Duration: 10/01/2014 to 09/30/2019

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

THE NEED AS INDICATED BY STAKEHOLDERS. The United States 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 growth 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 2012 was $38.1 billion, up 8 percent from $35.3 billion in 2011 and up 48 percent from the $25.8 billion in 2006 (USDA, ERS data, 2012). Based on survey conducted by National Chicken Council (2012), Americans consume more chicken than anyone else in the world  83.6 pounds per capita  the number one animal protein consumed in the United States. Consumers rate chickens value very highly and chicken consumption per capita has increased nearly every year since the mid 1960s, while red meat consumption has steadily declined.

U.S. egg operations produce over 90 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 250 eggs per person. Chicken eggs are an important source of high quality protein and other nutrients in the diet.

The United States has the largest broiler chicken industry in the world, and over 17 percent of production is exported to other countries in 2011. In 2011, approximately 9 billion broiler chickens weighing 50 billion pounds live-weight were produced. The U.S. turkey industry produces over 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 United States 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 food security.

IMPORTANCE OF THE WORK, AND WHAT THE CONSEQUENCES ARE IF IT IS NOT DONE. Respiratory diseases continue to be a major concern to producers. Consequently, losses induced by respiratory diseases 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 (2012), the following pathogens continue to be a problem in the industry: variant infectious bronchitis (IB), infectious laryngotracheitis (ILT), infectious bursal disease (IBD), aspergillosis, Newcastle disease (ND), infectious coryza, avian influenza (AI), swine influenza, and infections caused by E. coli, avian mycoplasmas, Ornithobacterium rhinotracheale (ORT), P. multocida, and avian metapneumovirus. Environmental factors may augment these pathogens to produce the clinically observed signs and lesions. Thus, management of poultry is also a critical factor in controlling respiratory disease.

The export market comprises 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. Although HPAI and END have not been reported in the U.S. since our last project was initiated, outbreaks continue to occur around the world and in countries bordering the U.S. Considering the potential impact of possible outbreaks, surveillance and rapid detection are critical. For example, a major epidemic of END occurred in California from September 2002-May 2003. Eradication of that END cost more than $162 million, a value that does not include lost export trade at the time.

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) strains of IBDV of varying pathogenicity. IBDV, while not a respiratory pathogen, has demonstrated the ability to increase the severity of several poultry respiratory diseases and reduce the efficacy of vaccines via its immunosuppressive effects. While IBDV is endemic in the U.S., vvIBDV and other unique newly identified IBDV strains pose a new and more difficult management threat to poultry health.

The agrobioterrorism 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 agrobioterrorism agents. Considering the environmental stability, virulence and transmissibility of the virus among poultry flock, vvIBDV might also be an attractive target for bioterrorist. Our project will develop strategies for rapid diagnosis and control of these and other diseases.

Considering the challenges outlined above, it is essential that the proposed studies proceed to help protect the nation's food supply and the economic well-being of farmers and the poultry industry.
THE TECHNICAL FEASIBILITY OF THE RESEARCH. The technical challenges posed by the goal of improved respiratory disease control are significant but feasible. Participants in the project have experience and training to undertake the work and complete the objectives. In addition, physical facilities, equipment, and other resources are committed by member institutions to guarantee the success of the proposed activities.

THE ADVANTAGES FOR DOING THE WORK AS A MULTISTATE EFFORT. Control of respiratory infectious diseases lends itself to collaborative multistate research. The diseases are endemic in many poultry producing states. Furthermore the challenges posed by the number of different disease etiologies and their complexities require a multistate effort.

Current NC1180 project 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 state of the art array techniques to differentiate genotypes or serotypes. 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 such as vvIBDV and re-emerging diseases such as variant IB, and endemic infections such as low path AIV in wild birds, have led to better understanding of the disease, development of diagnostic tools, and development of surveillance and vaccine strategies to control the disease. The combination of these and other findings have led to very productive years of the current project as indicated by the number of papers published or in press including joint publications from participating institutions (Please refer to the Composite Annual Reports attached to the proposal).

WHAT THE LIKELY IMPACTS WILL BE FROM SUCCESSFULLY COMPLETING THE WORK. The overall impact of a successful outcome will be improved diagnosis and control of respiratory diseases that will benefit the poultry industry. Impact of the research will be derived from identification of disease agent reservoirs such as wild birds, factors involved in agent transmission to poultry, the development and delivery of gene and protein based diagnostics, determination of infection status, rapid strain identification, evaluation and development of vaccines, and the design and implementation of eradication protocols for selected agents. The overall outcome of the project is to produce findings that enable the poultry industry to remain competitive and profitable.

