Bacteriophage therapy to combat bacterial infections in poultry

The therapeutic effectiveness of phages is determined by their high lytic titre, the form and type of application, and the application period. Long-term use of phages in poultry has proved to be moderately effective in reducing the number of Salmonella pathogens colonizing the digestive tract [27]. However as shown by Fiorentin et al. [28], single oral application of a cocktail of phages (CNPSA1, CNPSA3 and CNPSA4) at a dosage of 10¹¹ PFU decreased the occurrence of Salmonella Enteritidis strains by 3.5 log units. The authors confirmed that applying a single dose of a bacteriophage suspension with a high titre was highly effective in reducing the population of pathogenic bacteria in the digestive tract, in contrast with long-term application of a lower titre.

A positive effect of phage therapy was also observed in combating horizontal infections induced by strains of S. Gallinarum in flocks of laying hens. Treatment using bacteriophages as a feed additive for chickens having contact with infected individuals led to a mortality rate of only 5%, as compared to 30% in the group that did not receive phage therapy [32].

The effectiveness of phage therapy may also depend on the individual antibacterial properties of a given bacteriophage and on the adaptive mechanisms of the bacteria. A study by Andreatti Filho et al. [30] showed that the use of selected bacteriophages in an orally administered cocktail to prevent colonization by S. Enteritidis strains in poultry was only effective for a short time (about 48 h), with no long-term protective effect, which was partly due to acquisition of resistance to the bacteriophage by the bacteria. All treatments resulted in a significant, 6-log reduction in Salmonella Enteritidis strains recovered from caecal tonsils at 24 h as compared to untreated controls, but no significant differences were observed at 48 h following treatment.

It seems promising that a wide range of lytic activity against three serovars of Salmonella – Enteritidis, Typhimurium, and Hadar – were obtained in 36-day-old broiler chicks, in which a significant reduction in the concentration of bacteria was noted following experimental infection with these serovars, by 2–4 log units [29]. The authors suggest that adjustment of treatment conditions may make it possible to use just one or two bacteriophages rather than many. In another study, Ahmadi et al. [35] demonstrated 100% efficacy in eliminating S. Enteritidis strains from the tonsils of 33-day-old Salmonella-free quails, 6 h after oral application of 100 ml of a 10⁹ to 10¹⁰ PFU ml⁻¹ bacteriophage suspension. It should be noted that all birds received the bacteriophage suspension for 3 days, and the therapeutic effect was noticeable within 6 h after experimental infection. The authors also confirmed that this treatment has a prophylactic effect in quails receiving 100 μl of 10⁶ PFU ml⁻¹ bacteriophages via oral gavage for 3 days, once every 24 h, before oral challenge with 100 μl of 1.2 × 10⁹ CFU ml⁻¹ S. Enteritidis. Significant prevention of colonization by S. Enteritidis strains was observed over a period of 7 days at a rate of 20% in comparison to the control (100% colonization).

Other studies suggest that bacteriophages may be used in combined treatment with other preparations, as indicated by a significant (about 80%) synergistic antibacterial effect of a commercial oral probiotic preparation applied together with a bacteriophage ‘cocktail’ of phages S2a, S9, and S11 (5.4 × 10⁶ PFU/0.5 ml/bird) at 4, 5, and 6 days of age and at 8, 9, and 10 days of age to combat S. Typhimurium infections in broilers. The authors showed that chickens treated with a probiotic and bacteriophages showed 10 times fewer bacteria in the ileum, caecum, liver and spleen than did untreated challenged chickens. [31].

In another study, simultaneous application of three phages (MOI 103) at 10⁸ PFU/ml/dose at 6 days of age (two daily doses) by aerosol spray and probiotics administered at 1 day of age by coarse spray, followed by oral inoculation with 2.95 × 10⁵ CFU/ml in seven-day-old chickens, reduced Salmonella incidence and Salmonella intestinal colonization, leading to complete elimination of deaths in broiler chickens caused by infection with Salmonella Enteritidis [36]. Similar results were obtained in inhibiting horizontal infection with Salmonella following application of a bacteriophage suspension in the amount of 10⁵ and 10 PFU/g as a feed additive for chickens challenged with 5 × 10⁷ CFU of bacteria. Different groups of birds were treated with different titres of bacteriophage contained in the feed additive for 21 days after Salmonella Enteritidis challenge. These preventive measures significantly inhibited the replication of pathogens in the digestive tract of the chickens; however, this effect was observed mainly in chickens treated with bacteriophages at concentrations of 10⁹ PFU/mL, which were compared only with the positive control groups [37]. Authors also suggest the occurrence the horizontal transmission of Salmonella Enteritidis strains, which was confirmed by a substantial reduction in the number of chickens treated with bacteriophages at concentrations of 10⁷ and 10⁹ PFU/g 1 week after treatment in comparison with untreated chickens. However, there was no significant reduction in Salmonella counts after two and 3 weeks of treatment as compared with the positive control group. And in many cases, the efficacy of phage therapy should be maximized by using a high titre of bacteriophages in order to reduce colonization by Salmonella via passive transmission.

