Title: 
Brucellosis Vaccination: assessing the stability and safety of a Nisin inducible plasmid expressing Brucella antigen in Lactococcos lactis . 
Summary:
    Brucellosis is a worldwide bacterial disease that negatively affects the Wyoming bovine ranching industry. Current vaccines against the pathogen do not provide total immunity, and some interfere with diagnostic testing. We propose using live Lactococcus lactis, a probiotic bacteria, to express an adhesion protein found in wild type Brucella abortus. Experiments showing stability and safety in a plasmid-containing, live probiotic introduce the possibility of a viable vaccine. A stable food safe vector such as this, paves a road to quick, robust immunization. This project is aiming to gain confidence that this strain will have normal growth along with adequate antigen expression, and that antibiotic resistance present in the plasmid remains inside the probiotic. 
    Knowing how the vector responds to bovine rumen conditions is largely unexplored, and illumination will provide knowledge for the field of probiotic vaccines. Dangerous pathogens acquiring and expressing resistance acquired from the recombinant DNA is of great concern, and the extent of this problem is unknown. The rumen conditions may be unfavorable for the strain compared to the local residents, and various testing must show strain durability and plasmid accountability. Despite the challenges, we are expecting standard growth using this nisin-induced system. We aim to grow the vector-strain in conditions varying in temperature and microbial community, measuring if it has adequate survival capability. Rumen bacteria and pathogens grown alongside L. lactis will be reviewed for plasma expression, hopefully quantifying the rate of transfer. Testing these variables will give understanding of the pressures and limits affecting the vaccine-strain, and if successful, ultimately leading to novel innovation and broad application in vaccination methods. . 
 Statement of Problem and Significance:
    B. abortus is a huge issue in commercial livestock production, that if efforts to eradicate this bacteria were halted, the cost of producing beef and milk would increase by $80 million annually (2). This issue is worsened by the fact that B. abortus can infect humans and is found in wild ruminants like elk and bison, but also infects the bovine population causing calves to be aborted or become chronically infected, acting as reservoirs for further herd infection . Even with current vaccination methods for cattle, which are an injection based live attenuated strain, B. abortus is still an issue to livestock production . With these issues apparent, the scientific community is hard at work to find alternatives to vaccination of B. abortus. Probiotics are an avenue of investigation as a more widespread and efficient method of antigen delivery. If this work is successful, costs of livestock vaccination could significantly drop and human infections could be a thing of the past.  
 
Introduction:
Relevant literature:
 Brucellosis, caused by Brucella abortus results in spontaneous abortions in domestic animals, as well as wildlife (1). Two animals of importance for our study are domesticated cattle, and wild elk (3). With the prevalence of this pathogen in these species, and their interactions together, the cattle agricultural industry is under economic threat by the loss of fetal calves. Currently one vaccination is on the market for use, a live attenuated strain of B. abortus RB51(3). Although this vaccine is on the market for use in the agriculture industry it is only moderately efficacious as a cattle vaccine, and is not applicable to elk, serving as the wild reservoir for this pathogen(3). The elk infected with B. abortus develop a chronic infection, which spreads within their own population, and other ungulates that they come in contact with, damaging the populations (3). 
Preliminary data:
Our preliminary data is based on the possible immune activating capability of a Type V protein named Hia in Brucella Strain RB51, an adhesin homolog of Heamophilus. Our group is using this gene inside a nicin induced expression system (NICE) present in the Strain Lactococcus lactis.
Justification: 
Current methods involve inefficient intravenous procedures, and unnecessary risk to veterinarians administering the vaccine. There have been steps within the research community to advance an alternative vaccine using a non-pathogenic microbe that expresses the B. abortus surface antigens. This probiotic vaccine would reside in the gut of the rumens allowing safe protection from B. abortus.  The live vaccine in the form of Lactococcos lactis is a possible route to immunity without the risk of fetal abortion or human infection. From research we are able to gain knowledge of some conditions that will be faced, however there still lies many unknowns. Initially we will be seeking to better understand the growth curve of the transformed L. lactis in comparison to the wild type strain. In addition we are attempting to test the transformed L. lactis against rumen and gastrointestinal conditions.
 
Objectives: