Progress 10/01/10 to 09/30/14
Outputs
Target Audience:National and international poulty health scientists (industrial, government, and univeristy), and commercial poultry industry and Pharmaceutical employees and farmers. I have a web page on LinkedIn, a professional website. I have over 2,000 contacts from around the world that occasionally contact me with poultry health questions. On this site I follow over 50 poultry and allied industry related companies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Two commercial CEO vaccines (Trachivax and Laryngovac) were propagated in 10-day old SPF embryonating eggs. Viral DNAs were extracted from the harvested CAMs and the gB gene amplified by PCR using Phusion Flash High-Fidelity PCR Master mix and specific primers. PCR products (~3 Kb) were gel purified using Wizard PCR Preps DNA Purification System. T-vector ligation (pGEM-T Easy Vector System) of purified DNA were carried out and subsequently transformed in NEB 5- alpha Competent E. coli. Six clones from each transformation LB-agar plate of the 2 vaccines were picked and cultured for screening and identification of recombinants (ILTV-gB positive). Plasmids were extracted and run on a gel. Bands of about 6Kb were identified and presumed to be the T-vector (3Kb) containing the ILTV-gB (2.9 Kb) insert. Four purified recombinant plasmids were submitted for sequence analysis at the Auburn University Genomics & Sequencing Laboratory and are we anxiously waiting for these significant results. We are encouraged by our progress so far and have made great strides in meeting our objective to make an improved vaccine against ILT, which will aid the industry in the prevention of ILT.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Poultrydisease researchers, commercial and academic poultry health experts, commercial poultry live producion managers, poultry farmers, and the the general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A commerical poultry farmer in North Alabama was shown the proper way to clean and disinfect a house to prevent reoccurance of infectious laryngotracheitis virus (ILTV) disease on his farms. Two graduate students and one laboratory technician were shown how to take samples for ILTV detection on a farm. One undergradute student was shown how toisolate ILTV from farm samples and then use a molecular techniques to quanitate the virus. One undergraduate student and one laboratory technician were shown how to make a transgenic plant vaccine. How have the results been disseminated to communities of interest? One paper has been published in a refereedscientifc journal, one in an Alabama State Poultry magazine, one in a popular over the counter magazine for the general public, and twopapers were presented at internationalmeetings. What do you plan to do during the next reporting period to accomplish the goals? We will develop a recombinant vaccine against ILT using a live Newcastle Disease vaccine virus (NDV) as a vector. The immunogenic gene of ILTV, glycoprotein B (gB) will be cloned and inserted into the hemagglutinin (HN) gene of NDV. The pcDNA-HN-gB plasmid will be co-transfected with NDV whole genome RNA in fibroblast cell culture. The recombinant vaccine will be screened by plaque assay, amplified, and then purified. When optimum expression of rNDV-gB is achieved, its safety and ability to induce protection against challenge with virulent ILTV will be done in experimental studies in broilers having maternal antibody against both NDV and ILTV. Protection afforded by our vaccine will be evaluated and compared against currently available commercial ML and recombinant vaccines.
Impacts
What was accomplished under these goals?
A broiler farm in North Alabama suffered a mild infectious laryngotracheitis (ILT) outbreak as determined by clinical disease and the polymerase chain reaction. The poultry integrator sought help to control further outbreaks in subsequent flocks. Samples were collected from various areas of the poultry houses on the farm over an 8-week period. The first sampling was conducted 9 days after the infected farm was depopulated; the second was 2 days prior to subsequent flock placement; and the third was when the new flock was 5 weeks of age. Samples were examined for ILTV DNA by real-time PCR (rtPCR) and virus isolation in embryos. The infected houses were cleaned, disinfected, heated, litter composted, and curtains replaced after the first sampling and prior to placement of the next flock. Samples from all periods were positive for ILTV DNA. However, the number of positive samples and crossing point (Cp) values indicated a decrease in the amount of viral DNA, while virus isolation in embryos was successful only on the first sampling. The subsequent flock was vaccinated against ILTV by in ovo route using a commercial recombinant vaccine. Cleaning and sanitation after the disease outbreak reduced the amount of ILTV on the farm and together with in ovo vaccination of the new flock may have prevented a recurrence of another ILT outbreak. The HA gene of an H1N1 avian influenza virus (AIV) was expressed in a plant (Arabidopsis thaliana). Protein from the plant leaves was used to immunize SPF chickens. Birds given the transgenic vaccine had HI and ELISA antibody titers against AIV; and similarly upregulated levels of cell-mediated immune cytokines as birds given the commercial vaccine. All vaccinated birds had reduced viral shedding after challenge. Transgenic edible grains could be developed into vaccines and mixed in feed to immunize birds
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Dormitorio, T. V., K. S. Macklin, and J. J. Giambrone, 2013. Detection
and isolation of an infectious larygotracheitis virus on a broiler farm after a disease outbreak. Avian Dis. 57:803-807.
