Progress 10/01/09 to 09/30/11
Outputs
OUTPUTS: The goal of this project is to further our understanding of avian pathogenic Escherichia coli (APEC) pathogenesis and APEC's role as a primary or secondary pathogen of poultry. Isolates have been collected from cases of turkey colibacillosis to perform virulence factor genotyping to better understand if the APEC involved in systemic colibacillosis in Minnesota turkeys are similar to those classically characterized as APEC. We have also developed in vitro infection models using APEC of varying levels of virulence and turkey and chicken cell lines. We are isolating RNA from this model to compare the transcriptomic response of APEC to host cell exposure, and to identify and compare the transcriptomic response of APEC relative to their virulence capability. We are also defining the baseline transcriptomes of archetypic APEC and their plasmids. PARTICIPANTS: Timothy Johnson, University of Minnesota; Robert Porter, University of Minnesota; Lisa Nolan, Iowa State University; Kakambi Nagaraja, University of Minnesota; Zheng Xing, University of Minnesota TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The determination of APEC's role as a primary vs. secondary pathogen will increase our understanding of the manifestation of colibacillosis in commercial turkeys. Our results thus far suggest that APEC of lower virulence potential require predisposition for disease, such as immunosuppression due to a primary respiratory virus. In contrast, APEC of high virulence potential likely require little or no host immunosuppression enabling them to cause disease. An improved understanding of the APEC populations circulating in turkeys, and their propensity to cause disease with or without predisposing agents, will allow for better control of such diseases in the production setting.
Publications
- Fernandez-Alarcon, C., Singer, R.S., and Johnson, T.J. 2011. Comparative Genomics of Multidrug Resistance-Encoding IncA/C Plasmids from Commensal and Pathogenic Escherichia coli from Multiple Animal Sources. PLoS One 6:e2341.
- Li, G., Tivendale, K.A., Liu, P., Feng, Y., Wannemuehler, Y.M., Cai, W., Mangiamele, P., Johnson, T.J., Penn, C.W., and Nolan, L.K. 2011. Transcriptome analysis of avian pathogenic Escherichia coli O1:K1:H7 in chicken serum reveals adaptive responses to systemic infection. Infect Immun 79:1951-1960.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: 1. Screening of wild and domestic birds populations for avian influenza. Almost all possible subtype combinations of 16 H and 9 N subtypes of avian influenza virus (AIV) have been isolated from domesticated and wild birds. We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. We will continue to use this new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs. 2. Screening of waterfowl populations for Newcastle disease. In another study on detection of avian influenza viruses in waterfowl population in the Midwest, we isolated Newcastle disease virus (NDV) from several waterfowl. A total of 43 NDVs were isolated from 24 mallards, seven American green-winged teals, six northern pintails, four blue-winged teals, and two wood ducks. Partial sequences of fusion gene were analyzed to determine the pathotypes and genotypes involved. None of the isolates were phylogentically related to commonly used NDV vaccine strains. 3. Development of a degenerate primer set for amplification of avian influenza. Molecular methods such as reverse transcription-polymerase chain reaction (RT-PCR) and DNA microarray have been developed and used for the detection and/or typing of influenza viruses. We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are being performed to determine if this primer set can be used for subtyping of influenza virus isolates. 4. Development of diagnostic PCR assays for highly pathogenic Escherichia coli. We have utilized genome sequencing of multiple avian pathogenic E. coli strains and population-based prevalence studies to develop a diagnostic multiplex PCR predicting highly virulent strains of E. coli for poultry. We have also screened large collections of human and avian pathogenic E. coli, and have identified strains that have zoonotic potential. We have screened for the presence of these strains, and have identified them, in live birds and poultry meat. 5. Diagnostic identification of Ornithobacterium rhinotracheale (ORT) in turkeys. UMN continues to perform antigen-based testing for ORT in turkey populations. We have collaborated with the turkey industry to screen for the exposure to pathogenic ORT in turkeys. 6. Study of polymicrobial infection using ORT / aMPV co-infection model. We are examining the effects of varying age and stress levels in turkeys on their susceptibility to co-infection with ORT and avian metapneumovirus (aMPV), a model of co-infection in turkeys. PARTICIPANTS: Sagar Goyal, Kakambi Nagaraja, Srinand Sreevatsan, Timothy Johnson TARGET AUDIENCES: Target audiences include poultry producers, poultry scientists, and microbiologists. Work has been presented at the annual NC-1180 meeting and at numerous scientific meetings, including the annual meetings of the American Association of Avian Pathologists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The isolation, identification, and analysis of AIV from mixed infection will aid in the understanding of the ecology of influenza A virus populations. The understanding of NDV in wild populations will enhance our understanding of the transmission of this virus to domesticated poultry. The development of degenerate primer sets for detecting AI will ultimately result in the development of improved methods for detecting AI in birds. We have developed rapid mPCR assay that enable the rapid identification of highly virulent E. coli in poultry. The use of diagnostic assays for ORT and other respiratory pathogens enables our continued collaboration with the turkey industry. Study of polymicrobial infection will improve our understanding of etiology of polymicrobial respiratory infection in turkeys.
Publications
- Johnson TJ, Wannemuehler Y, Doetkott C, Johnson SJ, Rosenberger SC, Nolan LK. 2008. Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J Clin Microbiol. 46:3987-96.
- Johnson TJ, Wannemuehler Y, Johnson SJ, Stell AL, Doetkott C, Johnson JR, Kim KS, Spanjaard L, Nolan LK. 2008. Comparison of extraintestinal pathogenic Escherichia coli strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens. Appl Environ Microbiol. 74:7043-50.
- Jindal N, Patnayak DP, Chander Y, Ziegler AF, Goyal SM. 2010. Detection and molecular characterization of enteric viruses from poult enteritis syndrome in turkeys. Poult Sci. 89:217-26.
- Khatri M, O'Brien TD, Goyal SM, Sharma JM. 2009. Isolation and characterization of chicken lung mesenchymal stromal cells and their susceptibility to avian influenza virus. Dev Comp Immunol. Dec 28. [Epub ahead of print].
- Ramakrishnan MA, Gramer MR, Goyal SM, Sreevatsan S. 2009. A Serine12Stop mutation in PB1-F2 of the 2009 pandemic (H1N1) influenza A: a possible reason for its enhanced transmission and pathogenicity to humans. J Vet Sci. 10:349-51.
- Jindal N, Chander Y, Chockalingam AK, de Abin M, Redig PT, Goyal SM. 2009. Phylogenetic analysis of Newcastle disease viruses isolated from waterfowl in the upper midwest region of the United States. Virol J. 5:191.
- Ramakrishnan MA, Tu ZJ, Singh S, Chockalingam AK, Gramer MR, Wang P, Goyal SM, Yang M, Halvorson DA, Sreevatsan S. 2009. The feasibility of using high resolution genome sequencing of influenza a viruses to detect mixed infections and quasispecies. PLoS One. 4:e7105.
- Jindal N, Chander Y, de Abin M, Sreevatsan S, Stallknecht D, Halvorson DA, Goyal SM. 2009. Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method. J Virol Methods. 160:163-6.
- Johnson TJ, Nolan LK. 2009. Plasmid replicon typing. Methods Mol Biol. 551:27-35.
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