Biological properties of the Salmonella phage BF-1356 strain and its effectiveness in phage therapy of chicken pullorosis

Salmonella Pullorum is a rare but significant pathogen of the genus Salmonella, responsible for considerable damage to the poultry industry and economic losses due to high poultry mortality. The potential use of bacteriophage therapy as an adjunctive treatment for poultry has been the subject of considerable debate in recent times. This study demonstrates, for the first time, the therapeutic efficacy of virulent bacteriophage Salmonella phage BF-1356 administered 12 hours after oral infection of cross Cobb 500 chickens (Gallus gallus L.) with a highly virulent strain Salmonella Pullorum № 732-VIEV. The objective of this study was twofold: firstly, to characterize Salmonella phage BF-1356 and its interaction with the host bacterial strain in vitro; secondly, to evaluate the efficacy of phage therapy for pullorosis in vivo on the Cobb 500 chicken model. Salmonella strain (Salmonella Pullorum № 732-VIEV) was isolated in 2017 at a poultry farm in the Moscow region. The virulent bacteriophage Salmonella phage BF-1356 from the collection of the Gabrichevsky Research Institute of Epidemiology and Microbiology (Moscow, Russia), was used for phage therapy. The purified bacteriophage exhibited a titer of at least 1011 PFU/ml. The sterility of the phage preparation was verified through the incubation of 0.1 ml of phage lysate in 5 ml of fluid thioglycollate medium (HiMedia, India) at 32.5±2.5 and 22.5±2.5 °C for 14 days. The host range of bacteriophage lytic activity was determined using 164 bacterial strains of Salmonella enterica isolated in the Russian Federation between 2010 and 2018. The lytic activity was evaluated visually by a lysis zone at the points of bacteriophage application on indicator Petri dishes with 1.5 % Mueller-Hinton agar which contained 200 µl of a mid-log-phase culture of the tested strains (106 CFU). The adsorption of Salmonella phage BF-1356 on Salmonella Typhimurium strain B-1025 was evaluated. The latent period and burst size of Salmonella phage BF-1356 were determined by monitoring the dynamic changes in the number of phage particles during the replicative cycle on Salmonella Typhimurium B-1025. In order to evaluate the effect of pH on bacteriophage titer, known concentrations of purified bacteriophages were incubated in buffers at different pH values. Salmonella -free 14-day-old chickens of cross Cobb 500 ( n = 40) were used as laboratory objects. The chickens were randomly divided into three groups. Ten chickens were used to control housing conditions (I control group not exposed to infection and phage therapy), 20 chickens were used to determine the efficacy of the phage after its use for therapeutic purposes and to assess further Salmonella carriage (experimental group), and 10 chickens were used as infection control (II control group infected with Salmonella without bacteriophage administration). The 14 day-old chicks in the experimental and II control groups were orally infected with Salmonella Pullorum № 732-VIEV at a dose of 3.8½108 cells in 0.4 ml which corresponded to approximately 5 LD50 (the acute infectious dose that guaranteed the modelling of pullorosis infection with the complete death of control group subjects). The administration of bacteriophage was initiated upon the onset of symptoms indicative of the infectious process. The chicks in the experimental group were administered bacteriophage orally at a dose of 1010 PFU/ml in 0.2 ml. The presence of phages and Salmonella cells was promptly identified following the act of defecation. The quantity of phage particles was subsequently determined by the double-layer agar method. To confirm the efficacy of phage therapy, the birds in the experimental group were slaughtered for pathological and anatomical autopsy and collection of sectional material for laboratory studies and the identification of Salmonella cultures from organs. In the control group, pathological autopsy were conducted as the birds died. Salmonella phage BF-1356 demonstrated specific lytic activity against common Salmonella serovars, including Pullorum, Enteritidis, Typhimurium, Cholerasuis and Infantis. The mean latent period for bacteriophage was 40 minutes, with an average of 67 viral particles released per cell. The bacteriophage demonstrated stability at pH levels between 5 and 8, with a slight decrease in titer observed at pH 3.6 within the first hour. A single oral administration of bacteriophage BF-1356 in a titer of 1010 PFU/ml 12 hours after infection of Cobb 500 chickens with Salmonella Pullorum strain № 732 resulted in 0 % mortality in the population and a significant reduction in the consequences of the pathological process in the chickens after phage therapy. The chickens in the control group exhibited mortality within a period of 2-6 days. The structure of the pathological lesions was consistent with that observed in salmonellosis, a diagnosis that was subsequently confirmed in laboratory conditions.