Characterization of a novel bacteriophage φCJ22 and its prophylactic and inhibitory effects on necrotic enteritis and Clostridium perfringens in broilers

Abstract

High necrotic enteritis ( NE ) incidence and mortality rates in poultry can be caused by Clostridium perfringens ( CP ) coinfected with Eimeria spp., a causative agent of coccidiosis. Banning of prophylactic use of antibiotics in feed has been accompanied by increased NE outbreaks, resulting in economically devastating losses to the broiler industry. To determine alternatives for controlling NE, we isolated CP-specific bacteriophages (BP), characterized their properties, evaluated their inhibitory effects on pathogenic CP, selected a highly effective phage (φCJ22), and used φCJ22 as a dietary supplement in experimental NE-afflicted broiler chickens. Male broilers ( n = 780) were randomly assigned to 60 pens ( n = 13 broilers/pen) and into 5 groups [CP-uninfected negative control ( NC ), basal diet ( BD ) without CP and BP; CP-infected positive control ( PC ), BD + CP; and 3 BP groups receiving low- ( LP ; BD + CP+10 5 BP), medium- ( MP ; BD + CP+10 6 BP), and high-phage ( HP ; BD + CP+10 7 BP plaque-forming units/kg) concentrations]. The results showed that MP and HP groups presented an antimicrobial activity toward clinical CP isolate strains, and the groups decreased NE lesions and mortality rates without changes in chicken performance at the end of the experimental period. After CP-challenge body weight gain and feed efficiency were significantly lower in phage-fed groups than that in the PC group ( P < 0.05), and NE-associated mortality was the lowest in the HP group ( P < 0.001). Moreover, histopathology revealed lesser gastrointestinal mucosal damage in the NC and BP-treated (LP, MP, and HP) groups than that in the PC group, and MP and HP significantly lowered viable CP number in the cecum content by up to 1.24log10 relative to only CP-infected PC group ( P < 0.05). These findings suggest that addition of φCJ22 to chicken feed might effectively ameliorate NE, which is accompanied by reduced CP strains in the gut and compensate the performance of NE-afflicted broilers.

In Vivo Recovery of Bacteriophages and Their Effects on Clostridium perfringens-Infected Broiler Chickens

“…A corn and soybean meal-based diet was used as the control diet ( Table 1 ), and the experimental diets were formulated by mixing the control diet with either powder (10 6 pfu/g of diet) or encapsulated (10 6 pfu/g of diet) BPs. BPs were originally isolated from chicken feces [ 8 ] and encapsulated using hot-melt fluidized bed coating process. Encapsulated BP was found to be stable in harsh conditions, such as high humidity and temperature [ 19 ].…”

“…Sci. 2022, 9, 119 3 of 13 strain harbored the NE B-like (NetB) gene [8] and was anaerobically incubated in fluid thioglycolate (Sigma-Aldrich, St. Louis, MO, USA) broth at 37 • C before oral gavage. The scheme of the experimental schedule is shown in Figure 1.…”

“…Consequently, alternative veterinary and nutritional methods, such as the use of bacteriophages (BPs), vaccines, or probiotics, have been proposed as potential strategies for preventing NE [ 8 , 9 , 10 ]. Among these alternatives, BP is an obligate parasite of bacteria that uses bacterial cells to replicate and has long been used in the fields of human and veterinary medicine, agriculture, aquaculture, and the food industry, with no safety concerns [ 11 ].…”

“…Among these alternatives, BP is an obligate parasite of bacteria that uses bacterial cells to replicate and has long been used in the fields of human and veterinary medicine, agriculture, aquaculture, and the food industry, with no safety concerns [ 11 ]. BP-expressed endolysins can destroy the peptidoglycan layer of pathogenic bacteria as novel antimicrobial agents [ 12 ], and BPs exhibit antibacterial effects against C. perfringens in broiler chickens [ 8 , 9 ]. BPs show antibacterial specificity against various bacteria, including colibacillosis, Salmonella gallinarum, Campylobacter and C. perfringens , in chickens [ 8 , 13 , 14 , 15 ].…”

“…BP-expressed endolysins can destroy the peptidoglycan layer of pathogenic bacteria as novel antimicrobial agents [ 12 ], and BPs exhibit antibacterial effects against C. perfringens in broiler chickens [ 8 , 9 ]. BPs show antibacterial specificity against various bacteria, including colibacillosis, Salmonella gallinarum, Campylobacter and C. perfringens , in chickens [ 8 , 13 , 14 , 15 ].…”