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. 2026 Jun 13;105(10):107287. doi: 10.1016/j.psj.2026.107287

Effect of in ovo and oral bacteriophage administration on intestinal Salmonella colonization in Cobb broiler chicks

Shruti Kawthekar M 1, Ruchi Tiwari 1,⁎, Ajay Pratap Singh 1, Ambika Arun 1
PMCID: PMC13316619  PMID: 42341500

Abstract

Salmonellosis is a major zoonotic disease of poultry that causes substantial economic losses and poses a significant public health risk. Given the growing concern of antimicrobial resistance, this study evaluated the efficacy of lytic bacteriophages against Salmonella enterica serovar Typhimurium in Cobb broiler chicks following in ovo and oral administration. Bacteriophages isolated and maintained in the Department of Veterinary Microbiology, DUVASU, Mathura, were characterized by transmission electron microscopy, demonstrated a broad host range against multiple Salmonella serovars, and showed no lytic activity against non-target bacterial species.

The in vivo efficacy of the phages was assessed in 60 broiler chicks allocated to six groups (n=10): oral administration at multiplicity of infection (MOI) 1 and MOI 2, in ovo administration at MOI 1 and MOI 2, challenged control, and unchallenged control. Phage treatment significantly reduced fecal shedding of S. Typhimurium compared with the challenged control group throughout the experimental period. Oral administration at MOI 1 resulted in the greatest reduction in bacterial shedding, while in ovo administration at MOI 2 provided sustained suppression of bacterial colonization. Cecal bacterial counts were significantly lower in all phage-treated groups, with the most pronounced reductions observed at MOI 1. Histopathological examination further demonstrated reduced intestinal lesions and tissue damage in phage-treated birds compared with challenged controls.

These findings demonstrate that lytic bacteriophages effectively reduce intestinal colonization and fecal shedding of S. Typhimurium in broiler chickens and highlight their potential as a promising alternative to antibiotics for the control of poultry salmonellosis.

Keywords: Bacteriophage therapy, Salmonella Typhimurium, In ovo administration, Cecal colonization, Poultry health

Introduction

The poultry industry is one of the fastest-growing sectors of animal agriculture and plays a critical role in meeting the increasing global demand for affordable animal protein. In India, the poultry sector contributes substantially to food security, employment generation, and rural livelihoods (Mitra and Bose, 2005; Kler et al., 2022). However, the intensification of poultry production has been accompanied by an increased risk of infectious diseases and foodborne pathogens, which compromise bird health, productivity, and food safety.

Among bacterial pathogens, Salmonella enterica remains one of the most important zoonotic agents associated with poultry production worldwide. Poultry and poultry-derived products are recognized as major reservoirs of non-typhoidal Salmonella, contributing significantly to human salmonellosis through the consumption of contaminated meat and eggs or through direct contact with infected birds (Pal, 2007; Feasey et al., 2012; Dar et al., 2017). The zoonotic nature of Salmonella and its widespread occurrence in poultry production systems pose a substantial public health concern worldwide. Furthermore, invasive non-typhoidal Salmonella infections have emerged as an important cause of morbidity and mortality, particularly among vulnerable populations, highlighting the need for effective control strategies at the poultry production level (Gast et al., 2022). Among the numerous serovars, Salmonella enterica serovar Typhimurium is of particular concern because of its broad host range and its ability to cause gastroenteritis and invasive systemic infections in humans (Eng et al., 2015; Ferrari et al., 2019). In poultry, S. Typhimurium colonizes the intestinal tract, resulting in enteritis, reduced growth performance, and prolonged fecal shedding, thereby facilitating environmental dissemination and transmission within and between flocks (Cosby et al., 2015; Sohail et al., 2021).

The control of salmonellosis in poultry has traditionally relied on biosecurity measures and antimicrobial therapy. However, the extensive and often indiscriminate use of antibiotics in poultry production has contributed to the emergence and dissemination of multidrug-resistant (MDR) Salmonella strains, posing a serious threat to both animal and public health (Brands et al., 2005; Eng et al., 2015; Abreu et al., 2023). Increasing resistance to critically important antimicrobials has reduced therapeutic efficacy and intensified concerns regarding the transfer of resistant bacteria and resistance determinants through the food chain (Wajid et al., 2019; Hatfull et al., 2022). Consequently, there is an urgent need for effective and sustainable alternatives to conventional antibiotics for the control of Salmonella infections in poultry.

