Adding of bacteriophage in diet to treatment of pullorum infection induced experimentally in broiler chicken (Immunological and Histopathological study)

Introduction

Pullorum disease is a significant global poultry affliction in avian species because to its substantial economic repercussions, widespread prevalence, and challenges associated with disease management (Endris et al., 2013; Agapé et al., 2025) [8,2] . Significant economic detriment resulting from elevated death rates that may attain 100%, reduction in production (eggs and chicks), and expenses associated with medicine for both humans and animals (Netsanet et al., 2012) [21] . Including Iraq and other Asian nations, it also incurs significant economic losses in the poultry business. Antimicrobial medications are regrettably employed not only for therapeutic purposes but also to enhance animal production, growth rate, and feed conversion efficiency in food-producing animals across numerous developing nations (Snary et al., 2004) [25] . Numerous studies have demonstrated that incorrect antibiotic use enhances productivity while also elevating the selection pressure for antimicrobial-resistant microorganisms (Levy, 2014) [15] . Antibiotic resistance has no boundaries, as germs that have acquired resistance to certain antibiotics can swiftly disseminate from countries with robust surveillance systems to those lacking such measures. Consequently, a collaborative strategy among developing nations is essential (Galperina & Kyian, 2021) [9] . The prominent emergence of multidrug-resistant bacterial illnesses in poultry farms renders treatment with conventional antibiotics impractical, hence demanding the adoption of alternative therapies (Abreu et al., 2023) [1] . Phage treatment is an efficacious method for addressing drug-resistant bacterial infections in animals while preserving their normal gut microbiota (Mohsen et al., 2024) [18] .

Bacteriophages possess several attributes that render them potentially advantageous as therapeutic agents for bacterial infections. One of them is the remarkable specificity and efficiency in lysing particular pathogenic microorganisms (Nabil et al., 2018) [20] . Furthermore, several investigations have documented effective decrease in Salmonella spp. (Wernicki et al., 2022; Zeng et al., 2024; García et al., 2024) [26, 29, 10] . Rydman and Bamford (2002) [22] elucidated the strategy employed during phage-bacterium interactions, which involves the degradation of extracellular polymeric substances in the biofilm through the production of polysaccharide depolymerases, thereby facilitating phage access to encased bacterial cells and inducing their lysis. The objective of this study was to evaluate the beneficial effects of bacteriophage on bodily performance and the gut microbiome in the context of S. pullorum challenge.

Materials and Methods

Drugs and vaccines

Florfenicol 100 mg/ml was purchased from (Cenavisa-Spain). Bacteriophage E. coli Bacteriophage 3.2×10 8 PFU/g was procured from EASY BIO-Korea and used in the current study as described by (Aljuhaishi and Albawi, 2024) [4] .

Birds

The chicks in this study were weighed at one day of age (mean weight 43 g±2) and subsequently brought to the poultry hall. The chicks are housed in enclosures separated by wooden barriers, each measuring 1 x 1.5 m. The hen coop bedding consisted of sawdust and measured 7 cm in thickness. Access to potable water and sustenance is provided gratuitously and facilitated by a continuous illumination system. No clinical manifestations of illness were observed prior to the commencement of the investigation. This study involved 250 one-day-old Ross308 broilers, which were randomly assigned to five treatments, with two replication pens containing 25 birds each. This research was conducted at the broiler farm of the College of Veterinary Medicine, Al-Qasim Green University, located in Babylon City, Iraq.

Experimental design 250 broiler chicks divided into 5 groups as follow:

 G1: Using vaccine program and bacteriophage as feed additive from one day to the end of experiment as (dosage 3.2* 10^10 PFU/g)  G2: Same vaccination but without use bacteriophage as feed additive with treated at one day to five days by using Florfenicol and after challenge  G3: Using bacteriophage as feed additive after challenge (from 14 days until the end of experiment).  G4: Challenged 14 days without any treatment (control positive)  G5: Control group without challenge and without using any treatment.

All groups vaccinated with Traditional Vaccines against viral diseases like (ND, AI, IB, and IBD).

Vaccination of chicks

The birds received vaccinations against infectious bronchitis at one day old with the IB QX strain administered via eye drop, and at ten days old with the IB H120 strain provided through drinking water. Newcastle disease was inoculated at one day old with the LaSota attenuated strain via eye drop, at ten days old with the Clone attenuated strain through drinking water, and at twenty days old with the LaSota attenuated strain via drinking water. Additionally, avian influenza was administered on day one and infectious bursal disease on day fourteen.

