ABSTRACT

Though recently considered a therapeutic treatment for commercial broilers, little is known about the effects of bacteriophages on native, slow-growing birds. This study evaluated their efficacy against Salmonella enterica subsp. enterica serovar Typhimurium infected Noi chicken, a native Vietnamese broiler breed. In total, 420 birds were used in a completely randomized design consisting of seven treatments and four replicates of 15 birds. The treatments were NC (negative control), PC (positive control, S. Typhimurium challenged); NC+B1 and NC+B2 (negative control plus B1 or B2 bacteriophage, respectively); PC+B1, PC+B2 (positive control plus B1 or B2 bacteriophage, respectively) and PC+B1B2 (positive control plus both B1 and B2 bacteriophages). After four weeks of infection, the mortality rate in the PC group was 51.1% compared with 11.1% in the PC+B1B2 treatment. Bacteriophage administration had resulted in increased weight gain and decreased feed conversion ratio, particularly when both phages were included in the treatment (p<0.001). Moreover, the relative percentage of carcass weight was lowest in the PC treatment (66.9%) (p<0.001), whereas the other treatments registered similar carcass weight values. Regarding the internal organs, liver weight percentage was higher in the non-treated Salmonella group, and enlarged spleens were also noted in infected chickens even when treated with bacteriophages. The correlation between phage administration and blood parameters was unclear. Although the use of two bacteriophages for therapy was determined to be preferable for the majority of the criteria examined, further genetic characterization of the phages will be required before they can be widely used in chicken farms.

Keywords:
Growth; indigenous chickens; mortality; phage therapy; Salmonella

INTRODUCTION

Salmonella is a significant poultry pathogen that is of concern for public health and can potentially cause losses in poultry production (Berchieri Jr et al., 2001Berchieri Jr A, Murphy C, Marston K, Barrow P. Observations on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background. Avian Pathology 2001;30(3):221-231.). In Vietnam, salmonellosis is one of the most frequent bacterial infections in poultry farms, with 8.81 percent of chickens in the Mekong Delta being infected with the disease in 2010 (Khai et al., 2010Khai LTL, Phan TT, Chuc NT. Determination of transmitted sources of salmonellosis caused by Salmonella as zoonosis in some provinces in the Mekong Delta. Can Tho University Journal of Science 2010;(16b):69-79.). So far, antibiotics have been used extensively to treat and prevent bacterial infections, resulting in antibiotic-resistant microorganisms. It is estimated that roughly 148 mg of antimicrobial are required to raise one kilogram of live chicken worldwide; however, in the Mekong Delta region of Vietnam, a higher level of antimicrobials in chicken production (260 mg/kg of chicken, excluding medicated feed) has been documented (Van Boeckel et al., 2015Van Boeckel TP, Brower C, Gilbert M, Grenfell BT, Levin SA, Robinson TP, et al. Global trends in antimicrobial use in food animals. Proceedings of the National Academy of Sciences 2015;112(18):5649-5654.; Nguyen et al., 2015Nguyen VT, Carrique-Mas JJ, Ngo TH, Ho HM, Ha TT, Campbell JI, et al. Prevalence and risk factors for carriage of antimicrobial-resistant Escherichia coli on household and small-scale chicken farms in the Mekong Delta of Vietnam. Journal of Antimicrobial Chemotherapy 2015;70(7):2144-2152.; Carrique‐Mas et al., 2015Carrique-Mas JJ, Trung NV, Hoa NT, Mai HH, Thanh TH, Campbell JI, et al. Antimicrobial usage in chicken production in the Mekong Delta of Vietnam. Zoonoses and Public Health 2015;62:70-78.). Salmonellosis control at the farm level can be accomplished through a multi-factorial approach, and when broilers get Salmonella, several simultaneous or sequential procedures may be applied (Clavijo et al., 2019Clavijo V, Baquero D, Hernandez S, Farfan J, Arias J, Arévalo A, et al. Phage cocktail SalmoFREE(r) reduces Salmonella on a commercial broiler farm. Poultry Science 2019;98(10):5054-5063.), of which bacteriophage therapy has been described as a promising biological control for salmonellosis in poultry (Thanki et al., 2021Thanki AM, Hooton S, Gigante AM, Atterbury RJ, Clokie MR. Potential roles for bacteriophages in reducing Salmonella from poultry and swine. In: Lamas A, Regal P, Franco CM, editor. Salmonella spp. London: IntechOpen; 2021.).

Bacteriophages, or phages, are predators of bacteria that are naturally present in the environment. According to some estimates, they are abundant in the environment, with an estimated 10:1 ratio compared to their bacterial counterparts (O’Flaherty et al., 2009O'Flaherty S, Ross RP, Coffey A. Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiology Reviews 2009;33(4):801-819.). Bacteriophages are defined as bacterial viruses that can infect and subsequently kill their host bacteria via bacteriolysis. Due to their availability, non-toxicity, and specificity to the bacterial hosts, lytic phages have been utilized as efficient tools for various purposes such as improving food safety or preventing and treating bacterial pathogens (Brovko et al., 2012Brovko LY, Anany H, Griffiths MW. Bacteriophages for detection and control of bacterial pathogens in food and food-processing environment. Advances in Food and Nutrition Research 2012;67:241-288.; Sulakvelidze, 2013Sulakvelidze A. Using lytic bacteriophages to eliminate or significantly reduce contamination of food by foodborne bacterial pathogens. Journal of the Science of Food and Agriculture 2013;93(13):3137-3146.). In addition, bacteriophages are extremely specific by infecting only a single species of bacteria, and phage therapy is regarded as safer and more effective than antibiotics (Upadhaya et al., 2021Upadhaya SD, Ahn JM, Cho JH, Kim JY, Kang DK, Kim SW, et al. Bacteriophage cocktail supplementation improves growth performance, gut microbiome and production traits in broiler chickens. Journal of Animal Science and Biotechnology 2021;12(1):1-12.). Previously, several reports have mentioned the positive effects of bacteriophage in reducing pathogens and improving the production performance of broiler chickens (Atterbury et al., 2007Atterbury RJ, Van Bergen M, Ortiz F, Lovell M, Harris J, De Boer A, et al. Bacteriophage therapy to reduce Salmonella colonization of broiler chickens. Applied and Environmental Microbiology 2007;73(14):4543-4549.; Lim et al., 2012; Wang et al., 2013Wang J, Yan L, Lee J, Kim IH. Evaluation of bacteriophage supplementation on growth performance, blood characteristics, relative organ weight, breast muscle characteristics and excreta microbial shedding in broilers. Asian-Australasian Journal of Animal Science 2013;26(4):573-578.). According to a recent study (Nabil et al., 2018Nabil NM, Tawakol MM, Hassan HM. Assessing the impact of bacteriophages in the treatment of Salmonella in broiler chickens. Infection Ecology and Epidemiology 2018;8(1):1539056.), bacteriophage treatment can rapidly reduce S. Typhimurium and S. Enteritidis in broiler chickens and could be used instead of antibiotics. The investigation of Clavijo et al. (2019Clavijo V, Baquero D, Hernandez S, Farfan J, Arias J, Arévalo A, et al. Phage cocktail SalmoFREE(r) reduces Salmonella on a commercial broiler farm. Poultry Science 2019;98(10):5054-5063.) also demonstrated that phage treatment on commercial broiler farms had no adverse effects on production parameters and significantly reduced Salmonella infection in the chickens.

The majority of the research appears to have been concentrating on commercial broilers, with little information on the use of bacteriophage for indigenous breeds with slower growth and superior meat quality, which is an important component in the economies of rural and underdeveloped countries (Padhi, 2016Padhi MK. Importance of indigenous breeds of chicken for rural economy and their improvements for higher production performance. Scientifica 2016;2604685.). Therefore, the present study was undertaken in Noi chickens, one of the native breeds mainly reared in the Southern regions of Vietnam, aiming to evaluate the effects of bacteriophage on treating S. Typhimurium infection and further identify its effectiveness in the growth and carcass characteristics of the birds.