Related, Current and Previous Work

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 (18, 125). However, some strains adapt to poultry and other mammals including humans and cause severe disease. Recently, a brand new HA and NA subtypes of influenza virus (H18N11) was first discovered in little yellow-shouldered bats in Guatemala (118).

The U.S. government spent $60 million in 1983-84 to eradicate a highly pathogenic (HP) H5N2 virus in poultry in Pennsylvania (75). There have also been several costly outbreaks of low pathogenic (LP) AI affecting poultry flocks in Minnesota from 2000-2002 (41) and flocks in California (124). LPAIV has regularly been found in live bird markets (LBMs) in the northeastern U.S. (114) 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 (129). Additionally, the 2009 pandemic H1N1 virus was detected in turkeys with a severe drop in egg production (10). SIVs replicates both in respiratory and digestive tracts of both juvenile and layer turkeys and also in the reproductive tract of layer turkeys (4). Compared to SIVs, a limited replication of seasonal human H1N1 and no replication of both recent human-like swine H1N2 and pandemic H1N1 viruses were observed. 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.

Recent outbreaks of AI in different species including humans have highlighted the necessity to improve existing diagnostic tests and to develop new more rapid and specific methods to detect future outbreaks. With conventional methods of virus isolation and serologic testing (116), the laboratory confirmation of AI takes a relatively long time because of the lack of capacity for providing high throughput, sensitive and specific assays. In the last decade, new-generation assays based on molecular techniques have become available and applied successfully to disease diagnostics and active surveillance programs (13). The majority of the new techniques are based on the amplification of specific nucleic acid sequences by polymerase chain reaction (PCR), or other similar methods (NASBA) (76). Real-time RT-PCR is widely used because it offers high specificity and sensitivity. However, the main disadvantage is the cost and the lack of multiplexing capability. To overcome the limit of multiplexing, microarray technology is being developed rapidly and has been applied to diagnosis of influenza A virus (103). However, due to its solid phase format in nature, cost and flexibility have been an issue for practical application. The microsphere-based assay is an emerging technology capable of multiplexing up to 100 different assays from a single sample (87, 89). Considering the flexibility, ability to multiplex, and automated high throughput capabilities at an affordable price, microsphere-based assay has great potential in rapid AIV diagnostics.

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 (122) and swine-origin H3N2 influenza (32) and recent outbreak of H7N9 avian influenza in China which killed more than 40 people (74) 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 (77). 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 (77). 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 (15, 47). 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 (85, 109, 110, 120). 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 (97). A reverse genetics technique was used for the rapid generation of reassortant viruses that may serve as candidate vaccine strains for AI (78, 83). In addition, live-virus vaccines were developed for AI (113, 123). In broilers, vaccines are often administered during later stages of embryonation, usually at 17-18 days of incubation (104). 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. (70). 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 (91, 120). 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 investigation.

With the development of highly efficient vector (or carrier molecule) and adjuvant, the conserved protein-based universal vaccine approach also warrants further investigation (102). 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 (31, 94). The M2e specific antibody can also bind to and prevent the influenza virus progeny release from infected cells (27). 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.

AVIAN PARAMYXOVIRUS-1 (NEWCASTLE DISEASE VIRUS (NDV)). Newcastle disease (ND) is a contagious disease of various species of wild and domestic birds (2, 3) that can have severe economic consequences for poultry producers, including a serious impact on the international trade of poultry 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) (43), mesogenic (intermediate virulence) (9), and velogenic (high virulence) strains (8, 28). Regardless of the severity of the pathotype, vaccination usually protects birds from clinical disease (11).

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 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 (Miller PJ, unpublished data). 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 (Miller PJ, unpublished data). 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.

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 (12, 98). It is thought that most APEC infections are secondary in nature, affecting an immunocomprimised 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 immunocomprimised host (62).

