Novel Recombinant Newcastle Disease Virus-Based In Ovo Vaccines Bypass Maternal Immunity To Provide Full Protection From Early Virulent Challenge Part 2

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4. Discussion

Despite the wide use of vaccination programs in commercial flocks, virulent Newcastle disease viruses continue to cause disease outbreaks globally [11]. The frequent occurrence of outbreaks and lack of complete efficacy of the currently available vaccines indicate that the development of alternative approaches for vaccine generation is probably needed. Here, the immunogenicity of two recombinant NDV-vectored experimental vaccines, containing an antisense avian IL4 insert and their backbone viruses for in-ovo vaccination of 18-day-old commercial eggs with high maternal immunity, and their protective efficacy against challenge with vNDV were evaluated. These recombinant vaccines efficiently overcame maternal immunity and elicited a strong post-vaccination immune response. The experimental vaccines provided 100% protection after early challenges with vNDV. Of note, the live LaSota low virulent virus, widely used as a conventional ND vaccine in chickens, induced humoral immune response and protected from clinical disease after early vNDV challenge in the ovo-vaccinated birds that survived post-hatch. 

However, the post-hatch mortality after in-ovo vaccination with LaSota was very high—100% in the LS-105.5 group and 43% in the LS-104.5 group. In addition, there were significant differences in body weights compared with the non-vaccinated control groups and, although low, 8.4% morbidity after the challenge. The high mortality and differences in body weights render this vaccine not applicable for ovo vaccination, as these adverse effects will result in significant economic losses when scaled to industrial poultry settings.

The recombinant ZJ1*L and ZJ1*L-IL4R vaccines administered in ovo at 18 DOE efficiently induced a strong humoral immune response in the presence of maternally derived anti-NDV antibodies. In most vaccine groups, the titers of post-vaccination HI antibodies reached or surpassed the titer of maternal immunity at hatch, while the titers in the control groups decreased by 35%. The ability of ND vaccines to overcome maternal immunity is critical, as vaccine efficacy in birds is correlated with the level of maternally derived antibodies, which neutralize the vaccine and thereby reduce the effectiveness of vaccination [18,47–49]. The presence of maternal antibodies is known to interfere with the development of active immunity when ND vaccines are administered intramuscularly, subcutaneously, via the intranasal route, in drinking water, and by aerosol [9,12,18,50]. The protective efficiency of live ND vaccines after administration in chickens with maternal immunity through conjunctival and intranasal routes has been less impacted compared with the other routes, perhaps due to the development of local immunity induced by these vaccines [18,51]. 

The immunity induced by inactivated vaccines has also been less affected by the presence of maternal antibodies [52], specifically when high doses are used, but the limitation of these vaccines is their inability to induce mucosal immunity. It has also been demonstrated that the efficacy of recombinant NDV vector-based vaccines expressing avian influenza proteins was reduced when the vaccines were administered in birds with pre-existing anti-ND and anti-AIV antibodies [53–55]. In this study, as suggested by the increase in HI-antibody titers in the birds vaccinated with the experimental rNDV vaccines, a strong host humoral immune response was generated despite the presence of high maternal antibody levels.

The recombinant ND vaccines are protected from early virulent NDV challenges. While the birds in all groups vaccinated with ZJ1*L, ZJ1*L-IL4R, or LS-IL4R attained 100% survival, the morbidity and mortality in the control groups were above 80% and 33%, respectively. The birds in the Hatch and BHI control groups were not fully protected when challenged with virulent ZJ1 NDV, displaying a high level of morbidity and mortality with lower after-challenge body weights compared with the rNDV vaccine groups. Although antibody titers above log2 3 are generally considered protective [56,57], the high morbidity and mortality observed in the control birds (antibody titers log2 4.5 at 13DPH) in the present study are suggestive that additional immune factors must play a role in the protection of birds from a virulent challenge. It is possible that in absence of mucosal immunity, only maternally derived antibodies, even in titers above the protective cutoff, do not afford full protection from clinical disease and mortality. It has been reported that the in-ovo route of vaccination presents the viral antigens to the mucosal surfaces of the respiratory tract and digestive tract favoring the development of mucosal immunity [58]. 

