Duration: 10/01/2019 to 09/30/2024

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

THE NEED AS INDICATED BY STAKEHOLDERS. The United States (U.S.) is the world's largest poultry producer and the second-largest egg producer and exporter of poultry meat. The poultry industry is a highly integrated and constantly growing industry and is a major contributor to animal agriculture. The combined value of production from broilers, eggs, turkeys, and the value of sales from chickens in 2017 was $42.7 billion, up 10 percent from $38.7 billion in 2016 (USDA, ERS data, 2018).

 

Based on the figures published by the National Chicken Council (2018), Americans consume more poultry than anyone else in the world – 108.6 pounds per capita – being chicken the number one animal protein consumed in the U.S. Consumers rate chicken’s value very highly and chicken consumption per capita has increased nearly every year since the mid 1960’s, while red meat consumption has steadily declined.

 

U.S. egg operations produce over 105 billion eggs annually. Over 75% of egg production is for human consumption (the table- egg market). The remainder of production is for the hatching market. These eggs are hatched to provide replacement birds for the egg-laying flocks and to produce broiler chicks for grow out operations. U.S. per capita consumption of eggs and egg products is around 278.9 eggs per person. Chicken eggs are an important source of high quality protein and other micro nutrients in the diet.

 

The U.S. has the largest broiler chicken industry in the world, and over 16 percent of the production was exported to other countries in 2017. In 2017, approximately 8.9 billion broiler chickens weighing 55.6 billion pounds live-weight were produced. The U.S. turkey industry produces approximately one-quarter of a billion birds annually, with the live-weight of each bird averaging over 25 pounds. The U.S. is by far the world's largest turkey producer, followed by the European Union. Even though exports are a major component of U.S. turkey use, the U.S. consumes more turkey per capita than any other country.

 

Protection of poultry by effective control and prevention of diseases is critical in order to maintain wholesome poultry and poultry product markets. Such efforts will also make a significant contribution towards national and international food security.

 

IMPORTANCE OF THE WORK, AND WHAT THE CONSEQUENCES ARE IF IT IS NOT DONE. Respiratory diseases continue to be a major concern for the poultry industry. Consequently, losses induced by respiratory diseases in poultry are of major economic impact on the producer, the local and U.S. economies. Various pathogens may initiate respiratory disease in poultry including a variety of viruses, bacteria, and fungi. Many endemic respiratory infectious diseases in the U.S. continue to decrease the profitability of commercial poultry production. Losses are realized from mortality and morbidity via condemnation at processing and poor performance (increased feed conversion and medication costs, decreased growth). In addition, endemic, emerging and re-emerging respiratory diseases add to the economic loss to the poultry industry. Based on the USAHA report (2017), the following respiratory pathogens continue to be a problem in the industry: variant infectious bronchitis (IB), infectious laryngotracheitis (ILT), lentogenic Newcastle disease (ND), infectious coryza, avian influenza (AI), and infections caused by Gallibacterium anatis, avian mycoplasmas, Ornithobacterium rhinotracheale (ORT), Escherichia coli, and Pasteurella multocida. In addition, severe cases of infectious coryza across the U.S. and velogenic Newcastle disease virus during 2018-2019 are an alert call for researchers to focus on understanding upper respiratory dynamics in poultry.  Environmental factors and novel production systems may augment the incidence of these pathogens producing the clinical signs and lesions. Thus, preventative strategies including poultry management are critical to control the diseases mentioned above and other respiratory diseases. Because these diseases are multifactorial in nature, improved understanding of the underlying factors driving the ‘respiratory disease complex’ are necessary, including understanding the polymicrobial relationships occurring between pathogens and the normal microbiota during the disease process. This group has shifted focus in recent years towards these endeavors.

 

The export market comprises a large portion of the U.S. poultry meat and egg production. Export markets are subject to immediate restrictions when poultry respiratory diseases such as highly pathogenic AI (HPAI) or exotic Newcastle disease (END) are diagnosed and reported in commercial flocks. HPAI and END have been reported in the U.S. since our last project was initiated, which adds another level of complexity in understanding and preventing these exotic diseases. The magnitude of losses caused by these diseases are well illustrated by the 2015-2016 HPAI which was the most severe outbreaks in the history of the U.S. poultry industry decimating 9 and 6% of the layer and turkey population, respectively, and the 2002 outbreak of END in California affected commercial poultry and costed more than $160 million in eradication and control efforts. In 2018, virulent Newcastle disease virus (vNDV) has been detected in backyard flocks in Southern California. These viruses apparently entered through illegal bird movement as shown by molecular epidemiology tracing the outbreak virus to Central American viruses (Belize). Over 350 confirmed cases have been reported including at least 4 commercial poultry farms. An additional case in Utah has been reported and is believed to be linked to Southern California. More than a million backyard and commercial birds have been euthanized in this on-going outbreak. In terms of endemic diseases, the losses they cause are less profound but accumulate throughout an extended period of time. Researchers have calculated poultry endemic disease losses in the U.S. to be 2.31 billion in US dollars, an 8.2% of the gross value of poultry production in 2005.The agri-bioterrorism threat to the U.S. food supply is a reality after the terrorist attacks of September 11, 2001. HPAI and END viruses are classified as agri-bioterrorism agents considering the virulence, environmental stability, and transmissibility of these viruses among poultry flocks. Our project will develop strategies for rapid diagnosis and control of newly emerging and endemic diseases. Considering the challenges posed by endemic and exotic diseases, it is essential that the proposed studies proceed to help protect the world and our nation's food supply and the economic well-being of the poultry industry and our country.

 

THE TECHNICAL FEASIBILITY OF THE RESEARCH. The technical challenges posed by the goal of improved respiratory disease control are complex and significant, but feasible. Participants in the project have experience and training in a range of disciplines including: immunology, infectious diseases, genetics, genomics, virology, poultry medicine, physiology, biochemistry, microbiology, bioinformatics, and molecular biology. That expertise will be used to undertake the work and complete the objectives. In addition, the technical committee of this project work on the leading-edge of science and have demonstrated their expertise with the requisite infrastructure to successfully complete the described work, if sufficient financial support is made available.

 

THE ADVANTAGES FOR DOING THE WORK AS A MULTISTATE EFFORT. Control of respiratory infectious diseases lends itself to collaborative multistate research. The multistate effort is required for the synergistic and collaborative conduct of research that is based upon the combination of biological materials facilities, equipment and expertise from multiple stations. No single station possesses all of these to address the major scientific issues for the project. The use of a team approach enables us to perform cross-disciplinary, multi-industry research with translatable value. Proof of this are our ongoing collaborations and the Poultry Respiratory Diseases (PRD-CAP) project funded by USDA in which several of our members collaborate.  

 

The current NC1180 project has served as a venue for gathering, discussing, and providing critical information on disease status at the state and national level which helped to develop research and prevention strategies for the researchers, industry and state and federal government officials. The understanding of respiratory diseases of poultry has been advanced considerably through the current project. Of notable mention are the advances made in rapid diagnostics for all major respiratory pathogens which include; practical field-based tests to third generation sequencing use for rapid identification and rapid prediction of pathogenicity of pathogens exotic to the U.S. e.g. the use of this technique in the 2018-2019 vNDV outbreak shows effective collaboration between stations in GA, IA and CA. DNA and recombinant vector-based vaccine technologies have also moved forward at a dramatic pace and hold much promise for improved control of respiratory diseases. Studies with newly recognized diseases and re-emerging diseases in addition to endemic infections have led to better understanding of the disease, development of diagnostic tools, and surveillance and vaccine strategies to control these diseases. The combination of these and other findings have led to very productive years of the current project as indicated by the number of published papers including joint publications from participating institutions.

 

In addition, this project synergizes and collaborates closely with other projects. There is a strong cooperation between NC1180 and another multi-state project: NE1834, Genetic Bases for Resistance and Immunity to Avian Diseases. The participation of some of the members in both projects allows for excellent coordination between the two multi-state projects, ensuring good communication and collaboration while avoiding duplicative efforts.

 

Finally, the evolution of this collaborative group has enabled large funding to pursue deeper research questions. Through this group, a multi-million dollar research grant was obtained from USDA, the Poultry Respiratory Disease Coordinated Agricultural Project (http://www.prdcap.com). This project includes 38 investigators from all states in the NC1180 group. The goal of this project, now in year 5, is to develop knowledge-based integrated approaches to detect, control and prevent endemic, emerging and re-emerging poultry respiratory diseases in the US. Numerous projects have been funded through this effort, with significant progress towards better understanding of the respiratory disease complex and the continued development of tools by which to identify and predict disease (http://www.prdcap.com/research/).

 

WHAT THE LIKELY IMPACTS WILL BE FROM SUCCESSFULLY COMPLETING THE WORK. The overall impact of a successful outcome will be improved understanding of the ecology, pathobiology, epidemiology, diagnosis and control of respiratory diseases. These outcomes will not only benefit the poultry industry but also the food security of our country. Impact of this research will be derived from identification and characterization of disease agents, their reservoirs, factors involved in agent transmission to poultry, development and delivery of novel, fast and accurate molecular and protein-based diagnostics, determination of infection status, rapid strain identification or characterization, evaluation and development of vaccines, and the design and implementation of eradication protocols for select agents. The overall outcome of the project is to produce findings that enable the poultry industry to remain competitive and profitable reaching the ultimate goal of feeding the population of our country.

Related, Current and Previous Work

The below section highlights some of the successes achieved during the past project periods.

 

AVIAN INFLUENZA VIRUS (AIV). Avian Influenza (AI) is an economically important disease of domestic poultry and has caused considerable economic losses in many countries including the U.S. Wild aquatic birds are the natural reservoir of influenza A viruses which do not cause lethal disease in these hosts. However, some strains adapt to poultry and other mammals including humans and cause severe disease. The NC1180 group has worked extensively towards understanding the ecology and evolution of AI, and developing novel vaccine platforms

Low Pathogenicity (LP)AIV was regularly found in live bird markets in the northeastern U.S., which were the source of outbreaks in commercial poultry in Virginia and Delmarva. In addition to avian-origin influenza virus, since the first reported isolation of swine influenza viruses (SIVs) in turkeys in the 1980s, transmission of SIVs to turkeys has been repeatedly documented. NC group has conducted extensive pathogenesis studies and demonstrated a limited replication of seasonal human H1N1 and no replication of both recent human-like swine H1N2 and pandemic H1N1 viruses in turkeys compared to SIVs. In turkey toms, swine H3N2 virus was detected in semen and reproductive tract of infected toms. The finding shows that turkey hens could be affected more severely from SIVs. Moreover, the results also indicate a potential risk of venereal transmission of influenza viruses in turkeys.

 

Influenza virus continues to evolve due to its genetic nature and the emergence of novel strains is inevitable. The continuing outbreaks of highly pathogenic H5N1 avian flu and swine-origin H3N2 influenza and recent outbreak of H7N9 avian influenza in China which killed more than 600 people since February 2013 speak to the necessity of developing vaccines with greater efficacy and protection against a broader range of antigenic variants and subtypes. However, commercially available vaccines are in general directed specifically towards the strain contained in the vaccine and careful selection of vaccine strain should be made every few years so that they match with the circulating strain. Thus, it is of high veterinary and public health importance to explore novel ways to develop more broadly reactive vaccines without compromising their safety. The most commonly used AI vaccines are produced from chick embryo-propagated virus. Oil-adjuvanted inactivated vaccines, if properly prepared and administered, provide high levels of neutralizing antibody and protection from clinical disease. A recombinant fowlpox virus (rFPV) vaccine expressing the HA protein has also been commercialized and approved for use by several countries as one of the first viral-vectored vaccines. Experimental recombinant vaccines for AI are currently being developed to improve and overcome the drawbacks of current vaccines. Live viral vectors have been engineered to express either the H5 or H7 antigens. Using a recently developed plasmid-based reverse genetics system for influenza, viruses can be rescued from cloned cDNA for the purposes of producing reassortant influenza viruses. A reverse genetics technique was used for the rapid generation of reassortant viruses that may serve as candidate vaccine strains for AI. In addition, live-virus vaccines were developed for AI. In broilers, vaccines are often administered during later stages of embryonation, usually at 17-18 days of incubation. In ovo vaccine delivery is cost-effective and has largely replaced post-hatch injection of Marek’s disease vaccine in broilers in the U.S. More recently, protective immunity against AI virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA)-free human adenovirus (Ad) vector encoding an H7 HA. Chickens vaccinated in ovo with an Ad vector encoding an H5 HA were subsequently vaccinated intramuscularly with AdChNY94.H7 post-hatch, responded with robust antibody titers against both the H5 and H7 AI proteins. Replication-defective live influenza virus that induces strong immune responses but does not generate infectious virus in hatched chicks warrants further investigation.

