Salmonella Enteritidis (S. Enteritidis) CICC10467 [The strain CICC10467 is a common S. Enteritidis strain. Wang et al. (27) have proved through research that the amino acid sequence of FliC protein of strain CICC10467 and S. Gallinarum is identical, which will cause obvious Salmonella symptoms in chicks and increase the number of Salmonella colonization in chicks' intestines] was obtained from the China Industrial Microorganism Culture Collection Center, and phage SP4, isolated from sewage, was stored in the laboratory of Hebei Engineering University. Wastewater samples were collected from the wastewater treatment station of Hebei University of Engineering for phage separation. Approximately 100 mL of the sample was stored in sterile bottles and stored in a refrigerator (4°C) during transport to the laboratory for analysis. Centrifuge the wastewater at 6,000 rpm at 4°C for 15 min, and incubate it with the host bacteria at 37°C. A single plaque was chosen for purification for three passages with a specific host that showed a positive result, using a double-layer agar technique. Isolated plaque from the third purification passage was used to prepare 10-fold serial dilutions in SM buffer. Appropriate dilutions were used to prepare the overlay with the given host to yield the semi-confluent lysis and then harvest with 5 mL of SM buffer followed by centrifugation at 6,000 rpm for 15 min at 4°C. The supernatant was filtrated through 0.22-μm syringe filters, and phage lysates were kept at 4°C (28).

120 one-day-old Jingfen No. 1 laying hens (male and female 1:1) were purchased from Handan Xinguan Poultry Industry Co., Ltd (Handan, China). All animals were raised in an animal house with ad libitum feed and water. The study protocols (BER-YXY-2022027) were approved by the Institutional Animal Care and Use Committee and the Animal Research Ethics Committee of Hebei University of Engineering, China.

S. Enteritidis CICC10467 was cultured in Luria-Bertani (LB) liquid medium at 37°C for 12 h (log-growth phase). For phage propagation, 20 μL of phage suspension was mixed with 500 μL of the Salmonella strain suspension in the logarithmic growth phase and 5 mL of LB medium containing 7 g/L agar; the mixture was incubated at 37°C overnight. The phage solution was filter sterilized using a 0.22 μm membrane filter.

Microencapsulation was performed as described by Ma et al. (29) with appropriate modifications for phages. Briefly, 1 g/100 mL XG solution was prepared by dissolving XG granules in deionized water. 2 g/100 mL SA solution was prepared in 50 mmol/L Tris-HCl buffer (pH7.5). The XG solution, SA solution, and propagated phage solution were mixed well in a beaker with stirring and vacuum degassing to remove air bubbles at 4°C. Next, the above-mentioned mixed solution was added dropwise using a sterile syringe to 2 g/100 mL CaCl₂ solution (in deionized water) at 10 mL/min, and the reaction was allowed for 30 min at room temperature (RT). The phage microspheres were collected by filtration, washed with deionized water, and then moved to the 0.6 g/100 mL COS solution (in deionized water) for 30 min. After the COS treatment, the phage microspheres were collected by filtration, cleaned with deionized water, and some were stored in a closed container at 4°C. The other part of the microspheres was air dried in an oven at 30°C for 24 h and then stored in a closed container at 4°C. Phage titer was calculated as follows: Phage titer (PFU/mL) = plaque number × dilution factor × 100 (30). The phage encapsulation efficiency was calculated as follows: Encapsulation efficiency (%) = (amount of phage released from the dissolved microspheres/number of phages initially used to prepare the microspheres) × 100 (31). The results are expressed as the mean encapsulation efficiency ± standard deviation (SD) and each experiment had two replicates.

Microspheres were examined for size and surface morphologies by scanning electron microscopy (SEM) at 5.0 kV (Hitachi S-4500; Hitachi Co. Ltd., Tokyo, Japan). The dried microspheres were mounted on metal grids using double-sided tape and coated with Au/Pd under vacuum. The diameter of phage microspheres was measured using an eyepiece micrometer and the calculation formula d¯ = ∑d_i/n (In the formula, d¯ is the average particle size, ∑d_i is the sum of each particle size, and n is the number).

