Toll-like Receptor Ligands Enhance Vaccine Efficacy against a Virulent Newcastle Disease Virus Challenge in Chickens.

To enhance the efficacy of the current Newcastle disease vaccine, we have selected potential adjuvants that target well-characterized pattern recognition receptors: the toll-like receptors (TLRs). Imiquimod is a small-molecule activator of TLR7, which is a sensor of dsDNA. ODN-1826 is a mimetic of CpG DNA and ligates TLR21 (a chicken homologue of TLR9 in mammals). The activation of TLRs leads to antiviral responses, including the induction of type I interferons (IFNs). In this study, birds were vaccinated intranasally with a live LaSota strain with or without imiquimod or ODN-1826 (50 µg/bird). Two weeks after vaccination, the birds were challenged with a virulent Newcastle disease virus (chicken/CA/212676/2002). Both adjuvants (imiquimod or ODN-1826) induced higher and more uniform antibody titers among vaccinated birds compared with the live vaccine-alone group. In addition, adjuvanted vaccines demonstrated greater protective efficacy in terms of the reduction in virus-shedding titer and the number of birds shedding the challenge virus at 2 and 4 days post-challenge. A differential expression of antiviral and immune-related genes was observed among groups from tissues (Harderian gland, trachea, cecal tonsil, and spleen) collected 1 and 3 days after treatment. These results demonstrate the potential of TLR-targeted adjuvants as mucosal vaccine enhancers and warrant a further characterization of immune correlates and optimization for efficacy.

Novel Recombinant Newcastle Disease Virus-Based In Ovo Vaccines Bypass Maternal Immunity to Provide Full Protection from Early Virulent Challenge.

Newcastle disease (ND) is one of the most economically important poultry diseases. Despite intensive efforts with current vaccination programs, this disease still occurs worldwide, causing significant mortality even in vaccinated flocks. This has been partially attributed to a gap in immunity during the post-hatch period due to the presence of maternal antibodies that negatively impact the replication of the commonly used live vaccines. In ovo vaccines have multiple advantages and present an opportunity to address this problem. Currently employed in ovo ND vaccines are recombinant herpesvirus of turkeys (HVT)-vectored vaccines expressing Newcastle disease virus (NDV) antigens. Although proven efficient, these vaccines have some limitations, such as delayed immunogenicity and the inability to administer a second HVT vaccine post-hatch. The use of live ND vaccines for in ovo vaccination is currently not applicable, as these are associated with high embryo mortality. In this study, recombinant NDV-vectored experimental vaccines containing an antisense sequence of avian interleukin 4 (IL4R) and their backbones were administered in ovo at different doses in 18-day-old commercial eggs possessing high maternal antibodies titers. The hatched birds were challenged with virulent NDV at 2 weeks-of-age. Post-hatch vaccine shedding, post-challenge survival, challenge virus shedding, and humoral immune responses were evaluated at multiple timepoints. Recombinant NDV (rNDV) vaccinated birds had significantly reduced post-hatch mortality compared with the wild-type LaSota vaccine. All rNDV vaccines were able to penetrate maternal immunity and induce a strong early humoral immune response. Further, the rNDV vaccines provided protection from clinical disease and significantly decreased virus shedding after early virulent NDV challenge at two weeks post-hatch. The post-challenge hemagglutination-inhibition antibody titers in the vaccinated groups remained comparable with the pre-challenge titers, suggesting the capacity of the studied vaccines to prevent efficient replication of the challenge virus. Post-hatch survival after vaccination with the rNDV-IL4R vaccines was dose-dependent, with an increase in survival as the dose decreased. This improved survival and the dose-dependency data suggest that novel attenuated in ovo rNDV-based vaccines that are able to penetrate maternal immunity to elicit a strong immune response as early as 14 days post-hatch, resulting in high or full protection from virulent challenge, show promise as a contributor to the control of Newcastle disease.

A Novel Recombinant Newcastle Disease Vaccine Improves Post- In Ovo Vaccination Survival with Sustained Protection against Virulent Challenge.

In ovo vaccination has been employed by the poultry industry for over 20 years to control numerous avian diseases. Unfortunately, in ovo live vaccines against Newcastle disease have significant limitations, including high embryo mortality and the inability to induce full protection during the first two weeks of life. In this study, a recombinant live attenuated Newcastle disease virus vaccine containing the antisense sequence of chicken interleukin 4 (IL-4), rZJ1*L-IL4R, was used. The rZJ1*L-IL4R vaccine was administered in ovo to naïve specific pathogen free embryonated chicken eggs (ECEs) and evaluated against a homologous challenge. Controls included a live attenuated recombinant genotype VII vaccine based on the virus ZJ1 (rZJ1*L) backbone, the LaSota vaccine and diluent alone. In the first of two experiments, ECEs were vaccinated at 18 days of embryonation (DOE) with either 104.5 or 103.5 50% embryo infectious dose (EID50/egg) and chickens were challenged at 21 days post-hatch (DPH). In the second experiment, 103.5 EID50/egg of each vaccine was administered at 19 DOE, and chickens were challenged at 14 DPH. Chickens vaccinated with 103.5 EID50/egg of rZJ1*L-IL4R had hatch rates comparable to the group that received diluent alone, whereas other groups had significantly lower hatch rates. All vaccinated chickens survived challenge without displaying clinical disease, had protective hemagglutination inhibition titers, and shed comparable levels of challenge virus. The recombinant rZJ1*L-IL4R vaccine yielded lower post-vaccination mortality rates compared with the other in ovo NDV live vaccine candidates as well as provided strong protection post-challenge.