 

With the development of highly efficient vector (or carrier molecule) and adjuvant, the conserved protein-based universal vaccine approach also warrants further investigation. The extracellular domain of the M2 protein (M2e) consists of 23 amino acids which are remarkably conserved among influenza subtypes. The anti-M2e antibodies showed the ability to opsonize infected cells inducing cell killing by complement and/or natural killer cells. The M2e specific antibody can also bind to and prevent the influenza virus progeny release from infected cells. For this reason, M2e has been considered a promising target for the development of universal influenza vaccines. However, most of the experimental studies have been done using lab animals and limited research has been done in chickens or other farm animals to determine the protective efficacy and applicability of the M2e-based vaccines.

 

SEPRL and OH groups continue to evaluate the efficacy of commercially available vaccines against emerging strains which is critical for the preparation of potential HPAI outbreaks. In addition, SEPRL, OH, AL and CT group has been developing new experimental vaccines and vaccination regimens that can either replace or complement the existing vaccines.  These projects demonstrate coordination and collaboration among the named stations.    

 

Avian Paramyxovirus-1 (NEWCASTLE DISEASE VIRUS (NDV)). Newcastle disease (ND) is a contagious disease of various species of wild and domestic birds that can have severe economic consequences for poultry producers, including a serious impact on the international trade of poultry genetics, meat and eggs. NDV is also called avian paramyxovirus serotype-1. ND varies in its degrees of severity, ranging from a subclinical infection to severe disease causing very high mortality. NDV strains are categorized into three main pathotypes for chickens: lentogenic (low virulence), mesogenic (intermediate virulence), and velogenic (high virulence) strains. Regardless of the severity of the pathotype, vaccination usually protects birds from clinical disease.

NDV has a high capacity to mutate, allowing the development of multiple virulent NDV (vNDV) genotypes evolving simultaneously at different locations. Large gaps in existing knowledge in the areas of epidemiology and evolution limit the possibilities to control the disease. vNDV are found in most countries of the world and the U.S. has strict rules to prevent their entry. However, it is important to continuously monitor and characterize viruses that are a potential threat to the U.S. poultry industry.

 

The latest introductions of NDV in the U.S. have occurred in California. The outbreak in 2002, affecting backyard and commercial premises causing losses that exceeded $200 million. A current outbreak in Southern California, starting in May 2018, has been affecting exhibition birds and has driven the attention to biosecurity and depopulation as control measures to avoid its spread to commercial premises. Unfortunately, this outbreak has affected 4 commercial premises causing the depopulation of more than 1 million birds. The latest confirm that NDV is a real risk for our poultry production validating work on prevention, diagnostic and biosecurity. During the latest outbreak in CA, third generation sequencing techniques have been deployed and used in collaborative efforts between members of this multistate effort from GA (SEPRL) and CA. Surprisingly other than fast characterization, these techniques have allowed characterization and sequencing of NDV’s that were not characterized by conventional sequencing. The performance of fast and accurate diagnostic methods is often affected by the evolution of viral genomes. Therefore, there is a need for the validation of multiple recently developed experimental tests and a need to develop additional fast and inexpensive diagnostic tests to be used in the field. There are many avian paramyxoviruses that can infect avian species but never get characterized because there are no defined systems to do so. As a result of a surveillance program in wild birds a hemagglutinating virus that was not recognized with current diagnostic tests, was identified. The virus was sequenced using a random approach and serologically characterized. It was determined that the viruses corresponded to a new serotype (serotype 10). Characterization of new types of paramyxoviruses is important from the diagnostic perspective, as they may be confused with NDV.

 

Current commercial ND vaccines, when given correctly, protect birds from dying or getting sick after infection with virulent viruses, but they do not protect vaccinated birds from being infected and from shedding viruses. The relationship between the amount of antibodies produced by vaccinated animals and the capacity to transmit challenge viruses was assessed. Birds vaccinated with live ND vaccines were challenged at different times post vaccination to test vaccine efficacy to prevent transmission. Vaccinated birds that were challenged before 21 days post vaccination were able to transmit viruses to vaccinated and non-vaccinated birds and shed significantly more viruses in oral secretions than birds challenged after 21 days. This finding suggests that an earlier onset of immunity may help protect against transmission and spread of the disease. The effectiveness of new vaccines using NDV as a vector against challenges with the 2018 CA strain is being currently tested. This is a collaborative effort between SEPRL and CA multistate members in addition to private laboratories.  

Our SERPL group and CA members of this multistate project have been directly involved with the recent outbreaks SEPRL as a government agency and CA members as local experts. They have provided critical services related to diagnostics, epidemiology, biological characterization, outreach and evaluation of vaccines. This has been in collaboration with the California Department of Food and Agriculture (CDFA).

 

AVIAN ESCHERICHIA COLI (E. coli). Avian pathogenic Escherichia coli (APEC) involved in respiratory disease is a major cause of economic loss for the poultry industry. It is well established that certain APEC serogroups are most commonly associated with colibacillosis in poultry, including O1, O2, and O78 strains. It is thought that most APEC infections are secondary in nature, affecting an immunocompromised bird previously infected with a primary viral or bacterial agent. However, recent genotyping data suggests that some APEC isolates appear to be primary pathogens, while others appear to be commensal isolates taking advantage of an immunocompromised host. The ecology and evolution of APEC is being studied to determine the changing landscape of APEC in the face of selective pressures such as commercial and autogenous vaccination. Also, the relationships between E. coli load, gut colonization, respiratory colonization, and clinical outcome are being studied. The MN group has been utilizing whole genome sequencing-based approaches combined with mathematical modeling to predict and respond to population shifts in efforts to prevent disease. In collaboration with MN group, OH group is developing antibiotic alternatives for the control and prevention of APEC.

 

INFECTIOUS BRONCHITIS VIRUS (IBV). IB is an economically important disease of commercial chickens. IBV, a coronavirus, is highly contagious, causing respiratory, kidney disease and egg production losses. The S1 subunit of S induces neutralizing antibodies and is one of the least conserved segments of the genome. Variability within the S gene is largely responsible for the vast genotypic and serotypic diversity. Field strains differing from vaccines may not cross-protect, creating a challenge when developing vaccine programs. Monitoring IBV by isolating, identifying and characterizing field isolates is important in commercial industry and this has been accomplished by NC1180 members. Understanding antigenic diversity, pathogenicity, persistence and other important properties of IBV will require study of the complete genome.

The viruses in Coronaviridae family are divided into three groups based on serological and genetic properties. Groups 1 and 2 comprise mammalian coronaviruses that differ extensively with respect to genome organization and gene sequences from group 3, which is largely restricted to birds. IBV is the most important pathogen in group 3, which affects chickens. Reservoirs for mammalian coronaviruses have been found in bats. However, the existence of a reservoir for avian coronaviruses is still unresolved. Group 3 coronaviruses with genetic similarities to IBV were reported in healthy wild bird species in Europe and China. These findings suggest the possibility of a wild bird reservoir for the avian coronaviruses and the potential of those viruses to infect commercial poultry. There are limited data regarding the presence of the coronaviruses in wild bird species in the U.S., and more surveillance as well as pathogenicity studies using wild bird isolates in commercial poultry is needed.

 

Conventional diagnostic methods to differentiate IBV serotypes include virus isolation in SPF embryonated eggs followed by virus neutralization tests, hemmaglutination inhibition tests, or ELISA using monoclonal antibodies. However, genetic based tests to identify IBV types have become the test of choice since the discovery that sequences in the S1 gene are correlated with different serotypes of IBV. RT-PCR targeting the S1 portion of the spike protein, followed by sequencing of the RT-PCR product, restriction enzyme fragment length polymorphism (RFLP), or hybridization with specific probes have been developed for differentiating serotypes and variants of the virus. Microsphere- based suspension array is a relatively new diagnostic platform that enables high throughput detection of nucleic acids as well as other analytes. Microspheres contain two internal fluorochromes with different intensities giving each microsphere a unique spectral character. This unique spectral character theoretically allows up to 500 different microspheres to be combined and used in the microsphere-based assay. The test involves direct hybridization between PCR amplified DNA products from clinical samples and target specific oligonucleotide probes coupled to the microspheres. Microsphere-based assays have been used for the diagnosis of many infectious pathogens including, but not limited to Salmonella, avian influenza, and human respiratory disease associated viruses. The majority of IBVs isolated from commercial chickens are vaccine type viruses and rapidly distinguishing them from each other and from variant viruses is critical for control of IB. Because multivalent vaccines are routinely used to vaccinate broilers, it is important to have a multiplexed assay that can detect more than one IBV vaccine type in a single sample. This multiplexed microsphere assay for IBV vaccine types can provide accurate data on the persistence of modified live vaccines in commercial broilers. In order to establish preventative measures molecular surveillance is needed in order to detect variants, characterize them and select vaccines for prevention, currently this is done by RT-PCR and sequencing or by targeted qRT-PCR’s. These techniques have been developed by members of our multistate project NC1180 and shared to be used by all members and the scientific community.     

 

Control of the IB is extremely important because IBV replicates in the upper-respiratory tract, which predisposes infected birds to potentially lethal secondary pathogens like E. coli. Previous studies have shown that vaccinating with two different types of IBV vaccine can provide broad protection against different IBV types. But, apparently not all vaccine combinations provide the same breadth of protection, and it is not clear which vaccine combinations provide the best protection against specific variant viruses. Thus, these so called protectotype vaccine combinations must be evaluated for each new variant virus in vivo. Also complicating the picture are the different criterion used to evaluate protection. Vaccine efficacy in the U.S. is evaluated by detection of the challenge virus as specified by the Code of Federal Regulations, Title 9 whereas in Europe, ciliostasis in the trachea is evaluated as specified by the European Pharmiocopia. To develop universal criteria which can be used in different countries and to better understand the relationship between protection defined by challenge virus detection and ciliostasis in the trachea, it is important to examine both parameters in vaccinated chickens challenged with homologous and heterologous strains of IBV.

 

Finally, the immune response against IBV is not fully understood. Since IBV was first described almost 90 years ago we have not been able to control it despite the use of vaccination programs. The chicken major histocompatibility complex (MHC) has been linked with genetic resistance to several poultry infectious diseases, including IBV. While the mammalian MHC is large, complex and encoded by multiple genes, the chicken MHC genes encode single dominantly expressed molecules that confer a strong association of resistance or susceptibility to particular infectious agents. We previously challenged different MHC-B congenic chicken lines with an IBV Massachusetts 41 (M41) strain with the goal of selecting the lines that presented the most divergent immune responses to IBV. Based on viral loads at 2 days post-infection (dpi), 335/B19 chickens showed a higher degree of susceptibility, while based on humoral responses at 14 dpi chickens of line 331/B2 were better immune responders. Our goals will align to this research trying to determine which components of the immune response, innate or adaptive, relate with relative resistance to IBV.

Our NC committee include all the key IBV experts in the U.S. AL, CA, CT, DE, GA group have been actively involved in the all area of research described above and providing critical information to our stakeholders. Other than sharing results and targeting research there are collaborative efforts like research on variant strains in CA conducted by CA and AL members. 