Several non-antibiotic interventions, including phytogenic feed additives, probiotics, prebiotics, organic acids, and antimicrobial peptides, have been investigated with varying degrees of success (Hashemi and Davoodi, 2011; Pourabedin and Zhao, 2015; Nazeer et al., 2021). Among these alternatives, bacteriophages have attracted considerable attention because of their high host specificity, self-replicating nature, and minimal disruption of the normal gut microbiota. Lytic bacteriophages specifically infect and lyse bacterial cells and have demonstrated promising efficacy in reducing intestinal colonization and fecal shedding of Salmonella in poultry under both experimental and field conditions (Atterbury et al., 2003; Kollanoor-Johny et al., 2012; Lin et al., 2017; Nicolas et al., 2023; Olawade et al., 2024).

Recent advances in poultry technology have also highlighted the potential of in ovo delivery systems for administering vaccines, probiotics, and other biologically active agents during embryonic development. In ovo administration enables early establishment of protective interventions before hatch and may enhance post-hatch health and disease resistance (Uni et al., 2005; Saeed et al., 2019). Although in ovo approaches have been successfully applied for vaccines and antimicrobial compounds (Cuperus et al., 2016; Duan et al., 2021; Oliveira et al., 2024), studies evaluating in ovo-delivered bacteriophages against S. Typhimurium in poultry remain relatively scarce. Recent work demonstrated that in ovo phage administration via the amniotic fluid significantly reduced S. Typhimurium colonization and prevented bird-to-bird transmission, highlighting its potential as a hatchery-level intervention strategy (Torres-Boncompte et al., 2026).

Therefore, the present study was undertaken to evaluate the therapeutic potential of lytic bacteriophages specific for S. Typhimurium administered through oral and in ovo routes in Cobb broiler chicks. The study assessed the ability of bacteriophage treatment to reduce intestinal colonization, fecal shedding, and pathological lesions associated with experimental S. Typhimurium infection.

Rationale: Despite increasing evidence supporting bacteriophage therapy as a viable alternative to antibiotics, comparative information on the effectiveness of oral and in ovo administration of anti-Salmonella bacteriophages in broiler chickens is limited. Understanding the relative efficacy of these delivery routes is essential for developing practical and sustainable phage-based interventions for controlling salmonellosis in poultry production systems.

Hypothesis: It was hypothesized that administration of lytic bacteriophages, either orally or in ovo, would significantly reduce intestinal colonization, fecal shedding, and pathological lesions caused by S. Typhimurium in broiler chicks compared with untreated challenged birds, with early in ovo delivery providing sustained protection against infection.

Materials and methods

The study was conducted in the Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Uttar Pradesh Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU), Mathura, India, after approval from the Institutional Animal Ethics Committee (IAEC/25/58). A reference strain of Salmonella enterica serovar Typhimurium and other departmental isolates were used.

Sewage and wastewater samples from different regions of Mathura were processed for isolation of lytic bacteriophages using log-phase host enrichment, followed by centrifugation and membrane filtration. Phages were detected, purified, and enumerated by spot assay and double agar overlay methods. Selected isolates were characterized for host range, pH and temperature stability, and examined by transmission electron microscopy. High titer lysates were prepared and combined as a six phage cocktail for in vivo studies.

Seventeen-day-old (ECE) embryonated chicken eggs (n = 80) were randomly divided into six groups (10 eggs/group). On day 18 of ECE incubation, 0.1 ml of phage cocktail (MOI 1 and 2) was administered via the intra-allantoic route under aseptic conditions in Group E and F. Upon hatching, chicks were reared under standard management. Experimental infection was carried out using phage administration in Group A and B on day one old chicks, while they were challenged with log-phase S. Typhimurium (10⁵ CFU/ml) via oral routeon second day post hatch (in two day old chicks) in Group A and B.Experimental groups were comprised of Group A Oral administration of phage cocktail (MOI 1), Group B oral administration of phage cocktail (MOI 2), Group C Control (NSS as placebo), Group D Challenged (only bacteria, no phage treatment), Group E in ovo administration of phage cocktail (MOI 1), Group F in ovo administration of phage cocktail (MOI 2).

Cloacal swabs were collected from all the groups periodically to assess phage recovery and bacterial shedding. Samples were processed and plated for enumeration, with counts expressed as log₁₀ CFU/ml. Salmonella confirmation was performed by molecular technique, PCR targeting the invA gene. At seven days post-hatch, birds were sacrificed, and liver, intestine, and cecum samples were collected for bacterial counts and histopathological studies. Tissues were stained with hematoxylin and eosin for microscopic examination.

Results and discussion

Lytic activity and phage Titration

Transmission electron microscopy revealed phages with icosahedral heads and tail structures. Based on morphology, five phages (SR1, SR2, SR3, SR7, SR8) were classified under the Myoviridae family (Fig. 1), characterized by contractile tails, while SR5 belonged to the Siphoviridae family with a long non-contractile tail, were used in cocktail preparation. The predominance of Myoviridae phages, which are often associated with strong lytic activity and broader host range, suggests their suitability for therapeutic applications, consistent with previous reports on Salmonella bacteriophages (Lopez-Cuevas et al., 2011).

Fig. 1.

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Transmission electron Microscopic Image of SR1.

The turbidity reduction assay demonstrated strong antibacterial activity of all phages, with maximum clearing observed at higher phage concentrations (100 µL lysate), indicating dose-dependent bacterial lysis. Serial dilution assays confirmed high lytic potency, with phages remaining active up to 10⁻¹¹ dilution and maintaining stability across three passages. Final phage titers ranged from 1.2 × 10¹¹ to 9.8 × 10¹¹ PFU/ml, which are considered adequate for therapeutic applications and phage cocktail preparation (Abedon, 2016). Comparable titers have been reported in previous Salmonella phage studies in poultry (Atterbury et al., 2007).

Multiplicity of infection (MOI)

MOI is a critical parameter influencing bacteriophage efficacy, as it determines the initial phage–bacteria interaction dynamics (Abedon, 2016). The selection of MOI-1 and MOI-2 levels for in vivo evaluation was consistent with previous studies demonstrating effective bacterial control within this range (Atterbury et al., 2007; Wernicki et al., 2017).The multiplicity of infection values varied among the phages, with SR1 showing the highest MOI and SR3 the lowest. High-titer lysates (∼10¹¹ PFU/ml) ensured adequate phage availability for cocktail formulation and in vivo administration.

Experimentation on birds

Phage persistence and recovery

Viable bacteriophages were consistently recovered from cloacal swabs and cecal contents of birds receiving oral and in ovo phage administration throughout the experimental period, indicating successful survival and persistence of the phages within the gastrointestinal tract. Recovery of phages from post-mortem cecal contents further confirmed their ability to remain biologically active in vivo. This persistence is likely attributable to the presence of susceptible bacterial hosts, which support phage replication and maintenance within the intestinal environment. Similar observations have been reported by Huff et al. (2006) and Atterbury et al. (2007), who demonstrated prolonged persistence of bacteriophages in the poultry gut following administration.

Reduction of Salmonella Shedding

Quantitative analysis of cloacal swab samples demonstrated a clear reduction in Salmonella Typhimurium shedding in all phage-treated groups compared with the challenged control group (Group D). The challenged birds consistently exhibited the highest bacterial load during the observation period, whereas phage-treated birds showed progressive declines in bacterial shedding following challenge.

Among the treatment groups, oral administration of the phage cocktail at MOI 1 (Group A) resulted in a reduction in Salmonella counts from 5.70 log₁₀ CFU/g on day 5 to 3.48 log₁₀ CFU/g on day 7 post-challenge, corresponding to an approximate 39% decrease. Similarly, birds receiving in ovo phage administration at MOI 1 (Group E) exhibited a reduction from 4.48 to 3.48 log₁₀ CFU/g, representing a 22% decrease. Phage administration at MOI 2 also reduced bacterial shedding, with reductions of approximately 40% and 23% observed in Groups B and F, respectively. These findings indicate that both oral and in ovo administration of lytic bacteriophages were effective in lowering intestinal Salmonella burden and subsequent fecal shedding (Table 1).

Table 1.

The quantitative bacterial load of Salmonella enterica serovar Typhimurium in cloacal swab samples expressed as log₁₀ CFU/g on days 3, 5, and 7 post-challenge for Groups A to F.

