Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Target audience included researchers from universities and research institutes working on the avian diseases in the USA and international level (China, Taiwan). Science based knowledge was delivered at the annual meeting of American Veterinary Medical Association in USA and lectures on avian diseases and laboratory based techniques in China and Taiwan. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project allowed for the training of graduate students at UConn and lectures on avian diseases and laboratory based techniques in China and Taiwan. How have the results been disseminated to communities of interest? Immunogenicity studies of nanoparticle based vaccine for bird flu was presented at the World Innovation Conference & Expo, June 15-18 2014, Washington, D.C. A talk, "Global impact of avian influenza: Its diagnostic surveillance and preventive approvaches" was presented to poultry industry research workers in China. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective I: Identify reservoirs of infectious respiratory disease agents in wild birds and poultry. Isolation and characterization of avian influenza viruses (AIV) from wild birds and commercial poultry flocks which include live bird markets and backyard flocks were accomplished. The surveillance data obtained from different states (AL, CT, DE) were shared. No AIV activity using USDA NAHLN-approved agent detection (real time RT-PCR and antigen capture on oropharyngeal swabs) were seen in commercial flocks (CT, DE). Objective II. Develop improved diagnostic capabilities including real-time PCR as well as other rapid on-farm tests for economically important respiratory diseases. CT in collaboration with Guangxi Veterinary Institute, China developed loop-mediated isothermal amplification (LAMP) assays to detect the H3 subtype AIVs visually and rapid detection of group I avian adenoviruses. The newly developed H3-RT and group I avian adenoviruses LAMP assays are simple, sensitive, rapid and can identify H3 subtype AIVs and group I avian adenoviruses visually. Consequently, they will be very useful screening assays. Objective IV. Develop new prevention and control strategies for poultry respiratory diseases. CT evaluated the level of protection of M2e-nanopartle based vaccine using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Li,J., P.Burkhard, J. Gelb, M.I. Khan. Immunogenicity studies of nanoparticle based vaccine for bird flu. World Innovation Conference & Expo, June 15-18, 2014 Washington, D.C.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Mazhar I. Khan, Jianping Li, Sharareh Emadi, Anmin Tan, Peter Burkhard, Brian S. Ladman, Jack Gelb, Jr. Immunogenicity Sutdies in Nanoparticle Vaccine constructs for avian influenza virus. Proceeding AVMA Annual Convention, July 25-29, 2014. Denver, CO,
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zhixun Xiea, Sisi Luoa, Liji Xiea, Jiabo Liua, Yaoshan Panga, Xianwen Denga, Zhiqin Xiea, Qing Fana, Mazhar I. Khanb. Simultaneously Typing Nine avian respiratory pathogens by a novel GeXP Analyzer-Based Multiplex PCR Assay. J Virol Meth 207: 188-195
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Target audiences includeresearchers from the universities and research institutesworking onavian diseasesin the USA and international level (China, Pakistan).Delivered science-based knowledge at the International symposium on bioinformatics Research and Application in USA and Mycoplasma workshop in Pakistan and lectures on avian diseases and laboratory based techiniquesinstructions in China. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project allowed for the training of graduate students at Uconn and lectures on avain diseases and laboratory based techiniques in China. How have the results been disseminated to communities of interest? Results published in the Journal of Animal and Plant Sciences and presented at the International Symposium on Bioformatices Research and Application,a Mycoplasma workshop in Pakistan, and lectureson avian diseases and laboratory techiniques in China. What do you plan to do during the next reporting period to accomplish the goals? Work Planned for next year High Path challenge study will be performed at the UDEL biosecurity containment facilityafter selection from the immunogenicity studies for the suitable nanoparticle construct. Development of rapid diagnostic tests for respiratory diseases. Developing of Bioinformatics tools for viral quasispecies reconstruction from next generation sequencing and vaccine optimization
Impacts
What was accomplished under these goals?