- Type:
Other
Status:
Published
Year Published:
2013
Citation:
Hess, J. B., K.S. Macklin, and J. J. Giambrone. 2013. Alabama Poultry.
Reducing disease-proper management and antibodies: a growers
guide. May/June/pp. 24-25
- Type:
Other
Status:
Published
Year Published:
2013
Citation:
Giambrone, J. J. and H. Sellers. 2013. AMI magazine. A virus is
causing trifus in chickens, is there a solution in the Verizon? March 6/
pp. 2 4-25.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Dormitorio, J. J. Giambrone, and K. S. MacKlin. 2012. Detection of
infectious laryngotracheitis virus from poultry house environments. PSA Annual Meeting. Athens, Ga. July 9-12.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Giambrone, J. J., C. Barnes, N. Singh, H. Wu, and N. K. S. Gunn. 2013.
Transgenic plant vaccine against avian influenza. WVPSA, Nantes, France. August 19-23.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Infectious laryngotracheitis (ILT) remains an important viral (V) respiratory disease of poultry, despite the development of new recombinant vaccines and improved management practices. Heating the house at 100 deg F for 3 days, cleaning, disinfection, biosecurity, and litter treatments are all important steps to elliminate the virus from an infected poultry house. Continuous surveillance and characterization of ILTV's from poultry house environments would help in the understanding of the origin, evolution, transmission and control of present and future ILTV outbreaks. To that end we performed surveillance of ILTV and examined the effect of cleaning and disinfection on a farm in North Alabama. This farm was identified to have a mild ILT outbreak as determined by the Alabama State Veterinary Diagnostic laboratory. Eight days after the broilers from the farm were processed; we obtained swab samples from the water lines, brooders, house curtains, mud outside the doors, beetles, and stray non-caught chickens outside the house. We took the samples back to our laboratory to determine if they contained ILTV DNA using real-time (RT) polymerase chain reaction (PCR). All positive samples were examined for live virus by isolation in embryos. Embryos showing lesions indicative of ILTV were again tested for ILTV DNA using the RT-PCR test. After the flock was processed, the litter was composted and all equipment was cleaned and disinfected. We again took samples from the cleaned house before the house was repopoulated with chicks from the same sights as before to determine if the cleaning and disinfection process was successfull in elliminating live ILTV. PARTICIPANTS: The following individuals helped in obtaining the samples from the farm: Dr. Ken Mcklin (an Associate Professor), Teresa Dormitorio (a laboratory technician 5, Amnuay Segrest (a laboratory technician 2) and Matthew Bailey (a MS candidate). Teresa Dormitorio performed all the RT-PCR and viral isolation work. TARGET AUDIENCES: Our work was the first to show the presence of live ILTV in the curtains and brooders in a poultry houses, and mud outside the houses. We are the first to show that windrow composting and the cleaning and disinfection techniques, which are commonly used on farms after a disease outbreak, completely elliminated ILTV from the farm. This knowledge is important for poultry health experts and poultry farmers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Samples from all sights were positive for ILTV DNA by RT-PCR. Beetles and stray chickens had the highest viral DNA concentration based on Cp value. All RT-PCR+ samples were determined to contain live virus. All samples taken after cleaning and disinfection of the houses and all where RT-PCR negative. Some of these samples were also tested for virus isolation in embryos. None of the samples contained live virus. Therefore, the management methods initiated after processing were succesful in removing ILTV from the houses. The newly placed chicks were also vaccinated in the hatchery using a new recombinant vaccine by in ovo route. This flock did not break with ILTV. The combination of cleaning and disinfection and a new vaccine prevented the return of clinical ILT in this flock. Continuous surveillance and characterization of ILTV's from poultry house environments would help in the understanding of the origin, evolution, transmission and control of present and future ILT outbreaks. Composting litter, a thorough cleanout out and disinfection of a house, and possibly the use of commercial recombinant vaccines given in ovo will reduce the incidence and severity of subsequent ILTV outbreaks.
