Performing Department
Pathology, Microbiology & Immunology
Non Technical Summary
Parjaroellobacter abortibovis (P.a.) is the causative agent of epizootic bovine abortion (EBA or foothill abortion), a devastating and costly disease that results in late term abortions or weak calves in pregnant naïve cattle pastured in dry foothill and mountainous areas of CA, NV, OR and AZ. Incubation periods in animals suggest a slow replication rate. Limited in vitro replication has been achieved in splenic white blood cells harvested from P.a.-infected immunodeficient (SCID) mice. Evaluating growth and determining replication rates in a slow-growing intracellular pathogen is difficult. Our approach is to use techniques for enumerating intracellular bacteria with immunofluorescence (IFA). This IFA-based assay was used in the current project to demonstrate its utility in the study of microbial pathogenesis. The techniques are now providing critical insight into establishing optimal media and atmospheric conditions for bacterial growth. Upon optimization of P.a. culture conditions, research will focus on identifying a susceptible primary or established cell line such as McCoy cells and uterine epithelial cells, respecitively.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Pajaroellobacter abortibovis is an intracellular bacterial pathogen and the etiologic agent of epizootic bovine abortion (commonly referred to as foothill abortion). The bacteria has only been propogated in the developing bovine fetus and immunodeficient mice. Project goals include the following: Phenotype Pajaroellobacter abortibovis-infected murine host cells using murine leukocyte-specific monoclonal antibodies. Compare bacterial replication in infected murine splenocytes using 8% vs. 5% CO2. Bacterial replication will be optimized in two basal media (RPMI-1640 and DMEM) by varying concentrations of glycine (Glc), sodium pyruvate (Pyr), L-glutamine (L-Gln) and non-essential amino acids (NEAA). Apply optimized culture conditions to study P.a. pathogenesis in Mφs and identify alternate cell culture systems suitable for bacterial passage and propagation. Analyze available genome information to identify the presence or absence of P.a. genes within the glycolysis pathway and utilize the knowledge to test the hypothesis that P.a. will replicate more efficiently with alternate carbon sources due to the apparent replacement of PfkA enzyme with pfkB in P.a..
Project Methods
Splenocytes will be recovered from SCID mice infected with P.a.. Single cell suspensions will be applied to 8-well glass tissue culture slides at 1 x 10^6 cells/well and allowed to adhere over a 2-day period. Cells will be acetone-fixed at 3-day intervals up to Day 12; slides will be stored at -20 °C. Slide wells will be probed with a combination of AF488 anti- P.a., AF594 anti-mouse CD68 and DAPI to identify bacteria, Mφ cytoplasm and cell nuclei, respectively; analysis will be by 3-color fluorescence microscopy. A matching well for each treatment will be probed with appropriate AF488- and AF594- irrelevant antibodies. Each well will be divided into 9 quadrants and representative fields in each quadrant will be photographed followed by cell and bacteria enumeration. All experiments will be performed in triplicate.Adherent cells will be probed with α-Ms CD68, α-Ms F4/80, α-Ms CD11b and α-Ms CD11c to better define macrophage type and origin (Hey et al, 2012).Splenocytes infected with P.a. will be cultured in 5% vs. 8% CO2.Splenocytes infected with P.a. will be cultured with 10% FBS in a variety of media combinations to compare bacterial replication in RPMI vs. DMEM; 1 g/L vs. 4.5 g/L Glc, 0.11 vs. 0.22 g/L Pyr, 0.3, 0.6 and 0.9 g/L L-Gln and the addition of 1% non-essential amino acids (NEAA)..Splenocytes infected with P.a. will be cultured in the presence of IFNg at two concentrations (0.8 and 8.0 ng/ml) to determine if this cytokine promotes bacterial clearance. Controls will consist of media with 1.0ng/ml IL-10 and media lacking cytokines.McCoy cells (ATCC 1696) and primary murine uterine epithelial cells will be cultured in optimal culture conditions and infected by addition of either cryopreserved P.a.-infected murine spleen supernatants or disrupted P.a.-infected Mφs after passage through 3.5" x 22g needles. Hypotonic shock with KCL or addition of peptide Arg-Gly-Asp may be used in an attempt to improve cell uptake of bacteria.Utilize UniProt to identify key genes associated with glycolysis in the P.a. genomeSubstitute gluconate, glucose-6-P, arabinose or glycerol for glucose in cultures for optimization of bacterial growth