Objective 2. Surveillance, occurrence and consequences of agent and host variation on disease susceptibility.
Avian Influenza
No work in pathogenesis and interactions of the avian influenza
virus with any other specific agent is planned during the proposed
study period.
Avian Pneumovirus
Minnesota and Iowa will continue to use and improve serum
antibody detection assays for surveillance of avian pneumovirus
infections in turkey flocks. Epidemiologic investigations will
also be initiated to determine the source of avian pneumovirus
to turkey flocks, with special emphasis on the role of wild birds
and rodents in disease transmission. To identify the natural hosts
of APV and elucidate the mechanisms of virus transmission, we
will initiate a comprehensive program of trapping and screening
wild bird (e.g. starlings, crows, sparrows, and migratory waterfowl,
etc.) populations in and around turkey barns in Minnesota turkey
farms.
Minnesota will continue the molecular characterization by DNA sequence analysis of clinical isolates of the avian pneumovirus in order to determine the presence of sequence and strain variation among isolates in the United States. Minnesota will also use a polymerase chain reaction and automated DNA sequencing based strategy to characterize all seven structural genes of the avian pneumovirus to determine estimates of total nucleotide diversity in each of the viral genes. The levels of nucleotide and amino acid diversity will be determined and the pattern of nucleotide variation examined to understand the molecular mechanisms of evolution among US isolates of the avian pneumovirus. Minnesota will provide Indiana and other stations with primer sequence information for the characterization of the avian pneumovirus and its variants.
Ohio will supply Iowa with viruses adapted to tissue culture for determination of antigenic relatedness. Iowa and Ohio will collaborate on epidemiologic studies on naturally occurring pneumovirus infections in chicken and turkey flocks. The antigenic relatedness of isolates of pneumovirus from field cases will be studied.
Escherichia coli
Alabama, Michigan and Minnesota will conduct surveillance
and determine the occurrence and consequences of E. coli variability
and susceptibility. Alabama will obtain suspect respiratory
and non-respiratory Escherichia coli isolates from commercial
poultry integrators throughout the United States. These isolates
will be definitively identified and analyzed by pulsed field gel
electrophoresis (PFGE). Whole cell protein lysates will also be
analyzed by 2-D electrophoresis. Dendrograms of relatedness will
be generated. Results will be compared to environmental and feed
E .coli isolates taken from farms that are experiencing
significant disease with those farms having no problems.
Michigan will compare and contrast the effects of a non-serotypable E.coli versus the effects of serotype 078 E.coli on turkeys. The effects will be determined by clinical signs, gross lesions and mortality rate.
Minnesota will continue to develop fluorophore enhanced repetitive element PCR (FERP) and other related techniques for the rapid and unambiguous DNA fingerprinting of E. coli and other bacterial pathogens. FERP employs oligonucleotide primers with a 5' covalently bound fluorescent dye for the amplification and fluorescent detection of repetitive elements in bacterial DNA. Subsequent to amplification by PCR, the products of amplification are resolved on a polyacrylamide or equivalent gel matrix, and the separated DNA fragments sized and quantitated with the ABI DNA fragment analyzer (ABI 377) equipped with GENESCAN software.
Fowl pox
In order to determine the antigenic and genetic relatedness of
avian poxviruses from Hawaiian forest birds to fowl poxvirus strains
that infect commercial poultry, their genomes and antigens will
be compared by Illinois. In addition, as a continuation
of the examination of the epidemiology of avian pox, its impact
on commercial poultry will be examined. New poxvirus isolates
will be characterized genetically, antigenically and biologically
by Illinois.
Infectious Bronchitis Virus
Indiana will continue to collect turkey corona virus from
field outbreaks and characterize the isolates by IFA and RT-PCR.
The S gene of PCR untypable isolates will be sequenced and compared
to other respiratory and enteric coronavirus.
Infectious Bursal Disease
Indiana and Ohio will collaborate on studying the molecular
basis for increased pathogenicity of the highly virulent IBDV.
Ohio will provide Indiana with viruses characterized
for pathogenicity for study of molecular basis for variability.
Ohio will use molecular diagnostic assays to determine
the genetic variability among IBDV isolates from commercially
reared poultry. The host susceptibility of selected IBDV isolates
with unique molecular characteristics will be examined.