This, in addition to the high post-vaccination titers in the birds vaccinated with the recombinant experimental vaccines, could explain, in part, the 100% survival observed in these groups. Further studies to evaluate the level of mucosal immunity in ovo-vaccinated birds are necessary to support this hypothesis. The rHVT-ND vaccine did not provide full protection, and relatively high percentages of morbidity (83.3%) and mortality (33.4) were observed. This is not surprising, as it has been reported that it takes up to four weeks before full immunity is reached with some rHVT-ND vaccines [31,59]. This delayed immunity has been attributed to the slower in vivo replication of the vector [19]. As anti-NDV maternal antibodies decline rapidly after hatch [1,60], this slower development of protective immunity induced by rHVT-ND vaccines may present a window of exposure, during which chickens are susceptible to vNDV infection. The clinical signs (predominantly neurological) that were observed in the Hatch, BHI, and rHVT-ND groups are indicative of suboptimal protection, likely afforded by the maternal antibodies.

The absence of significant changes in pre- and post-challenge HI antibody titers in the rNDV vaccinated groups is suggestive of low replication of the challenge virus and effective neutralizing immunity induced by the rNDV vaccines in these birds. The ZJ1*L, ZJ1*L-IL4R, and LS-IL4R vaccine group titers were all within one log difference compared with the challenge titers. The decreased replication efficacy of the challenge virus in the vaccinated groups compared with the Hatch, BHI, and rHVT-ND control groups is additionally supported by the low amount of viral RNA detected from the challenged birds inoculated with rNDV vaccines. This would strongly suggest that although humoral immunity has an important protective role, with these recombinant ND vaccines, the innate mucosal immunity, in association with the primed cell-mediated immunity, is likely responsible for neutralizing the challenge virus. To highlight this point, birds vaccinated with the rNDV experimental vaccines had significantly less viral RNA detected at 2 DPC and 4 DPC. Indeed, at 2 DPC and 4 DPC, 25% to 50% of the DV experimentally vaccinated birds had no detectable viral RNA in the samples. 

In contrast, significant post-challenge seroconversion was observed in the control Hatch, BHI, and rHVT-ND groups in which the HI antibody titers doubled as compared with pre-challenge, suggesting high replication of the challenge virus. Accordingly, the shedding of high amounts of the virus through the oral route at 2 DPC and both routes at 4 DPC from the control birds was observed. In addition, the post-challenge body weights in all rNDV vaccinated groups were uniform and significantly higher compared with the Hatch, BHI, and rHVT-ND control groups. 

Although not significantly different, the titers of pre-challenge antibodies elicited by the LS-IL4R vaccines were one log lower than those elicited by the ZJ1*L-IL4R vaccines. It has been previously reported that anti-NDV-antibodies more efficiently neutralize homologous viruses [16,17,61]. It is therefore possible that the differences in pre-challenge HI antibody titers are a result of more efficient inactivation of the LS-IL4R vaccine by maternal antibodies (LaSota was used to vaccinate the parents) compared with the ZJ1*L-IL4R vaccine.

The post-hatch survival in the groups vaccinated at 18 DOE with rNDV vaccines containing IL4R insert, although not significantly different, was dose-dependent. While some post-hatch mortality was observed in the groups vaccinated with the rNDV experimental vaccines, mortality decreased with decreasing vaccine doses. In the LS-IL4R 103.5 group, the post-hatch survival was as high as the rHVT-ND control group. The post-hatch body weights in some of the vaccine groups were significantly lower at 8 and 13 DPH. However, the body weights in the ZJ1*L-IL4R 104.5 and 105.5 and LS-IL4R 103.5 groups were only slightly and insignificantly lower compared with the control groups. These minimal post-hatch adverse effects could be mitigated by additionally decreasing the vaccine dose. The LS-IL4R vaccine provided 100% survival after early virulent challenge at the low 103.5 doses, and evaluation of the protective efficacy of the recombinant ZJ1*L-IL4R vaccines using this and even lower doses is warranted. Moreover, the recombinant vaccines could be further attenuated by inserting an additional foreign gene [62].

The insertion of the antisense avian cytokine IL4 in the low virulent ZJ1*L and LS strains further attenuated these viruses, as was evident in the differences in post-hatch mortality and post-hatch body weights between the backbones and the recombinant experimental constructs. Insertion of foreign genes in the genome of NDV of low virulence has been shown to attenuate the virus with no adverse effects (e.g., the recombinant virus is not becoming more virulent or pathogenic) [62,63]. The expression of interferon-gamma (IFNγ) by a virulent NDV attenuated the virus and decreased morbidity and mortality in SPF chickens [40]. Chicken IFNγ, which modulates macrophage activation and inhibits viral replication, has been shown to improve protection and enhance immune responses against different avian pathogens [39], including NDV [26]. A dangerous gain-of-function, such as increased virulence, has been observed with inserting IL4 in certain poxviruses [64]. In contrast, our study indicates a decrease in the virulence of the vector, which highlights the differences between viral vectors and probably the main pathways involved in the infection they cause. 

Although cytokine-expressing avian vaccines have been suggested to improve protective immunity [12], no vaccine product has yet become commercially available. Cytokines are components of a well-balanced system of immune responses with multiple feedback loops [19]. Modifying the balance in this system by up-or down-regulation of certain cytokines may direct the immune response in a desired direction [39], but additional studies to quantify the broad spectrum of interleukins, chemokines, and interferons are needed to properly evaluate the overall impact of vaccines containing cytokine inserts.

The results from the SPF control groups demonstrate that the observed differences in induced humoral immunity, shedding, and survival from early challenge with a virulent NDV do not appear to be influenced solely by the maternal antibody titers in these commercial egg-hatched birds. While there was significant morbidity, mortality, and seroconversion in the Hatch, BHI, and rHVT-ND groups, as well as marked clinical signs, the vaccinated challenged SPF groups had 100% survival, no morbidity, and minimal post-challenge seroconversion. 

These findings would appear to support the conclusion that the observed protection in the experimentally vaccinated groups is linked to the in ovo immunization (vaccination with rNDV vaccines) and not linked to the presence or titer of maternally derived antibodies. Furthermore, the observed active shedding of the virus in all the vaccinated birds demonstrates the successful uptake of the administered novel vaccines.

In summary, due to the global use of the in-ovo method of vaccine administration and its multiple advantages to the poultry industry [19–21], the development of in-ovo NDV-based vaccines holds great promise for the future control of this disease. Our results demonstrate the development of vaccines able to bypass maternal immunity elicits a strong immune response denoted by antibody titer as early as two weeks post-hatch (which coincides with the decay of maternal antibodies), provides full protection from clinical disease, and significantly decreases viral shedding after virulent challenge. 

This is the first report describing the in-ovo use of live ND vaccines with high post-hatch survival rates and full protection from clinical signs after early virulent challenge. These novel vaccines are excellent candidates for further testing at lower doses and additional attenuation through reverse genetics and the insertion of more genes. Due to their low cost and convenient production, storage, and transportation, such live rNDV vaccines may present an efficient alternative to current ovo ND vaccines.

Supplementary Materials:

Supplementary Figure S1: (A) Schematic representation of recombinant constructs and (B) schematic and (C) table representation of the study, Supplementary Figure S2: Survival of chickens post-hatch after inoculation of commercial eggs at 18 days of embryo nation with experimental in ovo NDV vaccines and controls, Supplementary Figure S3: (A) Post-hatch survival, (B) mean post-hatch vaccine shedding titers, (C) pre- and post-challenge HI antibody titers, (D) post-challenge survival, and (E) mean post-challenge viral shedding titers after inoculation of SPF eggs at 18 days of embryo nation with experimental in ovo vaccines and challenge with virulent Newcastle disease virus at 14 days post-hatch. SPF eggs were inoculated with ZJ1*L-IL4R 103.5 and ZJ1*L 103.5 EID50/per egg.