Statistical analysis

The analysis was conducted using the pre-existing statistical software SAS. The data were presented as mean±standard deviation (STD) unless specified otherwise. SAS (2018) was utilized for data analysis using the Randomized Completely Block Design (RCBD). The disparities among the coefficients of the initial experiment were evaluated utilizing Duncan's multilevel test, with a significance threshold of 0.05 (Duncan, 1955; Dirwal et al., 2020) [6,7] .

Results and Discussion

A. Live body weight in experimental study

Table (1) shows the live body weight in experimental groups. At 7 days, group G1 (bacteriophage-treated) had significantly higher body weight (205.3 g) compared to G2 (201.4 g), G3 (201.9 g), and G4 (203.1 g), while G5 (208.2 g, negative control) recorded the highest weight (p<0.05). At 14 days, G1 remained the highest (517.8 g) compared to other groups, with G5 (505.2 g) having the lowest. At 21 days, G1 and G3 showed higher body weights compared to G2, G4, and G5, with significant differences (p<0.05). At 28 days, G3 (1631.2 g) showed the best growth, followed closely by G1 (1629 g), whereas G4 (1429 g, positive control) had the lowest weight, indicating significant improvement by bacteriophage and combined treatments.

B. Mortality rates

The results of mortality rate illustrated in Table 2, demonstrated a clear group difference. G1 (bacteriophagetreated) and G5 (negative control) recorded 0% mortality, indicating full protection. G2 (antibiotic-treated) showed 32% mortality, while G3 (combined treatment) recorded only 10%, significantly lower than G2 (p<0.05). The highest mortality was observed in G4 (positive control), reaching 70%, which was significantly higher than all other groups.

D. Antibody titers against Infectious Bronchitis disease

Table (4) shows antibody titers against infectious bronchitis. At day 7, G1 (3209) and G3 (2875) were significantly higher than G2 (2377), with G4 and G5 having the lowest titers. At day 14, G1 (3950) and G3 (3659) maintained higher antibody responses compared to G2, G4, and G5 (p<0.05). By day 21, G1 (2957) and G3 (2093) were significantly better than G2, while G4 and G5 showed very low responses (1221 and 731, respectively). At day 28, G1 (6763) and G3 (5342) remained significantly higher than G2 (3424), while G4 (1353) and G5 (789) recorded the lowest antibody titers, confirming the superior immune response in bacteriophage and combined treatments. showing the crypts of Lieberkühn (black arrow) with villi (yellow arrow) projecting above the crypt layer (Figure 1A).