Groups log₁₀ CFU/g (day 3) log₁₀ CFU/g (day 5) log₁₀ CFU/g (day 7)
A 5.40 5.70 3.48
B 6.60 6.70 4.00
C ND ND ND
D 6.90 7.49 4.28
E 5.78 4.48 3.48
F 4.60 4.78 3.70
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The reduction in bacterial shedding observed in phage-treated birds is likely attributable to active phage replication within the gastrointestinal tract, resulting in targeted lysis of Salmonella cells and interruption of bacterial colonization. Reduced fecal shedding is particularly important from an epidemiological perspective because it decreases environmental contamination and may limit horizontal transmission within poultry flocks. Similar reductions in Salmonella shedding following bacteriophage treatment have been reported in broiler chickens by Atterbury et al. (2007) and Wernicki et al. (2017), supporting the potential of phage therapy as a biological control strategy against salmonellosis. The ability of bacteriophages to selectively target bacterial pathogens while sparing beneficial microbiota further supports their potential as an alternative to conventional antibiotics in poultry production systems (Lin et al., 2017).

Cecal salmonella colonization

Evaluation of cecal bacterial counts revealed marked differences between phage-treated and challenged birds. The challenged control group (Group D) exhibited the highest cecal Salmonella load (9.11 log₁₀ CFU/g), indicating extensive intestinal colonization. In contrast, all phage-treated groups demonstrated lower bacterial counts, suggesting that phage administration effectively limited bacterial persistence within the gastrointestinal tract (Table 2).

Table 2.

The quantitative bacterial load of Salmonella enterica serovar Typhimurium in Cecum.

Group Treatment log₁₀ CFU/g
A Oral phage (MOI 1) + Salmonella challenged 5.70
B Oral phage (MOI 2) + Salmonella challenged 6.48
C Control (NSS placebo, no bacteria no phage) Not detected
D Salmonella challenged only (No phage) 9.11
E In ovo phage (MOI 1) + Salmonella challenged 5.60
F In ovo phage (MOI 2) + Salmonella challenged 6.70
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The greatest reduction in cecal colonization was observed in Group E (in ovo phage administration, MOI 1) and Group A (oral phage administration, MOI 1), which showed reductions of approximately 38.5% and 37.4%, respectively, compared with the challenged control group. Groups B and F (MOI 2) also exhibited reductions of approximately 28.9% and 26.5%, respectively. No Salmonella was detected in the unchallenged control group. These findings indicate that phage administration at MOI 1 provided greater suppression of intestinal colonization than MOI 2 under the conditions of the present study.

The enhanced efficacy observed at MOI 1 may reflect a more favorable balance between phage replication and bacterial availability within the intestinal environment. Effective reduction of cecal colonization is particularly important because the cecum serves as a major reservoir for Salmonella persistence and fecal dissemination in poultry. Therefore, lowering cecal bacterial loads may contribute to reduced contamination of poultry products and the farm environment. Similar reductions in intestinal Salmonella colonization following bacteriophage administration have been reported by Atterbury et al. (2007) and Wernicki et al. (2017). Furthermore, recent studies have highlighted the potential of early-life phage administration, including in ovo delivery, to establish protection before hatch and reduce subsequent bacterial colonization and transmission (Nicolas et al., 2023; Torres-Boncompte et al., 2026).

Molecular confirmation

PCR amplification targeting the invA gene produced the expected 284 bp amplicon, confirming the identity of the bacterial isolate as Salmonella Typhimurium. Molecular confirmation validated the presence of S. Typhimurium and ensured experimental accuracy (Fig. 2a, 2b).

Fig. 2a.

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Picture showing agarose gel electrophoresis of PCR amplification of the invA gene, 100bp ladder (M), S1 to S9 presumptive Salmonella colonies on different Days isolated from XLD agar. All representative isolates showed the expected amplicon size of 284bp.

Fig. 2b.

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Picture showing agarose gel electrophoresis of PCR amplification of the invA gene, 100bp ladder (M). P represents positive sample, S10 to S12 shows Salmonella Typhimurium colonies on different days obtained on XLD agar. All representative isolates showed the expected amplicon size of 284bp.

Gross pathological findings

Post-mortem examination revealed substantial differences in lesion severity among experimental groups. Birds in the challenged control group exhibited severe pathological alterations, including hepatomegaly, hepatic congestion, intestinal wall thickening, and catarrhal to hemorrhagic enteritis. In contrast, phage-treated groups displayed markedly reduced lesion severity, with only mild intestinal congestion and focal hepatic changes observed. Notably, Group C did not show any gross pathological lesions, and all examined organs appeared normal (Fig. 3) whereas severe gross lesions were observed in the Group D (Salmonella-challenged group) indicating systemic infection and intestinal inflammation typical of salmonellosis

Fig. 3.