Author Contributions:

Conceptualization, K.M.D., T.L.T., D.W.-C., T.L.O., Q.Y., V.C.M., R.M.G.J., D.L.S., and C.L.A.; methodology, K.M.D., T.L.T., C.L.A., and D.L.S.; formal analysis, K.M.D., T.L.T., T.L.O., and D.W.-C.; investigation, K.M.D., T.L.T., Q.Y., D.L.S., and C.L.A.; resources, funding acquisition, and project administration, D.L.S. and C.L.A.; writing—original draft preparation, K.M.D. and T.L.T.; writing—review and editing, all authors. All authors reviewed and modified this manuscript, and read and agreed to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding:

This research was funded by the U.S. Department of Agriculture, ARS CRIS Project 6612-32000-072-00D.

Institutional Review Board Statement:

All animal experiments were approved and performed under the regulations of the USNPRC’s Institutional Animal Care and Use Committee under Animal Use Proposal Afonso USNPRC-2020-020 approved on 20 November 2017.

Informed Consent Statement:

Not applicable.

Acknowledgments:

We would like to thank Leonardo Susta for his help in the preliminary evaluation of the experimental vaccines. We would also like to thank Roger Brock and Keith Crawford for their assistance with the animal studies and R. Gayman Helman for his useful comments on the manuscript. We are also thankful to Hy-Line Rockside for kindly donating the commercial eggs for this study. The mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.

Conflicts of Interest:

The authors declare no conflict of interest.

References

1. Suarez, D.L.; Miller, P.J.; Koch, G.; Mundt, E.; Rautenschlein, S. Newcastle disease, Other Avian Paramyxoviruses, and Avian Metapneumovirus infections. In Diseases of Poultry, 13th ed.; Swayne, D.E., Ed.; Wiley-Blackwell: Hoboken, NJ, USA, 2013; pp. 89–138. [CrossRef]

2. International Committee on Taxonomy of Viruses. Virus Taxonomy: 2018 Release.

3. Amarasinghe, G.K.; Ayllon, M.A.; Bao, Y.; Basler, C.F.; Bavari, S.; Blasdell, K.R.; Briese, T.; Brown, P.A.; Bukreyev, A.; BalkemaBuschmann, A.; et al. Taxonomy of the order Mononegavirales: Update 2019. Arch. Virol. 2019, 164, 1967–1980. [CrossRef]

4. Lamb, R.A.; Parks, G.D. Paramyxoviridae. In Fields Virology, 6th ed.; Knipe, D.M., Howley, P.M., Eds.; Lippincott, Williams & Wilkins: Philadelphia, PA, USA, 2013; pp. 957–995.

5. Czeglédi, A.; Ujvári, D.; Somogyi, E.; Wehmann, E.; Werner, O.; Lomniczi, B. Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications. Virus Res. 2006, 120, 36–48. [CrossRef] [PubMed]

6. The World Bank; The TAFS forum. World Livestock Disease Atlas. A Quantitative Analysis of Global Animal Health Data (2006–2009); The International Bank for Reconstruction and Development: Washington, DC, USA, 2011.

7. Dimitrov, K.M.; Ramey, A.M.; Qiu, X.; Bahl, J.; Afonso, C.L. Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus). Infect. Genet. Evol. 2016, 39, 22–34. [CrossRef] [PubMed]

8. Mayers, J.; Mansfield, K.L.; Brown, I.H. The role of vaccination in risk mitigation and control of Newcastle disease in poultry. Vaccine 2017, 35, 5974–5980. [CrossRef] [PubMed]

9. Dimitrov, K.M.; Afonso, C.L.; Yu, Q.; Miller, P.J. Newcastle disease vaccines—A solved problem or a continuous challenge? Vet. Microbiol. 2017, 206, 126–136. [CrossRef] [PubMed]

10. Senne, D.A.; King, D.J.; Kapczynski, D.R. Control of Newcastle disease by vaccination. Dev. Biol. 2004, 119, 165–170. [PubMed]

11. OIE. World Animal Health Information Database (WAHIS Interface)—Version 1.

12. Armour, N.K.; García, M. Current and Future Applications of Viral-Vectored Recombinant Vaccines. In The Poultry Informed Professional; Department of Population Health, University of Georgia: Athens, GA, USA, 2014; pp. 1–9.