 Control positive: The Histopathological findings showed severe necrosis of epithelial cells in the crypts of Lieberkühn and villi is observed, with infiltration of inflammatory cells and epithelial cells hyperplasia (black arrow) occupying the necrotic areas, leading to fusion of villi and loss of normal intestinal architecture. Focal necrotic areas (yellow arrow) are also present within the affected regions. Destruction of villi (blue arrow) is noted as adjacent to the necrotic zones (Figure 1B).  Florfenicol group: Photomicrograph of small intestine of Florfenicol group broiler chicken. A and B/ Mild to moderate necrosis was observed in the villi, accompanied by infiltration of inflammatory cells in the villus core, leading to increased villus thickness. In addition, destruction of villus tips (yellow arrow) was noted in the affected areas (Figure 1C).  14 days bacteriophage group: Photomicrograph of small intestine of day 14 bacteriophage group broiler chicken. A and B/ Marked thickening of the affected villi is observed due to infiltration of inflammatory cells (black arrow) in the villus core, with significant hyperplasia of epithelial cells (yellow arrow). In addition, destruction of villus tips is noted in the affected areas, with necrotic cell debris (blue arrow) present in the intestinal lumen (Figure 1D).  1 day bacteriophage group: Photomicrograph of small intestine of day 1 bacteriophage group broiler chicken. A and B/ Moderate thickening of the affected villi is observed due to infiltration of inflammatory cells (black arrow) in the villus core, with moderate hyperplasia of epithelial cells (yellow arrow), (Figure 1D). [5,13] . Bacteriophagetreated groups (G1 and G3) exhibited superior body weight gain compared to antibiotic-treated (G2) and untreated challenged groups (G4), indicating that phages enhanced feed efficiency and nutrient utilization. This outcome is consistent with reports that phages restore gut microbial balance and reduce pathogenic colonization (Kakasis & Panitsa, 2019; Seo et al., 2025) [12,24] . Interestingly, G3 showed comparable growth at 28 days to G1, suggesting that even post-infection phage application can mitigate disease severity. Mortality rates further support the therapeutic value of phages, with G1 and the negative control showing 0% mortality, while the antibiotic group had 32% and the positive control reached 70%. These results emphasize that florfenicol alone may not provide full protection under severe infection pressure, corroborating earlier findings that antibiotics have limited efficacy against resistant Salmonella strains (Lee et al., 2021) [14] . The immunological assessments revealed that bacteriophage groups had significantly higher antibody titers against Newcastle disease and infectious bronchitis, particularly at later stages of the experiment. This suggests that phages not only reduce pathogen load but may also enhance systemic immunity, potentially through preservation of gut integrity and modulation of host immune signaling [17,19,4] . Histopathological analysis confirmed these outcomes, as phage-treated bird's maintained better villus architecture compared to antibiotic and positive control groups, where necrosis, villus fusion, and epithelial hyperplasia were evident. These findings underscore the gutprotective effect of phages, consistent with recent reports describing their role in maintaining intestinal barrier function and reducing inflammatory responses. Overall, the results provide strong evidence that bacteriophage supplementation, either prophylactically or therapeutically, offers superior protection against S. pullorum compared to conventional antibiotic treatment. This supports growing international efforts to reduce antibiotic use in animal production and adopt sustainable alternatives to combat antimicrobial resistance (WHO, 2022) [27] .

Conclusion

Bacteriophage supplementation effectively protected broiler chickens against Salmonella pullorum, improving growth, survival, immunity, and gut health compared to antibiotics. Florfenicol offered limited protection, while phages provided strong prophylactic and therapeutic benefits. These results support bacteriophages as a sustainable alternative to antibiotics in poultry production and a valuable tool against antimicrobial resistance.