Objective I. Identify reservoirs of infectious respiratory disease agents in wild birds and poultry Avian influenza (AI) surveillance in backyard and commercial poultry flocks in New England states were conducted at the Connecticut Veterinary Medical Diagnostic Laboratory, Department of Pathobiology and Veterinary Science. We have used real time RT-PCR assays specific for AI matrix, H5 and H7 genes on tracheal swab samples of 2014 from LBM birds, 244 birds from domestic poultry and 14 from wild birds. The real time RT-PCR assays was unable to detect AI matrix, H5 and H7 specific genes from LBM and domestic birds. However, fewer than 5 samples from wild birds were PCR positive for AI matrix gene but were negative for H5 subtypes gene. 5200 serum samples from domestic and commercial poultry were tested using AI-AGID mostly from CT, MA and some RI. No significant AI-AGID positive samples were identified. Summary/Impact: Avian influenza subtype H5 and H7 were negative from the LBM and domestic poultry birds in New England states. There is a need to perform virus isolation studies to confirm and identify other subtypes in LBM, domestic and wild birds. Objective II. Develop improved diagnostic capabilities including real time PCR as well as other rapid on-farm tests for economically important respiratory diseases. GeXP-multiplex PCR-assay for simultaneous differentiation of avian influenza subtypes H5, H7, H9 and six chicken respiratory pathogens (Collaborative work in China) A new, rapid, and high-throughput Gene Expression (GeXP)-multiplex PCR method was developed for simultaneous detection and differentiation of avian influenza subtypes (H5, H7, H9) and six avian respiratory pathogens. Respiratory pathogens included in study were Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Infectious laryngtracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Haemophillus paragallinarum (HPG). Ten pairs of primers were designed according to the conserved and specific sequences of genes of AIV subtypes and respiratory pathogens from the GenBank. Single and mixed pathogens cDNA/DNA templates were used to evaluate specificity and accuracy of the GeEX-multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The specific DNA product amplification peaks were observed on the GeXP analyzer: The specific DNA product amplification peaks of AIV subtypes and six respiratory pathogens were observed. No amplification of DNA products peaks on non-respiratory pathogens was observed. The detection limit of GeXP-multiplex assay was 100 copies/μL with 10 pre-mixed plasmids containing known target genes of respiratory pathogens. All known positive plasmids and reference cDNA/DNA of respiratory pathogens were tested positive , GeXP-multiplex PCR assay provide new tool for simultaneous detection and differentiation of important avian influenza subtypes (H5, H7,H9) and six avian respiratory diseases pathogens. Therefore, GeXP-multiplex PCR described here will be very useful for differentiation and diagnosis AIV subtypes and of avian respiratory diseases in one test. Summary/Impact: The newly developed GeXP-multiplex PCR-assay for simultaneous differentiation of avian influenza subtypes H5, H7, H9 and six chicken respiratory pathogens is simple, sensitive, rapid and can identify subtypes of AIVs and 6 avian respiratory pathogens. Consequently, it will be very useful screening assay. Objective III. No work was assigned. Objective IV. Develop new preventive and control strategies for poultry respiratory diseases We have designed novel vaccine constructs representing HA cleavage, B cell epitope, and M2e (in the core) genes as a monomeric forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with these constructs. Specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased IgG responses for each of the constructs as compared to a non-vaccinated group. Summary/Impact: The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against AI. Further studies are in progress. High path challenge studies will be carried out at the University of Delaware's BSL III animal containment facilityin near future after selection of the nanoparticles construct with higher immunogenicity and neutralizing antibody responses.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Tork, B., A. Zelikovsky, I. Mandoiu, E. Nenastyeva, A. Artyomenko, R. ONeil, M. I. Khan and N. Mancuso. Reconstruction of infectious bronchitis virus quasispecies from NGS Data. Proc. 9th International Symposium on Bioinformatics Research and Application. Charlotte, North Carolina, May 20-22, 2013. P20-23.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Gondal, M. A., M. Rabbani, K. Muhammad, T. Yaqub, M. E. Babar, A. A. Sheikh, A. Ahmad, M. Z. Shabbir and M. I. Khan. Antibodies response of broilers to locally prepared oil based Mycoplasma gallisepticum vaccine. Journal of Animal and Plant Sciences, 23(4). 