Publications
- Ou, Shan-Chia, J. J. Giambrone, and K. S. Macklin. 2012. Comparison of a TAQMAN real Time PCR with a loop-mediated isothermal amplication assay for detection of infectious laryngotracheitis. J. Vet.Biotech research. 3:27-36.
- Ou, Shan-Chia, J. J. Giambrone, and K. S. Macklin. 2012. Detection of infectious laryngotracheitis virus from darkling beetles and their immature stage (lesser mealworms) by quantitative polymerase chain reaction and virus isolation. J. Appl. Poult. Res. 21:33-38.
- Gunn, N. K. S., N. Singh, J. J. Giambrone, and H. Wu. 2012. Using transgenic plants as bioreactors to produce bioreactors to produce edible vaccines. Journal of Biotech.4:92-99.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: A simple, rapid, and sensitive method to detect avian influenza virus (AIV) is needed. Cloacal swabs were collected from hunter-killed ducks in wildlife refuges in the SE USA. Swabs were inoculated into embryonated eggs followed by hemagglutination (HA) test. Out of 229 swabs, 23 were HA positive (HA+). Rapid Immuno-Migration (RIM) based commercial kit and real-time RT-PCR (RRT-PCR) were used for detection of AIV from both allantoic fluids (AF) and swab specimens of HA+ samples. The RIM kit did not detect AIV directly from swabs, whereas RRT-PCR detected 16 out of 23. RRT-PCR showed 23 AIV positives on AF samples in contrast to 8 by the RIM kit. The RIM kit was not as good as RRT-PCR in detecting AIV AFs samples. Nevertheless, the RIM kit is rapid and simple, can be stored at room temperature, there is no need for expensive equipment or technical expertise. Four of our AIV isolates were of the H1N1 subtype using the HA inhibition test and sequence analysis. Phylogenetic comparison of the HA gene of these isolates revealed that they were 92-97% similar to previously published H1N1 isolates including one, which came from swine. The influenza A viral genome consists of eight (HA, NA, M, PB2, PB1, PA, NP and NS) segments of single-negative strand RNAs. The virus responsible for the 2009 pandemic is a unique reassortant virus containing NA and matrix genes from the Eurasian swine influenza virus lineage and the other 6 gene segments are derived from the North American triple reassortant swine influenza virus lineage. This particular genetic combination of influenza virus segments had not been seen before in the United States or elsewhere. We have determined the sequences of the HA gene of our H1N1 viruses using PCR products. We compared the HA genes of these isolates with that of several pandemic strains and they ranged from 65% (A/Catalonia/NS5408/2009-H1N1) to 75% (A/Mexico City/013/2009-H1N1) relatedness. In order to fully characterize our H1N1 isolates, we need to determine the sequences of the rest of the other viral genome segments. We developed 2 AIV vaccines. In the first the HA gene of an AIV subtype HlN1 was expressed in yeast, Saccharomyces pombe, and the recombinant protein used as the vaccine. It was able to immunize chickens when given via the oral route at 1, 7 and 14 days of age. The second AIV vaccine was cloned into a DNA plasmid and the DNA vaccine immunized chickens when given by intramuscular (IM) injection at 1 and 3 weeks of age. Both vaccines induced both humoral and cell-mediated immunity similar to a commercially available killed vaccine, designed for turkey breeders, when given IM at day of age. These vaccines could be farther developed for use in controlling AIV in chickens. Infectious laryngotracheitis (ILTV) is an important disease of chickens. Live ILTV vaccines may cause a mild form of ILTV. Management techniques were developed to reduce the ILTV reservoir viruses from rodents, beetles, and drinking waterers. A new RT-PCR and loop-mediated isothermal amplification assay (LAMP) were developed and were specific, sensitive, and reproducible for ILTV detection. PARTICIPANTS: Collaborators: Narendra Singh of Biological