Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Population Health and Pathobiology
Non Technical Summary
High pathogenic strains of Enterococcus cecorum (EC) continue to cause economic losses to the broiler chicken industry in all of the top 5 broiler producing states including North Carolina. Routine farm hygiene procedures and antimicrobial therapy have proven insufficient to control outbreaks of pathogenic EC; therefore, it is apparent that a safe and efficacious vaccine will be essential to control this important emerging pathogen of broilers. Through previous CVM Research Grants, we have identified 4 virulence gene targets, cbp2, lpxtg3, capC and capD which are 1) homologous to known virulence genes of other Gram positive organisms and 2) significantly enriched in pathogenic EC. We hypothesize that these genes contribute to virulence in pathogenic EC.
Animal Health Component
30%
Research Effort Categories
Basic
20%
Applied
30%
Developmental
50%
Goals / Objectives
Our overall goal is to produce a safe and effective, modified live vaccine to control EC-associated disease. This proposal will produce attenuated strains of pathogenic EC which are the necessary first step in vaccine development.
Project Methods
We have recently developed an embryo lethality assay (ELA). As this bioassay successfully identifies virulent strains; we will employ the ELA to screen our mutant strains for attenuation. Once attenuation is confirmed in the ELA, we will assess the ability of attenuated strains to 1) bind collagen and 2) evade macrophage killing. A collagen binding assay will be utilized which allows for quantification of bacteria attached to collagen in microtiter plate wells by staining with crystal violet (CV) as previously described.15 Chicken type II collagen (Sigma-Aldrich) is commercially available and will be used to coat flat bottom microtiter plates. Wells will be blocked with BSA, washed with PBS, inoculated with 107 cfu/per well of each bacterial strain and incubated at 37°C and 5% CO2 overnight. Adhered bacteria will be stained with crystal violet; washed twice with PBS and adhered bacteria will be quantified by solubilizing the crystal violet in ethanol/acetone (80:20) and measuring the absorbance at 595nm. Absorbance values will be compared using a Student's t-test. A macrophage killing assay will be performed using established methods.16-19 Chicken peritoneal phagocytes will be infected in vivo by inoculating 108 cfu of EC into the coelom of healthy 6-9 week old chickens. Phagocytes will be collected 4 hr later by flushing the coelomic cavity with sterile heparinized (.5U/ml) saline and this coelomic wash will be centrifuged at 100Xg for 25 min. Collected phagocytes will be washed with PBS, re-suspended in RPMI with vancomycin (1 μg/ml), mutanolysin (400 U/ml) and lysozyme (0.94mg/ml) and incubated at 37°C and 5% CO2 for 2 hr to kill extracellular bacteria.19 Following trypan-blue staining to enumerate live leukocytes, equal numbers of phagocytes will be seeded into a 96 well plate at 0.5x106 WBC/well. One set of triplicate wells will be immediately lysed (T0) with TritonX-100, and viable EC enumerated (CFUs) using serial dilutions and plate counts. The remaining wells will be incubated in RPMI without antibiotics for up to 72 hr with sets of wells collected for intracellular bacterial counts at 6, 24, 48 and 72 hr. Percent survival will be calculated and compared between wild-type and deletion mutants using a Student's t-test.