Phage therapy has shown promise as an effective pre-harvest intervention by controlling foodborne pathogens in animals before they enter processing plants,2–4
with several studies indicating that phage therapy is effective against a broad range of foodborne pathogens belonging to the genera Salmonella, Campylobacter, Listeria
Phage therapy has been investigated for efficacy in red meat producing animals (cattle, sheep, swine) and white meat producing animals (poultry). Several studies have investigated the use of phages either alone, or in a cocktail, to control foodborne pathogens in sheep and cattle.2–4,10
Generally, the use of only one phage in some experiments has led to resistance,2,10
while the use of multiple phages in a cocktail has decreased the chance of developing resistance to a single phage.4,7,11
Phage based interventions have been aimed at controlling Escherichia coli
serotype O157:H7 in cattle and other ruminants. This bacterial pathogen causes a myriad of foodborne disease manifestations, including diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura.12,13
Cattle and other ruminants are considered to be the principal reservoirs of E. coli
and the contents of the intestines and fecal material on the hide may contaminate meat during slaughter. Animals that shed high levels of this pathogen may pose an elevated risk of contaminating the food chain if presented to slaughter, and phage-based approaches to reduce fecal shedding of this pathogen have been designed to limit both the duration of shedding and concentration of E. coli
O157 in the bovine gastrointestinal tract. For example, Callaway et al.2
inoculated sheep with phages specific against E. coli
O157:H7. This treatment caused a decrease in the concentration of E. coli
O157:H7 throughout the gastrointestinal tract, but the differences were not statistically significant.2
Bach et al.10
evaluated the ability of a single phage (DC22) to eliminate E. coli
O157:H7 in experimentally inoculated sheep. After introduction of phage DC22 to the sheep, there was no observed effect on the fecal shedding of E. coli
O157:H7, probably due to nonspecific binding of the phage to food particles and other debris present in the rumen and gastrointestinal tract which may have ultimately limited their efficacy. The use of one phage in the study by Bach et al.10
strengthens the idea that a mixture of phages might be more effective at controlling E. coli
O157:H7 in livestock than a single phage.
As such, other research groups have also evaluated the use of phage cocktails to decrease various bacterial pathogens in livestock during brief time periods. Callaway and coworkers15
anaerobically isolated phage that targeted E. coli
O157:H7 from fecal samples collected from commercial feedlot cattle in the central US. The host range of the phages was determined, and the phages were combined to form a cocktail of phage for in vivo studies. When a 21-phage cocktail was inoculated into sheep artificially contaminated with E. coli
O157:H7, intestinal populations of E. coli
O157:H7 were decreased (p < 0.05) in the cecum and rectum, a result that indicates that properly selected phages can be used to reduce E. coli
O157:H7 in food animals. The authors concluded that phage therapy approaches could be an important part of an integrated foodborne pathogen reduction program. Raya et al.16
isolated a bacteriophage, CEV1, from sheep that were resistant to E. coli
O157:H7 colonization, and used this phage to reduce populations of E. coli
O157 in sheep. In vitro, CEV1 efficiently infected E. coli
O157:H7 grown both aerobically and anaerobically. Four sheep were treated once orally with 1011
PFU of phage CEV1, 3 d after challenge with E. coli
O157. Sheep receiving a single oral dose of CEV1 showed a 2–3 log-unit reduction in cecal and rectal E. coli
O157:H7 levels within 2 d compared with levels in the controls, although rumen concentrations remained unchanged.
This same research group later combined phage CEV1 with a newly isolated phage, CEV2, which had high specificity for E. coli
O157:H7, and showed that cocktail was very effective at reducing E. coli
O157:H7 by 3 log units compared with the untreated phage-free control.17
The authors concluded the phage cocktails are more effective than individual phages at reducing E. coli
O157:H7 populations in the ruminant gastrointestinal tract.17
These studies show that phage therapy may be useful to decrease E. coli O157:H7 counts in adult livestock, and may provide a viable method to reducing E. coli O157:H7 in live animals immediately before slaughter. Other studies have addressed the question of using phages to control shedding of pathogenic bacteria in younger animals.