Sciences Department, Auburn University and Hongzhuan Wu of the Department of Biological Sciences, Alabama State University (AIV vaccine). Drs. Wu and Singh developed the yeast derived and DNA vaccines. Dr. Giambrone tested the vaccines in chickens. All 3 scientists had graduate students involved in the study. Dr. Giambrone used money from and an AEES grant to fund the work on the surveillance and testing of AIV methods for the detection in wild ducks. Dr. Macklin of Auburn University's Poultry Science Department and Drs. Fred Hoerr and Lanqing Li of the Alabama State Veterinary Diagnostic Laboratory helped with the ILTV work in Alabama Poultry flocks. The diagnostic lab determined which flocks had field cases of ILTV. Dr. Macklin helped developed techniques for prevention of ILTV in commercial farms. TARGET AUDIENCES: Veterinarians from commercial poultry companies need to know if there are AIVs in wild birds and swine farms close to their facilities, which may represent a threat to their flocks. New AIV vaccines need to be developed for use in foreign countries to prevent the spread of AIV to US poultry flocks. Live production managers need to know if there can be live ILTVs in the drinking water, beetles, rats, dust, and litter on their farms so that they can make appropriate plans to clean and disinfect waterers, floors, walls, and compost the litter, and rid their farms of external parasites. PROJECT MODIFICATIONS: Clones containing the neuraminidase (NA) gene of our H1N1 isolate have already been produced and gene sequence determined, but not yet analyzed. We propose to take environmental samples from inside and outside ILTV positive farms. The positive farms to be tested will be commercial poultry farms that have been confirmed positive for ILTV by the Alabama State Veterinary Diagnostic Laboratory. Samples to be taken from outside the house will include: beetles and rodents (if possible), cool cell (pad and water), and from the inside of the house; feathers, dander, fan louvers, litter, soil, and drinkers. Samples will be taken from these ILTV positive houses before and after cleaning and disinfection. Samples will be taken back to our lab to be tested for ILTV DNA with real time-PCR. If RT-PCR positive the same samples will be used to infect chicken embryos to determine if the virus is viable. Ideally three ILTV positive farms from different complexes in Alabama will be sampled. A transgenic plant vaccine expressing the HA protein of Avian influenza virus in Arabidopsis thaliana, a laboratory plant, will be developed. We proposal to orally immunize chickens against AIV using this transgenic plant vaccine.
Impacts
Objective 1. Wild ducks carry AIVs and orthomyxoviurses. Water fowl should continue to be tested for these viruses. Continuous surveillance and characterization of AIVs from its natural reservoir would help in the understanding of the origin, evolution, transmission and control of present and future human influenza outbreaks. Objective 2. We developed two AIV vaccines against the H1N1 virus. The first vaccine was cloned in the yeast Saccharomyces pombe and the recombinant protein was able to to immunize chickens. The second vaccine was cloned into a DNA plasmid and the DNA vaccine was also able to immunize chickens. Both vaccines could be farther developed and used as commercial products. Safer AIV vaccines need to be developed to immunize poultry in foreign countries where AIV is endemic, otherwise the viruses, if left unchecked could spread to US poultry flocks. Objective 3. New management techniques were developed to eliminate the ILTV reservoir viruses from rodents, beetles, and drinking waterers. A new RT-PCR and loop-mediated isothermal amplification assay (LAMP) were developed and were specific, sensitive, and reproducible for ILTV detection. This data can provide information on evolution, persistence, and elimination of ILTV in flocks.
