Isolation and identification of the broad-spectrum high-efficiency phage vB_SalP_LDW16 and its therapeutic application in chickens

Abstract

Background: Salmonella infection in livestock and poultry causes salmonellosis, and is mainly treated using antibiotics. However, the misuse use of antibiotics often triggers the emergence of multi-drug-resistant Salmonella strains. Currently, Salmonella phages is safe and effective against Salmonella, serving as the best drug of choice. This study involved 16 Salmonella bacteriophages separated and purified from the sewage and the feces of the broiler farm. A phage, vB_SalP_LDW16, was selected based on the phage host range test. The phage vB_SalP_LDW16 was characterized by the double-layer plate method and transmission electron microscopy. Furthermore, the clinical therapeutic effect of phage vB_SalP_LDW16 was verified by using the pathogenic Salmonella Enteritidis in the SPF chicken model.

Results: The phage vB_SalP_LDW16 with a wide host range was identified to the family Siphoviridae and the order Caudoviridae, possess a double-stranded DNA and can lyse 88% (22/25) of Salmonella strains stored in the laboratory. Analysis of the biological characteristics, in addition, revealed the optimal multiplicity of infection (MOI) of vB_SalP_LDW16 to be 0.01 and the phage titer to be up to 3 × 10¹⁴ PFU/mL. Meanwhile, the phage vB_SalP_LDW16 was found to have some temperature tolerance, while the titer decreases rapidly above 60 ℃, and a wide pH (i.e., 5-12) range as well as relative stability in pH tolerance. The latent period of phage was 10 min, the burst period was 60 min, and the burst size was 110 PFU/cell. Furthermore, gastric juice was also found to highly influence the activity of the phage. The clinical treatment experiments showed that phage vB_SalP_LDW16 was able to significantly reduce the bacterial load in the blood through phage treatment, thereby improving the pathological changes in the intestinal, liver, and heart damage, and promoting the growth and development of the chicken.

Conclusions: The phage vB_SalP_LDW16 is a highly lytic phage with a wide host range, which can be potentially used for preventing and treating chicken salmonellosis, as an alternative or complementary antibiotic treatment in livestock farming.

Keywords: Biological characteristics; Phage therapy; Phages vB_SalP_LDW16; Recovery rate; Salmonella.

Fig. 1

Isolation and purification of the Salmonella phage vB_SalP_LDW16. A Sample filtrate displays the clear phage plaques on the double-layer plate overlays with suspensions of Salmonella after incubation for 24 h, depicting lysed bacteria, 1–8 represent partial filtrate samples. B The phage vB_SalP_LDW16 crude filtrate. After 24 h of incubation, clear and different sized plaques were displayed on the Salmonella lawn. C The purified phage vB_SalP_LDW16 displays clear and uniform patched plaques on a Salmonella lawn after incubation for 24 h

Fig. 2

Micrograph and categorization of the phage vB_SalP_LDW16. A The transmission electron micrograph of the phage vB_SalP_LDW16 virus particles was negatively stained with 2% uranyl acetate. The scale bar represents 100 nm. B The agarose gel electrophoresis analysis of the nucleic acid of the phage vB_SalP_LDW16 after restriction endonuclease digestion. Lane 1, M, 15 kb DNA ladder; Lane 2, vB_SalP_LDW16, undigested; Lane 3, vB_SalP_LDW16 digested with DNase Ι; Lane 4, vB_SalP_LDW16 digested with RNase A; Lane 5, vB_SalP_LDW16 digested with Mung Nuclease

Fig. 3

Biological characteristics of the phage vB_SalP_LDW16. A MOI of the phage vB_SalP_LDW16. Phage titers were measured at different MOIs. B One-step growth curve of the phage vB_SalP_LDW16. The titer of the phage vB_SalP_LDW16 was determined every 10 min. C The thermal stability of the phage vB_SalP_LDW16. The phages were cultured in a water bath at different temperatures. **p < 0.01, ***p < 0.001 versus samples at 40 ℃ under the same incubation time, ^###p < 0.001 versus sample at 20 min at the same incubation temperature. D The pH stability of the phage vB_SalP_LDW16. The phages were incubated in different acid–base environments for 1 h. A, B, C, D The phage titers were determined using the double-layer agar method and each data point represented the mean values ± standard deviations (SD) from at least three replicate experiments

Fig. 4

Recovery rate of the phage vB_SalP_LDW16. A The intestinal juice recovery rate of the feeding phage in the chickens in vivo; B Intestinal juice recovery rate of phage in the chickens in vitro; C Gastric juice recovery rate of phage in the chickens in vitro. A, B, and C The phage titers were determined using the double-layer agar method. Each data points represent the mean values ± standard deviations (SD) from at least three replicate experiments

Fig. 5

Therapeutic effect of the phage vB_SalP_LDW16 on the chicken model infected with Salmonella. A Survival rates. The chickens were intraperitoneally injected with 10⁸ CFU/mL of S. Enteritidis S64. Two hours later, the chickens were orally fed with 10⁶ PFU/mL of phage, amoxicillin (2.5 mg/mL), florfenicol (2 mg/mL), and neomycin (100 mg/mL). Chickens injected with bacteria, phage or saline only were set as a control group; B Dynamic changes in the bacteria in the blood in different groups. C Dynamic changes in the phage in the feces in different groups. D Histopathological images of the representative tissues and organs. The red arrow represents the lesion area. Intestine (HE, 100 ×), Heart (HE, 400 ×), Liver (HE, 400 ×), Kidney (HE, 400 ×). E The average weight of the different groups at the end of the experiment. The error bars indicate the standard deviations. Asterisks and pound signs indicate a statistically significant difference (P < 0.05), in which “#” (#p < 0.05, ##p < 0.01) are compared with the blank control group and “*” (*p < 0.05, **p < 0.01) are compared with the phage-treated group