group. Group 2: combined (Lasota-genotype VIId NDV) vaccinated group. Group 3: genotype VIId NDV vaccinated group. Group 4: unvaccinated challenged group. Group 5: unvaccinated, not challenged group. NS: no survivors

Results of virus isolation and titration of PCR-positive tracheal swab samples shown in Table 1 revealed that group1 was negative for NDV in swabs collected on the 2nd and 4th day post challenge, but the virus shedding was positive in 2/3 of the tested chicks on the 7th and 10th day post challenge. In case of group3, the virus shedding in the swabs collected on the 2nd, 4th, 7th and 10th day post challenge was positive in 2/3, 2/3, 1/3 and 0/3 of the tested chicks, respectively. While in group2, the shedding results revealed that samples collected on the 2nd, 4th, 7th and 10th days post challenge were negative. A significant reduction in the virus titer was observed in all vaccinated groups when compared to group 4 on the 2nd and 4th day post challenge. While on the 2nd and 4th day post challenge there was a difference between group3 and other vaccinated groups as shown in Table 1. On the other hand, there was no virus isolated in the tested samples taken from group2 along all days of sampling.

Histopathological examination

On the 4th day post challenge the non-vaccinated challenged birds kidneys exhibited moderate hyperemic blood vessels with degeneration of renal tubules in addition to focal mononuclear cells infiltration (Fig. 2A2). The Spleen showed moderate depletion and necrobiotic changes of lymphocytes (Fig. 2B2). While the cecal tonsils showed sloughed epithelium with hemorrhage in tips of villi and pronounced depletion and degeneration of lymphocytes also were observed as shown in (Fig. 2 C2). On the other hand, Brain showed pronounced perineural and perivascular edema in addition to focal cerebral spongiosis and focal cerebellar demyelination (Fig. 3A2). The proventriculus exhibited focal necrobiotic changes in glandular epithelium with focal mononuclear cells infiltration accompanied with interglandular congestion (Fig. 3 B2) and the lungs showed focal interstitial fibrinous pneumonia (Fig. 3C2). All vaccinated groups showed mild lesion scores Figs. 2 and 3A1, B1, C1.

Fig. 2.

Fig. 2

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Histopathological sections from kidney, spleen and cecal tonsils. A Kidney, B spleen and C cecal tonsils. (1) Representative of organs collected from groups 1, 2 and 3. (2) Representative of the group 4 positive control. (3) Representative of the group 5 negative control

Fig. 3.

Fig. 3

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Histopathological sections from Brain, Proventriculus and Lungs. A Brain, B proventriculus and C lungs (1) Representative of organs collected from groups 1, 2 and 3. (2) Representative of the group 4 positive control. (3) Representative of the group 5 negative control

Discussion

Control of Newcastle disease (ND) centers on biosecurity application in poultry flocks and vaccination [33]. In Egypt the available commercial vaccines are supposed to confer protection and limit viral transmission between vaccinated flocks, but ND outbreaks continue to occur.

The present study was designed to prepare three different formulations of inactivated Newcastle disease virus vaccines using genotype VII; the causative agent of recent outbreaks and genotype II lasota strain that mainly used in manufacturing of inactivated classical vaccines. The study design was conducted to compare between both vaccines and also a mix of two strains vaccines to determine their ability to protect against NDV and prevent virus shedding post challenge with the current circulating strain of NDV.

The potency of the prepared vaccines was evaluated by challenge trials according to OIE regulations [33]. Despite the genetic and antigenic diversity between both strains of the prepared Lasota vaccine and the challenge virus, full protection was observed in all vaccinated groups. It is reasonable to expect that after NDV vaccination, homologous antibodies elicit and can neutralize common and similar antigenic sites between the antigenically different NDV strains that can fully protect the vaccinated birds this is similar to which was reported previously [23]. In contrast, some previous studies reported that any antigenic modification in the antigenic sites proteins (HN and F) can influence the vaccine efficacy and thus overcome the vaccine barrier and cause the disease [17]. In case of non-vaccinated challenged group, typical signs and post mortem findings for NDV appeared clearly after the 4th day post challenge with 100% mortalities within 4–5 days post challenge.

Detectable antibody titers were observed in the first week post vaccination in all vaccinated groups. However, comparing the results of antibody titers in genotype VII vaccinated group with Lasota vaccinated group, the results showed that in genotype VII vaccinated group, early and slightly higher titers were induced than in Lasota vaccinated group in the first week post vaccination, which is in accordance with a previous study [10]. Cross HI titer can vary greatly depending on the antigen used in HI test; each vaccinated group showed higher antibody titers when tested against its homologous antigen than the heterologous one Fig. 1 and this is parallel to previous studies [12, 22, 29]. Such observation was also confirmed with virus neutralization test, the highest neutralization indices were observed in combined vaccinated group with Lasota and genotype VII in the 3rd and 4th weeks post vaccination with using genotype VII in VN test. Our results indicated that the combined vaccine formula has the ability to induce maximum neutralization for the circulating strain of NDV genotype VII. The high level of antibodies which was shown in HI and VN results was sufficient to induce protection through homologous and/or heterologous vaccines suggesting that the protection depends on the level of antibodies at the time of challenge, this is in accordance with the previous studies which reported that heterologous vaccines can prevent transmission if sufficient time is allowed for birds to mount a proper immune response [15, 29].