Ornithobacterium rhinotracheale (ORT)
Minnesota will continue to use and improve the serum antibody
detection assays for surveillance of O. rhinotracheale infections
in turkey flocks. Epidemiologic investigations will also be initiated
to determine the source of O. rhinotracheale to turkey
flocks, with special emphasis on the role of wild birds and rodents
in disease transmission. Assays such as repetitive-element PCR
and DNA sequencing for the fingerprinting of O. rhinotracheale
will continue to be refined and employed for O. rhinotracheale
strain tracking and epidemiology. Minnesota will continue
to share reagents and other information regarding the fingerprinting
of ORT isolates recovered from diseased poultry with the other
stations.
In addition, Minnesota will continue to use and improve the killed bacterins that have been shown to protect birds from O. rhinotracheale clinical signs and infection on subsequent challenge. In addition, future investigations will continue to improve diagnostic tests for O. rhinotracheale and develop a live temperature-sensitive mutant vaccine that will protect turkeys against disease caused by O. rhinotracheale.
Pasteurella multocida
Minnesota will collaborate with NADC in initiating
a program with the long-term goal 16 of determining the role of
host genetic variation among turkeys on susceptibility to bacterial
and viral respiratory diseases. In the first instances, a comprehensive
molecular genetic linkage maps of the turkey genome and a program
to identify potential molecular genetic markers for disease resistance
and susceptibility against Pasteurella multocida and Escherichia
coli will be established. Minnesota will continue to
work with NADC to determine specific virulence traits in
P. multocida isolates recovered form avian sources.
Objective 3: Develop new and improved methods for the diagnosis, prevention and control of avian respiratory diseases.
Avian Influenza
Minnesota will continue to work on various aspects of
Avian Influenza disease detection, prevention, and control. Minnesota
will continue to produce and evaluate AI antigens with broad cross-specificity
for disease diagnosis. These include continued development of
assays for the rapid diagnosis of AI virus infected flocks using
recombinant nucleoprotein-based assays. These assays will enable
the rapid flock-side screening of infected birds. The diagnostic
assays developed will be for both antigen (virus) detection as
well as for routine screening of antibodies in serum samples.
Minnesota will also continue research on the development of a recombinant nucleoprotein (NP)-based vaccine against avian influenza. Research will be focused on the isolation of stable NP recombinants in herpes virus of turkeys (HVT) and characterizing their genetic and phenotypic properties. This research will be carried out with the ultimate aim of studying the immunogenic properties of this recombinant by vaccinating chickens and turkeys. Various parameters of immune response including humoral and cell mediated immunity will be evaluated and the potential of rHVT-NP to protect against virulent AIV challenge will be investigated.
Minnesota will share with investigators at the other stations reagents and information regarding the use of recombinant NP-based plasmid and viral constructs for the detection and control of avian influenza.
Avian Pneumovirus
Minnesota and Iowa will continue with the development
of rapid molecular diagnostic assays for the avian pneumovirus.
The assays to be developed and refined include both serological
(traditional whole-virus and recombinant fusion-protein based
ELISA assays) and molecular (PCR-based) assays. Analytical and
clinical sensitivities of these assays will be determined, and
the tests employed for the rapid and unambiguous diagnosis of
avian pneumovirus infected flocks. Iowa will work with
the National Veterinary Services Laboratory (NVSL) to evaluate
the new assays for the detection of non-USA (Foreign) APV isolates.
Minnesota and Iowa will also continue to work on developing
vaccines to prevent avian pneumovirus infections in turkey flocks.
The vaccines that will be evaluated include traditional inactivated
virus vaccines as well as recombinant fusion-protein based vectored
vaccines. Iowa has received USDA permission to conduct
in vivo challenge studies using the Colorado strain of APV under
BL3 animal facilities. The challenge studies can be conducted
in the BL3 facilities located in Iowa. Minnesota will provide
scientists at the other stations with primer sequence information
for the detection of the avian pneumovirus and its variants.
Iowa and Ohio will collaborate on development, validation, and use of serologic tests and development of standardized reagents for pneumovirus infections. Since Iowa has already obtained the USDA permit and Colorado APV, Iowa will develop reference antigen and antisera initially to provide to Ohio.
Escherichia coli
Alabama will develop new and improved methods for respiratory
disease diagnosis and attempt to streamline the procedures for
the collection and initial screening of E. coli isolates from
commercial poultry operations. Restriction endonucleases will
be optimized for analysis of poultry isolates using pulsed field
gel electrophoresis. A database will be set up to store restriction
fragment profiles for avian E.coli.
Since E. coli is one of the major concerns of the Michigan poultry industry, Michigan will determine the optimal time for antibiotic administration for the control of postvaccinal E. coli septicemia by infecting 4-week-old turkeys with E. coli to produce postvaccinal E. coli septicemia in an E. coli septicemia model. Appropriate antibiotics will be administered 2 days before infection with E. coli, the day of infection only, at infection, and for 2 days following infection, as well as when clinical signs of illness are noted after infection. Effectiveness of antibiotic timing will be determined by reduction in mortality rate and lesion score. To determine the most effective antibiotic choice, 3 different antibiotics will be used at the appropriate time, as determined in the study above. Effectiveness will be evaluated based on reduction in mortality rate and lesion score.
Minnesota will determine the virulence of common E. coli clones by experimental infection of three-week-old turkeys to test the hypothesis that the ability of a bacterial isolate to cause disease is non-randomly distributed amongst the clones. In addition, Minnesota will attempt transformation of non-pathogenic E. coli (npEc) with the fragment of the ColEl plasmid found to be unique to pathogenic E. coli (pEc). The transformed npEc will be tested for a gain of pathogenicity in order to determine the role and relevance of ColEl fragment as an E. coli virulence factor. Other plans include transformation of npEc with kpsM followed by testing for a gain of pathogenicity and sequencing one of the smaller plasmids of the pEc in order to look for additional virulence-associated genes.
Fowlpox
Since fowl poxvirus has one of the largest viral genomes, it can
accommodate sufficient foreign genetic material to encode for
more than one gene from an avian pathogen(s) and still remain
viable. Illinois will continue to create recombinant fowl
poxviruses expressing such foreign genes. For this purpose, collaboration
will be established with investigators, located at Indiana
and Ohio, who have identified gene(s) that encode for protective
antigens of other avian pathogens. Indiana will collaborate
with Illinois on study of fowl pox as a vector to express
IBDV genes for protection of chickens against challenges with
virulent IBDV. Ohio will provide Illinois with gene
constructs from IBDVs characterized antigenically and immunogenically
for insertion in vectors developed by Illinois for the
production of recombinant vaccines.
Since quail poxvirus does not provide protection against subsequent infection by fowlpox virus, Illinois plans to generate quail pox virus recombinants which can be used in the presence of antibodies recognizing fowlpox virus. Initially, the potential of quail pox virus as a recombinant expression vector for genes from Newcastle Disease and avian influenza viruses will be evaluated. Thus, the continuing molecular studies on avian pox viruses will focus on the use of fowlpox and quail pox viruses as expression vectors for genes from poultry pathogens and on the identification of genes that could be used as insertion sites and/or diagnostic probes.
Infectious Laryngotracheitis
Illinois plans to investigate the feasibility of using
ILTV as an expression vector for genes from respiratory pathogens
in collaboration with investigators at Indiana. Illinois
will continue to use PCR for the differential diagnosis of fowlpox
and infectious laryngotracheitis as well as for the identification
of gene(s) of interest. For the diagnosis of these two avian diseases,
the simultaneous use of pairs of primers specific for either the
genome of fowlpox or infectious laryngotracheitis virus will continue
to be evaluated by Indiana.
Infectious Bursal Disease
Ohio will concentrate on the molecular characterization
and comparison of IBDV serotypes. IBDV strains will be cloned
and the VP2 gene sequenced to develop diagnostic assays. These
tests will employ specific cDNA probes in a dot blot test, a reverse
transcriptase polymerase chain reaction restriction endonuclease
(RT/PCR-RE) assay. The VP2 proteins from IBDV will be expressed
in the baculovirus expression system for the purposes of developing
a highly strain-specific enzyme-linked immunosorbent assay. Recombinant
viruses expressing the VP2 protein from IBDV will be tested for
specific neutralizing epitopes using defined monoclonal and polyclonal
antibodies. Ohio will cooperate with Illinois to express
IBDV genes in avian pox viruses for the purpose of developing
a recombinant vaccine.
Ohio imported highly virulent strains of IBDV from The Netherlands, Turkey and Taiwan. These strains have caused major epidemics in the old world in the last 10 years but have not been detected in the US. The strains will be adapted to tissue culture and chicken embryos and used as inactivated preparations to produce antibodies in chickens. Sera from immunized birds will be used to conduct in vitro cross neutralization studies with variant and classic American strains to establish relatedness values. Chickens will be immunized with the inactivated IBDV and will be challenged with American strains (classic and variant) to study the characteristics of these viruses. Results from the above studies should provide a rational basis for prevention of these infections should they appear in the US at some later time. We compared these strains to the American strains using RT/PCR/RE test and a set of restriction enzymes were identified that distinguished the highly virulent strains from the classic, variant, and serotype 2 American strains.
Indiana will identify and differentiate infectious bursal disease virus (IBDV) field isolates and vaccine viruses by PCR, PCR/sequencing, or PCR/RFLP. In addition to published primers (Wu et. al., 1991; Lin et. al., 1994), new primers flanking the entire VP2 or hypervariable region of VP2 will be designed for PCR amplification, sequencing, or RFLP.
Indiana will improve detection and differentiation of IBDV in poultry flocks by PCR using PCR amplification coupled with IBDV-specific DNA probes in a colorimetric assay.
Indiana will explore the potential use of antisense nucleic acids and ribozymes to inhibit IBDV replication. Antisense nucleic acids and ribozymes targets will be selected and used in vitro transcription and translation. Viral RNA levels will be determined by quantitative competitive PCR and viral proteins will be analyzed by ELISA and western blotting.
Ornithobacterium rhinotracheale (ORT)
Minnesota will develop an ELISA for detection of ORT.
We will examine various fractions of the ORT to determine the
suitable antigen for use in ELISA and develop the assay for use.
ELISA will enhance our ability to detect birds exposed to ORT
in situations where the serum plate test may not be sensitive.
Minnesota will produce a killed or live vaccine for protection against ORT infection. We will produce a killed vaccine and evaluate its ability to protect turkeys against ORT infection. Immune response and length of protection will be investigated. We will continue our studies in the development of a live vaccine through chemical mutagenesis.
Minnesota will fingerprint ORT isolates from different geographic locations. Isolates of ORT will be analyzed by multi-locus enzyme electrophoresis to estimate chromosomal genotypic diversity and relationship among the field isolates.
Minnesota will continue to share reagents and other information regarding the diagnosis and fingerprinting of ORT isolates recovered from diseased poultry with the other stations.
Pasteurella multocida
Vaccines and methods to determine vaccinal immunity to fowl cholera
are being studied at the National Animal Disease Center.
The National Animal Disease Center will evaluate anti-idiotype
vaccines for their ability to induce cross-protective immunity
in turkeys. A blocking ELISA will be evaluated to measure the
immune response of vaccinated turkeys to a cross-protective antigen
of Pasteurella multocida. The Ohio State University
will supply field sera from vaccinated and non-vaccinated commercial
chickens and turkeys to be evaluated in the blocking ELISA test.
The National Animal Disease Center will develop and conduct
the tests and evaluate the serums for passive immune cross-protection.
Minnesota will make available DNA-chips containing the
P. multocida genome for scientists at Indiana and
other stations to better detect and develop methods of
preventing and controlling P. multocida infections in poultry.
Riemerella anatipestifer
The National Animal Disease Center will provide serotyping
and DNA fingerprinting for isolates of Riemerella anatipestifer
for other members of the project. This will depend on outbreaks
in the different states.
Source of Funding for the Proposed Work
| Alabama | Alabama Agricultural Experiment Station Industrial Grant |
| Arkansas | Arkansas Agricultural Experiment Station Arkansas Allied Poultry Industrial Fund |
| Indiana |
Purdue Agricultural Experiment Station Purdue School of Veterinary Medicine Indiana State Value Added Grant USDA-Special Grant |
| Illinois | San Diego Zoo US Poultry and Egg Association Illinois Agricultural Experiment Station. |
| Iowa |
Iowa Livestock Health Advisory Council (ILHAC) Iowa Agricultural Experiment Station |
| Michigan | Michigan State Animal Disease Diagnostic Laboratory
Fund Michigan Allied Poultry Industries Fund |
| Minnesota | Minnesota Agricultural Experiment Station USDA-NRI NIH Industrial Funds and others NVSL NVSL Research Fund |
| Ohio | US Poultry and Egg Association Ohio Agricultural Research and Development Center Agency for International Development |
Dissemination of Research
Findings
Through collaborative effort, this Committee will create a web
site to electronically publish their annual project report (or
an edited version that is more producer-oriented). This site will
be in conjunction with an existing site of the American Association
of Avian Pathologists (AAAP). The NC web page will be at http://www.vetmed.iastate.edu/AAAP/
under the "Other Organizations" section. This page,
commonly used by poultry veterinarians and those involved with
poultry health, makes an ideal place to publish and disseminate
our information to our colleagues and the allied industries.