1094-1098. 2013.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Objective I. Identify reservoirs of infectious respiratory disease agents in wild birds and poultry. Avian influenza (AI) surveillance in backyard and commercial poultry flocks in New England states were conducted. We have used real time RT-PCR assays specific for AI matrix, H5 and H7 genes on tracheal swab samples from live bird market (LBM) birds, backyard poultry and wild birds. The real time RT-PCR assays was unable to detect AI matrix, H5 and H7 specific genes from LBM, backyard and wild birds. Objective II.Development of loop-mediated isothermal amplification (LAMP) assay for detection of Mycoplasma gallisepticum (MG). MG infection is commonly designated as chronic respiratory disease in chickens and as infectious sinusitis in turkeys. Diagnosis of MG infection is based on agglutination reactions, ELISA and PCR assays. These tests are mainly performed under ideal and sophisticated laboratories. Recently, reported a novel nucleic acid amplification method termed LAMP, which can amplify DNA under isothermal conditions with high specificity, efficiency, and speed. The most significant advantage of LAMP is its ability to amplify specific DNA sequences at 63 -65 C without thermo cycling. LAMP assay for the detection of MG was developed. Cytadhesion gene of MG strain was used for generating primers for LAMP assay. This LAMP assay was able to detect MG DNA at the level of 10 pg. Further studies are under way to optimize and tests its specificity. Objective IV. Develop new prevention and control strategies for poultry respiratory diseases. Bioinformatics Methods for Reconstruction of Infectious Bronchitis virus(IBV) Quasi-species from Next Generation Sequencing Data. Viral infections cause a significant burden on animal health, reducing yields and increasing production costs due to expensive control programs. Vaccination is a vital part of control programs; however, its effectiveness is reduced by the quick evolution of escape viral quasi-species in animal hosts. Existing techniques for studying quasi-species evolution and response to vaccines have severely limited sensitivity and often require prior knowledge of sequence polymorphisms. By generating millions of short reads per run, with no need for culture or cloning, next-generation sequencing (NGS) technologies enable comprehensive identification of viral quasi-species infecting an animal. However, analysis of NGS data is challenging due to the huge amount of data on one hand, and to the short read lengths and high error rates on another. To address these shortcomings we have developed computational methods for reconstructing quasi-species sequences and estimate their frequencies from both shotgun and amplicon NGS data. Preliminary analysis of 454 sequencing reads generated from synthetic pools of infectious bronchitis virus (IBV) clones and field isolates collected at various intervals after vaccination with attenuated live IBV vaccine has defined the specific area where mutation occurs frequently at the S1 gene target. Further sequencing data are being generated using field samples from the IBV vaccinated flocks at various stages of production. PARTICIPANTS: 1) Mazhar I. Khan, PhD, Professor, supervise the research program. 2) Dr.Hongjun Wang,and Qianlian Su were responsible for isolation and propagating the IBV field isolates and amplifying the S1 genes. 3) Connecticut Veterinary Medical Diagnostic laboratory (CVMDL)were instrumental in testing the field samples for the avian influenza surveillance program. TARGET AUDIENCES: Research findings were presented at the regional and international conferences. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1). Avian influenza subtype H5 and H7 were negative from the LBM and domestic poultry birds in New England states. There is a need to perform virus isolation studies to confirm and identify other subtypes in LBM, domestic and wild birds. 2). LAMP assay will provide rapid tool to identify MG infection without the use of sophisticated and expensive thermal cycler. 3). We will be able to define the quasispecies phenomenon in the IBV strains occurring in the field. This identification and characterization will be helpful for designing the better vaccines against IBV infections for poultry.
Publications
- Rabbani, M., Muhammad, K., Sheikh, A.A., Ahmad, A., Ahmad, M., Khalid, R.K., Muhammad,J., Asim, M., and Khan, M.I. 2012. Isolation and identification of Mycoplasma gallisepticum from commercial poultry flocks in Pakistan.19th Congress of the International Organisation of Mycoplasmology IOM, Toulouse, France, July 15-20, 2012. Poster,68.
