This study investigated the effects of dietary treatments of isolated phage cocktail at different dosages and their combinations with probiotic on growth performance, health and gut microbial communities of chickens. The assessment on phages as dietary supplementation at various dosages and their combinations with probiotic in broiler chickens has never been reported. However, similar experiments have been conducted on pigs in which commercial phages and probiotic were used, and phages were found to be more effective [14, 16]. This study showed that chickens fed with 0.1% phage cocktail and probiotic combination (1ɸP) can positively modulate the gut microbiota based on the significant reduction of E. coli.
The selection of phage-target bacteria was based on the previous study where both genera of Escherichia and Shigella were observed at a moderate concentration in ilea (9–32%) and caeca (1–5%) at different ages [34]. E. coli was selected as the target bacteria due to their consistent representations in various ages in ilea and caeca, the ease of handling (e.g., aerobic, fast growing bacteria), and also due to their non-pathogenic nature for some of the strains.
Chicken has a range of body temperature between 40.5 to 42°C [59]. The survivability of phage within this temperature is required to ensure the efficacy of phage application. In the current study, all phages had an optimal lytic activity at 37°C. Although phage titre for all phages remained high at a high temperature, phage lytic activity (host reduction) was reduced (at 42°C compared to at 37°C). This observation suggested that phage activity will still persist at 42 °C, but phage may take a longer time to clear targeted hosts. The use of an additive such as skim milk might be useful to preserve the phage activity at a high temperature, where it has been demonstrated in Lactococcus phage [53]. At a lower temperature, phage lytic activity was slightly reduced but was stable at 25°C, especially for C2, C3 and C4 phages when compared to C1 phage. This showed that they were stable at room temperature. The E. coli host was nullified at 60°C and hence replications of phages were not possible.
The results of the present study showed that chickens fed with a combination of 0.1% phage cocktail and probiotic (1ɸP) had significantly better BW (35 d), BWG (22–35 d, 1–35d), and lower FCR compared to the control. However, the BW (35 d) and BWG (22–35 d, 1–35 d) of chickens supplemented with phage cocktail singly (1ɸ, 2ɸ) or probiotic (P) singly, or a combination of 0.2% phage cocktail and probiotic (2ɸP) were not significantly different from those of control chickens. This showed that the use of 0.1% phage cocktail with a combination of probiotic (1ɸP) has the potential to improve the growth performance of broiler chickens. Although supplementation of phage cocktail alone did not significantly improve growth performance in broiler chickens, studies on laying hens reported that supplementation of phage cocktail significantly increased their growth performance and egg production [70]. Studies in pigs had also shown that supplementation of phage cocktail singly or in combinations with probiotic significantly improved the growth performance [14, 15].
The results of the present study revealed that 0.2% phage cocktail (2ɸ), probiotic (P) and a combination of 0.1% or 0.2% phage cocktail and probiotic (1ɸP, 2ɸP) significantly reduced serum triglycerides in chickens, but the reduction was not significantly different between the treatments. This suggests that the addition of a phage cocktail to the probiotic did not further reduce the serum triglycerides level. The reduction of serum triglycerides by probiotic has been widely reported in chickens [5, 65, 31, 66, 38], but there is very little information on the effects of phages or their combinations with probiotic on serum triglycerides.
The exact mechanism(s) on the reduction of triglycerides by probiotic is still not well understood. Taranto [67] suggested that reduction of triglycerides in mice supplemented with Lactobacillus reuteri could be due to hypolipemic effect, which involved the reduction of lipid assimilation or increase of lipid catabolism. Santoso [65], on the other hand, suggested that reduction of triglycerides by Bacillus subtilis in broiler chickens was due to their ability to decrease the acetyl coenzyme A carboxylase activity, which has a crucial function on restricting the synthesis of fatty acids. As the fatty acids decrease, it will in turn reduce the triglycerides esterification process [38].
None of the dietary treatments in the present study had any significant effects on serum total cholesterol, HDL and LDL of chickens at 35 d, though LDL was significantly reduced by 0.1% and 0.2% phage cocktails at 21 d and the serum total cholesterol was numerically the lowest in the 1ɸP group. This is rather surprising as many studies have shown the ability of probiotic to reduce serum cholesterol in broiler chickens [5, 38, 44, 35].
Immunoglobulins are antibodies that are released by plasma cells in response to foreign materials with antigens such as bacteria or viruses. Chickens immunoglobulins can be classified into three main classes: IgA, IgY (IgG) and IgM [47]. IgA is found mainly in bile and intestines of chickens [47]. IgG is the most dominant immunoglobulins found in chickens and it carries out an important function of protecting the host from infection [47]. IgM, on the other hand, is present in the first week post-infection and also appears during immunisation [64].
In the current study, the IgA and IgM concentrations of chickens were not significantly different among all the dietary treatment groups. However, IgG was significantly lower in the 1ɸ, 1ɸP and 2ɸP groups when compared with the control at 21 d. The current study also showed that there were no significant differences in IgG between the probiotic (P) group and the rest of the supplemented groups. This showed that supplementation of probiotic singly or in combination with a phage cocktail did not improve antibody titres. These findings are in contrast to those of other studies, in which probiotic either enhance [63, 39, 40] or have no effects [36] on the chicken humoral immune response. Beneficial effects of probiotic in modulating immune response at the cellular and humoral levels have also been reported in chickens [56]. Immunomodulatory activities of probiotics can be influenced by various factors such as type of probiotic strains, host genetics and types of antigens present [39, 62]. There were also no significant effects on immunoglobulin levels when phage cocktail was fed to pigs [15]. Probiotics are known to enhance immunomodulatory activities to the host by improving antibody response, but this was not observed in this study. It is however important to note that Mountzouris [36] had pointed out that studies which highlighted on enhancement of antibody response with probiotic supplementation [63, 39, 40] were performed against specific model antigens. The immunoglobulin levels in this study showed a general overview of systemic antibody response with supplementation of different diets, but not against specific antigens.
Phage supplementation has been known to initiate an immune response in chickens. This had been demonstrated by Huff [13] who reported that chickens supplemented with phages had significantly higher IgG due to activation of immune responses. Repetitive supplementations of phages would lead to an increase in mortality of birds challenged with pathogenic E. coli [13]. This shows that repetitive doses of phages would limit phage action due to the immune response that was initiated. In the current study, significantly lower IgG was observed in birds supplemented with phage cocktails or their combinations with probiotic (1ɸ, 1ɸP and 2ɸP), as compared to the control at 21 d. This indicated that IgG was not increased even with ad libitum supplementation of phages, suggesting that supplementation of very high titre of phage cocktail at 1010 PFU/g might have overwhelm the host antibody response on the phages. However, this can only be confirmed by measuring the antibodies at short intervals after supplementation of phages.
A study of intestinal villus height and crypt depth would provide valuable information on the changes of chicken intestinal epithelial cells in response to supplementation of phage cocktail, probiotic and their combinations. An increase in villus height is associated with greater nutrient absorption due to the larger surface area for efficient nutrient uptake [55]. Villus crypt is recognised as an important site for generation of intestinal villus [69]. A shorter villus is correlated with accumulation of toxins and a high rate of tissue turnover of villus production due to pathogens, while deeper crypt is associated with toxins that cause inflammation or sloughing [69, 52, 33].
In the current study, supplementation of phage cocktail and probiotic combination to chickens significantly improved in villus height (35 d) and crypt depth (21 d) in the 1ɸP and 2ɸP groups, and villus to crypt ratio (35 d) in the 2ɸP group, when compared to the probiotic (P) group. However, chickens supplemented with probiotic alone or in combination with phage cocktail did not show any significant improvements in villus height, crypt depth or villus to crypt ratio when compared to the control. These results are in contrast with previous reports that chickens with probiotic supplementation showed an increase in intestinal villus height and villus to crypt ratio in broilers [44, 52, 56, 58, 61, 41, 8]. Recently, Ledezma [46] have reported that villus height was significantly increased in the jejunum and ileum, but not duodenum in probiotic-supplemented chicken. Aliakbarpour [51] also found that Lactobacillus supplementation increased the intestinal villus height, but this was not demonstrated in chickens that were supplemented with Bacillus subtilis. To date, there has been no previous study on the effect of phage supplementation on intestinal villus height and crypt depth study. These findings show that the effects of probiotic on intestinal villi and crypts are influenced by the types of probiotic used and the part of the intestinal studied.
E. coli population was significantly lower in chickens fed with phage cocktail and probiotic combination (1ɸP) than the control in ilea of 21 d chickens and also lower in phage cocktail singly groups (1ɸ and 2ɸ), although this was not significant. This showed that E. coli phage cocktail employed was potentially effective in reducing E. coli, especially in combination with probiotic. Based on the current study, it was difficult to deduce the phage cocktail alone which was responsible for the gut modulation as the concentration of phage titer was not quantified. The caveat is due to the challenge in quantifying C2, C3 and C4 phages as they can cross-infect between each target bacterial hosts. Nevertheless, the qPCR results showed that the E. coli population in the phage singly group was significantly reduced, compared to the control. This result potentially suggests that phage cocktail and probiotic worked synergistically in ablating E. coli in the gut. The milder reduction of E. coli population was also detected in the 1ɸP group at 21 d caeca and 35 d caeca than the control, although this was not significant. The lack of difference may be explained by the fact that gut microbiota became more complexed as chicken aged, thus was more resistant to modulation. Indeed, several studies have shown that gut modulation was effectively performed in chickens of young age between 3 to 25-day-old, where these chickens still harboured transient gut microbiota than the adult chickens that had complex gut microbial communities [45, 50, 68]. Furthermore, E. coli population was abundant in ilea compared to caeca, where it was expected that greater reduction of the bacterium was mainly detected in ilea [34].
The PrimaLac® probiotic used in this study was comprised of Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium termophilum, Enterococcus faecium and Aspergillus oryzae. In the current study, gut population study based on real-time qPCR showed that there was no significant different for Lactobacillus spp. population between dietary treatments for all sampling (age and part of intestine). The populations of Lactobacillus spp. and Bifidobacterium spp. were expected to be higher in probiotic supplemented groups (P, 1ɸP and 2ɸP), but Bifidobacterium population was found to be higher in chickens fed with phage cocktail singly instead (1ɸ and 2ɸ). This suggests that the indigenous gut commensals replaced the niche created by the clearance of E. coli in phage cocktail singly groups. However, this was not observed in probiotic supplemented groups. It could be due to the inefficacy of PrimaLac® probiotic to colonise in the intestines. This result was in contrast with the previous studies, where probiotic supplementation was found to increase the level of probiotic bacteria in chicken intestines [36, 49, 54]. It also need to be highlighted that in the current study, PrimaLac® probiotic microbes such as Enterococcus faecium and Aspergillus oryzae were not quantified, where their populations might be elevated in probiotic supplemented groups.
In the current study, chickens from probiotic supplemented groups (P, 1ɸP and 2ɸP) had significantly lower population of C. perfringens compared to the control at 21 d caeca. This result was consistent with previous studies, in which C. perfringens population was significantly lower in chickens supplemented with probiotic [49]. This was due to the protective roles of probiotic in reducing pathogens based on the reported mode of actions of competitive exclusion [48, 42] and antagonistic activity [44, 43, 32, 60].