Performing Department
(N/A)
Non Technical Summary
Bovine Respiratory Disease (BRD) is the most devastating disease affecting U.S. cattle producers. Although many etiological agents have been implicated in BRD, a bacterial infection with the opportunistic pathogen Mannheimia haemolytica is most often observed. Although much research has focused on BRD, morbidity and mortality have not declined in the past 45 years, and antimicrobial resistance, especially in M. haemolytica, has been observed. As a result, there is a pressing need for novel preventatives. As it is known that the respiratory microbiota of calves with BRD differs from that of healthy calves, it is possible that probiotics from the bovine respiratory microbiota may be the answer. Our preliminary data indicate that commensal Corynebacterium from the bovine respiratory tract can inhibit M. haemolytica growth in vitro, potentially due to their ability to produce secondary metabolites. However, the metabolites, the manner of inhibition, and other probiotic properties possessed by these isolates are unknown. As a result, the overall objective of this project is to further characterize the probiotic capabilities of our commensal Corynebacterium isolates, providing valuable data regarding the role within the bovine respiratory microbiota, as well as their potential to serve as effective probiotics for BRD prevention. The development of a probiotic for BRD prevention would be highly valuable as it will improve cattle health while reducing antimicrobial usage, increasing producer profitability, and ensuring a sustainable and safe food supply.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of this project is to develop BRD probiotics from health-associated microbes isolated from the bovine respiratory tract. Our objectives are to evaluate the ability of commensal Corynebacterium's (ASV19 and ASV39) probiotic characteristics of pathogen growth inhibition and immunomodulation. The objectives can be broken down into 1) evaluate commensal ASV19's and ASV39's mechanism for inhibiting opportunistic pathogen growth in vitro, and 2) evaluate ASV19's and ASV39's ability to colonize the nasal cavity, modulate the bovine immune system, and provide colonization resistance against M. haemolytica. Furthermore, an additional goal of this project is to prepare the PD for a career in academia by affording her diverse scientific and professional opportunities. Specifically, this will be accomplished by providing the PD with experience in project management, increasing her scientific skills, communicating research to both scientific and lay audiences, and strengthening her scientific writing skills.
Project Methods
For objective 1, a co-culture assay will be designed to determine if ASV19 and ASV39 act synergistically to increase each other's growth and if co-culture with the supernatant from the other increases their anti-M. haemolytica (MH) effect. Briefly, the following will be co-cultured (n=3/group) using a 24-well cell culture plate and 0.1 µM inserts comprised of a semi-permeable membrane that sits within the well. Using this method, the bacterial cells will be co-cultured one on top of the other, allowing for the diffusion of metabolites, but not bacterial cells, between the membrane (conditions depicted as (co-culture microbe + OD600 microbe): 1) ASV19 + ASV39, 2) ASV39 + ASV19, 3) ASV19 + MH; 4) ASV39 + MH; 5) ASV19 and ASV39 + MH; 6) ASV19 alone (control); 7) ASV39 alone (control); 8) MH alone (control). The plate will be incubated at 37ºC, and opportunistic pathogen OD600 readings will be taken at plate insertion (0h) into an HTS Synergy plate reader (BioTek) and every subsequent hour for 36 hours. The plate will be incubated at 37ºC while shaking, and OD600 readings will be taken at 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, and 36h using an HTS Synergy plate reader (BioTek). Growth curves will then be created to visualize the effect of the various co-culture conditions on cell growth. Therefore, this will be repeated 5 times (n = 15/group total). Another co-culture assay will be designed to determine if ASV19 and ASV39 inhibit MH in a bactericidal or bacteriostatic manner. Briefly, the following will be co-cultured in quadruplicate (n = 4/group) using a 12 well cell culture plate