Host-pathogen interactions during E. coli infection in the broiler chicken has been studied. DNA microarray technology is being used (both E. coli and chicken genomes) to study genes differentially expressed during initial versus late infection, moderate versus severe infection, and initial colonization versus systemic infection (62). In addition, transcriptome analysis of avian E. coli is being performed to identify genes that are differentially expressed during primary versus secondary infection (108). An improved understanding of the nature of E. coli infection in poultry will elucidate its role as a primary or secondary respiratory pathogen, and the genetic traits used by E. coli to cause disease in the bird.

Full genome sequencing of avian E. coli in a reverse genetics approach has been utilized to identify promising antigens that might stimulate an effective immune response in the bird, protecting it against the highly diverse avian E. coli strains that cause colibacillosis. A subset of putative antigens has been computationally identified, and these antigens are being screened against our E. coli collections to identify the most promising antigens for future vaccine development. This study will identify the most promising antigens that will elicit heterologous protection against the diverse E. coli involved in poultry diseases.

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 (16). The S1 subunit of S induces neutralizing antibodies (17) 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 (35-38, 126, 130). 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 (16). 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 (118). However, the existence of a reservoir for avian coronaviruses is still unresolved (22). 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 (14, 48, 127). 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 (60, 66, 93). RT-PCR targeting the S1 portion of the spike protein, followed by sequencing of the RT-PCR product (79), restriction enzyme fragment length polymorphism (RFLP) (73, 80), or hybridization with specific probes (52, 65, 72) 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 (29). 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 (30), avian influenza (19, 71), and human respiratory disease associated viruses (86). 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.

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 (23). 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.

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 (26), California (82), the Delmarva peninsula region (67, 105, 106, 115), Georgia (5), Mississippi (92), North Carolina (40) and Pennsylvania (25, 68). An outbreak that occurred on the Delmarva peninsula from 1998-2000 cost the broiler industry $4 million (106).

ILT is characterized by moderate to severe respiratory distress, conjunctivitis, and tracheitis (39). 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 (39).

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 (39). 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 (34). 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 (24). 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 (64). Ritter (105) described two modes of spread of ILT. 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.

INFECTIOUS BURSAL DISEASE VIRUS (IBDV). IBD is characterized by destruction of the lymphoid cells of the bursa of Fabricius (33). Severe B-cell suppression (69) 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 (90). 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 (50). Antigenic variant strains break through the immunity provided by vaccination (49, 112). 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 (56, 58, 59, 60, 81, 99, 121).
The genome of IBDV consists of two segments of double-stranded RNA. The smaller segment B encodes VP1, a 97-kDa multifunctional protein with polymerase activity (69). The larger segment A encodes a 110-kDa-precursor protein in a single large open reading frame (ORF), which is processed into mature VP2, VP3 and VP4 proteins (46). VP2 and VP3 are major structural proteins of the virion, whereas VP4 is a minor protein involved in the processing of the precursor protein (6, 61). VP2 is the major protective immunogen and is responsible for inducing neutralizing antibody. Antigenic determinants also reside in VP2 and variability within the gene is noted (7, 42 121). VP3, as a group specific antigen, forms a complex with VP1, which may have an essential role for the morphogenesis of the virion (84). Segment A also encodes a 17-kDa nonstructural VP5 protein from a small ORF, which is found in infected cells (95). VP5 is not essential for replication and a reverse genetics-generated VP5-deficient mutant did not induce bursal lesions in chickens (128). Expression of VP2,4,3 polyprotein suppressed proliferation of bursal lymphocytes independent of infection as well as suppressed cell growth of transiently transfected DT40 B lymphocytes, suggesting that IBDV polyprotein is a mediator of immunosuppression (101).

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 (55). Since then, several other California backyard and commercial flocks have been affected by the same vvIBDV strain and other unique (previously undiscovered) 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 (53). 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 (54). 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 (1) and DNA vaccination (20, 21), and also reverse genetics (96) 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 (45). Nevertheless, chickens primed with a DNA plasmid carrying the VP2 gene and subsequently, boosted with killed IBD vaccine achieved 100% protection (44). Recent study on DNA vaccine shows 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.