13. Alexander, D.J. Newcastle disease virus—An avian Paramyxovirus. In Newcastle Disease; Kluwer Academic Publishers: Boston, MA, USA, 1988; pp. 11–22.

14. Alexander, D.J.; Manvell, R.J.; Lowings, J.P.; Frost, K.M.; Collins, M.S.; Russell, P.H.; Smith, J.E. Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies. Avian Pathol. 1997, 26, 399–418. [CrossRef]

15. Miller, P.J.; King, D.J.; Afonso, C.L.; Suarez, D.L. Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge. Vaccine 2007, 25, 7238–7246. [CrossRef]

16. Miller, P.J.; Estevez, C.; Yu, Q.; Suarez, D.L.; King, D.J. Comparison of viral shedding following vaccination with inactivated and live Newcastle disease vaccines formulated with wild-type and recombinant viruses. Avian Dis. 2009, 53, 39–49. [CrossRef]

17. Xiao, S.; Nayak, B.; Samuel, A.; Paldurai, A.; Kanabagatte Basavarajappa, M.; Prajitno, T.Y.; Bharoto, E.E.; Collins, P.L.; Samal, S.K. Generation by reverse genetics of an effective, stable, live-attenuated Newcastle disease virus vaccine based on a currently circulating, highly virulent Indonesian strain. PLoS ONE 2012, 7, e52751. [CrossRef]

18. Kapczynski, D.R.; Martin, A.; Haddad, E.E.; King, D.J. Protection from clinical disease against three highly virulent strains of Newcastle disease virus after in ovo application of an antibody-antigen complex vaccine in maternal antibody-positive chickens. Avian Dis. 2012, 56, 555–560. [CrossRef] [PubMed]

19. Schat, K. Back to the Past: Do Vector Vaccines Represent the Future? Cornell University: Ithaca, NY, USA, 2015; pp. 1–12.

20. Johnston, P.A.; Liu, H.; O’Connell, T.; Phelps, P.; Bland, M.; Tyczkowski, J.; Kemper, A.; Harding, T.; Avakian, A.; Haddad, E.; et al. Applications in ovo technology. Poult. Sci. 1997, 76, 165–178. [CrossRef] [PubMed]

21. Williams, C.J.; Zedek, A.S. Comparative field evaluations of in ovo applied technology. Poult. Sci. 2010, 89, 189–193. [CrossRef]

22. Wen, G.; Li, L.; Yu, Q.; Wang, H.; Luo, Q.; Zhang, T.; Zhang, R.; Zhang, W.; Shao, H. Evaluation of a thermostable Newcastle disease virus strain TS09-C as an in-ovo vaccine for chickens. PLoS ONE 2017, 12, e0172812. [CrossRef]

23. Avakian, A.P.; Poston, R.M.; Kong, F.K.; Van Kampen, K.R.; Tang, D.C. Automated mass immunization of poultry: The prospect for nonreplicating human adenovirus-vectored in ovo vaccines. Expert Rev. Vaccines 2007, 6, 457–465. [CrossRef]

24. Wakenell, P.S.; Bryan, T.; Schaeffer, J.; Avakian, A.; Williams, C.; Whitfill, C. Effect of in ovo vaccine delivery route on herpesvirus of turkeys/SB-1 efficacy and viremia. Avian. Dis. 2002, 46, 274–280. [CrossRef]

25. Karaca, K.; Sharma, J.M.; Winslow, B.J.; Junker, D.E.; Reddy, S.; Cochran, M.; McMillen, J. Recombinant Fowlpox Viruses Coexpressing Chicken Type I IFN and Newcastle Disease Virus HN and F Genes: Influence of IFN on Protective Efficacy and Humoral Responses of Chickens Following in ovo or Post-Hatch Administration of Recombinant Viruses. Vaccine 1998, 16, 1496–1503. [CrossRef]