and 0.1 µM inserts (conditions depicted as co-culture microbe + imaged microbe): 1) ASV19 + MH; 2) ASV39 + MH; 3) MH alone (control). The plate will be incubated at 37ºC for 24 hours while shaking. The 24-hour MH growth will undergo live-dead cell staining and imaging with a Leica confocal laser scanning microscope to determine if co-culture with ASV19 and ASV39 inhibits MH in a bacteriostatic or bactericidal manner. A final co-culture assay will be designed to determine if ASV19 and ASV39 produce a secondary metabolite responsible for their anti-MH properties. The following will be co-cultured a 24-well cell culture plate with 0.1 µM inserts: 1) ASV19 + MH; 2) ASV39 + MH; 3) ASV19 alone, 4) ASV39 alone, 5) MH alone. The plate will be incubated at 37ºC for 24 hours while shaking. This will be repeated twice (n=8/group total). Samples will be filter sterilized and sent to the University of Arkansas for Medical Sciences (UAMS) Metabolomics and Analytical Chemistry Core for untargeted metabolomic analysis.For objective 2, a cell adherence assay will be designed to determine if ASV19 and ASV39 adhere to bovine turbinate cells based on methods from Amat et al. (2019). Briefly, bovine turbinate cells will be inoculated into a 6-well co-culture plate and incubated at 37C. Once cells have formed a monolayer, ASV19 and ASV39 will be inoculated into a 6-well plate in duplicate (with two wells free of any bacteria to serve as a negative control). Cells will be incubated for 3 hours at 37ºC. Cell growth media (containing unbound ASV19 and ASV39) will then be removed, and cells will be washed with media. Cells will be lysed, serially diluted, and plated on tryptic soy + 5% blood agar plates. After 24 hours of incubation, colonies will be counted, and the colony-forming units (CFU)/ mL will be calculated. Isolated colonies will be Sanger sequenced to ensure that the isolated colonies are ASV19 or ASV39. Next, a colonization resistance assay will be designed to determine if ASV19 and ASV39 provide colonization resistance against MH based on methods from Amat et al. (2019). Briefly, bovine turbinate cells will be inoculated into three 2-well Nunc Lab-Tek II chamber slide systems. Bovine turbinate cells will be stained with DAPI, ASV19, and ASV39 will be labeled with Alexa Fluor 594 microscale protein, and MH will be stained with Alexa Fluor 488 microscale protein. Equal concentrations of the following will be inoculated onto the bovine turbinate cells (ASV19, ASV39, MH, ASV19 + MH, ASV39 + MH) and incubated for 3 hours at 37C. Cell growth media (containing unbound bacterial cells) will then be removed, and cells will be washed with media and imaged using a Leica laser scanning confocal microscope. Simultaneously, bovine turbinate cells will be inoculated into a 24-well co-culture plate and incubated at 37C. Once cells have formed a monolayer, Equal concentrations of the following conditions will be inoculated in the 24-well plate (n = 8/ group) (ASV19 + MH, ASV39 + MH, MH alone). After 3 hours of incubation at 37C, the cell growth media (containing unbound ASV19 and ASV39) will then be removed, and cells will be washed with media. Cells will be lysed, serially diluted, and plated on tryptic soy + 5% blood agar plates. After 24 hours of incubation, colonies will be counted, and CFU/ mL will be calculated. Isolated colonies will be Sanger sequenced to determine the final cell count for ASV19, ASV39, and MH. MH counts will be averaged for all conditions and calculated as follows: average MH control CFU - average MH co-culture CFU. This will be repeated for all co-culture conditions. Finally, to determine if the co-culture of bovine turbinate cells with ASV19 or ASV39 modulates the immune system, a co-culture assay will be designed based on the methods by Amat et al. (2019). Briefly, bovine turbinate cells will be inoculated into a 24-well cell culture plate (n=6/group) and incubated until they reach confluency. ASV19, ASV39, MH, and bovine turbinate cells with no bacteria will be inoculated, and cells will be co-cultured for 6 hours at 37ºC. Next, mRNA will be extracted and reverse-transcribed into cDNA. Then qPCR with RT² Profiler™ PCR Array Cow Innate & Adaptive Immune Responses (Qiagen) will be performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene.