Biogeography, Inter-animal variation and dynamic of Bovine upper airway Microbiome

The project or proposal was presented as a Ph.D. CEMB student at the University of Arkansas while I take the seminar Fall 2016 course which requires all master & doctoral student to present their projects under the supervision of their mentors. The presentation method can be used as guidance to the new graduate student.

Transcript

1. PIs: Dr. Jiangchao Zhao, Dr. Jeremy Powell, Dr. Beth Kegley, Dr. Paul Beck Presented by: Suad Algarni, doctoral student at CEMB program, UARK. Fall 2016
2. § In Arkansas, beef cattle production is very important. § The 5th largest agriculture commodity produced in Arkansas. § There are over 27,000 farms in Arkansas produced beef cattle.
3. Bovine Respiratory Disease (BRD) any disease of the upper or lower respiratory tracts. § Most common diseases effect the cattle industry in the US. § Approximately 75% of feedlot morbidity, and 50% to 70% of mortality. § It costs around $800 million to $900 million of economic losses every year. upper respiratory tract lower respiratory tract
4. § BRD is a complex multi-factorial disease . § Host factors: age, immune status, prior exposure to the pathogens, genetics, etc. § Environmental factors: transport, temperature fluctuations, crowding, ventilation.
5. § Serology § Bacterial isolation in culture § Gel based polymerase chain reaction (PCR) § Real time PCR § Multiplex PCR –real time § Microarrays § Sequencing of agent genomes
6. § Mannheimia haemolytica : Major bacterial agent of the BRD § Pasteurella multocida : Most prevalent and cause less acute respiratory disease § Mycoplasma Bovis : Found in healthy cattle and cause mild symptoms § Haemophilus somnus: Found mostly in fatal cases of BRD
7. § Proper early diagnosis. § Selection of an appropriate antibiotic. § Proper dosage, route, and treatment schedule.
8. §Can we use a nasal swab sampling ”Nostril” as a reliable and practical site for bovine upper airway microbiome study? Nasopharynx Nostril
9. § We hypothesize that a nasal swab sampling will be reliable and superior to the nasopharyngeal swab sampling approach which easier to perform, and could be applied to a large number of calves.
10. § Examine the biography inter-animal variation and dynamic of the bovine upper airway microbiome. § For upper airway microbiome study: identify the most practical and reliable sampling site in the bovine upper airway (nostrils vs nasopharynx).
11. § Animal study : Healthy calves (n=18) Swabs (nasal and nasopharyngeal) Time point on day 7 and day 14
12. § Sampling strategy: nasal sample § First nasal swab : § Respectively , the anterior , middle and posterior part of the left and right nostril § Second nasal swab: § Swab collected to the whole left and right nostril. Nasal swab
13. § Nasopharyngeal sample § Collected by using a double guarded culture swab. § Swab inserted into the nasal cavity through the right nostril protrude from the casing rotated 360 and withdrawn into the casing before removal from the nasal cavity. Nasal cavity Double guarded culture swab
14. § DNA extraction Nasal & Nasopharyngeal samples § by using a commercial kit § DNA concentration § Quantify and qualify DNA extracted samples
15. § Sample validation using by “Universal Bacterial Primer” sequences for the 16S rRNA 27F, 1492R
16. § Validation of amplification of all samples using gel electrophoresis (~1.5 kb band) M 1 2 3 4 5 6 PC NC Samples 16S rRNA (1,500 bp)
17. Illumina MiSeq 2 3 250 platform DNA nasal sample for library preparation and sequencing: PCR using dual barcoded primers and amplification of V4 hypervariable regions of the 16SrRNA gene. Validation of amplification using gel electrophoresis (~250 bp band) PCR cleanup/normalization of DNA concentration of all samples Sequencing MiSeq, 2X250 bp for V4
18. § Analyze the pyrosequencing data according to the MiSeq SOP. § By using software package mothur v. 1.21 (17) § Sequences normalized to the smallest number of reads to minimized the effect of sequencing depth on diversity measures.
19. § Alpha diversity (diversity within a sample or community) § OTU counts different type of microbes § Richness: the measure of a number species a sample. § Distribution of different microbes § Evenness : a measure of relative abundance of different species. Zhao et al,2012,PNAS,109:5809-5814
20. § Beta diversity a measure of the distance or dissimilarity between samples or community § Beta diversity metrics Bray-Curtis § Inter-animal variations in bovine upper airway microbiome will be evaluated based on Bray-Curtis distances.
21. § Principal Coordinates Analysis (PCoA) to visualize the data in the beta diversity distance matrix. § The community dynamics will be visualized on PCoA plots Bray-Curtis distances Zhao et al,2012,PNAS,109:5809-5814
22. § Investigate the metagenome and met transcriptome of the upper airway microbiome. § Conduct a pilot study of the upper airway microbiome from healthy calves and calves with BRD to determine the effect of host genetics, environment and diets on the airway microbiome to calculate the power and sample size needed § Identify BRD biomarkers and potential probiotics.
23. §Robert Story §Fanli Kong §Xiaoyuan Wei §Jianmin Chai