Phage suspensions applied directly to the air sac in 3-day-old birds in a range of titres from 10⁶ to 10³ PFU to treat E. coli infections substantially reduced mortality rates to 5% and 25%, respectively. Similar results were obtained after inoculation of a bacteriophage suspension in the drinking water of birds at 1 week of age (10³ or 10⁴ PFU of bacteriophages per mL) followed by air sac challenge with 10³ CFU of E. coli phages. Mortality was decreased to 25% and 5%, respectively. No mortality was observed in chickens treated with 10⁸ PFU of an E. coli bacteriophage mixture [38]. Bacteriophages have also been shown to be highly effective in treating sepsis and meningitis in newly hatched and 3-week-old chicks infected intramuscularly and intracranially with a strain of E. coli. Mortality in the untreated chicks was 100%, whereas intramuscular administration of phage R at titres of 10⁴ and 10⁶ PFU completely eliminated deaths in chickens in the group of treated birds. Another positive effect of the treatment was the absence of visible clinical symptoms. In the chickens intracranially infected with E. coli, application of a higher dose of the phage, at a titre of 10⁸ PFU, fully protected the birds against the development of infection. Intramuscular application (in different muscles) of phage R at a titre of 10⁶ PFU resulted in a lack of morbidity or mortality in all chickens. Administration of lower doses from 10⁴ PFU of the phage after challenge with E. coli also provided significant protection, indicating that the phage had multiplied in vivo. However, the application of phages in lower doses, e.g. 10² PFU, produced no statistically significant protection against E. coli infection.

The authors also demonstrated that the bacteriophages administered to the birds intramusculary had the ability to penetrate the blood-brain barrier, and confirmed that the bacteriophages had a prophylactic effect in addition to the therapeutic effect. In 3-week-old birds effective protection against morbidity and mortality following intracranial inoculation with E. coli was obtained only after administration of 10⁸ PFU of the phage. Only in younger birds was statistically significant protection obtained after administration of 10⁶ PFU of the phage. Application of a suspension 1–2 days prior to experimental infection with E. coli in chicks reduced the mortality rate by 70%, as well as the intensity of the course of infection [26]. The use of bacteriophages at titres of 10⁴–10² PFU in the form of an aerosol in chicks with symptoms of colibacillosis significantly reduced the mortality of the chicks and prevented infections in other birds. Aerosol administration of bacteriophage SPR02 at a titre of 10⁸ PFU/mL combined with challenge with 10⁴ CFU/mL of E. coli completely protected the birds against infection. When these phages at 10⁴ PFU/mL were mixed with 10⁴ CFU/mL of E. coli, mortality was significantly reduced to 35%^.

The authors suggest also that similar effects preventing early development of colibacillosis in chicks are obtained by applying a bacteriophage suspension in ovo [39]. The authors also demonstrated that the effect of this kind of bacteriophage treatment is comparable to enrofloxacin treatment, and suggest that a combination of enrofloxacin and bacteriophage treatments could be efficacious and beneficial in controlling colibacillosis.

Apart from bacteriolytic activity, the effectiveness of bacteriophages is also determined by the site and route of administration of the preparation. According to Huff et al. [40] bacteriophages should be applied directly to the site of infection, which was confirmed during treatment of E. coli infections in the air sacs of chickens. Application of bacteriophages per os with drinking water proved ineffective in treating the infection and reducing clinical symptoms. When a suspension was injected directly into the air sac, an effective protective effect was obtained, manifested as the absence of clinical symptoms. This treatment significantly reduced mortality from 50 to 20% when given immediately after challenge, but had little efficacy when administered 24 or 48 h after challenge. IM injection of bacteriophages significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24 h, or 48 h after challenge, respectively.