For example, Waddell et al.18
treated weaned 7- to 8-week old calves with a six phage cocktail up to 7 d before infection with E. coli
O157:H7. The results indicated that most of the untreated calves shed E. coli
O157:H7 in their feces for at least 12–16 d. In contrast, the treated calves stopped shedding E. coli
O157:H7 after day 8, which corresponded with a dramatic increase in the concentration of phages that were shed in the feces of the animals. The increase in the number of phages that were excreted was due to phage replication in the calves, as determined by the fact that such a result was not observed in uninfected control calves that were only inoculated with the phage cocktail. Chase et al.19
used a 37-phage cocktail in an attempt to reduce shedding in Black Angus calves ranging from 4–6 mo of age. The calves were orally inoculated with E. coli
O157:H7 in two separate trials. The first trial evaluated ileal samples and the second trial evaluated fecal samples for the presence of E. coli
O157:H7 and phages. In the first trial, a significant decline in the concentration of E. coli
O157:H7 was observed at 8 h (p < 0.05). However, the concentration of E. coli
O157:H7 increased back to the concentration of the control samples at 16 h. In the second trial, shedding of E. coli
O157:H7 decreased significantly in the treated group (p < 0.05) at 24 h. As with the ileal samples, an increase in the concentration of E. coli
O157:H7 was observed at 36 h. The increases in cell concentration were associated with a decrease in phage concentration. None of the E. coli
O157:H7 cultured from the ileal or fecal samples showed resistance to the phage cocktail. These results highlight the ability of the 37-phage cocktail to transiently reduce E. coli
O157:H7 in calves, without the formation of phage resistant mutants.
One concern that arises when phage therapy is applied to reduce pathogen concentration in ruminants is that fact that the viability of orally administered phage may be rapidly reduced under the acidic conditions of the abomasum20
and in the presence of enzymes and other digestive compounds such as bile. To address this issue, Sheng and colleagues21
evaluated the phagebased rectal treatment of ruminants based on a previous study22
which showed the recto-anal junction to be the primary site of E. coli
O157:H7 colonization in cattle. Two phages (designated KH1 and SH1) were tested, alone or in combination, for their ability to reduce intestinal E. coli
O157:H7 in animals. To optimize bacterial carriage and phage delivery in cattle, E. coli
O157:H7 was applied rectally to Holstein steers 7 d before the administration of 1010
PFU of SH1 and KH1. Phages were applied directly to the recto-anal junction mucosa at a multiplicity of infection (MOI) of 102
. In addition, phages were maintained at a concentration of 106
PFU/ml in the drinking water of the treatment group. Results showed that this approach reduced the average number of E. coli
O157:H7 CFU among phage-treated steers compared with control steers (p < 0.05), but did not eliminate the bacteria from the majority of steers. In a similar study, Rozema et al.23
compared oral and rectal administration of E. coli
O157-specific phages for efficacy in reducing or eliminating the fecal shedding of E. coli
O157:H7 by experimentally inoculated steers. Fecal shedding was monitored over 83 d after oral, rectal, both oral and rectal, or no treatment with a four-strain O157-specific phage cocktail delivered in multiple doses. Orally treated steers produced the fewest E. coli
O157:H7 culture-positive samples, but this number was not statistically significant when compared with control steers. Animals that received the phage cocktail rectally shed E. coli
O157:H7 at a higher concentration than steers from the other treatment groups, though there was no statistical difference in the number of E. coli
O157 positive samples among treatments. Phages were isolated from steers that did not receive the cocktail, showing that the phages could be acquired from the environment.
While these studies indicate the possibility of using phages to control E. coli O157:H7 colonization and shedding in ruminants, more studies need to be conducted to determine adequate phage dose, number of doses (a single dose vs. continuous dosing), standardized methods of phage delivery (water or feed delivery vs. rectal delivery), and the economics of phage therapy in food producing animals.
The majority of phage therapy applications to control foodborne pathogens in live animals have been conducted in poultry. Poultry and egg products are important sources of the human pathogens Salmonella
spp and Campylobacter
which cause many cases of foodborne illness globally. The scientific literature indicates that phage therapy-based attempts at controlling Salmonella
in poultry have reduced, but not eliminated the pathogen. Berchieri et al.26
showed that newly hatched chicks challenged with Samonella enterica
serovar Typhimurium followed by oral or feed-based introduction of a single phage for 7 d did not show decreased levels of S.
Typhimurium in cecal contents. The researchers reported a high percentage of phage resistant S.
Typhimurium isolates, which occurred in 34–82% of 50 bacterial isolates tested on each of days 2, 4, 7 and 10 post infection. However, when a second phage was used, significant reductions in mortality were observed over a 21-d period (from 56% to 20%). However, while the concentration of the challenge strain was reduced by more than 2 log units within 3–6 h, these reductions were transient.