Minnesota will lead an effort on behalf of the Committee to develop a web site regarding the fingerprints of common avian P. multocida isolates.. Through this web site, investigators at the other station can access information and analyze the similarities and differences.
This Committee will develop extension publications on avian respiratory diseases every two years. This will be accomplished by the collaboration of members with extension responsibilities.
Currently, a symposium on emerging respiratory diseases of poultry, co-sponsored by this Committee and the NCADC Planning Committee will be held simultaneously with the 50th NCADC in Minneapolis, MN in September of 1999. Once approved to be an NC group, it is the Committee's consensus to organize a Symposium on Avian Respiratory Diseases every four years in conjunction with North Central Avian Disease Conference (NCADC) as a mean to bring everyone up to speed with the current developments and research progress.
Other means of information delivery include: an editorial on the pitfalls in interpreting results of molecular testing prepared by members of this Committee
Through the above activities, the most current research information generated as a result of the collaboration of all the members will be disseminated efficiently to peers and the poultry industry in the United State as well as the world.
Collaborative Efforts
We have proposed the formulation of a regional research committee
(program) on avian respiratory diseases in the North Central region
with objectives of stimulating and facilitating co-operative research
among scientists from Experiment Stations at nine State Universities
and the National Animal Disease Center. The proposed research
focuses on what is by far the most important and significant health
problem associated with domesticated and wild birds: respiratory
diseases.
It is important to note that the proposed program involves research that is truly collaborative. The intent is to continue to promote the free-flow of ideas, scientific results, and sharing of resources between individual scientists located in several SAESs (and the NADC) that has been initiated and maintained under the auspices of NCR-191.
As expected for regional research projects of this nature and scope, we have developed a program that will accomplish the following: (i). bring together highly talented and competent scientists from different SAESs and the USDA to work on avian respiratory diseases. This will serve to stimulate the exchange of ideas, research results, and reagents among the co-operating scientists in order to achieve a common goal; (ii). promote team and collaborative efforts to address research problems in avian respiratory diseases that will be too costly or difficult for any single SAES or Government agency to accomplish individually; And, (iii). facilitate, through collaborations and consultation, the free-flow of ideas and sharing of resources and research results, and the rapid implementation and application of research results for the better detection, prevention, and control of avian respiratory diseases.
The table below summarizes the collaborative projects that will be carried out during the proposed project period. Detailed descriptions of the collaborative efforts among all Stations are described in pages 11 to 20.
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| Escherichia coli | Minnesota, Alabama, Michigan | Chickens, Pathogenesis Turkeys, Pathogenesis and surveillance |
| Infectious bursal disease virus | Ohio, Indiana, Illinois | Chicken, genetic variations of strains and molecular basis for increased resistance. Chickens, Immunosuppressive effect of different strains |
| Pasteurella multocida | NADC, Arkansas, Minnesota | Turkeys, Fingerprinting of isolates. Validate tests. Type isolates, Sequence genome, develop vaccines, understand role of host genetics on disease susceptibility |
| Coronaviruses | Indiana, Arkansas | Chickens & Turkeys, Antigenicity and molecular relatedness of isolates |
| Avian Penumovirus | Iowa, Ohio, Minnesota | Chickens & Turkeys Pathogenesis, epidemiology and antigenic relatedness studies. Development of serologic and molecular diagnostic tests. Vaccine development. |
| Avian influenza | Minnesota | Chickens and turkeys. Development of diagnostic tests and vaccines |
| Riemerella anatipestifer | Michigan, NADC | Ducks. Antigenic relatedness. |
| Ornithobacterium rhinotracheale | Minnesota | Interaction with other agents, transmission, surveillance, vaccination |
| Infectious laryngotracheitis | Illinois, Indiana | Use virus as an expression vector |
| Fowl Pox | Illinois, Indiana, Ohio | Recombinant viruses containing foreign genes. Characterization of field isolates from vaccinated birds. |