26. Rautenschlein, S.; Sharma, J.M.; Winslow, B.J.; McMillen, J.; Junker, D.; Cochran, M. Embryo vaccination of turkeys against Newcastle disease infection with recombinant fowlpox virus constructs containing interferons as adjuvants. Vaccine 1999, 18, 426–433. [CrossRef]

27. United States Department of Agriculture, Animal and Plant Health Inspection Service. Veterinary Biological Products in Licensed Establishments; Center for Veterinary Biologics: Ames, IA, USA, 2019; pp. 1–39.

28. Esaki, M.; Godoy, A.; Rosenberger, J.K.; Rosenberger, S.C.; Gardin, Y.; Yasuda, A.; Dorsey, K.M. Protection and antibody response caused by turkey herpesvirus vector Newcastle disease vaccine. Avian Dis. 2013, 57, 750–755. [CrossRef]

29. Palya, V.; Kiss, I.; Tatar-Kis, T.; Mato, T.; Felfoldi, B.; Gardin, Y. Advancement in vaccination against Newcastle disease: Recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions. Avian Dis. 2012, 56, 282–287. [CrossRef]

30. Toro, H.; Tang, D.C.; Suarez, D.L.; Zhang, J.; Shi, Z. Protection of chickens against avian influenza with non-replicating adenovirus vectored vaccine. Vaccine 2008, 26, 2640–2646. [CrossRef]

31. Schultz-Cherry, S.; Dybing, J.K.; Davis, N.L.; Williamson, C.; Suarez, D.L.; Johnston, R.; Perdue, M.L. Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with Hong Kong-origin H5N1 viruses. Virology 2000, 278, 55–59. [CrossRef]

32. Mebatsion, T.; Verstegen, S.; De Vaan, L.T.; Romer-Oberdorfer, A.; Schrier, C.C. A recombinant Newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos. J. Virol. 2001, 75, 420–428. [CrossRef]

33. Steel, J.; Burmakina, S.V.; Thomas, C.; Spackman, E.; Garcia-Sastre, A.; Swayne, D.E.; Palese, P. A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus. Vaccine 2008, 26, 522–531. [CrossRef]

34. Ahmad, J.; Sharma, J.M. Evaluation of a modified-live virus vaccine administered in ovo to protect chickens against Newcastle disease. Am. J. Vet. Res. 1992, 53, 1999–2004. [PubMed]

35. Ge, J.; Wang, X.; Tian, M.; Wen, Z.; Feng, Q.; Qi, X.; Gao, H.; Wang, X.; Bu, Z. Novel in-ovo chimeric recombinant Newcastle disease vaccine protects against both Newcastle disease and infectious bursal disease. Vaccine 2014, 32, 1514–1521. [CrossRef] [PubMed]

36. Ramp, K.; Topfstedt, E.; Wackerlin, R.; Hoper, D.; Ziller, M.; Mettenleiter, T.C.; Grund, C.; Romer-Oberdorfer, A. Pathogenicity and immunogenicity of different recombinant Newcastle disease virus clone 30 variants after in ovo vaccination. Avian Dis. 2012, 56, 208–217. [CrossRef] [PubMed]

37. Dilaveris, D.; Chen, C.; Kaiser, P.; Russell, P.H. The safety and immunogenicity of an in ovo vaccine against Newcastle disease virus differ between two lines of chicken. Vaccine 2007, 25, 3792–3799. [CrossRef] [PubMed]

38. Marcano, V.C.; Cardenas-Garcia, S.; Diel, D.G.; Antoniassi da Silva, L.H.; Gogal, R.M.; Miller, P.J.; Brown, C.C.; Butt, S.L.; Goraichuk, I.V.; Dimitrov, K.M.; et al. A Novel Recombinant Newcastle Disease Vaccine Improves Post-In Ovo Vaccination Survival with Sustained Protection against Virulent Challenge. Vaccines 2021, 9, 953. [CrossRef] [PubMed]