Fiorentin et al.3
isolated a cocktail of phages from free-range chickens and used them to reduce the concentration of Salmonella enterica
serovar Enteritidis phage type 4 (PT4) in the ceca of broilers. Five days post treatment, the concentration of S.
Enteritidis PT4 per gram of cecal content in the phage-treated group was reduced by 3.5 logs, and samples collected up to 25 d after treatment revealed that the treated birds still had lower colony-forming units of S.
Enteritidis PT4 per gram of cecal content compared with untreated broilers.
Both Andreatti-Filho et al.27
and Sklar and Joerger28
evaluated cocktails of phages for their ability to reduce S.
Enteritidis in experimentally infected chicks and young chickens. The results of both studies showed that the cocktails significantly reduced the concentrations of S.
Enteritidis recovered from the treated birds, but the reductions were not statistically significant.
A possible solution to the problem of transient bacterial reduction following phage therapy is the use of phage cocktails in combination with another biocontrol method such as competitive exclusion bacteria to sustain the ability of phages to control pathogens in the live animal. Toro et al.4
used a mixture of three Salmonella
-specific phages in combination with competitive exclusion bacteria to reduce Salmonella
colonization in experimentally infected chickens. The phages were administered orally to the chickens several days prior and after Salmonella
challenge. A competitive exclusion product consisting of a defined culture of seven different microbial species was used either alone or in combination with phage cocktail treatment, and was administered orally at hatch. Salmonella
counts in the intestine, ceca and a pool of liver/spleen samples were evaluated in Salmonella
-challenged chickens treated with the phage cocktail or with the cocktail and competitive exclusion bacteria. A reduction in Salmonella
counts was detected in the cecum and ileum of phage treated, competitive exclusion treated, and phage treated/competitive exclusion treated chickens as compared with nontreated birds.
Atterbury and colleagues29
individually administered three phages that had broad host ranges against Salmonella enterica
serotypes Enteritidis, Hadar and Typhimurium to birds experimentally colonized with their specific Salmonella
host strains. The first phage reduced S.
Enteritidis cecal colonization by more than 4.2 log CFU within 24 h compared with controls. Administration of the second phage reduced S.
Typhimurium by more than 2.19 log CFU within 24 h. The third phage was ineffective at reducing S.
Hadar colonization. Phage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant Salmonellae
. The researchers concluded that the selection of appropriate phages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of Salmonellae
in broiler chickens. To investigate the effect that method of delivery has on the efficacy of phage therapy, Borie et al.30
used three phages to reduce S.
Enteritidis colonization in experimentally infected 10-d old chickens. The chickens were treated by coarse spray or drinking water with a cocktail of the three phages 24 h before challenge with S.
Enteritidis. Chickens were euthanized at 20 d of age and sampled for S.
Enteritidis. The results showed that aerosol-spray delivery of the phage cocktail significantly reduced the incidence of S.
Enteritidis infection when compared with the control group. Also, phage delivery by both coarse spray and drinking water significantly reduced the intestinal S.
Enteritidis colonization. It was concluded that phage treatment, either by aerosol spray or drinking water, may be a plausible approach to reduce Salmonella
infection in poultry.30
The collective research conducted to investigate the use of phages to control foodborne pathogens in ruminants and poultry indicates the successes, but also challenges, that are present when this biocontrol method is applied to live animals. A main challenge is the need for regulatory approval of phage based products prior to their large scale investigation in animals. Currently, there are no phage-based products approved for use to reduce pathogens within the live animal. The United States Department of Agriculture (USDA) issued two no objection letters for the use of E. coli
O157:H7 and Salmonella
targeted bacteriophages developed by Omnilytics™ (Salt Lake City, UT, USA) for use as hide sprays on cattle prior to slaughter.31
The phages would aid in the reduction of E. coli
O157:H7 and Salmonella
spp on hides prior to further processing, to decrease transfer of these pathogens from the hide to meat. The hide sprays produced by Omnilytics™ are the only phage products currently approved for use in the animal industry.32–34
Elanco (Greenfield, IN) in conjunction with Omnilytics™ has produced two products called Finalyse to reduce E. coli
O157:H7 on cattle hides, and Armament for reduction of Salmonella
on poultry. Further approval of phage based products may become easier to achieve with the European Food Safety Authority Biohazards Panel endorsement of the use of phages as a treatment for foods of animal origin including carcasses, meat and dairy products.35