- Almeida D O, Tortelly, R, Nascimento E R,Chagas M A, Khan M I, Pereira V L. 2012. Avian infectious bronchitis and deep pectoral myopathy--A case control study. Poult Sci. Dec;91(12):3052-6.
- Mandoiu I., O'Neill, R., Khan, M.I., Zelikovsky, A., Tork*, B., Mancusoet, N. 2012. Bioinformatics methods for reconstruction of infectious bronchitis virus quasispecies from next generation sequencing data. 11th International Conference on Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases, October 30 - November 2, 2012. New Orleans, USA. 59.3.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Objective II. Develop improved diagnostic capabilities including real time PCR as well as other rapid on-farm tests for economically important respiratory diseases.Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect the H3 subtype AIVs visually. Specific primer sets target the sequences of the hemagglutinin (HA) gene of H3 subtype AIVs were designed, and assay reaction conditions were optimized. The established assay was performed in a water bath for 50 minutes, and the amplification result was visualized directly as well as under ultraviolet (UV) light reflections. The detection limit of the RT-LAMP assay was 0.1pg total RNA of virus, which was one hundred-fold higher than that of RT-PCR. The results on specificity indicated that the assay had no cross-reactions with other subtype AIVs or avian respiratory pathogens. Furthermore, a total of 176 clinical samples collected from birds at the various live-bird markets (LBMs) were subjected to the H3-subtype-specific RT-LAMP (H3-RT-LAMP). Thirty-eight H3 subtype AIVs were identified from the 176 clinical samples that were consistent with that of virus isolation.Objective IV. Develop new preventive and control strategies for poultry respiratory diseases. We have designed two novel vaccine constructs representing M2e in monomeric (Mono-M2e) and tetrameric (Tetra-M2e) forms. Groups of specific pathogen free (SPF) chickens were immunized intramuscularly with Mono-M2e or Tetra-M2e with and without an adjuvant. Two weeks after the second boost, chickens were challenged with 107.2 EID50 of H5N2 low pathogenicity avian influenza (LPAI) virus. M2e-specific antibody responses to each of the vaccine constructs were tested by ELISA. Vaccinated chickens exhibited increased M2e-specific IgG responses for each of the constructs as compared to a non-vaccinated group. However, the vaccine construct Tetra-M2e elicited a significantly higher antibody response when it was used with an adjuvant. On the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. The level of protection was evaluated using quantitative real time PCR at 4, 6, and 8 days post-challenge with H5N2 LPAI by measuring virus shedding from trachea and cloaca. The Tetra-M2e with adjuvant offered statistically significant (P < 0.05) protection against subtype H5N2 LPAI by reduction of the AI virus shedding PARTICIPANTS: Objective II. Yi Peng,Zhixun Xie, Jiabo Liu, Yoshan Pang as a collaborators, Guangxi Veterinary Research Laboratory. Objective IV. Sankhiros Babapoor, Toby Neef, graduate students, University of Connecticut Peter Burkhard,Collaborator, University of Connecticut TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective II. The newly developed H3-RT LAMP assay is simple, sensitive, rapid and can identify H3 subtype AIVs visually. Consequently, loop-mediated isothermal amplification (LAMP) is cost-effective nucleic acid amplification method that does not require any specialized equipment. Therefore, its application will be suitable at farm setting laboratories. Objective IV. The self-assembling polypeptide nanoparticle Tetra-M2e with adjuvant offered protection against subtype H5N2 LPAI by reduction of the AI virus shedding. The results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against high pathogenic AI.
