Microbial ecology of Lactobacillus strains in the chicken gastrointestinal tract : identification of persistent strains for use as live vectors

Modern poultry production conditions increase the susceptibility of birds to bacterial and viral infections, such as necrotic enteritis which is caused by the bacterium Clostridium perfringens. Currently disease control is facilitated through the use of antibiotic growth promoters, although their use has recently been banned within Europe based upon the potential spread of antibiotic resistance genes to potential human pathogens. The Australian poultry industry is seeking alternatives to antibiotic growth promoters which are able to control disease without compromising bird health or productivity. The aim of this study was to develop lactobacilli as live delivery vectors for the expression of therapeutic proteins in the chicken gastrointestinal tract. The use of lactobacilli as delivery vectors is a viable antibiotic-free strategy for the prevention of diseases such as necrotic enteritis. During the course of this study significant advances were made in the identification and development of Lactobacillus strains for use as delivery vectors. A high throughput strain typing system was optimised and used to analyse the Lactobacillus strain representation in the gastrointestinal tract of chickens, revealing great strain diversity within each Lactobacillus species, and illustrating the differences in the Lactobacillus ecology among chickens under different dietary conditions. A novel in vivo model was used to assess the ability of Lactobacillus strains to persist within the chicken gastrointestinal tract. Three strains, Lactobacillus agilis La3, Lactobacillus crispatus Lc9, and Lactobacillus vaginalis Lv5, persisted for up to 42 days in the chicken gastrointestinal tract regardless of diet and microbial competition. Persistence was confirmed in a second in vivo trial where all three strains persisted for up to 42 days in the chickens fed a commercial diet, while only the La3 and Lv5 persisted in chickens fed a high protein diet used to facilitate the development of necrotic enteritis. This finding was significant because these strains can be developed as therapeutic delivery vectors and appropriately tested using the necrotic enteritis disease model, and are likely to be successful when introduced into chickens grown in Australian commercial conditions. Protocols to efficiently transform La3 and Lv5 were developed and heterologous protein expression of the reporter gene gfp was observed. To our knowledge this is the first report of successful transformation and heterologous protein expression in L. agilis and L. vaginalis. Additionally, heterologous and homologous transcriptional promoters were tested in both strains, revealing that the greatest gfp expression occurred when the La3 ldhL promoter was used. Finally, the heterologous expression of the therapeutic antimicrobial protein piscicolin 126 was attempted in the La3 strain. The lack of success led to an in silico investigation of the genome to identify homologous genetic tools. Several Sec-dependent endogenous signal peptides, a putative bacteriocin with its native expression system, and m endogenous plasmids were identified. Each of these tools could potentially be utilised in developing the La3 strain as a delivery vector. Overall this study represents a major step forward in the development of lactobacilli as live delivery vectors as alternatives to antibiotic growth promoters for use within the Australian poultry industry