Progress 01/01/15 to 12/31/15
Outputs
Target Audience:The findings from this research will be presented in peer-reviewed scientific journals and at national scientific meetings. Additionally the findings will be presented at local and regional seminars which target faculty and students. Changes/Problems:Several challenges were faced with optimizing RNA extraction from dual samples. We overcame these challenges and have excellent sequencing data from prokaryotic and eukaryotic RNA which originated from one sample. What opportunities for training and professional development has the project provided?This project has trained a Ph.D. student and several undergraduate students in microbiology and RNA-Seq. How have the results been disseminated to communities of interest?Results have been presented as an abstract at an international meeting and are being prepared for manuscripts in peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?This project is coming to completion and the final results will be submitted for publication and used as preliminary data for future grant proposals.
Impacts
What was accomplished under these goals?
To date we have determined that plant-derived antimicrobials, trans-cinnamaldehyde (TC) and eugenol (EG), reduced adhesion to and invasion of Staphylococcus aureus (S. aureus) to bovine mammary epithelial cells (MEC) in vitro. Specifically, using 4 strains of S. aureus and MEC from two different cows, we observed a 1 to 2-log CFU/mL reduction in adhesion when S. aureus and MEC were pre-treated with TC or EG (P < 0.05). Pre-treatment of S. aureus and MEC with TC or EG reduced invasion 2 to 4-log CFU/mL (P < 0.05). To determine potential mechanisms that contribute to TC and EG reduction of adhesion and invasion, we determined mRNA expression of prokaryotic and eukaryotic genes by real-time RT-PCR. In S. aureus, treatment with EG and TC reduced expression of virulence genes nuclease, intercellular adhesion, human leukocyte antigen, fibronectin binding protein-B, and fibronectin binding protein-A (P < 0.05) and increased expression of hlB (P < 0.05). Treatment of MEC with EG and TC increased expression of inflammatory markers complement, toll-like receptor-2, and Interleukin-8 (P < 0.05). To further elucidate the mechanisms by which the pathogen infects the host and the plant molecules prevent this infection, we performed RNA-Seq analysis. In the first experiment, prokaryotic mRNA was sequenced using Illumina MiSeq and 174 and 53 genes were differentially expressed with TC and EG treatment, respectively (P < 0.05). The second experiment used dual RNA-Seq to determine the host-pathogen interaction by determining prokaryotic and eukaryotic mRNA expression using Illumina NextSeq. These data are currently being aligned and analyzed.
Publications
|
Progress 01/01/12 to 12/31/15
Outputs
Target Audience:The information obtained from this project has been shared with undergraduate students, graduate students, faculty and staff at the University of Connecticut. In addition, it has been shared with other scientists at the Annual PD workshop and at the Joint Annual Meetings of the American Society of Animal Science and American Dairy Science Association in July 2014. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The funds from this seed grant have provided training for graduate and undergraduate students in research in the fields of mammary gland physiology, microbiology, and statistics. Additionally, students have presented data at regional and national conferences allowing them to learn from experts in the field and improve written and oral communication skills. How have the results been disseminated to communities of interest?The results have been published in an abstract in the Journal of Animal Science, presented at regional scientific seminars, local stakeholder meetings and will be submitted for publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We determined that sub-inhibitory concentrations of two plant-derived antimicrobials, trans-cinnamaldehyde (TC; 0.006 %) and eugenol (EG; 0.03 %), reduce adhesion to and invasion of four strains (Thorn 17, DST 35, M9175 and Thorn 15) of Staphylococcus aureus (SA) to bovine primary mammary epithelial cells (MEC) using an in vitro culture system. To determine the potential mechanisms by which these plant molecules prevent adhesion and invasion, we explored changes in gene expression of virulence factors in SA and immune genes in MEC. As expected, when MEC are infected with SA, we observe increased expression of several virulence genes in SA and immune response genes in MEC. However, with treatment of TC and EG we observed altered expression of these immune and virulence factors, suggesting that these plant-derived antimicrobials may work to prevent SA infection of MEC through modulating gene expression. To further explore the effect of these plant molecules on SA gene expression, we sequences the entire transcriptome of SA in the absence and presence of TC or EG. Treatment of SA with TC resulted in 110 differentially expressed genes of which 3 were down-regulated greater than 2-fold and 10 were up-regulated greater than 2-fold. Treatment of SA with EG resulted in 222 differentially expressed genes of which 17 were down-regulated by greater than 2-fold and 13 were up-regulated greater than 2-fold. To gain a better understanding of the host-pathogen interaction during mastitis infection, we developed an in vitro model in which MEC were infected with SA and then RNA was isolated from both the MEC and SA for whole transcriptome analysis. RNA-Seq analysis determined that 706 genes in SA are differentially expressed when exposed to MEC and 1,260 genes were differentially expressed in MEC with infected with SA. These whole transcriptome analyses demonstrate that infection of MEC with SA causes several changes to both the host and pathogen, which contribute to infection. Additionally, TC and EG may be able to prevent infection by modulating the expression of these genes in SA. These studies demonstrate that novel methods to prevent or treat mastitis may be viable using TC or EG; however in vivo experiments are needed to confirm these findings. Lastly, there are several mechanisms at the host and pathogen level that contribute to mastitis infection and the host's immune response.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Jaganathan, D, Kollanoor-Johny, A, Venkitanarayanan, K, Kazmer, GW, Kuo, L, Wang, YB, and Govoni, KE. 2014. Plant-derived compounds, trans-cinnamaldehyde and eugenol, reduce adhesion and invasion of Staphylococcus aureus in bovine mammary epithelial cells in vitro. J Anim Sci Vol. 92, E-Suppl 2, p 182.
