In this period, to accomplish the production of commercial batches of phage cocktail, a new phage producer company was subcontracted produced different batches of the PhagoVet product used in the diverse tasks performed during this term. For this, it was necessary to conduct and provide the company with protocols related to phage lysate production, PCR detection of phages, and PCR detection of production strains DNA, among others, to ensure a product achieved EFSA requirements. In this sense, concerning the effectiveness of the PhagoVet biocide product, three different batches of the PhagoVet cocktail have been tested in experiments performed following the standard regulations of the evaluation of the bactericidal activity of chemical disinfectants and antiseptics (biocides) used in the veterinary area on nonporous and porous surfaces. To do this, the regulations were adapted to the specific nature of bacteriophages. Results demonstrated a significant reduction of Salmonella concentration in liquid and nonporous surfaces under two different soiling conditions, achieving the log10 reduction required by the corresponding standards. A low effect was observed on porous surfaces. Regarding "Assessment of Phagovet Zootechnical feed additive”, the efficacy and tolerance PhagoVet zootechnical feed additive against Salmonella has been assayed by in vivo trials in broilers and layer hens’ farms in two different geographical locations in Europe. A total of 10 independent animal trials have been performed, from which two are proof concept trials, seven are the efficacy trials, and one is the safety trial, performed following the Regulation (EC) 1831/2003. Two different serovars of Salmonella have been used for infection, and different conditions of infection and administration of phage cocktail via drinking water have been assayed. Altogether, results obtained in some trials demonstrated the efficacy of PhagoVet treatment in broilers. Significant differences were evidenced for salmonella counts in boot swabs at the endpoint of 35 days and positively impacted on zootechnical performance.
Significant effort was also put into the preparation of dossiers for evaluation of both Phagovet feed additive and biocide by corresponding agencies. For this, different studies including characterization and sequencing of bacteriophages and Salmonella host production strains and others associated with the safety of the product, including consumer and user safety, have been accomplished. Regarding the evaluation of the PhagoVet product as a biocide, competent authorities of almost 25 EU Member States were contacted to take care of the risk assessment and evaluation of the PHAGOVET biocidal product. Nevertheless, most of them declined the request alleging an excess workload and lack of expertise in the assessment of bacteriophages. We will continue working on preparing the register dossier and searching for an authority available to review it. The dossier of the PhagoVet feed additive is under preparation, and it is expected to send it for evaluation in the short term.
Finally, the final commercialization and marketing plan has been designed and focused on the development of the zootechnical feed additive and biocide to control Salmonella.
The development of a feed additive to control the presence of E. coli in poultry production was also contemplated in the project. Due to budget and regulatory constrainments (after the register pathway as feed additive for the E. coli PHAGOVET product would not be suitable or accepted by the EU authorities and should be registered as veterinary product) and thus the work. was focused in WP1, WP2 and WP3, aiming to gather knowledge and data to develop a product prototype and sustain a potential register of the E. coli phage solution as a veterinary product in the future. A total of 44 E. coli phages were isolated and characterized functionally morphologically where 32 phages were found to be either lysogenic or to have toxin or antibioresistance genes making them not suitable for therapy. The remaining 12 would be suitable for integrating a cocktail formulation. However, the lytic spectra of those phages were very narrow to cover the genetic diversity of E. coli isolated from the field, thus making it difficult to produce a fixed formulation cocktail that would work as a broad-spectrum solution. In the case o E. coli treatment we found that tailored formulations designed to kill the bacteria that is actually infecting the animals would be the best treatment option.
