Progress 09/15/15 to 09/30/16
Outputs
Target Audience:The target audience for this project was food animal veterinarians, extension agents, and cattle producers who regularly deal with cattle affected by bovine respiratory disease. Information gathered in the course of this project has been disseminated to each of these audiences through continuing education presentation, training meetings for extension agents, and cattlemen's meeting. In December 2016 an overview of the project and experimental design was delivered in a brief report to at the Conference of Research Workers in Animal Diseases. In the next year we anticipate also delivering this information at the annual AABP research summaries meeting and a completed summary of the data again at CRWAD 2017. Additional benefactors of this project were 2 summer scholar veterinary students, who through summer research exposure assisted in the animal phase of data collection. Changes/Problems:Primary changes that occurred during the course of the project were in regard to the approved protocol for the use and care of animals. The original protocol stated that sampling would be attempted without sedation. This proved to be very stressful for the calves and the personnel involved. Permission to sedate the calves prior to sampling was requested and granted. The method of euthanasia was also mildly altered with permission of the IACUC committee in order to increase the amount of information that could be gleaned from the animals to be sacrificed. At this time the significance of the data collected is unknown. At each microenvironmental sampling, in addition to collection of DNA for 16 S rRNA sequencing, samples were collectedby conventional bacterial culture. Despite the large challenge inoculum of approximately 6 x 109 CFUs, no live M. haemolytica were recovered from any samples. We demonstrated that live organism was delivered based on our post-inoculation CFU confirmations. We hypothesize that the organism was not evenly distributed within the selected areas of sampling or was rapidly cleared by the mucosal immune response. In the future we plan to extend the timing of colonization to reflect a more natural exposure such as nebulization or intranasal administrations over 12 - 36 hours and perhaps a lower infectious dose at each administration. What opportunities for training and professional development has the project provided?Information gathered in the course of this project has been disseminated through continuing education presentation, training meetings for extension agents, and cattlemen's meeting. In December 2016 an overview of the project and experimental design was delivered in a brief report to at the Conference of Research Workers in Animal Diseases. In the next year we anticipate also delivering this information at the annual AABP research summaries meeting and a completed summary of the data again at CRWAD 2017. Additional benefactors of this project were 2 summer scholar veterinary students, who through summer research exposure assisted in the animal phase of data collection. How have the results been disseminated to communities of interest?Information gathered in the course of this project has been disseminated through continuing education presentation, training meetings for extension agents, and cattlemen's meeting. In December 2016 an overview of the project and experimental design was delivered in a brief report to at the Conference of Research Workers in Animal Diseases. In the next year we anticipate also delivering this information at the annual AABP research summaries meeting and a completed summary of the data again at CRWAD 2017. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During the course of this study we collected microenvironmental samples from several sites within the nasal cavity of 12 calves over a time course of 15 days (Day -1 - Day 14). Eight calves were additionally exposed to 6 X 10 9 CFUs M. haemolytica in 5 ml PBS intranasally on day 0. Four calves served as non-exposed controls. Metagenomic sequencing of several conserved V regions of the 16 S rRNA genes was conducted and produced approximately 75,000,000 reads of sequenced DNA that is at this time being analyzed and mapped to identify keystone microbial species in the various microenvironments of the calf's upper respiratory tract. From this data we will draw broad patterns of microbiome diversity between microenvironments, individuals and if or how those populations change following the introduction of M. haemolytica. Conclusion of data analysis and preparation of manuscripts for peer reviewed publication is anticipated by June 2017.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Abstract presented: Microenvironmental sampling techniques of the nasal cavity of cattle and experimental colonization of Mannheimia haemolytica presented at CRWAD conference, December, 2016. (NIFA was acknowledged during the presentation)
