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Title: Development and use of differentiated airway epithelial cell cultures and co-cultures for studying the pathogenesis of bovine respiratory disease and assessing antibiotic activity and transport
Author: Jones, Claire Louise
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2020
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Respiratory tract infections of domesticated livestock is a global issue with a huge economic burden. Bovine respiratory disease presents one of the largest economic issues facing livestock industries and is responsible for the vast majority of illnesses and deaths in cattle. Many bacterial and viral agents are involved in the development of bovine respiratory disease, of which, Mannheimia haemolytica is one of the most prevalent bacterial agents. The interactions between M. haemolytica, respiratory epithelial cells and immune cells, and the factors governing lower respiratory tract colonisation of the pathogen are not well understood. This lack of understanding surrounding the specific events resulting in pathogenesis is hampering the development of successful new therapeutics and preventative measures. Hence, there is currently an urgent requirement for the development of in vitro models of the bovine respiratory tract which can mimic the in vivo environment to gain a more in depth understanding of the pathogenesis of bovine respiratory disease to develop improved strategies for managing this complex disease. In the present study, a co-culture model was developed, comprising differentiated BBECs grown at ALI and bovine neutrophils. The parameters required for successful attachment and migration of neutrophils were optimised including cell density of neutrophils, concentration of stimulants, appropriate pore density of transwell inserts, application of neutrophils into the culture and attachment time of neutrophils to cultures. Following optimisation, the effects of different stimulants on neutrophil migration was assessed. The pathogenic serotype A1 strain of M. haemolytica elicited greater migration of neutrophils across the bronchial epithelium compared to the non-pathogenic serotype A2 strain of M. haemolytica. A leukotoxin mutant strain of M. haemolytica failed to elicit substantial migration of neutrophils compared to the wild type strain. Stimulation of neutrophils with LPS resulted in lower neutrophil migration numbers compared with the pathogenic strain of M. haemolytica. However, migration was greater for LPS compared to the non-pathogenic strain and the leukotoxin mutant. Stimulation of neutrophil migration with BRSV resulted in lower neutrophil migration numbers compared to both pathogenic and non-pathogenic strains of M. haemolytica. The ability of the pathogenic strain of M. haemolytica to adhere to and colonise the bronchial epithelium in the presence of neutrophils and antiserum was also assessed. Colonisation of M. haemolytica to the epithelium was substantially reduced in the presence of either neutrophils or antiserum compared to M. haemolytica alone. Furthermore, substantially lower colonisation was observed in the presence of neutrophils and antiserum together within co-cultures. Experimental in vivo infection was conducted to assess the response of the entire respiratory tract to infection with M. haemolytica, BRSV and to co-infection with these two pathogens. Calves were infected via aerosol with either a pathogenic strain of M. haemolytica, BRSV or co-infected with both M. haemolytica and BRSV. Samples of tissue were removed from the nasal turbinate, pharyngeal tonsil, trachea, bronchi and lung post mortem for histological assessment. Infection with M. haemolytica and BRSV alone led to development of macroscopic lesions and symptoms of pneumonia. However, co-infection resulted in greater severity of symptoms with development of more extensive macroscopic lung lesions. Co-infected animals also exhibited extravasation of cells resulting in large airway obstructions, vascular congestion, epithelial necrosis and alveolitis. Assessment of the relationship between the infection status of the URT and the LRT highlighted inconsistencies, questioning the reliability of utilisation of the URT as a predictor of respiratory disease. Substantial invasion of both the URT and LRT was observed for the majority of infected animals. Conversely, invasion of the upper respiratory tract was observed for a number of mock infected control animals, although the lower respiratory tract exhibited no signs of invasion. Additionally, the applicability of the in vitro BBEC co-culture model was validated through comparison to in vivo assessment of the response to infection. The transport properties and antimicrobial activity of antibiotics frequently used in the prevention and treatment of bovine respiratory disease were assessed within cultures of differentiated BBECs grown at ALI. Five different classes of antibiotics were assessed, fluoroquinolones (marbofloxacin), macrolides (tilmicosin), β-lactams (amoxicillin), tetracyclines (oxytetracyline) and amphenicols (florfenicol). All five antibiotics exhibited differing rates of transport across the bronchial epithelium determined via bioassay analysis and MALDI-TOF MS. The lipophilic drugs, florfenicol, marbofloxacin and tilmicosin exhibited the fastest transport rates. Conversely, amoxicillin and oxytetracyline which have lower lipophilicities, were transported at relatively slower rates. Metaphylactic (preventative) and therapeutic (treatment) application of the antibiotics florfenicol and marbofloxacin to BBECs infected with a pathogenic strain of M. haemolytica was conducted to assess their antimicrobial efficacy. Marbofloxacin provided a more immediate antimicrobial effect with lower numbers of bacteria present within the wash samples than for florfenicol for metaphylactic application and lower colonisation efficiency as well as lower numbers of bacteria present within the wash samples than for florfenicol for therapeutic application. Metaphylactic treatment of infected cell cultures with either florfenicol or marbofloxacin resulted in an immediate reduction in bacterial colonisation of the epithelium. Colonisation of the epithelium was successfully prevented by 24 h, with complete elimination of bacteria from the cultures by 48 h. Therapeutic treatment of infected cell cultures with either florfenicol or marbofloxacin did not result in an immediate reduction in bacterial adherence where an initial increase in colonisation was observed. However by 48 h post treatment, prevention of bacterial colonisation and complete elimination of bacteria from the cultures was observed. The results herein demonstrate the development of an in vitro co-culture model of the bovine respiratory tract for studying the pathogenesis of infectious disease. Infections assessed in vivo validated this co-culture as a model which can successfully mimic the in vivo environment and response to infection which may provide new insight into the interactions between pathogens, epithelial cells and immune cells. In addition, this study demonstrated that cell cultures of the bovine bronchial epithelium can be utilised to study antibiotic transport and activity, presenting a tool for potentially screening new antibiotics.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General) ; QR Microbiology