Publications
- 1) Ou, Shan Chia. J. J. Giambrone, and K. S. Macklin. 2011. Infectious laryngotracheitis vaccine virus detection in water lines and effectiveness of sanitizers for inactivating the viruses. J. Appl. Poul. Res: 20:223-230.
- 2) Hongzhuyan,Wu., K. Williams, S. R. Singh, K. S. Gunn, N. K. Singh, T. Shan-Chia-oh, and J. J. Giambrone. 2011. Yeast derived avian influenza virus hemagglutinin protein induced immune response in SPF chickens. J. of Animal Sci. and Vet. Advances 10 (8) 999-1002.
- 3) Guo, K, T. Dormitorio, Shan-Chi OU, and J. J. Giambrone. 2011. Development of Taqman real-time RT-PCR for detection of avian reoviruses. J. Virol. Meth.:117:75-79.
- 4)Hongzhuyan, Wu, K. S. Gunn, S R. Singh, N. K. Singh, Shan-chia Oh, and J. J. Giambrone. 2011. A DNA vaccine against an H1N1 avian influenza induced humoral and cell-mediated in SPF chickens. J. of Biotech Research. 3:27-36.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Isolation and characterization of 6 avian influenza viruses (AIVs) from at total of 150 fecal swabs from wild water (hunter killed) ducks from Lake City in Florida was done. All were propagated in embryonating specific pathogen free chicken eggs, followed by hemmagglutination (HA) testing. HA positive, Newcastle disease HI negative, viruses were detected by Real Time (RRT)-polymerase chain reaction (PCR) and a new commercial antigen capture enzyme linked immunosorbent assay (AC-ELISA) (Synbiotics) for AIV. RRT-PCR used both matrix (+), and H5 and H7 probes (-). None were positive for H5 or H7. Detection of infectious laryngotracheitis viruses (ILTVs) from commercial farms was done. Swabs were from dust, drinking waterers, litter, beetles, and a live rat. Detection was by viral isolation in embryos followed by RRT-PCR. PCR- RFLP tests should they were related to vaccines. Chicken embryo origin (CEO) ILT vaccine virus attached to biofilm in the drinking water system and be released through the water to infect chickens. Real-time PCR polymerase reaction (RT-PCR) detected ILTV DNA and live virus were isolated in chicken embryos from water, containing biofilm, and tracheal samples from chickens, which drank the vaccine. We evaluated a portion of the S1 gene sequence of the dominant genotype/phenotype of an infectious bronchitis virus (IBV) Ark vaccine strain during passage in chickens infected with the immunosuppressive viruses chicken anemia virus (CAV) and/or infectious bursal disease virus (IBDV) as well as in immunocompetent chickens. The IBV-Ark vaccine was applied ocularly and tears were collected from infected chickens for ocular inoculation for the next passage. IBV was maintained through 9 passages in CAV-infected chickens, whereas it was lost by the 3rd or 4th passage in the other groups, consistent with the importance of T cell responses for IBV clearance. The dominant genotype in the vaccine strain was rapidly negatively selected in all chicken groups (CAV, IBDV, CAV+IBDV, Immunocompetent). Distinct vaccine subpopulations were selected during passage in immunodeficient and immunocompetent chickens. In two independent experiments, one specific viral subpopulation was associated with IBV being maintained through the highest number of passages in immunodeficient chickens. Results suggested that immunodeficiency caused by ubiquitous immunosuppressive viruses can have an effect on evolution and persistence of IBV in flocks. We vaccinated chickens with 3 doses of IBV-Ark vaccine over a 200-fold range and determined viral load in tears by qRT-PCR and vaccine subpopulation by S1 gene sequencing in samples from individual chickens. The incidence of selection of vaccine subpopulations differed among groups. Within a group, viral loads between chickens harboring different vaccine subpopulations differed up to 100-fold. An interpretation is that specific vaccine subpopulations of higher abundance, but lower fitness, were selected in chickens inoculated with the lowest dose, while, those more abundant