39. Cardenas Garcia, S.; Dunwoody, R.P.; Marcano, V.; Diel, D.G.; Williams, R.J.; Gogal, R.M.; Brown, C.C., Jr.; Miller, P.J.; Afonso, C.L. Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity. PLoS ONE 2016, 11, e0159153. [CrossRef] [PubMed]

40. Susta, L.; Cornax, I.; Diel, D.G.; Garcia, S.C.; Miller, P.J.; Liu, X.; Hu, S.; Brown, C.C.; Afonso, C.L. Expression of interferon-gamma by a highly virulent strain of Newcastle disease virus decreases its pathogenicity in chickens. Microb. Pathog. 2013, 61–62, 73–83. [CrossRef]

41. Estevez, C.; King, D.; Seal, B.; Yu, Q. Evaluation of Newcastle disease virus chimeras expressing the hemagglutinin-neuraminidase protein of velogenic strains in the context of a mesogenic recombinant virus backbone. Virus Res. 2007, 129, 182–190. [CrossRef] [PubMed]

42. Senne, D.A. Virus propagation in embryonated eggs. In A Laboratory Manual for the Isolation, Identification, and Characterization of Avian Pathogens, 5th ed.; Dufour-Zavala, L., Swayne, D.E., Glisson, J.R., Pearson, J.E., Reed, W.M., Jackwood, M.W., Woolcock, P.R., Eds.; The American Association of Avian Pathologists: Athens, GA, USA, 2008; pp. 204–208.

43. United States Department of Agriculture, Animal and Plant Health Inspection Service. Supplemental Assay Method for Titration of Marek’s Serotype 3; USDA/CVB, SAM 407.05; Center for Veterinary Biologics: Ames, IA, USA, 2014; pp. 1–10.

44. Das, A.; Spackman, E.; Pantin-Jackwood, M.J.; Suarez, D.L. Removal of real-time reverse transcription polymerase chain reaction (RT-PCR) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of Avian influenza virus by RT-PCR. J. Vet. Diagn Invest. 2009, 21, 771–778. [CrossRef] [PubMed]

45. Wise, M.G.; Suarez, D.L.; Seal, B.S.; Pedersen, J.C.; Senne, D.A.; King, D.J.; Kapczynski, D.R.; Spackman, E. Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. J. Clin. Microbiol. 2004, 42, 329–338. [CrossRef]

46. OIE. Newcastle Disease, Biological Standards Commission. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Mammals, Birds and Bees, 7th ed.; World Organisation for Animal Health: Paris, France, 2012; pp. 555–574

47. Vrdoljak, A.; Halas, M.; Suli, T. Vaccination of broilers against Newcastle disease in the presence of maternally derived antibodies. Tierarztl. Prax. Ausg. G Grosstiere/Nutztiere 2017, 45, 151–158. [CrossRef]

48. Westbury, H.A.; Parsons, G.; Allan, W.H. Comparison of the immunogenicity of Newcastle disease virus strains V4, Hitchner B1, and La Sota in chickens. 2. Tests in chickens with maternal antibodies to the virus. Aust. Vet. J. 1984, 61, 10–13. [CrossRef]

49. Kapczynski, D.R.; King, D.J. Protection of chickens against overt clinical disease and determination of viral shedding following vaccination with commercially available Newcastle disease virus vaccines upon challenge with highly virulent virus from the California 2002 exotic Newcastle disease outbreak. Vaccine 2005, 23, 3424–3433. [CrossRef]

50. Sonoda, K.; Sakaguchi, M.; Okamura, H.; Yokogawa, K.; Tokunaga, E.; Tokiyoshi, S.; Kawaguchi, Y.; Hirai, K. Development of an Effective Polyvalent Vaccine Against Both Marek’s and Newcastle Diseases Based on Recombinant Marek’s Disease Virus Type 1 in Commercial Chickens with Maternal Antibodies. J. Virol. 2000, 74, 3217–3226. [CrossRef]

51. Elazab, M.F.; Fukushima, Y.; Fujita, Y.; Horiuchi, H.; Matsuda, H.; Furusawa, S. Induction of immune suppression in the chick by an optimal dose of an immunizing antigen in the presence of its specific maternal antibody. J. Vet. Med. Sci. 2010, 72, 257–262. [CrossRef]

52. Lancaster, J.E. Chapter 3: Control Measures. Live and inactivated vaccines compared. In Newcastle Disease: A Review 1926–1964 Monograph No. 3; Canadian Department of Agriculture: Ottawa, ON, Canada, 1966; pp. 80–115.