|
Progress 01/01/14 to 12/31/14
Outputs
Target Audience: The information obtained from this project has been shared with undergraduate students, graduate students, faculty and staff at the University of Connecticut. In addition, it has been shared with other scientists at the Annual PD workshop and at the Joint Annual Meetings of the American Society of Animal Science and American Dairy Science Association in July 2014. Changes/Problems: We found the process of RNA-Seq challenging since we could not separate the eukaryotic and prokaryotic RNA before sequencing. So we spend a lot of time optimzing the dual-RNA-Seq procedures. We have promising preliminary results and will use this method for RNA-Seq analysis. What opportunities for training and professional development has the project provided? The funds from this seed grant have provided training for graduate and undergraduate students in research in the fields of mammary gland physiology, microbiology, and statistic. How have the results been disseminated to communities of interest? The results have been shared with scientists at the University of Connecticut, during the PD workshop and at national meetings. What do you plan to do during the next reporting period to accomplish the goals? We plan to repeat the experiments in a third animal, extract RNA from all three animals, complete RNA-Seq analysis, and publish the findings in a peer-reviewed journal.
Impacts
What was accomplished under these goals?
To date we have determined that sub-inhibitory concentrations of two plant-derived antimicrobials, trans-cinnamaldehyde (TC; 0.006 %) and eugenol (EG; 0.03 %), reduce adhesion to and invasion of four strains of Staphylococcus aureus (SA) in bovine primary mammary epithelial cells (MEC). Specifically, for strain Thorn 17, pre-exposure of SA (5 h) and MEC (12 h) to EG reduced SA adhesion to and invasion of MEC by ~1.6 ± 0.01 log10 CFU/mL (control = 6.67 ± 0.01 log10 CFU/mL; P < 0.0001) and 2.8 ± 0.11 log10 CFU/mL (control = 3.9 ± 0.02 log10 CFU/mL; P < 0.0001), respectively. Pre-exposure of SA and MEC to TC reduced SA adhesion to and invasion of MEC by ~2.2 ± 0.02 log10 CFU/mL (control = 6.7 ± 0.02 log10 CFU/mL; P < 0.0001) and 2.85 ± 0.08 log10 CFU/mL (control = 3.94 ± 0.01 log10 CFU/mL P<0.0001), respectively. Similar results were observed with strains DST 35, M9175 and Thorn 15. Real- time quantitative PCR data revealed that PDAs down-regulated critical virulent genes of SA (intercellular adhesion and nuclease) and modified immune genes of MEC (complement, toll-like receptor2, interleukin1-beta and interleukin6; P ≤ 0.05). Preliminary analysis of SA and MEC RNA from these experiments has been completed using the Dual RNA-Seq procedure using Hi-Seq 2000 from Illumina.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
54. Jaganathan, D, Kollanoor-Johny, A, Venkitanarayanan, K, Kazmer, GW, Kuo, L, Wang, YB, and Govoni, KE. 2014. Plant-derived compounds, trans-cinnamaldehyde and eugenol, reduce adhesion and invasion of Staphylococcus aureus in bovine mammary epithelial cells in vitro. J Anim Sci Vol. 92, E-Suppl 2, p 182.