MATERIALS AND METHODS

Bacteria strains and bacteriophage preparation

In this study, the virulent strain S. Typhimurium ATCC^®14028™ (American Type Culture Collection) was used for the in vivo infection in Noi chickens. Moreover, two bacteriophages, namely B1 and B2, were isolated and screened from chicken intestines and environmental sources at various poultry farms in the Mekong Delta of Vietnam (Souvannaty et al., 2019Souvannaty V, Loc HT, Anh LH, Ngu NT. Isolation and application of bacteriophages to reduce Salmonella colonization on chicken intestinal diseases (in Vietnamese). Journal of Animal Husbandry Sciences and Technics 2019;241:86-92.). Briefly, for phage isolation and pure culture isolation, the double agar-plaque assay (Kropinski et al., 2009Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of bacteriophages by double agar overlay plaque assay. Methods in Molecular Biology 2009,501:69-76.) and the method of Poxleitner et al. (2017Poxleitner M, Pope W, Jacobs-Sera D, Silvanathan V, Hatfull G. Phage discovery guide. Ashurn: Howard Hughes Medical Institute; 2017.) were applied. The phages were then tested for host spectrum, pH tolerance (Verma et al., 2009Verma V, Harjai K, Chhibber S. Characterization of a T7-like lytic bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent. Current Microbiology 2009;59(3):274-281.), and lysis capacity in vitro (Kropinski et al., 2009; Verma et al., 2009). Based on the criteria for host spectrum, pH tolerance, and ability to lyse S. Typhimurium, the B1 and B2 bacteriophages were selected for the experimental infection of chickens.

Phage proliferation: from the isolates available in the laboratory, the phages were proliferated in the ratio of phage: Salmonella: TSB being 1: 2: 30 with a Salmonella (Salmonella Typhimurium - ATCC^®14028™) population of 10⁸ CFU/ml. The phages were incubated at 37°C for 24 hours. After that, chloroform solution was added to the proliferated phage biomass at the rate of 1 chloroform: 10 phages. The solution was then vortexed, incubated for 2 hours followed by centrifuging at 6,000 rpm at 4^oC for 15 minutes for phage collection (Silva et al., 2014Silva YJ, Costa L, Pereira C, Mateus C, Cunha A, Calado R, et al. Phage therapy as an approach to prevent Vibrio anguillarum infections in fish larvae production. PloS One 2014;9(12):e114197.). The concentration of phages was determined as described Poxleitner et al. (2017Poxleitner M, Pope W, Jacobs-Sera D, Silvanathan V, Hatfull G. Phage discovery guide. Ashurn: Howard Hughes Medical Institute; 2017.) and Gonzalez-Menendez et al. (2018Gonzalez-Menendez E, Fernandez L, Gutierrez D, Rodriguez A, Martinez B, Garcia P. Comparative analysis of different preservation techniques for the storage of Staphylococcus phages aimed for the industrial development of phage-based antimicrobial products. PLoS One 2018;13(10): e0205728.).

Chickens care and experimental design

The study was carried out on the experimental farm of Can Tho University located at Campus IV, Phung Hiep district, Hau Giang province. The care and handling of chickens were performed following guidelines of Animal Husbandry Law (32/2018/QH14), and the experimental procedure was approved by the Council for Science and Education, College of Agriculture (A10-02-2019/KNN), Can Tho University. The indigenous Noi chickens were obtained from a breeding company based in Soc Trang near Hau Giang province. During the rearing period, chickens were vaccinated against Marek disease (one-day-old), Newcastle disease and infectious bronchitis (days 4, 14, and 60^th), Gumboro (days 7 and 12^th), Fowl Pox (day 10^th), and H5N1 avian influenza (day 45^th). All birds were kept in confinement houses at the density of 10 birds/m² in the finishing stage. Drinking water was available, and chickens were fed ad libitum of commercial broiler diets: starter from 1 to 28 days (16% crude protein, 4% crude fiber, metabolizable energy 2,800 kcal/kg) and grower and finisher from 29 to 98 days (14% crude protein, 5% crude fiber, metabolizable energy 2,800 kcal/kg).

In total, 420 birds were arranged in a completely randomized design with 7 treatments and 4 replicates of 15 birds. The treatments were NC (negative control, without S. Typhimurium, without bacteriophage); PC (positive control, S. Typhimurium challenged, without bacteriophages); NC+B1 and NC+B2 (negative control plus B1 or B2 bacteriophage, respectively); PC+B1, PC+B2 (positive control plus B1 or B2 bacteriophage, respectively) and PC+B1B2 (positive control plus both B1 and B2 bacteriophages). The chicks were challenged with 1 ml of S. Typhimurium on the 2^nd day at the dose of 10^8.5 cfu/ml via oral administration. In treatments with bacteriophage therapy, the phages were orally inoculated at the dose of 10¹⁰ pfu/ml starting at 24 hours after infection, followed for three consecutive days, and repeated weekly until day 63^rd.

Data collection and measurements

The experiment lasted for 100 days till the Noi chickens reached their mature weight. Feed intake and body weight were recorded weekly for each replicate, and feed conversion ratio (FCR) was calculated as g feed intake/g weight gain after correcting mortality. The mortality was recorded in the first four weeks after infection. During the experimental period, four birds per treatment (sex-balanced when applicable) were randomly selected for re-isolation of S. Typhimurium on days 7, 21, and 35 (Gomes et al., 2014Gomes A, Quinteiro-Filho W, Ribeiro A, Ferraz-de-Paula V, Pinheiro M, Baskeville E, et al. Overcrowding stress decreases macrophage activity and increases Salmonella Enteritidis invasion in broiler chickens. Avian Pathology 2014;43(1):82-90.), and organ relative weight calculation on days 7, 35, and 63.

At the end of the experiment, 56 chickens (8 birds/treatment, sex-balanced) with weights around the means were selected for slaughtering to evaluate carcass characteristics and organ measurements. The broilers were individually weighed and then killed via cervical dislocation and exsanguination. The breast, thigh and drumstick muscle and wings were collected as described by Goliomytis et al. (2003Goliomytis M, Panopoulou E, Rogdakis E. Growth curves for body weight and major component parts, feed consumption, and mortality of male broiler chickens raised to maturity. Poultry Science 2003;82(7):1061-1068.). In addition, the liver, spleen, heart, and gizzard were then removed and weighed, and the organ weights were expressed as a percentage of the body weight.

Statistical analysis

The data were subjected to analysis of variance using the General Linear Model procedure of the Minitab version 16.2.1 software (State College, PA, USA) (Minitab, 2010). The difference of means among treatments was measured through Tukey’s comparison test with p≤0.05.

RESULTS AND DISCUSSION

Mortality of chickens during four weeks after infection

The effect of treatment on mortality is shown in Table 1. In the negative control and treatments without S. Typhimurium challenged, no death were recorded. In contrast, the mortality was the highest in the positive control during four weeks post-infection, with an accumulative value of 51.1%. The bacteriophage administered treatments either in a single phage or cocktail phage demonstrated a reduction in mortality down to 11.1% over four weeks. It was also noted that a majority of birds’ death happened during the first week of inoculation. There is little evidence available on the effects of S. Typhimurium on the death rate of native chickens. However, in broiler chickens, Nabil et al. (2018Nabil NM, Tawakol MM, Hassan HM. Assessing the impact of bacteriophages in the treatment of Salmonella in broiler chickens. Infection Ecology and Epidemiology 2018;8(1):1539056.) reported on two-day old chicks infected with S. Typhimurium (10⁵ cfu/ml) that mortality appeared after three days of infection with an overall rate of 20%, which is lower than the dead rate of Noi chickens in the current study, most likely due to the different breeds and higher dose of bacteria used in our study (10^8.5 cfu/ml). When bacteriophages were added, the effectiveness of reducing diarrhea after two days of infection was demonstrated, and no dead chickens were recorded in these treatments (Nabil et al., 2018). In the present study, the death were also investigated for lesions, and the nine internal organs (liver, spleen, and heart) were used for bacteria re-isolation, and their sequence was confirmed as Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S in BLAST, NCBI (https://blast.ncbi.nlm.nih.gov) (data not shown).

In the current study, there were no death in the treatments inoculated only bacteriophages after four weeks of infection, showing that the existing phage application was practically safe for chickens or did not cause any other health problems. Similar findings were also reported elsewhere by Wójcik et al. (2020Wójcik EA, Stanczyk M, Wojtasik A, Kowalska JD, Nowakowska M, Lukasiak M, et al. Comprehensive evaluation of the safety and efficacy of BAFASAL(r) bacteriophage preparation for the reduction of Salmonella in the food chain. Viruses 2020;12(7):742.). Moreover, in our study, the therapeutic effectiveness has been shown with reduced mortality in treatments with bacteriophage usage. The clinical manifestations of salmonellosis were comparably mild, and the combination of the two phages received a better response from chicken. It was reported by Fischer et al. (2013Fischer S, Kittler S, Klein G, Glünder G. Impact of a single phage and a phage cocktail application in broilers on reduction of Campylobacter jejuni and development of resistance. PLoS ONE 2013;8(10):e78543.) that combinations of multiple phages might be more effective at phage bio-control because they provide a wider range of hosts and, as a result, help to keep resistances under control. In terms of reducing the Campylobacter jejuni population, the phage cocktails performed slightly better than the single phage following phage delivery. On the other hand, the cocktail slowed and delayed the formation of phage resistance (Fischer et al., 2013), and the use of a phage cocktail for the treatment of S. Typhimurium in chickens might bring desirable results.

The high mortality rate in the first week after infection could be attributed to the high concentration of bacteria in internal organs, as well as the fact that the birds were still young, with an immature immune system (Akhtar et al., 2013Akhtar A, Bejo M, Zakaria Z. Pathogenicity of Salmonella Enteritidis phage types 6A and 7 in experimentally infected chicks. Journal of Animal and Plant Science 2013;23:1290-1296.). Previously, Berchieri Jr et al. (1991Berchieri Jr A, Lovell M, Barrow P. The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella Typhimurium. Research in Microbiology 1991;142(5):541-549.) also mentioned that when newly hatched chicks were infected with S. Typhimurium, giving phage lytic solutions soon after infection was shown to reduce mortality. Still, the efficacy was reduced when offering phages a few hours later or in lower numbers. This is important because controlling Salmonella from its origin has been difficult since S. Typhimurium infection in young chicks generally causes no symptoms, and as a result, environmental contamination occurs, which contributes to the organism’s proliferation among the flock (Hooton et al., 2011Hooton SP, Atterbury RJ, Connerton IF. Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin. International Journal of Food Microbiology 2011;151(2):157-163.). In addition, Huff et al. (2006Huff W, Huff G, Rath N, Donoghue A. Evaluation of the influence of bacteriophage titer on the treatment of colibacillosis in broiler chickens. Poultry Science 2006;85(8):1373-1377.) concluded that to treat a systemic bacterial infection successfully, a sufficient number of bacteriophages need to be present when the infection begins. Taken together, a two-phage cocktail in this study could reduce the S. Typhimurium invasion in the internal organs, as well as the mortality of chickens.

Re-isolation of S. Typhimurium in the internal organs

Among the organs used to re-isolate S. Typhimurium, a slightly lower concentration of bacteria was found in the heart over 7, 21, and 35 days post-infection (Figure 1). As expected, no bacteria were found in the negative treatments and those without S. Typhimurium inoculation. The bacterial invasion decreased over time, indicating that bacteriophages aided in eradicating S. Typhimurium colonization in the birds’ liver, spleen, and heart. Moreover, a combination of two phages consistently outperformed a single phage therapy regardless of the checking point.

