(58f) Investigating Bacterial Biofilm Infection in a 3D Bovine Airway Model

Bovine Respiratory Disease (BRD) is a multifactorial condition where different genera of bacteria, such as Mannheimia haemolytica (M. Haemolytica), Histophilus somni (H. Somni), Pasteurella multocida, Mycoplasma bovis, and viruses, like bovine respiratory syncytial virus, bovine viral diarrhea virus, and bovine herpes virus-1, infect the respiratory system of cattle. This condition causes severe illness or death within livestock, resulting in a loss of approximately $1 billion to the cattle industry annually. Because of the multiple pathogens involved in this disease, antimicrobials and vaccines are difficult to administer.

Current models of the bovine airway system are primary submerged tissue cultures (STC) and air-liquid interface (ALI) models. STCs involve a monolayer, most commonly bovine epithelial cells, submerged in a bacterial concentrated cultured supernatant (CCS). ALIs are seeded on Transwell® plates where cells are exposed to the environment on the apical side, which allows cells to differentiate into goblet cells and ciliated epithelial cells. However, this process can take anywhere from 14 to 42 days. In order to recapitulate the bovine respiratory tract, we have designed a novel 3D co-culture model that incorporates bovine epithelial (B-Epis) and airway endothelial cells (BAECs). We focus our analysis on cultures exposed to CCS from H. Somni to understand signaling factors that could contribute to the pathogenesis of BRD.

Methods

The assembly of the co-culture 3D models began on day 1 and finished on day 2. In general, on day 1, B-Epis were encapsulated in a 1.1 mg/ mL type 1 collagen gel and seeded on a well-plate. On day 2, BAECs were encapsulated at a ratio of 3 B-Epis : 1 BAEC in type 1 collagen and then seeded on top of the B-Epis layer. B-Epis and BAEC monocultures served as controls. Three media conditions were then administered to all cultures on day 3, 24h after co-culture assembly, which included (1) cell media (or optimized media for co-cultures) (2) 50% cell media and 50% H. Somni CCS (50/50) and (3) H. Somni CCS. Cultures were ended for DNA analysis at 24h (day 4), 72h (day 6), and 108h (day 8) after CCS administration. For the cultures ended on day 6 and day 8, five media conditions were continued, which included (1) cell media, (2) 50/50 revert, where cell media was administered from day 4 onwards, (3) 50/50 (continuous), (4) H. Somni CCS revert, where cell media was also administered from day 4 onwards, and (5) H. Somni CCS (continuous).

Results

DNA analysis indicates that on day 8, B-Epi monocultures with cell media exhibited a 58% increase in cell proliferation compared to B-Epi monoculture controls at day 4. Co-cultures with cell media displayed a 73% increase in DNA on day 8 compared to co-cultures on day 4. However, both B-Epi monocultures and co-cultures with continuous H. Somni CCS administration do not exhibit significant changes in DNA from day 4 to day 8. B-Epi monocultures show a 67% increase in cell proliferation in 50/ 50 revert cultures on day 8 compared to 50/50 cultures on Day 4. Co-cultures also displayed a 73% increase in cell proliferation in H. Somni CCS revert cultures on day 8 compared to H. Somni CCS cultures on day 4. However, BAECs do not exhibit statistically significant changes in DNA under 50/50 and H. Somni CCS revert and continuous conditions from day 4 to day 8.

Conclusions and Future Work

H. Somni CCS results in cell death in B-Epi only and co-culture models. However, B-Epi monocultures and co-cultures exhibit cell proliferation in revert 50/50 media and revert H. Somni CCS cultures, potentially indicating cell recovery after initial bacterial media administration. However, BAECs do not display cell death upon H. Somni CCS administration. Further characterization of the monoculture and co-culture models will include examining changes in the epithelial barrier and tight junctions, as well as cytokine secretion with H. Somni CCS administration. Future investigations of bacterial biofilm interactions will expand to M. Haemolytica administration.