 

INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). ILT has become one of the most economically important diseases of commercial broiler chickens in the U.S. Numerous outbreaks have been reported in most poultry production areas; Alabama, California, the Delmarva peninsula region, Georgia, Mississippi, North Carolina and Pennsylvania. An outbreak that occurred on the Delmarva peninsula from 1998-2000 cost the broiler industry $4 million. ILT is characterized by moderate to severe respiratory distress, conjunctivitis, and tracheitis. Affected flocks experience increased mortality, poor feed conversion and reduced growth. ILTV belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. The virus does not survive for extended periods outside the host, and is sensitive to heat, drying, and disinfection.

 

Vaccination is effective in the prevention of ILTV infection. However, ILT vaccine viruses can create latent infected carrier chickens. These latent carriers are a source for spread of virus to non-vaccinated flocks. Therefore, it is recommended that ILT vaccines be used only in endemic areas. Currently, the most used ILT vaccine strains are attenuated modified-live TCOs or CEOs viruses. In general, the CEO vaccines induce better protection than TCO vaccines. However, the use of CEO vaccines has been associated with the spread of vaccine virus to non-vaccinates causing increasing vaccine reactions, a result of bird- to-bird passage and the production of latently-infected carriers. Recently, the genomic sequences of low and high passages of CEO and TCO vaccine strains were determined using hybrid next generation sequencing in order to define genomic changes associated with attenuation and reversion to virulence. Phylogenetic analysis of available full genomes grouped strains into three major clades: TCO, CEO, and Australian. Comparative genomics revealed that TCO attenuation is likely the result of an ORF C truncation.

 

A new generation of viral vector fowl poxvirus (FPV) and herpesvirus of turkey (HVT) vaccines carrying ILTV genes has been developed and such vaccines are commercially available. These vaccines are characterized by their lack of transmission, lack of ILTV-associated latent infections, and no reversion to virulence. HVT-vectored ILTV recombinant vaccines were originally approved for subcutaneous HVT or transcutaneous (pox) delivery. The increased incidence of ILTV outbreaks in broiler production sites encouraged the broiler industry to deliver the FPV-LT and HVT-LT recombinant vaccines in ovo. The protection induced by ILTV viral vector recombinant vaccines were evaluated after in ovo application in 18-day-old commercial broiler embryos. The protection induced by recombinant ILTV vaccines was assessed by their ability to prevent clinical signs and mortality; to reduce challenge virus replication in the trachea; to prevent an increase in body temperature; and to prevent a decrease in body weight gain after challenge. This study showed that both recombinant-vectored ILTV vaccines provided partial protection, thereby mitigating the disease, but did not reduce challenge virus loads in the trachea.

 

Epidemiologic investigations of recent outbreaks in the U.S. point to common sources of infection such as recovered or vaccinated birds, and the introduction of the virus via movement of contaminated humans/equipment or infected poultry at processing (live haul). Transmission from infected to non-infected farms within a wind vector has been implicated in recent outbreaks. Multiple modes of spread have been identified. Walk-in transmission occurs when clinical signs appear near the poultry house door, indicating spread by people. Blow-in transmission occurs when signs appear along sidewall air inlets of a poultry house located near a road, indicating spread by wind carrying the virus from live haul trucks. Although progress has been made in the areas of biosecurity, vaccination, and other prevention measures, knowledge gaps still exist in the persistence and recurrence of ILTV. To fully understand the pathogenesis and reasons for recurring outbreaks, additional research is needed on viral dose required, and its effects on incubation period, duration of shedding, latency, and survival of ILTV in the poultry house environment.

 

The DE and GA group have been key institutions providing ILTV surveillance and epidemiologic data. In addition, DE, GA, and SEPRL has been closely collaborating on genomic characterization of ILTV and development of new vaccines.

 

INFECTIOUS BURSAL DISEASE VIRUS (IBDV). IBD is characterized by destruction of the lymphoid cells of the bursa of Fabricius. Severe B-cell suppression leads to immunosuppression resulting in exacerbation of host susceptibility to respiratory disease agents, as well as a reduction of the ability of the chicken to respond to vaccines. Two distinct serotypes of IBDV exist. Serotype I strains are pathogenic for chickens, whereas serotype II viruses, mostly isolated from turkeys, are avirulent for chickens. Antigenic variant strains break through the immunity provided by vaccination. Ongoing monitoring-isolation, identification and characterization- of variants is critical to assess the effectiveness of vaccines and their ability to prevent/control immunosuppression. Challenge trials using broilers are an important tool to assess the effectiveness of breeder vaccination programs, the primary strategy in controlling IBD. Detection and differentiation of IBDV is accomplished using monoclonal antibodies, PCR-gene sequencing, RFLP, and real time RT-PCR.

 

In December 2008, a virulent strain of IBDV termed “very virulent” IBDV (vvIBDV), although prevalent in Asia, Africa, and South America, was identified for the first time in the U.S. in a commercial flock in northern California. Since then, several other California backyard and commercial flocks have been affected by the same vvIBDV strain and other unique (previously undiscovered) reassortant strains of IBDV of varying pathogenicity. For example, the CA-K785 virus was isolated from a California layer chicken flock in May 2009 and the CA-D495 virus was isolated from a backyard flock of Araucana chickens in October 2009. Nucleotide sequence analysis established their genome segment A was typical of vvIBDV. The nucleotide sequences of genome segment B however were most closely related to serotype 2 IBDV. Pathogenicity studies in chickens demonstrated the CA-K785 reassortant caused 20% mortality which was low compared to vvIBDV. The CA-K785 and a vvIBDV strain caused no morbidity or mortality in commercial turkey poults but they were infectious. Although these reassortants may have entered the U.S. independently of the vvIBDV, the genetic data suggest the vvIBDV may have reassorted with serotype 2 strains endemic to California. The reassortment of California vvIBDV with an endemic serotype 2 virus suggests vvIBDV may have entered California earlier than originally thought. The control of vvIBDV and now these newly emerging reassortant viruses will be economically important to the future of the U.S. broiler and layer industries.

 

Recent studies also indicate the ability of vvIBDV to infect chickens is not affected by maternal immunity to IBDV strains typically found in commercial U.S. chickens. However maternal immunity did reduce the severity of the clinical signs and macroscopic lesions. These data suggest vvIBDV might be infecting chickens in California and other regions of the U.S. but they are going unnoticed because maternal immunity affects the clinical picture which does not include mortality and macroscopic lesions typical of a vvIBDV infection.

 

In addition to recombinant subunit vaccines, other strategies to control IBDV infection include ribozyme and DNA vaccination, and also reverse genetics have been pursued for anti-viral infection or vaccination of chickens. However, DNA plasmid carrying VP2 gene of IBDV, co-administered with another DNA plasmid carrying chicken interferon-r gene, failed to achieve protection against IBDV challenge. Nevertheless, chickens primed with a DNA plasmid carrying the VP2 gene and subsequently, boosted with killed IBD vaccine achieved 100% protection. Recent studies on DNA vaccine showed that IBDV large segment gene-based DNA can elicit specific immune response and provide protection of broiler chickens with maternally derived antibody against infection challenge in a prime-boost approach. The kinetics for DNA vaccination to confer protection against IBDV challenge is attributed to delayed appearance and rapid clearance of the invading viruses. Thus, the IBDV large segment gene-based DNA vaccine has the potential for practical application to confer protection of chickens with maternal antibodies against IBD in the poultry industry.

 

DE and OH group has collaborated on IBDV epidemiology. AL and CA have collaborated in pathogenicity studies of these reassortant viruses. In addition, IL and OH group continues to work on pathogenesis of emerging IBDV and vaccine development.

 

POULTRY RESPIRATORY DISEASE CAP PROJECT. As mentioned above, this group secured a multi-million dollar research grant from USDA - the Poultry Respiratory Disease Coordinated Agricultural Project (http://www.prdcap.com). This project includes 38 investigators from all states in the NC1180 group. The collaborative goals and accomplishments of this project are listed below.

 

1. The coordinated and centralized effort is essential for disease prevention, control, and eradication. Experiences from avian influenza and Newcastle disease outbreaks in recent years emphasized the importance of reporting, coordination, and collaboration between industry and regulators in poultry disease control efforts. Unfortunately, apart from OIE reportable diseases, there is no centralized effort that coordinates data collection, control and eradication of other poultry diseases including most of the respiratory diseases. This multistate collaborative project in conjunction with NC1180 is facilitating a much needed coordinated and collaborative approach to research and disease control and establishing a strong basis for national poultry disease network.

 

2. We are continuing to define the microbiome in the respiratory tracts of broilers, layers, and turkeys in relation to health status and performance. This work will help us to understand the microbial communities that inhabit the respiratory tracts of poultry raised for meat and eggs. By understanding the microbial community, we can better predict and prevent disease in these animals, and identify alternatives to antibiotics. This work ultimately contributes to a sustained poultry supply in the United States in the face of increased demand and consumer pressures for change. In addition, we are collaborating with the investigators in this project to study the respiratory microbiome using models of respiratory infection. We are determining the changes that microbial communities undergo over the course of respiratory pathogen infection. We are also identifying specific microbial populations that might be favored, altered, or reduced during the infection, which can serve as critical information for development of intervention strategies. In the absence of antibiotics, alternative approaches are needed to maintain health and prevent disease, and probiotics have great promise as one such approach. Respiratory microbiome project which initially led by MN group in the current NC1180 created strong collaboration among MN, OH, and DE groups to cover different types of birds (broilers, layers, and turkeys) for comparative studies. In addition, the collaboration is essentially being expanded to all the groups working on pathogenesis to better understand the multifactorial etiology of respiratory diseases.

 

3. Respiratory diseases involve multiple pathogens, and they interact with each other. For example, our study highlighted the role of avian Mycoplasma in exacerbating the clinical outcome of poultry co-infected with respiratory viruses (LPAIV and IBV) and also inducing side effect from normal vaccination (eg. ILTV). Thus, researchers cannot study one pathogen but must look at how the host reacts; that can vary depending on the health condition of the host. The environment, including the air quality on the farm might affect the disease. Our co-infection studies in different environmental conditions provide important and much needed information on the interaction of respiratory pathogens in poultry, which will help improve diagnostics and vaccination strategies needed to control respiratory syndromes in poultry. Specifically, our studies provide practical information on what to expect in regards to clinical outcomes of co-infections with respiratory pathogens in different environmental and host conditions.  Future studies will address the role of co-infections on susceptibility and transmission of respiratory pathogens in poultry. SEPRL, GA, OH, PA groups have been the key groups addressing this challenging area in collaboration with microbiome group mentioned above.

 

4. The prompt identification of pathogens and antibodies in flocks is essential for early detection and the initiation of an appropriate response to limit the extent of the disease. Although the current detection methods are effective, respiratory pathogens continue to evolve and novel strains with changes in genetic sequences emerge. In addition, with the recent advent of new technologies, new diagnostic platforms continue to emerge. Thus, it is important to carefully validate and update the existing methods and compare them with new options applicable to poultry diseases. We are evaluating and developing new PCR, real-time PCR, next-generation sequencing, and other new techniques in collaboration with all the participants in the project including USDA-ARS, USDA-APHIS, and many diagnostic laboratories. Once bench validated, the test will be transferred to NVSL and other participating diagnostic laboratories. The improved and validated tests will be incorporated to the respiratory panel for molecular detection and diagnosis of different respiratory pathogens. SEPRL has been leading this effort as the government agency and all our NC participants affiliated with the University diagnostic labs are collaborating on this project.

 

5. Vaccination has been a widely used tool in the poultry industry to prevent or control diseases caused by infectious disease agents. Both inactivated and live vaccines have been successfully applied against major respiratory pathogens. However, in spite of extensive vaccination programs, respiratory pathogens continue to evolve and cause enormous economic losses. For this reason, generation of a broadly reactive vaccine that can confer protection across serotypes or variants has been a long sought goal for pathogens that continuously evolve. Four different vaccine platforms are being successfully developed which target Infectious Bronchitis (IB) which makes it easier for comparative evaluation using similar challenge protocols. AL, CT, GA, and TX groups are involved in this project and considering the flexibility of each system, once validated with IB, the platform can quickly be utilized to develop vaccines against different respiratory pathogens of interest which will create additional collaboration. In addition, MN group is developing vaccines for Ornithobacterium rhinotracheale which has been recognized as one of most important pathogens by our stakeholders that vaccine is not available. Several patents have been applied and agreement with commercial company is being made with some products to exploit commercialization of candidate vaccines.

