Evaluation of lyophilized bacteriophage cocktail efficiency against multidrug-resistant Salmonella in broiler chickens

Abstract

Currently, phage biocontrol is increasingly used as a green and natural technology for treating Salmonella and other infections, but phages exhibit instability and activity loss during storage. Therefore, in this study, the effects of lyophilization on the activity and stability of phage cocktails for the control of multidrug-resistant Salmonella in broiler chickens were determined. Eight serotypes of Salmonella were isolated and identified from broiler chicken farms, and bacteriophages against multidrug-resistant Salmonella enterica subsp. enterica serovar Kentucky, Salmonella enterica subsp. enterica serovar Typhimrium and Salmonella enterica subsp. enterica serovar Enteritidis were isolated. The bacteriophage cocktail was prepared and lyophilized, and it was subjected to in vitro and in vivo examinations. A reconstituted lyophilized bacteriophage cocktail was used for the oral treatment of chicks before and after challenge with multidrug-resistant S. Kentucky. The colonization of cecum by S. Kentucky was detected by using real-time PCR, and the serum levels of IgM, IgA and IL-4 and pathological changes in the different groups were detected. Three Caudovirales phages families were identified including Autographiviridae, Straboviridae and Drexlerviridae against multidrug-resistant S. Kentucky, S. Typhimrium and S. Enteritidis. The groups treated with the bacteriophage cocktail showed no clinical signs, no postmortem lesions, and a mortality rate of 0%, which improved the growth performance parameters. Additionally, the estimated serum levels of IgM, IgA and IL-4 were significantly greater in the bacteriophage cocktail-treated groups. Lyophilization effectively preserves the long-term storage stability of phages. Therefore, lyophilized bacteriophage cocktail therapy is a valuable approach for controlling multidrug-resistant Salmonella infections in broiler chickens.

Keywords: S. Kentucky; Biocontrol; Phage stability; Poultry farms.

Fig. 1

Transmission electron microscopy photograph of isolated bacteriophages. A & B: belong to Siphoviridae family, C: is belonging to Myoviridae family. D, E & F: belong to Podoviridae family

Fig. 2

Transmission electron microscopy photograph of isolated bacteriophages after lyophilization and rehydration

Fig. 3

Clinical signs and postmortem changes of the experimental chicks in 2nd group (positive control) which challenged with S. Kentucky. A, B and C: congested liver with petechial hemorrhages. D and E: unabsorbed and enlarged yolk sac. F and I: enlarged cecum filled with diarrhea. G and H: chicks showed reluctant to move with closed eyes

Fig. 4

Amplification plot of RT- PCR for S. Kentucky colonization in the examined cecal samples. S. Kentucky not detected in the cecal samples of the 1st group (samples) and 4th group (samples ). Meanwhile, cecal samples in 2nd group (samples) and 5th group (samples) showed S. Kentucky colonization with 1.160 × 10⁵ to 1.554 × 10⁵ and 1.407 × 10¹ to 5.068 × 10², respectively

Fig. 5

The curve of mean with standard error (mean ± SE) for IgM (ng/ml), IgA (ng/ml) and IL-4 (Pg/ml) results in the different 5 groups. 1st group: negative control, 2nd group: challenged with S. Kentucky without treatment, 3rd group: treated with bacteriophages cocktail without challenge, group 4: treated with bacteriophage before the challenge, 5th group: treated after the challenge

Fig. 6

Histopathological lesions in liver and cecal tissue for the negative control chicks (1st group; no challenge and no treatment) with H & E (hematoxylin and eosin), 100×. A: Liver with normal tissue architecture and cellular details. B: Cecum with normal mucosa, muscularis, submucosa and serosa

Fig. 7

Histopathological lesions in liver and cecal tissue for the challenged chicks without treatment (2nd group) with H&E (hematoxylin and eosin), 100×. A: liver with perivascular fibrosis and edema (thick arrow) with severe congestion of hepatic blood vessels (arrowhead) and proliferation of bile ductules (thin arrow). B: Liver with focal area of inflammatory mononuclear cells infiltration (arrow) and mild sinusoidal dilation (arrowhead). C: Liver with focal necrotic area (arrow) with perivascular fibrosis (arrowhead). D: Cecum with severe congestion of submucosal blood vessel (arrow). E: Cecum with fusion of some intestinal villi (arrowheads). F: caecum with edema beneath submucosa (arrow) with focal distortion and aplasia of submucosal glands(arrowhead)

Fig. 8

Histopathological lesions in liver and cecal tissue for the 3^rd* group which was treated without challenging and 4^th** group which was treated before challenging with H & E (hematoxylin and eosin), 100×. A*, D** and E**: liver with normal tissue architecture and cellular details. B*, C* and F**: Cecum with normal mucosa, muscularis, submucosa and serosa

Fig. 9

Histopathological lesions in liver and cecal tissue for the treated chicks after the challenge (5th group) with H&E (hematoxylin and eosin), 100×. A: liver with focal edema and atrophied hepatocytes. B: Liver with diffuse vacuolation of hepatocytes. C: Liver with focal mononuclear cells infiltration. D: Liver with perivascular coagulative necrosis and pyknotic nuclei. E: Cecum with diffuse congestion in submucosal blood vessel. F: Cecum with edema under submucosa