In the absence of Salmonella infection, re-isolated organs were free of S. Typhimurium, indicating that the control group from the current study was not infected either from other chickens or the feed used in the experiment, which is consistent with the report of Goliomytis et al. (2003Goliomytis M, Panopoulou E, Rogdakis E. Growth curves for body weight and major component parts, feed consumption, and mortality of male broiler chickens raised to maturity. Poultry Science 2003;82(7):1061-1068.). The presence of infected bacteria in the organs was expected because when predominant Salmonella serovars infect chickens, the germs can be found in the spleen, liver, heart, gallbladder, intestinal tissues, ceca, and various portions of the ovary and oviduct (Gast et al., 2007Gast RK, Guraya R, Guard-Bouldin J, Holt PS, Moore RW. Colonization of specific regions of the reproductive tract and deposition at different locations inside eggs laid by hens infected with Salmonella Enteritidis or Salmonella Heidelberg. Avian Diseases 2007;51(1):40-44.; Keller et al., 1995Keller LH, Benson CE, Krotec K, Eckroade RJ. Salmonella Enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infection and Immunity 1995;63(7):2443-2449.). Although the bacterial counts were reduced in the current investigation, their presence was still observed after 35 days. The phages applied in our work were not as effective as reported by Wong et al. (2013Wong CL, Sieo CC, Tan WS, Abdullah N, Hair-Bejo M, Abu J, et al. Evaluation of a lytic bacteriophage, ? st1, for biocontrol of Salmonella enterica serovar Typhimurium in chickens. International Journal of Food Microbiology 2013;172:92-101.) that within 12 hours, the Salmonella was wholly eradicated from the chicks with phage by a single intracloacal inoculation an hour after S. Typhimurium inoculation. The supplied route could be one reason, as intracloacal inoculation ensures caecal wall lining establishment and eliminates passage through the acidic conditions of the crop and gizzard (Wong et al., 2013). This intracloacal application, however, is impractical at the farm level. Another reason for the effectiveness of bacteriophage treatment may be the low ratio of bacteriophage to bacteria. Supporting this conclusion was the significant reduction in S. Typhimurium concentration achieved by first administering the phage cocktail before or just after bacterial infection, then twice daily for four days, and then weekly (Bardina et al., 2012Bardina C, Spricigo DA, Cortés P, Llagostera M. Significance of the bacteriophage treatment schedule in reducing Salmonella colonization of poultry. Applied and Environmental Microbiology 2012;78(18):6600-6607.).

Effects of bacteriophage treatments on production performance of broilers

Body weight, feed intake, and feed conversion ratio (FCR) of chickens were affected by treatments during the experimental period (Table 2). Bacteriophage administration has contributed to better weight gain and reduced FCR, especially in the treatment with both phages included. Chickens in the positive control treatment grew slowly (265 g/bird) in the first 35 days, which was significantly different (p<0.001) from the ones in other treatments without S. Typhimurium infection and tended to be lower than those with bacteriophage inoculation. In the next stages, although not significant, a superior growth rate was also documented in bacteriophage treatments, and this contributed to the difference in overall weight gain with a respective increase per bird of 1,117 g, 1,263 g, and 1,217 g for PC, NC, and PC+B1B2 (p<0.005). As a consequence of higher intake but low efficiency in feed conversion, the most considerable FCR increased was noted in the PC treatment. Among the infected chickens, those with phage applied provided lower FCR, ranging from 3.56 (phage cocktail) to 3.86 (phage B2). According to Khoa et al. (2019Khoa DAV, Tuoi NTH, Thuy NTD, Okamoto S, Kawabe K, Khang NTK, et al. Growth performance and morphology of in 28-84 day-old Vietnamese local Noi chicken. Biotechnology in Animal Husbandry 2019;35(3):301-310.), the BW of a Noi broiler at 84 days of age was 1,196 g for females and 1,424 g for males, with an average FCR of 3.52. Thus, the chickens used in this experiment developed normally, with an average body weight of 1.263 g in the NC treatment.

Body weight is an important characteristic in broiler production since lower body weight correlates to higher broiler meat production costs. A clear tendency of improving the growth rate of infected chickens treated with bacteriophages was identified in our study. The present findings corroborated the research outputs of a previous study (Toro et al., 2005Toro H, Price S, McKee S, Hoerr F, Krehling J, Perdue M, et al. Use of bacteriophages in combination with competitive exclusion to reduce Salmonella from infected chickens. Avian Diseases 2005;49(1):118-124.) that S. Typhimurium phage treatment resulted in a favorable effect on weight gain. These authors also pointed out that chicks gained less weight following the challenge and maintained lower weights throughout the study, which parallels our results.

Apart from being used as therapeutic bioagents to eradicate bacteria, bacteriophages have been used as feed additives to prevent pathogens, resulting in productivity advantage. For example, in experiments using diets containing phages against S. Gallinarum, S. Typhimurium, and S. Enteritidis, where broiler chickens were raised under normal physiological conditions (without bacterial challenge), improvements in body weight and FCR were observed (Lim et al., 2010; Kim et al., 2014Kim J, Kim J, Lee B, Lee G, Lee J, Kim G-B, et al. Effect of dietary supplementation of bacteriophage on growth performance and cecal bacterial populations in broiler chickens raised in different housing systems. Livestock Science 2014;170:137-141.; Wójcik et al., 2020Wójcik EA, Stanczyk M, Wojtasik A, Kowalska JD, Nowakowska M, Lukasiak M, et al. Comprehensive evaluation of the safety and efficacy of BAFASAL(r) bacteriophage preparation for the reduction of Salmonella in the food chain. Viruses 2020;12(7):742.). This is due to the fact that the bacteriophage inhibits the growth of bacteria in the broilers’ gastrointestinal tract (Lim et al., 2010), and the increase in body weight may be related to an increase in feed intake (Upadhaya et al., 2021Upadhaya SD, Ahn JM, Cho JH, Kim JY, Kang DK, Kim SW, et al. Bacteriophage cocktail supplementation improves growth performance, gut microbiome and production traits in broiler chickens. Journal of Animal Science and Biotechnology 2021;12(1):1-12.). However, not all cases of phage therapy or phage supplementation in diets resulted in enhanced broiler production traits. Kim et al. (2013) found that feeding S. enteritidis-targeted phages had little influence on broiler performance and it was additionally suggested that phages targeting several pathogenic bacteria have more favorable effects than phages targeting single pathogenic bacteria in boosting broiler performance (Kim et al., 2014). In another study, Noor et al. (2020Noor M, Runa N, Husna A. Evaluation of the effect of dietary supplementation of bacteriophage on production performance and excreta microflora of commercial broiler and layer chickens in Bangladesh. MOJ Proteomics & Bioinformatics 2020;9(2):27-31.) concluded that antibiotics and bacteriophages did not affect body weight gain, feed intake, or FCR during the 15-32 days of commercial broilers. The phage-host interaction in production traits of chickens might be dependent on phage types and genotypes, which can be found from poultry and environmental isolates.

Carcass characteristics and internal organs of Noi chickens

Carcass composition and internal organ measurements of chickens are presented in Table 3. The relative percentage of carcass weight was lowest at the PC treatment (66.9%) (p<0.001), while in the other treatments, these values were similar. The greatest breast muscle and thigh and drumstick ratios were shown in NC+B1 and NC+B2 treatments (p<0.001). As a consequence of low carcass content in the body, the percentage of breast, thigh, and drumstick in chickens from PC was significantly lower than those from others. There were also differences in wing weight ratio among treatments, with the highest being 11.0% in the PC+B1B2 treatment. The improved carcass weight percentage may benefit from the superior live weight as these two criteria are closely related (Tuoi et al., 2021Tuoi NTH, Giang NT, Thuy NTD, Loan HTP, Shimogiri T, Khoa DVA. Carcass characteristics of Vietnamese indigenous Noi chicken at 91 days. Asian Journal of Poultry Science 2021;15:53-59.).