53. Faulkner, O.B.; Estevez, C.; Yu, Q.; Suarez, D.L. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination. Vet. Immunol. Immunopathol. 2013, 152, 341–347. [CrossRef]

54. Sarfati-Mizrahi, D.; Lozano-Dubernard, B.; Soto-Priante, E.; Castro-Peralta, F.; Flores-Castro, R.; Loza-Rubio, E.; Gay-Gutierrez, M. Protective dose of a recombinant Newcastle disease LaSota-avian influenza virus H5 vaccine against H5N2 highly pathogenic avian influenza virus and velogenic viscerotropic Newcastle disease virus in broilers with high maternal antibody levels. Avian Dis. 2010, 54, 239–241. [CrossRef]

55. Schroer, D.; Veits, J.; Keil, G.; Romer-Oberdorfer, A.; Weber, S.; Mettenleiter, T.C. Efficacy of Newcastle disease virus recombinant expressing avian influenza virus H6 hemagglutinin against Newcastle disease and low pathogenic avian influenza in chickens and turkeys. Avian Dis. 2011, 55, 201–211. [CrossRef]

56. Van Boven, M.; Bouma, A.; Fabri, T.H.; Katsma, E.; Hartog, L.; Koch, G. Herd immunity to Newcastle disease virus in poultry by vaccination. Avian. Pathol. 2008, 37, 1–5. [CrossRef]
57. Alexander, D.J.; Bell, J.G.; Alders, R.G. A Technology Review: Newcastle Disease, with Special Emphasis on its Effect on Village Chickens; FAO: Rome, Italy, 2004; pp. 1–26.

58. Jochemsen, P.; Jeurissen, S.H. The localization and uptake of in ovo injected soluble and particulate substances in the chicken. Poult. Sci. 2002, 81, 1811–1817. [CrossRef] [PubMed]

59. Palya, V.; Tatar-Kis, T.; Mato, T.; Felfoldi, B.; Kovacs, E.; Gardin, Y. Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers. Vet. Immunol. Immunopathol. 2014, 158, 105–115. [CrossRef]

60. Alexander, D.J. Historical Aspects. In Newcastle Disease; Kluwer Academic Publishers: Boston, MA, USA, 1988; pp. 1–10.

61. Miller, P.J.; Afonso, C.L.; El Attrache, J.; Dorsey, K.M.; Courtney, S.C.; Guo, Z.; Kapczynski, D.R. Effects of Newcastle disease virus vaccine antibodies on the shedding and transmission of challenge viruses. Dev. Comp. Immunol. 2013, 41, 505–513. [CrossRef] [PubMed]

62. Park, M.S.; Steel, J.; Garcia-Sastre, A.; Swayne, D.; Palese, P. Engineered viral vaccine constructs with dual specificity: Avian influenza and Newcastle disease. Proc. Natl. Acad. Sci. USA 2006, 103, 8203–8208. [CrossRef] [PubMed]
63. Veits, J.; Wiesner, D.; Fuchs, W.; Hoffmann, B.; Granzow, H.; Starick, E.; Mundt, E.; Schirrmeier, H.; Mebatsion, T.; Mettenleiter, T.C.; et al. Newcastle disease virus expressing the H5 hemagglutinin gene protects chickens against Newcastle disease and avian influenza. Proc. Natl. Acad. Sci. USA 2006, 103, 8197–8202. [CrossRef]

64. Stanford, M.M.; McFadden, G. The ‘supervirus’? Lessons from IL-4-expressing poxviruses. Trends Immunol. 2005, 26, 339–345. [CrossRef]

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