Next, we performed an orthogonal test to explore the optimal concentration ratio of coating materials for the preparation of microencapsulated phages; the encapsulation efficiency was used as an index to evaluate the effects of XG, SA, CaCl₂ and COS amounts on the encapsulation process. Orthogonal test factors were as follows: Factor A, XG concentration 0.5, 1, and 1.5 g/100 mL; Factor B, SA concentration 1, 2, and 3 g/100 mL; Factor C, CaCl₂ concentration 1, 2 and 3 g/100 mL; Factor D, COS concentration 0.4, 0.6 and 0.8 g/100 mL (Table 1). All factor levels were selected based on previous literature (29). The experiment were repeated twice and the results are expressed as mean survival rate ± SD.

To determine the temperature stability, 1 mL of free phages (1 × 10⁸ PFU/mL) were placed in different microcentrifuge tubes, which were, respectively, incubated at 10, 20, 30, 40, 50, 60, and 70°C in a water bath. After 1 h of incubation, the samples were diluted 10-fold and used for phage titers determination using double-layer agar (29). For microencapsulated phages, 1 g of microencapsulated phages (1 × 10⁸ PFU/g) were placed in different microcentrifuge tubes, which were, respectively, incubated at 10, 20, 30, 40, 50, 60, and 70°C in a water bath. After 1 h of incubation, microsphere lysis solution (The microsphere lysis solution was composed of 14.7 g C₆H₅O₇Na₃·2H₂O and 16.8 g NaHCO₃ dissolved in 1 L SM buffer. Hebei University of Engineering, China) was added to each tube to crack open the microsphere, and the free particles were filtered through a 0.22 μm membrane filter. These free phage particles were then subjected to titer determination (29). The experiment were repeated twice and the results are expressed as mean survival rate ± SD.

The gastric stability of free and microencapsulated phages was assessed using simulated gastric fluid (SGF) (32), which consisted of 3.2 mg/mL pepsin (Sigma-Aldrich, Oakville, Ontario, Canada) in 0.2% (wt/vol) NaCl, pH 2.0 and 3.0. Briefly, 1 mL of free phages (1 × 10⁸ PFU/mL) were added to a tube containing 10 mL of pre-warmed (37°C) SGF, and the tube was then incubated at 37°C for 10, 20, 30, 40, 50, and 60 min. 100 μL of the sample was collected at the respective time point and diluted 10 times for phage titer determination as described above. Because of prolonged survival time of microencapsulated phages in SGF, 1 g of phage microspheres (1 × 10⁸ PFU/g) were added to a tube containing 10 mL of pre-warmed (37°C) SGF and incubated at 37°C for 30, 60, 90, 120, 150, and 180 min. At the respective time, microencapsulated phages were lyzed and subject to titer determination (29). SM buffer mainly containing magnesium sulfate, sodium chloride and gelatin was used as a control, which is sterilized and used for diluting and preserving phage stock. The experiments were repeated twice and the results are expressed as mean survival rate ± SD.

SIF was prepared as described in the US Pharmacopeia and consisted of 10 mg/mL pancreatin (Sigma-Aldrich, Oakville, Ontario, Canada) in 50 mM KH₂PO₄, pH 7.5 (33). Briefly, 1 mL of free phages (1 × 10⁸ PFU/mL) and 1 g of microencapsulated phages (1 × 10⁸ PFU/g) were added to a tube containing 10 mL of preheated (37°C) SIF, respectively, which were incubated at 37°C and 100 rpm. To investigate the release behavior of microencapsulated phages in SIF, 100 μL samples were collected at 0, 1, 2, 3, 4, 5, 6, 7, and 8 h, respectively. Like earlier, phage titer determination was conducted by the double-layer plate method (29). The experiments were repeated twice and the results are expressed as mean survival rate ± SD.