 

6. Antibiotics have been used to control mycoplasma and other bacterial pathogens in poultry. However, because of the rise in antibiotic resistant bacteria, antibiotics use in poultry production will phase out. To overcome these problems and reconcile the demand of antibiotic withdrawal with maintaining animal health and food security, there is urgent need to develop antibiotic-independent approaches to control respiratory diseases. We have identified several novel non-antibiotic compounds that inhibit avian pathogenic E. coli (APEC) and Mycoplasma gallisepticum which show low toxicity. These small molecules are highly suitable for commercial application because of their small size, specificity, and stability. In addition, since they target specific virulence mechanisms, the pathogen is less likely to develop resistance. The identified small molecules demonstrated good efficacy against APEC and Mycoplasma infections in chicken. The effort is led by OH group with collaborative support from IA and MN groups on Mycosplasma and APEC, respectively. Rapid progress is being made for commercial application.

 

7. A major obstacle in the control of infectious diseases in poultry is non-science based management practices and lack of proper understanding of the real issues and practical control strategies among the stakeholders including poultry industries and small backyard flock owners. Science-based information on control and prevention of poultry respiratory diseases needs to be disseminated effectively and in a timely manner to our stakeholders. Our extension group is conducting a comprehensive and effective educational training program on the importance of controlling respiratory diseases including HPAI for veterinarians, extension educators, gamebird producers, small organic and pastured poultry operations and backyard, hobby, and exhibition growers, state and federal government stakeholders, and the general public. In 2017, we also developed and held a Poultry Respiratory Health Seminar which directly introduced the science-based information obtained mainly from this project. Extension experts from CA, CT, DE, GA, IA, MN, NE, OH, PA groups are collaborating on this project and coordinated effort is made among participants for effective generation and validation of extension and education materials and approaches. This outreach efforts also include outreach in Southern California which is the zone where vNDV is causing severe outbreaks.

Objectives

1. Investigate the ecology of poultry respiratory diseases and their role in poultry flocks
   Comments: The new NC project will continue to build upon the foundation laid during previous NC projects. In the new NC project, state representatives will gather disease surveillance information from each state and share the data among participating institutions in annual meetings looking for further collaboration. A standardized data reporting system will be developed. Since the state representatives and other participants are actively involved in key national committees related to poultry diseases, the NC project will serve by gathering, discussing, and providing critical information on disease status at the state and national level which will help to identify research priorities and develop prevention strategies for the researchers, industry and state and federal government officials. This effort is obviously based on mutually beneficial, collaborative, multi-disciplined approaches among participants and collaborators in state and government agencies. The established surveillance efforts will continue to be coordinated and reassessed as needed at annual meetings and via e-mail communication. In addition to proposed surveillance plan described below, efforts will be made to monitor new or re-emerging respiratory pathogens as needed. AVIAN INFLUENZA VIRUS (AIV). Surveillance will be performed in commercial poultry, backyard poultry, live bird markets and auctions, wild aquatic birds, and swine in collaboration with industry, USDA, US Wildlife Services, and state diagnostic laboratories. AIV will be detected by real-time RT-PCR, virus isolation in eggs or MDCK cells. AIV specimens will be sent to USDA National Veterinary Services Laboratory in Ames, Iowa for HA and NA typing and standard pathotyping tests. The HA genes of the isolates, including all H5 and H7 subtypes will be sequenced. Other genes, depending on the significance of the isolates, may be sequenced. Third generation sequencing efforts are being tested with current surveillance methods in order to update surveillance strategies in the future. This effort is driven by our members in collaboration with the government and diagnostic laboratories. AVIAN PARAMYXOVIRUS-1 (APMV-1). Virological surveillance testing will be performed in commercial poultry, backyard and auction poultry, and wild birds. Using USDA NAHLN protocols, APMV-1 will be detected by virus isolation and/or real-time RT-PCR. APMV-1 real-time RT-PCR positive samples will be sent to USDA National Veterinary Services Laboratory for confirmation. The fusion (F) genes of recovered isolates will be sequenced and compared to sequences in GenBank. The pathogenicity of APMV-1 isolates will be evaluated in commercial poultry (refer to Objective 2). Likewise, third generation sequencing is being tested as described for AI. E. COLI. Avian E. coli from respiratory-diseased broiler chickens will be isolated and characterized by O antigen serotyping molecular virulence factors tsh, iss, iucC, IntI and TraT, and pulse field gel electrophoresis (PFGE) to establish diversity. MYCOPLASMA. Pathogenic avian mycoplasmas in poultry will be isolated and characterized using strain specific PCR, RT-PCR, and culture. INFECTIOUS BRONCHITIS VIRUS (IBV). Virological surveillance in commercial poultry will be performed. Real time RT-PCR positive samples will undergo virus isolation attempts in SPF embryonated eggs. The S1gene of the isolates will be identified by sequence analysis. Novel variant strains of IBV will be evaluated for their pathogenicity as well as for their ability to break through immunity provided by commercial vaccines (refer to Objective 3). Genetic changes in the nsp3 gene and in the spike gene of IBV occur at a much higher rate than other genes as the virus adapts to a new host which indicate that monitoring changes in both nsp3 and spike can be a useful indicator of mutations that could potentially lead to emergence of new IBV types. IBV viruses circulating in the field will be examined for genetic changes associated with genetic drift of the virus. This data will be collected for a variety of IBV types isolated over time and be correlated with pathogenicity and emergence of new types. In addition to commercial poultry, the possibility of a wild bird reservoir for the avian coronaviruses and the potential of those viruses to infect commercial poultry will be investigated. Using a universal avian coronavirus RT-PCR assay, different wild bird species will be monitored. If detected by RT-PCR, efforts will be made to isolate and test wild bird coronavirus isolates for their ability to infect and cause disease in commercial poultry. INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). ILTV virological surveillance in commercial chickens will be performed. In particular potential sources of infection in the poultry house environment will be identified such as dust, litter, water drinkers, feeders, walls, fans, and darkling beetles. Specimens will be tested by virus isolation and real time (RT)-polymerase chain reaction (PCR). Isolates will be characterized using PCR and restriction fragment length polymorphism (RFLP) to determine if they are wild type or vaccine origin. To evaluate the impact of vaccination in the persistence of the disease, trachea and environmental samples will be collected from ILTV positive and negative farms located in a vaccine buffer zone. INFECTIOUS BURSAL DISEASE VIRUS (IBDV). Since IBDV significantly affects both the development and outcome of respiratory infection and also vaccine efficacy, IBDV is included in our project. Surveillance for all IBDV strains including vvIBDV will be conducted on commercial and backyard poultry. An RT-PCR assay will be used to detect IBDV strains in bursal tissue from chickens. The nucleotide sequence of positive RT-PCR samples will be determined across the hypervariable sequence region of the VP2 and the VP1 genes. The nucleotide and predicted amino acid sequences will be compared to known strains of the virus. Both VP2 and VP1 sequences are needed to identify vvIBDV strains. The sharing of surveillance data, sequence information, and isolates will be critical to reduce the overlap of the work among institutions and expedite the selection of strains for in vivo characterization and vaccine development (Objectives 3 and 4). In order to understand complex upper respiratory diseases, a concerted effort will be carried out to understand the upper respiratory tract virome. Procedures, analysis and bioinformatic pipelines will be shared and discussed in order to expedite the obtention of useful data to understand the ecology of these viruses and how changes can derive into multifactorial diseases.
2. Develop new and improved diagnostic tools for poultry respiratory diseases
   Comments: The prompt identification of pathogens and antibodies in flocks is essential for early detection and the initiation of an appropriate response to limit the extent of the disease. Although the current methods are effective, there is need for improved diagnostic methods that allow more rapid detection and efficient classification of the pathogen. Respiratory pathogens continue to evolve and novel strains with changes in genetic sequences emerge. Thus, existing assays require frequent validation and update. For example, primer and probes for conventional PCR and real-time PCR should be tested with new strains. The improved tests will then be added to the respiratory panel for molecular detection and diagnosis of different pathogens including infectious bronchitis, Newcastle disease virus, avian influenza virus, and laryngotracheitis virus, etc. In addition to surveillance, the serologic test, which can be used in conjunction with vaccines to differentiate infected from vaccinated animals (DIVA) continue to be needed. The following summary describes some of the specific efforts to this end and may apply to other respiratory pathogens not specifically mentioned here. New tools also involve third generation sequencing strategies that allow the recognition of mixed infections in a fast and affordable way. During the project, the participating institutions will exchange reference and field samples for validation of the proposed tests. This objective is directly tied with the Objective 1. For example, sequences of new isolates obtained from Objective 1 will be critical in the development and improvement of many molecular assays. AVIAN INFLUENZA VIRUS (AIV). Methods for AIV detection will be evaluated by comparing results obtained using the HA test, antigen capture (AC) ELISA, and RRT-PCR. A suspension array system for detecting and subtyping AIV will be developed. A rapid multiplex assay for identifying HA subtypes using a microsphere-based assay in combination with branched DNA (bDNA) signal amplification technology will be developed. AIV serological tests for differentiating infected from vaccinated animals (DIVA) will be developed. The use of neuraminidase subtype specific ELISAs will allow differentiation of vaccinated from infected (DIVA) animals when a vaccine with an NA subtype different from that of the circulating virus is used. The overall objective of this project is to exploit conserved overlapping amino acid stretches of the N1 and N2 protein subtypes as the basis to develop NA specific ELISAs suitable for surveillance among avian, human and swine populations. INFECTIOUS BRONCHITIS VIRUS (IBV). The majority of IB viruses isolated from commercial chickens are vaccine type viruses and rapidly distinguishing them from each other and from pathogenic variant viruses is critical for control of IB. Microsphere-based suspension array is a relatively new diagnostic platform that enables high throughput detection of nucleic acids as well as specific antigen and antibodies. For this specific aim, we will develop and validate a multiplexed microsphere-based assay for identifying the major IBV vaccine serotypes used in the U.S. Because multivalent vaccines are routinely used to vaccinate broilers, new approaches to identify multiple IBV strains in the same sample will be investigated. The multiplexed microsphere-based assay will be analyzed, and evaluation of the assay as a potential diagnostic tool for IBV will be performed using previously identified clinical samples. The results will be compared to current tests utilizing RT-PCR amplification and nucleotide sequencing. In addition, real time RT PCR primers are being developed to strains commonly used as vaccines as well as relevant field strains. Real time RT-PCR is quantitative and will be used to evaluate the viral load in vaccinated as well as pathogenic variant virus infected poultry. It will also be used to evaluate the transmissibility of IBV types. This is important because continued replication of IBV in commercial poultry provides opportunities for this rapidly changing virus to emerge as new variants or serotypes with the potential to cause disease. INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). A new generation of recombinant vaccines consisting of viral vector fowlpox virus (FPV) that carries the glycoprotein B (gB) of ILTV or herpesvirus of turkey (HVT) expressing glycoproteins I or B of ILTV are currently used by the broiler and commercial layer industries. To develop better DIVA approach, we will validate the use of ILTV glycoprotein I, E, J, and B specific ELISAs as a tool to discriminate ILTV sero-conversion due to vaccination or infection. The use of serum samples from FPV-LT and HVT-LT vaccinated flocks located in endemic and non-endemic production areas will be tested with the different ILTV specific glycoproteins ELISAs in order to determine if these new tools can differentiate sero-conversion due to vaccination with viral vector vaccines from sero-conversion due to exposure to circulating virus
3. Elucidate the pathogenesis of poultry respiratory diseases
   Comments: This objective includes experimental characterization of field isolates. The selection of the isolates and poultry species will be coordinated among NC participants as described in Aim 1. Research on pathogenesis of respiratory diseases involves exploration of the intricate and complex interactions among pathogen, host, and environment. The collaborative efforts will emphasize contemporary approaches to understanding multifactorial interactions of infections impacting respiratory disease of poultry. AVIAN INFLUENZA VIRUS (AIV). Pathogenicity and transmission potential of avian, swine and human influenza viruses in chickens and turkeys will be continued. In addition, virus histochemistry will be validated to determine the attachment patterns of avian, swine, and human origin influenza viruses including recent H3N2 variant of swine-origin in turkey respiratory and reproductive tissues. We will also test tissues from chickens for comparison and also to apply this in vitro system to evaluate the potential risk of emerging strains in chickens. In vivo characterization of selected strains will be conducted in turkey poults and hens to validate the in vitro data. It is expected that the optimized and validated virus histochemistry will be a useful tool to screen different influenza viruses to evaluate their potential replication and host tropism in birds. The new tool will expedite identifying the strains for further investigation in vivo for pathogenesis and vaccine efficacy studies. Investigation of host-specific factors associated with the infectivity, pathogenicity and transmissibility in different poultry species of current and emerging avian influenza viruses will be continued. In addition, virus-specific factors and viral molecular markers associated with infectivity, pathogenicity and transmissibility of influenza viruses in poultry species will be investigated with the objective of elucidating the genetic basis for the differences in pathogenesis observed with influenza viruses in poultry species. Effect of co-infections of avian influenza viruses with common poultry respiratory viruses on the pathogenicity and detection of avian influenza in poultry species will be evaluated. Co-infection studies in SPF chickens and turkeys using low and highly pathogenic AIV, lentogenic, mesogenic and velogenic NDV, IBV, and ILTV strains will be conducted. The viruses will be given simultaneously or sequentially and in different combinations. Metrics that will be evaluated include the outcome of infection (clinical signs, lesions), presence of the viruses in tissues, duration and titer of virus shedding (for each virus), transmission to contacts, and seroconversion. Samples collected from these studies will be used to evaluate the impact of mixed infections on the detection of each virus when conducting virus isolation in embryonating eggs. These studies will provide important and much needed information on the interaction of respiratory viruses in poultry which will help improve diagnostics and control strategies. INFECTIOUS BRONCHITIS VIRUS (IBV). Novel IBV variant (S1 gene) viruses will be evaluated for their pathogenicity in chickens. Also vaccination-challenge of immunity studies will be performed to assess the potential protection provided by commercial vaccines. In addition, evolutionary pathways of IBV populations will continue to be evaluated. Both in vitro and in vivo experiments will be performed to understand the relevance of emerging subpopulations on pathogenicity and immunogenicity. Variants causing variety of clinical signs will be full genome characterized and the basis of the clinical outcomes investigated. Immune responses elicited from IBV genotypes will be also investigated using molecular, immunological, clinical and genetic tools. Special focus will be given to innate responses their cells and chemical signals. INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). In order to better characterize the virulence of ILTV isolates the minimum infective dose, latency, incubation period, and duration of shedding of currently circulating field strains will be determined and compared to standard challenge USDA strain. In addition, comparative genome analysis of vaccine strains and virulent isolates allowed a more accurate prediction of potential virulent determinants of ILTV. So far, vaccine strains or isolates derived from commercial poultry have been analyzed. To further identify additional determinants of ILTV virulence, full genome sequences of strains from non-commercial poultry (backyard flocks) will be obtained. The hypothesis is that sequencing the full genome of these isolates will more precisely identify determinants of virulence and possible hot spots in the GaHV-1 genome that may trigger recombination events. This information is pivotal to developing safer live attenuated vaccine candidates. INFECTIOUS BURSAL DISEASE (IBDV). The pathogenicity of new IBDV strains will be determined in specific-pathogen-free (SPF) chickens. Point mutations, reassorting of genome segments and genetic homologous recombination events have been shown to affect the virulence of IBDV. Inoculating SPF chicks at 4 weeks of age will be used to assess the relative virulence of IBDV strains that are found to have new or unique nucleotide sequence mutations. Pathogenic strains will then be inoculated into maternally immune broilers to determine if they can break through immunity produced from a typical breeder vaccination program in the U.S. In addition, effect of IBDV infection on the pathogenicity of avian respiratory infectious diseases, particularly avian influenza, infectious laryngotracheitis, or avian mycoplasmosis will be evaluated. Specific-pathogen-free (SPF) chickens or commercial broiler or layer chickens will be orally inoculated with variant IBDV or its mutants generated by reverse-genetic IBDV and followed by intra-nasal or intra-tracheal challenge of chickens with the respiratory pathogen, individually or in combination. Histopathology, immunohistopathology, ELISA, virus neutralization assay, cytokine gene mRNA profiling by real-time RT-PCR, and viral or bacterial loads by real-time PCR or RT-PCR will be performed on various immune and respiratory tissues. METAGENOMIC APPROACH. NC participants will continue collaborating to determine co-infecting viral and bacterial agents involved in respiratory disease using metagenomic approaches. This approach will first be optimized at UMN using models of colibacillosis in broilers and turkeys, then applied to other models of disease. Procedures for examining bacterial community composition using 16S rRNA amplicon sequencing on the Illumina MiSeq platform and examining DNA and RNA viral populations using shotgun metagenomic sequencing on the Illumina HiSeq platform will first be applied to study E. coli-caused colibacillosis in commercial birds and experimental models of infection. In commercial birds, samples will be collected from diseased birds that display signs of colibacillosis. Birds positively identified as affected by colibacillosis will be retained, and tissue samples from the respiratory tract will be collected for total DNA, viral DNA, and viral RNA extraction using published methods. The goal of this approach is to identify correlations between colibacillosis and common co-infecting microbial flora that may be missed due to culturing bias, inability to culture, or non-comprehensive culturing (all of which likely occur in commercial settings). Experimental models of infection using commercial-source birds will also be used to study the contributing factors in the establishment of colibacillosis in broiler chicks and turkey poults, since it is understood that even in controlled conditions the reproduction of disease is inconsistent. Using a well-characterized E. coli challenge strain, we will challenge healthy and stressed birds and subsequently examine their indigenous microbiota and correlations between existing respiratory microbiota and the manifestation of disease. Once again, viral metagenomics and bacterial microbiome analyses will be used to identify the correlations between presence/absence of clinical lesions and indigenous flora. Once the procedures for respiratory metagenomic analyses have been established using the colibacillosis model, they will be applied in a collaborative effort with other researchers in the group. Some examples of etiologic agents that will be studied include IBV, IBDV, ILTV, NDV, and mycoplasmas. In addition to correlating the respiratory microbiota with disease state, mathematical modeling will be used to determine generalized microbial markers of disease susceptibility. In addition, comprehensive culturing of bacterial flora inhabiting the respiratory tracts of healthy commercial birds will be performed. The goal of this approach is to generate genomic sequences of bacterial strains associated with “health.” This again will be a collaborative effort between UMN and other participating institutions. Samples from healthy turkeys and chickens of differing ages will be cultured to isolate organisms growing on non-selective aerobic media, or other media of interest for specific bacterial taxa. These colonies will be picked and archived using a Q-bot machine at UMN’s Biotechnology Institute. Archived samples will be speciated using 16S rRNA sequencing. Once dominant species inhabiting the respiratory tract have been identified, populations of these bacteria will be further characterized using molecular approaches such as MLST, PFGE, or MLVA. Representative isolates from major bacterial taxa associated with healthy birds will be sequenced using Illumina MiSeq technology or 454 Roche sequencing to generate draft genomic sequences. We will use 7-kb paired-end libraries where appropriate to effectively produce genomic scaffold that will be useful for the community of avian researchers. These isolates and genomic sequences will be publicly available for further use. The outcome of this approach is a community resource of sequenced isolates available to better understand how the respiratory microflora drives bird health.
4. Develop control and prevention strategies for poultry respiratory diseases
   Comments: We will continue to evaluate the existing control strategies and explore alternative strategies for vaccination and other prevention measures. Different recombinant vaccine technologies are being developed and the techniques described below for specific disease may apply to different respiratory pathogens. AVIAN INFLUENZA VIRUS (AIV). Broad-spectrum vaccines against influenza A viruses will be developed. In first approach, P particle of norovirus will be used as a vaccine platform to present the highly conserved protein (M2e and HA2 epitope) of viruses and immune-stimulatory proteins. In addition to the structural advantage as a vector, the P particle is highly immunogenic, easily produced in E.coli and extremely stable which may enable reducing production costs and less dependence on the cold - chain distribution which is critical for vaccination programs in remote areas and developing countries. Poultry (chicken), swine and mice challenge models will be used to study the mechanisms of the immune enhancement and develop polyvalent M2e-based vaccines to maximize the protection spectrum against all avian, swine and human flu viruses. In a similar approach, self-assembling peptide nanoparticles will be designed and evaluated. The resemblance of the peptide nanoparticles to virus capsids combines the strong immunogenic effect of live attenuated vaccines with the purity and high specificity in eliciting immune responses of peptide-based vaccines. Two vaccine constructs presenting peptide M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms will be evaluated, initially, for their ability to elicit serum antibody responses. Vaccine constructs eliciting antibody will then be tested for the ability to protect against AIV challenge. In second approach, a system to generate and select NS gene variants (delNS1) of influenza virus with potential use as Live Attenuated Influenza Vaccine (LAIV) will be established. It will demonstrate that most effective LAIV can be systematically selected and vaccine performance can be further potentiated by augmenting the induction of interferon (IFN). The study will include: 1) In vitro analysis of virus particle subpopulations and characterization of delNS1 LAIV candidates for systematic selection of the most effective vaccine; 2) In vivo analysis of selected LAIV candidate and their potential as broad-spectrum vaccines; and 3) Further development of vaccine to ensure the safety of delNS1LAIVs. INFECTIOUS BRONCHITIS VIRUS (IBV). IBV vaccination methods will be evaluated. The efficacy of IBV vaccines administered by spray at the hatchery will be assessed. Chicks will be examined by real time RT- PCR to assess vaccine coverage following spray administration using vaccines delivered in different volumes. Studies have shown that vaccinating with two different types of IBV vaccine can provide broad protection against different IBV types. These so called protectotype vaccine combinations will be evaluated for different variant virus in vivo. In addition, different criterion used to evaluate protection will be compared. Vaccine efficacy in the USA is evaluated by detection of the challenge virus as specified by the Code of Federal Regulations, Title 9 whereas in Europe, ciliostasis in the trachea is evaluated as specified by the European Pharmacopeia. To better understand the relationship between protection defined by challenge virus detection and ciliostasis in the trachea, both parameters in vaccinated chickens challenged with homologous and heterologous strains of IBV will be evaluated. Furthermore, clinical signs as well as macroscopic and microscopic lesions in the trachea will be evaluated to generate a more complete picture of parameters used to evaluate vaccine efficacy. The IBV S2 protein expressed from recombinant NDV has been shown to provide protection against challenge. New recombinant viruses encoding distinct S2 proteins will be produced to optimize protection. In addition, recombinant adenovirus type 5 (rAd) expressing S1 protein will continue to be evaluated for immune responses and protective efficacy in chickens. Finally, in order to boost the immune response generated by live commercial vaccines we will use adjuvants based on slow release chitosan encapsulated avian interferons (IFN), to boost innate and adaptive immune responses generated by these vaccines and test their efficacy with homoplogous and heterologous challenges. INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). Traditional and alternative methods used to attenuate other respiratory viruses will be used to develop new ILTV experimental vaccines. If successful, studies will characterize the attenuated strains in terms of their pathogenicity, transmissibility, and immunogenicity for chickens. The attenuated strains will also be sequenced to identify specific gene(s) mutations arising from the attenuation process. The use of potential live ILTV vaccines attenuated by deletion of virulence determinants for in ovo mass application of broilers will be studied. Recent full genome sequencing analysis of classically attenuated live vaccines indicated that a truncation of the ORF-C gene maybe responsible for the increased attenuation of the tissue culture origin (TCO) vaccine. It is hypothesized that the ORF-C protein is a virulent determinant of ILTV. Therefore, by deleting the ORF-C gene the USDA challenge strain will become attenuated in chickens without affecting in vitro replication. The specific aims of this objective are to determine if deletion of the ORF-C provides attenuation to the virulent USDA strain and whether an ORF-C deleted ILTV strain induce protection in specific pathogen free (SPF) chickens after in ovo vaccination. The development of an effective ILTV vaccine for in ovo vaccination will eliminate the need to vaccinate broilers during the grow-out, consequently reducing the circulation of chicken embryo origin vaccines. INFECTIOUS BURSAL DISEASE VIRUS (IBDV). DNA vaccination will be optimized for IBD. The chicken cytokine gene or chicken innate immunity gene will be used as the molecular adjuvant in the hope to enhance protective immunity. Specific-pathogen-free chickens or broiler chickens with maternally-derived antibody will be in ovo or intramuscularly injected with a chimeric DNA plasmid (carrying IBDV VP2 gene fused with molecular adjuvant) or a DNA plasmid carrying IBDV VP2 gene that will be co-administered in conjunction with a DNA plasmid carrying molecular adjuvant. Chickens will be challenged with homologous or heterologous classic or variant IBDV strain. ELISA, virus neutralization, gross pathology and histopathology, and IBDV viral RNA load assessment by real-time RT-PCR will be analyzed to determine immune response and protection efficacy. In addition, development of a reverse genetics-based IBDV will serve as a vaccine vector for protection against avian influenza and other emerging poultry respiratory diseases will be explored. The availability of a single vaccine capable of in ovo administration in broilers that could protect against IB, ND, IBD and Marek’s disease (MD) would be major breakthrough in sustainable agriculture and profitability. HVT vector vaccines will be generated by insertion, into one of the 2 genomic sites previously identified, of antigenic genes from IBDV, NDV and IBV into the HVT-BAC genome by the two-step Red recombination technique (86). The genes for the different immunogenic proteins will be cloned under the control of a cytomegalovirus (CMV) immediate early promoter and bovine growth hormone polyA signal sequences. Genes will be cloned in both loci and in different combinations to better identify the configuration that provides the best expression of the different proteins. In vitro characterization of the recombinant HVT viruses will be performed by multi-step growth kinetics in CEF and expression of the antigenic proteins determined by ELISA. In vivo characterization of the recombinant viruses will be performed by in ovo (18 day of embryonation) and at hatch inoculation with the different vaccines. Expression of the immunogenic proteins will be determined by examining levels of antibody production against the immunogenic proteins overtime using ELISA. Protection efficacy of the HVT vector vaccines will be examined by in ovo or day old vaccination with those vaccines that induce the best immune response followed by assessment of antibody levels and/or challenge with pathogenic viruses.

