CHARACTERIZATION OF LYTIC BACTERIOPHAGES WITH THERAPEUTIC POTENTIAL AGAINST SALMONELLA ENTERITIDIS IN POULTRY

Clarissa Silveira Luiz Vaz Vaz (CNPSA); Daiane Voss-rech Voss-rech (CNPSA); Luana Alves Alves (CNPSA); Diana Gritti Gritti (CNPSA); Iara Maria Trevisol Trevisol (CNPSA)

The oral administration of three genetically distinct lytic bacteriophages (LB) isolated from poultry feces by Embrapa Swine and Poultry has been able to reduce Salmonella Enteritidis in poultry caecum. These LB, which show tailed morphology and double-strand DNA as nucleic acid, have been tested as therapeutic agent against S. Enteritidis in broiler chickens. However, concerns have been raised about phage therapy safety. Bacteriophages chosen for therapeutic use need to be well characterized and the phage biology understood. The aim of the present study was improve the characterization of these LB. Analysis of the phage genome was carried out by the digestion of the extracted DNA with EcoRI, BamHI and HindIII restriction enzymes. Genomic DNA was also analyzed by random amplified polymorphic DNA (RAPD), whose typical fragments of each LB were purified and cloned for DNA sequencing. In order to assess the LB proteins patterns, SDS-PAGE was performed in proteins extracted from the LB pool containing 3X10⁷ PFU/mL of each BL, which was administrated by drink water to 3 days-old SPF broiler chickens for 4 successive days. Following that phage administration, the dynamic in the SDS-PAGE patterns was analyzed in proteins extracted from LB isolated from cloacal swabs collected from 7, 10, 13 and 16 days-old broiler chickens. Identity of LB isolated was confirmed by amplified fragment length polymorphism (AFLP) patterns. Phages DNA were digested only by HindIII. Sequencing of 375, 499 and 435 nucleotides from cloned DNA fragment chosen from each LB showed no significant homologies between other bacteriophages DNA sequences described, although higher similarities were identified against DNA sequences from families of tailed phages. Furthermore, all LB shared the same SDS-PAGE pattern, which displayed a major structural protein of 50kDa. On the other hand, there were no differences between the SDS-PAGE protein patterns from LB administrated to the broiler chickens and LB isolated from cloacal swabs, suggesting that these BL have not suffered protein changes into the avian gut during the period analyzed. The LB isolated from cloacal swabs showed the same AFLP pattern identified in LB before administration to the broiler chicken. More research involving the in-vivo activity of these LB might help to develop an effective phage therapeutic product against S. Enteritidis.