subpopulations were out-competed by specific rarer, more fit subpopulations in chickens inoculated with higher doses. PARTICIPANTS: Collaborators: K. Macklin (ILTV), Poultry Science Department; Auburn University H. Toro, Vicky van Santen, and F van Ginkle of the College of Veterinary Medicine, Auburn University (AIV, IBV) N. Singh of Biological Sciences Department, Auburn University AIV vaccines Partner organizations: H. Wu of the Department of Biological Sciences, Alabama State University (AIV vaccines); H. C. Tang of Vaxin, Inc. Birmingham Al, (AIV vaccine) Fred Hoerr and Lanqing Li of the Alabama State Veterinary Diagnostic Laboratory Histopathology and Serology D. Suarez SEPRL in Athens, Ga. (AIV), and E. Collison, UC Davis. AIV challenge infections TARGET AUDIENCES: Poultry Health and Disease workers Objective 1. Wild ducks carry AIVs and orthomyxoviurses in Florida. Water fowl should continue to be tested for these viruses. Objective 2. ILTV vaccines are wide spread in Northern Alabama and Georgia, causing a mild form of ILTV. New management techniques should be done to eliminate the reservoir viruses from rodents, beetles, and drinking waterers. A new RT-PCR and loop-mediated isothermal amplification assay (LAMP) were developed and found to be specific, sensitive, and reproducible for ILTV detection. Objective 3. Immunodeficiency caused by ubiquitous immunosuppressive viruses can have an effect on evolution and persistence of respiratory viruses in flocks. Objective 4. We have developed new AIV vaccines, which could be farther developed for use in controlling AIV in chickens. PROJECT MODIFICATIONS: None
Impacts
1) Wild ducks carry AIVs and orthomyxoviurses in Florida. Water fowl should be tested for the viruses. 2) ILTV vaccines are wide spread in Alabama and Georgia, causing a mild form of ILTV. New management techniques should be done to eliminate the reservoir viruses from rodents, beetles, and drinking waterers. A new RT-PCR and loop-mediated isothermal amplification assay (LAMP) were developed and found to be specific, sensitive, and reproducible for ILTV detection. 3)Immunodeficiency caused by ubiquitous immunosuppressive viruses can have an effect on evolution and persistence of respiratory viruses in flocks. 4)We have developed new AIV vaccines, which could be farther developed for use in controlling AIV in chickens.
Publications
- Mesonero, A.*, D. L. Suarez, E. van Santen, D. C. Tang, H. Toro. (2010).Avian Influenza In Ovo Vaccination with Replication-Defective Recombinant Adenovirus in Chickens: Vaccine Potency, Antibody Persistence, and Maternal Antibody Transfer Avian Diseases. Submitted 11/22/2010
- Toro, H,, D. L. Suarez, D. C. Tang, F.W. van Ginkel, C. Breedlove.(2010) Avian Influenza Mucosal Vaccination in Chickens with Replication-Defective Recombinant Adenovirus Vaccine Avian Diseases (in press).
- Toro, H., F.W. van Ginkel D.C. Tang, B. Schemera, S. Rodning, J. Newton (2010). Avian Influenza Vaccination with in chickens and pigs with replication competent adenovirus free human recombinant adenovirus 5. Avian Diseases, Supplement 54: 224-231
- Toro, H. (2010) Infectious Bronchitis Virus: Dominance of ArkDPI-type Strains in the United States Broiler Industry during the Last Decade Brazilian Journal of Poultry Science 12: 79-86
- Singh S*, Toro H., Tang DC, Briles WE, Yates LM, Kopulos RT, Collisson EW. (2010). Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8(+) T lymphocytes.405: 62-9
- Gallardo, R.A.*, F.J. Hoerr, W.D. Berry, V.L. van Santen, H. Toro. (2010.)Infectious Bronchitis Virus in Testicles and Venereal Transmission. Avian Diseases, (submitted 10/29/2010).
- Gallardo, R. A.*, V. L. van Santen, H. Toro (2010). Host Intraspatial Selection of Infectious Bronchitis Virus Populations. Avian Diseases 54: 807-813
- Shan-Chia OH. (2010) Improved detection and control of infectious laryngotracheitis virus on poultry farms. Auburn University PhD Dissertation.
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