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A cost-effective solution for controlling Salmonella and Escherichia coli in poultry production

Periodic Reporting for period 3 - PHAGOVET (A cost-effective solution for controlling Salmonella and Escherichia coli in poultry production)

Periodo di rendicontazione: 2020-11-01 al 2022-10-31

The Phagovet project is devised to satisfy market demand for a more secure and reliable solution to control Salmonella infection in poultry farms. To date, salmonellosis is considered the main bacterial infection in the poultry sector having an important economic impact worldwide. In addition, among other causes, the overuse of antibiotic in farming has caused a rise in antimicrobial resistance a major problem in human health nowadays. In response to the unmet need, PhagoVet proposes a cost-effective alternative to antibiotics against Salmonella. PhagoVet consists of a biocide and one technological feed additive products based on selected bacteriophages able to kill the target bacteria. PhagoVet will be the first phage-based solution to control bacteria naturally and effectively in poultry production while avoiding side effects and residues associated to antibiotic use. It will reduce Salmonella infections by 20% Thus, PhagoVet provides a promising effective control measure for a concerning problem for poultry farmers and for human health.
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.
Antibiotics have been routinely used in poultry to promote animal growth or prevent disease outbreaks. Their overuse has led to drug-resistant strains, increasing antimicrobial resistance, a major problem, both in animal and human medicine.
In 2006 the EU implemented a ban on antibiotics as growth promoters and will implement new restrictive legislation in 2018. In 2017 the American Food and Drug Administration (FDA) limited the use of antibiotics only for treating or preventing disease under veterinary prescriptions. So, the need for alternatives to the use of antibiotics in intensive farming is now mandatory.
PhagoVet solution is based on bacteriophages patented technology proprietary of a consortium partner, combined with years of lab research and experience in the microbiology field, to produce the 1st bacteriophage-based product for the effective control of Salmonella in poultry farms.
The project will tap into the global Animal Healthcare market as it will open the European and the global phage market not only by strengthening our industrial partners and boosting the research abilities, but also long-term impact on the EU economy and its employment. In addition, our goal is to provide poultry farmers with an efficient, cost-effective, easy-to-use and safe tool to combat Salmonella as an alternative to antibiotics. We are convinced that bacteriophages are the answer to this health and safety problem, and Phagovet will be a step towards an effective solution.
Broiler experimental trial, day 1 of life (Left) and Oral Salmonella challenge (right)
Bacteriophages morphology plaques
Phagovet rolldown
Laying hen’s experimental trial. Entry day (left) and after 20 wks (right)
Experimental facilities in Spain for Broiler (left) and laying hens (right)