To determine the storage stability, both kinds of phages were stored at 4 and 25°C, respectively. A small amount of free and microencapsulated phages was retrieved every 7 days and subjected to titer determination as described above (29). The experiments were repeated twice and the results are expressed as mean survival rate ± SD.

The animals were randomly divided into three experimental groups (Groups B, C, and D) and one control group (Group A). This was repeated twice for each group of 10 chicks. All experimental groups were infected with S. Enteritidis CICC10467 (3 × 10¹⁰ CFU/mL, 0.5 mL/chicks) by oral gavaging. On the contrary, Group A (control) was fed with normal saline (0.5 mL/chicks). Group B was without any phage treatment. Group C and D were, respectively, treated with free (3 × 10¹⁰ PFU/mL, 0.5 mL/chicks) and microencapsulated phages (3 × 10¹⁰ PFU/g, 0.5 g/chicks) immediately after S. Enteritidis infection. Being in solid form, 0.5 g of microencapsulated phage was first weighed and then administered to chicks. All animals were raised in an animal house with ad libitum feed and water. After 7 days of the experiment, all animals were euthanized with carbon dioxide (Animal euthanasia equipment, SHANGHAI BIOWILL CO., Ltd.). The duodenum, jejunum, ileum, and colorectal tissues were collected and washed to remove intestinal contents. Each segment of the intestinal tube was weighed, crushed, and then continuously dilute the intestinal tube grinding solution and conduct Salmonella culture on XLD medium (XLD medium is a selective medium, which can be used to isolate Salmonella). It is well known that Salmonella presents black colony and Shigella presents colorless colony on XLD medium. In order to avoid false positive colonies on XLD medium. Therefore, black colonies grown on XLD medium are selected, the API 20E Enterobacteriaceae G-bacilli identification kit (for the identification of Enterobacteriaceae and other non fastidious gram-negative bacilli, Shanghai Xinzhong Biotechnology Co., Ltd., China) was used to conduct biochemical identification of bacteria according to the method of the user manual, and further verified to be Salmonella. The therapeutic effect of free and microencapsulated phages on infected chicks was estimated as the number of Salmonella colonization, which was calculated based on the number of Salmonella colonies on the XLD medium and the weight of the intestinal tube.

The results were expressed as the means ± SD. All analyses were conducted using the GraphPad Prism 8 and SPSS Statistics 26 software. One-way ANOVA was employed to identify significant differences among multiple groups. ^nsp > 0.05, ^*p < 0.05, ^**p < 0.01, ^#p < 0.05, and ^##p < 0.01. A difference was considered statistically significant at p < 0.05.

The microencapsulated phage microspheres were analyzed by a digital camera (SONY, ILCE-7RM3, 24.2 million pixels, China). The microspheres with circular outer surfaces had a uniform average size of 950 μm (range 930–970 μm) (Figure 1A). Because the microspheres need to be dried during SEM scanning, after drying, the microspheres will lose water and shrink, so the average size of the microspheres will become smaller. Microscopic morphological analysis of the microspheres was performed by scanning electron microscopy. The microspheres were generally spherical with a wrinkled surface and collapsed center; the average microsphere size was reduced to 350 μm (range 330–370 μm) (Figure 1B).

The orthogonal analysis found that 1 g/100 mL XG, 2 g/100 mL SA, 2 g/100 mL CaCl₂, and 0.6 g/100 mL COS produced the best encapsulation efficiency of 85.62 ± 2.14%. The least microencapsulation efficiency was 24.12 ± 2.36% (Table 1).

Activity comparison revealed that microencapsulation improved the temperature stability of phages. At 10, 20, and 30°C, the activities of free and microencapsulated phages showed no difference, but a further increase in temperature (40–70°C) decreased their activities. However, compared with free phages, the activity of microencapsulated phages remained higher; at 70°C, the activity of free phages was 2 log₁₀PFU/mL (Figure 2A), while that of microencapsulated phages was 6 log₁₀PFU/g (p < 0.05) (Figure 2B). This data indicated that microencapsulated phages were more temperature resistant than the free phages.