Methods

Objectives

Our long-term goal continues to be the development of knowledge-based integrated approaches to detect, control and prevent the endemic, emerging and re-emerging poultry respiratory diseases in the United States. In our new NC project, the efforts of multiple institutions across the country will concentrate on four major specific objectives:

1. Understand the ecology of poultry respiratory diseases
2. Develop new and improved diagnostic tools for poultry respiratory diseases
3. Investigate the pathogenesis of poultry respiratory diseases
4. Develop control and prevention strategies for poultry respiratory diseases

 

OBJECTIVE # 1.

The new NC project will continue to build upon the foundation laid during previous NC projects. In the new NC project, state representatives will gather disease surveillance information from each state and share the data among participating institutions in annual meetings. A standardized data reporting system will be developed. Since the state representatives and other participants are actively involved in key national committees related to poultry diseases, the NC project will serve by gathering, discussing, and providing critical information on disease status at the state and national level which will help to identify research priorities and develop prevention strategies for the researchers, industry and state and federal government officials. This effort will obviously require mutually beneficial, collaborative, multi-disciplined approaches among participants and collaborators in state and government agencies. The established surveillance efforts will continue to be coordinated and reassessed as needed at annual meetings and via e-mail communication. The sharing of surveillance data, sequence information, and isolates will be critical to reduce the overlap of the work among institutions, develop diagnostic or detection methods (Objective 2), and expedite the selection of strains for in vivo characterization and vaccine development (Objectives 3 and 4). (All participating institutions)

Development of a poultry diseases control platform. We will build the infrastructure and create a platform (a safe working space) for the industry to allow for poultry disease control coordination among poultry professionals at the state level first. The platform will provide visualizations of data and serve as venue to discuss industry needs and poultry diseases of interest (high priority). The initial effort will be led by OH and expand regionally and nationwide in collaboration with other NC participants. (OH)

Avian influenza virus. Surveillance will be performed in commercial poultry, backyard poultry, live bird markets and auctions, wild aquatic birds, and swine in collaboration with industry, USDA, US Wildlife Services, and state diagnostic laboratories. AIV will be detected by real-time RT-PCR, virus isolation in eggs or MDCK cells. AIV specimens will be sent to USDA National Veterinary Services Laboratory in Ames, Iowa for HA and NA typing and standard pathotyping tests. The HA genes of the isolates, including all H5 and H7 subtypes will be sequenced. Other genes, depending on the significance of the isolates, may be sequenced. (All participating institutions)

Avian paramyxovirus-1 (APMV-1). Virological surveillance testing will be performed in commercial poultry, backyard and auction poultry, and wild birds. Using USDA NAHLN protocols, APMV-1 will be detected by virus isolation and/or real-time RT-PCR. APMV-1 real-time RT-PCR positive samples will be sent to USDA National Veterinary Services Laboratory for confirmation.  The fusion (F) genes of recovered isolates will be sequenced and compared to sequences in GenBank.  The pathogenicity of APMV-1 isolates will be evaluated in commercial poultry (refer to Objective 2). (GA)

Avian E. coli and Ornithobacterium rhinotracheale (ORT). From respiratory-diseased broiler chickens will be isolated and characterized by whole genome sequencing to understand changes in ecology. This will be studied in commercial broilers, layers, and turkeys. E. coli populations will be examined in the context of changes in antibiotic use, autogenous vaccine use, and changing management strategies. (IA, OH, MN)

Mycoplasma. Pathogenic avian mycoplasmas in poultry will be isolated and characterized using strain specific PCR, RT-PCR, and culture. Whole genome sequencing will be used to study mycoplasma diversity. Samples will be received from all participating institutions. (GA)

Infectious Bronchitis Virus (IBV). Virological surveillance in commercial poultry will be performed. Real time RT-PCR positive samples will undergo virus isolation attempts in SPF embryonated eggs. The S1gene of the isolates will be identified by sequence analysis. Novel variant strains of IBV will be evaluated for their pathogenicity as well as for their ability to break through immunity provided by commercial vaccines (refer to Objective 3).

In addition to commercial poultry, the possibility of a wild bird reservoir for the avian coronaviruses and the potential of those viruses to infect commercial poultry will be investigated. Using a universal avian coronavirus RT-PCR assay, different wild bird species will be monitored. If detected by RT-PCR, efforts will be made to isolate and test wild bird coronavirus isolates for their ability to infect and cause disease in commercial poultry.

Controlling infectious bronchitis virus (IBV), the coronavirus that causes the disease prevents secondary bacterial infections and significantly reduces the use of antibiotics in poultry. However, complete protection is difficult to establish because there are many different IBV types and newly emerging viruses that do not cross-protect. Therefore, it is important to quickly identify and characterize the strains of the virus causing disease in the field so the most effective vaccine strategy can be implemented. Work through our diagnostic laboratory focuses on the study of currently circulating, economically important strains of IBV. Research focuses on the most recent outbreaks and characterization of new and emerging strains. Molecular analysis of field strains is used to examine IBV evolution and vaccine or field origin of viruses. (DE, GA)

Infectious laryngotracheitis virus (ILTV). ILTV virological surveillance in commercial chickens will be performed.  In particular potential sources of infection in the poultry house environment will be identified such as dust, litter, water drinkers, feeders, walls, fans, and darkling beetles. Specimens will be tested by virus isolation and real time (RT)-polymerase chain reaction (PCR). Isolates will be characterized using PCR and restriction fragment length polymorphism (RFLP) to determine if they are wild type or vaccine origin. To evaluate the impact of vaccination in the persistence of the disease, trachea and environmental samples will be collected from ILTV positive and negative farms located in a vaccine buffer zone (CT, DE).

Infectious Bursal Disease Virus (IBDV). Since IBDV significantly affects both the development and outcome of respiratory infection and also vaccine efficacy, IBDV is included in our project. Surveillance for all IBDV strains including vvIBDV will be conducted on commercial and backyard poultry. An RT-PCR assay will be used to detect IBDV strains in bursal tissue from chickens.  The nucleotide sequence of positive RT-PCR samples will be determined across the hypervariable sequence region of the VP2 and the VP1 genes.  The nucleotide and predicted amino acid sequences will be compared to known strains of the virus. Both VP2 and VP1 sequences are needed to identify vvIBDV strains. (IN)

MICROBIOME AND METAGENOMICS. The primary goal of microbiome-based approaches in this Aim are to define the baseline poultry respiratory microbiome. To do so, we will sample from a variety of poultry flocks across the US. Tracheal swabs/washes and choanal swabs will be aseptically collected using standard approaches. Samples will be subsequently processed for nucleic acid extraction for microbiome analysis. Swabs will also be used for standard diagnostic assays to detect potential respiratory pathogens. The flock history including vaccination, daily mortality, etc. will be collected.

Respiratory disease complex (RDC) is caused by interactions between multiple environmental and biological (viruses, bacteria) elements. Metagenomic-based approaches will be used to understand the complex microbial ecology of the avian respiratory tract. Model systems using turkeys, layers, and broilers will be used to determine the microbial ecology of the avian respiratory tract during the life of the bird. The microbial community (animal RNA and DNA viruses, bacteria, bacteriophage, fungi/yeast) of the respiratory tract will then be compared to that of the intestinal tract. In addition, deviations from the normal respiratory microbial ecology will be determined for diseased commercial and backyard flocks of poultry. (DE, OH, MN)

The sharing of surveillance data, sequence information, and isolates will be critical to reduce the overlap of the work among institutions and expedite the selection of strains for in vivo characterization and vaccine development (Objectives 3 and 4).

 

OBJECTIVE #2

Infectious bronchitis virus (IBV).

It is important to rapidly identify IBV types causing disease in commercial poultry so that effective vaccine strategies can be applied. We have developed IBV type specific real time RT-PCR tests for common strains of IBV circulating in poultry. However, the nature of the virus to rapidly change requires constant evaluation of the suitability of those primers and probes as well as the development and design of new primer and probe sets to detect new emerging viruses. This work not only ensures the detection of all circulating types of IBV by real time RT-PCR but also studies the utility of novel tests based on new technologies. (GA, SEPRL). 

Improve genotyping of infectious laryngotracheitis virus (ILTV) by single allele MinION sequencing.

Previous nine genotypes (I-IX) of Infectious laryngotracheitis virus (ILTV) have been recognized using the polymerase chain reaction- restriction fragment length polymorphisms (PCR-RFLP) method with three alleles in the virus genome (gB, gM and U_L47/gG). a single allele (ORFA/ORFB) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partition based in full genome or the three alleles.  Ninety percent correlation between the genotyping results of the single allele assay using either Sanger or MinION sequencing and the multi-allele assay was obtained using archived samples. Therefore, genotyping of emerging ILTV strains could greatly simplified and expedited from real time amplicon sequencing using the single-allele assay and MinION sequencing. In collaboration with the Southeast Poultry Research laboratory (SEPRL)we will validate this method with diagnostic samples received at the Poultry Diagnostic and Research Center (PDRC) (UGA). We envision to transfer this technology to other poultry diagnostic laboratories that are currently establishing the MinION sequencing technology in there diagnostic laboratories for example Dr. Mohamed EL-Gazzar at Iowa State University (SEPRL, GA, OSU).

