Reducing Salmonella Horizontal Transmission During Egg Incubation by Phage Therapy Ana Henriques, 1 Rui Sereno, 1 and Adelaide Almeida 2 Abstract Salmonella is a serious problem for both animal production and public health worldwide. Contaminated poultry is the main vehicle of Salmonella and the most important serotype is Salmonella enterica serovar Enteritidis. In order to test the efficiency of bacteriophages to treat Salmonella enterica serovar Enteritidis infections in poultry, a cocktail of two phages, F1055S and F12013S, isolated from chicken litter was applied by aerosol spray on fertile eggs challenged with Salmonella Enteritidis. To the best of our knowledge, this is the first experiment in which phages were applied by spray on fertile eggs. Two groups of eggs were challenged with Salmonella (3 · 10 8 colony-forming units [CFU]/mL) and one of them was treated with the phage suspension (2 · 10 6 plaque- forming units [PFU]/mL). A third group was used as nonchallenged and nontreated control. The phage treatment of challenged Salmonella eggs reduced the disease symptoms in the chicks. The arthritis and pasting after 8 days in the challenged and treated group were similar to those normally occurring in chicks (non- challenged and nontreated chick control group) ( p = 1.000 and p = 0.828, respectively, for arthritis and pasting) and were significantly lower than the challenged but nontreated ones ( p = 0.017 and p = 0.002 for arthritis and pasting, respectively). The phage-treated group did not lose weight, showing an average weight similar to that of the nonchallenged control group and higher than that of the challenged nontreated group. The results of this study suggest that the application of phages by aerosol spray during the transfer of the eggs from incubators to hatchers may be an effective and inexpensive approach for reducing the horizontal transfer of Salmonella in poultry. Introduction S almonella is a major cause of foodborne disease in the developed world, causing gastrointestinal infections, commonly acquired through the consumption of under- cooked food products derived from poultry (Favrin et al., 2001). Salmonella may be introduced into poultry flocks hor- izontally from different sources, including direct transmis- sion between flocks, contaminated feeds, biologic vectors (insects, rodents, wild birds, humans), as well as via vertical transmission (Toro et al., 2005). Salmonella Enteritidis is one of the most important Salmonella serotypes associated with chicken meat and eggs (Fiorentin et al., 2005a). The factors that are responsible for its epidemic spread still remain un- clear. Epidemiological investigations indicate that laying flocks become infected directly from the farm environment (Van de Giessen et al., 1994; Omwandho and Kubota, 2010). However, it is also known that egg contamination by Sal- monella Enteritidis may occur by vertical transmission in the reproductive tract before deposition of the shell (Omwandho and Kubota, 2010). Traditional measures of salmonellosis prevention and control include the use of prebiotics and probiotics, safety measures, vaccination and, mainly, antibiotics (Borie et al., 2008). However, concerns related to antibiotic-resistant bac- teria have stimulated interest in alternative treatments for bacterial infections. Among these therapies, a special interest has been given to phage therapy (Fiorentin et al., 2005b). Recent research using bacteriophages has been focused on treating enteric infections in poultry (Bielke et al., 2007). The bacteriophages could be useful as therapeutic or prophylactic agents providing a natural, nontoxic, feasible, and inexpen- sive approach for Salmonella control in poultry (Borie et al., 2009). Nonetheless, additional work is needed in order to develop effective and practical phage therapy protocols as well as to select the appropriate method of phage delivery. In most studies undertaken to control Salmonella En- teritidis, phages were administered orally. Several studies have shown that phages thus administered significantly 1 Department of Research and Development, Controlvet, Seguranc ¸a Alimentar, SA, Tondela, Portugal. 2 Department of Biology and CESAM, University of Aveiro, Campus Universita ´rio de Santiago, Aveiro, Portugal. FOODBORNE PATHOGENS AND DISEASE Volume 10, Number 8, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2012.1363 718 For Review Only Not Intended for Distribution or Reproduction