To determine the effect of microencapsulation on the gastric stability of phages, the microencapsulated phages were tested in SGF. At pH 2.0 and 3.0, the activity of free phages declined rapidly reaching inactivation at 20 and 30 min, respectively (Figure 2C). This indicated less gastric (acidic environment) stability of free phages. Interestingly, the activity of microencapsulated phages also decreased gradually but took longer to reach inactivation than the free phages (60 and 180 min at pH 2.0 and 3.0, respectively) (Figure 2D). Overall, these data indicated that microencapsulated phages were more stable in an acidic environment than the free phages. Also, there was no significant change in the activity of free and microencapsulated phages in the SM buffer.

Microencapsulated phage microspheres should not only improve gastric stability but also allow viable phage release at the desired site (e.g., the small intestine). Accordingly, we tested phage release in SIF by determining phage activity. The results showed that the activity of free phages (indicating the release rate) did not change significantly in SIF and was relatively stable over time (Figure 3A). When the microencapsulated phages were put into SIF, the microspheres began to swell and disintegrate, and the release rate stabilized after the burst effect. The phage titer gradually increased with time and after 4 h became stable (Figure 3B). This indicating that all the microencapsulated phages were released in SIF.

Good storage temperature stability is necessary for long-term storage and application of microencapsulated phage microspheres. In this study, phages were examined for storage stability at refrigeration (4°C) and room incubation (25°C) temperatures. The results showed that the titer of free phage decreased at both 4 and 25°C, by 2.88 and 5.62 log₁₀PFU/mL, respectively (p < 0.05) (Figure 3C); the decrease was more significant at 25°C. On the contrary, the activity of microencapsulated phages did not significantly change at 4°C (p > 0.05) but decreased by 1.02 log₁₀PFU/g at 25 °C (p < 0.05) (Figure 3D). This data indicated better storage stability of microencapsulated phages over free phages; 4°C seems to be the good storage temperature.

Chicks infected with Salmonella showed obvious symptoms of salmonellosis. Necropsy showed fibrous exudation on the surface of the heart (Blue arrow), enlarged liver, which was earthy-yellow, and grayish white necrotic lesion on it (Yellow arrow) (Figure 4A). There was significant bleeding in the intestine (Black arrow) (Figure 4B).

The microencapsulated phages showed improved temperature stability, gastric stability, good SIF release, and storage stability. Next, we tested their in vivo therapeutic effect against Salmonella in chicks. As described in the method section, the animals were divided into four groups: Group A (control: not infected), Group B (infected), Group C (infected and treated with free phages), and Group D (infected and treated with microencapsulated phages). Since S. Enteritidis mainly invades the intestine, the chick intestines (duodenum, jejunum, ileum, and colorectal tissues) from the respective groups were examined for the therapeutic effect of phages. Compared with the Group A, the number of Salmonella colonization from the duodenum, jejunum, ileum, and colorectal samples increased by 1.29, 1.13, 1.56, and 1.75 log₁₀CFU/g in Group B (p < 0.05). Compared with the Group B decreased by 0.12, 0.13, 0.25, and 0.52 log₁₀CFU/g after treatment with free phages in Group C (p < 0.05). Compared with the Group B decreased by 0.55, 0.63, 0.66 and 1.24 log₁₀CFU/g after treatment with microencapsulated phages in Group D (p < 0.05). Compared with the Group C decreased by 0.43, 0.50, 0.41 and 0.72 log₁₀CFU/g after treatment with microencapsulated phages in Group D (p < 0.05) (Figure 5). The therapeutic effect of Group D is better than that of Group C. These results indicated a better in vivo therapeutic effect of microencapsulated phages against the Salmonella infection.