Project : USDA ARS

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Research Project: Intervention Strategies to Predict, Prevent and Control Disease Outbreaks Caused by Emerging Strains of Virulent Newcastle Disease Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

2018 Annual Report

Objectives
Objective 1. Identification of circulating and emerging Newcastle disease viruses, including conducting prevalence studies for NDV in poultry and in synanthropic birds from countries where virulent NDV strains are endemic to determine the presence of variant and emerging viruses in vaccinated poultry and in wild birds, and developing rapid identification assays for variant NDV strains. Objective 2. Identify agents that may cause NDV vaccine failures in endemic countries, including NDV variants and co-infecting agents that may immuno-compromise animals or enhance disease in vaccinated poultry flocks. Objective 3. Develop predictive biology strategies for risk assessment of virus evolution, including developing predictive biology strategies using NexGen (next generation) sequencing to evaluate the rate of change in different virulent NDV strains from unvaccinated, sub-optimally vaccinated, and well-vaccinated poultry. Objective 4. Develop improved NDV vaccines platforms, including identifying and evaluating effective and user friendly NDV vaccine platforms for in ovo or one-day old broilers, and identifying and characterizing protective immune responses for new vaccines platforms that are effective in ovo or in one-day old broilers.

Approach
Identification and characterization of new variants will be addressed by conducting active surveillance, and characterization of new isolates, and by developing rapid diagnostic assays that assures appropriate detection of these exotic samples (objective 1). Identifying immune suppressing agents and Newcastle disease viruses (NDV) variants that may cause disease in vaccinated animals will address the inadequate efficacy of commercial vaccines in endemic countries (Objective 2). Predictive strategies for identifying vaccines and vaccination practices that cause emergence of variant viruses will be addressed by Next Generation (NexGen) sequencing of viruses that emerge under different vaccination regimes (Objective 3). Reduced efficacy of commercial vaccines in young chickens with maternal antibodies will be addressed by developing an improved vaccine platform based in vector that is unknown to chickens (Objective 4).

Progress Report
Virulent NDV (VNDv) was detected in poultry in the United States (U.S.) for the first time since 2003 in California. The laboratory has committed to supporting the response effort and several challenge studies have been completed comparing the 2018 virus with the earlier California NDV isolate demonstrating the viruses are highly similar. Sequence analysis and literature reviews have been completed to support the control efforts. Additionally, virulent NDV remains present in cormorants and pigeons, but these viruses are not normally found in domestic poultry. During FY18, 23 complete NDV genomes from USA pigeon isolates, 7 complete genomes from Pakistani chicken isolates, 1 complete fusion gene Egypt chicken isolate and 21 complete fusion genes from Nigerian isolates have been completed and submitted to GenBank. In addition 8 genomes from bacteria co-infecting poultry in endemic countries have been sequenced. International collaborations essential to the study of Newcastle disease virus (NDV) have continued and an agreement with Boehringer Ingelheim animal health to study avian respiratory diseases from Latin America has begun. Virulent NDV is endemic in many countries commercially linked to the U.S., including Mexico, Peru, Colombia, and Venezuela. Whether in domestic poultry or wild birds, VNDv strains remain a threat to all producers of poultry. The longstanding collaboration between the Agricultural Research Service (ARS) Southeast Poultry Research Laboratory (SEPRL) and Latin American partners has been maintained as a result of those agreements and the U.S. has obtained important epidemiological information on the movement and evolution of the Newcastle disease virus. We have continued to disseminate information in international and national avian meetings attended by veterinarians across Latin America. They have improved our capabilities to rapidly detect, identify, and characterize new strains of viruses by the development of a rapid and sensitive sequencing protocol based on a new generation of portable sequencers (Oxford nanopore, Minion). In the area of epidemiology and diagnostics, our laboratory has contributed to the better characterization and classification of Newcastle disease viruses. A new diagnostic approach based on the analysis of paraffin embedded samples through next generation sequencing has allowed epidemiological and retrospective studies on pigeons infected with Newcastle disease virus across the U.S. As a part of an international team we have contributed for a new approach to phylogenetic characterization and nomenclature for Newcastle disease viruses. Development of new vaccine friendly platforms and in ovo vaccine containing an interleukin 4 gene is being evaluated. Experiments conducted in commercial chickens suggest significant attenuation in comparison to the current LaSota vaccine and improved protection after vaccination. A patent application for in ovo vaccines has been filled. In addition a new platform based on the use of APMV 10 as a vector designed to be used in animals with maternal immunity is being developed with the construction of a final plasmid expressing Newcastle disease virus antigens already complete.

Accomplishments
1. Identification of co-infecting agents in birds infected with virulent Newcastle disease viruses. To understand causes of vaccine failure in the field, ARS researchers at Athens, Georgia, have conducted random sequencing of nucleic acids obtained from poultry samples isolated in endemic countries. The outcome has been the identification of previously unrecognized infections with bacteria for the genus Ochrobactrum. The bacteria were isolated and determined to contain antibiotic resistance genes. The complete draft genome of a total of eight Ochrobactrum spp. bacteria has been published in two articles. These articles demonstrated that Newcastle disease infection is often accompanied by bacterial infection, and in this case multidrug-resistant novel Ochrobactrum species strains were isolated from a pigeon, a duck, and chickens. This work is important because it demonstrates that additional factors are yet to be identified that may be responsible for field vaccine failures, while re-assuring the producer on the efficacy of current vaccines.