Although complete protection (100%) and high antibody titers were reported in the present study, the causes of continuous outbreaks may be due to insufficient immune response induced by applied vaccines or failure in vaccination practice and/or bad storage of vaccine batches. Vaccine efficacy is not only affected by antigenic variation which is mainly responsible for outbreaks but it might be also a result of poor flock immunity due to inadequate vaccination practices, hence, leading to the spread of virulent NDV field strains [5]. Previous studies reported that poor NDV vaccine-induced immunity may be due to inappropriate vaccination doses and regimes, and/or due to the presence of infections, especially immunosuppressive diseases and nutritional deficiencies as well as mycotoxins in feed [25]. The biological properties of such a velogenic strain and the immuno-pathological patterns in early infection are different as previously reported [7, 9, 24]; where the immuno-pathological pattern of ND genotype VII virus was found to reach the spleen as early innate host response to this virus and splenocytes destruction occurs as a result of elevation of cytokines expression levels [7, 24]. In contrast, the lentogenic strain doesn’t reach the spleen but only replicates at the site of entry [30]. Also, the host response to the virus may contribute to the pathogenesis of this strain in chicken [24].

With setting aside the mortality and morbidity rates assessment, it is still a concern that NDV is mostly transmitted from an infected bird to a susceptible one through fecal–oral route which increases the opportunity of contamination of feed and water sources [1]. Therefore, the level of virus shedding in poultry house is also considered an important parameter for the evaluation of commercial vaccines and also for controlling ND [16].

The results of virus shedding revealed that the vaccinated group with combined vaccine Lasota-VII has shown a significantly reduced viral shedding titer on the 4th and 7th day post challenge compared to other vaccinated groups, which were either vaccinated with Lasota or genotype VII. Also, there was no virus isolated in all PCR positive samples of group 2; this may be because the combined formula has the property of generating a high level of heterologous antibody titers elicited by the Lasota strain, which can neutralize the challenge virus and the property of generating homologous antibodies which can neutralize the remaining viruses escaping from heterologous antibodies and thus limiting the transmission and infection to the minimum. Likewise, previous studies reported that high levels of heterologous antibodies can prevent mortality while the presence of more specific antibody levels is also necessarily required to significantly decrease viral replication. This increases the interest in developing vaccines formulated with genotypes homologous to the virulent NDV (vNDV) circulating in the field [15].

Recently, using genotype VII as HI antigen gives more specific results especially during genotype VII infection. The higher antibody titer that was observed in group2 than in group3 at the time of challenge (4th week post vaccination) using genotype VII as HI antigen may explain why birds in group2 (homologous + heterologous) exceeded group3 (vaccinated with homologous genotype VII) in reduction of viral shedding. Previous studies confirmed the correlation between serologic response and inhibition of virus shedding, so birds with higher antibody titers showed better inhibition of virus shedding [6, 29]. A similar study reported that combined (H5 + heterologous lasota strain) vaccine with higher HI titer reduced viral shedding than did homologous vaccine (genotype VII) with lower HI titer [26]. These findings were different than that reported by Miller et al. [12]. It can be suggested that the high level of antibody produced upon vaccination is more critical than the presence of homologous antigen used in vaccine preparation and/or it may be due to a special behavior of the velogenic genotype VII which does not necessarily depend on antigen type or serology [19].

Histopathological examination revealed characteristic lesions of NDV in challenged non-vaccinated group (positive control) that start to appear from the 2nd day post challenge and continue till the complete death of all chicks within 4–5 days post challenge. Lesions were severe,mainly in lymphoid organs such as the spleen and lymphoid aggregates of the intestine as cecal tonsils. Such observation has been also reported by Zenglei and Liu [34] who suggested that the severe immuno-pathology observed in immune organs caused by genotype VIId of NDV is due to high a replication level of this strain, which can induce more potent innate antiviral and inflammatory response, and causes more severe damages in lymphoid tissues, which was confirmed in the positive control group. In contrast, the vaccinated groups demonstrated mild to no lesion score.

In conclusion, all the prepared inactivated NDV vaccine formulations protect chicks from morbidity and mortality and they give 100% protection with challenge trials. Our results revealed that the inactivated combined Lasota-VII vaccine is a better vaccine formula than Lasota or VIId inactivated vaccine formulae, by inducing high immunity leading to prevention of virus shedding. Thus, may help in controlling outbreaks caused by recent circulating NDV strains reported in the Egyptian poultry sector.

Acknowledgements

We are grateful for all Staff members of Newcastle Department, Veterinary Serum and Vaccine Research Institute (VSVRI), Cairo, Egypt for their great help during the study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The use of animals and protocols were approved by the Animal Care and Use Committee of Veterinary Serum and Vaccine Research Institute, Egypt.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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