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
   

Methods

Objective 1. Ecology of poultry respiratory diseases 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 and expedite the selection of strains for in vivo characterization and vaccine development (Objectives 3 and 4). (All participating institutions) 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 (CT, DE, OH, SEPRL). 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) (DE, GA, SEPRL). 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 (DE, GA, MN). MYCOPLASMA. Pathogenic avian mycoplasmas in poultry will be isolated and characterized using strain specific PCR, RT-PCR, culture, and sequecing (GA, OH). INFECTIOUS BRONCHITIS VIRUS (IBV). Virological surveillance in commercial poultry will be performed. Real time RT-PCR positive samples will undergo virus isolation and the S1 gene sequenced. Novel variant strains of IBV will be evaluated for their pathogenicity and their ability to break through immunity provided by commercial vaccines (refer to Objective 3) (AL, CT, DE, GA). Genetic changes in NSP3 and Spike will be examined to correlate 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 (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 PCR. Isolates will be characterized using PCR and restriction fragment length polymorphism (RFLP) to determine if they are wild type or vaccine origin (DE, GA). 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 and sequence of hypervariable region of the VP2 and the VP1 genes will be determined to identify vvIBDV and variant strains (DE, GA, OH). Objective 2. New and improved diagnostic tools for poultry respiratory diseases 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. New and re-validated tests will be added to the respiratory panel for molecular detection and diagnosis of different pathogens including IBV, NDV, AIV, ILTV, etc. In addition to surveillance, the serologic test, which can be used in conjunction with vaccines to differentiate infected from vaccinated animals (DIVA) will be developed. During the project, the participating institutions will exchange reference and field samples for validation of the proposed tests (Objective 1, 3, 4). The works proposed in Objective 3 and 4 require standardized test in their complicated challenge and vaccine studies. Thus, collaborations among groups in different Objectives are mutually beneficial. 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 (DE, SEPRL). A suspension array system for detecting and subtyping AIV will be developed (CT, GA, OH). A surface-enhanced Raman spectroscopy based assay will be developed for detecting and subtyping AIV and tested for multiplexing capabilities (IL, SEPRL). DIVA serological test (NA specific ELISAs) will be developed (GA, OH, SEPRL). INFECTIOUS BRONCHITIS VIRUS (IBV). 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. We will develop and validate a multiplexed microsphere-based assay for identifying the major IBV vaccine serotypes used in the US. Because multivalent vaccines are routinely used to vaccinate broilers, new approaches to identify multiple IBV strains in the same sample will be investigated. The assay will be compared to current tests (GA). In addition, real time RT-PCR will be developed to strains commonly used as vaccines and 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 (AL, DE, GA). INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). A new generation of recombinant fowlpox virus (FPV) and herpesvirus of turkey (HVT) vaccines are currently used in commercial industries. ILTV glycoprotein I, E, J, and B specific ELISAs will be developed as a DIVA test. 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 (DE, GA). Objective 3. 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 understand 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 studied. 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 (OH, SEPRL). Virus- and host-specific factors and viral molecular markers associated with infectivity, pathogenicity and transmissibility of influenza viruses in different 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 (DE, OH, SEPRL). 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 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). 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 will be used in developing safer live attenuated vaccine candidates (DE, GA, SEPRL). INFECTIOUS BURSAL DISEASE (IBDV). The pathogenicity of new IBDV strains will be determined in 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. Pathogenic strains will be inoculated into maternally immune broilers to determine if they can break through immunity produced from a typical breeder vaccination program in the US. (DE, OH). CO-INFECTION STUDIES. Effect of co-infections of AIV with common poultry respiratory viruses on the pathogenicity and detection of AIV 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 (GA, SEPRL). In addition, effect of IBDV infection on the pathogenicity of avian respiratory infectious diseases, particularly AI, ILT or avian mycoplasmosis will be evaluated. 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. 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 co-infecting viral and bacterial agents involved in respiratory disease using metagenomic approaches. This approach will first be optimized at MN using models of colibacillosis in turkeys, then applied to other models of disease and species in collaboration with DE and OH groups. 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 MN 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 MN’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 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 (Initially MN, DE, and OH will lead the project and later participants conducting pathogenesis and vaccine studies will be involved – AL, CT, GA, IN, SEPRL) Objective 4. Control and prevention of poultry respiratory diseases We will evaluate the current control strategies and explore alternative strategies for vaccination and other prevention measures. 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 (OH). 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 (CT). 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 delNS1 LAIVs (OH). INFECTIOUS BRONCHITIS VIRUS (IBV). The efficacy of IBV vaccines administered by spray at the hatchery will be assessed. Specifically, different volumes of spray will be compared to investigate the degree of vaccine coverage that chicks receive in the hatchery. Chicks will be examined by real time RT- PCR to assess vaccine coverage following spray administration using vaccines delivered in different volumes (GA). 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 US 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 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 (AL, DE, GA). 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 and nanoparticle based IBV vaccine constructs containing S1, M and N proteins will also be evaluated for immune responses and protective efficacy in chickens (AL, CT, SEPRL). INFECTIOUS LARYNGOTRACHEITIS VIRUS (ILTV). New live attenuated ILTV vaccine will be developed using conventional and modified attenuation system 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 (DE, GA). 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. We will determine deletion of the ORF-C provides attenuation to the virulent USDA strain and whether an ORF-C deleted ILTV strain induces protection in 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 (DE, GA). 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. 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, OH). In addition, virus-like-particles (VLPs) will be produced using expression of IBDV proteins in the baculovirus system. These VLPs will be evaluated for their ability to induce immunity to IBDV in breeder chicken flocks. The progeny of the vaccinated breeders will be challenged and evaluated for protection to homologous and heterologous IBDV strains (DE, OH). It is expected that knowledge and experience acquired during this project will enable us to continue developing multidisciplinary cross collaborations with potential for funding by major funding agencies such as USDA-NIFA, NIH, NSF, DOD, U.S. Poultry & Egg Association, etc. The team will actively seek for extramural funding.