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Showing Gross lesions in Liver from different Groups.

The reduction in gross pathological lesions is likely associated with decreased bacterial colonization and reduced dissemination of Salmonella within host tissues. By lowering pathogen burden, bacteriophage treatment may attenuate inflammatory responses and tissue damage. Similar reductions in lesion severity following phage administration have been reported by Atterbury et al. (2003), supporting the protective effect of bacteriophage therapy against salmonellosis in poultry.

Histopathological findings

Histopathological examination further supported the protective effects of bacteriophage treatment. Birds receiving phage therapy exhibited substantially milder microscopic lesions than challenged controls. Microscopic examination of intestinal and hepatic tissue sections revealed marked differences in histopathological alterations among the experimental groups. Groups A and B showed only mild epithelial disruption and moderate inflammatory cell infiltration (Fig. 4), while Groups E and F maintained near-normal intestinal architecture with minimal inflammatory changes (Fig. 6 A,B). In contrast, tissues from Group C appeared histologically normal, with intact villous architecture and absence of any detectable lesions, confirming the healthy status of the control birds. In sharp contrast, Group D (Salmonella challenged) showed severe pathological lesions (Fig. 5)., including pronounced villous degeneration, epithelial desquamation, dense inflammatory cell infiltration in the lamina propria, and marked disruption of normal mucosal architecture (Fig. 7 A,B).

Fig. 4.

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Histopathological examination of Liver (Group A) Preserved hepatic architecture with well-arranged hepatocyte. Hepatocytes exhibit uniform cytoplasm and nuclei (200X, H&E).

Fig. 6.

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A. Gross lesion of Cecum (Group A) normal size and appearance B. Histopathological examination of Cecum (Group A) the cecal section showed well-preserved mucosal architecture Goblet cells are numerous and clearly visible the lamina propria appears compact. (1000X, H&E).

Fig. 5.

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Histopathological examination of Liver (Group D) Hepatocytes exhibit cytoplasmic vacuolation and cellular swelling, with loss of normal cord arrangement. There is severe vascular congestion. (200X, H&E).

Fig. 7.

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A. Gross lesion of Cecum (Group D) Cecal enlargement with excess fluid; B. Histopathological examination of Cecum (Group D) The cecum shows marked distortion of normal mucosal architecture, with irregular, thickened mucosal folds and separation of muscularis mucosa and lamina propria (1000X, H&E).

The preservation of intestinal and hepatic tissue architecture in phage-treated birds likely reflects effective bacterial clearance and reduced inflammatory stimulation. Since intestinal colonization is a key pathogenic mechanism of Salmonella infection, reductions in bacterial load directly contribute to maintenance of mucosal integrity. Similar histopathological improvements following bacteriophage therapy have been documented in previous experimental studies involving Salmonella-infected poultry.

Conclusion

The present study demonstrated that lytic bacteriophages administered through oral and in ovo routes effectively reduced Salmonella Typhimurium colonization, fecal shedding, and pathological lesions in Cobb broiler chicks. Among the evaluated treatments, MOI 1 produced the greatest reduction in bacterial load, while in ovo administration provided sustained protection against infection from an early stage of development. Recovery of viable phages throughout the experimental period confirmed their persistence and activity within the gastrointestinal tract.

These findings support the potential of bacteriophage therapy as a sustainable alternative to antibiotics for controlling salmonellosis in poultry. However, the study was conducted under controlled experimental conditions with a limited sample size and a single Salmonella serovar. Further large-scale field studies are required to evaluate long-term efficacy and assess performance under commercial production conditions.

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the preparation of this work the author(s) used [ChatGPT] in order to [Edit language]. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.

CRediT authorship contribution statement

Shruti Kawthekar M: Writing – review & editing. Ruchi Tiwari: Writing – review & editing, Visualization, Validation, Supervision, Methodology. Ajay Pratap Singh: Project administration, Formal analysis, Conceptualization. Ambika Arun: Formal analysis.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Contributor Information

Shruti Kawthekar M, Email: smkhere2255@gmail.com.

Ruchi Tiwari, Email: ruchi.tiwari@upvetuniv.edu.in.

Ajay Pratap Singh, Email: ajaypratap.singh@upvetuniv.edu.in.

Ambika Arun, Email: ambika.arun@upvetuniv.edu.in.

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