As for leg muscle percentage, the current outputs are in line with the report of (Kim et al., 2013Kim K, Lee G, Jang J, Kim J, Kim Y. Evaluation of anti-SE bacteriophage as feed additives to prevent Salmonella Enteritidis (SE) in broiler. Asian-Australasian Journal of Animal Science 2013;26(3):386-393.), according to which the anti-S. Enteritidis bacteriophage treatments had higher relative leg muscle weights than the control treatments. In the NC treatment, values of carcass cuts agreed with the report by Hung et al. (2020Hung LT, Lan LTT, Thu NTA, Phong NH, Nhan NTH, Ngu NT. Effects of dietary lysine on apparent amino acid digestibility and carcass characteristics of Noi broilers. Livestock Research for Rural Development 2020;32, Article #126.) in Noi chicken at the slaughter of 84 days. There is a scarcity of comparable data on the impact of bacteriophages on other carcass traits; however, positive responses to bacteriophage treatments have been demonstrated. Most internal organs were numerically higher in the PC treatment (p<0.005), mainly the relative weights of small intestines, liver, and gizzard. For spleen percentage, the highest values were on PC treatment for most of the time points (Table 3). In addition, throughout the experiment, a tendency to decrease liver weight percentage was shown, which could be attributed to an increase in final body weight (Figure 2a). In contrast, these figures demonstrated an upward trend in spleen to body weight ratio, with a notable figure in the PC treatment and other treatments with Salmonella infection (Figure 2b).

In the present study, liver weight percentage was higher in the non-treated Salmonella group, similar to that shown by Wang et al. (2013Wang J, Yan L, Lee J, Kim IH. Evaluation of bacteriophage supplementation on growth performance, blood characteristics, relative organ weight, breast muscle characteristics and excreta microbial shedding in broilers. Asian-Australasian Journal of Animal Science 2013;26(4):573-578.). The mechanism for this is unknown, but it could be hypothesized that bacteriophages may alter the population of gut intestinal bacteria, which are required for gut and liver immune system development (Wang et al., 2013). Regarding spleen weight ratio, the benefit of treating chicken with phages was not demonstrated compared to those from Kim et al. (2013Kim K, Lee G, Jang J, Kim J, Kim Y. Evaluation of anti-SE bacteriophage as feed additives to prevent Salmonella Enteritidis (SE) in broiler. Asian-Australasian Journal of Animal Science 2013;26(3):386-393.), whose results showed a drop in spleen weight relative to body weight, indicating that phage inclusion was better. The current outputs are consistent with the findings of Upadhaya et al. (2021Upadhaya SD, Ahn JM, Cho JH, Kim JY, Kang DK, Kim SW, et al. Bacteriophage cocktail supplementation improves growth performance, gut microbiome and production traits in broiler chickens. Journal of Animal Science and Biotechnology 2021;12(1):1-12.), who discovered that the relative weight of the gizzard increased more substantially in the positive control group than the control and phage groups, which could be explained by an increase in feed intake in the positive control group. On the other hand, supplementation with anti-S. Enteritidis bacteriophage did not affect the relative weight of organs after 35 days (Kim et al., 2013). Moreover, in E. coli-infected birds treated with bacteriophages, the heart, liver, and spleen sizes were not significantly different from the control groups (Lau et al., 2010Lau G, Sieo C, Tan W, Hair-Bejo M, Jalila A, Ho Y. Efficacy of a bacteriophage isolated from chickens as a therapeutic agent for colibacillosis in broiler chickens. Poultry Science 2010;89(12):2589-2596.). Nonetheless, on days 2, 3, and 21 post-infection, the livers of untreated E. coli-challenged birds were enlarged. These birds’ spleens were also enlarged on days 2, 3, and 7 post-infection but subsequently experienced a gradual reduction in size (Lau et al., 2010).

CONCLUSION

In this study, bacteriophage treatment resulted in a decrease in mortality in broilers infected with S. Typhimurium. Additionally, they tended to facilitate the growth of chickens and the improvement of feed conversion ratio. When bacteriophages were applied, the carcass proportion increased, yet no evident benefits were observed in internal organ weight. The use of two bacteriophages for therapy was preferable for most of the criteria investigated; nevertheless, a comprehensive genetic characterization of the phages and their host range needs to be undertaken as the next stage before they can be extensively used in chicken farms.

ACKNOWLEDGMENTS

This study is funded in part by the Can Tho University Improvement Project VN14-P6, supported by a Japanese ODA loan.

REFERENCES

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