2. Pathology and distribution of velogenic viscerotropic Newcastle disease virus in the reproductive system of vaccinated and unvaccinated laying hens. To better understand the phenomena of low egg production during laying in endemic countries ARS researchers at Athens, Georgia, have studied the pathological changes that accompany the drop in productivity in layers in Nigeria. The pathological changes in the reproductive system of laying hens that lead to the poor egg production and quality in Newcastle disease (ND) and the distribution of the virus in the system was presented. The study included vaccinated and non-vaccinated animals and the effect on vaccines on productivity at the peak time of egg production, was evaluated and compared with virus presence by immunohistochemistry. It was shown that both vaccinated and unvaccinated had a significant drop in egg production but only unvaccinated hens produced abnormal eggs significant damage in oviducts followed by atrophy and shortening of the reproductive tract. This study is important to demonstrate that abnormal eggs and damage in oviduct can be significantly reduced in well vaccinated animals.

3. Identification of previously unrecognized genetic diversity in avian paramyxovirus serotype 1 (APMV-1) isolated from wild birds in the U.S. To determine the presence of variant and emerging viruses in wild birds, formalin-fixed paraffin-embedded (FFPE) samples from North American pigeons were subjected to next-generation sequencing and compared to pathological observations. Next generation sequencing provided valuable genetic information from multiple organs (kidney, liver and spleen) from 10 mortality events in the U.S. between 2010 and 2016. ARS researchers at Athens, Georgia, in collaboration with the University of Georgia and the U.S. Geological Survey obtained the complete genetic sequence of the isolates recovered from mortality events across the U.S. A pigeon-adapted variant of genotype VI Newcastle disease virus, often termed pigeon paramyxovirus 1, was found in multiple columbids of the United States. The whole-genome sequence of the genotype VI Newcastle disease viruses obtained from formalin-fixed paraffin-embedded tissues revealed the utility of the technique to track virulent viruses in the U.S. to allow improved epidemiological findings and monitor evolution and genetic diversity of the virus.

4. Inactivation studies on cells of the immune system of chickens. To facilitate immunological studies of Newcastle disease, infected samples studies were conducted by ARS researchers at Athens, Georgia, to identify treatments that preserve immune surface receptors on the host cell while inactivating Newcastle disease virus. Inactivation of Newcastle disease virus was obtained in chicken allantoic fluids, macrophages and splenocytes was conducted using a commercial fixative that is normally used to preserve antigenic integrity. The study is important because it demonstrated that by allowing the capacity of fixation buffers to preserve surface markers while inactivating Newcastle disease virus, will allow the conduction immunological studies that are necessary for production of more effective vaccines.

5. Characterization of variant Newcastle disease virus (NDV) from recent outbreaks in countries in which the disease is endemic. To address the need to identify variants that may cause NDV vaccine failures in endemic countries, ARS researchers at Athens, Georgia, conducted isolation and characterization of recent NDV variants from farms that reported vaccine failures in Eastern Europe, Asia, and Africa. Highly virulent Newcastle disease viruses were found in poultry and captive non-poultry avian species. Six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) viruses were found to all be related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014-2016) that define a new sub-genotype (VIm) were isolated demonstrating that there is still unrecognized diversity in the field. The study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Understanding the evolution, current spread and methods of detection of these viruses is important to develop diagnostic reagents that are effective.

6. Identification of epidemiological risk factors of wild birds. To identify epidemiological risk factors between poultry and synantropic birds (wild birds living close to humans and farms), ARS researchers at Athens, Georgia, conducted field studies in collaboration with a team of Mexican scientist from SENASICA-DGSA Mexico, the Asociación de Avicultores de Tepatitlán, Jalisco, and the Department Avian Medicine from the Universidad Nacional Autónoma de México. These studies evaluated the risk that synantropic birds bring to the poultry farms in a highly densely productive area. The Altos de Jalisco region in west central Mexico is the location of the largest concentration of poultry farms and has witnessed the emergence of low pathogenic H5N2 and the highly pathogenic H7N3 influenza viruses recently. The survey identified 82 species of wild birds some which were linked to poultry farms using a network-theory model. The highest ranked species corresponded to the Mexican Great-tailed Grackle and the Barn Swallow; making those potential hosts for disease transmission of pathogens in the wild bird-poultry interface in the region of Jalisco. The ability to demonstrate epidemiological connections between wildlife and poultry is important to understand the risk to the poultry industry from wild birds. Because of the proximity with the U.S., these Mexican regions present a high risk of introduction through trade, wild birds and illegal transport of birds and are important to the U.S. poultry industry.

7. Characterization of the pathogenesis of Newcastle disease viruses in quail. The pathogenesis of Newcastle disease (ND) in quail is poorly documented and it is important to understand the risk of this species to be infected and transmit viruses. To characterize the ability of virulent Newcastle disease virus (NDV) strains to replicate and cause disease in quail, ARS researchers at Athens, Georgia, groups of 14 two-week-old Japanese quail (Coturnix japonica) were experimentally inoculated with four virulent NDV strains and 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 mortality ranging from 10 to 28%. Contact birds showed no clinical signs or lesions. This study demonstrates that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.

U.S. DEPARTMENT OF AGRICULTURE

Exotic & Emerging Avian Viral Diseases Research: Athens, GA