|
Progress 01/01/13 to 12/31/13
Outputs
Target Audience: The information obtained from this project has been shared with undergraduate students, graduate students, faculty and staff at the University of Connecticut. In addition, it has been shared with other scientists at the Annual PD workshop in October 2013. Changes/Problems: We have faced several challenges with isolating RNA for RNA-Seq analysis. In particular, obtaining enough prokaryotic RNA from samples in which S. aureus invasion has been reduced or prevented has posed a challenge. We are working with more samples to increase our yield. We also anticipate finding a challenge with mapping our RNA-Seq data to the bovine genome. However, more publications are available now with sequence data that we will use to map to our findings. What opportunities for training and professional development has the project provided? The funds from this seed grant have provided training for graduate and undergraduate students in research in the fields of mammary gland physiology, microbiology, and statistic. How have the results been disseminated to communities of interest? The results to date have been shared with students and faculty at the University of Connecticut and with other scientists at the annual PD workshop in October 2013. What do you plan to do during the next reporting period to accomplish the goals? We plan to repeat the expeirments using different strains of S. aureus. In addition, we will complete isolationf of RNA and whole transcriptome analysis using RNA-Seq. The current results are being presented at the ASAS meeting in July 2014 and a manuscript is in preparation.
Impacts
What was accomplished under these goals?
To date we have confirmed that our primary mammary gland cell population is primarily mammary epithelial cells using immuno-histochemistry staining for multi-cytokeratin. In addition, we have determined the sub-inhibitory concentrations of TC as 0.006% and 0.0125% and EG as 0.03%. These concentrations do not inhibit mammary epithelial cell growth in culture. Consistent with our preliminary findings in MAC-T cells, TC inhibits attachment of S. aureus (6.1 ± 0.2, 5.4 ± 0.3, and 4.6 ± 0.5 log CFU/mL; control, 0.006%, and 0.0125% TC, respectively) and invasion (3.4 ± 0.05, 2.4 ± 0.09, and 0.8 ± 0.2 log CFU/mL; control, 0.006%, and 0.0125% TC, respectively) when incubated with S. aureus alone. When both S. aureus and mammary epithelial cell are incubated with TC, attachment (5.1 ± 0.2, 4.2 ± 0.2, and 2.5 ± 0.3 log CFU/mL; control, 0.006%, and 0.0125% TC, respectively) and invasion (3.0 ± 0.02, 1.1 ± 0.16, and 0 log CFU/mL; control, 0.006%, and 0.0125% TC) are further reduced. Pre-treatment of S. aureus with EG reduced attachment (5.6 ± 0.1 vs. 4.3 ± 0.3 log CFU/mL; control vs. EG) and invasion (3.0 ± 0.3 vs. 1.4 ± 0.3 log CFU/mL; control vs. EG) of mammary epithelial cells. When both S. aureus and mammary epithelial cell were incubated with EG attachment (5.7 ± 0.04 vs. 3.8 ± 0.07 log CFU/mL; control vs. EG) and invasion (3.2 ± 0.03 vs. 0 log CFU/mL; control vs. EG) were further reduced. Statistical analysis is in progress. Data presented are the average of three separate experiments.
Publications
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: This project has provided collaboration between the Department of Animal Science and the Statistics Department at UConn. In addition, it has contributed to the training of a Ph.D. candidate in Animal Science and two Ph.D. candidates in Statistics. An undergraduate student in Animal Science has also received laboratory skills training through this project. PARTICIPANTS: Kristen Govoni, Kumar Venkitanarayan, Gary Kazmer, Devi Jaganathan, Cameron Smart; University of Connecticut, Department of Animal Science. Lynn Kuo, Patrick Harrington, Yu-Bo Wang; University of Connecticut, Statistics Department. TARGET AUDIENCES: This work targets scientists, faculty, graduate students and undergraduates. This work will be presented at national meetings, in peer-reviewed scientific journals and local and national seminars. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project is still in the preliminary stages of development. To date, we have established the primary mammary epithelial cell line from five different cows. These cells have been expanded and stored in cryo tanks for proposed experiments. In addition, we are in the process of optimizing RNA extraction from both prokaryotic and eukaryotic RNA from the same cell culture well. We have also repeated the attachment and invasion assays completed in our preliminary data (MAC-T cells) using primary mammary epithelial cells. We have confirmed successful attachment and invasion of S. Aureus using one strain. After several trial experiments with different MOI (eukaryotic to bacterial cell) of 1:10,1:20,1:30,1:100, we found that 1:100 i.e. 10^5 : 10^7 (eukaryotic to bacterial cell) showed adequate invasion of Staphylococcus aureus into the bovine mammary epithelial cells in cell culture. The invasion with 1:100 MOI was determined to be around 4 log CFU / ml. The invasion rate with other MOI was found to be < 3 log CFU/ml. We have decided to use an MOI of 1:100 using staphylococcus strain DST 35. Further this MOI will be checked with other strains of mastitis causing Staphylococcus aureus (DST 38 and DST 4) for its invasion ability into the bovine mammary epithelial cells.
Publications
- No publications reported this period
|
|