Next generation sequencing (NGS) are relatively new technologies that continue to evolve and become more powerful, more accurate and less expensive. As these technologies continue to drop in prices, they are becoming more available as diagnostic tools. However, the amount and the scope of data generated by these sequencing technologies is usually the challenge. Computing power and bioinformatics processing expertise are the bottle necks that determine their use in clinical diagnostic setup. There is a need for adopting and customizing these technologies to make them more user friendly in diagnostic laboratory. It is necessary to develop new assays that harness the power of these new technologies, but deliver these results to diagnosticians and clinicians in a way they can utilize. This has the potential to significantly increase our understanding of most diseases epidemiology and pathogenesis, which in turn will allow for developing better prevention, control and eradication practices. Two areas of investigation will be evaluated for NGS use as a diagnostic tool.  First, a comparison of targeted template amplification methods versus sequence independent amplification methods will be examined to determine both sensitivity of techniques and determine if bias is introduced towards certain pathogens.  Efforts to amplify four common poultry pathogens including avian influenza, Newcastle Disease virus, infectious bronchitis and mycoplasma will be the initial focus.  Second, determine optimized methods for sequence analysis with the goal of processing on a laptop computer.  Specific interest in the Minion sequencer, because of its low initial cost and ability to analyze the data produced in real time will be the focus.  Although de novo sequence assembly is more likely to identify novel disease pathogens, this technique is computer processor intensive.  Alternative faster and easier computer processing methods including sequence analysis using a template framework approach will be evaluated.  Using a minimal database of poultry pathogens to reduce Blast processing time, sequence reads will be matched to the closest sequence in the database to allow sequence contigs to be more easily developed.  The ultimate goal of the research is to test the sequence system in poultry diagnostic samples and compare this with conventional molecular diagnostic tools with the goal of technology transfer to diagnostic laboratories to perform routine diagnostic testing.  (GA, SEPRL, OSU)

Real-time RT-PCR diagnostic tests will be evaluated to provide optimal sensitivity and offer the possibility of multiplex testing.  Ongoing work to look at adding an internal positive control to avian influenza and Newcastle disease virus tests are ongoing. (SEPRL).

 

OBJECTIVE #3

Elucidate the pathogenesis of poultry respiratory diseases 

This objective includes experimental characterization of field isolates. The selection of the isolates and poultry species will be coordinated among NC participants as described in Aim 1. Research on pathogenesis of respiratory diseases involves exploration of the intricate and complex interactions among pathogen, host, and environment. The collaborative efforts will emphasize contemporary approaches to understanding multifactorial interactions of infections impacting respiratory disease of poultry.

Infectious Bronchitis Virus (IBV)

Avian infectious bronchitis (IB) is a highly contagious upper-respiratory tract disease in chickens that is worldwide in distribution and costs the U.S. poultry industry millions of dollars annually. Although viruses initially infect the upper-respiratory tract and cause respiratory signs, some strains of the virus can go on to affect the kidneys and cause nephritis while others can infect and cause damage to the reproductive tract of the hen. Evaluating the pathogenicity of viruses isolated from the field is important because vaccine strategies need to be designed to protect different systems in the bird. The pathogenesis of important IBV types that have become widespread in commercial poultry will be evaluated in specific pathogen free as well as maternal antibody positive chicks and in older birds. Where possible, the level of production losses will be quantitatively accessed to provide another measure of disease impact. (GA)

Novel IBV variant (S1 gene) viruses will be evaluated for their pathogenicity in chickens. Also vaccination-challenge of immunity studies will be performed to assess the potential protection provided by commercial vaccines. In addition, evolutionary pathways of IBV populations will be evaluated by serial passage of virus in different condition and sequence analysis. Both in vitro and in vivo experiments will be performed to understand the relevance of emerging subpopulations on pathogenicity and immunogenicity. (AL, DE, GA)

In order to understand better immune responses to IBV, we will improve TOC techniques using a cell-adapted IBV strain as a challenge antigen. We will culture primary TOC lines from B2 and B19 chickens using two different strategies: (1) rotating tubes allowing aeration of the TOC’s and (2) solid medium surrounding the TOC keeping the integrity of the tracheal lumen. Tracheas will be cultured for 7 days before challenge with IBV by that time we expect the inflammation caused by the processing to be resolved. Rings and supernatant will be collected at different times post-infection. IFN-β, IL-1β, IL-6 and IL-10 mRNA levels will be measured by RT-qPCR from tracheal rings and by ELISA from the supernatant. One ring per time point will be investigated by immunohistochemistry. (CA, AL, NY)

Studies on virus replication in avian thrombocytes. To determine if respiratory viruses replicate in avian thrombocytes we will isolate thrombocytes from blood taken from specific pathogen free (SPF) chickens.  We will then plate avian thrombocytes in tissue culture plates using a seeding density of 5 X 10⁶ per ml.  We will inoculate these cells with vaccine strains of NDV, IBV and IBD to determine if these viruses replicate in avian thrombocytes in vitro.  To explore AIVs we will collaborate with our colleagues in Ohio and ship them the cells and protocols for the AIV in vitro evaluation.  We will follow up on these studies with in vivo trials using SPF birds.  We will inoculate the birds with NDV, IBV and IBD vaccine strains, isolate the avian thrombocytes and determine by PCR if the viruses have infected the avian thrombocytes. (NE, OH)

Infectious laryngotracheitis virus (ILTV).

Minimum infective dose, latency, incubation period, and duration of shedding of currently circulating field strains will be determined and compared to standard challenge USDA strain. Comparative genome analysis of vaccine strains and virulent isolates will be done for more accurate prediction of potential virulent determinants of ILTV. To further identify additional determinants of ILTV virulence, full genome sequences of strains from non-commercial poultry (backyard flocks) will be obtained. This information is pivotal in the development of safer live attenuated vaccine candidates. (DE, GA, SEPRL)

Our collaborations have previously determined that ILTV vaccine and virulent strains have the ability to infect macrophages in  a semi-productive fashion in vitro while blocking apoptosis of these cells. In a collaborative effort we will develop two ILTV strains expressing a fluorescent reporter protein. One of the strains will be developed in the background of a well characterized virulent strain and the second will use the chicken embryo origin (CEO) vaccine as background. These reporter strains will be used as tools to investigate the interaction of ILTV with macrophages and dendritic cells in primary and secondary lymphoid organs. We will use both confocal microscopy and flow cytometry analysis to assess these interactions. This information will provide a better understanding of the role key immune cells play in supporting persistence of both vaccine and virulent strains and how these interactions correlate with pathogenicity.  To complement these work we will study the transcriptome of secondary lymphoid, after ocular inoculation of  chickens to analyze the host and viral genes expressed at early time points post-infection. We are also planning to evaluate the resistance of different chicken lines to ILTV (GA, SEPRL, IL, ADOL)

Avian influenza virus (AIV).

To better understand the epidemiology of avian influenza virus it’s important to identify virus- and host-specific factors associated with the infectivity, pathogenicity and transmissibility of the virus. We will characterize: virus infectivity, clinical presentation, gross and microscopic lesions, tissue tropism, virus shed titers and duration, antibody response, and transmission dynamics of AIV of concern.  The genetics of the virus will also be evaluated to correlate pathobiology with markers for virulence, transmission and host adaptation. To characterize how IAVs adapt as they pass in domestic and wild avian reservoir species we will analyze genome changes occurring in different lineages of AIV as they circulate in poultry and wild birds (e.g. United States H5N2 HPAIV, and Mexican H7N3 HPAIV). Whole genome sequence of viruses shed from chickens and mallards infected with different IAVs from our experimental studies will be examined to determine the genetic changes related to intra-host virus adaptation.

We will also study the effect of co-infections on the infectivity and transmissibility of AIV in chickens. The objective of these studies is to address the role of co-infections with respiratory pathogens (e.g. IBV, MS) in lowering the infectious dose needed to infect chickens with different AIV and to transmit them to other birds. (SEPRL)

Infectious bursal disease virus (IBDV)

The pathogenicity of new IBDV strains will be determined in specific-pathogen-free (SPF) chickens.  Point mutations, reassorting of genome segments and genetic homologous recombination events have been shown to affect the virulence of IBDV.  Inoculating SPF chicks at 4 weeks of age will be used to assess the relative virulence of IBDV strains that are found to have new or unique nucleotide sequence mutations. Pathogenic strains will then be inoculated into maternally immune broilers to determine if they can break through immunity produced from a typical breeder vaccination program in the U.S.

In addition, effect of IBDV infection on the pathogenicity of avian respiratory infectious diseases, particularly avian influenza, infectious laryngotracheitis, or avian mycoplasmosis will be evaluated.  Specific-pathogen-free (SPF) chickens or commercial broiler or layer chickens will be orally inoculated with variant IBDV or its mutants generated by reverse-genetic IBDV and followed by intra-nasal or intra-tracheal challenge of chickens with the respiratory pathogen, individually or in combination. Histopathology, immunohistopathology, ELISA, virus neutralization assay, cytokine gene mRNA profiling by real-time RT-PCR, and viral or bacterial loads by real-time PCR or RT-PCR will be performed on various immune and respiratory tissues (OH).

Co-Infection studies.  

The effect of co-infections with common poultry respiratory viruses on pathogenicity will be evaluated. Metrics that will be evaluated include the outcome of infection (clinical signs, lesions), presence of the viruses in tissues, duration and titer of virus shedding transmission to contacts, and seroconversion. Samples collected from these studies will be used to evaluate the impact of mixed infections. Histopathology, immunohistopathology, ELISA, virus neutralization assay, cytokine gene mRNA profiling by real-time RT-PCR, and viral or bacterial loads by real-time PCR or RT-PCR will be performed on various immune and respiratory tissues. These studies will provide important and much needed information on the interaction of respiratory viruses in poultry which will help improve diagnostics and control strategies. (DE, GA, OH, SEPRL) 

Metagenomic approach.

Participants will collaborate to determine the co-infecting viral and bacterial agents involved in respiratory disease using metagenomic approaches. Experimental models of infection using commercial-source birds will be used to study the contributing factors in the establishment of respiratory disease in broiler chicks and turkey poults. We will challenge healthy and stressed birds and subsequently examine their indigenous microbiota and correlations between existing respiratory microbiota and the manifestation of disease. Once again, viral metagenomics and bacterial microbiome analyses will be used to identify the correlations between the presence/absence of clinical lesions and indigenous flora. Some examples of etiologic agents that will be studied include IBV, IBDV, ILTV, NDV, and mycoplasmas. In addition to correlating the respiratory microbiota with disease state, mathematical modeling will be used to determine generalized microbial markers of disease susceptibility. (Initially, DE, OH, MN, later participants will be include – AL, CT, GA, IN, SEPRL, TX)

 

OBJECTIVE #4

We will evaluate recently developed vaccines and explore novel alternative strategies for prevention of respiratory disease. The research work will include development of novel vaccines using recombinant viruses, reverse genetics, recombinant proteins, as well as conventional and modified attenuation systems vaccine technologies. The efficacy of novel adjuvants and delivery systems, as well as enhancers of adaptive and innate immune responses will be tested and characterized both by conventional homologous and heterologous challenge studies as well as humoral, cell, and immune gene expression technologies. Finally, novel technologies to enhance biosecurity through in-house composting and new disinfectant treatments and procedures will be evaluated for primary avian disease control.

Avian Influenza.

• We will evaluate the current control strategies for avian influenza and explore alternative vaccines and strategies for vaccination. We will explore recombinant epitope-based vaccines and live attenuated vaccines in combination with different adjuvants, delivery systems, and vaccination approaches. Toward the development of broadly reactive vaccines (aka “universal flu vaccines”), we will focus on prime-boost vaccination strategy.

• We will also establish a practical approach to develop inactivated vaccines using reverse genetics in combination with site directed mutagenesis in the antigenic and potential glycosylation sites in the hemagglutinin (HA) protein.

Infectious Bronchitis.

• Currently, the best strategy for control of infectious bronchitis virus (IBV) is the use of live attenuated IBV vaccines. However, it can take up to a year to develop attenuated strains of IBV by passage of the virus in embryonated eggs, the traditional method of developing vaccines against IBV. Because IBV can change very rapidly, new strains can come and go from one year to the next, presenting an enormous challenge to quickly respond and produce live attenuated vaccines to the new strain. Novel vaccines are being developed using new methodologies for rapid vaccine synthesis in the laboratory. The efficacy of those vaccines against homologous and heterologous challenge will be accessed.