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 strategis 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

(2015): Each year - 20% increase in number of collaborative research efforts amongst NC-1180 members each year with the goal of 100% increase at the end of the project

(2015): Each year - 10% increase in number of presentations, publications, and funding of individual and collaborative projects each year

(2017): At least four diagnostic tests and three vaccines developed

(2018): At least two diagnostic tests and two vaccines validated

(2019): A better understanding of the ecology, disease, transmission, and factors involved in pathogenesis.

(2019): Positive contribution of the program to the prevention of respiratory disease outbreaks in the US.

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

Projected Participation

View Appendix E: Participation

Outreach Plan

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 efforts will focus on translating research findings into practices that will reach both industry and government stakeholders under real world conditions. We will work closely with producers to adopt and implement effective intervention strategies, train for management of respiratory diseases and evaluate their effect on overall respiratory disease prevalence. Extension effort will be lead by CT, DE, OH, and IA and development of extension material will involve all the NC1180 participants.

CONSTRUCTION OF POULTRY NETWORK IN STATES AND REGIONS.
Apart from reportable diseases, centralized effort that coordinates the control and eradication of poultry diseases including respiratory diseases is limited in most of the states. One of the very few good examples of functional bodies that undertake this task is the “Poultry Technical Advisory Committee” in the state of Georgia which is formed from a representative from major poultry producers in the state of Georgia, and the Georgia laboratory network from both the University of Georgia and the State lab. This committee coordinates all infectious poultry diseases control and eradication efforts. However, depending on the nature of the poultry industries which varies greatly by the type of poultry species dealing with and also by the region, the coordinated centralized effort on non-reportable poultry disease is limited in most of the poultry producing states.

The goal of the project is to generate a functional body that will effectively monitor poultry respiratory diseases and coordinate prevention, eradication and control efforts initially in the state of Ohio. This structure is scalable to the local region including the Tri-state organization (Ohio, Indiana and Michigan). We will first construct the “Ohio Poultry Network” which will be consisted of poultry production veterinarians, extension veterinarian, state laboratory veterinarians and other relevant professionals from Ohio Poultry Associations and NAHLN diagnostic lab. The objectives of the poultry network is to build and update a poultry production network providing data about the species, housing capacity and geographical location of poultry production in the state of Ohio. The main goals of this network are to monitor poultry infectious diseases, coordinate prevention, control and eradication efforts based on the information gathered and processed by this network. Quarterly reports will be generated to discuss the updated infectious poultry disease status including respiratory diseases and the proposed measures of prevention and control. In addition detailed emergency plans for different poultry diseases will be developed in first three years, including initial response, vaccination programs, zoning maps and poultry traffic. This obtained data will also be used to direct training courses and education for the poultry professionals, veterinarians and general public. The successful structure developed in this study will serve as an excellent model for other states to adopt or develop similar structure.