A similar effect eliminating disease symptoms in E. coli respiratory infections in poultry was obtained in broiler chickens aged 10 days to 2 weeks following repeated application of a two-phage (SPRO2 and DAF6) suspension in aerosol spray form after challenge with E. coli by injection of 10⁴ CFU into the thoracic air sac. The authors observed the best overall protection after aerosol treatment with phage titres of 2.6 × 10⁸ and 2.35 × 10⁹ PFU/mL for SPR02 and DAF6, respectively. The study found a significant decrease in mortality ranging from 20% and 27% in comparison to chickens untreated with bacteriophages, but mortality was still high [40]. In the septic form of colibacillosis intramuscular application proved more effective than aerosol application, particularly in the initial phase of sepsis. In another study on broiler chicks, Huff et al. [41] demonstrated a prophylactic effect of application of a bacteriophage suspension in the form of a single intramuscular injection of two different bacteriophages (10⁹ PFU/mL) in combination with enrofloxacin administered in drinking water immediately after E. coli challenge. Mortality decreased significantly to 15% in comparison with untreated birds challenged with E. coli (68%). The authors also demonstrated a significant synergistic protective effect in chickens that received both the bacteriophage and enrofloxacin. Because colibacillosis in poultry develops in the respiratory system, some studies suggest that bacteriophages should be applied in aerosol form as a preventive measure 1–3 days before anticipated infection, e.g. transport or transfer to a new environment [42]. The incidence of disease in 7-day old chicks treated with a phage aerosol in the first few days following experimental infection fell to under 10%, while the mortality rate in the chicks that did not receive the aerosol was 60%. A study by Oliveira et al. [43] confirmed that colibacillosis-induced high morbidity and mortality in poultry may be significantly reduced by aerosol spraying of housing systems with bacteriophage cocktails and oral administration of bacteriophages. In that study 1 ml of the phage suspension at a high titre of 1.0 × 10⁹ PFU/ml and a lower titre of 5.0 × 10⁷ PFU/ml of phages phi F78E, F258E and F61E was dispensed into the birds’ mouth with a syringe and by spraying directly into the beak, through a spray nozzle set to release 1 ml per fine drop scatter. Immediately after phage administration the chickens were challenged with a pathogenic E. coli suspension. The results also demonstrated a protective effect of the bacteriophages against new colonization by E.coli strains in the days following the challenge. The study confirmed the therapeutic effectiveness of one of the phages, phi F78E, administered orally and by spray at 1 × 10⁹ PFU/mL, resulting, on average, in a 25% decrease in mortality and a 41.7% reduction in morbidity in the chickens.

The direct or aerosol administration of bacteriophages in poultry and evaluation of their therapeutic effect has been a subject of study at many research centres. A study by El-Gohary et al. [44] demonstrated that bacteriophage treatment of litter by spraying 200 mL of a bacteriophage preparation at a titre of 8 × 10⁸ PFU/mL on the surface of 3.9 m² pens significantly reduced the mortality of male broiler chickens (about 2–3 weeks old) with colibacillosis resulting from exposure to E. coli in the litter, even when the birds were exposed to cold stress, and furthermore reduced shedding of the pathogen among flocks.

The potential uses of phage therapy against Campylobacter bacteria may offer an alternative means of eliminating bacteria in the digestive tract of birds. This pertains in particular to infections induced by Campylobacter jejuni and C. coli, which constitute 80% of the bacteria colonizing the digestive tract in poultry. One of the first attempts to use bacterial viruses against Campylobacter bacteria was a study by Wagenaar et al. [33], in which colonization by C. jejuni was inhibited in 10-day-old chicks and adult birds, first by 2 and then by 1 log unit in broiler caeca. The Ross broiler chickens received phages by oral gavage from day 7 to 16 at different titres varying from 4 × 10⁹ to 2 × 10¹⁰ PFU and were orally challenged with 1 × 10⁵ CFU of C. jejuni on day 10.

The authors confirmed that phage treatment prior to bacterial challenge does not prevent but may delay bacterial colonization. However, chicks receiving phages after C. jejuni colonization showed an immediate 3-log reduction in CFU counts. It should be emphasized that the bacteria were not completely eliminated in this study, which is the main problem in the use of phage therapy for elimination of Campylobacter strains in poultry [33].

In another study, conducted at the University of Nottingham in the UK on 25-day-old chickens, following application by oral gavage of bacteriophages CP34 or CP8 isolated from the environment against C. jejuni strains HPC5 and GIIC8 obtained from birds and humans, a substantial but short-lived reduction in the number of bacteria was obtained in the intestines of infected birds, ranging from 0.5 to 5 log units. A considerable reduction in the total number of Campylobacter bacteria in the upper and lower digestive tract and in the caecum was obtained when bacteriophages were applied at a concentration of 10^7–9 PFU [45]. A study evaluating the effect of bacteriophages on the number of Campylobacter jejuni in the caecum in broiler chickens also confirmed a significant (P < 0.001) reduction in the total number of bacteria, to a value of 10^5.1 CFU/g, in comparison with chickens that were not treated with bacteriophages (mean density of bacteria 10⁷ CFU/g) [46]. A similar reduction in the number of Campylobacter jejuni and Campylobacter coli bacteria in infected birds was obtained following application of a suspension of bacteriophage CP220 at titres of 10⁷ and 10⁹ PFU/ml for 5 days. A reduction in C. jejuni bacteria was observed just 48 h after injection of the phage, while in the case of C. coli a significant decrease in the number of infected birds was obtained following administration of a bacteriophage suspension with a density of 10⁹ PFU. It should be emphasized that the percentage of birds resistant to a second Campylobacter infection was very low, at about 2% [47].