• We will evaluate the use of AvAIFN as a genetic adjuvant. Plasmid encoding AvAIFN optimized sequence under CMV promoter will be used as a nanoparticle in combination with a commercial live vaccine against IBV. Vaccinated groups will be immunized via the oculo-nasal route at 1 and 14 days of age, then they were challenged at 7 days after the last immunization with a Mass strain. IBV specific IgG and IgA antibody titers will be measured by ELISA. Respiratory signs, viral load, tracheal histomorphometry, cilia score and quantification of AVAIFN, IL-6, IL-10 and IL-1B will be analyzed. These results will help to evaluate the use of AvAIFN as a genetic adjuvant in the poultry industry against IBV and other avian viral diseases.

• We previously demonstrated that adaptation of an extensively used embryo-attenuated IBV ArkDPI-derived vaccine to chicken embryo kidney (CEK) cells shifted the virus population towards homogeneity in spike and non-structural protein genes. More importantly, the typical ArkDPI vaccine virus subpopulations commonly emerging in chickens vaccinated with the commercial ArkDPI vaccines were not detected in chickens vaccinated with the CEK cell-adapted Ark virus (CEK-Ark). In addition, CEK-adapted ArkDPI vaccination drastically reduced the emergence of subpopulations from a wild Ark challenge strain. We will conduct studies aimed at understanding the immune responses induced in chicken populations to more homogeneous and stable IBV vaccine viruses.

• We previously demonstrated protection against IBV infection in chickens following subcutaneous vaccination with recombinant soluble trimeric recombinant spike (S)-ectodomain (e) protein. We also demonstrated proof-of-principle that vaccination with this recombinant protein delivered by a recombinant viral vector can also protect chickens against IBV infection. We will evaluate compliance of protection induced by recombinant LaSota strain Newcastle disease virus encoding Ark-type IBV S-ectodomain protein with criteria of the Code of Federal Regulation, title 9.

• Self-assembling peptide nanoparticles will be designed and evaluated. The resemblance of the peptide nanoparticles to virus capsids combines the strong immunogenic effect of live attenuated vaccines with the purity and high specificity in eliciting immune responses of peptide-based vaccines. The new vaccines we are proposing will be better for storage and transport with greater immunogenicity for multiple strains, delivered through a unique protein-nanoparticle. Currently available vaccines have to be stored at low temperature and mild conditions, in appropriate conditions such as warmer temperatures or UV light, can cause vaccines to become ineffective but the proposed vaccines do not need such a cold chain for storage or transport. Here, we propose to design and test novel vaccine carriers called FluoDots. FluoDots are bovine serum albumin based nanoparticles, with a size of around 10 nm and live viruses will be ligated around the nanoparticle to carry 3, 4 or 9 viral particles per nanoparticle, in specific ratios and geometries. This specific stoichiometry is derived from the trigonal, tetrahedral and hexagonal geometries for packing the carrier and the viral particles. Two vaccine constructs presenting peptide S1 plus S2 in monomeric and tetrameric forms will be evaluated, initially, for their ability to elicit serum antibody responses. Vaccine constructs eliciting antibody will then be tested for the ability to protect against IBV challenge. Studies carried out so far demonstrate that FluoDots themselves caused immunity in live chickens. ELISA studies showed the formation of antibodies corresponding to FluoDots. Real time RT-PCR results indicated that after injection of FluoDots, the viral copy number is much lower than the buffer control group by 66%. All these results show that FluoDots could be used as vaccines and as vaccine carriers for chickens.

Newcastle Disease Virus

• We hypothesize that a never before recognized way in which beta glucans enhance the innate and adaptive immune responses is through avian thrombocytes. We propose to prove this hypothesis by using both in vitro and in vivo approaches with avian thrombocytes and chickens. We propose to (1) Develop and optimize those assays that will characterize avian thrombocytes and will allow us to evaluate the innate immune response. (2) Perform in vitro assays that assess the innate immunity effects of beta glucan on avian thrombocytes.  (3) Assess the in vivo innate immunity effects of beta glucans on avian thrombocytes. (4) Determine the immune priming effects of in vitro and / or in vivo stimulated avian thrombocytes using a live virus vaccine (NDV), and an immune enhancer.

• Studies on enhancing biosecurity through in-house composting. The objective of this study is to evaluate biosecurity / disease transmission potential of a tumbler composter for use in in-house mortality disposal. We will evaluate the biocontainment of a Mantis composter by placing the unit in a chicken brooding room in our BL2 animal facility where we will place day-old SPF chicks for rearing for 4 weeks.  We will inoculate egg embryos with NDV vaccine and place 2 dozen infected eggs into the composter along with fresh litter.  We will add additional inoculated eggs three times a week (MWF) to the composter.  The birds will be tested weekly for serologic antibodies to NDV to determine if the virus has escaped the composters and infected the birds.  We will perform 2 trials. The first with day-old chicks and the second with 5-week-old birds.

Infectious Laryngotracheitis

• New live attenuated ILTV vaccine will be developed using conventional and modified attenuation systems including serial passage of viruses. Once potential candidates have been selected, we will characterize the attenuated strains in terms of their pathogenicity, transmissibility, and immunogenicity for chickens. The attenuated strains will also be sequenced to identify specific gene(s) mutations arising from the attenuation process.

• Biosecurity remains a primary control measure that has impact across multiple avian diseases. Research groups will evaluate, improve, and disseminate poultry biosecurity measures including cleaning and disinfection.  New disinfectant treatments and procedures for the primary avian diseases of interest will be evaluated in the laboratory and field.

 

 

Measurement of Progress and Results

Outputs

• Collect, share and maintain surveillance and epidemiologic data of respiratory disease
• Collect and maintain new isolates of respiratory disease agents for research.
• Characterize emerging disease agents in terms of their pathogenicity, antigenicity and genetic composition.
• Identify key microbial genes and antigens associated with the immune response for use as vaccine candidates.
• Develop new and improved diagnostic tools for field and laboratory use.
• Develop antisera, antigens, cloned genes, and primers and probes, etc. for diagnostic test applications.
• Develop new vaccine candidates and vaccination strategies
• Develop new strategies for prevention, control and disease management including specific biosecurity measures.
• Provide research updates via presentations at regional, national , and international meetings (e.g. Transmissible Disease of Poultry Committee meeting at United State Animal Health Association Meeting and Respiratory Disease Committee meeting at American Association of Avian Pathologists) to ensure information dissemination to all stake holders.
• Disseminate information through refereed publications. Sponsor symposia, workshops on research on poultry respiratory diseases, and informal discussions at national and international meetings.

Outcomes or Projected Impacts

• Collaborative network aimed at maximizing resources for respiratory disease research, and standardizing research protocols.
• A greater understanding of the natural ecology of respiratory pathogens in its natural and non-natural hosts.
• A greater understanding of the disease, pathogenicity, transmission, and molecular factors involved in pathogenesis.
• Reduced mortality and condemnation will result through the use of improved diagnostic tools, vaccines and biosecurity measures.
• As a long term impact, consumers will enjoy safe, healthy, and competitively priced eggs and poultry meat products.

Milestones

(2019):10% increase in number of collaborative research efforts amongst NC-1180 members each year

(2019):10% and 5% increase in number of presentations, publications, and funding of individual projects and collaborative projects, respectively

(2021):At least one diagnostic test and vaccine per pathogen developed and validated

(1):Positive contribution of the program to the prevention of respiratory disease outbreaks in the US.

(1):Positive contribution of the program to improved animal and public health and biosecurity.

Projected Participation

View Appendix E: Participation

Outreach Plan

• Post annual reports on project web page.
• Invite veterinarians or scientists working in the poultry industry to annual meeting.
• E-mail annual reports to industry (producers and biologics manufacturers).
• Present findings at industry-sponsored and professional society meetings.
• Publish findings in referred journals in a timely manner.
• Sponsor specific respiratory disease symposia at regional and national meetings.
• Strenghthen collaborations between participants and with other multistate projects  

Organization/Governance

GOVERNANCE The research technical committee shall consist of one technical committee representative from each cooperating agency as appointed or otherwise designated by the respective organization, and administrative advisor appointed by the Association of North Central Experiment Stations Directors, and a representative of the National Institute of Food and Agriculture (NIFA).

The Governance shall consist of an elected chair of this committee and a secretary. All officers are to be elected for (at least) renewable two-year terms to provide continuity. The assigned Administrative Advisor will provide administrative guidance.

All decisions by a committee will be made in an open and democratic process. To ensure fairness in decision making, voting is restricted to one vote per respective entity; an entity being a SAES, VMES, CES, federal agency, etc.

Meetings will be held annually at the time and place mutually agreed upon by the technical committee with the approval of the Administrative Advisor.

COMMITTEE GOVERNANCE: Chair: The Chair of the committee is responsible for organizing the meeting agenda, conducting the meeting, and assuring that task assignments are completed. The chair is elected for at least a two-year term to provide continuity. The Chair is eligible for re-election. The Chair will be responsible for continuity and coordination of the project, including such activities as the annual meetings, special meetings and/or workshops to address various aspects of the project, and communication with the administrative advisor and/or administering committees. Such coordination will occur continuously throughout the project period. The annual meeting of the technical committee will serve as a monitoring session for coordination.

Secretary: The Secretary is responsible for the distribution of documents prior to the meeting. The Secretary is also responsible for keeping records on decisions made at meetings (a.k.a. keeping the minutes), maintaining an updated roster of participants (as a list server), and assisting in the preparation of the accomplishments report (i.e., the SAES-422). The Secretary will serve as the chair in the absence of the elected chair.

Members: The Project Members are responsible for working to ensure the overall success of the NC-1180 by completing their proposed research, fostering collaborations, providing timely progress updates and information exchanges, and attending the annual meetings.

Literature Cited

All listed references were selected not only for their collaboration between participants of NC_1180, but also collaboration with other multistate projects e.g. NE1334 "Genetic bases for resistance to poultry diseases"

 

1. Balzli, C. L., K. Bertran, D. H. Lee, L. Killmaster, N. Pritchard, P. Linz, T. Mebatsion, and D. E. Swayne. The efficacy of recombinant turkey herpesvirus vaccines targeting the H5 of highly pathogenic avian influenza virus from the 2014-2015 North American outbreak. Vaccine 36:84-90. 2018.
2. Beltran, G., S. M. Williams, G. Zavala, J. S. Guy, and M. Garcia. The route of inoculation dictates the replication patterns of the infectious laryngotracheitis virus (ILTV) pathogenic strain and chicken embryo origin (CEO) vaccine. Avian Pathol 46:585-593. 2017.
3. Bertran, K., C. Balzli, D. H. Lee, D. L. Suarez, D. R. Kapczynski, and D. E. Swayne. Protection of White Leghorn chickens by U.S. emergency H5 vaccination against clade 2.3.4.4 H5N2 high pathogenicity avian influenza virus. Vaccine 35:6336-6344. 2017.
4. Bertran, K., D. H. Lee, C. Balzli, M. J. Pantin-Jackwood, E. Spackman, and D. E. Swayne. Age is not a determinant factor in susceptibility of broilers to H5N2 clade 2.3.4.4 high pathogenicity avian influenza virus. Vet Res 47:116. 2016.
5. Bertran, K., D. H. Lee, M. F. Criado, D. Smith, D. E. Swayne, and M. J. Pantin-Jackwood. Pathobiology of Tennessee 2017 H7N9 low and high pathogenicity avian influenza viruses in commercial broiler breeders and specific pathogen free layer chickens. Vet Res 49:82. 2018.
6. Bertran, K., C. Thomas, X. Guo, M. Bublot, N. Pritchard, J. T. Regan, K. M. Cox, J. R. Gasdaska, L. F. Dickey, D. R. Kapczynski, and D. E. Swayne. Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens. Vaccine 33:3456-3462. 2015.
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Iowa State University - College of Vet Med, Southeast Poultry Research Laboratory, University of Georgia, USDA-ARS/Georgia