Publications
- Peng Y, Xie Z, Liu J, Pang Y, Deng X, Xie Z, Xie L, Fan Q, Feng J, Khan MI. Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay. Virol. J. 8:337-341. 2011
- Babapoor, S., T. Neef, C. Mittelholzer, T. Girshick, A. Garmendia, H. Shang, M.I. Khan and P. Burkhard. A novel vaccine using nanoparticle platform to present immunogenic M2e against avian influenza Infection. Influenza Research & Treatment. Volume 2011, Article ID 126794, 12 pages. 2011. doi:10.1155/2011/126794.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: (1) Primer design tool for PCR-based virus subtype identification.Primer Hunter is a primer design tool for PCR-based virus subtyping. Previously we have validated the efficiency of Primer Hunter to design the real time RT-PCR (RRT-PCR) primers for hemagglutinin (HA) subtyping of Avain Influenza Virus (AIV). In this study, nine pairs of neuramidinase (NA) subtype-specific primers were designed and successfully used in real time RT-PCR with four primer-pool reactions to differentiate nine NA subtypes of AIV. The RRT-PCR assays are sensitive and can detect in vitro transcribed RNA of different NA subtypes ranging from 176 to 4000 copies per reaction, or 2-30fg of total RNA of AIV per reaction. The assays possess good specificity. There was no cross reaction detected between different NA subtypes in RRT-PCR with each subtype-specific primers. No amplification was identified when RNA of IBV, IBDV, NDV were tested with the primer pools. This study validated further the powerful function of Primer Hunter for the design of subtyping primers and also introduced a sensitive and specific method for NA subtyping of AIV. (2) Investigating the virucidal potential of caprylic acid, monocaprylin, and sodium caprylate for inactivating avian influenza virus. Avian influenza is an important disease, causing tremendous economic losses to the poultry industry. Further, transmission of the virus to humans highlights the public health significance of the disease. Since the virus is highly contagious, strict hygienic measures employed at the farm can greatly help to contain the virus. Contaminated food, water, and poultry manure are three important vehicles of the virus. Effective and safe antimicrobials that kill the virus in the aforementioned vehicles could greatly reduce spread of the virus. Caprylic acid is a natural fatty acid present in breast milk and coconut oil. From previously published research, it is known that caprylic acid and its other chemical forms, namely monocaprylin and sodium caprylate are highly effective in killing a variety of disease causing bacteria and viruses. In this study, low pathogenic AI viruses H5N1 and H5N2 were subjected to caprylic acid, sodium caprylate and monocaprylin. Concentrations of viruses were determined using TCID50 in the cell culture. Plaque assays were optimized and 100 PFU viruses were used to infect chicken embryo kidney cells. Serial dilutions of fatty acid compounds were made from 0.005% to 0.5% for each virus to observe their virucidal effects in plaque assays. In result, caprylic acid and its derivatives were able to inhibit plaque forming in chicken embryo kidney cells. The minimum inhibitory concentrations were 0.5% for caprylic acid, 0.1% for sodium caprylate and 0.05% for monocaprylin. Using transmission electron microscope, negative staining of avian influenza virus particles treated with 0.5% caprylic acid indicated disruption of the cell membrane and envelope of the viral particle. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Both studies were presented at the international conferences, the target audience were Infectious diseases and bioinformatic researchers. These studies will benefit poultry producers in the control of avian influenza infection in poultry flocks as well as protection of human from infections. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(1) Primer design tool for PCR-based virus subtype identification.This study validated further the powerful function of Primer Hunter for the design of subtyping primers and also introduced a sensitive and specific method for NA subtyping of AIV. In Conclusions: The primer pool test described in this study is a better choice for NA subtyping than using each NA-specific primer. Compared to the latter, primer pool method is labor-saving since it decreases the amount of reactions needed to differentiate NA subtypes from 9 to 4.(2) Investigating the virucidal potential of caprylic acid, monocaprylin, and sodium caprylate for inactivating avian influenza virus: In conclusion, low concentration of caprylic acid and its derivatives in-vitro can reduce or inhibit avian influenza virus. Therefore, caprylic acid and its derivates have the potential to be effective and safe antimicrobials that can be used in poultry feed in order to reduce transmission to human.
Publications
- Hariastuti,N.I.,Babapoor,S.,Grishick,T.,and Khan,M.I.(2010).In-vitro inactivation of avian influenza viruses using caprylic acid and its derivatives.Proceed 14th International Congress on Infectious Diseases, Miami, Florida, March 9-12,2010,CDROM.
- Huang,Y.,Khan,M.I., and Mandoiu,I.I. (2010). Development of real time RT-PCR assays for neuraminidase subtyping of avian influenza virus.Proceed 6th International Symposium on bioinformatics research and applications. Storrs, Connecticut. May 23-26,2010. P19.
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