The use of a suspension of bacteriophages specific for Campylobacter jejuni and C. coli bacteria in the water or feed of broiler chickens caused a significant decrease of nearly 2 log₁₀ CFU/g in colonization by both species of bacteria. Moreover, in contrast with earlier research, the bactericidal effect of the phages was maintained for over 7 days, enabling application of the suspension at each stage of the production cycle [48]. Preventive treatment delayed but did not prevent colonization. Levels of C. jejuni were initially 2 log units lower than in controls, and then stabilized at 1 log unit lower than in the controls.

On the other hand, the use of bacteriophages to prevent colonization by Campylobacter spp. bacteria in newly hatched broiler chicks was only partially successful. Application by oral gavage of a phage suspension with 0.4 to 2 × 10¹⁰ PFU/mL of phage 71 in 10-day-old broiler chickens initially reduced the total number of bacteria, but colonization by pathogens re-occurred within 24 h [33]. The studies cited also showed that resistance of Campylobacter spp. to particular phages was about 4%. For this reason the authors suggest creating a combination of several bacteriophages specific for Campylobacter, which in vitro research has shown to improve the effectiveness of phage therapy [49].

Phage therapy was shown to be effective in the case of infection of broiler chickens with anaerobic Clostridium perfringens inducing necrotic enteritis [34]. Bacterial toxins produced by this bacterium are responsible for generalization of the disease process, cause a decrease in feed intake, and inhibit growth. Oral administration to chickens of various ages of a suspension of a cocktail (INT-401) of 5 different C. perfringens phages (CPAS-7, CPAS-12, CPAS-15, CPAS-16, and CPLV-42) at titres of 10⁵ PFU/mL, with feed or water or by oral gavage and spray application, led to a significant decrease (P ≤ 0.05) in mortality during the 0-to-42-day of experiment in comparison with the group of untreated birds. These measures also improved weight gain in the chicks. It should also be emphasized that the treatment proved more successful in reducing mortality than a formalin-inactivated vaccine containing C. perfringens alpha toxin. However, the study cited confirmed the high efficacy of the bacteriophages in controlling necrotic enteritis in poultry.

Besides whole phages, phage enzymes (endolysins and mureolytic enzymes), including murein hydrolase, merit particular attention as an additive element in combating infections induced by C. perfringens. These enzymes, binding directly to the peptidoglycans of the cell walls of Gram-positive bacteria, cause rapid lysis of these bacteria, including cells infected with bacteriophages, which accelerates their destruction. Simultaneous use of bacteriophage preparations and endolysins against Gram-positive bacteria such as Clostridium spp. and Listeria monocytogenes seems to have a highly beneficial effect. This has been confirmed in the case of use of a bacteriophage (ϕ3626) against C. perfringens, whose spectrum of lysogenic activity was at a level of 22%, while the lytic effect was 8%. In the case of combined application of bacteriophages with murein hydrolase, a lytic effect was observed against all (n = 51) tested strains of C. perfringens [50, 51]. In view of the fact that bacteriophages eliminate C. perfringens bacteria mainly by lysogeny, supplementation of phage preparations with endolysins would seem to be necessary for continuation of successful treatment.

The bactericidal effectiveness of phages has also been confirmed in fighting infections induced by Listeria monocytogenes, which like Campylobacter spp. or Salmonella is included among zoonotic pathogens inducing food poisoning in humans, with a high mortality rate of 30%. Application of bacteriophages on the surface of poultry products ready for consumption reduced the number of bacteria by 2.5 log units on a product stored at 30 °C after just 5 h. Later testing for Listeria monocytogenes in food samples kept in cold storage also yielded positive results, as the pathogen was not detected for a period of 21 days or use of a bacteriophage mixture on poultry carcasses could completely eliminated L. monocytogenes [52, 53]. Due to the risk posed by the occurrence of poultry infections induced by L. monocytogenes, as well as their increasing drug-resistance and efforts to limit the use of antibiotics, international and American health organizations are attempting to replace antibiotics with other preparations. This resulted in FDA approval on 18 August 2006 of 102-LMP™, a suspension of bacteriophages specific for L. monocytogenes, as an antibacterial agent against L. monocytogenes. This product has been estimated to successfully kill over 170 strains of Listeria spp. [54].