EDUCATION OF STAKEHOLDERS FOR PREVENTION AND CONTROL OR RESPIRATORY DISEASE.
We will develop and implement an effective and comprehensive outreach program to educate and train poultry producers on recognizing and preventing endemic poultry respiratory diseases. The primary target audience will be commercial poultry industries and our effort will expand to reach backyard flocks and other non-commercial poultry. While the commercial poultry industry is the largest population, with high density in some regions, the backyard poultry sector is as also wide-spread geographically. This sector represents a significant challenge when it comes to poultry disease surveillance, diagnosis and control and also poses a threat to commercial poultry industries. Recently, the “urban chicken phenomenon” has resulted in large numbers of birds being kept in many households in cities, suburbs and rural areas. The birds are kept for meat and eggs or often as pets. Multiple bird species are often kept on small hobby farms, including game birds and waterfowl. This population does not have easy access to veterinary health care. Most small animal or mixed animal veterinary practices do not have much expertise in servicing poultry. Many calls are fielded daily by diagnosticians and cooperative extension professionals from veterinarians, county educators and the bird owners themselves describing concerns with illness in the flocks. Many of the described symptoms suggest respiratory diseases. Of those who do use the animal diagnostic laboratories to submit samples, a high incidence of respiratory diseases such as Mycoplasma gallisepticum, IB, Fowl Pox, ILT and others are often detected.

Knowledge and training in poultry health is greatly needed to help practitioners, county agents and the owners effectively prevent and/or manage these respiratory illnesses. This in turn would improve the safety of the meat and eggs consumed or sold (often by direct retail on site or in small markets) as only approved treatments would be utilized. Importantly, effective biosecurity measures and vaccination strategies would lessen the chance that these diseases would spill over into the commercial poultry industry sectors.

INFORMATION DEVELOPMENT, DISSEMINATION AND EVALUATION OF EFFECTIVENESS.

INFORMATION DEVELOPMENT. Information related to poultry respiratory diseases will be developed which includes: 1) general disease information, 2) recent disease outbreak information, 3) vaccine and other prevention strategies, 4) recent changes or new regulation or policies (FDA, USDA, etc) related to poultry health, 5) research findings from this project and also from literature, and 6) extension activities being conducted by our team. This information will be reviewed by all participants.

INFORMATION DISSEMINATION. The extension team will utilize multifaceted approaches to educate and train producers, owners, educators and veterinarians using tools that will most effectively reach the targeted groups. Local databases with large, medium and small scale poultry producers from the participating regions will be updated and the team will collaborate with other extension specialists to produce current mailing lists, including postal addresses and email addresses, to be used for inviting these producers to attend workshops/meetings/webinars. Notices of meetings will also be posted on Craigslist, and Facebook as successfully used in Alaska to increase program participation. Information will also be posted on various poultry association websites, popular press poultry publications, and on eXtension pages. Without requesting personal identification information, a survey of a minimum of 25 poultry producers will be conducted to assess the various practices/steps followed for identifying and reducing respiratory diseases poultry, and identify risk factors and critical control points for reducing respiratory disease prevalence in farms.

1) A planning group/advisory panel including stakeholders and extension personnel will be formed to develop the final educational materials to be disseminated. Peer advisory groups have proven to increase program participation and the development of relevant materials.
2) Veterinary extension newsletters, state and regional meetings and webinars, on-line courses that provide continuing education credits, extension bulletins, extension websites, lay journals, interest group websites, others will be utilized.
3) Other training efforts will include meetings in the homes of some of the producers themselves. This has shown to be very effective when working with certain farming groups such as the Amish or other low technology farmers who generally do not attend large centralized meetings.
4) Training and materials targeted to 4-H and FFA youth who participate in poultry programs will be developed based on a youth train-the-trainer program. Train-the-trainer programs have been proven successful in HACCP programs and would fit perfectly into our educational model. Emphasis on character development and decision-making skills will translate into positive responses about the responsibilities of a poultry producer, both to the animals and to consumers.


EVALUATION OF THE EXTENSION EFFORTS. Follow-up assessments will be distributed to participants at meetings and other direct contact programs to determine if their respiratory disease reduction practices have changed as a result of the educational effort. For those who learn about control of respiratory disease through our web based materials, a questionnaire will be included with the web materials for them to self-assess any changes they make as a result of accessing our educational materials. It is expected that producers would increase their knowledge of the risk factors associated with the sources and transmission of respiratory diseases. We also anticipate that targeted groups will adopt practices that will have a positive effect on reducing respiratory diseases in poultry.

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.

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Southeast Poultry Research Laboratory, University of Georgia, USDA-ARS/Georgia