Conflict of interest Not available

Financial Support

References

1. Abreu R, Lemsaddek ST, Cunha E, Tavares L, Oliveira M. Antimicrobial drug resistance in poultry production: Current status and innovative strategies for bacterial control. Microorganisms. 2023;11(4):953.
2. Agapé L, Boncompte TJ, García P. In ovo phage administration to mitigate Salmonella Typhimurium colonization in broiler chickens: A new firewall strategy for the poultry industry. Food Control. Advance online publication; 2025.
3. Aljuhaishi DA, Albawi FHK. Using bacteriophage in poultry diet as therapeutics against Escherichia coli in broiler chickens. J Anim. Health Prod. 2024;12(s1):2331.
4. Aljuhaishi DA, Albawi FHK. Investigating the presence of E. coli bacteria in broiler chickens and detecting virulence genes (rfbE, eaeA, fliCH7); Zoological and Entomological Letters. 2024;4(1):120-124.
5. Clavijo V, Morales T, Flores VM, Muñoz RA. The gut microbiota of chickens in a commercial farm treated with a Salmonella phage cocktail. Scientific Reports. 2023;12(1):991.
6. Duncan DB. Multiple range and multiple F-test Biometrice. 1955;11:1-42.
7. Dirwal AR, Falih IB, Layj DM. Manifestation of cysticercus tenuicolliscyst infected organs of slaughtered sheep with determination of reactive proteins of cyst fluids by SDS-Page. Plant Archives. 2020;20(1):10721076.
8. Endris M, Taddesse F, Geloye M, Degefa T, Jibat T. Sero and media culture prevalence of Salmonellosis in local and exotic chicken, Debre Zeit, Ethiopia. African Journal of Microbiology Research. 2013;7(12):1041-1044.
9. Galperina L, Kyian Y. Conceptual Principles of Global Coordination of Economic Development Strategies. International Economic Policy. 2021;(35);7-27.
10. García P, Boncompte TJ, Agapé L. Limited emergence of Salmonella enterica serovar Infantis variants with reduced phage susceptibility in PhagoVet-treated broilers. Animals. 2024;14(16):2352.
11. Hawal GR, Bakr TM. Isolation and Molecular Identification of Salmonella pullorum from Broiler Chicken in Iraqi Fields. Archives of Razi Institute. 2023;78(2):587-592.
12. Kakasis A, Panitsa G. Bacteriophage therapy as an alternative treatment for human infections. A comprehensive review. International journal of antimicrobial agents. 2019;53(1):16-21.
13. Kwaśnicka KK, Podlacha M, Grabowski Ł, Stasiłojć M, Zaleska NA, Ciemińska K, et al. Biological aspects of phage therapy versus antibiotics against Salmonella enterica serovar Typhimurium infection of chickens. Frontiers in Cellular and Infection Microbiology. 2022;12:941867.
14. Lee S, Park N, Yun S, Hur E, Song J, Lee H, Ryu S. Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019. Gut pathogens. 2021;13(1):35.
15. Levy S. Reduced antibiotic use in livestock: How Denmark tackled resistance; 2014.
16. Li D, Gao Y, Cui L, Li Y, Ling H, Tan X, et al. Integrative analysis revealed the role of glucagon-like peptide-2 in improving experimental colitis in mice by inhibiting inflammatory pathways, regulating glucose metabolism, and modulating gut microbiota. Frontiers in Microbiology. 2023;14:1174308.
17. Colom LP, Castillejos L, Sorrento RA, Puyalto M, Mallo JJ, Orúe MSM. Impact of in-feed sodium butyrate or sodium heptanoate protected with medium-chain fatty acids on gut health in weaned piglets challenged with Escherichia coli F4+. Archives of Animal Nutrition. 2020;74(4):271-295.
18. Mohsen AK, Jassim SE, Alaridhi TT, Al-Erjan AM, Lahhob QR, Mudhafar M. Revolutionizing infectious disease management in animals through advanced phage therapy: A comprehensive strategy for the eradication of multidrug-resistant bacterial pathogens. J Anim Health Prod. 2024;12(S1):157-167.
19. Nabil A, Elshemy MM, Uto K, Soliman R, Hassan AA, Shiha G, et al. Coronavirus (SARS-CoV-2) in gastroenterology and its current epidemiological situation: An updated review until January 2021. EXCLI J. 2021;20:366.
20. Nabil NM, Tawakol MM, Hassan HM. Assessing the impact of bacteriophages in the treatment of Salmonella in broiler chickens. Infect Ecol Epidemiol. 2018;8(1):1539056.
21. Netsanet B, Berihun A, Nigus A, Abreha T, Shewit K. Seroprevalence of Salmonella pullorum infection in local and exotic commercial chicken from Mekelle areas, northern Ethiopia. Rev Electrón Vet. 2012;13:091204.
22. Rydman PS, Bamford DH. The lytic enzyme of bacteriophage PRD1 is associated with the viral membrane. J Bacteriol. 2002;184(1):104-110.
23. SAS. Statistical analysis system, user's guide. Statistical. Version 9.6 th Ed. SAS. Inst. Inc. Cary. N.C. USA; 2018.
24. Seo S, Son B, Kong M. Characterization of Clostridium perfringens phage endolysin PlyDolk21. Antibiotics (Basel). 2025;14(1):81.
25. Snary EL, Kelly LA, Davison HC, Teale CJ, Wooldridge M. Antimicrobial resistance: A microbial risk assessment perspective. J Antimicrob Chemother. 2004;53(6):906917.
26. Wernicki A, Nowaczek A, Chamier UA. Oral administration of a phage cocktail to reduce Salmonella colonization in broiler gastrointestinal tract: A pilot study. Animals (Basel). 2022;12(22):3087.
27. World Health Organization (WHO), UNEP United Nations Environment Programme, & World Organisation for Animal Health. Implementing the global action plan on antimicrobial resistance: First quadripartite biennial report. World Health Organization; 2023.
28. World Health Organization (WHO), World Organisation for Animal Health, & UN Environment. Antimicrobial Resistance Multi-Partner Trust Fund annual report 2021: Annual report 2023. World Health Organization; 2022.
29. Zeng Q, Shen J, Zhang Z, et al. Evaluation of lyophilized bacteriophage cocktail efficiency against multidrugresistant Salmonella in broiler chickens. BMC Microbiology. 2024;24(1):467.