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Progress 09/15/15 to 09/30/15
Outputs
Target Audience:
Nothing Reported
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?Phase I: Six cadaver heads will be obtained from a local cull cow facility. A 20 cm pediatric endoscope will be used to visualize and confirm the anatomical placement of swabs and cytology brushes either through the indwelling instrument port or independent of the endoscope. Four sampling locations will be attempted; the cranial and caudal nasal turbinates, palatine tonsils and nasopharynx. Phase II: Animals. A total of 10 Holstein steer calves weighing approximately 300-400 pounds will be acquired from the University of Tennessee Research and Education Center Agricultural Units or purchased from private sources. Calves will be transported to a climate controlled room in the Johnson Animal Research and Teaching Unit (JARTU) at the University of Tennessee and appropriately cared for and fed according to NRC guidelines. The calves will be provided 4 days of acclimation to this new environment prior to initiation of the study and will be housed there until termination of the study. Antemortem Sampling. Following the acclimation period each calf will be sampled via nasal swab, pharyngeal swab, endoscopy guided swab of the palentine tonsils, and tonsillar scraping on day 0. Nasal swab will be performed by inserting a sterile cotton tipped applicator into each nostril and gently swabbing the mucosa. Pharyngeal swabs will be obtained very similarly to the nasal swab, however a guarded swab will be used so there is no contamination from the nasal cavity. The swab will be inserted to about the level of the medial canthus of the eye. Endoscopy guided swab of the palantine tonsils will be achieved by passing a pediatric scope through the nasal cavity to the level of the tonsil. The mouth will be held open with an oral speculum for the tonsillar scrapings. The sample itself will be obtained using a stainless steel spoon with an elongated handle. The palantine tonsil will be scraped with the edge of the spoon as decribed by Angen et al. Composition of bacterial communities. The composition of the bacterial communities will be determined by metagenomic analysis via Ion Torrent PGM sequencing of 16S rRNA gene amplicons. The V3-V4 regions of the 16 S rRNA genes will be amplified from extracted DNA with barcoded primers. From these libraries will be prepared and pooled then sequenced by the Ion Torrent PGM platform at a depth of approximately 150,000 reads per sample. Resulting sequence data will be uploaded to appropriate public databases (e.g. NCBI) and compared with Life Sciences curated library of bacterial taxonomic sequences. Sequences will be aligned to the Silva bacterial database, and clustered into operational taxonomic units (OTUs) based on >97% similarity (approximately species level). Ecological indices and alpha diversity metrics (richness, diversity, and rarefaction curves) will be calculated for each sample. Finally, each sample will be subjected to standard aerobic culture to identify the predominate cultivable isolates from each microenvironment and if possible confirm the sequencing results. Inoculation and serial sacrifice. After sampling calves will receive an intranasal nebulization of an inoculum containing approximately 3-5 x 109 CFUs of M. haemolytica suspended in 5 ml of PBS. Two calves will be serially sacrificed every 3 days beginning on Days 1 (24 hours post-inoculation), 4, 7, 10, and 13 post-inoculation. Prior to euthanasia each calf will be sampled as previously described for ante mortem sampling. Immediately following euthanasia the head of each animal will be removed and carefully transected in a sagittal plane in order to expose the cranial and caudal nasal turbinates, palantine tonsils and nasopharynx. The bacterial communities of each site will be determined from both ante- and post-mortem samples as well as standard aerobic microbial culture. In addition, samples from each site will be subjected to quantitative PCR using primers specific for M. haemolytica. Tissue sections from each site will be collected at the time of euthanasia and fresh frozen in liquid nitrogen for immunohistochemical straining. Tissue sections will be prepared and exposed to antibodies specific for M. haemolytica in an attempt to demonstrate in situ colonization of the pathogen. Data analysis. Samples collected from each microbiome will be analyzed using metogenomic techniques to determine the bacterial population within each microbiome. Cultures and PCR from each location at various time points will also give some insight to the chronological timeline of colonization of these microbiomes post intranasal inoculation.
Impacts
What was accomplished under these goals?
Work has not yet begun on the project, and no funds have been spent.
Publications
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