Phage endolysins to control clostridia in poultry: the lytic activity of PlyCP41 enzyme using an ex vivo method

Authors: TIMMONS, JENNIFER - University Of Maryland Eastern Shore (UMES); BARNAS, MIKE - Ahpharma, Inc; DONOVAN, DAVID - Morgan State University; Skory, Christopher; Hammond, Rosemarie; SIRAGUSA, GREG - Scout Microbiology Llc

Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 8/26/2022
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Objective: The endolysin PlyCP41 is known to exhibit lytic activity against Clostridium perfringens (CP) cells in vitro and, therefore, could potentially be used in vivo to achieve similar results within the gastrointestinal tract (GIT). The objective of this study was to measure the inhibitory capacity of naked PlyCP41 enzyme in an assay containing gut fluids from the upper (crop, proventriculus, and ventriculus), middle (duodenum, jejunum, and ileum), and lower (ceca) GIT of 21d broiler chickens. Methods: A positive control (PC) was developed using a pure strain of C. perfringens (CP509) incubated at 37°C for 24h in FTG broth. The pellet was resuspended in 2 mL of PBS, then split into two aliquots: a PC containing CP cells only; and PC+enzyme containing the CP cells + 5% PlyCP41 enzyme. The PlyCP41 enzyme had a stock concentration of 15 mg/ml and was added to 1.0 ml of the PC assay at a 5% inclusion rate (15 mg/mL * 0.05 = 0.75 mg of enzyme). Chicken gut fluids were harvested and pooled by region (upper, middle, and ceca) from 3 birds at 21d of age. Contents were homogenized, then PBS buffer was added to half the contents from each region at a rate of 5% and vortexed to combine. The contents+PBS were incubated anaerobically at 40°C for 20 min, then plated to quantify the existing CFU/g of Clostridium perfringens without enzyme treatment. The remaining half of the gut contents had naked PlyCP41 enzyme added at a rate of 5%, then were vortexed to combine. The contents+enzyme were cultured using the same method to calculate CFU/g after treatment with the enzyme. Results: PlyCP41 naked enzyme reduced CP509 by >1.0 log (7.08 to 5.89 log10 CFU/g) in a pure CP assay. 0.75 mg of PlyCP41 per gram of gut fluids reduced CP by >1.0 log in the upper GIT (4.81 to 3.78 log10 CFU/g). A less significant effect (0.2 log reduction) was observed in the small intestine, and there was no reduction of C. perfringens in the ceca. Conclusions: These results indicate that the naked enzyme may not be effective in cecal contents but does elicit lytic activity in the low pH and lower viscosity environment of the upper GIT.