Experimental Infection and Transmission of Newcastle Disease Vaccine Virus in Four Wild Passerines.

Our prior work has shown that live poultry vaccines have been intermittently isolated from wild birds sampled during field surveillance studies for Newcastle disease virus (NDV). Thus, we experimentally investigated the susceptibility of four native agriculturally associated wild bird species to the NDV LaSota vaccine and evaluated the shedding dynamics, potential transmission from chickens, and humoral antibody responses. To test susceptibility, we inoculated wild-caught, immunologically NDV-naïve house finches (Haemorhous mexicanus; n = 16), brown-headed cowbirds (Molothrus ater; n = 9), northern cardinals (Cardinalis cardinalis; n = 6), and American goldfinches (Spinus tristis; n = 12) with 0.1 ml (106.7 mean embryo infectious doses [EID50/ml]) of NDV LaSota vaccine via the oculo-nasal route. To test transmission between chickens and wild birds, adult specific-pathogen-free white leghorn chickens were inoculated similarly and cohoused in separate isolators with two to five wild birds of the species listed above. This design resulted in three treatments: wild bird direct inoculation (five groups) and wild bird exposure to one (two groups) or two inoculated chickens (six groups), respectively. Blood and oropharyngeal and cloacal swabs were collected before and after infection with the live vaccine. All wild birds that were directly inoculated with the LaSota vaccine shed virus as demonstrated by virus isolation (VI). Cardinals were the most susceptible species based on shedding viruses from 1 to 11 days postinoculation (dpi) with titers up to 104.9 EID50/ml. Although LaSota viruses were shed by all inoculated chickens and were present in the drinking water, most noninoculated wild birds cohoused with these chickens remained uninfected for 14 days as evidenced by VI. However, one American goldfinch tested positive for vaccine transmission by VI at 7 dpi and one house finch tested positive for vaccine transmission by real-time reverse-transcription PCR at 13 dpi. Only one directly inoculated cowbird (out of three) and two cardinals (out of two) developed NDV-specific hemagglutination inhibition antibody titers of 16, 16, and 128, respectively. No clinical signs were detected in the chickens or the wild birds postinoculation.

Infección experimental y transmisión del virus de la vacuna contra la enfermedad de Newcastle en cuatro paseriformes silvestres. Nuestras investigaciones anteriores han demostrado que las vacunas vivas utilizadas en avicultura se han aislado de forma intermitente de aves silvestres muestreadas durante los estudios de vigilancia en el campo para el virus de la enfermedad de Newcastle (NDV). Por lo tanto, se investigó experimentalmente la susceptibilidad a la vacuna contra la enfermedad de Newcastle cepa LaSota en cuatro especies de aves silvestres y nativas asociadas con se han asociado con la agricultura y se evaluó la dinámica de transmisión, la transmisión potencial desde el pollo y las respuestas de anticuerpos humorales. Para evaluar la susceptibilidad, se inocularon pinzones mexicanos (Haemorhous mexicanus; n = 16), tordos cabecicafés (Molothrus ater; n = 9), cardenales (Cardinalis cardinalis; n = 6) y jilgueros norteamericanos (Spinus tristis; n = 12), todos de origen silvestre y sin exposición previa al virus de Newcastle. Estas aves se inocularon con 0.1 ml (106.7 dosis medias infecciosas para embrión de pollo [EID50]/ml) de la vacuna de Newcastle cepa LaSota a través de la vía oculonasal. Para determinar la transmisión entre pollos y aves silvestres, se inocularon de igual forma aves adulta tipo Leghorn libres de patógenos específicos y se alojaron en unidades de aislamiento en cohabitación con dos a cinco aves silvestres de las especies mencionadas anteriormente. Este diseño dio como resultado tres tratamientos: inoculación directa de aves silvestres (cinco grupos), exposición de aves silvestres a un pollo inoculado (dos grupos), o exposición a dos pollos inoculados (seis grupos), respectivamente. Se recolectaron muestras de sangre e hisopos de la orofaringe y de la cloaca antes y después de la infección con la vacuna viva. Todas las aves silvestres que se inocularon directamente con la vacuna LaSota eliminaron el virus, como se demostró mediante el aislamiento viral (VI). Los cardenales fueron la especie más susceptible con base en el aislamiento viral de uno a 11 días después de la inoculación con títulos de hasta 104.9 EID50/ml. Aunque todos los pollos inoculados eliminaron el virus LaSota y este virus estaba presente en el agua de bebida, la mayoría de las aves silvestres no inoculadas que cohabitaron con estos pollos permanecieron sin infectar durante 14 días, como lo demuestra el aislamiento viral. Sin embargo, un jilguero norteamericano resultó positivo mediante aislamiento viral a la transmisión de la vacuna a los siete días después de la inoculación y un pinzón mexicano resultó positivo para la transmisión de la vacuna mediante transcripción reversa y PCR en tiempo real a los 13 días después de la inoculación. Solo un tordo cabecicafé inoculado directamente (de un total de tres) y dos cardenales (de un total de dos) desarrollaron títulos de anticuerpos de inhibidores de la hemaglutinación específicos contra la enfermedad de Newcastle de 16, 16 y 128, respectivamente. No se detectaron signos clínicos en los pollos ni en las aves silvestres después de la inoculación.

Newcastle Disease Virus Infection in Quail.

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The pathogenesis of ND in quail is poorly documented. To characterize the ability of virulent NDV strains to replicate and cause disease in quail, groups of 14 two-week-old Japanese quail ( Coturnix japonica) were experimentally inoculated with 108 EID50 (embryo infectious dose 50%) units of 1 of 4 virulent NDV strains: 2 isolated from quail ( N2, N23) and 2 from chickens ( Israel, Pakistan). At day 2 postinfection, noninfected quail (contact group) were added to each infection group to assess the efficacy of virus transmission. Tested NDV strains showed moderate pathogenicity, with highest mortality being 28% for the N2 strain and below 10% for the others. Two N2-inoculated birds showed neurological signs, such as head tremor and ataxia. Microscopic lesions were present in N2-, Israel-, and Pakistan-inoculated birds and consisted of nonsuppurative encephalitis. Contact birds showed no clinical signs or lesions. In both inoculated and contact birds, virus replication was moderate to minimal, respectively, as observed by immunohistochemistry in tissues and virus isolation from oropharyngeal and cloacal swabs. Strains originally isolated from quail resulted in higher numbers of birds shedding in the inoculation group; however, transmission appeared slightly more efficient with chicken-derived isolates. This study shows that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.

Repeated Challenge with Virulent Newcastle Disease Virus Does Not Decrease the Efficacy of Vaccines.

Globally, poultry producers report that birds well-vaccinated for Newcastle disease (ND) often present clinical disease and mortality after infection with virulent strains of Newcastle disease (vNDV), which is contrary to what is observed in experimental settings. One hypothesis for this discrepancy is that the birds in the field may be exposed to multiple successive challenges with vNDV, rather than one challenge dose, and that the repeated infection may overwhelm the immune system and neutralizing antibodies available to prevent clinical disease. In this study, we evaluated this hypothesis under highly controlled conditions. We challenged well-vaccinated chickens with high doses of vNDV daily for 10 days, and looked for signs of clinical disease, changes in antibody titers, and mortality. All sham-vaccinated birds died by the fourth day postchallenge. No morbidity or mortality was observed in any of the NDV-vaccinated birds up to 14 days postchallenge; repeated high-dose challenges of vNDV was not sufficient to overcome vaccine immunity.

Newcastle Disease Viruses Causing Recent Outbreaks Worldwide Show Unexpectedly High Genetic Similarity to Historical Virulent Isolates from the 1940s.

Virulent strains of Newcastle disease virus (NDV) cause Newcastle disease (ND), a devastating disease of poultry and wild birds. Phylogenetic analyses clearly distinguish historical isolates (obtained prior to 1960) from currently circulating viruses of class II genotypes V, VI, VII, and XII through XVIII. Here, partial and complete genomic sequences of recent virulent isolates of genotypes II and IX from China, Egypt, and India were found to be nearly identical to those of historical viruses isolated in the 1940s. Phylogenetic analysis, nucleotide distances, and rates of change demonstrate that these recent isolates have not evolved significantly from the most closely related ancestors from the 1940s. The low rates of change for these virulent viruses (7.05 × 10(-5) and 2.05 × 10(-5) per year, respectively) and the minimal genetic distances existing between these and historical viruses (0.3 to 1.2%) of the same genotypes indicate an unnatural origin. As with any other RNA virus, Newcastle disease virus is expected to evolve naturally; thus, these findings suggest that some recent field isolates should be excluded from evolutionary studies. Furthermore, phylogenetic analyses show that these recent virulent isolates are more closely related to virulent strains isolated during the 1940s, which have been and continue to be used in laboratory and experimental challenge studies. Since the preservation of viable viruses in the environment for over 6 decades is highly unlikely, it is possible that the source of some of the recent virulent viruses isolated from poultry and wild birds might be laboratory viruses.